EFFECT OF GIBBERELLIC ACID ON POLYPHENOL OXIDASE ACTIVITY IN DE EMBRYONATED WHEAT AND BARLEY GRAINS

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1 New Phytl. (1977) 78, EFFECT OF GIBBERELLIC ACID ON POLYPHENOL OXIDASE ACTIVITY IN DE EMBRYONATED WHEAT AND BARLEY GRAINS BY P. H. JENNINGS* and C. M. DUFFUS Department f Agricultural Bichemistry, University f Edinburgh, Schl f Agriculture, Edinburgh, Sctland. {Received 8 September 1976) SUMMARY De-embrynated grains f wheat and barley shwed varius respnses when incubated with gibberellic acid (GA3). Amylase activity in the three cultivars studied shwed similar respnses t GA3 cncentratin and perid f incubatin. Changes in -diphenlase and mnphenlase activities f ply phenl xidase (-Diphenl: xygen xidreductase, E.C.I ) which ccurred when de-embrynated grains were incubated in the absence f GA3, were enhanced by the hrmne. Activity was prmted in extracts frm wheat halfgrains, but inhibited in extracts frm the tw barley cultivars. Whereas mnphenlase activity was detected primarily in extracts f the wheat aleurne layer, it was nly detectable in the barley endsperm extracts. The inhibitry effect f GA3 n mnpheniase activity in the barley cultivars is discussed. INTRODUCTION Plyphenl xidase activity has been studied in a number f plants and plant tissues in relatin t plant disease develpment, wunding f plant tissue and hrmnal regulatin. N clear cut picture emerges as regards the first. In sme cases the amunt f detectable plyphenl xidase activity increases with infectin f the hst plant by the pathgen (Jennings, Branneman and Zscheile, 1969; Kammen and Bruwer, 1964; Lvrekvich, Lvrekvich and Stahmann, 1967; Maxwell and Bateman, 1967), while in thers the activity decreases (Farkas and Lvrekvich, 1965). Als, it is unclear whether these changes in enzyme activity are primary r secndary respnses t disease develpment. In additin, mechanical wunding f plant tissue may result in increased plyphenl xidase activity (Hulme and Rhdes, 1971; Hyd and Uritani, 1966). Of the hrmnes tested fr their effect in regulating plyphenl xidase activity in plants, bth ethylene (Abeles, 1972; Herrer and Hall, 1960; Stahmann, Clare and Wdbury, 1966) and indleacetic acid (Staffrd and Galstn, 1970; Vernn and Straus, 1972) have been shwn t cause an increase in enzyme activity in cttn leaves and tbacc pith cultures respectively. This by n means suggests a universal respnse r a direct cause and effect relatinship. Gibberellic acid (GA3) has been shwn t stimulate plyphenl xidase (mnphenlase) activity (Taneja and Sachar, 1974). As there are several ther well-knwn enzyme systems which have been shwn t be regulated by GA3 in cereal grains during germinatin, it was f interest t test the generality f the plyphenl xidase respnse using de-embrynated grains. Fr this purpse ne cultivar f spring wheat (Kleiber) and tw cultivars f spring * Present address: Department f Plant and Sil Science, University f Massachusetts, Amherst, USA. 383

2 384 P. H. JENNINGS and C. M. DUFFUS barley (NaJkta and Glden Prmise) were selected. It was necessary t dehusk nly the Glden Prmise since bth Nakta and Kleiber lack an adhering husk at maturity. Amylase activity was als mnitred in this study t serve as a basis fr cmparisn with a knwn GA3 effect. N attempt was made t distinguish between alpha- and beta-amylase. MATERIALS AND METHODS De-embrynated grains were surface-sterilized with 0.02% mercuric chlride fr 10 min and rinsed ten times with distilled water. The half-grains (twenty) were placed in 5-cm Petri dishes with 3.5 ml f incubatin medium cntaining the required cncentratins f GA3, incubated at 25 C in the dark, then remved fr hmgenizing; the residual incubatin medium (RIM) was saved fr enzyme assay. Cntrls were treated similarly, but in the absence f GA3. Enzyme extract A mdified methd f Taneja and Sachar (1974) was used where ten half-grains were hmgenized with a chilled mrtar and pestle using 4 ml f 0.05 M sdium phsphate buffer (ph 6.6) and centrifuged at 20,000^ fr 15 min. The supernatant was used as the crude enzyme extract fr all