aba in roots and leaves of flooded pea plants

Journal of Experimental Botany, Vol. 38, No. 189, pp. 649-659, April 1987

ABA in Roots and Leaves of Flooded Pea Plants

JIANHUA ZHANG AND W. J. DAVIES1

Department of Biological Sciences, University of Lancaster, Bailrigg, Lancaster LAI 4YQ,U.K.

Received 9 September 1986

A B S T R A C TZhang, J. and Davies, W. J. 1987. ABA in roots and leaves of flooded pea plants.—J. exp. Bot. 38:649-659.

Roots of potted pea (Piston sativum L. cv. Feltham First) seedlings were flooded with tap water.Within a few hours of the start of the flooding treatment the content of free ABA in roots increasedcompared to contents of roots of unflooded control plants but this increase was not statisticallysignificant until the beginning of the second day after flooding. Approximately 36 h after first floodingsignificant increases in the free-ABA content of leaves were detected. This was 14 h after significantincreases in the amount of ABA in the roots of the same plants. There was marked diurnal variation infree-ABA content of leaves and roots of plants that had been flooded for several days, with maximumcontents recorded 3 h or more after the beginning of the light period. Very rapidly after the lights wereswitched oft ABA contents declined. On day 3 of the flooding treatment, there was more than a 5-folddecrease in the free-ABA content of leaves within a few hours of the beginning of the dark period.Radio-immunoassay suggested that a very large proportion of the total ABA in the plant was in abound form. This form of ABA increased substantially as the flooding period progressed.

The importance of variation in ABA content for the control of water relations and gas exchange offlooded plants is discussed.

Key words— Flooding, Pisum sativum, ABA, water relations.

Correspondence to: Department of Biological Sciences, University of Lancaster, Bailrigg, LancasterLAI 4YQ, U.K.

INTRODUCTIONIt is well-known that flooding can have marked effects on the growth and physiology ofplants. Careful observation shows that stomatal apertures and leaf growth rates may bereduced following inundation of the soil, even though leaf water potentials and turgors arenot significantly reduced (Pereira and Kozlowski, 1977; Jackson, Gales, and Campbell, 1978;Coutts, 1981; Bradford and Hsiao, 1982; Jackson and Kowalewska, 1983; Zhang and Davies,1986). There are several possible explanations for a chemical limitation of leaf growth andstomatal opening in flooded plants. In a previous paper investigating flooding effects onpea plants (Zhang and Davies, 1986), we discounted the importance of reduced cytokinintransport from flooded roots (Burrows and Carr, 1969) and suggested that under somecircumstances reduced uptake of potassium from flooded soils may restrict leaf growth andconductance of peas. Incubation of leaves from flooded plants in solutions of K.C1 causedstomata to reopen (Zhang and Davies, 1986). In that work, we were not able to discount thepossibility that stomatal opening was actually restricted by increased ABA content of leavesas it is known that a limitation of stomatal opening of Commelina by ABA can be overcome

1 To whom correspondence should be addressed.

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650 Zhang and Davies—ABA in Flooded Pea Plants

by increasing K+ concentration in the incubation medium (Wilson, Ogunkanmi, andMansfield, 1978; Snaith and Mansfield, 1982) and similar treatments can over-ride thelimiting effects of ABA on leaf growth of Phaseolus (Van Volkenburgh and Da vies, 1983).

There are a number of recent reports of increased ABA content in leaves of flooded plants(Hiron and Wright, 1973; Sivakumaran and Hall, 1978; Jackson, 1985; Wadman-van-Schravendijk and van Andel, 1985). The stimulus for this increase is apparently not always adecrease in leaf turgor (Jackson and Hall, 1987). Jackson (1985) and Jackson and Hall (1987)have suggested that ABA accumulating in leaves of flooded plants would normally betransported to roots but that transport out of the leaf is restricted as a result of flooding. It isimportant to substantiate this hypothesis.

