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Effects of Light and of Growth Regulators on the Reversibility of Senescence in Attached Leaves of Barley
Effects of Light and of Growth Regulators on the Reversibility of Senescence in Attached Leaves of Barley M. 1. ESCRIBANO GARAIZABAL and M. T. RODRIGUEZ Depanment of Plant Physiology, Faculty of Biology, Complutense University, Madrid -3-, Spain Received June 6, 1983 . Accepted September 25, 1983
Summary Senescence of intact barley seedlings (Hordeum vulgare) judged by the loss of chlorophyll, was induced by putting them in the dark. Reversal of senescence by application of light was also observed. The chlorophyll content recovered after two days in the light, but seedlings were unable to recover their initial level of pigment after four days in the dark, This suggests the onset of an irreversible stage in senescence from the founh day. Supply of kinetin to the leaf after two or four days in the dark induces a slight increase in chlorophyll content. Whereas reversion of the senescence is obtained immediately after the dark period, the same treatment with abscisic acid accelerates the loss of chlorophylls and retards the reversion by light treatment.
Key words: Hordeum vulgare, abscisic acid, chlorophyll loss, kinetin.
Introduction
The main advances in the study of plant senescence with special emphasis on its regulation and characterization as a genetically directed process have been achieved on leaves. The first visible symptom of senescence is the yellowing of leaves caused by the degradation of chlorophylls. Chlorophyllase seems to be the enzyme responsible for the degradation of chlorophylls in detached leaves of barley and oat (Sabater and Rodriguez, 1978). Degradation is often preceded by a decrease in the amount of chloroplastic fraction I protein and also by loss of photosynthetic activity (Woolhouse, 1967; Woolhouse and Batt, 1976). Catabolism of chlorophyll and proteolysis are not synchronous processes since the loss of protein precedes the decay in the rate of photosynthesis and RNA degradation (Woolhouse, 1967; Tetley and Thimann, 1974). A direct consequence of these occurrences in the loss of chloroplast integrity which leads to an increase in the amount of free lipids produced by desintegration of tylakoids (Barton, 1966) this being related to chlorophyll degradation (Holden, 1972). Catabolism of chlorophylls seems to be directed by a nuclear gene, the expression of which is restricted to senescence (Thomas and Stoddart, 1977). The loss of chloroAbbreviation: ABA, abscisic acid. 2. Pjlanzenphysiol. Ed. 112. S. 435-442. 1983.
436
M. I. ESCRIBANO GARAIZABAL and M. T. RODRIGUEZ
phyll is usually prevented by cytokinins, whereas ABA accelerates its degradation (Osborne, 1967; Naito et aI., 1978). In wheat leaves, cytokinins act during a reversible phase of senescence whereas they have no effect when the growth regulators are applied to the leaves during a second, irreversible phase of the process (Witten bach, 1977). Choudhury and Biswal (1980) found that, in maize leaves, reversibility implies the re-establishment of the original level of chlorophylls. In this work, we attempt to obtain new evidence on the reversibility of senescence of leaves, as well as, on the role that different zones of the leaf play in this process.
Material and Methods The barley seeds employed throughout this work were supplied by the Servicio Nacional de Cereales (Spain). They were grown on Perlite in Cron medium (Bond, 1951) for two weeks, in natural light, in a greenhouse at 25 0c. The seedlings were kept for varying periods in the dark and then transferred to natural conditions. Parallel, two controls were always performed: one in which the natural conditions were maintained and the other in continuous dark. Solutions containing 30 mg .1- 1 kinetin or ABA (both from Sigma) in 0.02 % Tween 20 were applied as droplets along the longitudinal axis of leaves in apical, medium, and basal positions. Controls were always performed by applying 0.02 % Tween 20 as above. The application of growth regulator solutions was carried out on seedlings kept in natural conditions as well as in continuous or discontinuous darkness. This pattern of supply of growth regulators has been reported to be the most effective for attached leaves (Thimann et a!., 1974). Two applications of 10 f.tl hormone solution were made at each site. When it is indicated 300 mM glucose was added to the culture medium. Chlorophyll content was measured in the primary leaves. When it is indicated chlorophyll content was measured in the apical or basal zone of leaves (Fig. 1). Whole leaves as well as cut were macerated in a mortar with a sufficient volume of 80 % acetone. The homogenates were kept in the dark at room temperature. When extraction was completed, homogenates were centrifuged at 5,000 xg, for 20 min and chlorophylls were determined according to Strain et a!. (1971). Relative values of total chlorophyll were refered to that in untreated leaves, the amount
2-3 em
1.5-2 em I
basal
apical
~ Segments in which chlorophyll contf"nt was determined Fig. 1: Scheme of barley leaf showing basal and apical zones in which chlorophyll content were determined. Circles indicate where the application of hormone solution were made (2 drops at each).
