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Regulation of senescence in detached rice leaves by light, benzimidazole and kinetin
Exp. Geront. Vol. 8, pp. 153-155. Pergamon Press 1973. Printed itx Great Britain.
REGULATION OF SENESCENCE IN DETACHED RICE LEAVES BY LIGHT, BENZIMIDAZOLE AND KINETIN* D. MISHRAand P. PRADHAN Plant Biochemistryand EnzymologyLaboratory, Department of Botany, Utkal University,Bhubaneswar-4, Orissa, India (Received 20 February 1973)
INTRODUCTION THE SENESCENCEof detached leaves is characterised by the loss of chlorophyll and protein (Vickery et al., 1937; Gregory and Sen, 1937). The rate of sensecence is retarded by an exogenous application of benzimidazole (Mishra and Waygood, 1968; Mishra and Misra, 1968; Mishra and Samal, 1971; Mishra and Ka, 1973) or kinetin (Mishra and Waygood, 1968; Sugiura, Umemura and Oota, 1962). Very few reports have, however, brought to light the effect of red and far-red (R and FR) light in regulating the senescence of leaves. A recent report (De Greef and Fredericq, 1972) has emphasized the enhancement of senescence in mature, green tissue of intact thalli of Marchantia polymorpha by FR light. As relatively little work has been done on the effect of R and FR light in regulating the senescence of detached leaves, this work was undertaken to analyse the effect of benzimidazole and kinetin in regulating the senescence of detached leaves under dark, R and FR illumination. EXPERIMENTAL Seven-week-old rice (Oryza sativa Vat. BBS 873) plants were selected and the upper 5 cm leaf segments were thoroughly washed in water and randomized. A sample of 5 leaf segments weighed ca. 200 rag. The leaves were floated with adaxial side up in sterile 10 cm Petri dishes containing 30 ml of distilled water (control) or aqueous solutions of benzimidazole (10- aM) or kinetin (2"5 x 10- 5M).All manipulations of the sample were performed aseptically. The dishes were kept in the dark at 27°C or under continuous red (Witham, Blaydes and Devlin, 1971) (590-700 nm) or far-red (Witham, Blaydes and Devlin, 1971) (700-735 rim) illumination at 32°C. Samples were taken initially and after periods of incubation for the estimation of chlorophyll (Osborne, 1962) and protein (Gornall, Bardawill and David, 1949). Data are expressed as the percentage of the initial amount remaining after the experimental treatment. The initial values of the 5 apical leaf segments weighing ca. 200 mg for (i) chlorophyll: extracted in 80 per cent ethanol (25 ml) averaged Ae65 nm ---1"4 _+ 0"05 (ii) protein: 18"8 mg as bovine serum albumin equivalent. The criteria of senescence in this experiment are the net changes in chlorophyll and protein over time. The experiment was repeated thrice. RESULTS AND DISCUSSION As shown in the figure, FR illumination speeded the rate of chlorophyll breakdown but R illumination retarded the process compared to the dark. The rate of protein breakdown *Part IV of the series "Studies on Leaf Senescence".Listed as paper No. 5 of the Plant Biochemistryand Enzymology Laboratory of the Department of Botany, Utkal University. 153
154
D. MISHRA AND P. PRADHAN
Chlorophyll
Profein
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60
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20
I
I
BZI
I00
I BZl
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I
60
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ne,,n o
IOC
It
60
2C 0
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8
I0 0
[
I
[
I
I
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I0
Days- after detachment