enzyme assays. Amylase activity The assay mixture cntaining 1 ml f a sluble starch substrate (0.3 g/50 ml cntaining 1.5 ml f 1 M acetate buffer, ph 5.2), 0.4 ml enzyme and 0.1 ml water. The amunt f enzyme was adjusted accrding t the activity f the extracts and the difference made up with water t maintain a cnstant vlume f 1.5 ml in the assay mixture. AO.l-ml aliqut was withdrawn after 2 and 4 min f incubatin at 30 C and added t 5 ml f idine slutin (0.254 g I2 and 4 g KI/1). Absrbance was measured at 608 nm and activity expressed as change in absrbance per half-grain under the cnditins f assay. Plyphenl xidase activity The assay f mnphenlase and -diphenlase activity was a mdified methd f Taneja and Sachar (1974). Mnphenlase activity was assayed using 1 ml crude enzyme extract, 2 ml L-tyrsine 0.5 mg/ml) and 1 ml 0.05 M phsphate buffer, ph 6.6. The mixture was incubated at 37 C fr 3 h and absrbance measured at 430 nm. Fr -diphenlase activity the reactin invlved 0.5 ml crude enzyme extract, 2 ml catechl (10 mg/ml) and 0.5 M phsphate buffer, ph 6.6. The change in absrbance was measured at 430 nm and was linear fr at least 1 min. Reactin mixtures lacking substrate served as cntrls fr bth assays. Nitrgen and -diphenl determinatins The nitrgen cntent f the dry de-embrynated grains was determined by the micrkjeldahl prcedure. Fr the -diphenl determinatin, 5 g f dry, de-embrynated grains were extracted with 20 ml biling 80% ethanl fr 5 min. After decanting, the residue was re-extracted 3 times, each with 20 ml biling 80% ethanl fr 10 min. The extracts were cmbined, stred fr at least 24 h at 4 C and then centrifuged fr 20 min at 3,000 rev/min. The supernatant was made up t 100 ml with 80% ethanl and aliquts taken fr determinatin f -diphenl cntent by a mdified prcedure f Mapsn, Swain and Tmalin,

3 Gibberellic acid and plyphenl xidase 385 (1963). T 10 ml f extract r a suitable dilutin, was added 2 ml f 4% sdium mlybdate in 50% ethanl. Anther 10 ml sample was treated with 2 ml f 50% ethanl t serve as a cntrl and the absrbance measured at 370 nm. When the efficiency f the extractin prcedure was checked by adding a knwn amunt f catechl t the sample befre extractin, 100% recvery was attained. A standard curve was prepared using catechl and results are expressed as mg -diphenl in terms f catechl equivalents. Absrbance was linear between 0.01 and 0.08 mg catechl/ml. All experiments were cnducted at least twice and unless stated therwise the results presented represent the average f duplicate experiments. RESULTS The effect f GA3 n amylase activity extracted frm the de-embrynated grains is characteristic f this well-dcumented respnse. A slight enhancement f activity is bserved at 10'^ M GA3 with maximum respnse between 10"^ and 10"^ ivi GA3(Fig. 1). Althugh the level f activity is quite similar in the three cereals. Glden Prmise releases mre than twice as much amylase t the incubatin medium as the ther tw cultivars. The time curse f activity is als quite similar amng the three cereals tested (Fig. 2). The 24-h lag in Kleiber may refiect a slwer uptake f GA3 due t its thicker pericarp. Little, if any, change in activity ccurs in the absence f added GA3. The effect f GA3 n -diphenlase activity is mre cmplex and differences in respnse are seen between the wheat and the tw barleys. Significant levels f enzyme activity are detectable at zer time in all three cereals with Nakta having the highest (Fig. 3). Activity in Kleiber increases ver the whle f the 72-h incubatin perid being enhanced by GA3 after 48 h. In cntrast, activity in Nakta decreased with time f incubatin and the presence f 400r 400r 300- k / V ^ J lg GA cncentratin Fig Hurs incubatin at 25 C Fig. 2 Fig. 1. Effect f GA3 cncentratin n amylase activity (AA^OS) extracted frm deembrynated grains incubated 48 h at 25 C and activity secreted t the residual incubatin medium (RIM), (n) Kleiber; () Nakta; (A) Glden Prmise., half-grain;, RIM. Fig. 2. Effect f 10"* M GA3 n amylase activity (^Ajg) extracted frm de-embrynated grains, (a) Kleiber; () Nakta; (A) Glden Prmise., Cntrl;, 10"^ MGA.

