Plant Growth Regulation28: 187–197, 1999.© 1999Kluwer Academic Publishers. Printed in the Netherlands.

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Effects of abscisic acid (ABA) on grain filling processes in wheat

A. Ahmadi∗ & D.A. Baker∗∗Department of Biological Sciences, Wye College, University of London, Wye, Ashford, Kent, TN25 5AH,U.K. (∗Present address: Department of Agronomy and Plant Breeding, College of Agriculture, University ofTehran, Karaj, Iran (∗∗Author for correspondence: phone: +44 1233812401; fax: +44 1233813140; e-mail:d.baker@wye.ac.uk)

Received 29 December 1998; accepted in revised form 5 June 1999

Key words:ABA, grain filling, water stress, wheat

Abstract

The effect ofin situ water stress on the endogenous abscisic acid (ABA) content of the endosperm and thein vitroapplication of ABA on some important yield regulating processes in wheat have been studied. Water stress resultedin a marked increase in the ABA content of the endosperm at the time close to cessation of growth. Applicationof ABA to the culture medium of detached ears reduced grain weight. Exogenously applied ABA, at the highestconcentration (0.1 mM) reduced transport of sucrose into the grains and lowered the starch synthesis ability ofintact grains.In vitro sucrose uptake and conversion by isolated grains was stimulated by low ABA concentrations(0.001 mM) in the medium but was inhibited by higher concentrations. ABA application had no effect on sucrosesynthase (SS) and uridine diphosphate glucose pyrophosphorylase (UDP-Gppase) activities, whereas adenosinediphosphate glucose pyrophosphorylase (ADP-Gppase), soluble starch synthase (SSS), and granule-bound starchsynthase (GBSS) activities were reduced. These results raise the possibility that water stress-induced elevatedlevels of endogenous ABA contribute to reduced grain growth.

Abbreviations:ABA – abscisic acid; ADP-Gppase – adenosine diphosphate glucose pyrophosphorylase; ES –ethanol soluble; EINS – ethanol insoluble; UDP-Gppase – uridine diphosphate glucose pyrophosphorylase; SS –sucrose synthase; SSS – soluble starch synthase; GBSS – granule-bound starch synthase

1. Introduction

The adverse effect of water stress on grain yield incereals is well documented. Depending on the stageof development, this effect can be via reduced grainnumber or grain weight, the latter being due to adecreased rate and/or duration of dry matter accumu-lation. The rate of dry matter accumulation (importrate) by grain (i.e. sink) is determined by sink strength[17] which is a product of sink size, determined byendosperm cell number, and sink activity.

Abscisic acid (ABA), generally regarded as aninhibitory growth hormone [39, 43], increasesmarkedly in leaves [26, 47], floral organs [32, 47] anddeveloping grains [14, 15, 25] of water stressed plants.Water stress-induced reduction in grain set in wheat

[23, 32] and a decreased rate of endosperm cell divi-sion in water stressed maize [24, 25] have been attrib-uted to elevated levels of ABA. However, observedeffects of ABA on the capacity of the sink to accumu-late dry matter are not consistent; both inhibition andstimulation have been reported. The increase in ABAlevels towards the end of grain filling and its rapidfall during maturation have raised questions about therole of ABA in controlling dry matter accumulation[3, 18, 20]. Indeed in several cases the applicationof ABA to the medium has enhanced accumulation ofreserves, particularly storage proteins in legumes, andproduction of mRNA [18]. Stimulatory effects of ABAon assimilate unloading [33,39] andin vitro sucroseuptake [6, 33] have also been reported. Nevertheless,these promoting effects are not always observed, and

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depending on the concentration and the timing of ABAapplication, inhibitory effects may predominate. It hasbeen reported that on media with low ABA concentra-tions, the sugar consumption by carrot embryos wasrelatively slow, and on a high ABA medium sugaruptake completely ceased [40]. Earlier grain matura-tion in wheat ears treated with ABA during late graindevelopment [19] and partial inhibition of the stim-ulatory effects of ABA on transport of14C-sucrose,number and mass of kernels following ABA appli-cation to the culture medium of detached wheat ears[4] have been reported. Reduced grain yield in waterstressed wheat has been attributed to an increasedABA content and decreased cytokinin levels in thegrains [44]. It has been proposed that ABA reducesgrain yield by sealing the conducting tissues, acceler-ating grain water loss and affecting starch synthesizingenzymes [29].