FIG. l.Time course of chlorophyll or protein loss during senescenceunder dark, red or far-red light. Detached leaves floated on water, benzimidazole (BZI) or kinetin. Days of incubation indicated on abscissa and percentage of the initial content on the ordinates. was also retarded by R illumination compared to the dark. Treatment with benzimidazole retarded the senescence under all conditions. There was less than 20 per cent loss of chlorophyll under R light due to benzimidazole treatment even after 8 days of detachment. This treatment was the best effective in retarding the senescence. When the leaves were floated on benzimidazole and kept under R or F R light the senescence was also retarded considerably compared to the water floated leaves. Kinetin treatment initially decreased the protein level under dark but the protein level was maintained thereafter up to 10 days of detachment and this finding is in close agreement to a report for excised barley leaves (Atkin and Srivastava, 1969). R light treatment maintained the level of protein in the leaves floated on kinetin. However, there was no effect of kinetin in retarding the chlorophyll breakdown either under dark, R or F R light. The results of the present study indicate that light is of importance in regulating the senescence of excised leaves, there being an enhancement by F R and a retardation under R light. There is a report (Sugiura, 1963) for a phytochrome mediated control of protein and chlorophyll loss in tobacco leaf discs. In view of the F R enhancement and R light retardation of chlorophyll loss in detached rice leaves a phytochrome mediated control of senescence may be suggested. The light effect on the chlorophyll level may be involved in the breakdown steps since the presence of active chlorophyll synthesis in mature leaves is in question (Kirk and Tilney-Bassett, 1967). It is suggested (Parthier, 1961; Parthier, Malavia and
REGULATION OF SENESCENCE IN DETACHED RICE LEAVES
155
Mothes, 1964) that light effect is caused by A T P p r o d u c t i o n f r o m photosynthetic phosphorylation. Benzimidazole or kinetin m a i n t a i n s the level of A T P a n d N A D P i n wheat leaf chloroplasts (Mishra a n d Waygood, 1968) d u r i n g senescence. Since protein synthesis is itself d e p e n d e n t u p o n a high energy phosphate c o m p o u n d it m a y be suggested that the R light induced r e t a r d a t i o n of senescence in rice leaves is dependent o n photosynthesis a n d is also u n d e r p h y t o c h r o m e control. Acknowledgements--The authors are grateful to Prof. B. Padhi for providing the necessary facilities. This work was supported in part by a grant from the Board of Scientific and Industrial Research, Government of Orissa, to one of us (D.M.) REFERENCES ATKIN, R. K. and SRIVASTAVA,B. I. S. (1969) PhysioL Planta 22, 742. DE GREEF,J. A. and FREDERICQ,H. (1972) Planta 104, 273. GORNALL,A. G., BARDAWILL,C. S. and DAVID, M. M. (1949) J. biol. Chem. 177, 751. GREC_,ORY,F. G. and SEN, P. K. (1937) Ann. Bot. N.S. 1, 521. KIRK, J. T. O. and TILNEV-BAssErr,R. A. E. (1967) The Plastids, p. 482. Freeman, London. MxsrmA, D. and WAYC,OOD, E. R. (1968) Can. J. Biochem. 46, 167. MISrmA, D. and MISRA,B. (1968) Z. PflanzenphysioL 58, 207. MtS.RA, D. and SAMAL,B. (1971) Z. Naturforsch. 26h, 1377. MtSnRA, D. and KAR, M. (1973) Phytochem. 