Grapevine carbohydrate and nitrogen allocation during berry maturation:
Implications of source-sink relations and water supply
Gerhard C. Rossouw
BSc (Agric), University of Stellenbosch
MSc (Agric), University of Stellenbosch
Submitted in total fulfilment of the requirements of the degree of
Doctor of Philosophy
March 2017
National Wine and Grape Industry Centre
School of Agricultural and Wine Sciences - Charles Sturt University
Faculty of Science
Wagga Wagga, NSW, Australia
Table of contents
Table of contents
Certificate of authorship ........................................................................................................ i
Editorial note ........................................................................................................................ii
Acknowledgements.............................................................................................................. iii
Statement of contribution to publications ...............................................................................iv
Abstract ..............................................................................................................................vi
Chapter 1: General introduction and research aims ................................................................. 1
1.1. Introduction and aims ............................................................................................ 1
1.2. References ............................................................................................................ 7
Chapter 2: Literature review ................................................................................................. 9
2.1. Introduction .......................................................................................................... 9
2.2. Grapevine non-structural carbohydrates................................................................... 10
2.2.1. Roles of carbohydrate reserves in grapevines ................................................... 12
2.2.2. Mobilisation of carbohydrate reserves ........................................................... 13
2.2.3. Soluble sugar specific roles .......................................................................... 15
2.2.4. Minor sugars and sugar alcohols ................................................................... 19
2.2.5. Factors influencing carbohydrate reserve distribution in grapevines .................. 23
2.3. Grapevine nitrogen............................................................................................... 30
2.3.1. Roles of N in grapevines ............................................................................... 32
2.3.2. Fruit N accumulation .................................................................................... 33
2.3.3. Factors influencing N distribution .................................................................. 34
2.3.4. Amino acids ................................................................................................. 37
Table of contents
2.4. Concluding remarks .............................................................................................40
2.5. Literature cited ....................................................................................................41
Chapter 3: Paper 1 ............................................................................................................... 52
3.1. Main objective for paper 1 .....................................................................................52
Carbohydrate distribution during berry ripening of potted grapevines: Impact of water
availability and leaf-to-fruit ratio ......................................................................................53
Chapter 4: Paper 2 ............................................................................................................... 64
4.1. Main objective for paper 2 .....................................................................................64
Implications of the presence of maturing fruit on carbohydrate and nitrogen distribution in
grapevines under postveraison water constraints ..................................................................65
Chapter 5: Paper 3 ............................................................................................................... 79
5.1. Main objective for paper 3 .....................................................................................79
5.2. Supporting information ........................................................................................79
Vitis vinifera root and leaf metabolic composition during fruit maturation: Implications of
defoliation ......................................................................................................................80
Chapter 6: Paper 4 ............................................................................................................. 122
6.1. Main objective for paper 4 ................................................................................... 122
6.2. Supplementary material..................................................................................... 122
Impact of post-véraison leaf source limitation on the metabolic profile of Vitis vinifera cv.
Shiraz berries................................................................................................................ .123
Chapter 7: General conclusions and future work .................................................................... 158
Appendix ..........................................................................................................................166
Certificate of authorship
i
Certificate of authorship
I, Gerhard Rossouw, hereby declare that this submission is my own work and that, to
the best of my knowledge and belief, it contains no material previously published or
written by another person nor material which to a substantial extent has been accepted
for the award of any other degree or diploma at Charles Sturt University or any other
educational institution, except where due acknowledgment is made in the thesis.Any
contribution made to the research by colleagues with whom I have worked at Charles
Sturt University or elsewhere during my candidature is fully acknowledged.
I agree that this thesis be accessible for the purpose of study and research in accordance
with the normal conditions established by the Executive Director, Library Services,
Charles Sturt University or nominee, for the care, loan and reproduction of thesis,
subject to confidentiality provisions as approved by the University.
Gerhard Rossouw
10 March 2017
Editorial note
ii
Editorial note
Thesis structure
This thesis contains two accepted and two submitted peer review publications. The
English style in the thesis is mainly Australian English in accordance with Charles Sturt
University`s academic manual available at http://www.csu.edu.au/acad_sec/academic-
manual/hcontm.htm (section 4: Regulations for presentations of print theses, other
examinable print works and the written component of examinable multi-media work).
However, American English is used in three of the publications (chapters 3, 4 and 6), in
accordance with the preference of the respective journals.
Chapters 1 and 2 have been referenced based on the American Psychological
Association (APA 6th edition), in line with Charles Sturt University`s referencing
style. The four publications (chapters 3, 4, 5 and 6) are referenced in accordance to
the respective journal’s preferred referencing style.
The APA 6th edition is available at:
http://www.csu.edu.au/division/library/ereserve/pdf/apa-6ed.pdf.
Acknowledgements
iii
Acknowledgements
I would like to acknowledge my principle supervisor, Dr Bruno Holzapfel, for
mentoring me and offering me guidance during my PhD studies. I also thank my
co-supervisors, Dr Jason Smith, Dr Celia Barril and Prof Alain Deloire for their
valuable support and contributions during this research.
I thank Robert Lamont, David Foster and Peter Carey for providing me with technical
assistance during the three pot experiments. I also thank Beverley Orchard for her
contributions toward the statistical planning of my experiments and for conducting
statistical analyses. I thank Katja Suklje for her contributions to the GC/MS analysis.
I would also like to acknowledge Wine Australia for financially supporting my PhD
project through stipend and operating fund contributions. Likewise, I would like to
thank the National Wine and Grape Industry Centre, Charles Sturt University, for
providing me with a postgraduate scholarship.
Finally, I would like to thank my family and friends for continuously supporting me
throughout my studies.
Statement of contribution to publications
iv
Statement of contribution to publications
The following publications are included in this thesis:
Rossouw, G. C.,Smith, J. P., Barril, C., Deloire, A., & Holzapfel, B. P. (2017).
Carbohydrate distribution during berry ripening of potted grapevines: Impact of water
availability and leaf-to-fruit ratio. Scientia Horticulturae, 216, 215-225.
Rossouw, G. C.,Smith, J. P., Barril, C., Deloire, A., & Holzapfel, B. P. (2017).
Implications of the presence of maturing fruit on carbohydrate and nitrogen distribution
in grapevines under postveraison water constraints. Journal of the American Society for
Horticultural Science, 142(2), 71-84.
Rossouw, G. C.,Orchard, B. A.,Suklje, K., Smith, J. P., Barril, C., Deloire, A., &
Holzapfel, B. P. (2107). Vitis vinifera root and leaf metabolic composition during fruit
ripening: Implications of defoliation. Physiologia Plantarum (Under review).
Rossouw, G. C.,Suklje, K., Orchard, B. A., Smith, J. P., Barril, C., Deloire, A., &
Holzapfel, B. P. (2107). Impact of post-véraison leaf source limitation on the metabolic
profile of Vitis vinifera cv. Shiraz berries. Plant Physiology and
Biochemistry (Under review).
Statement of contribution to publications
v
All papers were written by Gerhard Rossouw as first author and contributions by co-
authors during the writing process were made on the understanding that these papers
would contribute to this thesis and should therefore represent the work of Gerhard
Rossouw. As such the papers do represent the first author's development of concepts,
hypothesis formation, experimental design and implementation, data analysis and
interpretation. All co-authors support the use of the papers as experimental chapters of
this thesis.
Date
Prof Alain Deloire
Dr Jason Smith Date
Dr CeliEi Barril Date
Dr Katja Suklje
Ms Beverley Orchard Date
V
( Dr Bruno Holzapfel
Date
Date
Abstract
vi
Abstract
The post-véraison period is characterised by rapid berry sugar accumulation, and
therefore, a substantial carbon (C) sink demand. In contrast to sugar accumulation, berry
nitrogen (N) incorporation is often variable at distinct stages of the season, and does not
necessarily predominate after véraison. Nevertheless, important alterations in fruit N
content and composition likely occur during the post-véraison period. The fruit sugar
requirement is sourced from leaf photoassimilation, however, total non-structural
carbohydrate (TNC) remobilisation from perennial tissues may provide an alternative C
source when photoassimilation is insufficient. N is translocated from the roots, leaves
and shoots to the berries after véraison, when soil N uptake is expected to be restricted.
The grapevine leaf-to-fruit ratio and water availability are major factors influencing
canopy photoassimilation, and subsequently, the allocation of TNC among perennial
organs and the fruit. Likewise, the leaf area and water supply also affect N distribution
between the perennial and reproductive organs. Restricting the post-véraison leaf area
and/or vine water availability may induce reserve TNC and N utilisation, and could
subsequently be detrimental toward TNC and N storage. Therefore, the overall aim of
this study was to evaluate the post-véraison distribution and partitioning of TNC and N
among the different grapevine organs, as influenced by source-sink relationships and
water supply.
Three distinct pot experiments, using three-year-old own rooted Vitis vinifera
grapevines, were conducted during the post-véraison period. For the first experiment
(2013-14), within each leaf-to-fruit ratio treatment (full and 50% leaves), grapevines
were grown under full or 50% reduced irrigation. Changes in dry biomass, and starch
Abstract
and total sugar concentrations were monitored in the roots, trunks, shoots and leaves.
Berry sugar and anthocyanin accumulation were also assessed. During the second
experiment (2014-15), grapevines were grown with or without fruit from véraison, with
water constraints sustained throughout the experiment. The root, trunk, shoot and leaf
structural biomass, starch, total soluble sugar, total N, and amino N concentrations were
determined, while the fruit sugar and N accumulation were also assessed. The root
glucose, fructose and sucrose contents were additionally measured through high
performance liquid chromatography. The final experiment (2015-16) consisted of a full
leaf area control (100 primary shoot leaves and all laterals) and two defoliation
treatments (25 primary leaves only and no leaves), and the vines were well watered
throughout. Changes in fruit sugar, anthocyanin and N content, and juice yeast
assimilable N (YAN) concentration were monitored. The root and leaf starch and N
concentrations were also determined, and primary metabolite abundance in the fruit,
roots and leaves was measured via untargeted gas chromatography/mass spectrometry
analysis.
A sustained post-véraison water constraint caused root starch depletion, concurrent with
the phase of rapid fruit sugar accumulation. As soon as fruit sugar accumulation slowed,
roots accumulated starch reserves. When water constraints were sustained, root sucrose
accumulation coincided with starch hydrolysis during peak fruit sugar accumulation.
Leaf N depletion corresponded with fruit N accumulation, while the roots of defruited
vines stored N reserves. Fruit sugar and anthocyanin accumulation continued during the
post-véraison period in completely defoliated vines, albeit at reduced rates. However,
defoliation had little impact on the fruit total N content, although the juice YAN
increased after complete and partial defoliation. Most sugars and organic acids depleted
vii
Abstract
in the roots after defoliation, while many amino acids accumulated in roots and
remaining leaves. Defoliation suppressed the root and leaf myo-inositol concentration,
and similarly affected its sugar and organic acids derivatives. Shikimate pathway-
derived amino acids accumulated in roots after full defoliation, while organic acids
derived from this pathway depleted in remaining leaves after partial defoliation.
Although restricted, glucose, fructose and sucrose, and most minor sugars and sugar
alcohols still accumulated in the fruit under leaf source restriction or absence. Fruit
arginine accumulated after partial or full defoliation, while the content of various
shikimate pathway products (such as anthocyanins and phenolic acids) increased or
decreased to different extents in response to leaf area availability.
Among the different grapevine organs, TNC reserves were most abundant in the roots.
Root TNC reserves subsequently supported the post-véraison fruit sugar content when
canopy photoassimilation restriction was induced by water constraints and/or a reduced
leaf area. The root starch reserves were replenished as soon as fruit sugar accumulation
slowed, even occurring from a few weeks before harvest. During sustained post-
véraison water constraints, root starch hydrolysis during peak fruit sugar accumulation
yielded sucrose, which is subsequently transportable to the berries to support the fruit
sugar content. The concentration of myo-inositol and its derivatives (e.g. galactinol and
raffinose), in addition to that of amino acids derived from the shikimate pathway (e.g.
phenylalanine), were significantly affected in roots during a post-véraison leaf C source
limitation or absence. Myo-inositol metabolism seemingly played a distinct role during
root starch remobilisation while berry sugar accumulation occurred. In contrast to the
roots being an important TNC source when photoassimilation was restricted by limited
water supply, leaf N seemed to be a significant contributor to fruit N content during the
viii
Abstract
ix
sustained water constraints. The presence of ripening fruit in conjunction with water
constraints, subsequently prevented root N storage between véraison and fruit maturity.
While root total N concentration was little affected by defoliation, root amino N
composition was altered, for example prompting arginine accumulation. Arginine also
subsequently accumulated in the fruit, effectively increasing the juice YAN of
defoliated vines. This study provided a novel illustration of post-véraison TNC and N
partitioning and distribution under differing water availability and/or source-sink
relationships. The results contribute to the understanding of grapevine reserve TNC
utilisation during a period of substantial fruit C demand. Furthermore, although the
extent of fruit N sink requirement after véraison is not as clear as the corresponding fruit
C demand, the study contributes to understanding grapevine N reserve utilisation during
fruit maturation.
Chapter 1: General introduction and research aims
1
Chapter 1: General introduction and research aims
1.1. Introduction and aims
The period between the onset of berry softening (véraison) and fruit maturity of
grapevines is characterised by berry sugar accumulation and cell expansion. Berry sugar
accumulation occurs rapidly from the start véraison, while slower accumulation could
take place towards the latter stages of berry maturation (McCarthy & Coombe, 1999).
Nitrogenous and phenolic compounds, such as amino acids and anthocyanins, are some
of the other major metabolites incorporated into the berries during berry maturation.
Some organic acids (predominantly malic acid) which accumulates earlier in berry
development, are in contrast degraded after véraison (Iland, Dry, Proffitt, & Tyerman,
2011). During this period of berry sugar accumulation, the grapevine water supply and
the relationship between the vegetative and reproductive organ biomasses, are major
determinants of non-structural carbohydrate (TNC) (Holzapfel, Smith, Field, & Hardie,
2010) and nitrogen (N) (Roubelakis-Angelakis & Kliewer, 1992) allocation between the
perennial (mainly the roots) and reproductive (fruit) grapevine structures.
Carbon (C) assimilated during leaf photosynthesis is primarily translocated to the
ripening berries (Williams, 1996). However, when the canopy leaf area is low or
deliberately reduced in relation to the total fruit biomass, TNC reserves are thought to
be remobilised to support the fruit sugar content (Candolfi-Vasconcelos, Candolfi, &
Koblet, 1994). Grapevines store a large proportion of their TNC in the roots (Holzapfel
et al., 2010), and the root system could provide an alternative source of C to supplement
the supply from canopy photosynthesis. However, as the major perennial structure,
TNC reserve storage in the roots is also essential prior to dormancy, and the roots
Chapter 1: General introduction and research aims
2
therefore start to replenish TNC reserves from flowering (Holzapfel et al., 2010). These
reserves are subsequently utilised towards vegetative and reproductive development
from budburst the next season (Bennett, Javis, Creasy, & Throught, 2005; J. P. Smith &
Holzapfel, 2009). Water constraints (Escalona, Flexas, & Medrano, 1999) and/or a low
leaf-to-fruit ratio (Petrie, Trought, & Howell, 2000) are detrimental to overall canopy
photoassimilation and can, therefore, increase the allocation of the restricted available C
to the fruit in the expense of root reserve replenishment. Root TNC are mainly stored as
immobile starch molecules, and in order to facilitate the remobilisation of carbohydrate
reserves, the starch is hydrolysed, resulting in the accumulation of phloem mobile
soluble sugars (A. M. Smith, Zeeman, & Smith, 2005). Conditions leading to TNC
reserve remobilisation, therefore, reduce the ratio of starch to soluble sugars in the
storage tissues, and subsequently alter the composition of carbohydrates within the
related organs, such as the roots.
Amino acid incorporation enables the accumulation of organic N in the berries. During
the post-véraison period, the soil N uptake is likely restricted or absent, and
translocation of N occurs from the roots, leaves and shoots towards the berries
(Conradie, 1991). The mature leaves and roots are, however, the major sources of amino
acids in higher plants, from where the amino acids are translocated to sink organs to
support metabolism or development (Rentsch, Schmidt, & Tegeder, 2007). Therefore,
similar to TNC, the source requirements placed upon the roots and leaves to supply
towards the fruit N sink, may be affected by the biomass ratio of the perennial and
annual structure. Furthermore, water supply during berry maturation can influence the
allocation of N between the perennial and annual structure (Holzapfel, Watt, Smith,
Suklje, & Rogiers, 2015). Also similar to TNC, the perennial structure of grapevines
Chapter 1: General introduction and research aims
3
stores N reserves, and trunk and root N storage start before berry maturation
(Roubelakis-Angelakis & Kliewer, 1992). These N reserves are also utilised during
early shoot growth after budburst the following season. Storage proteins are degraded in
the N source organs of plants, resulting in the accumulation of free amino acids
(Masclaux-Daubresse et al., 2010), which are subsequently transportable to sink organs
such as the ripening fruit. The grapevine source-sink biomass ratio and water status,
could therefore alter the composition of N containing metabolites in the source organs
(especially mature leaves and the roots) and in the berries.
Ultimately, the grapevine leaf-to-fruit ratio and water supply are major determinants of
important fruit quality parameters during berry maturation. In addition to the fruit sugar
and N content, intermediates of C metabolism (e.g. organic acids ) are also likely
impacted (López-Bucio, Nieto-Jacobo, Ramırez-Rodrıguez, & Herrera-Estrella, 2000).
These compounds are important precursors for the biosynthesis of secondary
metabolites, such as phenolic compounds (e.g. anthocyanins). These secondary
metabolites are important fruit quality parameters, and contribute to wine colouration
and aromatic potential. For the winemaking process and desired wine style, berry sugar
content is crucial for alcoholic fermentation, while berry N is assimilated by the yeasts
and contribute to the development of aromatic compounds. The partitioning, allocation
and distribution of TNC and N during the period between véraison and fruit maturity
are essential for both, their accumulation in the fruit and in the storage tissues.
The main aim of this research project was to determine the impact of both grapevine
water status, and the ratio between the sizes of vegetative and reproductive organs, on
the allocation of TNC and N between the reserve organs and the fruit during berry
Chapter 1: General introduction and research aims
4
maturation. In the context of this study, the allocation includes compound partitioning
and distribution. The current literature regarding this research topic is discussed in
chapter 3. However, the outcomes of the present study contribute to the following gaps
in the literature:
Gap 1:
Carbohydrate reserve remobilisation from perennial tissues (roots and trunks) towards
post-véraison berries, when the canopy leaf area is restricted, has been illustrated
through 14
C studies. However, the extent or importance of the contribution from TNC
reserves towards the fruit sugar content when leaf photoassimilation is restricted, still
needs to be quantified. To the best of our knowledge, the contribution of TNC reserves
to berry sugar accumulation has only been shown when canopy photoassimilation is
limited by a restricted leaf area. Inhibition of grapevine canopy photoassimilation by
water constraints, however, also commonly occurs during berry maturation, especially
in warmer climate regions. Furthermore, to investigate the effects of the grapevine crop
load or post-véraison water constraints on TNC reserve dynamics, prior studies only
evaluated the TNC reserve abundance on a concentration basis, and exclusively at a
particular stage of the season (mostly at budburst). The kinetics of TNC allocation
between the different grapevine organs during berry maturation still requires
investigation. In order to contribute to the above mentioned gaps in the literature, the
following research objective was addressed:
1. To investigate the interactive effects of the leaf-to-fruit ratio and grapevine water
status during two phases of berry sugar accumulation (rapid and slow) on the
carbohydrate distribution between the different grapevine organs (Chapter 3).
Chapter 1: General introduction and research aims
5
Gap 2:
Grapevine berries are a post-véraison sink for both, TNC and N. The extent of the
utilisation of TNC and N reserves during berry maturation, especially sourced from the
roots, largely determines starch and N reserve replenishment by fruit maturity. The
kinetics of TNC and N content development in the different grapevine organs during
berry maturation, have not yet been studied. Sustained water constraints during berry
maturation are likely, a major determinant of the extent of root TNC and N reserve
contribution towards berry sugar and N content. Water constraints may influence the
partitioning of TNC (starch and soluble sugars) and N (total and amino N) in source and
sink organs, which contributes to their mobilisation between the different organs. More
work is required to better understand the implications of a sustained post-véraison water
constraint on the composition of TNC and N within the perennial (especially the roots)
grapevine organs. The subsequent consequences on reserve starch and N replenishment
by fruit maturity, additionally, needs clarification. The following research objective was
aimed at contributing to the above mentioned gaps:
2. To determine how the presence or absence of fruit during sustained post-véraison
water constraints influences the allocation of carbohydrates and N between the
different grapevine organs (Chapter 4).
Gap 3:
Carbon and N metabolism in grapevine source organs are crucial during the process of
TNC and N distribution to sink organs. The root system and leaves of grapevines
represent two major sources of TNC and N. When the leaf area is restricted during berry
Chapter 1: General introduction and research aims
6
maturation, the source demand placed upon the roots is likely amplified. The
composition of primary metabolites within the roots and leaves during the post-véraison
period could regulate C and N translocation towards sink organs, such as the berries. To
the best of our knowledge, no previous studies have evaluated the profiles of primary
grapevine metabolites in C and N source organs during the berry maturation period.
Yet, an untargeted evaluation of the change in source organ primary metabolite
abundance may be beneficial towards understanding the underlying functioning of
primary metabolism during reserve TNC and N utilisation. Restricting the C and N
source availability through defoliation, may enable a novel assessment of root C and N
metabolism and the subsequent utilisation of these compounds during berry maturation.
The following objective was established in order to enhance the understanding of
grapevine source organ C and N metabolism during berry maturation:
3. To assess the implications of defoliation on fruit sugar and N accumulation in
conjunction with the carbohydrate, N and primary metabolite composition of the
major grapevine source organs (roots and leaves) (Chapter 5).
Gap 4:
The post-véraison canopy leaf area availability affects the development of berry
composition. As an important C and N source, limiting the grapevine leaf area can be
detrimental towards berry sugar, N and anthocyanin accumulation. Although a
few previous studies observed primary berry metabolism during the post-véraison
period, these studies were mostly aimed at comparing different genotypes. More work is
needed to observe the implications of limiting or eliminating the leaf C and N
source, on primary metabolism in the berries. Studying alterations in the contents of
Chapter 1: General introduction and research aims
7
untargeted primary metabolites within maturing berries, may contribute to
understanding the changing berry composition as influenced by a restricted leaf
area availability. The following objective is intended to contribute to the above
mentioned gaps:
4. To study the implications of defoliation on the post-véraison metabolic composition
of grapevine berries (Chapter 6).
Overall, this study is intended to improve the understanding of TNC and N reserve
utilisation during berry maturation, and how the extent of this utilisation ultimately
affects berry composition. The implications of water supply, and the relationship
between reproductive and vegetative grapevine organ sizes during berry maturation are
of particular interest.
1.2. References
Bennett, J., Javis, P., Creasy, G. L., & Throught, M. C. T. (2005). Influence of
defoliation on overwintering carbohydrate reserves, return bloom, and yield of
mature Chardonnay grapevines. American Journal of Enology and Viticulture,
56(4), 386-393.
Candolfi-Vasconcelos, M. C., Candolfi, M. P., & Koblet, W. (1994). Retranslocation of
carbon reserves from the woody storage tissues into the fruit as a response to
defoliation stress during the ripening period in Vitis vinifera L. Planta, 192(4),
567-573.
Conradie, W. (1991). Distribution and translocation of nitrogen absorbed during early
summer by two-year-old grapevines grown in sand culture. American Journal of
Enology and Viticulture, 42(3), 180-190.
Escalona, J. M., Flexas, J., & Medrano, H. (1999). Stomatal and non-stomatal
limitations of photosynthesis under water stress in field-grown grapevines.
Australian Journal of Plant Physiology, 26, 421-433.
Holzapfel, B. P., Smith, J. P., Field, S. K., & Hardie, W. J. (2010). Dynamics of
carbohydrate reserves in cultivated grapevines. Horticutural Reviews, 37, 143-
211.
Holzapfel, B. P., Watt, J., Smith, J. P., Suklje, K., & Rogiers, S. Y. (2015). Timing of N
application and water constraints on N accumulation and juice amino N
concentration in Chardonnay grapevines. Vitis, 54(4), 203-211.
Chapter 1: General introduction and research aims
8
Iland, P., Dry, P., Proffitt, T., & Tyerman, S. D. (2011). The grapevine: from the
science to the practice of growing vines for wine. Adelaide, Australia: Patrick
Iland Wine Promotions Pty Ltd.
López-Bucio, J., Nieto-Jacobo, M. A. F., Ramırez-Rodrıguez, V., & Herrera-Estrella, L.
(2000). Organic acid metabolism in plants: from adaptive physiology to
transgenic varieties for cultivation in extreme soils. Plant Science, 160(1), 1-13.
McCarthy, M. G., & Coombe, B. G. (1999). Is weight loss in ripening grape berries cv.
Shiraz caused by impeded phloem transport? Australian Journal of Grape and
Wine Research, 5(1), 17-21.
Masclaux-Daubresse, C., Daniel-Vedele, F., Dechorgnat, J., Chardon, F., Gaufichon, L.,
& Suzuki, A. (2010). Nitrogen uptake, assimilation and remobilization in plants:
Challenges for sustainable and productive agriculture. Annals of Botany, 105,
1141-1157.
Petrie, P., Trought, M., & Howell, S. (2000). Influence of leaf ageing, leaf area and crop
load on photosynthesis, stomatal conductance and senescence of grapevines
(Vitis vinifera L. cv. Pinot Noir) leaves. Vitis, 39(1), 31-36.
Rentsch, D., Schmidt, S., & Tegeder, M. (2007). Transporters for uptake and allocation
of organic nitrogen compounds in plants. FEBS letters, 581(12), 2281-2289.
Roubelakis-Angelakis, K. A., & Kliewer, W. M. (1992). Nitrogen metabolism in
grapevine. Horticultural Reviews, 14, 407-452.
Smith, A. M., Zeeman, S. C., & Smith, S. M. (2005). Starch degradation. Annual
Review of Plant Biology, 56, 73-98.
Smith, J. P., & Holzapfel, B. P. (2009). Cumulative responses of Semillon grapevines to
late season perturbation of carbohydrate reserve status. American Journal of
Enology and Viticulture, 60(4), 461-470.
Williams, L. E. (1996). Grape. In E. Zamski & A. Schaffer (Eds.), Photoassimilate
distribution in plants and crops. Source-sink relationships (pp. 851-881). New
York, NY: Marcel Dekker.
Chapter 2: Literature review
9
Chapter 2: Literature review
2.1. Introduction
The period of grapevine berry maturation (from véraison to fruit maturity) coincides
with strong competition between the different grapevine organs for the utilisation of
photoassimilates and nutrients. During this period, temporary sinks (e.g. the fruit) and
permanent sinks (e.g. the roots) are competing for the accumulation of both non-
structural carbohydrates (TNC) (Holzapfel, Smith, Field, & Hardie, 2010) and nitrogen
(N) (Cheng, Xia, & Bates, 2004). Abiotic conditions, such as water constraints, and
vegetative and reproductive organ biomass balances, are major determinants of the
utilisation and distribution of TNC and N within grapevines (Cheng et al., 2004;
Escalona, Flexas, & Medrano, 1999; Petrie, Trought, & Howell, 2000; Roubelakis-
Angelakis & Kliewer, 1992).
The overall aim of this review is to summarise the existing literature regarding
grapevine TNC and N allocation between the different organs during berry maturation.
In the context of this review, the allocation of these compounds includes their respective
distribution, partitioning and utilisation within the different organs. This review is
focussed on grapevine related literature, however, information from other plant species
is mentioned when available evidence from grapevine related literature is insufficient.
In this review, a separate emphasis is placed upon the distributions of TNC and N in
grapevines. It is, nevertheless, important to note that inorganic N is required to allow
carbon (C) to be utilised for structural tissue growth, while TNC breakdown provides
adenosine triphosphate (ATP) and C skeletons to support the accumulation of inorganic
Chapter 2: Literature review
10
N (Stitt & Krapp, 1999). Although both TNC and N have distinct and essential roles
during berry ripening in plants, it is clear that the metabolisms of these compounds are
strongly linked.
As the grapevine water supply (Escalona et al., 1999), crop load (Poni, Lakso, Turner,
& Melious, 1994) and leaf area (Petrie, Trought, Howell, & Buchan, 2003) are major
determinants of leaf C assimilation, and because of the impact of vine water supply
(Araujo, Williams, & Matthews, 1995) and cropping (Rodriguez-Lovelle & Gaudillere,
2002) on N distribution, specific emphases are placed upon the effects of grapevine
water status and source-sink relations within this review. The goals are to summarise,
during fruit ripening: a) the physiological roles and utilisation of TNC, b) the
distribution of TNC, c) the functions of N, and d) the distribution of N within
grapevines. Information on TNC and N distribution during other stages of the annual
grapevine development cycle is included to enable a thorough description of the roles of
TNC and N in grapevines. The overall intention is to clarify the existing information,
and to identify literature gaps, thereby improving the knowledge regarding the effects of
TNC and N distribution on short- and long-term grapevine development and
productivity.
2.2. Grapevine non-structural carbohydrates
Carbohydrates in plants consist of structural carbohydrates (cellulose and
hemicellulose) and total non-structural carbohydrates (TNC, predominantly starch and
soluble sugars). Carbohydrates are synthesised during the day through the process of
photosynthesis in the leaves (photoassimilation), when atmospheric carbon dioxide
(CO2) is converted into carbohydrates. In grapevines, most assimilated C is allocated
Chapter 2: Literature review
11
towards the biosynthesis of structural cellulose (Winkler & Williams, 1938), which
cannot be further utilised, as Ccannot be recovered from cellulose (Kozlowski &
Pallardy, 1997). TNC are therefore accumulated, and utilised towards C distribution and
as a resource for reproductive and vegetative development. In the leaves, sucrose is
vastly biosynthesised during photoassimilation, and then becomes available for
distribution from the leaves to the rest of the plant, via the osmotic gradient of the
phloem vascular system. Some photoassimilates are also stored in the leaves during the
day as starch and, at night, this stored C is hydrolysed and further distributed within the
plant to continue the mobilisation of leaf assimilates in the absence of photosynthesis
(A. M. Smith & Stitt, 2007). Most of the TNC in plants are present as starch and stored
in different parts of the plant, especially in the roots, while soluble sugars generally only
represent a small part of total TNC (Scholefield, Neales, & May, 1978; Zufferey et al.,
2012). The various types of TNC found in plant tissues include sugar alcohols (e.g.
inositol and mannitol), monosaccharides (e.g. fructose, glucose and galactose),
disaccharides (e.g. sucrose and maltose), oligosaccharides (e.g. raffinose) and
polysaccharides (e.g. starch).
Fruit sugar accumulation initiates rapidly at the start of berry maturation, when the
berries start to soften (véraison) (Davies & Robinson, 1996). However, the
replenishment of TNC reserves in the permanent grapevine structure (especially the
roots) could start around flowering, and may continue throughout berry maturation,
dependent on the grapevine crop load (Holzapfel et al., 2010).
Chapter 2: Literature review
12
2.2.1. Roles of carbohydrate reserves in grapevines
The distribution of TNC in plants provides resources that are used towards generating
energy and structure, or are stored as TNC reserves (mainly as starch) in perennial
tissues (roots and trunks). In deciduous plants like the grapevine, TNC reserves play a
role in cold hardiness and the maintenance of metabolism during dormancy (Loescher,
McCamant, & Keller, 1990). TNC reserves also aid in the replenishment of damaged
tissues, contribute to fruit ripening, and improve the defence of plants against pests and
diseases (Holzapfel et al., 2010). TNC reserves in grapevines are also important for the
annual reestablishment of the vegetative growth of leaves and shoots, and can contribute
in determining the reproductive capacity (fruit yield) of grapevines for the following
season (Bennett, Javis, Creasy, & Throught, 2005; J. P. Smith & Holzapfel, 2009;
Zufferey et al., 2012).
During early season grapevine reproductive development, TNC reserves are utilised
during inflorescence development (J. P. Smith & Holzapfel, 2009) and aid in flower
induction, while TNC are also utilised during the processes of pollination, fertilisation,
and fruit set (Zapata, Deléens, Chaillou, & Magné, 2004). The TNC reserve availability
will therefore contribute to the fruitfulness and quality of fruiting canes in grapevines,
with the reserve status therefore playing an important role in determining the
fruitfulness of grapevines (Bennett et al., 2005). Later in the season, TNC reserves
could also contribute to fruit sugar accumulation. It was, for example, shown through
14C labelling that TNC reserves are directed from perennial plant organs towards the
fruit, when vines are excessively defoliated at the onset of fruit maturation. This
mobilisation of TNC reserves peaks during mid fruit ripening, a period when the berry
Chapter 2: Literature review
13
TNC sink requirement is especially strong (Candolfi-Vasconcelos, Candolfi, & Koblet,
1994).
The contribution of TNC reserves towards early season vegetative development (shoot
and leaf expansion), and towards root growth, is crucial until leaf photosynthesis
becomes the primary source of C later in the season (as they only contribute little to C
accumulation early on). In fact, it was previously suggested that new shoots are
generally dependent on TNC reserves until newly developing leaves are about half their
full size, when they start to act as sources for photoassimilates (Hale & Weaver, 1962).
Insufficient abundance of TNC reserves leads to reduced shoot and root growth
(Candolfi-Vasconcelos et al., 1994), and to reduced leaf size and lateral shoot
development (J. P. Smith & Holzapfel, 2009). The roles of soluble sugars in the early
vegetative and reproductive development of grapevines are further discussed later in
this review.
2.2.2. Mobilisation of carbohydrate reserves
Starch is the predominant TNC in woody grapevine tissues, and is especially abundant
in the roots (J. P. Smith & Holzapfel, 2009). Starch is a large molecule, and is not
osmotically active, and can therefore not be transported between different parts of the
plant, while sugars can, by generating a reduction in osmotic potential when it
accumulates (Gibson, 2005; Zufferey et al., 2012). When a TNC deficiency develops in
plants, starch reserve deposits are degraded (solubilised), yielding C containing
intermediates, such as glucans, maltose and glucose, which are ultimately restructured
as sucrose (A. M. Smith, Zeeman, & Smith, 2005). Sucrose can then be transported in
the phloem from source to sink organs (Ruan, Jin, Yang, Li, & Boyer, 2010).
Chapter 2: Literature review
Insufficient leaf photoassimilation leads to an undersupply of assimilated C towards the
requirements of TNC sinks, and thereby creates a TNC deficiency in the plant. Such
a deficiency can easily be generated during the berry maturation period, as the berries
are a strong sink for TNC during this stage. The degradation of starch reserves during
berry maturation therefore leads to increased concentrations of soluble sugars in the
source organ, allowing for TNC remobilisation towards the maturing berries
(Candolfi-Vasconcelos et al., 1994).
Plant sugar abundance is suggested to be involved in the signalling towards the
regulation of reserve starch degradation or storage. The expression of numerous plant
genes responds to TNC abundance or shortage (Eveland & Jackson, 2012; Rolland,
Moore, & Sheen, 2002). Many of these genes encode enzymes involved in
photosynthesis, and in sugar and starch metabolism (S. M. Smith et al., 2004). When a
plant sugar deficiency occurs, genes involved in photosynthesis, TNC remobilisation
and export, and in N metabolism, tend to be up-regulated (Eveland & Jackson, 2012).
Low sugar status therefore leads to enhanced photosynthesis, as well as reserve TNC
mobilisation and exportation from source tissues (Gupta & Kaur, 2005). However,
when sugars are abundant, altered gene expression enhances typical sink organ
activities, like TNC importation, and further utilisation (for e.g., vegetative growth or
storage) (Eveland & Jackson, 2012).
High grapevine crop load, insufficient canopy leaf area, and/or sustained water
constraints, are major conditions that increase the sink (fruit) TNC requirement, relative
to the supply from canopy photoassimilation. Such an imbalance of C availability then
induces the remobilisation of reserves from perennial vine parts during berry maturation
14
Chapter 2: Literature review
15
(Candolfi-Vasconcelos et al., 1994). The TNC reserve mobilisation is one adaptive
mechanism for grapevines to adjust to limitations in photoassimilation during fruit
maturation, while compensatory photosynthesis by the intact leaves is also another
regulatory feature to adapt to the insufficient C assimilation, especially after defoliation
treatments (Petrie et al., 2000; Scholefield et al., 1978). The increased photosynthetic
rate of the remaining leaves does not always compensate for the reduced leaf area, as
excessive leaf removal could still easily reduce the overall canopy photosynthesis rate
(Poni, Bernizzoni, & Civardi, 2008).
2.2.3. Soluble sugar specific roles
Apart from the mentioned role in TNC transportation between the source and sink
organs of grapevines, soluble sugars are also involved in various other functions within
the plant. These roles include the utilisation of sugars as structural components, as cell
nutrients, and as potential signals in plant growth and development (Çakir et al., 2003).
Sugars are also used in the biosynthesis of polysaccharides, such as starch and cellulose,
in plants (Gupta & Kaur, 2005). Sucrose is the major TNC transported from
photosynthetic tissue to sinks, where it can be degraded to hexoses or their derivatives,
which are useful for various metabolic or biosynthetic processes (Ruan et al., 2010).
However, raffinose family oligosaccharides (e.g. raffinose) and sugar alcohols (e.g.
mannitol) also play important transport roles in many plants (Noiraud, Maurousset, &
Lemoine, 2001).
2.2.3.1. Signalling and gene expression
As briefly mentioned earlier, sugars can act as signalling molecules that control distinct
aspects of plant development. These signals provide information on the prevailing
Chapter 2: Literature review
16
internal and external conditions, and contribute to signalling networks in response to the
abundance of metabolites (e.g. sugars or amino acids) (Smeekens, Ma, Hanson, &
Rolland, 2010). Such signalling networks create a link between C assimilation, C
storage, and plant growth (A. M. Smith & Stitt, 2007), and can significantly contribute
to yield and crop quality in plants (Smeekens et al., 2010).
Sugar signals can either be generated by TNC abundance and ratios to other metabolites
(e.g., C-to-N ratios), or by the flux through sugar specific sensors and/or transporters in
the plant (Eveland & Jackson, 2012). Sensor proteins are responsible for the sensing of
plant sugar status. The interaction between a sugar molecule and a sensor protein causes
a signal to be generated (Gupta & Kaur, 2005), and this allows plants to modulate site-
specific and whole-plant growth, potentially to coordinate developmental programmes
with available TNC (Eveland & Jackson, 2012). Plants therefore monitor their own
sugar status in order to optimally utilise available sugar for growth and development
(Smeekens et al., 2010).
Sucrose can be sensed as a signal directly or through an indirect signal that arises via its
hexose cleavage products, i.e., glucose and fructose (Eveland & Jackson, 2012; Koch,
2004). Invertase and sucrose synthase are enzymes responsible for sucrose cleavage
(Koch, 2004), and the activities of these enzymes contribute to sugar signalling. Hexose
sugars generally have greater signalling potential in promoting organ growth and cell
proliferation, while sucrose is associated with cell differentiation and maturation
(Eveland & Jackson, 2012). Various minor sugars and sugar alcohols, e.g., myo-inositol
(Valluru & Van den Ende, 2011), trehalose (Iturriaga, Suárez, & Nova-Franco, 2009;
Smeekens et al., 2010), raffinose (Valluru & Van den Ende, 2011) and galactinol
Chapter 2: Literature review
17
(Valluru & Van den Ende, 2011), were also previously indicated as being involved in
plant sugar signalling networks, and are discussed later.
2.2.3.2. Osmotic regulation and protection
As osmotically active solutes, plant sugars can play distinct roles during abiotic stress
conditions, such as a drought (Rodrigues et al., 1993). In fact, the biosynthesis and
accumulation of osmotic regulators (e.g. sugars) is a well-known adaptive mechanism
for plants in response to osmotic constraints (Shulaev, Cortes, Miller, & Mittler, 2008).
Sugars are also important osmoprotectants, as they stabilise proteins and cell
membranes (Rontein, Basset, & Hanson, 2002). Indeed, many sugars interact with
proteins and cell membranes through hydrogen bonding, and thereby importantly
prevent protein denaturation (Devi & Sujatha, 2014). The accumulation of soluble
solids corresponds to the increase of drought tolerance in plants (Hoekstra, Golovina, &
Buitink, 2001), and certain plant species, or even within specie varieties with a higher
abiotic stress tolerance, are known to accumulate higher volumes of sugar metabolites
related to osmotic regulation and cell membrane stabilisation (Morsy, Jouve, Hausman,
Hoffmann, & Stewart, 2007).
During abiotic stress conditions (e.g., dry or saline conditions), accumulated sugars can
raise the osmotic pressure in plant cells at the site of their accumulation (e.g., the roots
or leaves) (Rontein et al., 2002). Sugar accumulation in plant organs thereby contributes
to the maintenance of a cell turgor potential, and thus generates a gradient for water
uptake into the plant cells (Rhodes & Samaras, 1994). Grapevine leaves (Patakas &
Noitsakis, 1999) and roots (Düring & Dry, 1995) are known to accumulate sugars in
order to undergo osmotic adjustment during water constraints. However, as it provides
Chapter 2: Literature review
18
the point of contact between the plant and the soil, the osmotic regulation of grapevine
roots cells is especially important, and root sugar accumulation could be associated with
increased root osmolarity when the soil moisture content decreases (Rogiers, Holzapfel,
& Smith, 2011). Root soluble sugar accumulation can also play a role in embolism
repair, stimulating the osmotic movement of water into embolised vessels, to refill and
restore water flow (Canny, 1998; Salleo, Trifilò, Esposito, Nardini, & Lo Gullo, 2009).
Sucrose accumulation is often suggested to be related to osmotic regulation in many
plants that are exposed to water stress (Quick, Siegl, Neuhaus, Feil, & Stitt, 1989;
Rogiers, Holzapfel, et al., 2011). Sucrose is also thought to play a role in the protection
of cellular macromolecules or cell membranes (Bray, 1997; Leopold, 1990).
Nevertheless, sucrose may be cleaved into fructose and glucose during abiotic stress
conditions, and invertase activity is related to genetic variation, where more stress
tolerant varieties may have greater invertase activity (Morsy et al., 2007). In fact, high
hexose (glucose and fructose) levels are suggested to drive lower cell water potential
and maintain turgor pressure during water stress (Sturm, 1999). Many other sugars (e.g.
raffinose) and sugar alcohols (e.g. myo-inositol), that are usually present in relatively
lower abundance in plants, also play crucial roles in plant osmotic regulation and
osmoprotection (Krasensky & Jonak, 2012; Morsy et al., 2007; Rontein et al., 2002).
These aspects are discussed later.
2.2.3.3. Vegetative and reproductive development
As briefly mentioned earlier, reserve TNC plays important roles in the reestablishment
of vegetative spring growth. When the abundance of sugars that were synthesised in the
leaves exceeds the TNC requirement of temporary sink organs (e.g., the fruit), sugars
are translocated to woody plant parts (e.g., the trunks and roots), where they can be
Chapter 2: Literature review
19
converted into starch (Scholefield et al., 1978). This happens especially late in the
growing season, when fruit sugar accumulation slows down. This reserve starch is then
the first form of TNC used for new shoot growth the following season (Scholefield et
al., 1978).
Sugars utilised for reproductive development are either derived from TNC reserves or
synthesised through photoassimilation in the leaves or inflorescence, depending on the
stage of annual grapevine development (Lebon et al., 2008). However, inadequate sugar
supply due to insufficient reserve TNC availability at budburst could inhibit flower
initiation and, subsequently, also shoot fruitfulness (J. P. Smith & Holzapfel, 2009).
Sugars therefore play an important role in flowering and in the formation of plant
reproductive organs and structures, by providing essential energy resources (Lebon et
al., 2008). Low sugar abundance in the grapevine may cause flower abortion, especially
when this limited sugar availability is found during female meiosis (Lebon et al., 2008).
Furthermore, fruit set, and ultimately the fruit yield, is suppressed by low sugar
availability (Bennett et al., 2005; Intrieri, Filippetti, Allegro, Centinari, & Poni, 2008).
A further role of sugars towards the reproductive development is the possible
contribution towards berry sugar accumulation, as discussed earlier.
2.2.4. Minor sugars and sugar alcohols
Apart from sucrose and the hexose sugars, a range of other sugars and sugar alcohols
were previously reported as being present within grapevine tissues. However, most of
the early studies related to the presence of minor sugars in grapevines were only
conducted during the cold winter periods. In these studies, the minor sugars that were
reported to be present in grapevines included raffinose (Hamman, Dami, Walsh, &
Chapter 2: Literature review
20
Stushnoff, 1996; Jones, Paroschy, McKersie, & Bowley, 1999; Kliewer, 1966; Koussa,
Cherrad, Bertrand, & Broquedis, 1998), stachyose (Hamman et al., 1996; Kliewer,
1966; Panczel, 1962), maltose (Kliewer, 1966; Panczel, 1962), galactose (Kliewer,
1966), inositol (McArtney & Ferree, 1999; Ndung'u, Shimizu, Okamoto, & Hirano,
1997) and melibiose (Kliewer, 1966; Panczel, 1962). With more recent developments in
the methods to profile the metabolic composition of plant tissues, various late studies
have successfully identified the presence of various other minor sugars in grapevines
(Cuadros-Inostroza et al., 2016; Degu et al., 2014; Hochberg, Batushansky, Degu,
Rachmilevitch, & Fait, 2015; Hochberg, Degu, Cramer, Rachmilevitch, & Fait, 2015;
Hochberg et al., 2013). Most of these studies, however, focussed on the metabolic
profile of grapevine berries. However, as the abundance of various minor sugars and
sugar alcohols leads to improved tolerance towards different plant stress conditions,
e.g., water and cold stress (Gupta & Kaur, 2005; Rontein et al., 2002), and could play a
crucial role in plant metabolism (Valluru & Van den Ende, 2011), more work is needed
to improve the knowledge of an environmentally stress related change of minor sugar
abundance in different grapevine organs.
Sugar alcohols, i.e., non-reducing carbohydrates that are a class of polyols, include
sorbitol, mannitol, galactinol and myo-inositol, and are present in plants, commonly
synthesised as primary photosynthetic products (Moing, 2000). These compounds can
act as transport substances and storage compounds, sometimes appearing as minor
compounds in the roots of woody plants (Loescher et al., 1990). The abundance of these
compounds in plant tissues can increase when plants are exposed to abiotic constraints,
and could thereby promote resistance towards various abiotic plant stresses, including
low temperature, water constraints, and high salinity, as well as biotic stress (Gupta &
Chapter 2: Literature review
Kaur, 2005; Moing, 2000). In a variety of plant species, it is possible that hexoses and
other TNC (e.g. sucrose and starch) can be converted into sugar alcohols during dry
conditions (Wang, Quebedeaux, & Stutte, 1996). These compounds can then also act as
osmoprotectants (Rontein et al., 2002), and are therefore involved in the stabilisation of
cell membranes and enzymes (Krasensky & Jonak, 2012). The accumulation of sugar
alcohols can, furthermore, induce the importation of water into plant cells during
osmotic stress, by causing an adjustment in cell osmotic potential (Wang et al., 1996).
Myo-inositol is an important example of a plant sugar alcohol, and functions as a potent
osmoprotectant (Silva et al., 2011). It plays a role in structuring cell membranes, and
can also act as a signalling molecule in plants (Valluru & Van den Ende, 2011). Myo-
inositol accumulation was previously found to correlate with salt tolerance in tomato
plants (Cuartero & Fernández-Muñoz, 1998), while genes involved in myo-inositol
synthesis were also previously found to be upregulated during water stress in various
other plant species (Ishitani et al., 1996). Furthermore, myo-inositol serves as a
precursor of various other metabolites in plants. It serves as a precursor in the synthesis
of galactinol, and subsequently also the synthesis of the raffinose family
oligosaccharides (Valluru & Van den Ende, 2011). It is also proposed that myo-inositol
could provide an alternative substrate for a pathway for ascorbic acid synthesis
(Lorence, Chevone, Mendes, & Nessler, 2004). Ascorbic acid, on the other hand, is a
major substrate for the synthesis of tartaric acid, threonic acid, glyceric acid and oxalic
acid (Loewus, 1999).
Raffinose family oligosaccharides (RFOs) consist of raffinose, stachyose
and verbascose, and are suggested to play a role in plant osmoprotection under water
21
Chapter 2: Literature review
stress (Taji et al., 2002). RFOs were therefore previously indicated to contribute to cell
membrane protection (Krasensky & Jonak, 2012). RFOs can also be used as
transport compounds in plants, accumulate in response to a range of abiotic stresses, and
also as reserve compounds (Sengupta, Mukherjee, Basak, & Majumder, 2015; Valluru
& Van den Ende, 2011). Raffinose is known to accumulate in plant tissues during
chilling conditions, and may thereby increase the tolerance against chilling due to its
role in membrane stabilisation via interactions with phospholipid headgroups (Morsy et
al., 2007). It was also found that drought, high salinity, and cold conditions could
induce the accumulation of high levels of raffinose, along with its precursor, galactinol,
but not of stachyose. Raffinose and galactinol are therefore suggested to play a role in
stress tolerance in plants (Taji et al., 2002), and were found to play roles as
osmoprotectants in the leaves of Arabidopsis plants (Nishizawa-Yokoi, Yabuta, &
Shigeoka, 2008). It is also suggested that raffinose and galactinol could act as signals to
mediate stress responses in plants (Valluru & Van den Ende, 2011). While raffinose is
therefore perhaps an important osmoprotector, the role of stachyose is suggested to
rather be more related to the storage and transport of TNC in various woody plants,
cucurbits, and legumes (Dey & Harborne, 1997).
Other minor sugars include trehalose, which only accumulates in certain plants, and
mainly in so-called resurrection plants (which can survive extreme dehydration for
extended periods), where it can lead to increased drought tolerance, improved
photosynthesis and dry matter accumulation under water stressed conditions (Gupta &
Kaur, 2005). Trehalose has also been proposed to act as an osmoprotectant during water
constraints (Penna, 2003; Rontein et al., 2002), as it can protect proteins and cell
membranes against denaturation (Silva et al., 2011). Trehalose can also be an important
22
Chapter 2: Literature review
23
signalling metabolite, involved in the regulation of plant growth and development in
response to C availability (O’Hara, Paul, & Wingler, 2013; Silva et al., 2011). The Vitis
vinifera genome contains genes encoding the enzymes responsible for trehalose
synthesis and degradation, and trehalose was found to be present in grapevine tissue,
after exposure to excessive chilling (Fernandez et al., 2012).
2.2.5. Factors influencing carbohydrate reserve distribution in grapevines
2.2.5.1. Seasonal development
Seasonal developmental stage or environmental conditions usually play a larger role in
the grapevine TNC reserve dynamics, than viticultural practices (Holzapfel & Smith,
2012). TNC allocation towards different vine parts fluctuates throughout the season,
mainly due to changes in the grapevine developmental stage, and the size and activity of
TNC sinks (Holzapfel & Smith, 2007). The berries, for example, become sinks for
carbohydrate assimilates at fruit set (Hale & Weaver, 1962), although the woody
tissues, e.g. the roots, also start to accumulate TNC around flowering (Holzapfel et al.,
2010). Furthermore, the berries are also strong TNC sinks between véraison and harvest
(Davies & Robinson, 1996). TNC reserve levels in the roots and trunks are usually the
highest during dormancy (at leaf fall), and the lowest after their utilisation for the
reestablishment of vegetative growth (just before flowering) (Zufferey et al., 2012).
TNC levels are then usually restored again and can be as high as at budbreak, around
two weeks after flowering (Mullins, Bouquet, & Williams, 1992).
The translocation of TNC, synthesised in the leaves, towards woody vine parts, usually
starts at the 10-leaf stage and becomes prominent four weeks after flowering (Yang &
Hori, 1979). However, the rates of leaf C assimilation can vary throughout the season,
Chapter 2: Literature review
24
with the peak rates of individual leaf photosynthesis attained about 40 days after
unfolding, with the rates declining gradually thereafter (Kriedemann, Kliewer, & Harris,
1970). Maximum canopy photosynthesis rates are often found at around véraison (Poni,
Intrieri, & Magnanini, 2000). Photosynthesis rates decline after harvest, but a
considerable amount of assimilates can still be accumulated during the post-harvest
period (Williams, 1996). After harvest, it is possible for the leaves to assimilate C for
two to three months prior to leaf fall, if conditions permit it (Scholefield et al., 1978).
The duration and conditions of the post-harvest period are therefore important for
reserve accumulation and for the next season’s early vegetative growth and reproductive
development (Holzapfel, Smith, Mandel, & Keller, 2006), and should be maintained by
good viticultural practices (e.g. irrigation management and disease spray programs to
retain the leaves).
2.2.5.2. Abiotic conditions
Any factor that affects leaf photoassimilation will impact on reserve TNC accumulation
(Holzapfel & Smith, 2012). The reserves are utilised whenever the source TNC supply
cannot meet the demand of the sinks. Abiotic conditions, such as water availability,
atmospheric temperature, and leaf CO2 supply, have a major influence on leaf (source)
photoassimilation.
The leaf photosynthesis of higher plants is strongly affected by atmospheric temperature
(Berry & Bjorkman, 1980), and fluctuations in the temperature therefore affect
grapevine leaf photosynthesis, and subsequently impact on TNC accumulation and
distribution. High atmospheric temperatures can inhibit starch accumulation in the
leaves, as this can also cause starch to convert to lipid-like material within the
Chapter 2: Literature review
25
chloroplasts (Buttrose & Hale, 1971). TNC reserve concentrations in grapevine organs
in warmer climate regions can generally be much higher than in the cooler regions
(Zufferey et al., 2012). In warmer regions, such as the Riverina in Australia
(Holzapfel et al., 2006), there is a much longer period between fruit maturity and leaf
fall than in cooler regions, such as Canterbury in New Zealand (Bennett et al., 2005),
leading to prolonged post-harvest TNC reserve accumulation in warmer climates.
The longer post-harvest period in warmer regions is also important to sustain the higher
fruit yields that are generally associated with such regions (Holzapfel et al., 2006).
In cool climates, because there is usually no substantial accumulation period
after harvest, the maintenance of reserves before harvest should be prioritised (Bennett
et al., 2005). Soil temperature also affects the utilisation and depletion of reserve
TNC from the roots (Field, Smith, Holzapfel, Hardie, & Emery, 2009; Rogiers,
Hardie, & Smith, 2011). Warmer soil temperatures between budbreak and flowering
can, for example, lead to depleted root starch (Field et al., 2009), and soil
temperature can therefore modulate root TNC mobilisation.
Leaf stomatal conductance is sensitive to atmospheric CO2 conditions, and the CO2
abundance in the atmosphere therefore affects photosynthesis (Farquhar & Sharkey,
1982). Elevated atmospheric CO2 concentrations have been shown to be a cause of
increased assimilate supply and subsequent greater plant TNC concentrations (Schultz,
2000). A combination of high temperatures and CO2 in the atmosphere can increase
plant photoassimilation and, except for the subsequent increased shoot and root growth
rates, it also leads to increased leaf starch concentrations (Kriedemann, Sward, &
Downton, 1976). However, long term exposure to increased CO2 may result in
decreased photosynthesis due to the likely sink saturation by assimilates (Samarakoon
& Gifford, 1995).
Chapter 2: Literature review
26
Plants perceive and respond to water constraints through a rapid alteration in gene
expression in conjunction with physiological and biochemical changes, and this can
even start occurring under mild to moderate stress conditions (Chaves, Flexas, &
Pinheiro, 2009; Gambetta, Matthews, Shaghasi, McElrone, & Castellarin, 2010). Under
mild to moderate water constraints, one of the first plant responses is a reduction in
stomatal conductance, thereby reducing water loss through transpiration, also restricting
photosynthesis and C assimilation (Chaves, Maroco, & Pereira, 2003). Stomatal closure
due to water stress is associated with increased abscisic acid (ABA) in the petiole xylem
and leaf blades, and an increased xylem pH, while decreased plant hydraulic
conductance could also be involved (Lovisolo et al., 2010). Under water constraints,
chemical compounds such as ABA, synthesised in the roots, act as long-distance
signals, inducing stomatal closure and/or restricting leaf growth (Chaves et al., 2010).
Water constraints thereby limit photosynthesis, and can cause photo-oxidative damage
in grapevines, which reduces the photosynthetic rate and weakens plant growth
(Hochberg, Degu, Fait, & Rachmilevitch, 2012). Grapevine C assimilation is therefore
limited by water constraints due to restricted canopy photoassimilation (Escalona et al.,
1999) and restricted canopy size (Hochberg et al., 2012).
