THE CHARACTERISATION OF CANAS2
AND BIOFORTIFICATION OF CHICKPEA
Grace Zi Hao TAN
BSc Applied Science (Biotechnology)
BSc Applied Science (Biotechnology). Hons
Principal supervisor:
Prof Sagadevan Mundree (QUT)
Associate supervisors:
Dr Brett Williams (QUT)
Dr Sudipta Das Bhowmik (QUT)
Dr Alex Johnson (University of Melbourne)
Submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy
Centre for Tropical Crops and Biocommodities
Science and Engineering Faculty
Queensland University of Technology
November, 2016
The Characterisation of CaNAS2 and Biofortification of Chickpea i
Keywords
Agrobacterium-mediated transformation, bioavailability, biofortification, chickpea,
ferritin, iron, iron deficiency, iron content, manganese, mineral accumulation,
nicotianamine synthase, transgenic, translocation, zinc.
ii The Characterisation of CaNAS2 and Biofortification of Chickpea
Abstract
Iron deficiency in humans is a significant global problem, contributing to the
bulk of global anaemia cases and afflicting both developed and developing nations.
Its effects are far-reaching, affecting people not just on a personal but also national
scale. Existing methods of alleviating this problem include supplementation, food
fortification and dietary diversification. Such measures however, are limited by the
economic status of the targeted demographics. An alternative and more sustainable
method is the enhancement of the inherent iron content and bioavailability in crops
through biofortification.
Iron biofortification through genetic modification has been done in several
important crops like rice and wheat, but there is no known precedent in legumes. The
crop of interest to this project is chickpea, the second most important pulse crop in
the world that is widely consumed where anaemia is prevalent. This study documents
the first known attempt at biofortifying chickpea via genetic modification. The genes
of interest are rice nicotianamine synthase 2 (NAS) and soybean ferritin (FER),
which have been successfully used to enhance plant iron content and bioavailability
in rice. In this project, the novel chickpea NAS2 gene was also characterised and
investigated for its potential in a cisgenic biofortification approach.
This research was conducted in three main stages. First, commercial chickpea
cultivars were surveyed to determine the existing iron content and identify the factors
influencing it. The desi cultivar, PBA HatTrick, was then used for further work.
Second, was the characterisation of CaNAS2 in chickpea and the model species,
tobacco. The NAS-GmFER transgenic approach was then applied to chickpea in the
third stage, and its effectiveness as a biofortification strategy assessed.
For the survey of commercial chickpea cultivars conducted in the first stage,
samples were obtained for six cultivars from five locations within Eastern Australia.
Trace element content was assessed via ICP-OES (inductively coupled plasma
optical emission spectroscopy). Iron content was in chickpea found to range from
3.36 to 5.2mg/100g. HatTrick, the cultivar of interest, had 3.97 to 4.37g/100g of iron,
most of which was stored in the cotyledons. Principal component analysis of the
mineral profiles indicated that seed iron content was influenced by both genotype
and the environment.
The Characterisation of CaNAS2 and Biofortification of Chickpea iii
In the second stage, chickpea (cv HatTrick) were grown under iron-sufficient
and iron-deficient conditions, and CaNAS2 expression measured via qPCR. The
results showed CaNAS2 to be systemically expressed under iron-sufficient
conditions, and downregulated under iron-deficiency. To determine the effect of
CaNAS2 on iron accumulation, the gene was then cloned and overexpressed in
tobacco alone or together with GmFER. The well-characterised OsNAS2 was used in
these gene combinations as a positive control. Transgene presence and expression
was confirmed via PCR and RT-PCR. A total of three CaNAS2, seven OsNAS2, ten
GmFER-CaNAS2 and six GmFER-OsNAS2 transgenic lines were generated and
grown to the T1 generation. Assessment of leaf iron content showed the transgenic
lines to be mostly similar to the vector control, with only one CaNAS2 and OsNAS2
line being significantly higher, with approximately 1.3-fold increase in iron content.
The NAS-GmFER gene combinations were then applied to chickpea in the
third stage of this project, where optimisation of the chickpea transformation
protocol allowed for up to three-fold increase in transformation efficiency. Several
transgenic lines were generated, of which a total of three OsNAS2-GmFER and two
CaNAS2-GmFER transgenic lines were carried down to the T3 and T4 generations.
Transgene presence, expression and copy number was confirmed via PCR, qPCR and
Southern analysis respectively. Glasshouse evaluation showed no significant
differences between the transgenic lines and non-transgenic control in terms of
morphology, biomass, harvest index, or yield. Seed iron concentrations of up to
9mg/100g were achieved; as with the transgenic tobacco however, only one
OsNAS2-GmFER lines was significantly higher than the control with a 1.3-fold
increase. Assessment of leaf iron content yielded similar results, despite preliminary
data showing enhanced iron accumulation.
Based on these results, the NAS-GmFER biofortification approach appears to
be capable of enhancing iron in tobacco or chickpea, albeit not to the same
effectiveness as in rice. The precise mechanism behind this was unclear, though
differing physiologies, and limitations in plant uptake were suspected to be possible
causes. Such factors can be investigated in future studies.
Being the first known attempt at transgenic biofortification of a pulse crop, this
project provides insight which can serve to advise future attempts and pinpoint areas
for further development. In addition, the improvement of the chickpea transformation
iv The Characterisation of CaNAS2 and Biofortification of Chickpea
protocol in this study presents an opportunity for advancing both basic and applied
research, which has considerable implications for chickpea industry as a whole.
The Characterisation of CaNAS2 and Biofortification of Chickpea v
Table of Contents
Keywords .................................................................................................................................. i
Abstract .................................................................................................................................... ii
Table of Contents ......................................................................................................................v
List of Figures ....................................................................................................................... viii
List of Tables ............................................................................................................................x
List of Abbreviations ............................................................................................................. xii
Statement of Original Authorship ...........................................................................................xv
Acknowledgements ............................................................................................................... xvi
Chapter 1: Introduction ...................................................................................... 1
1.1 Aims and Objectives .......................................................................................................2
Chapter 2: Literature Review ............................................................................. 3
2.1 World population and malnutrition ................................................................................3 2.1.1 Iron deficiency anaemia .......................................................................................3
2.2 Biofortification ...............................................................................................................5 2.2.1 Approaches to biofortification ..............................................................................6 2.2.2 Target crops ..........................................................................................................7
2.3 Pulses as a vehicle for biofortification ............................................................................7 2.3.1 Pulse production and market ................................................................................8 2.3.2 Challenges ..........................................................................................................10 2.3.3 Chickpea .............................................................................................................13
2.4 Iron metabolism in plants .............................................................................................16 2.4.1 Strategy I – The reduction-based strategy ..........................................................19 2.4.2 Strategy II – The phytosiderophore chelation strategy .......................................21 2.4.3 Translocation ......................................................................................................23 2.4.4 Storage ................................................................................................................24 2.4.5 Regulation ..........................................................................................................27
2.5 Engineering for enhanced iron content .........................................................................28
2.6 Summary and Implications ...........................................................................................29
Chapter 3: General Materials and Methods ................................................... 31
3.1 General materials ..........................................................................................................31 3.1.1 Sources of specialised reagents ..........................................................................31 3.1.2 Iron metabolism genes ........................................................................................31 3.1.3 Bacterial strains ..................................................................................................31 3.1.4 Plant material ......................................................................................................32 3.1.5 General solutions: Abbreviations and composition ............................................32
3.2 General methods ...........................................................................................................34 3.2.1 General molecular techniques ............................................................................34 3.2.2 Bacterial transformation .....................................................................................35 3.2.3 Plant transformation ...........................................................................................36 3.2.4 Plant growth conditions ......................................................................................40 3.2.5 Verification and molecular characterisation of transgenic plants.......................40
vi The Characterisation of CaNAS2 and Biofortification of Chickpea
3.2.6 Trace element analysis ....................................................................................... 45
3.3 Data analysis ................................................................................................................ 47
Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick ................. 49
4.1 INTRODUCTION ....................................................................................................... 49
4.2 MATERIALS AND METHODS ................................................................................. 51 4.2.1 Seed material and locations ............................................................................... 51 4.2.2 Measurement of trace element distribution within the chickpea seed ............... 53 4.2.3 Statistical analysis .............................................................................................. 54
4.3 RESULTS .................................................................................................................... 54 4.3.1 Trace element composition of Australian-grown chickpea ............................... 54 4.3.2 Relationships between location and cultivar on seed trace elemental
composition ........................................................................................................ 58 4.3.3 Cotyledons the primary storage for iron in PBA HatTrick seeds ...................... 60
4.4 DISCUSSION .............................................................................................................. 63
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes .. 67
5.1 INTRODUCTION ....................................................................................................... 67
5.2 MATERIALS AND METHODS ................................................................................. 69 5.2.1 Designation of chickpea NAS2 .......................................................................... 69 5.2.2 Assessment of NAS amino acid sequence and protein properties ..................... 69 5.2.3 Phylogenetic analysis of NAS proteins .............................................................. 70 5.2.4 Assessment of CaNAS2 expression ................................................................... 70 5.2.5 Isolation and cloning of chickpea NAS2 and other genes of interest ................ 72 5.2.6 Generation of expression plasmids .................................................................... 73 5.2.7 Generation and molecular characterisation of transgenic tobacco ..................... 76 5.2.8 Assessment of iron accumulation in transgenic tobacco leaf ............................ 76
5.3 RESULTS .................................................................................................................... 76 5.3.1 Designation and sequence analysis of CaNAS2 ................................................ 76 5.3.2 Phylogenetic analysis of CaNAS2 ..................................................................... 79 5.3.3 CaNAS2 expression is downregulated in response to iron deficiency .............. 82 5.3.4 Generation of expression plasmids .................................................................... 87 5.3.5 Generation and molecular characterisation of transgenic tobacco ..................... 88 5.3.6 Transgenic tobacco exhibit to significant increase in leaf iron or zinc
contents .............................................................................................................. 91
5.4 DISCUSSION .............................................................................................................. 93
Chapter 6: Generation and Characterisation of Transgenic Chickpea ........ 99
6.1 INTRODUCTION ....................................................................................................... 99
6.2 MATERIALS AND METHODS ............................................................................... 100 6.2.1 Generation of transgenic chickpea ................................................................... 100 6.2.2 Molecular characterisation of transgenic chickpea .......................................... 102 6.2.3 Glasshouse trial for T3, T4 plants .................................................................... 103 6.2.4 Assessment of agronomic parameters .............................................................. 104 6.2.5 Assessment of iron content in transgenic chickpea plants ............................... 104
6.3 RESULTS .................................................................................................................. 105 6.3.1 Optimisation of chickpea transformation procedure ........................................ 105 6.3.2 Generation and molecular characterisation of transgenic chickpea ................. 107 6.3.3 Morphology and agronomic properties of transgenic chickpea ....................... 112 6.3.4 Iron content in transgenic chickpea ................................................................. 115
The Characterisation of CaNAS2 and Biofortification of Chickpea vii
6.4 DISCUSSION .............................................................................................................117
Chapter 7: General Discussion ....................................................................... 122
7.1 The physiological role of CaNAS2 in the subcellualr and systemic context ..............123
7.2 NAS-FER transgene combination has limited effect on iron accumulation in tobacco
and chickpea..........................................................................................................................125
Chapter 8: Concluding remarks ..................................................................... 131
Appendices .............................................................................................................. 133
Bibliography ........................................................................................................... 141
viii The Characterisation of CaNAS2 and Biofortification of Chickpea
List of Figures
Figure 2.1. Global burden of anaemia across all ages .................................................. 5
Figure 2.2. Average pulse production by region (FAO, 2016a). ............................... 10
Figure 2.3. Fe acquisition strategies in higher plants: Strategy I in
nongraminaceous plants (left) and Strategy II in graminaceous plants
(right). .......................................................................................................... 18
Figure 2.4. Biosynthetic pathway of mugeneic acid (MA) family of
phytosiderophores (Sharma and Dietz, 2006). ............................................. 21
Figure 2.5. Possible rate-limiting steps for grain iron accumulation (Sperotto et
al., 2012). ..................................................................................................... 28
Figure 4.1. Chickpea cultivars used in this study. ...................................................... 52
Figure 4.2. PCA of trace element composition of chickpea grown in QLD and
NSW based on overall mineral composition. ............................................... 59
Figure 4.3. Clustering analysis of chickpea grown in QLD and NSW based on
mineral composition of samples. ................................................................. 60
Figure 4.4. Relative distribution of trace elements within PBA HatTrick seeds. ...... 61
Figure 5.1. Schematic diagram of the mini-hydroponics system. .............................. 71
Figure 5.2. Expression plasmids generated for plant transformation. ........................ 75
Figure 5.3. Predicted 3D structures of OsNAS and CaNAS2 proteins. ..................... 77
Figure 5.4. Predicted biochemical properties of CaNAS2. ........................................ 78
Figure 5.5. Amino acid sequence of CaNAS2. .......................................................... 79
Figure 5.6. Phylogenetic relationship between CaNAS2 and NAS proteins from
other plants. .................................................................................................. 81
Figure 5.7. Qualitative assessment of CaNAS2 expression in different chickpea
tissues via PCR. ............................................................................................ 82
Figure 5.8. Qualitative assessment of the expression of other CaNAS family
members in different chickpea tissues via PCR. .......................................... 83
Figure 5.9. Morphology of iron-sufficient (+Fe) and iron-deficient (-Fe)
chickpea grown under hydroponics conditions. ........................................... 84
Figure 5.10. Representative photos of verification of RNA and cDNA for
qPCR analysis. ............................................................................................. 85
Figure 5.11. Expression of CaNAS2 in different tissues under iron-sufficient
(+Fe) and iron-deficient conditions (-Fe)..................................................... 86
Figure 5.12. PCR detection of the genes of interest in the Agrobacterium
strains AGL1 and LBA4404 used for plant transformation work. .............. 87
Figure 5.13. Representative photos of T0 tobacco PCR screening with gene-
specific primers. ........................................................................................... 89
The Characterisation of CaNAS2 and Biofortification of Chickpea ix
Figure 5.14. Detection of transgene expression in GM tobacco lines via PCR. ........ 90
Figure 5.15. Average A) iron and B) zinc content in non-transgenic (n=10) and
transgenic tobacco leaves (n=4 to 7). ........................................................... 92
Figure 6.1. Overview of the chickpea transformation process. ............................... 101
Figure 6.2. Morphology of emerging putative transgenic shoots. ........................... 106
Figure 6.3. Representative photo of PCR screening of T0 plants. ........................... 108
Figure 6.4. Detection of transgene expression in transgenic chickpea lines via
PCR. ........................................................................................................... 109
Figure 6.5. Relative expression of transgenes in transgenic chickpea. .................... 110
Figure 6.6. Southern analysis of transgenic chickpea lines used in the
glasshouse trial. .......................................................................................... 111
Figure 6.7. Morphology of 9 week old transgenic chickpea at the
flowering/pod-filling stage. ........................................................................ 113
Figure 6.8. Agronomic properties of transgenic chickpea under glasshouse
conditions. .................................................................................................. 114
Figure 6.9. Preliminary study on leaf iron, zinc and manganese contents in 7
week old transgenic chickpea at the T1 generation. .................................. 116
Figure 6.10. Leaf iron, zinc and manganese contents in 7 week old transgenic
chickpea. .................................................................................................... 116
Figure 6.11. Iron, zinc and manganese contents in transgenic chickpea seeds. ....... 117
Figure 8.1. Representative photos of T1 tobacco screening via PCR using gene-
specific primers. ......................................................................................... 137
Figure 8.2. Concentrations of A) iron, and B) zinc in T1 transgenic tobacco
leaves.......................................................................................................... 138
Figure 8.3. GUS staining of transiently transformed chickpea ................................ 139
Figure 8.4. Iron, zinc, manganese and phosphorus content of GM chickpea
seeds. .......................................................................................................... 140
x The Characterisation of CaNAS2 and Biofortification of Chickpea
List of Tables
Table 2.1. Recommended Dietary Allowances (RDAs) for iron (Trumbo et al.,
2001). ............................................................................................................. 4
Table 2.2. Growth in production of major pulse producers (FAO, 2014, 2016b). ..... 10
Table 2.3. Factors affecting bioavailability of some trace elements (House,
1999). ........................................................................................................... 12
Table 2.4. Nutritional values per 100g of chickpea and important staple crops
(USDA, 2013). ............................................................................................. 14
Table 2.5. Information and assumptions used to set target levels for iron
biofortification in chickpea. ......................................................................... 16
Table 4.1. Description of chickpea cultivars used in this study. ................................ 52
Table 4.2. Locations from which seed samples were obtained. ................................. 53
Table 4.3. Cultivation conditions for each location. .................................................. 53
Table 4.4. Summary of Fe, Zn and P concentrations in kabuli and desi cultivars
grown at different locations. ........................................................................ 56
Table 4.5. Pearson’s correlation coefficient between the different trace
elements in PBA HatTrick. .......................................................................... 57
Table 4.6. Representation quality of a variable for each axis. ................................... 59
Table 4.7. Relative mass distribution within chickpea seeds of PBA HatTrick. ....... 61
Table 4.8. Concentrations of macro-elements in the different chickpea parts. .......... 62
Table 4.9. Concentrations of micro-elements in the different chickpea parts. ........... 62
Table 5.1. List of characterisation studies done on NAS from selected species. ....... 68
Table 5.2. List of primers used in qPCR. ................................................................... 72
Table 5.3. List of cloning primers. ............................................................................. 73
Table 5.4. List of primers used for screening. ............................................................ 76
Table 5.5. Similarity between the CaNAS and OsNAS amino acid sequences. ........ 77
Table 5.6. Summary of transgenic tobacco lines generated and progressing to
the T1 generation. ........................................................................................ 90
Table 6.1. Summary of modifications made to the original protocol. ..................... 102
Table 6.2. List of primers used for qPCR. ............................................................... 103
Table 6.3. Summary of transgenic lines generated. ................................................. 108
Table 8.1. Profile of ferrosol soil from Kingaroy (Chauhan, 2015). ........................ 133
Table 8.2. Concentration of macro-elements in dry chickpea seed. ........................ 134
Table 8.3. Concentration of micro-elements in dry chickpea seeds. ........................ 135
Table 8.4. List of proteins used in the phylogenetic analysis. ................................. 136
The Characterisation of CaNAS2 and Biofortification of Chickpea xi
Table 8.5. Germination rates and segregation of transgenic chickpea lines. ........... 139
xii The Characterisation of CaNAS2 and Biofortification of Chickpea
List of Abbreviations
Abbreviations
aa = Amino acids
BAP = 6-benzylaminopurine
BLAST = Basic Logical Alignment Tool
bp = Base pairs
Ca = Cicer arietinum (chickpea)
CaMV = Cauliflower mosaic virus
cDNA = Complementary DNA
CTAB = Cety trimethyl ammonium bromide
C-terminal = Carboxyl- terminal
DEPC = diethylpyrocarbonate
dH2O = Distilled water
DIG = Digoxygenin
DMSO = Dimethyl sulphoxide
dNTPs = Deoxyribonucleotide triphosphates
DTT = 1, 4-dithiothreitol
2, 4,-D = 2, 4-dichlorophenoxyacetic acid
EDTA = Ethylenediaminetetraacetic acid
E.coli = Escherichia coli
GUS = Β-glucoronidase
HDPE = High-density polyethylene
ICP-OES = Inductively coupled plasma atomic emission
spectroscopy
IPTG = Iso-propyl-β-D-thiogalatopyranoside
LA-ICP-MS = Laser-abalation inductively coupled plasma mass
spectroscopy
LB = Luria-Bertani
MES = 2-(N-morpholino)ethanesulfonic acid
MS = Murashige and Skooge media
NAA = α-napthalene acetic acid
NCBI = National Centre for Biotechnology Information
nos = Nopaline synthase
N-terminal = Amino terminal
The Characterisation of CaNAS2 and Biofortification of Chickpea xiii
ORF = Open reading frame
Os = Oryza sativa (rice)
PCR = Polymerase chain reaction
PP = Polypropylene
qPCR = Quantitative real-time polymerase chain reaction
RNase = Ribonuclease
RT-PCR = Reverse transcription polymerase chain reaction
SDS = Sodium dodecyl sulphate
SSC = Saline sodium citrate buffer
TAE = Tris acetate EDTA
Tris = Tris(hydroxymethyl)aminomethane
TPS = Tris-phosphate buffer
Tween20 = Polyoxyethylene (20) sorbitan monolaurate
UC = University of California
uidA = Reporter gene encoding β-glucuronidase
X-gal = 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside
xiv The Characterisation of CaNAS2 and Biofortification of Chickpea
Units
°C = Degrees Celcius
d = days
Da = Daltons(s)
g = Gram(s)
g = Relative centrifugal force in units of gravity
h = Hour(s)
L = Litre(s)
M = Molar
m = Metre(s)
MW = Molecular weight
min = Minute(s)
mol = Mole(s)
rpm = Revolutions per minute
s = Second(s)
V = Volt(s)
vol = Volume(s)
v/v = Volume per volume
W = Watt
w/v = Weight per volume
Prefixes
G = Giga (109)
M = Mega (106)
k = Kilo (103)
c = Centi (10-2)
m = Milli (10-3)
µ = Micro (10-6)
n = Nano (10-9)
p = Pico (10-12)
The Characterisation of CaNAS2 and Biofortification of Chickpea xv
Statement of Original Authorship
The work contained in this thesis has not been previously submitted to meet
requirements for an award at this or any other higher education institution. To the
best of my knowledge and belief, the thesis contains no material previously
published or written by another person except where due reference is made.
Signature:
Date:
QUT Verified Signature
January 2018
xvi The Characterisation of CaNAS2 and Biofortification of Chickpea
Acknowledgements
Alas, it is time. It has been a long, tumultuous journey in so many ways, and to
have come thus far would have been impossible alone. All glory to my Lord and
Saviour, Jesus Christ, who has brought me to where I am despite my constant
failings and flaws, who never gave up on me even when I gave up on myself. Thanks
be to Him too, for the many people with whom these past years have been shared.
For all its difficulties and challenges, it has been a time most precious and blessed.
To the Fantastic Four (aka my wonderful supervisory team): Saga, Brett,
Sudipta and Alex – words can scarce express my gratitude. One could not ask for
better mentors or role models, and my growth as a scientist would not have been
possible without all of you. Beyond the science, you all have also taught me more
about being a better person. Thank you for this chance to do this PhD, for seeing in
me more than I saw in myself, and for challenging me to reach new heights which I’d
otherwise never dare aspire to. I would not have come so far without your
immeasurable patience or constant encouragement and guidance.
To the Tropical Pulses group and the greater Abiotic Stress group as a whole,
thank you for being not just an amazing team, but also a family away from home. To
my friends and colleagues in the CTCB, thank you too for your guidance, friendship
and support around the lab. You guys are an incredible bunch, and it’s been an
honour to be able to work alongside all of you.
I would also like to thank following people for their invaluable contributions in
the various components of this project.
Dr Alex Johnson, for providing the genes used in this study.
Hao, for his guidance and help with the molecular work.
TJ Higgins, for his guidance and mentorship in the chickpea transformation
process, as well as getting our presentations up to shape.
Sudipta and Alam, for their guidance and assistance in the tissue culture and
glasshouse work.
The Characterisation of CaNAS2 and Biofortification of Chickpea xvii
Charlotte, Karine, Aarshi and Sunny, for their advice, assistance and support
in the elemental analysis. Special shout-out to Bulu as well, for teaching and
watching over me during extraction process.
Tom, Col, Rex and Yash, for provision of the chickpea seeds and
environmental data used in Chapter 4. Many thanks for humouring me in
this spontaneous endeavour that somehow evolved into a chapter of its own.
Julien, for the crash course on Geneious and phylogenetics.
Rob and JY, for sharpening up my writing and presentation skills during
Honours, and setting the foundation for my PhD life.
Associate Prof Terry Walsh and Professor Martin Sillence, for reviewing
my major milestones in this PhD. Their feedback at the different stages of
this PhD was most constructive and invaluable.
Dani, the ever reliable manager of the CTCB labs, for her meticulous work
in managing the day-to-day business in the lab. Thank you also for teaching
me how to be more organised, a trait I sorely needed improvement in. Your
teachings have saved my hide more times than I care to count.
I would also like to thank the Queensland Government for their funding for the
Tropical Pulses for Queensland project, without which this PhD would not exist.
To my family and friends, thank you for being there for me. A lot of things
have happened over these years and your support and encouragement has kept me
afloat in the storms. Though geography separates us, you all are ever in my heart and
prayers.
To all of you who have walked with me one way or another, thank you. Thank
you for your love and kindness, for your patience and constant guidance. Thank you
for putting up with me even as I fumbled through life trying to be a better human
than I was yesterday. This would’ve not been possible without you. God bless.
Chapter 1: Introduction 1
Chapter 1: Introduction
Despite food production keeping up with the burgeoning global population, the
problem of micronutrient deficiency has yet to be eradicated. Iron deficiency in
humans in particular, is a worldwide problem in both developed and developing
nations, affecting approximately 30% of the global population. The shifting focus
from quantity to quality and the development of management and food processing
methods aim to enhance the nutritional value of food. However, while effective, such
measures are not always feasible and may be limited by the economic status of the
targeted demographics.
Breeding for self-fortifying plants, also known as biofortification is a
sustainable means for the delivery of deficient nutrients; the one-time cost of
development is negated by the long-term benefits. Biofortification can be achieved
through conventional breeding or genetic modification. Conventional breeding is
extremely time-consuming as the lack of specificity may result in the loss of
desirable traits over successive generations, requiring many years and generations of
plants before a product is ready for use. The degree of nutritional enhancement is
also dependent on the available gene pool, and this can be particularly problematic in
plants with low fertility and diversity. In contrast, genetic modification is highly
specific, allowing for the addition of desired traits without loss of existing agronomic
qualities and effectively reducing the time required for product development.
Additionally, versatility of the techniques used allows for the expansion of the range
of available traits by tapping into the genetic resources of other species.
This project focused on the iron biofortification of the important leguminous
crop, chickpea, through genetic modification. Two components involved in iron
homeostasis, nicotianamine synthase (NAS) and ferritin, have been effectively used
to enhance plant iron content and bioavailability in other crop species. The effect on
legumes remains unknown. This project is the first known attempt at the iron
biofortification of legumes, specifically chickpea, through genetic modification. It is
hypothesized that transformation with NAS and ferritin will enhance the iron content
and bioavailability of chickpea, and thus provide a sustainable and affordable means
to alleviate global iron deficiency.
2 Chapter 1: Introduction
1.1 AIMS AND OBJECTIVES
The project aimed for the biofortification of chickpea for enhanced iron content
through genetic engineering. Considering that chickpea naturally has higher iron
content than rice, from which most existing iron biofortification genes have been
sourced, there exists a possibility that more efficient components can be found in
chickpea. As such, this project included a study of the currently uncharacterised
NAS2 homologue in chickpea with the purpose of assessing its effectiveness in
biofortification strategies in comparison to the well-characterised OsNAS2.
The aims of this project were accomplished through the following objectives:
1. Molecular characterisation of chickpea NAS2
2. Design and construction of vectors for plant transformation
3. Generation of transgenic chickpea expressing OsNas2, CaNas2 and
soybean ferritin
The outline of the project is as illustrated below:
Chapter 2: Literature Review 3
Chapter 2: Literature Review
2.1 WORLD POPULATION AND MALNUTRITION
The current world population stands at an estimated 7.3 billion (United
Nations, 2015) and is projected to increase by 2 billion over the next four decades.
Concomitant to this growth is the challenge of providing sustenance amidst
dwindling resources. Currently food production is adequate at approximately four
billion metric tonnes per annum, yet in spite of this, about 870 million people still
suffer from chronic malnutrition due to factors like unequal distribution, wastage and
poor diets (FAO, 2012; IMECHE, 2013).
Malnutrition, as defined by the World Health Organization (WHO), is “the
cellular disparity amid the supply of energy, nutrients and the body’s demand for
them to ascertain maintenance, growth and specific functions” (Batool et al., 2013).
It refers to both the insufficient and excessive intake of nutrients (both macro and
micro) and as such covers not only food shortage but also obesity. Undernourishment
can be classified categories: protein-energy malnutrition and micronutrient
deficiency. As the names suggest, the former refers to inadequate calorie or protein
intake while the latter to the lack of essential micronutrients such as vitamin A,
iodine, zinc and iron (Batool et al., 2013).
While both pose significant risks to health and negatively affect overall
productivity and quality of life, micronutrient deficiency, also known “hidden
hunger”, is perhaps the more pervasive and lethal due to the lack of visible effects. It
is consequently more difficult to identify and tackle, and afflicts both developing and
developed nations
2.1.1 Iron deficiency anaemia
Among the various kinds of micronutrient deficiencies in humans, iron
deficiency is the most prevalent, afflicting more than two billion individuals
worldwide (WHO, 2008). It has been identified as the greatest contributor to
anaemia, accounting for 66.2% of cases globally (Alvarez-Uria et al., 2014). The
extent of its impact is such that the terms are used interchangeably and the
prevalence of anaemia is used as a measure for the more specific iron deficiency
4 Chapter 2: Literature Review
anaemia (IDA) (WHO, 2001). IDA can be attributed to three main factors –
increased iron requirement (e.g. growth and pregnancy), poor absorption, and
inadequate dietary intake. The recommended values for daily iron intake for human
adults are 8 mg for males and 18 mg for females (Trumbo et al., 2001), and
insufficient intake impedes the formation of biologically important compounds, most
notably haeme, resulting in anaemia. Symptoms include fatigue, loss of energy, and
dizziness, all of which diminish the work capacity of the individual. Iron deficiency
also results in poor pregnancy outcomes and impediment of physical and cognitive
development, thereby increasing the risk of morbidity in children (WHO, 2008).
Table 2.1. Recommended Dietary Allowances (RDAs) for iron (Trumbo et al.,
2001).
mg/day
Age Male Female Pregnancy Lactation
Birth to 6 months 0.27 0.27
7–12 months 11 11
1–3 years 7 7
4–8 years 10 10
9–13 years 8 8
14–18 years 11 15 27 10
19–50 years 8 18 27 9
51+ years 8 8
This presents a problem of great economic and social significance, particularly
in developing countries where approximately 50% of pregnant women and 40% of
preschool children suffer from IDA (WHO, 2008). The consequences of this
manifest not only in the form of lives lost, but also in a rising generation of
individuals afflicted with developmental complications. While considerable progress
to reduce IDA has been made in several countries, it remains a significant a problem
given that a prevalence rate below 10% has yet to be seen in any country (see Figure
2.1).
Out of the three major risk factors contributing to IDA, the issue of dietary
intake is the most feasible to address on a large scale. Efforts to remedy the problem
include food-based strategies like dietary diversification, food fortification and
Chapter 2: Literature Review 5
supplementation. However, while such measures have proven to be effective in
alleviating the problem of iron deficiency, they also incur a recurring cost and the
beneficiaries are limited to those who can afford it, namely those in developed
countries. Consequently such measures are unfeasible for the low-income
demographics that, incidentally, have the greatest need. The challenge then is to
develop a cost-effective means to deliver the required nutrients to the vulnerable
parties.
Figure 2.1. Global burden of anaemia across all ages in A) 1990 and B) 2013
(Kassebaum, 2016).
2.2 BIOFORTIFICATION
One such means of nutrient delivery is biofortification, which can generally be
defined as the enhancement of nutritional quality in the edible portions of food crops
during plant growth (HarvestPlus, 2015b; WHO, 2016). The precise definition of the
term “biofortification” may vary depending on the scope of the means (HarvestPlus,
2015b; WHO, 2016); for the purpose of this review it shall be used to refer solely to
the generation of self-fortifying plants, to the exclusion of agronomic interventions
(e.g. fertiliser application, management practices etc.).