Z. Pjlanzenphysiol. Ed. 112. s. 435-442. 1983.
Senescence in attached leaves of barley
437
of leaf chlorophyll before the senescence-inducing treatment being taken as 100 % of pigment content. Seedlings grown for 15 days under natural photoperiodic conditions were used. Senescence was induced by a period in the dark which varied from 1 to 5 days. Then the seedlings were returned to photoperiodic conditions to complete a total period of 9 days. Two controls were performed: one lot of seedlings was kept under photoperiod for the whole time of the experiment whereas a second lot was grown in continuous darkness.
Results Results on the chlorophyll content of the first leaf of each seedling is shown in Fig. 2. A period in the dark induces a rapid loss of chlorophyll although it can be reversed by transferring the seedlings to the light but more than 4 days in the dark cannot be reversed by light. The recovery was however delayed when the seedlings were transferred from the dark to the light. Applications of kinetin at three different places on the longitudinal axis of the first leaf of seedlings kept in the dark for two (Fig. 3 A) or four (Fig. 3 B) days induces a slight increase in the chlorophyll content (statistical treatment is shown on each figure). However, recovery after a two or four day period in the dark could be made to occur immediately by applying kinetin. It has also been observed that, in the light, kinetin has no effect on chlorophyll content. However, in excised leaves of barley,
days
Fig. 2: Time-course of total chlorophyll in the attached primary leaves of barley seedlings during dark-induced senescence for 1 (_), 2 (.), 3 (6), 4 (0) and 5 (V) days and subsequent recovery in the light (t). Control in the light is taken to be 100 %; control in the dark (0). The values are the average of four different experiments. The median variation coefficient was defined for: 1 day in dark as 7.7 %; 2 days in dark as 9.0 %; 3 days in dark as 7.2 %; 4 days in dark as 4.4 % and 5 days in dark as 10.6 %.
Z. Pjlanzenphysiol. Bd. 112. S. 435-442. 1983.
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Fig. 3: Time-course of total chlorophyll in the attached primary leaves of barley seedlings during dark-induced senescence for 2 (A) or 4 (B) days. Applications to the leaf with (.) or without (b.) 30 mg .1- 1 kinetin was given. Arrows indicate transfer to the light. Controls in the light with (_) or without (0) 30 mg .1- 1 kinetin supply, and controls in the dark with (.) or without (0) 30 mg .\-1 kinetin supply were performed. The values are the average of four different experiments. The median variation coefficient was defined for: 2 days in dark as 9.8 % and 4 days in dark as 9.8 %.
floated on kinetin in the light, a joint effect occurs between kinetin and light (Sabater and Rodriguez, 1978). A similar treatment with ABA accelerates the loss of chlorophyll (Figs. 4 A and B) and retards the response to transfer back to light. Application of ABA has not any effect on the recovery of the senescence during the first two days in the dark. Moreover, ABA has no effect on whole plants which have been kept under natural photoperiodic conditions (Figs. 4 A and B). When analyzing both apical and basal zones from ABA-treated leaves separately, it is observed that the former zone does not recover its chlorophyll content in the light but the latter zone does. The basal portion appears to be responsible, therefore, for the recovery found in whole leaves kept in the dark for two days (Fig. 4 C). A supply of 300mM glucose (as has been suggested by Tetley et al., 1974) in the medium partially reverses the effect of ABA. Although glucose has no effect on the loss of chlorophyll during the period in the dark, the decrease in pigment of seedlings Z. Pf/anzenphysiol. Bd. 112. S. 435-442. 1983.