This experiment was conducted to investigate further the basis of limited stomatal openingby flooded pea plants. ABA contents of leaves and roots were measured to try to gain someunderstanding of the plant's initial responses to inundation of the roots.

MATERIALS AND METHODSPea (Pisum sativum L. cv. Feltham First) seeds were germinated in moist sand and when the first leaveswere visible plants were transplanted into 95 mm diameter pots containing Levington compost. Plantswere grown for a further 2 weeks in a greenhouse at a minimum temperature of 25 °C. During this time,pots were watered daily to the drip point. When four leaves had developed, plants were moved to agrowth cabinet and allowed to acclimate for several days prior to the start of the experimentaltreatment (23 °C, 14 h day and 200 /anol nT2 s~* PAR). At this time, half of the plants were flooded byplacing pots inside plastic beakers filled with water to the level of the soil surface. Other plants remainedwell-watered during the period of the experiment.

In the first experiment leaf discs were removed from the third youngest leaves of control plants andincubated for manipulation of stomatal aperture using the technique described by Rodriguez andDavies (1982). Leaf discs were floated abaxial side down in 1 x 10~3 m"3 of 10 mol m~3 MES buffer,pH 6-15, with or without 10 mmol m"3 ABA. KC1 concentration in the buffer was in the range0-150 mol m"3.

Solutions were contained in 50 mm Petri dishes which were placed on a water bath at 25 °C andilluminated with a photon flux density of 130 /miol m~2 s~' (PAR). Illumination was provided withfluorescent and tungsten tubes located beneath the water bath. CO2-free air was bubbled through theincubation solutions at a rate of 6 x 10~3 m"3 min~'. After 4 h incubation, abaxial epidermis wasstripped from leaf discs and stomatal apertures were determined immediately under a projectionmicroscope.

In the second experiment, plants were flooded 2 h after the lights were switched on and leaves androots of four plants were sampled for ABA assay every 3 h for the next 4 d. Leaves (not includingaxillary stipules) were plunged immediately into liquid nitrogen while roots were washed and blotteddry before freezing. All samples were freeze-dried for at least 48 h and then stored in a desiccator forlater analysis. Determinations of free ABA in the plant were made with the gas chromatograph fittedwith the electron capture detector (GC-ECD). For each sample, approximately 100 mg dry weight ofplant material were used for one assay. Each sample was ultrasonified in 5 x 10 "6 m 90% acetone.From this extract, subsamples of 250-300 mm3 were loaded onto one T.L.C. plate and suspected ABAfrom this plate was methylated and redissolved in 100 mm3 cyclohexane. Extraction and purificationprocedures were as described by Quarrie (1978). The Pye Unicam 104 instrument proved sufficientlysensitive to detect 5-0 pg ABA and, therefore, only 1-0 mm3 of each sample was injected. A glass column(150 x 0-4 cm) containing 1-5% SE-30 on 80-100 mesh diatomite CQ was used. Column, injector anddetector oven temperatures were 230 °C, 245 °C and 250 °C respectively. Oxygen-free nitrogen was usedas the carrier gas at a rate of 40 x 10~6m3min~1 and detector purge gas at 20 x 10~6m3 min"1. Ethylabscisate was added as an internal standard and quantification was accomplished as described byQuarrie (1978). The recovery of ABA using this technique was consistently between 75-80%. ABAcontents presented are the result of at least duplicate assays.

Total (free plus bound) ABA content of leaves was determined using radio-immunoassay with anantibody developed against free and conjugated ABA (Zhou, Xu, and Cheng, 1985). This antibody,produced using the techniques described by Weiler (1979), was a gift from Professor Xie Zhou, NanjingAgricultural University, China. Tests against ABA metabolites show it to be specific to ABA and its



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Zhang and Davies—ABA in Flooded Pea Plants 651

t o

0.8

0.6

oCO

0.4

0 21 10

ABA pmol

10 1 0 0ABA omol

FIG 1 Standard curve for ABA determination by radio-immunoassay in a sigmoid and a linear plot.B = Antibody radioactivity in the presence of unlabelled antigen. Bo = Antibody rad.oactmty in the

absence of unlabelled antigen. Points are means ±s.e.

