Effect of Water Stress and Ethylene on Osmoregulation in the Subhook Region of Pea Epicotyls

Biochem. Physiol. Pflanzen 182, 41-48 (1987) VEB Gustav Fischer Verlag Jena

Effect of Water Stress and Ethylene on Osmoregulation in the Subhook Region of Pea Epicotyls KENSUKE MIYAMOTO and SEIICHIRO KAMISAKA Department of Biology, Faculty of Science, Osaka City University, Osaka, Japan Key Term Index: ethylene, epicotyls, osmoregulation, water stress; Pisum sativum L.

Summary Possible involvement of ethylene in osmoregulation under water stress was studied. Osmotic stress caused by 100 mM maunitol inereased osmotic coneentration of cell sap in the subhook region of the second internode of pea cuttings. Either exogenous ethylene or ACC (1-aminocyc1opropane-1carboxylic acid) increased osmotic coneentration. The increase in the osmotic concentration was well correlated with the amount of ethylene released from ACe-treated cuttings, suggesting that osmotic concentration is under the control of the endogenous level of ethylene. )Iannitol treatment inereased the eoncentration of both soluble sugars and potassium ions in cell sap obtained from the subhook region, while ethylene treatment increased only the concentration of soluble sugars. Neither osmotic stress eansed by mannitol nor drought stress stimulated ethylene production in pea cuttings. These results suggest that in the subhook region of the pea second internode, ethylene is not the effector in osmoregulation under water stress.

Introduction

In response to water stress, plants increase osmotic concentration of the cell by increasing the concentration of osmotica in the vacuolar sap (see e.g. HELLEBUST 1976). The increased osmotic concentration enhances the ability of the cell to absorb water and thus results in continued stem elong'ation under water stress (MEYER and BOYER 1981; ZHAO ct al. 1983, 1985). A rapid increase in ethylene production under water stress has been reported in intact cotton petioles (McMICHAEL et al. 1972), in excised orange leaves (BEN-YEHOSHL\ and ALOXI 1974; AHARONI 1978) and in excised wheat leaves (WRIGHT 1977, 1979, 1980; APELBAUM and YANG 1981; McKEON et al. 1982). Exogenously applied ethylene is known to increase osmotic concentration of stem cells in pea seedlings (EISINGER et al. HJ83). These findings suggest that ethylene is responsible for the control of osmotic potential under water stress. In this study, however, evidence is presented that in pea cuttings, ethylene is not involved in osmoregulation under water stress. Materials and Methods Plant material

Seeds of Pisum sativum L. cv. Alaska were soaked at 25 DC for 1.5 d in running tap water, then germinated at 25 DC in the dark under water-saturated atmosphere in a plastic basket. After 1 d,

Abbreviation,' ACC, 1-aminocyclopropane-1-carboxylic acid

4

41

seedlings with 2-4 em radicles were planted in moistened vermiculite in a basket which was held on the surface of water, then grown at 25°C for 3 d in the dark. Five-day-old seedlings were selected for epicotyl size (55-60 mm), then roots were cut off leaving 5 mm of the main root. The subhook region (the elongation zone) of the second internode (5 mm below the hook) was marked with India ink, then a lot of five cuttings was placed in a 10 ml-conical flask containing 5 ml of distilled water and grown at 25°C in the dark. Water-stress treatment was done by placing cuttings in the conical flask containing varying concentrations of mannitol solution. Ethylene treatment was done by placing the conical flask with cuttings in a sealed I-liter bottle containing varying concentrations of ethylene. In some experiments, cuttings were placed in a 10 ml-conical flask containing of 5 ml of ACC solution and incubated in the sealed I-liter bottle. All manipulations were done under dim green light.

Determination of ethylene production Ethylene produetion in pea cuttings subjected to either osmotic stress or drought stress was measured. In the case of osmotic stress, 3 sets of 5 cuttings placed in a 10 ml-conical flask containing 5 ml of 0.1 M mannitol solution were incubated at 25°C in the dark in a sealed I-liter bottle. In the case of drought stress, cuttings (15 g in fresh weight) were kept at 25°C in the dark in a I-liter bottle equipped with inlet and outlet openings. Dry or water-saturated air was supplied to cuttings through the inlet and the air from the outlet was introdueed into a gas collection trap containing 50 ml of mercuric perchlorate and 5 drops of n-butanol as a foaming agent. After 4 h-incubation, trapped ethylene was released by adding 4 M LiCI to mercuric perchlorate (YoUNG et al. 1951, 1952). The measurement of ethylene was done with a gas-liquid chromatograph (Hitachi model 163, Japan) equipped with 1-m glass column packed with activated aluminum and a flame ionization detector. The assay conditions were as follows: column temperature, 80°C; injection port temperature, 150°C; deteetor temperature, 150°C; He flow rate, 30 ml min-I.