Senescence in attached leaves of barley
439
A
4
• ...• ..- •.. . . B
40 20 6
8
10
days Fig. 4: Time-course of total chlorophyll in the attached primary leaves of barley seedlings during dark-induced senescence in 2 (A) or 4 (B) days. An application to the leaf with (.) or without (.6.) 30 mg .1- 1 ABA was given. Arrows indicate transfer to the light. Controls in the light with (_) or without (0) 30mg·l- 1 ABA, and controls in the dark with (e) or without (0) 30 mg .1- 1 ABA supply were performed. In (C) chlorophyll content in the apical zone of the leaf with (_) or without (0) 30 mg .1- 1 ABA supply, and in the basal zone with (.) or without (.6) 30 mg .1- 1 ABA application during 2 days in the dark. The values are the average of four different experiments. The median variation coefficient was defined for: 2 days in dark as 8.8 %; 4 days in dark as 7.4 % and in the apical and basal zones as 8.2 %. fed with glucose is about 20 % less than that observed in the abscence of glucose (Fig. 5).
Discussion Senescence of attached leaves of barley induced by one or two days in the dark is totally reversed by light although the rate of degradation of chlorophyll in whole leaves after these first two days of treatment reaches its maximum. This pattern of senescence and its reversal is similar to that reported by Wittenbach (1977) for wheat Z. Pjlanzenphysiol. Bd. 112. S. 435-442. 1983.
440
M. I. ESCRIBANO GARAIZABAL and M. T.
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~--~--~--~--~--~--~--~--~--~
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Fig. 5: Time-course of total chlorophyll in the attached primary leaves of barley seedlings during dark-induced senescence in 4 days with 300 mM glucose in the medium and 30 mg .1- 1 kinetin supply to the leaf (~), 300 mM glucose in the medium and 30 mg .1- 1 ABA applied to the leaf (.), 300 mM glucose in the medium (.). Arrows indicate transfer to the light. Control in the light with 300 mM glucose in the medium (0) and control in the dark with 300 mM glucose (0). The values are the average of four different experiments. The median variation coefficient was defined as 7.1 %.
leaves. In addition, chlorophyll loss in attached leaves of barley is lower than that in leaf discs (Sabater and Rodriguez, 1978). Senescence induced by the dark, on barley, is not reversed by light after four days treatment whereas wheat leaves need almost six days in the dark to reach an irreversible status. This can be explained in terms of the degree of chlorophyll loss during the two days of treatment, since in wheat, degradation of chlorophylls is lower than in barley (Witten bach, 1977). It is also possible that synthesis of chlorophyll does not reach an adequate level to produce a complete re-establishment of the functional lamellar structure, as indicated by Choudhury and Biswal (1980). Obviously, the reversal of senescence by kinetin does not differ from that described in previous reports (Even-Chen et al., 1978) but there is an interesting difference with regard to the application of growth regulators. Whereas Tetley and Thimann (1974) show that kinetin applied to leaf discs only reverses the chlorophyll loss at the site of application, its application to whole leaves produces a homogenous effect along the whole length of the organ. This can be explained by the maintenance of actived translocation when the leaf is joined to the seedling, whereas transport is abolished in discs of leaves. The effect of kinetin of reversal of chlorophyll loss has been explained by Naito et al. (1978) as a re-cycling process of low molecular weight metabolites produced during the first stages of senescence. Z. Pjlanzenphysiol. Bd. 112. s. 435-442. 1983.
Senescence in attached leaves of barley
441
This is also in agreement with the enhancing effect of glucose on chlorophyll production when it is added to the medium. As has been seen, kinetin and light do not show an additive effect. Perhaps, this is related to the discontinuous supply of growth regulators (Tetley and Thimann, 1974) or to high levels of endogenous cytokinins (Choudhury and Biswal, 1980). Therefore, the action of kinetin is observed when the leaf kept in the light begins its natural senescence. The immediate response can be explained in terms of prevention of the translocation of low molecular weight metabolites by kinetin which implies rapid synthesis, a condition which does not occur in untreated leaves. The greatest loss of chlorophyll in leaves treated with ABA in the dark may indicate that the rate of reversal is not only dependent on the average of degraded chlorophyll. In the light ABA has no effect, perhaps due to the high levels of endogenous cytokinins (Even-Chen et al., 1978). Abscisic acid applied to leaves does not support senescence but accelerates the loss of chlorophyll in the dark. Moreover, the recovery of chlorophyll in the basal zone, but not in the apex may reflect the sequential senescence from the apex to the base of the leaf which is a general process in cereal leaves. Acknowledgements We wish to thank Dr. Carlos Vicente and Dr. M a Estrella Legaz of the Lichen Team of this Department, for their help on the preparation of this manuscript.