Apart from the above reports on the effects ofABA application on grain growth and assimilate trans-port in various plants, there are no other detailed orsystematic studies on the effects of ABA on yield regu-lating processes, particularly those involved with sinkactivity and the starch synthesis process, in wheat. Theobjective of the present investigation was to elucidatethe role of ABA in influencing some important sinkrelated yield regulating factors.

2. Materials and methods

2.1 Plant material and water stress imposition

Spring wheat plants (Triticum aestivumvar. Cadenza)were grown in a glasshouse under natural light supple-mented to provide 300µmol m−2 s−1 PAR at plantheight by 400W mercury vapour lamps extending thelight period to 16 h. Plants were watered twice dailyand supplied with 1 g l−1 Sangral 211 fertiliser weeklythrough watering. The plants were then transferredto a growth chamber at the early jointing stage. Airtemperature was held at 20/15◦C during the light/darkperiods and air relative humidity was 60 to 70%. Aphotoperiod of 16h was obtained with a mixture of400W Sodium and Halide lamps supplemented with25W tungsten lamps providing 850µmol m−2 s−1

PAR at ear level. Plants were watered daily to nearfield capacity and supplied with 1 g l−1 Sangral 211fertiliser twice weekly through watering.

Water was withheld from treated pots and the soilwater content allowed to fall to 15% of field capacity at

15 days after anthesis and the pots then weighed everyday. Sufficient water was applied on each occasionto return the soil moisture to these original levels. Incontrol treatments the soil water status was maintainedat 50% field capacity by weighing the pots every dayand adding sufficient water to bring the soil moistureto its original value. Preliminary experiments showedthat compost moistures above 15% had no detrimentaleffects on the plants.

2.2 Abscisic acid (ABA) assay

Grains were sampled at 15, 23 and 30 days afteranthesis (d.a.a.) from control and water stressedplants. 6 grains were removed, immediately frozenin liquid nitrogen and kept at−20 ◦C for the assay.Corresponding grains were used to estimate graindry weight. The outer pericarp, embryo and thetissue making up the furrow were removed fromfrozen grains. These peeled grains were weighed andhomogenised with a pestle and mortar in 1 ml coldextraction solvent (80% methanol containing 10 mg/lbutylated hydroxy toluene, pH 7.0). Homogenateswere kept on crushed ice, extracted on a shaker for24 h in the dark and centrifuged at 10000 rpm for 10min. The pellet was washed with an additional oneml extraction solvent and centrifuged as above. Super-natants were combined and evaporated under vacuumto remove methanol. Volumes of all samples werereadjusted to 2 ml with Tris saline buffer, TBS, (25mM Tris, 150 mM NaCl, and 2 mM MgCl2, pH 7.5)and used for the assay.

ABA was analysed by an indirect enzyme-linkedassay (ELISA) employing a commercially availablePhytodetek assay Kit (Idetek) supplied by SigmaLtd. Colour absorbency following reaction with thesubstrate was read at 405 nm using a microplateauto reader (EL 311 Bio-Tek instruments). Percentagebinding was calculated using established procedures[33].