6 Chapter 2: Literature Review
Biofortification emerged within the last two decades as an approach to combat
micronutrient deficiency. While it cannot be considered a cure-all to micronutrient
deficiency, it alleviates the problem by complementing existing strategies like the
aforementioned ones of dietary diversification, fortification and supplementation.
With the one-time cost of development negated by the long term benefits,
biofortification presents a sustainable means of delivering the needed micronutrients
across large spatial and temporal scales (Nestel et al., 2006; Horton et al., 2008; De
Moura et al., 2014; HarvestPlus, 2015b).
Currently there exists two means of generating biofortified crops. The first is
conventional breeding, in which the desired traits are selected for and traditionally
bred into successive generations. The second is genetic modification (GM), in which
the genetic material of the host is altered in a manner that does not occur naturally.
Each method has its own advantages and disadvantages which will be discussed in a
later section. Irrespective of the means however, is the underlying principle of
manipulating the associated metabolic pathways which, in this case, is iron. Some
measure of understanding in that aspect is therefore required for effective
biofortification. With regards to that, a wealth of information has been gleaned and
reviewed extensively over the last 30 years in particular, fuelled by advances in
technology and analytical methods and growing interest in biofortification and
bioremediation (e.g. Briat et al., 1995; Hell and Stephan, 2003; Kim and Guerinot,
2007; Jeong and Guerinot, 2009; Thomine and Lanquar, 2011; Hindt and Guerinot,
2012; Kobayashi and Nishizawa, 2012).
2.2.1 Approaches to biofortification
Generation of biofortified crops can be done through two ways: conventional
breeding and/or genetic modification. Selection of desired traits by conventional
breeding is a practice that has existed since the advent of agriculture. Traditionally a
long-term process requiring much investment of time and effort, advances in
technology and molecular biology has since shortened the process and increased its
precision when targeting specific traits. Several quantitative trait loci (QTLs) for iron
accumulation has been identified in rice (Norton et al., 2010; Anuradha et al., 2012),
wheat (Xu et al., 2012), maize, (Jin et al., 2013) and bean (Blair et al., 2009; Blair et
al., 2010). Already, several crops have been developed through conventional
breeding under the HarvestPlus program and their success has been demonstrated in
Chapter 2: Literature Review 7
several feeding trials. Consumption of biofortified pearl millet improved iron
adsorption and iron stores in women and children (Cercamondi et al., 2013; Kodkany
et al., 2013; Finkelstein et al., 2015), while biofortified rice have been found to help
maintain the iron stores of non-anaemic women (Haas et al., 2005).
Despite its effectiveness, the extent to which biofortification can be done
through conventional breeding is limited to the diversity in the gene pool and fertility
of the species. In cases where such limitations prevail, genetic modification provides
an alternative pathway.
2.2.2 Target crops
To date, starchy staples that contain little micronutrients like cereals, root
crops, and banana have the primary targets for iron biofortification (Namanya, 2011;
HarvestPlus, 2015a; Banana21, 2016). The advantages of such targets is that they
form the bulk of local diets and given proper processing, have a long shelf-life,
allowing for efficient delivery of the biofortified micronutrient over a large spatial
and temporal scale. A wealth of information has been generated concerning these
crops as a consequence of extensive focus. Biofortification works using genetic
modification in particular, have largely concentrated on major graminaceous crops
like rice, wheat and maize. In contrast, aside from banana (Matovu, 2016) and lettuce
(Goto et al., 2000), existing studies in non-graminaceous plants were conducted
mainly in model species like tobacco or Arabidopsis for characterisation purposes.
Given the physiological differences between the non-graminaceous and
graminaceous plants, it is difficult to extrapolate the effectiveness of iron
biofortification approaches in the latter to the former. As it stands, there remains
much to be explored in terms of iron biofortification of non-graminaceous crops.
2.3 PULSES AS A VEHICLE FOR BIOFORTIFICATION
Aside from the aforementioned starchy staples, another group of crops have
been targeted for biofortification, albeit to a lesser degree. Pulses, as defined by the
FAO (1994), are leguminous crops harvested for solely for dry grain. Like cereals,
they have a long history of cultivation and have been a significant constituent in
human diets since around 10, 000 BC (Fuller et al., 2001; Caracuta et al., 2015). As a
crop, pulses present two main benefits, both of which are complementary to cereals.
The first is their agronomic characteristics. By virtue of their nitrogen fixing
8 Chapter 2: Literature Review
properties, pulses are often grown as an intercrop or as a mixed crop to replenish soil
nitrogen levels, thereby reducing the need for fertilisers. Cultivation with pulse crops
have also been shown to increase the uptake of nitrogen, sulphur and phosphorus by
cereals, resulting in an enhanced yield and grain quality (Li et al., 2003; Li et al.,
2004b; Agegnehu et al., 2006; Banik et al., 2006; Gooding et al., 2007). Yield
stability in also increased (Rao and Willey, 1980).
The second benefit of pulses is their nutritional density. Pulses are a rich source
of carbohydrates and fiber. Their most prominent feature however, is their high
protein content of 21–26% and an amino acid profile complementary to that of
cereals, being rich in lysine, leucine and arginine (Phillips, 1993; Iqbal et al., 2006;
Pulse Canada, 2016). Its excellence as a vegetarian source of protein and
affordability in contrast to livestock products has earned it the famous moniker of
‘poor man’s meat’. Pulses are also rich in micronutrients like folate, thiamine,
riboflavin, niacin, calcium, magnesium, iron and zinc (Phillips, 1993; Iqbal et al.,
2006; Jukanti et al., 2012). Other than contributing to the macro- and
micronutritional needs, several health benefits have been associated with inclusion of
pulses in the diet. Their low glycemic index (GI) has been linked to the management
of diabetes and diabetes-related diseases (Rizkalla et al., 2002; Sievenpiper et al.,
2009) while bioactive components have been investigated for their health potential –
e.g. lectins for their immunomodulatory effect, protease inhibitor for anti-
inflammatory effect, and angiotensin I-converting enzyme (ACE) inhibitory peptides
for their anti-hypertensive properties (Rochfort and Panozzo, 2007; Roy et al., 2010).
2.3.1 Pulse production and market
Despite their agronomic and nutritional benefits, pulses have not received the
same amount of attention or development as the main starchy staples. Between 1961
and 2014, pulse yield and production values increased by 42.3% and 90.4%
respectively, a small fraction compared to the increase of 187.2% and 219.4% in
cereals (FAO, 2016b). Much of this disparity can be attributed to developments made
during the Green Revolution, in which the focus on productivity and protein-calorie
malnutrition led to the shift from cultivation of traditional micronutrient-rich crops to
the more productive and profitable starchy cereals (Pinstrup‐Andersen and Hazell,
1985; Pingali, 2012). Poor policy and diversion of land to cereal cultivation has led
to a reduction in pulse supply, effectively driving prices up and decreasing
Chapter 2: Literature Review 9
consumption per capita (Kennedy and Bouis, 1993; Kataki, 2002; Akibode and
Maredia, 2012).
As highlighted in the special feature on pulses in the 2014 Food Outlook (FAO,
2014), recent years have seen several key changes in pulse production and trade.
Asia remains the region with the highest pulse production, with India continuing as
the largest pulse producing country, contributing at least 20% towards global pulse
production (Figure 2.2, Table 2.2). Production in other regions except Europe has
also increased, fuelled by domestic and international demand. In contrast to these
countries is China, whose production has decreased due to a number of factors such
as population increase and decreasing availability of arable land. Despite the shift in
preference for animal-based products and protein that accompanies growing
affluence, India and China remain as major importers, consuming approximately
40% of the world’s pulse production as food, and 30% as feed. Much of this is
provided by major exporters like Canada and Australia. With other major producers
like Myanmar and Brazil, pulse consumption is primarily domestic.
Pulse production, consumption and trade are expected to increase alongside
population growth, particularly with increasing promotion from government
campaigns (Akibode and Maredia, 2012) and the declaration of 2016 as the
“International Year of Pulses” by the UN General Assembly. Increasing awareness
and concern over nutritional composition of food, particularly by food
manufacturers, has attracted greater interest in pulses, which will likely translate into
further support and development of pulses and the industry (FAO, 2014).
10 Chapter 2: Literature Review
Figure 2.2. Average pulse production by region (FAO, 2016a).
Table 2.2. Growth in production of major pulse producers (FAO, 2014, 2016b).
Production (mmt)
Country 1961 1981 2001 2014 Principle pulse crops
India 12.9 10.8 12.2 19.98 Chickpeas, beans, pigeon peas
Myanmar 0.2 0.4 2.0 5.0 Beans, pigeon peas, chickpeas
Canada 0.1 0.2 3.4 5.8 Peas, lentils
China 8.5 6.4 5.1 4.5 Beans, broad beans, peas
Brazil 1.8 2.4 2.5 3.3 Beans
Nigeria 5 0.6 2.3 2.2 Cowpeas
Ethiopia 0.6 0.9 1.2 2.6 Broad beans, beans, chickpeas,
peas
Australia 0 0.3 2.7 3.0 Lupines, lentils, chickpeas
USA 1.1 1.7 1.3 2.4 Beans, peas
Tanzania, U.
Rep. 0.1 0.3 0.8 1.8 Beans
Rest of the world 15.0 17.5 22.6 27.02
Total 40.8 41.6 55.9 77.6
2.3.2 Challenges
Pulses are a diverse group featuring a wide variety of species and cultivars, and
this genetic richness is a treasure trove that lends itself to crop improvement.
However this diversity has also contributed to the lack of a concerted global effort,
with production and development being dispersed across various localities (FAO,
2014). Such development thus far have been on yield, disease tolerance and
macronutrient quality, though in the last decade there has been a growing interest in
micronutrient content, with increasing numbers of genotypes and cultivars being
Chapter 2: Literature Review 11
assayed for their iron and zinc composition (Blair et al., 2013; Thavarajah et al.,
2014). In spite of this there has been little published work on pulse biofortification.
Currently the only known example of a biofortified pulse is the high iron common
bean generated from the HarvestPlus breeding program; to date, several varieties
have been produced with improvements in iron content ranging from 47 – 94%
(Katsvairo, 2015). Despite such success, bioavailability of this iron remains a
problem.
Bioavailability, as defined by Carpenter and Mahoney (1992), is the
“proportion of a nutrient present in food that the body is able to absorb and utilise by
incorporation into physiologically functional pools”. As with other plant-based
foods, iron found in pulses is non-heme iron which has lower bioavailability
compared to its heme counterpart (Björn-Rasmussen et al., 1974). This
bioavailability is further subject to other factors such as those listed in Table 2.3.
Amongst these, higher inherent levels of antinutritional factors like polyphenols and
phytate have been identified as major contributors to the poor iron bioavailability in
pulses.
This trait has presented a particular challenge to pulse biofortification efforts.
Despite the success of increasing overall iron content, feeding trials conducted in
Rwanda have indicated iron bioavailability of biofortified beans were to be similar, if
not lower, compared to the unfortified beans (Petry et al., 2012, 2014). This has been
ascribed to the influence of phytic acid, and whose reduction in concentration was
recommended by the authors as a means to improve the effectiveness of
biofortification (Petry et al., 2012, 2014).
This recommendation has been applied in several cereal crops (Larson et al.,
1998; Larson et al., 2000; Raboy et al., 2000; Guttieri et al., 2004) and more recently
in bean (Campion et al., 2009). The effectiveness of the low-phytic acid bean lines is
currently inconclusive however, as bioavailability assessments have yielded
conflicting results due to differences in experimental design (Petry et al., 2013; Petry
et al., 2016). Poor cooking quality was also observed in the low-phytic acid seeds,
which may have contributed the adverse gastrointestinal side-effects in the
participants in one of the studies (Petry et al., 2016). The relationship between phytic
acid and cooking quality have been alluded to in other studies on lentil and bean
(Kon and Sanchuck, 1981; Bhatty and Slinkard, 1989). Interestingly, no such effect
12 Chapter 2: Literature Review
was reported in low-phytic acid maize lines (Mendoza et al., 1998); whether this this
is a legume-specific issue remains to be confirmed. Aside from influencing cooking
quality, phytic acid is also known to have antioxidant properties and protective
effects against heart disease and cancer (Sharma, 1986; Nelson et al., 1988; Vucenik
and Shamsuddin, 2003). It is unknown if reduction in phytic acid content would
affect such properties. Similarly, the subsequent long-term effect on human health is
unknown.
Currently the bulk of existing knowledge is limited to the work done on the
common bean as it is currently the forefront in pulse iron biofortification efforts.
Despite the aforementioned challenges, the iron biofortified bean has proven to be a
successful means of alleviating iron deficiency, promising much for work in other
pulses.
Table 2.3. Factors affecting bioavailability of some trace elements (House, 1999).
Host factors Dietary factors
Dietary composition Food preparation
Age
Sex
Ethnic background
o Types of food
selected
o Geographic
living area
Economic status
o Type, quality
and quantity of
selected food
Physiological status
o Pregnancy
o Lactation
o Physical
activity
Nutritional status
o Moderate or
frank
deficiency
o Lean body
mass
Disease (including
parasitism)
Protein quality
o Protein source
o Animal vs plant protein
o Amino acid balance
Protein quantity
Trace element quantity
Physiochemical form of trace
element
Nutrient interactions
o Element–element
o Element–organic compounds
Promoters
o Meat
o Ascorbate
o Citrate
o Vitamin D
o Some amino acids
o Some sugars
Inhibitors
o Phytate
o Oxalate
o Polyphenols
o Fiber
o Goitrogens
o Excess ascorbate or folate
Micronutrient deficiencies
o Ascorbate
o Riboflavin
o Vitamin E
Raw
Cooking (various
methods)
Fermentation
Malting
Milling
Extraction
Soaking
Chapter 2: Literature Review 13
2.3.3 Chickpea
Chickpea (Cicer arietinum) is an important pulse crop that has been cultivated
by humans since the Stone Age. As of 2009, it is the second most important pulse
crop in the world after the common bean, having overtaken peas as the pulse crop
with the second highest global production values. Global production has climbed
steadily since 2008 to exceed 14.2 million tonnes in 2014, of which approximately
96 % is grown in developing countries (FAO, 2016b). India in particular, has
historically been the largest producer and consumer of chickpea; in 2013 alone it
contributed approximately 65% and 33% to total chickpea production and import
respectively (FAO, 2016b). In terms of consumption, it is difficult to obtain precise
statistics due to the lack of available data. However based on calculations using
production and trade values, the global average for chickpea consumption was
estimated to be around 1.3kg/year per person between 2006 and 2008, with South
Asia and the Middle East-North Africa regions being the biggest consumers at 4.25
kg/person and 2.11 kg/person per year respectively (Akibode and Maredia, 2012).
The demand is predicted to grow, particularly in Africa and Asia, due to population
increase and increasing support from the governments in encouraging pulse
consumption (Rao et al., 2010; Akibode and Maredia, 2012). This increase in
demand is not limited to those regions; in the USA for instance, net domestic use of
chickpea nearly doubled from 199.6g in 2010 to 322.1g in 2014 (Wells, 2016).
Much like other pulses, the nutritional qualities of chickpea have long been
recognised and documented. In addition to high protein content (20-22%), chickpeas
are also rich in micronutrients like folate, magnesium, zinc and iron (Table 2.4)
(USDA, 2013). Studies conducted by different authors have found iron content to
range from 2.4 to 11 mg/100g (e.g. USDA; Meiners et al., 1976; Wood and Grusak,
2007). Likewise, various studies have reported differing values for phytic acid and
other antinutrients (e.g. Chitra et al., 1995; Ghavidel and Prakash, 2007; Hemalatha
et al., 2007b), indicating a possible effect of genotype and environmental factors on
overall iron bioavailability. When measured as dialyzable iron generated from a
simulated gastrointestinal digest, bioavailability has been found to vary widely across
different studies, ranging from about 6% to 25% (Chitra et al., 1997; Ghavidel and
Prakash, 2007; Hemalatha et al., 2007b). The reason behind this disparity is as yet
unclear, though analytical procedures and variations in samples have been suggested
14 Chapter 2: Literature Review
Table 2.4. Nutritional values per 100g of chickpea and important staple crops (USDA, 2013).
Nutrient Unit Chickpea,
dried
Chickpea, boiled
without salt
Corn,
yellow
Wheat,
durum
Rice, white, medium-
grain, cooked Potato, raw
Cassava,
raw
White
sorghum,
raw
Proximates
Water G 11.53 60.21 10.37 10.94 68.61 83.29 59.68 9.2
Energy kcal 364 164 365 339 130 58 160 339
Protein G 19.3 8.86 9.42 13.68 2.38 2.57 1.36 11.3
Total lipid (fat) G 6.04 2.59 4.74 2.47 0.21 0.1 0.28 3.3
Carbohydrate, by
difference G 60.65 27.42 74.26 71.13 28.59 12.44 38.06 74.63
Fiber, total dietary G 17.4 7.6 7.3 - 0.3 2.5 1.8 6.3
Sugars, total G 10.7 4.8 0.64 - -
1.7 3.39
Minerals
Calcium, Ca mg 105 49 7 34 3 30 16 28
Iron, Fe mg 6.24 2.89 2.71 3.52 1.49 3.24 0.27 4.4
Magnesium, Mg mg 115 48 127 144 13 23 21 190
Phosphorus, P mg 366 168 210 508 37 38 27 287
Potassium, K mg 875 291 287 431 29 413 271 350
Sodium, Na mg 24 7 35 2 0 10 14 6
Zinc, Zn mg 3.43 1.53 2.21 4.16 0.42 0.35 0.34 1.54
Vitamins
Vitamin C, total
ascorbic acid mg 4 1.3 0 0 0 11.4 20.6 0
Thiamin mg 0.477 0.116 0.385 0.419 0.167 0.021 0.087 0.237
Riboflavin mg 0.212 0.063 0.201 0.121 0.016 0.038 0.048 0.142
Niacin mg 1.541 0.526 3.627 6.738 1.835 1.033 0.854 2.927
Vitamin B-6 mg 0.535 0.139 0.622 0.419 0.05 0.239 0.088 0.59
Folate, DFE µg 557 172 19 43 97 17 27 20
Chapter 2: Literature Review 15
as a possible cause (Platel and Srinivasan, 2016). Given the multifaceted nature
of nutrient bioavailability, the values obtained are at best relative.
In light of this, it would be prudent for biofortification efforts to first target
total seed iron content before progressing to bioavailability. Considerable progress
has been made to that end, particularly with the growing interest in chickpea as a
target for iron biofortification. While a concerted global effort has yet to materialise,
pockets of development have emerged with India and Canada at the forefront. To
date, the chickpea genome has been sequenced (Varshney et al., 2013). Chickpea
populations in those countries have also been screened for genetic diversity and iron
accumulation traits, allowing for identification of the associated QTLs (quantitative
trait loci) (Diapari et al., 2014; Upadhyaya et al., 2016). In terms of biofortification
via genetic modification, no work done has been yet. It is however, a viable option –
while chickpea can be considered a recalcitrant species, successful transformation
protocols have been established (Sarmah et al., 2004; Indurker et al., 2010).
Given the relative youth of this endeavour to biofortify chickpea for iron, no
biofortification targets have yet been set. As stated by Bouis and Welch (2010),
several factors need be considered in the setting of such targets. Unlike the common
bean, which is a staple, chickpea is a secondary staple and depending on the type and
cultivar, may be processed into various forms for consumption. This would in turn
affect iron content and bioavailability. Consequently, the consumption profile for
chickpea is lower and potentially more varied compared to the common bean,
particularly across different age and cultural demographics.
As with other crops, the challenge in setting biofortification targets lies
primarily in the lack of information concerning the different variables (Bouis and
Welch, 2010). Until more detailed and specific information is obtained, only gross
assumptions may be made.
Table 2.5 illustrates a crude estimate of the iron biofortification target for
chickpea, calculated using a formula modified from Bouis and Welch (2010). The
targeted demographic in this case was adult, non-pregnant, non-lactating females
from the South Asian region.
16 Chapter 2: Literature Review
Table 2.5. Information and assumptions used to set target levels for iron
biofortification in chickpea.
*EAR (µg/day) 1460
10% EAR 146
Per capita consumption (g/day) 10
Baseline Fe content (µg/g) 5
Bioavailability (%) 6
Fe retention after processing (%) 85
Additional Fe required (µg/100g) 121
Final target as dry weight (µg/100g) 286
* – Estimated Average Requirement
2.4 IRON METABOLISM IN PLANTS
Prior to attempting any biofortification strategies, the significance of iron in the
plants, as well as the underlying mechanisms governing its metabolism, must first be
understood. Iron is the fourth most common element in the Earth’s crust and can
exist in a wide range of oxidation states, of which the most common are the ferrous
(Fe2+) and ferric (Fe3+) forms. By virtue of its high redox potential, it forms a key
component of biological processes involving electron exchange such as DNA
synthesis, oxygen transport, cellular respiration and photosynthesis, where it
participates in the form of a cofactor in iron complexes. Examples of such complexes
include haemoglobin, chlorophyll, DNA helicases, and catalase.
For all its biological significance however, iron metabolic pathways can be
summarised with the imagery of a precarious transfer of a nuclear material between
containment facilities. Biologically, free iron may result from iron overload and/or
insufficient sequestration capacity of the organism (Pietrangelo, 2003). Left alone,
free Fe2+ catalyses the formation of hydroxyl (OH) radicals through the Fenton
reaction, and the process repeats when Fe2+ is regenerated from the resultant Fe3+
through reduction by the superoxide radical (O2-) (Haber and Weiss, 1934). The
summation of this self-perpetuating reaction is known as the Haber-Weiss reaction:
𝑂2− + 𝐹𝑒3+ → 𝑂2 + 𝐹𝑒2+
𝐹𝑒2+ + 𝐻2𝑂2 → 𝐹𝑒3+ + 𝑂𝐻− + ∙ 𝑂𝐻
Chapter 2: Literature Review 17
𝑂2− + 𝐻2𝑂2 → 𝑂2 + 𝑂𝐻− + ∙ 𝑂𝐻
Reactive oxygen species (ROS) generated as a consequence of this reaction can
react with cellular components to cause oxidative damage (Kehrer, 2000; Aisen et
al., 2001; Papanikolaou and Pantopoulos, 2005; Jeong and Guerinot, 2009;
Kobayashi and Nishizawa, 2012); however on the other hand, they also serve as
important signalling molecules and are an integral part of the stress response (Apel
and Hirt, 2004). The fine line between cytotoxicity and biological function, and the
intimate association between iron and ROS production, highlights the significance of
proper regulation of iron metabolic pathways.
Iron metabolic pathways can be divided into three main processes: uptake,
translocation and storage. Despite its abundance iron has poor solubility under
aerobic conditions, particularly in high pH and calcareous soils, necessitating its
solubilisation before uptake can occur. This process is mostly accomplished via root
exudates, the composition of which varies in response to the plant’s physiological
state and needs. In response to iron deficiency, the plant triggers the production of
factors that directly or indirectly aid iron solubilisation. Enhanced concentrations of
glutamate, ribitol and glucose were observed in the root exudates of iron-deficient
maize, which were suggested to attract and support siderophore-producing bacterial
communities to aid iron solubilisation (Carvalhais et al., 2011). Notable increases
were also observed in the production of organic acids like malate and citrate, which
increase the availability of iron through dissolution of insoluble iron compounds
(Jones et al., 1996; Sánchez-Rodríguez et al., 2014).
In addition to the aforementioned means, different plant species have adopted
specific approaches toward solubilise and acquire iron. These have been categorised
as Strategy I and Strategy II (Römheld and Marschner, 1986) (see Figure 2.3). It
should be noted that while differences between both strategies primarily affect the
uptake process, the involvement of molecular components in the translocation
process has further implications on the overall physiology; this will be discussed in a
later section.
In summary, Strategy I is a reduction-based strategy in which insoluble iron is
reduced via acidification of the rhizosphere. Phenolics are also secreted to chelate the
iron. Chelated iron is reduced at the root surface and the resulting Fe (II) ions are
18 Chapter 2: Literature Review
absorbed across the plasma membrane by specialised transporter proteins. Strategy I
is used by non-graminaceous plants, which includes all plants except for grasses.
Strategy II on the other hand, is used by graminaceous plants (grasses) and revolves
around the chelation of insoluble iron with secreted phytosiderophores, the
production and uptake of which is specific to Strategy II plants. The resulting Fe
(III)-phytosiderophore complex is subsequently taken up via specialised transporters.
Unlike the reduction-based approach used in Strategy I, phytosiderophore uptake is
not limited by high pH, thereby conferring an advantage where such conditions are
present (Römheld and Marschner, 1986).
The use of both strategies may be present in a single species, of which the only
known example is rice (Ishimaru et al., 2006). This combination may represent an
adaptation to the submerged conditions in which rice and its wild relatives grow,
where iron is more readily available in ferrous than ferric form; whether a similar
occurrence may be found in other species remains to be seen.
Figure 2.3. Fe acquisition strategies in higher plants: Strategy I in
nongraminaceous plants (left) and Strategy II in graminaceous plants (right).
Ovals represent the transporters and enzymes that play central roles in these
strategies, all of which are induced in response to Fe deficiency. Abbreviations:
DMAS, deoxymugineic acid synthase; FRO, ferric chelate reductase oxidase; HA,
H+ -ATPase; IRT, iron-regulated transporter; MAs, mugineic acid family
phytosiderophores; NA, nicotianamine; NAAT, nicotianamine aminotransferase;
NAS, nicotianamine synthase; PEZ, PHENOLICS EFFLUX ZERO; SAM, S-
adenosyl –L-methionine; TOM1 transporter of mugineic acid phytosiderophores 1;
YS1/YSL1, YELLOW STRIPE1/YELLOW STRIPE 1-like (Kobayashi and
Nishizawa, 2012).
Chapter 2: Literature Review 19
2.4.1 Strategy I – The reduction-based strategy
2.4.1.1 Proton extrusion
One of the components of Strategy I uptake is the acidification of the
rhizosphere via proton extrusion. While this directly encourages the protonation and
solubilisation of insoluble hydroxides (Schwertmann, 1991), a decrease in pH
facilitates uptake by enhancing Fe(III) reduction at the root surface (Wei et al., 1997)
and may also serve to promote release of other reducing (Römheld and Marschner,
1983) while stabilising organic acid-iron compounds (Jones et al., 1996).
Proton extrusion occurs through the action of H+-ATPases (HA), whose
expression has been found to increase upon detection of iron deficiency (Rabotti and
Zocchi, 1994; Dell'Orto et al., 2000; Santi et al., 2005; Santi and Schmidt, 2009). A
higher capacity for H+ release and rhizosphere acidification has been associated with
increased tolerance to iron deficiency (Wei et al., 1997; Mahmoudi et al., 2007).
However, studies in Arabidopsis and cucumber have found that not all isoforms are
involved with the iron deficiency response; some are serve as housekeeping genes or
are tissue-specific (Santi et al., 2005; Santi and Schmidt, 2009).
2.4.1.2 Phenolic production and exudation
Another component of the Strategy I uptake is the production, accumulation
and exudation of phenolics, particularly under iron deficiency (Römheld and
Marschner, 1981). Phenolics contribute directly to uptake by dissolving and
chelating insoluble iron in the soil (Dakora and Phillips, 2002). In addition, they also
facilitate remobilisation of the otherwise inaccessible apoplastic iron by stripping it
from cell walls in a process independent of proton extrusion and ferric-chelate
reductase oxidase (FRO) activity (Jin et al., 2007).
A more indirect effect on iron uptake is through management of the
rhizospheric microbial community, where their properties as both an attractant and a
form of plant defence allow for the selection for beneficial plant-microbial
interactions (Peters and Verma, 1990; Bhattacharya et al., 2010). Phenolics secreted
by iron-deficient red clover, for instance, select for siderophore and auxin producing
microbes, thereby enhancing soil iron bioavailability and root growth (Jin et al.,
2006).
20 Chapter 2: Literature Review
2.4.1.3 Reduction and absorption
Prior to uptake into the root cell, Fe3+ chelates must first be reduced to Fe2+ at
the root surface (Chaney et al., 1972). This is done through the activity of ferric-
chelate reductase oxidase (FRO) at the root surface, which reduces Fe3+ chelates to
Fe2+ by transferring electrons across the plasma membrane (Robinson et al., 1999;
Waters et al., 2002).
FRO is a family of membrane-bound metalloreductase that transfers electrons
from cytosolic NADPH across membranes to electron-accepting substrates on the
other side. In addition to facilitating acquisition of iron from the soil, this capability
is also utilised in localisations where iron reduction is required for transport and/or
assimilation, such as in the mesophyll (Brüggemann et al., 1993), reproductive
tissues (Waters et al., 2002; Li et al., 2004a) and chloroplast membranes (Jeong and
Connolly, 2009). Several members of this family has been identified and
characterised in various species, namely Arabidopsis (Robinson et al., 1997;
Mukherjee et al., 2006), pea (Waters et al., 2002), and tomato (Li et al., 2004a). Not
all isoforms are involved in iron acquisition from the soil; this is exemplified in the
study conducted by Wu et al. (2005), in which AtFRO5, AtFRO6, AtFRO7 and
AtFRO8 were found to be shoot specific, while only AtFRO2 and AtFRO3 were
expressed in the roots.
Following reduction at the root surface, the resulting Fe(II) ions are absorbed
across the plasma membrane via the iron-regulated transporter (IRT) (Eide et al.,
1996; Vert et al., 2002). IRT is a member of the zinc-regulated transporter, iron-
regulated transporter-like protein (ZIP) family that functions as membrane-bound
uptake transporter for Zn and Fe (Lin et al., 2009). In Arabidopsis and tomato, IRT1
has been identified as responsible for uptake from the soil (Bereczky et al., 2003;
Vert et al., 2009), with loss of function producing a severely stunted and chlorotic
phenotype (Varotto et al., 2002; Vert et al., 2002). Co-regulated with IRT1 is the
AtIRT2 homologue, which facilitates subcellular transport of iron and localises to
vesicle membranes instead of plasma membranes (Vert et al., 2009).
Both ferric chelate reduction and IRT activity is regulated in response to iron
concentrations and increases in response to iron deficiency (Robinson et al., 1999;
Connolly et al., 2003; Vert et al., 2003). Enhanced ferric reduction in particular, has
been considered a hallmark indicator of iron deficiency (Römheld and Marschner,
Chapter 2: Literature Review 21
1981; Higuchi et al., 1995) and increased capacity for FRO activity confers increased
tolerance to low iron (Connolly et al., 2003; Peng et al., 2015).
2.4.2 Strategy II – The phytosiderophore chelation strategy
Unlike Strategy I, Strategy II revolves around the use of the mugineic acid
(MA) family phytosiderophores in iron acquisition. The MA biosynthetic pathway
starts with the conversion of three units of S-adenosylmethionine (SAM) into
nicotianamine (NA) by nicotianamine synthase (NAS) (Higuchi et al., 1994; Higuchi
et al., 1999a). NA is converted to a 3” keto-intermediate by nicotianamine
aminotransferase (NAAT), before being reduced to deoxymugeneic acid (DMA) by
deoxymugeneic acid synthase (DMAS) (Kanazawa et al., 1994; Bashir et al., 2006).
DMA can subsequently be converted to other MAs through a series of
hydroxylations (Mori and Nishizawa, 1989; Ma and Nomoto, 1993), increasing
levels of which improves the affinity for Fe3+ and chelate stability under acidic
conditions (von Wirén et al., 2000). An overview of the MAs synthetic pathway is as
illustrated in Figure 2.4.
Figure 2.4. Biosynthetic pathway of mugeneic acid (MA) family of
phytosiderophores (Sharma and Dietz, 2006).
22 Chapter 2: Literature Review
Synthesized MAs are secreted into the soil through the phytosiderophore efflux
transporter TOM1 (Nozoye et al., 2011) and the resulting ferric complexes are then
taken up by the roots through specialized transporters like YELLOW STRIPE 1
(YSL1) and YELLOW STRIPE 1-like (YSL) (Curie et al., 2001; Murata et al., 2006;
Inoue et al., 2009; Kobayashi and Nishizawa, 2012).
