REVERSAL BY PHENOLIC COMPOUNDS OF ABSCISIC ACID-INDUCED INHIBITION OF IN VITRO ACTIVITY OF AMYLASE FROM SEEDS OF TRITICUM AESTIVUM 1..

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1 New Phytol. (1986) 103, REVERSAL BY PHENOLIC COMPOUNDS OF ABSCISIC ACID-INDUCED INHIBITION OF IN VITRO ACTIVITY OF AMYLASE FROM SEEDS OF TRITICUM AESTIVUM 1.. BY SHASHI SHARMA, S. S. SHARMA AND V. K. RAI* Department of Biosciences, H.P. University, Shimla , India {Accepted 5 December 1985) SUMMARY Effects of abscisic acid and certain phenolic compounds have been studied, individually and in combination, on in vitro amylase activity from Triticum aestivum L. seeds. ABA suppressed the amylase activity, in vitro, while salicylic acid, cinnamic acid and ferulic acid applied alone promoted it; vanillic acid caused a slight inhibition. Irrespective of their individual effects, all the phenolic compounds studied reverted the inhibitory action of ABA on amylase activity. This behaviour suggests that phenolic compounds could have a role in regulating mobilization of carbohydrates through degradation of starch, and in turn some role in tbe regulation of seed germination. Key words: Phenolic compounds, abscisic aid, amylase, Triticum aestivum. INTRODUCTION The inhibitory action of abscisic acid on seed germination of various plant species is well established (Milborrow, 1974; Karssen, 1982). Phenolic compounds have also been recognized as inhibitors of plant growth (Kefeli & Kadyrov, 1971), for example they inhibit seed germination in a number of plant species (Stom, 1982). Reversal of ABA-induced inhibition of seed germination and hypocotyl growth by phenolic compounds has, however, been demonstrated (Ray, Guruprasad & Laloraya, 1980; Pawar, Ray & Laloraya, 1985). With the aim of finding a working mechanism for such an interaction, we have studied the effect of ABA and certain phenolic compounds, alone and in combination, on in vitro amylase activity which is responsible for starch degradation, and which in turn determines the availability of mobilizable carbohydrates necessary for germination and subsequent seedling growth. MATERIALS AND METHODS Source of seeds Seeds of Triticum aestivum L. cv. Lok-1 procured from the National Seeds Corporation, New Delhi were used. Enzyme extraction Seeds of T. aestivum which had been selected for uniformity were soaked in distilled water for 24 h at 20 C. Twenty imbibed seeds were homogenized in X/86/ $03.00/0 * Reprint requests to V. K. Rai The New Phytologist

