Dipartimento di Coltivazioni e Difesa delle Specie Legnose ‘G. Scaramuzzi’, Università di Pisa, I-56124 Pisa, ItalyDipartimento di Scienze e Tecnologie per l’Agricoltura, le Foreste, la Natura e l’Energia, Via S. C. DeLellis s.n.c, Università della Tuscia, Viterbo 01100, Italy
a r t i c l e i n f o
rticle history:eceived 8 November 2012eceived in revised form 9 March 2013ccepted 13 March 2013
Plants are aerobic organisms that require the availability of O2 for nutrient uptake, so excess watersurrounding roots can generate lethal conditions. Therefore a new flood-tolerant stone-fruit rootstockwas used in a grafted combination with peach cv Suncrest (Prunus persica (L.) Batsch.) used as scion, totest its tolerance strength during a twenty-one day flooding period. Potted plants, from graft combinationof cv Suncrest with rootstock Mr.S 2/5 wild type (Prunus cerasifera Erhr) and its clone variant (S.4), weresubmitted to normoxic and flooded stress for 21 days, under open conditions. Suncrest/S.4 plants had thehighest plant growth under normoxic condition and the highest plant survival under flooded conditions.Under flooded conditions a halt in plant growth and the appearance of severe damage were detected inthe Suncrest/wt plants. In the latter, symptoms of flooding were desiccation of the shoot apex, strong
omaclonal variant reddening of leaves followed by appearance of necrotic areas and senescence of almost all leaves.The responses observed in all organs of the grafted plant could be linked to relevant morpho-
physiological adaptations to flooding that would ensure survival during short periods of anoxia. Resultsalso provided evidence that flooding tolerance was conferred by the S.4 clone to the scion. The availabil-ity of a novel rootstock tolerant to short-term waterlogging conditions will offer new possibilities to thestone-fruit industry located in various adverse environments.
Anaerobic stress is one of the physical stresses that a plant canxperience due to waterlogged soil conditions in areas where rainun-off is impeded or bad irrigation practices occur, bringing waterp onto the soil surface. Waterlogging imposes constraints on culti-ation of many agricultural crops, due to low availability of oxygeno root systems (Drew, 1997; Dennis et al., 2005), leading to highconomic losses (Sullivan et al., 2001). Prolonged flooding oftennhibits plant growth and induces leaf senescence, and compro-
ises crop productivity and plant survival (Pereira and Kozlowski,977; Tsukahara and Kozlowski, 1986; Visser et al., 2003; Fukaond Bailey-Serres, 2004), because it blocks the transfer of free oxy-
en and other gases between soil and atmosphere and gas diffusionround roots (Dennis et al., 2005; Steffens and Sauter, 2010). Plant
response to hypoxia depends on the species, genotype, age, andduration of flooding (Bailey-Serres and Voesenek, 2008).
Stone-fruit tree species are usually grown in Mediterraneanclimates, which are characterized by irregular and concentratedrainfalls lasting a few days (Alpert et al., 2002). It is becomingimportant to have rootstocks that improve the tolerance of trees tohypoxic and anoxic conditions. The genus Prunus includes speciesthat produce fruit of great commercial importance but they arefrequently intolerant to waterlogging, compared to many othertemperate woody horticultural plants, including apple, pear andquince (Andersen et al., 1984). Among the different species ofPrunus, Myrobalan plum (Prunus cerasifera Erhr) and Europeanplum (P. domestica L.) are considered flood tolerant (Ranney, 1994;Kozlowski, 1997). A rootstock breeding program for Prunus speciesis being developed through interspecific crosses of the Myrobalanplum (P. cerasifera Ehrh.) and almond × peach hybrids [P. amyg-dalus Batsch × P. persica (L.) Batsch], generating hybrid lines thatare still under evaluation (Xiloyannis et al., 2007). However very
little research has been conducted in Prunus, or other fruit treespecies, into helping to unravel the flooding tolerance mechanisms(Arbona et al., 2008, 2009; Amador et al., 2012; Pistelli et al., 2012).In short-term trials carried out to study early antioxidant response
Amador et al., 2012), enzyme activities of superoxide dismutaseSOD), guaiacol peroxidase (POD) and catalase (CAT) were highern the sensitive genotype ‘Felinem’, an interspecific hybrid betweenlmond x peach [P. amygdalus Batsch × P. persica (L.) Batsch], thann the tolerant ‘P.2175’ Myrobalan genotype (P. cerasifera Erhr).lthough the set of adaptive responses was quite complex, theuthors suggested that the higher tolerance of Myrobalan plumso flooding cannot be ascribed to these enzyme activities (Amadort al., 2012). Peach seedlings used as rootstock have been reportedo be intolerant of flooding, as indicated by slow growth, plantecline, or both, on poorly drained soils (Rutto et al., 2002). Graft-
ng peach cultivar onto an appropriate rootstock could generate aore adaptable tree by combining the high quality production of
he cultivar (scion) with a flood-tolerant rootstock.Rootstock Mr.S 2/5, selected from a half-sib progeny from a P.
