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

ABSTRACT Zhang, 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 increased compared to contents of roots of unflooded control plants but this increase was not statistically significant until the beginning of the second day after flooding. Approximately 36 h after first flooding significant increases in the free-ABA content of leaves were detected. This was 14 h after significant increases in the amount of ABA in the roots of the same plants. There was marked diurnal variation in free-ABA content of leaves and roots of plants that had been flooded for several days, with maximum contents recorded 3 h or more after the beginning of the light period. Very rapidly after the lights were switched oft ABA contents declined. On day 3 of the flooding treatment, there was more than a 5-fold decrease 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 a bound 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 of flooded plants is discussed. Key words Flooding, Pisum sativum, ABA, water relations. Correspondence to: Department of Biological Sciences, University of Lancaster, Bailrigg, Lancaster LAI 4YQ, U.K.

Downloaded from jxb.oxfordjournals.org at Lancaster University on August 30, 2011

INTRODUCTION It is well-known that flooding can have marked effects on the growth and physiology of plants. Careful observation shows that stomatal apertures and leaf growth rates may be reduced following inundation of the soil, even though leaf water potentials and turgors are not 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 and stomatal opening in flooded plants. In a previous paper investigating flooding effects on pea plants (Zhang and Davies, 1986), we discounted the importance of reduced cytokinin transport from flooded roots (Burrows and Carr, 1969) and suggested that under some circumstances reduced uptake of potassium from flooded soils may restrict leaf growth and conductance of peas. Incubation of leaves from flooded plants in solutions of K.C1 caused stomata to reopen (Zhang and Davies, 1986). In that work, we were not able to discount the possibility that stomatal opening was actually restricted by increased ABA content of leaves as it is known that a limitation of stomatal opening of Commelina by ABA can be overcome
1

To whom correspondence should be addressed.

Oxford University Press 1987

650
+

Zhang and DaviesABA in Flooded Pea Plants

by increasing K concentration in the incubation medium (Wilson, Ogunkanmi, and Mansfield, 1978; Snaith and Mansfield, 1982) and similar treatments can over-ride the limiting 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-vanSchravendijk and van Andel, 1985). The stimulus for this increase is apparently not always a decrease 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 be transported to roots but that transport out of the leaf is restricted as a result offlooding.It is important to substantiate this hypothesis. This experiment was conducted to investigate further the basis of limited stomatal opening by flooded pea plants. ABA contents of leaves and roots were measured to try to gain some understanding of the plant's initial responses to inundation of the roots. MATERIALS AND METHODS Pea (Pisum sativum L. cv. Feltham First) seeds were germinated in moist sand and when thefirstleaves were visible plants were transplanted into 95 mm diameter pots containing Levington compost. Plants were 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 a growth cabinet and allowed to acclimate for several days prior to the start of the experimental treatment (23 C, 14 h day and 200 /anol nT 2 s~* PAR). At this time, half of the plants werefloodedby placing pots inside plastic beakersfilledwith water to the level of the soil surface. Other plants remained well-watered during the period of the experiment. In thefirstexperiment leaf discs were removed from the third youngest leaves of control plants and incubated for manipulation of stomatal aperture using the technique described by Rodriguez and Davies (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 range 0-150 mol m" 3 . Solutions were contained in 50 mm Petri dishes which were placed on a water bath at 25 C and illuminated with a photon flux density of 130 /miol m~2 s~' (PAR). Illumination was provided with fluorescent and tungsten tubes located beneath the water bath. CO2-free air was bubbled through the incubation solutions at a rate of 6 x 10~3 m" 3 min~'. After 4 h incubation, abaxial epidermis was stripped from leaf discs and stomatal apertures were determined immediately under a projection microscope. In the second experiment, plants wereflooded2 h after the lights were switched on and leaves and roots of four plants were sampled for ABA assay every 3 h for the next 4 d. Leaves (not including axillary stipules) were plunged immediately into liquid nitrogen while roots were washed and blotted dry before freezing. All samples were freeze-dried for at least 48 h and then stored in a desiccator for later analysis. Determinations of free ABA in the plant were made with the gas chromatograph fitted with the electron capture detector (GC-ECD). For each sample, approximately 100 mg dry weight of plant 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 ABA from this plate was methylated and redissolved in 100 mm3 cyclohexane. Extraction and purification procedures were as described by Quarrie (1978). The Pye Unicam 104 instrument proved sufficiently sensitive 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 and detector oven temperatures were 230 C, 245 C and 250 C respectively. Oxygen-free nitrogen was used as the carrier gas at a rate of 40 x 10~ 6 m 3 min~ 1 and detector purge gas at 20 x 10~6m3 min"1. Ethyl abscisate was added as an internal standard and quantification was accomplished as described by Quarrie (1978). The recovery of ABA using this technique was consistently between 75-80%. ABA contents presented are the result of at least duplicate assays. Total (free plus bound) ABA content of leaves was determined using radio-immunoassay with an antibody 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, Nanjing Agricultural University, China. Tests against ABA metabolites show it to be specific to ABA and its