The inhibition of leaf stomatal conductance is often observed following deficit
irrigation, causing a reduction in root TNC concentrations and also, to a lesser extent,
the TNC concentrations in the trunks and shoots (Holzapfel et al., 2010). Furthermore,
as mentioned, water stress can induce the enzymatic degradation of starch in plant
tissues, as previously observed in the leaves (Jacobsen, Hanson, & Chandler, 1986; Li
& Li, 2005) and roots (Regier et al., 2009) of various plant species. This subsequently
leads to sugar accumulation in those tissues, and water constraints were therefore
Chapter 2: Literature review
27
previously linked to the accumulation of soluble sugars in grapevine roots and trunks,
while the starch concentrations in these organs are decreased (Dayer, Prieto, Galat, &
Perez Peña, 2013; Rogiers, Holzapfel, et al., 2011). A reduction in starch-to-sugar ratios
therefore regularly corresponds with water constraints, and this could therefore induce
reduced starch reserve availability at budburst.
2.2.5.3. TNC Source and sink organ relations
The translocation of TNC requires sugar export from a site of production/synthesis (the
source organ, e.g. the photoassimilating leaves or the starch abundant roots), phloem
transport via an osmotic gradient, and a site of sugar export where the sugar is further
utilised (the sink organ, e.g. the sugar accumulating fruit or the starch storing roots).
The canopy leaf-to-fruit ratio of grapevines is a frequently quantified measurement,
used when studying the effects of source-sink relations on grapevine physiological
development (Kliewer & Dokoozlian, 2005; Santesteban & Royo, 2006; Zufferey et al.,
2015). It was previously suggested, following a comparative study of grapevines with a
wide range of leaf-to-fruit ratios in a given climatic region, that a leaf area-to-fruit ratio
of 8-12 cm2
leaf area/g fruit fresh weight is required for field grown wine grapes to
reach the maximum level of soluble solids, fruit weight, and skin colour when cultivated
on a single canopy type trellis system (Kliewer & Dokoozlian, 2005). However,
environmental conditions during the berry maturation period impact on how these ratios
affect the accumulation of quality parameters in the berries (e.g., juice soluble solid
concentration and fruit yield) (Howell, 2001). Furthermore, the abundance of TNC
reserves, and their availability to be utilised towards fruit sugar accumulation, will also
Chapter 2: Literature review
28
help determine the required leaf-to-fruit ratio that would promote maximum fruit sugar
accumulation.
Alterations in the grapevine leaf-to-fruit ratio at different stages of the season differently
affect the TNC reserve distribution and partitioning within grapevines and,
subsequently, also impact on vine vegetative and reproductive development. Early
defruiting may, for example, increase the total TNC concentrations in the roots, and lead
to improved bud fruitfulness in subsequent seasons (J. P. Smith & Holzapfel, 2009).
However, defoliation at harvest may decrease the root TNC concentration, and can
cause reduced fruit yields in subsequent seasons (J. P. Smith & Holzapfel, 2009).
Although reduced leaf-to-fruit ratios can lead to stimulated leaf level photosynthesis
(Candolfi-Vasconcelos & Koblet, 1991), this can also cause a reduction in whole-vine
photoassimilation due to the subsequent restricted canopy leaf area. Furthermore, the
exportation of leaf TNC benefits from the presence of sink organs, such as the fruit, as
well as a low leaf-to-fruit ratio (Zufferey & Murisier, 2005). Cropped grapevines were
previously found to have higher or similar stomatal conductance and photassimilation
rates than vines that were defruited (Downton, Grant, & Loveys, 1987; Naor, Gal, &
Bravdo, 1997). However, it is also possible that a larger crop load could increase leaf
transpiration, leading to enhanced root signalling, and a subsequent reduction in leaf
stomatal conductance (Naor et al., 1997). These impacts of crop load on stomatal
conductance, can subsequently affect the TNC reserve accumulation.
Defoliation can increase the net leaf stomatal conductance and CO2 exchange rate per
leaf area basis (Poni et al., 2008). However, defoliation can also cause the
remobilisation of TNC reserves from the perennial structure towards the fruit during
Chapter 2: Literature review
29
berry maturation, as described earlier (Candolfi-Vasconcelos et al., 1994). Excessive
defoliation causes a reduction in canopy photosynthesis, causing lower TNC reserve
levels in grapevine tissues at the end of the growing season (Bennett et al., 2005;
Candolfi-Vasconcelos et al., 1994; Holzapfel et al., 2006). Root TNC abundance is
more sensitive to grapevine source-sink balances than the TNC levels in other woody
grapevine parts (e.g. the trunk) (J. P. Smith & Holzapfel, 2009). Starch concentrations
in woody grapevine parts usually decrease due to defoliation, while there can be an
increase in soluble sugar content in these tissues. However, sugar concentrations in the
roots can also be unresponsive towards defoliation and fruit removal (J. P. Smith &
Holzapfel, 2009), or can even reduce because of early defoliation (Bennett et al., 2005).
High grapevine crop loads may cause a delay in fruit maturation, and a subsequently
shorter post-harvest period, which can reduce reserve TNC accumulation (Holzapfel &
Smith, 2012). In fact, high crop loads reduce the starch reserve concentration in
grapevine trunks (Dayer et al., 2013). It was also previously found that the starch and
soluble solid concentrations in grapevine roots and wood were the highest by fruit
maturity in defruited vines, and that partially defruited vines have higher concentrations
than higher yielding vines (J. P. Smith & Holzapfel, 2009).
Defruiting of grapevines at the onset of fruit maturation increases TNC concentrations
in the roots and, to a lesser extent in the trunks, and this can result in fruit yield
increases the following season (J. P. Smith & Holzapfel, 2009). Crop load reductions
before véraison can induce increased starch and sugar reserve concentrations in roots in
the following winter, and the requirement of less TNC reserves to support fruit sugar
accumulation perhaps contributes to this (Holzapfel et al., 2010). Very low sink
Chapter 2: Literature review
30
demands following excessive early defruiting, might cause increased vegetative growth
because of reduced competition for TNC between vegetative and reproductive sinks,
and this frequently happens mainly through lateral shoot growth (Mattii & Orlandini,
2004). The duration of the storage and accumulation periods of TNC throughout the
season, and the length of the post-harvest period for reserve accumulation, can vary due
to climatic and canopy conditions, but are also influenced by grapevine crop load, and
the subsequent fruit yield (Holzapfel et al., 2006).
2.3. Grapevine nitrogen
The supply of grapevine nitrogen (N) plays an essential role in sustaining vine growth
and development (Cheng et al., 2004). The status of N reserve availability early in the
season could largely determine the fruit yield and the vegetative growth of grapevines
(Cheng et al., 2004).
N is taken up from the soil as nitrate ions and ammonium. However, nitrate is thought
to be the preferred form of N uptake by grapevine roots, and ammonium can be toxic
when taken up at high concentrations (Roubelakis-Angelakis & Kliewer, 1992). Nitrates
enter root cells, and are reduced and incorporated into organic molecules, stored, or
translocated to other vine parts for further utilisation (Roubelakis-Angelakis & Kliewer,
1992). The reduction of nitrates to ammonia results in a useable form of N, and nitrate
reductase (NR) catalyses the first step in nitrate reduction in higher plants (Roubelakis-
Angelakis & Kliewer, 1992). The Glutamine synthetase/glutamate synthase pathway
(GS/GOGAT) is the primary route of ammonia assimilation, while glutamate
dehydrogenase may also play a role. Amino acids are the first products of ammonia
assimilation, and are the building blocks of proteins, also playing roles in the regulation
Chapter 2: Literature review
of metabolism, N transport, and the storage of N (Roubelakis-Angelakis & Kliewer,
1992). Ammonium is first assimilated into glutamine and glutamic acid, and then,
through aminotransferase reactions, into other amino acids, such as aspartic acid and
asparagine.
N fertilisation, and the timing thereof, not only affects N reserve accumulation, but also
the partitioning and concentration of fruit N. Soil application of N at bloom, for
example, promotes the allocation of N towards annual grapevine tissues, while the
application after set could promote N accumulation in perennial vine parts (Holzapfel,
Watt, Smith, Suklje, & Rogiers, 2015). Furthermore, N application two weeks after
véraison could lead to increased berry juice yeast assimilable N (YAN) content,
which benefits the fermentation process during winemaking, and improves must
quality (Holzapfel et al., 2015).
The spring growth of grapevines depends on N reserve remobilisation from the roots,
as N uptake is usually still insufficient during this time of the season (Zapata et al.,
2004). This remobilisation during early spring growth accounts for most N
distribution until flowering (Zapata et al., 2004). The fruit, leaves and shoots are all
considerable N sinks between flowering and véraison (Conradie, 1991; Roubelakis-
Angelakis & Kliewer, 1992), and sufficient soil N availability during this period is
therefore important. Between véraison and harvest, when N uptake is reduced or absent,
redistribution of N from the roots, shoots and leaves towards the bunches take place
(Conradie, 1991). The assimilation of root N for the overwintering reserve N storage is
important late in the growing season, as these reserves are, as mentioned, crucial for
early vine development the next season, when N soil uptake is still insufficient (Cheng et
31
Chapter 2: Literature review
al., 2004). Root N reserve replenishment can start from about one month after
flowering (Bates, Dunst, & Joy, 2002).
2.3.1. Roles of N in grapevines
N plays a central role in plant metabolism, as it is a constituent of proteins, nucleic
acids, chlorophyll, co-enzymes, phytohormones, and secondary metabolites
(Hawkesford et al., 2012). The photosynthetic capacity of leaves is related to its N
content, as proteins involved in photosynthesis represent the majority of leaf N (Evans,
1989). N availability is required for plant growth, and sufficient N availability during
vine vegetative growth leads to increased leaf area, fruit yield, and overall shoot
development, while it also enhances leaf CO2 assimilation (Cheng et al., 2004). Higher
N availability can lead to increased vegetative growth (Smart & Robinson, 1991);
although it could also induce increased fruit yield (Benz, Bogdanoff, & Kliewer, 1991),
given that the N supply is not excessive (Ahmedullah & Roberts, 1991). Low N
availability around bloom causes reduced fruit set, and N availability at bloom is a
major determinant of fruit yield (Keller, Arnink, & Hrazdina, 1998).
N reserves are predominantly stored in the roots of grapevines, and consist of a range of
amino acids (mainly arginine), and proteins (Xia & Cheng, 2004; Zapata et al.,
2004). Proteins are used for structure, metabolism and N storage, and storage
proteins are therefore important for N metabolism, and play a role in the overwintering
N storage in deciduous plants (Roubelakis-Angelakis & Kliewer, 1992). N reserves are
essential for spring growth, as the early vegetative growth might even be more
responsive towards N reserve availability, than the availability of TNC reserves (Cheng
et al., 2004).
32
Chapter 2: Literature review
Shoot growth before flowering depends on N mobilisation from root reserves,
and the accumulation of N into the storage sinks of perennial organs prior to harvest is
therefore important for vine development the following season (Weyand & Schultz,
2006; Zapata et al., 2004). The vegetative and reproductive growth of young
Concord vines were found to be largely determined by the N reserve status, and N
supply sustains shoot and leaf growth, and contributes to fruit development from fruit
set (Cheng et al., 2004).
2.3.2. Fruit N accumulation
Fruit N content is influenced by scion and rootstock genetics, fruit maturity,
atmospheric temperature, mineral nutrition availability, fruit crop level, canopy
management practices, and disease prevalence (Roubelakis-Angelakis & Kliewer,
1992). However, it is suggested that fruit N accumulation peaks at two stages, one
pre-véraison (just before pea-size) and one just after véraison (Roubelakis-
Angelakis & Kliewer, 1992). The availability of N during the growing season can
therefore directly impact berry nitrogenous compounds, and can also
indirectly impact berry juice composition through the effects of N availability on
canopy vegetative growth and fruit yield (Smart & Robinson, 1991).
Fruit N accumulation is crucial for juice YAN concentrations, which
impact fermentation and wine composition. YAN in the must is composed of free
assimilable amino N (FAN) and ammonium. Low N supply reduces YAN, but
also leads to undesirable thiols and higher alcohols and lower concentrations of esters
and long chain volatile fatty acids in wine (Bell & Henschke, 2005).
33
Chapter 2: Literature review
34
However, excessively high fruit N content leads to increased ethyl acetate and acetic
acid, ethyl carbamate and biogenic amines in wine (Bell & Henschke, 2005). Arginine,
phenylalanine, histidine, valine, and glutamic acid in berry juice usually contribute the
towards berry juice amino N, useful towards fermentation by the yeasts
(Roubelakis-Angelakis & Kliewer, 1992). It is suggested that about a minimum of
130 mg YAN is required per litre of must, in order for the yeast to complete must
fermentation during winemaking (Agenbach, 1977). The composition of amino acids
in the must is also important for the development of aromatic compounds during
winemaking, as various amino acids provide C skeletons, utilised in the biosynthesis
of the aromatic compounds (Bell & Henschke, 2005).
2.3.3. Factors influencing N distribution
Any factor affecting N sink and source functioning, e.g. abiotic factors and management
practices (including crop load, leaf area, water availability, and disease prevalence), will
likely affect the N status of the grapevine, and subsequently the vine development the
following season (Cheng et al., 2004).
2.3.3.1. Seasonal grapevine development
No remarkable root N uptake from the soil takes place prior to budbreak, however,
significant uptake starts soon after budbreak, and peaks at four weeks post-flowering.
Soil N uptake can also take place soon after véraison (Löhnertz, 1991), and another
peak period of N uptake takes place shortly after harvest (Roubelakis-Angelakis &
Kliewer, 1992). Total N reserve content in permanent grapevine tissues (e.g. the roots)
decreases at budbreak, as it is utilised towards spring growth, and some reserve
accumulation starts again from around flowering (Zapata et al., 2004). When vegetative
Chapter 2: Literature review
35
growth slows down at the end of the season, the N taken up from the soil is primary
stored as reserves (Cheng et al., 2004), and the perennial vine parts therefore usually
start accumulating N reserves before berry maturation (Roubelakis-Angelakis &
Kliewer, 1992).
The highest concentrations and greatest fluctuations in N are thought to be found in the
roots, where N content stays constant early in the season and increases thereafter
(Roubelakis-Angelakis & Kliewer, 1992). Early in the season, N is remobilised from the
roots to support the vegetative growth in the shoots and leaves (Peacock, Christensen, &
Broadbent, 1989). In the shoots, N concentrations increase after budbreak and stay
constant towards the end of the vegetative growth period, and then increase again due to
retranslocation of N from senescing leaves (Roubelakis-Angelakis & Kliewer, 1992).
From budbreak, abundant N accumulation takes place in the leaves, and maximum leaf
N is found at full leaf expansion, while leaf N decreases towards the end of the growing
period (Roubelakis-Angelakis & Kliewer, 1992). Shoot and leaf N can be intermediate
N reservoirs between the roots and berries (Conradie, 1986).
2.3.3.2. Abiotic conditions
Water availability, and N fertilisation are the main drivers of N uptake, and water
constraints can limit N uptake by the roots (Keller, 2005). However, the metabolism and
distribution of nitrate and ammonium ions, and the partitioning of N in plants, are also
affected by other abiotic factors, such as light intensity and temperature, which may
particularly affect N partitioning related enzymes (Roubelakis-Angelakis & Kliewer,
1992).
Chapter 2: Literature review
36
Reduced water supply can result in the higher allocation of N to perennial structures,
and less to annual components of the vine (Holzapfel et al., 2015). Water stress also
reduces the activity of nitrate reductase in the leaves, due to a decreased nitrate flux
(Thomas & Stoddart, 1980), and this can have repercussions on N assimilation by the
plant. A number of N containing compounds accumulate in plants following abiotic
stress conditions (such as high salinity). These include amino acids, amids, imino acids,
proteins, quarternary ammonium compounds, and polyamines. The accumulation of
these compounds is suggested to be involved in cell osmotic adjustment, protection of
cellular macromolecules, storage of N, detoxification of cells, and the scavenging of
free radicals under stressful conditions (Mansour, 2000). The impact of abiotic
conditions on the abundance of certain amino acids within plants, is discussed later.
2.3.3.3. N Source and sink organ relations
The distribution of N requires a site of N storage (e.g. the leaves and roots) or the site of
soil N uptake (the roots) (i.e. the source organ), organic N transport, e.g., amino acid
xylem and phloem transport, and a site of N importation (e.g. N accumulating fruit or N
storing roots).
Vines developing a large vegetative vigour, depend less on N reserves and more on
current N supply (Cheng et al., 2004). However, early vine vegetative defoliation can
cause decreased total N content in grapevines at dormancy, and this happens as
mobilisation of N from leaves to storage tissues is interrupted, along with a decreased
root N uptake (Cheng et al., 2004).
Chapter 2: Literature review
37
The presence of fruit is also described to cause reduced N assimilation in grapevine
roots (Morinaga, Imai, Yakushiji, & Koshita, 2003), while higher crop loads cause
reduced N storage, at least as found in grapevine canes (Balasubrahmanyam, Eifert, &
Diofasi, 1978). The fruit of vines with low crop loads tends to have higher
concentrations of total N, arginine, proline, and total free amino acids (Kliewer & Ough,
1970).
Source-sink relations can greatly impact leaf N dynamics, and leaf senescence is often
associated with leaf N deficiency as a consequence of the remobilisation of leaf N
towards the development of reproductive organs (i.e., the fruit) (Thomas & Stoddart,
1980). N from the leaves that accumulated early in the season, is later used for
reproductive development, however, if the inflorescence or the developing fruit are
removed, leaf senescence can be delayed, or even reversed in some plant species
(Thomas & Stoddart, 1980). N remobilisation from the leaves of higher plants is related
to the biomasses of the source and sink organs (Diaz et al., 2008).
2.3.4. Amino acids
Major roles of amino acids in plants include their involvement in the regulation of N
metabolism, N transport, osmotic regulation, regulation of ion transport, and the storage
of N (Lam, Coschigano, Oliveira, Melo-Oliveira, & Coruzzi, 1996; Rai, 2002;
Roubelakis-Angelakis & Kliewer, 1992). Amino acids are also involved in modulating
stomatal opening, and the detoxification of heavy metals, while they also affect the
synthesis and activity of some enzymes, and impact on gene expression and redox
homeostasis (Rai, 2002). Amino acids can accumulate in high levels in the leaves and
Chapter 2: Literature review
38
roots of higher plants, and are transported in the xylem and phloem to sink organs
(Fischer et al., 1998).
One of the major pathways involved in amino acid metabolism in higher plants involves
α-ketoglutaric acid metabolism, and this pathway yields important plant amino acids
with well-defined functioning, i.e., glutamic acid, glutamine, arginine, and proline
(Verma, Zhang, & Singh, 1999). Glutamine and glutamic acid have known transport
roles (Coruzzi & Last, 2000) and are, in fact, major transporters of organic N from
source to sink tissues in many plants (Lam et al., 1996). Glutamic acid is also the
intermediate product of nitrate reduction and ammonium assimilation, and a key amino
donor in the synthesis of many other amino acids (Fritz, Mueller, Matt, Feil, & Stitt,
2006). In fact, glutamic acid is the substrate for the synthesis of glutamine from
ammonia and, in addition, the α-amino group of glutamic acid is transferred to all other
amino acids, and the C skeleton of glutamic acid forms the basis for the synthesis of γ-
aminobutyric acid (GABA), arginine and proline (Forde & Lea, 2007).
Arginine is one of the more abundant amino acids in grapevines, and is a major N-
storage compound, and participates in the biosynthesis of other amino acids
(Roubelakis-Angelakis & Kliewer, 1992). Arginine is actually the main form of storage
N in grapevines, and is the most abundant amino acid in both free amino acids and
protein in grapevine roots (Xia & Cheng, 2004). It is due to arginine’s high N:C ratio
(4:6) that it acts as a major N storage compound in higher plants. Besides the known N
transport role of glutamine in higher plants, it is also suggested that arginine and
asparagine could be involved as N transport compounds in various plants (Lea, Sodek,
Parry, Shewry, & Halford, 2007).
Chapter 2: Literature review
39
Proline accumulation in plants is suggested to frequently occur in response to biotic and
abiotic stresses. Proline is involved in cellular osmotic adjustment, the stabilisation of
subcellular structures, and the scavenging of free radicals (Hare & Cress, 1997). Free
proline reduces cellular water potential, and thereby serves as an osmotic regulator to
maintain turgor pressure in plant cells (Hare & Cress, 1997). Plants subjected to abiotic
stresses therefore typically accumulate certain amino acids, such as proline. Proline
accumulation is frequently described to coincide with plant water constraints in many
species, thereby promoting plant abiotic stress tolerance (Bertamini, Zulini,
Muthuchelian, & Nedunchezhian, 2006; Rai, 2002; Singh, Aspinall, & Paleg, 1972).
Arginine and proline (proline is not assimilated by yeast), are usually the predominant
amino acids in the must during winemaking (Bell & Henschke, 2005). Seasonal water
deficits elevate proline concentration, and also that of other amino N in the must, and
pre véraison water constraints can increase YAN in the berries (Hannam, Neilsen,
Forge, & Neilsen, 2013). Water constraints between flowering and harvest also increase
the YAN concentration of berry juice at harvest (Holzapfel et al., 2015). However,
partial rootzone drying (PRD) and regulated deficit irrigation can lower berry proline
and arginine concentrations (Wade, Holzapfel, Degaris, Williams, & Keller, 2002). The
composition and abundance of amino acids in the berries also affect flavour
development during winemaking (Bell & Henschke, 2005).
Another important pathway essential for amino acid biosynthesis in plants involves
shikimic acid metabolism (Haslam, 1993). This pathway yields the aromatic amino
acids, i.e., phenylalanine, tryptophan and tyrosine, which are essential compounds for
protein synthesis in plants, and serve as precursors for various secondary metabolites,
Chapter 2: Literature review
40
essential for plant growth (Maeda & Dudareva, 2012; Tzin & Galili, 2010).
Phenylalanine act as a precursor for phenylpropanoids, flavonoids, ligin, and
anthocyanins. Tyrosine is a precursor of various secondary metabolites, including
alkaloids, and several non-protein amino acids. Tryptophan is catabolised into indole-
containing secondary metabolites, such as auxin, and tryptamine derivatives (Tzin &
Galili, 2010). The metabolic pathway facilitating the synthesis of the aromatic amino
acids, is a major determinant of the phenolic content in the berries, affecting crucial
quality parameters (e.g., colour intensity and flavour). In fact, phenylalanine ammonia-
lyase enzyme activity plays a major role in this pathway, and is involved in the
catabolism of phenylalanine, thereby ultimately contributing to the biosynthesis of
anthocyanins in the berries (Boss, Davies, & Robinson, 1996).
Other amino acids that are present in different tissues of grapevines include alanine, β-
alanine, aspartic acid, asparagine, glycine, serine, threonine, valine, histidine,
methionine and lysine (Cuadros-Inostroza et al., 2016; Hochberg, Batushansky, et al.,
2015; Holzapfel et al., 2015).
2.4. Concluding remarks
The berry maturation period (between véraison and fruit maturity) is an important stage
in the annual grapevine developmental cycle, a period when strong sink organ
competition for plant metabolites and nutrients exists. The maturing fruit are strong
sinks for TNC and N, and the roots and leaves are potential sources of both these
compounds during berry maturation. However, the roots are also sinks for TNC and N
before dormancy, as these compounds are redistributed from the roots early in the next
season, to be utilised for spring vegetative and reproductive development.
Chapter 2: Literature review
41
Water availability and grapevine leaf-to-fruit relations are major determinants of the
availability, distribution, and partitioning of TNC and N reserves in the grapevine.
These factors could contribute to determining the abundance of TNC and N in both the
fruit and the storage organs, throughout berry maturation.
The existing literature in regards to grapevine TNC and N dynamics, is centred around
the concentrations of TNC and N at distinct stages of the annual growth cycle, in
specific organs. In addition to the existing literature, more work is required to
understand the kinetics of the allocation and distribution of TNC and N between
different grapevine organs, and the partitioning thereof within the organs, during the
berry maturation period. Such work will be essential in explaining the links between the
availability of reserves of TNC (especially in the roots) and N (especially in the leaves
and roots), and the accumulation of these compounds in the fruit. As major drivers of
TNC and N metabolism in higher plants, the water status, crop load, and leaf area
variability of grapevines during the berry maturation period, could supply additional
value in understanding the allocation of TNC and N between storage and temporary
grapevine sinks.
2.5. Literature cited
Agenbach, W. A. (1977, November). A study of must nitrogen content in relation to
incomplete fermentations, yeast production and fermentation activity.
Proceedings of the South African Society for Enology and Viticulture (pp. 66-
88). South African Society of Enology and Viticulture: Stellenbosch, South
Africa.
Ahmedullah, M., & Roberts, S. (1991, June). Effect of soil-applied nitrogen on the yield
and quality of Concord grapevines. Proceedings of the International Symposium
on Nitrogen in Grapes and Wine (pp. 200-201). American Society for
Enology and Viticulture: Seattle, WA.
Araujo, F., Williams, L. E., & Matthews, M. A. (1995). A comparative study of young
‘Thompson Seedless’ grapevines (Vitis vinifera L.) under drip and furrow
Chapter 2: Literature review
42
irrigation. II. Growth, water use efficiency and nitrogen partitioning. Scientia
Horticulturae, 60(3–4), 251-265.
Balasubrahmanyam, V., Eifert, J., & Diofasi, L. (1978). Nutrient reserves in grapevine
canes as influenced by cropping levels. Vitis, 17(1), 23-29.
Bates, T. R., Dunst, R. M., & Joy, P. (2002). Seasonal dry matter, starch, and nutrient
distribution in 'Concord' grapevine roots. HortScience, 37(2), 313-316.
Bell, S.-J., & Henschke, P. A. (2005). Implications of nitrogen nutrition for grapes,
fermentation and wine. Australian Journal of Grape and Wine Research, 11(3),
242-295.
Bennett, J., Javis, P., Creasy, G. L., & Throught, M. C. T. (2005). Influence of
defoliation on overwintering carbohydrate reserves, return bloom, and yield of
mature Chardonnay grapevines. American Journal of Enology and Viticulture,
56(4), 386-393.
Benz, M., Bogdanoff, C., & Kliewer, W. M. (1991, June). Responses of Thompson
seedless grapevines trained to single and divided canopy trellis systems to
nitrogen fertilization. Proceedings of the International Symposium on Nitrogen
in Grapes and Wine (pp. 282-289). American Society for Enology and
Viticulture: Seattle, WA.
Berry, J., & Bjorkman, O. (1980). Photosynthetic response and adaptation to
temperature in higher plants. Annual Review of Plant Physiology, 31(1), 491-
543.
Bertamini, M., Zulini, L., Muthuchelian, K., & Nedunchezhian, N. (2006). Effect of
water deficit on photosynthetic and other physiological responses in grapevine
(Vitis vinifera L. cv. Riesling) plants. Photosynthetica, 44(1), 151-154.
Boss, P. K., Davies, C., & Robinson, S. P. (1996). Analysis of the expression of
anthocyanin pathway genes in developing Vitis vinifera L. cv Shiraz grape
berries and the implications for pathway regulation. Plant Physiology, 111(4),
1059-1066.
Bray, E. A. (1997). Plant responses to water deficit. Trends in Plant Science, 2(2), 48-
54.
Buttrose, M., & Hale, C. (1971). Effects of temperature on accumulation of starch or
lipid in chloroplasts of grapevine. Planta, 101(2), 166-170.
Çakir, B., Agasse, A., Gaillard, C., Saumonneau, A., Delrot, S., & Atanassova, R.
(2003). A grape ASR protein involved in sugar and abscisic acid signaling. The
Plant Cell, 15(9), 2165-2180.
Candolfi-Vasconcelos, M. C., Candolfi, M. P., & Koblet, W. (1994). Retranslocation of
carbon reserves from the woody storage tissues into the fruit as a response to
defoliation stress during the ripening period in Vitis vinifera L. Planta, 192(4),
567-573.
Candolfi-Vasconcelos, M. C., & Koblet, W. (1991). Influence of partial defoliation on
gas exchange parameters and chlorophyll content of field-grown grapevines-
Mechanisms and limitations of the compensation capacity. Vitis, 30, 129-141.
Canny, M. (1998). Transporting Water in Plants Evaporation from the leaves pulls
water to the top of a tree, but living cells make that possible by protecting the
stretched water and repairing it when it breaks. American Scientist, 152-159.
Chaves, M. M., Flexas, J., & Pinheiro, C. (2009). Photosynthesis under drought and salt
stress: regulation mechanisms from whole plant to cell. Annals of Botany,
103(4), 551-560.
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43
Chaves, M. M., Maroco, J. P., & Pereira, J. S. (2003). Understanding plant responses to
drought - from genes to the whole plant. Functional Plant Biology, 30(3), 239-
264.
Chaves, M. M., Zarrouk, O., Francisco, R., Costa, J., Santos, T., Regalado, A., . . .
Lopes, C. (2010). Grapevine under deficit irrigation: Hints from physiological
and molecular data. Annals of Botany, 105(5), 661-676.
Cheng, L., Xia, G., & Bates, T. (2004). Growth and fruiting of young 'Concord'
grapevines in relation to reserve nitrogen and carbohydrates. Journal of the
American Society for Horticultural Science, 129(5), 660-666.
Conradie, W. (1980). Seasonal uptake of nutrients by Chenin blanc in sand culture: I.
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Williams, L. E. (1996). Grape. In E. Zamski & A. Schaffer (Eds.), Photoassimilate
distribution in plants and crops. Source-sink relationships (pp. 851-881). New
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Winkler, A. J., & Williams, W. O. (1938). Carbohydrate metabolism of Vitis vinifera:
Hemicellulose. Plant Physiology, 13(2), 381-390.
Xia, G., & Cheng, L. (2004). Foliar urea application in the fall affects both nitrogen and
carbon storage in young `Concord' grapevines grown under a wide range of
nitrogen supply. Journal of the American Society for Horticultural Science,
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Zufferey, V., & Murisier, F. (2005, August). Leaf to fruit ratio and photosynthetic
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Chapter 3: Paper 1
Carbohydrate distribution during berry ripening of potted
grapevines: Impact of water availability and leaf-to-fruit ratio
(Paper 1 has been published in Scientia Horticulturae as in the format below.)
3.1. Main objective for paper 1
To investigate the interactive effects of the leaf-to-fruit ratio and grapevine water status
during two phases of berry sugar accumulation (rapid and slow) on the carbohydrate
distribution between the different grapevine organs.
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Scientia Horticulturae 216 (2017) 215–225
Contents lists available at ScienceDirect
Scientia Horticulturae
journa l homepage: www.e lsev ier .com/ locate /sc ihor t i
Carbohydrate distribution during berry ripening of potted grapevines:Impact of water availability and leaf-to-fruit ratio
Gerhard C. Rossouwa,b,∗, Jason P. Smitha,1, Celia Barril a,b, Alain Deloirea,2,Bruno P. Holzapfela,c
a National Wine and Grape Industry Centre, Wagga Wagga 2678, New South Wales, Australiab School of Agriculture and Wine Sciences, Charles Sturt University, Wagga Wagga 2678, New South Wales, Australiac New South Wales Department of Primary Industries, Wagga Wagga 2678, New South Wales, Australia
a r t i c l e i n f o
Article history:Received 24 May 2016Received in revised form 3 January 2017Accepted 6 January 2017Available online 16 January 2017
Keywords:Carbon translocationWater stressLeaf areaCrop loadStarch reserveFruit maturation
a b s t r a c t
Insufficient leaf photoassimilation could allow mobilized carbohydrate reserves to contribute to berrysugar accumulation. However, the extent of this contribution during rapid and slow berry sugar accumu-lation is undefined. The potential effect of leaf-to-fruit ratio and water availability on carbohydrate reservedistribution in potted Tempranillo grapevines was examined during berry maturation. Within each leaf-to-fruit ratio treatment (full and 50% leaves), vines were grown under full or 50% reduced irrigationregimes. Dry biomass development, and the starch and soluble sugar concentrations were determinedin the roots, trunks, stems and leaves. Berry sugar and anthocyanin accumulation were also assessed.Under full irrigation, no starch remobilization from roots was observed, regardless of the leaf-to-fruitratio. Under reduced water supply, starch remobilization from roots was concurrent with rapid berrysugar accumulation, especially in grapevines with low leaf-to-fruit ratio. Soluble sugar accumulationcoincided with starch depletion in the roots of grapevines under reduced water availability. When berrysugar accumulation slowed, an increase in carbohydrates was observed in the roots. Sustained waterconstraints during rapid berry sugar accumulation resulted in a forced reliance on stored carbohydratesto support berry sugar accumulation, but did not significantly alter the tempo of berry sugar and antho-cyanin accumulation. A reduced leaf-to-fruit ratio intensified the reliance of fruit sugar accumulation onstored carbohydrates. Besides the importance of post-harvest carbohydrate reserve replenishment whenroot carbohydrate reserves are depleted during berry maturation, the reserves are also refilled duringmaturation when berry sugar accumulation slows. This study showed distinctly that root carbohydratereplenishment could already start a few weeks before harvest, and this replenishment could be importantwhen the post-harvest carbon assimilation period is ineffective.
© 2017 Elsevier B.V. All rights reserved.
1. Introduction
Leaf-to-fruit ratio and vine water status are parameters likelyto influence vine carbon balance during berry maturation (i.e. theberry sugar accumulation phase). Abiotic factors such as tempera-ture, light intensity, and water could limit vine carbon assimilationby restricting leaf photoassimilation (Escalona et al., 1999), whilereduced leaf-to-fruit ratios, up to a point, could result in an increase
∗ Corresponding author at: National Wine and Grape Industry Centre, WaggaWagga 2678, New South Wales, Australia.
E-mail address: [email protected] (G.C. Rossouw).1 Present address: Institut für Allgemeinen und ökologischen Weinbau,
Hochschule Geisenheim University, Geisenheim 65366, Germany.2 Present address: Montpellier SupAgro, Montpellier 34060, France.
of leaf photosynthetic activity (Candolfi-Vasconcelos and Koblet,1991). However, the importance of the contribution of root car-bohydrate reserves to support berry sugar accumulation underdiffering leaf-to-fruit ratios and grapevine water status is still aresearch question.
Carbohydrates are synthesized by plants through leaf photosyn-thesis and the effect of the abiotic factors in association with thevine internal competition for carbon, can affect the dynamics ofnon-structural carbohydrate reserve storage within the grapevine(Holzapfel and Smith 2012). These reserves are distributed tothe different plant organs, and the concentration and partitioningwithin different organs vary throughout the growing season. Thedistribution of carbohydrates could be affected by soil water avail-ability (soil depth, root implementation and functioning) and soil
http://dx.doi.org/10.1016/j.scienta.2017.01.0080304-4238/© 2017 Elsevier B.V. All rights reserved.
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temperature (Dayer et al., 2013; Field et al., 2009; Rogiers et al.,2011a).
Studies have shown that grapevines with higher crop load, andthose subjected to elevated water constraint, exhibit reduced car-bohydrate reserve concentrations at budburst the following season.Water constraints during the growing season (pre-dawn leaf waterpotential values below −0.6 MPa), and high crop loads (leaf-to-fruitratios below 8 cm2 leaf area per gram of fruit), have been reportedto cause reduced starch concentrations in grapevine trunks duringthe dormancy period (Dayer et al., 2013). Furthermore, defruiting atthe onset of fruit ripening increases total non-structural carbohy-drate (TNC) concentration in the roots in subsequent seasons, whilea complete defoliation at harvest reduces it (Smith and Holzapfel2009). No previous studies, to the best of our knowledge, have how-ever investigated the potential effect of the interaction betweenwater availability and leaf-to-fruit ratio on the distribution of non-structural carbohydrate content between the different grapevineorgans, during the berry sugar accumulation phase.
Non-structural carbohydrates provide energy and carbon forgrapevine growth, and/or are stored as reserves in perennial plantorgans. The stored carbohydrates are used for early season veg-etative growth until leaf photosynthesis becomes the primarysource of carbon, generally around flowering for the grapevine(Zapata et al., 2004). Carbohydrate reserves are also utilizedtowards the reproductive development, including supporting berrysugar accumulation, as confirmed by 14C tracing studies (Candolfi-Vasconcelos et al., 1994). Woody tissues, especially perennial roots,also start to accumulate carbohydrates from anthesis, and the cropload influences the continuation of the perennial reserve accumu-lation during grape maturation (Holzapfel et al., 2010). Due to theinvolvement of carbohydrate reserves in these various functions,strong competition is expected to exist between the different sinksfrom véraison (berry softening) and the end of berry sugar accu-mulation (Davies and Robinson 1996; Wang et al., 2003a).
Carbohydrates are mainly stored as starch in grapevine roots,and this starch can be hydrolyzed to form soluble sugars. Duringdry conditions, the activity of starch-degrading enzymes, such as�-amylase, is often found to increase in plant tissues, resulting instarch breakdown, and an increase in soluble sugar concentrations(Jacobsen et al., 1986; Li and Li 2005). The ratio of starch to solublesugars has been reported to decrease in grapevine perennial organsduring water constraints (Rogiers et al., 2011b), as well as follow-ing early (Bennett et al., 2005) or late season (Smith and Holzapfel2009) defoliation.
Similar to sugar accumulation, fruit anthocyanin accumulationalso commences at véraison, and normally continues throughoutberry maturation (Boss et al., 1996). The accumulation of antho-cyanins in the berries is an important contributor to the qualityof the fruit from a wine quality perspective. The grapevine waterstatus is one of the major factors known to affect sugar (Wanget al., 2003b) and anthocyanin (Ojeda et al., 2002) accumulationin ripening berries.
The aim of this study was to investigate the interactive effectsof leaf-to-fruit ratio and vine water status during the berry sugaraccumulation stage on carbohydrate partitioning in perennial andannual grapevine organs. Although starch and/or soluble sugar con-centrations at certain stages of the annual grapevine growth cycle(mainly at dormancy, budburst or harvest) have been predomi-nantly reported for the roots and trunks, the kinetic of whole-vineTNC content distribution during the berry sugar accumulationphase is still a research question. The first goal was to determine thecombined effect of water constraint and limited leaf-to-fruit ratioon the TNC allocation to perennial and vegetative organs duringthe berry sugar accumulation phase. The second goal was to quan-tify the contribution of remobilized starch reserves towards berrysugar content when whole vine leaf photoassimilation becomes
insufficient for sink demands during berry maturation. The last goalwas to investigate how the accumulation of fruit sugar and antho-cyanins responds when a greater reliance is placed on the starchreserves to support berry sugar accumulation. Experiments wereconducted on grapevines grown in large pots, allowing the analy-sis of whole grapevines and individual organ biomass, including thewhole root systems, where carbohydrate distribution was deter-mined as affected by the different treatments (leaf-to-fruit ratioand water availability).
2. Materials and methods
2.1. Experimental design and treatments
Forty own-rooted Vitis vinifera L. cv Tempranillo (clone D8V12)grapevines were used in the 2013/2014 growing season, plantedin commercial potting mix soil in 50 L pots. The grapevines weregrown in an outside bird proof cage in the warm to very warmclimate of the Riverina region (Wagga Wagga, New South Wales,Australia). The three-year-old grapevines were spur pruned to fourtwo-bud spurs in the winter, left with eight primary shoots each,and distributed in four rows of ten vines each, with a three-wiretrellis system installed to support the vegetative growth. At fruitset, the total amount of bunches and berries per vine were counted,and vines were crop thinned just after fruit set so that all grapevineswere left with six to seven bunches, totaling 400 berries per vine.Prior to the application of the treatments at the onset of véraison(very first sign of berry softening), four randomly selected vines,one per row, were destructively harvested in order to representT0 for the population of grapevines. After removal of the four ini-tial vines through destructive harvesting, the nine remaining vinesper row were evenly spaced out in the row, resulting in a fourrow by nine column array, containing three treatment replicates.Two irrigation treatments, two defoliation treatments, and threedestructive harvest dates were randomized in the block design.Pressure compensated drip emitters (4 L/hr each) were used for irri-gation during the experiment. Rainfall, atmospheric temperature,and relative humidity were recorded and collected from an on-siteweather station and vapor pressure deficit (VPD) was calculated.Environmental conditions were summarized for three fortnightlyintervals during the experiment, referred to as intervals 1, 2 and 3.
In order to study the interaction between either low or high leaf-to-fruit ratios, and either low or high water availability throughoutthe berry sugar accumulation phase, four distinct treatments wereapplied, i.e., low leaf-to-fruit ratio and low water availability(LowL/F:50%); low leaf-to-fruit ratio and high water availability(LowL/F:100%); high leaf-to-fruit ratio and low water availability(HighL/F:50%); high leaf-to-fruit ratio and high water availability(HighL/F:100%). Vines with a low leaf-to-fruit ratio were left with50% less leaves than those with a high ratio (40 vs 80 leaves). Everysecond leaf from the base of each shoot was removed until each vinehad the desirable amount of leaves. Vines were irrigated three timesa day (0730, 1400 and 1800 h), with equal water volume appliedeach time of the day, ranging between 12 and 20 min of irrigationapplication time per irrigation event. The higher water availabilitytreatment was conducted with the aim of watering pots each dayjust to the point of first visual free draining during the midday irri-gation, via two irrigation emitters located to the left and right ofa vine near the middle of each pot. In the lower water availabilitytreatment, 50% of the water was delivered over the same period,through one irrigation emitter in the middle of the pot. Vines ofboth leaf-to-fruit ratio treatments received the same water volumewithin each irrigation treatment. Secondary shoots (laterals), andany newly formed leaves from primary shoots during the course of
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the experiment, were removed daily as soon as the regrowth wasobserved.
At the fortnightly destructive harvest dates, i.e., véraison (27 Dec2013, i.e., V), V + 14 (10 Jan 2014), V + 27 (23 Jan 2014) and V + 40(5 Feb 2014), the pre-selected grapevines were dismantled. Wholeroot systems, trunks, spurs, stems, petioles, and leaves were sep-arated, collected and washed with phosphate-free detergent andrinsed with deionized water. Leaves were collected in the morn-ing between 0800 and 1000 h on each of the destructive harvestdates. The fresh weights of these organs were determined, and thesamples were oven dried at 60 ◦C until a constant dry weight wasreached.
2.2. Leaf-to-fruit ratio and berry composition
The total leaf area of all the leaves that were sampled from eachindividual vine at the respective destructive harvest dates was mea-sured using a leaf area meter (LLI-3100C, LI-COR Biosciences Inc.,Lincoln, Nebraska, USA). The total fruit weight of each grapevinewas also recorded, and the leaf-to-fruit ratios determined. A 50berry subsample per vine was oven dried at 60 ◦C until constantweight, and total vine fruit dry weight determined. The averagesoluble solid content per berry per vine was determined in a sub-sample of 50 representative berries, on the basis of berry freshweight and juice soluble solid concentration (◦Brix).
Berry anthocyanin concentration was analyzed from a 50 berrysubsample per vine. The whole berries were homogenized (Ultra-Turrax T25, IKA, Selangor, Malaysia), and the phenolic compoundsextracted from 1 g homogenate in 10 mL 50% ethanol (pH 2)for two hours. The samples were centrifuged at 3000 rpm for10 min, and 1 mL supernatant was added to 9 mL HCl and leftfor 3 h at room temperature. The absorbance was measured at520 nm (�Quant universal spectrophotometer MQX200, Bio-Tek,Winooski, VT, USA), to determine berry total anthocyanin concen-trations (Iland et al., 2000).
2.3. Grapevine water status and leaf gas exchange
Weekly measurements of soil water content were made directlyunder an irrigation emitter in the midday period (between 1200and 1400 h), using a time-domain reflectometry (TDR) probe(Trime
®-FM3, Imko GmbH, Ettlingen, Germany). Measurements of
stem water potential (SWP) were started one week after the treat-ments were initiated, and undertaken once a week, with a singleleaf removed per vine per measurement to minimize the impacton vine leaf-to-fruit ratio. Following the method outlined by Chonéet al. (2001), one leaf from each vine on a main shoot was enclosed ina zip-lock plastic bag covered with aluminum foil, and left coveredfor 30 min in the midday period (between 1200 and 1400 h) to allowstomatal closure. The leaves were then removed by a single cut ofthe petiole using a sharp blade, the bags removed, and the leaveswere immediately placed in a pressure chamber for measurement(Model 1000, PMS instruments, Albany, Oregon, USA).
A portable photosynthesis system instrument (LCA-4, ADC Bio-scientific Ltd., Hoddesdon, Hertfordshire, UK) was used to measurecomparison values of leaf temperature, stomatal conductance (gs)and photosynthesis rates (A). Two fully intact leaves were chosenweekly on each vine between the 4th and 7th shoot node positionfrom the base, to be used for measurements during midday periods(between 1200 and 1400 h) on clear, non-cloudy days.
2.4. Non-structural carbohydrates
Whole, dried plant material, collected during the destructiveharvest dates (roots, trunks, spurs, stems, petioles and leaves),were ground through a heavy duty cutting mill (Retsch SM2000,
Hann, NRW, Germany) to 5 mm and a subsample was then groundthrough a 0.12 mm sieve using an ultracentrifugal mill (RetschZM200, Hann, NRW, Germany).
Following the method outlined in Smith and Holzapfel (2009),the starch concentration was determined in a 20 mg subsample bya commercial enzymatic assay (K-TSTA, Megazyme International,Bray, Ireland). In short, soluble solids were first extracted usingthree 1 mL portions of 80% (v/v) aqueous ethanol, two at 80 ◦C andone at room temperature for ten minutes. The extracts were cen-trifuged after each wash, the supernatants collected together, andthen used for later soluble sugar analysis. The remaining dried plantmaterial was suspended in 200 �L dimethyl sulfoxide and heatedat 98 ◦C for 10 min. Starch was then hydrolyzed with �-amylase(30 units) and amyloglucosidase (33 units), and the starch contentwas calculated from the concentration of released glucose in thesample.
For the soluble sugar analyses, the combined extracts preparedduring the starch analyses were diluted to 10 mL with deion-ized water, and used for determination of the concentrations ofsucrose, D-glucose, D-fructose and total sugars, with a commercialenzymatic assay (K-SUFRG, Megazyme International, Bray, Ireland),as outlined in Smith and Holzapfel (2009). In this method, eachsugar was converted into glucose-6-phosphate (G6P), and quan-tification of reduced nicotinamide-adenine dinucleotide phosphate(NADPH) was performed, following oxidation in the presence ofnicotinamide-adenine dinucleotide phosphate (NADP+) and G6P-dehydrogenase.
2.5. Statistical analysis
Data were analyzed using Statistica 12 (Statsoft Inc., Tulsa, OK,USA), with the analysis of variance (ANOVA) used to test the sig-nificance of each variable. Fisher’s least significant difference (LSD)test was used to identify significant differences between means(P < 0.05). Significant differences in table columns and rows areindicated by upper case and lower case letters, respectively.
3. Results
The fortnightly periods between the four destructive harvestdates are referred to as intervals 1, 2 and 3. Intervals 1 and 2 rep-resent the rapid berry sugar accumulation period, while interval 3represents the slow berry sugar accumulation period.
3.1. Environmental conditions
The average daily temperature and vapor pressure deficit (VPD),and the total rainfall data collected during each interval of theexperiment are shown in Table 1.
3.2. Overview of non-structural carbohydrate assimilation andallocation
For all treatments, the soluble sugar content (SSC) per berryinitially accumulated significantly between V and V + 27 (rapidaccumulation), and slowed down between V + 27 and V + 40, whenthere were no significant changes in berry SSC (slow accumulation)(Fig. 1). The tempo (mg/berry/day) of berry sugar accumulation didnot differ significantly between the treatments during both, therapid and slow berry sugar accumulation periods (Fig. 1).
The leaf stomatal conductance (gs) of all treatments decreasedsignificantly between intervals 1 (V to V + 14) and 2 (V + 14 toV + 27) for all treatments, while leaf photoassimilation rates (A)also significantly decreased during this stage, except for treatmentLowL/F:100% (Fig. 1B). Further reduction in gs took place duringinterval 3 (slow berry sugar accumulation) for grapevines under
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Table 1Periodic rainfall, daily mean, minimum and maximum atmospheric temperature and vapor pressure deficit (VPD) averages, and berry juice soluble solid concentration (◦Brix)during the experimental period.
V to V + 14 (Interval 1) V + 14 to V + 27 (Interval 2) V + 27 to V + 40 (Interval 3)
Daily mean 22.9 29.6 28.0Mean min 15.6 21.8 20.1Mean max 29.3 36.7 35.0
Daily mean 2.1 3.3 2.9Mean min 0.8 1.5 1.1Mean max 3.4 5.4 4.9Total 1.9 1.2 14.8
Temperature (◦C)
VPD (kPa)
Rainfall (mm)
Total soluble solids (◦Brix) Mean change 7.3–15.4 15.4–21.6 21.6–23.2
Fig. 1. Effects of the different irrigation and leaf-to-fruit ratio treatments (A: LowL/F:50%, B: LowL/F:100%, C: HighL/F:50% and D: HighL/F:100%) on the soluble solid contentaccumulation per berry, leaf stomatal conductance (gs) and photosynthesis (A), and root starch, soluble sugar and total non-structural carbohydrate (TNC) content evolutionper vine (n = 3).
higher water availability, while A also reduced significantly for alltreatments except LowL/F:50% (Fig. 1A), during slow fruit sugaraccumulation.
Under higher water supply, the root starch content per vinewas not significantly affected during rapid berry sugar accumu-lation (Fig. 1B + D). However, under reduced irrigation, significantdepletion in root starch content occurred during rapid berry sugar
accumulation (Fig. 1A + C). During slow berry sugar accumulation,the starch content in these roots increased significantly, and backto their initial levels at V. The root soluble sugar content of treat-ment HighL/F:50% increased significantly during rapid berry sugaraccumulation, and was significantly higher than that of vines withhigh water availability at V + 27 (Fig. 1C). The root sugar content
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of treatment LowL/F:50% showed a similar increasing trend duringrapid berry sugar accumulation (Fig. 1A) (P = 0.07).
Reduced irrigation induced significant root TNC content deple-tion during rapid berry sugar accumulation (Fig. 1A + C). These TNCcontents then increased significantly during slow berry sugar accu-mulation.
3.3. Soil water content and vine water status
Reduced water supply induced significantly lower soil watercontent (Table 2). However, the leaf-to-fruit ratio treatments alsoaffected the soil water content of vines that received more irri-gation, where the high leaf-to-fruit ratio significantly caused 30%lower average soil water content compared to the treatment withlower leaf-to-fruit ratio, despite receiving the same water volume.Under reduced irrigation, the leaf-to-fruit ratio did not significantlyimpact the soil water content.
The stem water potential (SWP) was significantly affected bythe irrigation regime and the defoliation treatments (Table 2).Higher water supply resulted in less negative SWP values, andcorresponded to a moderate to weak overall grapevine waterconstraint according to published classification thresholds (VanLeeuwen et al., 2009); however, higher leaf-to-fruit ratio treat-ments showed more negative SWP values. During interval 2 (V + 14to V + 27), SWP values became significantly more negative for treat-ment lowL/F:100%. Reduced water supply resulted in a moderateto severe grapevine water constraint, according to previously sug-gested thresholds (Van Leeuwen et al., 2009).
3.4. Leaf and fruit structural development
Vines with a high leaf-to-fruit ratio lost 19 and 24% of their leafarea between V and V + 40 under higher and lower irrigation supply,respectively (Fig. 2A). No significant differences in leaf area amongthe different destructive harvests were observed for grapevineswith low leaf-to-fruit ratio, irrespective of the irrigation regime.
Total fresh fruit weight per vine increased significantly from Vto a maximum at V + 27 for all treatments, except HighL/F:100%,where the maximum fresh weight was observed at the end of inter-val 1 (V + 14) (Fig. 2B). After the initial increase in fresh weight, totalfresh fruit weight decreased for all treatments to varying degrees(significantly for treatments with high leaf-to-fruit ratio), withgrapevines from treatment HighL/F:50% showing the largest totalfresh fruit weight loss towards the end of interval 3 (25% of overalldecrease). There was no significant difference in leaf-to-fruit ratiosat the final destructive harvest date (V + 40) (Fig. 2C).
3.5. Leaf gas exchange
Leaf stomatal conductance (gs) and photosynthesis rates(A) were significantly affected by both water availability, andgrapevine leaf-to-fruit ratio (Table 2). Grapevines generallyexposed to the highest seasonal water constraints, according tothe SWP values (treatment HighL/F:50%), showed the lowest gs
and A. Under both irrigation regimes, the low leaf-to-fruit ratiotreatments showed significantly higher gs and A values thanthe corresponding high leaf-to-fruit ratio treatments. TreatmentLowL/F:100% resulted, on average, in 34% higher A values thantreatment HighL/F:100%, while treatment LowL/F:50% resultedin 43% higher A values than treatment HighL/F:50%. Vines fromtreatment LowL/F:100% showed the highest A, and treatmentHighL/F:50% the lowest A (P < 0.05).
The average mid-day leaf temperature per interval was con-stantly measured above 35 ◦C during the experiment (Table 2).
Fig. 2. Effects of the different irrigation and leaf-to-fruit ratio treatments on totalgrapevine leaf area (A), total berry fresh weight per vine (B), and leaf-to-fresh fruitweight ratio per vine (C) (mean ± SE; n = 3).
3.6. Berry composition
Treatment HighL/F:50% induced significantly lower berry sol-uble solid content (SSC) at V + 40 than the grapevines underhigh water availability, furthermore, the berries of treat-ment LowL/F:50% had significantly lower SSC than treatmentHighL/F:100% at V + 40 (Fig. 3A). The berry juice soluble solidconcentrations at V + 40 ranged from 22.3◦Brix for treatmentLowL/F:100% to 24.6◦Brix for treatment HighL/F:100%, but did notsignificantly differ between any of the treatments at this stage (datanot shown).
Seasonal berry dry weight evolution followed a similar patternthan that observed with berry sugar accumulation (Fig. 3A). Rapidberry dry weight increase was observed during rapid berry sugaraccumulation (V to V + 27), with a slower berry dry weight increaseduring slow fruit sugar accumulation (V + 27 to V + 40). The averagefresh weight per berry at V + 40 ranged from 1.62 g for treatmentHighL/F:50% to 1.93 g for treatment HighL/F:100%, and the differ-ences were significant between these treatments. There were noother significant differences in fresh weights per berry betweenany treatments (data not shown).
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Table 2Average leaf temperatures during the different intervals of the experiment, and the influence of different irrigation and leaf-to-fruit ratio treatments on soil water content,stem water potential, and leaf stomatal conductance and photosynthesis rates.
Treatment V to V + 14 V + 14 to V + 27 V + 27 to V + 40 Treatment main effect
Rapid berry sugar accumulation Slow berry sugar accumulation
Average 35.1 36.0 35.8 –Leaf temperature (◦C)Soil water content (%) LowL/F:50% C a 9.0 a b B 9.2 a B 9.0 a C 9.08
LowL/F:100% A 14.7 a A 16.6 a A 14.6 a A 15.32HighL/F:50% C 7.9 a B 8.9 a B 8.8 a C 8.40HighL/F:100% B 10.8 a B 10.7 a B 10.3 a B 10.68
Stem water potential (-MPa) LowL/F:50% C 1.21 a BC 1.34 a B 1.24 a C 1.26LowL/F:100% A 0.66 a A 0.97 b A 0.87 a A 0.80HighL/F:50% D 1.38 ab C 1.57 b 1.32B aHighL/F:100% B 1.00 a 1.15AB a A 0.93 a B 1.04
Stomatal conductance (mol/m2/s) LowL/F:50% B 0.03 a B 0.02 b B 0.02 b B 0.02LowL/F:100% A 0.05 a A 0.03 b A 0.02 c A 0.04HighL/F:50% C 0.02 a C 0.01 b C 0.01 b C 0.01HighL/F:100% B 0.03 a B 0.02 b BC 0.01 c B 0.02
Photosynthesis rate (�mol/m2/s) LowL/F:50% B 4.90 a B 4.18 b B 3.40 b B 4.39LowL/F:100% A 6.42 a A 6.09 a A 4.43 b A 5.94HighL/F:50% C 3.73 a C 2.86 b C 1.77 c C 3.06HighL/F:100% B 5.52 a 3.89B b 2.68 cB B 4.44
a Means separated within columns using Fisher’s LSD test, significant differences are indicated at P < 0.05. Where the same capital letter appears in a column, values donot differ significantly.
b Means separated within rows using Fisher’s LSD test, significant differences are indicated at P < 0.05. Where the same lower case letter appears in a row, values do notdiffer significantly.
Fig. 3. Impact of irrigation and leaf-to-fruit ratio on A: soluble solid content (SSC, left axis) and dry weight (DW, right axis) per berry, and B: anthocyanin content per berry(left axis) and berry anthocyanin concentration (right axis) during the experimental period (mean ± SE; n = 3).
The berry anthocyanin concentration increased significantlyfor all treatments during intervals 1 and 2, when rapid berrysugar accumulation also took place (Fig. 3B), and continued toincrease significantly during interval 3 for all treatments, exceptHighL/F:100%. At V + 27, the fruit of treatment HighL/F:100%had significantly higher anthocyanin concentration than that ofall other treatments. The total anthocyanin content per berryincreased significantly during rapid berry sugar accumulation,and especially accumulated rapidly for treatment HighL/F:100%during interval 2 (Fig. 3B). The anthocyanin content per berry con-tinued to increase significantly during interval 3 for treatmentLowL/F:100%. At V + 40, the anthocyanin content per berry did notdiffer significantly between the treatments, although the berries oftreatment HighL/F:100% tended to have higher anthocyanin con-tent than that of treatments HighL/F:50% (P = 0.06) and LowL/F:50%(P = 0.08).