erasifera Erhr clone, is also considered to be fairly tolerant (Dichiot al., 2004). The somaclonal variant, S.4, of rootstock Mr.S 2/5 withncreased tolerance to waterlogging has been generated in vitrorom leaf explants (Muleo et al., 2006). Although the relevant vari-tion in the S.4 genome has yet to be determined, morphological,hysiological and gene expression alterations clearly conferred aood-tolerant phenotype (Pistelli et al., 2012).
In the work described here, grafting experiments were per-ormed with the aim to evaluate whether the flood-tolerantomaclone S.4 used as rootstock confers tolerance to the peachv Suncrest. In addition, a short-term flooding stress experimentas carried out to evaluate for how long flood-tolerance could be
onferred to the scion by the rootstock under persistently stressedonditions. The changes observed were probably responsible forhe plant’s adaptation to anaerobic conditions.
. Materials and methods
.1. Plant materials
Micropropagated plantlets of S.4 and Mr.S 2/5 wild type (wt)lones were acclimatized and forced to grow vigorously in a green-ouse for at least 6 months, during winter–summer 2008. Thelants were grown in 3.8-L plastic containers with a soil mediumomposed of 45% clay, 45% sand and 10% silt, at a relative humid-ty of 35–85%, a temperature between 18 and 30◦ C, a dailyapour pressure deficit (VPD) of 4–10.2 kPa and a maximum Pho-osynthetically Active Radiation (PAR) of 1240 �M m−2 s−1. In lateugust, once scion stem diameter of both clones was sufficient,
arge, dormant cv Suncrest peach buds were grafted, approximately0–15 cm above soil level, onto the before mentioned rootstockssing a T-budding (shield budding) grafting technique and tied with
budding rubber. The plants were transplanted on first February009 into 15-L plastic containers filled with the medium previ-usly described, and moved outside. Once buds of the peach cvuncrest began to swell and started their outgrowth, tops of thenderstock were then pruned to 25 cm high. After 1 month theemaining portion of the understock was removed to force theigorous outgrowth of the shoot, thus obtaining the scion/stocklant combination, Suncrest/wt and Suncrest/S.4. Pots were irri-ated once a day, and a routine fertilization program was applieduring the whole vegetative period in which the plants were grow-
ng until the waterlogging treatments were imposed. No symptomsue to graft-incompatibility was observed on grafted plants.
.2. Stress conditions
At the end of June 2009, plants were moved into trenchesFig. 1a and b) coated with a double layer of plastic to preventrainage, and treatments began on 20 July 2009. The design of the
ulturae 156 (2013) 1–8
long-term trial was a two (flooded and normoxic condition) × two(scion/rootstock combinations) factorial arrangement in a random-ized complete-block design with 9 grafted plants as replications pereach factorial combination. Flooding was imposed on each groupof plants by placing plant containers into the trenches (Fig. 1b)that were submerged, the water level maintained at 3–5 cm abovesoil level. Plants grown under normoxic conditions were used ascontrol and irrigated daily. Midday air temperature was between26 and 34 ◦C, daily relative humidity 25–93%, vapour pressuredeficit (VPD) 1.5–10.3 kPa and Photosynthetically Active Radiation(PAR), measured between 12.00 and 14.00 HR, 1680 �M m−2 s−1
PAR. Oxygen depletion measurements were periodically recordedusing a portable dissolved Oxygen meter (Hanna Instruments,Lansing, MI, USA) inserted into the soil at 5 cm depth. The diffu-sion of O2 in the soil of non-flooded plants was always constant(903.3 ± 40.4 ppm).