Downloaded from jxb.oxfordjournals.org at Lancaster University on August 30, 2011

Zhang and DaviesABA in Flooded Pea Plants to

651

0.8

0.6

CO

o
0.4

02

10 ABA pmol
Downloaded from jxb.oxfordjournals.org at Lancaster University on August 30, 2011

10 ABA omol

100

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 antibody Sonstrates its reliabSSS A 20-30 mm* aliquot from the acetone ABA extract: w, ptoced m a 20 cm3 glass tube and evaporated down with N 2 gas. 150 mm3 10 mol m 3 PBS buffer (100 mol m phosphate buffer, pH 7-4, containing 100 mol m'^NaCl, 0-1% NaN3 and 0-1% gelatin was added to S o l v e the res due on a rotary mUer for lmin. 100 mm3 of [ 3 H]() -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 unspeenfic binding, 50 mm3 water were added instead of antiserum. The tubes were incubated at 4 C in the dark for 90 min a l then 400 mm3 saturated ammonium sulphate added to stop the reaction Tube, were then centrifuged at 2 500 g for 15 min. Following this, 500 mm3 supernatant was taken and mixed with 4 r S " S m f f l o n cocktail W (BDH). Tubes were placed in the dark for 1 h and then counted for 5 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 antigen 5 = precipitation radioactivity in the presence of sample antigen or standard antigen^ ABA concentrations were calculated according to the logit (B/Bo) versus log [ABA] linear regression S 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 a viscous flow porometer (Zhang and Da vies, 1986). RESULTS Figure 2 shows the results of a simple preliminary experiment conducted to determine whether the effect of increasing potassium concentration could over-ride the effects of ABA on the stomata of peas. It is clear that at KC1 concentrations above 120 mol m treatment with 10 mmol m ~ 3 ABA had no influence on stomatal aperture. In a previous paper (Zhang and Davies, 1986) we have shown that increasing KC1 concentrations in the incubation solution around pea leaves could overcome a limitation in stomatal aperture imposed by

652

Zhang and DaviesABA in Flooded Pea Plants

flooding. This experiment suggests that such a limitation may have been caused by enhanced ABA contents in the leaves. GC analysis of ABA contents of leaves and roots over the first 4 d of the flooding period showed that flooding greatly enhanced the amount of free ABA found in both leaves and roots (Fig. 3). Interestingly, small increases in ABA in roots were detectable within hours of the beginning of the flooding treatment (Fig. 3b) and these increases were statistically significant by the beginning of the second day of treatment. There were no statistically significant increases in free ABA in leaves of the same plants for another 14 h (Fig. 3a). This increase occurred at the end of the second day of the experiment and ABA contents of flooded leaves 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.

E a

Downloaded from jxb.oxfordjournals.org at Lancaster University on August 30, 2011

re 4 *

E o

IB

50
KCI
(mol

100
m-3)

1 50

FIG. 1 Effect of increasing KCI concentration on the stomatal aperture of leaves of a pea plant incubated with 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 a function of time of day. After 3 d of flooding, ABA contents increased markedly within a few hours of the beginning of the light period and decreased rapidly again when the lights were switched off. There was an approximately 5-fold variation in the ABA content of leaves of flooded 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 had been 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) than did lower fully expanded leaves. Leaves from flooded plants showed relatively high ABA contents during the light period. The total ABA content of leaves and roots of pea plants which were watered normally was 10 to 20 times higher than the content of free ABA (Fig. 5), presumably indicating that there

Zhang and DamesABA in Flooded Pea Plants

4> U

653

O 3 o

c o u
0

120-

Downloaded from jxb.oxfordjournals.org at Lancaster University on August 30, 2011

Days FIG. 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 flooded plants 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.

