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Effect of Water Stress on the Photosynthetic Activity of Greening Wheat Seedlings
Bioehem. Physio!. Pflanzen 178, 409-412 (1983)
Short Communication
Effect of Water Stress on the Photosynthetic Activity of Greening Wheat Seedlings NIKHIL KUMAR') and PRASANNA MOHANTY School of Life Science, J.N.U., New Delhi, India Key T er mIn d ex : water stress, photochemical activity, uncoupling, ageing; Triticum aestivum
Summary RWC, total chrolorophyll and the activities of PS II and PS I was inhibited by water stress, the effect being morc in PS II (H 2 0 -+ FeCN, 0 2 1) as compared to PS I (DCPIPH2' -+ MV, 02~)' pH response of PS II (H2 0 -+ DCPIP) reaction showed the maximum activity around pH 6.0 to 6.5 in control which was shifted to pH 7.0 due to water stress. Th e extent of uncoupling was also higher in plants subjected to water stress.
The development of photochemical activies during greening of etiolated plants is inhibited due to water stress (VIRGIN 1966). In a fully grown leaf, water stress inhibit various partial reactions of photosynthesis and net photosynthesis (KECK and BOYER 1974; MOHANTY and BOYER 1976). Prolonged high water stress is known to cause certain structural changes in the chloroplasts which are found to be correlated with the inhibition in PS II activity (FELLOWS and BOYER 1976). Accumulation of osmophilic granules in the chloroplasts due to water stress has been documented in several cases (OKAMOTO and KAToH 1977; POLJAKOFF-MAYBER 1981). The effect of water stress on different photochcmical activities has been well worked out, howcver, there appears to bc lack of information regarding the pH characteristics of these reactions. In this report we communicate our findings on the development of photochemical activities especially the pH characteristics in etiolated wheat seedling subjected to water stress. Wheat seedlings (Triticum aestivum C.V.) were grown in dark at 28 °0 for 7 d after soaking. On the 7th d roots were flooded with PEG solution (30 g PEG/1OO ml H,O) and the seedlings kept in the dark for another 24 h. Greening was carried out in white light for 12 and 24 h. Relative water content has been calculated according to BARRS (1968). Chloroplasts were isolated at 0- 4"C in the medium containing 0.331\1 sucrose, 10 mM NaCI, 20 mM HEPES·KOH (pH 7.8) and 0.2 % BSA (w/v) by homogenising the leaves in a mortar pestle. The homogenate was passed through double layer of mira cloth. The pellets containing chloroplasts, obtained after centrifugation at 5,000 g for 10 min, suspended in the isolation buffer to give ca. 1 rug Chl/ml. Chlorophyll estima-
1) Present address: National Botanical Resea.rch Institute, Lucknow - 226001, V.P., India. Abbreviations: RWC - relative water content, FeCN - ferrieyanide, DCPIP - 2, 6 dichloro~ phenol indophenol, MV - methyl viologen, PS I - photosystem It PS II - photosystem II
410
N.
KUMAR
and P.
MOHANTY
Table 1. RWC, Total chlorophyll and photochemical activities of isolated chloroplasts of greening wheat seedlitlgs under water stress, The reaction mixture for oxygen evolution contained 0.15 M sucrose, 10 mM NaCl 5 rnM MgC1 2 • 20 mM HEPES·KOH (pH 7.0), 10 roM K, Fe(CN),. For uptake assay, the reaction mixture con· sisted 01 0.15 M sucrose, 10 mM NaCl, 5 mM MgCl 2, 20 mM HEPES·KOH (pH 8.0), 0.3 mM DCMU, 1 mM DCPIP, 0.2 M Sodium ascorbate, 0.5 mM Sodium azide. Total reaction volume was 3 ml with chloroplast equivalent to 5- 10ttg Chi/ml. Reaction mixtures were sparged with N2 or O2 for monitoring oxygen evolution and uptake, respectively
Hours of greening
12
RWC (%) Total chlorophyll mg!g d.w. PS II (H2 0 ~ FeCN) pmoles O2 evolved/rug Chl!h PS I (DCPIPH2 ~ MV) ,umoles O2 uptake/ mg Chl!h
24
Control 94 2.56
Stress 84 1.74
Control 92 6.34
Stress 73 4.10
67.90
39.60
80.89
50.13
476.30
390.10
706.00
560.21
tion was done according to ARNON (1949). Oxygen evolution and uptake were measured polarographically using YS I Oxygen electrode. DCPIP reduction was measured at 590 nrn as described by K UMA R and 1\{OIiANTY (1982).