3.7. Non-structural carbohydrates
Whole vine (excluding the fruit) and perennial tissue TNC con-tent evolution is represented in Fig. 4. In Table 3, organ-specificstarch and soluble sugar concentrations are reported, while organ-
specific total dry biomass and TNC content development is reportedin Table 4.
3.7.1. Whole-vine and perennial TNC contentThe TNC content for the combined perennial and vegetative
seasonal organs (total vine TNC) (Fig. 4A) was reflective of theoverall perennial tissue TNC content (Fig. 4B). Under higher wateravailability, the total vine and perennial tissue TNC content didnot significantly change during rapid berry sugar accumulation(V to V + 27), but increased significantly during slow berry sugaraccumulation (V + 27 to V + 40) when vines also had a high leaf-to-fruit ratio. Under lower water availability, total vine and perennialTNC contents reduced significantly during rapid berry sugar accu-mulation (Fig. 4). This was especially obvious in vines with lowleaf-to-fruit ratio, where both TNC contents decreased by 30%.The decrease was most pronounced during interval 2 (V + 14to V + 27), and especially for vines with low leaf-to-fruit ratio.During slow berry sugar accumulation, significant TNC replen-ishment took place, regaining the initial TNC contents observedat V.
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Table 3Influence of different irrigation and leaf-to-fruit ratio treatments on the starch and sugar concentrations in the roots, trunks, stems and leaves (% DW) at the various destructiveharvest dates (n = 3).
Rapid berry sugaraccumulation
Slow berry sugaraccumulation
Rapid berry sugaraccumulation
Slow berry sugaraccumulation
Treatment V V + 14 V + 27 V + 40 V V + 14 V + 27 V + 40
Starch% Roots LowL/F:50% 25.3 a Ba 20.7 bb B 18.4 b B 25.1 a Trunk 7.1 ab A 9.3 a B 6.2 b A 8.2 abLowL/F:100% 25.3 a A 26.8 a A 25.3 a AB 29.3 a 7.1 a A 8.6 a A 9.3 a A 9.5 aHighL/F:50% 25.3 a AB 22.7 ab B 18.9 b B 24.7 a 7.1 a A 8.0 a B 6.7 a A 8.1 aHighL/F:100% 25.3 b AB 25.5 b AB 20.3 b A 34.0 a 7.1 b A 7.4 b A 10.2 ab A 11.9 a
Sugar% LowL/F:50% 2.5 b B 2.4 b AB 2.9 ab A 3.8 a 1.5 a BC 1.3 a A 1.6 a A 2.3 aLowL/F:100% 2.5 b 1.7B c 2.3B bc A 3.1 a 1.5 a 0.8C c B 0.9 bc A 1.4 abHighL/F:50% 2.5 b A 3.1 ab A 4.0 a A 4.0 a 1.5 b A 2.1 a A 2.0 ab A 1.7 abHighL/F:100% 2.5 b B 2.4 b B 2.4 b A 3.4 a 1.5 a AB 1.6 a AB 1.4 a A 1.4 a
Starch% Stems LowL/F:50% 3.4 b A 4.8 a B 3.8 ab B 4.7 a Leaves 0.14 a C 0.06 b B 0.06 b B 0.15 aLowL/F:100% 3.4 b A 4.7 a B 4.7 a B 4.8 a 0.14 a AB 0.10 a B 0.14 a B 0.22 aHighL/F:50% 3.4 b A 4.6 ab B 3.5 b B 5.4 a 0.14 a BC 0.05 b B 0.06 b B 0.14 aHighL/F:100% 3.4 c A 5.5 b A 6.7 ab A 8.0 a 0.14 b A 0.12 b A 1.32 a A 0.55 ab
Sugar% LowL/F:50% 2.1 a A 1.9 a A 2.2 a B 1.9 a 4.9 a A 2.6 b B 2.4 b A 3.6 abLowL/F:100% 2.1 ab B 1.2 b B 1.2 b AB 2.8 a 4.9 a A 2.6 b AB 2.9 b A 3.3 bHighL/F:50% 2.1 b 2.5A a 2.5A a 2.4AB ab 4.9 a A 2.7 b AB 3.0 b A 3.2 bHighL/F:100% 2.1 b A 2.0 b A 2.2 b A 3.1 a 4.9 a A 3.4 b A 4.4 ab A 3.6 b
a Means separated within columns using Fisher’s LSD test, significant differences are indicated at P < 0.05. Where the same capital letter appears in a column, values donot differ significantly.
b Means separated within rows using Fisher’s LSD test, significant differences are indicated at P < 0.05. Where the same lower case letter appears in a row, values do notdiffer significantly.
Table 4Influence of different irrigation and leaf-to-fruit ratio treatments on the dry biomass of total fruit per vine, and the dry biomass and total non-structural carbohydrate (TNC)content per organ.
Rapid berry sugar accumulation Slow berry sugar accumulation
Treatment V V + 14 V + 27 V + 40
Biomass (g) Fruit LowL/F:50% 55.0 c Ba 106.2 bb B 170.4 a AB 179.7 aLowL/F:100% 55.0 c B 118.4 b AB 185.6 a AB 180.7 aHighL/F:50% 55.0 d B 118.9 c AB 182.5 a B 161.0 bHighL/F:100% 55.0 c A 157.0 b A 196.3 a A 194.0 a
Biomass (g) Roots LowL/F:50% 191.5 a A 197.6 a B 171.0 b A 181.8 abLowL/F:100% 191.5 a A 168.7 a B 182.0 a A 182.6 aHighL/F:50% 191.5 a A 190.5 a AB 192.7 a A 183.2 aHighL/F:100% 191.5 b 163.3A b 236.1 aA A 207.2 ab
TNC content (g) LowL/F:50% 49.8 a A 42.2 a A 33.2 b B 47.9 aLowL/F:100% 49.8 a A 44.2 a A 48.1 a B 55.2 aHighL/F:50% 49.8 a A 45.1 a A 38.7 b B 48.9 aHighL/F:100% 49.8 b A 42.9 b A 49.3 b A 71.6 a
Biomass (g) Stems LowL/F:50% 54.7 a AB 69.2 a A 58.2 a A 63.6 aLowL/F:100% 54.7 a B 55.8 a A 70.8 a A 67.9 aHighL/F:50% 54.7 a AB 61.1 a A 55.2 a A 57.4 aHighL/F:100% 54.7 b A 77.9 a A 74.8 ab A 77.4 a
TNC content (g) LowL/F:50% 3.3 a AB 4.6 a B 3.5 a B 4.2 aLowL/F:100% 3.3 a B 3.3 a B 4.2 a B 5.0 aHighL/F:50% 3.3 b B 4.2 ab B 3.3 b B 4.5 aHighL/F:100% 3.3 c A 5.9 b A 6.6 ab A 8.6 a
Biomass (g) Trunk LowL/F:50% 84.7 a A 106.7 a A 81.8 a A 93.3 aLowL/F:100% 84.7 a AB 94.5 a A 87.8 a A 99.6 aHighL/F:50% 84.7 a B 78.7 a A 86.3 a A 85.4 aHighL/F:100% 84.7 a AB 97.9 a A 94.5 a A 95.9 a
TNC content (g) LowL/F:50% 7.3 b BC 11.4 a A 6.4 b A 9.8 abLowL/F:100% 7.3 b C 9.0 ab B 8.8 ab A 10.8 aHighL/F:50% 7.3 a A 7.9 a A 7.5 a A 8.4 aHighL/F:100% 7.3 c AB 8.8 bc AB 11.0 ab A 12.6 a
Biomass (g) Leaves LowL/F:50% 0.7 bc AB 0.9 a A 1.0 a A 1.0 aLowL/F:100% 0.7 b AB 0.9 a A 0.9 a A 1.0 aHighL/F:50% 0.7 b B 0.8 a A 0.8 a B 0.8 aHighL/F:100% 0.7 b A 1.0 a A 0.9 a AB 0.9 a
TNC content (g) LowL/F:50% 0.03 ac A 0.02 a B 0.02 a A 0.04 aLowL/F:100% 0.03 a A 0.02 a AB 0.03 a A 0.04 aHighL/F:50% 0.03 a A 0.02 a AB 0.02 a A 0.03 aHighL/F:100% 0.03 b A 0.04 ab A 0.05 a A 0.04 ab
a Means separated within columns using Fisher’s LSD test, significant differences are indicated at P < 0.05. Where the same capital letter appears in a column, values donot differ significantly.
b Means separated within rows using Fisher’s LSD test, significant differences are indicated at P < 0.05. Where the same lower case letter appears in a row, values do notdiffer significantly.
c Leaf biomass and TNC content indicated as per leaf per vine.
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Fig. 4. Total non-structural carbohydrate (TNC) content per vine in combined grapevine roots, trunks, spurs, shoots, leaves and petioles (A) and in total perennial tissues (B),as affected by the irrigation and leaf-to-fruit ratio treatments (mean ± SE; n = 3).
3.7.2. Starch and soluble sugar concentration distributionUnder higher water supply, the total root starch concentration
was not significantly affected during rapid berry sugar accumula-tion (V to V + 27) (Table 3). However, the root starch concentrationdid significantly increase by V + 40 for the high leaf-to-fruit ratiotreatment. Under reduced irrigation, the root starch concentra-tions of treatments with low and high leaf-to-fruit ratio reducedsignificantly by 27 and 25% respectively, during rapid berry sugaraccumulation. During slow berry sugar accumulation (V + 27 toV + 40), the starch concentrations in these roots increased sig-nificantly. The root soluble sugar concentration of treatmentHighL/F:50% increased significantly during rapid berry sugar accu-mulation, and were significantly higher than that of vines with highwater availability at V + 27 (Table 3).
The trunk starch concentration of treatment HighL/F:100% wassignificantly higher at V + 40 than at V (Table 3). The trunk solu-ble sugar concentrations reduced significantly during rapid berrysugar accumulation for treatment LowL/F:100%, and was signifi-cantly lower than that of the reduced irrigated grapevines at V + 27(Table 3).
In the stems, vines with a higher water availability exhibiteda significant increase in starch concentration during rapid berrysugar accumulation, and this resulted in significantly higher stemstarch concentration for treatment HighL/F:100%, compared to theother treatments at V + 27 (Table 3). All treatments showed signif-icantly higher stem starch concentrations at V + 40 than at V. Stemsoluble sugar concentration increased significantly for treatmentHighL/F:50% during rapid berry sugar accumulation (Table 3).
The leaf starch concentrations for treatment HighL/F:100%increased significantly during rapid berry sugar accumulation(Table 3), and this treatment also induced significantly higher leafstarch concentrations at V + 40 than any of the other treatments.The leaf starch concentration of vines receiving reduced irriga-tion depleted significantly during rapid berry sugar accumulation.The leaf soluble sugar concentration also reduced significantly forall treatments, except treatment HighL/F:100% during rapid berrysugar accumulation (Table 3).
3.7.3. Organ dry biomass and TNC contentThe total fruit dry weight per vine increased significantly for
all treatments during rapid berry sugar accumulation (V to V + 27)(Table 4). The total fruit dry weight per vine remained constant dur-ing slow berry sugar accumulation for all treatments, apart from
treatment HighL/F:50% where it significantly reduced. At V + 40,treatment HighL/F:100% had significantly higher total fruit dryweight than treatment HighL/F50%.
The total root dry weight decreased significantly during rapidberry sugar accumulation for vines of treatment LowL/F:50%,while treatment HighL/F:100% induced significantly larger root dryweights at V + 27 than both of the low leaf-to-fruit ratio treatments(Table 4). Reduced irrigation induced significant root TNC con-tent depletion during rapid berry sugar accumulation (Table 4). AtV + 40, treatment HighL/F:100% exhibited significantly higher rootTNC content than any other treatments.
The trunk total dry weights did not significantly change duringthe experiment for any of the treatments (Table 4). The trunk TNCcontent of treatment HighL/F:100% increased significantly duringrapid berry sugar accumulation (Table 4).
The total stem dry weight of treatment HighL/F:100% was sig-nificantly higher at V + 40 than at V, but stem dry weights did notchange significantly during the experiment for any of the othertreatments (Table 4). Stem TNC contents increased significantlyfor treatment HighL/F:100% during rapid berry sugar accumulation(Table 4).
The dry weight per leaf increased significantly between Vand V + 14 for all treatments, and remained constant thereafter(Table 4). Both low leaf-to-fruit ratio treatments had significantlyhigher dry weight per leaf than treatment HighL/F:50% at V + 40.The TNC content per leaf of treatment HighL/F:100% increased sig-nificantly during rapid berry sugar accumulation.
4. Discussion
To study the contribution of non-structural carbohydrate (TNC)reserves towards berry dry matter accumulation, two distinct treat-ments of leaf-to-fruit ratio in combination with two vine watersupply regimes were implemented at véraison (onset of berry soft-ening). The reduced water supply and/or leaf area treatments wereaimed to reduce canopy photoassimilation enough so as for berrysugar accumulation to rely on remobilized stored carbohydratesfrom the perennial structure.
The relationship between the tempo of berry sugar accumu-lation and root TNC content, in potted grapevines subjected tomoderate to severe water constraints, has been illustrated in thepresent study. Although it has previously been confirmed by 14Ctracing studies that root carbohydrates can be relocated towards
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the berries during berry sugar accumulation (Candolfi-Vasconceloset al., 1994), this is to the best of our knowledge, the first studyto indicate an inverse relationship between the contents of rootTNC and berry sugar, when leaf photoassimilation is limited dur-ing rapid fruit sugar accumulation. The clear replenishment of rootTNC as the berry sugar accumulation tempo slowed down, is anadditional original result. The roots, therefore, became a comple-mentary source of TNC to supply towards the sink TNC demand ofmaturing fruit.
When comparing the TNC contents and concentrations betweenthe roots, trunks, stems and leaves: the roots had the highestvalues, and the root TNC content represented, on average, 73%of the total TNC content in these organs. The net loss of com-bined root, trunk, stem and leaf TNC content (Fig. 4A) during rapidberry sugar accumulation, in grapevines under reduced water sup-ply, can be attributed to root starch depletion. In fact, root starchremobilization accounted for 89% of the whole-vine (excluding thefruit) TNC loss during rapid berry sugar accumulation in treatmentLowL/F:50%. An apparent hydrolysis of root starch took place duringrapid fruit sugar accumulation in the reduced irrigated grapevines,corresponding to the soluble sugar accumulation in these roots. Thestarch hydrolysis was induced by a sugar deficit, prompted by thefruit (temporary TNC sink) sugar demand outweighing the leaf (TNCsource) photoassimilate supply (Eveland and Jackson, 2012). Solu-ble sugars are transported from the roots in the phloem, and TNCcan thereby be mobilized from the roots to the berries, contributingto the sink TNC demand. Further investigation is, however, neededto quantify the absolute amount of sugars relocated from the rootstoward the fruit during rapid berry sugar accumulation.
The net loss of TNC in the roots, trunks, stems and leaves duringrapid berry sugar accumulation, and in grapevines under moder-ate to severe water constraints, potentially contributed up to 18and 10% to total fruit dry biomass accumulation per vine for treat-ments LowL/F:50% and HighL/F:50%, respectively. As there wereno significant biomass increases for any other organs in grapevinesfrom these treatments during this period, it implies that significantamounts of stored TNC were unlikely used towards the struc-tural development of other organs. While not quantified in thepresent study, it must however also be noted that some of the TNCcould have been lost through respiration, although the whole-vinerespiration rate is likely reduced under limited water availabil-ity (Escalona et al., 2012). In addition, the amount of carbon lostthrough respiration in relation to the total pool of carbohydratesis also thought to be very limited, as previously illustrated ingrapevine berries (Romieu et al., 1992). It, therefore, seems likelythat root reserve TNC made a significant contribution to the berrysugar content for the grapevines that received reduced irrigation,to compensate the limited leaf assimilation (Candolfi-Vasconceloset al., 1994). To further clarify the relative contribution of rootrespiration to the change in TNC content during berry ripening,future studies could include the determination of root respirationrates.
When berry sugar accumulation slowed down, starch accumu-lated in the roots as the berry carbohydrate sink strength wasreduced. The content in TNC and especially starch, at the end ofberry maturation is an indicator of the starch reserve availability atbudburst for the following season (Smith and Holzapfel 2009). Thereserve TNC at budburst is utilized for early season vegetative andreproductive growth and development. Previous studies suggestthat low carbohydrate reserve content at budburst is detrimen-tal towards vegetative growth (Loescher et al., 1990), inflorescenceand flower initiation and development, fruit set, and overall fruityield (Bennett et al., 2005; Smith and Holzapfel 2009). It is, there-fore, probable that the vegetative and reproductive developmentof grapevines from treatments with low water availability couldbe affected in the following season, especially if further reserve
accumulation is impaired due to a short post-harvest period. Morework is however needed to quantify the post-harvest recovery ofroot TNC content following the depletion thereof during berry sugaraccumulation.
The leaf-to-fruit ratio at the final destructive harvest date(V + 40) of grapevines with low leaf-to-fruit ratios, was found to bewithin a range (8–12 cm2 leaf area per gram of fruit) estimated to,in a comparison of grapevines with a wide range of leaf-to-fruitratios in a given climatic region, allow the maximum accumu-lation of berry soluble solids, as well as maximum developmentof berry fresh weight and skin anthocyanins, on a single canopytrellis-system (Kliewer and Dokoozlian, 2005). However, whenusing potted vines, the present study indicates that the water sta-tus of grapevines with the same leaf-to-fruit ratios can significantlyimpact on the berry soluble solid content (SSC) of mature fruit, asgrapevines under reduced water supply and a high leaf-to-fruitratio had inferior berry SSC than those under higher water sup-ply, and the same leaf-to-fruit ratio. Nevertheless, the consistentpattern and tempo of berry sugar content accumulation betweenthe treatments, and the lack of significant treatment differences inberry anthocyanin content and soluble solid concentration (◦Brix)at V + 40, suggests that no treatment caused an inhibition of berrysugar import or skin anthocyanin biosynthesis. Water stress cancause alterations in the expression of genes involved in regulat-ing plant sugar transportation between source and sink organs(Williams et al., 2000), as well as those involved in berry skin antho-cyanin biosynthesis (Castellarin et al., 2007). A sustained waterstress, for example, causes an up- or down-regulation of the genesencoding hexose transporters in grapevines (Medici et al., 2014),and may thereby affect the tempo of berry sugar accumulation.Likewise, water stress can inhibit or promote anthocyanin biosyn-thesis in grapevine berries (Ojeda et al., 2002). Because there wereno significant alterations in the tempo of berry sugar accumula-tion and no significant differences in the berry anthocyanin contentof mature berries in the present study, it can be assumed thatthe reduced water supply treatments (treatments lowL/F:50% andHighL/F:50%) induced a sustained water constraint rather than astress. The water constraints, therefore, affected leaf photosynthe-sis (A), although not altering berry ripening in terms of sugar andanthocyanin accumulation.
Increased water availability and decreased leaf-to-fruit ratioimproved mid-day leaf gas exchange rates during the present study.Although leaf stomatal conductance (gs) and A declined as theexperiment progressed, the gas exchange rates in the present studywere determined by the treatments, rather than the variation inatmospheric temperatures and vapor pressure deficits (VPD) dur-ing the different intervals of the experiment. The gas exchangerates recorded in the present study, especially for grapevines undermoderate to weak water constraints, were however, relativelylow in comparison to values reported for field-grown Tempranillograpevines (Medrano et al., 2003). Constantly high atmosphericVPD, and air and leaf temperatures during the midday periods whenthese measurements were conducted could attribute to these lowvalues. Another contributor to the low gas exchange values is thefact that the scheduling of the irrigation events caused the soil mois-ture content in the pots to presumably reach its lowest volumes atthe time of the day when these measurements were conducted,and thereby promoted stomatal closure. Furthermore, the mea-surements were conducted late in the growing season, and on olderleaves towards the basal parts of the shoot, when leaf aging likelyalready impaired maximum leaf gas exchange rates (Poni et al.,1994).
Based on the observations from the present study, it is, however,important to note that limitations in whole-vine photoassimilationduring berry ripening is thought to be overcome through TNC remo-bilization from storage tissues (Candolfi-Vasconcelos et al., 1994).
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Under moderate to weak water constraints, no net whole-vine TNCremobilization was observed throughout the experiment (Fig. 4A),indicating sufficient canopy photoassimilation. However, undermoderate to severe water constraints, reserve TNC remobilizationtook place during rapid berry sugar accumulation, suggesting thatthe net photoassimilation of these grapevines was insufficient tomeet sink demands during berry maturation. The reduced leaf areaof the low leaf-to-fruit ratio grapevines likely suppressed canopytranspiration, causing lower water constraints compared to thatof the grapevines with full leaf area. However, the combinationof reduced leaf area and low water supply induced the greatestsuppression of canopy photoassimilation, resulting in the high-est relative remobilization of TNC from the roots of treatmentLowL/F:50%. This indicates that under limited water supply duringrapid berry sugar accumulation, a higher leaf-to-fruit ratio couldbe beneficial towards maintaining root carbohydrate reserves priorto harvest. This would be important when a post-harvest reserveaccumulation period is absent or insufficient, as often observed, forexample, in cooler climate areas.
5. Conclusion
The effects of water availability and grapevine sink-source rela-tions on carbohydrate reserve storage by dormancy, expressed ona concentration basis, have been studied previously. However, anovel approach was undertaken in the present study to inves-tigate carbohydrate content distribution, specifically during theactive berry sugar accumulation phase, and to quantify the contri-bution of carbohydrate reserves towards berry sugar accumulation.Moderate to severe water constraints resulted in less carbohydrateallocation to the perennial grapevine organs, although not alteringthe evolution of berry sugar and anthocyanin accumulation. Carbo-hydrate reserves were remobilized in reduced-irrigated grapevinesto contribute to the berry sugar content. When berry carbohydratesink demand decreased, carbohydrates were redirected towardsthe roots, and root starch accumulated. The largest relative con-tribution (up to 18%) of total perennial and vegetative seasonalorgan carbohydrate mobilization towards berry dry matter accu-mulation, occurred for vines with low leaf-to-fruit ratios and underreduced irrigation. In these grapevines, root starch mobilizationaccounted for up to 89% of the loss of total perennial and vegeta-tive seasonal organ carbohydrate content during rapid berry sugaraccumulation. Moderate to severe water constraints can cause agreater reliance on TNC reserves to support berry dry matter accu-mulation, although seemingly not impacting on the effectiveness ofberry sugar import or anthocyanin biosynthesis. Although reservecarbohydrate replenishment starts as soon as the berry sugar accu-mulation tempo slows (possibly even a few weeks prior to harvest),restricted water availability during berry maturation can causelower carbohydrate reserve content in the roots by fruit matu-rity. In vineyards where no, or an ineffective post-harvest periodoccurs, the impact of this prevention of root carbohydrate reserveaccumulation by fruit maturity is more severe.
Acknowledgements
This work was supported by the National Wine and GrapeIndustry Centre, and the Australian grapegrowers and winemak-ers through their investment body, Wine Australia, with matchingfunds from the Australian Government. The authors thank RobertLamont and David Foster for technical assistance, and BeverleyOrchard for statistical advice.
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Rogiers, S.Y., Hardie, W.J., Smith, J.P., 2011a. Stomatal density of grapevine leaves(Vitis vinifera L.) responds to soil temperature and atmospheric carbondioxide. Aust. J. Grape Wine Res. 17, 147–152.
Rogiers, S.Y., Holzapfel, B.P., Smith, J.P., 2011b. Sugar accumulation in roots of twogrape varieties with contrasting response to water stress. Ann. Appl. Biol. 159,399–413.
Romieu, C., Tesniere, C., Than-Ham, L., Flanzy, C., Robin, J.-P., 1992. An examinationof the importance of anaerobiosis and ethanol in causing injury to grapemitochondria. Am. J. Enol. Vitic. 43, 129–133.
Smith, J.P., Holzapfel, B.P., 2009. Cumulative responses of Semillon grapevines tolate season perturbation of carbohydrate reserve status. Am. J. Enol. Vitic. 60,461–470.
Van Leeuwen, C., Tregoat, O., Choné, X., Bois, B., Pernet, D., Gaudillère, J.-P., 2009.Vine water status is a key factor in grape ripening and vintage quality for redBordeaux wine: how can it be assessed for vineyard management purposes. J.Int. Sci. Vigne Vin. 43, 121–134.
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Wang, Z., Deloire, A., Carbonneau, A., Federspiel, B., Lopez, F., 2003a. An in vivoexperimental system to study sugar phloem unloading in ripening grapeberries during water deficiency stress. Ann. Bot. 92, 523–528.
Wang, Z., Deloire, A., Carbonneau, A., Federspiel, B., Lopez, F., 2003b. Study of sugarphloem unloading in ripening grape berries under water stress conditions. J.Int. Sci. Vigne Vin. 37, 213–222.
Williams, L.E., Lemoine, R., Sauer, N., 2000. Sugar transporters in higher plants −adiversity of roles and complex regulation. Trends Plant Sci. 5, 283–290.
Zapata, C., Deléens, E., Chaillou, S., Magné, C., 2004. Partitioning and mobilisationof starch and N reserves in grapevine (Vitis vinifera L.). J. Plant Physiol. 161,1031–1040.
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Chapter 4: Paper 2
Implications of the presence of maturing fruit on
carbohydrate and nitrogen distribution in grapevines under
post-veraison water constraints
(Paper 2 has been published in the Journal of the American Society for
Horticultural Science as in the format below.)
4.1. Main objective for paper 2
To determine how the presence or absence of fruit during sustained post-véraison water
constraints influences the allocation of carbohydrates and N between the different
grapevine organs.
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J. AMER. SOC. HORT. SCI. 142(2):71–84. 2017. doi: 10.21273/JASHS03982-16
Implications of the Presence of Maturing Fruiton Carbohydrate and Nitrogen Distributionin Grapevines under Postveraison Water ConstraintsGerhard C. Rossouw3
National Wine and Grape Industry Centre, School of Agricultural and Wine Sciences, Charles SturtUniversity, Wagga Wagga, New South Wales 2678, Australia
Jason P. Smith1
National Wine and Grape Industry Centre, Charles Sturt University, WaggaWagga, New SouthWales2678, Australia
Celia BarrilNational Wine and Grape Industry Centre, School of Agricultural and Wine Sciences, Charles SturtUniversity, Wagga Wagga, New South Wales 2678, Australia
Alain Deloire2
National Wine and Grape Industry Centre, Charles Sturt University, WaggaWagga, New SouthWales2678, Australia
Bruno P. HolzapfelNational Wine and Grape Industry Centre, New South Wales Department of Primary Industries,Wagga Wagga, New South Wales 2678, Australia
ADDITIONAL INDEX WORDS. starch mobilization, nitrogen allocation, amino acids, water deficit, sucrose translocation
ABSTRACT. Grapevine (Vitis vinifera) berries are sugar and nitrogen (N) sinks between veraison and fruit maturity.Limited photoassimilation, often caused by water constraints, induces reserve total nonstructural carbohydrate (TNC)remobilization, contributing to berry sugar accumulation, while fruit N accumulation can be affected by vine watersupply. Although postveraison root carbohydrate remobilization toward the fruit has been identified through 14C tracingstudies, it is still unclear when this remobilization occurs during the two phases of berry sugar accumulation (rapid andslow). Similarly, although postveraison N reserve mobilization toward the fruit has been reported, the impact of waterconstraints during berry N accumulation on its translocation from the different grapevine organs requires clarification.Potted grapevines were grown with or without fruit from the onset of veraison. Vines were irrigated to sustain waterconstraints, and fortnightly root, trunk, shoot, and leaf structural biomass, starch, soluble sugar, total N, and amino Nconcentrations were determined. The fruit sugar and N accumulation was also assessed. Root starch depletion coincidedwith root sucrose and hexose accumulation during peak berry sugar accumulation. Defruiting at veraison resulted incontinuous root growth, earlier starch storage, and root hexose accumulation. Leaf N depletion coincided with fruitN accumulation, while the roots of defruited vines accumulated N reserves. Root growth, starch, and N reserveaccumulation were affected by maturing fruit during water constraints. Root starch is an alternative source to supportfruit sugar accumulation, resulting in reserve starch depletion during rapid fruit sugar accumulation, while root starchrefills during slow berry sugar accumulation. On the other hand, leaf N is a source toward postveraison fruit Naccumulation, and the fruit N accumulation prevents root N storage.
Grapevine berries are sinks for the incorporation of bothcarbohydrates (Davies and Robinson, 1996) and N (Roubelakis-Angelakis and Kliewer, 1992) between veraison and fruitmaturity. Restricted TNC availability, induced by limited leafphotoassimilation, can cause starch redistribution from the rootsduring berry sugar accumulation (Candolfi-Vasconcelos et al.,1994), while N concentrations in the berries and roots are
affected by abiotic conditions, such as vine water availability,during the growing season (Araujo et al., 1995). Furthermore,apart from also being affected by vine N supply, the N reserveaccumulation in the roots is restricted by the presence of fruitbefore and after veraison (Rodriguez-Lovelle and Gaudillere,2002). It is still unclear how the postveraison distribution ofTNC and N reserves among the different organs, are affectedby a combination of fruit presence and sustained water con-straints. A further question remains on how this distributioncontributes to, or inhibits, TNC and N reserve storage, or thecontents of TNC and N in the fruit during berry maturation.
In plant roots, TNC are mainly stored as starch, which can behydrolyzed, yielding osmotic active soluble sugars (Regieret al., 2009). Apart from the possible remobilization of sugarsvia phloem sucrose transportation (Ruan et al., 2010) between
Received for publication 23 Nov. 2016. Accepted for publication 9 Jan. 2017.This work was supported by the National Wine and Grape Industry Centre, andthe Australian grapegrowers and winemakers through their investment body,Wine Australia, with matching funds from the Australian Government.1Current address: Institut f€ur Allgemeinen und €okologischen Weinbau,Hochschule Geisenheim University, Geisenheim, 65366, Germany2Current address: Montpellier SupAgro, Montpellier, 34060, France3Corresponding author. E-mail: [email protected].
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the perennial vine parts (the roots and trunk) and the ripeningberries, thereby contributing to berry sugar accumulation(Candolfi-Vasconcelos et al., 1994), sugars also accumulate invarious tissues of water stressed plants, to aid in osmotic regu-lation (Regier et al., 2009). Therefore, upon water constraintsduring berry sugar accumulation, the accumulation of solublesugars in different vine parts could theoretically contribute tovarious functions; e.g., to facilitate TNC remobilization, and toimprove abiotic stress tolerance. As grapevines are perennialplants, the storage of starch reserves at the end of the season isessential for reserve TNC utilization the following season,required for vegetative and reproductive development frombudburst (Holzapfel et al., 2010). Depleted root starch concen-tration can then lead to limited initiation and development of theinflorescence, and decreased fruit set and fruit yield the followingseason (Smith and Holzapfel, 2009).
The N allocation to grapevine berries, and subsequent accu-mulation during berry maturation is, from a wine qualityperspective, essential as it determines the juice yeast assimilableN content, influencing fermentation and wine composition.However, root N accumulation late in the growing season isimportant for its overwintering storage (Cheng et al., 2004).Similar to TNC, N reserves are used toward the initiation of earlyseason vegetative growth, where their mobilization regulatespring growth and account for most of the N distribution untilaround flowering, as N soil uptake is usually still insufficient atthis stage (Zapata et al., 2004). Nitrogen accumulation in theperennial vine parts usually initiates before berry maturity, andthe reserves continue to increase until leaf fall (Roubelakis-Angelakis and Kliewer, 1992). The presence of fruit reduces Nassimilation in grapevine roots (Morinaga et al., 2003). Nitrogenis mostly stored in the roots, and these reserves consists of a rangeof amino acids and proteins (Zapata et al., 2004). Amino acids inplants are involved in the regulation of N metabolism, and playessential roles in N transport and storage (Roubelakis-Angelakisand Kliewer, 1992). The metabolic pathway related to thea-ketoglutarate family of amino acids, yields glutamic acid,glutamine, arginine, and proline. These amino acids are abundantin plants, and have distinct roles in N metabolism (Verma et al.,1999). Glutamic acid is the intermediate product of nitrate andammonium assimilation, and a precursor for the synthesis ofglutamine, arginine, and proline (Berg et al., 2002). Arginine isconsidered the main N-storage amino acid in grapevines (Xiaand Cheng, 2004), while glutamine is an essential N transporter(Coruzzi and Last, 2000). Proline accumulation is linked toosmotic adjustment following abiotic plant stresses (Hare andCress, 1997). The metabolism of the a-ketoglutarate-derivedamino acids is therefore essential to regulate plant N partitioningand distribution, particularly during abiotic constraints.
The aim of this experiment was to determine the effect offruit presence during sustained postveraison water constraints,on the TNC and N distribution within grapevines. The first goalwas to investigate the response in the structural development ofthe leaves, shoots, trunk, and roots, based on the presence offruit, during sustained water constraints. The second goal wasto determine how fruit presence affects TNC accumulation inthe different organs, during the sustained water constraints, andto assess which individual sugars accumulate in the grapevineroots during the two phases of berry maturation (rapid and slowsugar accumulation). The final goal was to determine how thepresence of maturing fruit affects the allocation of N betweenthe grapevine organs, and to identify potential contributions of
amino N, and especially the amino acids yielded from a-keto-glutarate metabolism, toward N storage or translocation.
Materials and Methods
EXPERIMENTAL DESIGN. Own-rooted ‘Shiraz’ grapevines,grown in 50-L pots containing commercial potting mix, wereused for this experiment in the 2014–15 growing season. Thegrapevines were grown in an outside bird-proof cage in thewarm to very warm climate Riverina region of New SouthWales, Australia. The 3-year-old grapevines were winterpruned to 10 spurs with two buds each, and arranged in fourrows of nine vines each. From just after budburst, thegrapevines were fertilized every 3 weeks with 250 mL of50:1 diluted complete liquid fertilizer (MEGAMIX Plus;Rutec, Tamworth, Australia). In total, �2.6 g N was appliedto each vine through fertilization, and the fertilization eventswere ceased 1 month before the start of the experiment, aimingto limit soil N uptake during the experiment. The grapevineswere well watered between budburst and veraison, whenirrigation was supplied three times a day to the point of visualfree drainage from the pots.
Vines were shoot thinned so as to leave 17 shoots per vinefrom fruit set, and at the onset of veraison, 2 d after the first signof berry softening was observed, the treatments were initiated.Four randomly selected vines, one from each row, weredestructively harvested on the day when then the treatmentswere initiated, to represent the population of grapevines beforethe implementation of the treatments. After removal of the fourinitial vines, the eight remaining vines per row were evenlyspaced out in the row. All bunches were removed on half thevines, to have 16 vines with, and 16 vines without fruit. Twovines in each row (one with fruit and one without fruit) wereused as a visual reference to control irrigation scheduling, andreceived double the irrigation volume than the other vines.Irrigation was scheduled three times per day (0800, 1400, and1800 HR) for all vines, and the vines receiving double theirrigation, were rewatered each day just to the point of visualfree drainage from the pots during the 1400 HR irrigation event,through two irrigation emitters per pot. The remaining 24 vineswere irrigated through one irrigation emitter, aiming to inducesustained water constraints. Selected vines were destructivelyharvested fortnightly from after the start of veraison, over fourdistinct harvest dates, as described later. Three vines with andwithout fruit each, and that received reduced irrigation, wereharvested during each of these dates. Vines were distributedin triplicate for each treatment and harvest date. Pressure-compensated drip emitters (4 L�h–1 each) were used to supplythe irrigation during the experiment, and the irrigation timeranged between 15 and 22min per irrigation event (the same foreach irrigation event per day) to reach free drainage from thepots receiving double irrigation, as described above. Beforeforecasted rainfall events, the top of the pots, around the trunksof the grapevines, were covered with plastic to avoid rain waterfrom entering the soil.
At the destructive harvest dates; i.e., veraison [V (22 Dec.2014)], V + 14 (5 Jan. 2015), V + 29 (20 Jan. 2014), V + 42(2 Feb. 2015), and V + 56 (16 Feb. 2014), the preselectedgrapevines were dismantled. Whole root systems, trunks,shoots, and leaves were separated, collected, and washed withphosphate-free detergent and rinsed with deionized water.Leaves were collected between 0800 and 1000 HR on each of
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modified from Reed et al. (2004), and used to calculate the roottotal sugar concentrations. Extractions were conducted, once in2 mL of a 200 mg�L–1 mannitol (internal standard) solution,followed by twomore extractions in 2mL ultrapure water. Extractsolutions were homogenized with the root tissue, before incuba-tion in an 80 �Cwater bath for 15 min, before being centrifuged at3000 gn for 5 min. The three supernatants were collected together,and purified through a solid-phase extraction cartridge, containingreverse phase C18 packing. The cartridges were first preequili-brated with 4 mL methanol followed by 8 mL ultrapure water,and the sample was finally eluted with 1.5 mL ultrapure water. Acentrifugal evaporator (CentriVap 7812014; Labconco, KansasCity, MO) was used to evaporate the purified supernatants todryness, before being resuspended in 1 mL ultrapure water. Thesuspensions were placed in an ultrasonic waterbath (FX14;Unisonics, Sydney, Australia) for 30 min, and filtered througha 0.2-mm cellulose acetate syringe filter. Final samples of 60 mLwere injected into a high-performance liquid chromatography(HPLC) system (600 series; Waters, Milford, MA), with ultrapurewater used as mobile phase, pumped at a flow rate of 0.4mL�min–1. Sugars in the samples were separated with a mono-saccharide column [300 · 7.8 mm, 8 mm (REZEX 8% Pb2+;Phenomenex, Torrance, CA)]. The column was heated to 75 �C,and sugars were detected with a refractive index detector (model2414; Waters). Standard solutions of sucrose, glucose, fructose,and mannitol were used to determine the retention times and toestablish calibration curves.
TOTAL N CONCENTRATION. N concentrations were determinedin the finely ground, dried samples of roots, trunks, shoots, andcombined leaf blades and petioles. For the fruit, 50 frozen berrieswere ground to a fine powder under liquid N with an analyticalmill (A11; IKA, Selangor, Malaysia), freeze-dried (Gamma 1-16LSC), and used for the determination of fruit N concentration. Nconcentration in 200 mg of a representative sample were deter-mined by the LECO method (Standard methods of Rayment andLyons, Soil chemical methods, Australasia, Dumas CombustionMethod 6B2b), using an elemental analyzer (CNS TruMAC;LECO Corp., St. Joseph, MI).
AMINO N CONCENTRATION. Subsamples of the fruit, roots,trunks, shoots, leaf blades, and petioles were taken from–80 �C storage and ground to a powder under liquid N, usingan analytical mill (A11; IKA). Free amino acids wereextracted from a 100 mg subsample of the ground tissue,using 100 mL of an 80% (v/v) methanol solution. Thesamples were vortexed for 1 min, and sonicated for 15 minat room temperature, before centrifugation at 12,000 gn for10 min. The supernatant (20 mL) was mixed with 475 mL 0.25 M
borate buffer (pH 8.5) and 5 mL internal standard (10 mM Lhydroxyproline), and 100 mL of the mixture used in thederivatization of the amino groups, according to Hayneset al. (1991), using 9-fluoreonylethyl chloroformate. Aminoacids were analyzed by a HPLC system (600 series; Waters),and were separated with a C18 column [4.6 · 150 mm, 5 mm(Zorbax Eclipse plus; Agilent, Santa Clara, CA)], and quan-tified with a fluorescence detector (model 2475; Waters)according to Haynes et al. (1991). The N concentration offree amino acids was determined in relation to the amino Natoms of each amino acid. Total free amino N concentrationswere determined from the amino N atoms of 17 free aminoacids.
STATISTICAL ANALYSIS. Data were analyzed using Statistica12 (Statsoft, Tulsa, OK), with the analysis of variance used to
the harvest dates. The fresh weights of these organs weredetermined, and the samples were oven-dried at 60 �C untilconstant dry weight. During the destructive harvests, root,trunk, shoot, leaf blade, and petiole subsamples were collected.The root subsamples consisted of full length roots taken within10 cm from the basal part of the trunk, always between 2 and6 mm in diameter, with at least 50 g in total fresh weight. Soilparticles were shaken off and the roots rapidly rinsed withdeionized water, before the samples being frozen in liquid N.Trunk subsamples, 10 cm in length, were taken from 20 cmabove soil level. One whole shoot per vine represented the shootsubsamples, whereas 20 leaves from the base of one shoot pervine represented the leaf blade and petiole subsamples. Berries(50 per vine) were also collected between 0800 and 1000 HR eachday, and all subsamples were immediately frozen in liquid N andstored at –80 �C. Rainfall, atmospheric temperature, and relativehumidity were recorded and collected from an on-site weatherstation, and the vapor pressure deficit was calculated (Castellvı�et al., 1996).
WATER STATUS AND LEAF GAS EXCHANGE. Measurements ofstem water potential (SWP) were conducted weekly accordingto Chon�e et al. (2001), selecting one healthy leaf from eachvine on a main shoot and enclosing it with aluminum foil bagsfor 30 min between 1200 and 1400 HR. The leaves were thenplaced in a pressure chamber to measure SWP (model 1000;PMS instruments, Albany, OR). A portable photosynthesis systeminstrument (LCA-4; ADC Bioscientific, Hoddesdon, UK) wasused to measure leaf surface temperature, stomatal conductance(gS), and photosynthesis (Pn). Two healthy, fully intact leaveswere chosen weekly on each vine between the fourth and seventhshoot node position, to be used for these measurements between1200 and 1400 HR on clear, noncloudy days.
VEGETATIVE AND REPRODUCTIVE DEVELOPMENT. The total fruitfresh weight of each grapevine was recorded at each destructiveharvest. Subsamples of 50 berries per vine were used to deter-mine the fresh weight per berry, and were juiced to measure thejuice total soluble solid (TSS) concentration with a digital benchrefractometer (PR-101; Atago, Tokyo, Japan). Berry solublesolid content (SSC) was calculated on the basis of berry freshweight and TSS concentration.
The total tissue dry weight of whole root systems, trunks,shoots, and leaves and petioles were calculated for each vine, bycombining the weights measured from the dried samples andthe estimated dry weights of the subsamples. The subsampledry weights were estimated through the percentage weight lossduring drying of the main samples. The structural biomass ofthese tissues was determined by subtracting the TNC contentfrom the total biomass. Ground berry powder (5 g) from eachvine was freeze-dried (Gamma 1-16 LSC; Christ, Osterode amHarz, Germany), and the berry dry weight estimated from theweight loss during drying.
TNC. Whole, dried plant parts, collected during the de-structive harvest dates (roots, trunks, shoots, and combined leafpetioles and blades), were ground through a heavy duty cuttingmill (SM2000;Retsch, Haan, Germany) to 5mmand a subsamplewas then ground to 0.12 mm by using a ultracentrifugal mill(ZM200; Retsch). Starch (in the roots, trunks, shoots, and leaves),and total sugar (in the trunk, shoots, and leaves) concentrations ina 20 mg subsample were determined following the methodsoutlined in Smith and Holzapfel (2009).
The concentrations of sucrose, glucose, and fructose in theground root tissue were measured from a 200 mg subsample, as
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test the significance of each variable. Fisher’s least significantdifference test was used to identify significant differencesbetween means (P < 0.05).
Results
Atmospheric conditions, leaf gas exchange, and SWPThe average daily temperature and VPD data collected
during each interval of the experiment are shown in Table 1.The leaf surface temperatures were significantly the lowest
during Interval 3, and significantly higher for the defruitedvines during Interval 4 (Table 2). Fruit absence or presence didnot induce significant differences in leaf gS and Pn at any stageof the experiment (Table 2). Stomatal conductance and Pn,however, reduced significantly between Intervals 1 and 2, andgS of the fruited vines reduced further between Intervals 2 and4. The fruited vines had significantly more negative SWPvalues than the defruited vines, albeit receiving the sameamount of irrigation water. The SWP values generally becamemore negative as the experiment progressed, and weresignificantly more negative than during Interval 1 by Interval2 for the fruited vines, and by Interval 3 for the defruited vines.Defruited vine SWP values also reduced significantly betweenIntervals 3 and 4.
Dry weight, TNC, and N accumulationThe fruit SSC per vine increased significantly during In-
tervals 1 and 2 (Table 3) at rates of 9.2 and 9.7 g�d–1respectively, and rapid berry sugar accumulation therefore tookplace between V and V + 29. The fruit SSC accumulation pervine slowed down during Interval 3 (P > 0.05) at a rate of 7 g�d–1,and no accumulation took place during Interval 4. The fruit Ncontent per vine increased significantly during Interval 1, andbetween V + 14 and V + 42 (Table 3) at rates of 111 and 32mg�d–1, respectively. The fruit dry weight per vine increasedsignificantly between V and V + 29 (Table 3). The fresh weightper berry increased significantly between V and V + 29, andthen decreased during Interval 4 (Table 3).
Combined roots, trunk, shoots, and leaves per vine exhibiteda significant increase in the TNC content of both treatmentsduring Interval 4 (Table 3). These TNC contents also increasedsignificantly in the defruited vines during Interval 3. Further-more, the defruited vines had higher TNC content than thefruited vines as showed by the treatment main effect. Root TNCcontent accounted for most (53% on average) of the total vine(excluding the fruit) TNC content (Table 3), and decreasedsignificantly between V and V + 29 in the fruited vines, beforeincreasing to the initial level by V + 56. The root TNC contentin the defruited vines increased significantly between V + 14
Table 1. Summary of the periodic atmospheric temperature, vapor pressure deficit (VPD) and the total irrigation volume applied per grapevineduring the different experimental intervals. Intervals 1, 2, 3, or 4 refer to the periods of V (veraison) to V + 14 (14 d after veraison), V + 14 toV + 29 (29 d after veraison), V + 29 to V + 42 (42 d after veraison), or V + 42 to V + 56 (56 d after veraison), respectively.
Treatment
Time after veraison (d)
V to V + 14(Interval 1)
V + 14 to V + 29(Interval 2)
V + 29 to V + 42(Interval 3)
V + 42 to V + 56(Interval 4)
Atmospheric temp (�C) Daily mean 25.4 24.1 23.7 26.2Mean minimum 18.1 18.3 17.3 18.9Mean maximum 33.3 31.6 31.0 34.4
VPD (kPa) Daily mean 2.3 1.6 1.7 2.2Mean minimum 0.8 0.5 0.6 0.7Mean maximum 4.3 3.5 3.4 4.4
Irrigation (L/vine) Periodic total 50.4 54 46.8 50.4
Table 2. The influence of grapevine fruiting during sustained water constraints on leaf surface temperature, stomatal conductance (gS), netphotosynthetic rate (Pn), and stem water potential (SWP) averaged during the different experimental intervals. Intervals 1, 2, 3, or 4 refer to theperiods of V (veraison) to V + 14 (14 d after V), V + 14 to V + 29 (29 d after V), V + 29 to V + 42 (42 d after V), or V + 42 to V + 56 (56 d after V),respectively. Themain effect indicates themeanmeasured values betweenV + 14 andV + 56 for each treatment. The fruited treatment consisted ofvines with their fruit intact between V and V + 56, whereas the defruited treatment consisted of vines without fruit during the same period.
Treatment
Time after veraison (d)
V to V + 14(Interval 1)
V + 14 to V + 29(Interval 2)
V + 29 to V + 42(Interval 3)
V + 42 to V + 56(Interval 4) Main effect
Fruited 33.6 a 33.8 a 30.2 b 33.0Defruited 33.7 a 33.7 a 31.0 b 33.3Fruited 27.7 a 8.9 b 7.1 b 7.5Defruited 29.3 a 12.2 b 6.3 bc 8.8Fruited 4.71 a 3.01 b 3.80 ab 3.71Defruited 4.91 a 3.44 b 3.59 b 3.81
Leaf surface temp (�C)
gS (mmol�m–2�s–1)
Pn (mmol�m–2�s–1)
SWP (–MPa) FruitedDefruited
*z1.47 ay
*1.37 a*1.71 b*1.41 ab
*1.65 b*1.48 b
*33.3 a*35.3 a
5.8 b1.7 c4.19 ab3.18 b1.68 b1.64 c
*1.62*1.43
zMeans separated within columns using Fisher’s least significant difference (LSD) test, significant differences between the treatments (*) areindicated at P < 0.05.yMeans separated within rows using Fisher’s LSD test, significant differences are indicated at P < 0.05.Where the same lower case letter appears ina row, values do not differ significantly.
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Furthermore, the leaf N concentra-tions (Table 3) in vines of bothtreatments were, at veraison, withina range considered to indicate ade-quate vine N supply, and the Nstatus of these vines could thereforebe considered sufficient for ‘Shiraz’vines after veraison (Holzapfel andTreeby, 2007). By estimation, andalthough not significantly affectingthe total N content in a combinationof the fruit, leaves, shoots, trunk,and roots per vine (P > 0.05), thefruited vines absorbed 0.6 and 0.4 gsoil N per vine during respectively,Intervals 1 and 2. However, andwhile also not significantly contrib-uting to the total vine N content (P >0.05), the defruited vines absorbedan estimated 0.8 and 0.5 g N pervine from the soil during Intervals 2and 3, respectively.
The total dry weight of com-bined roots, trunk, shoots, andleaves per vine did not changesignificantly in the fruited vines,whereas the defruited vines exhibiteda significant increase during Interval 3(Table 3). The defruited vines also hadsignificantly higher dry weights pervine than the fruited vines, as a treat-ment main effect.
Organ structural developmentThe leaf and shoot structural
dry weight per vine did not signifi-cantly differ among the treatments,and did not change significantly(Fig. 1A and B). There was nosignificant difference in trunk struc-tural dry weight for both treatments,apart from the defruited vinesexhibiting a larger trunk dry weightat V + 42 (Fig. 1C). The rootstructural dry weight did not signif-icantly change in the fruited vines,
whereas that of the defruited vines significantly increased fromV + 42 (Fig. 1D).
TNCSTARCH CONCENTRATION PER ORGAN. The leaf starch concen-
tration was never significantly different between the treatments,and was similar to that at V until Interval 4 where it significantlyincreased (Fig. 2A). The shoot starch concentration in the bothtreatments increased significantly during Intervals 3 and 4 (Fig.2B), and was significantly higher at V + 56 than at V. The shootstarch concentration was significantly higher by V + 42 in thedefruited vines.
The trunk starch concentration in the fruited vines wassignificantly higher at V than by V + 42, whereas that in thedefruited vines was significantly higher by V + 29 (Fig. 2C).The root starch concentration in the fruited vines decreased
Table 3. Influence of sustained grapevine water constraints on the fruit total soluble solidconcentration (TSS), total soluble solid content (SSC), N content, total fruit dry weight (DW)per vine, fresh weight (FW) per berry and the impact of grapevine fruiting on the nonstructuralcarbohydrate (TNC) content, N content, and DW of vegetative annual and perennial tissues pervine at the different destructive harvests. Intervals 1, 2, 3, or 4 refer to the periods of V (veraison)to V + 14 (14 d after V), V + 14 to V + 29 (29 d after V), V + 29 to V + 42 (42 d after V), or V + 42to V + 56 (56 d after V), respectively. The main effect indicates the mean measured valuesbetween V + 14 and V + 56 for each treatment. The fruited treatment consisted of vines with theirfruit intact between V and V + 56, whereas the defruited treatment consisted of vines without fruitduring the same period.
Treatment
Time after veraison (d)
Maineffect
Interval 1 Interval 2 Interval 3 Interval 4
V V + 14 V + 29 V + 42 V + 56
Fruit4.9 d 9.5 c 16.4 b 18.2 b 22.6 a —58.5 c 187.9 b 333.1 a 423.7 a 409.5 a —2.42 c 3.98 b 4.03 ab 4.87 a 4.54 a —
161.0 c 315.6 bc 435.9 ab 562.1 a 581.7 a —
TSS (%) FruitedSSC (g/vine) FruitedN content Fruited
(g/vine)DW (g/vine) FruitedFW (g/berry) Fruited 0.8 c 0.9 bc 1.3 a 1.3 a 1.0 b —
Rest of vineTNC
Total content(g/vine)x
Fruited 65.5 b 52.6 b *78.2 b 129.4 a *75.2Defruited 65.5 c 64.3 c 202.3 a *111.6
Root content(g/vine)
Fruited 41.4 ab 36.1 bcDefruited 41.4 c 37.9 c
*z53.4 by
*93.7 c*28.7 c*54.7 bc
*59.8 a*108.9 a
NTotal content
(g/vine)xFruited 10.3 a 9.3 a 9.7 a 8.7 aDefruited 10.3 a 9.9 a 10.7 a 10.6 a
Leaf content(g/vine)
Fruited 6.3 a 5.0 ab 5.5 ab 4.5 bDefruited 6.3 a 5.3 a 5.5 a 5.2 a
Leaf concn(% DW)
Fruit 2.64 a 2.50 a 2.51 a
*147.6 b*34.3 bc*79.6 b
*8.6 a*11.2 a
4.8 b5.8 a2.26 b 1.99 c
No fruit 2.64 a 2.47 ab 2.41 ab 2.38 b 2.03 c
*39.7*70.3
*9.1*10.6*4.9*5.52.322.32
DWTotalx
(g/vine)Fruit 767.3 a 759.2 a *751.8 a 864.7 a *785.0No fruit 767.3 b 804.6 b
*765.3 a*865.6 b *1,022.8 a 1,055.0 a *937.0
zMeans separated within columns using Fisher’s least significant difference (LSD) test, significantdifferences between the treatments (*) are indicated at P < 0.05.yMeans separated within rows using Fisher’s LSD test, significant differences are indicated atP < 0.05. Where the same lower case letter appears in a row, values do not differ significantly.xIndicate the combination of roots, trunks, shoots, and leaves per vine.
and V + 42, and also during Interval 4. The root TNC content indefruited vines was significantly higher than that in the fruitedvines from V + 29 onward.
The total N content in combined roots, trunks, shoots, andleaves per vine (vine N content) did not change significantly invines of both treatments (Table 3). However, the defruited vineshad significantly higher N content in combined roots, trunks,shoots, and leaves per vine, than the fruited vines as showed bythe treatment main effect (Table 3). Most of the vine N contentwas present in the leaves (61% of total at veraison), and the leafN content per vine in the fruited vines tended to decrease duringInterval 1 (P = 0.07) (Table 3). The leaf N content of those vinesdecreased significantly between V and V + 42, whereas that ofthe defruited vines did not change significantly. The defruitedvines had significantly higher leaf N content per vine than thefruited vines as demonstrated by the treatment main effect.
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significantly between V and V + 29, followed by a signifi-cant increase during Interval 4 back to the initial concentration(Fig. 2D). The root starch concentration in the defruited vineswas significantly higher at V + 56 than at V, and wassignificantly higher than that of the fruited vines at V + 29.
Total soluble sugar concentration per organThe leaf sugar concentration in the fruited vines signifi-
cantly increased between V + 14 and V + 42, whereas that inthe defruited vines significantly increased between V and V +42 (Fig. 2E). The shoot sugar concentration in the defruitedvines remained stable throughout the experiment (Fig. 2F).Although the shoot sugar concentration of the fruited vinessignificantly decreased during Interval 1 and then signifi-cantly increased until V + 42, the concentration at V + 56 wasnot different to that at V.
The trunk sugar concentration significantly increased duringInterval 2 and remained higher than at V until V + 56 in thefruited vines, whereas that in the defruited vines was signifi-cantly higher than at V at all stages (except V + 29) (Fig. 2G).The root total sugar concentration significantly decreasedduring Interval 1, and then significantly increased duringInterval 2 in the fruited vines (Fig. 2H). At V + 29, the rootsugar concentration in the fruited vines was significantly higherthan at V. The fruited vine root sugar concentration thenreduced significantly during Interval 4. There were no signif-icant changes in root sugar concentration in the defruited vines;however, the fruited vines had significantly higher root sugarconcentration at V + 29 and V + 42, than the defruited vines.
Individual root sugar concentrationsThe root sucrose concentration in the fruited vines signifi-
cantly decreased during Interval 1, and significantly increasedduring Interval 2 (Fig. 3A). By V + 42, these root sucroseconcentrations were significantly higher than at V, beforedecreasing to the concentration observed at V + 14 duringInterval 4. The root sucrose concentration in the defruited vineswas significantly lower than at V by V + 42. The sucroseconcentration of the roots was the same for the fruited anddefruited vines except at V + 29 and V + 42, where it wassignificantly higher in the fruited vines.