Under iron deficiency, the phytosiderophore biosynthetic pathway is
upregulated (Kanazawa et al., 1994; Takahashi et al., 1999; Inoue et al., 2003;
Bashir et al., 2006), leading to an increase in the MAs secretion (Higuchi et al.,
1996; Fan et al., 1997). The pattern of secretion also switches from a constant one to
a diurnal rhythm (Mori et al., 1987; Walter et al., 1995; Nozoye et al., 2011).
Expression of YS1 and YSL is also upregulated (Koike et al., 2004; Murata et al.,
2006).
2.4.2.1 Nicotianamine synthase (NAS) and nicotianamine (N A)
NA is a non-proteogenic amino acid synthesised from three units of
adenosylmethionine in a reaction catalysed by NAS. In addition to serving as a
precursor for the mugeneic acid family of phytosiderophores (Higuchi et al., 1999b),
it is also a common component in the translocation process in both Strategy I and II,
where it functions primarily as a chelator of divalent transition metals (Scholz et al.,
1992).
NA has mainly been associated with iron, zinc and, to a lesser extent,
cadmium, nickel, manganese and copper (Stephan et al., 1996; Inoue et al., 2003;
Takahashi et al., 2003a; Weber et al., 2004; Mari et al., 2006), forming stable
complexes at pH >6 (Stephan et al., 1996). Like citrate, it is a key chelator of metals
in plants (von Wirén et al., 1999; Takahashi et al., 2003a; Klatte et al., 2009) and
contributes to the translocation of metals in the vascular tissue (Stephan et al., 1994a;
Mari et al., 2006). However, unlike citrate-metal complexes which deliver to older
leaf tissues, NA-metal complexes are able to transport metals out of the vascular
bundle into the interveinal regions of leaves and reproductive organs (Takahashi et
al., 2003a; Schuler et al., 2012a).
As a result of this role, NA exerts considerable influence on the regulation of
the iron deficiency response mechanism in both graminaceous and non-graminaceous
plants. Loss of NAS function or depletion of NA results in the accumulation of iron
Chapter 2: Literature Review 23
in the roots and stem, but as it is unable to enter the sites of requirement (i.e. young
leaves and reproductive organs) there is a perpetual induction of iron deficiency
response even in the face of excess iron. A prime example of this is the tomato
mutant chloronerva, in which a single base change in the only NAS gene has resulted
in a loss of function (Ling et al., 1999), making it the only known NA-auxotroph
amongst higher plants. It is characterised by retarded growth and sterility, and
exhibits iron deficiency symptoms despite high accumulation of iron and other heavy
metals and exposure to high concentrations of Fe-EDTA (>10µM) (Stephan and
Grün, 1989; Scholz et al., 1992). Such symptoms include intercostal chlorosis of
young leaves, and enhancement of citrate accumulation in the roots and the Strategy
I uptake process i.e. proton extrusion, Fe3+ reduction, and exudation of phenolics and
other iron chelating compounds (Scholz et al., 1992). The activity of iron-containing
enzymes was also deregulated (Pich and Scholz, 1993).
Similar effects were observed in Arabidopsis NAS-knockout mutants (Klatte et
al., 2009; Schuler et al., 2012b) and transgenic tobacco overexpressing HvNAAT-A
(Takahashi et al., 2003b). In all studies, normal growth, development, and
physiology were restored upon NA resupply, be it by application of exogenous NA
or grafting onto a wild-type or NAS-overexpressing rootstock.
2.4.3 Translocation
Following acquisition into the root symplast, iron is transported across the root
to the vascular tissue and subsequently to the rest of the plant. The translocation
process itself is a multi-step process, involving symplastic movement across the
Casparian strip and to the desired site; the loading, unloading and transport through
the vascular tissue; as well as remobilisation from source tissue (Kim and Guerinot,
2007).
During transport, iron is maintained as a chelated complex with ascorbate,
citrate or NA (Brown and Chaney, 1971; Stephan and Scholz, 1993; Pich et al.,
1994; Grillet et al., 2014). With graminaceous plants, iron may also be complexed to
DMA or MAs for transport (Koike et al., 2004; Ishimaru et al., 2010). As
demonstrated by von Wirén et al. (1999), such complexes are pH-dependent, as are
their interactions with other iron chelators. NA for instance, chelates both Fe(II) and
Fe(III) at a higher pH but will preferentially bind for the former at pH 7.0. When
bound to Fe(III) at equilibrium, the NA complex dominates at pH 7.0 to 9.0 while
24 Chapter 2: Literature Review
the structurally similar DMA complex dominates at pH 3.0 to 6.0. Citrate removes
iron from NA at pH 5.5; and it must be converted to Fe(III)-citrate even if Fe(II) is
the major form in which Fe is loaded into xylem.
As with the uptake process, non-graminaceous plants seem to rely on
reduction-based strategy while graminaceous plants utilise a chelation-based one in
which Fe(III) undergoes little to no change in redox state. A marked difference in
stable iron isotope fractionation was observed between several graminaceous and
non-graminaceous species, with the former exhibiting a near consistent isotope
composition across all tissues, while the latter showed increasing depletion of heavy
isotopes in the younger parts corresponding with growth (Guelke and Von
Blanckenburg, 2007; Guelke et al., 2010).
2.4.4 Storage
Upon reaching the sink tissue, iron is reallocated as a cofactor in various
complexes, or bound to the iron storage molecule ferritin and stored in the apoplastic
space and vacuoles (Briat and Lobréaux, 1997). Subsequent translocation and
remobilisation may occur in response to developmental and physiological needs,
such as during iron deficiency (Waters and Troupe, 2012), seed filling (Hocking and
Pate, 1977; Burton et al., 1998; Garnett and Graham, 2005), senescence (Shi et al.,
2012; Maillard et al., 2015), and nodulation (Strozycki et al., 2007).
2.4.4.1 Ferritin
Ferritins are a superfamily of iron storage proteins found in all living things
except yeast. They consist of 24 subunits which form a spherical protein shell with a
central cavity capable of holding between 2000 and 4500 ferric iron atoms. Despite
significant structural conservation, the localisation of ferritin and much of their
structural and functional properties is organism specific. Plant ferritins are distinct in
that they straddle the middle ground between animal and bacteria ferritins – they
share conserved regions with a 39 to 49% amino acid sequence similarity with
animal ferritins though, like bacterial ferritins, plant ferritins are amorphous and have
mineral cores with high phosphate content (Briat et al., 1999). Also, unlike animal
ferritins plant ferritins are localised within the plastids and the apoplasmic space
rather than in the cytosol (Briat et al., 2010).
Chapter 2: Literature Review 25
This localisation into apoplasmic space and plastids has been attributed to the
optimal conditions (low pH, high organic acid concentrations) which facilitate iron
deposition (Briat et al., 1999). This is at odds with the results of Laulhere and Briat
(1993) which found iron uptake into ferritin to be faster at pH 8.5 compared to pH 5
and 6. On the other hand, it was also mentioned that iron release is facilitated at low
pH, which may have hindered the sensitivity of specific radioactive detection of
exogenous iron. This is likely to be the case if one were to consider the necessity of
the presence of a reductant in the loading/unloading of iron into ferritin (Laulhere
and Briat, 1993), as it is at below pH 6 that NA-iron complexes dissociate, promoting
for iron transfer to ferritin (Stephan et al., 1996).
The exact means of uptake remains ambiguous, though it has been
hypothesised that the channels in apoferritin shells allow for some degree of access
to the core for small reducing and chelating agents/complexes (Harrison et al., 1974).
In the core, iron-binding sites allow for the initial formation of stable hydrous ferric
oxide nuclei, unto which more iron is subsequently loaded. Whether iron is
converted to an intermediate form during the transfer from chelated complex to the
core is unknown (Harrison et al., 1974).
When stored in ferritin, iron is maintained in a soluble and bioavailable form.
In plants, accumulation of ferritin mostly occurs in non-green plastids present in seed
and meristematic tissues (Seckback, 1982). Its notable absence in mature
photosynthetic tissue is indicative of its function in providing developing tissue with
the necessary stores for synthesis of iron-containing proteins, such as those required
in photosynthesis (Briat et al., 1999). A similar deduction can be made in nodule
formation in leguminous plants. Iron is required for nodule initiation and function in
a quantity exceeding that of the host roots (Tang et al., 1990); the initiation stage in
particular, is sensitive to iron deficiency (O'Hara et al., 1988). Ferritin is present in
the initial stages to supply the necessary iron stores, after which is it succeeded by
heme as the nodule matures (Bergersen, 1963; Ko et al., 1987).
Senescing tissues also accumulate ferritin, possibly as a means to safely
contain iron released from the breakdown of the photosynthetic machinery
(Seckback, 1982; Briat et al., 1999). While bound to ferritin, iron is unable to react
with oxygen and generation of reactive oxygen species (ROS) through the Fenton
reaction is avoided (Ravet et al., 2009). Consequently, it is imperative that the
26 Chapter 2: Literature Review
release of iron from ferritin be tightly regulated and the iron quickly chelated, as
reductive release of free iron from ferritin contributes to oxidative stress and toxicity
(Thomas and Aust, 1986). Due to this ability to sequester iron and nullify its toxicity
in vivo, ferritins serve not only as a form of iron storage but also as homeostatic
rheostats against oxidative stress. Overexpression of ferritin has been reported to
confer tolerance to oxidative stress and, consequently, necrotrophic pathogens which
utilise that mechanism for infection (Deák et al., 1999; Van Wuytswinkel et al.,
1999). Loss of function results in a decreased ability to deal with excess iron, leading
to overaccumulation and altered development, with mutants having reduced
vegetative growth, defective flower development, and reduced fertility (Ravet et al.,
2009).
For the purpose of iron biofortification, ferritin has traditionally been used to
increase iron content and bioavailability. Plant ferritin-bound iron has a
bioavailability comparable to that of ferrous sulfate, a ferrous salt used in iron
supplements (Davila-Hicks et al., 2004; Lönnerdal et al., 2006), and has been used
successfully to treat anemia (Beard et al., 1996; Murray-Kolb et al., 2003). However,
the mechanism through which it is absorbed by the body has been subject to some
contention. It has been proposed that ferritin is resistant to gastrointestinal digestion
and remains largely intact when absorbed (Lönnerdal et al., 2006), with uptake
occurring through endocytic pathways (San Martin et al., 2008; Antileo et al., 2013).
Others have disagreed, asserting that ferritin is degraded during digestion and
absorbed as non-heme iron (Bejjani et al., 2007; Hoppler et al., 2008). With the
exception of Hoppler et al. (2008), most studies from both camps utilized pure
ferritin in their experiment. This makes it difficult to confirm the structural and
chemical influence of surrounding tissue on the digestion of plant ferritin (e.g.
protection by hulls, cell walls, plastid membrane). In addition, the effect of various
food processing and cooking techniques on ferritin breakdown is currently unknown;
it is possible that a mixture of degraded and undegraded ferritin may result from that,
requiring both absorption mechanisms.
Use of ferritin in monogenic approaches for iron biofortification has provided
increases in seed iron content ranging from 1.2-3 times (Goto et al., 1999;
Vasconcelos et al., 2003). On the other hand, deleterious effects have also been
observed by various authors. Iron-deficiency symptoms were found to manifest in
Chapter 2: Literature Review 27
ferritin-transformed rice when grown under both iron-sufficient and high iron
conditions (Qu et al., 2005; Masuda et al., 2013b). Similar symptoms were also
manifested in tobacco when ferritin was expressed under a strong constitutive
promoter; additionally, ferritin over accumulated in the leaves, leading to illegitimate
iron sequestration and increased root ferric reductase activity (Van Wuytswinkel et
al., 1999). It is generally agreed that in ferritin transformants, iron storage capacity
exceeds that of uptake and that iron uptake becomes the limiting factor to iron
accumulation (Van Wuytswinkel et al., 1999; Qu et al., 2005; Masuda et al., 2013b).
This limitation can be compensated for by using iron uptake genes in conjunction
with ferritin, as proven by Masuda et al. (2013).
2.4.5 Regulation
Regulation of iron metabolism in response to internal and external iron
concentrations is achieved at both the transcriptional and post-transcriptional levels.
In non-graminaceous plants, the family of basic helix-loop-helix (bHLH)
transcriptional regulators serve as key regulators by moderating the expression of
IRT and FRO (Colangelo and Guerinot, 2004; Brumbarova and Bauer, 2005; Bauer
et al., 2007; Yuan et al., 2008). A similar mechanism present in rice affects
phytosiderophore biosynthesis and transport pathways in addition to IRT and FRO
(Ogo et al., 2006; Ogo et al., 2007; Ogo et al., 2011; Itai et al., 2013). In
graminaceous plants, the transcription factor IDEF (IDE-binding factor) binds to IDE
(cis-acting iron deficiency-responsive element) and triggers expression of genes
involved in uptake and translocation (Kobayashi et al., 2003; Kobayashi et al.,
2007). Another transcription factor is NAC (NO APICAL MERISTEM Arabidopsis
transcription factor, and CUP-SHAPED COTYLEDON), which regulates
remobilisation of iron from vegetative tissues (Waters et al., 2009).
Regulation also occurs on a hormonal level. Ferritin expression has been linked
to abscisic acid (ABA) concentrations (Lobréaux et al., 1993; Briat et al., 1999),
while ethylene have been associated with upregulation of iron uptake genes (Lucena
et al., 2006; Garcia et al., 2010) Nitric oxide (NO), a bioactive compound that serves
as a signalling molecule, has also been implicated in the transcriptional and post-
transcriptional regulation of the iron uptake, translocation and storage genes (Murgia
et al., 2002; Graziano and Lamattina, 2005; Garcia et al., 2010). Recently
epigenetics has emerged as an additional layer of iron metabolic regulation; however,
28 Chapter 2: Literature Review
further work is required to elucidate the molecular mechanisms driving regulation
(Xing et al., 2015).
2.5 ENGINEERING FOR ENHANCED IRON CONTENT
In the interest of iron biofortification, five rate-limiting steps to grain iron
accumulation have been identified by Sperotto et al. (2012) (see Figure 2.5). These
can be classified into three main processes: uptake, translocation, and storage.
Figure 2.5. Possible rate-limiting steps for grain iron accumulation (Sperotto et
al., 2012).
Genes associated with these processes have been identified as promising
candidates for iron biofortification. Over the past decade several of them have been
used in both monogenic and multigenic approaches in different model plants, most
notably rice (Masuda et al., 2013a). These approaches and their varying levels of
success have been covered in the review by Masuda et al. (2013a), and of the
combinations investigated thus far, those containing NAS and ferritin have yielded
the most promising results. Iron accumulation was increased by approximately three
times in rice expressing with the rice YSL2, barley NAS1, and soybean ferritin genes
(Masuda et al., 2012). A four-fold increase in seed iron content was observed in
Chapter 2: Literature Review 29
transgenic rice expressing soybean ferritin in conjunction with barley NAS1, two
nicotianamine aminotransferase genes and a mugineic acid synthase gene (Masuda et
al., 2013b). Constitutive expression of Arabidopsis NAS1 together with barley
ferritin and a fungal phytase, both under the regulation of a rice seed storage globulin
promoter, enhanced iron accumulation in rice endosperm by up to six times (Wirth et
al., 2009).
Aside from the choice of genes themselves, a key feature in these studies is the
promoters used to drive the genes. While having too weak a promoter may not
produce the desired level of iron accumulation, one too strong may lead to adverse
effects. Ferritin in particular, is liable to such side effects when overexpressed under
the strong constitutive promoter like the 35S promoter (Qu et al., 2005; Masuda et
al., 2013b). For such cases the use of tissue-specific promoters, like the seed-specific
globulin promoter, appears to negate the issue. With NAS, high expression levels
appear to be more well tolerated physiologically, giving no apparent side effects
(Johnson et al., 2011).
It should be noted that that these studies were conducted in rice, which utilises
both Strategy I and II mechanisms. Whether using similar genes and promoters will
have the same effect on iron accumulation in non-graminaceous grain crop like
chickpea is currently unknown. The effect on bioavailability is likewise unknown;
unlike rice, chickpea contains higher levels of bioavailability inhibitors (Hemalatha
et al., 2007b). Hypothetically, improving the translocation and storage capabilities
using NAS and ferritin would drive the flux in favour of iron accumulation. As both
NAS and ferritin also promote bioavailability (Davila-Hicks et al., 2004; Zheng et
al., 2010), bioavailability may also be expected to increase.
2.6 SUMMARY AND IMPLICATIONS
In summary, iron deficiency is global problem which may be alleviated
through the use of biofortified crops. Transgenic biofortification efforts, as well as
most studies on iron metabolism, thus far have largely been directed at cereal crops
like rice. As members of the Gramineae family, their molecular biology and
physiology are differ significantly from their non-graminaceous counterparts.
Consequently, biofortification strategies successfully applied in a graminaceous
species like rice may behave differently in a non-graminaceous species.
30 Chapter 2: Literature Review
In this study, the species of interest is chickpea, the second most important
pulse crop globally. As pulse crop, chickpea is rich in protein, making it an ideal
complement to the traditional starchy staples in biofortification efforts. To date, there
have been no recorded attempts to biofortify pulse crops via a transgenic approach.
The effectiveness of the different transgenic strategies therefore remains to be
explored.
Chapter 3: General Materials and Methods 31
Chapter 3: General Materials and Methods
3.1 GENERAL MATERIALS
3.1.1 Sources of specialised reagents
All reagents used in this project were sourced from scientific companies
including Bio-Rad (USA), Invitrogen (USA), Promega (USA), Qiagen
(Netherlands), Roche Diagnostics (Switzerland), and Sigma Aldrich (USA).
Oligonucleotides were synthesized and purchased from GeneWorks (Australia)
at a concentration of 100µM. All primers were diluted to form a 10µM working
stock.
Powdered MS media (with vitamins) was obtained from PhytoTechnology
Laboratories (USA).
Antibiotics used in this project were purchased from the following sources:
Antibiotic name Source
Ampicillin Roche
Kanamycin monosulfate Austratec (Australia)
Rifampicin
Timentin (also known as ticarcillin) Pure Science (New Zealand)
Merrum (also known as meropenem) Clifford Hallam (Australia)
3.1.2 Iron metabolism genes
The soybean Ferritin H2 gene was kindly provided by Dr Alexander Johnson
from the University of Melbourne, NSW, Australia.
3.1.3 Bacterial strains
Escherichia coli strain XL1-Blue was used for general plasmid cloning. For
plant transformation, Agrobacterium tumefaciens strains AGL1 and LBA4404 were
used for chickpea and tobacco transformation respectively.
32 Chapter 3: General Materials and Methods
3.1.4 Plant material
Seeds of Cicer arietinum (chickpea cv. HatTrick) were obtained from the
Australian seed company Grainland, Moree, NSW. Wild-type Nicotiana tabacum
plants were kindly provided by Ms Maiko Kato from the CTCB.
3.1.5 General solutions: Abbreviations and composition
3.1.5.1 Media recipes and related additives
Bacterial culture
IPTG: isopropyl-β-D-thiogalactopyranoside, 20% (w/v) prepared in dimethyl
sulphoxide
LB liquid growth media: 1% (w/v) bacto-tryptone, 0.5% (w/v) bacto-yeast extract,
170mM sodium chloride
LB agar: LB media solidified with 1.5% bacto agar
X-gal: 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, 2% (w/v) prepared in
dimethyl sulphoxide
X-gluc: 5-bromo-4-chloro-3-indolyl-β-D-glucoronide-cyclohexylamine salt
Plant tissue culture
All media and solutions in this section are prepared in a total volume of 1L unless
otherwise stated.
Chickpea tissue culture
B5 co-cultivation media: 100mL B5 Macro Stock (10x), 1mL B5 Micro Stock
(1000x), 10mL Fe-EDTA Stock (100x), 10mL B5 Vitamins Stock (100x), 3%
sucrose, 1.95g MES monohydrate, 1mL BAP (0.5mg/L), 1mL NAA (0.5mg/L), pH
5.8, 8g Difco agar, 1mL acetosyringone (100mM)
B5 Macro stock (10x): 25g KNO3, 1.5g CaCl2.2H2O, 2.5g MgSO4.7H2O, 1.4g
NaH2PO4.2H2O, 1.34g (NH4)2SO4 per 100mL
B5 Micro stock (1000x): 0.3g H3BO3, 1.36g MnSO4, 0.2g ZnSO4, 75mg KI, 25mL
NaMoO4.H2O (1mg/mL), 2.5mL CuSO4 (1mg/mL), 2.5mL CoCl2 (1mg/mL)
Fe-EDTA stock (100x): 37.25g Na2EDTA.2H2O, 27.85g FeSO4.7H2O
Chapter 3: General Materials and Methods 33
B5 Vitamins stock: 100mg thiamine HCl, 10mg nicotinic acid, 10mg pyridoxine
HCl per 100mL
RS0 media: 4.43g MS powder, 30g sucrose, 1.95g MES monohydrate, 8g Difco
agar, pH 5.8
RS1 media: 500µL BAP (1mg/mL), 500µL kinetin (1mg/mL), 50µL NAA
(1mg/mL) per L of RS0 media
RS2 media: 500µL BAP (1mg/mL), 500µL kinetin (1mg/mL) per L of RS0 media
RS3 media: 100µL BAP (1mg/mL), 100µL kinetin (1mg/mL) per L of RS0 media
Water agar: 0.8g Difco agar
Tobacco tissue culture
MS0 media: 4.43g MS powder, 30g sucrose, 8g Difco agar, pH 5.7
MS104 media: 1mL BAP (1mg/mL), 100µL NAA (1mg/mL) per L of MS0 media
Plant hydroponics
All media and solutions in this section are prepared in a total volume of 1L unless
otherwise stated.
Hoagland nutrient solution
Hoagland nutrient solution: 3mL Hoagland Macro stock 1, 2mL Hoagland Macro
stock 2, 0.5mL Hoagland Micro stock, 2.5mL Hoagland Fe-EDTA stock, 2.5mL
Hoagland buffer
Hoagland Macro stock 1: 101.1g KNO3, 18.99g NH4H2PO4, 81.31g MgSO4.7H2O
Hoagland Macro stock 2: 236.2g Ca(NO3)2.4H2O
Hoagland Micro stock: 2.86g H3BO3, 1.81g MnCl2.4H2O, 0.22g ZnSO4, 0.08g
CuSO4.5H2O, 0.03g NaMoO4.H2O
Hoagland Fe-EDTA stock: 3.72g Na2EDTA.2H2O, 2.78g FeSO4.7H2O
Hoagland Buffer: 39.06g MES monohydrate, pH 6.0
34 Chapter 3: General Materials and Methods
3.1.5.2 Solutions for molecular work
Solutions for nucleic acid extraction
CTAB buffer: 2% CTAB (cetyltrimethylammonium bromide), 2M NaCl, 25mM
EDTA pH 8, 100mM Tris-HCl, 2% polyvinylpyrrolidone (PVP
40)
CHCl3:IAA: chloroform: isoamyl alcohol (24:1)
TPS buffer (adapted from Thomson and Henry (1995)): 100mM Tris-HCl (pH
9.5), 1M KCl, 10mM NaEDTA
Solutions for gel electrophoresis
Agarose gel loading dye (6X): 0.25% (w/v) bromophenol blue, 50% TE, 50%
glycerol
TAE buffer: 10mM Tris-acetate, 0.5mM EDTA, pH7.8
TE: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA
Solutions for Southern analysis
Denaturation solution: 0.5M NaOH, 1.5M NaCl
Depurination solution: 0.25M HCl
Detection buffer: 0.1M Tris-HCl, 0.1M NaCl, pH 9.5
High stringency buffer: 0.1x SSC, 0.1% SDS
Low stringency buffer: 2x SSC, 0.1% SDS
Maleic acid buffer: 0.1M maleic acid, 0.15M NaCl, pH 7.5
Neutralisation buffer: 0.5M Tris-HCl (pH 7.0), 1.5 NaCl, 1mM EDTA
SSC (10x): 30mM sodium citrate, 0.3M NaCl, pH 7.5
Washing buffer: 0.1M maleic acid, 0.15M NaCl, pH 7.5, 0.3% (v/v) Tween 20
3.2 GENERAL METHODS
3.2.1 General molecular techniques
3.2.1.1 Polymerase chain reaction (PCR)
Each reaction consisted of 5µL of 2X GoTaq green (Promega), 0.25µL of each
of 10µM forward and reverse primers, and up to 1µL of the template DNA. MilliQ
Chapter 3: General Materials and Methods 35
water added to reach a final volume of 10µL. Where required, 0.6µL of DMSO was
added to reach a final volume of 10.6µL.
The PCR program used was as such: initial denaturation at 95⁰C for three
minutes, followed by 30 cycles of denaturation at 95⁰C for 30 seconds, annealing at
48–60⁰C (depending on primers) for 30 seconds, and extension at 72⁰C. Extension
time was set at one minute per 1 kbp of the final product size. A final extension was
done at 72⁰C for five minutes.
3.2.1.2 Gel electrophoresis
Agarose gels with concentrations ranging from 0.8–2% (w/v) were prepared by
dissolving agarose (Roche Diagnostics – USA) in 1X TAE buffer, and adding 0.5X
SYBR Safe DNA gel stain (Invitrogen – USA). Gels were cast and run in an
EasyCast Mini Gel System. A 2-Log molecular weight marker (New England
Biolabs, USA) was loaded in at least one lane in each run to allow for determination
of size and concentration of nucleic acids. Electrophoresis was carried out in TAE
buffer at 100V for 40 minutes. Visualisation and photography of the electrophoresis
results were done using a Syngene Geldoc system (G-box and GenSnap Version
6.07) (Syngene – UK).
Purifying of electrophoresis products, if desired, was done by excising the
appropriate band and extracted using Freeze ‘N Squeeze DNA Gel Extraction Spin
Columns (Biorad) as per the manufacturer’s instructions.
3.2.1.3 DNA sequencing
All DNA sequencing was performed using the BigDye 3.1 standard methods at
the genomics facility located at the Queensland University of Technology (QUT),
Gardens Point Campus.
3.2.2 Bacterial transformation
3.2.2.1 Escherichia coli transformation
Transformation was done on chemically-competent XL1 Blue E.coli using a
heat-shock method. A 200µL aliquot of competent cells was first thawed on ice, and
1 to 5µL of the plasmid of interest mixed in. After approximately 30 minutes of
incubation on ice, the cells were heat-shocked by placing the tube in 42⁰C water bath
for 30 seconds, after which the tubes were immediately placed on ice. When cooled,
36 Chapter 3: General Materials and Methods
the cells were transferred to a 2mL microfuge tube containing 250µL of pre-warmed
LB broth (with no selection) and incubated at 37⁰C, 200 rpm for one hour. A 50µL
aliquot of the outgrowth culture was then spread onto an LB plate containing the
relevant selection agent, and the plates incubated overnight at 37⁰C or for two nights
at room temperature.
3.2.2.2 Agrobacterium transformation
A 100µL aliquot of electro-competent competent cells was first thawed on ice
and 1µL of the plasmid of interest added. The mixture was then mixed and
transferred to a cooled electrocuvette and electroporated using an EC100
electroporator (Thermo EC). Following this, 400µL of pre-warmed LB broth (with
no selection) was added to the cuvette. The cuvette contents were then transferred to
a 2mL microfuge tube and incubated at 28⁰C, at 200 rpm for one to two hours. A
50µL aliquot of this culture was then spread onto an LB plate containing 25µg/L of
rifampicin and 100µg/L of kanamycin, and the plates incubated for two days at 28⁰C.
3.2.3 Plant transformation
3.2.3.1 Tobacco transformation
3.2.3.1.1 Preparation of Agrobacterium culture
A starter culture was prepared by inoculating approximately 5mL of LB media
containing 25mg/L rifampicin and 100mg/L kanamycin with 100µL of LBA4404
Agrobacterium glycerol stock. The culture was incubated overnight at 28⁰C at
200rpm. To prepare the final working culture, 10µL of the starter culture was used to
inoculate 5mL of LB media containing 100mg/L kanamycin; this final culture was
then incubated overnight at 28⁰C at 200rpm.
3.2.3.1.2 Transformation, selection and regeneration
Nicotiana tabacum (cv Samsun) leaves were cut into 1 – 1.5cm2 pieces and
immersed in an overnight culture of Agrobacterium (strain: LBA4404) diluted 1:10
with liquid MS0 medium (pH 5.7). The mixture was shaken vigorously. The leaf
discs were then blotted dry on filter paper and placed adaxial side down on MS104
media. The plates were incubated at 25⁰C with moderate light for three days.
Following that, the leaf discs were transferred to MS104 media containing 200mg/L
kanamycin and incubated at 25⁰C with a 16 hour photoperiod. Leaf discs were
transferred to fresh media every two weeks. When well-defined stems have formed,
Chapter 3: General Materials and Methods 37
the plantlets were excised and placed onto fresh MSO selection media to allow for
further growth and rooting
3.2.3.1.3 Acclimatisation of transgenic plants
Upon sufficient rooting, plantlets were washed to remove all traces of media
from the roots, then planted in 150 mm pots filled with Plugger 222 potting mix
(Australian Growing Solutions). The soil was gently compressed around the roots
and the plantlets watered well. The plantlet was kept in a tub sealed with a clear
plastic film and placed in a growth room set at 25 ± 1⁰C, with 16 hour light/ 8 hour
dark cycle. After three days, the plastic film was loosened, then completely removed.
Hardened plants were maintained in the same growth conditions until seeds were
obtained.
3.2.3.2 Chickpea transformation
3.2.3.2.1 Seed sterilisation
Approximately 50 – 60g of dry seed was weighed out into a plastic canister and
rinsed with water before vigorous shaking in 70% (v/v) ethanol for two minutes. The
ethanol was then replaced with a freshly prepared 1.5% (v/v) sodium hypochlorite
solution (diluted with sterile MilliQ water) and the canister agitated for 7 – 8
minutes. Following this, the sodium hypochlorite was decanted and the seeds washed
5 – 7 times with sterile MilliQ water. Damaged and discoloured seeds were removed
with a pair of sterile forceps. The remaining seeds were imbibed overnight at room
temperature in sterile MilliQ water.
3.2.3.2.2 Preparation of Agrobacterium culture
A starter culture was prepared by inoculating approximately 5mL of LB media
containing 25mg/L rifampicin and 100mg/L kanamycin with 100µL of AGL1
Agrobacterium glycerol stock. This culture was incubated overnight at 28⁰C at
200rpm. To prepare the final working culture, 100µL of the starter culture was used
to inoculate 100mL of LB media containing 100mg/L kanamycin. This final culture
was then incubated overnight at 28⁰C at 200 rpm, until an OD600nm of 0.6 – 1.2 was
reached.
3.2.3.2.3 Co-cultivation of explants with Agrobacterium
An aliquot of 100µL of freshly prepared 100mM acetosyringone solution was
added to 100mL of overnight Agrobacterium culture. The culture was then incubated
38 Chapter 3: General Materials and Methods
at room temperature for approximately three hours, during which the explants are
prepared for infection. Approximately 1 mm was trimmed off the ‘beak’ of the
overnight imbibed seeds, and the seeds bisected along the longitudinal axis. The seed
coat was removed and the radicle of each half embryonic explant was pricked
between six to eight times with a sterile 26 gauge needle dipped in Agrobacterium
culture. The injured explants were then immersed in Agrobacterium culture for one
to two hours, during which they were incubated in a growth cabinet set at 24⁰C with
fluorescent light. Following this, the culture was drained and the explants placed cut
side-down on B5 media. The plates were then sealed with Micropore tape. The
explants were co-cultivated with Agrobacterium culture for 72 hours at 24 ± 1⁰C,
under fluorescent lights with a 16 hour light/ 8 hour dark cycle.