2.3 Detached ear culture

At 16 or 18 days after anthesis uniform ears were cutbelow the penultimate node and divided into groups(treatments) of four replicates. The flag leaves wereremoved, the stem of the detached ears’ surface steril-ised with 10% sodium hypochlorite and recut understerilised distilled water 2 cm below the flag leaf node[34]. The explants were then placed in sterilised 10× 2.5 glass vessels containing culture medium [12]and sealed with cotton wool. In the culture medium

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glutamine (0.02 M) was used as the sole source ofnitrogen and the sucrose concentration was 40 g l−1

[1]. Abscisic acid (+,−, cis, trans, Sigma Chem-ical Ltd) was added to the culture medium at 0.1and 0.001 mM. The cultured ears were maintainedat 20/15 ◦C day/night temperature in a controlledenvironment cabinet with air relative humidity of 55 to60%. A photoperiod of 16 h was provided by a mixtureof sodium and tungsten lamps providing 180µmolm−2 s−1 PAR at ear level. The media vessels werecovered with aluminium foil and, to reduce micro-bial contamination, immersed in a shallow water bathsystem maintained at 3–5◦C. Ears were cultured for10 days (from 16 or 18 d.a.a to 26 or 28 d.a.a). Mediawere changed after 4 days and consumption measuredvolumetrically.

2.4 Grain growth,14C sucrose import anddetermination of starch synthesis

After four days, media were replaced with 25 ml [U-14C] sucrose labelled media (7.8125µCi g−1sucroseradioactive concentration). The following treatmentswere employed: control, continuous ABA application(0.001 and 0.1 mM), pre-treated with ABA (0.1 mM)during the first half of the culture period followed byno hormone in the media. At the beginning of theexperiment 8 grains from one side of each spike weresampled and used for initial grain dry weight. Afterterminating the experiment, 8 other grains were usedto determine grain fresh weight, water content anddry weight. 6 grains from the opposite side of the earwere frozen in liquid nitrogen and freeze dried. Thesefreeze-dried grains were then used to estimate [14C]sucrose import and [14C] starch synthesis by intactgrains. The proportion of radioactivity of the kernelsin the ES and EINS (starch) fractions was determinedas described by Felker et al. [13].

2.5 Determination ofin vitro sucrose uptake andconversion

Twenty grains were removed from the middle of eachear from well-watered plants, mixed and divided intofour groups of five and each group allocated to onetreatment within each replicate. Grains were halvedtransversely and incubated in labelled sucrose solu-tions for 24 h at 25◦C. Ten mM (±) 2-cis, 4-transABA stock solution was prepared in Taps buffer (pH9.0) and the pH adjusted to 8.5. Following preliminaryexperiments with peeled grains, three concentrationsof 0.1, 0.01, and 0.001 mM ABA were employed.

After incubation the grains were rinsed and placedin 25 ml conical flasks each containing 5 ml ofdeionised water, the containers were embedded incrushed ice and then shaken gently. The rinsing waterwas replaced after 30 minutes and after a further 60minutes with fresh water. The fractions remaining inthe tissue after this rinsing treatment were presumedto be located within the symplastic compartment. Theamount of14C-sucrose taken up into ES and EINS(starch) was determined as above.

2.6 Enzyme assay

Kernels from one side of detached ears treated withzero, 0.001 and 0.1 mM ABA were used for grain dryweight and water content estimation. Ten grains fromthe other side of the ear were sampled, frozen imme-diately in liquid nitrogen and maintained at−80 ◦Cfor enzyme assays; five grains were used for solubleand granule-bound starch synthases (SSS and GBSS,respectively) and the other 5 grains were assayed forsucrose synthase (SS), ADP-G and UDP-G pyrophos-phorylase (ADP-Gppase and UDP-Gppase, respec-tively) activities. Enzymes were extracted as describedby Hawker and Jenner [16]. For GBSS, the pelletobtained from centrifugation was resuspended in 1.5ml extraction medium (EM), filtered through two layerof muslin and washed twice with EM. The final pelletwas suspended in 1 ml EM and used for assay. SSSactivity was assayed after using the anion exchangeresin described in [16]. 1.5 ml cocktail T with aToluene/Triton×-100 base along with 400 ml distilledwater were added to the column eluent and counted byliquid scintillation spectrometry. GBSS was assayedby the method employed for SSS except that thereaction was terminated with cold methanol-KCl andstarch was precipitated by the addition of solid carrierstarch and centrifugation. The pellet was washed twicewith EM and distilled water and the final pellet resus-pended in distilled water and counted as describedfor SSS. The activity of SS was determined in thedirection of sucrose-dependent UDP-glucose forma-tion [22]. PPi dependent activities of ADP-Gppase andUDP-Gppase were assayed spectrophotometrically byincubating ADP (or UDP)-glucose and PPi with thesupernatant enzyme preparation [30].