References BARTON, R.: Fine structure of mesophyll cells in senescing leaves of Phaseolus. Planta 71, 314-325 (1966). BOND, G.: The fixation of nitrogen associated with root nodules of Myrica gale L., with special reference to its pH relation and ecological significance. Ann. Bot. 15,446-459 (1951). CHOUDHURY, N. K. and U. C. BISWAL: Recovery of loss in chlorophyll and 2,6-dichlorophenol indophenol Hill reaction of isolated chloroplast during dark induced aging of intact maize seedlings. Physiol. Plant. 49, 43-48 (1980). EVEN-CHEN, Z., D. ATSMON, and C. H. ITAI: Hormonal aspects of senescence in detached tobacco leaves. Physiol. Plant. 44, 377-382 (1978). HOLDEN, M.: Effects of EDTA and other compounds on chlorophyll breakdown in detached leaves. Phytochemistry 2,2393-2402 (1972). NAITO, K., H. TSUJI, and 1. HATAKEYAMA: Effect of benzyladenine on DNA, RNA, protein and chlorophyll contents in intact bean leaves: differencial responses of benzyladenine according to leaf age. Physiol. Plant. 43, 367-371 (1978). OSBORNE,].: Hormonal regulation of leaf senescence. Symp. Soc. Exp. BioI. 21,305-322 (1967). RICHMOND, A. E. and G. LANG: Effect of kinetin on protein content and survival of detached Xanthium leaves. Science 125, 610-651 (1957). SABATER, B. and M. T. RODRIGUEZ: Control of chlorophyll degradation in detached leaves of barley and oat through effect on kinetin on chlorophyllase levels. Physiol. Plant. 43, 274-276 (1978). - - Hormonal and other effects on chlorophyllase of detached barley and oat leaves. I Asamblea General y Conferencia Cientifica de la Federation European of Societies of Plant Physiology (FESPP). Edimburgo. Abstracts p. 456-457 (1978).
Z. Pjlanzenphysiol. Bd. 112. S. 435-442.1983.
442
M. I. ESCRIBANO GARAIZABAL and M. T. RODRIGUEZ
STRAIN, H. H., B. T. COPE, and W. A. SVEC: Analytical procedures for the isolation, identification, estimation and investigation of the chlorophylls. In: A. SAN PIETRO (Ed.): Methods in Enzymology, 23, 452-457. Academic Press, New York and London, 1971. TETLEY, R. M. and K. V. THIMANN: The metabolism of oat leaves during senescence. I Respiration, carbohydrate metabolism, and the action of cytokinins. Plant. Physio!. 54, 294-303 (1974). THIMANN, K. V., R. R. TETLEY, and T. VAN THANH: The metabolism of oat leaves during senescence. II Senescence in leaves attached to the plant. Plant. Physio!. 54, 859-862 (1974). THOMAS, H. and J. L. STODDART: Biochemistry of leaf senescence in grasses. Ann. App!. Bio!. 85,461-463 (1977). WITTENBACH, V. A.: Induced senescence of intact wheat seedling and its reversibility. Plant. Physio!. 59, 1039-1042 (1977). WOOLHOUSE, H. W.: The nature of senescence in plants. Symp. Soc. Exp. Bio!. 21, 179-214 (1967). WOOLHOUSE, H. W. and T. BATT: The nature and regulation of senescence in plastids. In: N. SUNDERLAND (Ed.): Perspectives in Experimental Biology, Volume 2-Botany, 163-175. Pergamon, Oxford, 1976.