4 386 P. H. JENNINGS and C. M. DUFFUS GA3 resulted in even lwer activity cmpared with the cntrl after 48 and 72 h. The - diphenlase activity f Glden Prmise, which is lwest f the three grains at zer time, increased slightly during incubatin as with Kleiber, but was reduced by GA3 after 48 h. Whereas GA3 had an inhibitry effect n -diphenlase activity ver the whle range f cncentratin tested n the barleys, activity appears t be stimulated in Kleiber between 10""^ and 10"^MGA3(Fig.4). An inhibitry effect f GA3 n mnphenlase activity is als bserved with the tw barley cultivars, but nt s with Kleiber (Fig. 5). Mnphenlase activity is present at zer time in all three cereals, but in this case Kleiber has the highest initial level and this increases during incubatin being enhanced slightly after 48 h incubatin with GA3 (Fig. 6). Activity remained fairly cnstant in the absence f GA3, but Glden Prmise exhibited a marked reductin as early as 24 h when GA3 was present. _ -D 70 - c I'60 " x: ^ 50 - \v. ^ n 7- / ^^ 0 ^ 5 70 D-// ^ 60 ILU/ S40 K f 30 - > B 20 A-* - " ^ ^ ^ ' ^ ' ^ ~ ^.- x>~.- ^ - ^ -0 S 40 TO f 30 I "~" -A- - A 10-1 U Hurs incubatin at 25 C Fig _L lg GA cncentratin Fig. 4 Fig. 3. Effect f 10"' M GAj n -diphenlase activity extracted frm de-embrynated grains. ( ) Kleiber; () Nakta; (A) Glden Prmise., Cntrl;, 10'^ MGA. Fig. 4. Effect f GA, cncentratin n -diphenlase activity extracted frm de-embrynated grains incubated 48 h at 25 C. ( ) Kleiber; () Nakta; (A) Glden Prmise X S 150 p a 1? lg GA cncentratin Fig Hurs incubatin at 25 C Fig. 6 Fig. 5. Effect f GA3 cncentratin n mnphenlase activity (AA^J^) extracted frm deembrynated grains incubated 48 h at 25 C. ( ) Kleiber; () Nakta, (A) Glden Prmise. 72 Fig. 6. Effect f 10"^ M GA3 n mnphenlase activity extracted frm deembrynated grains. ( ) Kleiber; () Nakta; (A) Glden Prmise., Cntrl;,

5 Gibberellic acid and plyphenl xidase 387 In rder t lcate the enzyme activities under study, the aleurne layer and endsperm were separated after 24 h f incubatin. In this case the aleurne layer als included the pericarp. The results indicate that -diphenlase and amylase activities are present in extracts f bth the aleurne and endsperm f all three cereal half-grains (Table 1). Mnphenlase activity, hwever, was nt detectable in aleurne extracts f the tw barley cultivars but was detectable in the endsperm extracts. Kleiber, n the ther hand, exhibited a high level f mnphenlase activity in aleurne extracts and very little in the endsperm extracts. The -diphenl cntent f the dry half-grains des nt appear t be crrelated with the initial levels f -diphenlase activity (Table 2). Kleiber, which develps the highest level f enzyme activity during incubatin, cntained the least -diphenl in the dry grain, while Glden Prmise, which maintained the lwest level f activity during incubatin, was fund t cntain the highest -diphenl cntent. Table 1. Enzyme activities in extracts f aleurne and endsperm separated frm deembrynated grains after 24 h f incubatin. Cultivar Kleiber Nakta Glden Prmise Aleurne Endsperm -diphenlase Mnphenlase Amylase -diphenlase Mnphenlase Amylase Mg caiecni catechl -^^430/ ^^^^J ^KJ ^^tj Mg catechl ^k^j AA / AA ^\J/ xidized/min half-grain half-grain xidized/min half-grain half-grain half-grain (X 10'') (X 10"') half-grain (X 10'') (X 10"') Table 2. Nitrgen