12, in press. OSBORNE,D. J. (1962) Plant PhysioL 37, 595. PARTnmR,B. (1961) Flora 151, 368. PARrnmR, B., MALAVIYA,B. and MoTnES, K. (1964) Plant Cell PhysioL 5, 104. SOGIURA,M., UME~gJRA,K. and OOTA,Y. (1962) Physiol. Planta 15, 457. SOGn_rRA,M. (1963) Botan Mag. (Tokyo) 76, 174. VICKER¥, H. B., PUO-mR, G. W., WAKEMAN,A. J. and LEAVENWORTH,C. S. (1937) Conn. agr. expt. Sta. New Haven Bull. No. 399. WITHAM, F. H., Bt~AYDES,D. F. and DEVLIN, R. M. (1971) In Experiments in Plant Physiology, p. 236. Van Nostrand-Reinhold, New York. Summary--The senescence of excised leaves of Oryza sativa Var. BBS 873 was followed by measuring the net breakdown of chlorophyll and protein with respect to the time of detachment. Kinetin was relatively ineffective but benzimidazole was effective in retarding the senescence. Red light was also effective in senescence retardation but on the other hand, far-red light speeded the senescence. Benzimidazole was the most effective treatment under red light in retarding leaf senescence. The red light induced retardation and the far-red enhancement of senescence is suggested to be under phytochrome control and dependent on photosynthesis. R~sumf----On a observ6 la s6nescence de feuilles d'Oryza sativa Var. BBS 873 excis6es en mesurant la d6gradation nette de la chlorophylle et des prot6ines par rapport au moment du d6tachement. La kin6tine 6tait relativement inefficace, mais le benzimidazole agissait en retardateur de la s6nescence. La lumi6re rouge avait le m6me effet, mais la lumi6re ultrarouge acc616rait la s6nescence. Le benzimidazole 6tait le plus efficace sous la lumi6re rouge pour retarder la s6nescence. On suppose que la retardation de la s6nescence sous l'effet de la lumi6re rouge et son acc61&ationsous celui de la lumi~re ultrarouge sont contr616espar le phytochrome et d6pendent de la photosynthSse. Zusammenfasstmg--Das Altern exzidierter Bl/itter von Oryza sativa Var. BBS 873 wurde durch Messung des Nettoabbaus yon Chlorophyll und Protein in Abhfingigkeit yon der Entnahmezeit verfolgt. Kinetin war relativ wenig wirksam, aber Benzimidazol war wirksam bei der Verlangsamung des Alterns. Rotes Licht war ebenfalls wirksam bei der Alterns-Verlangsamung, aber auf der anderen Seite beschleunigte langwelligeres Rot das Altern. Benzimidazol war die wirksamste Behandlung bei Rotlicht zur Verlangsamung des Alterns. Die durch Rotlicht bedingte Retardation und durch langwelligeres Rot bedingte Beschleunigung des Alterns dOrfte unter Phytochromkontrolle stehen und abh~ingigvonder Photosynthese sein.
REGULATION OF SENESCENCE IN DETACHED RICE LEAVES BY LIGHT, BENZIMIDAZOLE AND KINETIN* D. MISHRAand P. PRADHAN Plant Biochemistryand EnzymologyLaboratory, Department of Botany, Utkal University,Bhubaneswar-4, Orissa, India (Received 20 February 1973)
INTRODUCTION THE SENESCENCEof detached leaves is characterised by the loss of chlorophyll and protein (Vickery et al., 1937; Gregory and Sen, 1937). The rate of sensecence is retarded by an exogenous application of benzimidazole (Mishra and Waygood, 1968; Mishra and Misra, 1968; Mishra and Samal, 1971; Mishra and Ka, 1973) or kinetin (Mishra and Waygood, 1968; Sugiura, Umemura and Oota, 1962). Very few reports have, however, brought to light the effect of red and far-red (R and FR) light in regulating the senescence of leaves. A recent report (De Greef and Fredericq, 1972) has emphasized the enhancement of senescence in mature, green tissue of intact thalli of Marchantia polymorpha by FR light. As relatively little work has been done on the effect of R and FR light in regulating the senescence of detached leaves, this work was undertaken to analyse the effect of benzimidazole and kinetin in regulating the senescence of detached leaves under dark, R and FR illumination. EXPERIMENTAL Seven-week-old rice (Oryza sativa Vat. BBS 873) plants were selected and the upper 5 cm leaf segments were thoroughly washed in water and randomized. A sample of 5 leaf segments weighed ca. 200 rag. The leaves were floated with adaxial side up in sterile 10 cm Petri dishes containing 30 ml of distilled water (control) or aqueous solutions of