3.2.3.2.4 Regeneration and selection of transformed plants
After co-cultivation, the explants were transferred on RS1 media, with up to 16
explants per plate. After 14 days, the explants were subcultured to RS2 media, during
which the roots, and any dead or dying tissue were trimmed off. Two-thirds of the
cotyledon was also removed, and a small incision made at the cotyledonary node, to
an approximate depth approximately 1mm, with a size 10 scalpel blade. The explants
were incubated for 14 to 21 days before transferral to fresh RS2 media for the third
round of selection. Bleached, dead and dying tissues were trimmed off, as was the
remaining cotyledon. The explants were arranged with 9 explants per plate. The base
of another deep Petri dish (90 x 25mm) was used as a lid and the plates sealed with
Micropore tape. The explants were subcultured for up to eight rounds of selection,
with each round lasting up to 21 days. Clumps of secondary shoots that emerged
from the base of the explants during the selection process were isolated and
transferred to separate RS2 plates for further multiplication. Each clump was
numbered and considered a single putative transgenic event. Upon reaching an
appropriate size, shoots from such clumps were then grafted onto non-transgenic
rootstocks. Throughout the selection process, culture conditions were maintained at
24 ± 1⁰C, with full light and a 16 hour light/ 8 hour dark cycle.
3.2.3.2.5 In vitro grafting
Non-transgenic chickpea seeds were sterilised and germinated for four days on
deep petri dishes containing ½ MS media with no sucrose. After four days, the most
of the hypocotyl was removed with a transverse cut above the first node. A silicon
Chapter 3: General Materials and Methods 39
ring was placed over the decapitated hypocotyl and an incision made along the
longitudinal axis to prepare for insertion of the scion. Healthy looking shoots were
selected for use as scions; they were removed from shoot clumps and the base cut
into a V-shape. The scion was then inserted into the incision in the rootstock and the
silicon ring pulled up to secure the graft. The base of another deep Petri dish was
inverted to serve as a lid for the plate, and the setup was sealed with Micropore tape.
The grafted plants were incubated in a growth chamber at 24 ± 1⁰C, with 16 hour
light/8 hour dark cycle, for eight to ten days to allow the graft to seal. Any side
shoots emerging from the rootstock were removed to promote growth of the grafted
scion.
3.2.3.2.6 Acclimatisation of grafted plants
Grafted plantlets were carefully removed from the medium and the roots
washed to remove all agar. The seed coats were removed to reduce fungal growth.
The grafted plantlets were then transferred to 150mm diameter pots half filled with
sterilised University of California (UC) mix and the roots covered with additional
UC mix. The soil was then gently compressed around the roots and the plantlets
watered. During the transfer, care was taken to keep the plantlet upright and the graft
above the soil. The plantlet was then covered with a transparent plastic jar and placed
in a growth cabinet set at 22 ± 1⁰C, with 16 hour light/8 hour dark cycle. Checks
were performed every day, during which the transparent plastic jars were wiped free
of condensation. After 15 – 20 days, or when the plants had grown big enough to
nearly touch the walls of the jar, the jars were left partially open for two to three days
to reduce humidity, and then completely removed. The plants were transplanted to
400mm x 250mm pots and transferred to a glasshouse upon successful establishment
in the soil.
3.2.3.2.7 Generation of transgenic seed material
Seeds were harvested from T0 plants and sown into potting mix in 100mm tube
stocks. The soil was kept damp, but not saturated. After emergence, samples were
collected from fully expanded leaves for PCR screening. PCR-positive progeny were
transplanted to 400mm x 250mm pots with slow release fertiliser for further growth
and seed production. The process of harvesting, screening and transplanting was
repeated with subsequent generations. Generation time was approximately 90 days
from sowing to harvest. In all harvests, seeds were collected only from dried pods
40 Chapter 3: General Materials and Methods
and the quantity obtained from each plant was recorded. Collected seeds stored in
small labelled envelopes and allowed to dry further at room temperature for at least
three days, after which they were stored at 4⁰C until ready for planting. At least five
PCR-negative siblings from the same generation were also maintained to serve as
controls.
3.2.4 Plant growth conditions
Conditions for the growth chamber and glasshouse were set at 23⁰C and 28⁰C
respectively, with a 16 hour photoperiod.
3.2.5 Verification and molecular characterisation of transgenic plants
3.2.5.1 Nucleic acid extraction
3.2.5.1.1 Quick release method (for rapid screening)
A piece of tissue at least 2 mm2 was homogenised in 100µL of TPS buffer and
incubated at 65⁰C for ten minutes, then cooled on ice. When cool, 100µL of
choloroform/IAA (24:1) was added and mixed by vortex or inversion. The mixture
was then centrifuged at 18, 000 rcf for ten minutes at room temperature. A 10µL
aliquot of the upper phase was removed and diluted in 40µL of MilliQ water for use
as template in PCR.
3.2.5.1.2 Small scale DNA extraction
Fresh leaf samples were homogenised with a TissueLyser or with mortar and
pestle. The homogenised tissue was mixed with 800µL of pre-warmed CTAB
solution and incubated at 65⁰C for thirty minutes. An 800µL aliquot of
choroform:IAA (24:1) was added and the mixture mixed by inversion. Samples were
then centrifuged at 18, 000 rcf for five minutes. The resulting upper phase was
transferred to a fresh tube and 2µL of RNAse A added. The mixture was then
incubated at 37⁰C for 15 minutes, following which 800µL of choroform:IAA (24:1)
was mixed in and the mixture centrifuged at 18, 000 rcf for five minutes. The upper
phase was transferred to a fresh tube and an equal volume of choroform:IAA (24:1)
mixed in. The mixture was centrifuged at 18, 000 rcf for five minutes and the
resulting upper phase was transferred to a fresh tube, to which 2.5 times the volume
of 100% ethanol was added. DNA allowed to precipitate at 4 ⁰C for at least 30
minutes. The samples were centrifuged at 18, 000 rcf for ten minutes and the
supernatant discarded. A 1mL aliquot of 70% ethanol was added to the remaining
Chapter 3: General Materials and Methods 41
DNA pellet and the samples centrifuged again at 18, 000 rcf for five minutes. The
supernatant was discarded and the DNA pellet allowed to dry on the benchtop at
room temperature, after which it was resuspended in 30µL of MilliQ water.
3.2.5.1.3 Large scale DNA extraction
Fresh leaf samples were homogenised with a TissueLyser or with mortar and
pestle. The homogenised tissue was mixed with 2 mL of pre-warmed CTAB solution
and incubated at 65⁰C for ten minutes. A 2 mL aliquot of choroform:IAA (24:1) was
added and the mixture mixed by inversion. Samples were then centrifuged at 3000
rcf for ten minutes. The resulting upper phase was transferred to a fresh tube and 3µL
of RNAse A added. The mixture was then incubated at 37⁰C for 15 minutes,
following which 2mL of choroform:IAA (24:1) was mixed in and the mixture
centrifuged at 3000 rcf for ten minutes. The upper phase was transferred to a fresh
tube with 2.5 times the volume of 100% ethanol, and the DNA allowed to precipitate
at 4⁰C for at least 30 minutes. Precipitated DNA threads were harvested via spooling
with a glass hook, on which it was dried on the bench at room temperature. Upon
drying, the spooled DNA was resuspended in a 400µL of 1M NaCl solution, then re-
precipitated with 2 times the volume of 100% ethanol. The precipitated samples were
centrifuged at 18, 000 rcf for ten minutes and the supernatant discarded. A 1mL
aliquot of 70% ethanol was added to the remaining DNA pellet and the samples
centrifuged again at 18, 000 rcf for five minutes. The supernatant was discarded and
the DNA pellet allowed to dry on the benchtop at room temperature, after which it
was resuspended in 100µL of MilliQ water.
The samples were centrifuged at 18, 000 rcf for ten minutes at 4⁰C and the
supernatant discarded. The resulting DNA pellet was allowed to dry on the benchtop
at room temperature, then resuspended in 30µL of MilliQ water.
3.2.5.1.4 RNA extraction
RNA extraction was performed using RNeasy Mini kits (QIAGEN). All
reagents were prepared as per manufacturer’s instructions. Briefly, up to 100mg of
frozen fresh tissue or 30mg of freeze-dried tissue was used as starting material. All
tissues were homogenised using a TissueLyser until a fine powder was obtained.
Fresh tissues were first snap-frozen in liquid nitrogen prior to homogenisation, and
cooled between 20 second milling rounds to prevent thawing of samples. An aliquot
of 450µL of Buffer RLT with 1% β-mercaptoethanol was added to each sample and
42 Chapter 3: General Materials and Methods
the tubes vortexed vigorously to mix. The lysate was then transferred to a
QIAshredder spin column and centrifuged at 18, 000 rcf for two minutes. The
supernatant was transferred to a fresh 2mL microfuge tube and 0.5X volume of
100% ethanol mixed in by pipetting. The sample was then transferred to an RNeasy
spin column and centrifuged for 15 seconds at 18, 000 rcf. The flowthrough was
discarded and 700µL of Buffer RW1 added to wash the column. The samples were
again centrifuged and the flowthrough discarded. This was followed by two washes
with 500µL of Buffer RPE; in the final wash, the samples were centrifuged for two
minutes to dry the membrane. The flowthrough was again discarded and the samples
centrifuged for an additional one minute to remove all traces of Buffer RPE. The
column was then transferred to a 1.5mL microfuge tube. Elution was done by adding
30µL of RNAse-free water to the column and centrifuging at 18, 000 rcf.
3.2.5.1.5 Quantification of nucleic acids
Estimation of nucleic acid quantity was performed using a NanoDrop2000
spectrophotometer (Thermo Scientific – USA). Absorbance was measured at 230,
260, and 280nm. The purity of gDNA and RNA was evaluated based on the ratios of
absorbance at 260 and 280nm, and 260 and 230nm respectively. The quantity and
quality was confirmed through electrophoresis of 500 ng of nucleic acids on a 1% gel
for gDNA, and a 1.5 – 2% gel for RNA.
3.2.5.2 Gene expression analysis
3.2.5.2.1 DNAse treatment
Prior to cDNA synthesis, contaminating DNA was removed from RNA
samples using RQ1 DNAse (Promega). One unit of RQ1 RNAse-free DNAse was
used per 1µg of RNA. Up to 8µL of RNA was mixed with 1µL of RQ1 RNAse-free
DNAse 10X reaction buffer and 1µL of RNAse-free DNAse, and nuclease-free water
added to a final volume of 10µL. The reaction was incubated at 37⁰C for at least 30
minutes, then transferred to -20⁰C for temporary storage. A 0.5µL aliquot of the
treated RNA was used as a template for PCR to check for the presence of DNA.
Upon confirming the absence of DNA, the reaction was incubated at 65⁰C for 10
minutes to deactivate the DNAse. The treated RNA was stored at -80⁰C.
3.2.5.2.2 cDNA synthesis
cDNA synthesis was performed using SuperScript™ IV Reverse Transcriptase
(ThermoFisher Scientific) as per the manufacturer’s instructions. Briefly, 1µL of
Chapter 3: General Materials and Methods 43
50µM oligod(T), 1µL of 10mM dNTP mix was added to 500ng of RNA, and
nuclease-free waster was added to a final volume to 13µL. The reaction was mixed,
heated at 65⁰C for five minutes, then incubated on ice for one minute.
Following this, 7µL of RT reaction mix was added to the annealed RNA. The
RT reaction mix consisted of 4µL of SSIV buffer, 1µL of 100mM DTT, 1µL of
RNseOUT™ Recombinant RNase Inhibitor, and 1µL of SuperScript® IV Reverse
Transcriptase (200U/µL). The combined reaction mixture was incubated at 55⁰C for
10 minutes, then inactivated at 80⁰C for 10 minutes. The resulting cDNA was stored
at -20⁰C.
3.2.5.2.3 Qualitative analysis of gene expression
Qualitative analysis of gene expression was done via PCR (see Chapter
3.2.1.1), using 1µL of cDNA as the template.
3.2.5.2.4 Quantitative real-time PCR (qPCR)
Measurement of transgene expression was performed on a CFX384 Touch™
Real-Time PCR Detection System (BIO-RAD) using the SYBR Green PCR Master
Mix kit (Applied Biosystems). A 1:30 dilution of cDNA was used for gene
expression analysis. Primers for three housekeeping genes were included in each run
to serve as internal controls.
3.2.5.2.5 Primer design.
Primers targeting the transgenes were designed the using the Primer3 software
(Koressaar and Remm, 2007; Untergasser et al., 2012). The length of all primers
ranged from 60 to 200bp, with a GC content of 40-60% and a melting temperature of
58 - 63⁰C. All primers were tested against gDNA and cDNA. The products generated
were sequenced as per section 3.2.1.3 to ensure amplification of the right product.
Primer sequences for reference genes were selected based on the publication by
Garg et al. (2010). The housekeeping genes actin 1, elongation factor 1-α (EF1α) and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were selected for use as
internal references. The stability of these genes was confirmed in all tissue types
following the guidelines recommended by Hellemans et al. (2007). The
recommended values are as listed below, where CV refers to the coefficients of
variation, and M to the gene stability value.
44 Chapter 3: General Materials and Methods
Sample type CV M
Homogenous <0.25 <0.5
Heterogeneous <0.5 <1.0
Gene expression was calculated using the following formula:
𝐹𝑜𝑙𝑑 𝑐ℎ𝑎𝑛𝑔𝑒 = 𝑙𝑜𝑔2[𝐺𝑂𝐼 − (𝐻𝐾1 × 𝐻𝐾2 × 𝐻𝐾3 … )1𝑛]
GOI represents the gene of interest, HK represents the housekeeping gene, and
n represents the number of housekeeping genes.
3.2.5.3 Southern analysis
To determine transgene copy number, 15µg of gDNA was initially digested
overnight with an appropriate restriction enzyme which cut once between the T-DNA
borders of the respective plasmids used for the generation of the transgenic events.
Digested DNA was electrophoresed through a 0.8% agarose gels in TAE buffer
containing 0.5X SYBR Safe (Invitrogen) at 45V for 5 hours. Gels were prepared for
transfer by incubation for 10 minutes in depurination solution, followed by 30
minutes in denaturation solution and finally twice for 30 minutes in neutralisation
solution; gels were rinsed in double distilled water between each treatment. Gels
were then equilibrated in 10X SSC for 5 minutes before transfer to a positively
charged nylon membrane (Roche) overnight in 10X SSC according to the capillary
method of Southern (1975). Following transfer, the membrane was washed twice in
2X SSC for two minutes to remove salt residues and the DNA fixed to the membrane
by baking at 80˚C for 2 hours. Pre-hybridisation of the nylon membrane was done for
1 hour at 42ºC in DIG-easy Hyb Solution (Roche) with agitation. The DIG-labelled
PCR probe was then added and allowed to hybridise to the membranes overnight
with rotation at 42ºC. The following day, the membrane was washed twice for 5
minutes with low stringency buffer at room temperature. This was followed by two
rounds of washing with pre-warmed high stringency buffer, each done for 15 minutes
at 68 ºC. The membrane was rinsed briefly in maleic acid buffer and blocked in
maleic acid buffer containing 3% skim milk powder. After blocking, the membrane
was incubated in a solution containing 1:20,000 diluted mouse-derived anti-DIG
antibody (Roche) in maleic acid buffer with 3% skim milk powder for 30 minutes at
room temperature. Unbound antibody was removed by two 15 minutes rounds of
Chapter 3: General Materials and Methods 45
washing in washing buffer. Prior to detection, all membranes were equilibrated in
detection buffer. Detection of DIG-labelled DNA was achieved using CDP-star
(Roche), as per manufacturer’s instructions.
3.2.5.3.1 Preparation of DIG-labelled probe
DIG-labelled probes used in for Southern analysis were generated via PCR.
The components for the labelling reaction are as listed below. An unlabelled
reaction, in which the DIG labelling mix was replaced with unlabelled dNTPs, was
run done simultaneously as a control.
10X buffer 3µL
25mM MgCl 1µL
Forward primer (10µM) 1µL
Reverse primer (10µM) 1µL
DIG labelling mix (10X) 3µL
Plasmid DNA (1:10 dilution of miniprep) 1µL
HybriPol DNA polymerase 1µL
dH2O added to final volume of 30µL
The PCR conditions for the reactions are as follows: initial denaturation at
94⁰C for two minutes, 35 cycles of denaturation at 94⁰C for ten seconds, annealing at
55⁰C for 55 seconds, extension 72⁰C for one minute, and a final extension at 72⁰C
for five minutes. The resulting PCR product was separated via electrophoresis,
purified, then stored at -20⁰C.
3.2.6 Trace element analysis
To avoid trace element contamination, washed nitrile gloves were worn and
only non-metal implements (e.g. plastic, wood) were used to handle samples.
Likewise, only PP tubes with HDPE screw caps from Greiner Bio-One were used for
extraction or storage purposes. Following appropriate preparation, leaf samples were
analysed via laser ablation inductively coupled plasma mass spectrometry (LA-ICP-
MS). Seed samples were analysed via inductively coupled plasma optical emmision
spectrometry (ICP-OES).
46 Chapter 3: General Materials and Methods
3.2.6.1 Sample preparation
To avoid contamination from external sources of iron, all large foreign
particulate matter (e.g. soil, agar) was removed. Seeds were briefly brushed down
while leaf samples were rinsed twice in MilliQ water. All samples were freeze-dried
for at least 48 hours using a BenchTop Pro with Omnitronics freeze-dryer (SP
Scientific).
3.2.6.1.1 Preparation of samples for trace element analysis by laser-ablation
inductively coupled plasma mass spectroscopy (LA-ICP-MS)
Leaf samples were transferred to 1.5mL microfuge tubes containing a 3mm
tungsten carbide bead (QIAGEN) and milled using a TissueLyser II for ten minutes
at 30/s. To ensure complete and even milling, tubes were filled to a maximum of a
third of the total volume. Milled samples were then pressed into 5mm diameter
pellets, which were then stuck onto a hard plastic sheet using double-sided tape.
3.2.6.1.2 Preparation of samples for trace element analysis by inductively coupled
plasma optical emission spectroscopy (ICP-OES)
For analysis of seed material by ICP-OES, a minimum of five freeze-dried
whole seeds were ground with an IKA Tube Mill (IKA®, Germany) at 25, 000 rpm
for five rounds of 30 seconds. Milling was repeated as needed until a fine powder
was obtained. All processed material was stored in labelled 50mL PP tubes.
3.2.6.1.3 Extraction of trace elements for ICP-OES
When ready for analysis, no more than 350mg of processed freeze-dried tissue
was transferred to a labelled 50mL tube. Three technical replicates were prepared for
each sample. Digestion was initiated by the addition of 2mL HNO3 and 0.5mL H2O2.
The tubes were vortexed to ensure complete wetting of samples and allowed to stand
overnight at room temperature. The following day, the tubes were shaken at 200 rpm
for 20 minutes and heated first to 80⁰C for 30 minutes, then to 125⁰C for two hours.
Prior to shaking and increasing the temperature, the caps were briefly loosened to
release the accumulated pressure. The sample was allowed to cool to room
temperature before addition of MilliQ water to a total volume of 25mL. The caps
were then sealed and the samples agitated at 300 rpm for five minutes. Undissolved
material (e.g. silicates) was allowed to settle for 60 minutes. Filtering was performed
as required, such as with leaf tissue. The settled extract was then decanted into 15mL
Chapter 3: General Materials and Methods 47
falcon tube, capped and sealed with Parafilm, and stored at room temperature until
analysis.
3.2.6.2 Trace element quantification
All quantification of trace elements was performed in the analytical
laboratories located at the Queensland University of Technology (QUT), Gardens
Point Campus unless otherwise specified.
LA-ICP-MS was done using an Agilent 8800 Inductively Coupled Plasma
Mass Spectrometer attached with an ESI 193nm Excimer Laser. The laser ablation
settings used were as follows:
Laser ablation
Pulse width = 4 ns
Laser energy = 2.00 J/cm2
Repetition rate = 10 Hz
Laser helium flow = 515 mL/min
Speed of each line scan = 10 microns/sec
Spot size = 85 microns
ICP-OES was done on a Perkin ElmerOptima 8300 DV Inductively Coupled
Plasma Optical Emission Spectrometer.
3.3 DATA ANALYSIS
All graphs and standard errors were prepared using Microsoft Excel. Statistical
analysis was performed using one-way ANOVA, and comparison of means done
using Tukey’s HSD test or Dunnett’s test. All statistical analysis was done using
Minitab statistical software (Arend, 2010).
Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick 49
Chapter 4: Assessment of Iron Content in
Chickpea cv Hattrick
4.1 INTRODUCTION
Cicer arietinum, or more commonly known as chickpea, is an annual self-
pollinating diploid (n=16) pulse crop belonging to the Fabaceae family that serves as
an important secondary staple. It is cool season legume that can be cultivated in a
wide range of soils and environments across both tropical and sub-tropical region,
and can been utilised in a variety of cropping systems (Saxena, 1987).
As a food crop, chickpea can be utilised in a variety of ways. Green pods,
immature seeds and young leaves can be consumed as a vegetable while the stover
and pod husks can be used as animal feed (Ibrikci et al., 2003; Yadav et al., 2007).
The primary commodity however, is the dried mature seed which can be used as
animal feed or for human consumption. With the latter, the long history of
consumption in various regions such as India, the Middle East and Europe has given
rise to a diversity of dishes in which chickpea can be utilised. Chickpeas are
consumed on their own or with other foods; seeds may be eaten whole, hulled, or
ground into flour from which other products may be derived. Preparation for
consumption can be done via various processing methods such as soaking, sprouting,
fermenting, boiling, steaming, roasting, extrusion and puffing (Yadav et al., 2007),
all of which exert different effects on the overall nutritional quality (Poltronieri et al.,
2000; Sebastiá et al., 2001; Ghavidel and Prakash, 2007; Hemalatha et al., 2007a).
Most of the chickpea in the global market can be classified into two main types
which are primarily distinguishable by their seed morphology, specific aspects of
which influence their end-use. The first type is the kabuli, also known as garbanzos.
Kabuli seeds are large and round, weighing approximately 400 mg per seed (Pulse
Australia, 2016). The seed coat is thin and light-coloured, ranging from shades of
white to cream and the seeds are typically consumed whole or made into hummus
(Gaur et al., 2015; Pulse Australia, 2016). Kabuli cultivation areas are mostly located
in Southern Europe, Northern Africa, Afghanistan, Pakistan, Chile and India (Gaur et
al., 2015).
50 Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick
The second type is the desi, which forms the bulk of the international export
market (Rao et al., 2010). Desi seeds are small, wrinkled and angular, with an
approximate weight of 120 mg per seed (Pulse Australia, 2016). The seed coat is also
1.2 to 3 times thicker than the kabuli (Umaid et al., 1984; Wood et al., 2011) and can
be found in a greater variety of colours ranging from brown to yellow, as well as
orange, black and green. Desi seeds are commonly dehulled and split to obtain the
cotyledons, which are then known as chana dhal and can in turn be milled to flour,
known as besan or gram flour. Australian-grown desi chickpea, in particular, are
known to be superior quality for dhal production, with up to 90% of total export used
for that purpose (Agbola et al., 2000).
Desi and kabuli types are generally similar in terms of nutritional profiles with
comparable starch and amino acid contents. However there are differences in a few
key aspects which have been linked to seed coat morphology – kabuli seeds for
instance, tend to have a higher fat content while desi seeds are higher in fibre and
tannin contents (Petterson and Mackintosh, 1994; Khan et al., 1995; Rincón et al.,
1998). However considerable variations can be found across different cultivars and
sites, particularly when it comes to the mineral composition and distribution within
the seed (Wood and Grusak, 2007). Iron content for instance, have been reported to
range from 2.4 to 11 mg/100g (e.g. Jambunathan and Singh, 1981; Petterson and
Mackintosh, 1994; Bueckert et al., 2011; Thavarajah and Thavarajah, 2012; Nobile
et al., 2013). Even where overall iron content of the seeds may be similar,
accumulation patterns may vary. As demonstrated in the study by Jambunathan and
Singh (1981), the seed coat of kabuli cultivars was found to be significantly richer in
iron compared to the desi. A significant difference was also found in the phosphorus
content from seeds grown in two different locations; this is of especial interest as that
it may reflect phytate localisation and may thus be an indicator of iron bioavailability
of the end product. For that reason, both total iron content and distribution within the
seed require due consideration in biofortification approaches, particularly with
downstream processing methods in mind.
The significance of the interactions between genotype and environment (and by
extension, management practices) on the grain micronutrient profile has been
documented in several crop species including wheat (Ficco et al., 2009), rice (Norton
et al., 2014), sorghum, bean, pea, lentil, and chickpea (Ray et al., 2014). However up
Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick 51
until recently, crop development has primarily focused on productivity with little
consideration to the nutritional aspect. Current efforts to improve micronutrient
content of chickpeas are few (e.g. Diapari et al., 2014; Ray et al., 2014; Upadhyaya
et al., 2016), with no existing program within Australia. Consequently information
on the micronutrient-accumulating behaviour of recently developed Australian
cultivars is relatively sparse, if not non-existent, making it difficult to ascertain the
degree to which various factors may influence the micronutrient profile.
The aim of this chapter therefore, is to determine the range of iron
accumulation in the seeds of the Australian commercial desi cultivar PBA HatTrick
and identify potential factors that influence it. As part of this study, other commercial
cultivars from a range of field locations were assessed for comparison. The
approximate distribution of trace elements within PBA HatTrick seed was also
examined to establish a baseline for comparison in later chapters.
4.2 MATERIALS AND METHODS
4.2.1 Seed material and locations
The three kabuli and three desi cultivars were used in this study (Figure 4.1).
The details of each cultivar are as described in
Table 4.1, and the sites from which samples were obtained are listed in
Table 4.2. Seeds from field trials at Billa Billa, Warra, Roma, and Kingaroy
were provided by Dr Yash Chauhan and the Queensland Department of Agriculture
and Fisheries (DAF). Seeds from New South Wales (NSW) were obtained from the
seed company Grainland, Moree.
All seeds used in this study were grown in the 2014 growing season. Sowing
was done during winter between June and July, 2014. Harvesting was done during
summer between December, 2014, and January, 2015. Information on the cultivation
sites and conditions during the growing period, where available, are listed in Table
4.3. The soil types of the field locations were provided by Dr Yash Chauhan from the
Agricultural Production Systems sIMulator (APSIM) database. Whole undamaged
seeds were selected and the samples were sent to Waite Analytica Laboratories
(University of Adelaide) for trace element analysis.
52 Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick
Figure 4.1. Chickpea cultivars used in this study. Top row, from left: Kabuli
culitvars Genesis090™, Kalkee™ and PBA Monarch. Bottom row, from left:
Desi cultivars PBA Boundray, CICA0912 and PBA HatTrick.
Table 4.1. Description of chickpea cultivars used in this study.*
Genotypes Description Seed weight
(g/100 seeds)
Genesis090™ Small seeded kabuli. Cream coloured seed coat. 31.3
Kalkee™ Medium to large seeded kabuli. Cream coloured
seed coat. 45.0
PBA
Monarch
Medium sized kabuli. Cream coloured seed
coat. 40.5
PBA
Boundary
Medium sized desi. Dark brown coloured seed
coat. Suitable for splitting and direct
consumption.
19.5
CICA0912 Medium sized desi. Dark brown coloured seed
coat. 22.6
PBA
HatTrick
Medium sized desi. Dark brown coloured seed
coat. Suitable for splitting and direct
consumption.
20.1
*Details as published by Pulse Australia (2016)
Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick 53
Table 4.2. Locations from which seed samples were obtained.
Genotypes Billa
Billa Roma Warra Kingaroy NSW
Kabuli
Genesis090™ Yes Yes Yes n/a n/a
Kalkee™ Yes Yes Yes n/a n/a
PBA Monarch Yes Yes Yes n/a n/a
Desi
PBA Boundary Yes Yes Yes Yes n/a
CICA0912 Yes n/a Yes Yes n/a
PBA HatTrick Yes Yes Yes Yes Yes
Table 4.3. Cultivation conditions for each location.
Site Australian soil
classification Total fertiliser used
Approximate rainfall
during growing period
(mm)
Billa Billa Gray vertosol 25kg/ha Zinz star 25 64.5
Roma Black vertosol 25kg/ha Zinz star 25 85
Warra Gray vertosol 25kg/ha Zinz star 25 80.2
Kingaroy Ferrosol n/a 594
NSW n/a n/a n/a
4.2.2 Measurement of trace element distribution within the chickpea seed
A total of 100 undamaged seeds were rinsed and imbibed for 20 hours in
MilliqQ water at 4 ⁰C. Following imbibition, seeds were rinsed again in MilliqQ
water and carefully dissected to separate into three parts: seed coat, cotyledons, and
radicle. To determine the approximate distribution of mass, the weights of individual
components of ten randomly selected seeds were measured and calculated as a
percentage of the total seed weight. To determine trace element profile of each
component, tissues (from all 100 seeds) of the same type were then pooled and
prepared as described in Chapter 3.2.6.1. Following milling, three subsamples of up
to 250 mg per tissue type were analysed by ICP-OES following the protocol in
Chapter 3.2.6.1.3. The exception to this was the radicle tissue – due to the low
quantity of the material only a single sample was analysed. The relative distribution
of trace elements proportional to the mass of each tissue was calculated using the
following formula:
54 Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick
𝑇𝑜𝑡𝑎𝑙 𝑒𝑙𝑒𝑚𝑒𝑛𝑡 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑖𝑛 𝑡𝑖𝑠𝑠𝑢𝑒
= 𝐸𝑙𝑒𝑚𝑒𝑛𝑡 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 (𝑚𝑔
100𝑔) × 𝑀𝑎𝑠𝑠 (𝑔)
𝑅𝑒𝑙𝑎𝑡𝑖𝑣𝑒 𝑒𝑙𝑒𝑚𝑒𝑛𝑡 𝑑𝑖𝑠𝑡𝑟𝑖𝑏𝑢𝑡𝑖𝑜𝑛
=𝐸𝑙𝑒𝑚𝑒𝑛𝑡 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑖𝑛 𝑡𝑖𝑠𝑠𝑢𝑒
𝑆𝑢𝑚 𝑜𝑓 𝑒𝑙𝑒𝑚𝑒𝑛𝑡 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑜𝑓 𝑎𝑙𝑙 𝑡𝑖𝑠𝑠𝑢𝑒𝑠 × 100%
4.2.3 Statistical analysis
Data was analysed using one-way ANOVA and Tukey’s HSD test (MINITAB
17 Statistical Software, 2010). The elements of interest expressed as mean ± standard
deviation (SD). Pearson correlation analysis and Principal component analysis (PCA)
were performed on seed trace element concentrations using the XLSTAT statistical
package (Addinsoft, 2016).
4.3 RESULTS
4.3.1 Trace element composition of Australian-grown chickpea
The macro and micro-elemental profile of the desi cultivar PBA HatTrick was
assessed to determine the approximate range within which different elements
accumulated in the dry seeds. Seeds were sourced from five different locations
within Eastern Australia to determine the extent of variation within the cultivar. The
profiles of five other commercial cultivars were also assessed for comparison.
In general, the seeds used in this study were found to be rich in calcium (105–
257 mg/100g), magnesium (118–180 mg/100g), potassium (836–1117 mg/100g),
phosphorus 186–420 mg/100g), iron (3.36–5.20 mg/100g), zinc (2.60–4.35mg/100g)
and copper (0.44–1.10 mg/100g) (Table 4.8 and Table 4.9).