2. Pjlanzenphysiol. Bd. 112. s. 435-442. 1983.
Summary Senescence of intact barley seedlings (Hordeum vulgare) judged by the loss of chlorophyll, was induced by putting them in the dark. Reversal of senescence by application of light was also observed. The chlorophyll content recovered after two days in the light, but seedlings were unable to recover their initial level of pigment after four days in the dark, This suggests the onset of an irreversible stage in senescence from the founh day. Supply of kinetin to the leaf after two or four days in the dark induces a slight increase in chlorophyll content. Whereas reversion of the senescence is obtained immediately after the dark period, the same treatment with abscisic acid accelerates the loss of chlorophylls and retards the reversion by light treatment.
Key words: Hordeum vulgare, abscisic acid, chlorophyll loss, kinetin.
Introduction
The main advances in the study of plant senescence with special emphasis on its regulation and characterization as a genetically directed process have been achieved on leaves. The first visible symptom of senescence is the yellowing of leaves caused by the degradation of chlorophylls. Chlorophyllase seems to be the enzyme responsible for the degradation of chlorophylls in detached leaves of barley and oat (Sabater and Rodriguez, 1978). Degradation is often preceded by a decrease in the amount of chloroplastic fraction I protein and also by loss of photosynthetic activity (Woolhouse, 1967; Woolhouse and Batt, 1976). Catabolism of chlorophyll and proteolysis are not synchronous processes since the loss of protein precedes the decay in the rate of photosynthesis and RNA degradation (Woolhouse, 1967; Tetley and Thimann, 1974). A direct consequence of these occurrences in the loss of chloroplast integrity which leads to an increase in the amount of free lipids produced by desintegration of tylakoids (Barton, 1966) this being related to chlorophyll degradation (Holden, 1972). Catabolism of chlorophylls seems to be directed by a nuclear gene, the expression of which is restricted to senescence (Thomas and Stoddart, 1977). The loss of chloroAbbreviation: ABA, abscisic acid. 2. Pjlanzenphysiol. Ed. 112. S. 435-442. 1983.
436
M. I. ESCRIBANO GARAIZABAL and M. T. RODRIGUEZ
phyll is usually prevented by cytokinins, whereas ABA accelerates its degradation (Osborne, 1967; Naito et aI., 1978). In wheat leaves, cytokinins act during a reversible phase of senescence whereas they have no effect when the growth regulators are applied to the leaves during a second, irreversible phase of the process (Witten bach, 1977). Choudhury and Biswal (1980) found that, in maize leaves, reversibility implies the re-establishment of the original level of chlorophylls. In this work, we attempt to obtain new evidence on the reversibility of senescence of leaves, as well as, on the role that different zones of the leaf play in this process.
Material and Methods The barley seeds employed throughout this work were supplied by the Servicio Nacional de Cereales (Spain). They were grown on Perlite in Cron medium (Bond, 1951) for two weeks, in natural light, in a greenhouse at 25 0c. The seedlings were kept for varying periods in the dark and then transferred to natural conditions. Parallel, two controls were always performed: one in which the natural conditions were maintained and the other in continuous dark. Solutions containing 30 mg .1- 1 kinetin or ABA (both from Sigma) in 0.02 % Tween 20 were applied as droplets along the longitudinal axis of leaves in apical, medium, and basal positions. Controls were always performed by applying 0.02 % Tween 20 as above. The application of growth regulator solutions was carried out on seedlings kept in natural conditions as well as in continuous or discontinuous darkness. This pattern of supply of growth regulators has been reported to be the most effective for attached leaves (Thimann et a!., 1974). Two applications of 10 f.tl hormone solution were made at each site. When it is indicated 300 mM glucose was added to the culture medium. Chlorophyll content was measured in the primary leaves. When it is indicated chlorophyll content was measured in the apical or basal zone of leaves (Fig. 1). Whole leaves as well as cut were macerated in a mortar with a sufficient volume of 80 % acetone. The homogenates were kept in the dark at room temperature. When extraction was completed, homogenates were centrifuged at 5,000 xg, for 20 min and chlorophylls were determined according to Strain et a!. (1971). Relative values of total chlorophyll were refered to that in untreated leaves, the amount
2-3 em
1.5-2 em I
basal
apical
~ Segments in which chlorophyll contf"nt was determined Fig. 1: Scheme of barley leaf showing basal and apical zones in which chlorophyll content were determined. Circles indicate where the application of hormone solution were made (2 drops at each).