Total hexose concentration; i.e., combined fructose andglucose concentrations, increased significantly during Interval2 in the roots of the fruited vines (Fig. 3D). Glucose (Fig. 3C),and the total hexose concentrations, decreased significantlyduring Interval 4 in the roots. At V + 56, both the total hexoseand the fructose (Fig. 3B) concentrations in the roots of thefruited vines, were significantly higher than at V. The roothexose concentration in the defruited vines increased graduallyduring the experiment, and from V + 42, both the total hexoseand fructose concentrations were significantly higher than at V.At V + 42, the fruited vines had significantly higher root hexose,fructose and glucose concentrations per vine, than thosewithout fruit.
NitrogenTOTAL N CONTENT PER ORGAN. The development of total leaf
N content per vine between V and V + 56 (Table 3) wasdescribed earlier. The shoot N content in vines of bothtreatments did not alter significantly (Fig. 4A), although theshoot N content in defruited vines was significantly higher thanthat in fruited vines, as determined by the treatment main effect.
Fig. 1. Effects of grapevine fruiting during sustained water constraints on thetotal (A) leaf (combination of leaf blades and petioles), (B) shoot, (C) trunk,and (D) root structural dry weight accumulation per vine. Time after veraisonrefers the different destructive harvest dates [V (veraison), V + 14 (14 d afterV), V + 29 (29 d after V), V + 42 (42 d after V), and V + 56 (56 d after V)]. Thefruited treatment consisted of vines with their fruit intact between V and V +56, whereas the defruited treatment consisted of vines without fruit during thesame period [mean ± SE (n = 3)].
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harvests (except V + 42) than at V (Fig. 4B). In the defruitedvines, the trunk N content was significantly higher during therest of the experiment than at V. Furthermore, the defruited
Fig. 2. Effects of grapevine fruiting during sustained water constraints on the leaf (combination of leaf blades and petioles), shoot, trunk, and root starch (A, B, C,andD, respectively) and total sugar (E,F,G, andH, respectively) concentration dryweight) per vine. Time after veraison refers to the different destructive harvestdates [V (veraison), V + 14 (14 d after V), V + 29 (29 d after V), V + 42 (42 d after V), and V + 56 (56 d after V)]. The fruited treatment consisted of vines with theirfruit intact between V and V + 56, whereas the defruited treatment consisted of vines without fruit during the same period [mean ± SE (n = 3)].
The N content in vine trunks of both treatments increasedsignificantly during Interval 1. The trunk N content in thefruited vines was significantly higher at all the destructive
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vines had significantly higher trunk N content than those withfruit, as determined by the treatment main effect. The root Ncontent in the fruited vines did not change significantly
Fig. 3. Effects of grapevine fruiting during sustained water constraints on theroot (A) sucrose, (B) fructose, (C) glucose, and (D) total hexose concen-tration dry weight per vine. Time after veraison refers to the differentdestructive harvest dates [V (veraison), V + 14 (14 d after V), V + 29 (29 d afterV), V + 42 (42 d after V), and V + 56 (56 d after V)]. The fruited treatmentconsisted of vines with their fruit intact between V and V + 56, whereas thedefruited treatment consisted of vines without fruit during the same period[mean ± SE (n = 3)].
Fig. 4. Effects of grapevine fruiting during sustained water constraints on thenitrogen content per vine in the (A) shoots, (B) trunks, and (C) roots. Time afterveraison refers to the different destructive harvest dates [V (veraison), V + 14(14 d after V), V + 29 (29 d after V), V + 42 (42 d after V), andV + 56 (56 d afterV)]. The fruited treatment consisted of vines with their fruit intact betweenV and V + 56, whereas the defruited treatment consisted of vines without fruitduring the same period [mean ± SE (n = 3)].
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(Fig. 4C); however, the root N content in the defruited vineswas significantly higher than at V by V + 29. The defruitedvines also had significantly higher root N content than thosewith fruit, as determined by the treatment main effect.
TOTAL AMINO N CONCENTRATION PER ORGAN. In the fruitedvines, the fruit amino N concentration was significantly higherby V + 29 than at V (Fig. 5A). The fruit amino N concentrationalso increased significantly during Interval 4.
The leaf blade total amino N concentration in vines of bothtreatments increased significantly during Interval 1, but de-creased significantly during Interval 2, and did not differbetween the treatments (Fig. 5B). In the petioles of the fruitedvines, the total amino N concentration increased significantly
during Interval 1 (Fig. 5C), and was significantly higher thanthat in the defruited fruited vines at V + 14 and V + 29.Furthermore, during Interval 3, the petiole amino N concen-tration in the fruited vines decreased significantly. The totalshoot amino N concentration increased significantly in thefruited vines during Interval 1 (Fig. 5D), and was significantlyhigher than that in the defruited vines at V + 14. The shootamino N concentration of the fruited vines decreased signif-icantly during Interval 2.
In the trunks of both treatments, the total amino N concentra-tion increased significantly during Interval 1 (Fig. 5E). The trunkamino N concentration in the defruited vines was significantlylower than at V + 14 by V + 42, whereas that in the fruited vines
Fig. 5. Effects of grapevine fruiting during sustained water constraints on total amino nitrogen concentration fresh weight in the (A) fruit, (B) leaf blades, (C) leafpetioles, (D) shoots, (E) trunks, and (F) roots. Time after veraison (d) refers to the different destructive harvest dates [V (veraison), V + 14 (14 d after V), V + 29(29 d after V), V + 42 (42 d after V), and V + 56 (56 d after V)]. The fruited treatment consisted of vines with their fruit intact between V and V + 56, whereas thedefruited treatment consisted of vines without fruit during the same period [mean ± SE (n = 3)].
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decreased significantly during Interval 4. In the roots, the totalaminoN concentration did not change significantly (Fig. 5F), anddid not significantly differ between the treatments.
INDIVIDUAL AMINO ACID CONCENTRATIONS. The concentrationsof arginine, glutamic acid, glutamine and proline, and aminoacids derived from a-ketoglutarate metabolism, are presentedfor the different organs in Fig. 6.
Arginine was the most abundant amino acid in the fruit, whereits concentration increased significantly between V and V + 29.The fruited vines had higher leaf blade arginine concentration thanthe defruited vines at V + 14 and V + 29. Likewise, in the petioles,the arginine concentration in the fruited vines was significantlyhigher at V + 14 than that in the defruited vines. The shootarginine concentration in the fruited vines increased significantlyduring Interval 1, and was significantly higher than that in thedefruited vines at V + 14. In the defruited vines, the shoot arginineconcentration was significantly higher at V + 56 than at V. Therewere no significant differences in trunk and root arginineconcentrations between the treatments, although between all ofthe perennial and vegetative annual organs, the roots had thehighest arginine concentrations.
The fruit glutamic acid concentration was higher by V + 42than at V, whereas that in the leaf blades, petioles, and shootsnever differed between the treatments. The glutamic acid con-centration in leaf blades and petioles increased significantlyduring Interval 1 for both treatments. The trunk glutamic acidconcentration in the defruited vines was significantly higher thanthat in the fruited vines at V + 14; however, it was significantlyhigher in the trunks of fruited vines at V + 42. There were nosignificant differences in root glutamic acid concentration.
The fruit glutamine concentration did not change signifi-cantly, whereas that in the leaf blades, petioles, shoot, and trunkincreased significantly for both treatments during Interval 1.The glutamine concentration then decreased significantly in theleaf blades and shoots of the vines from both treatments duringInterval 2, and also in the petioles and trunk in the defruitedvines, whereas that in the petioles and trunk of the fruited vinesdecreased by V + 42. The glutamine concentration in thepetioles of fruited vines was significantly higher than that of thedefruited vines at V + 29, and at V + 14 in the shoots. The trunkand root glutamine concentrations never significantly differedbetween the treatments.
The fruit proline concentration increased significantly dur-ing Interval 4. There was no significant difference in the prolineconcentration between the treatments in the leaf blades (exceptat V + 56 where the defruited vines had higher concentration)and petioles, although these concentrations increased signifi-cantly during Interval 1, and also during Interval 4 in the leafblades. Likewise, the proline concentration in both, shoots andtrunks, did not differ significantly between the treatments,although it increased significantly during Interval 1 in thetrunks, and then decreased during Interval 2. The root prolineconcentration in the fruited vines increased significantly duringInterval 1 before decreasing significantly during Interval 2, butwas significantly higher in the roots of defruited vines at V + 29.
Discussion
Grapevines were grown with and without fruit betweenveraison and fruit maturity, and irrigation limited to createa sustained postveraison water constraint. The aim was to reduceleaf photoassimilation, and force greater reliance on stored
carbohydrates for berry sugar accumulation in the fruiting vines.For the nonfruiting vines, the absence of the strong reproductivesink allowed vegetative growth and partitioning responses towater constraints to be examined in more detail. The TNCcontent in different organs, and the concentration changes of themajor root sugars were investigated. The water constraints alsoaimed to alter the contribution of the different N sources towardfruit N accumulation, and the concentrations of amino N wasdetermined in the different organs.
Themore negative SWP values seenwith the presence of fruitis not uncommon in deciduous fruit species. Berman and DeJong(1996), for example, described the higher crop load of peach(Prunus persica) trees under reduced irrigation to induce morenegative SWP values. The crop load induced SWP differences inthat study were attributed to either increased leaf transpiration ofthe higher cropping trees, or the reduced root growth of thosetrees and a subsequent inferior soil water uptake. In the presentstudy, leaf gS and Pn were unaffected by fruit presence. However,if higher leaf transpiration rates of the fruited vines induced themore negative SWP values, it is probable that the leaf transpi-ration rate differences between the treatments occurred due to gSvariations during periods other than the middle of the day; e.g.,midafternoon (Downton et al., 1987). The low midday Pn valuesin the present study are, however, consistent with of the impact ofthe imposed grapevine water constraints on net photoassimila-tion (Medrano et al., 2003). In addition to the water constraints,and as illustrated in apple (Malus domestica) trees, the highmidday leaf surface temperatures likely also contributed to thecorresponding low gS values (Greer, 2015). Classification of themidday SWP values, according to published thresholds, confirmsthat water constraints were sustained from veraison to berrymaturity (Van Leeuwen et al., 2009).
A depletion of root TNC coincided with rapid fruit sugaraccumulation (Table 3), and root TNC reserves were seeminglynot used toward structural development in the fruited vines (Fig.1). In the absence of fruit, the vines stored the available photo-assimilate in the roots as starch, and as implied by the gain in rootstructural biomass, also used carbohydrates toward root devel-opment. When there was no more fruit sugar accumulation, thefruited vines also stored starch. The reduced demand from thefruit therefore caused surplus carbohydrates to be stored as starch,predominantly in the roots. The absence of fruit as a carbohydratesink induced the earlier storage of starch reserves in the roots,trunks, and shoots. Although the reduction in TNC content duringrapid fruit sugar accumulation was only observed in the roots, theTNC content in the leaves, shoots, and trunks only accumulatedonce fruit sugar accumulation slowed.
Root TNC depletion in the fruited vines was caused bystarch reduction, but coincided with increased total sugarconcentrations in these roots during the maximum rates of fruitsugar accumulation. This suggests that starch was hydrolyzed,resulting in the root sugar accumulation (Regier et al., 2009).The root TNC depletion during the phase of rapid berry sugaraccumulation resulted from TNC remobilization and/or rootrespiration. Candolfi-Vasconcelos et al. (1994) illustratedthrough 14C that perennial TNC reserves are directed towardthe sugar-accumulating berries when a C source limitation wasinduced by grapevine defoliation.
The water constraints of the present study restricted mid-day leaf Pn, with values comparable to field-grown grapevinessubjected to severe water constraints (Medrano et al., 2003).Restricted leaf-level Pn may limit canopy photoassimilation
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2012). As the demand from the sugar-accumulating berries inthe present study was apparently greater than could be met bycurrent photosynthesis, the abundant root starch reserveslikely provided an alternative carbohydrate source. The roots
Fig. 6. Effects of grapevine fruiting during sustained water constraints on the concentration fresh weight of arginine, glutamic acid, glutamine, and proline in the(A) fruit, (B) leaf blades, (C) petioles, (D) shoots, (E) trunks, and (F) roots. Time after veraison (d) refers to the different destructive harvest dates [V (veraison),V + 14 (14 d after V), V + 29 (29 d after V), V + 42 (42 d after V), and V + 56 (56 d after V)]. The fruited treatment consisted of vines with their fruit intact betweenV and V + 56, whereas the defruited treatment consisted of vines without fruit during the same period [mean ± SE (n = 3)].
enough to induce a C deficiency during a period of intensefruit C demand. When a plant sugar deficiency occurs, geneexpression related to TNC remobilization, and its exportationfrom source tissues is upregulated (Eveland and Jackson,
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had the highest TNC concentration, possibly explaining whysignificant starch depletion during rapid fruit sugar accumu-lation was only observed in the roots. In fact, root TNC isusually more affected by abiotic constraints and grapevineseasonal development, than the TNC in other vine parts(Holzapfel et al., 2010). As mentioned, respiration lossesmay also partly account for the root starch depletion duringrapid fruit sugar accumulation. However, the root respirationrates of fruited pot-grown grapevines have been found to beless than defruited vines during berry maturation (Morinagaet al., 2003). Furthermore, water constraints may reducerespiration in comparison with well-watered potted vines(Escalona et al., 2012). Therefore, although root respirationmay account for some of the depleted root TNC during rapidberry sugar accumulation, it also remains probable that theroots served as an alternative TNC source. Future studies mayinclude measuring root respiration rates to quantify therelated C expense.
The concentration of root total sugars initially decreased atthe start of berry sugar accumulation. The root sugar con-centration reduction, caused by sucrose depletion, suggeststhat existing free sugars were initially translocated out of theroot system. In the subsequent sampling root sugar concen-tration increased resulting from the apparent starch hydroly-sis (Fig. 2). The breakdown of starch through enzymaticdegradation, yields C-containing intermediates such as glu-cans, which is subsequently restructured as sugars (Smithet al., 2005). It was during the period of maximum fruit sugaraccumulation, when the root sugar concentration also rapidlyincreased. Sugars are osmotically active, and facilitatesource-to-sink C translocation via the pressure flow of thephloem. Through 14C labeling, Yang et al. (2002) observedrice (Oryza sativa) stem TNC reserves to be remobilized tothe grains during grain filling, when these plants weresubjected to water constraints. Furthermore, they describedstarch remobilization to coincide with stem sucrose accumu-lation, which was suggested to sustain the C flux to the grainswhen leaf photoassimilation was restricted. Therefore, thebiosynthesis of root sugars resulting from the starch hydro-lysis in the present study, suggests that these sugars becameavailable for translocation to the ripening berries underconditions of limited C availability. Concurrent with theslowdown of berry sugar loading, root sugar accumulationstopped, and total root sugar concentration then reduced whenberry sugar accumulation ceased. The lack of a change in thetotal sugar concentrations in the roots of the defruited vines isfurther evidence that the rate of berry sugar accumulationimpacted on the root sugar abundance.
The root sucrose depletion during Interval 1 suggests thatTNC translocation from the roots mainly took place throughsucrose export. Grapevines, like most other plants, transportcarbohydrates as sucrose (Ruan et al., 2010). The rapid rootsucrose accumulation during peak berry sugar accumulationsuggests that sucrose synthesis resulted from substratesprovided by starch hydrolysis (Smith et al., 2005). Thissucrose accumulation could create a concentration gradientbetween the root tissues and upper vine parts, as the berrysugar demand outweighed canopy photoassimilation, drivingsucrose availability for phloem translocation to the berries,where it is hydrolyzed into glucose and fructose (Davies andRobinson, 1996). In the roots of the defruited vines, thegradual sucrose decline coincided with increased hexoses,
suggesting sucrose cleavage. Furthermore, an increase inroot structural biomass was observed in these vines, inagreement with Wang et al. (2010), who concluded that roothexose accumulation contributes to root growth. In fact, theaccumulation of glucose and fructose in growing plant organssupports the generation of an osmotic gradient to regulatecell expansion, especially, during abiotic stresses, such asdrought (Roitsch and Gonz�alez, 2004). As significant rootsucrose was presumably exported during rapid berry sugaraccumulation, it is further possible that the hexose accumu-lation in the roots of the fruited vines occurred to maintaincell osmotic potential (Sturm, 1999).
Similar to vine TNC, defruiting also induced higher reserveN accumulation, although the abundance of fruit N was muchlower than that of its sugar. Leaves exhibited the highest Ncontent at veraison, and also the only significant postveraisonN depletion. The trend in leaf N reduction during Interval 1and the significant leaf N depletion by V + 42 coincided withperiods of fruit N accumulation, suggesting that leaf N re-distribution took place toward the fruit (Verdenal et al., 2016).The accumulation of root N in the defruited vines suggests, inagreement with Morinaga et al. (2003), that the roots were analternative N sink due to fruit absence. The lack of root Ndepletion indicates that the roots could not be considereda considerable N source. Root, shoot, and leaf N mobilizationis thought to take place toward the bunches during berrymaturation (Conradie, 1991). However, the water constraintsof the present study likely inhibited root N reserve mobili-zation, as preharvest water constraints are thought to promoteN allocation toward the roots (Holzapfel et al., 2015). Thereason for the lack of root N translocation under waterconstraint during fruit maturation, while root TNC utilizationwas apparently not limited, needs further exploration.
One possibility for the differing TNC and N mobilizationresponses is that the fruit was not a strong enough N sink tocause a substantial root N loss. Furthermore, the leaves were thelargest N source, and with almost three times more leaf thanroot N available at veraison, may have been sufficient to meetthe fruit demand.Water constraints, as well as reducing the flowof water to the shoot, can induce a loss of xylem transportfunctioning in grapevines, by restricting xylem hydraulic con-ductivity (Lovisolo and Schubert, 1998). As N translocation fromthe roots during the period of berry maturation can take placethrough both the xylem and phloem (Roubelakis-Angelakisand Kliewer, 1992), the question is raised whether root Nmobilization could have been limited due to a potential waterconstraint induced restricted xylem functioning, an aspect thatrequires further investigation. Nevertheless, despite the sug-gested leaf N contribution toward fruit N, and although Nfertilization was ceased 1 month before the experiment, thefruited vines seemingly absorbed N from the soil during thefirst two intervals. Grapevine soil N absorption is not unusualduring the period soon after the start of veraison (L€ohnertz,1991). This newly absorbed N did not significantly affect tothe total vine N content, but potentially contributed, togetherwith the exported leaf N, toward fruit N. With the absence offruit, the implied soil N uptake likely contributed to root Naccumulation.
The elevated concentrations of amino N in the petioles andshoots of the fruited grapevines within Intervals 1 and 2, arepossibly related to N translocation from the vegetative tissuestoward the fruit (Conradie, 1991). Elevated petiole and shoot
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glutamine concentrations in the fruited vines were majorcontributors toward these amino N increases, and coincided(during Interval 1) with rapid fruit N accumulation. Glutamineis a known N transporter from source to sink organs in plants(Coruzzi and Last, 2000). On the other hand, the highest overallamino N concentrations were found in the roots of bothtreatments, present as arginine. Arginine is the major N storagecompound in grapevines (Xia and Cheng, 2004), and most ofthe amino N in the perennial structures of both treatments wastherefore likely related to N storage.
In summary, this study was performed to understand therelationship, during sustained postveraison water con-straints, between the distributions of TNC and N, and therespective accumulation of sugar and N in the fruit. Fruitinginhibited root structural development, whereas defruiting atveraison prompted continuous root growth. During theperiod of rapid fruit sugar accumulation, root TNC remobi-lization occurred, where starch depleted, and sugars accu-mulated. The results suggest that root sucrose accumulationcreated a concentration gradient to drive sucrose transporttoward the fruit. In the absence of fruit, starch accumulated,and the sucrose-to-hexose ratio decreased, indicating a po-tential role for hexoses as important osmotic regulators bypromoting a gradient to attract water into expanding rootcells. Leaf N depletion in the fruited vines coincided withfruit N accumulation, suggesting the leaves to be an impor-tant N source. In the absence of fruit, the roots became analternative N sink. Amino N accumulation in the leaf petiolesand shoots, largely attributed to glutamine accumulation,peaked during the first half of the experiment in the fruitedvines, suggesting a role in N transport from the vegetativetissues toward the fruit.
Therefore, during sustained water constraints, TNC can besourced from the roots during the rapid berry sugar accumula-tion phase. This has repercussions on starch storage, and mayaffect the vegetative and reproductive development of grape-vines the following season if starch reserve replenishment isunsatisfactory. On the other hand, leaf N translocation cansupport berry N accumulation during sustained water con-straints, causing reduced root N accumulation. This too couldhave repercussions on spring growth the following season,especially if postharvest root N replenishment is insufficient.
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Chapter 5: Paper 3
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Chapter 5: Paper 3
Vitis vinifera root and leaf metabolic composition during fruit
ripening: Implications of defoliation
(Paper 3 has been accepted for publication in Physiologia Plantarum, subject to a
minor revision. The revised manuscript was submitted to the journal in the format
below. The table and figures are shown after the main manuscript text.)
5.1. Main objective for paper 3
To assess the implications of defoliation on fruit sugar and N accumulation in
conjunction with the carbohydrate, N and primary metabolite composition of the major
grapevine source organs (roots and leaves).
5.2. Supporting information
Supporting information, as referred to throughout Paper 3 (Tables S1 and S2, and
Figures S1, S2 and S3), is included in appendix A.
Chapter 5: Paper 3
Vitis vinifera root and leaf metabolic composition during fruit maturation: Implications of defoliation
Gerhard C. Rossouwa,b,*, Beverley A. Orchardc, Katja Šukljea,†, Jason P. Smitha,‡, Celia
Barrila,b, Alain Deloirea,§ and Bruno P. Holzapfela,c
aNational Wine and Grape Industry Centre, Wagga Wagga 2678, New South Wales, Australia. bSchool of Agricultural and Wine Sciences, Charles Sturt University, Wagga Wagga 2678, New South
Wales, Australia. cNew South Wales Department of Primary Industries, Wagga Wagga 2650, New South Wales,
Australia.
*Corresponding author email: [email protected]†Present address: Wine Research Centre, University of Nova Gorica, Lanthieri Palace, Glavni trg 8,
5271 Vipava, Slovenia.‡Present address: Department of General and Organic Viticulture, Hochschule Geisenheim University,
Geisenheim 65366, Germany.§Present address: Montpellier SupAgro, Montpellier 34060, France.
Abstract
Grapevine (Vitis vinifera) roots and leaves represent major carbohydrate and nitrogen
(N) sources, either as recent assimilates, or mobilised from labile or storage pools. This
study examined the response of root and leaf primary metabolism following
defoliation treatments applied to fruiting vines during ripening. The objective was to
link alterations in root and leaf metabolism to carbohydrate and N source functioning
under conditions of increased fruit sink demand. Potted grapevine leaf area was
adjusted near the start of véraison to 25 primary leaves per vine compared to 100 leaves
for the control. An additional group of vines were completely defoliated. Fruit sugar
and N content development was assessed, and root and leaf starch and N
concentrations determined. An untargeted GC/MS approach was undertaken to
evaluate root and leaf primary metabolite concentrations. Partial and full defoliation
increased root carbohydrate source contribution towards berry sugar accumulation,
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evident through starch remobilisation. Furthermore, root myo-inositol metabolism
played a distinct role during carbohydrate remobilisation. Full defoliation induced
shikimate pathway derived aromatic amino acid accumulation in roots, while arginine
accumulated after full and partial defoliation. Likewise, various leaf amino acids
accumulated after partial defoliation. These results suggest elevated root and leaf
amino N source activity when leaf N availability is restricted during fruit ripening.
Overall, this study provides novel information regarding the impact of leaf source
restriction, on metabolic compositions of major carbohydrate and N sources during
berry maturation. These results enhance the understanding of source organ carbon and
N metabolism during fruit maturation.
Abbreviations
25L, 25 leaves treatment; ANOVA, analysis of variance; C, carbon; FL, full leaf area
treatment; GABA, γ-amino-n-butyric acid; GC/MS, gas chromatography/mass
spectrometry; LSD, least significant difference; N, nitrogen; NL, no leaf treatment;
PCA, principal component analysis; SCC, soluble solid content; TCA, tricarboxylic
acid; TSS, total soluble solids; V, véraison; V+9, 9 days after véraison; V+18, 18 days
after véraison; V+27, 27 days after véraison; V+37, 37 days after véraison; V+46, 46
days after véraison
Introduction
Maturing post-véraison grapevine (Vitis vinifera) berries are sinks for non-structural
carbohydrates and N (Davies and Robinson 1996, Roubelakis-Angelakis and Kliewer
1992). Carbohydrates are ultimately derived from leaf photoassimilation, but reserve
carbohydrate remobilisation from perennial tissues can provide an alternative carbon
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(C) source if photosynthetic supply is limited (Candolfi-Vasconcelos et al. 1994).
Among the different grapevine organs, root starch is often the largest pool of storage
carbohydrates, and this rapidly depletes if the period of high berry sugar demand after
véraison coincides with restricted canopy photoassimilation (Rossouw et al. 2017a,
2017b). After the slowing of fruit sugar accumulation in the later ripening period, the
roots instead become a C sink, and root starch storage initiates (Rossouw et al. 2017a).
Soil N uptake is limited during the berry maturation period, and redistribution of N
from roots, shoots, and leaves is thought to contribute to fruit N (Conradie 1991).
Mature leaves and the roots are, additionally, the major sources of amino N in higher
plants (Rentsch et al. 2007). Grapevine roots and leaves are, therefore, important
sources of C and/or N during fruit sugar and N accumulation. The extent of the
contribution of primary compound metabolism towards root and leaf C and N source
activity requires further research.
While starch is often the predominant non-structural carbohydrate, sucrose, glucose,
fructose, and less abundant (minor) sugars and sugar alcohols (e.g. raffinose and myo-
inositol) also contribute to the carbohydrate pool of higher plants (Noiraud et al. 2001,
Valluru and Van den Ende 2011). Conditions of high carbohydrate demand, such as
those created by reducing the leaf area of fruiting grapevines, induce enzymatic starch
breakdown, and subsequent carbohydrate exportation from reserve storage (Eveland
and Jackson 2012). Breakdown of starch reserves is therefore expected to alter the
relative composition of root non-structural carbohydrates, such as the different sugars,
and influence the metabolism of other C containing compounds (e.g. organic acids).
Organic acids, such as malic and citric acid, are important intermediates during C
metabolism (López-Bucio et al. 2000). The C skeletons of these tricarboxylic acid
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(TCA) cycle intermediates, are also utilised during N assimilation and amino acid
biosynthesis (Popova and Pinheiro de Carvalho 1998). Restricting the leaf C source
may cause altered root source activity by impacting on C flux through different
pathways of primary root metabolism. The C flux through the shikimate pathway,
which can represent up to 20% of the available C in plants (Haslam 1993), is for
example, likely affected by limited C availability. As the origin of many amino acids
and secondary metabolites (e.g. phenolic compounds), changes in C flux through the
shikimate pathway could have crucial consequences on plant C and N source organ
metabolic composition.
Grapevine N reserves are stored as proteins and amino acids (mainly arginine), with
the largest proportion located in the root system and mature leaves (Roubelakis-
Angelakis and Kliewer 1992). Amino acids provide a soluble source of organic N
which can be transported between sources (leaves and roots) and sinks (the fruit) (Lam
et al. 1996). As the fruit normally accumulates N during the post-véraison period
(Roubelakis-Angelakis and Kliewer 1992), the metabolism of N containing
compounds (e.g. amino acids) in N source organs are potentially altered by limited
post-véraison N availability. By subsequently restricting the availability of organic N,
defoliation may induce protein degradation in remaining N sources such as the roots
(Volenec et al. 1996). Protein degradation in the roots will subsequently promote root
amino N accumulation, which becomes available for further N translocation to sinks.
Monitoring changes in the abundance of primary metabolites in grapevines may
provide an insight into metabolic pathway responses to abiotic conditions, and to
canopy or crop load manipulations that are commonly used in commercial viticulture.
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84
Methods such as gas chromatography/mass spectrometry (GC/MS) allow the profiling
of a wide range of plant metabolites, including soluble sugars, sugar alcohols, organic
acids and amino acids from a single sample preparation (Lisec et al. 2006). In relation
to C and N containing storage compounds and their associated metabolism, the
responses of vegetative tissues is of particular interest. However, recent literature
concerning grapevines has focused more on comparative studies between genotypes.
Where the implications of abiotic conditions such as water or heat constraints have
been examined, most of the related analyses were conducted in fruit samples (Cuadros-
Inostroza et al. 2016, Hochberg et al. 2013, 2015a, 2015b). To the best of our
knowledge, no previous studies have conducted a detailed profiling of grapevine root
and leaf metabolite composition during the post-véraison period.
This study assessed the effects of post-véraison defoliation on the contents of non-
structural carbohydrates and N in the fruit and in major C and N sources (roots and
leaves), in conjunction with the source organ abundance of primary metabolites. By
changing leaf C and N source availability, the main objective was to profile the
primary metabolic composition (including soluble sugars, amino acids and organic
acids) of remaining leaves and/or the roots. A further objective was to link specific
alterations in source organ primary metabolite concentrations to carbohydrate or N
distribution between the source and sink organs, as influenced by leaf source
availability.
Chapter 5: Paper 3
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Material and methods
Experimental design and sample collection
Forty own-rooted V. vinifera L. cv. Shiraz (clone EVOVS12) grapevines, grown in 30
litre pots containing commercial potting mix, were used for the study during the
2015/16 growing season. The grapevines were enclosed in a bird-proof cage in the hot
climate Riverina grape growing region of New South Wales, Australia. The three-year-
old grapevines were spur-pruned to five two-bud spurs in the winter and distributed in
four rows with ten vines each. Shortly after budburst, the grapevines were fertilised
every three weeks with 250 ml of 1:50 diluted complete liquid fertiliser (MEGAMIX
Plus, Rutec, Tamworth, Australia). In total, approximately 3.2 g N was applied to each
vine from after budburst, and the fertilisation events were ceased one month prior to
the start of the experiment, aiming to avoid excessive soil N uptake during the
experiment. After budburst the vines were trained where possible to 10 shoots, and the
number of bunches per vine was counted at fruit set. All vines naturally contained
between 13 and 19 bunches, and individual vines were subsequently classified as either
containing low (13-15), medium (15-16) or high (16-19) number of bunches. This
classification was later only used to minimise vine cropping variability among
treatments and harvest dates. Nine days after the first sight of berry softening (i.e.
véraison + 9 days; V+9), four vines, one out of each row, and from each of the bunch
classes (two from the medium class) to ensure the collection of vines to be as unbiased
as possible, were destructively harvested to represent the population of grapevines
prior to the start of the experiment. The remaining nine vines per row were separated
into three replicates, each representing a bunch number class, and randomly allocated
a specific treatment and harvest date. This resulted in three replicates, spread over a
four row, nine column randomised block design.
Chapter 5: Paper 3
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The three experimental treatments: full leaf (i.e., control, 100 primary shoot leaves per
vine and all laterals, FL), 25% leaf (25 primary shoot leaves, with no lateral leaves,
25L), and no leaves (NL), were established at a stage (V+9) when berry sugar
accumulation was expected to occur rapidly. All the leaves were removed on NL vines,
while the 25L vines were left with 25 primary leaves each, adjacent to a bunch, and
additionally on one node above or below a bunch when required. When more than 100
primary shoot leaves were present, the leaves on FL vines were reduced to 100 per
vine. The leaf-to-fruit ratio of FL vines was adjusted to approximately 8 cm2 leaf area
per g fresh fruit weight, while that of 25L vines was adjusted to 2 cm2 g-1. The adjusted
FL ratio is on the lower end of a range (8-12 cm2 g-1) suggested to, in a given climatic
region, contribute towards maximum grapevine fruit sugar accumulation capacity
(Kliewer and Dokoozlian 2005). For NL and 25L vines, any new vegetative growth
was removed as soon as the regrowth of leaves and lateral shoots was observed.
A pressure compensated drip emitter (4 l h-1) was installed in the middle of each pot,
close to the vine trunk, and irrigation events were scheduled four times daily (08:00,
11:30, 14:30 and 18:00 h). All vines received the same amount of water during each
irrigation event. The irrigation event duration was the same at each application per
day, and ranged between 13 and 20 min per event throughout the experiment
(depending on daily atmospheric conditions), aiming to always allow visual free water
drainage from all pots during each irrigation event. Three vines from each treatment
were destructively harvested every 9-10 days after the start of the experiment. At the
destructive harvest dates, i.e., 28 Dec 2015 (V+9), 6 Jan 2016 (V+18), 15 Jan 2016
(V+27), 25 Jan 2016 (V+37), and 3 Feb 2016 (V+46), the pre-selected grapevines were
dismantled. Whole root systems, leaf blades and all fruit were collected from each
Chapter 5: Paper 3
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vine, collected and washed with phosphate-free detergent and rinsed with deionised
water. The fresh weights of these organs were determined, and the root and leaf
samples were oven-dried at 60 °C until a constant dry weight was reached. During the
destructive harvests, subsamples of the roots, leaf blades and berries were collected.
The root subsamples consisted of full-length root parts taken from within 10 cm from
the basal part of the trunk, always between 2 and 6 mm in diameter, with at least 50 g
in total tissue fresh weight. Soil particles were shaken off and the roots rapidly rinsed
with deionised water, prior to freezing in liquid N. Leaf subsamples consisted of 20
leaves, taken adjacent to a bunch, or from the shoot node directly above or below a
bunch when required, and frozen in liquid N. Berry subsamples consisted of 100
berries per vine, immediately frozen in liquid N after their removal from the vine. The
snap-frozen subsamples were stored at -80 °C until further processing. The periods
between the different destructive harvest dates are referred to as Intervals 1, 2, 3 and
4, respectively.
Vegetative and reproductive development
The total area of the leaves collected from each individual vine at the respective
destructive harvest dates was measured using a leaf area meter (LI-3100C, LI-COR
Biosciences Inc., Lincoln, NE, USA). The leaf subsample areas were measured
immediately after removal of the leaves, prior to the snap-freezing of these leaves.
Total fruit weight of each grapevine was recorded, and subsamples of 50 berries per
vine were used to determine the fresh weight per berry and juice total soluble solid
(TSS) concentration. Berry soluble solid content (SSC) was calculated on the basis of
berry fresh weight and TSS. The total fruit sugar content per vine basis was
subsequently calculated and is henceforth referred to as the fruit sugar content.
Chapter 5: Paper 3
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Total tissue dry weight of whole root systems and leaves were calculated for each vine
by combining the weights measured from the dried main samples with estimated dry
weights of the sub-samples. A subsample of 50 frozen berries per vine was ground to
a fine powder under liquid N using an analytical mill (A11 basic analytical mill, IKA,
Selangor, Malaysia), and freeze-dried (Gamma 1-16 LSC, Christ, Osterade am Harz,
Germany) until a constant weight was reached. Total fruit weight per vine was
estimated from the weight loss during drying. Root and leaf structural biomass per vine
were estimated by subtracting the non-structural carbohydrate content (total starch and
soluble sugar content) of these tissues from their total dry weight.
Non-structural carbohydrate determination
The root and leaf subsamples for each vine were taken from -80 °C storage and ground
to a fine powder under liquid N, using an analytical mill (A11 basic analytical mill,
IKA, Selangor, Malaysia). Frozen ground tissues of each sample were then freeze-
dried (Gamma 1-16 LSC, Christ, Osterade am Harz, Germany) until a constant weight
was reached. Starch and total soluble sugar concentrations in a 20 mg freeze-dried
sample of ground tissue were determined following the methods outlined in Smith and
Holzapfel (2009).
Total Nitrogen (N) determination
Nitrogen concentrations were determined in finely ground, freeze-dried samples of
roots, leaves and fruit. N concentration in 200 mg of a representative sample was
determined by the LECO method (Standard methods of Rayment and Lyons, Soil
chemical methods, Australasia, Dumas Combustion Method 6B2b), using a LECO
CNS TruMAC analyser (LECO corporation, St. Joseph, MI, USA).
Chapter 5: Paper 3
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Metabolite extraction and analysis
Extraction and derivatisation of untargeted metabolites in freeze-dried root and leaf
subsamples were performed using the method outlined in Lisec et al. (2006) with some
modifications. Firstly, 100 mg ground tissue were homogenised with 1.4 ml 100%
(v/v) methanol and 30 µl internal standard solution (1 g l-1 of each, adonitol, L-
hydroxyproline, and adipic acid, dissolved in 50% v/v methanol). The homogenate
was shaken at 70 ºC for 10 min (Thermomixer 5436, Eppendorf, North Ryde, NSW,
Australia), before being centrifuged at 11 000 g for 10 min. The supernatant was
transferred to a glass vial, and mixed with 0.75 ml chloroform and 1.4 ml ultrapure
water. The mixture was centrifuged at 2200 g for 15 min, and 150 µl of supernatant
(polar phase) collected and dried under a constant stream of pure N2 gas.
Derivatisation of the extracted metabolites was initiated by adding 40 µl of 20 mg ml-
1 methoxyamin hydrochloride in pure pyridine, to the dried extracts. Samples were
then shaken at 37 ºC for 2 h, before being centrifuged at 5000 g for 2 min. N-Methyl-
N-(trimethylsilyl) trifluoroacetamide (MSTFA, 70 µl) was added, and samples were
shaken again for 30 min at 37 ºC, before being centrifuged at 5000 g for 2 min.
Solutions of analytical grade standards (10 µg l-1 in 50% v/v methanol), obtained from
Sigma-Aldrich (Sigma, St. Louis, MO, USA), consisted of soluble sugars: Sucrose,
D(+)-glucose, D(-)-fructose, D(+)-mannose, L-rhamnose, D(-)-mannitol, galactinol
dehydrate, D(+)-raffinose, melibiose, D(+)-turanose, D(+)-melezitose, D(+)-
cellobiose, D(-)-ribose, D(-)-arabinose, D(+)-trehalose, maltose monohydrate, D(+)-
galactose, D(+)-xylose, dulcitol (galactitol), L-fucose, and myo-inositol; amino acids:
L-glutamic acid, L-arginine, L-proline, L-glutamine, γ-amino-n-butyric acid (GABA),
L-threonine, L-methionine, β-alanine, L-lysine, L-asparagine, L-aspartic acid, L-
Chapter 5: Paper 3
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leucine, L-isoleucine, L-valine, L-alanine, L-serine, glycine, L-tyrosine, L-
phenylalanine, L-tryptophan, L-histidine, L-cysteine and L-cysteine), and
miscellaneous compounds (L-ascorbic acid and protocathechuic acid), and were
prepared in order to assist in the retention index and spectra identification of these
compounds.
Extraction and analysis of samples were randomised, and after every tenth sample, a
quality control root sample was injected. GC/MS analyses were conducted by injecting
1 µl into the GC column (30 m ×0.25 mm, 0.25 µm HP-5MS, Agilent, Santa Clara,
CA, USA), in both split-less and split mode (300:1, to allow the measurement of more
abundant metabolites). The GC/MS system consisted of a 7683B series autosampler,
7890A gas chromatograph and 5975C mass spectrometer with an electron impact
ionisation source and a quadruple analyser (all from Agilent, Santa Clara, CA, USA).
The injection port was set at 250 ºC, the transfer line at 280 ºC, the ionisation source
at 230 ºC, and the quadrupole at 150 ºC. The helium carrier gas was set at a constant
flow rate of 1.3 ml min-1. The column temperature program was set at 65 ºC for 2 min,
followed by a 6 ºC min-1 ramp to 300 ºC, where it was held for 25 min. The ionisation
energy was set at 70 eV. Mass spectra were recorded in full mode at 2.66 scans s-1 with
a mass-to-charge ratio of 50 to 600 amu. Spectral deconvolution (signal-to-noise ratio
threshold = 10; mass absolute height ≥ 2000; compound absolute area ≥ 10000)
allowed the identification of co-eluting chromatographic peaks, and was conducted
through the MassHunter Workstation software (Qualitative Analysis, version B.07.00,
Agilent, Santa Clara, CA, USA). Acquired MS spectra were searched for, and
identified by using the National Institute of Standards and Technology algorithm
(NIST, Gaithersburg, MD, USA). The retention index for each compound in the
Chapter 5: Paper 3
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analysed samples was calculated by using the retention times of a series of alkanes
(C8-C28) in an injected retention index solution (Fluka, Buchs, Switzerland).
Statistical analysis
Datasets regarding grapevine vegetative and reproductive development (Table 1), non-
structural carbohydrate (Fig. 1) and total N distribution (Fig. 2), and primary
metabolite concentration (Figs. 3 and 4, with Supporting Information in Tables S1 and
S2) were analysed using Statistica 13 (Dell Inc., Tulsa, OK, USA). For each variable,
both treatment differences at a single time and how a treatment changed over time
were of interest. It was also recognised that residual variance at each time may differ
and that the interventionist nature of the treatments may also lead to reduced residual
variance for some treatments. To facilitate these comparisons, univariate analysis of
variance (ANOVA) at each date (all treatments) and for each treatment (all dates) were
conducted. An average Fisher’s least significant difference (LSD) test was used to
identify significant differences between means (P < 0.05). Significant differences in
table and heat map columns and rows are indicated by upper case letters (between
treatments) and lower case letters (between dates), respectively.
For each of the grapevine organs (roots and leaf blades), at each harvest date after the
initial harvest (V+18, V+27, V+37 and V+46), a linear mixed model was fitted for
every unequivocally identified primary metabolite (78 for roots and 75 for leaves)
using ASReml-R (Butler et al. 2007). Each model included Treatment as a fixed effect
and Replicate as a random effect. For the roots, Treatment included FL, 25L and NL,
while for the leaves Treatments included FL and 25L. The significance of treatment
effects was assessed using approximate F-tests using the techniques of Kenward and
Chapter 5: Paper 3
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Roger (1997). For each vine organ, primary metabolites which had significant
treatment effects (P < 0.05) for any of V+18, V+27, V+37 and V+46 were retained for
a Principal Component Analysis (PCA). The PCA was only conducted as an initial
data analysis step to interpret the interactions between metabolites, treatment and
harvest dates. Predicted means from the ASReml-R analysis were used in a PCA based
on the correlation matrix, conducted in Genstat 17th Edition (VSN International Ltd.,
Hemel Hempstead, Hertfordshire, UK). Biplots were drawn using R statistical
software plus the add-in package ‘shape’ (developed by Karline Soetaert, Royal
Netherlands Institute of Sea Research Yerseke, The Netherlands). These biplots and
further PCA information are provided in the Supplementary Information (Figs. S1 and
S2).
To test the relationship between the concentration of root starch and myo-inositol, a
cubic smoothing spline was fitted using the linear mixed model methods of Verbyla et
al. (1999). The fixed effects included intercept effects for the overall mean and
treatments, and effects for linear trend including overall linear trend and trend due to
treatment. The random effects included overall spline curvature and curvature due to
treatment as well as effects due to replicates at each time of measurement. The
significance of fixed treatment effects was assessed by the approximate F-tests using
the techniques of Kenward and Roger (1997) and the significance of spline curvature
was assessed by examining 0.5(1-Pr( χ 2 ≤ d)) where d refers to models which differ in
a single spline curvature term. This linear model was fitted using ASReml 3.0
(Gilmour et al. 2009).
Chapter 5: Paper 3
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Results
Vegetative and reproductive development
The total leaf area (data not shown) and corresponding leaf-to-fruit ratio per control
(FL) vine were initially (at V+9) adjusted to 1.4 m2 and 8 cm2 leaf area g-1 fresh fruit
(Table 1) respectively, and these values did not change significantly during the
experiment. The leaf area and leaf-to-fruit ratio of the 25 leaf treatment (25L) was
adjusted to 0.6 m2 and 1.9 cm2 g-1 respectively, with the latter being significantly lower
than those of FL from V+18.
The root structural biomass per FL vine increased significantly during Interval 1 (Table
1), while that of 25L and no leaf (NL) did not alter significantly. FL had significantly
larger root structural biomass at V+46 than 25L or NL. The FL leaf structural biomass
per vine increased significantly between V+18 and V+46 (Table 1), while that of 25L
decreased during Interval 1 due to the defoliation. After treatment implementations,
FL leaf structural biomass was significantly larger than that of 25L at all harvest dates.
The FL and 25L total fruit dry weight per vine increased significantly during Interval
1 (Table 1). The FL total fruit dry weight continued to increase between V+18 and
V+37, while that of 25L increased significantly between V+18 and V+46. The NL
total fruit dry weight per vine increased significantly between V+9 and V+27. FL
significantly induced the largest fruit dry weight per vine from V+27.
Carbohydrate distribution
Fruit sugar accumulation: FL total fruit sugar content per vine increased rapidly during
Intervals 1 (12 g d-1) and 2 (14 g d-1) (Fig 1A). It continued to increase significantly
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during Interval 3 although at a reduced rate (7 g d-1), and did not change significantly
during Interval 4 (3 g d-1). Although at rates lower than that of the control, 25L fruit
sugar content per vine increased significantly during Intervals 1 (7 g d-1) and 2 (8 g d-
1). The NL total fruit sugar content per vine increased at a slow but significant rate
during Interval 1 (4 g d-1) and between V+18 and V+37 (3 g d-1). Among treatments,
FL fruit had significantly higher sugar content from V+27, and 25L fruit contained
significantly more sugar per vine than those of NL at V+27.
Root carbohydrate abundance: Starch concentration in FL roots decreased
significantly between V+9 and V+27, and then increased back to its original
concentration during Interval 4 (Fig. 1B). The 25L and NL root starch concentrations
reduced significantly during Intervals 1 and 3. Among the treatments, FL root starch
concentration was highest at V+18, V+37 and V+46. The FL root total sugar
concentration was significantly higher at V+46 than at V+9 and V+27, and was
significantly higher than that of 25L at V+46 (Fig. 1B). The NL root total sugar
concentration was significantly higher from V+37 than at V+18.
Leaf carbohydrate abundance: FL leaf starch concentration decreased significantly
during Interval 2, and increased significantly during Intervals 3 and 4 (Fig. 1C). The
25L leaf starch concentration reduced significantly during Intervals 1 and 2. Among
treatments, FL significantly induced the highest leaf starch concentration from V+37.
The only significant leaf sugar concentration changes occurred where the FL
concentration increased during Intervals 3 and 4 (Fig. 1C). No leaf sugar concentration
treatment differences occurred.
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Nitrogen (N) distribution
Fruit N accumulation: FL total fruit N content per vine increased significantly between
V+9 and V+46 (0.02 g d-1) (Fig. 2A). The 25L and NL total fruit N contents per vine
increased significantly between V+9 and V+27 (0.04 g d-1). No significant treatment
differences occurred in total fruit N content.
Root N abundance: The only significant root N concentration change occurred where
25L N increased during Interval 3 (Fig. 2B). Among treatments, root N concentrations
of 25L and NL was significantly higher than that of FL at V+46.
Leaf N abundance: FL leaf N concentration reduced significantly during Interval 3,
and was significantly lower at V+46 than before V+37 (Fig. 2C). The 25L leaf N
concentration increased significantly during Interval 1, and reduced significantly
during Intervals 2 and 3. Among the treatments, 25L leaf N concentration was the
highest at V+18.
Metabolic adjustments
Primary metabolites from the roots and leaves were categorised as sugars, sugar
alcohols, amino acids, miscellaneous acids, or others (including flavonoids and
stilbenoids). Further information regarding the metabolite abundance and MS spectra
are indicated in Tables S1 and S2 (Supporting Information). Simplified listings of all
metabolites which had significant treatment effects for any of the destructive harvest
dates, and the associated biosynthetic pathway of each metabolite, are indicated for
roots (Table 2) and leaves (Table 3).
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Figures 3 and 4 illustrate the effects of the defoliation treatments on root and leaf
metabolite concentrations respectively. The concentrations of metabolites that
exhibited significant treatment differences, and are involved in major C and N
metabolic pathways, are indicated in the figures. For ease of interpretation, treatment
effects and notable metabolite responses are described in further detail below. This
description is structured in accordance to the simplified metabolic pathways for roots
(myo-inositol metabolism, amino acid metabolism including shikimate pathway
derived amino acids, and the TCA cycle) and leaves (sugar alcohol and further myo-
inositol metabolism, the shikimate pathway including aromatic amino acids, the TCA
cycle, and amino acid metabolism other than those related to the shikimate pathway).
Root metabolism
Myo-inositol metabolism: The FL root myo-inositol concentration decreased between
V+9 and V+27, and then increased between V+27 and V+46. For the 25L and NL
treatments root myo-inositol concentrations decreased during both the first two
intervals. FL roots subsequently contained more myo-inositol than those of 25L and
NL at V+18, and again from V+37 (Fig. 3). While FL root galactinol decreased
between V+9 and V+46, that in 25L and NL roots reduced during Interval 1. Among
treatments, FL roots contained the most galactinol at V+37, and additionally more
galactinol than those of 25L and NL at V+18 and V+46, respectively. FL root raffinose
increased during Interval 3, while that of 25L and NL reduced between V+9 and V+27.
Among treatments, FL roots exhibited the highest raffinose concentration from V+27.
FL root melibiose increased during Interval 2 before decreasing during Interval 3,
while that in 25L and NL roots increased between V+9 and V+37. Melibiose was
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subsequently more abundant in 25L and NL roots than those of FL at V+37, while NL
roots additionally contained more melibiose than those of FL at V+46.
FL root ascorbic acid increased between V+18 and V+46, while that in 25L and NL
roots reduced during Interval 1. Among treatments, FL roots contained the most
ascorbic acid at V+27 and V+46. While FL root tartaric acid did not change
significantly, that in 25L and NL roots increased between V+9 and V+37. 25L and NL
roots subsequently contained more tartaric acid than FL roots from V+37 and at V+46,
respectively.
Amino acid metabolism: The FL root glutamic acid concentration never changed
significantly, however, that in 25L and NL roots decreased during Interval 1. FL roots
subsequently contained more glutamic acid than those of NL and 25L from V+27 and
at V+37, respectively. Furthermore, 25L roots contained more glutamic acid than those
of NL at V+46. The FL root arginine concentration also did not change during the
experiment, however, that in 25L and NL roots accumulated during Interval 3 and
between V+18 and V+37, respectively. Among treatments, 25L contained most
arginine at V+46, when NL roots contained more arginine than FL roots.
For shikimate pathway derived amino acids, FL and 25L root tryptophan
concentrations did not change during the experiment, however, that in NL roots
accumulated during Interval 3. NL roots subsequently exhibited more tryptophan than
those of FL at V+37. Like with tryptophan, FL and 25L root tyrosine concentrations
did not change significantly, however, NL roots accumulated tyrosine between V+9
and V+27. Among treatments, NL roots contained most tyrosine at V+27. FL root
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phenylalanine increased between V+18 and V+37, before decreasing during Interval
4. While 25L root phenylalanine did not change significantly, that in NL roots
increased between V+9 and V+27. NL roots subsequently contained more
phenylalanine than those of FL and 25L at V+27.
Aspartic acid reduced in FL roots between V+9 and V+27, before increasing towards
V+46. 25L aspartic acid reduced between V+9 and V+27, while NL root aspartic acid
concentration did not alter significantly. FL roots contained more aspartic acid than
those of NL at V+46. FL root lysine did not change significantly, while that of 25L
and NL increased during Interval 3. At V+46, 25L roots contained the most lysine,
while NL roots exhibited higher lysine concentration than FL roots.
TCA cycle intermediate metabolism: FL root citric acid increased during Interval 4,
while that in 25L and NL roots accumulated during Interval 3, the NL citric acid then
decreased during Interval 4. Among treatments, 25L and NL roots contained more
citric acid than those of FL from V+37, while 25L roots also contained more citric acid
than NL roots at V+46. No significant maleic acid concentration changes occurred,
however, 25L roots contained more maleic acid than those of FL at V+37.
Leaf metabolism
Sugar alcohol and further myo-inositol metabolism: While the FL leaf mannitol
concentration did not change significantly, that of 25L leaves decreased during Interval
1. As a result, FL leaves contained more mannitol than those of 25L at V+46 (Fig. 4).
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FL myo-inositol decreased between V+9 and V+27, while that in 25L leaves gradually
decreased from V+9 to V+46. Among treatments, myo-inositol concentration was
lower in 25L than FL leaves, from V+27. FL leaf ascorbic acid concentration did not
change significantly, while that in 25L leaves decreased between V+9 and V+27. The
25L leaf ascorbic acid concentration was subsequently higher than that of FL leaves
from V+37. Tartaric acid decreased in FL leaves during Interval 2 and between V+27
and V+46. The 25L leaf tartaric acid decreased during Intervals 1 and 2, and among
treatments, FL leaves contained more tartaric acid at V+27 and V+37. While the FL
leaf threonic acid concentration did not change significantly, that in 25L leaves
decreased during Interval 1. 25L leaves subsequently exhibited more threonic acid
from V+27. FL leaf glyceric acid increased between V+9 and V+27, before decreasing
towards V+46. 25L leaf glyceric acid decreased during Interval 1, and among
treatments, FL leaves contained more glyceric acid from V+27.
Shikimate pathway derivatives: For amino acids, leaf phenylalanine accumulated
during Interval 2 regardless of the treatments, before decreasing during Interval 3.
However, 25L leaves contained more phenylalanine than those of FL at V+37. Leaf
tyrosine also accumulated during Interval 2, before depleting with no significant
concentration differences among treatments. Likewise, tryptophan accumulated during
Interval 2 before depletion in 25L leaves at V+37, while increasing between V+9 and
V+27 in FL leaves. In FL leaves, 5-hydroxytryptophan decreased between V+9 and
V+37, while reducing during Interval 1 and between V+18 and V+37 in 25L leaves.
Among treatments, FL leaves contained more 5-hydroxytryptophan at V+27 and
V+37.
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Caffeic acid depleted in the leaves of both treatments between V+9 and V+46, without
significant treatment differences. On the other hand, gallic acid was significantly less
abundant at V+27 than at V+9 in FL leaves, while 25L leaf gallic acid reduced during
Interval 1. FL leaf gallic acid was less abundant than that of 25L leaves from V+37.
Arbutin accumulated in leaves of both treatments between V+9 and V+37, and
additionally during Interval 4 in those of 25L. 25L leaves subsequently contained most
arbutin at V+46. While FL (+)-Catechin did not change significantly, it decreased
between V+9 and V+37 in 25L leaves, before increasing during Interval 4. The 25L
leaves contained more (+)-catechin than those of FL at V+46.
TCA cycle intermediates: Citric acid reduced in leaves of both treatments during
Interval 1 and between V+18 and V+37. However, among treatments, FL leaves
contained more citric acid from V+18.
Amino acid metabolism: FL and 25L leaf glutamic acid decreased during Interval 1,
before increasing in FL leaves during interval 2. However, no leaf glutamic acid
treatment differences were observed. While FL leaf GABA concentration did not
change significantly, that in 25L leaves accumulated during Interval 3. 25L leaves
subsequently exhibited more GABA at V+18, and from V+37.
FL leaf aspartic acid decreased between V+9 and V+37, while that in 25L leaves
decreased both, during Interval 1, and between V+18 and V+37. However, no leaf
aspartic acid treatment differences were observed. Threonine decreased in leaves of
both treatments during Interval 1, before accumulating in those of 25L during Interval
3. Among treatments, 25L leaves contained most threonine from V+18. While FL leaf
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isoleucine concentration did not change significantly, that of 25L leaves accumulated
during Interval 4. 25L leaves subsequently contained most isoleucine at V+27 and
V+46. FL leaf leucine concentration did not alter significantly, however, 25L leaf
leucine accumulated between V+9 and V+46. Among treatments, 25L leaves
contained most leucine at V+46. Although no significant valine concentration changes
occurred for both treatments, 25L leaves contained more valine at V+27 and V+46.
Serine decreased in FL leaves between V+9 and V+27, while increasing in 25L leaves
during Intervals 1 and 3. The 25L leaves contained most serine among treatments from
V+18. Cysteine accumulated in FL leaves during Interval 2, while it accumulated in
25L leaves between V+18 and V+37. The 25L leaves contained more cysteine than
those of FL at V+37.
Relationship between root starch and myo-inositol
Changes in root starch and myo-inositol concentrations were similar over time (Fig.
5). In FL roots, both starch and myo-inositol significantly declined from V+9 to V+37
but recovered to the abundance at V+9, by V+46. For 25L and NL, root starch and
myo-inositol declined significantly between V+9 and V+37, and plateaued during
Interval 4. The relationship between starch and myo-inositol is additionally illustrated
in the Supplementary Information (Fig. S3).
Discussion
The current study evaluated implications of reduced carbohydrate and N source-sink
biomass ratios during berry maturation for metabolite concentrations of the major
source organs. Specific defoliation induced changes in root and leaf carbohydrate and
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N utilisation, and linkages to primary metabolism, are explained below. Leading into
the discussion, it is particularly noteworthy that decreasing source-sink ratios during
berry ripening distinctly increased root carbohydrate source activity. Conversely,
removing the leaf N source only had minor effects on total N re-distribution from
leaves and roots. Defoliation did, however, alter N composition of roots and leaves,
suggesting increased amino N source activity when the total vine N source-sink
biomass ratio was reduced.