2 294 S. SHARMA, S. S. SHARMA AND V. K. RAI chilled 0-05 M Tris-HCl buffer (ph 7-2) in a chilled mortar and pestle. Homogenate was centrifuged at 3000 g for 10 min at 2 C. The volume of the clear supernatant was raised to 50 ml with buffer and this was used as crude enzyme extract. (+) ABA (99 %), salicylic acid, ferulic acid, vanillic acid and cinnamic acid were procured from the Sigma Chemical Co., USA. Solutions of various concentrations of these compounds were prepared in cold or hot buffer (ph 72). Crude enzyme extract treated in the following manner was incubated for 3 h in the dark at 20 C followed by the assay for amylase activity. (1) 4 ml extract+ 8 ml buffer (control). (2) 4 ml extract + 4 ml solution of various concentrations of phenolic compounds 4-4 ml buffer. (3) 4 ml extract+ 4 ml solution of ABA 10"* M + 4 ml of solution containing the phenolic compounds (various concentrations). The quoted concentrations of ABA or phenolics are those in the final incubation mixtures. Enzyme assay Amylase activity was measured by the method of Filner & Varner (1967) and was expressed as //g starch degraded min"^ mg~^ protein. Protein estimation was accomplished according to Lowry et al. (1951). Experiments were done in triplicate and repeated twice. The values presented are arithmetic means together with standard deviations indicated by vertical bars. RESULTS AND DISCUSSION Increasing concentrations of ABA inhibited the activity of amylase in extracts from the seeds of T. aestivum (Eig. 1), 10~* M ABA causing a decrease of approximately 40%. Different phenolic compounds variably infiuenced the amylase activity (Table 1). Salicylic, cinnamic, and ferulic acids promoted the activity, ferulic acid being the most effective. Vanillic acid, on the other hand inhibited the enzyme activity. The interaction studies with ABA (10~* M) and the phenolic compounds (10~*, 10~^, 10^^, 10~^ M) revealed that all the phenolics, namely, vanillic, salicylic, cinnamic and ferulic acids, irrespective of their individual effects, reversed the ABA-induced inhibition of amylase activity (Eigs 2 and 3). Total restoration of enzyme activity was achieved by every phenolic compound at one or other concentration. Eerulic acid again proved to be the most effective in reverting the effects of ABA. Concentration effects were, however, not clear. The action of ABA in inhibiting the synthesis and activity of a-amylase has been shown by earlier workers, for example the inhibition of GAg-induced a-amylase synthesis in barley aleurone layers (Cornforth et al., 1965; Chrispeels & Varner, 1966). Ho & Varner (1976) showed that ABA decreased the synthesis of a-amylase, whereas the synthesis of most other proteins remained unaltered. Inhibition of a-amylase activity in rice endosperm and of the germination of rice seeds has been shown by Roy, Ghose & Sircar (1973). Promotion of a-amylase activity by certain phenols in the seeds of Cicer arietinum has also been observed (Sharma & Tayal, 1984). The antagonistic action of phenolic compounds on ABA-induced inhibition of seed germination and hypocotyl growth has already been shown (Ray et al., 1980; Pawar etal.,\9^s). The present experiments suggest that ABA inhibits germination

3 Phenols., ABA and amylase from wheat seeds "" ABA concentration (M) Fig. 1. Effect of ABA concentrations on amylase activity from Triticurn aestivum. (Vertical bars represent SD. Table 1. Effect of some phenolic acids on amylase activity from Triticum aestivum (the control value was ) Phenolic compound (M) //g starch degraded mg ' protein min " Vanillie acid Salicylic acid Cinnamic acid Ferulic acid 5-2 ±0-3** 6-5±0-4NS 7-2 ±0-4* 7-5 ±0-3** 5-4±0-3NS 6-2±O-5NS 7-7 ±0-6** 8-3±O-l** 5-2 ±0-3** 7-O±O-3* 7-1 ±0-3* 8-8 ±0-4** 5-8±0-4NS NS 7-6 ±0-3** 8-8 ±0-4** **P<0-01; *P ^ NS, Not significant (based on student's 't' test). (a) (b) ABA(M) 10"'' 10"^ io"^ io'" io"' ABA4 vaniilic acid (M) ABA(M) I M i,1 i ''" 10' 10"* lo"" io-' ABA + solicyiic acid (M) Fig. 2. Restoration of ABA (10 ' M) induced suppression of amylase activity by vanillie acid (a) and salicylic acid (b) in Triticum aestivum.