conjugates (X Zhou-paper in the press). The standard curve (Fig. 1) obtained with this antibodySonstrates its reliabSSS A 20-30 mm* aliquot from the acetone ABA extract: w», ptoced m a20 cm3 glass tube and evaporated down with N2 gas. 150 mm3 10 mol m 3 PBS buffer (100 mol mphosphate buffer, pH 7-4, containing 100 mol m'^NaCl, 0-1% NaN3 and 0-1% gelatin was added toS o l v e the res due on a rotary mUer for lmin. 100 mm3 of [3H](±) -ABA (about 10 pmol ABA,S oactiv ty 15 000 dpm) containing 0-25% BSA (bovine serum albumin) and 50 mm3 ant.serum at a

dilution of 1 1000 (with PBS buffer) were mixed into each tube. For the determination of unspeenficbinding, 50 mm3 water were added instead of antiserum. The tubes were incubated at 4 C in the darkfor 90 min a l then 400 mm3 saturated ammonium sulphate added to stop the reaction Tube, werethen centrifuged at 2 500 g for 15 min. Following this, 500 mm3 supernatant was taken and mixed with4 r S " S m f f l o n cocktail W (BDH). Tubes were placed in the dark for 1 h and then counted for5 rZ Counts were corrected for unspecific binding. Antigen responses were expressed as log t(B/Bo) = In ZB/Bo/(\-B/BoQ (Bo = precipitation radioactivity in the absence of unlabelled antigen5 = precipitation radioactivity in the presence of sample antigen or standard antigen^ ABAconcentrations were calculated according to the logit (B/Bo) versus log [ABA] linear regressionS a r d curve (Fig. 1). The counting efficiency was 45% ± 14%. All standards were assayed in triplicate

TSilT^Llt^ and unflooded pea plants was monitored continuously using aviscous flow porometer (Zhang and Da vies, 1986).

R E S U L T SFigure 2 shows the results of a simple preliminary experiment conducted to determinewhether the effect of increasing potassium concentration could over-ride the effects of ABAon the stomata of peas. It is clear that at KC1 concentrations above 120 mol m treatmentwith 10 mmol m ~3 ABA had no influence on stomatal aperture. In a previous paper (Zhangand Davies, 1986) we have shown that increasing KC1 concentrations in the incubationsolution around pea leaves could overcome a limitation in stomatal aperture imposed by



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flooding. This experiment suggests that such a limitation may have been caused by enhancedABA contents in the leaves.

GC analysis of ABA contents of leaves and roots over the first 4 d of the flooding periodshowed that flooding greatly enhanced the amount of free ABA found in both leaves androots (Fig. 3). Interestingly, small increases in ABA in roots were detectable within hours ofthe beginning of the flooding treatment (Fig. 3b) and these increases were statisticallysignificant by the beginning of the second day of treatment. There were no statisticallysignificant increases in free ABA in leaves of the same plants for another 14 h (Fig. 3a). Thisincrease occurred at the end of the second day of the experiment and ABA contents of floodedleaves then declined to the levels found in leaves of unflooded plants during the dark period.ABA contents of flooded roots were still significantly enhanced during this dark period.

Ea

re 4•»*IBEo

50 100KCI (mol m-3)

1 50

FIG. 1 Effect of increasing KCI concentration on the stomatal aperture of leaves of a pea plant incubatedwith ABA (10 mmol m"3) (• •) or without ABA (o o). Points are means ±s.e.