Root carbohydrate reserve remobilisation
Following the removal of all leaves in the full defoliation treatment, root starch
concentrations rapidly declined (Fig 1B). However, the continued accumulation of
berry sugar in the corresponding period, albeit at a lower rate than control vines,
indicates a clear contribution of reserve carbohydrates to berry ripening (Fig 1A). Such
contributions from stored carbohydrates has previously been demonstrated by 14C
labelling, when carbohydrates from perennial tissues were translocated to fruit after
defoliation during fruit sugar accumulation (Candolfi-Vasconcelos et al. 1994). The
retention of some leaves on partially defoliated vines did not alter the rate of root
carbohydrate mobilization relative to the fully defoliated vines, but the availability of
extra carbohydrates from concurrent photosynthesis did allow an increased rate of
berry sugar accumulation. Under full leaf area, root starch concentration reduced only
during the phase of rapid fruit sugar accumulation, and then increased when fruit sugar
accumulation slowed. This starch reduction may imply reserve remobilisation towards
the sugar-accumulating berries as these vines carried a substantial crop load. However,
root reserves could also be utilised for respiration and structural development
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(Holzapfel et al. 2010), of which latter was observed during Interval 1 under full leaf
area.
Total N distribution was unaffected by defoliation
Under full leaf area, fruit sugar content per vine increased by 252% in the 37-day
period following the start of the experiment (Fig. 1A). During the same period fruit N
content only increased by 39% (Fig. 2A), implying a proportionally greater importance
of the véraison to harvest period for berry carbohydrate than N accumulation. The leaf
N concentrations of 2.1% (Fig 2C) were adequate according to published levels
(Holzapfel and Treeby 2007), and suggest that the lower N accumulation reflected
lower fruit sink demand rather than reduced availability of N from the vegetative parts
of the vine.
Although fruit N accumulation rates varied somewhat across the five sampling dates,
the fruit did not exhibit any significant N content treatment differences by the final
harvest. Therefore, despite the reduction or complete removal of leaves as an N source
(Rossouw et al. 2017b), fruit N accumulation was maintained. The lack of change in
root N concentration after partial or full defoliation (Fig. 2B), suggests the roots did
not become a significant net source during fruit N accumulation (Conradie 1991).
Although N fertilisation was ceased a month prior the experiment, it is likely that soil
N uptake contributed to maintaining fruit N accumulation. Soil N uptake is not unusual
shortly after véraison (Löhnertz 1991), and the limitation or absence of the leaf N
source did not interfere with N allocation towards the fruit by the final harvest.
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A central role for myo-inositol during root carbohydrate remobilisation
The parallel changes of root starch and myo-inositol concentrations with treatment and
time (Fig. 5), suggests a role for myo-inositol during root carbohydrate remobilisation.
The roles of myo-inositol in plants include signalling, involvement in the synthesis of
cell wall polysaccharides, and precursor for other metabolites including galactinol,
raffinose, and ascorbic acid (Valluru and Van den Ende 2011). However, while myo-
inositol has been found in the phloem sap of various plants, suggesting a potential
long-distance transport role (Noiraud et al. 2001), myo-inositol is not generally
considered a major C transport compound. The close similarities between starch and
myo-inositol concentration profiles in the grapevine roots of the present study is an
original result, and further investigation is needed to determine the underlying
connection. The impact of defoliation on the decreased root concentrations of myo-
inositol and its derivatives, i.e., galactinol and raffinose (Loewus and Murphy 2000)
(Fig. 3), suggests that these metabolites play a role during carbohydrate reserve
remobilisation.
Similar to myo-inositol, root ascorbic acid depleted shortly after partial or full
defoliation (Fig. 3). It has been established that myo-inositol metabolism provides an
alternative pathway for ascorbic acid biosynthesis (Lorence et al. 2004). The
similarities in root myo-inositol and ascorbic acid concentrations over time, in terms
of both, for example, exhibiting higher concentration in FL roots by V+46, is therefore
potentially related to myo-inositol providing the initial substrate towards an ascorbic
acid biosynthetic route (Lorence et al. 2004, Valpuesta and Botella 2004).
Furthermore, ascorbic acid is a precursor for tartaric acid, and defoliation led to a
depletion of root ascorbic acid, while tartaric acid accumulated (Fig. 3). Therefore,
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root ascorbic acid catabolism potentially resulted in tartaric acid accumulation (DeBolt
et al. 2006). Additionally, root citric acid also accumulated under reduced leaf area
(Fig. 3). Intermediates of the TCA cycle, such as citric acid, play an essential role
during C metabolism, supplying C skeletons for the biosynthesis of various
metabolites, such as phenolic compounds and amino acids (Popova and Pinheiro de
Carvalho 1998).
Defoliation induced root amino acid accumulation
Glutamic acid was the only root amino acid that depleted under partial and full
defoliation (Fig. 3). The biosynthesis of amino acids in higher plants mainly occurs in
roots and mature leaves, from where it is transportable to sinks, thereby facilitating the
distribution of organic N between plant organs (Rentsch et al. 2007). Although
defoliation did not affect the root total N concentration, it did impact on root amino N
composition. As an essential amino-group donor during the synthesis of many other
amino acids, glutamic acid plays a crucial role during N partitioning (Forde and Lea
2007). The depletion of root glutamic acid after partial and full defoliation may
indicate its involvement in amino N repartitioning within these roots.
Various other amino acids accumulated in the roots of partially or fully defoliated
vines, including arginine, lysine, phenylalanine, tryptophan, and tyrosine (Fig. 3). It
can thus be proposed that glutamic acid metabolism was involved in the accumulation
of these amino acids in the roots, either directly by providing a C skeleton for arginine
synthesis (Berg et al. 2002), or as an amino donor towards the synthesis of the others.
The accumulation of arginine in roots, only after partial or full defoliation may relate
to a N transport role. In fact, arginine is characterised by a high N:C ratio, and is known
Chapter 5: Paper 3
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to be transported in the xylem and phloem between source and sink organs, facilitating
organic N distribution (Lea et al. 2007). When the leaves as a N source was restricted
or limited, root amino acid accumulation presumably contributed to the root amino N
source activity.
The aromatic amino acids (i.e., phenylalanine, tryptophan and tyrosine) only
accumulated in the roots after full defoliation (Fig. 3). These amino acids originate
from the shikimate pathway, and are precursors for many secondary metabolites,
including phenolic compounds (Maeda and Dudareva 2012). Various secondary
metabolites derived from the aromatic amino acids, including anthocyanins (Boss et
al. 1996), accumulate in post-véraison grapevine berries. The removal of leaves as an
amino acid source seemingly induced the biosynthesis of root amino acids through the
shikimate pathway. Genes related to the aromatic amino acids are expressed in post-
véraison grapevine berries (Berdeja et al. 2015). However, many amino acids,
including phenylalanine, are also present in the vascular tissues of higher plants (as for
example, indicated in Trifolium repens and Lupinus albus), where they accrete after
defoliation (Hartwig and Trommler 2001). The possibility is, therefore, raised that
grapevine leaves are important aromatic amino acid sources, from where they may be
phloem translocated to the fruit to contribute to secondary metabolism. The roots may
become an alternative aromatic amino acid source after exclusion of the leaf source.
Leaf sugar alcohols and organic acids depleted rapidly after partial defoliation
Myo-inositol and mannitol, some of the most prevalent sugar alcohols in higher plants
(Noiraud et al. 2001), depleted rapidly in remaining leaves after partial defoliation
(Fig. 4). Apart from sucrose as the principal plant transported sugar, some sugar
Chapter 5: Paper 3
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alcohols and raffinose family oligosaccharides can also be important C transporters
(Noiraud et al. 2001). Mannitol, unlike myo-inositol, is a known primary
photosynthetic product in mature leaves, and a recognised transport compound
(Noiraud et al. 2001). During the present study, mannitol was, therefore, presumably
synthesised in leaves during photosynthesis, and its rapid depletion in leaves after
partial defoliation suggests an important C transport role during limited canopy
photoassimilation. Although there has been ambiguity around the transport role of
myo-inositol (Noiraud et al. 2001), the current study suggests that root myo-inositol
plays a central role during carbohydrate remobilisation, and perhaps similarly in
leaves.
In addition to the sugar alcohols mentioned above, various leaf organic acids rapidly
depleted after partial defoliation (Fig. 4). Citric acid was among the organic acids
depleted under reduced leaf area, and like other TCA intermediates, it is a vital
metabolic branch point as its conversion provides C skeletons for N assimilation, in
addition to playing an important role in plant energy and C metabolism (Popova and
Pinheiro de Carvalho 1998). Leaf ascorbic acid concentration, like that of myo-inositol,
was negatively impacted by partial defoliation (Fig. 4). As a potential precursor, leaf
myo-inositol metabolism may have affected the ascorbic acid concentration. Likewise,
leaf tartaric acid, threonic acid and glyceric acid also depleted after partial defoliation
(Fig. 4). These organic acids are derived from ascorbic acid (Loewus 1999), further
indicating a change in ascorbic acid metabolism in remaining source leaves. The
phenolic acids, caffeic acid and gallic acid, depleted towards the end of berry ripening
in remaining leaves after partial defoliation, while arbutin and (+)-catechin increased
(Fig. 4). These compounds are products of the shikimate pathway (Balasundram et al.
Chapter 5: Paper 3
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2006, Siegler 1998) and, therefore, like in roots, leaf metabolites yielded from the
shikimate pathway were affected by post-véraison leaf source limitation.
Partial defoliation induced leaf amino acid accumulation
Various amino acids accumulated in remaining leaves after partial defoliation (Fig. 4).
Amino acid synthesis in plants, as mentioned above, mainly occurs in roots and mature
leaves, where they are utilised or stored, or exported to sinks to contribute to growth
and secondary metabolism (Rentsch et al. 2007). Leaf proteins (e.g. Rubisco and
chloroplast proteins) are extensively degraded during leaf ageing, thereby producing
free amino acids, outsourceable to sinks (Masclaux-Daubresse et al. 2010). In the
present study, GABA, leucine, isoleucine, as well as cysteine and serine were among
the amino acids that accumulated in the remaining leaves after partial defoliation (Fig.
4). After defoliation in the present study, the ratio of the leaf N source to the fruit N
sink was drastically reduced. It could, therefore, be argued that the increased source
requirement placed upon the remaining leaves of treatment 25L advanced its ageing
process, prompting amino acid accumulation and its subsequent exportation.
Conclusion
A study was conducted to determine the implications of reduced leaf carbohydrate and
N source availability, during a period of considerable fruit C and N sink demand, on
remaining leaf or root source activity. A focus was placed upon primary metabolite
abundance responses in the source organs, ultimately with the goal of identifying
metabolites that contribute to carbohydrate and N source functioning in roots and
leaves. In terms of carbohydrate distribution, post-véraison leaf source absence
slowed, but did not completely stop fruit sugar accumulation. In the absence of leaf C
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assimilation, root starch provided an alternative C source for berry ripening. Root myo-
inositol concentrations were directly related to starch concentration, suggesting an
important, if yet to be elucidated role, in starch metabolism. Furthermore, the depletion
of myo-inositol metabolism derived metabolites (galactinol, raffinose and ascorbic
acid) after defoliation, illustrates the contribution of this proposed pathway towards
root carbohydrate source functioning. Compared to carbohydrates, defoliation did not
have a considerable effect on fruit N content. In fact, vegetative N pools did not
contribute to post-véraison fruit N requirements, which instead appear to have been
met by root uptake from the soil and/or regulation of amino N composition in the
leaves and/or roots. However, arginine and shikimate pathway derived root aromatic
amino acids did accumulate after full defoliation, indicating there is a least a response
in this pool in roots during leaf N absence. The remaining leaves also accumulated
various amino acids (including GABA, leucine, isoleucine and serine) after partial
defoliation, suggesting protein degradation could make a small N contribution to the
fruit. Overall, this study has shown that myo-inositol metabolism and the flux through
the shikimate pathway play central roles in grapevine carbohydrate and N source
organs during fruit ripening. The findings of this study contribute to understanding leaf
and root C and N metabolism and utilisation during fruit maturation.
Author contributions
GCR conducted the experiment, wrote the body of the paper, and carried out sample
preparations, and laboratory and data analyses. KŠ contributed to sample preparation
and led the GC/MS analysis. BAO contributed to the experimental layout and
conducted statistical analyses. BPH coordinated the project and supported the
experimental planning. AD contributed to treatment planning and experimental design.
Chapter 5: Paper 3
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JPS and CB reviewed the methods and results. All authors reviewed, edited and
approved the final version of the manuscript.
Acknowledgements - This work was supported by the National Wine and Grape
Industry Centre, and the Australian grapegrowers and winemakers through their
investment body, Wine Australia, with matching funds from the Australian
Government. The authors thank Robert Lamont and Peter Carey for technical
assistance.
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Supporting Information
Additional Supporting Information may be found in the online version of this article:
Table S1. Root metabolite concentration and GC/MS information.
Table S2. Leaf metabolite concentration and GC/MS information.
Fig. S1. Root primary metabolite PCA.
Chapter 5: Paper 3
113
Fig. S2. Leaf primary metabolite PCA.
Fig. S3. Linear and curvilinear trends of root starch concentration and myo-inositol
concentration.
Tab
le 1
Eff
ect o
f def
olia
tion
treat
men
ts (f
ull l
eaf –
FL,
25%
leav
es –
25L
and
no
leaf
– N
L) o
n gr
apev
ine l
eaf-
to-fr
esh
frui
t wei
ght r
atio
, roo
t and
leaf
stru
ctur
al
biom
ass
per v
ine,
and
tota
l fru
it dr
y w
eigh
t per
vin
e at
the
dest
ruct
ive
harv
ests
(V+9
, V+1
8, V
+27,
V+3
7 an
d V
+46 ;
mea
n ±
SE, n
=3).
Mea
ns a
re s
epar
ated
w
ithin
row
s an
d co
lum
ns u
sing
Fish
er’s
LSD
test
, sig
nific
ant d
iffer
ence
s ar
e in
dica
ted
at P
< 0
.05.
Whe
re d
iffer
ent l
ower
cas
e le
tter a
ppea
rs in
a ro
w, v
alue
s di
ffer
sign
ifica
ntly
bet
wee
n da
tes.
Whe
re d
iffer
ent u
pper
cas
e le
tter a
ppea
rs in
a c
olum
n, v
alue
s diff
er si
gnifi
cant
ly b
etw
een
treat
men
ts.
Inte
rval
1
Inte
rval
2
Inte
rval
3
Inte
rval
4
Tre
atm
ent
V+9
V
+18
V+2
7 V
+37
V+4
6
Lea
f-to-
fres
h fr
uit
wei
ght r
atio
(c
m2 /g
)
FL
11 ±
2
a A
8
± 1
ab
A
6.9
± 0.
6 b
A
7.0
± 0.
8 b
A
7.5
± 0.
4 b
25L
11 ±
2
a B
1.
9 ±
0.2
b B
1.
9 ±
0.2
b B
1.
6 ±
0.1
b B
1.
5 ±
0.1
b N
L 11
± 2
-
- -
-
Roo
t st
ruct
ural
bi
omas
s (g
/vin
e)
FL
172
± 6
b A
22
6 ±
19
a A
22
6 ±
19
a A
22
4 ±
19
a A
20
1 ±
5 a
25L
172
± 6
a A
18
2 ±
17
a A
18
2 ±
17
a A
16
8 ±
16
a B
14
7 ±
13
a N
L 17
2 ±
6 a
A
197
± 19
a
A
197
± 19
a
A
199
± 24
a
B
155
± 13
a
Lea
f st
ruct
ural
bi
omas
s (g
/vin
e)
FL
76 ±
3
b A
76
± 2
b
A
82 ±
3
ab
A
82 ±
4
ab
A
91 ±
3
a 25
L 76
± 3
a
B
19 ±
1
b B
19
± 3
b
B
22.8
± 0
.4
b B
22
± 2
b
NL
76 ±
3
- -
- -
Tot
al fr
uit
dry
wei
ght
(g/v
ine)
FL
203
± 20
c
A
338
± 53
b
A
457
± 25
ab
A
54
4 ±
46
a A
57
5 ±
51
a 25
L 20
3 ±
20
c A
29
2 ±
19
b B
35
6 ±
11
ab
B
365
± 7
ab
B
408
± 49
a
NL
203
± 20
b
A
272
± 37
ab
B
30
1 ±
24
a B
31
6 ±
17
a B
32
2 ±
8 a
Chapter 5: Paper 3
114
Chapter 5: Paper 3
115
Fig. 1. Impact of defoliation (full leaf – FL, 25% leaves – 25L and no leaf – NL) on total fruit sugar content per vine (A), root starch and total sugar (total non-structural carbohydrates, TNC) concentrations (B), and leaf starch and total sugar (TNC) concentrations (C) during the experimental period (mean ± SE; n=3). Significant differences (P < 0.05) between harvest dates for each treatment are indicated by different lower case letters. Significant differences (P < 0.05) between treatments at each harvest date are indicated by different numerals [1: FL > (25L and NL) and 2: FL > 25L > NL]. To allow clarity of the most important results, these significant differences are indicated for fruit sugar content (A), and root (B) and leaf (C) starch concentrations only.
Chapter 5: Paper 3
116
Fig. 2. Impact of defoliation (full leaf – FL, 25% leaves – 25L and no leaf – NL) on total fruit nitrogen (N) content per vine (A), root N concentration (B), leaf N concentration (C) during the experimentalperiod (mean ± SE; n=3). Significant differences (P < 0.05) between harvest dates for each treatmentare indicated by different lower case letters. Significant differences (P < 0.05) between treatments ateach harvest date are indicated by different numerals [1: FL < (25L and NL) and 2: FL < 25L].
Chapter 5: Paper 3
117
Table 2 Proposed metabolic pathways related to the biosynthesis of significantly treatment affected root metabolites. All metabolites significantly differing among the defoliation treatments (full leaf, 25% leaves and no leaf) for any of the destructive harvest dates after treatment implementation (V+18, V+27, V+37 and V+46) are listed. The metabolites are categorised based on their compound properties (sugars, sugar alcohols, amino acids or miscellaneous acids).
Classification Metabolite Proposed primary pathway
Sugars
Sucrose Raffinose Melibiose Arabinose
Sugar alcohols
Myo-inositol Galactinol Mannitol Arabitol Glycerol
Primary carbohydrate metabolism Myo-inositol metabolism Myo-inositol metabolism Glucose-6-phosphate
Glucose-6-phosphate Myo-inositol metabolism Fructose metabolism Glucose-6-phosphate Glycerate
Amino acids
α-Ketoglutarate α-Ketoglutarate α-Ketoglutarate Shikimate Shikimate Shikimate
Miscellaneous acids
Glutamic acid Arginine Glutamine Tryptophan Phenylalanine Tyrosine Glycine Lysine Threonine Valine
Ascorbic acid Tartaric acid Citric acid Maleic acid 3-Hydroxyanthranilic acidProtocatechuic acid2-Keto-gluconic acid
3-PhosphoglycerateOxaloacetateOxaloacetatePyruvate
Myo-inositol metabolism Myo-inositol metabolism Tricarboxylic acid cycle Tricarboxylic acid cycle Shikimate Shikimate Gluconate
Chapter 5: Paper 3
118
Table 3 Proposed metabolic pathways related to the biosynthesis of significantly treatment affected leaf metabolites. All metabolites significantly differing among the defoliation treatments (full leaf and 25%) for any of the destructive harvest dates after treatment implementation (V+18, V+27, V+37 and V+46) are listed. The metabolites are categorised based on their compound properties (sugars, sugar alcohols, amino acids, miscellaneous acids and others).
Classification Metabolite Proposed principle pathway
Sugars
Glucose Raffinose Melibiose Rhamnose Melezitose Ribose
Primary carbohydrate metabolism Myo-inositol metabolism Myo-inositol metabolism Fructose metabolism Sucrose metabolism Glucose-6-phosphate
Sugar alcohols Mannitol
Amino acids
Fructose metabolism Glucose-6-phosphate
α-Ketoglutarate α-Ketoglutarate 3-Phosphoglycerate3-PhosphoglyceratePyruvatePyruvatePyruvateShikimateShikimateShikimate
Miscellaneous acids
Other compounds
Myo-inositol
Arginine GABA Serine Cysteine Valine Leucine Isoleucine Phenylalanine Tryptophan 5-HydroxytryptophanThreonine
Ascorbic acid Tartaric acid Threonic acid Glyceric acid Caffeic acid Gallic acid Lactic acid Citric acid Fumaric acid 2-Keto-glutaric acidPhosphoric acidGluconic acidRibonic acidNonanoic acidPalmitic acid
Arbutin Catechin Glycerol monostearate
Oxaloacetate
Myo-inositol metabolism Myo-inositol metabolism Myo-inositol metabolism Myo-inositol metabolism Shikimate Shikimate Pyruvate Tricarboxylic acid cycle Tricarboxylic acid cycle Tricarboxylic acid cycle Tricarboxylic acid cycle Glucose metabolism Glucose-6-phosphate Glycerol metabolism Glycerol metabolism
Shikimate Shikimate Glycerol metabolism
Chapter 5: Paper 3
119
Fig.
3. S
impl
ified
pat
hway
resp
onse
of t
he ro
ot p
rimar
y m
etab
olite
s si
gnifi
cant
ly a
ffec
ted
by th
e tre
atm
ents
(ful
l lea
f: FL
; 25%
leav
es: 2
5L; n
o le
af: N
L) a
nd
othe
r met
abol
ites
dire
ctly
invo
lved
in th
e pa
thw
ays
(1).
Sign
ifica
nt d
iffer
ence
s ar
e in
dica
ted
at P
< 0
.05,
hea
tmap
col
umns
indi
cate
the
thre
e tre
atm
ents
, whi
le
heat
map
row
s in
dica
te th
e ha
rves
t dat
es (V
+9, V
+18,
V+2
7, V
+37
and
V+4
6). W
here
diff
eren
t upp
er c
ase
lette
rs a
ppea
r in
heat
map
col
umns
(2),
valu
es d
iffer
sig
nific
antly
bet
wee
n tre
atm
ents
. Whe
re d
iffer
ent l
ower
cas
e le
tters
app
ear
in a
row
, val
ues
diff
er s
igni
fican
tly b
etw
een
harv
est d
ates
. Ave
rage
met
abol
ite
abun
danc
e is
colo
ur c
oded
acc
ordi
ng to
the
scal
e on
the
left
(3).
3-PG
A: 3
-pho
spho
glyc
eric
aci
d; P
EP: p
hosp
hoen
olpy
ruvi
c ac
id; T
CA
: tric
arbo
xylic
aci
d.
Chapter 5: Paper 3
120
Fig.
4. S
impl
ified
pat
hway
resp
onse
of t
he le
af p
rimar
y m
etab
olite
s sig
nific
antly
aff
ecte
d by
the
treat
men
ts (f
ull l
eaf:
FL; 2
5% le
aves
: 25L
) and
oth
er
met
abol
ites
dire
ctly
invo
lved
in th
e pa
thw
ays (
1). S
igni
fican
t diff
eren
ces a
re in
dica
ted
at P
< 0
.05,
hea
tmap
col
umns
indi
cate
the
thre
e tre
atm
ents
, whi
le
heat
map
row
s ind
icat
e th
e ha
rves
t dat
es (V
+9, V
+18,
V+2
7, V
+37
and
V+4
6). W
here
diff
eren
t upp
er c
ase
lette
rs a
ppea
r in
heat
map
col
umns
(2),
valu
es d
iffer
sig
nific
antly
bet
wee
n tre
atm
ents
. Whe
re d
iffer
ent l
ower
cas
e le
tters
app
ear i
n a
row
, val
ues
diff
er si
gnifi
cant
ly b
etw
een
harv
est d
ates
. Ave
rage
met
abol
ite
abun
danc
e is
colo
ur c
oded
acc
ordi
ng to
the
scal
e on
the
left
(3).
3-PG
A: 3
-pho
spho
glyc
eric
aci
d; P
EP: p
hosp
hoen
olpy
ruvi
c ac
id; T
CA
: tric
arbo
xylic
aci
d.
Chapter 5: Paper 3
121
Fig. 5. Impact of defoliation (full leaf – FL, 25% leaves – 25L and no leaf – NL) on root starch (A) and myo-inositol (B) concentration during the experimental period (mean ± SE; n=3). Significant differences (P < 0.05) between harvest dates for each treatment are indicated by different lower case letters. Significant differences (P < 0.05) between treatments at each harvest date are indicated by a numeral [1: FL > (25L and NL)].
Chapter 6: Paper 4
122
Chapter 6: Paper 4
Impact of post-véraison leaf source limitation on the
metabolic profile of Vitis vinifera cv. Shiraz berries
(Paper 4 has been submitted for publication in Plant Physiology and Biochemistry.
The tables and figures are shown after the main manuscript text.)
6.1. Main objective for paper 4
To study the implications of defoliation on the post-véraison metabolic composition of
grapevine berries.
6.2. Supplementary material
Supplementary material, as referred to in Paper 4 (Supplementary table S1 and figure
S1), is included in appendix B.
Chapter 6: Paper 4
123
Impact of post-véraison leaf source limitation on the metabolic profile of Vitis vinifera cv. Shiraz berries
Gerhard C. Rossouw a,b,*, Katja Šuklje a,1, Beverley A. Orchard c, Jason P. Smith a,2, Celia Barril a,b, Alain Deloire a,3, Bruno P. Holzapfel a,c
a National Wine and Grape Industry Centre, Wagga Wagga 2678, New South Wales, Australia. b School of Agricultural and Wine Sciences, Charles Sturt University, Wagga Wagga 2678, New South Wales, Australia. c New South Wales Department of Primary Industries, Wagga Wagga 2650, New South Wales, Australia. 1 Present address: Wine Research Centre, University of Nova Gorica, Lanthieri Palace, Glavni trg 8, 5271 Vipava, Slovenia. 2 Present address: Institut für Allgemeinen und ökologischen Weinbau, Hochschule Geisenheim University, Geisenheim 65366, Germany. 3 Present address: Montpellier SupAgro, Montpellier 34060, France.
*Corresponding author (email [email protected])
Keywords: Amino acids, carbon allocation, defoliation, fruit ripening, grape metabolism, nitrogen partitioning, sugar
Abstract
Leaves are an important contributor toward berry sugar and nitrogen (N)
accumulation during fruit maturation. The post-véraison grapevine (Vitis vinifera L.)
leaf area may, therefore, affect the development of fruit composition. The aim of this
study was to investigate the impact of leaf source limitation or absence on key berry
quality attributes in conjunction with the accumulation of primary berry metabolites.
Shortly after the start of véraison, potted grapevines were defoliated (total defoliation
and 25% of the control), and the accumulation of berry soluble solids, N and
anthocyanins were compared to that of a full leaf area control. An untargeted approach
was undertaken to measure the content in primary metabolites by Gas
Chromatography/Mass Spectrometry. Defoliation resulted in reduced berry sugar and
anthocyanin accumulation, while total berry N content was unaffected. The juice yeast
assimilable N (YAN), however, increased upon partial and full defoliation.
Remobilized carbohydrate reserves allowed accumulation of the major berry sugars
during source limitation. Berry anthocyanin biosynthesis was strongly inhibited by
Chapter 6: Paper 4
defoliation, which could relate to the carbon source limitation and/or increased bunch
exposure. Arginine accumulation, likely resulting from reserve translocation,
contributed to increased YAN upon defoliation. Furthermore, assessing the
implications on various products of the shikimate pathway, suggests the carbon flux
through this pathway to be largely affected by leaf source limitation during fruit
maturation. This study provides a novel investigation of impacts of carbon and N
source limitation during berry maturation on the development of key berry quality
parameters as underlined by alterations in primary metabolism.
1. Introduction
The post-véraison period is essential for the development of key grapevine berry
quality parameters. During this berry maturation period, the accumulation of large
quantities of sugars takes place (Davies and Robinson, 1996). Furthermore, berry
Nitrogen (N) (Roubelakis-Angelakis and Kliewer, 1992) and anthocyanin (Boss et al.,
1996) incorporation also occurs, while the organic acid content declines (Degu et al.,
2014). Leaf photoassimilation yields soluble sugars which are translocated to the fruit,
mostly as sucrose, where it is hydrolyzed into glucose and fructose (Davies and
Robinson, 1996). The grapevine leaves are also a major source of organic N toward
the fruit (Rossouw et al., 2017b), and amino acid export from mature leaves provides
a soluble form of N, transportable to sink organs (Rentsch et al., 2007). Canopy
defoliation during berry maturation, therefore, removes a potential source of both,
carbon (C) and N, which can induce a severe reduction in berry sugar and N
accumulation. To the best of our knowledge, the implications of a post-véraison C and
N source limitation, as induced by severe defoliation, on the kinetic development of
fruit C and N containing primary metabolites are yet to be determined. 124
Chapter 6: Paper 4
Developments in gas chromatography/mass spectrometry (GC/MS) allow the
detection and measurement of complex mixtures of plant metabolites (Lisec et al.,
2006). These metabolites include organic acids, amino acids, sugars, sugar alcohols,
phosphorylated intermediates and lipophilic compounds. Although glucose and
fructose predominate, some minor sugars and sugar alcohols, in addition to sucrose,
are also accumulated in the grapevine berries (Cuadros-Inostroza et al., 2016). Apart
from being essential for alcoholic fermentation during winemaking, berry sugars are,
amongst other things, utilized as structural components and cell nutrients (Çakir et al.,
2003). In addition to sucrose, raffinose family oligosaccharides (e.g. raffinose) and
sugar alcohols (e.g. mannitol) can also play important roles in transporting C from
source to sink organs in higher plants (Noiraud et al., 2001). Sink organs, such as the
fruit, have little to no capacity to synthesize sugar alcohols, which are generally
produced as primary photosynthetic products (Loescher and Everard, 1996). In Olea
europaea fruit, sugar alcohols have been found to act as storage compounds and are
important during metabolic transformation (Marsilio et al., 2001). Some minor sugars,
such as trehalose, can play a role in signaling of the plant C status or as a cell
membrane constituent (O’Hara et al., 2013).
The content and composition of berry amino acids are essential from a wine
quality perspective, as it determines the juice yeast assimilable N (YAN)
concentration, influencing fermentation and wine aroma potential (Bell and Henschke,
2005). The major amino acids in berries are usually arginine and proline, the latter not
assimilable by yeasts (Bell and Henschke, 2005). Furthermore, the C skeletons of
different amino acids are utilized during the biosynthesis of secondary flavor
125
Chapter 6: Paper 4
compounds in berries (Bell and Henschke, 2005). Phenolic compounds in berries, such
as anthocyanins, are also derived from C skeletons provided by amino acid metabolism
(Boss et al., 1996). One of the major organic acids in the grapevine berries, malic acid,
is catabolized after véraison and provides a vital source of C, utilized during secondary
metabolism (Sweetman et al., 2009). Other organic acids, such as citric acid, do not
accumulate in large quantities in grapevine berries, while tartaric acid can be abundant
in the berries, but has ambiguous roles during plant metabolism (Sweetman et al.,
2009). Observing changes in the content of different berry primary metabolites during
maturation, as influenced by excessive defoliation, can provide a novel indication of
the impact of source limitation on the accumulation of key berry quality attributes
(e.g., soluble solids, N, anthocyanins and YAN).
Leaves are, however, not the only source of C and N during berry maturation.
When grapevine leaf area is limited, carbohydrate reserves, mostly stored in the roots,
are remobilized towards the fruit to support the fruit sugar content (Candolfi-
Vasconcelos et al., 1994). In addition to leaves, grapevine roots, trunks and shoots are
also transitional reservoirs of soil-absorbed N, from where it is translocated to the fruit
during maturation as soil N uptake is usually limited or absent during this period
(Roubelakis-Angelakis and Kliewer, 1992). The removal of leaf C and N source,
therefore limits, but not necessarily inhibits, fruit sugar and N accumulation during
berry maturation. A complete defoliation would also be detrimental towards sap flow
through the xylem, thereby potentially limiting root N exportation. Profiling berry
primary metabolite accumulation between véraison and fruit maturity can, however,
indicate which metabolites are likely leaf-derived and synthesized in the berries, or
derived from source organs other than the leaves.
126
Chapter 6: Paper 4
127
The aim of this experiment was to determine how defoliation near the onset of
fruit maturation affects key berry quality attributes (soluble solids, pH, TA, total N
and YAN), and especially how these parameters relate to the berry primary metabolite
composition development. The first goal was to better understand the leaf contribution
to fruit maturation and composition. It was, therefore, evaluated if a partial or complete
defoliation near véraison inhibits berry sugar, N and anthocyanin accumulation, and
the juice YAN concentration. The second goal was to conduct an untargeted profiling
of the primary metabolites in the berries during the berry maturation period. These
metabolites include sugars, sugar alcohols, organic acids, amino acids and fatty acids.
By profiling the metabolic changes in maturing grapevine berries due to defoliation,
information was gathered to better understand how primary berry metabolism is
affected by limited leaf area availability during fruit maturation.
2. Materials and methods
2.1. Experimental design and treatments
Three-year-old Vitis vinifera L. cv Shiraz (clone EVOVS12) grapevines, planted
in 30 L pots containing commercial potting mix, were grown in a bird-proofed
enclosure and used for an experiment during the 2015-16 growing season. The
experiment was conducted in the warm to very warm Riverina region, New South
Wales, Australia. Forty vines were distributed in four rows of ten vines each, and were
spur-pruned during the winter preceding the experiment to five, two-bud spurs each.
Vines were thinned to ten primary shoots after budburst. Between budburst and
approximately one month prior to the start of véraison, the grapevines were fertilized
once every three weeks with 250 mL 1:50 diluted liquid fertilizer (MEGAMIX Plus,
Chapter 6: Paper 4
Rutec, Tamworth, Australia), and an approximate total of 3.2 g N was subsequently
applied per vine. Vines were well-watered throughout the experiment to avoid any
water constraints.
At fruit-set, the bunch amount per vine was counted, and each vine was classified
as naturally containing low (13 – 15), medium (15 – 16) or high (16 – 19) bunch
numbers. This classification was later only used to minimise natural vine cropping
variability among treatments and harvest dates, and vines from the different bunch
number classes were thus equally distributed among the treatments and harvest dates.
The experiment was initiated on 28 Dec 2015 (V+9), nine days after the very first
indication of berry softening (véraison). On V+9, all the fruit from four vines, one out
of each row, were harvested in order to represent the population of grapevines prior to
the start of the experiment. To ensure an unbiased selection of vines, these vines
consisted of one vine from each of the low and high bunch number classes and two
from the medium class. Additionally, a 50 berry subsample from each of the above
mentioned vines was collected and immediately frozen in liquid N and stored at -80
ºC. To assess the vine N status, 20 petioles from each of the four vines were also
collected (from adjacent a bunch or from the shoot node directly above or below a
bunch when required). The petioles were frozen in liquid N and ground to a fine
powder (IKA A11, Selangor, Malaysia) before being freeze dried (Gamma 1-16 LSC,
Christ, Osterade am Harz, Germany). The petiole N concentration was subsequently
determined by the method also used to determine berry N, described in section 2.2.
The remaining nine vines per row were spread over a four row, nine column
randomized block design, consisting of three treatment replicates. The experimental
treatments were implemented on V+9, with the control treatment (FL) consisting of
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vines with 100 primary shoot leaves and all the intact laterals. The partial defoliation
treatment (25L) consisted of vines with 25 primary shoot leaves, and the full
defoliation treatment (NL) consisted of vines without leaves. The leaves of treatment
25L were left adjacent to a bunch or on a node directly above or below a bunch. To
eliminate any new vegetative growth, any newly developing buds were removed from
the shoots of treatments 25L and NL daily, as soon as the growth was observed. Every
9 - 10 days after the start of the experiment, all the fruit from three vine replicates per
treatment were harvested and 50 berries per vine were selected and immediately frozen
in liquid N and stored at -80 ºC to later be used for primary metabolite analysis. Fruit
was harvested on 6 Jan 2016 (V+18), 15 Jan 2016 (V+27), 25 Jan 2016 (V+37) and 3
Feb 2016 (V+46). The periods between each of the harvest dates are referred to as
Interval 1, 2, 3 and 4, respectively. All leaves per vine were removed at the time of
fruit harvest, and the leaf area determined with a leaf area meter (LI-3100C, LI-COR
Biosciences Inc., Lincoln, Nebraska, USA).
2.2. Berry weight and composition
At each harvest, the total fresh fruit weight per vine was recorded, and a
subsample of 50 fresh berries per vine was collected, weighed, and the fresh weight
per berry determined. These berries were juiced by hand in a plastic bag and the juice
total soluble solid concentration (TSS) measured using a bench refractometer (PR-
101, Atago, Tokyo, Japan). The soluble solid content per berry (SSC) was
subsequently calculated based on the berry fresh weight and TSS concentration. The
remaining fruit from each vine was stored at -20 ºC until further processing as
described below. A subsample of 50 frozen berries per vine was ground to a fine
powder under liquid N (IKA A11, Selangor, Malaysia) and freeze-dried until constant
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weight (Gamma 1-16 LSC, Christ, Osterade am Harz, Germany) to determine the
weight loss percentage during the drying process. The dry weight per berry was
subsequently calculated. To determine the berry total N concentration, a 200 mg
freeze-dried sample was analyzed using the LECO method (Standard methods of
Rayment and Lyons, Soil chemical methods, Australasia, Dumas Combustion Method
6B2b), through a LECO CNS TruMAC analyzer (LECO corporation, St. Joseph, MI,
USA).
Another subsample of 50 frozen berries per vine was thawed in a shaking water-
bath at 30 ºC for 30 min, juiced and vortexed, again left at 30 ºC for 30 min and then
centrifuged for 5 min at 3000 × g. The juice titratable acidity (TA) and pH was
determined by sodium hydroxide (0.1 M) titration using an automatic titrator
(Metrohm Fully Automated 59 Place Titrando System, Metrohm AG, Herisau,
Switzerland) to an end point of pH 8.2. The ammonium and α-amino acid
concentrations of the juice were determined by using a commercially available
enzymatic assay kit, designed for an Arena discrete analyzer (Thermofisher, Scoresby,
Australia). The yeast assimilable N (YAN) concentration was subsequently calculated
from the ammonium and free amino N (FAN) (Iland et al., 2004). Fruit total
anthocyanin concentration was analyzed from a third 50 berry subsample per vine.
Whole berries were thawed at 4 °C and homogenized (Ultra-Turrax T25, IKA,
Selangor, Malaysia), and the total anthocyanin concentration determined as described
in Iland et al., (2004).
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2.3. Primary metabolite analysis
The subsample of 50 berries per vine, stored at -80 ºC, was ground to a fine
powder under liquid N and freeze-dried as described above. Sample extraction
and derivatization from a 100 mg freeze-fried sample was conducted according to
the method of Lisec et al. (2006) with some modifications. In summary, the berry
tissue of each vine was homogenized with 1.4 mL methanol and 30 µL of an internal
standard solution (1 g.L-1 of each, adonitol, L-hydroxyproline, and adipic acid, in
50% v/v methanol). The homogenate was shaken at 70 ºC for 10 min
(Thermomixer 5436, Eppendorf, North Ryde, NSW, Australia), and centrifuged at
11000 × g for 10 min. The supernatant was transferred to a glass vial, and mixed
with 0.75 mL chloroform and 1.4 mL ultrapure water. The mixture was
centrifuged at 2200 × g for 15 min, before the polar phase supernatant was
collected. In order to avoid saturation of samples with highly abundant metabolites,
the supernatant was diluted with 50% (v/v) methanol. To measure less abundant
metabolites, the dilution factor (DF) was 10, and to measure more abundant metabolites
(e.g., glucose, fructose and malic acid), the DF was 100. The diluted supernatant
(150 µL) was subsequently dried under a constant flow of pure N gas.
Methoxyamin hydrochloride (40 µL of 20 mg.mL-1) in pure pyridine, was added
to the dried extracts. The samples were then shaken at 37 ºC for 2 h, before being
centrifuged at 5000 × g for 2 min. N-Methyl-N-(trimethylsilyl) trifluoroacetamide
(70 µL) was added, and the samples were shaken at 37 ºC for 30 min, before being
centrifuged at 5000 × g for 2 min, after which the supernatant was collected. The
sample extraction, derivatization and measurement order was randomized.
Gas chromatography/mass spectrometry (GC/MS) analyses of the samples were
conducted by injecting 1 µL into the GC column (30 m × 0.25 mm, 0.25 µm HP-5MS,
Agilent, Santa Clara, CA, USA). The GC/MS system consisted of a
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7683B series autosampler, 7890A gas chromatograph, and 5975C mass spectrometer
with an electron impact ionization source and a quadrupole analyser (all from Agilent,
Santa Clara, CA, USA). The injection port was set at 250 ºC, the transfer line at 280
ºC, the ionization source at 230 ºC, and the quadrupole at 150 ºC. Helium gas, set at a
constant flow rate of 1.3 mL.min-1, was the carrier gas. The column temperature
program was set at 65 ºC for 2 min, followed by a 6 ºC.min-1 ramp to 300 ºC, where it
was held for 25 min. The ionization energy was set at 70 eV. Mass spectra were
recorded in full scan mode at 2.66 scans per second with a mass-to-charge ratio of 50
to 600 amu. Spectral deconvolution (signal-to-noise ratio threshold = 10; mass
absolute height ≥ 2000; compound absolute area ≥ 10000) allowed the identification
of co-eluting chromatographic peaks, and was conducted using MassHunter
Workstation software (Qualitative Analysis, version B.07.00, Agilent, Santa Clara,
CA, USA). Acquired MS spectra were searched for, and compounds identified using
the National Institute of Standards and Technology algorithm (NIST, Gaithersburg,
USA). The retention index for each compound in the analyzed samples was calculated
by using the retention times of a series of alkanes (C8-C28) in an injected retention
index solution (Fluka, Buchs, Switzerland).
Analytical grade standards, obtained from Sigma-Aldrich (Sigma, St. Louis, MO,
USA), were prepared in order to assist in the identification of these compounds. These
standards (10 mg.L-1 in 50% v/v methanol) consisted of various soluble sugars, i.e.,
sucrose, D(+)-glucose, D(-)-fructose, D(+)-mannose, L-rhamnose, D(-)-mannitol,
galactinol dehydrate, D(+)-raffinose, melibiose, D(+)-cellobiose, D(-)-ribose, D(-)-
arabinose, D(+)-trehalose, maltose monohydrate, D(+)-galactose, D(+)-xylose,
dulcitol (galactitol), L-fucose, and myo-inositol; amino acids, i.e., L-glutamic acid, L-
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arginine, L-proline, L-glutamine, γ-amino-n-butyric acid (GABA), L-threonine, L-
methionine, β-alanine, L-lysine, L-asparagine, L-aspartic acid, L-leucine, L-
isoleucine, L-valine, L-alanine, L-serine, glycine, L-tyrosine, L-phenylalanine, L-
tryptophan, L-histidine, L-cysteine and L-cysteine; and the organic acids, L-ascorbic
acid and protocathechuic acid.
2.4. Statistical analysis
The datasets in regard to the grapevine leaf area and basic berry composition
(Table 1), berry size and sugar, N and anthocyanin content (Fig. 1), and primary
metabolite abundance (Fig. 2, Supplementary Table S1) were analyzed using Statistica
13 (Dell Inc., Tulsa, OK, USA). For each variable, both treatment differences at a
single time and how a treatment changed over time were of interest. It was also
recognized that residual variance at each time may differ and that the interventionist
nature of the treatments may also lead to reduced residual variance for some
treatments. To facilitate these comparisons univariate analysis of variance (ANOVA)
at each date (all treatments) and for each treatment (all dates) were conducted. An
average Fisher’s least significant difference (LSD) test was used to identify significant
differences between means (P < 0.05). Significant differences in table and heat map
columns and rows are indicated by upper case letters (between treatments) and lower
case letters (between dates), respectively.
For each harvest date (V+18, V+27, V+37 and V+46) after the initial pre-
treatment harvest (V+9), a linear mixed model was fitted for every identified primary
metabolite (79 metabolites) using ASReml-R (Butler et al., 2007). Each model
included Treatment as a fixed effect and Replicate as a random effect. The significance
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of treatment effects was assessed by approximate F-tests, using the techniques of
Kenward and Roger (1997). For each vine organ, primary metabolites which had
significant treatment effects (P < 0.05) for any of V+18, V+27, V+37 and V+46 were
retained for a Principal Component Analysis (PCA). The PCA was only conducted as
an initial data analysis step to interpret the interactions between metabolites, treatment
and harvest dates. Predicted means from the ASreml-R analysis were used in a PCA
based on the correlation matrix, conducted in Genstat 17th Edition (VSN International
Ltd., Hemel Hempstead, Hertfordshire, UK). Biplots were drawn using R statistical
software plus the add in package ‘shape’ (developed by Karline Soetaert, Royal
Netherlands Institute of Sea Research Yerseke, The Netherlands). These biplots and
further PCA information (latent vector loadings and contributions of the measured
metabolites to principal components 1 and 2) are provided in Supplementary Fig. S1.
3. Results
3.1. Leaf area, petiole N and basic berry juice composition
The defoliation of 25L and NL caused FL vines to exhibit significantly larger leaf
area from V+18 (Table 1). While partial or full defoliation did not cause any
significant differences in the fruit fresh weight per vine, the FL fresh fruit weight
increased between V+9 and V+27, while that of 25L and NL increased during Interval
1 (Table 1). The petiole N concentration at the start of the experiment was 0.54% (±
0.02) (data not shown), and therefore, within a range indicating adequote N supply to
Shiraz vines by véraison (Holzapfel and Treeby, 2007).
Under full leaf area, an increase in berry juice TSS was noticed during Intervals
1 to 3 (Table 1). The reduced leaf area of 25L resulted in a slower TSS accumulation
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compared to FL, whereas a small increase in NL juice TSS was only noticed during
Interval 2. Therefore, the FL juice TSS was significantly higher than that of 25L and
NL at all sampling dates after V+9. In addition, 25L resulted in significantly higher
juice TSS than NL from V+37 onwards. Reduced leaf area had no significant effect
on the juice TA and pH (Table 1). The juice TA of all treatments decreased during
Intervals 1 to 3, while the juice pH increased during the same intervals, and that of FL
and NL further increased during Interval 4.
Defoliation resulted in significant alterations of the juice nitrogenous
composition. Under full leaf area, juice FAN concentrations were significantly lower
from V+18 compared to the defoliated treatments, while no significant differences in
juice FAN concentrations occurred between 25L and NL (Table 1). Generally, the
FAN concentration increased from the first to the last sampling date irrespective of
the treatment. The FL juice FAN concentration, however, decreased during Interval 1,
then increased during Interval 2, and was significantly higher at V+46 than at V+9
(Table 1). The 25L juice FAN increased during Intervals 2 and 3, while that of NL
increased during Interval 2 and between V+27 and V+46. The FL juice ammonium
concentration was significantly lower than that of 25L at V+18 and V+46. Similar to
the juice FAN, the YAN concentrations of FL were generally lower than those of 25L
and NL after V+9, with an exception at V+37 when there was no significant difference
amongst FL and NL (Table 1). Furthermore, no significant YAN differences occurred
between 25L and NL.
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3.2. Berry dry weight, SSC, N and anthocyanin content
The dry weight per FL berry was significantly larger than those of the defoliation
treatments from V+18 (Fig. 1A). Similarly, among the treatments, SSC per FL berry
was the highest from V+18, while the SSC per 25L berry was significantly higher than
that of NL from V+37 (Fig. 1B). The FL berry SSC increased significantly during
Intervals 1 to 3, while that of 25L increased more slowly, albeit significantly during
every interval. The SSC per NL berry increased significantly between V+9 and V+27,
and V+27 and V+46. Comparable to the SSC content per berry, the anthocyanin
content was generally superior in FL berries, where it was significantly the highest
from V+27 (Fig. 1C). The anthocyanin content per berry generally increased for all
treatments between V+9 and V+46, although at a much slower rate in the 25L and NL
berries.
Although no significant berry N content differences occurred at the final harvest
date, the N content per 25L berry was significantly superior to that of FL at V+37 (Fig.
1D). Furthermore, although the N content per berry increased between V+9 and V+46
irrespective of the leaf area, FL berry N content increased between V+18 and V+46,
while that of 25L increased during Interval 3. The N content per NL berry increased
between V+9 and V+27, and also between V+18 and V+37.
3.3. Metabolic adjustments
Inspection of the metabolite spectra, obtained from the GC/MS analyses, resulted
in the identification of 75 metabolites, categorized according to their chemical classes
(Table 2). In addition, three unidentified metabolites with sugar-like spectra were
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classified as unknown sugars 1, 2 and 3. Another metabolite with a spectrum similar
to that of raffinose was classified as unknown oligosaccharide 1.
Figure 2 illustrates the changes in the metabolite content per berry as induced by
partial or full defoliation. The contents of the metabolites that exhibited significant
treatment differences (listed in Supplementary Fig. S1), are indicated in the figure and
are briefly described below. In addition, metabolites that are directly involved in the
proposed metabolic pathways are also shown in the figure. Table 3 additionally lists a
simplified summary of the metabolites that accumulated and/or depleted during berry
maturation.
3.3.1. Accumulative metabolites
The majority of sugars accumulated in the berries between V+9 and V+46.
Glucose, fructose and sucrose accumulated irrespective of the treatments, and were
more abundant under full leaf area at V+37. A number of less abundant (minor) sugars
also accumulated as the berries matured, and mostly irrespective of the leaf area. These
include cellobiose, trehalose, tagatose, arabinofuranose, fucose, 3α-mannobiose and
unknown sugars 1, 2 and 3. These sugars were, however, generally more abundant
under full leaf area. In fact, at V+46 the FL fruit contained more cellobiose, trehalose,
arabinofuranose, 3α-mannobiose and unknown sugars 1, 2 and 3 than those of the
defoliated treatments (with the exception of unknown sugar 1, which did not
significantly differ between FL and 25L at V+46). The sugar alcohols myo-inositol
and dulcitol accumulated in FL berries, which had a superior myo-inositol content
compared to NL berries from V+27. Dulcitol also accumulated in the other treatments,
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however, FL berries contained the most dulcitol at V+46. Arabitol, on the other hand,
only accumulated after full defoliation.
A number of amino acids accumulated as the berries matured. Included among
these were glutamic acid, which accumulated irrespective of the leaf area, but was
more abundant in FL berries compared to those of NL from V+27. In addition, the 25L
berries contained more glutamic acid than those of NL at V+46. Similarly, berry
proline accumulated especially under full leaf area, where it was most abundant at
V+46. The 25L berry proline content was additionally superior to that of NL at V+46.
β-Alanine accumulated irrespective of the treatment, but was more abundant in FL
berries compared to those of NL from V+27. Arginine also accumulated irrespective
of the treatment, however, the NL berries contained more arginine than those of FL
from V+27. In addition, the 25L berries contained more arginine than those of FL from
V+37. Tyrosine accumulated in the berries of treatments FL between V+9 and V+46,
but the content did not differ among the treatments.
Unlike most other organic acids, pyruvic acid and lactic acid generally
accumulated in the berries. In fact, pyruvic acid was the most abundant in FL berries
at V+46, and additionally more abundant than in those of NL from V+18. Lactic acid
was more abundant in FL berries, compared to those of 25L at V+46. The phenolic
compounds, gallic acid and benzoic acid, also generally accumulated as the berries
matured. Gallic acid accumulated in FL and 25L berries, and was more abundant in
those of FL at V+37. Benzoic acid accumulated irrespective of leaf area, and was more
abundant in FL and 25L berries compared to those of NL from V+37 and at V+46,
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respectively. The other compounds that exhibited accumulating trends during berry
maturation were glycerol monostearate, 1-monopalmatin and hydroxylamine.
3.3.2. Metabolites exhibiting accumulative and depletive trends
Galactinol accumulated between V+9 and V+27, irrespective of the leaf area, and
then depleted between V+27 and V+46. However, 25L and NL berries contained more
galactinol than FL berries at V+27. Similarly, raffinose accumulated between V+9 and
V+27 before depleting. The FL berries contained more raffinose than those of 25L at
V+27 and more than those of NL at V+37. Unknown oligosaccharide 1 increased
between V+9 and V+27 in NL berries, and was significantly the most abundant in
these berries at V+37.
Aspartic acid accumulated until V+18 in NL berries and until V+27 in FL and
25L berries. The 25L berries contained the most aspartic acid from V+37.
Additionally, 25L and NL berries contained more aspartic acid at V+18. Asparagine
accumulated in NL berries during Interval 1, but depleted irrespective of the leaf area,
towards V+46. Asparagine was more abundant in the NL berries, than in the berries
of other treatments at V+37. Although the phenylalanine content never significantly
differed among the treatments, it accumulated in 25L berries during Interval 3, while
depleting during Interval 4. Likewise, the phenylalanine content in NL berries
increased during Interval 2.
3.3.3. Depletive metabolites
Rhamnose was the only sugar that depleted under full leaf area as the berries
matured. Among the amino acids, threonine depleted in the FL berries during Interval
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140
1, however, it accumulated in those of 25L during Interval 3. Tryptophan depleted in
the FL berries, and was lower than in 25L and NL at V+27, and lower than in 25L at
V+46.
Various organic acids depleted as the berries matured. Although malic acid
accumulated during Interval 1, the contents subsequently decreased in all treatments
towards V+46, and malic acid was less abundant in NL berries at V+37. Citric acid
also depleted over time, and at V+27, FL berries contained more citric acid than in
25L and NL, while NL berries contained less than those of the other treatments at
V+37. Fumaric acid depleted irrespective of treatments, and the FL berries contained
less fumaric acid than those of 25L and NL from, and at, V+37 respectively. Gluconic
acid only reduced upon partial and full defoliation, and the FL berries contained more
gluconic acid than those of 25L and NL from V+18 and V+27, respectively. Ascorbic
acid reduced in the berries, irrespective of leaf area, while tartaric acid only reduced
in 25L and NL berries, where it was significantly less abundant than in those of FL at
V+27. Threonic acid reduced irrespective of leaf area, and was more abundant in FL
berries from V+27. Glyceric acid reduced in the berries of all treatments, but was the
least abundant in those of FL from V+27. In terms of the phenolic acids, the berry
protocatechuic acid depleted irrespective of leaf area, but was less abundant in those
of FL at V+46, while caffeic acid only depleted upon partial or full defoliation. In
addition to the acids, (+)-catechin also reduced irrespective of leaf area, as the berries
matured.
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4. Discussion
Grapevines were partially (25L) or fully (NL) defoliated nine days after the start
of véraison, and the berry sugar, nitrogen (N) and anthocyanin accumulation, and juice
yeast assimilable N (YAN) concentration was compared to that of grapevines with full
leaf area (FL). The defoliation treatments were aimed to force a partial or full reliance
on carbohydrate and N reserves during restricted or no leaf availability towards canopy
photoassimilation. An untargeted investigation of the primary metabolite content in
the berries was conducted, aiming to emphasize the impact of C and N source
limitation on the different metabolites.
After treatment implementation, partially defoliated vines had a leaf-to-fresh fruit
ratio of around 0.2 m2.kg-1, while the ratio of the control treatment was 0.8 m2.kg-1.
According to published threshold levels (0.8 – 1.2 m2.kg-1), the leaf-to-fruit ratio of
the control vines should be sufficient to allow maximum accumulation of berry sugar
and color in a given climatic region (Kliewer and Dokoozlian, 2005). In contrast, the
partially and fully defoliated vines exhibited an insufficient leaf C source to support
its fruit sugar and color development. The 35 and 49% reduction in berry soluble solid
content (SSC) at the final harvest under partial and full defoliation, respectively, is
therefore not surprising. However, in the absence of leaf photosynthesis (full
defoliation, NL), there was still an 86% increase in SSC per berry at the last harvest.
The remobilization of carbohydrate reserves from the perennial structure (mostly the
roots) therefore contributed to the fruit sugar content after excessive defoliation
(Candolfi-Vasconcelos et al., 1994; Rossouw et al., 2017a). Furthermore, partial and
full defoliation caused a 71 and 79% reduction in berry anthocyanin content,
respectively by the final sampling date. Carbon limitation due to the defoliation
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treatments, may have caused a greater down regulation of berry anthocyanin
biosynthesis than sugar accumulation, however, an intensified sunlight exposure after
defoliation is likely to have inhibited anthocyanin metabolism (Haselgrove et al.,
2000). In their study, Bobeica et al. (2015) concluded that under C limitation, the
grapevine can manage the metabolic fate of C in such a way that sugar accumulation
is maintained at the expense of secondary metabolites. In fact, grapevine defoliation
may inhibit anthocyanin biosynthesis at protein (Wu et al., 2013) and transcription
(Pastore et al., 2013) levels.
While berry sugar and anthocyanin contents were highly affected by defoliation,
the total N content per berry was unaffected by the final harvest date. During berry
maturation, leaves provide a source of organic N towards fruit N accumulation
(Rossouw et al., 2017b), while soil N absorption could be restricted or absent.