4 296 S. SHARMA, S. S. SHARMA AND V. K. RAI II 2-10"" 10"^ 10"^ 10^" 10"' ABA+cinnamic ocid (M) ABA4 ferulic ocid ( M) Fig. 3. Restoration of ABA (10"" M) induced suppression of amylase activity by cinnamic acid (a) and ferulic acid (b) in Triticum aestivum, and seedling growth by inhibiting amylase activity, thus checking the availability of mobilizable carbohydrates essential for these processes. Phenolic compounds are able to remove this check on amylase activity, and they can restore germination and seedling growth by increasing the availability of mobilizable carbohydrates. It is possible, therefore, that these phenolic compounds could have a regulatory role in the processes of germination and seedling growth. Under natural conditions, phenolic compounds are present in the seed coat, cotyledons etc. and have been imphcated in dormancy (Bewley & Black, 1982). They are also available through soil from where they produce allelochemic effects. Reversal of ABA-induced stomatal closure (Rai, Sharma & Sharma, 1986) and abscission (Apte & Laloraya, 1982) by phenolic compounds has been observed earlier, and possibilities of ABA-inactivation by these compounds have been eliminated, (Ray et al., 1980). The mechanism by which they antagonize the inhibitory action of ABA, however, remains to be examined. However, since the effects are observed in vitro, they seem to be at allosteric level. Binding of ABA to proteins in the plasma membranes of guard cells has been reported, and this could be the point at which the phenolic compounds interfere with the action of ABA in other cell types (Hornberg & Weiler, 1984). ACKNOWLEDGEMENTS This work was supported by the Department of Environment, Government of India through Himalayan Eco-Development projects. REFERENCES APTE, P. V. & LALORAYA, M. M. (1982). Inhibitory action of phenolic compounds on ahscisic acid induced abscission. Jourrtal of Experimental Botany, 33, BEWLEY, J. D. & BLACK, M. (1982). Physiology artd Biochemistry of Seeds in Relation to Germination vol. 2, Viability, dormancy and environmental control. Springer-Verlag, Berlin. CHRISPEELS, M. J. & VARNER, J. E. (1966). Inhibition of gibberellic acid induced formation of a-amylase by abscisin II. Nature, 212,

5 Phenols, ABA and amylase from wheat seeds 297 CORNFORTH, J. W., MILBORROW, B. V., RYBACK, G. & WAHEING, P. F. (1965). Chemistry atid physiology of 'dormins' in sycamore. Identity of sycamore 'dormin' with abscisin I, Nature, 205, FILNER, P. & VARNER, J. E. (1967). A test for de novo synthesis of enzymes; Density labeling with HjO" of barley a-amylase induced by gibberellic acid. Proceedings of National Academy of Sciences, 58, Ho, D. T. & VARNER, J. F. (1976). Response of barley aleurone layers to abscisic acid. Plant Physiology, 57, HoRNDERG, C. & WEILER, E. W. (1984). High affinity binding sites for ABA on the plasmalemma of V.faba guard cells. Nature, 310, KARSSEN, C. M. (1982). Indirect effect of ABA on the induction of secondary dormancy in lettuce seeds. Physiologia Plantarum, 54, KEFELI,V. L&KADyROV,C. S.( 1971). Natural growth inhibitors, their chemical and physiological properties. Annual Review of Plant Physiology 22, Lo-WRY, O. H., RosEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with Folin phenol ntagent. Journal of Biological Chemistry, 193, MILBORROW, B. V. (1974). Chemistry and Physiology of abscisic acid. Annual Review of Plant Physiology 25, PAWAR, M. S., RAY, S. D. & LALORAYA, M. M. (1985). Antagonistic action of phenolic compounds on abscisic acid - induced itihihition of seed germination. Biochemische und Physiologische der Pflanzen, 60, RAI, V. K., SHARMA, S. S. & SHARMA, S. (1986). Reversal of ABA induced stomatal closure by phenolic compounds. Journal of Experimental Botany 39, RAY, S. D., GURUPRASAD, K. N. & LALORAYA, M. M. (1980). Antagonistic action of phenolic compounds on abscisic acid induced inhibition of hypocotyl growth. Journal of E.xperimental Botany, 31, ROY, T., GHOSE, B. & SIRCAR, S. M. (1973). Cyclic AMP promotion an abscisic acid inhibition of a-amylase activity in the seeds of rice (Orysa sativa h,.) Journal of Experimental Botany. 24, SHARMA, S. M. & TAYAL, M. S. (1984). EtTect of some phenols and GA^ on germination, early seedling growth and a-amylase and protease activity of Cicer arietinum L. Journal of Indian Botanical Society, 63, STOM, D. J. (1982). EfTect of polyphenols on shoot and root growth and on seed germination. Biologia Plantarum, 24, 1-6.

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