EFFECT OF WATER STRESS UPON ENDOGENOUS ETHYLENE LEVELS IN VICIA FABA

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1 New Phytol (1974) 73, EFFECT OF WATER STRESS UPON ENDOGENOUS ETHYLENE LEVELS IN VICIA FABA BY A. S. EL-BELTAGY* AND M. A. HALL Department of Botany and Microbiology, The University College of Wales, Aberystwyth {Received 23 July 1973) SUMMARY Conditions of drought or waterlogging lead to greatly increased internal ethylene concentrations in Vida faba L. These increases appear to be due partly to decreased diffusion and partly to increased synthesis and are superimposed on normal diurnal fluctuations in internal ethylene concentration. The higher concentrations observed are correlated with a reduction in growth rate and increased leaf and flower abscission and senescence. The role of ethylene in the mediation of developmental responses to water stress is discussed. INTRODUCTION Mesophytes, unlike plants growing at one or the other of the extremes of water availability, do not possess anatomical adaptations that cope with conditions of water stress; nevertheless, many plants are exposed to drought, or flooding at some stage. Since the mesophyte group includes most economically important crops, considerable attention has been focused on physiological responses of such plants to water imbalance. Recent work has indicated that changes in the levels of abscisic acid occur in plants subjected to drought (Wright, 1969) or waterlogging (Wright and Hiron, 1970; Wright, 1972). This phenomenon appears to have an adaptive significance in that the increased levels lead to stomatal closure and reduced transpiration (Jones and Mansfield, 1970). In addition to these findings many workers have observed changes in endogenous levels of other hormones as a consequence of water imbalance. For example, Itai and Vaadia (1965) found reduced cytokinin activity in the root exudate of droughted sunflower plants and Hatcher (1959) observed an increase in difl"usible auxin in plum and apple under these conditions. Waterlogging may lead to an increase in auxin levels (Phillips, 1964) and a marked reduction in gibberellins (Reid, Crozier and Harvey, 1969; Reid and Crozier, 1971) and cytokinins (Burrows and Carr, 1969). Many of the symptoms of water imbalance such as leaf and flower abscission, epinasty, reduction of shoot growth and enhanced senescence are also typical plant responses to ethylene and it seemed profitable to explore the possibility of a causal connection. The purpose of these studies, therefore, has been to determine the effect of water imbalance upon internal ethylene concentration and to attempt to relate the changes observed to the physiological and developmental responses occurring as a result of stress imposition. After the initiation of this work McMichael, Jordan and Powell (1972) and Kawase (1972) have shown that enhanced ethylene production by plants may occur as a consequence of drought or waterlogging. * Permanent address: Department of Horticulture, Ain Shams University, Cairo, Egypt. D 47

2 48 A. S. EL-BELTAGY AND M. A. HALL MATERIALS AND METHODS Broad bean {Viciafaba L.) cv. Sutton's exhibition and tomato {Lycopersicon esculentum Mill.) cv. Ailsa Craig were grown in 7-in (178-mm) plastic pots in John Innes No. 2 compost in unheated glasshouses. For droughting experiments bean plants 85 days old were used. Beans and tomatoes for waterlogging experiments were 60 and 50 days old respectively. At the appropriate time plants were transferred to growth rooms with a 16 h photoperiod at 23 C + 2. The light intensity was ^^^ cm"^ min"^ at the midpoint of the plants. Plants were allowed to acclimatize for 5 days prior to the commencement of experiments. After the results of preliminary experiments became available, control and treated plants were placed in separate growth rooms in order to eliminate possible effects upon controls of ethylene evolved from treated plants. Drought conditions were imposed by withholding water from zero time onwards. Waterlogging was initiated by placing the potted plants in plastic buckets and adding water until it was level with the soil surface. Ethylene and ethane in the internal air spaces of stems and leaves were extracted by the vacuum method of Beyer and Morgan (1970), the only modification being the use of a 300 mm Hg vacuum for 3 min instead of 100 mm Hg for 2 min. In addition gas samples were obtained directly from the central lacunae of bean plants with a hypodermic syringe. In diurnal rhythm experiments, for samples taken during the dark period, all operations between and including leaf excision and extraction of ethylene were carried out in the dark or under dim green light. Ethylene and ethane were separated and measured by gas chromatography on glass columns 6 ft in length packed with activated alumina ( mesh). The nitrogen flow rate was 40 ml min~^ and the oven temperature 120 C. Chlorophyll (a and b) was extracted from ten leaf discs 0.7 cm in diameter, taken at random from the eighth to eleventh internode upward, with boiling 80 (, ethanol for 30 min. Absorbance of the resulting extract was measured at 650 and 665 nm. Results given are the mean of three replicate determinations and are expressed as a percentage of controls. Water saturation deficit was measured by the leaf disc method as described by Barrs (1968); no more than two discs were taken from any one leaf. Discs were taken from the same leaves as those used for chlorophyll determinations. A total of twelve 1.5-cm discs were used per treatment, results being the average of three replicate determinations in each case. Relative stomatal aperture was measured using an infiltration method based on that described by Alvim and Havis (1954). This involves a graded series of xylene/nujol mixtures. The series was numbered from i (pure xylene) to 11 (pure Nujol) and these are the figures referred to in Table i. Statistical analyses were carried out where appropriate by an analysis of variance program using a Hewlett-Packard calculator. RESULTS As no information is available on internal ethylene concentrations in leaves at different times of day, experiments were first carried out to investigate this problem. Data from such experiments were particularly desirable to serve as controls for later work on