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Table 1. Influence of ABA in the culture medium on the volumeof medium absorbed by detached ears, grain final dry weight(dw), water content and grain dry weight gain over the cultureperiod. Values are the means of four replicates± SEM. Abbre-viations: c, continuous presence of ABA;p, pre-treated withABA

Treatments Medium used mg.grain−1

ABA (mM) (ml. day−1) dw water dw.gain

Control 14.2 43.7± 0.94 54.6± 2.4 17.50± 0.640.001c 13.75 43.6± 0.47 56.62± 0.64 16.98± 0.690.1c 11.65 42.5± 0.81 58.85± 2.3 15.74± 1.160.1p 11.75 42.3± 0.48 53.36± 1.4 16.30± 0.75

3. Results

3.1 Effect of long termin situwater stress on abscisicacid (ABA) content of the endosperm

A simplified ABA extraction procedure omitting thepurification stage [37] minimised ABA loss and thusthe estimate of ABA recovery was omitted. Datareported here are not corrected for ABA loss whichwould have been relatively small. Under water-limited conditions, ABA content remained unchangedbetween days 15 and 23, but rose substantially by day30 (Figure 1). A comparison between the ABA contentof water stressed grains and that of the control clearlyindicated the response of this hormone to water stress.At 15 d.a.a, the effect of stress on the hormone levelwas barely detectable but by day 23 the ABA contentof the stressed grains increased significantly. With 30days water stress a further increase in the ABA contentof the grain was observed.

3.2 Effects of ABA in the culture medium of detachedears on grain growth and water content,14C-sucroseimport and starch synthesis

The rate of consumption of the medium per day wasnot affected by 0.001 mM ABA, whereas in the pres-ence of 0.1 mM ABA consumption of the mediumshowed an 18% reduction compared with that of thecontrol. Grain dry weight showed a small but insignifi-cant reduction over the 10 day period of culture, in thepresence of 0.1 mM ABA compared with the control.In contrast to grain dry weight the grain water contentwas highest under this treatment (Table 1).

Application of ABA to the medium tended toinhibit the transport of sucrose into the grains. Theeffect was statistically significant when ABA wasused at 0.1 mM continuously throughout the culture

period. Sucrose import by grains was also notice-ably, although not significantly, inhibited in the earswhich had been pre-treated with ABA during thefirst half of the culture and then transferred to thelabelled medium without ABA (Figure 2(A)). Thepartitioning of the imported labelled sucrose by intactgrains into ES and EINS (starch) was also affected by0.1 mM ABA, the inhibitory effect being greater withcontinuous hormone application than when pre-treated(Figure 2(B & C)). The continuous presence of ABAresulted in a significant reduction in the conversionof labelled sucrose to starch (EINS). For both sucroseimport and its partitioning to starch, when results wereexpressed on a grain dry weight basis the same patternof response to treatments was evident. The percentageof conversion, i.e., starch synthesis efficiency, was alsoslightly reduced by continuous treatment with 0.1 mMABA (Figure 2(D)).

3.3 In vitrosucrose uptake and conversion byisolated grains in the presence of ABA

Total sucrose uptake was stimulated by the low ABAconcentration (i.e. optimum concentration) but inhib-ited by higher ABA concentrations; the higher theABA concentration, the greater the inhibition (Table2). Increases in ABA concentration caused a signifi-cant reduction in the total uptake relative to theoptimum uptake. The inhibitory effects of ABA (0.1mM) on the partitioning of labelled sucrose into EINS(starch) was greater than into ES. The decline inboth ES and EINS at higher ABA concentrationswas statistically significant when compared with theircounterparts at the optimum ABA concentration. Thepercentage conversion, as a proportion of total uptake,was also reduced at the high ABA concentration indi-cating a direct effect of ABA on the starch synthesisprocesses.