benzimidazole (10- aM) or kinetin (2"5 x 10- 5M).All manipulations of the sample were performed aseptically. The dishes were kept in the dark at 27°C or under continuous red (Witham, Blaydes and Devlin, 1971) (590-700 nm) or far-red (Witham, Blaydes and Devlin, 1971) (700-735 rim) illumination at 32°C. Samples were taken initially and after periods of incubation for the estimation of chlorophyll (Osborne, 1962) and protein (Gornall, Bardawill and David, 1949). Data are expressed as the percentage of the initial amount remaining after the experimental treatment. The initial values of the 5 apical leaf segments weighing ca. 200 mg for (i) chlorophyll: extracted in 80 per cent ethanol (25 ml) averaged Ae65 nm ---1"4 _+ 0"05 (ii) protein: 18"8 mg as bovine serum albumin equivalent. The criteria of senescence in this experiment are the net changes in chlorophyll and protein over time. The experiment was repeated thrice. RESULTS AND DISCUSSION As shown in the figure, FR illumination speeded the rate of chlorophyll breakdown but R illumination retarded the process compared to the dark. The rate of protein breakdown *Part IV of the series "Studies on Leaf Senescence".Listed as paper No. 5 of the Plant Biochemistryand Enzymology Laboratory of the Department of Botany, Utkal University. 153
154
D. MISHRA AND P. PRADHAN
Chlorophyll
Profein
I00
60
-
-\
20
I
I
BZI
I00
I BZl
1
I
60
2C
ne,,n o
IOC
It
60
2C 0
2
4
6
8
I0 0
[
I
[
I
I
2
4
6
8
I0
Days- after detachment
FIG. l.Time course of chlorophyll or protein loss during senescenceunder dark, red or far-red light. Detached leaves floated on water, benzimidazole (BZI) or kinetin. Days of incubation indicated on abscissa and percentage of the initial content on the ordinates. was also retarded by R illumination compared to the dark. Treatment with benzimidazole retarded the senescence under all conditions. There was less than 20 per cent loss of chlorophyll under R light due to benzimidazole treatment even after 8 days of detachment. This treatment was the best effective in retarding the senescence. When the leaves were floated on benzimidazole and kept under R or F R light the senescence was also retarded considerably compared to the water floated leaves. Kinetin treatment initially decreased the protein level under dark but the protein level was maintained thereafter up to 10 days of detachment and this finding is in close agreement to a report for excised barley leaves (Atkin and Srivastava, 1969). R light treatment maintained the level of protein in the leaves floated on kinetin. However, there was no effect of kinetin in retarding the chlorophyll breakdown either under dark, R or F R light. The results of the present study indicate that light is of importance in regulating the senescence of excised leaves, there being an enhancement by F R and a retardation under R light. There is a report (Sugiura, 1963) for a phytochrome mediated control of protein and chlorophyll loss in tobacco leaf discs. In view of the F R enhancement and R light retardation of chlorophyll loss in detached rice leaves a phytochrome mediated control of senescence may be suggested. The light effect on the chlorophyll level may be involved in the breakdown steps since the presence of active chlorophyll synthesis in mature leaves is in question (Kirk and Tilney-Bassett, 1967). It is suggested (Parthier, 1961; Parthier, Malavia and
REGULATION OF SENESCENCE IN DETACHED RICE LEAVES
155