The flooding tolerance of plants was evaluated as the numberof days elapsed between the beginning of submersion and the wil-ting of all of the expanded leaves of each plant, coinciding withthe appearance of epinasty. Plant growth and phytomer develop-ment was measured. Number of abscised leaves was also measuredand the data were reported as cumulative percentage. Leaf androot fresh and dry weight, and chlorophyll content were also ana-lyzed. For root inspection and weight determination, the soil wasremoved, and the fine roots were gently washed with tap water.
2.3. Chlorophyll determination
During the waterlogging period, the leaf chlorophyll concentra-tion was estimated using an SPAD 502 meter (Minolta Co., Osaka,Japan) on a set of four replicates on days 0, 6, 9, 12, 15, 18, 21 ofthe waterlogging treatment. The measurements were made in twowell-developed leaves for each replicated plant, one at the top andthe second in the middle of plant. During the treatment, when theleaf dropped, the measurements were made in the immediatelyabove.
2.4. TTC reactivity test
The triphenyltetrazolium chloride (TTC) reactivity test was usedto measure tissue vitality and respiratory activity of leaves androots. The colorless TTC accepts electrons from the electron trans-port chain of mitochondria reducing it to the red colored triphenylformazane (TF), which can be quantified at 520 nm (Richter et al.,2007). Analyses were carried out on 100 mg of fresh tissues, usingthe same protocol as described in Pistelli et al. (2012). The reactiv-ity of the samples with TTC was measured as absorption of TF perg dry mass (A520 g D.W.−1).
2.5. Anthocyanin assay
Anthocyanin’s assay (AA) in the leaves was conducted accordingto the Mancinelli et al. (1975) procedure. Fresh sample (100 mg)was placed in a test tube, and anthocyanins were extracted with100 mL of 1% (w/v) HCl in methanol. The mixture was placed in thedark at 4 ◦C for 24 h, with continuous shaking. Thus, the extractswere cleared by filtration, and their absorbance at 532 nm wasdetected with a spectrophotometer (UV/VIS Spectrometer, Lambda25, Perkin Elmer, Milan, Italy). Total anthocyanin concentration wascalculated using a molar excitation coefficient of 3.43 × 104 as nM100 mg−2.
2.6. Statistical analysis
Statistical analysis was carried out using SigmaStat 3.1 package(SYSTAT software Inc., Chicago, IL, USA). Analysis of variance was
C. Iacona et al. / Scientia Horticulturae 156 (2013) 1–8 3
Fig. 1. Plant behavior. View of the trenches containing the grafted plants under normoxic (a) and anoxic (b) conditions. Suncrest/wt (d) and Suncrest/S.4 (e) plants as seenat the end of 21 days of flooding stress. Shoot apex (c) of Suncrest/S.4 plant at the twenty-first day of flooding stress. Growth of the organ is maintained. Root system as seenafter 21 days of flooding stress of the Suncrest/wt (f) and Suncrest/S.4 (g) plants. Aerial part (h) and root system (j) of Suncrest/wt (left side) and Suncrest/S.4 (right side)after 21 days of growth under normoxic conditions. Rootstock stems cross sections (i), of S.4 variant (left) and Mr.S 2/5 wt (right), after long-term flooding stress.
4 C. Iacona et al. / Scientia Horticulturae 156 (2013) 1–8
Fig. 2. Effect of flooding on node development in the stem of the peach cv Suncrest,determined as number of new nodes formed and detected at the end of exposure toflt(
ptartmsatTt
3
3
pgp(rwplStait
3
a(eanSa
Fig. 3. Plant stem elongation of the peach cv Suncrest grafted on the two rootstockclones after 21 days of exposure to flooding and normoxic conditions. Histograms
clone for the node formation and elongation of stem (Table 1), con-firming the good performance of Suncrest/S.4 under short floodingstress period.
Table 1ANOVA of the parameters detected for both the Suncrest/wt and Suncrest/S.4 graftedcombinations under normoxic and flooding conditions. Factorial ANOVA highlightsthe presence of significant interaction between plant grafted combinations andtreatment.