In terms of average iron content, higher values were measured in kabuli
samples compared to the desi ones from the same site though the difference was not
significant (Table 4.4). Iron concentrations also corresponded with location, with the
highest concentrations being found the samples from Roma, followed by Warra and
Billa Billa (Table 4.4 and Appendix A, Table 8.3). This pattern was not observed
amongst the desi cultivars sourced from those sites. However a location-specific
effect was apparent upon inclusion of the Kingaroy desi samples in the assessment,
which had lower average iron concentrations compared to the samples from sites
with vertosol-type soils (Table 4.4 and Appendix A, Table 8.3). Also unique to the
Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick 55
Kingaroy samples was the higher manganese content to iron ratio, which was likely
due to the soil type. Within the cultivars themselves, no significant differences were
found between samples from different sites. The exception to this was the Kingaroy-
grown Boundary and CICA0912, which had significantly lower iron compared to
those grown on vertosol-type soils. Little significant difference was also seen
between the iron contents of different cultivars from the same location. The
exception to this was Genesis090™, which was significantly higher compared to
PBA HatTrick at Warra and Roma. Genesis090™ was in general the richest in iron
even amongst the kabuli cultivars, while PBA HatTrick was similar to the other desi
types in terms of low iron content (Appendix A, Table 8.3).
As with iron, Genesis090™ generally had the highest zinc content while PBA
HatTrick ranked amongst the lowest of the cultivars. However comparison of all
samples found zinc accumulation to be primarily influenced by location rather than
genotype. The distinction between the kabuli and desi types was not as distinct, and
significant differences could be observed within the same cultivar across different
locations (Appendix A, Table 8.3). The trend resulting from this was similar to that
of iron, with highest zinc accumulation in the seed from Roma, followed by Warra,
Billa Billa and Kingaroy (Table 4.4and Appendix A, Table 8.3).
Similar to the trend observed in zinc content, grain phosphorus content
appeared to be primarily influenced by location, albeit in a different pattern. Despite
have having higher iron and zinc concentrations compared to the other locations,
samples from Roma were distinctly lower in phosphorus concentration (Table 4.4
and Appendix A, Table 8.2). Even lower still were the phosphorus contents of
Kingaroy-grown samples. No significant differences were found between the desi
and kabuli types except at Warra.
An examination of the relationship between iron and other trace elements in
PBA HatTrick revealed a moderate negative correlation only with manganese, while
positive correlations were observed with zinc, phosphorus, sulphur, potassium and
magnesium (Table 4.5). Zinc and phosphorus in particular, shared the strongest
correlation with seed iron content.
56 Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick
Table 4.4. Summary of Fe, Zn and P concentrations in kabuli and desi cultivars
grown at different locations. Data are expressed as mg/100g and presented as a
mean of all cultivars collected for that site. For each cultivar from each site, n>3.
Significance was calculated using Tukey’s HSD test, and values sharing the same
superscript letters indicate no significant difference (p-value>0.05).
Kabuli Desi PBA HatTrick
Location Range Mean Range Mean Range Mean
Fe
Billa Billa 4.12 - 4.67 4.42 bc 3.97 - 4.53 4.20 c 3.97 - 4.33 4.10
Roma 4.43 - 5.22 4.87 a 4.10 - 4.91 4.45 abc 4.10 - 4.30 4.23
Warra 4.13 - 5.54 4.63 ab 4.23 - 4.65 4.46 bc 4.23 - 4.36 4.31
Kingaroy n/a n/a 3.31 - 4.24 3.63 d 3.57 - 4.24 4.31
NSW n/a n/a n/a n/a 4.08 - 4.37 4.23
Zn
Billa Billa 2.99 - 3.37 3.23 c 2.78 - 3.19 3.02 c 3.01 - 3.10 3.06
Roma 3.86 - 4.42 4.19 a 3.85 - 4.33 4.02 a 3.85 - 3.93 3.06
Warra 3.18 - 4.12 3.61 b 3.82 - 3.96 3.88 ab 3.84 - 3.88 3.86
Kingaroy n/a n/a 2.53 - 3.74 2.96 c 2.53 - 2.68 2.60
NSW n/a n/a n/a n/a 3.39 - 3.61 3.49
P
Billa Billa 370 – 410 389 ab 340 - 410 382 abc 340 - 390 363
Roma 270 – 350 322 d 300 - 400 343 cd 300 - 310 303
Warra 310 – 440 367 bc 400 - 430 412 a 410 - 430 420
Kingaroy n/a n/a 183 - 248 204 e 194 - 202 199
NSW n/a n/a n/a n/a 400 - 420 408
Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick 57
Table 4.5. Pearson’s correlation coefficient between the different trace elements in PBA HatTrick. Analysis was performed on PBA
HatTrick samples collected from all locations. Values marked with an * indicate a significant correlation between two elements (p-value <0.05).
Ca Mg Na K P S Fe Mn Zn B Cu
Ca 1
Mg 0.618* 1
Na -0.352 -0.026 1
K 0.544* 0.844* 0.120 1
P 0.388 0.831* 0.453 0.735* 1
S 0.061 0.644* 0.523* 0.469 0.905* 1
Fe 0.439 0.498* 0.385 0.542* 0.673* 0.554* 1
Mn -0.542* -0.839* 0.171 -0.745* -0.691* -0.498* -0.474 1
Zn 0.638* 0.439 0.192 0.329 0.626* 0.471 0.692* -0.529* 1
B 0.654* 0.945* -0.295 0.816 0.669* 0.467 0.361 -0.886* 0.342 1
Cu 0.095 -0.255 0.602* -0.178 0.155 0.139 0.362 0.312 0.574* -0.443 1
58 Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick
4.3.2 Relationships between location and cultivar on seed trace elemental
composition
Identification of factors contributing to trace element composition was done
using principal component analysis (PCA) and agglomerative hierarchical clustering
(AHC). Both methods serve to determine relationships between samples within the
data set with no reference to prior knowledge. PCA is a bilinear modelling method
which reduces multivariate data to a few principal components in which maximum
data variation is found, allowing for the visualisation of data structure. Cluster
analysis serves to group a set of objects based on similarity.
Amongst the elements, iron, manganese, magnesium, and calcium were
identified as the ones in which the greatest variations were found (see Table 4.6).
The results of both PCA and AHC on seed trace elemental composition showed a
segregation of samples into three main groups based on cultivation location (Figure
4.2 and Figure 4.3). The first group consisted of the samples from Kingaroy which
were grown in ferrosol soil, while the second group comprised of the sole sample
from NSW whose cultivation conditions are unknown. These outgroups had a strong
correlation to iron and manganese contents and magnesium and calcium contents
respectively (Figure 4.2, Table 4.6).
The third and largest group consisted of samples grown in Billa Billa, Warra
and Roma. These locations had vertosol-type soils and similar environmental
conditions (see Table 4.3). This group was further segregated into the desi and kabuli
types along the Y-axis, to which magnesium and calcium had the strongest
correlation (Table 4.6); this corresponds to the higher average calcium and
magnesium concentrations in the desi types compared to the kabuli (see Appendix A,
Table 8.2). No such distinction was observed in the distribution along the X-axis
where the range of both types overlapped. Within both the desi and kabuli groups,
samples appeared to cluster loosely based on source location (Figure 4.3).
Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick 59
Figure 4.2. PCA of trace element composition of chickpea grown in QLD and
NSW based on overall mineral composition.
Table 4.6. Representation quality of a variable for each axis. The greater the
value, the greater the association with the axis.
Fe Mn P S Zn Cu B K Mg Ca Na
PC1 0.84 0.61 0.57 0.56 0.53 0.51 0.44 0.35 0.04 0.02 0.10
PC2 0.01 0.01 0.12 0.00 0.01 0.09 0.23 0.02 0.73 0.68 0.23
60 Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick
Figure 4.3. Clustering analysis of chickpea grown in QLD and NSW based on
mineral composition of samples. The dotted line indicates the level at which
truncation had been carried out to generate homogenous groups.
4.3.3 Cotyledons the primary storage for iron in PBA HatTrick seeds
To determine the storage site of the various trace elements, PBA HatTrick
seeds from NSW from the same stock as those used in later plant transformation
work were imbibed and split into the seed coat, cotyledon and radicle. The weight of
each individual part was measured. Each component was pooled and three
subsamples taken to determine trace element concentration via ICP-OES. The
exception to this was the radicle tissue, where the low volume allowed for only one
sample to be taken.
The weights of whole, imbibed seeds ranged from 361.3 – 511.2 mg. When
split into the three parts, a large variation was observed in the mass across the ten
replicates, ranging from 60.1 – 81.4 mg for the seed coat, 293 – 422 mg for the
cotyledons, and 5.3 – 9.2 mg for the radicle (see Table 4.7). When converted to a
percentage of the total seed mass, the cotyledons was found to constitute 82.28 ±
1.58%, followed by the seed coat and radicle at16.07 ± 1.54% and 1.65 ± 0.21%
respectively.
Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick 61
In terms of mineral concentrations, the results obtained for PBA HatTrick were
found to be consistent with range reported by Jambunathan and Singh (1981). The
seed coat was found to have the highest values for calcium, magnesium, and
manganese while being low in all other elements assayed (see Table 4.8 and Table
4.9). The opposite was seen in the elemental profile of the radicle, which was the
richest in sodium, potassium, phosphorus, sulphur, iron, zinc, boron and copper. The
cotyledons were not exceptionally high in any particular element, but were
moderately rich in potassium, phosphorus, sulphur, iron and zinc while being low in
calcium, magnesium, sodium, boron, copper and manganese. However due to their
large mass, they contained the bulk of the total content of all elements except
calcium and manganese (see Figure 4.4). Calcium was predominantly held in the
seed coat (75%), while and manganese was split between the cotyledons and seed
coat at 55% and 43% respectively. Despite having high concentrations of several
elements, the radicle contributed less than 5% of the total content of any of the
elements studied due to its small mass.
Table 4.7. Relative mass distribution within chickpea seeds of PBA HatTrick.
n=10. Data are presented as a mean ± SD.
Component Weight (mg) Average (mg) Weight (%) Average (%)
Seed coat 60.1 – 81.4 68.67 ± 6.24 13.60 – 18.95 16.07 ± 1.54
Cotyledon 293.0 – 422.0 354.15 ± 44.15 79.29 – 84.89 82.28 ± 1.58
Radicle 5.3 – 9.2 7.08 ± 1.08 1.33 – 1.99 1.65 ± 0.21
Total 361.3 – 511.2 429.89 ± 48.25
Figure 4.4. Relative distribution of trace elements within PBA HatTrick seeds.
62 Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick
Table 4.8. Concentrations of macro-elements in the different chickpea parts. Data are presented as a mean ± SD. All samples had n>3 except
the radicle, where n=1 due to small volume.
Ca
(mg/100g)
Mg
(mg/100g)
Na
(mg/100g)
K
(mg/100g)
P
(mg/100g)
S
(mg/100g)
Whole seed (dry) 242 (± 11) 180 (± 4.6) 6.34 (± 0.63) 1016 (± 23) 408 (± 8.4) 182 (± 2.8)
Seed coat 934 (± 50) 265 (± 14) 9.74 (± 0.62) 665 (± 48) 26.13 (± 1.2) 26.33 (± 1.1)
Cotyledon 58.4 (± 0.87) 145 (± 2.2) 9.25 (± 0.11) 913 (± 25) 520 (± 11) 246 (± 6.8)
Radicle 88.7
204
11.24
1081
926
374
Table 4.9. Concentrations of micro-elements in the different chickpea parts. Data are presented as a mean ± SD. All samples had n>3 except
the radicle, where n=1 due to small volume.
Fe
(mg/100g)
Zn
(mg/100g)
Mn
(mg/100g)
B
(mg/100g)
Cu
(mg/100g)
Whole seed (dry) 4.23 (± 0.1) 3.49 (± 0.09) 2.54 (± 0.07) 1.41 (± 0.02) 0.44 (± 0.03)
Seed coat 1.60 (± 0.7) 0.46 (± 0.03) 5.56 (± 0.3) 1.38 (± 0.09) 0.05 (± 0.01)
Cotyledon 4.96 (± 0.3) 4.44 (± 0.1) 1.40 (± 0.02) 1.11 (± 0.04) 0.54 (± 0.02)
Radicle 9.52
7.40
1.84
2.07
1.28
Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick 63
4.4 DISCUSSION
HatTrick is one of the highest yielding desi varieties across all chickpea
growing areas in NSW and southern Australia, and is widely grown due to its
resistance to ascochyta blight and phytopthera root rot (Queensland Department of
Agriculture and Fisheries, 2015). Despite its widespread use, there is no information
concerning its nutritional value. In order to determine the baseline from which the
iron content of the commercial chickpea cultivar PBA HatTrick may be improved,
the elemental profiles of samples from five different locations were assessed. Other
commercial cultivars were also included in this study determine the extent of the
influence exerted by genotype and environment and/or management practices.
Mineral compositions of the seeds used in this study fell within the ranges
previously reported in the literature (Wood and Grusak, 2007). Iron was found to be
positively correlated to phosphorus as well as another important micronutrient, zinc.
Comparison of the overall mineral composition showed segregation of samples into
distinct groups based firstly by location and cultivar types. Specifically, that
distribution was found to be particularly influenced by variations in iron, manganese,
calcium and magnesium contents. Comparison between the cultivars studied
generally found little significant difference between them, though the cultivar of
interest, PBA HatTrick, ranked among the lowest in terms of iron and zinc contents.
An examination of its seed confirmed the localisation of almost all iron and zinc to
the cotyledons, which was also the primary site of phosphorus storage.
Effect of location and soil type on grain micronutrient content
In this study, seeds obtained from various sites exhibited distinct mineral
profiles which corresponded to their source location. The locational effect was
particularly pronounced in the zinc contents of the grain. This is in agreement with
the findings of Ray et al. (2014) and Diapari et al. (2014), who have also reported
significant year to year variation even within the same site, further highlighting the
impact on environmental and management factors on zinc accumulation in the seed.
Where the management is concerned however, the effects of zinc fertilisation on
grain zinc content are unpredictable and may differ between seasons (Akay, 2011).
This is worth noting given that majority of the soil types within the Australian Soil
64 Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick
Classification (ASC) have been identified to be at risk of zinc deficiency and
administration of zinc fertilisers is a recommended practice in chickpea cultivation
(Norton, 2013; Pulse Australia, 2016).
Iron was identified to be partially subject to environmental influence, which
corresponds with the results obtained by Diapari et al. (2014) and Jambunathan and
Singh (1981). Accounting for genotype effect, grain iron content was not markedly
different amongst seeds grown on similar soil types. However while one might
expect enhanced iron accumulation in plant tissue where soil conditions are
favourable for uptake, this was not necessarily the case, as demonstrated by the
samples grown in Kingaroy. The high levels of iron oxides and low pH characteristic
of ferrosol soils, both of which favour plant uptake, did not translate to enhanced
seed iron content. Ironically the opposite was observed, with those samples having
the lowest iron contents amongst the locations. Considering the reversal of
manganese to iron ratios within those samples and the high manganese content in
Kingaroy soils (Appendix A, Table 8.1), it is likely that iron accumulation was
inhibited by the manganese concentrations. Similar cases of iron-manganese
antagonism have also been documented in other crops like rice, lettuce, tomato and
oat (Twyman, 1951; Tanaka and Navasero, 1966; Alvarez-Tinaut et al., 1980). As
both elements share components of the uptake and translocation processes (e.g.
Stephan et al., 1996; Vert et al., 2002), competition may occur where the metabolic
pathways overlap. How high iron-genotypes would respond to such conditions
cannot be concluded in this study due to lack of available material, but further
investigation should be conducted to guide grower decisions in this aspect.
Effect of genotype on trace element profile of seeds
When compared to the maximum levels reported in chickpea, the iron and zinc
levels of all cultivars in this study can at best be considered mid-range (Petterson and
Mackintosh, 1994). The highest average values were found in the kabuli cultivar
Genesis090™, while desi cultivars tended to be at the bottom of the spectrum for
iron. Whether this is biotype specific currently cannot be confirmed due to the
insufficient information on these traits. Studies in wheat and more recently in
Canadian chickpea have reported a negative correlation between seed micronutrient
content and yield, though the extent is subject to environmental effects (Garvin et al.,
Chapter 4: Assessment of Iron Content in Chickpea cv Hattrick 65
2006; Ficco et al., 2009; Diapari et al., 2014). In light of this, it is possible that
cultivars developed with productivity at the main objective would suffer from a
trade-off in terms of micronutrient content. Further work is required to verify this in
the Australian context.
In terms of other elements, a clear distinction in calcium content was observed
between the kabuli and desi cultivars used in this study. The higher concentrations in
the desi cultivars were consistent with the findings of Jambunathan and Singh (1981)
and is likely to be attributed to the differing morphologies of the seed coat in which
most of the total calcium is stored. Unless the seeds are consumed whole however,
this calcium is likely to be lost as seed coats are discarded in the dhal-making
process. Most of the nutrition and trace elements are otherwise retained due to their
localisation to the cotyledons (Lal et al., 1963), a trait which can be exploited for
biofortification efforts.
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 67
Chapter 5: Characterisation of Chickpea
Nicotianamine Synthase Genes
5.1 INTRODUCTION
Nicotianamine synthase (NAS) is the enzyme responsible for the biosynthesis
of the nicotianamine (NA). NA is a common component of Strategy I and II plants
that serves as a chelator for the systemic translocation of divalent cations, and is also
a precursor of mugineic acids (MAs) in Strategy II plants.
As a consequence of its role in iron transport, NAS holds significant influence
over iron content in plant tissue. For that reason, it has been extensively investigated
for use in iron biofortification. When expressed constitutively at elevated levels,
NAS has consistently increased the accumulation of iron and other transition metals
like zinc in plant tissues (Douchkov et al., 2005; Johnson et al., 2011; Lee et al.,
2011). The enhancement of seed iron content in transgenic rice overexpressing
endogenous NAS genes has been reported to be as high as 2.9 to 4 times (Lee et al.,
2009; Johnson et al., 2011) with a concomitant enhancement in bioavailability
(Zheng et al., 2010; Trijatmiko et al., 2016). In addition, NAS transformants have
also been demonstrated across various studies to have greater tolerance to heavy
metals (Douchkov et al., 2005; Kim et al., 2005; Pianelli et al., 2005) and iron
deficiency (Douchkov et al., 2005) compared to untransformed plants. No penalty on
general plant growth has been reported in these studies. Sensitivity to iron starvation
however, may be affected depending on the amount of accumulated NA – severe
overaccumulation (up to 100 times) may enhance sensitivity to iron deficiency
(Cassin et al., 2009). No such characteristic was noted in when NA accumulation
was increased by 4.8 fold (Wirth et al., 2009). It should be noted that the species
used by Cassin et al. (2009) and Wirth et al. (2009) differ in iron uptake strategies;
as such, the threshold for NAS accumulation remains inconclusive.
Despite extensive investigation into its function, there is a general lack of
information on the behaviour of the NAS family. Consequently, the potential of
different members for biofortification purposes remains largely unexplored save for a
select few. The number of NAS genes may range from one, such as in tomato (Ling
68 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes
et al., 1999), to twenty-one such as in wheat (Bonneau et al., 2016). To date, the
most well-characterised homologues are those from Arabidopsis (e.g. Schuler et al.,
2012b; Koen et al., 2013), tomato (Ling et al., 1999), rice (e.g. Inoue et al., 2003;
Nozoye et al., 2014b), and barley (e.g. Herbik et al., 1999; Higuchi et al., 1999b).
While not as extensively studied, NAS homologues from other species have also
been characterised to some degree (see Table 5.1).
Table 5.1. List of characterisation studies done on NAS from selected species.
Species Gene ID Source
Arabidopsis halleri AhNAS
Weber et al. (2004)
Deinlein et al. (2012)
Cornu et al. (2014)
Lotus japonicas LjNAS Hakoyama et al. (2009)
Thlaspi caerulescens TcNAS Mari et al. (2006)
Wheat (Triticum aestivum) TaNAS Bonneau et al. (2016)
For the purposes of biofortification, the native expression patterns of a gene
requires due consideration. Given that the physiological role of NAS in Strategy I
and II is fundamentally different, the behaviour of the different homologues is
expected to vary. In species utilising the Strategy II uptake mechanism, NAS
expression and activity are generally upregulated in the roots during iron deficiency
(e.g. Higuchi et al., 1996; Inoue et al., 2003; Mizuno et al., 2003). The reverse is true
for Strategy I plants however, where downregulation is observed instead (Stephan
and Scholz, 1990; Higuchi et al., 1995).
Tissue-specific expression also occurs under various iron regimes. In the study
conducted on rice by Inoue et al. (2003), constitutive expression of OsNAS1 and
OsNAS2 was confined to the root cells involved in long-distance transport (e.g. stele,
pericycle and xylem parenchyma cells). Under iron deficient conditions however,
expression of both was induced in all tissues including the leaf sheaths and vascular
bundle of leaves. In contrast, OsNAS3 expression in the same tissues was lowered in
response to iron deficiency. On top of external iron concentrations, NAS expression
is also subject to specific developmental needs. In the legume Lotus japonicas,
LjNAS2 expression was nodule-specific while LjNAS1 served a more systemic
housekeeping role (Hakoyama et al., 2009).
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 69
The degree to which these specific expression patterns can be extrapolated to
homologues in other species is currently unclear, given the varying number of NAS
genes as well as differing iron requirements. Leguminous plants in particular, may
have specific iron requirements due to nodulation, which may in turn influence
overall NAS behaviour.
The aim of this chapter therefore, was to characterise chickpea NAS2 for use in
iron biofortification. As part of this study, the native expression of CaNAS2 was
investigated. Expression plasmids were also generated for overexpression studies in
tobacco and chickpea, the former of which is covered in this chapter.
5.2 MATERIALS AND METHODS
5.2.1 Designation of chickpea NAS2
The sequences of annotated chickpea NAS genes were obtained from NCBI
and the coding sequences translated to a hypothetical protein. A BLAST check of the
putative protein sequence was performed on the resulting protein to ensure correct
annotation and open reading frames (ORFs). Upon confirmation, a progressive
pairwise alignment was done to determine the degree of similarity between the rice
and chickpea NAS sequences. The chickpea NAS protein with the highest similarity
to OsNAS2 was designated as CaNAS2.
5.2.2 Assessment of NAS amino acid sequence and protein properties
Following the designation of CaNAS2, a range of bioinformatics tools were
used to predict the biochemical properties and localisation of the enzyme. The
theoretical isoelectric point (pI) and molecular weight were calculated using the
Compute pI/Mw tool on ExPASY (Bjellqvist et al., 1993; Bjellqvist et al., 1994;
Gasteiger et al., 2005b). The hydrophobicity profile of the protein was assessed using
ProtScale (Gasteiger et al., 2005a) and potential transmembrane sections identified
using TMpred (http://www.ch.embnet.org/software /TMPRED_form.html). A check
for motif sequences was conducted using ScanProsite (De Castro et al., 2006) and
MOTIF Search (GenomeNet). Phobius (Käll et al., 2004) and iPSORT (Bannai et al.,
2001, 2002) was used to identify potential signalling peptides. Hypothetical 3D
structures were generated using Phyre2 (Kelley et al., 2015).
70 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes
5.2.3 Phylogenetic analysis of NAS proteins
A progressive pairwise alignment was performed on full length protein
sequences of chickpea and other species to determine the degree of homology at the
amino acid level. Of the latter, only NAS genes and/or proteins whose activity or
expression patterns have been experimentally verified were used. The alignment was
done using the default settings of Geneious alignment (global alignment with free
end gaps, Blosum62, gap open penalty 12, gap extension penalty 3). An unrooted
neighbour-joining tree was constructed with Juke-Cantor as the genetic distance
model. No outgroups were selected.
All sequences were obtained from NCBI and the accession numbers are as
listed in Appendix B, Table 8.4. Amino acid sequences used were checked against a
translated cDNA sequence to confirm the correct open reading frames (ORFs). All
alignments and phylogenetic analysis were done using Geneious 7.1.9.
5.2.4 Assessment of CaNAS2 expression
5.2.4.1 Preliminary study
A preliminary study was conducted to determine approximate spatial and level
of expression of CaNAS2 and other CaNAS genes. Samples were taken from a
healthy, non-transgenic chickpea plant. The plant was approximately one month old
at the time of sampling, and had been grown on potting mix under iron-sufficient
conditions. The following tissues were sampled: mature green leaf, senescing leaf,
stem, cotyledon, and root. All samples were snap-frozen in liquid nitrogen
immediately after collection and stored in -80⁰C. RNA extraction and cDNA
synthesis was done following the method described in Chapter 3.2.5.1 and 3.2.5.2.
Gene expression was assessed qualitatively using end-point PCR with cDNA as the
template. Primers used for CaNAS2 and other CaNAS family members are as
described in Table 5.4 and 5.2 respectively.
5.2.4.2 Iron deficiency experiment
Chickpea seeds cv Hattrick were sterilised and germinated on half MS media
as described in Chapter 3.2.3.2.1. One week post-germination, the seed coats were
removed and the seedlings were acclimatised for four days in tap water in a loosely
covered beaker. Healthy seedlings of approximately the same size and developmental
stage were selected and transferred to the mini hydroponics set-up illustrated below
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 71
(Figure 5.1). A total of twenty seedlings were chosen, with ten per set-up. Both set-
ups were placed in a growth cabinet set at 23⁰C, with a 16 hour photoperiod.
All plants were grown on tap water for four weeks from the time of
acclimatisation. Full-strength Hoagland solution with or without Fe-EDTA (Chapter
3.1.5.1) was then provided in the subsequent weeks until sampling. All solutions
used were topped up every two to three days as required. During the treatment,
MilliQ water was used every third top-up instead of Hoagland solution to dilute
accumulated salts. Sampling was done two weeks following the onset of chlorosis in
the iron-deprived plants. Three plants of similar conditions and growth stage were
selected from each treatment for analysis of gene expression. The selected plants
were rinsed in tap water, photographed, and the following tissue types were
collected: mature leaf, stem, cotyledon, and root. Senescent leaf and chlorotic leaf
were also collected from the iron-sufficient and iron-deficient plants respectively. All
samples were snap-frozen in liquid nitrogen immediately after collection and stored
in -80⁰C. RNA extraction and gene expression analysis was done following the
method described in Chapter 3.2.5. Primers used in qPCR are as listed in Table 5.2.
Figure 5.1. Schematic diagram of the mini-hydroponics system.
5.2.4.3 Measurement of CaNAS2 expression level
Measurement of CaNAS2 expression was done as described in Chapter 3.2.5.
RNA was extracted from the sampled tissues and the quality confirmed via gel
electrophoresis. DNase treatment was then performed on 1 µg of RNA, from which
cDNA was generated. The cDNA was checked via PCR using a housekeeping gene
72 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes
to confirm successful synthesis, following which it was used for qPCR. Primers used
in qPCR are as listed in Table 5.2.
Table 5.2. List of primers used in qPCR.
Gene Sequences (5’-3’)
Expected
amplicon
size (bp)
Actin 1 Fw GCCTGATGGA CAGGTGATCA C
62 Rv GGAACAGGAC CTCTGGACAT C
EF1α Fw TCCACCACTT GGTCGTTTTG
64 Rv CTTAATGACA CCGACAGCAA CAG
GAPDH Fw CCAAGGTCAA GATCGGAATC A
65 Rv CAAAGCCACT CTAGCAACCA AA
CaNAS2 Fw AGTAGTGCCT TTCTAAATGG CC
116 Rv CATGTCACCA ATCCCCAACA T
CaNAS
(XP_004487761.1)
Fw GTCACTCAAG TCTGATTCGA CC 172
Rv TGAGGTGGTG CATGTTGTTA C
CaNAS
(XP_004488704.1)
Fw TAGCAAGATC GTGGCATCGG 135
Rv CCTCTACTCA TACCAACAAG TGC
CaNAS
(XP_004494544.1)
Fw AGTGCTTTGT ATCTCATGGA GC 133
Rv TGCATGCCCT TATATACGGC T
5.2.5 Isolation and cloning of chickpea NAS2 and other genes of interest
The OsNAS2 gene was kindly provided by Dr Alex Johnson from the
University of Melbourne. The GmFER gene was synthesized. CaNAS2 was isolated
from chickpea genomic DNA using primers designed from the predicted sequence
from Genbank database, accession number XM_004495601.
The genes of interest were amplified and restriction sites added to the ends via
site-directed mutagenesis using high fidelity PCR (Phusion®, NEB) with the primers
listed in Table 5.3. For OsNAS2 and CaNAS2, AscI and PacI restriction sites were
added to the 5’ and 3’ ends respectively. For GmFER, SalI and BstEII restriction
sites was added to the 5’ and 3’ end respectively. Following this, the mix was
incubated at 72⁰C for one hour with 5µL of GoTaq (Promega) to restore the A
overhang in the ends blunted by high fidelity PCR.
The resulting PCR products were separated by gel electrophoresis, purified
using Freeze ‘N Squeeze DNA Gel Extraction Spin Columns (Biorad) and ligated
overnight into pGEM®-T Easy vectors (Promega). A 2µL aliquot of ligation reaction
was transformed into E.coli using an in-house heat shock method, and the culture
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 73
spread onto Luria-Bertani (LB) plates containing 14% IPTG, 2% X-gal and 100mg/L
of ampicillin. White colonies were selected and their DNA sequenced to verify gene
integrity.
Table 5.3. List of cloning primers. Restriction sites have been highlighted in gray.
Gene Restriction
site Sequences (5’-3’)
Expected
amplicon
size (bp)
OsNAS2
Fw AscIF GGCGCGCCAT GGAGGCTCAG
AACCAAGAG 997
Rv Pac1R TTAATTAATC AGACGGATAG
CCTCTTGG
CaNAS2
Fw AscIF GGCGCGCCAT GGTTTGCAAG
GAAGATATAT TAATC 939
Rv Pac1R TTAATTAATC ATTCTTCAAT
GACCAATTCC TC
GmFER
Fw SalIF GTCGACCCTA GGATGGCCCT
TTCTTGCTCC 793
Rv BstEIIR GGTGACCTTA TACATGATCT
TCATCGTGAA GAA
5.2.6 Generation of expression plasmids
The pOpt-EBX expression vector was obtained from the Gates research group
from the Centre for Tropical Crops and Biocommodities (CTCB). The pOpt-EBX-
35s-UidA expression vector and pGEM-T cloning vector with a cassette containing a
Nos promoter and CaMV 3’ UTR was obtained from Hao Long from the Abiotic
Stress research group in the CTCB.
Upon verification of sequence, the genes of interest were excised from
sequence verified pGEM-T® Easy vectors. OsNAS2 and CaNAS2 were digested
using AscI and PacI. GmFER was digested using SalI and BstEII. The digested
products were separated by gel electrophoresis and the genes ligated into their
respective backbones/sub-backbones in a 5’ to 3’ direction. GmFER was ligated into
the pOpt-EBX backbone, in between the CaMV 35s promoter and Nos terminator.
To add the promoter and terminator sequences to OsNAS and CaNAS, those genes
were ligated with a pGEM-T cloning vector with a cassette containing a Nos
promoter and CaMV 3’ UTR. The constructs were sequenced verified to determine
gene integrity and orientation, following which the NosP-CaNAS/OsNAS-CaMV 3’
UTR cassette was digested and ligated with the pOpt-EBX-GmFER backbone to
74 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes
form the complete vector. The final constructs were sequence verified and
transformed into Agrobacterium tumefaciens strains Agl1 and LBA4404.
To generate the NAS-only constructs, the verified pOpt-EBX-OsNas2/CaNas2-
GmFER constructs were digested with BamHI and StuI to remove GmFER and its
CaMV 35s promoter and Nos terminator. The digested constructs (without ferritin
and its promoter and terminator) were then separated by gel electrophoresis. As
BamHI produces a sticky end incompatible to the blunt end from StuI, blunting of
the sticky end was done using a high-fidelity polymerase (Phusion®, NEB).
Following this, self-ligation was performed to join the now compatible ends of the
construct to each other. The newly generated NAS2-only constructs were cloned into
E.coli for amplification, then sequenced to confirm the integrity of the remaining
genes and complete removal of GmFER and its promoter and terminator. The
constructs (Figure 5.2) were subsequently transformed into Agrobacterium
tumefaciens strains Agl1 and LBA4404.
To confirm the presence of the gene/s of interest in Agrobacterium cultures
used for transformation, genomic DNA was isolated from the respective AGL1 and
LBA4404 cultures for PCR verification. PCR was performed using GoTaq
(Promega) and gene-specific primers (Table 5.4). The resulting products were
separated via electrophoresis in a 1% agarose gel and made visible by staining with
SYBR® Safe (Life Technologies).