Determination of osmotic concentration The subhook region of the second internode was excised from cuttings. The epicotyl segments were immediately wrapped in aluminum foil, then frozen with liquid nitrogen. The cell sap of the segments was prepared by the modified method (Zn AO et al. 1983) of the centrifugation method developed by TERRY and BOXNER (1980). Frozen segments were placed on the stainless mesh set in the barrel of a 12 ml-plastic syringe (Top Surgical MFG. Co. Ltd., Japan) cut off at the 4 lUI-mark to form a small tube, and thawed at room temperature. The tube with epicotyl segments was placed on the top of a centrifugation tube and centrifuged at 1,000 X g at 5°C for 10 min to obtain the cell sap. Osmotic coneentration of the cell sap was determined with a vapor pressure osmometer (Wescor model 5100C, USA) (HOYElt and KNIPLING U1Gfl).

Analysis of solute in cell sap The amount of sugars in the cell sap obtained from the subhook region of the second internodes of pea cuttings was determined by the phenol-sulfurie acid method (DenoIs et al. 195G), and expressed as the glucose equivalent. The amount of amino acids in the cell sap was determined by the ninhydrin reaction (GARItEL et al. 1972) and expressed as the homoserine equivalent. The amount of potassium ions in the cell sap was determined using an atomic absorption speetrophotometer (Hitaehi model 208, Japan).

Results

Effect of mannitol and ethylene on osmoregulation Both mannitol and ethylene substantially increased osmotic concentration in the subhook region of the second internode of pea cuttings (Figs. 1 and 2). Like ethylene, ACC also increased osmotic concentration (Fig. 3). This effect of ACC seems to be due to ethylene that was released from ACC-treated cuttings, because exogenously applied ACC induced an active production of ethylene in the present experimental conditions

42

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Mannitol, mM

50

100

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0.1

0.3

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Fig. 1. Effect of mannitol on osmotic concentration in the subhook region of pea cuttings. Cuttings were grown in the dark for 24 h with and without varying concentrations of mannitol, then the cell sap was obtained from the subhook region of the second internode and assayed for osmotic concentration. Vertical lines represent standard errors (n = 3). Fig. 2. Effect of ethylene on osmotic concentration in the subhook region of pea cuttings. Cuttings were grown in the dark for 24 h with and without varying concentrations of ethylene. Vertieal lines represent standard errors (n = 3). Otherwise as for Fig. 1.

(Fig. 4). In ACC-treated cuttings, a good correlation was found between the amount of ethylene produced and osmotic concentration in the subhook region (Fig. 5). These results suggest that water stress stimulates the production of ethylene, resulting in an increase in osmotic concentration in the subhook region of pea cuttings.

Effect of water stress on ethylene production Ethylene production in cuttings under osmotic stress was examined. The amount of ethylene produced during 24 h-incubation was not influenced by incubating cuttings with 100 mM mannitol (Table 1), although osmotic concentration in the subhook region substantially increased in response to mannitol treatment (Fig. 1). Further, kinetics of thu rate of ethylene production during 24 h-incubation was not affected by 100 mM mannitol (data not presented). Drought treatment induced about 10 % decrease in fresh weight of cuttings in the present experimental conditions (Table 2). The amount of ethylene produced, however, was not enhanced by drought stress.

Effect of mannitol and ethylene on the concentration of solutes Fig. 6 shows the effect of mannitol and ethylene on the concentration of soluble sugars, amino acids and potassium ions in the cell sap obtained from the subhook 4*

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Fig. 3. Effect of ACC on osmotic concentration in the subhook region of pea cuttings. Cuttings were grown in the dark for 24 h with and without varying concentrations of ACC. Vertical lines represent standard errors (n = 3). Otherwise as for Fig. 1. Fig. 4. Effect of ACC on ethylene production in pea cuttings. Each lot of 15 cuttings was incubated for 24 h with and without varying coneentrations of ACC, then the amount of ethylene produced dnring 24 h·incubation was determined. Vertical lines represent standard errors (n = 4).

Table 1. Rffect of osmotic stress on ethylene production in pea cuttings Concentration of mannitol (m:VI)

Ethylene produced (n1/24 h/15 cuttings) 32.9 ± 2.4 34.8 ± 2.2

o 100

Fifteen cuttings were ineubated for 24 h in a sealed l-liter bottle with and without 0.1 .M mannitol.