3 Water stress and ethylene 49 changes in internal ethylene concentration over longer periods. Ethylene concentrations in leaves over a 24-h period are shown in Fig. i. The experiments were carried out over a 2-day period but as the results were consistent they have been presented as changes in a single day. In the leaves there were significant diurnal changes in the concentrations of ethylene and ethane. Since it is well established that diurnal fluctuations in water potential occur in leaves (e.g. Gardner and Nieman, 1964), water saturation deficit (WSD) [iark l.lqtil Fig. I. Diurnal changes in internal ethylene and ethane concentration in broad bean leaves. Plants were given a i6-h photoperiod., Ethylene concentration; L^, ethane concentration. a, L.S.D. (1%) ethylene; b, L.S.D. (i%) ethane. 50r Dark Light J Fig. 2. Diurnal changes in water saturation deficit in broad bean leaves. Plants were given a i6-h photoperiod. Samples taken from the same experiment as shown in Fig. i. was also measured under the conditions of Fig. i and the results of these experiments are shown in Fig. 2. Comparison of the two figures shows that the rise in ethylene concentration just before and for some hours after stomatal opening was to some extent correlated with increased WSD; later in the light period, however, internal ethylene concentration fell whilst WSD increased. Because of these results, all sampling during experiments on drought or waterlogging was done at the same time of day as far as possible.

4 50 A. S. EL-BELTAGY AND M. A. HALL The effect of drought upon internal ethylene concentration is shown in Fig. 3. Plants remained unwatered from the start of the experiment. The ethylene concentration increased seven-fold over the first 24 h but fell back to about 50% above the control level after 3 days. Then followed a further more gradual increase up to 9 days. Ethylene in control plants (watered twice daily) did not change significantly. WSD rose from 20% to 47% in the first 24 h and increased by a further 8% over the next 8 days. In contrast, ethylene concentrations in the central lacunae of stressed plants differed little from those in controls; the concentrations were about o.oi /il/1, i.e. much lower than those observed in the leaves i; Time (days) Fig. 3. Effect of drought on internal ethylene concentration, water saturation deficit and senescence in broad bean leaves. Plants unwatered from zero time. Ethylene concentration: C, droughted;, control. Water saturation deficit: L, droughted; A, control., Chlorophyll {a and h) in droughted plants (/ of control). L.S.D. (l" ) refers to ethyk-ne values only. The first rise in ethylene concentration appears to be correlated with increased WSD rather than with senescence since measurable leaf senescence compared to control plants did not begin until the third day (note chlorophyll levels in Fig. 3). The second phase of ethylene production did occur, however, during a period of advanced senescence. Relative stomatal aperture was measured at each of the sampling times in this experiment and the results are shown in Table i. Although some closure occurred during the first 12 h of stress it seems unlikely that the marked increase in ethylene concentration was due to this factor. Further closure occurred over the next 12 h and part of this period was also a night break so ethylene accumulation rather than increased production may have contributed to the higher concentration observed. Nevertheless, the high concentration must be due partly to increased production of ethylene since calculations

