AXIAL CONTROL OF PROTEASE DEVELOPMENT IN COTYLEDONS OF HORSE GRAM (MACROTYLOMA UNIFLORUM LAM.) SEEDS DURlNG GERMINATION

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1 indian J. Piant Physlol \Tot xxxin, No 3, pp (Sept., i99o) AXIAL CONTROL OF PROTEASE DEVELOPMENT IN COTYLEDONS OF HORSE GRAM (MACROTYLOMA UNIFLORUM LAM.) SEEDS DURlNG GERMINATION D. KARUNAGARAN* AND P. RAMAKRISHNA RAO Department of Biochemistry, Sri Krishnadevaraya University, Anantapur Received on 20 Sept., 1990 SUMMARY An intact axis was found to be essential for the maximal rates of increase in protease activity, protein degradation and amino acid utilization in the cotyledons of horse gram during gennination. Inhibition of protease activity by cycloheximide was suggestive of dt! novo synthesis of the enzyme. Gibberellic acid and benzyladenine did not replace the effect ofthe axis on protease. The amino acid content increased continuously in detached cotyledons thongh the protease development was lower. Casein hydrolysate inhibited the protease development and its withdrawl from the medium stimulated the activity. The results suggest that the axis acts as a sink for the products of hydrolysis and in the absence ofthe axis, the amino acids accumulate restricting further enzyme activity. INTRODUCTION The embryonic axis is known to infl.uence the mobilization of food reserves through the development of hydrolytic enzyme activities in the cotyledons of germinating seeds of dicots. However, the axial control of food reserve mobilization, in general, and protein mobilization, in particular, is not well understood (Bewley and Black, 1978). Cytokinins probably act as mediators of axial control in beans (Gepstain and Ilan, 1980). In peas, the axis acts as a sink for the products of enzyme action and in the absence of the axis, the end products accumulate thereby inhibiting the enzyme activity, by a feedback regulatory mechanism (Yomo and Varner, 1973). However, this has been found non-functional in mung beans (Morohasbi, 1982). Probably the mechanism (s) of axial control differ from species to species and an extensive examination is needed to confirm/discern from unified * Praent address PSG College of Arts and Science, Coimbatore

2 PllOTEASE IN COTY~NS OP HOltSB OllAM 233 mechanism of axial control of protein reserve mobilization in this group of higher plants (Davies and Slack, 1981). The changes in protease activity over a 4-day period of germination in the cotyledons of horse gram in the presence and absence of the axis, and the involvement of hormonal and/or feedback regulatory mechanism (s) were investigated. MATERIALS AND METHODS Seeds of Co-I variety of horse gram (Macro tyloma unijlorum Lam.) were obtained from the Tamil Nadu Agricultural University, Coimbatore. Seeds were surface sterilized with 0.1 % mercuric chloride for 5 min, rinsed and anowed to imbibe distilled water for 4 hr. Seed coats were removed and the cotyledons separated from each other, so that the axis remained attached to one of them. The cotyledons with (attached) or without (detached) axis were incubated in Petri dishes (10 em diameter) lined with 2 layers of filter paper kept moistened with 8 ml of distilled water or test solution. The Petri disbes were placed in a tray, covered with polythene sheets and germination was carried out in the dark at 28 2 C. Fresh additions of water or test solution were made, every day and sterile conditions were maintained by including streptomycin sulfate (20 ppm) in the incubation medium. The period of germination was measured from the time when the plant materials were transferred to the Petri dishes. The experiments were carried out in duplicate and each experiment was repeated atieast three times. Protease assay: Cotyledons (5 pairs) were homogenized using 6 ml of 0.05 M Tris-HCI buffer containing M cysteine (ph 7.2) and filtered tbrough 4 layers of cbeese cloth. Centrifuged at 10,000 X g for J5 min in a refrigerated centrifuge and the supernatant was used as the enzyme source. All operations were carried out at 2-4"C and completed within 5 min. Protease was assayed by the method of Beevers (1968). The reaction mixture contained 1 mt of extract, 1 ml of casein (l% w/v prepared in 0.1 N NaOH and the ph was adjusted to 7.0 using O.t N HCI) and 1 ml of 0.2 M citrate buffer (ph 5.5). Incubation was carried out for 60 min at 40"C. The reaction was arrested by the addition of 1 ml of 20% TCA. The mixture was centrifuged (1000 X g, 15 min) and the supernatant was used for the determination of amino acid content by the method of Rosen (1957). Estimation ofprotein and amino acid content: Cotyledons were plunged into boiling 80% (v/v) ethanol. and homog..nized The homogenate was centrifuged (1000 X g. 15 min), the residue reextracted with ethanol, the supernatants made up to a known volume and used for the estimation of amino acids (Rosen, 1957). Proteins were precipitated from the residue by ]0% TCA, dissqtv~4 in, O~ t ~ ~aofl ~n4 4et~:PC?~ of its protei~ ~~t~n~,lowry et' al., 195] ~~

3 234 D. KARUNAOAltAN AND P. RAMAKRISHNA RAO RESULTS AND DISCUSSION The increase in protease activity was 9 fold in the attached cotyledons and 3 fold in the detached cotyledons from day 0 to day 3. The activity decjined thereafter in either case (Fig. 1). The rate of protein decline in the attached cotyledons during 4-day period was greater than that in the detached cotyledons (Fig. 2). The amino acids did not accumulate after day 2 in the attached cotyledons, where the protease development was higher, contrarily they accumulated continuously in the detached cotyledons where as protease development was lower (Fig. 3). These results indicate that the maxima] rates of increase in protease activity. protein degradation and amino acid utilization in horse gram cotyledons depended upon the presence of the axis. Axis excision restricted the protease increments, similar to reported in mung bean (Morohashi. 1982). and buckwheat (Dunaevsky and Belozersky, 1988), but not in beans. (Yomo and Srinivasan, 1973). The inhibition of protease development by cycloheximide in horse gram (Table I) suggested the need for de novo synthesis of proteins for the process, similar to ones reported in other systems (Bose t Fig. 1. Effect of axis excision on the development of protease activity in the cotyledons of horse gram during germination., ~.orc--'--:--'---'-.,-+-...l---l...~-,t-,,...j-..-l.--..l...,...j.---l~