Table 2. Effect of drought stress on ethylene production and fresh weight of pea cuttings Experiment number

II

Ethylene produced (nl/4 hlg cuttings)

% Decrease in fresh weight

Control

Stress

Control

Stress

7.4

G.7

0.0

9.3

4.7

G.1

1.3

9.0

Pea cuttings were incubated in a l·liter bottle. Dry or water-saturated air was supplied to cuttings through the inlet of the bottle and ethylene gas from the outlet was trapped with mercuric perchlorate. After 4 h-incubation, changes in fresh weight of cuttings was determined.

44

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Fig. 5. Quantitative relationship between the osmotic concentration of the cell sap and the amount of ethylene released from ACC-treated cuttings. Data in Figs. 3 and 4 were plotted. The correlation coefficient (0.992) is statistically significa.nt (better than the 1 % level). Fig. 6. Effect of 'manl!1:tol and ethylene on the concentrat·ion of wgafs, amino acids and potassium ions in the cell sap. Pea cuttings were incubated for 24 h with and without varying concentrations of mannitol or ethylene, then th e comentration of sugars (A), amino acids (B) and potassium ions (C) in the cell sap of the subhook region was determined. Verti callin(~s represent standard errors (n = 3).

Table 3. Correlation coefficients betu;eel! osmotic cOllcentration and the concentration of solutes in the cell sap of tlte subhook regl:on in pea cuttings Treatment

Correlation ('oefficient Sugars

Amino acids

Potassium ions

}fannito]

0.989 a -0.155

0.999a

Ethylene

0.999a

0.807

0.G56

Correlation coefficients were determined from the experimcnt of :Fig. 6. Values followed by "a" are statistically significant (better than thc ] % level).

region of the second internode in pea cuttings. The concentration of soluble sugars increased in response to both mannitol and ethylene treatments, while the concentration of free amino acids was not. Mannitol increased the concentration of potassium ions, but ethylene did not. For mannitol-treated cuttings, good correlations existed between osmotic concentration and the concentration of soluble sugars and of potassium ions BPP 182 (1987) 1

45

(Table 3). For ethylene-treated cuttings, on the other hand, only the concentration of free sugars was well correlated with osmotic concentration. These results indicate that ethy lene affects osmotic concentration in the subhook region in a different way from mannitol. Discussion

The effect of ethylene on osmotic concentration in pea stem cells is controversial. EISINGER et al. (1983) found an increase in osmotic concentration in response to ethylene treatment, while RIDGE (1973) found a decrease in osmotic concentration. The present study revealed that osmotic concentration in the subhook region of the second internode of pea cuttings substantially increased in response to ethylene treatment (Fig. 2). Judging from these results, ethylene seems to have the action of increasing osmotic concentration of the cell sap. This conclusion may be supported by the finding that ethylene increased osmotic concentration in rice coleoptile segments (IsHIzAwA and ESAsHI 1984). Experiments with ACC revealed that an increase in osmotic concentration caused by ACC treatmcnt is well correlated with the amount of ethylene released from ACCtreated cuttings (Fig. 5). This result strongly suggests that osmotic concentration in the subhook region of the pea second internode is under the control of the endogenous level of ethylene. Mannitol increased osmotic concentration in the subhook region of the second internodes: control = 356 mmol kg- I, 100 mM mannitol = 514 mmol kg-l (Fig. 1). If this effect of mannitol on osmotic concentration is brought about by an increase in the endogenous level of ethylene in pea cuttings, about 100-fold increase in ethylene production should occur in response to treatment with 100 mM mannitol in the present experimental conditions (Fig. 5). Under non-watcr stress conditions, pea cuttings produced ca. lnl h-l g fresh weight- 1 (Tables 1 and 2). The observed rate of ethylene production well corresponds to that reported by OSBOR~E (1988). Ethylene production was not stimulated by the mannitol treatment (Table 1). Drought stress also had no substantial effect on ethylene production (Table 2). These results indicate that in pea cuttings, the increase in osmotic concentration caused by water stress is not mediated by a change in the endogenous level of ethylene. Mannitol treatment increased the concentration of soluble sugars and potassium ions in the cell sap obtained from the subhook region of the pea second internodes, but did not affect that of free amino acids (Fig. 6). On the other hand, ethylene treatment increased the concentration of soluble sugars but did not that of free amino acids and potassium ions. These results further support the idea that water stressinduced increase in osmotic concentration of the cell sap of the pea subhook region is not mediated by increased production of endogenous ethylene. Acknowledgements We wish to thank Prof. YOSHIO MASUDA, Dr. RYOICHI YAMAMOTO and TAKA YUKI HOSON for their invaluable suggestions and discussions. This work was partly supported by a Grant-in-Aid for Scientific Research (#57340039) from the Ministry of Education, Science and Culture, Japan.