6S4
160 _

Zhang and DaviesABA in Flooded Pea Plants

Downloaded from jxb.oxfordjournals.org at Lancaster University on August 30, 2011

7:30

2130

0:30

FIG. 4. Diurnal variation in free ABA content of pea leaves either watered normally ( ) or kept flooded for 10 d prior to ABA determinations ( o). Closed symbols indicate contents of lower, older leaves, open symbols 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 but substantial increase in total ABA in shoots, while the content of total ABA in roots also increased for 3 d after the plants werefirstfloodedbut then declined to pre-flooding levels. At this time most roots were almost dead and had apparently completely lost turgidity. About 5 d after flooding, new adventitious roots were formed near the surface of the soil. There was no 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. DISCUSSION We report here on substantial increases in the ABA content of leaves of peas rooted in waterlogged soil. This accumulation, which can presumably account for some restriction in

Zhang and DaviesABA in Flooded Pea Plants

300 -

700

600

Downloaded from jxb.oxfordjournals.org at Lancaster University on August 30, 2011

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.

656

Zhang and DaviesABA in Flooded Pea Plants

leaf conductance, occurred under conditions where there was no indication of the development of leaf water deficit (Zhang and Da vies, 1986). Jackson (1985) has reported a similar result but other workers have linked increases in ABA content of flooded plants with an increase in leaf water deficit (Wadman-van-Schravendijk and van Andel, 1985). In our experiment, free ABA content of leaves was not enhanced for 36 h after the first waterlogging of the soil (Fig. 3) whereas some indications of enhanced ABA contents in roots of the same plants were detected within a few hours of soil flooding and this increase was statistically significant after 22 h (Fig. 3). Despite some reports to the contrary (Hartung, Gimmler, and Heilmann, 1982), it now seems that roots of some plants can produce increased amounts of ABA when stressed (Walton, Harrison, and Cote, 1976; Cornish and Zeevaart, 1985) and the results described here suggest that increased production by roots may be among the plant's first responses to waterlogging of the soil. We have noted that flooding reduces the turgor of roots, presumably because of effects on membrane properties. It is possible that this provides the stimulus for enhanced ABA synthesis. In our previous work (Zhang and Da vies, 1986) we have confirmed the observation of others that flooding of the soil around pea roots results in restriction of stomatal opening within a few hours (Fig. 3). This restriction correlates in time with the first detectable increase in ABA content of the roots but ABA content of leaves is apparently not enhanced at this time. The transpiration stream provides a direct link between roots and stomata since the epidermal cells adjacent to the guard cells and even the guard cell walls themselves are important evaporating sites (Meidner, 1975). Because the epidermis constitutes only a small proportion of the total volume of the leaf, it is possible for the ABA content of the epidermis to rise substantially, presumably to influence stomata even though the ABA content of the whole 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 of action for ABA in closing the stomata. ABA, therefore, may act as a direct link between the effects 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 of initial flooding and that this increase precedes any increase observed in the leaves conflicts with the suggestion of Jackson (1985) and Jackson and Hall (1987) that ABA accumulation in leaves of flooded plants arises as a result of reduced transport out of leaves. We propose that roots themselves produce an increased amount of ABA immediately after first flooding and that this ABA moves to the leaves as a signal of root perturbation. We cannot, however, rule out the possibility that subsequently, some ABA accumulating in leaves does so as a result of reduced transport to roots. Indeed this seems likely, since the total ABA content of roots does decline during the third day after first flooding (Fig. 5) probably reflecting the poor condition of many roots at this time. We confirm the suggestion of Jackson and Hall (1987) that, in peas, the stimulus for the build-up of ABA in leaves of flooded plants is not a decrease in leaf turgor. One very interesting observation is the large diurnal fluctuation in free ABA in roots and leaves of flooded plants (Fig. 3a, b and Fig. 4). When the lights are switched off after 3 d of flooding there is a more than 5-fold decline in the free ABA content of leaves. It seems likely that this rapid decline is the result of metabolism which is known to proceed more rapidly in the dark than in the light (Zeevaart and Boyer, 1982). This may be because leaves in darkness produce 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,

Downloaded from jxb.oxfordjournals.org at Lancaster University on August 30, 2011