However, other grapevine organs, such as roots and shoots, are also sources of N
during this period (Roubelakis-Angelakis and Kliewer, 1992), and soil N uptake by
the potted grapevines of the present study seems likely, even though N fertilization
was ceased a month prior the experiment. Redistribution of N from source organs other
than the leaves, and/or from soil N uptake, therefore, supported the accumulation of
berry N, irrespective of the leaf area. Nevertheless, the juice YAN concentration was
increased by partial and full defoliation, and was at a level (close to 130 mg.L-1)
thought to be just sufficient to allow the completion of must fermentation (Bell and
Henschke, 2005). Without must N supplementation, the lower juice YAN of the
control treatment could result in a sluggish fermentation, and inferior wine aroma
development (Bell and Henschke, 2005). The underlying aspects contributing to the
increased YAN after defoliation are discussed later, however, although defoliation had
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an insignificant influence on the total berry N content, the amino N content increased
after the defoliation treatments.
Most sugars, including many minor sugars, accumulated during berry maturation,
but the accumulation was reduced after partial and full defoliation. Accumulation of
the major berry sugars (glucose, fructose and sucrose) occurred faster under full leaf
area during the period of rapid berry SSC accumulation (until V+37), but as the berry
SSC accumulation slowed, accumulation of these sugars ceased. The resulting lack in
significant differences in the respective contents of berry glucose, fructose and sucrose
among the treatments at V+46, indicates the contribution of mobilized carbohydrate
reserves toward the fruit sugar content (Rossouw et al., 2017a), and in contrast to the
significantly higher content of most minor sugars (e.g. trehalose) under full leaf area
at V+46. Therefore, it seems that berry sugar metabolism, sourced from reserve starch
hydrolysis (Smith et al., 2005) rather than leaf photoassimilation, favored the major
sugars. In grapevines, like most other plants, sucrose is the major transport sugar
through the phloem (Ruan et al., 2010). Sucrose is translocated from the leaves or
reserve organs, such as the roots, towards the fruit where it is hydrolyzed into glucose
and fructose (Davies and Robinson, 1996). Carbon limitation, however, restricted the
accumulation of many minor sugars, although most still accumulated.
Galactinol, raffinose and unknown oligosaccharide 1 did not, like most other
sugars, accumulate progressively, but rather typically accumulated until midway
through the experiment, before depleting. Among their functions, the raffinose family
oligosaccharides (RFOs) can serve as transport compounds between plant sources and
sinks (Sengupta et al., 2015). Galactinol is derived from myo-inositol, and is a
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precursor for raffinose and its subsequent higher molecular weight RFOs (e.g.
stachyose and verbascose) (Sengupta et al., 2015). The accumulation pattern of
galactinol suggests that, especially after partial and full defoliation, it was transported
to the berries while the fruit was presumably a strong C sink. Although little is known
about their transport roles in grapevines, through studying Solanum tuberosum,
Hannah et al. (2006) found that both, galactinol and raffinose, are transported in the
phloem. It is therefore possible that either, galactinol and the RFOs were transported
to the berries, or that after translocation, galactinol was metabolized in the berries, and
the RFOs subsequently accumulated. The accumulation of galactinol, raffinose and
unknown oligosaccharide 1 after full defoliation, suggests that these compounds were
sourced from carbohydrate reserves during a period of strong berry C demand. On the
other hand, instead of accumulating during berry maturation like most other sugars,
rhamnose depleted under full leaf area. A similar observation was made by Cuadros-
Inostroza et al. (2016), who reported the post-véraison depletion of berry rhamnose.
Apart from being a cell wall pectic polysaccharide component, rhamnose is also
present in many secondary metabolites, such as anthocyanins and flavonoids (Watt et
al., 2004). As the only sugar that clearly depleted during berry maturation, further
work is needed to determine the functioning of rhamnose during berry maturation.
Unlike most sugars that progressively accumulated during berry maturation, the
different amino acids had diverse patterns of accumulation or depletion. Volenec et al.
(1996) described root and stem protein degradation to occur rapidly after forage plant
species, such as Medicago sativa, are defoliated, when vegetative regrowth occurs.
The degradation of storage proteins results in amino acid accumulation, which
becomes available to be translocated from the source to sink organs. As mentioned
144
Chapter 6: Paper 4
earlier, there was an increase in juice YAN after defoliation. Typically, arginine and
proline are the most abundant amino acids in mature grapevine berries (Bell and
Henschke, 2005). Proline, however, is not yeast assimilable, and arginine is therefore
a major contributor to juice free amino N (FAN). The increased accumulation of
arginine after partial and full defoliation explains the increased FAN and subsequent
YAN, in the berry juice of those treatments. Apart from being the major storage amino
acid in grapevine roots as a free amino acid or protein component (Xia and Cheng,
2004), arginine also plays an important role as a N transport compound (Lea et al.,
2007). The elimination or restriction of the leaf N source, therefore, presumably
induced N translocation as arginine from the storage tissues towards the berries,
contributing to berry N accumulation, and effectively raising juice YAN. Asparagine
only accumulated after a complete defoliation and has, like arginine, a high N:C ratio
making it an effective N storage compound, while also a major transport amino acid
in plants (Lea et al., 2007). Like arginine, the accumulation of berry asparagine in the
absence of leaves, therefore, implies its mobilization from N storage tissues (e.g. the
roots), subsequently also contributing to the increased YAN (Bell and Henschke,
2005). Furthermore, the defoliation treatments of the present study, especially full
defoliation, would force a greater reliance on phloem amino acid translocation, as the
xylem flow would likely be interrupted. Tromp and Ovaa (1971) described arginine
and asparagine as being major phloem translocated amino acids in Malus domestica,
and likely similarly so in the grapevines of the present study. The bulk phloem flow
solution, moving from source to sink organs contains sugars and amino N, and is
propelled by osmotically generated hydrostatic pressure differences (Lalonde et al.,
2003). In the present study, the elimination of the leaf C source enforced root
carbohydrate reserves to be become a sugar source towards the maturing berries, a
145
Chapter 6: Paper 4
strong C sink (Candolfi-Vasconcelos et al., 1994). If root N repartitioning and
subsequently, amino acid accumulation, occurred after removal of the leaf N source,
these amino acids could follow the sugar regulated osmotic phloem flow towards the
berries. The results of the present study, therefore, suggest amino N translocation from
reserve tissues to the berries.
Although the berry phenylalanine content never significantly differed among
treatments, it only accumulated after partial and full defoliation. The transport
mechanisms of phenylalanine in grapevines has not yet been studied, however,
phenylalanine accumulated in the xylem of Trifolium repens and the phloem of
Lupinus albus after defoliation (Hartwig and Trommler, 2001). Such accumulation
likely implies the possibility of phenylalanine transport between source and sink
organs in plants. Phenylalanine is an important precursor for anthocyanin biosynthesis
in grapevine berries (Boss et al., 1996), and the results of the present study suggest
that the grapevine regulates its berry phenylalanine accumulation under source (leaf)
limitation. Roots and mature leaves are the major sources of amino acids in higher
plants, from where they are translocated to sink organs (Rentsch et al., 2007). In the
present study, the accumulation of amino acids like arginine, asparagine and
phenylalanine after partial and full defoliation, suggests that they were sourced from
N reserve tissues (likely the roots) when the leaf source was limited or unavailable.
On the other hand, many amino acids, e.g., glutamic acid, proline and β-alanine
accumulated more in the berries under full leaf area, implying the leaves to be an
important source of these amino acids. More work is, however, needed to determine
the extent of amino acid biosynthesis within the grapevine berries. In Solanum
lycopersicum fruit, peptidases are very active during ripening, and they are able to
146
Chapter 6: Paper 4
release free amino acids from endogenous proteins (Sorrequieta et al., 2010).
Therefore, although the results of the present study suggest berry amino acids being
sourced from the leaves or alternatively, reserve tissues (e.g. the roots), the occurance
of amino acid biosynthesis within the berries needs further exploration.
Defoliation did not affect the juice titratable acidity (TA) and pH, implying that
organic acids are less responsive to C limitation, than sugars (Bobeica et al., 2015).
This lack of TA differences was thus expected to yield minimal differences in berry
malic acid, tartaric acid and citric acid content among the treatments. In fact, at the
final harvest, there were no significant differences in the contents of any of these major
berry acids. The intermediates of the tricarboxylic acid (TCA) cycle usually decline in
the berries after véraison, which likely matches the increased demand of precursors in
the synthesis of carbohydrates, amino acids and subsequent flavonoids (Degu et al.,
2014). The initial accumulation of malic acid, before its decline was thus surprising,
however, the other TCA cycle intermediates generally depleted with berry maturation.
These organic acids were therefore likely catabolized, and were useful precursors for
energy production and further C metabolism (López-Bucio et al., 2000).
The abundance of many berry metabolites derived from the shikimate pathway
were affected by defoliation. The implication of the defoliation treatments on the
aromatic amino acids (e.g. phenylalanine) has already been described above, however
arbutin, (+)-catechin and various phenolic acids (gallic acid, benzoic acid, caffeic acid
and protocatechuic acid) were also affected in different ways. Furthermore,
anthocyanins are also secondary products of the shikimate pathway, and the berry
anthocyanin content was, as mentioned, greatly decreased by defoliation. The flux
147
Chapter 6: Paper 4
through the shikimate pathway can represent up to 20% of the total C in plants
(Haslam, 1993), and the diverse reactions of products of this pathway illustrate how C
limitation greatly impacts this pathway in grapevine berries. In addition, the
intensified berry sunlight exposure as associated with the defoliation treatments, may
have affected the expression of genes involved in encoding enzymes that play key
roles in the shikimate pathway of grapevine berries (Zhang et al., 2012). Therefore,
further investigation is needed to separate the implications of C source availability and
sunlight exposure during the post-véraison period, on berry products of the shikimate
pathway.
In summary, the objective of this study was to illustrate the impact of an imposed
reliance on stored carbohydrate and N reserves during berry maturation on the
accumulation of berry soluble solids, N and anthocyanins in conjunction with the
content of primary berry metabolites. The study was additionally aimed to better
understand the role of the leaves towards berry maturation and composition. Carbon
source limitation, induced by defoliation from near the start of berry maturation,
limited fruit sugar accumulation and anthocyanin biosynthesis. The major berry
sugars, and most minor sugars and sugar alcohols, accumulated during maturation and
the accumulation was stimulated under full leaf area. Glucose, fructose and sucrose
accumulation was maintained under C source limitation at the expense of anthocyanin
biosynthesis. Defoliation did not influence the N content of mature berries, but
increased the juice YAN. Arginine, asparagine and phenylalanine accumulated after
defoliation, while various other amino acids accumulated under full leaf area. Arginine
accumulation contributed to the increased juice YAN after defoliation, and was likely
148
Chapter 6: Paper 4
149
sourced from reserve tissues. Anthocyanins and various other shikimate pathway
products, such as phenolic acids, were largely affected by defoliation.
Therefore, excessive limitation of canopy leaf area had severe implications on
sugar and anthocyanin accumulation during berry maturation, and also altered the
berry sugar profile. Defoliation, may however, increase juice YAN through enhanced
allocation of certain amino acids to the berries, supposedly originating from N
repartitioning in alternative N source organs. This study provides a novel indication
of the effects of leaf source limitation on development of key berry quality
characteristics in conjunction with underlying metabolic alterations.
Acknowledgements
This work was supported by the National Wine and Grape Industry Centre, and
the Australian grapegrowers and winemakers through their investment body, Wine
Australia, with matching funds from the Australian Government. The authors thank
Robert Lamont, Peter Carey and Viera Mendoza Huallanca for technical assistance.
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153
Tabl
e 1 L
eaf a
rea
and
fresh
frui
t wei
ght p
er v
ine,
and
juic
e to
tal s
olub
le so
lid c
once
ntra
tion,
titra
tabl
e ac
idity
(TA
), pH
, fre
e am
ino
nitro
gen
(FA
N)
conc
entra
tion,
am
mon
ium
con
cent
ratio
n an
d ye
ast a
ssim
ilabl
e ni
troge
n (Y
AN
) co
ncen
tratio
n, a
t the
diff
eren
t har
vest
dat
es (V
+9, V
+18,
V+2
7,
V+3
7 an
d V
+46)
as a
ffect
ed b
y th
e de
folia
tion
treat
men
ts (f
ull l
eaf:
FL; 2
5% le
aves
: 25L
; no
leaf
: NL;
mea
n ±
SE, n
=3).
Inte
rval
1
Inte
rval
2
Inte
rval
3
Inte
rval
4
Trea
tmen
t V
+9
V+1
8 V
+27
V+3
7 V
+46
Leaf
are
a pe
r vi
ne (m
2 ) FL
1.
46 ±
0.0
6 A
1 1.
43 ±
0.0
6 A
1
.44
± 0.
04
A
1.47
± 0
.05
A
1.56
± 0
.03
25L
1.46
± 0
.06
a2 B
0.
35 ±
0.0
2 b
B
0.3
4 ±
0.04
b
B
0.36
± 0
.01
b B
0.
34 ±
0.0
3 b
NL
1.46
± 0
.06
- -
- -
Fres
h fr
uit
wei
ght p
er
vine
(kg)
FL
1.34
± 0
.1
b 1.
85 ±
0.3
ab
2.1
2 ±
0.1
a 2.
16 ±
0.2
a
2.08
± 0
.2
a 25
L 1.
34 ±
0.1
b
1.93
± 0
.2
a 2
.21
± 0.
1 a
2.10
± 0
.1
a 2.
17 ±
0.1
a
NL
1.34
± 0
.1
b 1.
90 ±
0.3
a
1.8
4 ±
0.1
a 2.
00 ±
0.1
a
1.92
± 0
.1
a
Juic
e TS
S (º
Bri
x)
FL
10.0
± 0
.4
d A
13
.5 ±
0.8
c
A
17.
5 ±
0.8
b A
20
.6 ±
0.8
a
A
22.4
± 0
.3
a 25
L 10
.0 ±
0.4
c
B
10.4
± 0
.3
c B
1
2.3
± 0.
2 b
B
13.7
± 0
.2
a B
14
.5 ±
0.5
a
NL
10.0
± 0
.4
bc
B
9.0
± 0
.3
c B
1
1.0
± 0.
3 ab
C
11
.3 ±
0.1
ab
C
12
.1 ±
0.7
a
Juic
e TA
(g
.L-1
) FL
24
.5 ±
0.9
a
11.9
± 0
.6
b 6
.7 ±
0.3
c
4.5
± 0
.1
d 4.
0 ±
0.1
d 25
L 24
.5 ±
0.9
a
11.6
± 0
.4
b 7
.0 ±
0.3
c
5.3
± 0
.1
d 4.
6 ±
0.1
d N
L 24
.5 ±
0.9
a
12.0
± 0
.2
b 6
.8 ±
0.2
c
5.1
± 0
.1
d 4.
3 ±
0.1
d
Juic
e pH
FL
2.
80 ±
0.0
2 e
3.09
± 0
.06
d
3.4
8 ±
0.03
c
3.8
4 ±
0.06
b
3.97
± 0
.03
a
25L
2.80
± 0
.02
d 3.
20 ±
0.0
3
c 3
.55
± 0.
02
b 3
.90
± 0.
02
a 3.
94 ±
0.0
1
a N
L 2.
80 ±
0.0
2 e
3.13
± 0
.02
d
3.5
9 ±
0.02
c
3.8
7 ±
0.02
b
3.98
± 0
.04
a
Juic
e FA
N
(mg.
L-1)
FL
36.3
± 2
.6
b B
22
.7 ±
3.8
c
B
45.
0 ±
1.7
ab
B
46.
3 ±
5.8
ab
B
48.
3 ±
1.3
a 25
L 36
.3 ±
2.6
c
A
47.7
± 5
.5
c A
7
5.7
± 3.
8 b
A
110.
7 ±1
1.9
a A
10
4.0
± 8.
2 a
NL
36.3
± 2
.6
c A
43
.3 ±
5.2
c
A
80.
3 ±
10.2
b
A
85.
3 ±
8.7
ab
A
103.
3 ±
6.8
a
Juic
e A
mm
oniu
m
(mg.
L-1)
FL
28.0
± 3
.6
B
8.7
± 2
.8
17.
7 ±
9.7
21.
3 ±
12.5
B
16
.7 ±
5.7
25
L 28
.0 ±
3.6
A
39
.7 ±
11.
5 3
1.0
± 0.
6 3
8.7
± 5.
2 A
39
.3 ±
7.8
N
L 28
.0 ±
3.6
A
B
25.7
± 3
.8
36.
0 ±
3.8
35.
7 ±
3.7
AB
36
.7 ±
4.5
Juic
e Y
AN
(m
g.L-1
) FL
57
.9 ±
4.6
a
B
27.3
± 5
.8
b B
5
4.7
± 11
.0
ab
B
59.
5 ±
17.1
a
B
56.
5 ±
5.1
ab
25L
57.9
± 4
.6
c A
78
.9 ±
8.6
bc
A
9
3.3
± 2.
8 b
A
129.
9 ±
13.7
a
A
125.
0 ±
2.1
a N
L 57
.9 ±
4.6
b
A
61.4
± 7
.6
b A
10
2.2
± 12
.1
a A
B
106.
0 ±
10.8
a
A
121.
8 ±
9.6
a
1 Mea
ns s
epar
ated
with
in c
olum
ns u
sing
Fis
her’
s LSD
test,
sign
ifica
nt d
iffer
ence
s are
indi
cate
d at
P <
0.0
5. W
here
diff
eren
t upp
er c
ase
lette
rs a
ppea
r in
a co
lum
n, v
alue
s diff
er si
gnifi
cant
ly b
etw
een
treat
men
ts.
2 Mea
ns s
epar
ated
with
in ro
ws u
sing
Fis
her’
s LSD
test,
sign
ifica
nt d
iffer
ence
s are
indi
cate
d at
P <
0.0
5. W
here
diff
eren
t low
er c
ase
lette
rs a
ppea
r in
a ro
w, v
alue
s diff
er si
gnifi
cant
ly b
etw
een
harv
est d
ates
.
Chapter 6: Paper 4
154
Fig. 1. Impact of leaf area (full leaf: FL; 25% leaves: 25L; no leaf: NL) during berry maturation on the development of the dry weight (A), soluble solid content (SSC) (B), anthocyanin content (C) and nitrogen (N) content per berry (D) (mean ± SE; n=3). Significant differences (P < 0.05) between harvest dates for each treatment are indicated by different lower case letters. Significant differences (P < 0.05) between treatments at each harvest date are indicated by different numerals [1: FL > (25L and NL), 2: FL > 25L > NL and 3: FL < 25L].
Chapter 6: Paper 4
155
Table 2 Classification of the different metabolites in the berries by GC/MS analysis.
Compound class Number of metabolites
20
9
20
4
4
10
Sugars1
Sugar alcohols (polyols)
Amino acids
Phenolic acids
TCA intermediates (acids)
Other acids2
Fatty acids 6
2
2
Flavonoids
Glycerides
Glycosides 1
1 Unclassified
Total 79
3 Includes four unidentified compounds with sugar-like spectra. 4 Includes sugar acids, dicarboxylic acids, keto acids and α-hydroxy acids.
Chapter 6: Paper 4
156Fig.
2. A
sim
plifi
ed p
athw
ay r
espo
nse
of th
e be
rry
prim
ary
met
abol
ites
signi
fican
tly a
ffect
ed b
y tre
atm
ents
(fu
ll le
af: F
L; 2
5% le
aves
: 25L
; no
leaf
: NL)
and
oth
er m
etab
olite
s dire
ctly
invo
lved
in th
e pa
thw
ays (
3). T
he si
mpl
ified
pat
hway
s inc
lude
suga
r and
suga
r alc
ohol
met
abol
ism
, am
ino
acid
met
abol
ism
, fat
ty a
cid
met
abol
ism
, phe
nolic
aci
d m
etab
olis
m, t
he tr
icar
boxy
lic a
cid
(TC
A)
cycl
e an
d th
e sh
ikim
ate
path
way
. Sig
nific
ant
diffe
renc
es a
re in
dica
ted
at P
< 0
.05,
hea
tmap
col
umns
indi
cate
the
thre
e tre
atm
ents
, whi
le h
eatm
ap ro
ws
indi
cate
the
harv
est d
ates
(V+9
, V+1
8,
V+2
7, V
+37
and
V+4
6). W
here
diff
eren
t upp
er c
ase
lette
rs a
ppea
r in
heat
map
col
umns
(1),
valu
es d
iffer
sig
nific
antly
bet
wee
n tre
atm
ents
. Whe
re
diffe
rent
low
er c
ase
lette
rs a
ppea
r in
a r
ow, v
alue
s di
ffer
sig
nific
antly
bet
wee
n ha
rves
t da
tes.
Ave
rage
met
abol
ite a
bund
ance
is c
olou
r co
ded
acco
rdin
g to
the
scal
e on
the
right
(2).
TCA
: tric
arbo
xylic
aci
d; G
-6-P
: glu
cose
-6-p
hosp
hate
; PEP
: pho
spho
enol
pyru
vic
acid
.
Chapter 6: Paper 4
157
Table 3 Summary of accumulation patterns of the different berry metabolites during the experiment.
Late accumulative (until V+37/V+46)
More abundant under FL
Sugars: Glucose, fructose, sucrose, cellobiose, trehalose, tagatose, arabinofuranose, fucose, 3α-mannobiose, unknown sugars 1, 2 and 3, ribose, arabinose. Sugar alcohols: Dulcitol, myo-inositol Amino acids: Glutamic acid, proline, pyroglutamic acid, β-alanine, tyrosine1 Organic acids: Pyruvic acid, lactic acid Phenolic acids: Gallic acid, benzoic acid Other: Glycerol monostearate, 1-monopalmitin, hydroxylamine
More abundant under 25L/NL Sugar alcohols: Arabitol Amino acids: Arginine
Early accumulative (until V+27), late depletive (after V+27)
More abundant under FL
More abundant under 25/NL
Sugars: Raffinose
Sugars: Unknown oligosaccharide 1 Sugar alcohols: Galactinol Amino acids: Aspartic acid, asparagine, phenylalanine1
Late depletive (until V+37/V+46)
More abundant under FL
More abundant under 25L/NL
Organic acids: Malic acid, citric acid, threonic acid, gluconic acid Phenolic acids: caffeic acid Sugars: Rhamnose Amino acids: Tryptophan Organic acids: Fumaric acid, glyceric acid Phenolic acids: Protocatechuic acid
Early depletive (until V+27)
More abundant under FL Organic acids: Tartaric acid Other: Catechin
More abundant under 25L/NL Sugar alcohols: cis-inosi tol
Amino acids: ThreonineOrganic acids: Ascorbic acid
5 No significant differences occurred between the treatments, although there were some changes over time.
Chapter 7: General conclusions and future work
158
Chapter 7: General conclusions and future work
Rapid berry sugar accumulation distinctly occurs during the post-véraison period, a
substantial sink demand for photoassimilates, therefore, exists during berry maturation.
In comparison, the extent of fruit nitrogen (N) incorporation often varies at distinct
stages of the growing season, the berries are nevertheless expected to be notable N sinks
after véraison. Grapevine water supply, and the relationship between the vegetative and
reproductive organ sizes, are likely major determinants of carbon (C) and N allocation
between the perennial and reproductive structures during the post-véraison period. Four
key research objectives were evaluated in order to better understand grapevine total
non-structural carbohydrate (TNC; starch and soluble sugars) and N (total N and amino
acids) partitioning and distribution during berry maturation. The effect of vine water
supply and/or the relationship between the leaf area and crop load on TNC and N was
also investigated.
Objective 1:
To investigate the interactive effects of the leaf-to-fruit ratio and grapevine water status
during two phases of berry sugar accumulation (rapid and slow) on the carbohydrate
distribution between the different grapevine organs (chapter 3).
The extent of TNC reserve contribution towards fruit sugar accumulation, when leaf
photoassimilation is restricted during the post-véraison period, was investigated in
chapter 3. Previous studies largely focussed on TNC concentrations in roots and trunks
at distinct stages of the season (predominantly budburst), on the other hand, the results
Chapter 7: General conclusions and future work
159
of this chapter are novel in demonstrating the changes of TNC content in whole
grapevine organs during berry ripening.
Sustained water constraints during the rapid berry sugar accumulation phase enforced a
reliance on root TNC reserves to support the berry sugar content. In fact, root starch
reserves accounted for 89% of the total perennial and vegetative organ TNC content
loss during rapid berry sugar accumulation. On the other hand, the perennial and
vegetative TNC content at véraison contributed to up to 18% of the berry dry matter
accumulation. The contribution from root starch reserves, as induced by water
constraints, may maintain the rate of berry sugar accumulation relative to that of well-
water vines. In addition, a reduced leaf-to-fruit ratio intensified the reliance of fruit
sugar accumulation on stored TNC. Besides the well documented replenishment of root
TNC reserves during the post-harvest period, the reserves were also stored before
harvest during the phase of slower berry sugar accumulation.
Root TNC reserve storage can significantly start occurring a few weeks prior to berry
maturity. In a practical sense, where grapegrowing regions only experience a short or no
substantial post-harvest period, the maintenance of a functional canopy leaf area during
the final few weeks before harvest could be essential in order to ensure sufficient
reserve TNC replenishment. Late season irrigation management could especially be
beneficial to allow reserve storage when post-véraison water constraints prevailed.
Chapter 7: General conclusions and future work
160
Objective 2:
To determine how the presence or absence of fruit during sustained post-véraison water
constraints influences the allocation of carbohydrates and N between the different
grapevine organs (chapter 4).
Chapter 4 evaluated the content development of both TNC and N in the different
grapevine organs during berry maturation. This study is novel in terms of demonstrating
the partitioning of both TNC and N within the different organs, thereby enhancing the
understanding of TNC and N reserve utilisation during post-véraison water constraints.
The study additionally focussed on root reserve utilisation and replenishment, and
investigatedthe implications of post-véraison water constraints on TNC and N storage
by berry maturity.
Root TNC reserves were remobilised throughout the phase of rapid berry sugar
accumulation during sustained post-véraison water constraints. However, root N
reserves, in terms of total content, were less affected during the corresponding fruit N
accumulation. Root starch hydrolysis, and subsequently root sucrose accumulation,
occurred when rapid fruit sugar accumulation coincided with sustained water
constraints. The accumulated root sucrose presumably became available for
translocation towards the fruit, as leaf photoassimilation was restricted. The
accumulation of root hexoses after defruiting may have played a role in regulating root
osmotic potential during structural root development, which also occurred after
defruiting. During sustained post-véraison water constraints, leaf N was likely exported
to the fruit, contributing to the N content. However, subsequent to defruiting, the roots
were an alternative N sink, prompting the storage of root N reserves.
Chapter 7: General conclusions and future work
When the post-véraison canopy photoassimilation is limited enough due to water
constraints, the fruit C requirement are sourced from the roots. On the other hand, leaf N
could support the post-véraison fruit N requirements. Root starch depletion during berry
sugar accumulation is subsequently detrimental towards reserve carbohydrate storage.
Furthermore, the presence of post-véraison fruit during sustained water constraints is
also detrimental towards root N storage by fruit maturity. Therefore, when water
constraints are sustained between véraison and fruit maturity, the post-harvest
replenishment of TNC and N reserves are especially crucial in order to ensure sufficient
reserve availability by budburst the next season.
Objective 3:
To assess the implications of defoliation on post-véraison fruit sugar and N
accumulation in conjunction with the carbohydrate, N and primary metabolite
composition of the major grapevine source organs (roots and leaves) (chapter 5).
In chapter 5, an untargeted approach was undertaken to determine the profiles of root
and leaf primary metabolites during berry maturation. These analyses were not intended
to describe full metabolic pathways, but were rather conducted to enable a description
of the compounds (sugars, sugar alcohols, amino acids, organic acids, etc.) involved in
source organ C and N metabolism. This study is thereby original in terms of
demonstrating the implications of C and N source limitations during berry maturation,
on subsequent C and N metabolism in the remaining source organs.
A complete defoliation shortly after the start of véraison enabled confirmation of the
post-véraison contribution of remobilised root TNC reserves towards fruit sugar
161
Chapter 7: General conclusions and future work
162
content. In fact, after the leaves, a C assimilation source, were completely removed,
some continuation of fruit sugar accumulation occurred, in conjunction with an intense
root starch depletion. The absence or limitation of the leaf source was detrimental
towards the rate of fruit sugar accumulation, but did not affect the total N content of the
mature fruit. Sugars and organic acids generally depleted in the roots and remaining
leaves after defoliation, while root and leaf amino acids accumulated.
Two metabolic pathways were identified as being largely affected in the roots by leaf
source limitation during berry maturation. Firstly, defoliation induced a rapid decrease
in root myo-inositol concentration, and likewise, the depletion of known myo-inositol
derivatives, including galactinol, raffinose and ascorbic acid. Changes in root starch and
myo-inositol concentrations were also closely related, and the results suggest myo-
inositol to play an underlying role during root starch remobilisation. Secondly,
defoliation induced the accumulation of shikimate pathway-derived aromatic amino
acids, i.e., phenylalanine, tyrosine and tryptophan, in the roots. The biosynthesis of
shikimate pathway products in the roots is, therefore, likely enhanced during a C source
limitation during berry ripening. Furthermore, as the only amino acid that clearly
depleted in the roots after defoliation and a known amino-group donor, glutamic acid is
likely involved in modulating the root amino acids composition when the leaf N source
is restricted or absent. The accumulated amino acids in the roots after defoliation, which
in addition to the aromatic amino acids also included arginine, may play a role in root to
fruit amino N translocation. As observed in the roots, amino acids accumulated in the
remaining leaves after partial defoliation, suggesting leaf protein degradation, and a
likely intensified amino N export from the remaining leaves to the fruit sink.
Chapter 7: General conclusions and future work
163
Myo-inositol metabolism and shikimate pathway derivatives are largely affected in
major TNC and N source organs (roots and leaves) during berry maturation, when a leaf
area restriction prevails. A qualitative untargeted evaluation of the primary root and leaf
metabolic composition was conducted during this study, and the results contribute to the
understanding of source organ TNC and N reserve utilisation during berry ripening.
Objective 4:
To study the implications of defoliation on the post-véraison metabolic composition of
grapevine berries (chapter 6).
Restricting the leaf area during fruit maturation limits C and likely N supply towards the
berries. Chapter 6 illustrates the implications of limiting canopy leaf area on berry
compositional parameters (soluble solids, total N, yeast assimilable N and
anthocyanins), as underlined by the contents in primary berry metabolites. The study
enhances the understanding of the contribution of primary berry metabolites towards
overall berry composition during fruit ripening.
Apart from restricting berry sugar accumulation, post-véraison leaf source limitation
strongly suppressed the berry anthocyanin accumulation rate. The inhibition of berry
anthocyanin accumulation was likely related to a C source limitation and/or an
intensified bunch exposure subsequent to defoliation. Although the total N content per
berry remained largely unaffected by the vine leaf area, the yeast assimilable N (YAN)
content increased when grapevines were partially or fully defoliated. Defoliation,
therefore, did not affect the total berry N content, but did influence N composition in the
berries through a preference towards certain amino acids.
Chapter 7: General conclusions and future work
164
Berry arginine accumulation largely explained the increased YAN after defoliation, and
was likely sourced from the roots. Glucose, fructose and sucrose content in mature
berries were less affected by post-véraison leaf source limitation, however, the
accumulation of many minor sugars and sugar alcohols, such as trehalose and myo-
inositol, were inhibited by defoliation. Galactinol and its sugar derivatives (e.g.
raffinose) accumulated early on during berry maturation, before subsequently depleting,
suggesting important transport roles for these compounds during a period of intense
fruit sugar demand, especially after defoliation. The C flux through the shikimate
pathway, as implied by the impacts on its products in the berries (e.g. anthocyanins and
phenolic acids), was largely affected by leaf source restriction during fruit maturation.
Excessive limitation of canopy leaf area had severe implications on sugar and
anthocyanin accumulation during berry maturation, and additionally altered the berry
sugar composition. Defoliation may, however, increase the juice YAN through a
subsequent increased amino acid allocation to the berries.
Synopsis of the general conclusions and future work:
This research provides an original perspective on the post-véraison utilisation of TNC
reserves towards the fruit when canopy photoassimilation is restricted by water
constraints and/or an insufficient leaf area. In comparison to TNC, the effects of vine
water supply or vegetative and reproductive organ size relations on the post-véraison
total N distribution between the different grapevine organs is less obvious. The
difference is likely attributable to the post-véraison period being the predominant stage
of fruit C demand, while fruit N incorporation may be more variable over time.
Nevertheless, the study still contributes to the understanding of post-véraison N
Chapter 7: General conclusions and future work
165
partitioning in the different grapevine organs, as demonstrated by the N compositional
changes occuring in source and sink organs during berry N accumulation.
In order to quantify TNC reserve recovery during the post-harvest period, future studies
involving post-véraison water constraints and/or restricted leaf area could include the
determination of root TNC reserve content at budburst the following season. Thereby,
the implications of restricted vine photoassimilation during berry ripening on reserve
TNC availability at the start of the next season could be revealed to a greater extent.
Furthermore, as root respiration is an additional carbohydrate expense, future studies
could include the quantification of post-véraison root respiration rate to determine its
contribution towards root TNC depletion. Carbon isotopic labelling could also be
considered in future studies in order to accurately trace TNC translocation between the
roots and fruit during the post-véraison period. Likewise, N isotopic labelling could be
utilised in order to better illustrate N distribution between the different grapevine organs
during berry ripening.
In terms of metabolic studies, future work could include the targeted monitoring of
certain metabolites in source organs and fruit during berry ripening. In fact, a particular
focus on myo-inositol and its derivatives, and compounds yielded from the shikimate
pathway, would enhance the understanding of the roles these metabolites play in
grapevine source organs during berry ripening. The relationship between myo-inositol
metabolism and root starch remobilisation especially requires further investigation.
Appendix
166
Appendix
Appendix A: Supporting information (Chapter 5 - Paper 3)
Tables S1 and S2; Figures S1, S2 and S3.
Appendix B: Supplementary material (Chapter 6 - Paper 4)
Supplementary table S1 and figure S1.
Appendix A – Table S1
167 Days
aft
er v
erai
son
Root
met
abol
ite a
bund
ance
Trea
tmen
tva
lue
SDEV
valu
eSD
EVva
lue
SDEV
valu
eSD
EVva
lue
SDEV
RT (m
in)
m/z
Kegg
IDst
anda
rd id
entif
icat
ion
NIS
T lib
rary
com
poun
dRe
tent
ion
inde
x
FL0.
009
0.01
90.
060
0.00
60.
064
0.05
60.
055
0.02
30.
032
0.05
625
L0.
009
0.01
90.
376
0.57
90.
110
0.03
80.
165
0.01
40.
196
0.02
8N
L0.
009
0.01
90.
000
0.00
00.
084
0.08
80.
378
0.10
50.
225
0.03
9FL
0.06
10.
041
0.07
30.
024
0.06
10.
021
0.06
50.
020
0.07
70.
067
25L
0.06
10.
041
0.14
90.
028
0.10
50.
023
0.09
40.
085
0.09
50.
026
NL
0.06
10.
041
0.10
20.
013
0.14
30.
060
0.06
00.
050
0.10
80.
011
FL0.
377
0.05
80.
465
0.03
00.
624
0.26
50.
580
0.26
90.
506
0.08
825
L0.
377
0.05
80.
469
0.16
40.
511
0.14
71.
045
0.29
30.
794
0.18
5N
L0.
377
0.05
80.
537
0.10
10.
399
0.21
61.
097
0.25
90.
703
0.21
1FL
31.7
196.
219
41.6
9915
.954
27.3
671.
237
47.0
206.
867
68.4
4526
.144
25L
31.7
196.
219
33.7
1012
.955
42.7
528.
251
34.5
617.
973
45.6
215.
719
NL
31.7
196.
219
25.2
6512
.532
41.4
6721
.694
41.5
0517
.838
44.8
3721
.004
FL1.
645
0.73
10.
972
0.40
60.
757
0.77
31.
043
0.60
70.
555
0.07
525
L1.
645
0.73
10.
273
0.17
50.
004
0.00
60.
160
0.15
60.
225
0.28
7N
L1.
645
0.73
10.
336
0.33
90.
076
0.13
20.
171
0.18
00.
027
0.02
6FL
0.67
60.
130
1.04
90.
739
0.73
00.
187
1.30
60.
164
1.10
10.
397
25L
0.67
60.
130
1.12
40.
862
0.74
10.
088
1.09
60.
100
1.36
60.
264
NL
0.67
60.
130
0.64
20.
246
0.51
00.
141
1.29
80.
315
1.29
10.
200
FL21
.246
6.18
831
.543
16.8
0918
.364
1.16
835
.554
8.42
037
.702
13.5
6325
L21
.246
6.18
826
.564
12.3
9131
.565
6.63
626
.294
3.56
533
.711
2.49
6N
L21
.246
6.18
820
.883
9.93
626
.837
10.7
3435
.589
7.10
140
.119
6.67
3FL
0.70
80.
552
0.46
40.
121
0.30
50.
255
0.26
70.
154
2.38
52.
675
25L
0.70
80.
552
1.65
90.
597
0.47
50.
199
0.24
90.
431
0.65
60.
967
NL
0.70
80.
552
0.82
90.
646
1.72
42.
700
0.20
20.
349
0.12
00.
141
FL8.
287
1.26
56.
322
0.82
34.
346
1.79
84.
652
0.68
26.
725
1.36
325
L8.
287
1.26
54.
198
1.05
42.
124
0.77
21.
511
0.30
02.
132
0.38
5N
L8.
287
1.26
54.
205
0.56
22.
381
0.75
61.
709
0.24
71.
860
0.14
9FL
0.96
90.
347
0.86
50.
075
0.99
80.
269
1.23
10.
168
1.22
90.
430
25L
0.96
90.
347
0.90
10.
161
0.94
50.
094
1.26
60.
654
0.79
40.
512
NL
0.96
90.
347
1.05
00.
152
0.84
90.
271
0.59
80.
336
0.83
40.
265
FL0.
542
0.46
41.
005
1.45
31.
141
0.68
60.
593
0.14
80.
580
0.43
525
L0.
542
0.46
40.
738
0.17
50.
521
0.24
30.
278
0.09
10.
587
0.28
7N
L0.
542
0.46
40.
461
0.22
90.
349
0.07
60.
770
0.05
60.
357
0.08
6FL
0.44
20.
119
0.41
60.
361
0.36
90.
322
0.37
60.
483
0.91
80.
614
25L
0.44
20.
119
0.44
60.
207
0.58
90.
067
0.68
80.
698
0.23
30.
191
NL
0.44
20.
119
0.51
60.
220
0.47
60.
240
0.31
90.
172
0.81
71.
228
FL0.
004
0.00
90.
008
0.00
70.
012
0.02
00.
035
0.04
20.
000
0.00
025
L0.
004
0.00
90.
000
0.00
00.
000
0.00
00.
013
0.02
30.
012
0.01
2N
L0.
004
0.00
90.
008
0.01
40.
002
0.00
20.
030
0.01
80.
001
0.00
3FL
0.14
70.
157
0.12
10.
105
0.51
30.
293
0.11
50.
137
0.04
30.
074
25L
0.14
70.
157
0.35
80.
066
0.26
00.
232
0.74
10.
182
0.36
10.
317
NL
0.14
70.
157
0.21
40.
198
0.30
80.
273
0.75
40.
302
0.60
60.
214
FL1.
695
1.13
41.
024
0.13
51.
530
0.61
43.
410
1.58
41.
993
0.46
625
L1.
695
1.13
40.
663
0.59
60.
032
0.00
80.
082
0.07
80.
044
0.03
9N
L1.
695
1.13
40.
795
0.64
70.
021
0.02
40.
125
0.12
30.
005
0.00
8FL
0.12
20.
075
0.05
60.
049
0.05
50.
009
0.06
10.
006
0.29
50.
248
25L
0.12
20.
075
0.13
30.
044
0.06
80.
036
0.13
70.
066
0.08
90.
038
NL
0.12
20.
075
0.10
60.
057
0.19
50.
212
0.15
60.
088
0.08
60.
009
FL34
6.33
248
.470
364.
187
129.
338
187.
393
18.3
9039
8.26
593
.810
671.
744
194.
295
25L
346.
332
48.4
7018
4.68
254
.999
278.
863
77.6
1527
1.10
517
.301
239.
451
57.6
19N
L34
6.33
248
.470
244.
579
48.4
8129
5.98
165
.031
368.
200
93.4
9145
4.56
521
3.55
0FL
1.00
10.
216
1.11
30.
144
0.88
20.
356
1.17
20.
625
1.62
00.
625
25L
1.00
10.
216
1.19
70.
347
1.52
70.
373
1.48
11.
021
1.27
00.
328
NL
1.00
10.
216
1.20
50.
176
1.43
00.
299
0.72
40.
538
1.16
00.
044
FL3.
964
0.88
55.
670
3.36
18.
444
3.16
57.
922
1.29
14.
596
1.64
325
L3.
964
0.88
54.
652
3.15
04.
544
0.58
28.
208
0.79
56.
508
1.58
8N
L3.
964
0.88
55.
633
2.54
05.
122
1.76
99.
371
0.52
56.
780
1.57
527
98
1792
2705
1292
1749
V+9
V+18
V+27
V+37
V+46
3647
2944
3834
1933
1968
2060
2123
1939
1927
3066
1915
2769
1800
1814
Suga
rs (i
nclu
ding
suga
r alc
ohol
s):
ARAB
ITO
L22
.210
3; 2
17; 3
07C0
0532
Ar
abito
l (5T
MS)
ARAB
INO
SE21
.610
3; 1
47; 2
17C0
0216
*
DL-A
rabi
nose
, tet
raki
s(tr
imet
hyls
ilyl)
ethe
r, tr
imet
hyls
ilylo
xim
e (is
omer
2)
FRUC
TOSE
25.6
; 25.
814
7; 1
03C1
0906
*Fr
ucto
se, (
5TM
S)
CELL
OBI
OSE
38.1
204;
217
; 361
C001
85
*D-
(+)-
Cello
bios
e, o
ctak
is(t
rimet
hyls
ilyl)
ethe
r (is
omer
2)
GALA
CTO
SE25
.831
9; 2
05; 2
17C0
0124
*D-
Gala
ctos
e, 2
,3,4
,5,6
-pen
taki
s-O
-(tr
imet
hyls
ilyl)-
GALA
CTIN
OL
41.5
103;
305
; 361
C012
35*
Gala
ctin
ol, n
onak
is(t
rimet
hyls
ilyl)
ethe
r
myo
-INO
SITO
L29
.314
7; 2
17; 3
05C0
0137
*m
yo-In
osito
l (6T
MS)
GLUC
OSE
26.0
; 26.
314
7; 3
19C0
0031
*D-
Gluc
ose,
2,3
,4,5
,6-p
enta
kis-
O-(
trim
ethy
lsily
l)-
MAN
NIT
OL
26.5
319;
205
C003
92*
Man
nito
l, (6
TMS)
-
scyl
lo-IN
OSI
TOL
28.2
318
C061
53In
osito
l, 1,
2,3,
4,5,
6-he
xaki
s-O
-(tr
imet
hyls
ilyl)-
, scy
llo-
MEL
EZIT
OSE
50.6
361
C082
43*
Mel
ezito
se, 1
1TM
S
MAN
NO
SE25
.920
4C0
0159
*D-
(+)-
Man
nose
, pen
taki
s(tr
imet
hyls
ilyl)
ethe
r, tr
imet
hyls
ilylo
xim
e (is
omer
1)
RAFF
INO
SE48
.436
1C0
0492
*Ra
ffin
ose,
11T
MS
MEL
IBIO
SE40
.120
4C0
5402
*M
elib
iose
, oct
akis
(trim
ethy
lsily
l)-
SUCR
OSE
37.2
361;
217
C000
89*
Sucr
ose,
oct
akis
-O-(
trim
ethy
lsily
l)-
RHAM
NO
SE22
.611
7C0
0507
*D-
(-)-
Rham
nose
, tet
raki
s(tr
imet
hyls
ilyl)
ethe
r, m
ethy
loxi
me
(ant
i)
TREH
ALO
SE38
.536
1; 2
04; 1
91C0
1083
*D-
(+)-
Treh
alos
e, o
ctak
is(t
rimet
hyls
ilyl)
ethe
r
TAGA
TOSE
23.4
217
C007
95D-
(-)-
Taga
tose
, pen
taki
s(tr
imet
hyls
ilyl)
ethe
r
GLYC
ERO
L12
.914
7; 2
05; 1
17C0
0116
Glyc
erol
, tris
-TM
S
Tabl
e S1
: Im
pact
of d
efol
iatio
n tr
eatm
ents
(ful
l lea
f: FL
; 25
leav
es: 2
5L; n
o le
af: N
L) o
n m
easu
red
prim
ary
root
met
abol
ite a
bund
ance
dur
ing
the
expe
rimen
tal p
erio
d. T
he G
C/M
S re
tent
ion
time,
maj
or fr
agm
ent m
/z, r
eten
tion
inde
x, a
nd th
e N
IST
libra
ry co
rres
pond
ing
com
poun
d in
form
atio
n is
incl
uded
.
Appendix A – Table S1
168
FL0.
238
0.07
30.
191
0.07
90.
241
0.11
30.
334
0.09
20.
362
0.21
025
L0.
238
0.07
30.
321
0.11
90.
270
0.09
30.
475
0.11
80.
337
0.09
4N
L0.
238
0.07
30.
372
0.13
30.
357
0.17
50.
421
0.06
90.
492
0.27
7FL
0.00
70.
014
0.00
00.
000
0.00
60.
007
0.00
30.
006
0.00
00.
000
25L
0.00
70.
014
0.00
00.
000
0.00
00.
000
0.01
70.
028
0.01
00.
017
NL
0.00
70.
014
0.00
00.
000
0.01
40.
021
0.02
10.
004
0.02
40.
021
FL1.
406
0.32
91.
246
0.28
10.
876
0.52
11.
374
0.31
32.
598
1.44
825
L1.
406
0.32
91.
447
0.75
41.
490
0.36
41.
487
0.49
51.
145
0.44
9N
L1.
406
0.32
91.
182
0.13
92.
925
2.69
61.
046
0.71
11.
138
0.69
6FL
0.91
30.
779
0.52
00.
797
0.43
60.
206
1.19
31.
147
0.64
70.
354
25L
0.91
30.
779
2.47
23.
799
1.57
51.
325
16.9
4316
.480
20.1
850.
901
NL
0.91
30.
779
0.45
30.
279
3.78
73.
670
14.7
1013
.139
9.00
86.
854
FL0.
000
0.00
00.
000
0.00
00.
000
0.00
00.
000
0.00
00.
000
0.00
025
L0.
000
0.00
00.
000
0.00
00.
000
0.00
00.
000
0.00
00.
050
0.04
4N
L0.
000
0.00
00.
000
0.00
00.
017
0.02
90.
072
0.06
90.
043
0.03
7FL
0.28
10.
098
0.16
00.
121
0.11
50.
040
0.14
80.
022
0.27
90.
109
25L
0.28
10.
098
0.16
00.
165
0.07
60.
012
0.16
90.
097
0.22
10.
042
NL
0.28
10.
098
0.16
50.
152
0.23
40.
169
0.16
00.
090
0.13
80.
010
FL1.
561
0.77
20.
973
0.24
60.
507
0.17
10.
936
0.31
82.
911
2.52
225
L1.
561
0.77
21.
654
0.75
30.
974
0.28
00.
943
0.84
01.
190
0.46
3N
L1.
561
0.77
21.
330
0.42
72.
423
2.76
10.
949
0.70
30.
986
0.24
6FL
0.42
70.
128
0.30
50.
162
0.30
70.
106
0.35
50.
068
0.32
60.
084
25L
0.42
70.
128
0.14
40.
034
0.18
70.
040
0.19
90.
092
0.26
30.
015
NL
0.42
70.
128
0.23
70.
153
0.06
80.
065
0.11
70.
042
0.10
50.
005
FL0.
280
0.07
40.
274
0.16
00.
271
0.14
70.
216
0.01
70.
494
0.45
025
L0.
280
0.07
40.
290
0.19
90.
166
0.04
10.
240
0.09
10.
220
0.06
9N
L0.
280
0.07
40.
312
0.20
50.
331
0.20
60.
603
0.18
80.
338
0.12
2FL
0.07
70.
101
0.02
80.
048
0.07
80.
136
0.03
40.
059
0.00
00.
000
25L
0.07
70.
101
0.00
00.
000
0.05
70.
099
0.00
00.
000
0.00
00.
000
NL
0.07
70.
101
0.23
40.
083
0.14
70.
131
0.00
00.
000
0.00
00.
000
FL0.
004
0.00
70.
000
0.00
00.
000
0.00
00.
000
0.00
00.
000
0.00
025
L0.
004
0.00
70.
006
0.01
10.
000
0.00
00.
000
0.00
00.
000
0.00
0N
L0.
004
0.00
70.
036
0.03
50.
019
0.03
30.
000
0.00
00.
000
0.00
0FL
0.00
20.
004
0.00
20.
004
0.00
00.
000
0.00
00.
000
0.00
00.
000
25L
0.00
20.
004
0.00
00.
000
0.00
00.
000
0.00
00.
000
0.00
00.
000
NL
0.00
20.
004
0.01
20.
011
0.00
50.
008
0.00
80.
014
0.00
00.
000
FL0.
028
0.03
80.
018
0.03
10.
000
0.00
00.
016
0.02
80.
017
0.03
025
L0.
028
0.03
80.
074
0.12
80.
016
0.02
80.
527
0.60
60.
642
0.08
5N
L0.
028
0.03
80.
023
0.04
00.
101
0.10
60.
441
0.36
00.
214
0.12
3FL
0.24
70.
123
0.17
60.
039
0.00
00.
000
0.16
10.
142
0.54
10.
409
25L
0.24
70.
123
0.36
30.
084
0.09
10.
158
0.19
50.
146
0.19
10.
170
NL
0.24
70.
123
0.17
30.
150
0.40
20.
591
0.13
60.
236
0.13
60.
147
FL0.
079
0.01
60.
062
0.04
20.
067
0.02
50.
110
0.01
70.
013
0.02
325
L0.
079
0.01
60.
114
0.15
90.
058
0.05
70.
225
0.17
90.
136
0.09
9N
L0.
079
0.01
60.
157
0.04
90.
418
0.18
60.
321
0.26
20.
318
0.12
0FL
0.32
30.
195
0.22
10.
043
0.10
60.
048
0.23
00.
085
0.84
50.
752
25L
0.32
30.
195
0.45
30.
253
0.25
30.
163
0.35
20.
146
0.31
90.
172
NL
0.32
30.
195
0.32
00.
154
0.65
40.
868
0.24
00.
246
0.18
60.
107
FL1.
329
0.38
21.
161
0.88
10.
872
0.10
21.
312
0.34
70.
996
0.11
025
L1.
329
0.38
20.
661
0.48
50.
603
0.12
70.
833
0.14
20.
862
0.12
4N
L1.
329
0.38
21.
239
0.83
90.
870
0.23
20.
929
0.40
91.
025
0.30
2FL
0.12
00.
023
0.11
50.
060
0.05
90.
036
0.13
20.
045
0.14
70.
029
25L
0.12
00.
023
0.12
40.
055
0.07
80.
024
0.13
30.
032
0.10
70.
071
NL
0.12
00.
023
0.34
30.
386
0.12
10.
071
0.10
60.
089
0.05
20.
014
FL0.
093
0.04
50.
082
0.07
30.
062
0.00
70.
060
0.02
70.
045
0.04
125
L0.
093
0.04
50.
076
0.05
40.
063
0.01
40.
068
0.06
10.
076
0.02
8N
L0.
093
0.04
50.
188
0.10
40.
096
0.01
90.
157
0.07
00.
145
0.06
3FL
0.35
50.
235
0.21
60.
284
0.19
00.
044
0.27
50.
153
0.24
40.
207
25L
0.35
50.
235
0.32
00.
333
0.16
40.
143
0.67
80.
541
0.54
50.
195
NL
0.35
50.
235
0.29
50.
145
0.54
70.
299
1.25
60.
581
0.80
80.
403
2798
2230
1336
1787
GLYC
INE
13.6
174
C000
37
GLUT
AMIN
E23
.315
6C0
0064
LEUC
INE
12.8
158
C001
23
ISO
LEUC
INE
13.3
158
1433
ASPA
RTIC
ACI
D18
.523
2; 1
47; 2
18C0
0049
*
L-As
part
ic a
cid,
(3TM
S)-
ASPA
RAGI
NE
21.6
116;
231
C001
52
*As
para
gine
, O,O
',N-t
ris(t
rimet
hyls
ilyl)-
1836
1130
1674
Amin
o ac
ids:
XYLO
SE21
.221
7; 3
07C0
0181
*D-
(+)-
Xylo
se, t
etra
kis(
trim
ethy
lsily
l) et
her,
met
hylo
xim
e (a
nti)
1414
1532
1313
1635
1532
1944
1288
1313
*Gl
y, O
,N,N
-tris
-TM
S
*L-
Glut
amin
e, N
,N2-
bis(
trim
ethy
lsily
l)-, t
rimet
hyls
ilyl e
ster
*L-
Leuc
ine,
N-(
trim
ethy
lsily
l)-, t
rimet
hyls
ilyl e
ster
1635
1542
1542
1696
TURA
NO
SE38
.820
4; 3
61; 3
07C1
9636
*D-
(+)-
Tura
nose
, oct
akis
(trim
ethy
lsily
l) et
her,
met
hylo
xim
e (is
omer
1)
ARGI
NIN
E 2
0.3;
24.
2;
142
C000
62
*Ar
gini
ne, (
3TM
S)
ALAN
INE
8.9
147
C000
41
*l-A
lani
ne, N
-(tr
imet
hyls
ilyl)-
, trim
ethy
lsily
l est
er
GLUT
AMIC
ACI
D20
.424
6C0
0025
*Gl
utam
ic a
cid
(3TM
S)
GABA
(γ-A
min
obut
yric
aci
d)18
.517
4C0
0334
*
GABA
3TM
S
C004
07*
Isol
euci
ne, d
i-TM
S
MET
HIO
NIN
E18
.312
8C0
0073
*M
et, (
2TM
S)
LYSI
NE
26.1
156
C000
47*
l-Lys
ine,
N2,
N6-
bis(
trim
ethy
lsily
l)-, t
rimet
hyls
ilyl e
ster
PRO
LIN
E13
.321
6; 1
47; 1
58C0
0148
*Pr
olin
e, d
i-TM
S
PHEN
YLAL
ANIN
E20
.421
8C0
0079
*Ph
enyl
alan
ine
(N,O
-TM
S)
SERI
NE
14.9
204;
218
C000
65*
Serin
e, (3
TMS)
PYRO
GLUT
AMIC
ACI
D (5
-oxo
-Pro
line)
18.3
156
C018
79Py
rogl
utam
ic a
cid,
(N,O
-TM
S)
Tryp
toph
an, N
,N,O
-triT
MS
THRE
ON
INE
15.5
218
C001
88*
Thre
onin
e (N
,O,O
-TM
S)
TRYP
TOPH
AN30
.829
1; 2
02C0
0078
*
Appendix A – Table S1
169
FL0.
076
0.04
20.
068
0.02
70.
105
0.09
70.
064
0.05
60.
060
0.01
925
L0.
076
0.04
20.
071
0.03
20.
027
0.02
30.
117
0.00
50.
127
0.03
2N
L0.
076
0.04
20.
115
0.00
70.
042
0.04
70.
087
0.08
50.
054
0.04
0FL
0.10
10.
042
0.09
30.
080
0.10
10.
031
0.11
50.
024
0.12
40.
215
25L
0.10
10.
042
0.08
50.
147
0.07
70.
026
0.17
70.
060
0.09
70.
041
NL
0.10
10.
042
0.17
80.
066
0.23
30.
052
0.27
40.
138
0.16
70.
065
FL0.
022
0.00
90.
013
0.02
20.
007
0.01
30.
011
0.01
00.
003
0.00
525
L0.
022
0.00
90.
000
0.00
00.
007
0.01
20.
011
0.01
90.
000
0.00
0N
L0.
022
0.00
90.
071
0.04
50.
066
0.02
80.
013
0.01
10.
029
0.02
8
FL0.
270
0.09
00.
127
0.10
80.
222
0.07
20.
263
0.10
80.
342
0.05
225
L0.
270
0.09
00.
116
0.05
90.
076
0.04
70.
134
0.05
80.
100
0.03
9N
L0.
270
0.09
00.
133
0.04
30.
099
0.02
50.
144
0.06
10.
091
0.04
6FL
12.2
037.
095
12.7
283.
233
1.31
51.
930
9.89
54.
236
28.0
7925
.404
25L
12.2
037.
095
10.6
902.
991
7.88
66.
181
12.1
9711
.198
12.6
947.
372
NL
12.2
037.
095
11.4
343.
879
24.2
5029
.199
10.2
1311
.017
8.90
15.
862
FL0.
054
0.02
20.
064
0.02
40.
031
0.02
90.
085
0.02
40.
095
0.03
625
L0.