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 75
Figure 5.2. Expression plasmids generated for plant transformation. The
combinations of genes of interest and the backbone are as illustrated in (A) and (B)
respectively. NosT represents the nopaline synthase terminator, NPTII represents the
neomycin phosphotransferase II gene, S1 Pro represents the S1 promoter, GUS
represents the β-glucuronidase gene, CaNAS2 represents the Cicer arietinum
nicotianamine synthase 2 gene, OsNAS2 represents the Oryza sativa nicotianamine
synthase 2 gene, GmFER represents the Glycine max ferritin H1 gene, CaMV 35sP
represents the cauliflower mosaic virus 35s promoter, CaMV 3’UTR represents the
cauliflower mosaic virus 3’UTR terminator, and NosP represents the nopaline
synthase promoter.
A)
B)
76 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes
Table 5.4. List of primers used for screening.
Gene Sequences (5’-3’)
Expected
amplicon
size (bp)
uidA (GUS) Fw TGAACATGGC ATCGTGGTGA
507 Rv GCTAACGTAT CCACGCCGTA
OsNAS2 Fw CTGAGCAAGC TGGAGTACGA
660 Rv TCAGACGGAT AGCCTCTTGG
CaNAS2 Fw GCATGTCACC AATCCCCAAC
568 Rv CGCAGCATCA AAAGTGCTCC
GmFER Fw ATGGCCCTTT CTTGCTCCAA
604 Rv GTTCTGCCAC ACTGTGAACG
Neomycin
phosphotransferase II
Fw ATTCGGCTAT GACTGGGCAC 675
Rv TAAAGCACGA GGAAGCGGTC
5.2.7 Generation and molecular characterisation of transgenic tobacco
Transgenic tobacco was generated following the procedure outlined in Chapter
3.2.3.1. The resulting lines were screened for the genes of interest and RT-PCR was
performed on PCR-positive plants to check for gene expression. All lines were
acclimatised and grown as per Chapter 3.2.3.1.3, and seeds collected upon maturity.
5.2.8 Assessment of iron accumulation in transgenic tobacco leaf
T1 tobacco seeds were sterilised and germinated on half-strength MS with
100mg/L of kanamycin for two weeks. Germinated seedlings were transferred to
half-strength MS with no selection for two weeks.
Selected plants were subcultured to fresh MS media containing 150µM Fe-
EDTA and after eight days, the second, third and fourth youngest fully expanded
leaves were harvested for trace element analysis. Samples were rinsed twice in DI
water and prepared for LA-ICP-MS following the protocol outlines in Chapter 3.2.6.
5.3 RESULTS
5.3.1 Designation and sequence analysis of CaNAS2
Depending on the species, the number of NAS genes may range from one to
twenty-one. In chickpea, four hypothetical NAS proteins are annotated on NCBI. A
progressive pairwise alignment was done to compare the sequences to OsNAS2,
which had been successfully applied in other biofortification efforts. Hypothetically,
similarity in amino acid sequence would translate into a comparable activity and
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 77
effectiveness for biofortification. Of the four CaNAS sequences, XP_004495658.1
had the highest sequence similarity to OsNAS2, as well as the other OsNAS proteins
(see Table 5.5). Visual inspection of the predicted 3D structure however, showed
XP_004487761.1 to be more similar to OsNAS2 – this observation was arrived at by
several independent parties (see Figure 5.3). Given potential inaccuracies in
predictive 3D modelling however, sequence similarity was set as the criteria for
designation of CaNAS2. XP_004495658.1 was therefore selected and designated as
CaNAS2.
Table 5.5. Similarity between the CaNAS and OsNAS amino acid sequences.
Comparison was done using a progressive pairwise alignment. Similarity values are
expressed as a percentage.
OsNAS1 OsNAS2 OsNAS3
CaNAS2 XP_004495658.1
44.089 44.66 46.326
XP_004487761.1 41.009 40.317 41.195
XP_004488704.1 42.908 42.199 41.176
XP_004494544.1 42.547 41.304 45.652
Figure 5.3. Predicted 3D structures of OsNAS and CaNAS2 proteins. Structures
are coloured based on progression from the N (red) to C terminus (dark blue).
78 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes
Upon designation of CaNAS2, predictive modelling was done to identify the
potential biochemical properties of the enzyme, from which the subcellular
localisation and behaviour may be alluded. CaNAS2 was predicted to have an
approximate molecular weight of 34.36kDA and a pI of 5.52. Assessment of the
hydrophobicity profile and transmembrane topology showed the enzyme to be
primarily hydrophilic (Figure 5.4.A) with a potential transmembrane domain at
position 126 to 151 (Figure 5.4.B). The lack of significant hydrophobic regions
suggests that membrane association, if any, occurs only at a peripheral level. A
subsequent check for motifs revealed a single NAS motif from position 5 to 275
which occupied approximately 88% of the entire protein (see Figure 5.5). A
methyltransferase domain was detected within the NAS motif from positions 151 to
250, just outside the potential transmembrane domain.
Figure 5.4. Predicted biochemical properties of CaNAS2. A) Hydrophobicity plot,
and B) predicted transmembrane topology. The potential transmembrane site has
been highlighted in blue.
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 79
1 MVCKEDILIE QVCDLYNQIS NLDTLKPCKI VNTLFTKLVL TCMSPIPNID VTKLATNVQE
61 IRSKLIILCG EAEGHLESHY STILASHNNP LNHLNIFPYY TNYLKLSLLE FNILNQHITN
121 NVPKNVAFIG SGPLPLTSIV LATNYLPSTI FHNYDIDPLA NSKASCLISS NPDLSNRMLF
181 HTNDILNVTN DLKEFEVVYL AALVGMNNEE KNKIIDHLGK YMAHGALLML RSAHGARAFL
241 YPVVDTSDLR GFEVLSIFHP TDEVINSVLI ARKYNPIVLL PNKCCGDEIQ VFKPLNNMIE
301 ELVIEE
Figure 5.5. Amino acid sequence of CaNAS2. Red text marks out the predicted
NAS motif, underlined text marks out the methyltransferase domain, and highlighted
section is the YXXΦ and LL motifs.
Scans using Phobius and iPSORT predicted a non-cytoplasmic localisation and
detected no signal peptides, mitochondrial targeting or chloroplast transit signals.
However, a cross-check with existing studies on other NAS proteins (Nozoye et al.,
2014a; Bonneau et al., 2016) pointed to the presence of two motifs at the N-terminus
region. The YXXΦ (Y refers to tyrosine, X to any amino acid residue, and Φ to
bulky hydrophobic residues) and di-leucine (LL; leucines may be substituted with
isoleucines) motifs were located at the position 103 and 118 respectively. Both are
conserved in the NAS superfamily, with the former implicated in vesicular
localisation and movement, and the latter with maintenance of enzyme structure
(Nozoye et al., 2014a).
However, whether this culminates in actual vesicular localisation is uncertain.
Despite the conservation of YXXΦ and LL motifs, studies with GFP-tagged NASes
from Arabidopsis, rice and maize have reported localisation to both vesicles and
cytoplasm; this has been suggested to reflect physiological roles (Mizuno et al.,
2003; Zhou et al., 2013a; Nozoye et al., 2014b). To verify the results obtained with
CaNAS2, the OsNAS, AtNAS and ZmNAS sequences (accession numbers as listed
in Appendix B, Table 8.4) were also assessed. The results obtained were similar to
that of CaNAS2 despite the reported cytoplasmic localisation of all the inputs except
OsNAS2, ZmNAS1 and ZmNAS2.
5.3.2 Phylogenetic analysis of CaNAS2
To determine the relationship between CaNAS2 and other NAS proteins, a
pairwise multiple alignment was done on the amino acid sequences and a
phylogenetic tree generated. Consistent with the findings of other authors (Filipe de
80 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes
Carvalho et al., 2012; Zhou et al., 2013b), the result show a clear segregation
between the graminaceous and non-graminaceous sequences (Figure 5.6). Amongst
the non-graminaceous plants, two groups could be observed. The first is a legume-
specific outgroup containing a NAS from chickpea and Medicago truncatula (Group
1). The second group comprises of all other non-graminaceous NAS, within which is
another legume-specific cluster containing the remaining legume NAS sequences.
This cluster contained two distinct groups (Figure 5.6, Group 2 and 3). In
Group 3 an additional duplication event appears to have taken place in the Medicago
and chickpea lineages giving rise to CaNAS2 and CaNAS (XP_04494544.1), and
MtNAS XP_003591220.1 and XP_13450461.1 respectively. Based on existing
studies, it is unclear if each group fulfils specialised functions. Differing patterns of
expression have been exhibited in Group 2 members LjNAS2 and MtNAS
(XP_003594753.1), with the former being nodule-specific (Hakoyama et al., 2009),
and the latter having low expression in all tissues (Medicago truncatula Gene
Expression Atlas).
On the other hand, the tissues in which Group 3 are expressed or upregulated
appear to be more uniform. LjNAS1 was reported to be expressed in leaves, stem and
cotyledons (Hakoyama et al., 2009). Similar patterns were also observed with
MtNAS XP_003591220.1 and XP_013450461.1, which showed general upregulation
in the various aerial tissues as well as the roots (Medicago truncatula Gene
Expression Atlas). Amongst these three genes, the stem appears to be a common site
of expression. It is likely that the CaNAS homologues would behave in a comparable
manner as the genomes of both M. truncatula and chickpea share approximately 95%
synteny, and MtNAS (XP_003591220.1) was the most similar to CaNAS2 with
74.8% sequence similarity.
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 81
Figure 5.6. Phylogenetic relationship between CaNAS2 and NAS proteins from
other plants. The scale bar and branch labels represent the number of substitutions
per site. The legume-specific clades are as indicated by the green boxes and the red
box shows the locations of CaNAS2 in the unrooted phylogenetic tree. Species
included in this tree are Arabidopsis thaliana (AtNAS), Arabidopsis halleri
(AhNAS), barley (HvNAS), Lotus japonicus (LjNAS), Medicago truncatula
(MtNAS), rice (OsNAS), Thlaspi caerulescens (TcNAS), tomato (SlNAS) and maize
(ZmNAS).
82 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes
5.3.3 CaNAS2 expression is downregulated in response to iron deficiency
NAS expression has been reported to vary between species, particularly
between the graminaceous and non-graminaceous ones (Higuchi et al., 1995). This is
further subject to the number of homologues, which may be differentially expressed
in various tissues in response to external and internal iron supply. A preliminary
study was done on a one month-old non-transgenic chickpea (cv HatTrick) to
determine the approximate pattern of expression for CaNAS2. Five different tissue
types were surveyed: mature green leaf, senescing leaf, stem, cotyledons, and root.
Qualitative assessment of gene expression showed CaNAS2 to be expressed in
almost all tissues except senescing leaf (see Figure 5.7). The strongest expression
occurred in the stem and cotyledons, while a fainter signal was detected in the green
leaf and root samples. The same assessment was carried out for the other CaNAS
family members showed differing patterns of expression (see Figure 5.8).
XP_004487761.1 was not expressed in any of the tissues tested, while
XP_004488704.1 was expressed in the leaf, cotyledons, and roots. XP_00449454.1
was expressed in all tissues.
Figure 5.7. Qualitative assessment of CaNAS2 expression in different chickpea
tissues via PCR.
M = 2-log ladder (NEB)
“-” = No template control
“+” = gDNA positive control
Leaf (GR) = Mature green leaf
Leaf (SC) = Senescing leaf
Stem = Stem
Coty = Cotyledon
Root = Root
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 83
Figure 5.8. Qualitative assessment of the expression of other CaNAS family
members in different chickpea tissues via PCR.
M = 2-log ladder (NEB)
“-” = No template control
“+” = gDNA positive control
Leaf (GR) = Mature green leaf
Leaf (SC) = Senescing leaf
Stem = Stem
Coty = Cotyledon
Root = Root
84 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes
To confirm these results and to determine the effect of iron status on CaNAS2
expression, plants were grown in a hydroponics set-up under iron-sufficient and iron-
deficient hydroponic conditions. Chlorotic symptoms were allowed to develop for
two weeks after onset before tissues were sampled for expression analysis. Initial
germination and establishment was done in a nutrient-free solution did not produce
visible symptoms of iron deficiency. Subsequent treatment with nutrient solution
allowed for further vegetative growth, however it still took approximately four weeks
before visible symptoms of iron deficiency, chlorosis, were observed. Including an
additional two weeks to allow for further development of symptoms, the treated
plants were deprived of iron for a total of 9½ weeks. By the time of harvest, young
leaves of the treated plants were severely chlorotic (see Figure 5.9). The whole plant
in general was also more yellowish compared to their iron-sufficient counterparts,
though no apparent senescing leaves were present. Consequently, the chlorotic leaves
were harvested in place of senescing leaves. In addition to the leaves, the roots of the
treated plants were also paler compared to the iron-sufficient controls. No nodules
were observed in either treated or untreated plants.
Following harvest, RNA was extracted from the sampled tissues and treated
with DNAse. Upon confirmation of gDNA removal, cDNA was synthesized. Good
quality RNA was obtained from all tissues except the roots, which were excluded in
this analysis. The removal of gDNA and subsequent synthesis of cDNA was verified
by PCR (Figure 5.10).
Figure 5.9. Morphology of iron-sufficient (+Fe) and iron-deficient (-Fe)
chickpea grown under hydroponics conditions.
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 85
Figure 5.10. Representative photos of verification of RNA and cDNA for qPCR
analysis. A) Quality of extracted chickpea RNA, B) Removal of contaminating
gDNA, and C) confirmation of cDNA synthesis using GAPDH.
M = 2-log ladder (NEB)
“-” = No template control
“+” = gDNA positive control
1, 2, 3 = Biological replicates
Leaf (GR) = Mature green leaf
Leaf (SC) = Senescing leaf
Stem = Stem
Coty = Cotyledon
Root = Root
86 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes
Evaluation of CaNAS2 expression in the sampled tissues found it to be
generally upregulated under iron-sufficient conditions, with expression
predominantly occurring in the stem and roots (see Figure 5.11). Under iron-deficient
conditions however, an overall downregulation of CaNAS2 was observed. No
expression was observed in the cotyledons of treated plants, possibly due to the
exhaustion of iron stores and resulting lack of iron for export to the rest of the plant.
Lower levels of CaNAS2 expression was observed in the senescing and chlorotic
leaves compared to the mature green leaves (see Figure 5.11).
Figure 5.11. Expression of CaNAS2 in different tissues under iron-sufficient
(+Fe) and iron-deficient conditions (-Fe). n=3. Error bars represent standard error.
Expression was measured via qPCR and calculated as relative to that of the
housekeeping genes GAPDH and EF1α. No viable RNA could be extracted from the
iron-deficient root samples and they were therefore excluded from this analysis.
Leaf (GR) = Mature green leaf
Leaf (SC) = Senescing leaf
Leaf (CHL) = Chlorotic leaf
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 87
5.3.4 Generation of expression plasmids
Following the characterisation of CaNAS2 in the native host, transgenic
vectors were generated for overexpression studies in the model plant tobacco
(Nicotiana tabacum cv Samson). These vectors were also used in Chapter 6 for
overexpression studies in chickpea. OsNAS2 was used as a positive control for
comparison. All Agrobacterium cultures containing transgenic constructs were
screened prior to use in plant transformation, and the presence of their respective
genes of interest confirmed (see Figure 5.12).
Figure 5.12. PCR detection of the genes of interest in the Agrobacterium strains
AGL1 and LBA4404 used for plant transformation work.
M = 2-log ladder (NEB)
“H2O” = No template control
“WT” = Untransformed Agrobacterium control
“+” = Positive plasmid control
GUS = Agrobacterium transformed with pEBX-UidA
FO = Agrobacterium transformed with pEBX-GmFER-OsNAS2
O = Agrobacterium transformed with pEBX-OsNAS2
FC = Agrobacterium transformed with pEBX-GmFER- CaNAS2
C = Agrobacterium transformed with pEBX- CaNAS2
88 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes
5.3.5 Generation and molecular characterisation of transgenic tobacco
Following determination of CaNAS2 expression profile in chickpea, transgenic
tobacco overexpressing CaNAS2 was generated to determine its influence on iron
concentrations. To also determine its effect in a multigenic approach like the one
used by Trijatmiko et al. (2016), transgenic tobacco plants expressing both CaNAS2
and soybean ferritin (GmFER) were generated. OsNAS2, a homologue from rice that
has successfully been used in other biofortification studies, was used as a positive
control in both the monogenic and multigenic constructs.
Putative transgenic lines were screened via PCR using gene-specific primers
(see Figure 5.13). Amongst the PCR positive lines, one GUS, 12 GmFER-CaNAS2
and 11 GmFER-OsNAS2, CaNAS2, and OsNAS2 lines were randomly selected for
further work. Expression of the genes of interest was confirmed via end-point RT-
PCR (see Figure 5.14). All lines were subsequently grown to seed to obtain the T1
generation. Not all lines flowered successfully; amongst those that did, at least three
lines per construct did not produce viable seed; this occurrence was consistent across
all the clones from the same line. This failure to produce flowers or seed was not
limited to any particular construct – at least two lines per construct were affected.
The final summary of transgenic events progressing to the T1 generation is as listed
in Table 5.6.
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 89
Figure 5.13. Representative photos of T0 tobacco PCR screening with gene-
specific primers. The following genes were screened: A) GmFER, B) CaNAS2, and
C) OsNAS2. Each lane bearing FO, O, FC, or C represents an individual transgenic
event.
M = 2-log ladder (NEB)
“-” = No template control
“+” = Positive plasmid control
NT = Untransformed tobacco control
FO = pEBX-GmFER-OsNAS2
O = pEBX-OsNAS2
FC = pEBX-GmFER-CaNAS2
C = pEBX- CaNAS2
90 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes
Figure 5.14. Detection of transgene expression in GM tobacco lines via PCR.
The constructs are as follows: A) pEBX-Ferr-CaNAS2, B) pEBx-Ferr-OsNAS2, C)
pEBX-CaNAS2, and D) pEBX-OsNAS2. The target genes are as listed in each
image. Each lane bearing a number represents an individual transgenic event.
M = 2-log ladder (NEB)
“-” = No template control
“+” = Positive plasmid control
NT = Untransformed tobacco control
GUS = pEBX-UidA transgenic tobacco lines
Table 5.6. Summary of transgenic tobacco lines generated and progressing to
the T1 generation.
Construct RT-PCR +
lines
Lines progressing to
T1 generation
pEBX-GUS 1 1
pEBX-GmFER-CaNAS2 12 10
pEBX- GmFER-OsNAS2 11 6
pEBX-CaNAS2 11 3
pEBX-OsNAS2 8 7
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 91
5.3.6 Transgenic tobacco exhibit to significant increase in leaf iron or zinc
contents
To assess the impact of the genes of interest on iron accumulation, the leaves
of one month old T1 plants were assessed for iron content using LA-ICP-MS. Prior
to commencement of the experiment, all plants used were screened for the presence
of the transgenes (Appendix B, Figure 8.1). Only plants testing positive for the genes
of interest were used in this study. Of the lines tested, FC6 was positive for GmFER,
but not for CaNAS2. It was included in this study as a Ferr-only control.
To determine viability of further work, an initial assessment was done on the
CaNas2 and Ferr-CaNAS2 line. Amongst those samples, leaf iron content ranged
from 132 to 177ppm (see Figure 5.15). While considerable variability was observed
between different lines within the same construct, there was no significant difference
between them. Similarly, no significant difference was found when compared to the
non-transgenic and vector controls. The same result was obtained after including
three randomly chosen OsNAS2 and Ferr-OsNAS2 lines for further comparison
(Figure 5.15). Examination of individual transgenic plants showed that, save for a
few individuals within each line, iron contents were comparable to that of the non-
transgenic controls (Appendix B, Figure 8.2).
A similar observation was made with zinc content where, despite more
variability between lines, the average values were not markedly different from the
non-transgenic control (see Figure 5.15 and Figure 8.2). However, the difference was
significant compared to the GUS vector control line, which contained the lowest iron
and zinc content.
92 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes
Figure 5.15. Average A) iron and B) zinc content in non-transgenic (n=10) and
transgenic tobacco leaves (n=4 to 7). Gray bars represent the non-transgenic (NT)
and GUS control lines, dark orange bars represent the GmFER-CaNAS2 (FC)
transgenic lines, light orange bars represent the CaNAS2 (C) transgenic lines, dark
blue bars represent the GmFER-OsNAS2 (FO) transgenic lines, and light blue bars
represent the OsNAS2 (O) transgenic lines. Error bars indicate standard error.
Statistical significance was calculated using Dunnett’s test, and p-values<0.05 were
considered statistically significant.
NT = Non-transgenic tobacco control
GUS = pEBX-UidA transgenic tobacco lines
FO = pEBX-GmFER-OsNAS2 transgenic tobacco lines
O = pEBX-OsNAS2 transgenic tobacco lines
FC = pEBX-GmFER-CaNAS2 transgenic tobacco lines
C = pEBX-CaNAS2 transgenic tobacco lines
* = Statistically significant compared to GUS line
= Statistically significant compared to non-transgenic control
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 93
5.4 DISCUSSION
Compared to their counterparts in the Gramineae family, relatively little is
known about the NAS orthologues in non-graminaceous species. Chickpea has four
NAS genes, and this chapter focuses on the characterisation of the one of the
homologues. Dubbed CaNAS2, it was selected due to its sequence similarity to
OsNAS2, which has been successfully applied in biofortification work by other
authors (e.g. Johnson et al., 2011; Trijatmiko et al., 2016). Using a combination of
modelling and phylogenetic analyses, we predicted the subcellular localisation and
expression profile of CaNAS2. Transgenic tobacco overexpressing CaNAS2 were
subsequently generated to determine its effect on iron accumulation.
Predicted subcellular location of CaNAS2
To determine potential subcellular behaviour, predictive modelling was
performed on the CaNAS2 amino acid sequence. CaNAS2 was predicted to be a
hydrophilic protein with a single transmembrane domain. No other signalling
sequences were detected aside from the YXXΦ and LL vesicular translocation
motifs. Whether this is reflective of actual vesicular localisation however, is
contentious. Studies on NAS homologues from other species have showed
conflicting results – despite conservation of the YXXΦ and LL motifs in the NAS
family, only OsNAS2, ZmNAS1 and ZmNAS2 were confirmed to localise to
vesicles (Mizuno et al., 2003; Nozoye et al., 2014a). Others, like ZmNAS3 and the
AtNAS family, localised to the cytoplasm (Mizuno et al., 2003; Nozoye et al.,
2014b). For comparison, the same analysis performed on CaNAS2 was repeated on
these NAS homologues. The results obtained were inconclusive – no other
localisation signals were detected despite the proven vesicular localisation of some
homologues (data not shown). Whether this discrepancy can be attributed to
limitations on existing databases or post-translation modifications is uncertain, and
further study required.
94 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes
Systemic expression of Group 3 NAS homologues and their potential housekeeping
role
Consistent with the findings of other authors (Higuchi et al., 2001; Hakoyama
et al., 2009; Filipe de Carvalho et al., 2012), phylogenetic analysis of NAS proteins
showed a clear distinction between monocots and dicots. This corresponds to the
differing physiological roles of NAS between the two lineages. Incidentally all the
monocots used in this study belonged to the Gramineae family, and it is unclear if
this clustering will change upon inclusion of non-graminaceous monocots. No
conclusive statements can be made in that regard due to a current lack of
experimentally verified NAS of non-graminaceous monocot origins. While this lack
of inputs may mask potential phylogenetic links, the results thus far indicate that
specific physiological capabilities such as MAs synthesis can exert a selective
pressure on NAS evolution.
With that in mind, it is possible that symbiosis with Rhizobium may provide
sufficient selective pressure to produce NAS specific for that function. LjNAS2 for
instance, was reported to be nodule-specific (Hakoyama et al., 2009). However,
including the closely related MtNAS (XP_003594753.1), no other genes used in this
study were known to share that trait. This can partly be attributed to the lack of
information on dicot NAS homologues as a whole, and future studies may uncover
more nodule-specific homologues. In the interim, existing information only permits
reasonable allusion to the potential function of the one of the three NAS groups
observed within the leguminous lineage.
Group 3 of leguminous NAS homologues are characterised by their widespread
expression in various tissues, ranging from roots, to leaves and cotyledons. This
expression pattern was also exhibited by CaNAS2 and XP_00449454.1, both
members of Group 3. While the range of expression sites was found to vary between
the homologues, the stem remained a common site of expression. As suggested by
Hakoyama et al. (2009), such an expression pattern points to a housekeeping role in
the systemic redistribution of iron. Given that NA is involved in both long and short-
distance translocation (Stephan et al., 1994b; Takahashi et al., 2003b; Schuler et al.,
2012b), the NAS expressed in the various sites may operate at different scales.
Expression in the stem for instance, may serve to feed NA into the vascular tissue
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 95
and symplast for systemic transport, while expression in the other locations may
provide NA for more localised translocation.
Differential expression of CaNAS2 in response to external iron conditions
In this study, CaNAS2 transcripts were detected in a wide range of tissues,
particularly in the stem and roots. Higher expression was also noted in the iron-
sufficient plants. This supports the idea of a housekeeping role in systemic
translocation of iron, particularly from the roots to the leaves. That a general
downregulation was observed in the iron-deficient plants suggests that CaNAS2 is
not heavily involved in the iron deficiency response, and this housekeeping role
occurs mostly under iron-sufficient conditions.
This downregulation was also observed by Higuchi et al. (1995), who reported
decreased NAS activity in tomato, soy, and several tobacco species in response to
iron deficiency. Douchkov et al. (2005) on the other hand, reported enhanced NA
accumulation in Arabidopsis under iron-deficient but not iron sufficient conditions.
These accounts hint at potential species-specific responses even amongst the non-
graminaceous species, though the regulatory mechanisms underlying this disparity or
the levels at which they occur is unknown. Given that the role of NA in the iron
metabolism of chickpea is also not fully understood, it is plausible that other CaNAS
homologues may be involved in the iron deficiency response even is CNAS2 is not.
With NA being a key ligand for iron translocation, it was expected that NAS
expression would be upregulated in the cotyledons to facilitate iron export during
iron deficiency. The absence of CaNAS2 transcripts in that tissue under iron-
deficiency was therefore quite unusual. Tiffin et al. (1973) had previously
demonstrated the significance of the cotyledons as a nutritional support and buffer
during both germination and later vegetative growth. They had found iron to be
exported from the cotyledons during iron deficiency, while the reverse was observed
under iron sufficient conditions. Assuming a similar phenomenon had occurred in
this study, there are two probable reasons behind the expression pattern of CaNAS2
in the cotyledons. First, is the exhaustion of iron reserves in the cotyledons and the
resulting lack of iron to transport. Second, is that CaNAS2 is not involved in the
96 Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes
export of iron from the cotyledons during iron-deficiency. Further work is required
for confirmation.
Overexpression of CaNAS2 in N.tabacum leads to enhancement of zinc, but not
iron, content in the leaf
Overexpression of NAS have been demonstrated to enhance iron content in
both monocot and dicot species (Douchkov et al., 2005; Masuda et al., 2009;
Johnson et al., 2011). To determine if overexpression of CaNAS2 would produce a
similar result, several transgenic tobacco lines overexpressing CaNAS2 on its own or
in combination with GmFER were generated. The same expression constructs, but
with OsNAS2 instead of CaNAS2, were used as positive controls.
Between the OsNAS2 and CaNAS2 overexpressing lines, no notable
differences were observed in the leaf iron and zinc contents. Compared to the non-
transgenic and GUS vector control, no significant differences were also observed in
the average leaf iron contents of the NAS and NAS-GmFER lines. However,
considerable variability was also observed amongst the replicates in each line. In
contrast, consistent enhancements were seen in the zinc contents, which were
significantly higher in most transgenic lines compared to the GUS vector control.
This increased zinc accumulation was unsurprising. NA has previously been
implicated in the zinc homeostasis, particularly in the zinc hyperaccumulating
species Arabidopsis halleri (Pich and Scholz, 1996; Deinlein et al., 2012; Tsednee et
al., 2014). NAS has also been successfully used in zinc biofortification efforts in rice
(Johnson et al., 2011; Trijatmiko et al., 2016). However increases in zinc and iron
levels have typically been observed to occur in tandem (Douchkov et al., 2005;
Johnson et al., 2011), and the absence of such a phenomenon in this study was
unforeseen. This was particularly so for the NAS-GmFER transgenic lines in which
GmFER was driven by the 35s promoter. Unlike NA, which interacts with a range of
divalent cations, GmFER is known to be specific to iron. Also, the positive effect of
FER overexpression on iron accumulation has been consistently demonstrated by
several authors (Van Wuytswinkel et al., 1999; Drakakaki et al., 2000; Goto et al.,
2000).
Chapter 5: Characterisation of Chickpea Nicotianamine Synthase Genes 97
Considering that NAS was a common factor in all the expression cassettes, a
possible reason behind this discrepancy might be external iron concentrations. When
grown under iron-limiting conditions, elevated iron contents have been observed in
transgenic tobacco and Arabidopsis overexpressing AtNAS2 and TcNAS1
respectively (Douchkov et al., 2005; Cassin et al., 2009). With the latter, shoot iron
content was also noted to be lower than the wild-type under iron-sufficient
conditions (Cassin et al., 2009). In this chapter, all plants were grown under iron
sufficient conditions and the response to iron deficiency was not tested. Whether a
similar response to that reported by Cassin et al. (2009) and Douchkov et al. (2005)
will be evoked bears further investigation.
Chapter 6: Generation and Characterisation of Transgenic Chickpea 99
Chapter 6: Generation and
Characterisation of Transgenic
Chickpea
6.1 INTRODUCTION
Iron deficiency is one of the most common micronutrient deficiencies in the
world. More than 60% of global anaemia cases are attributed to iron deficiency, and
it afflicts both developing and developed nations (Alvarez-Uria et al., 2014). Several
strategies have been developed to combat this problem, amongst which the
generation of ‘self-fortifying’ crops (also known as biofortification) demonstrates
significant promise. Recently, pulses have gained recognition as suitable targets for
biofortification. Despite this pique in interest, there remains a paucity of information
in this aspect compared to the more well-studied crops like cereals.
Considerable progress has been made in the past five years, particularly with
chickpea. Latest advancements include the sequencing of the chickpea genome and
mapping of quantitative trait loci (QTLs) associated with iron and zinc accumulation
(Varshney et al., 2013; Diapari et al., 2014; Upadhyaya et al., 2016). Several genes
involved in iron metabolism have also been identified amongst the QTLs
(Upadhyaya et al., 2016). Although these genes remain uncharacterised, this
information has shed light on key mechanisms, thereby allowing for more targeted
approaches.
While such information accelerates the development of elite micronutrient-rich
cultivars, the breeding process itself is laborious and time-consuming. This selection
of traits may be expedited through the use of transgenic technologies. As an added
benefit, closer investigation of the underlying molecular mechanisms may be done
due to the specificity of this approach. The generation of transgenic chickpea,
however, remains a challenging undertaking. Chickpea transformation has been
attempted since 1993, and several protocols have been reported in the literature.
Most involve Agrobacterium-mediated transformation of explants like axillary
meristems (Bhatnagar-Mathur et al., 2009), cotyledonary nodes (Sanyal et al., 2005;
Indurker et al., 2010), embryos and its derivative tissues (Polowick et al., 2004;
100 Chapter 6: Generation and Characterisation of Transgenic Chickpea
Sarmah et al., 2004; Tewari-Singh et al., 2004). As observed by Sarmah et al.