3.4 Effect of ABA on the activity of the enzymesinvolved in the sucrose to starch pathway

There was no effect of hormone application to theculture medium of detached ears on the SS activityand the response of UDP-Gppase activity to hormoneapplication was a slight reduction (Figure 3). ABAapplication had a significant effect on the ADP-Gppase activity, on a grain unit, grain fw, and dwbasis, the effect being doubled (on a grain basis) whenthe hormone concentration in the culture mediumincreased from 0.001 mM to 0.1 mM (Figure 4). A

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Figure 1. Effect of water stress on the abscisic acid (ABA) content of wheat endosperm. water stress commenced 10 days after anthesis at 15%of field capacity level. (A) expressed as ng.grain−1 and (B) expresed as ng.g−1 dw. Data are means of four replicates each consisting of sixgrains from the middle part of each ear. Bars indicate SEM; solid columns, control; open columns, water stressed.

Table 2. Effects of ABA application on totalin vitro sucrose uptake,expressed asµg equivalent sucrose grain−1 h−1, and percentageconversion into EINS (A) and the partitioning of sucrose taken upinto ES and EINS fractions (B). Halved grains were incubated for24 h at 25◦C in labelled sucrose solutions containing different ABAconcentrations. Values are means of four replicates± SEM. Meansfollowed by the same letter are not statistically different

A:

Treatments Total % Decrease (−)/ % ConversionABA (mM) uptake increase(+)

Control 31.37± 1.64ab 0.00 280.1 26.36± 1.61bc −16 250.01 27.34± 1.87bc −13 280.001 36.67± 0.42a +17 27P values 0.002

B:

Treatments ES EIN % Decrease (−)ABA (mM) ES EIN

Control 22.72± 1.09ab 8.65± 0.744ab 0.00 0.00.1 19.66± 1.25bc 6.70± 0.643b −14 −220.01 19.71± 1.21bc 7.60± 0.686ab −13 −120.001 26.76± 0.47a 9.91± 0.539a +18 +15P values 0.001 0.026

nonsignificant reduction of 18% (across ABA treat-ments), on a grain basis, was observed in the activityof GBSS of grains from ABA treated ears (Figure 4).The response of SSS was similar to that of the ADP-Gppase in that the reduction in the enzyme activity

became greater when ABA concentration increasedfrom 0.001 mM to 0.1mM. This pattern of responsewas evident on both a grain dw and fw basis (Figure4).

4. Discussion

The increase in endosperm ABA content observedhere as a result ofin situwater stress confirms previousreports for wheat, barley and maize [13, 15, 25, 26].ABA in the grain may result from autosynthesis withinthe grain [20], and partly by translocation from leavesand/or roots [25, 26]. Since water stress increasesthe ABA level of the flag leaf [10] which providesassimilates via the phloem to the grain and ABA ishighly mobile in the phloem, translocation of ABA inthe phloem could have contributed to the higher ABAcontent of grains in the stress treatment.

The ABA level of water stressed grains showeda substantial increase at 30 d.a.a. when grainsapproached their maximum dry weight. Althoughthe ABA level of control grains remained almostunchanged, that of the stressed grains was consistentwith the previously reported pattern of ABA changesduring late grain fill under stress conditions [20, 29,31]. Although an increase in grain ABA content withan increase in grain weight is taken as an indica-tion of the involvement of ABA in grain development[3, 18, 20], there is evidence implying an inhibitory

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Figure 2. Effects of ABA at 0.001 and 0.1 mM on sucrose import (A), and its partitioning into ethanol soluble (B) and ethanol insoluble (C),and the percentage of conversion (D) by intact grains of detached ears. Data, expressed on a grain (top panels) and grain dry weight (bottompanels) basis, are means of four replicates. Bar marks indicate SEM. Abbreviations:C, continuous presence of ABA in the culture medium;P,pre-treated with ABA during the first half of the culture period followed by no hormone in the medium.