and -diphenl cntent f dry, de-embrynated grains Cultivar Kleiber Nakta Glden Prmise Nitrgen (mg N/half-grain) DISCUSSION -diphenl (as Mg catechl/half-grain) Amylase activity extracted frm the three cereals studied exhibited a very similar respnse t GA3 cncentratin and time f incubatin; little change ccurred when the de-embrynated grains were incubated in the absence f the hrmne (Figs. 1 and 2). The activities f -diphenlase and mnphenlase, n the ther hand, varied widely amng the three grains even at zer time and changes in activity ccurred during incubatin in the absence f GA3 (Figs. 3 and 6). Fr the mst part, GA3 nly enhanced thse changes in activity which ccurred during incubatin and in the case f the tw barley cultivars, lwered the level f detectable activity extracted frm the half-grains (Figs. 4 and 5). In cntrast t these results, Taneja and Sachar (1974) reprted an inhibitin f -diphenlase and stimulatin f mnphenlase activities by GA3 with the wheat cultivar Sharbati Snra. These differences in respnse t GA3 suggest that the regulatin f -diphenlase and mn-phenlase in incubated, de-embrynated cereal grains is different frm that shwn fr alpha-amylase and prtease (Chrispeels and Varner, 1967; Glasziu, 1969; Jacbsen and Varner, 1967; Paleg, 1960).

6 388 P. H. JENNINGS and C. M. DUFFUS The inhibitry effect f GA3 n -diphenlase and mnphenlase activities when the tw barley cultivars were incubated with the hrmne might pssibly be the result f GA3stimulated prtease activity. A similar interpretatin was made in the case f nitrate-induced nitrate reductase activity f barley aleurne layers in which GA3 was shwn t have an inhibitry effect (Ferrari and Varner, 1969). It was als shwn that nitrate had n effect n GA3-stimulated amylase activity. The difference in nitrgen cntents f the three cereal grains suggests that Kleiber has a higher prtein cntent than the tw barley cultivars (Table 2). This may serve as a suitable substrate fr prtease activity, thus diverting the enzyme and thereby sparing the plyphenl xidases frm prtelytic attack. The lwer prtein cntents f the tw barley cultivars may nt be as effective in this regard and thus plyphenl xidase activity is lwered thrugh GA3-stimulated prtelysis. The lcatin f enzyme activities within the de-embrynated grains appears t differ between the three cereal grains. Taneja and Sachar (1974) lcated mnphenlase activity in the aleurne layer f the wheat cultivar used in their study and this agrees with ur results fr Kleiber (Table 1). Hwever, in the present wrk, mnphenlase activity was detectable nly in the endsperm extracts'f the tw barley cultivars tested. Thus, if mnphenlase is subject t prtelytic attack and GA3 prmtes prtease activity which is then secreted t the endsperm, then mnphenlase activity in Nakta and Glden Prmise might be expected t decrease with time f incubatin with GA3 and increasing GA3 cncentratin (Figs. 5 and 6). Bth -diphenlase and amylase activities, n the ther hand, were fund in extracts f the aleurne and endsperm f all three grains. Althugh it is pssible that sme mvement f enzyme activity between the tw tissues may have ccurred at the time f tissue separatin (24 h f incubatin) and that the tissues were nt cmpletely separated, it is felt that the lcalizatin f activity bserved represents fairly clsely the rigins f the enzymes in