(2004), early protocols (Fontana et al., 1993; Kar et al., 1996; Krishnamurthy et al.,
2000) were fraught by three major hurdles, namely 1) lack of reproducibility, 2) low
transformation efficiencies, and 3) poor transmission of transgenes to subsequent
generations. Such issues appear to have been somewhat addressed in later
publications (Polowick et al., 2004; Sarmah et al., 2004). The protocol used by
Sarmah et al. (2004) and Acharjee et al. (2010) for instance, reported transformation
efficiencies of 0.3 – 0.72% and transmission of transgenes up to the T3 generation.
Transgenic work in chickpea thus far has revolved around development of
drought tolerance (Bhatnagar-Mathur et al., 2009) and insect resistance (Acharjee et
al., 2010; Ganguly et al., 2014). To date, no known attempts at biofortification of
chickpea have been made. In addition, while several iron biofortification strategies
have been developed (e.g. Masuda et al., 2013a), the focus has been on cereals.
General information on the effectiveness of these strategies in dicots is lacking.
Consequently, it is difficult to reliably estimate the influence of these strategies on
seed iron accumulation.
This chapter aims to fill that gap. In this study, chickpea (cv HatTrick) was
transformed with a combination of rice nicotianamine synthase 2 (OsNAS2) and
soybean ferritin H1 (GmFER). This combination was recently proven to be highly
effective in rice when overexpressed under a constitutive and seed-specific promoter
respectively (Trijatmiko et al., 2016). However due to the lack of prior study in
chickpea, both genes were constitutively overexpressed to determine the
effectiveness of these genes in chickpea. To also evaluate the effectiveness of a
cisgenic approach, a chickpea nicotianamine synthase (CaNAS2) and GmFER
combination was assessed. The progeny of transformed plants were assessed for iron
accumulation in the leaf and seed tissues.
6.2 MATERIALS AND METHODS
6.2.1 Generation of transgenic chickpea
Plants were transformed with the pEBX-GmFER-CaNAS and pEBX-GmFER-
OsNAS2 constructs generated in Chapter 5.2.6. The chickpea transformation process
was initially done following the protocol developed by Sarmah et al. (2004).
However, modifications were later made to adapt and optimise it for laboratories in
Chapter 6: Generation and Characterisation of Transgenic Chickpea 101
the Queensland University of Technology (QUT). The modified protocol is as
outlined in Section 3.2.3.2 and an overview of the transformation process is as
illustrated in Figure 6.1. The modifications made are listed in Table 6.1.
Figure 6.1. Overview of the chickpea transformation process. A) Half-embryonic
axis explant, B) micro-injury with needle in Agrobacterium culture, C) explants after
3 days of co-cultivation, D) explants after first selection, E) shoot multiplication, and
F) putative transgenic shoot grafted onto non-transgenic rootstock in vitro.
102 Chapter 6: Generation and Characterisation of Transgenic Chickpea
Table 6.1. Summary of modifications made to the original protocol.
Step Original protocol
(Sarmah et al., 2004) Modified protocol
Explant
preparation Bisection along embryonic
axis only
Bisection along embryonic
axis and additional injury to
radicle using needle
Elimination of explant
washing step after co-
cultivation.
Co-
cultivation
Explants placed directly onto
B3 media after incubation
with Agrobacterium
Explants washed after 3 days
co-cultivation, before
transfer to selection media
Explants placed on a layer of
sterile filter paper on B3
media
Direct transfer of explants to
selection media (elimination
of washing step)
Selection
media
Single round on
multiplication media,
followed by successive
rounds on B3 (elongation)
media
Timentin used as selection
against Agrobacterium
Successive rounds on
multiplication media and
eliminated use of R3
(elongation) media
Injury to the cotyledonary
node after first selection
round
Replacement of timentin with
merrem as selection against
against Agrobacterium
Culture
vessels Tissue culture pots
Replacement of 250mL pots
with deep Petri dishes (90 x
25mm)
6.2.2 Molecular characterisation of transgenic chickpea
Shoots emerging from the same explant were considered as a single
independent transgenic event. Following successful acclimatisation, each T0 plant
was screened for the genes of interest following the protocol described in Chapter
3.2.3.2. Seeds from the PCR-positive individuals were collected, sown, and
screening was performed on all germinated seedlings. This process was repeated for
every generation produced.
Expression of the genes of interest was assessed via the method outlined in
Chapter 3.2.5.2. A qualitative analysis was performed on the T1 generation to
confirm gene expression, while a quantitative analysis performed on seven week old
plants used in the final glasshouse trial. RNA was extracted from the leaf tissue of
Chapter 6: Generation and Characterisation of Transgenic Chickpea 103
three biological replicates per line and the quality confirmed by gel electrophoresis.
DNAse treatment was performed on 1 µg of RNA, from which cDNA was generated.
The cDNA was checked via PCR using a housekeeping gene to confirm successful
synthesis, following which it was used for qPCR. Gene expression was normalised to
the same housekeeping genes used in Chapter 5.2.4.3. Primers used in qPCR are as
listed in Table 6.2.
Table 6.2. List of primers used for qPCR.
Gene Sequences (5’-3’)
Expected
amplicon
size
Actin 1 Fw GCCTGATGGA CAGGTGATCA C
62 Rv GGAACAGGAC CTCTGGACAT C
EF1α Fw TCCACCACTT GGTCGTTTTG
64 Rv CTTAATGACA CCGACAGCAA CAG
GAPDH Fw CCAAGGTCAA GATCGGAATC A
65 Rv CAAAGCCACT CTAGCAACCA AA
CaNAS2 Fw AGTAGTGCCT TTCTAAATGG CC
116 Rv CATGTCACCA ATCCCCAACA T
OsNAS2 Fw TGATCAACTC CGTCATCGTC
175 Rv TCAGACGGAT AGCCTCTTGG
GmFER Fw GTGCAATCGG AACAGCAAGA
138 Rv TTGGGTCTTT CTAAGGGTGT TG
To determine transgene copy number, DNA was extracted from leaf tissue and
Southern analysis carried out following the protocols described in Chapter 3.2.5.3.
The DNA was digested with KpnI, and a DIG-labelled probe against the NPTII
selectable marker was used to detect for the transgene. The probe was generated
using the NPTII-specific primers listed in Chapter 5, Table 5.4.
6.2.3 Glasshouse trial for T3, T4 plants
All plant material was germinated and screened following the protocols
described in Chapter 3.2.5.1.1. At 4 weeks of age, verified plants were moved to the
Queensland Crop Development Facility (Redlands, Queensland, Australia and
transplanted to 10 inch pots. The potting mix used was a 1:1 mixture of University of
California (UC) mix and regular potting mix. Plants were watered twice a day with
100 mL of water. To ensure sufficient quantities of iron in the soil, 16 mL of 50 µM
Fe-EDTA was added to the base of each plant at the 7 and 10 week stage. When the
plants began to senesce, watering was reduced by half to 50mL per dose for three
104 Chapter 6: Generation and Characterisation of Transgenic Chickpea
days, then cut completely in preparation of harvest. The plants were dried for two
weeks before harvesting and measurement of agronomic parameters.
6.2.4 Assessment of agronomic parameters
After drying, the aerial portion of the plants was harvested to measure the
following parameters: biomass, yield, 100 seed weight, harvest index and pod
abortion rate. Biomass was measured as the dry weight of the entire aerial portion of
the plants including the pods. Yield was calculated as the total seed count, in which
only filled seeds were considered. The 100 seed weight, harvest index, and pod
abortion rates were calculated using the following formulas:
100 𝑠𝑒𝑒𝑑 𝑤𝑒𝑖𝑔ℎ𝑡 = 𝑇𝑜𝑡𝑎𝑙 𝑠𝑒𝑒𝑑 𝑤𝑒𝑖𝑔ℎ𝑡
𝑇𝑜𝑡𝑎𝑙 𝑠𝑒𝑒𝑑 𝑐𝑜𝑢𝑛𝑡× 100
𝐻𝑎𝑟𝑣𝑒𝑠𝑡 𝑖𝑛𝑑𝑒𝑥 (𝐻𝐼) = 𝑇𝑜𝑡𝑎𝑙 𝑠𝑒𝑒𝑑 𝑤𝑒𝑖𝑔ℎ𝑡
𝑇𝑜𝑡𝑎𝑙 𝑏𝑖𝑜𝑚𝑎𝑠𝑠× 100
𝑃𝑜𝑑 𝑎𝑏𝑜𝑟𝑡𝑖𝑜𝑛 𝑟𝑎𝑡𝑒 = 𝑇𝑜𝑡𝑎𝑙 𝑝𝑜𝑑 𝑐𝑜𝑢𝑛𝑡 − 𝑛𝑜. 𝑜𝑓 𝑓𝑖𝑙𝑙𝑒𝑑 𝑝𝑜𝑑𝑠
𝑇𝑜𝑡𝑎𝑙 𝑝𝑜𝑑 𝑐𝑜𝑢𝑛𝑡× 100%
6.2.5 Assessment of iron content in transgenic chickpea plants
A preliminary study was conducted on the leaves of T1 plants to determine
iron accumulation. The third and fourth youngest leaves were collected from a single
branch of seven week old plants grown in the P9 glasshouse at QUT Gardens Point
campus. These plants were grown on Plugger 222 potting mix (Australian Growing
Solutions) and fertilised with full-strength Hoagland solution every two weeks.
Samples were prepared for analysis via LA-ICP-MS following the procedure
described in Chapter 3.2.6.
For the final glasshouse trial, iron accumulation in both leaf and seed were
assessed. For leaf iron content, the third, fourth and fifth youngest leaves of 7 week
old plants were pooled and prepared for LA-ICP-MS analysis as described in
Chapter 3.2.6. For seed iron content, three to four plants were selected to represent
each line, with each plant representing a single biological replicate. Ten seeds
Chapter 6: Generation and Characterisation of Transgenic Chickpea 105
harvested from each plant were used for the analysis. Sample preparation was done
as described in Chapter 3.2.6 and the trace element content measured by ICP-OES.
6.3 RESULTS
6.3.1 Optimisation of chickpea transformation procedure
Various chickpea transformation protocols have been reported since 1992
(Anwar et al., 2010). Amongst these, the protocol developed Sarmah et al. (2004)
was selected for use in this study. Initial attempts were unsuccessful, and over the
course of work several factors were identified as major contributors to this outcome,
namely humidity, Agrobacterium overgrowth, and poor induction of transgenic
shoots. A few key modifications were therefore made to address these challenges and
adapt the protocol to local conditions.
One of the major problems was excessive condensation within the plant tissue
culture vessels, which led to tissue vitrification and contributed to Agrobacterium
overgrowth. This was particularly prevalent during the selection steps, resulting in
poor quality of shoots for grafting as well as the complete loss of explants. To
overcome this challenge, pots were replaced with petri dishes as culture vessels,
which helped to promote airflow and reduce the humidity and condensation. Despite
this change, Agrobacterium overgrowth remained a recurring problem. Attempts to
salvage affected explants by removing contaminated sections and washing in sterile
water supplemented with timentin were unsuccessful. This problem was resolved
through two modifications. Firstly, a layer of sterile filter paper was placed on top of
the media during the co-cultivation step. Secondly timentin (200mg/L) was replaced
with merrem (25mg/L) as the selection agent. The combination of these two
modifications eliminated the incidence of recurring Agrobacterium contamination
and also allowed for omission of the washing and drying steps following co-
cultivation.
The next challenge was the induction of putative transgenic shoots. Explants
produced a single primary shoot that was able to withstand successive rounds of
selection. Despite this, such shoots did not harbour the genes of interest. Preliminary
studies with plants transformed with the GUS vector control showed expression to be
weak with a patchy distribution (Appendix C, Figure 8.3), and scions generated from
these primary shoots tested negative for the genes of interest (data not shown).
106 Chapter 6: Generation and Characterisation of Transgenic Chickpea
Figure 6.2. Morphology of emerging putative transgenic shoots. The region
highlighted with black is the primary shoot, and the region highlighted with red is the
putative transgenic shoot.
True transgenic shoots on the other hand, typically stemmed from the
secondary shoots emerging from the base of the explant near the cotyledonary node.
A common characteristic of such shoots was their emergence through the selection
media with varying degrees of shoot multiplication (see Figure 6.2). Induction and
multiplication of such shoots rarely occurred using the original protocol (less than
1% induction rate).
To increase exposure to Agrobacterium and allow deep-seated transgenic cells
access to the growth hormones in the media, additional injury was applied at two
stages. The first was to the radicle before incubation with Agrobacterium, using a
needle dipped in the Agrobacterium culture. The second injury was inflicted during
the first subculture to multiplication media, where a shallow incision was made in the
cotyledonary node using a scalpel blade. Successive rounds of selection on
multiplication media was also done to improve shoot proliferation. This was found to
greatly enhance the number of putative lines obtained per transformation experiment,
as most secondary shoots emerged after the second cycle on multiplication media.
This modification also allowed for the continuous proliferation of these shoots and
thus increased supply of material for grafting. With these modifications, induction
rate of these secondary shoots was increased to approximately 3%.
Chapter 6: Generation and Characterisation of Transgenic Chickpea 107
6.3.2 Generation and molecular characterisation of transgenic chickpea
Several rounds of transformation were performed to optimise the procedure
and later produce the number of lines required for later trials. Each transformation
attempt started off with approximately 500 explants. Attempts to further scale-up
operations made unfeasible by logistical constraints (i.e. lack of space).
Following grafting and acclimatisation, putative transgenic plants were
screened for the genes of interest. This screening was also performed on every plant
in successive generations to confirm the inheritance of the genes of interest (see
Figure 6.3). Testing of the acclimatised T0 plants showed the transformation
efficiency to be approximately 1%. In total, eight OsNAS2 lines and seven CaNAS2
lines were generated. Of these, four of the former and two of the latter were
successfully carried down to the T1 and subsequent generations (see Table 6.3).
Despite several attempts, no GUS transgenic plants were successfully generated; as
negative control, a line established from a PCR negative sibling from the T1
generation was used instead. Expression of the genes of interest was confirmed in all
lines at the T1 generation via end-point RT-PCR (see Figure 6.4).
Transgenic lines 7.1, 7.2, 1.1, 6.6, and 6.14 were selected for the glasshouse
trial. Due to low number of seeds, line 7.15 was not included in this trial. All seeds
used were at the T3 generation. The exception was Line 1.1, which was assessed at
the T4 generation. T3 seeds from a PCR-negative sibling were included in this trial
as the null control.
108 Chapter 6: Generation and Characterisation of Transgenic Chickpea
Figure 6.3. Representative photo of PCR screening of T0 plants. Each number
represents an individual transgenic line, within which each lane represents a single
plant.
M = 2-log ladder (NEB)
“-” = No template control
“+” = Plasmid positive control
WT = Untransformed chickpea
Table 6.3. Summary of transgenic lines generated.
Genes of interest
GmFER-OsNAS2 GmFER-CaNAS2 GUS
No. of explants 5508 4840 2727
Lines generated: 8 7 0
No. of lines passing
to T1 generation 4 2 0
Line ID
7.1
7.2
7.15
1.1
6.6
6.14 -
Chapter 6: Generation and Characterisation of Transgenic Chickpea 109
Figure 6.4. Detection of transgene expression in transgenic chickpea lines via
PCR. A) GmFER expression, B) CaNAS2 expression, C) OsNAS2 expression in T1
plants. Each number represents an individual transgenic line. All lines were at the T1
generation; the exception was Line 1.1, which was at the T2 generation.
M = 2-log ladder (NEB)
“-” = No template control
“+” = Positive plasmid control
WT = Untransformed chickpea
Null = Non-transgenic sibling
FO = pEBX-GmFER-OsNAS2
FC = pEBX-GmFER-CaNAS2
110 Chapter 6: Generation and Characterisation of Transgenic Chickpea
To determine the levels of transgene expression in the material used for the
glasshouse trial, qPCR was performed on leaf tissue harvested from seven week old
plants. The results confirmed the continued expression of the genes of interest in
most lines (see Figure 6.5). Relative expression of GmFER was at least 18-fold in
Lines 1.1, 6.6, and 6.14. With the CaNAS2 overexpressing lines 6.6 and 6.14,
CaNAS2 expression was enhanced by 49 and 100 times respectively compared to the
null control. With the OsNAS2-overexpressing line 1.1, relative expression of
OsNAS2 was approximately 0.032.
In lines 7.1 and 7.2, negligible expression of OsNAS2 was detected (Figure
6.5). GmFER expression was similarly affected, with relative expressions of 0.01 and
0.11 respectively. Southern analysis using a probe for the selection marker showed
multiple integration sites for those two lines. With the other line, only a single copy
was found (see Figure 6.6).
Figure 6.5. Relative expression of transgenes in transgenic chickpea. A) GmFER,
B) CaNAS2, and C) OsNAS2. The gray bar represents the non-transgenic segregant
(Null), the orange bars represent CaNAS2-GmFER lines, while blue bars represent
OsNAS2-GmFER lines. n=3. Error bars indicate standard error. Expression was
measured using qPCR calculated as relative to that of the housekeeping genes
GAPDH and EF1α. All lines were at the T3 generation; the exception was Line 1.1,
which was at the T4 generation.
Chapter 6: Generation and Characterisation of Transgenic Chickpea 111
Figure 6.6. Southern analysis of transgenic chickpea lines used in the glasshouse
trial. gDNA was digested using KpnI and 15µg loaded onto the gel. A probe for the
selection marker, NPTII, was used to detect for presence of the integrated transgenes.
All lines were at the T3 generation; the exception was Line 1.1, which was at the T4
generation.
Null = Non-transgenic sibling
FO = pEBX-GmFER-OsNAS2 transgenic chickpea lines
FC = pEBX-GmFER-CaNAS2 transgenic chickpea lines
Restriction enzyme = KpnI
DNA quantity = 15 µg
Probe target = NPTII
Exposure time = 15 minutes
The lanes containing the 2-log ladder (NEB) and the plasmid control were
covered during the exposure period due to excessively strong signal strength.
112 Chapter 6: Generation and Characterisation of Transgenic Chickpea
6.3.3 Morphology and agronomic properties of transgenic chickpea
To establish sufficient numbers for a glasshouse trial, transgenic plants were
grown to the T3 and T4 generation. Lines 7.1 and 7.2 consistently yielded the highest
seed count (data not shown) and germination rate (appendix C, Table 8.5). Lines
7.15 and 6.14 produced low yields with poor seed viability, and the former being
dropped for the glasshouse trial due to insufficient numbers.
In the final trial, no morphological differences were observed between the
transgenic lines when compared to the null controls during the vegetative and
flowering states (see Figure 6.7). Flower and pod morphology were similarly
unaffected. In terms of agronomic performance, the average biomass of the null
controls was noticeably higher than that of line 7.2, 6.6, and 6.24 (see Figure 6.8 A).
The difference was insignificant and did not appear to affect the other parameters
measured. No significant differences were observed between the transgenic lines and
null controls in other parameters measured (see Figure 6.8).
Chapter 6: Generation and Characterisation of Transgenic Chickpea 113
Figure 6.7. Morphology of 9 week old transgenic chickpea at the flowering/pod-filling stage.
114 Chapter 6: Generation and Characterisation of Transgenic Chickpea
Figure 6.8. Agronomic properties of transgenic chickpea under glasshouse
conditions. A) Biomass, B) harvest index (HI), C) seed count, D) seed weight, and
E) pod abortion rate. n= 3 to 20. The gray bar represents the non-transgenic
segregant (Null), blue bars represent the GmFER-OsNAS2 transgenic lines, and the
orange bars represent the GmFER-CaNAS2 transgenic lines. Error bars indicate
standard error. Statistical significance was calculated using Dunnett’s test and p-
values<0.05 were considered significant. No significant differences were found
between the null control and transgenic lines. All lines were at the T3 generation; the
exception was Line 1.1, which was at the T4 generation.
Chapter 6: Generation and Characterisation of Transgenic Chickpea 115
6.3.4 Iron content in transgenic chickpea
A preliminary assessment was conducted at the T1 generation to determine the
effect of the transgenes on iron accumulation in the plants. Line 1.1 was not included
in this analysis due to insufficient numbers in this generation. In general, all
transgenic lines except line 7.1 were found to have higher average leaf iron content
than the null controls (see Figure 6.9). A similar observation was made with the zinc
and manganese contents; the exception was line 7.2, which had a manganese content
similar to the null control (see Figure 6.9). Amongst the lines tested in this
preliminary assessment, line 7.15 in particular, was significantly higher than the null
in terms of leaf iron, zinc, and manganese contents. Due to insufficient numbers
however, subsequent data for later generations are unavailable.
Following this preliminary assessment, a large scale glasshouse trial was
conducted using plants at the T3 and T4 generation. The trends observed were
similar to that of the preliminary study, in that the average iron content of all
transgenic lines (except 7.1) was higher than the null (see Figure 6.10). As with the
preliminary study, the difference was not significant. Zinc and manganese levels in
the leaves were also not significantly different except for line 1.1, which had the
highest manganese contents compared to all other lines and the null (see Figure
6.10).
This higher manganese content was not reflected in the subsequent analysis of
the seed. Examination of the micronutrient content in the seeds generated from this
trial showed a trend unlike that observed in the leaf. Of the lines, line 7.1 was shown
to be the highest in terms of average seed iron and zinc contents at 9.3mg/100g,
despite consistently low concentrations of the said elements in the leaf. However, as
observed with the leaves, there were no significant differences between the
transgenic lines and null controls (see Figure 6.11). The lower manganese to iron
content also indicated that there was, at least, no inhibition of iron accumulation by
manganese. Total seed phosphorus were also similar between the null and transgenic
lines (Appendix C, Figure 8.4).
116 Chapter 6: Generation and Characterisation of Transgenic Chickpea
Figure 6.9. Preliminary study on leaf iron, zinc and manganese contents in 7
week old transgenic chickpea at the T1 generation. n=3. The gray bar represents
the non-transgenic segregant (NULL), blue bars represent the GmFER-OsNAS2
transgenic lines, and the orange bars represent the GmFER-CaNAS2 transgenic lines.
Error bars indicate standard error. Statistical significance was calculated using
Dunnett’s test and p-values<0.05 were considered significant. “*” indicate a
significant difference compared to the null control. Line 1.1 was excluded from this
preliminary study due to insufficient replicates.
Figure 6.10. Leaf iron, zinc and manganese contents in 7 week old transgenic
chickpea. n=3 to 23. The gray bar represents the non-transgenic segregant (NULL),
blue bars represent the GmFER-OsNAS2 transgenic lines, and the orange bars
represent the GmFER-CaNAS2 transgenic lines. Error bars indicate standard error.
Statistical significance was calculated using Dunnett’s test and p-values<0.05 were
considered significant. “*” indicate a significant difference compared to the null
control. Line 7.15 was excluded from this preliminary study due to insufficient
Chapter 6: Generation and Characterisation of Transgenic Chickpea 117
replicates. All lines were at the T3 generation; the exception was Line 1.1, which was
at the T4 generation.
Figure 6.11. Iron, zinc and manganese contents in transgenic chickpea seeds.
n=4. The gray bar represents the non-transgenic segregant (NULL), blue bars
represent the GmFER-OsNAS2 transgenic lines, and the orange bars represent the
GmFER-CaNAS2 transgenic lines. Error bars indicate standard error. Statistical
significance was calculated using Dunnett’s test, and p-values<0.05 were considered
significant. “*” indicate a significant difference compared to the null control. Line
7.15 was excluded from this preliminary study due to insufficient replicates. All lines
were at the T3 generation; the exception was Line 1.1, which was at the T4
generation.
6.4 DISCUSSION
Crop biofortification efforts to date have primarily been focused on primary
staple crops such as rice and wheat, with pulses only garnering attention recently. As
such, knowledge on the pulse biofortification can be considered to still be in its
infancy. With transgenic work in particular, there is currently no known precedent.
In this chapter, transgenic chickpea expressing iron metabolism genes were
generated. In this strategy, soybean ferritin was used in combination with the rice
nicotianamine synthase 2 (NAS2) gene. A variation of this strategy was also used in
this study, where OsNAS2 was replaced by CaNAS2 – this was done to compare the
effects of a trans-genic versus a cis-genic approach. The effect of the transgenes was
assessed in terms of the leaf and seed iron content at the T3 and T4 generation.
118 Chapter 6: Generation and Characterisation of Transgenic Chickpea
Optimisation of chickpea transformation protocol
Despite the availability of several published protocols, transformation of
chickpea remains a challenge with low reported efficiencies, ranging from 0.2 to 5%
(e.g. Krishnamurthy et al., 2000; Polowick et al., 2004; Senthil et al., 2004). Most
protocols also are tend to lack reproducibility, as evident in the general lack of
reported downstream work using the transgenic chickpea.
One of the most successful protocols thus far is the one developed by Sarmah
et al. (2004), which has been successfully replicated by other authors (Acharjee et
al., 2010; Ganguly et al., 2014). As such, this was the protocol of choice used in this
study. Concurrently, attempts were also made to use calli as explants instead (data
not included). These proved fruitless – despite reported successes by other authors
(Barna and Wakhlu, 1993; Sagare et al., 1995), no plants could be regenerated from
the calli.
In the course of work, the original procedure by Sarmah et al. (2004) was
generally found to be insufficient in generating transgenic material. Several
modifications were required for effective implementation within the CTCB
laboratories.
One of the key modifications was the infliction of additional injuries to the
explant. Despite the extensive injury caused by bisection of the along the embryonic
axis, poor induction of putative transgenic shoots was observed when using the
original protocol. This was suspected to be due to either poor penetration of
Agrobacterium into the tissue, or lack of contact between transformed cells and the
growth hormones in the media, or both. To test this hypothesis, additional injury was
applied during the infection step and transfer to multiplication media. This was
observed to increase both shoot induction rate and overall transformation efficiency
at the T0 generation.
This use of micro-injury to enhance plant transformation efficiency is not a
new development, having been applied in other plants transformation systems (Trick
and Finer, 1997; Trick and Finer, 1998; Bakshi et al., 2011). In addition to increasing
the surface area for Agrobacterium colonisation and interaction, this technique
allows access to and infection of deeply-embedded germ layers. In other chickpea
transformation protocols, sonication of explants increased the number of transient
Chapter 6: Generation and Characterisation of Transgenic Chickpea 119
transformants by at least three-fold (Sanyal et al., 2005; Tripathi et al., 2013). Stable
transformation frequency at the T0 generation was reported to range from 0.32 –
1.12% (Sanyal et al., 2005) and 1.60 – 2.08% (Mishra et al., 2013; Tripathi et al.,
2013). In this study, transformation efficiency was found to be approximately 1%.
This was higher than the 0.72% reported in the original protocol by Sarmah et al.
(2004). Given that PCR screening was only performed on acclimatised plants and
accounting for loss of lines during the grafting and acclimatisation processes, actual
transformation efficiency is likely to be higher.
Currently, the grafting and acclimatisation processes are the main bottlenecks
in this chickpea transformation system. Despite reports of successful root induction
(Sanyal et al., 2005; Tripathi et al., 2013), attempts to induce rooting in this project
were unsuccessful. Grafting, while laborious, was found to be more efficient though
success was directly affected by the condition of the material and handler skill. As
such, the availability of good quality scions is vital. The supply of healthy grafted
plants in turn increases the chances of survival of the lines past the acclimatisation
process. This is particularly significant given the humidity of local conditions, which
is conducive for Botrytis cinerea growth (Pande et al., 2006). Deterioration of shoot
health inevitably led to infection and loss of the plants. Repeated use of the same
growth cabinet or room also increased spore load and chance of subsequent losses.
In addition to its significance in the acclimatisation process, humidity was also
a key factor in vitro. Vitrification of shoots was an ongoing problem, and similar
issues have been reported by other authors (Brandt and Hess, 1994; Sarker et al.,
2005). However this problem was not observed in the CSIRO laboratory in Canberra,
where the same transformation and regeneration protocol by Sarmah et al. (2004)
was used. Attempts to replicate their growth conditions (e.g. light, temperature,
culture vessels) were unfruitful, though a solution was eventually found by using
different culture vessels which increased airflow.
Collectively, one may suggest variations in local environmental conditions to
be a major contributor to the successful implementation of this chickpea
transformation system. Whether this can be said of other protocols is uncertain. It is
clear however, that adoption of this protocol in a different laboratory may require
further modification to adapt it to local conditions.
120 Chapter 6: Generation and Characterisation of Transgenic Chickpea
Iron accumulation on transgenic chickpea is not consistent between generations
This chapter documents the first example of a transgenic biofortification
attempt in chickpea. This strategy consisting of a combination of NAS and FER
genes had been applied with great success in rice (Johnson et al., 2011; Trijatmiko et
al., 2016). In this chapter, a total of three GmFER-OsNAS2 lines and two GmFER-
CaNAS2 lines were generated and iron accumulation in their leaf and seed assessed.
Initial measurements at the T1 generation showed higher average leaf iron content in
most transgenic lines compared to the null control. However such differences were
considerably reduced in the subsequent assessment of leaf and seed of the T3 and T4
generation. Zinc content, which have been demonstrated in other studies to be
correlated to iron content (e.g. Johnson et al., 2011; Trijatmiko et al., 2016), shared a
similar pattern.
This discrepancy between generations was unexpected, though it likely evolved
as an artefact of experimental design and the environment in addition to genetic
influence. This can primarily be attributed logistical limitations early in the project,
which necessitated the use of different growth facilities and potting mixes between
the trials. Also, only a small population was assessed in the preliminary trial due to
the limited number of plants in the early generations. Consequently the transgenic
lines therefore not have been sufficiently or accurately represented. The results
obtained from this preliminary trial should therefore be interpreted with caution.
As both NAS and GmFER have individually been demonstrated to enhance
leaf iron content in other non-graminaceous species (Van Wuytswinkel et al., 1999;
Douchkov et al., 2005; Cassin et al., 2009), a similar effect was expected when they
were used in conjunction. However, results from the second trial showed the
transgenic chickpea lines to be similar to the controls in iron content, contrasting
with the observations in biofortified rice transformed with the same genes. This
disparate result might have arisen due to the different physiologies of Strategy I and
II plants – rice uses a combination of both Strategy I and II (Ishimaru et al., 2006),
while chickpea uses only Strategy I. Interestingly, the results also showed that iron
contents in the leaf and seed were not necessarily correlated, as was most evident in
Lines 7.1 and 6.6. This was unusual as iron remobilisation from vegetative tissues to
seeds occurs during development; higher leaf iron content was therefore expected to
produce similarly high seed iron content. Several factors may have contributed to this
Chapter 6: Generation and Characterisation of Transgenic Chickpea 121
discrepancy. For instance, the extent of iron contribution to the seeds differs between
tissues (Hocking and Pate, 1977; Burton et al., 1998), and variations in iron
accumulation in said tissues may affect seed iron levels. Another possible reason
may be overaccumulation of iron in a way that exceeds the plant’s sequestration
ability. Excessive accumulation has been known to result in the formation of
insoluble precipitates in the apoplast as a means of avoiding toxicity (Becker et al.,
1995; Garnett and Graham, 2005), though the iron pool available for seed loading
would be simultaneously reduced. Given that NAS was overexpressed however, such
a scenario is improbable as the NA produced would aid in solubilising such
precipitates. Perhaps another unknown mechanism was at work, and further
investigation is required to determine what it is.
Regardless, it is clear that a measure of physiological disruption had occurred
in the transgenic lines, as evident in the reduced yield and seed viability of the early
generations. This was most prominent in the high expressing lines like 7.15, 1.1 and
6.14. Such physiological penalties may have inadvertently selected for individuals
with reduced expression and/or iron accumulation, which may somewhat explain the
recovery of the yield during the glasshouse trial. This may also explain why the
transgenic seeds did not contain that much more iron than the controls – this is
difficult to prove though, as the early generations lacked sufficient seeds to allow for
elemental analysis. Additional trials are required to determine if this is a consistent
trend across the generations.
A consideration for such future trials is the setting in which they are conducted.