The amount of free ABA in leaves and roots of flooded peas varied enormously as afunction of time of day. After 3 d of flooding, ABA contents increased markedly within a fewhours of the beginning of the light period and decreased rapidly again when the lights wereswitched off. There was an approximately 5-fold variation in the ABA content of leaves offlooded pea plants between the days and nights of days 3 and 4 of the experimental period.Figure 4 shows detailed variation in the free ABA content of pea leaves in plants which hadbeen flooded for 10 d compared to leaves of plants which had been watered normally. Upper,growing leaves of normally watered plants contained more ABA (on a dry weight basis) thandid lower fully expanded leaves. Leaves from flooded plants showed relatively high ABAcontents during the light period.

The total ABA content of leaves and roots of pea plants which were watered normally was10 to 20 times higher than the content of free ABA (Fig. 5), presumably indicating that there



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Zhang and Dames—ABA in Flooded Pea Plants 653

4>U

O3

•ocou

0

120-

DaysFIG. 3. Effects of flooding on relative stomatal conductance and ABA content of leaves (a) and roots (b)of pea plants. Plants were first flooded at point shown by arrow. Stomatal conductance of control plants( ) and flooded plants ( ) was measured with a viscous flow porometer. ABA contents of floodedplants are shown by closed symbols and open symbob show ABA contents of control plants. Points are

means ± s.e. Dark bars indicate periods when lights are off.



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6S4 Zhang and Davies—ABA in Flooded Pea Plants

160 _

7:30 2130 0:30

FIG. 4. Diurnal variation in free ABA content of pea leaves either watered normally (• •) or kept floodedfor 10 d prior to ABA determinations (• o). Closed symbols indicate contents of lower, older leaves, opensymbols show contents of upper, growing leaves. Points are means ± s.e. Dark, horizontal bar shows the

dark period.

was a very large pool of conjugated ABA in the plant. Flooding caused a gradual butsubstantial increase in total ABA in shoots, while the content of total ABA in roots alsoincreased for 3 d after the plants were first flooded but then declined to pre-flooding levels. Atthis time most roots were almost dead and had apparently completely lost turgidity. About5 d after flooding, new adventitious roots were formed near the surface of the soil. There wasno easily-definable diurnal variation in total ABA contents of flooded plants.

Leaf conductance of pea plants was limited within a few hours of the flooding treatment(Fig. 3) and remained restricted, when compared to leaf conductance of control plants.

DISCUSSIONWe report here on substantial increases in the ABA content of leaves of peas rooted inwaterlogged soil. This accumulation, which can presumably account for some restriction in



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Zhang and Davies—ABA in Flooded Pea Plants

300 -

700

600

180

160

Days

FIG. 5. Total ABA contenu of leaves (a) and roots (b) of pea plants flooded on day 1 (indicated by arrow)(closed symbols) or watered normally (open symbols) for the duration of the experimental period. Points

are means ±s&. Dark bars indicate periods when lights are off.



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leaf conductance, occurred under conditions where there was no indication of thedevelopment of leaf water deficit (Zhang and Da vies, 1986). Jackson (1985) has reported asimilar result but other workers have linked increases in ABA content of flooded plants withan increase in leaf water deficit (Wadman-van-Schravendijk and van Andel, 1985). In ourexperiment, free ABA content of leaves was not enhanced for 36 h after the first waterloggingof the soil (Fig. 3) whereas some indications of enhanced ABA contents in roots of the sameplants were detected within a few hours of soil flooding and this increase was statisticallysignificant after 22 h (Fig. 3). Despite some reports to the contrary (Hartung, Gimmler, andHeilmann, 1982), it now seems that roots of some plants can produce increased amounts ofABA when stressed (Walton, Harrison, and Cote, 1976; Cornish and Zeevaart, 1985) and theresults described here suggest that increased production by roots may be among the plant'sfirst responses to waterlogging of the soil. We have noted that flooding reduces the turgor ofroots, presumably because of effects on membrane properties. It is possible that this providesthe stimulus for enhanced ABA synthesis.