4 P~OT.eASB IN COTYLEDONS OF HORSE GRAM 235, LU <Ii a z III ~ 4 ~ 1&1 t o tlc Q. 10 " TI.14E (DAYS)_ Fig. 2. Effect of axis excision on protein eontent in the cotyledons of horse gram during germination. Table I. Effect of cycloheximide on protease activity in attached and detach~ cotyledons. Each value is the mean of 6 replicates followed by SE Protease activity (mg amino acid/hr/io cotyledons) Time Attached cotyledons Detached cotyledons (days) Control Treated Control Treated O2 O.64O.O SO O.OS 1.65O.O2 1.35O O O O.99O S OS O.92O.02

5 236 D. KARU~AGAllAN AND '.llamaklushna UO I ' U U tao ;; 0 co ~ " Ii u Ii!... 1~! '" co ụ. I.. i 0 iii 12 iii II. Fig. 3. Bffect of axis excision on. amino acid content in the cotyledons of horse gram during germination. 1 Q '.8 8.0, Q 2 TIME (DAYS)_ Table II. Effect of casein hydrolysate on protease development and amino acid content in attached and detached cotyledons Protease activity (mg amino acid/hr/l0 cotyledons) Amino acid content (mg/l0 cotyiedons) Time Attached Detached Attached Detached (days) cotyledons cotyledons cotyledons cotyledons I Control Treated Control Treated Control Treated Control Treated ])

6 PROtEASE in cot~ledons OF BoRsa GitAM et at., 1982; Ihie and Dure, 1969; Srivastava et at., 1972). The effect of the axis on protease development in horse gram (data not given), could not be replaced by GA & BA, such finding is in confirmity with others (Yomo and Varner, Dunaevsky and Belozersky, 1988; Sharma, 1988), Treatment with 1% casein hydrolysate (to simulate amino acid accumulation) showed a marked inhibitory effect on protease activity in the attached and detached cotyledons of horse gram, the extent of increase in amino acids being higher in the detached cotyledons (Table 11). Inhibition of the protease activity by casein hydrolysate has also been observed in buckwheat (Dunaevsky and Belozersky, 1988). peas (Yomo and Varner, 1973), cucumber (Davies and Chapman, 1980) and mung bean (Kern and Chrispeels, 1978). Withdrawal of casein hydrolysate from the medium after 2 days and replacing it with water for another 2 days stimulated the protease activity (data not shown). Thus, the embryonic axis acting seems to be as a sink for the products of hydrolysis by protease and in the absence of th: axis the end products accumulated restricting further development of the enzyme in the cotyledons of horse gram during germination. REFERENCES Beevers, L. (1968). Protein degradation and proteolytic activity in the cotyledons of germinating pea seeds. Phytochemistry,7: Bewley, J.D. and Black, M. (1978). Physiology and Biochemistry of Seeds in Relation to Germination 1. Development, Germination and Growth. Springer-Verlag, Heidelberg, pp Bose, B., Srivastava, H.S. and Mathur, S.N. (1982). Effect of antibiotics on the germination and protease activity of maize seeds. Indian J. Plant Physiol., 25: Davies, H.V. and Chapman, J.M. (1980). The control offood mobilization in seeds ofcucumis sativus L. III. The control of protein degradation. Planta, 149: Davies, H.V. and Slack, P.T. (1981). The control of food mobilization in seeds of dicotyledon ous plants. New Phytol., S8 : Dunaevsky, Y.E. and Belozersky, M.A. (1988). The role of the embryogenic axls in degradation of the buckwheat seed storage protein. Fiziologiya Rasternii.3S: 1()() Gepstain, S. and llan, I. (1980). Evidence for involvement of cytokinins in the regulation of proteolytic activity in cotyledons of germinating beans. Plant Cell PhysJol., 21 : Ihle, J.N. and Dure. L.S. (1969). Synthesis of a protease in germinating cotton cotyledons catalysed by mrna synthesized during embryogenesis. Biochem. Biophys. Res. Comm.,.36: Kern, R. and Chrispeels, M.J. (1978). Influence of the axis on the enzymes of protein and amide metabolism in the cotyledons of mung bean seedlings. Plant Physiol., 62 : 81S-819. Lowry O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951). Protein measurement with the Folin phenol reagent. J. Bioi. Chern., 19.3 : Morohashi, Y. (1982). Control of development of amylolytic and proteolytic activities of germinating black gram seeds. Physiol. Plant., 56:

7 Rosen, R. (1957). A modified ninhydrin colorimetric analysis for amino acids. Arch. Biochem. Blophys.,67: 1()"15. Sharma, S.O. (1988). Regulation of reserve protein mobilization during germination and early seedling establishment in chickpea. Indian J. Plant Physiol., 31 : Srivastava, A.K., Azhar, S. and Krishna Murti, C.R. (1972). Inhibition of germination in Cieer arietinum. Phytochemistry,11 : Yomo, H. and Srinivasan, K. (1973). Protein breakdown and formation of protease in attached and detached cotyledons of Phaseolus 'Vulgaris (L) Plant Physiol., 52 : Yomo,lI. and Varner, l.e. (1973). Control of the formation of amylases and proteases in the Cotyledons of germinating peas. Plant Physioi, 51 :