5 Water stress and ethylene 51 from the previous experiment on diurnal changes show that the magnitude of the observed increase could only be accounted for by decreased diffusion if the stomata closed completely for the whole 24 h. Ethane concentration rose between 24 h and 48 h (Fig. 4) when ethylene concentration was falling. It is possible that ethylene was converted to ethane although so far we have been unable to demonstrate such a conversion. Under waterlogging a somewhat similar picture emerged. There was a rise in concentration in the stems and leaves over the first 24 h of stress imposition (Fig. 5) which Table i. Relative stomatal aperture in broad bean during the water stress experiments shown in Figs. 3 and 5 Days from start Relative stomatal aperture* of stress Control Drought Waterlogged o 0-5 I I I I 50 I I I * Measured by using a graded series of xylene/ Nujol mixtures after Alvim and Havis (1954). 2.0 r Time (days) Fig. 4. Kffect of drought on internal ethane concentration in broad bean leaves. Ethane concentration:, droughted;, central. The same experiment as shown in Fig. 3. was correlated with a 20",, rise in WSD in the leaves (Fig. 6). However, whereas concentration in the leaves continued to rise until the second day, that in the stem fell back to a level of ^^0% above controls at this time. A similar drop in concentration occurred in the leaves by the third day. It is interesting to note that the internal ethylene concentration in stems was several-fold higher than that in the leaves both in control and stressed plants; similar observations have been made in cotton (McAfee and Morgan, 1971). Relative stomatal apertures during the experiment were measured at each sampling time and the results are shown in Table i. As in the previous experiments some stomatal closure occurred quite rapidly and the stomata were completely closed by the second day; nevertheless the rate of closure under conditions of waterlogging was slower than with drought.

6 A. S. EL-BELTAGY AND M, A, HALL Time (days) Fig. 5. Effect of waterlogging on internal ethylene concentrations in broad bean stems and leaves. Plants waterlogged from zero time. Ethylene concentration: stems (, waterlogged;, control); leaves (, waterlogged; 2, control), a, L.S.D. (i",,) etbylene from leaves, b, L.S.D. (i/u) ethylene from stems. = 100 -i50 5; I Time (doys) Fig. 6. Effect of waterlogging on water saturation deficit and senescence in broad bean leaves. Same experiment as shown in Fig. s- Water saturation deficit: :', waterlogged; A, control. G, Chlorophyll {a and b) in waterlogged plants (" of control).

7 Water stress and ethylene p CJ ' 0.02 O.I B 0 'ime (days) Fig. 7. Effect of waterlogging on internal ethylene and ethane concentrations in lacunae of broad bean plants. The same experiment as shown in Fig. 5. Ftbylene concentration:, waterlogged;, control. Ethane concentration: A, waterlogged; A, control, a, L.S.D. (1%) ethylene; b, L.S.D. (i'',,) ethane ii80 Fig. 8. Effect of waterlogging on internal ethane concentration in leaves and stems of broad bean. The same experiment as shown in Fig. 5. Leaves:, waterlogged;, control. Stems: A, waterlogged;, control, a, L.S.D. (1",,) leaves; b, L.S.D. (!" ) stems. The subsequent pattern of ethylene levels in the leaves was similar to that already described for drought conditions, i.e. a continued increase correlated with senescence. In the stem, however, levels continued to rise up to the seventh day; thereafter they began to decline.

8 54 A. S. EL-BELTAGY AND M. A. HALL i ^ I I s "S O O N 00 c o u o o o fn o O V f^oo \D E ^ ; 00»O -I C "S N I C w o O c fi"^ o u u B il o rorr ^^ O U moo en 3 V _3 a M ^ S r- N 00 C N upo 1 O: N 0 Tabl Con C U en CIS Q c U K