Z. Pjlanzenphysiol. Ed. 112. s. 435-442. 1983.
Senescence in attached leaves of barley
437
of leaf chlorophyll before the senescence-inducing treatment being taken as 100 % of pigment content. Seedlings grown for 15 days under natural photoperiodic conditions were used. Senescence was induced by a period in the dark which varied from 1 to 5 days. Then the seedlings were returned to photoperiodic conditions to complete a total period of 9 days. Two controls were performed: one lot of seedlings was kept under photoperiod for the whole time of the experiment whereas a second lot was grown in continuous darkness.
Results Results on the chlorophyll content of the first leaf of each seedling is shown in Fig. 2. A period in the dark induces a rapid loss of chlorophyll although it can be reversed by transferring the seedlings to the light but more than 4 days in the dark cannot be reversed by light. The recovery was however delayed when the seedlings were transferred from the dark to the light. Applications of kinetin at three different places on the longitudinal axis of the first leaf of seedlings kept in the dark for two (Fig. 3 A) or four (Fig. 3 B) days induces a slight increase in the chlorophyll content (statistical treatment is shown on each figure). However, recovery after a two or four day period in the dark could be made to occur immediately by applying kinetin. It has also been observed that, in the light, kinetin has no effect on chlorophyll content. However, in excised leaves of barley,
days
Fig. 2: Time-course of total chlorophyll in the attached primary leaves of barley seedlings during dark-induced senescence for 1 (_), 2 (.), 3 (6), 4 (0) and 5 (V) days and subsequent recovery in the light (t). Control in the light is taken to be 100 %; control in the dark (0). The values are the average of four different experiments. The median variation coefficient was defined for: 1 day in dark as 7.7 %; 2 days in dark as 9.0 %; 3 days in dark as 7.2 %; 4 days in dark as 4.4 % and 5 days in dark as 10.6 %.
Z. Pjlanzenphysiol. Bd. 112. S. 435-442. 1983.
438
M. I. ESCRIBANO GARAIZABAL and M. T. RODRIGUEZ
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Fig. 3: Time-course of total chlorophyll in the attached primary leaves of barley seedlings during dark-induced senescence for 2 (A) or 4 (B) days. Applications to the leaf with (.) or without (b.) 30 mg .1- 1 kinetin was given. Arrows indicate transfer to the light. Controls in the light with (_) or without (0) 30 mg .1- 1 kinetin supply, and controls in the dark with (.) or without (0) 30 mg .\-1 kinetin supply were performed. The values are the average of four different experiments. The median variation coefficient was defined for: 2 days in dark as 9.8 % and 4 days in dark as 9.8 %.
floated on kinetin in the light, a joint effect occurs between kinetin and light (Sabater and Rodriguez, 1978). A similar treatment with ABA accelerates the loss of chlorophyll (Figs. 4 A and B) and retards the response to transfer back to light. Application of ABA has not any effect on the recovery of the senescence during the first two days in the dark. Moreover, ABA has no effect on whole plants which have been kept under natural photoperiodic conditions (Figs. 4 A and B). When analyzing both apical and basal zones from ABA-treated leaves separately, it is observed that the former zone does not recover its chlorophyll content in the light but the latter zone does. The basal portion appears to be responsible, therefore, for the recovery found in whole leaves kept in the dark for two days (Fig. 4 C). A supply of 300mM glucose (as has been suggested by Tetley et al., 1974) in the medium partially reverses the effect of ABA. Although glucose has no effect on the loss of chlorophyll during the period in the dark, the decrease in pigment of seedlings Z. Pf/anzenphysiol. Bd. 112. S. 435-442. 1983.
Senescence in attached leaves of barley
439
A
4
• ...• ..- •.. . . B
40 20 6
8
10
days Fig. 4: Time-course of total chlorophyll in the attached primary leaves of barley seedlings during dark-induced senescence in 2 (A) or 4 (B) days. An application to the leaf with (.) or without (.6.) 30 mg .1- 1 ABA was given. Arrows indicate transfer to the light. Controls in the light with (_) or without (0) 30mg·l- 1 ABA, and controls in the dark with (e) or without (0) 30 mg .1- 1 ABA supply were performed. In (C) chlorophyll content in the apical zone of the leaf with (_) or without (0) 30 mg .1- 1 ABA supply, and in the basal zone with (.) or without (.6) 30 mg .1- 1 ABA application during 2 days in the dark. The values are the average of four different experiments. The median variation coefficient was defined for: 2 days in dark as 8.8 %; 4 days in dark as 7.4 % and in the apical and basal zones as 8.2 %. fed with glucose is about 20 % less than that observed in the abscence of glucose (Fig. 5).