As previously mentioned, the use of different glasshouses may contributed to the
difference in leaf iron contents between the generations. The effect however, is
unlikely to be limited to leaf iron content. Several authors have reported an
environmental influence on both yield and seed micronutrient content, though the
specific factor (or combination of factors) has yet to be identified (Garvin et al.,
2006; Ficco et al., 2009; Diapari et al., 2014). It is possible that aside from the
aforementioned genetic and physiological selection, the environmental conditions
have contributed to the recovery of yield in the glasshouse trial. Again, more trials
are required for confirmation. For future reference though, glasshouse trials should
ideally be conducted within the same compound to maximise comparability.
122 Chapter 7: General Discussion
Chapter 7: General Discussion
Iron deficiency is one of the major micronutrient deficiencies worldwide.
Several methods have been developed to alleviate this global problem. Amongst
them, biofortification has gained considerable attention over the past decades. Iron
biofortification via genetic modification (GM) typically target the three main
processes that make up iron metabolism: uptake, translocation, and storage. Several
strategies have been developed using one or more components from these processes,
and varying levels of success have been achieved in several species (Drakakaki et al.,
2000; Kumar et al., 2011; Ihemere et al., 2012). The effectiveness of these strategies
however, is best illustrated in rice, a species used extensively in iron biofortification
studies (Masuda et al., 2013a). Targeting of grain sink strength using soybean ferritin
(GmFER) for instance, increased seed iron content by 1.5 to 3.7-fold (Vasconcelos et
al., 2003; Qu et al., 2005), while a 2.9 to 4.5-fold increase was attained when the
uptake and translocation processes were targeted using nicotianamine synthase
(NAS) (Lee et al., 2009; Masuda et al., 2009). Recently, a strategy combining both
NAS and GmFER was reported to have achieved up to 7.5-fold increase in iron
content (Trijatmiko et al., 2016).
Whether a similar phenomenon may be observed when applied to a dicot grain
crop like chickpea remains unknown. Being a non-graminaceous species, chickpea
differs significantly from rice on both the molecular and physiological levels. This is
of interest particularly where nicotianamine synthase (NAS) is concerned – its
product, nicotianamine (NA), is a precursor for mugineic acid (MA) synthesis and
thus contributes to iron uptake in addition to translocation in graminaceous plants.
While overexpression of NAS has been demonstrated to enhance iron content in both
graminaceous and non-graminaceous species, the effect is more pronounced in the
former (Masuda et al., 2009; Johnson et al., 2011) than the latter (Douchkov et al.,
2005; Cassin et al., 2009). With the latter, the effectiveness of multigenic
biofortification approaches such as the one using NAS and FER is also currently
unknown.
The aim of this project therefore, was the biofortification of chickpea using a
combination of NAS and GmFER. To compare between the effectiveness of cisgenic
Chapter 7: General Discussion 123
and transgenic approaches, the novel chickpea NAS2 (CaNAS2) gene was also
characterised and investigated for its use in the biofortification of chickpea. The
well-characterised rice NAS2 (OsNAS2) gene was used as positive control, and
homology with its amino acid sequence was the basis on which CaNAS2 was
designated.
7.1 THE PHYSIOLOGICAL ROLE OF CANAS2 IN THE SUBCELLUALR
AND SYSTEMIC CONTEXT
Members of the Fabaceae family are unique in that most are capable of
nitrogen fixation via symbiosis with Rhizobium. This relationship imposes an
additional demand for iron (Tang et al., 1990; Strozycki et al., 2007), which may
result in different homeostatic behaviour compared to other non-graminaceous
species. The extent of this difference, if any, on NA homeostasis is unknown, and
examination of NAS subcellular localisation may reveal more about downstream
usage and regulation.
In this project, predictive modelling was performed to determine the protein
properties and localisation of CaNAS2. However, aside from its hydrophilic nature,
the results obtained did not allow for other conclusive statements. Much of this could
be attributed to discrepancies in the existing literature concerning predicted
localisation sites and signals (Mizuno et al., 2003; Nozoye et al., 2014b). Using the
AtNAS homologues as an example (Nozoye et al., 2014b), CaNAS2 may be
speculated to be a cytoplasmic protein. However actual localisation studies are
required for further verification – this was unable to be accomplished within the time
constraints of this project, and can be investigated in future works.
Based on that hypothesis however, some degree of physiological function can
be guessed. Vesicular localisation of NAS has only been reported in some
graminaceous homologues, and this has been linked to the synthesis and secretion of
DMA and MAs (Nozoye et al., 2014a; Nozoye et al., 2014b). Cytoplasmic
localisation on the other hand, has been confirmed in both graminaceous and non-
graminaceous NAS homologues (Mizuno et al., 2003; Nozoye et al., 2014b).
Interestingly, the examples of such graminaceous homologues like OsNAS3 and
ZmNAS3 are known to be downregulated during iron deficiency (Inoue et al., 2003;
Zhou et al., 2013b). As pointed out by Inoue et al. (2003), such behaviour is contrary
to expectations as MAs production, and thus iron uptake, is directly linked to NA
124 Chapter 7: General Discussion
production. It may be inferred implied that such homologues serve other
physiological functions. Such functions may be shared with the non-graminaceous
counterparts, and may include systemic transport or regulation of subcellular iron
levels to prevent toxicity (Pich et al., 2001).
Alternatively, or in addition to the above, NA may serve in iron uptake under
certain limiting conditions. Recently, Tsednee et al. (2014) discovered the presence
of NA in the root exudates of Arabidopsis halleri and Arabidopsis thaliana. This was
linked to zinc tolerance, as the secreted NA served to prevent toxicity by regulating
zinc bioavailability in the soil. Notably, this phenomenon was not seen in any of the
graminaceous species examined by the authors. It is unclear if NA secretion is a trait
shared by all non-gramineae or is limited to the Arabidopsis species; nonetheless it
points to a secretory pathway unique from that of MAS. However, outside of aiding
iron uptake in the presence of excess zinc, there is no indication of the secreted NA
affecting iron uptake under other circumstances like iron deficiency.
Concerning the general role of NA and NAS in the non-gramineae under iron
deficiency, reports by other authors have been conflicting. As previously mentioned
in Chapter 5.4, the response appears species-specific (Higuchi et al., 1995;
Douchkov et al., 2005). Interestingly, while NAS activity in iron-deficient tobacco
was reduced (Higuchi et al., 1995), overexpression of AtNAS1 conferred tolerance
to iron deficiency (Douchkov et al., 2005). This tolerance is unlikely to be due to NA
secretion, if any, facilitating uptake – in the latter study, iron was present as a chelate
and content, rather than bioavailability, was limited. Tolerance may instead be due to
the increased remobilisation capability, as iron from senescing leaves would be more
efficiently moved to younger tissues. Whether increased remobilisation led to
enhanced iron uptake is unknown, as the authors did not report on plant iron content
under iron-sufficient conditions. The higher iron accumulation in our CaNAS2 and
OsNAS2 tobacco lines suggests that there might be a slight increase in uptake. As to
whether NAS overexpression will lead to iron deficiency tolerance, it is difficult to
say. While most studies typically simulate iron deficiency through removing or
reducing iron in the media, iron deficiency in plants is caused by limited
bioavailability in the soil rather than content. Replicating such conditions within this
project was not logistically feasible. Nonetheless this environmental interaction is
important to biofortification efforts, and should be investigated in future studies.
Chapter 7: General Discussion 125
For the purpose of investigating gene expression within the plant however,
manipulation of external iron contents would suffice and was thus used to assess
changes in NAS expression in chickpea. Consistent with the results of studies done
in other species (Inoue et al., 2003; Schuler and Bauer, 2011; Bonneau et al., 2016),
differential expression across various tissues was observed in the CaNAS family.
Specifically, the expression profile of CaNAS2 indicated that it was not involved in
the iron deficiency response, but may have a systemic housekeeping role under iron-
replete conditions. Similar expression profiles were noted in the Medicago truncatula
and Lotus japonicas orthologues that share a common root, hinting at a conserved
function within this group of legume-specific NAS. At this point, this cannot be
confirmed until additional studies are performed due to the general lack of
characterised NAS genes. Also based on observations in Arabidopsis (Schuler et al.,
2012b), considerable functional overlap may exist between the homologues given
their small number.
7.2 NAS-FER TRANSGENE COMBINATION HAS LIMITED EFFECT ON
IRON ACCUMULATION IN TOBACCO AND CHICKPEA
To characterise the CaNAS2 gene and determine the effectiveness of the NAS-
FERR biofortification approach on chickpea, transgenic tobacco and chickpea lines
overexpressing NAS and FERR were generated. Interestingly, despite confirmation
of gene expression in both species, no notable enhancements in iron content
observed. This was unexpected, given previous reports on the positive effect of both
NAS and GmFER on leaf and seed iron content (Van Wuytswinkel et al., 1999; Goto
et al., 2000; Douchkov et al., 2005). The precise reason behind this is difficult to
ascertain; there are, however, several factors that may have contributed to this
outcome. These include, but are not limited, to the following: 1) the differing roles of
NA between graminaceous and non-graminaceous species, 2) promoter strength, 3)
external trace element concentrations, and 4) insufficient uptake capacity.
As mentioned previously, NAS and NA serve different physiological roles in
graminaceous and non-graminaceous species, which translates into different
regulatory mechanisms. However, while the general pattern of NA production
appears to be conserved in the former, conflicting results have been reported in the
latter. Differing patterns of NA accumulation in response to iron deficiency have
been observed in Arabidopsis compared to tomato, soy and tobacco (Higuchi et al.,
126 Chapter 7: General Discussion
1995; Douchkov et al., 2005), and it is unclear if these differences arise from
regulatory mechanisms at the transcriptional, translational or feedback level.
Given that CaNAS2 is upregulated in chickpea during iron-sufficient
conditions, transcript abundance in the overexpressing plants may lead to
suppression of the iron deficiency response and thus iron uptake. This, however, is
unlikely. Firstly, the Nos promoter driving CaNAS expression produced only a small
increase in expression. Secondly, this phenomenon was also observed in tobacco, as
well as transgenic chickpea and tobacco overexpressing OsNAS2. It is possible that
the NAS expression, and thus NA production, was too low to produce a noticeable
effect. A certain threshold may need to be reached for iron content to be significantly
enhancement, and even so the effect is dependent on external iron concentrations.
Such a phenomenon was evident in the study by Cassin et al. (2009) – despite having
100-fold higher NA levels, iron content was not significantly higher in the leaves of
transgenic plants compared to the wild type under iron-sufficient conditions. With
this in mind, it is possible that a stronger promoter than the Nos promoter may be
used without incurring any detrimental effects.
An additional consideration is the concomitant overexpression of GmFER with
the NAS genes. Overexpressing NAS may elicit differing responses between
graminaceous and non-graminaceous species. However no such distinction has been
reported for FER. While ferritin accumulation is not necessarily paralleled by the
iron accumulation of the same extent (Qu et al., 2005), the positive effect of FER
overexpression on iron accumulation has been extensively documented in several
species (e.g. Goto et al., 1999; Van Wuytswinkel et al., 1999; Goto et al., 2000).
Such an effect was noticeably absent in the transgenic tobacco and chickpea lines
used in this project.
The reason behind this is unclear, though there are a few plausible scenarios.
One such scenario is that rather than promoting iron accumulation, concurrent
overexpression of NAS with GmFER may serve instead to redistribute internal
stores. Such a mechanism may temper the added sink strength afforded by GmFER,
thereby avoiding the side-effects of excessive sequestration observed by Van
Wuytswinkel et al. (1999) and Masuda et al. (2013b). For such a purpose, a low
NAS expression may suffice. An alternative scenario is that the iron uptake ability is
unable to match up to the increased translocation and storage capacities. Iron
Chapter 7: General Discussion 127
entering the system is therefore limited, translating to a similar restriction on iron
accumulation. Like the previous scenario, the enhanced translocation capability may
sidestep the physiological problems associated with high FER expression.
It is uncertain which of these scenarios is accurate and in this regard, the results
of this project raise more questions than answers. Some degree of confirmation may
be obtained via quantification of NA and ferritin proteins. Measuring the expression
of genes associated with iron uptake such as IRT (iron-regulated transport) or FRO
(ferric chelate reductase) would also help verify the impact on the overall iron
metabolism. Such assessments could not be performed within this project due to time
and logistical limitations, but can be included in future studies.
Nonetheless, for the purposes of biofortification, the general increase in iron
content produced by this strategy is promising. Further optimisation can be done,
perhaps through the use of different promoters. As plants seem to tolerate higher
levels of NA rather than ferritin, the same strategy employed by Trijatmiko et al.
(2016) may be used instead. Their strategy, unlike the one used in this study, had
NAS constitutively expressed using the 35s promoter, while FER was driven by an
endosperm-specific promoter. By limiting ferritin overaccumulation to the seed, the
side effects on general growth appear to be bypassed. However whether revising the
promoters in chickpea will be as effective remains to be seen; again due to the lack
of MAS biosynthesis in chickpea.
Other aspects may also be examined in such future studies. One of this is iron
bioavailability. While the increase in iron content in the transgenic chickpea seeds
was not as dramatic as in rice (Trijatmiko et al., 2016), the bioavailability of that
iron is uncertain. Hypothetically, improved bioavailability is expected since NA is an
promoter of bioavailability (Zheng et al., 2010), while ferritin-bound iron has been
touted to have a bioavailability equivalent to ferrous sulfate (Davila-Hicks et al.,
2004; Lönnerdal et al., 2006). Should it prove true, then the NAS-FER strategy may
be considered to be somewhat effective, though the effects of downstream processing
still need to be considered. Experiments with cell cultures or animals are required to
verify this hypothesis. Based on existing results however, the effect of inhibitors,
specifically phytic acid (PA), on iron bioavailability may be inferred. PA is a major
inhibitor of iron bioavailability and is often present as a phytate salt of mineral
cations like potassium, magnesium, calcium, manganese and zinc (Sandberg et al.,
128 Chapter 7: General Discussion
1989). It serves as the principal form of phosphorus storage in seeds and depending
on the species, constitutes 40–84% of total phosphorus (Lolas et al., 1976; Griffiths
and Thomas, 1981; Ravindran et al., 1994). As such, phosphorus content was used as
an indication of bioavailability in this project. To this end, the transgenic and non-
transgenic chickpea seeds were found to be similar, which suggests transgene
expression to have no particular impact on phytate accumulation. However such a
method does not account for other bioavailability inhibitors and verification in a
biological system is required.
Another aspect that may be explored in later studies is the influence of the
environmental conditions, particularly soil mineral composition. Relatively little
work has been done in this area concerning transgenic crops, though the effect of the
environmental on crop micronutrient profile has been well documented in several
species (Diapari et al., 2014; Matovu, 2016). This was effect also exemplified in this
project, where a large disparity was seen between the field-grown (Chapter 4) and
glasshouse-grown samples (Chapter 6). In the field-grown samples, levels of iron,
zinc and manganese in the seed mineral were subject to locational variations and
mineral-mineral interactions. These three elements have been associated with NA
(Stephan et al., 1996) and their accumulation in planta may be affected by NAS
overexpression. This is particularly relevant if potential for concurrent zinc
biofortification is to be explored, as zinc accumulation was dependent on
environmental conditions. Assessing the response of the transgenic lines to different
soil types would therefore serve in advising managerial decisions to maximise the
effectiveness of biofortified crops.
Such information would also applicable in the non-transgenic content,
particularly in light of the recent interest in breeding for seed micronutrient content
(Diapari et al., 2014; Upadhyaya et al., 2016). The results obtained in Chapter 4
showed most cultivars surveyed to be fairly similar in trace element composition.
While this is no representative of diversity amongst Australian chickpea, it provides
an idea on the current state of the common germplasm. It is plausible that
micronutrient-accumulating traits may have been bred out of the more current
cultivars due to focus on yields and stress tolerance, and potential yield penalties
accompanying micronutrient accumulation (Garvin et al., 2006; Ficco et al., 2009;
Chapter 7: General Discussion 129
Diapari et al., 2014). At the moment, the extent to which seed micronutrient content
in chickpea can be improved via breeding is yet unexplored.
131
Chapter 8: Concluding remarks
The work presented in this thesis illustrates the first known attempt at the
biofortification of chickpea using a GM approach. The body of work was divided
into three main sections, the summaries of which are described below.
In Chapter Four, the iron contents of modern Australian chickpea cultivars
were assessed. This served to determine existing iron contents in common cultivars
and to identify the factors influencing it. Cultivar was found to exert a greater
influence on seed iron content, though an environmental effect was apparent where
soil iron bioavailability was limited. The environmental effect was also more
prominent in zinc and phosphorus levels, which were correlated to iron and are of
nutritional significance. For further work, HatTrick, a low iron desi cultivar, was
selected. Examination of the distribution of iron within its seed showed that iron was
mainly stored in the cotyledons; this was ideal for biofortification as iron would not
be lost during downstream processing.
Chapter Five focused on the characterisation of the novel chickpea NAS2 gene.
CaNAS2 was found to be closely related to MtNAS, and to be systemically
expressed and upregulated during iron-sufficient conditions, suggesting that it may
have a housekeeping role under iron-sufficient conditions. The gene was then
isolated and cloned into transgenic vectors for overexpression in tobacco, whether on
its own or in conjunction with GmFER. The transgenic lines exhibited slight
increases in leaf iron content compared to the GUS control, though the difference
was mostly insignificant. Similar results were obtained with the OsNAS2 and
OsNAS2-GmFER lines, which were also generated to serve as positive controls.
In Chapter Six, the NAS-GmFER vectors from the previous chapter were used
to transform chickpea. Despite challenges of chickpea transformation, various
modifications allowed for efficiency to be raised to 1% from the 0.7% reported in the
original protocol. A total of four OsNAS2-GmFER and two CaNAS2-GmFER lines
were successfully generated. Preliminary measurement of leaf iron content noted
enhanced levels in the transgenic lines compared to the non-transgenic control,
though later generations showed a less marked increase. While the difference was
132
mostly statistically insignificant, enhanced seen iron content was also observed with
up to a 1.3-fold increase attained.
Collectively, this study demonstrates that the NAS-FER approach is capable of
increasing the iron accumulation in non-graminaceous species like chickpea and
tobacco. However whether this increase of around 1.3-fold can be considered
sufficient is uncertain, as biofortification targets have yet to be set for chickpea.
Regardless, it is likely that further increase of seed iron content, along with
elimination of the physiological penalties, can be attained upon revision of the
promoters used. The use of other genes, such as those targeting the uptake process, is
also viable option – this may confer an added benefit of tolerance to suboptimal iron
concentrations (Connolly et al., 2003). All these can be explored in future studies.
Several other things may also be examined in future studies. For instance, to
determine the precise physiological impact of the transgenes, the expression of genes
and transcription factors associated with iron metabolism can be explored. The effect
of transgenes on nodulation and the response to different soils, particularly those
with limited iron bioavailability, would also be of great interest to growers. Last but
not least, the bioavailability of the seed iron should be assessed to determine the
actual effectiveness of the transgenic approach.
All in all, while exceptional improvements in iron content was not attained
within this project, this study is the first of its kind in chickpea. The information
gleaned can therefore provide a foundation on which future iron biofortification work
can be built on, not just for chickpea, but also other pulses. Ultimately, this is but a
step forward in alleviating global iron deficiency.
Appendices 133
Appendices
Appendix A
Chapter 4 supplementary figures
Table 8.1. Profile of ferrosol soil from Kingaroy (Chauhan, 2015).
Location Memerambi
Depth 0 – 10 cm
pH 6.4
C.E.C. 31 m.eq/100g
Macro elements Micro-elements
Organic C 2.9% Fe 24 ppm
N 0.33% Mn 186 ppm
P 0.073% Cu 3.8 ppm
K 0.28% Zn 4.0 ppm
S 0.053%
134 Appendices
Table 8.2. Concentration of macro-elements in dry chickpea seed. Data are presented as a mean ± SD (n= >3). Values sharing the same
superscript letters indicate groups that are not significantly different at p<0.05.
Ca
(mg/100g)
Mg
(mg/100g)
Na
(mg/100g)
K
(mg/100g)
P
(mg/100g)
S
(mg/100g)
Genesis090
Billa Billa efg 142 (± 8.33) cdefg 134 (± 1.73) cde 16.72 (± 3.10) bcde 1007 (± 11.55) abc 400 (± 10.00) abcd 193 (± 1.73)
Roma cdef 160 (± 20.50) fg 124 (± 1.00) cde 17.08 (± 2.73) bcde 1010 (± 45.83) efg 333 (± 11.55) abcd 187 (± 1.53)
Warra cdef 164 (± 23.52) defg 132 (± 5.29) cde 15.91 (± 3.88) fgh 913 (± 30.55) ab 410 (± 26.46) a 204 (± 14.00)
Kalkee
Billa Billa g 105 (± 4.93) efg 131 (± 1.00) cde 15.53 (± 1.84) ab 1080 (± 10.00) abc 393 (± 5.77) abcd 191 (± 3.61)
Roma fg 124 (± 11.15) efg 128 (± 0.00) cde 12.14 (± 1.89) a 1117 (± 5.77) def 343 (± 11.55) bcd 186 (± 4.51)
Warra fg 121 (± 10.21) fg 122 (± 12.49) cde 14.89 (± 10.56) defg 940 (± 60.83) cde 360 (± 26.46) abcd 190 (± 8.72)
Monarch
Billa Billa fg 127 (± 11.59) bcdef 138 (± 2.08) a 63.33 (± 14.57) ab 1077 (± 25.17) bcde 373 (± 5.77) cdef 180 (± 1.53)
Roma cdef 160 (± 20.03) fg 122 (± 7.00) b 33.00 (± 6.56) bcde 1020 (± 10.00) g 290 (± 20.00) defg 176 (± 6.66)
Warra def 157 (± 11.72) fg 124 (± 3.46) bc 24.67 (± 3.06) gh 863 (± 37.86) efg 330 (± 26.46) ab 198 (± 10.58)
Boundary
Billa Billa defg 148 (± 26.21) bcde 144 (± 10.5) cde 12.41 (± 3.63) abc 1047 (± 25.17) abcd 387 (± 11.55) abcd 189 (± 4.36)
Roma ab 217 (± 5.77) bcde 143 (± 5.51) cde 14.34 (± 1.55) ab 1097 (± 25.17) abcd 383 (± 15.28) abcd 191 (± 7.57)
Warra bcde 192 (± 9.17) b 152 (± 6.56) bcd 20.48 (± 4.20) fgh 903 (± 23.09) abc 403 (± 5.77) abc 196 (± 4.04)
Kingaroy cdef 165 (± 1.08) fg 124 (± 2.10) de 8.31 (± 0.32) gh 857 (± 6.08) k 186 (± 3.54) fg 161 (± 1.79)
CICA0912
Billa Billa def 159 (± 6.24) b 152 (± 5.00) de 7.32 (± 1.24) abc 1057 (± 45.09) abc 397 (± 11.55) cdef 181 (± 2.52)
Warra abc 210 (± 35.50) bcd 149 (± 10.15) cde 10.82 (± 1.73) fgh 890 (± 40.00) ab 413 (± 15.28) abc 196 (± 5.13)
Kingaroy bcde 185 (± 6.80) g 118 (± 4.18) e 4.00 (± 0.11) gh 871 (± 19.68) k 229 (± 17.65) def 178 (± 6.94)
HatTrick
Billa Billa bcd 196 (± 16.37) bc 151 (± 2.00) cde 16.02 (± 6.22) cdef 977 (± 55.08) cde 363 (± 25.17) cdef 180 (± 2.08)
Roma a 257 (± 15.28) bcde 144 (± 0.58) de 7.10 (± 1.45) efg 930 (± 10.00) fg 303 (± 5.77) efg 168 (± 3.06)
Warra bcd 196 (± 31.53) b 153 (± 8.50) cde 16.56 (± 1.49) fgh 903 (± 5.77) a 420 (± 10.00) abcd 190 (± 1.00)
Kingaroy bcdef 167 (± 12.96) fg 124 (± 4.78) e 5.15 (± 0.04) h 836 (± 6.00) k 199 (± 4.40) fg 165 (± 3.05)
NSW a 242 (± 10.95) a 180 (± 4.62) e 6.34 (± 0.63) bcd 1016 (± 23.02) ab 408 (± 8.37) cde 182 (± 2.79)
Appendices 135
Table 8.3. Concentration of micro-elements in dry chickpea seeds. Data is presented as a mean ± SD (n= >3). Values sharing the same
superscript letters indicate groups that are not significantly different at p<0.05.
Fe
(mg/100g)
Zn
(mg/100g)
Mn
(mg/100g)
B
(mg/100g)
Cu
(mg/100g)
Genesis090
Billa Billa abcd 4.60 (± 0.07) fgh 3.33 (± 0.04) defgh 2.98 (± 0.14) bcdef 1.01 (± 0.01) fg 0.74 (± 0.00)
Roma a 5.20 (± 0.02) a 4.35 (± 0.07) fgh 2.73 (± 0.09) bcd 1.07 (± 0.05) abc 0.99 (± 0.03)
Warra ab 5.00 (± 0.46) bcd 3.92 (± 0.21) cdefg 3.25 (± 0.18) b 1.11 (± 0.11) abcd 0.95 (± 0.08)
Kalkee
Billa Billa cdef 4.22 (± 0.11) fghi 3.25 (± 0.04) cdefg 3.23 (± 0.09) bcd 1.06 (± 0.04) efg 0.79 (± 0.01)
Roma abc 4.83 (± 0.04) ab 4.22 (± 0.12) efgh 2.85 (± 0.10) bcd 1.05 (± 0.05) a 1.01 (± 0.01)
Warra bcde 4.42 (± 0.49) efg 3.50 (± 0.35) bcd 3.68 (± 0.35) bcd 1.06 (± 0.04) abcde 0.89 (± 0.08)
Monarch
Billa Billa bcde 4.43 (± 0.09) ghij 3.09 (± 0.09) bcdef 3.42 (± 0.09) fghi 0.86 (± 0.06) g 0.69 (± 0.02)
Roma abcd 4.58 (± 0.17) abc 3.98 (± 0.20) gh 2.66 (± 0.18) bcde 1.04 (± 0.05) abcd 0.95 (± 0.05)
Warra bcd 4.47 (± 0.10) fgh 3.40 (± 0.19) abc 3.86 (± 0.19) bc 1.10 (± 0.10) abcde 0.90 (± 0.06)
Boundary
Billa Billa def 4.15 (± 0.08) ghi 3.12 (± 0.11) bcdef 3.41 (± 0.41) defghi 0.91 (± 0.02) fg 0.76 (± 0.02)
Roma abcd 4.66 (± 0.25) ab 4.14 (± 0.16) defgh 3.09 (± 0.09) bcd 1.05 (± 0.01) ab 1.01 (± 0.05)
Warra bcd 4.53 (± 0.06) bcde 3.85 (± 0.04) abc 3.88 (± 0.23) bcdefg 0.95 (± 0.03) def 0.85 (± 0.03)
Kingaroy g 3.36 (± 0.06) jk 2.70 (± 0.05) ab 4.11 (± 0.11) hi 0.84 (± 0.01) gh 0.67 (± 0.01)
CICA0912
Billa Billa cdef 4.32 (± 0.18) ijk 2.89 (± 0.10) bcd 3.61 (± 0.21) efghi 0.88 (± 0.01) efg 0.79 (± 0.02)
Warra bcd 4.55 (± 0.11) bcd 3.93 (± 0.03) bcd 3.66 (± 0.49) bc 1.10 (± 0.13) abcde 0.90 (± 0.03)
Kingaroy fg 3.70 (± 0.15) cdef 3.58 (± 0.15) ab 4.04 (± 0.05) gc0.78 (± 0.02) hi 0.55 (± 0.03)
HatTrick
Billa Billa def 4.14 (± 0.18) hij 3.06 (± 0.04) ab 4.12 (± 0.29) bcdefg 0.96 (± 0.07) fg 0.76 (± 0.11)
Roma cdef 4.23 (± 0.11) bcde 3.90 (± 0.04) bcd 3.60 (± 0.06) bcdefg 0.97 (± 0.03) bcdef 0.88 (± 0.01)
Warra cdef 4.31 (± 0.07) bcde 3.86 (± 0.02) bcde 3.54 (± 0.48) cdefgh 0.94 (± 0.05) cdef 0.86 (± 0.01)
Kingaroy efg 3.82 (± 0.36) k 2.60 (± 0.08) a 4.44 (± 0.20) i 0.76 (± 0.01) i 0.45 (± 0.01)
NSW def 4.23 (± 0.12) def 3.49 (± 0.09) h 2.54 (± 0.07) a 1.41 (± 0.02) i 0.44 (± 0.03)
136 Appendices
Appendix B
Chapter 5 supplementary figures
Table 8.4. List of proteins used in the phylogenetic analysis.
Organism Gene Accession no.
Sequence
length
(aa)
Thale cress
(Arabidopsis thaliana)
AtNAS1 NP_196114.1 320
AtNAS2 NP_200419.1 320
AtNAS3 NP_176038 320
AtNAS4 NP_176038 324
Arabidopsis halleri AhNAS2 AFH08366.1 320
Barley
(Hordeum vulgare)
HvNAS1 Q9ZQV9 328
HvNAS2 Q9ZQV7.1 335
HvNAS3 Q9ZQV8.1 335
HvNAS4 Q9ZQV6.1 329
HvNAS5 BAA74584.1 267
HvNAS6 Q9ZQV3.1 328
HvNAS7 Q9ZWH8.1 329
HvNAS8 Q9XFB6.1 329
HvNAS9 Q9XFB7.1 340
Chickpea
(Cicer arietinum)
CaNAS2 XP_004495658.1 306
CaNAS XP_004487761.1 311
CaNAS XP_004488704.1 285
CaNAS XP_004494544.1 318
Lotus japonicas LjNAS1 BAH22562.1 318
LjNAS2 BAH22563.1 312
Barrel medic
(Medicago truncatula)
MtNAS XP_003591220.1 341
MtNAS XP_003594753.1 282
MtNAS XP_013450461.1 320
MtNAS XP_013464357.1 284
Rice
(Oryza sativa)
OsNAS1 BAA74588.2 332
OsNAS2 BAB17823.1 325
OsNAS3 BAB17824.1 343
Alpine pennygrass
(Thlaspi caerulescens) TcNAS CAC82913.1 321
Tomato
(Solanum lycopersicum) SlNAS NP_001296307.1 317
Maize
(Zea mays)
ZmNAS1 AFW88604.1 327
ZmNAS2 DAA45018.1 601
ZmNAS3 XP_008665178.1 359
Appendices 137
Figure 8.1. Representative photos of T1 tobacco screening via PCR using gene-
specific primers. The following genes were screened: A) GmFER, B) CaNAS2, and
C) OsNAS2.
M = 2-log ladder (NEB)
“-” = No template control
“+” = gDNA positive control
NT = Untransformed chickpea
138 Appendices
Figure 8.2. Concentrations of A) iron, and B) zinc in T1 transgenic tobacco
leaves. Each bar represents an individual plant.
Appendices 139
Appendix C
Chapter 6 supplementary figures
Figure 8.3. GUS staining of transiently transformed chickpea A) after co-
cultivation and B) after the first selection round.
Table 8.5. Germination rates and segregation of transgenic chickpea lines.
LINES
GmFER-OsNAS2 GmFER-
CaNAS2
Generation 7.1 7.2 7.15 1.1 6.6 6.14
T1 Germination rate (%) 74.82 84.21 80.65 - 74.29 73.68
PCR + progeny (%) 69.23 75.00 72.00 - 17.31 85.71
T2 Germination rate (%) 46.74 15.38 8.99 72.73 46.58 27.52
PCR + progeny (%) 93.02 75.00 100.00 87.50 67.65 68.42
T3 Germination rate (%) 96.15 59.38 - 26.42 38.10 21.43
PCR + progeny (%) 100 57.89 - 100.00 87.50 100.00
T4 Germination rate (%) - - - 80.65 - -
PCR + progeny (%) - - - 100.00 - -
140 Appendices
Figure 8.4. Iron, zinc, manganese and phosphorus content of GM chickpea
seeds.
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