9 Water stress and ethylene 55 In contrast to the results with droughted plants marked fluctuations in ethylene concentration in the lacunae occurred under conditions of waterlogging (Fig. 7). No significant differences between stressed and control plants were observed up to 3 days but between the third and fifth day there was a twenty-fold increase in levels. This was followed by a subsequent decline up to the ninth day and then a further increase. The effect of waterlogging upon internal ethane concentration is shown in Fig. 8. In the leaves there was no significant increase in waterlogged plants above controls until Table 4. Ejfect of drought or waterlogging on ethylene levels and leaf abscission in tomato Treatment Drought Waterlogging Ethylene Treated I.IO 3.66 air) Control Leaf abscission (%) Treated Control Figures given are for plants stressed for 7 days Water stress Lowered transpiration Fig. 9. Possible inter-relationships between ethylene and other factors associated with water imbalance. after the fifth day but in the stem there was a marked rise within the first 24 h the level remaining high over the next 6 days and reaching a maximum on the seventh day. The fall in ethylene concentration in the stem after the seventh day of treatment was accompanied by a similar drop in ethane concentration. In the lacunae the change in internal ethane concentration resembled that of ethylene (Fig. 7). The effect of drought or waterlogging upon growth and development was also investigated. Both treatments reduced growth in height and internode production and both promoted abscission of leaves and fiowers (Tables 2 and 3); the effects on flower abscission were particularly rapid. Epinastic curvature was observed in the petioles of waterlogged plants after 5 days. That the phenomena described above are not unique to Vicia faba is demonstrated by the results shown in Table 4 on the effect of drought or waterlogging on internal ethylene levels and leaf abscission in tomato plants. Here again, both leaf abscission and ethylene concentration increased under water stress. DISCUSSION It is clear from the results described that both waterlogging and drought lead to increased internal ethylene concentrations. In both treatments there appear to be three distinct phases. An initial rise in ethylene concentration correlated with increased WSD followed

10 56 A. S. EL-BELTAGY AND M. A. HALL by a fall and a further subsequent increase correlated with a phase of rapid senescence. The principal difference in response between the two treatments lies in the greatly increased ethylene levels observed in the lacunae of waterlogged plants. Possibly the high ethylene concentrations observed in the lacunae of waterlogged plants arose not from the plant itself but from its environment, since Smith and Russell (1969) have shown that under waterlogging soil ethylene levels may rise to as much as 10 ppm; thus some uptake of ethylene from the soil may have occurred in our experiments. The high concentration in the lacuna later in the experiment is correlated with a drop in concentration in the stem. Since the plants were in an advanced state of senescence by the ninth day it is probable that these changes were a consequence of leakage of gases from the intercellular spaces of the stem into the lacuna. It seems probable that the fluctuations observed in leaves and stems are a consequence of changes within the plant; furthermore, the rapidity with which the initial rises occur, and their magnitude, is comparable to that observed with abscisic acid under similar conditions (Wright, 1972). Hitherto it has been assumed that internal ethylene concentration is controlled by a balance between biosynthesis and outward diffusion. Two observ^ations in our work appear to be inconsistent with this hypothesis. Firstly, the diurnal fluctuations in ethylene concentration in leaves were not entirely correlated with the degree of opening of the stomata which presumably constitute the chief diffusion pathway. Thus, whereas there was a rise in concentration associated with nocturnal stomatal closure, this rise began before the stomata began to close and the subsequent fall which occurred during the dark period was of suflicient magnitude to make it unlikely that this was due to decreased synthesis alone. Similar results have been obtained with other plants including those such as Bryophyllum which have a different pattern of stomatal behaviour (J. A. Kapuya and M. A. Hall, unpublished). Secondly, under conditions of drought or waterlogging a rapid drop in levels in the leaves was observed at a time when the stomata were closed as a consequence of stress imposition. It thus seems likely that mechanisms exist either for the metabolism of ethylene in this tissue or, possibly, for its translocation about the plant. Jansen (1964) has shown that ethylene is metabolized to a number of different components in avocado fruit although the amounts incorporated were rather small. Nevertheless this work clearly demonstrated that systems exist in higher plants capable of metabolizing ethylene and other unsaturated hydrocarbons. There are as yet no data available concerning movement of ethylene within the plant. Our observations that the pattern of ethane concentration to some extent varies inversely with that for ethylene under conditions of waterlogging or drought, although not for diurnal changes, has led us to suggest that some conversion of one to the other may occur. So far, however, we have not been able to demonstrate such a mechanism. It is not clear why the imposition of water stress leads to increased ethylene concentrations. Phillips (1964) has observed increases in auxin levels in waterlogged sunflowers and similar increases have been reported by Hatcher (1959) in droughted plums and apples. In view of the findings by several workers that high auxin levels may lead to increased ethylene biosynthesis (Abeles and Rubenstein, 1964; Burg and Burg, 1968) it seems possible that a relationship between these two factors may exist in the system studied here. On the other hand, the first increases in ethylene concentration observed in our work occurred after i day whereas in the work of Phillips auxin levels had not increased significantly at this time. It is known that water imbalance leads to increased rates of protein breakdown in leaves (Shah and Loomis, 1965; Dove, 1971; Naylor, 1972) and