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Figure 3. Effects of ABA at 0.001 and 0.1 mM on SS (top panels) and UDP-Gppase (bottom panels) activities, expressed on a grain (A), grainfresh weight (B), and dry weight (C) basis of grains from detached ears. Data are means of four replicates each consisting of five grains. Barsindicate SEM.

effect of ABA on grain growth [19, 20, 29, 45].However, attributing the reduced grain weight underin situ water stress conditions to an elevated level ofABA may not be sufficiently convincing as other waterstress-induced factors could be responsible.

Addition of ABA into the culture medium ofdetached ears during rapid grain fill resulted in a slightreduction in medium consumption and an increase ingrain water content implying a closure of stomata inthe ear. Despite the availability of substrate and water,ABA caused reduced grain growth. In detached earsof wheat cultured in sucrose solutions exogenouslyapplied ABA inhibited grain growth [20] and the endo-sperm of ABA-treated maize kernels had a reduced dryweight [24]. Similarly addition of ABA to detachedcultured ears of wheat prior to anthesis resulted in asignificant reduction in grain weight [4]. Reductionsin the grain weight of maize and wheat kernels couldhave been by reduction in endosperm cell number, thuslimiting the maximal storage capacity of the kernels.In the present study ears were cultured after the celldivision period and thus any reduction in grain dryweight resulting from fewer cells was ruled out, and

the results here indicate the inhibitory effect of ABAon reserve deposition processes only.

The concentrations of applied ABA were consid-erably higher than estimated concentrations of ABAin water stressed grains to compensate for the rateof ABA degradation in the culture medium and therapid ABA catabolism within the plants. Myers etal. [24], reported that over 5 days, 260 pmol ofABA was transported into the endosperm, yet kernelscultured in ABA accumulated only 11 pmol. Thus,the limited effect of ABA on grain growth possiblyreflects an inability of the kernel to establish high ABAconcentrations in the endosperm.

There are several potential sites of action for ABAto inhibit grain growth. The most important of these,at least in detached ear culture systems can be thesites of unloading of assimilates from sieve tubes, thesite of assimilate uptake by endosperm cells and theconversion of the sucrose taken up by the endospermcells to form starch. ABA is reported to have a stim-ulatory effect on the unloading processes rather thanan inhibitory one [33, 39]. The decreased percentageof conversion in ABA treated grains observed here

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Figure 4. Effects of ABA at 0.001 and 0.1 mM on ADP-Gppase (top panels) GBSS (middle panels), and SSS (bottom panels) activities,expressed on a grain (A), grain fresh weight (B), and dry weight (C) basis of grains from detached ears. Data are means of four replicates eachconsisting of five grains. Bars indicate SEM.

provides evidence of a direct ABA effect on theprocesses converting sucrose to starch within theendosperm. Alternatively, the reduced import oflabelled sucrose into ABA treated kernels might beattributed to a decreased transport of solutes in thexylem, as a result of decreased transpiration by theears [46]. However, this did not seem to be the maincause of the reduced import observed here as evenwhen pre-treated with ABA, import of labelled sucroseinto the kernels was reduced.

A slight stimulatory effect of low ABA concentra-tions and an inhibitory effect of high ABA concen-trations on total sucrose uptake by halved grainswas observed. Such a concentration dependent stim-

ulatory/inhibitory effect of ABA has been reported onassimilate import by barley grains [42] and growth oftomato plants [38]. The effect of ABA on total sucroseuptake can be either direct, acting via the uptakemechanism, or indirect by altering sucrose conversioninside the cells and thus controlling the sucrose move-ment through diffusional mechanisms (or both). ABAstimulates the accumulation of sucrose in a range oftissues and a correlation has been observed betweendry matter accumulation and endogenous ABA levelsin the sink regions of some plant species [41].In vitrosucrose uptake by soybean cotyledons, for instance,was enhanced by low ABA concentrations and wascorrelated with the endogenous ABA levels [33].

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Soybean cotyledons fromin situ water stressed plantshad a higher ABA concentration and took up sucroseat a higher rate compared with those from well wateredplants with a lower ABA content [6].