Source of variation df Sum of squares F Prob. > F
Number of developed nodesTreatment 1 34.549 98.100 0.001Clone 1 120.513 342.189 0.001Interaction 1 7.230 20.529 0.001Error 32 11.270Total 35 173.561
ooding and normoxic conditions. Histograms represent the mean of nine plants andhe bars ± two-fold Standard Error. Different letters indicate statistical differenceP < 0.01).
erformed for morphological measurements, anthocyanin concen-ration, and leaf and root TTC activity. Differences were accepteds statistically significant when P < 0.05, and Duncan’s test was car-ied out to identify significant differences among the samples. Forhe stem elongation, node formation, leaf abscission, colorimetric
easurements (SPAD index), and anthocyanin concentration eachtatistical sample included nine plants, while for leaf and root TTCctivity, and for fresh and dry weight of each plant organ the statis-ical sample included five biological replicates (individual plants).he percentage of leaf abscission was transformed by arcsin prioro ANOVA analysis.
. Results
.1. Oxygen depletion
The plot of oxygen concentration against time of depletionrovided a straight decrease with high R2 values (0.99) in allrafted plant combinations (data not shown). In normoxic exposedlants, the diffusion of O2 in the soil was always constant903.3 ± 40.4 ppm), while in the waterlogged soil the O2 decreasedapidly during the experiment; after three days the average valueas 333.4 ± 22.7 ppm and 368.7 ± 17.1 ppm in soil of Suncrest/wtlants and Suncrest/S.4 plants, respectively. At the sixth day the O2
evel decreased at 60.7 ± 10.9 ppm and 96.2 ± 8.4 ppm in the soil ofuncrest/wt and Suncrest/S.4 plants, respectively. Flooding condi-ions further reduced the level of O2 in the soil, by the ninth dayverage values detected were 17.3 ± 6.2 ppm and 55.2 ± 10.3 ppmn the soil of Suncrest/wt and Suncrest/S.4 plants, respectively; athis point anoxic conditions were established.
.2. Plant growth
In plants exposed to normoxic conditions growth was observedfter 21 days of treatment, irrespective of grafting combinationFig. 1h and j). In fact the formation of new nodes (Fig. 2) andlongation of stem (Fig. 3) were detected in all plants, although
significantly greater elongation of the stem and development ofodes were observed in the plants grafted onto rootstock clone.4. Exposure of Suncrest/wt plants to waterlogging induced therrest of stem growth and phytomer formation (Fig. 1d); conversely
represent the mean of nine plants and the bars ± two-fold Standard Error. Differentletters indicate statistical difference (P < 0.01).
sustained stem growth and development of nodes were observedin the Suncrest/S.4 plants (Fig. 1c and e). In these latter plants,the development of phytomers resulted to be around 4.5 per plant(Fig. 2). There was a significant interaction between treatment and
C. Iacona et al. / Scientia Horticulturae 156 (2013) 1–8 5
Table 2Fresh weight and dry weight of plants sampled after 21 days of flooding treatment.Values represent the averages of five sampled plants. ANOVA two way factorial anal-yses resulted to be significant for both factors (clone and treatment) and interactionexcept for the stem. Different letters, along the row, for each organ and parameterindicate statistical differences (P < 0.01).
Suncrest/wt Suncrest/S.4
Normoxic Flooding Normoxic Flooding
Fresh weight (g)Leaf 20.85 c 4.89 a 19.67 c 17.23 bStem 21.26 ns 21.45 ns 22.11 ns 20.86 nsRoot system 36.59 c 24.85 a 36.67 c 33.81 bTotal plant mass 78.69 c 51.20 a 78.44 c 71.69 b
Dry weight (g)Leaf 7.02 b 1.71 a 6.54 b 5.93 bStem 11.74 ns 11.85 ns 12.45 ns 11.47 ns
cd(dpaFre
3
bncSoc
l
FcSEta
Fig. 5. Anthocyanin content in the blade leaf tissues of the peach cv Suncrest grafted
Root system 16.33 b 11.09 a 16.32 b 15.31 bTotal plant mass 35.09 b 24.64 a 35.08 b 32.91 b
Plant stress imposed by flooding affected mass growth of Sun-rest/wt plants (Table 2). The reduction of fresh and dry weight wasue to the abscission of leaves and the reduction of the root massFig. 1f and g). Statistical differences were detected for leaf fresh andry weight in Suncrest/S.4 between normoxic and flooding stressedlants (Table 2), indicating that the stressed plants were affected,lthough not so severely as those of Suncrest/wt (Table 2 and Fig. 1).or the stem fresh and dry weight the statistical analysis did noteveal significant differences between the two factors and insideach factor.