11 Water stress and ethylene 57 since methionine appears to be the precursor of ethylene in higher plants (Yang and Baur, 1969) it may be that increased availability of this amino acid leads to the elevated ethylene levels observed in this work. What is the significance of the observed results in relation to the response of plants to the imposition of drought or waterlogging? Under both treatments there is a general inhibition of growth both in terms of height and internode production. This is accompanied by increased senescence and abscission of leaves and flowers. In addition marked epinasty is observed in waterlogged plants. It is clear that these effects are not a consequence of any single factor, nevertheless it is remarkable that almost all the symptoms of water imbalance mentioned above may also be induced by external application of ethylene or ethylene-releasing chemicals such as 2-chloroethyl phosphonic acid. For example, ethylene is known to retard stem elongation (Fuchs and Lieberman, 1968) and to accelerate senescence (Burg, 1968; Dela Fuente and Leopold, 1968) and abscission (Jackson and Osborne, 1970). A number of workers have suggested that senescence is accelerated by water imbalance via an inhibition of the biosynthesis or translocation of growth regulators (Reid and Crozier, 1971; Burrows and Carr, 1969). However, such effects alone cannot explain the observed symptoms and Burrows and Carr (1969) have invoked the production of unspecified senescence accelerating substances in the leaves in response to stress. This suggestion appears to be consistent with our findings. The relationship between ethylene and abscisic acid in this system, if any, remains to be elucidated. Pallaghy and Raschke (1972) have reported that ethylene, unlike abscisic acid, has no immediate effect on stomatal aperture. In this connection we found no effect of ethylene (10 ppm) on stomatal aperture in bean even after a 24-h exposure. This aspect may, however, repay further study. Of further interest is the work of Abeles (1967) and Cooper et al. (1968) who showed that ethylene production by bean explants and citrus leaves was increased by treatment with abscisic acid. It may be significant that abscisic acid also enhances abscission (Eagles and Wareing, 1964; Smith et al., 1968) and senescence (El-Antably, Wareing and Hillman, 1967; Aspinall, Paleg and Addicott, 1967). Do the observed changes in internal ethylene levels have an adaptive significance for the plant as appears to be so for abscisic acid? Under water stress stomata will close and hence transpiration will be reduced. Nevertheless in those species possessed of thin cuticles or relatively insensitive stomata (Brown and Pratt, 1965) this reduction may not be sufliciently rapid or effective in preventing the onset of severe and damaging water deficits. In addition older leaves which may have cracked cuticles and insensitive stomata (H. Veen, personal communication) may represent a transpirational 'drag' under these conditions. Since it is accepted that the loss of leaves by deciduous species is advantageous because, amongst other things, it reduces water loss in winter, it is possible that leaf abscission under conditions of water imbalance is a similar type of response. If, as seems likely, the high ethylene concentrations in stressed plants are responsible for the observed increase in leaf and flower abscission, then this may represent a favourable adaptation of the plant to water imbalance. This idea is supported by the observation that ethylene accelerates abscission of older leaves more readily than young ones (Leopold, 1970) and indeed it is these which are the first to abscind in the work reported here. The latter part of the above discussion is summarized in Fig. 9. Although continuous lines have been inserted where processes have been shown to occur, more work needs to be performed to show that they will all occur in the same system. In particular the