In our previous work (Zhang and Da vies, 1986) we have confirmed the observation ofothers that flooding of the soil around pea roots results in restriction of stomatal openingwithin a few hours (Fig. 3). This restriction correlates in time with the first detectable increasein ABA content of the roots but ABA content of leaves is apparently not enhanced at thistime. The transpiration stream provides a direct link between roots and stomata since theepidermal cells adjacent to the guard cells and even the guard cell walls themselves areimportant evaporating sites (Meidner, 1975). Because the epidermis constitutes only a smallproportion of the total volume of the leaf, it is possible for the ABA content of the epidermisto rise substantially, presumably to influence stomata even though the ABA content of thewhole leaf shows no appreciable increase (Davies, Metcalfe, Schurr, Taylor, and Zhang,1987). ABA from the roots arrives in the apoplast immediately adjacent to the guard cells.Hartung (1983) has shown that this (the external surface of the plasmalemma) is the site ofaction for ABA in closing the stomata. ABA, therefore, may act as a direct link between theeffects of the environment on roots and the responses of the stomata.

Clearly, our observation that ABA contents of pea roots increase within a few hours ofinitial flooding and that this increase precedes any increase observed in the leaves conflictswith the suggestion of Jackson (1985) and Jackson and Hall (1987) that ABA accumulation inleaves of flooded plants arises as a result of reduced transport out of leaves. We propose thatroots themselves produce an increased amount of ABA immediately after first flooding andthat this ABA moves to the leaves as a signal of root perturbation. We cannot, however, ruleout the possibility that subsequently, some ABA accumulating in leaves does so as a result ofreduced transport to roots. Indeed this seems likely, since the total ABA content of roots doesdecline during the third day after first flooding (Fig. 5) probably reflecting the poor conditionof many roots at this time. We confirm the suggestion of Jackson and Hall (1987) that, inpeas, the stimulus for the build-up of ABA in leaves of flooded plants is not a decrease inleaf turgor.

One very interesting observation is the large diurnal fluctuation in free ABA in roots andleaves of flooded plants (Fig. 3a, b and Fig. 4). When the lights are switched off after 3 d offlooding there is a more than 5-fold decline in the free ABA content of leaves. It seems likelythat this rapid decline is the result of metabolism which is known to proceed more rapidly inthe dark than in the light (Zeevaart and Boyer, 1982). This may be because leaves in darknessproduce more ethylene than when in the light (Bassi and Spencer, 1982; Grodzinski, Boesel,and Horton, 1982). Presumably, high levels of ethylene will build up when stomata are closed.Of course ethylene production is particularly enhanced when plants are flooded (Jackson,1982). High ethylene production seems to promote ABA metabolism (Zeevaart and Boyer,



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1982) and, therefore, one might predict rapid metabolism in flooded plants in the dark whenstomata are closed.

It is clear from Fig. 4 that maximum free-ABA contents are not recorded in leaves offlooded plants until 3 h or more after the beginning of the light period. It is possible that thisis due to de novo synthesis although it is not clear why the rate of synthesis should be higher inthe light than in the dark. The substantial increase in free ABA content of leaves of floodedplants during the first part of each day may simply be caused by a decline in the rate of ABAmetabolism imposed upon a relatively high rate of synthesis which remains constant duringboth night and day. Because a very high proportion of total ABA apparently exists in abound form (Fig. 5) in flooded plants, it is tempting to suggest that free ABA might be derivedfrom the conjugated form. Zeevaart and Boyer (1982) suggest, however, that conjugation ofABA is irreversible although there is at least one suggestion in the literature that free-ABAmight be derived from a bound form (Weiler, Schnabl, and Hornberg, 1982). The fact that pealeaves and roots contain a very large amount of conjugated ABA relative to free-ABAconfirms results obtained by Weiler (1980) for Hyoscyamus niger. Apart from this result andthe general observation that total ABA increases in leaves and roots of flooded pea plants, itwould seem unwise to draw too many conclusions from data obtained from the RIA.Although the antibodies have been tested for cross-reactivity with many ABA metabolitesthey have not been tested with the glucose ester of ABA (ABA-GE). This is an importantmetabolic product of ABA and it is possible that the increase in total ABA in leaves and rootswhich seems to occur particularly in the dark does reflect some metabolism of free-ABA toABA-GE.