Discussion Senescence of attached leaves of barley induced by one or two days in the dark is totally reversed by light although the rate of degradation of chlorophyll in whole leaves after these first two days of treatment reaches its maximum. This pattern of senescence and its reversal is similar to that reported by Wittenbach (1977) for wheat Z. Pjlanzenphysiol. Bd. 112. S. 435-442. 1983.
440
M. I. ESCRIBANO GARAIZABAL and M. T.
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RODRIGUEZ
• I
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days
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Fig. 5: Time-course of total chlorophyll in the attached primary leaves of barley seedlings during dark-induced senescence in 4 days with 300 mM glucose in the medium and 30 mg .1- 1 kinetin supply to the leaf (~), 300 mM glucose in the medium and 30 mg .1- 1 ABA applied to the leaf (.), 300 mM glucose in the medium (.). Arrows indicate transfer to the light. Control in the light with 300 mM glucose in the medium (0) and control in the dark with 300 mM glucose (0). The values are the average of four different experiments. The median variation coefficient was defined as 7.1 %.
leaves. In addition, chlorophyll loss in attached leaves of barley is lower than that in leaf discs (Sabater and Rodriguez, 1978). Senescence induced by the dark, on barley, is not reversed by light after four days treatment whereas wheat leaves need almost six days in the dark to reach an irreversible status. This can be explained in terms of the degree of chlorophyll loss during the two days of treatment, since in wheat, degradation of chlorophylls is lower than in barley (Witten bach, 1977). It is also possible that synthesis of chlorophyll does not reach an adequate level to produce a complete re-establishment of the functional lamellar structure, as indicated by Choudhury and Biswal (1980). Obviously, the reversal of senescence by kinetin does not differ from that described in previous reports (Even-Chen et al., 1978) but there is an interesting difference with regard to the application of growth regulators. Whereas Tetley and Thimann (1974) show that kinetin applied to leaf discs only reverses the chlorophyll loss at the site of application, its application to whole leaves produces a homogenous effect along the whole length of the organ. This can be explained by the maintenance of actived translocation when the leaf is joined to the seedling, whereas transport is abolished in discs of leaves. The effect of kinetin of reversal of chlorophyll loss has been explained by Naito et al. (1978) as a re-cycling process of low molecular weight metabolites produced during the first stages of senescence. Z. Pjlanzenphysiol. Bd. 112. s. 435-442. 1983.
Senescence in attached leaves of barley
441
This is also in agreement with the enhancing effect of glucose on chlorophyll production when it is added to the medium. As has been seen, kinetin and light do not show an additive effect. Perhaps, this is related to the discontinuous supply of growth regulators (Tetley and Thimann, 1974) or to high levels of endogenous cytokinins (Choudhury and Biswal, 1980). Therefore, the action of kinetin is observed when the leaf kept in the light begins its natural senescence. The immediate response can be explained in terms of prevention of the translocation of low molecular weight metabolites by kinetin which implies rapid synthesis, a condition which does not occur in untreated leaves. The greatest loss of chlorophyll in leaves treated with ABA in the dark may indicate that the rate of reversal is not only dependent on the average of degraded chlorophyll. In the light ABA has no effect, perhaps due to the high levels of endogenous cytokinins (Even-Chen et al., 1978). Abscisic acid applied to leaves does not support senescence but accelerates the loss of chlorophyll in the dark. Moreover, the recovery of chlorophyll in the basal zone, but not in the apex may reflect the sequential senescence from the apex to the base of the leaf which is a general process in cereal leaves. Acknowledgements We wish to thank Dr. Carlos Vicente and Dr. M a Estrella Legaz of the Lichen Team of this Department, for their help on the preparation of this manuscript.
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