054
0.02
20.
101
0.04
30.
055
0.01
10.
129
0.07
60.
140
0.07
7N
L0.
054
0.02
20.
080
0.02
90.
122
0.12
20.
078
0.11
40.
066
0.02
4FL
0.17
90.
109
0.20
10.
154
0.28
50.
131
0.06
10.
023
0.27
90.
470
25L
0.17
90.
109
0.27
90.
408
0.63
20.
584
0.21
20.
274
0.19
10.
224
NL
0.17
90.
109
0.09
80.
062
0.20
50.
243
0.02
40.
016
0.30
40.
105
FL0.
039
0.00
70.
022
0.01
10.
032
0.01
50.
021
0.01
80.
018
0.03
125
L0.
039
0.00
70.
035
0.03
10.
045
0.03
10.
046
0.04
30.
042
0.02
3N
L0.
039
0.00
70.
020
0.01
90.
025
0.02
20.
022
0.01
90.
050
0.00
4FL
1.57
20.
344
1.11
80.
332
1.12
80.
604
1.32
40.
464
2.21
20.
490
25L
1.57
20.
344
2.86
73.
696
1.40
60.
278
7.07
64.
451
8.53
21.
370
NL
1.57
20.
344
0.91
80.
056
2.34
62.
446
6.70
31.
122
4.36
31.
067
FL0.
121
0.04
90.
088
0.00
30.
036
0.02
20.
056
0.05
10.
307
0.27
525
L0.
121
0.04
90.
173
0.04
90.
126
0.09
10.
156
0.01
50.
131
0.03
1N
L0.
121
0.04
90.
129
0.06
90.
222
0.27
20.
065
0.08
30.
096
0.03
0FL
2.23
40.
861
2.27
00.
517
1.53
50.
425
2.92
50.
467
3.69
61.
533
25L
2.23
40.
861
3.04
01.
310
2.61
40.
433
3.21
11.
758
3.29
41.
126
NL
2.23
40.
861
2.83
80.
503
3.48
11.
796
3.03
81.
674
3.05
00.
311
FL0.
453
0.10
50.
189
0.11
40.
104
0.05
00.
150
0.03
00.
531
0.39
725
L0.
453
0.10
50.
134
0.12
10.
067
0.06
00.
099
0.09
00.
178
0.04
5N
L0.
453
0.10
50.
115
0.04
80.
000
0.00
00.
060
0.05
90.
125
0.01
4FL
0.10
20.
044
0.07
90.
009
0.02
50.
022
0.03
30.
040
0.13
00.
096
25L
0.10
20.
044
0.10
50.
025
0.00
70.
012
0.00
30.
006
0.05
60.
049
NL
0.10
20.
044
0.10
10.
031
0.01
80.
032
0.08
50.
025
0.06
50.
025
FL0.
143
0.17
00.
108
0.02
40.
000
0.00
00.
058
0.10
10.
538
0.58
525
L0.
143
0.17
00.
257
0.17
70.
098
0.08
80.
112
0.13
00.
000
0.00
0N
L0.
143
0.17
00.
179
0.15
60.
330
0.57
10.
093
0.12
60.
060
0.06
1FL
0.06
30.
051
0.05
80.
013
0.05
10.
016
0.04
00.
026
0.41
80.
534
25L
0.06
30.
051
0.05
20.
032
0.06
00.
025
0.06
50.
019
0.06
60.
013
NL
0.06
30.
051
0.03
20.
009
0.30
30.
428
0.12
10.
021
0.05
90.
010
FL1.
365
0.46
11.
532
0.66
80.
734
0.09
71.
643
0.96
42.
164
1.85
425
L1.
365
0.46
11.
282
0.58
01.
184
0.65
21.
566
0.70
11.
146
0.52
1N
L1.
365
0.46
11.
347
0.59
92.
489
2.76
20.
960
0.77
61.
047
0.50
3FL
0.12
50.
118
0.13
10.
037
0.11
60.
011
0.06
30.
023
0.11
10.
040
25L
0.12
50.
118
0.14
50.
079
0.14
00.
063
0.18
10.
035
0.15
10.
069
NL
0.12
50.
118
0.05
80.
024
0.17
00.
043
0.12
60.
091
0.18
60.
067
FL2.
643
0.72
42.
598
0.57
71.
768
0.65
32.
098
1.01
31.
738
0.24
325
L2.
643
0.72
42.
186
0.78
61.
947
0.63
34.
467
1.34
74.
828
1.27
3N
L2.
643
0.72
42.
294
1.06
92.
051
0.52
14.
167
0.89
93.
083
1.38
5FL
3.93
61.
389
2.96
90.
072
2.46
01.
305
3.45
50.
677
7.35
94.
632
25L
3.93
61.
389
4.57
02.
263
3.54
60.
566
5.36
02.
385
3.85
91.
925
NL
3.93
61.
389
3.61
50.
825
7.22
45.
475
3.23
43.
187
3.06
23.
313
FL8.
448
0.70
45.
592
1.70
05.
513
0.82
05.
967
0.85
97.
724
1.01
125
L8.
448
0.70
48.
096
2.42
79.
370
1.42
816
.193
4.92
113
.188
4.55
7N
L8.
448
0.70
49.
063
1.13
68.
161
1.34
313
.483
0.55
112
.080
1.52
1
1175
1508
PHO
SPHO
RIC
ACID
12.9
299
C000
09Ph
osph
oric
aci
d, tr
iTM
S12
92
MAL
IC A
CID
17.8
233
C001
49M
alic
aci
d (3
TMS)
1847
1328
1088
1991
1242
1956
2323
1382
1898
2049
1980
1397
1944
2130
1635
1260
1870
Mis
cella
neou
s aci
ds:
TYRO
SIN
E26
.321
8; 2
80C0
0082
*Ty
rosi
ne, (
3TM
S)
5-hy
drox
y-TR
YPTO
PHAN
32.1
290;
218
; 146
C010
17L-
5-Hy
drox
ytry
ptop
han,
trim
ethy
lsily
l eth
er, t
rimet
hyls
ilyl e
ster
ASCO
RBIC
ACI
D24
.814
7C0
0072
*As
corb
ic a
cid
(4TM
S)
VALI
NE
11.5
144;
218
C001
83*
Valin
e, d
i-TM
S
4-HY
DRO
XY-B
ENZO
IC A
CID
20.4
267
C001
564-
Hydr
oxyb
enzo
ic a
cid
(2T
MS)
BEN
ZOIC
ACI
D12
.017
9C0
0180
Benz
oic a
cid,
TM
S
P-CO
UMAR
IC A
CID
26.1
219
C008
11p-
Coum
aric
aci
d (T
MS)
CAFF
EIC
ACID
29.4
396
C011
97Ca
ffei
c aci
d (3
TMS)
FUM
ARIC
ACI
D14
.424
5C0
0122
Fum
aric
aci
d (2
TMS)
CITR
IC A
CID
24.4
273
C001
58Ci
tric
aci
d, (4
TMS)
GLUC
ON
IC A
CID
28.0
292;
333
C002
57Gl
ucon
ic a
cid,
(6TM
S)
GALL
IC A
CID
26.7
281;
458
C014
24Ga
llic a
cid,
tetr
aTM
S
GLYC
ERIC
ACI
D14
.214
7; 1
89; 2
92C0
0258
Glyc
eric
aci
d, (3
TMS)
2-KE
TO-G
LUCO
NIC
ACI
D23
.729
2; 1
03C0
6473
2-Ke
to-l-
gluc
onic
aci
d, p
enta
(O-t
rimet
hyls
ilyl)-
LACT
IC A
CID
8.1
147;
117
C001
86La
ctic
aci
d, (2
TMS)
3-HY
DRO
XYAN
THRA
NIL
IC A
CID
26.9
354
C006
323-
Hydr
oxya
nthr
anill
ic a
cid,
(3TM
S)
MAL
EIC
ACID
13.5
147;
245
C01
384
Mal
eic a
cid,
(2TM
S)
OXA
LIC
ACID
9.8
147;
133
C002
09O
xalic
aci
d (2
TMS)
Appendix A – Table S1
170
FL0.
711
0.36
10.
607
0.08
20.
362
0.26
20.
501
0.11
10.
878
0.11
425
L0.
711
0.36
10.
744
0.17
00.
905
0.56
60.
517
0.35
50.
474
0.19
8N
L0.
711
0.36
10.
759
0.05
30.
866
0.67
40.
320
0.33
60.
462
0.20
8FL
0.04
70.
035
0.03
20.
056
0.33
70.
512
0.05
10.
046
0.04
60.
079
25L
0.04
70.
035
0.17
10.
187
0.06
90.
060
0.00
00.
000
0.04
80.
043
NL
0.04
70.
035
0.10
00.
012
0.37
40.
531
0.02
30.
040
0.11
60.
061
FL4.
028
0.30
23.
415
0.75
512
.095
16.5
562.
748
1.41
15.
257
2.70
325
L4.
028
0.30
24.
840
4.87
76.
575
2.40
520
.539
7.41
224
.883
7.85
2N
L4.
028
0.30
23.
651
0.87
48.
062
5.46
814
.699
5.65
114
.777
5.23
5FL
1.14
50.
936
0.29
30.
271
0.07
60.
030
0.08
50.
027
0.11
70.
107
25L
1.14
50.
936
0.33
90.
351
0.08
00.
078
0.01
00.
018
0.05
80.
051
NL
1.14
50.
936
0.55
80.
316
0.00
00.
000
0.03
90.
051
0.38
10.
615
FL0.
064
0.06
10.
031
0.01
70.
039
0.03
10.
056
0.03
30.
270
0.36
925
L0.
064
0.06
10.
115
0.13
00.
019
0.00
50.
080
0.06
80.
054
0.03
0N
L0.
064
0.06
10.
097
0.07
70.
190
0.29
90.
076
0.04
90.
027
0.01
7FL
0.44
80.
319
0.34
30.
066
0.20
10.
066
0.32
50.
087
1.24
11.
297
25L
0.44
80.
319
0.59
20.
373
0.29
00.
109
0.47
50.
325
0.51
80.
233
NL
0.44
80.
319
0.52
90.
289
1.01
61.
303
0.35
90.
285
0.26
20.
132
FL0.
089
0.02
40.
136
0.04
60.
042
0.01
40.
087
0.04
20.
167
0.13
725
L0.
089
0.02
40.
125
0.06
20.
128
0.07
10.
112
0.00
80.
086
0.03
2N
L0.
089
0.02
40.
122
0.08
00.
128
0.08
40.
073
0.07
90.
103
0.07
2FL
0.06
10.
068
0.00
90.
014
0.00
20.
003
0.03
90.
013
0.22
70.
265
25L
0.06
10.
068
0.22
80.
278
0.03
30.
050
0.03
50.
060
0.04
00.
042
NL
0.06
10.
068
0.33
80.
482
0.10
60.
175
0.05
10.
088
0.00
20.
002
FL2.
294
1.46
81.
636
0.53
10.
742
0.25
41.
924
0.48
35.
661
5.11
425
L2.
294
1.46
83.
873
2.99
61.
789
1.11
42.
706
1.01
72.
461
1.26
7N
L2.
294
1.46
82.
829
1.94
25.
212
6.78
31.
942
2.38
11.
329
0.71
8FL
0.00
30.
006
0.00
00.
000
0.00
00.
000
0.00
00.
000
0.02
40.
042
25L
0.00
30.
006
0.01
50.
026
0.00
00.
000
0.00
00.
000
0.00
00.
000
NL
0.00
30.
006
0.05
70.
071
0.02
40.
042
0.00
00.
000
0.00
00.
000
FL3.
949
3.03
82.
823
0.84
11.
037
0.20
43.
167
1.20
110
.874
10.5
5825
L3.
949
3.03
86.
832
5.27
42.
485
1.54
84.
064
2.55
94.
501
2.55
9N
L3.
949
3.03
84.
339
2.70
79.
160
12.0
543.
237
3.99
71.
961
1.06
8
FL0.
264
0.06
80.
302
0.04
00.
405
0.08
30.
462
0.03
60.
445
0.12
225
L0.
264
0.06
80.
352
0.24
60.
250
0.09
30.
712
0.19
50.
477
0.05
3N
L0.
264
0.06
80.
272
0.07
10.
508
0.27
60.
656
0.05
30.
463
0.08
0FL
5.77
22.
439
5.63
20.
372
6.11
22.
538
6.47
90.
452
6.00
90.
211
25L
5.77
22.
439
5.24
13.
321
4.88
70.
667
9.48
43.
401
7.62
10.
917
NL
5.77
22.
439
7.33
50.
926
7.37
22.
506
7.91
54.
534
6.55
81.
118
FL17
.075
11.0
1213
.641
3.19
19.
191
5.16
615
.227
2.24
441
.513
36.8
2725
L17
.075
11.0
1223
.170
14.8
9312
.503
2.49
518
.053
12.4
8617
.881
7.31
1N
L17
.075
11.0
1218
.522
7.88
239
.290
47.8
0215
.556
10.7
8710
.065
5.94
5FL
0.41
20.
097
0.35
00.
053
0.24
00.
104
0.36
80.
054
0.77
90.
453
25L
0.41
20.
097
0.36
90.
076
0.50
90.
145
0.44
00.
132
0.39
20.
120
NL
0.41
20.
097
0.33
80.
033
0.59
20.
367
0.21
20.
286
0.26
70.
218
FL0.
007
0.00
50.
009
0.00
80.
008
0.00
60.
006
0.00
50.
004
0.00
625
L0.
007
0.00
50.
009
0.01
60.
013
0.01
10.
005
0.00
80.
006
0.01
0N
L0.
007
0.00
50.
002
0.00
30.
025
0.02
40.
002
0.00
30.
008
0.00
3FL
8.17
45.
032
6.79
51.
447
4.16
22.
297
6.97
50.
991
22.3
6921
.796
25L
8.17
45.
032
11.1
496.
728
6.49
91.
708
8.59
76.
101
9.16
73.
262
NL
8.17
45.
032
8.72
73.
693
19.1
5822
.971
6.80
05.
225
5.16
82.
967
FL0.
220
0.31
60.
193
0.02
20.
685
0.59
60.
688
0.27
00.
081
0.12
425
L0.
220
0.31
60.
319
0.39
40.
180
0.08
80.
509
0.23
20.
331
0.14
5N
L0.
220
0.31
60.
419
0.38
10.
353
0.29
51.
642
0.93
30.
238
0.18
3FL
0.13
00.
104
0.10
60.
033
0.08
20.
053
0.12
10.
050
0.06
80.
041
25L
0.13
00.
104
0.23
60.
076
0.24
20.
251
0.13
10.
090
0.13
70.
117
NL
0.13
00.
104
0.13
30.
074
0.14
90.
153
0.19
90.
103
0.12
80.
100
Adip
ic a
cid
1814
7; 2
75C0
6104
*Ad
ipic
aci
d, (2
TMS)
1528
Adon
itol (
Ribi
tol)
22.8
147;
217
; 103
C00474
*Ri
bito
l, 5T
MS
1760
L-hy
drox
ypro
line
18.5
230
C011
57*
3-Hy
drox
ypro
line,
N,O
,O'-t
ris(t
rimet
hyls
ilyl)-
1542
C000
42
1831
Succ
inic
aci
d (2
TMS)
2237
1940
2039
2209
1405
2407
2990
2734
4300
2602
3108
1135
2791
2889
2626
Mis
calle
neou
s com
poun
ds:
NO
NAN
OIC
ACI
D14
.611
7; 2
15C0
1601
Non
anoi
c aci
d, T
MS
este
r
MYR
ISTI
C AC
ID
L-Th
reon
ic a
cid,
tris
(trim
ethy
lsily
l) et
her,
trim
ethy
lsily
l est
er
Fatt
y ac
ids:
1586
PRO
TOCA
TECH
UIC
ACID
24.1
193;
370
C002
30*
Prot
ocat
echu
ic a
cid
(tm
s)
SUCC
INIC
ACI
D13
.714
7; 2
47
1666
1344
TART
ARIC
ACI
D21
.029
2C0
0898
Tart
aric
aci
d, T
MS
33.3
343
C064
24M
yris
tic a
cid,
2,3
-bis
(trim
ethy
lsilo
xy)p
ropy
l est
er
EICO
SAN
OIC
ACI
D40
.642
7C0
6425
Eico
sano
ic a
cid,
2,3
-bis
[(tr
imet
hyls
ilyl)o
xy]p
ropy
l est
er
THRE
ON
IC A
CID
19.4
147;
292
; 220
C016
20
PALM
ITIC
ACI
D27
.811
7; 3
13C0
0249
Palm
itic a
cid,
TM
S
OLE
IC A
CID
30.5
339
C007
12O
leic
aci
d, tr
imet
hyls
ilyl e
ster
STEA
RIC
ACID
30.9
117;
341
C015
30St
earic
aci
d, tr
imet
hyls
ilyl e
ster
PEN
TADE
CAN
OIC
ACI
D26
.211
7; 2
99C1
6537
Pent
adec
anoi
c aci
d, T
MS
este
r
GLYC
ERO
L MO
NO
STEA
RATE
38.4
399
N/A
Glyc
erol
mon
oste
arat
e, 2
tms d
eriv
ativ
e
CATE
CHIN
39.5
; 39.
836
8C0
6562
Cate
chin
e, p
enta
-TM
S-et
her,
(2R-
cis)
-
ARBU
TIN
36.2
254
C061
86Hy
droq
uino
ne-β
-d-g
luco
pyra
nosi
de,p
enta
kis(
trim
ethy
lsily
l)-
KAEM
PFER
OL
42.0
559
C059
03Ka
empf
erol
, 4TM
S
HYDR
OXY
LAM
INE
9.0
133
C001
92Hy
drox
ylam
ine,
N,N
,O-t
ris-T
MS
CIS-
RESV
ERAT
ROL
37.6
444;
147
; 207
C035
82ci
s-Re
sver
atro
l, 3T
MS
Inte
rnal
stan
dard
s:
CIS-
PICE
ID56
.144
4; 3
61; 2
17C1
0275
cis-
Pice
id, 6
TMS
1-M
ON
OPA
LMIT
IN35
.937
1N
/A1-
Mon
opal
miti
n tr
imet
hyls
ilyl e
ther
Appendix A – Table S2
171
Day
s af
ter
vera
iso
n
Ro
ot
me
tab
oli
te a
bu
nd
ance
Tre
atm
en
tva
lue
SDEV
valu
eSD
EVva
lue
SDEV
valu
eSD
EVva
lue
SDEV
RT
(min
)m
/zK
egg
IDst
and
ard
ide
nti
fica
tio
nN
IST
lib
rary
co
mp
ou
nd
Re
ten
tio
n in
de
x
FL0.
215
0.0
67
0.23
60.1
31
0.16
30.0
68
0.06
00.0
68
0.05
10.0
88
25L
0.21
50.0
67
0.15
60.0
98
0.12
00.0
53
0.13
40.0
09
0.20
20.1
18
FL0.
166
0.0
90
0.20
70.0
33
0.15
50.0
86
0.21
00.0
97
0.18
80.0
92
25L
0.16
60.0
90
0.16
30.0
61
0.22
40.0
79
0.32
50.0
27
0.09
30.0
21
FL18
.198
3.9
14
16.9
902.8
08
19.0
494.8
41
19.0
193.2
55
19.3
220.6
27
25L
18.1
983.9
14
17.6
991.5
60
18.3
192.3
54
20.2
532.5
40
23.2
047.0
08
FL0.
457
0.1
96
0.18
00.1
56
0.57
70.5
98
0.32
40.1
08
0.25
70.0
42
25L
0.45
70.1
96
0.00
00.0
00
0.15
60.1
66
0.10
30.1
79
0.05
30.0
92
FL53
.722
4.2
39
50.8
7230.5
98
29.5
357.2
26
74.0
103.7
46
76.0
0121.3
11
25L
53.7
224.2
39
38.5
0211.2
85
26.9
727.4
35
47.5
537.4
41
54.2
9619.5
74
FL3.
848
1.5
22
3.97
50.2
78
5.69
01.2
73
4.61
60.4
62
2.99
40.7
43
25L
3.84
81.5
22
3.17
90.9
65
5.17
11.1
90
3.68
00.6
26
4.20
41.6
05
FL1.
041
0.0
71
1.13
70.3
16
0.94
10.0
97
1.05
70.0
65
1.11
00.2
56
25L
1.04
10.0
71
0.77
20.0
20
0.89
80.2
24
1.01
10.1
79
1.55
90.7
33
FL40
.426
3.8
01
34.7
4918.0
70
22.9
354.8
26
53.6
605.3
61
45.4
089.7
09
25L
40.4
263.8
01
33.0
089.7
16
25.6
764.4
47
45.6
252.6
59
50.8
0123.1
69
FL1.
581
3.0
37
0.00
00.0
00
0.01
30.0
23
0.00
00.0
00
1.92
62.3
48
25L
1.58
13.0
37
0.00
00.0
00
0.42
30.7
33
0.43
10.7
47
0.00
00.0
00
FL24
7.90
614.2
94
232.
271
37.8
21
198.
083
9.7
08
183.
468
20.2
60
179.
005
13.8
46
25L
247.
906
14.2
94
186.
784
9.2
52
137.
310
22.1
76
116.
523
5.7
60
103.
425
13.8
78
FL1.
523
0.1
21
0.86
10.5
08
1.13
50.1
78
0.68
60.2
90
0.63
30.3
09
25L
1.52
30.1
21
0.93
50.0
83
1.00
20.1
96
0.48
00.0
47
0.32
50.1
56
FL2.
094
0.8
56
2.82
21.7
02
0.78
60.4
17
1.10
10.1
88
2.48
31.3
32
25L
2.09
40.8
56
4.16
01.0
21
1.25
70.5
55
1.63
30.7
58
4.13
20.2
86
FL8.
427
0.8
54
7.67
81.3
99
6.98
60.8
44
8.99
32.1
26
8.60
10.3
98
25L
8.42
70.8
54
6.07
20.9
51
5.80
91.2
67
6.40
01.2
35
6.02
00.8
34
FL0.
220
0.3
09
0.06
40.0
57
0.05
70.0
50
0.21
20.3
68
0.08
60.0
78
25L
0.22
00.3
09
0.07
70.0
69
0.25
40.3
89
0.11
80.0
53
0.12
40.0
83
FL0.
019
0.0
28
0.00
30.0
05
0.02
20.0
30
0.00
40.0
07
0.03
30.0
43
25L
0.01
90.0
28
0.01
40.0
18
0.01
20.0
20
0.04
10.0
18
0.13
90.0
41
FL3.
367
0.8
45
4.08
50.7
96
5.03
51.8
12
4.74
70.8
81
4.64
71.4
91
25L
3.36
70.8
45
4.89
61.6
45
5.99
60.3
02
5.85
51.1
28
6.90
80.9
29
FL1.
299
0.4
11
0.86
40.0
84
1.77
41.2
26
2.35
30.2
57
3.58
43.1
38
25L
1.29
90.4
11
1.03
00.6
51
0.98
30.6
32
2.30
00.6
86
7.20
33.6
08
FL0.
103
0.0
23
0.07
10.0
26
0.07
90.0
11
0.05
40.0
47
0.06
20.0
20
25L
0.10
30.0
23
0.07
70.0
20
0.13
70.0
68
0.20
90.0
35
0.62
30.4
31
FL1.
058
0.1
89
0.86
80.0
56
0.66
60.0
70
0.87
90.2
88
0.98
30.2
06
25L
1.05
80.1
89
0.54
00.1
45
0.47
80.0
95
0.54
30.1
35
0.51
00.0
98
FL74
2.82
866.5
34
807.
892
44.7
75
716.
296
70.5
17
764.
979
10.1
08
780.
693
52.2
92
25L
742.
828
66.5
34
784.
238
38.2
96
809.
772
36.6
14
723.
916
222.2
36
773.
685
49.1
73
FL3.
954
1.2
01
4.95
80.7
45
3.81
20.9
70
4.59
41.2
29
3.64
11.0
98
25L
3.95
41.2
01
4.01
11.4
63
4.50
02.1
58
5.65
61.1
20
3.34
10.1
97
FL7.
692
0.7
19
6.25
90.7
86
7.09
73.1
75
6.51
80.4
33
5.63
60.7
64
25L
7.69
20.7
19
5.58
90.8
05
6.37
80.3
34
5.94
81.2
37
6.52
91.1
64
FL0.
148
0.0
97
0.06
60.0
84
0.12
40.0
35
0.11
00.0
72
0.07
80.0
86
25L
0.14
80.0
97
0.07
60.0
44
0.14
20.0
21
0.09
70.0
15
0.06
00.0
30
FL0.
488
0.1
30
0.41
50.0
10
0.40
60.1
55
0.48
70.0
28
0.49
70.0
56
25L
0.48
80.1
30
0.51
50.0
49
0.56
90.1
17
0.53
70.0
78
0.51
00.1
40
Tab
le S
2: Im
pac
t o
f d
efo
liat
ion
tre
atm
en
ts (
full
leaf
: FL;
25
leav
es:
25L
) o
n m
eas
ure
d p
rim
ary
leaf
me
tab
oli
te a
bu
nd
ance
du
rin
g th
e e
xpe
rim
en
tal p
eri
od
. Th
e G
C/M
S re
ten
tio
n t
ime
, maj
or
frag
me
nt
m/z
, re
ten
tio
n in
de
x, a
nd
th
e N
IST
lib
rary
co
rre
spo
nd
ing
com
po
un
d in
form
atio
n is
incl
ud
ed
.
GLY
CER
OL
12.9
147;
205
; 117
C00
116
Gly
cero
l, t
ris-
TMS
ALA
NIN
E8.
914
7C
0004
1 *
l-A
lan
ine
, N-(
trim
eth
ylsi
lyl)
-, t
rim
eth
ylsi
lyl e
ste
r
XYL
OSE
21.2
217;
307
C00
181
* D
-(+)
-Xyl
ose
, te
trak
is(t
rim
eth
ylsi
lyl)
eth
er,
me
thyl
oxi
me
(an
ti)
Am
ino
aci
ds:
1130
TREH
ALO
SE38
.536
1; 2
04; 1
91C
0108
3*
D-(
+)-T
reh
alo
se, o
ctak
is(t
rim
eth
ylsi
lyl)
eth
er
1674
2798
TAG
ATO
SE23
.421
7C
0079
5D
-(-)
-Tag
ato
se, p
en
taki
s(tr
ime
thyl
sily
l) e
the
r
SUC
RO
SE37
.236
1; 2
17C
0008
9*
Sucr
ose
, oct
akis
-O-(
trim
eth
ylsi
lyl)
-
RIB
OSE
21.8
103;
217
; 307
C00
121
*d
-Rib
ose
, 2,3
,4,5
-te
trak
is-O
-(tr
ime
thyl
sily
l)-,
O-m
eth
ylo
xim
e
RH
AM
NO
SE22
.611
7C
0050
7*
D-(
-)-R
ham
no
se, t
etr
akis
(tri
me
thyl
sily
l) e
the
r, m
eth
ylo
xim
e (
anti
)
RA
FFIN
OSE
48.4
361
C00
492
*R
affi
no
se, 1
1TM
S
MEL
IBIO
SE40
.120
4C
0540
2*
Me
lib
iose
, oct
akis
(tri
me
thyl
sily
l)-
MEL
EZIT
OSE
50.6
361
C08
243
*M
ele
zito
se, 1
1TM
S
MA
NN
OSE
25.9
204
C00
159
*D
-(+)
-Man
no
se, p
en
taki
s(tr
ime
thyl
sily
l) e
the
r, t
rim
eth
ylsi
lylo
xim
e (
iso
me
r 1)
MA
NN
ITO
L26
.531
9; 2
05C
0039
2*
Man
nit
ol,
(6T
MS)
-
MA
LTO
SE38
.620
4; 2
17; 3
62; 5
98C
0020
8*
Mal
tose
, oct
akis
(tri
me
thyl
sily
l) e
the
r, m
eth
ylo
xim
e (
iso
me
r 2)
scyl
lo-I
NO
SITO
L28
.231
8C
0615
3In
osi
tol,
1,2
,3,4
,5,6
-he
xaki
s-O
-(tr
ime
thyl
sily
l)-,
scy
llo
-
myo
-IN
OSI
TOL
29.3
147;
217
; 305
C00
137
*m
yo-I
no
sito
l (6T
MS)
GLU
CO
SE26
.0; 2
6.3
147;
319
C00
031
*D
-Glu
cose
, 2,3
,4,5
,6-p
en
taki
s-O
-(tr
ime
thyl
sily
l)-
GA
LAC
TOSE
25.8
319;
205
; 217
C00
124
*D
-Gal
acto
se, 2
,3,4
,5,6
-pe
nta
kis-
O-(
trim
eth
ylsi
lyl)
-
GA
LAC
TIN
OL
41.5
103;
305
; 361
C01
235
*G
alac
tin
ol,
no
nak
is(t
rim
eth
ylsi
lyl)
eth
er
FRU
CTO
SE25
.6; 2
5.8
147;
103
C10
906
*Fr
uct
ose
, (5T
MS)
*D
L-A
rab
ino
se, t
etr
akis
(tri
me
thyl
sily
l) e
the
r, t
rim
eth
ylsi
lylo
xim
e (
iso
me
r 2)
DU
LCIT
OL
(gal
acti
tol)
26.7
217;
147
C01
697
*D
ulc
ito
l, (
6TM
S)
CEL
LOB
IOSE
38.1
204;
217
; 361
C00
185
*
D-(
+)-C
ell
ob
iose
, oct
akis
(tri
me
thyl
sily
l) e
the
r (i
som
er
2)
V+9
V+1
8V
+27
V+3
7V
+46
Suga
rs (
incl
ud
ing
suga
r al
coh
ols
):
1939
1927
3066
1915
1980
2769
1800
1814
AR
AB
ITO
L22
.210
3; 2
17; 3
07C
0053
2
Ara
bit
ol (
5TM
S)
AR
AB
INO
SE21
.610
3; 1
47; 2
17C
0021
6
2060
2123
1792
2705
1706
1749
3647
2944
3834
1933
1968
2806
1292
Appendix A – Table S2
172
FL0.
018
0.0
36
0.07
50.0
80
0.06
10.0
57
0.05
60.0
52
0.08
10.0
14
25L
0.01
80.0
36
0.01
80.0
30
0.06
70.0
29
0.05
90.0
52
0.01
50.0
26
FL0.
000
0.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
25L
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
0.00
80.0
14
FL0.
630
0.1
98
0.43
30.2
19
0.43
00.1
21
0.22
50.0
85
0.22
00.0
22
25L
0.63
00.1
98
0.40
40.0
62
0.24
70.0
26
0.16
30.0
33
0.21
00.0
17
FL0.
037
0.0
26
0.04
10.0
37
0.10
20.0
24
0.09
20.0
26
0.10
30.0
14
25L
0.03
70.0
26
0.06
00.0
03
0.10
30.0
19
0.15
70.0
19
0.21
20.1
06
FL0.
596
0.1
66
0.52
50.0
90
0.74
10.1
47
0.78
30.1
07
0.57
90.1
78
25L
0.59
60.1
66
0.81
10.1
17
1.11
40.2
80
2.08
00.2
60
2.48
80.5
90
FL1.
292
0.2
21
0.75
60.2
06
1.18
10.2
44
0.85
50.2
16
0.90
00.0
60
25L
1.29
20.2
21
0.97
80.0
67
0.84
50.2
21
0.75
10.0
45
0.85
60.0
65
FL0.
603
0.0
67
0.53
70.0
65
0.52
30.0
25
0.60
80.1
25
0.71
80.1
08
25L
0.60
30.0
67
0.51
50.0
80
0.56
50.1
56
0.76
70.0
48
0.85
00.1
21
FL0.
000
0.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
25L
0.00
00.0
00
0.00
00.0
00
0.03
00.0
07
0.03
10.0
36
0.07
30.0
33
FL0.
000
0.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
25L
0.00
00.0
00
0.00
00.0
00
0.01
70.0
19
0.01
10.0
19
0.02
10.0
06
FL0.
000
0.0
00
0.00
00.0
00
0.26
60.1
10
0.00
00.0
00
0.00
00.0
00
25L
0.00
00.0
00
0.00
00.0
00
0.45
30.3
34
0.09
50.0
08
0.07
70.0
68
FL1.
736
0.1
96
1.23
00.3
13
1.55
10.1
57
1.39
40.2
21
1.14
60.2
17
25L
1.73
60.1
96
1.26
80.3
96
1.16
30.3
20
1.16
80.1
45
1.32
80.0
94
FL0.
280
0.1
83
0.12
30.1
19
0.06
30.0
24
0.00
50.0
09
0.08
30.0
21
25L
0.28
00.1
83
0.83
00.0
75
0.25
80.1
17
0.61
60.1
89
0.57
70.1
09
FL0.
438
0.0
57
0.15
10.0
27
0.17
30.0
02
0.13
50.0
72
0.15
10.0
09
25L
0.43
80.0
57
0.28
70.0
41
0.25
10.0
35
0.35
40.0
05
0.36
90.0
15
FL0.
048
0.0
96
0.17
10.1
78
0.35
10.1
27
0.24
20.2
04
0.23
60.1
36
25L
0.04
80.0
96
0.10
90.0
95
0.60
70.2
03
0.18
90.0
08
0.41
40.2
47
FL0.
405
0.0
59
0.28
10.1
49
0.29
30.0
62
0.24
30.0
17
0.18
70.0
33
25L
0.40
50.0
59
0.21
00.0
97
0.11
90.0
16
0.04
30.0
39
0.08
30.0
95
FL0.
000
0.0
00
0.00
00.0
00
0.10
30.0
40
0.00
00.0
00
0.00
00.0
00
25L
0.00
00.0
00
0.01
60.0
28
0.10
40.0
30
0.01
90.0
32
0.00
00.0
00
FL0.
042
0.0
84
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
25L
0.04
20.0
84
0.00
00.0
00
0.05
60.0
13
0.01
80.0
19
0.07
00.0
28
FL15
.380
1.7
02
13.4
622.1
25
11.5
742.6
21
13.8
183.2
03
15.3
921.5
79
25L
15.3
801.7
02
12.9
174.5
11
9.34
91.5
00
7.30
00.3
82
9.32
60.2
46
FL3.
711
1.5
32
4.74
00.7
41
4.50
61.1
69
4.65
30.9
09
3.95
32.3
47
25L
3.71
11.5
32
2.57
32.6
73
3.95
22.5
25
5.02
31.9
14
4.54
21.8
11
FL0.
010
0.0
20
0.02
80.0
24
0.02
50.0
22
0.03
70.0
13
0.03
30.0
28
25L
0.01
00.0
20
0.02
30.0
21
0.03
10.0
32
0.04
80.0
09
0.01
30.0
22
FL4.
508
1.5
84
5.12
30.4
95
3.53
61.0
04
3.32
50.5
19
2.60
31.1
83
25L
4.50
81.5
84
3.30
61.1
52
3.03
21.4
02
2.75
40.5
02
1.25
90.3
64
FL0.
220
0.1
52
0.37
50.0
56
0.21
80.1
88
0.23
00.0
39
0.09
10.1
57
25L
0.22
00.1
52
0.11
00.1
90
0.17
60.1
52
0.11
80.1
02
0.00
00.0
00
FL3.
780
0.5
86
2.19
40.3
99
1.53
90.1
90
1.31
40.2
22
0.94
30.2
84
25L
3.78
00.5
86
1.04
60.2
77
0.71
50.0
16
0.33
00.0
34
0.37
50.0
98
FL0.
313
0.2
23
0.24
10.1
05
0.24
10.2
09
0.26
90.1
56
0.36
40.2
52
25L
0.31
30.2
23
0.14
00.0
97
0.30
70.3
20
0.34
40.1
43
0.10
80.0
59
FL2.
624
0.4
60
2.00
10.4
06
1.65
00.9
31
2.32
60.4
37
2.59
90.5
01
25L
2.62
40.4
60
1.46
30.1
94
1.28
10.3
72
1.13
50.2
39
1.44
50.3
16
FL3.
678
0.8
42
2.43
50.4
79
3.07
50.5
76
3.48
40.6
54
2.68
10.6
09
25L
3.67
80.8
42
1.32
70.2
54
1.31
20.2
91
0.73
80.3
95
0.51
60.0
61
GLU
CO
NIC
AC
ID28
.029
2; 3
33C
0025
7G
luco
nic
aci
d, (
6TM
S)
GA
LLIC
AC
ID26
.728
1; 4
58C
0142
4G
alli
c ac
id, t
etr
aTM
S
FUM
AR
IC A
CID
14.4
245
C00
122
Fum
aric
aci
d (
2TM
S)
CIT
RIC
AC
ID24
.427
3C
0015
8C
itri
c ac
id, (
4TM
S)
P-C
OU
MA
RIC
AC
ID26
.121
9C
0081
1p
-Co
um
aric
aci
d (
TMS)
CA
FFEI
C A
CID
29.4
396
C01
197
Caf
feic
aci
d (
3TM
S)
4-H
YDR
OX
Y-B
ENZO
IC A
CID
20.4
267
C00
156
4-H
ydro
xyb
en
zoic
aci
d
(2TM
S)
BEN
ZOIC
AC
ID12
.017
9C
0018
0B
en
zoic
aci
d, T
MS
ASC
OR
BIC
AC
ID24
.814
7C
0007
2*
Asc
orb
ic a
cid
(4T
MS)
VA
LIN
E11
.514
4; 2
18C
0018
3*
Val
ine
, di-
TMS
TYR
OSI
NE
26.3
218;
280
C00
082
*Ty
rosi
ne
, (3T
MS)
5-h
ydro
xy-T
RYP
TOP
HA
N32
.129
0; 2
18; 1
46C
0101
7L-
5-H
ydro
xytr
ypto
ph
an, t
rim
eth
ylsi
lyl e
the
r, t
rim
eth
ylsi
lyl e
ste
r
TRYP
TOP
HA
N30
.829
1; 2
02C
0007
8*
Tryp
top
han
, N,N
,O-t
riTM
S
THR
EON
INE
15.5
218
C00
188
*Th
reo
nin
e (
N,O
,O-T
MS)
SER
INE
14.9
204;
218
C00
065
*Se
rin
e, (
3TM
S)
C01
879
Pyr
ogl
uta
mic
aci
d, (
N,O
-TM
S)
ISO
LEU
CIN
E13
.315
8C
0040
7*
Iso
leu
cin
e, d
i-TM
S
18.3
156
GLU
TAM
INE
23.3
156
C00
064
*L-
Glu
tam
ine
, N,N
2-b
is(t
rim
eth
ylsi
lyl)
-, t
rim
eth
ylsi
lyl e
ste
r
GLU
TAM
IC A
CID
20.4
246
C00
025
*G
luta
mic
aci
d (
3TM
S)
GA
BA
(γ-
Am
ino
bu
tyri
c ac
id)
18.5
174
C00
334
*G
AB
A 3
TMS
CYS
TEIN
E32
.014
7; 2
04C
0009
7*
Cys
tein
e, N
,S,O
-tri
s-TB
DM
S
ASP
AR
TIC
AC
ID18
.523
2; 1
47; 2
18C
0004
9 *
L-A
spar
tic
acid
, (3T
MS)
-
ASP
AR
AG
INE
21.6
116;
231
C00
152
*
Asp
arag
ine
, O,O
',N-t
ris(
trim
eth
ylsi
lyl)
-
AR
GIN
INE
; 20.
3; 2
4.2;
14
2C
0006
2
*A
rgin
ine
, (3T
MS)
1313
1787
1635
1542
2316
1542
1696
1836
1635
1397
1847
1944
2130
1635
1260
1870
1288
Mis
cell
ane
ou
s ac
ids:
PH
ENYL
ALA
NIN
E20
.421
8C
0007
9*
Ph
en
ylal
anin
e (
N,O
-TM
S)
LEU
CIN
E12
.815
8C
0012
3*
L-Le
uci
ne
, N-(
trim
eth
ylsi
lyl)
-, t
rim
eth
ylsi
lyl e
ste
r
PYR
OG
LUTA
MIC
AC
ID (
5-o
xo-P
roli
ne
)
1956
2323
2230
1433
1414
1532
2049
1980
1242
Appendix A – Table S2
173
FL2.
195
1.0
31
1.56
10.1
63
1.51
40.9
67
1.64
00.2
11
2.03
20.8
37
25L
2.19
51.0
31
1.60
41.0
43
1.03
50.4
51
0.61
60.1
89
1.50
00.4
07
FL1.
041
0.3
63
1.11
00.6
63
1.72
90.2
35
1.58
20.1
66
0.96
10.2
69
25L
1.04
10.3
63
0.55
20.1
74
0.82
00.2
23
0.38
90.2
21
0.29
90.0
88
FL0.
142
0.0
31
0.09
30.0
10
0.08
20.0
35
0.07
90.0
02
0.05
60.0
52
25L
0.14
20.0
31
0.10
30.0
54
0.09
30.0
07
0.09
20.0
13
0.15
90.1
93
FL0.
786
0.2
63
0.67
50.2
95
0.94
40.1
59
0.64
60.5
64
0.48
70.1
31
25L
0.78
60.2
63
0.42
20.2
64
0.61
10.3
27
0.80
50.4
01
0.50
20.0
41
FL0.
887
0.3
76
1.05
90.3
45
0.76
70.3
64
0.89
50.2
45
0.96
40.5
64
25L
0.88
70.3
76
0.44
50.1
98
0.89
60.8
16
1.13
20.4
76
0.81
30.1
27
FL25
.754
3.5
34
17.9
002.5
25
19.9
372.6
77
19.4
824.4
18
21.2
753.1
81
25L
25.7
543.5
34
13.9
112.4
05
20.2
236.8
70
19.9
092.0
61
18.0
674.0
27
FL1.
435
0.0
29
1.35
20.1
98
1.71
50.1
63
1.39
20.3
44
1.57
40.1
12
25L
1.43
50.0
29
1.65
60.0
24
1.82
20.2
26
1.95
30.2
68
2.15
20.3
47
FL14
.145
5.1
11
8.34
21.5
32
11.8
354.1
28
11.5
052.0
01
15.3
954.8
75
25L
14.1
455.1
11
14.6
023.6
37
14.7
782.5
72
12.7
191.8
37
10.7
064.5
30
FL0.
194
0.0
48
0.17
50.0
27
0.20
50.1
87
0.28
00.0
21
0.17
30.0
58
25L
0.19
40.0
48
0.15
50.1
48
0.36
10.1
65
0.16
00.1
66
0.33
30.0
77
FL0.
699
0.3
35
0.28
80.1
68
0.40
90.1
62
0.33
60.0
43
0.22
50.0
95
25L
0.69
90.3
35
0.13
80.0
53
0.19
90.0
47
0.16
90.0
38
0.19
60.1
37
FL0.
723
0.0
61
0.45
50.1
06
0.45
00.0
21
0.48
00.0
72
0.75
50.2
18
25L
0.72
30.0
61
0.39
80.0
20
0.55
20.4
97
0.46
20.0
54
0.44
40.0
50
FL14
7.05
714.4
11
159.
382
21.2
28
88.6
0323.3
41
66.9
337.9
55
40.6
9012.9
12
25L
147.
057
14.4
11
78.5
8546.1
04
36.6
1713.8
28
16.0
613.5
08
14.9
999.8
76
FL0.
912
0.6
87
0.45
70.2
77
0.65
80.2
53
0.87
60.2
23
0.81
60.2
60
25L
0.91
20.6
87
0.15
20.0
29
0.21
10.0
98
0.20
70.1
10
0.11
20.0
51
FL0.
025
0.0
18
0.01
20.0
11
0.02
10.0
25
0.03
20.0
24
0.01
90.0
02
25L
0.02
50.0
18
0.00
40.0
08
0.03
50.0
13
0.03
10.0
26
0.04
00.0
10
FL0.
216
0.0
41
0.22
60.0
85
0.38
00.2
41
0.22
70.0
66
0.22
60.0
98
25L
0.21
60.0
41
0.22
10.0
90
0.31
20.1
67
0.12
30.0
24
0.10
90.0
12
FL0.
032
0.0
42
0.03
40.0
31
0.02
80.0
25
0.05
40.0
22
0.06
30.0
22
25L
0.03
20.0
42
0.00
90.0
16
0.03
40.0
30
0.04
10.0
08
0.01
20.0
20
FL0.
006
0.0
12
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
25L
0.00
60.0
12
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
FL0.
689
0.1
27
0.65
50.1
67
0.64
60.3
34
0.80
70.2
31
0.64
00.2
92
25L
0.68
90.1
27
0.52
50.3
14
0.86
10.5
48
0.68
00.2
10
0.80
40.3
06
FL0.
993
0.4
68
0.99
40.2
06
0.98
50.4
61
1.22
30.4
30
0.89
20.4
28
25L
0.99
30.4
68
0.73
90.5
71
1.16
10.8
13
1.08
10.4
00
1.36
40.7
17
FL0.
238
0.0
95
0.37
40.0
72
0.47
30.1
92
0.64
70.2
59
0.65
90.1
28
25L
0.23
80.0
95
0.45
70.1
87
0.77
90.3
78
1.99
00.8
93
3.96
21.6
58
FL6.
388
1.4
00
5.61
51.1
68
4.59
31.4
56
4.62
70.4
70
5.02
30.9
94
25L
6.38
81.4
00
5.35
40.5
67
5.20
60.7
52
4.52
30.9
81
7.93
70.9
93
FL6.
281
2.2
88
5.35
20.7
01
5.89
81.6
65
6.13
41.1
85
5.00
30.4
42
25L
6.28
12.2
88
5.15
00.5
31
5.60
90.6
72
5.75
21.2
30
9.31
71.9
29
FL0.
063
0.0
47
0.06
10.0
61
0.09
50.0
17
0.10
40.0
46
0.03
10.0
44
25L
0.06
30.0
47
0.06
60.0
13
0.07
50.0
46
0.07
10.0
21
0.07
70.0
33
FL0.
250
0.0
90
0.27
90.1
03
0.24
10.0
54
0.20
50.0
19
0.19
60.0
58
25L
0.25
00.0
90
0.28
30.0
85
0.24
50.0
38
0.22
90.0
93
0.22
90.1
55
FL13
.704
0.6
89
15.0
932.8
89
14.3
424.1
56
12.6
621.8
80
13.9
601.4
76
25L
13.7
040.6
89
15.0
603.4
60
13.6
453.1
22
15.8
351.5
69
16.1
543.4
44
FL0.
101
0.1
67
0.02
50.0
13
0.14
20.2
19
0.04
90.0
41
0.15
30.2
35
25L
0.10
10.1
67
0.17
20.2
59
0.22
60.3
63
0.02
10.0
14
0.41
00.3
97
Ad
ipic
aci
d18
147;
275
C06
104
*A
dip
ic a
cid
, (2T
MS)
1528
Ad
on
ito
l (R
ibit
ol)
22.8
147;
217
; 103
C00474
*R
ibit
ol,
5TM
S17
60
L-h
ydro
xyp
roli
ne
18.5
230
C01
157
*3-
Hyd
roxy
pro
lin
e, N
,O,O
'-tr
is(t
rim
eth
ylsi
lyl)
-15
42
QU
ERC
ETIN
57.3
575
C00
389
Qu
erc
eti
n, (
5TM
S)
Inte
rnal
sta
nd
ard
s:
1-M
ON
OP
ALM
ITIN
35.9
371
N/A
1-M
on
op
alm
itin
tri
me
thyl
sily
l eth
er
KA
EMP
FER
OL
42.0
559
C05
903
Kae
mp
fero
l, 4
TMS
HYD
RO
XYL
AM
INE
9.0
133
C00
192
Hyd
roxy
lam
ine
, N,N
,O-t
ris-
TMS
GLY
CER
OL
MO
NO
STEA
RA
TE38
.439
9N
/AG
lyce
rol m
on
ost
ear
ate
, 2tm
s d
eri
vati
ve
CA
TEC
HIN
39.5
; 39.
836
8C
0656
2C
ate
chin
e, p
en
ta-T
MS-
eth
er,
(2R
-cis
)-
AR
BU
TIN
36.2
254
C06
186
Hyd
roq
uin
on
e-β
-d-g
luco
pyr
ano
sid
e,p
en
taki
s(tr
ime
thyl
sily
l)-
STEA
RIC
AC
ID30
.911
7; 3
41C
0153
0St
ear
ic a
cid
, tri
me
thyl
sily
l est
er
OLE
IC A
CID
30.5
339
C00
712
Ole
ic a
cid
, tri
me
thyl
sily
l est
er
2039
2209
1405
NO
NA
NO
IC A
CID
14.6
117;
215
C01
601
EIC
OSA
NO
IC A
CID
40.6
427
C06
425
Eico
san
oic
aci
d, 2
,3-b
is[(
trim
eth
ylsi
lyl)
oxy
]pro
pyl
est
er
THR
EON
IC A
CID
19.4
147;
292
; 220
C01
620
L-Th
reo
nic
aci
d, t
ris(
trim
eth
ylsi
lyl)
eth
er,
tri
me
thyl
sily
l est
er
Fatt
y ac
ids:
1586
OX
ALI
C A
CID
9.8
147;
133
C00
209
Oxa
lic
acid
(2T
MS)
MA
LIC
AC
ID17
.823
3C
0014
9M
alic
aci
d (
3TM
S)
MA
LEIC
AC
ID13
.514
7; 2
45 C
0138
4M
ale
ic a
cid
, (2T
MS)
LAC
TIC
AC
ID8.
114
7; 1
17C
0018
6La
ctic
aci
d, (
2TM
S)
3-H
YDR
OX
YAN
THR
AN
ILIC
AC
ID26
.935
4C
0063
23-
Hyd
roxy
anth
ran
illi
c ac
id, (
3TM
S)
GLY
CER
IC A
CID
14.2
147;
189
; 292
C00
258
Gly
ceri
c ac
id, (
3TM
S)
2-K
ETO
-GLU
TAR
IC A
CID
25.3
157;
173
C00
026
α-K
eto
glu
tari
c ac
id, t
rim
eth
ylsi
lyl e
ste
r
1175
1508
1328
1088
1991
1382
1898
2407
2990
4402
2602
3108
1135
2791
2889
2626
Mis
call
en
eo
us
com
po
un
ds:
2237
No
nan
oic
aci
d, T
MS
est
er
MYR
ISTI
C A
CID
33.3
343
C06
424
Myr
isti
c ac
id, 2
,3-b
is(t
rim
eth
ylsi
loxy
)pro
pyl
est
er
PA
LMIT
IC A
CID
27.8
117;
313
C00
249
Pal
mit
ic a
cid
, TM
S
RIB
ON
IC A
CID
23.7
292;
217
C01
685
Tart
aric
aci
d, T
MS
SUC
CIN
IC A
CID
13.7
147;
247
C00
042
Succ
inic
aci
d (
2TM
S)
TAR
TAR
IC A
CID
21.0
292
C00
898
Rib
on
ic a
cid
, pe
nta
kis-
TMS
PR
OTO
CA
TEC
HU
IC A
CID
24.1
193;
370
C00
230
*P
roto
cate
chu
ic a
cid
(tm
s)
1666
1344
1808
PH
OSP
HO
RIC
AC
IDP
ho
sph
ori
c ac
id, t
riTM
S12
92C
0000
929
912
.9
1831
Appendix A – Figure S1
174
Roots PCA Analysis (using only data where treatment was significant at 5% for at least one time)
Principal component analysis of root primary metabolites for the three defoliation treatments (full leaf –
FL, 25% leaves – 25L and no leaf – NL), at each destructive harvest after the implementation of the
treatments (V+18, V+27, V+37 and V+46). A minimum convex polygon or convex hull (red
polygon) is included to define the treatment score space.
Appendix A – Figure S1
175
1. Input: Analysis based on the Correlation Matrix.
2. Eigenvalues or latent Roots (non-zero to 3 decimal places):
Eigenvalues 12 to 26 not included
1 2 3 4 5 6 7 8 9 10 11
Eigenvalue 9.819 4.688 3.638 2.855 1.711 1.092 0.752 0.602 0.449 0.275 0.120
3. Percentage Variation accounted for
1 2 3 4 5 6 7 8 9 10 11
%Variation 37.76 18.03 13.99 10.98 6.58 4.20 2.89 2.31 1.73 1.06 0.46
Appendix A – Figure S1
176 4.Ei
genv
ecto
rs o
r Lo
adin
gs
Clas
sifica
tion:
M
etab
olite
: 1
2 3
4 5
6 7
8 9
10
11
Suga
rs
Sucr
ose
-0.0
906
-0.0
006
0.40
03
-0.1
044
0.19
63
0.09
43
-0.4
566
-0.3
248
0.10
98
0.03
74
0.17
13
Raffi
nose
-0
.254
7 0.
1348
0.
0959
0.
1119
0.
1345
0.
0956
-0
.161
9 0.
4835
0.
0551
0.
0893
0.
1760
Mel
ibio
se
0.26
98
0.11
27
0.01
06
0.11
43
-0.0
581
0.22
55
0.11
48
0.00
99
-0.2
365
0.50
63
-0.4
199
Arab
inos
e 0.
2123
0.
0927
0.
0911
-0
.090
7 -0
.465
6 0.
0703
-0
.112
8 0.
3416
-0
.093
3 -0
.111
7 0.
0583
Suga
r alco
hols
Myo
-inos
itol
-0.2
894
0.02
45
0.13
12
-0.0
140
-0.1
263
-0.0
784
0.10
05
-0.0
866
0.32
65
0.09
15
-0.3
612
Gala
ctin
ol
-0.2
442
0.20
22
0.02
20
0.17
09
0.02
57
0.08
20
0.07
83
0.14
87
0.46
81
-0.1
447
-0.2
462
Man
nito
l -0
.135
1 0.
2254
-0
.012
7 0.
1397
-0
.391
4 0.
1691
0.
4981
-0
.209
5 0.
0605
-0
.195
3 0.
2573
Arab
itol
0.08
53
-0.3
150
-0.1
519
-0.2
798
-0.1
770
-0.0
503
-0.0
183
0.33
15
0.22
03
0.20
18
0.04
87
Glyc
erol
-0
.113
3 -0
.232
1 0.
2153
-0
.332
6 -0
.063
8 -0
.130
4 0.
3208
0.
2318
0.
0161
0.
0745
0.
0319
Amin
o ac
ids
Glut
amic
_aci
d -0
.241
1 0.
2495
-0
.006
8 0.
0470
0.
1922
-0
.240
9 0.
0486
-0
.036
8 0.
0285
0.
0297
-0
.108
5
Argi
nine
0.
2659
0.
1787
0.
0577
-0
.064
6 0.
1530
-0
.270
0 0.
0468
0.
0571
0.
0838
-0
.142
2 -0
.056
2
Glut
amin
e 0.
0542
-0
.048
4 0.
4780
0.
1168
-0
.166
8 0.
0688
0.
1363
-0
.103
6 -0
.184
9 -0
.061
4 -0
.045
0
Tryp
toph
an
0.26
73
0.03
50
0.23
95
0.14
30
-0.0
264
0.08
12
-0.0
952
0.05
61
0.03
04
-0.1
704
0.00
35
Phen
ylal
anin
e 0.
2391
-0
.195
7 0.
0889
0.
1367
0.
1104
0.
2493
0.
0184
0.
1998
0.
3570
-0
.068
9 0.
2120
Tyro
sine
0.18
29
-0.1
711
0.25
95
0.25
76
0.13
81
0.14
46
0.11
96
0.11
46
-0.0
314
-0.1
927
-0.3
546
Glyc
ine
-0.0
695
-0.3
207
-0.1
492
0.29
13
0.13
30
-0.1
846
0.30
47
-0.0
054
-0.1
370
0.05
85
0.06
65
Lysin
e 0.
2554
0.
1815
0.
0576
-0
.066
9 0.
1517
-0
.322
2 0.
1065
0.
0791
0.
0896
-0
.180
2 -0
.157
9
Thre
onin
e 0.
1308
-0
.193
2 0.
0785
0.
3990
-0
.171
4 -0
.268
6 -0
.188
8 -0
.168
5 0.
0347
0.
1385
0.
1123
Valin
e 0.
0287
-0
.380
3 -0
.029
3 0.
2705
0.
1769
-0
.087
0 0.
0942
-0
.012
5 0.
2565
0.
1163
0.
0023
Misc
ella
neou
s acid
s
Asco
rbic
_acid
-0
.209
7 0.
1311
0.
2922
0.
0044
0.
1994
0.
0704
0.
0825
0.
2487
-0
.223
5 0.
3751
-0
.005
8
Tart
aric
_acid
0.
2458
0.
2022
-0
.010
2 -0
.083
1 0.
2007
-0
.183
6 0.
2450
-0
.116
9 0.
0247
0.
2888
0.
3292
Citr
ic_ac
id
0.25
80
0.18
11
0.10
61
-0.1
141
0.09
49
-0.2
797
0.05
87
0.10
18
0.06
73
-0.0
954
-0.0
401
Mal
eic_
acid
0.
2183
-0
.008
0 -0
.046
0 -0
.316
0 -0
.010
0 0.
2957
0.
0619
-0
.309
7 0.