Large increases in free-ABA in leaves of flooded plants within hours of the beginning of thelight period may explain why leaf conductance apparently overshoots when stomata firstopen following lights-on (Zhang and Davies, 1986). Stomata open comparatively widelywithin minutes of the beginning of the light period and then close partially, presumably inresponse to accumulation of high ABA contents. This type of stomatal behaviour iscommonly seen in the field.

The important conclusions from the results presented here would seem to be that earlystomatal closure in response to flooding may result from ABA production in roots whichprecedes ABA increases in leaves by several hours. Subsequently, a very large amount of ABAaccumulates in leaves and roots of flooded plants and the stimulus for this increase in the rateof synthesis of ABA is not a decrease in leaf turgor. There is substantial diurnal variation inthe free-ABA content of leaves and roots.

In a previous paper we have suggested that stomatal opening in pea plants may be limitedby restricted uptake and transport of K+ to the leaves. This conclusion was reached partly asa result of the reversal of partial stomatal closure with incubation of leaves at high K+

concentrations. We have shown here that K+ application may simply be reversing anABA-imposed limitation in stomatal opening (Fig. 2). Despite the fact that ABA contents ofleaves are enhanced by flooding we cannot rule out the importance of effects on stomata ofreduced K+ concentrations in leaves, since these will undoubtedly enhance the sensitivity ofstomata to elevated ABA contents (Snaith and Mansfield, 1982).

ACKNOWLEDGEMENTWe are grateful to the Government of the People's Republic of China for providing funds tosupport the work of J.Z.



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L I T E R A T U R E CITEDBASSI, P. K., and SPENCER, M. S., 1982. Effect of carbon dioxide and light on ethylene production in

intact sunflower plants. Plant Physiology, 69, 1222-5.BRADFORD, K. J., and HSIAO, T. C , 1982. Stomatal behaviour and water relations of waterlogged

tomato plants. Ibid. 70, 1508-13.BURROWS, W. J., and CARR, D. T., 1969. Effect of flooding the root system of sunflower plants on

the cytokinin content of the xylem sap. Physiologia plantarum, 22, 1105-12.CORNISH, K., and ZEEVAART, J. A. D., 1985. Abscisic acid accumulation by roots of Xanthium

strumwium L. and Lycopersicon esculentum Mill, in relation to water stress. Plant Physiology, 79,653-8.

COUTTS, M. P., 1981. Effects of waterlogging on water relations of actively growing and dormantSitka spruce seedlings. Annals of Botany, 47, 747-53.

DAVIES, W. J., METCAUE, J. C , SCHURR, U., TAYLOR, G., and ZHANG, J., 1987. Hormones as chemical

signals involved in root-to-shoot communications of effects of changes in the soil environment.In Hormone action in plant development—a critical appraisal. Eds G. V. Hoad, J. R. Lenton,M. B. Jackson and R. Atkin. Butterworths, London (in press).

GRODZINSKI, B., BOESEL, I., and HORTON, R. F., 1982. Ethylene release from leaves of Xanthiumstrumarium L. and Zea mays L. Journal of Experimental Botany, 33, 344-54.

HARTUNG, W., 1983. The site of action of abscisic acid at the guard cell plasmalemma of Valerianellalocusta. Plant Cell and Environment, 6, 427-8.GIMMLER, H., and HEILMANN, B., 1982. The compartmentation of ABA-biosynthesis, ABA-metabolism and ABA conjugation. In Plant growth substances 1982. Ed. P. F. Wareing. AcademicPress, London. Pp. 325-33.

HIRON, R. W. P., and WRIGHT, S. T. C , 1973. The role of endogenous abscisic acid in the responseof plants to stress. Journal of Experimental Botany, 24, 769-81.