questin. The evidence presented suggests that there are varietal differences in the respnse f diphenlase and mnphenlase activities t GA3 and that the hrmne in part serves t enfrce changes in activity which take place in its absence. Further studies n the effect f GA3 n -diphenlase are being pursued and will be reprted in anther paper. REFERENCES ABELES, F.B. (1972). Bisynthesis and mechanism f actin f ethylene,/4nn. Rev. Plant, Physil., 23, 259. CHRISPEELS, M.J. & VARNER, J.E. (1967). Gibberellic acid enhanced synthesis and release f alphaamylase and ribnuclease by islated aleurne layers./*/. Physil, Lancaster, 42, 398. FARKAS, G.L. & LOVREKOVICH, L. (1965). Enzyme levels in tbacc leaf tissues affected by wildfire txin. Phytpath., 55,519. FERRARI, T.E. & VARNER, J.E. (1969). Substrate inductin f nitrate reductase in barley aleurne layers. PI. Physil., Lancaster, 44, 85. GLASZIOU, K.T. (1969). Cntrl f enzyme frmatin and inactivatin in plants. Ann. Rev. PI. Physil., 20,63. HERRERO, F.A. & HALL, W.C. (1960). General effects f ethylene n enzyme systems in the cttn leaf. Physil. Plant, 13, 736. HULME, A.C. & RHODES, M.J.C. (1971). Pme fruits. In: The Bichemistry' f Fruits and their Prducts, (Ed. by A. C. Hulme), Vl. 2, pp Academic Press, New Yrk and Lndn. HYODO, H. & URITANI, 1. (1966). A study n increase in -diphenl xidase activity during incubatin f sliced sweet ptat tissue. Plant and Cell Physil., 7, 137. JACOBSEN, J.V. & VARNER, J.E. (1967). Gibberellic acid induced synthesis f prtease by islated aleurne layers f barley. PL Physil., Lancaster, 42, 1596.

7 Gibberellic acid and plyphenl xidase 389 JENNINGS, P.H., BRANNEMAN, B.L. & ZSCHEILE, P.P. Jr. (1969). Perxidase and plyphenl xidase activity assciated with Helminthsprium leaf spt f maize. Phytpath., 59, 963. KAMMEN, A.V. & BROUWER, D. (1964). Increase in plyphenl xidase activity by a lcal virus infectin in uninculated parts f leaves. Virlgy, 22, 9. LOVREKOVICH, L., LOVREKOVICH, H. & STAHMANN, M.A. (1967). Inhibitin f phenl xidatin by Erwinia cartvra in ptat tuber tissue and its significance in disease resistance. Phytpath., 57, 737. MAPSON, L.W., SWAIN, T. & TOMALIN, A.W. (1963). Influence f variety, cultural cnditins and temperature f strage n enzyme brwning f ptat tubers. /. 5c/. Fd Agric, 14, 673. MAXWELL, D.P. & BATEMAN, D.F. (1967). Changes in the activities f sme xidases in extracts f Rhizctnia-irvf&cX&d bean hypctyles in relatin t lesin maxar^xxn. Phytpath., 57, 132. PALEG, L.G. (1960). Physilgical effect f gibberellic acid: I. On carbhydrate metablism and amylase activity f barley endsperm. P/. Physil, Lancaster, 35, 293. STAFFORD, H.A. & GALSTON, A.W. (1970). Ontgeny and hrmnal cntrl f plyphenl xidase iszymes in tbacc pith. PI. Physil, Lancaster, 46, 763. STAHMANN, M.A., CLARE, B.G. & WOODBURY, W. (1966). Increased disease resistance and enzyme activity induced by ethylene and ethylene prductin by black rt infected sweet ptat tissue. PI Physil, Lancaster, 41,1505. TANEJA, S.R. & SACHAR, R.C. (1974). Stimulatin f plyphenl xidase (mnphenl xidase) activity in wheat endsperm by gibberellic acid, cyclheximide and actinmycin D. Planta (Berl), 116,133. VERNON, S.L. & STRAUS, J. (1972). Effects f IAA and 2, 4-D n plyphenl xidase in tbacc tissue cultures. Phytchem., 11, 2723.

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