46

EPP 182 (1987) 1

References AHARONI, N.: Relationship between leaf water status and endogenous eth ylene in detached leaves. Plant Physiol. 61, 658- 662 (1978). APELBAUlII, A. , and YANG, S. F.: Biosynthesis of stress ethylene induced b y water deficit. Plant Physiol. 68, 594-596 (1981). BEN- YEHOSHUA, S., and ALO NI, B.: Effect of water stress on ethylene production by detached leaves of Valencia Orange (Citrus sinensis OSBECK). Plant Physiol. 03, 863-865 (1974). BOYER, J. S., and KNIPLING, E. n.: Isopiesti(' technique for measuring leaf water potentials with a t hermocouple psychromet er. Proe. Nat. Acad. Sci. 04, 1044- 1051 (1965). DeBoIs, :\1., GIL ES, K. A., HAMILTON, J . K., REB US , P. A., and SMITH, F. : Colorimetric method for detf'rmination of sugars and relat ed substances. Anal. Chern. 28, 350- 356 (1956). E rSIKGER, W. , CRO:O
~Ic KED:\',

Mc)hClHEL, B. L., JORDAN, W. R., and POWELL, R. D.: An effect of water stress on ethylene produttion by intact cotton petioles . Plant Physiol. 49, 658- 660 (1972). MEYER, R. F., and BOYER, J. S.: Osmoreg ulation, solute distribution, and gro wth in soybean seedlings having low water potentials. Planta Hi], 482-489 (1981). OSBORNE , D. J.: Ethylene. In: LETHAM, D. S., GOODWIN, P. B., and HIGGINS, T. J. V. (Eds.). Phytohormones and Related Compounds -- A Co mprehensive Treatis e, Vol. 1, pp. 265-294. Elsevierj ~orth-Holland Biochemical Press 1978. RIDG E, I. : The control of cell shape and rate of cell expansion b y ethylene: effe cts on microfibril oripn tation and cell wall extensi bility in etiolated peas. Act a Bot. Neerl. 22, 144-158 (1973). TERRY, M. E., and BONNER, B. A.: An examination of centrifugation as a m ethod of extracting a n extracellular solution from peas, and its use for the study of indol eacetic acid-induced growth. Plant Physiol. 66, 321- 325 (1980). WRIGHT, S. T. C.: The relationship betw een leaf water potential and the levels of abscisic acid and ethylene in excised wheat leaves . Planta 134, 183-189 (1977). WRIGHT, S. T. C.: The effect of 6-benzyladenine and aging treatment on the levels of stress-induced ethylene emanating from wilted wheat leaves. Planta 144, 179- 188 (1979). WRIGHT, S. T. C.: The effect of plant growth regulator treatments on the levels of ethylene emanating fr om excised turgid and wilted wheat leaves. Planta 148, 381-388 (1980). YOUXG, R. E., PRATT, H. K., and BLUE, J. B.: Identification of ethylene as a volatile product of the fun gus, Pem:cillium digitatum. P lant Physiol. 26, 304-310 (1951). YOUNG, R. E., PRATT, H. K, and BULE, J. B.: l\fanometri c determination of low concentrations of ethylene. Anal. Chern. 24, 551-555 (1952). ZHAO, Y. J., KAMISAKA, S., and MASUDA, Y.: Osmoregulation in hypocotyJs of etiolated mung bean seedlings with or without cotyledons in response to water-defici ent stress. Bot. Mag. Tokyo 96, 211-222 (1983). BPP 182 (1987) 1

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ZHAO, Y. J., KAMISAKA, S., and MASUDA, Y.: Quantitative relationships between osmotic potential and epicotyl growth in Vigna radiata as affected by osmotic stress and cotyledon excision. Physiol. Plant 61,431-437 (1985).

Received April 28, 1986; accepted June 11, 1986 Author's address: SEIICHIRO KA1IISAKA, D. Sc., Department of Biology, Faculty of Science, Osaka City University, Sumiyoshi-ku, Osaka 558, Japan.

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BPP 182 (1987) 1