JACKSON, M. B., 1982. Ethylene as a growth promoting hormone under flooded conditions. In Plantgrowth substances. Ed. P. F. Wareing. Academic Press, London. Pp. 291-301.1985. Responses of leafed and leafless peas to soil waterlogging. In The pea crop. A basis forimprovement. Eds P. B. Hebblethwaite, M. C. Heath and T. C. K. Dawkins. Butterworths, London.Pp. 163-72.GALES, K., and CAMPBELL, D. J., 1978. Effect of waterlogged soil conditions on the productionof ethylene and on water relationships in tomato plants. Journal of Experimental Botany, 29,183-93.and HALL, K. C , 1987. Early stomatal closure in waterlogged pea plants is mediated by abscisicacid in the absence of foliar water deficits. Plant, Cell and Environment (in press).and KOWALEWSKA, A. K. B., 1983. Positive and negative messages from roots induce foliardesiccation and stomatal closure in flooded pea plants. Journal of Experimental Botany, 34,493-506.

MEIDNER, H., 1975. Water supply, evaporation and vapour diffusion in leaves. Ibid. 26, 666-73.PEREIRA, J. S., and KOZLOWSKJ, T. T., 1977. Variations among woody angiosperms in response to

flooding. Physiologia plantarum, 41, 184-92.QUARRIE, S. A., 1978. A rapid and sensitive assay for abscisic acid using ethyl abscisate as an internal

standard. Analytical Biochemistry, 87, 148-56.RODRIGUEZ, J. L., and DAVIES, W. J., 1982. The effects of temperature and ABA on stomata of Zea

mays L. Journal of Experimental Botany, 33, 977-87.SIVAKUMARAN, S., and HALL, M. A., 1978. Effects of age and water stress on endogenous levels of

plant growth regulators in Euphorbia lathyrus L. Ibid. 29, 195-205.SNAJTH, P. J., and MANSFIELD, T. A., 1982. Stomatal sensitivity to abscisic acid: can it be defined?

Plant, Cell and Environment, 5, 309-11.VAN VOLKENBURGH, E., and DAVIES, W. J., 1983. Inhibition of light-stimulated leaf expansion by

abscisic acid. Journal of Experimental Botany, 34, 835-45.WADMAN-VAN-SCHRAVENDIJK, W., and VAN ANDEL, O. M., 1985. Interdependence of growth, water

relations and abscisic acid level in Phaseolus vulgaris during waterlogging. Physiologia plantarum,63, 215-20.

WALTON, D. C , HARRISON, M. A., and COTE, P., 1976. The effects of water stress on abscisic acidlevels and metabolism in roots of Phaseolus vulgaris L. and other plants. Planta, 47, 595-602.

WHLER, E. W., 1979. Radio-immunoassay for the determination of free and conjugated abscisic acid.Ibid. 144, 255-63.



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1980. Radio-immunoassays for the differential and direct analysis of free and conjugated abscisicacid in plant extracts. Ibid. 148, 262-72.SCHNABL, H., and HORNBERG, C., 1982. Stress-related levels of abscisic acid in guard cell proto-plasts of Viciafaba L. Ibid. 154, 24-8.

WILSON, J. A., OGUNKANMI, A. B., and MANSFIELD, T. A., 1978. Effects of external potassium supplyon stomatal closure induced by abscisic acid. Plant, Cell and Environment, 1, 199-201.

ZEEVAART, J. A. D., and BOYER, G. L., 1982. Metabolism of abscisic acid in Xanthium strumariumand Ricinus communis. In Plant growth substances 1982. Ed. P. F. Wareing. Academic Press,London. Pp. 335-42.

ZHANG, J., and D A VIES, W. J., 1986. Chemical and hydraulic influences on the stomata of floodedplants. Journal of Experimental Botany, 37, 1479-91.

ZHOU, X., Xu, Y.-J., and CHENG, W.-F., 1985. A radio-immunoassay for abscisic acid. Journal ofNanjing Agricultural University, 1, 89-91.



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