3747
0.
2472
-0
.156
4
3 Hy
drox
yant
hran
ilic a
cid
-0.0
592
-0.1
704
0.36
02
-0.2
502
0.21
57
0.13
17
0.29
87
-0.0
871
-0.0
315
-0.1
132
0.19
56
Prot
ocat
echu
ic_a
cid
-0.1
242
-0.3
304
-0.0
365
-0.2
965
0.02
95
-0.0
986
-0.0
728
-0.0
867
-0.2
399
-0.2
881
-0.3
113
2-Ke
to G
luco
nic
acid
-0
.089
6 -0
.054
5 0.
3283
0.
0021
-0
.400
0 -0
.436
9 -0
.087
7 -0
.060
4 0.
1516
0.
2316
-0
.008
6
Appendix A – Figure S1
177
5. Scores
Time_Treatment 1 2 3 4 5 6 7 8 9 10 11
T1_NL -1.5765 -3.2855 -0.6909 2.9007 0.0141 -1.9999 -0.0482 -0.0671 -0.3186 0.308 -0.1563
T2_NL 1.5895 -4.5492 0.0581 -0.596 1.1963 1.1299 1.0445 0.4201 0.5977 -0.3814 0.1591
T3_NL 4.4383 1.1417 3.1937 2.1682 -1.1573 0.3971 0.1549 0.0337 -0.6242 -0.6453 -0.0239
T4_NL 3.4055 -0.5346 0.4077 0.2756 -0.2211 0.6286 -1.4933 -0.7066 0.6207 0.8464 0.3655
T1_25L -0.2412 -0.6291 -0.6867 -1.9752 -3.0871 -0.1197 -0.0054 1.2281 0.0618 0.2578 -0.1359
T2_25L -0.5221 -0.8472 -2.7482 -1.4665 0.0055 0.4973 -0.7676 -0.8286 -1.2867 -0.5589 0.1093
T3_25L 3.7753 1.4292 -0.7637 -0.8197 1.7096 0.0926 -0.0049 0.243 -0.117 0.3794 -0.8061
T4_25L 2.7684 2.4369 -0.8056 -1.3935 0.6289 -2.0584 0.5956 0.0225 0.3906 -0.2949 0.4814
T1_FL -3.1123 1.0608 -0.5584 0.4658 -0.9542 0.175 0.0367 -1.1674 1.2463 -0.5847 -0.3735
T2_FL -2.1867 2.2389 -1.3433 1.3851 -0.1576 1.1241 1.6085 -0.2292 -0.3933 0.7152 0.2327
T3_FL -3.8363 1.7876 -0.2734 1.3461 1.2999 0.4971 -1.1781 1.4512 0.1938 -0.3241 0.1625
T4_FL -4.5019 -0.2496 4.2106 -2.2905 0.723 -0.3637 0.0574 -0.3998 -0.371 0.2825 -0.0148
Appendix A – Figure S2
178
Leaves PCA Analysis (using only data where treatment was significant at 5% for at least one time)
Principal component analyses of leaf primary metabolites for the three defoliation treatments (full leaf – FL, 25% leaves – 25L and no leaf – NL), at each destructive harvest after the implementation of thetreatments (V+18, V+27, V+37 and V+46). A minimum convex polygon or convex hull (red polygon)is included to define the treatment score space.
Appendix A – Figure S2
179
1. Input: Analysis based on the Correlation Matrix.
2. Eigenvalues or latent Roots (non-zero to 3 decimal places): 3. Eigenvalues or latent Roots (non-zero to 3 decimal places):
Eigenvalues 8 to 37 not included
1 2 3 4 5 6 7
Eigenvalue 19.558 5.339 4.557 3.593 2.027 1.290 0.635
4. Percentage Variation accounted for
1 2 3 4 5 6 7
%Variation 52.86 14.43 12.32 9.71 5.48 3.49 1.72
Appendix A – Figure S2
180
5. Eigenvectors or Loadings
Classification Metabolite 1 2 3 4 5 6 7 8
Sugars
Glucose 0.05381 0.33079 -0.01594 -0.19866 0.2327 0.10283 -0.38217 0.00219
Raffinose 0.14445 0.31981 -0.00858 0.02357 -0.01447 -0.10421 0.21392 0.07497
Melibiose 0.21706 0.00865 0.10766 0.04541 -0.05201 -0.09406 -0.03922 0.32557
Rhamnose 0.20081 0.15728 -0.04225 0.12726 0.03481 0.03101 0.12026 0.225
Melezitose 0.18479 0.21014 -0.05118 0.07387 0.01329 -0.08501 0.29454 -0.16781
Ribose -0.1763 0.24392 0.02686 -0.08148 0.04943 0.14339 0.15772 -0.00666
Sugar alcohols
Mannitol -0.16594 0.26431 0.07983 -0.08949 0.09189 0.03578 -0.13668 0.01946
Myo-inositol -0.20782 -0.02238 -0.14147 0.11935 -0.02417 0.05843 0.08528 -0.03326
Amino acids
Arginine -0.14035 -0.02498 0.28823 -0.06419 0.08223 0.28606 0.39431 -0.12982
GABA 0.21236 0.05229 0.02996 0.00068 0.22011 -0.02834 -0.01031 -0.05467
Serine 0.15167 -0.14713 -0.2695 -0.15336 0.05333 -0.08247 -0.0839 0.07941
Cysteine 0.19223 0.15413 0.12181 -0.00158 0.11636 -0.18285 0.13889 -0.00422
Valine 0.19677 0.01756 0.11437 0.11965 -0.18852 0.21141 -0.02349 -0.28563
Leucine 0.20738 -0.01456 0.13461 0.06387 -0.06596 0.20018 -0.00815 -0.24495
Isoleucine 0.21527 0.08121 0.04996 0.08633 0.01458 0.11818 0.05443 -0.01778
Phenylalanine 0.05423 -0.23828 0.29301 0.17414 -0.25373 -0.05964 0.07974 0.11112
Tryptophan 0.09496 -0.04163 0.30051 0.20041 -0.35261 0.07059 0.00999 0.0784
5-Hydroxytryptophan -0.19364 0.02586 -0.10521 0.22118 -0.08687 -0.12867 -0.01621 0.09981
Threonine 0.21026 -0.0946 -0.09164 -0.07118 0.11621 -0.05281 0.01478 -0.00915
Miscellaneous acids
Ascorbic_acid -0.17796 0.18059 -0.13327 -0.0621 -0.23382 0.02032 0.02313 0.40895
Tartaric_acid -0.16398 -0.0643 -0.18205 0.24326 0.07487 0.23911 0.07188 -0.03773
Threonic_acid -0.17535 0.21123 0.15868 -0.02677 0.00161 -0.1807 -0.03965 -0.04779
Glyceric_acid -0.17847 0.04999 0.15241 0.21036 -0.01914 -0.24949 -0.16762 -0.44368
Caffeic_acid -0.17726 -0.16657 -0.07047 0.13546 0.14613 0.28517 -0.03017 0.07324
Gallic_acid -0.15784 0.28989 0.01702 -0.0878 -0.11277 0.07493 0.05566 -0.16274
Lactic_acid -0.04948 -0.12734 0.23024 0.21894 0.40448 -0.27656 0.18375 0.08809
Citric_acid -0.18736 -0.0085 -0.10568 0.24807 0.04173 0.16515 0.05987 0.04003
Fumaric_acid -0.08571 -0.04889 0.33218 -0.26462 0.11965 0.13496 0.24001 0.26634
2-Ketoglutaric acid -0.11325 0.26538 -0.16072 0.01885 -0.33159 -0.09349 0.17722 -0.06427
Phosphoric_acid 0.01702 -0.1137 0.08621 -0.36855 -0.39039 -0.26848 0.04802 -0.05084
Gluconic_acid -0.20098 0.12582 0.09876 0.08837 -0.03167 -0.1723 -0.14101 0.1549
Ribonic_acid -0.13394 0.09879 0.15856 0.31908 0.10198 -0.2721 0.00143 0.12845
Nonanoic_acid -0.11691 0.14679 0.28151 -0.24567 0.09127 0.13187 0.02173 -0.04756
Palmitic_acid 0.08669 0.10435 0.31532 0.15049 -0.10445 0.26767 -0.4792 0.19256
Other compounds
Arbutin 0.20182 0.169 -0.01144 0.05224 0.13144 0.00489 0.09239 0.11068
Catechin 0.15011 0.1835 -0.17142 0.18226 -0.15247 0.21928 0.16397 0.13006
Glycerol monostearate 0.16757 0.21801 -0.01884 0.22606 0.02691 -0.06087 -0.0933 -0.12568
Appendix A – Figure S2
181
6. Scores
Time_Treatment 1 2 3 4 5 6 7
T1_25L -0.1092 -2.1327 -4.1121 -1.2603 -0.9652 -0.6701 -0.4453
T2_25L 2.2436 -2.8131 2.7636 0.3146 -1.926 0.9644 -0.2882
T3_25L 3.5286 -1.7166 1.0721 -2.0486 2.659 -0.1031 0.1135
T4_25L 8.3158 2.9697 -0.9956 1.5239 -0.2611 0.0392 0.1574
T1_FL -4.4244 -0.2239 -1.5675 1.7802 1.0028 1.9414 0.418
T2_FL -2.868 -1.0011 1.0163 2.4751 0.1714 -1.9522 0.6254
T3_FL -3.5698 2.4148 1.0846 -0.0186 0.4157 -0.2368 -1.5929
T4_FL -3.1167 2.5029 0.7387 -2.7662 -1.0965 0.0172 1.0121
Appendix A – Figure S3
182
Figure S3: Linear and curvilinear relationship between root starch and myo-inositol concentrations as
estimated by the linear mixed model. While the model indicates that the spline curvature is not
statistically significant, when a linear trend is fitted, the residuals do not appear to be random with fitted
values in the range 0.884 to 1.587 consistently smaller than observed values, and fitted values in the
range 3.076 to 4.211 consistently larger than observed values. NL: no leaf treatment, 25L: 25 leaves
treatment, FL: full leaf treatment, V+9: 9 days after the onset of véraison.
Appendix B – Supplementary table S1
183
Day
s af
ter
vera
iso
n
Me
tab
oli
te a
bu
nd
ance
pe
r b
err
yTr
eat
me
nt
valu
eSD
EVva
lue
SDEV
valu
eSD
EVva
lue
SDEV
valu
eSD
EVR
T (m
in)
m/z
Ke
gg ID
stan
dar
d id
en
tifi
cati
on
NIS
T li
bra
ry c
om
po
un
dR
ete
nti
on
ind
ex
FL0.
313
0.0
54
0.5
55
0.08
00.6
41
0.08
10.
850
0.16
60.
982
0.1
69
25L
0.31
30.0
54
0.42
00.
064
0.45
70.
062
0.52
40.
013
0.65
50.0
80
NL
0.31
30.
054
0.40
30.
058
0.39
50.
075
0.51
70.
035
0.53
40.144
FL0.
336
0.0
49
0.6
23
0.11
80.
599
0.1
79
0.8
34
0.39
80.
922
0.2
34
25L
0.33
60.0
49
0.42
90.
032
0.32
70.2
88
0.51
90.
107
0.67
50.1
42
NL
0.33
60.0
49
0.42
60.
075
0.46
10.1
24
0.52
10.
031
0.56
20.1
06
FL0.
008
0.00
90.0
17
0.01
30.
005
0.00
50.0
23
0.02
80.
018
0.030
25L
0.00
80.0
09
0.00
90.
012
0.01
50.0
10
0.03
30.
030
0.02
50.0
40
NL
0.00
80.
009
0.02
50.
038
0.03
10.
022
0.01
80.
019
0.05
10.024
FL0.
146
0.02
60.
346
0.05
90.
473
0.20
40.
843
0.27
10.
977
0.4
17
25L
0.14
60.
026
0.10
20.
016
0.14
50.
036
0.29
60.
091
0.26
50.1
30
NL
0.14
60.
026
0.12
70.
064
0.17
10.
010
0.19
90.
010
0.30
70.1
24
FL0.
154
0.1
48
0.1
99
0.22
70.
274
0.1
00
0.7
27
0.48
40.
931
0.0
28
25L
0.15
40.1
48
0.23
60.
093
0.53
30.0
62
0.56
20.
066
0.56
20.0
95
NL
0.15
40.1
48
0.23
90.
129
0.39
30.0
58
0.37
40.
124
0.50
60.0
61
FL53
.542
11.2
0613
2.03
413
.685
145.
049
31.8
2222
1.27
829
.647
178.
004
61.664
25L
53.5
4211.2
06
86.0
5122
.836
116.
024
40.0
04
146.
025
4.75
317
2.44
524.7
53
NL
53.5
4211
.206
100.
544
26.8
0211
2.70
324
.937
99.1
6612
.697
144.
098
42.146
FL1.
808
0.20
62.
614
0.10
62.
938
0.44
53.
770
0.53
33.
971
0.900
25L
1.80
80.2
06
1.93
60.
176
2.28
50.0
77
2.78
80.
132
2.70
60.8
84
NL
1.80
80.
206
2.28
20.
423
2.14
50.
242
2.01
20.
142
2.52
40.109
FL0.
067
0.01
90.
202
0.12
30.
425
0.03
60.
052
0.09
00.
000
0.0
00
25L
0.06
70.
019
0.10
70.
064
2.41
60.
396
1.84
32.
555
0.03
60.0
32
NL
0.06
70.
019
0.01
80.
031
3.20
50.
625
0.30
30.
084
0.17
40.0
46
FL0.
156
0.12
40.1
21
0.07
70.
435
0.52
81.7
76
1.62
50.
349
0.227
25L
0.15
60.1
24
0.63
90.
516
0.73
80.9
90
0.28
60.
272
0.25
10.1
78
NL
0.15
60.
124
0.08
90.
023
0.15
50.
043
0.13
40.
128
0.21
00.1
66
FL64
.326
17.1
11104.6
42
8.28
711
5.79
424
.842
171.1
29
26.1
7117
3.05
864.312
25L
64.3
2617.1
11
79.2
4469
.393
93.4
1218.5
77
119.
212
14.0
5413
9.61
428.3
07
NL
64.3
2617
.111
106.
039
13.5
3310
0.21
05.
005
104.
763
20.7
1112
7.23
422.407
FL0.
909
0.4
65
1.1
69
0.76
40.
414
0.1
72
1.2
57
1.00
31.
207
1.2
46
25L
0.90
90.4
65
1.06
90.
840
2.27
31.3
79
0.65
20.
107
0.74
70.9
18
NL
0.90
90.4
65
0.48
90.
564
0.34
20.1
50
1.05
70.
096
3.94
96.3
76
FL0.
105
0.0
22
0.0
00
0.00
00.0
00
0.00
00.
097
0.16
90.
119
0.1
04
25L
0.10
50.0
22
0.07
30.
020
0.03
30.
057
0.11
30.
100
0.09
80.0
85
NL
0.10
50.0
22
0.08
90.
015
0.02
50.0
43
0.09
40.
015
0.07
70.0
69
FL9.
726
3.5
52
13.9
25
1.24
515
.321
2.3
74
17.0
94
4.58
221
.456
9.5
90
25L
9.72
63.5
52
9.88
02.
659
8.97
30.7
34
13.3
851.
289
11.6
570.4
13
NL
9.72
63.5
52
11.9
473.
559
7.95
50.9
51
6.56
81.
540
7.65
92.5
63
FL0.
142
0.03
30.1
96
0.06
10.
184
0.06
70.2
45
0.06
00.
277
0.100
25L
0.14
20.0
33
0.17
40.
029
0.21
20.0
06
0.19
70.
047
0.22
40.0
26
NL
0.14
20.0
33
0.20
80.
054
0.21
30.0
38
0.18
70.
017
0.24
10.0
09
FL0.
410
0.21
90.5
09
0.15
60.
641
0.29
40.7
91
0.15
70.
486
0.269
25L
0.41
00.2
19
0.46
30.
241
0.46
30.0
99
0.54
70.
058
0.57
50.1
62
NL
0.41
00.
219
0.35
20.
162
0.38
40.
102
0.58
20.
070
0.38
10.019
FL1.
265
0.4
07
2.8
32
0.24
33.
477
1.0
55
4.8
65
0.98
46.
005
2.1
60
25L
1.26
50.4
07
1.30
00.
044
2.58
40.6
37
2.62
80.
613
2.85
50.3
43
NL
1.26
50.
407
2.17
40.
618
1.79
70.
108
1.75
40.
216
2.63
00.413
FL0.
024
0.02
00.
023
0.00
70.
020
0.01
80.
000
0.00
00.
000
0.0
00
25L
0.02
40.
020
0.00
90.
008
0.00
00.
000
0.00
60.
010
0.01
00.0
17
NL
0.02
40.
020
0.02
30.
023
0.00
00.
000
0.00
00.
000
0.00
00.0
00
FL0.
003
0.00
20.
001
0.00
20.
021
0.00
50.
008
0.00
30.
000
0.0
00
25L
0.00
30.
002
0.00
00.
000
0.00
90.
003
0.00
60.
002
0.00
20.0
02
NL
0.00
30.
002
0.00
00.
000
0.01
70.
005
0.00
00.
000
0.00
00.0
00
FL0.
068
0.02
50.0
23
0.04
00.
046
0.07
90.0
00
0.00
00.
000
0.000
25L
0.06
80.0
25
0.08
70.
012
0.03
50.0
61
0.05
60.
096
0.05
20.0
90
NL
0.06
80.
025
0.12
50.
036
0.08
50.
076
0.08
20.
071
0.09
50.083
FL0.
086
0.0
16
0.1
03
0.09
00.
048
0.0
84
0.2
12
0.06
40.
118
0.1
05
25L
0.08
60.0
16
0.10
90.
038
0.11
60.0
08
0.1
71
0.01
10.
161
0.0
22
NL
0.08
60.0
16
0.10
70.
047
0.10
90.0
42
0.07
30.
063
0.13
80.0
15
DU
LCIT
OL
(gal
acti
tol)
26.7
217;
147
C01
697
*D
ulc
ito
l, (
6TM
S)19
80
3647
RH
AM
NO
SE22
.611
7
GLY
CER
OL
12.9
147;
205
; 117
C00
116
Gly
cero
l, t
ris-
TMS
1292
C00
507
*D
-(-)
-Rh
amn
ose
, te
trak
is(t
rim
eth
ylsi
lyl)
eth
er,
me
thyl
oxi
me
(an
ti)
1749
RA
FFIN
OSE
48.4
361
C00
492
*R
affi
no
se, 1
1TM
S
MEL
IBIO
SE40
.120
4C
0540
2*
Me
lib
iose
, oct
akis
(tri
me
thyl
sily
l)-
2944
RIB
OSE
21.8
103;
217
; 307
C00
121
*d
-Rib
ose
, 2,3
,4,5
-te
trak
is-O
-(tr
ime
thyl
sily
l)-,
O-m
eth
ylo
xim
e17
06
1968
3α-M
AN
NO
BIO
SE26
.921
7; 3
61C
0172
83-
α-M
ann
ob
iose
, oct
akis
(tri
me
thyl
sily
l) e
the
r (i
som
er
2)19
92
MA
NN
ITO
L26
.531
9; 2
05C
0039
2*
Man
nit
ol,
(6T
MS)
-
1927
GLU
CO
SE26
.0; 2
6.3
a14
7; 3
19C
0003
1*
D-G
luco
se, 2
,3,4
,5,6
-pe
nta
kis-
O-(
trim
eth
ylsi
lyl)
-19
39
GA
LAC
TOSE
25.8
319;
205
; 217
C00
124
*D
-Gal
acto
se, 2
,3,4
,5,6
-pe
nta
kis-
O-(
trim
eth
ylsi
lyl)
-
1915
GA
LAC
TIN
OL
41.5
103;
305
; 361
C01
235
*G
alac
tin
ol,
no
nak
is(t
rim
eth
ylsi
lyl)
eth
er
3066
FRU
CTO
SE25
.6; 2
5.8
a14
7; 1
03C
1090
6*
Fru
cto
se, (
5TM
S)
FUC
OSE
22.8
118;
277
C01
019
*L-
(-)-
Fuco
se, t
etr
akis
(tri
me
thyl
sily
l) e
the
r, m
eth
ylo
xim
e (
syn
)17
60
1800
CEL
LOB
IOSE
38.1
204;
217
; 361
C00
185
*
D-(
+)-C
ell
ob
iose
, oct
akis
(tri
me
thyl
sily
l) e
the
r (i
som
er
2)27
69
AR
AB
ITO
L22
.210
3; 2
17; 3
07C
0053
2
Ara
bit
ol (
5TM
S)
Suga
rs a
nd
su
gar
alco
ho
ls:
AR
AB
INO
SE21
.610
3; 1
47; 2
17C
0021
6
*D
L-A
rab
ino
se, t
etr
akis
(tri
me
thyl
sily
l) e
the
r, t
rim
eth
ylsi
lylo
xim
e (
iso
me
r 2)
1814
V+9
V+1
8V
+27
V+3
7V
+46
AR
AB
INO
FUR
AN
OSE
20.5
191;
217
C06
115
Ara
bin
ofu
ran
ose
, 1,2
,3,5
-te
trak
is-O
-(tr
ime
thyl
sily
l)-
1640
Sup
ple
me
nta
ry T
able
S1:
Imp
act
of
de
foli
atio
n t
reat
me
nts
(fu
ll le
af: F
L, 2
5le
ave
s: 2
5Lan
d n
o le
af: N
L) o
n m
eas
ure
d b
err
y p
rim
ary
me
tab
oli
te a
bu
nd
ance
du
rin
g th
e e
xpe
rim
en
tal p
eri
o.
d T
he
GC
/MS
rete
nti
on
ti
,m
e m
ajo
r fr
agm
en
t m
/,z r
ete
nti
on
ind
e,x a
nd
th
e N
IST
lib
rary
corr
esp
on
din
g co
mp
ou
nd
info
rmat
ion
are
incl
ud
ed
.
cis-
INO
SITO
L18
.221
7; 3
05N
/AIn
osi
tol,
1,2
,3,4
,5,6
-he
xaki
s-O
-(tr
ime
thyl
sily
l)-,
cis
-15
28
2123
scyl
lo-I
NO
SITO
L28
.231
8C
0615
3In
osi
tol,
1,2
,3,4
,5,6
-he
xaki
s-O
-(tr
ime
thyl
sily
l)-,
scy
llo
-20
60
myo
-IN
OSI
TOL
29.3
147;
217
; 305
C00
137
*m
yo-I
no
sito
l (6T
MS)
Appendix B – Supplementary table S1
184
FL16
.162
4.15
928.5
61
5.31
238
.052
3.24
755.9
20
15.1
4844
.963
8.784
25L
16.1
624.1
59
22.2
084.
673
32.7
419.2
15
31.5
572.
959
34.5
190.9
17
NL
16.1
624.
159
23.6
206.
345
31.2
506.
705
20.8
082.
468
34.9
977.436
FL1.
562
0.42
03.
705
0.71
34.
764
0.92
26.
121
1.89
06.
405
1.4
48
25L
1.56
20.
420
2.21
40.
199
3.04
50.
143
3.90
00.
053
3.75
20.068
NL
1.56
20.
420
2.28
80.
431
2.47
30.
444
2.24
70.
206
2.85
30.288
FL0.
445
0.05
60.
638
0.06
00.
763
0.16
11.
025
0.33
31.
170
0.2
44
25L
0.44
50.
056
0.65
80.
029
0.64
70.
104
0.84
60.
076
0.85
50.0
13
NL
0.44
50.
056
0.74
40.
137
0.56
50.
068
0.59
80.
049
0.76
20.117
FL0.
094
0.01
60.
121
0.04
30.
206
0.04
40.
510
0.21
70.
539
0.2
84
25L
0.09
40.
016
0.06
10.
016
0.07
40.
033
0.14
50.
032
0.09
70.0
62
NL
0.09
40.
016
0.12
40.
036
0.09
00.
009
0.06
80.
015
0.12
10.0
15
FL0.
110
0.0
13
0.1
17
0.11
50.
000
0.0
00
0.1
10
0.19
10.
062
0.1
08
25L
0.11
00.0
13
0.07
40.
066
0.08
90.0
80
0.09
30.
082
0.04
00.0
70
NL
0.11
00.0
13
0.08
10.
072
0.00
00.0
00
0.12
50.
026
0.09
20.0
81
FL0.
000
0.00
00.
000
0.00
00.
000
0.00
00.
000
0.00
00.
011
0.0
19
25L
0.00
00.
000
0.00
00.
000
0.00
00.
000
0.00
00.
000
0.01
00.0
17
NL
0.00
00.
000
0.00
20.
004
0.03
30.
029
0.01
40.
008
0.00
90.0
16
FL6.
393
1.42
115
.236
3.45
321
.117
4.31
928
.340
7.87
235
.711
17.844
25L
6.39
31.4
21
8.82
50.
672
13.0
050.7
43
17.6
890.
943
16.2
910.7
42
NL
6.39
31.
421
10.9
263.
125
10.3
431.
499
9.57
81.
226
12.2
991.6
13
FL0.
171
0.0
19
0.32
60.
126
0.54
00.
103
0.83
60.
197
0.92
80.4
27
25L
0.17
10.
019
0.25
30.
079
0.30
40.
118
0.41
30.
088
0.37
00.0
31
NL
0.17
10.
019
0.21
40.
013
0.21
20.
066
0.20
90.
029
0.23
80.061
FL0.
245
0.03
00.
528
0.05
10.
685
0.12
21.
275
0.42
61.
340
0.500
25L
0.24
50.0
30
0.29
40.
022
0.37
20.0
63
0.53
60.
029
0.50
10.0
25
NL
0.24
50.
030
0.36
00.
084
0.37
80.
060
0.26
00.
015
0.44
50.039
FL0.
200
0.05
70.2
43
0.10
70.
289
0.09
10.3
76
0.10
10.
411
0.157
25L
0.20
00.0
57
0.28
60.
145
0.27
40.0
64
0.26
60.
187
0.44
60.1
30
NL
0.20
00.
057
0.18
60.
036
0.23
10.
070
0.30
00.
071
0.18
80.049
FL1.
169
0.1
81
1.64
70.
092
1.8
86
0.24
32.
372
0.45
52.
790
0.9
05
25L
1.16
90.
181
1.32
60.
185
1.44
60.
074
1.87
20.
050
1.93
10.178
NL
1.16
90.
181
1.43
10.
235
1.33
70.
077
1.26
60.
081
1.61
90.168
FL0.
052
0.0
21
0.0
90
0.01
70.
130
0.0
20
0.1
81
0.07
90.
155
0.0
27
25L
0.05
20.0
21
0.10
90.
025
0.23
10.0
25
0.57
80.
139
0.39
60.0
31
NL
0.05
20.0
21
0.10
40.
054
0.33
90.1
22
0.28
60.
017
0.42
40.1
34
FL0.
048
0.02
10.0
56
0.04
10.
000
0.00
00.
000
0.00
00.
000
0.0
00
25L
0.04
80.0
21
0.03
20.
028
0.00
90.0
16
0.00
00.
000
0.00
00.0
00
NL
0.04
80.
021
0.10
00.
051
0.03
30.
029
0.05
50.
015
0.00
60.010
FL0.
230
0.0
53
0.1
67
0.05
20.
460
0.0
47
0.1
80
0.04
40.
126
0.0
12
25L
0.23
00.0
53
0.28
50.
031
0.41
20.0
50
0.33
40.
056
0.37
20.0
08
NL
0.23
00.
053
0.35
00.
062
0.30
90.
074
0.15
70.
013
0.20
40.075
FL0.
241
0.0
97
0.2
54
0.07
10.
382
0.0
96
0.5
72
0.19
50.
466
0.0
56
25L
0.24
10.0
97
0.25
40.
077
0.29
20.0
29
0.54
80.
102
0.46
80.1
46
NL
0.24
10.
097
0.30
60.
133
0.29
00.
056
0.23
60.
048
0.32
60.049
FL0.
189
0.0
30
0.2
34
0.03
60.
324
0.0
64
0.4
64
0.18
60.
453
0.0
33
25L
0.18
90.0
30
0.19
00.
028
0.27
90.0
26
0.38
70.
020
0.38
50.0
54
NL
0.18
90.0
30
0.22
60.
033
0.18
10.0
74
0.23
00.
004
0.28
60.0
33
FL0.
047
0.03
70.0
51
0.08
70.
000
0.00
00.0
00
0.00
00.
000
0.000
25L
0.04
70.0
37
0.02
70.
046
0.03
10.0
54
0.00
00.
000
0.00
00.0
00
NL
0.04
70.
037
0.00
00.
000
0.00
00.
000
0.00
00.
000
0.00
00.000
FL0.
026
0.0
30
0.0
38
0.06
50.
141
0.0
14
0.1
39
0.16
10.
094
0.0
82
25L
0.02
60.0
30
0.00
00.
000
0.06
30.0
55
0.14
90.
023
0.13
80.0
33
NL
0.02
60.0
30
0.04
50.
078
0.10
40.0
32
0.06
20.
054
0.10
90.0
02
FL0.
000
0.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
25L
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
0.03
60.
062
0.00
00.0
00
NL
0.00
00.0
00
0.00
00.0
00
0.03
10.0
54
0.02
10.0
18
0.04
10.0
70
FL0.
000
0.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
25L
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
0.00
00.0
00
NL
0.00
00.0
00
0.00
00.0
00
0.00
40.
006
0.04
00.
036
0.00
00.0
00
FL0.
007
0.01
50.0
00
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
25L
0.00
70.
015
0.0
00
0.0
00
0.0
00
0.0
00
0.18
60.
204
0.0
00
0.0
00
NL
0.00
70.0
15
0.0
12
0.02
20.
215
0.1
93
0.0
47
0.08
20.
067
0.1
15
1674
2798
2705
SUC
RO
SE37
.2
*D
-(+)
-Tre
hal
ose
, oct
akis
(tri
me
thyl
sily
l) e
the
r
XYL
OSE
21.2
217;
307
C00
181
*D
-(+)
-Xyl
ose
, te
trak
is(t
rim
eth
ylsi
lyl)
eth
er,
me
thyl
oxi
me
(an
ti)
217;
437
N/A
N/A
2755
1288
LEU
CIN
E12
.815
8C
0012
3*
L-Le
uci
ne
, N-(
trim
eth
ylsi
lyl)
-, t
rim
eth
ylsi
lyl e
ste
r
PH
ENYL
ALA
NIN
E20
.421
8C
0007
9*
Ph
en
ylal
anin
e (
N,O
-TM
S)16
35
1336
ISO
LEU
CIN
E13
.315
8C
0040
7*
Iso
leu
cin
e, d
i-TM
S13
13
GLY
CIN
E13
.617
4C
0003
7*
Gly
, O,N
,N-t
ris-
TMS
1635
GLU
TAM
INE
23.3
156
C00
064
*L-
Glu
tam
ine
, N,N
2-b
is(t
rim
eth
ylsi
lyl)
-, t
rim
eth
ylsi
lyl e
ste
r17
87
GLU
TAM
IC A
CID
20.4
246
C00
025
*G
luta
mic
aci
d (
3TM
S)
β-A
LAN
INE
16.3
133
C00
099
*A
lan
ine
, be
ta-
(3TM
S)14
58
1542
GA
BA
(γ-
Am
ino
bu
tyri
c ac
id)
18.5
174
C00
334
*G
AB
A 3
TMS
1542
ASP
AR
TIC
AC
ID18
.523
2; 1
47; 2
18C
0004
9 *
L-A
spar
tic
acid
, (3T
MS)
-
1836
ASP
AR
AG
INE
21.6
116;
231
C00
152
*
Asp
arag
ine
, O,O
',N-t
ris(
trim
eth
ylsi
lyl)
-16
96
AR
GIN
INE
16.9
; 20.
3; 2
4.2;
24.
3 a
142
C00
062
*
Arg
inin
e, (
3TM
S)
Am
ino
aci
ds:
ALA
NIN
E8.
914
7C
0004
1 *
l-A
lan
ine
, N-(
trim
eth
ylsi
lyl)
-, t
rim
eth
ylsi
lyl e
ste
r11
30
UN
KN
OW
N S
UG
AR
128
.221
8; 3
19N
/AN
/A20
60
UN
KN
OW
N S
UG
AR
237
.9
UN
KN
OW
N S
UG
AR
3
UN
KN
OW
N O
LIG
OSA
CC
HA
RID
E 1
50.6
361
C08
243
N/A
361;
217
C00
089
*Su
cro
se, o
ctak
is-O
-(tr
ime
thyl
sily
l)-
THR
EITO
L18
.314
7; 3
07C
1688
4L-
Thre
ito
l, t
etr
akis
(tri
me
thyl
sily
l) e
the
r15
32
38.8
361;
437
N/A
N/A
2798
TREH
ALO
SE38
.536
1; 2
04; 1
91C
0108
3
3834
TAG
ATO
SE23
.721
7; 4
50C
0079
5D
-(-)
-Tag
ato
fura
no
se, p
en
taki
s(tr
ime
thyl
sily
l) e
the
r (i
som
er
2)18
08
Appendix B – Supplementary table S1
185
FL0.
000
0.0
00
0.0
00
0.00
00.
092
0.0
81
0.2
40
0.29
30.
230
0.0
81
25L
0.00
00.0
00
0.00
00.
000
0.02
50.0
43
0.09
30.
081
0.12
40.0
20
NL
0.00
00.0
00
0.00
00.
000
0.01
00.0
17
0.09
20.
113
0.00
00.0
00
FL0.
095
0.06
60.0
00
0.00
00.
345
0.15
80.2
08
0.18
40.
299
0.321
25L
0.09
50.0
66
0.00
00.
000
0.12
60.1
09
0.27
00.
019
0.24
80.0
17
NL
0.09
50.0
66
0.00
00.
000
0.06
40.1
11
0.03
50.
061
0.15
70.0
17
FL0.
084
0.0
35
0.0
12
0.00
30.
056
0.0
47
0.0
77
0.03
10.
061
0.0
28
25L
0.08
40.0
35
0.04
90.
039
0.05
20.0
20
0.10
00.
054
0.10
80.0
38
NL
0.08
40.0
35
0.02
90.
019
0.04
90.0
25
0.05
80.
027
0.05
60.0
49
FL0.
157
0.03
20.0
74
0.01
80.
084
0.00
40.0
57
0.04
90.
118
0.074
25L
0.15
70.0
32
0.17
20.
062
0.19
50.0
69
0.44
40.
120
0.29
10.1
36
NL
0.15
70.
032
0.15
30.
045
0.22
60.
091
0.15
70.
047
0.18
80.059
FL0.
193
0.0
35
0.1
08
0.09
30.
032
0.0
56
0.0
00
0.00
00.
023
0.0
41
25L
0.19
30.0
35
0.14
80.
022
0.20
50.0
33
0.17
30.
154
0.19
00.0
47
NL
0.19
30.0
35
0.18
90.
022
0.22
90.0
42
0.15
70.
038
0.12
20.1
06
FL0.
073
0.01
50.0
47
0.04
20.
076
0.00
70.0
66
0.05
80.
000
0.000
25L
0.07
30.0
15
0.04
60.
008
0.06
20.0
19
0.07
60.
066
0.03
50.0
61
NL
0.07
30.0
15
0.08
30.
019
0.06
10.0
17
0.06
80.
024
0.06
00.0
53
FL0.
001
0.00
30.0
23
0.04
00.
062
0.03
50.0
38
0.03
30.
059
0.039
25L
0.00
10.0
03
0.03
40.
025
0.03
00.0
33
0.02
50.
013
0.03
70.0
64
NL
0.00
10.
003
0.02
30.
040
0.03
60.
032
0.05
80.
029
0.00
00.000
FL0.
000
0.0
00
0.00
00.0
00
0.00
00.
000
0.00
00.
000
0.00
00.0
00
25L
0.00
00.
000
0.00
00.
000
0.00
00.
000
0.0
19
0.0
32
0.02
00.0
20
NL
0.00
00.
000
0.0
03
0.00
60.
030
0.02
60.0
56
0.04
50.
021
0.018
FL0.
267
0.0
41
0.3
54
0.17
00.
024
0.0
41
0.0
80
0.13
80.
097
0.1
68
25L
0.26
70.0
41
0.17
10.
039
0.16
40.0
75
0.07
70.
025
0.09
30.0
82
NL
0.26
70.
041
0.17
00.
070
0.08
80.
031
0.14
10.
023
0.13
40.031
FL3.
061
0.3
85
5.9
55
1.99
55.
843
2.1
99
8.7
11
2.98
87.
440
0.9
70
25L
3.06
10.3
85
3.73
20.
611
4.65
11.3
01
6.45
80.
430
6.38
80.5
06
NL
3.06
10.3
85
3.74
51.
157
4.66
50.4
19
4.20
70.
097
4.15
10.4
76
FL0.
062
0.0
21
0.0
48
0.01
60.
044
0.0
18
0.0
52
0.01
20.
047
0.0
27
25L
0.06
20.0
21
0.05
00.
013
0.03
30.0
20
0.03
90.
002
0.02
90.0
04
NL
0.06
20.0
21
0.06
60.
006
0.03
20.0
01
0.02
50.
004
0.03
40.0
07
FL4.
208
0.9
25
2.7
21
0.06
32.
238
0.3
69
2.1
18
0.56
43.
328
1.8
75
25L
4.20
80.9
25
1.92
20.
182
1.28
90.1
77
1.79
60.
141
1.63
30.5
66
NL
4.20
80.
925
3.56
81.
295
1.25
80.
101
1.06
20.
251
1.27
20.380
FL0.
493
0.11
30.2
80
0.02
70.
108
0.00
80.0
53
0.02
00.
054
0.017
25L
0.49
30.1
13
0.28
60.
072
0.16
50.0
48
0.14
70.
025
0.10
90.0
22
NL
0.49
30.
113
0.31
10.
019
0.13
20.
025
0.13
60.
047
0.10
00.035
FL0.
724
0.1
00
1.1
99
0.26
01.
116
0.3
65
1.5
75
0.40
71.
534
0.8
73
25L
0.72
40.1
00
0.84
60.
043
0.90
60.1
10
0.82
30.
081
1.05
10.0
54
NL
0.72
40.1
00
0.94
10.
228
0.67
70.1
09
0.67
90.
175
0.86
10.1
97
FL2.
718
0.34
13.
716
1.11
03.
752
0.84
63.
525
0.75
23.
417
0.7
85
25L
2.71
80.
341
2.17
90.
213
1.87
10.
364
1.68
10.
039
1.07
20.1
45
NL
2.71
80.
341
2.34
10.
533
1.39
10.
260
0.76
10.
044
0.78
50.1
51
FL0.
328
0.0
58
0.2
36
0.10
00.
076
0.0
67
0.0
00
0.00
00.
000
0.0
00
25L
0.32
80.0
58
0.37
10.
036
0.26
50.0
12
0.22
00.
007
0.14
70.0
12
NL
0.32
80.0
58
0.34
80.
134
0.20
10.0
51
0.10
10.
011
0.07
70.0
24
FL0.
331
0.07
10.5
01
0.28
70.
645
0.23
40.9
36
0.48
00.
838
0.220
25L
0.33
10.0
71
0.54
80.
029
0.33
50.0
74
0.55
20.
460
0.42
60.1
34
NL
0.33
10.
071
0.41
40.
203
0.43
10.
128
0.56
10.
158
0.58
70.186
FL2.
022
0.29
71.4
39
0.11
30.
701
0.08
80.4
95
0.07
70.
490
0.103
25L
2.02
20.2
97
1.43
10.
269
0.80
20.2
79
0.66
60.
101
0.56
30.1
06
NL
2.02
20.2
97
1.40
40.
147
0.68
30.1
44
0.66
00.
183
0.56
80.0
90
FL20
.745
7.37
345
.157
1.58
329
.455
3.62
222
.853
3.47
319
.620
7.1
15
25L
20.7
457.
373
31.4
750.
714
26.0
522.
191
26.5
052.
483
20.2
081.8
00
NL
20.7
457.
373
34.6
348.
671
24.1
692.
572
17.3
551.
568
16.4
831.3
25
FL0.
770
0.11
91.
053
0.31
81.
016
0.17
81.
417
0.33
61.
282
0.0
50
25L
0.77
00.1
19
1.05
90.
521
1.10
30.0
72
1.06
40.
103
1.19
00.1
22
NL
0.77
00.1
19
0.8
13
0.03
61.
060
0.1
29
0.9
63
0.07
10.
891
0.3
82
FL3.
830
1.09
05.
716
1.10
46.
402
0.23
88.
710
3.39
08.
537
4.9
92
25L
3.83
01.0
90
4.01
20.
235
7.57
05.0
46
6.96
70.
514
9.44
85.1
99
NL
3.83
01.0
90
5.17
01.
146
4.42
00.1
97
4.63
20.
462
7.19
12.4
63
1175
PH
OSP
HO
RIC
AC
ID12
.929
9C
0000
9P
ho
sph
ori
c ac
id, t
riTM
S12
92
OX
ALI
C A
CID
9.8
147;
133
C00
209
Oxa
lic
acid
(2T
MS)
1328
MA
LIC
AC
ID17
.823
3C
0014
9M
alic
aci
d (
3TM
S)15
08
MA
LEIC
AC
ID13
.514
7; 2
45 C
0138
4M
ale
ic a
cid
, (2T
MS)
GLY
CER
IC A
CID
14.2
147;
189
; 292
C00
258
Gly
ceri
c ac
id, (
3TM
S)13
82
LAC
TIC
AC
ID8.
114
7; 1
17C
0018
6La
ctic
aci
d, (
2TM
S)10
88
1980
GLU
CO
NIC
AC
ID28
.029
2; 3
33C
0025
7G
luco
nic
aci
d, (
6TM
S)20
49
GA
LLIC
AC
ID26
.728
1; 4
58C
0142
4G
alli
c ac
id, t
etr
aTM
S
1847
FUM
AR
IC A
CID
14.4
245
C00
122
Fum
aric
aci
d (
2TM
S)13
97
CIT
RIC
AC
ID24
.427
3C
0015
8C
itri
c ac
id, (
4TM
S)
1260
BEN
ZOIC
AC
ID12
.017
9C
0018
0B
en
zoic
aci
d, T
MS
2130
CA
FFEI
C A
CID
29.4
396
C01
197
Caf
feic
aci
d (
3TM
S)
Mis
cell
ane
ou
s ac
ids:
ASC
OR
BIC
AC
ID24
.814
7C
0007
2*
Asc
orb
ic a
cid
(4T
MS)
1870
1956
VA
LIN
E11
.514
4; 2
18C
0018
3*
Val
ine
, di-
TMS
1242
TYR
OSI
NE
26.3
218;
280
C00
082
*Ty
rosi
ne
, (3T
MS)
2230
5-h
ydro
xy-T
RYP
TOP
HA
N32
.129
0; 2
18; 1
46C
0101
7L-
5-H
ydro
xytr
ypto
ph
an, t
rim
eth
ylsi
lyl e
the
r, t
rim
eth
ylsi
lyl e
ste
r23
23
TRYP
TOP
HA
N30
.829
1; 2
02C
0007
8*
Tryp
top
han
, N,N
,O-t
riTM
S
1414
THR
EON
INE
15.5
218
C00
188
*Th
reo
nin
e (
N,O
,O-T
MS)
1433
SER
INE
14.9
204;
218
C00
065
*Se
rin
e, (
3TM
S)
1313
PYR
OG
LUTA
MIC
AC
ID (
5-o
xo-P
roli
ne
)18
.315
6C
0187
9P
yro
glu
tam
ic a
cid
, (N
,O-T
MS)
1532
PR
OLI
NE
13.3
216;
147
; 158
C00
148
*P
roli
ne
, di-
TMS
Appendix B – Supplementary table S1
186
FL2.
394
0.51
72.6
89
0.17
01.
589
0.33
61.0
57
0.36
20.
525
0.392
25L
2.39
40.5
17
2.40
90.
328
2.07
20.2
45
1.26
60.
204
1.10
40.0
70
NL
2.39
40.5
17
3.10
40.
637
2.17
00.1
44
1.45
10.
148
1.33
80.1
36
FL0.
094
0.01
30.1
41
0.00
80.
155
0.02
80.2
00
0.06
70.
272
0.071
25L
0.09
40.0
13
0.12
00.
015
0.13
10.0
14
0.14
20.
021
0.15
10.0
27
NL
0.09
40.
013
0.11
30.
016
0.11
50.
015
0.11
40.
013
0.15
30.032
FL0.
104
0.03
60.0
00
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
0.0
00
25L
0.10
40.
036
0.03
80.
037
0.00
00.
000
0.00
00.
000
0.00
00.000
NL
0.10
40.0
36
0.0
11
0.01
90.
000
0.0
00
0.0
00
0.00
00.
000
0.0
00
FL12
.147
2.29
912
.242
7.35
312
.794
5.68
318
.448
5.71
915
.865
10.8
85
25L
12.1
472.
299
4.26
91.
260
3.84
32.
229
11.4
142.
884
8.58
72.4
42
NL
12.1
472.
299
12.9
449.
394
2.20
20.
872
5.23
31.
134
7.49
53.4
76
FL1.
343
0.13
90.
364
0.23
00.
112
0.02
50.
154
0.04
70.
135
0.0
43
25L
1.34
30.
139
0.16
60.
049
0.02
30.
020
0.02
80.
048
0.02
20.0
19
NL
1.34
30.
139
0.14
80.
045
0.01
80.
016
0.01
30.
023
0.00
00.0
00
FL0.
025
0.00
60.0
36
0.01
00.
045
0.01
70.0
56
0.01
10.
046
0.008
25L
0.02
50.0
06
0.04
90.
032
0.04
60.0
28
0.05
60.
011
0.04
20.0
04
NL
0.02
50.
006
0.02
80.
008
0.04
20.
025
0.03
90.
012
0.03
70.007
FL0.
102
0.0
20
0.1
42
0.03
80.
154
0.0
78
0.2
41
0.11
30.
129
0.0
46
25L
0.10
20.0
20
0.17
50.
075
0.16
00.0
79
0.19
30.
053
0.13
20.0
26
NL
0.10
20.
020
0.1
01
0.01
30.
131
0.05
80.1
40
0.04
40.
093
0.021
FL0.
046
0.03
40.0
52
0.02
00.
028
0.03
00.0
25
0.00
60.
014
0.025
25L
0.04
60.0
34
0.10
30.
062
0.03
70.0
28
0.02
30.
026
0.03
60.0
13
NL
0.04
60.0
34
0.04
40.
034
0.03
90.0
18
0.05
00.
037
0.02
10.0
20
FL0.
023
0.02
60.0
09
0.00
90.
049
0.03
50.0
60
0.04
60.
033
0.021
25L
0.02
30.0
26
0.10
20.
072
0.01
00.0
13
0.02
80.
004
0.01
20.0
17
NL
0.02
30.
026
0.04
50.
041
0.02
20.
013
0.00
80.
009
0.01
80.019
FL1.
255
1.2
06
1.7
95
1.77
00.
895
0.3
58
1.8
31
0.48
91.
436
0.3
73
25L
1.25
51.2
06
2.27
61.
597
1.08
20.2
35
1.16
20.
096
1.01
20.2
61
NL
1.25
51.2
06
1.07
40.
460
0.90
70.3
00
1.27
00.
667
0.63
30.3
07
FL1.
430
0.9
80
1.9
13
1.42
41.
383
0.5
52
2.4
39
0.80
21.
723
0.3
93
25L
1.43
00.9
80
2.77
71.
753
1.63
10.7
42
1.74
70.
309
1.48
40.4
07
NL
1.43
00.9
80
1.45
60.
715
1.26
80.4
39
2.00
91.
388
0.89
40.3
65
FL0.
048
0.02
10.0
43
0.02
90.
092
0.01
80.0
91
0.10
60.
042
0.073
25L
0.04
80.0
21
0.01
40.
019
0.01
40.0
25
0.09
60.
011
0.09
10.0
32
NL
0.04
80.
021
0.00
90.
008
0.03
20.
021
0.03
00.
026
0.03
40.028
FL8.
041
1.3
72
6.3
17
1.05
33.
488
0.4
19
3.1
15
0.58
12.
889
1.2
19
25L
8.04
11.3
72
5.76
40.
552
2.65
80.4
15
2.21
30.
352
1.89
90.3
16
NL
8.04
11.
372
6.60
62.
026
2.96
50.
235
1.61
60.
272
2.05
80.238
FL7.
397
0.35
911.4
90
1.82
814
.009
2.11
716.7
81
1.26
318
.887
5.380
25L
7.39
70.3
59
10.2
692.
235
11.4
061.2
37
14.9
950.
936
13.6
231.8
57
NL
7.39
70.3
59
9.22
21.
683
9.49
31.7
41
9.19
90.
855
10.6
660.5
11
FL0.
186
0.06
80.2
81
0.06
60.
363
0.07
10.4
00
0.09
70.
518
0.293
25L
0.18
60.0
68
0.16
00.
057
0.19
00.0
26
0.32
40.
075
0.28
00.0
37
NL
0.18
60.0
68
0.28
60.
083
0.20
60.0
14
0.09
40.
126
0.28
60.0
70
FL0.
001
0.00
30.0
01
0.00
10.
001
0.00
10.0
00
0.00
10.
000
0.001
25L
0.00
10.0
03
0.00
00.
000
0.00
10.0
01
0.00
10.
000
0.00
80.0
13
NL
0.00
10.
003
0.00
00.
000
0.00
00.
000
0.00
10.
000
0.00
00.001
FL3.
729
0.2
40
5.5
81
0.97
36.
597
1.6
82
8.6
52
1.94
48.
219
1.8
16
25L
3.72
90.2
40
5.21
21.
520
5.51
80.8
80
6.93
60.
773
6.16
00.4
97
NL
3.72
90.2
40
4.27
90.
428
4.71
21.1
05
4.55
80.
683
4.64
60.2
25
Ad
ipic
aci
d18
147;
275
C06
104
*A
dip
ic a
cid
, (2T
MS)
1528
Ad
on
ito
l (R
ibit
ol)
22.8
147;
217
; 103
C00474
*R
ibit
ol,
5TM
S17
60
L-h
ydro
xyp
roli
ne
18.5
230
C01
157
*3-
Hyd
roxy
pro
lin
e, N
,O,O
'-tr
is(t
rim
eth
ylsi
lyl)
-15
42
Inte
rnal
sta
nd
ard
s:
3108
1-M
ON
OP
ALM
ITIN
35.9
371
N/A
1-M
on
op
alm
itin
tri
me
thyl
sily
l eth
er
2602
KA
EMP
FER
OL
42.0
559
C05
903
Kae
mp
fero
l, 4
TMS
2791
HYD
RO
XYL
AM
INE
9.0
133
C00
192
Hyd
roxy
lam
ine
, N,N
,O-t
ris-
TMS
1135
GLY
CER
OL
MO
NO
STEA
RA
TE38
.439
9N
/AG
lyce
rol m
on
ost
ear
ate
, 2tm
s d
eri
vati
ve
2889
CA
TEC
HIN
39.5
; 39.
8 a
368
C06
562
Cat
ech
ine
, pe
nta
-TM
S-e
the
r, (
2R-c
is)-
Mis
call
en
eo
us
com
po
un
ds:
AR
BU
TIN
36.2
254
C06
186
Hyd
roq
uin
on
e-β
-d-g
luco
pyr
ano
sid
e,p
en
taki
s(tr
ime
thyl
sily
l)-
2626
STEA
RIC
AC
ID30
.911
7; 3
41C
0153
0St
ear
ic a
cid
, tri
me
thyl
sily
l est
er
2237
2209
PA
LMIT
IC A
CID
27.8
117;
313
C00
249
Pal
mit
ic a
cid
, TM
S20
39
OLE
IC A
CID
30.5
339
C00
712
Ole
ic a
cid
, tri
me
thyl
sily
l est
er
2407
NO
NA
NO
IC A
CID
14.6
117;
215
C01
601
No
nan
oic
aci
d, T
MS
est
er
1405
MYR
ISTI
C A
CID
33.3
343
C06
424
Myr
isti
c ac
id, 2
,3-b
is(t
rim
eth
ylsi
loxy
)pro
pyl
est
er
Fatt
y ac
ids:
EIC
OSA
NO
IC A
CID
40.6
427
C06
425
Eico
san
oic
aci
d, 2
,3-b
is[(
trim
eth
ylsi
lyl)
oxy
]pro
pyl
est
er
2990
1666
THR
EON
IC A
CID
19.4
147;
292
; 220
C01
620
L-Th
reo
nic
aci
d, t
ris(
trim
eth
ylsi
lyl)
eth
er,
tri
me
thyl
sily
l est
er
1586
TAR
TAR
IC A
CID
21.0
292
C00
898
Tart
aric
aci
d, T
MS
1831
SUC
CIN
IC A
CID
13.7
147;
247
C00
042
Succ
inic
aci
d (
2TM
S)13
44
PR
OTO
CA
TEC
HU
IC A
CID
24.1
193;
370
C00
230
*P
roto
cate
chu
ic a
cid
(tm
s)
PYR
UV
IC A
CID
13.6
73; 1
47C
0002
2P
yru
vic
acid
oxi
me
, bis
(tri
me
thyl
sily
l)-
de
riv.
1335
a M
ore
th
an o
ne
pe
ak c
hro
mat
ogr
aph
ic p
eak
s e
lute
d f
or
eac
h t
he
se m
eta
bo
lite
s.Th
e c
om
bin
ed
pe
ak a
reas
we
re u
sed
to
cal
cult
e t
he
me
tab
oli
te a
bu
nd
ance
.
Figure S1 Principle component analysis of the significantly affected berry primary metabolites for the three defoliation treatments (full leaf – FL, 25% leaves – 25L and no leaf – NL), at each destructive harvest after the implementation of the treatments (V+18, V+27, V+37 and V+46). A minimum convex polygon or convex hull (red polygon) has been included to define the treatment score space.
Appendix B – Supplementary figure S1
187
Appendix B – Supplementary figure S1
188
Pri
nci
pal
co
mp
on
ent
anal
ysis
(P
CA
) la
ten
t ve
cto
r lo
adin
gs o
f th
e d
iffe
ren
t b
erry
met
abo
lites
sig
nif
ican
tly
affe
cted
by
the
trea
tmen
ts d
uri
ng
the
exp
erim
enta
l per
iod
.
PC
1P
C2
1-M
onop
alm
itin
0.18
178
-0.0
0041
3α-M
anno
bios
e0.
1871
0.02
784
Ara
bino
fura
nose
0.18
744
-0.0
0168
Ara
bito
l-0
.015
79-0
.179
52A
rbut
in0.
1142
-0.0
9782
Arg
inin
e-0
.016
73-0
.292
44A
spar
agin
e-0
.114
860.
1908
8A
spar
tic a
cid
-0.0
6385
-0.0
3286
Ben
zoic
aci
d0.
1761
3-0
.011
45C
affe
ic a
cid
0.03
690.
2813
5C
atec
hin
-0.0
5034
0.31
738
Cel
lobi
ose
0.18
795
0.02
869
cis
-Inos
itol
0.06
622
-0.1
1093
Citr
ic a
cid
0.07
354
0.25
846
Dul
cito
l0.
1507
6-0
.151
18Fr
ucto
se0.
1743
8-0
.058
95Fu
cose
0.19
126
0.01
827
Fum
aric
aci
d-0
.131
270.
2272
3G
alac
tinol
-0.0
6606
-0.1
4315
Gal
lic a
cid
0.17
321
0.12
873
Glu
coni
c ac
id0.
1118
10.
2484
1G
luco
se0.
1787
5-0
.056
74G
luta
mic
aci
d0.
1774
6-0
.076
72G
lyce
ric a
cid
-0.1
4901
0.12
735
Gly
cero
l mon
oste
reat
e0.
1846
1-0
.019
77H
ydro
xyla
min
e0.
1708
60.
0550
4La
ctic
aci
d0.
1641
10.
0275
8M
alic
aci
d-0
.051
0.28
287
myo
-Inos
itol
0.16
940.
1282
Pro
line
0.17
55-0
.058
95P
roto
cate
chui
c ac
id-0
.142
870.
2091
8P
yrog
luta
mic
aci
d0.
1409
9-0
.135
51P
yruv
ic a
cid
0.18
045
0.00
619
Raf
finos
e0.
0133
4-0
.053
3R
ham
nose
-0.1
5514
-0.0
231
Suc
rose
0.17
284
-0.0
4049
Taga
tose
0.19
032
0.02
443
Tarta
ric a
cid
0.15
558
0.13
678
Thre
onic
aci
d0.
0325
30.
3059
7Th
reon
ine
-0.0
5206
-0.2
1073
Treh
alos
e0.
1822
30.
0462
8Tr
ypto
phan
-0.1
594
-0.1
0485
Unk
now
n ol
igos
acch
arid
e 1
-0.0
382
-0.1
651
Unk
now
n su
gar 1
0.18
948
0.02
395
Unk
now
n su
gar 2
0.18
905
0.03
217
Unk
now
n su
gar 3
0.18
854
0.03
777
β-a
lani
ne0.
1890
30.
0018
4