12 58 A. S. EL-BELTAGY AND M. A. HALL possible relationships between the biosynthesis of both ethylene and abscisic acid merit close attention. In addition levels of other growth regulators in the same system need to be investigated; our current studies on these aspects will be published elsewhere. Since ethylene levels vary even with normal diurnal fluctuations in water balance it becomes important to determine whether the response of plants is strictly related to a given level of the growth regulator or whether this will vary. A number of workers have shown that carbon dioxide is a competitive inhibitor of ethylene action in a number of systems (Burg and Burg, 1967; Abeles, Craker and Leather, 1971) and since ethylene may enhance respiration rates (Pratt and Goeschl, 1968) the observation by a number of workers that such rates increase in response to the imposition of water stress (Heath and Meidner, 1961; Brix, 1962) together with our observations on enhanced ethylene levels may indicate a close relationship. Such a connection might suggest that the absolute concentration of ethylene may be of less importance than its concentration relative to that of carbon dioxide. In order to provide an answer to these questions it will be necessary to investigate changes in internal levels of both carbon dioxide and ethylene under a wide range of intermediate conditions of water balance and relate these to associated developmental responses. In addition it is imperative that the point at which control is exercised on ethylene levels be clarified by a thorough investigation of the relative contributions of biosynthesis, degradation and diffusion to the maintenance of these levels. ACKNOWLEDGMENTS We wish to thank Mr J. A. Kapuya, Mr A. M. Salama and Mrs D. Redmond for assistance. One of us (A.S.B.) wishes to thank Ain Shams University, Cairo, Egypt for granting study leave. REFERENCES ABELES, F. B. (1967). Mechanism of action of abscission accelerators. Phyiohgia PL, 20, 442. ABELES, F. B., CRAKER, L. E. & LE.'\THER, G. R. (1971). Abscission: tbe pbytogerontological effect of ethylene. PI. PhysioL, Lancaster, 47, 7. ABELES, F. B. & RUBINSTEIN, B. (1964). Regulation of ethylene evolution and leaf abscission by auxin. PL PhysioL, Lancaster, 39, 963. ALVIM, D. DE T. & HAVIS, J. R. (1954). An improved infiltration series for studying stomatal openings as illustrated with coffee. PL PhysioL, Lancaster, 29, 97. AspiNALL, D., PALEG, L. G. &.\DDICOTT, F. T. (1967)..-Xbscisin II and some hormone-regulated plant responses. Aust.J. bid. Sci., 20, 869. B.ARRS, H. D. (1968). Determination of water deficits in plant tissues. In: Water Deficits and Plant Growth (Ed. by T. T. Kozlowski), Vol. 1, p Academic Press, New York and London. BEYER, E. M. JR & MORGAN, P. W. (1970). A method for determining tbc concentration of etbylenc in gas pbase of vegetative plant tissues. PL PhysioL, Lancaster, 46, 354. BRIX, H. (1962). Tbe effect of water stress on tbe rates of photosynthesis and respiration in tomato plant and lobolly pine seedlings. Physiologia PL, 15, 10. BROWN, W. V. & PRATT, G. A. (1965). Stomatal inactivity in grasses. 5. West. Nat., 10, 48. BURG, S. P. (1968). Etbylene, plant senescence and abscission. PL PhysioL, Lancaster, 43, BURG, S. P. & BURG, E. A. (1967). Molecular requirements for the biological activity of ethylene. PL PhysioL, Lancaster, 42, 144. BURG, S. P. & BURG, E. A. (1968)..Auxin stimulated etbylene formation. Its relationship to auxin inhibited growtb, root geotropism and other plant processes. In: Biochemistry and Physiology of Plant Growth Substances (Ed. by F. Wigbtman & G. Setterfield), p Rungt Press, Ottawa. BURROWS, W. J. & CARR, D. J. (1969). Effect of flooding tbe root system of sunflower plants on tbe cytokinin content in xylem sap. Physiologia PL, 22, COOPER, W. C, RASMUSSEN, G. K., ROGERS, B. J., REECI:, P. C. & HENRV, W. H. (1968). Control of abscission in agricultural crops and its pbysiological basis. PL PhysioL, Lancaster, 43, DELA FUENTE, R. K. & LEOPOLD, A. C. (1968). Senescence processes in leaf abscission. PL PhysioL, Lancaster, 43, 1496.

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