.3. Physiological and morphological adaptation
Colorimetric measurements showed significant differencesetween the plant exposed to waterlogging and those exposed toormoxic conditions (Fig. 4). In the latter, SPAD values were almostonstant during the treatment period, while in the flooded plantsPAD values started to decrease significantly from the twelfth day
f treatment, and the highest decrement was statistically signifi-ant in the Suncrest/wt plants.
During the stress period, the leaves of the Suncrest/wt plantsost their green color and turned yellowish to red, starting from
ig. 4. Colorimetric measurements in the peach cv Suncrest grafted on Mr.S 2/5 wtlone and S.4 variant clone, during plant exposure to a period of waterlogging stress.ymbols represent the average of nine plants. Bars represent ± two-fold Standardrror. Averages were separated on the basis of each detection time, using the Fisher’sest (** = 0.01, *** = 0.001). Different letters within each time indicate differencesmong the cultivar/rootstock combinations between the treatments.
on the two rootstock clones after 21days of exposure to flooding and normoxicconditions. Histograms represent the mean of nine plants and the bars ± two-foldStandard Error. Different letters indicate statistical difference (P < 0.01).
the lower leaves of the stem (Fig. 1d). These stressed leaf bladesaccumulated almost 3 times more anthocyanins than leaf blades ofboth normoxic plant and of the Suncrest/S.4 flooded-plants (Fig. 5).The interaction between treatment and genotype was significant(P = 0.001) (Table 1).
After that, an extensive necrosis appeared on the entire sur-face, and an accentuated defoliation was detected along the stem(Fig. 1d). In contrast to this latter plant combination, plants of theSuncrest/S.4 combination did not show any stress effects underflooding, the leaf cell turgor was retained, and only 20% of the totalleaves dropped, after 21 days of flooding treatment (Fig. 6).
Morphological leaf features of the peach cv Suncrest and theroots of wt and S.4 clone showed marked differences between plant
scion/stock combinations: in particular, some leaf tissues and manyroots of the Suncrest/wt were compromised (Fig. 1d and f), so themitochondrial activity was tested in the two plant combinationsafter 21 days of flooding treatment. The TTC activity, carried out in
Fig. 6. Leaf abscission in the peach cv Suncrest grafted on Mr.S 2/5 wt clone and S.4variant clone, during plant exposure to flooding stress, determined as cumulativepercentage. Plants under normoxic conditions have not lost leaves, during the flood-ing period. Symbols represent the average of nine plants. Bars represent ± two-foldStandard Error. Averages were separated on the basis of each detection time, usingthe t test (* = 0.05, *** = 0.001).
6 C. Iacona et al. / Scientia Hortic
Fig. 7. TTC reactivity as detected in the roots of the two somaclonal rootstock (a),and in the leaves of the peach cv Suncrest (b), after 21 days from the beginning oftS
tte(d(ottaptg
SaopshcacSorfll
ethanol produced in the roots (Kreuzwieser et al., 2001). High lev-
he treatment. Histograms represent the mean of five plants and the bars ± two-foldtandard Error. Different letters indicate statistical difference (P < 0.01).
he roots and leaves, remained high in Suncrest/S.4 plants exposedo flooding stress (Fig. 7), but declined in Suncrest/wt plants, coher-ntly with the significant interaction found between the two factorTable 1). Under submergence conditions, differences in mitochon-rial activity in the roots was the most apparent between clonesFig. 7a), where the roots of Suncrest/S.4 maintained a high activityf approximately 88% of that observed under normoxic condi-ions, whereas the activities in the Suncrest/wt plants decreasedo around 68% (Fig. 7a). Flooding strongly affected mitochondrialctivity in the leaves (Fig. 7b), although the decrement was lessronounced in leaves of the Suncrest/S.4 plants (63%) with respecto those of the Suncrest/wt plants (48.1%). The interaction betweenenotype and treatment was highly significant (P < 0.000) (Table 1).