Mothes, 1964) that light effect is caused by A T P p r o d u c t i o n f r o m photosynthetic phosphorylation. Benzimidazole or kinetin m a i n t a i n s the level of A T P a n d N A D P i n wheat leaf chloroplasts (Mishra a n d Waygood, 1968) d u r i n g senescence. Since protein synthesis is itself d e p e n d e n t u p o n a high energy phosphate c o m p o u n d it m a y be suggested that the R light induced r e t a r d a t i o n of senescence in rice leaves is dependent o n photosynthesis a n d is also u n d e r p h y t o c h r o m e control. Acknowledgements--The authors are grateful to Prof. B. Padhi for providing the necessary facilities. This work was supported in part by a grant from the Board of Scientific and Industrial Research, Government of Orissa, to one of us (D.M.) REFERENCES ATKIN, R. K. and SRIVASTAVA,B. I. S. (1969) PhysioL Planta 22, 742. DE GREEF,J. A. and FREDERICQ,H. (1972) Planta 104, 273. GORNALL,A. G., BARDAWILL,C. S. and DAVID, M. M. (1949) J. biol. Chem. 177, 751. GREC_,ORY,F. G. and SEN, P. K. (1937) Ann. Bot. N.S. 1, 521. KIRK, J. T. O. and TILNEV-BAssErr,R. A. E. (1967) The Plastids, p. 482. Freeman, London. MxsrmA, D. and WAYC,OOD, E. R. (1968) Can. J. Biochem. 46, 167. MISrmA, D. and MISRA,B. (1968) Z. PflanzenphysioL 58, 207. MtS.RA, D. and SAMAL,B. (1971) Z. Naturforsch. 26h, 1377. MtSnRA, D. and KAR, M. (1973) Phytochem. 12, in press. OSBORNE,D. J. (1962) Plant PhysioL 37, 595. PARTnmR,B. (1961) Flora 151, 368. PARrnmR, B., MALAVIYA,B. and MoTnES, K. (1964) Plant Cell PhysioL 5, 104. SOGIURA,M., UME~gJRA,K. and OOTA,Y. (1962) Physiol. Planta 15, 457. SOGn_rRA,M. (1963) Botan Mag. (Tokyo) 76, 174. VICKER¥, H. B., PUO-mR, G. W., WAKEMAN,A. J. and LEAVENWORTH,C. S. (1937) Conn. agr. expt. Sta. New Haven Bull. No. 399. WITHAM, F. H., Bt~AYDES,D. F. and DEVLIN, R. M. (1971) In Experiments in Plant Physiology, p. 236. Van Nostrand-Reinhold, New York. Summary--The senescence of excised leaves of Oryza sativa Var. BBS 873 was followed by measuring the net breakdown of chlorophyll and protein with respect to the time of detachment. Kinetin was relatively ineffective but benzimidazole was effective in retarding the senescence. Red light was also effective in senescence retardation but on the other hand, far-red light speeded the senescence. Benzimidazole was the most effective treatment under red light in retarding leaf senescence. The red light induced retardation and the far-red enhancement of senescence is suggested to be under phytochrome control and dependent on photosynthesis. R~sumf----On a observ6 la s6nescence de feuilles d'Oryza sativa Var. BBS 873 excis6es en mesurant la d6gradation nette de la chlorophylle et des prot6ines par rapport au moment du d6tachement. La kin6tine 6tait relativement inefficace, mais le benzimidazole agissait en retardateur de la s6nescence. La lumi6re rouge avait le m6me effet, mais la lumi6re ultrarouge acc616rait la s6nescence. Le benzimidazole 6tait le plus efficace sous la lumi6re rouge pour retarder la s6nescence. On suppose que la retardation de la s6nescence sous l'effet de la lumi6re rouge et son acc61&ationsous celui de la lumi~re ultrarouge sont contr616espar le phytochrome et d6pendent de la photosynthSse. Zusammenfasstmg--Das Altern exzidierter Bl/itter von Oryza sativa Var. BBS 873 wurde durch Messung des Nettoabbaus yon Chlorophyll und Protein in Abhfingigkeit yon der Entnahmezeit verfolgt. Kinetin war relativ wenig wirksam, aber Benzimidazol war wirksam bei der Verlangsamung des Alterns. Rotes Licht war ebenfalls wirksam bei der Alterns-Verlangsamung, aber auf der anderen Seite beschleunigte langwelligeres Rot das Altern. Benzimidazol war die wirksamste Behandlung bei Rotlicht zur Verlangsamung des Alterns. Die durch Rotlicht bedingte Retardation und durch langwelligeres Rot bedingte Beschleunigung des Alterns dOrfte unter Phytochromkontrolle stehen und abh~ingigvonder Photosynthese sein.