Increased ABA concentrations in the mediumreduced sucrose uptake and, since the percentage ofconversion of sucrose was also reduced, the lowerconsumption of labelled sucrose taken up by thecells reduced diffusional movement. The stimulatoryeffects of ABA application into the culture mediumof soybean cotyledons disappeared when endogenousABA content of the cotyledons increased due toinsitu growth conditions [33]. If ABA decreases theATPase proton extrusion [39] a reduced proton co-transport of sucrose into the endosperm cells wouldresult. In developing carrot embryos high concen-trations of ABA stopped sugar uptake and reducedrespiration [40].

Apart from the effect at the site of uptake, ABA athigh concentrations reduced the conversion of sucroseto starch whereas at low concentrations such an effectwas not evident. The site of ABA action in this respectappears to be one or more of the key enzymes of starchsynthesis [29]. The inhibitory effects of ABA on thegrowth of isolated wheat kernels observed here and ofmaize kernels [24] imply a specific role for ABA ingrowth and related regulatory parameters and not inassimilate import.

Among the enzymes of starch synthesis, ADP-Gppase in maize [11], GBSS [7] and SSS [16]in wheat are considered as the most important ratelimiting enzymes. Reduced activity of ADP-Gppasehas been reported to be one possible importantfactor involved in growth cessation in wheat [7].The inhibitory effect of ABA on any one of theseenzymes would provide a convincing explanation forthe effect of hormone application in reducing grainyield. Adverse effects of ABA on SS activity andgrowth level as well as on soluble protein content inpotato cell culture [36], invertase, SS and SPS activ-ities and growth rate of chicory suspension [9] andF1,6 Bpase activity in soybean [8] have been reported.In rice kernels ABA completely inhibitedβ-amylaseactivity, corresponding with a complete inhibition ofβ-amylase synthesis indicating that the ABA inhibi-tion of enzymes was not a result of enzyme inacti-vation [44]. The level of enzyme mRNA correlatedpositively with the level of enzyme protein. Similarly,inhibitory effects of ABA at the transcript level werereported for nitrate reductase of barley plants [21].ABA may inhibit enzyme synthesis by blocking the

transcription or alternatively it may be possible that theinhibition of enzymes is post-transcriptionally regu-lated; it may be that an ABA-induced ribonucleasedecreases enzyme mRNA [44]. ABA may exert itseffect on enzyme protein synthesis at the translationlevel by interfering with the polysome content of thecell. Application of ABA to well-watered seedlings ofsoybean reduced growth rate and polysome content ofthe hypocotyl [2].

Endogenous ABA levels increase with water stressand exogenously supplied ABA mimics many of theplant responses to water stress. Genes that are simi-larly regulated – by drought and exogenous ABA havebeen identified [5, 35]. Genes encoding enzymes ofcarbohydrate metabolism may respond similarly toboth water stress and ABA application. F1,6 Bpaseactivity was reduced by both water stress and ABAapplication [8].

The relationship between ABA content and yieldprobably has an optimum ABA content which islikely to differ for each environment and crop. Belowtheir optima, increases in ABA content might bereflected in higher yields, but too much ABA perhapsadversely affects grain filling and reduces yield [27].The positive relationship between grain growthin vivoand grain ABA content is likely to be via the effectof ABA on stomatal aperture, enhancing water useefficiency (WUE) and leading to a better yield underfield conditions. The negative response to ABA mayreflect adverse effects on source activity (e.g. reducingcurrent photosynthesis and accelerating senescence)and a decreased sink ability to attract and utilize avail-able carbohydrates. The maximum ABA content ofwheat grains [28] is reported to be approximately1000-fold lower than the levels reported for legumes[20]. This implies that wheat grains are better adaptedto low ABA than legumes and thus the optimum ABAcontent may be achieved at lower values. IncreasedABA levels under water stress conditions are likelyto be above this optimum and thus negatively affectphysiological processes including grain fill.

Acknowledgement

This research was supported by a postgraduate schol-arship provided by the Ministry of Culture and HigherEducation of the Islamic Republic of Iran

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