Over the course of the experiment, disintegration of roots inuncrest/wt plants occurred due to prolonged anoxic conditionsnd a subsequent decrease in size of the whole root system wasbserved (Fig. 1f, g and j). The transverse cross section of the stemortion of the two rootstocks (Fig. 1i), under anoxic conditionshowed that in the wt clone, the inner part of the xylem and theeartwood were severely compromised, as seen from the browningolor, without any distinguishable structures of tissue layers, prob-bly generated by damaged cells. On the contrary, under the sameonditions a regular structure was present in the stem portion of the.4 clone. Moreover, the living phloem and the vascular cambiumf the wt stem portion exhibited different structure and color, with
espect to the same tissues of the S.4 stem portion (Fig. 1i). Manyooded plants of the Suncrest/wt combination were severely defo-
iated at the end of the treatment period, yet the stem tissue under
ulturae 156 (2013) 1–8
the bark was typically green. No late-formed roots were observedon the stem of the rootstock for any of the graft combinations, after21 days of waterlogging.
4. Discussion
Plants try to avoid and adapt themselves to flooding throughflood-tolerance responses, for maintaining or reviving their growthand survival (Visser et al., 2003; Bailey-Serres and Voesenek, 2008).Tree roots require not only water and minerals, but also air: in fact,they need to breathe just as much as the leaves do. A lack of O2in the rhizosphere affects the maintenance of numerous pathwaysof plant growth and development (Drew, 1997; Vartapetian et al.,2003; Visser et al., 2003; Colmer and Voesenek, 2009). Under ourexperimental conditions, free oxygen in the soil fell very rapidlywithin a few days, and establishing anoxic conditions that couldindicate the different adaptive capacity to the stress of the twografted plant combinations, highlighting the properties of the tworootstock clones. The submerged Suncrest/S.4 plants continued togrow and develop new nodes and leaves, during the stress period,and very few leaves turned from green to yellow and red, andfinally abscised. On the contrary, under the same conditions, theSuncrest/wt plants blocked growth, new phytomers developmentand a very large number of leaves finally abscised.
Plum is a species considered tolerant to flooding (Domingo et al.,2002; Amador et al., 2012); however, the range of physiological andmorphological responses observed in the peach cv Suncrest graftedonto the S.4 clone indicates that flooding tolerance is increasedwith respect to the Mr.S 2/5 wt rootstock. Under the pressure offlood stressors, Suncrest/S.4 plants did not display signs of rootnecrosis; in contrast, the root system of Suncrest/wt plants dis-played signs of necrosis in the root tips and a lower root masswas evident after 21 days of flooding. In addition, the Suncrest/wtstressed plants’ SPAD values were observed to decrease signifi-cantly. SPAD values indicate that the same flooding period, 21 days,had a different effect on plant tolerance in the two grafted plantcombinations.
Under a flooding stress of 6 days, low levels of ethylene weredetected in S.4 plants, in a previous work (Pistelli et al., 2012),consistent with the low level of Aminocyclopropane-1-carboxylateoxidase and 1-amino-cyclopropane-1-carboxylic acid synthasetranscripts observed in leaves and roots of S.4, while high levels ofethylene and gene transcripts were detected in the wt plants. Ethyl-ene synthesis in flooding-sensitive plants induces strong epinastyand leaf senescence (Jackson, 2002), as was observed in the Sun-crest/wt plants. Under hypoxic and anoxic conditions, the S.4 clonewas apparently able to transport O2 and its roots maintained well-organized tissue layers, whereas in the roots of wt the tissuestructure was completely disaggregated (Pistelli et al., 2012). Thereduction of the root system in Suncrest/wt plants observed in thiswork, therefore, can be ascribed to the disorganization of the tissuelayers of the roots.
Although the flooding did not apparently increase the diameterof the rootstock stem between above-water level and below-grafting point, the stem portion of the wt clone was however largelycompromised from an contemporary increase of bark and phloemtissues, and the deconstruction of xylem and pith tissues. The dam-age of tissues observed in the stem of the rootstock could findpossible explanations in different type of polyphenol synthesis andalcohol dehydrogenase (ADH) activity, as previously observed inthe two clones (Pistelli et al., 2012), and in the xylem-transported
els of ethylene is deleterious to cell membranes for its “fluidizing”effect (Kiyosawa, 1975), and its association with low antioxidantactivity detected in a similar genotype (Amador et al., 2012) could
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ffect the stability of tissue organization in stem and roots of root-tock wt.