Zhang and DaviesABA in Flooded Pea Plants

657

1982) and, therefore, one might predict rapid metabolism infloodedplants in the dark when stomata are closed. It is clear from Fig. 4 that maximum free-ABA contents are not recorded in leaves of flooded plants until 3 h or more after the beginning of the light period. It is possible that this is due to de novo synthesis although it is not clear why the rate of synthesis should be higher in the light than in the dark. The substantial increase in free ABA content of leaves of flooded plants during the first part of each day may simply be caused by a decline in the rate of ABA metabolism imposed upon a relatively high rate of synthesis which remains constant during both night and day. Because a very high proportion of total ABA apparently exists in a bound form (Fig. 5) infloodedplants, it is tempting to suggest that free ABA might be derived from the conjugated form. Zeevaart and Boyer (1982) suggest, however, that conjugation of ABA is irreversible although there is at least one suggestion in the literature that free-ABA might be derived from a bound form (Weiler, Schnabl, and Hornberg, 1982). The fact that pea leaves and roots contain a very large amount of conjugated ABA relative to free-ABA confirms results obtained by Weiler (1980) for Hyoscyamus niger. Apart from this result and the general observation that total ABA increases in leaves and roots offloodedpea plants, it would 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 metabolites they have not been tested with the glucose ester of ABA (ABA-GE). This is an important metabolic product of ABA and it is possible that the increase in total ABA in leaves and roots which seems to occur particularly in the dark does reflect some metabolism of free-ABA to ABA-GE. Large increases in free-ABA in leaves offloodedplants within hours of the beginning of the light period may explain why leaf conductance apparently overshoots when stomata first open following lights-on (Zhang and Davies, 1986). Stomata open comparatively widely within minutes of the beginning of the light period and then close partially, presumably in response to accumulation of high ABA contents. This type of stomatal behaviour is commonly seen in the field. The important conclusions from the results presented here would seem to be that early stomatal closure in response to flooding may result from ABA production in roots which precedes ABA increases in leaves by several hours. Subsequently, a very large amount of ABA accumulates in leaves and roots offloodedplants and the stimulus for this increase in the rate of synthesis of ABA is not a decrease in leaf turgor. There is substantial diurnal variation in the free-ABA content of leaves and roots. In a previous paper we have suggested that stomatal opening in pea plants may be limited by restricted uptake and transport of K + to the leaves. This conclusion was reached partly as a 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 an ABA-imposed limitation in stomatal opening (Fig. 2). Despite the fact that ABA contents of leaves are enhanced byfloodingwe cannot rule out the importance of effects on stomata of reduced K + concentrations in leaves, since these will undoubtedly enhance the sensitivity of stomata to elevated ABA contents (Snaith and Mansfield, 1982). ACKNOWLEDGEMENT We are grateful to the Government of the People's Republic of China for providing funds to support the work of J.Z.

Downloaded from jxb.oxfordjournals.org at Lancaster University on August 30, 2011

658

Zhang and DaviesABA in Flooded Pea Plants

LITERATURE CITED BASSI, 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 dormant Sitka 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 developmenta 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 Xanthium strumarium 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 Valerianella locusta. Plant Cell and Environment, 6, 427-8.
GIMMLER, H., and HEILMANN, B., 1982. The compartmentation of ABA-biosynthesis, ABA-

Downloaded from jxb.oxfordjournals.org at Lancaster University on August 30, 2011

metabolism and ABA conjugation. In Plant growth substances 1982. Ed. P. F. Wareing. Academic Press, London. Pp. 325-33. HIRON, R. W. P., and WRIGHT, S. T. C , 1973. The role of endogenous abscisic acid in the response of plants to stress. Journal of Experimental Botany, 24, 769-81. JACKSON, M. B., 1982. Ethylene as a growth promoting hormone under flooded conditions. In Plant growth 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 for improvement. 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 production of 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 abscisic acid 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 foliar desiccation 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 acid levels 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.

Zhang and DaviesABA in Flooded Pea Plants

659

1980. Radio-immunoassays for the differential and direct analysis of free and conjugated abscisic acid in plant extracts. Ibid. 148, 262-72. SCHNABL, H., and HORNBERG, C., 1982. Stress-related levels of abscisic acid in guard cell protoplasts of Viciafaba L. Ibid. 154, 24-8.
WILSON, J. A., OGUNKANMI, A. B., and MANSFIELD, T. A., 1978. Effects of external potassium supply

on 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 strumarium and 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 flooded plants. Journal of Experimental Botany, 37, 1479-91. ZHOU, X., Xu, Y.-J., and CHENG, W.-F., 1985. A radio-immunoassay for abscisic acid. Journal of Nanjing Agricultural University, 1, 89-91.

Downloaded from jxb.oxfordjournals.org at Lancaster University on August 30, 2011