Mitochondrial respiration is one of the basic cellular pathwaysecessary to generate energy, and is dependent on O2 avail-bility. If this availability decreases, mitochondrial respiration isompromised and plants switch from respiration to fermentativeetabolism, to allow regeneration of NAD+ in the absence of respi-
ation, thereby maintaining glycolysis and the generation of ATPnder anaerobic conditions (Bailey-Serres and Voesenek, 2008).his metabolic activity is vital, because in the absence of NAD+,lycolysis ceases (Kennedy et al., 1992; Robertson et al., 1994).olorimetric results and measurements of mitochondrial activity
ndicate that the leaves of Suncrest/S.4 plants performed betterhan the Suncrest/wt plants, with a probably favorable impact onarbon flow in the branches of fermentative pathway. Moreover,ince no difference in ADH activity has been previously detectedn the roots of the two clones, after six days of flooding (Pistellit al., 2012), we consider that a variation on level of Pyruvateecarboxylase (PDC) activity could take place in the roots of S.4ariant, leading to an alternative mechanism to avoid toxicity fromhe accumulation of acetylaldehyde during the exposure to anoxicoil conditions, as it is suggested by the research carried out inrabidopsis transgenic plants, where the overexpression of eitherDC1 or PDC2 improved plant survival under low oxygen conditionsIsmond et al., 2003). Therefore, the challenge of the next investi-ation will be that to evaluate the PDC activity in roots and shootsf grafted plants.
The O2 concentration in the soil was nil from the sixth day ofooding; thus, O2 was probably more effectively transported to theoots in the S.4 clone than in the plants of the wt. This hypothesiss also supported by the TTC-test activity measured in the rootsf both clones. During the stress period, the tissue organization ofhe stem was maintained in the S.4 clone, which probably allowedhe carbohydrate supply to the roots and their metabolic activityo be maintained. In fact the carbohydrate supply is correlated tohe formation of energy, and the level of carbohydrate reserves orhe capacity to maintain their transport may be a crucial factorn the tolerance of short-term flooding (Parent et al., 2008). Ourypothesis is that the Suncrest/S.4 plants were able to maintainetabolism efficiently during flooding conditions: even though itight be reduced, it was sufficient to maintain energy production.
common feature in woody species subjected to flooding stress ishe reduction of photosynthesis, which is associated with a declinen the chlorophyll content (McLeod et al., 1999) that in turn reduceshe availability of carbohydrates. The growth and development ofew leaves in the Suncrest/S.4 did not change during the stresseriod, whereas the SPAD index underwent just a slight reduc-ion, and the TTC activity never dropped below 60% of the initialalue. Moreover, the high TTC levels observed in the roots could bettributed to the slight loss in oxygen supply, such that an activeetabolism might still occur.The reduced abscission of leaves, the greater capacity to retain
eaf greenness and the capacity to preserve mitochondrial activ-ty in Suncrest/S.4 plants confirm the hypothesis that exogenousarbohydrate supply becomes more stable in this respect to Sun-rest/wt plants (Perata et al., 1992; Loreti et al., 2005). In this work,e have observed that the root system of exposed plant combi-ations is preserved in short-term flooding experiments, probablyue an alternative mechanism for coping with low oxygen soilnvironments. However, it is important to note that we did notxamine the growth of plants under recovery from flooding stress,nd it is possible that the different tissue structures within roots
ay be disadvantaged when water is a more severe limiting fac-
or. Further studies may assess whether the S.4 clone has acquiredhenotypic plasticity that induces rapid changes in the shorterm.
ulturae 156 (2013) 1–8 7
In this work, it has been shown that between the two graftedbionts a positive cross talk occur which improves flooding toler-ance in the scion. Therefore, it may be that a transmissible signalacropetally moves up from the roots of the S.4 clone to the scionSuncrest, inducing adaptation to the low oxygen, which in turnactivates supply of O2 via the leaves. Plant growth behavior, mor-phology and few sign or even absence of visible abiotic stresssymptoms in Suncrest/S.4 plant parts such as leaves, stem androots, provide a clear indication of the induced tolerance to floodingstress in peach cultivar, with a high metabolic flexibility in supply-ing oxygen to the roots. This paper presents clear evidence that theS.4 clone can be considered as the rootstock plus variant againsthypoxia and anoxia, probably due to novel strategies of avoidanceand adaptation to oxygen deficits at the genetic, physiological andmorphological levels.
Acknowledgements
The University of Tuscia is gratefully acknowledged for financialsupport. We wish to thank Dr. Giuliano Dradi, Dr. Romano Ron-casaglia and Dr. Paolo Laghi of Battistini Vivai srl for the accuratepropagation of the plants of the rootstock Mr.S 2/5 and its variantclone S.4.
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