Results presented in the Table 1 show that water stress leads to water loss by the tissue as evident by the decline in RWC. This is also accompanied by the inhibition in the accumulation of chlorophyll in the seedlings. Water stress is known to inhibit some of the key enzymes of chlorophyll bio·synthesis (VIRGIN 1966). Therefore, the low ac· cumulation of chlorophyll might be due to the inhibition in biosynthesis. However, it should be mentioned that activity of many hydrolases is increased due to water stress (HSIAO 1973), which may also lead to the break·down of the pigment in the greening leaves. Data in the Table 1 show that PS I and PS II rates are lowered due to water stress. The extent of inhibition is more for PS II than PS I showing that the organisation of PS II is more labile than PS 1. FELLOWS and BOYER (1976) have shown that pro· longed severe water stress leads to conformational (lamellar thickness) and configura· tional changes (intra thylakoid spacing) in thylakoids in situ but in the isolated chloro· plasts, only conformational changes persist. They could find a correlation between the decrease in lamellar thickness and loss in PS II activity. Accumulation of osmophilic granules in the chloroplast due to water stress has been demonstrated in many cases (FELLOWS and BOYER 1976; POLJAKOFF-MAYBER 1981)
Effect of Water Stress on the Photosynthetic Activity
411
Table 2. pH profile of the dye reduction by isolated chloroplasts at 12 h of greening under water stress. Reaction mixture: 0.15 M sucrose. 10 mM Nacl. 5 mM Mgcl 2 • 20 mM MES-KOH (pH 6.0 and 6.5), HEPES-KOH (pH 7.0 and 8.0) and chloroplast equivalent 5- 10l'g Chl/ml and with or without
20 mM NH,CI) pH
I'moles DCPIP reduced' mg Chi· h Water stress
Control
6.0 6.5 7.0 8.0
NH,CI
138.00 ± 10.5 136.00 ± 17.7 112.67 ± 15.87 99.76 ± 10.85
+ NH,CI
- NH,CI
+ NH,CI
95.00
150.72 ± 22.00 160.10 ± 28.00 147.44 ± 18.00
± 14.86
99.27 17.43 106.00 ± 13.90 91.55 ± 12.10
±
109.60 ± 14.90 114.94 ± 10.26 115.5 ± 19.00
which may be a source for the release of free fatty acids. Inhibition of various photochemical activities due to ageing of isolated chloroplasts and fatty acid induced ageing is well known (OKAMOTO and KATOH 1977; SIEGENTHALER and RAWLER 1977). Unsaturated fatty acids like linolinic acid have been shown to cause a loss in Mn, thus, inactivating PS II. BHARDWAJ and SINGHAL (1981), by using artificial elcctron donors of PS II, have shown that the oxidising side of PS II near oxygen evolving site is most affected due to water stress. It is, therefore, quite possible that a part of the inhibition in photochemical activities (especially PS II) may be due to the release of fatty acids from the lipid droplets formed during water stress. It is worthy of note that fatty acid inhibition is also more pronounced for PS II and PS I, as is the case with water stress_ Uncoupling of photophosphorylation due to water stress has been reported by KEK and BOYER (1974) in sun-flower leaves. Table 2 shows coupled and uncoupled rates of DCPIP reduction by the chloroplasts after 12 h of greening. At this stage, photosynthetic units are small as evident by light saturation curve (data not presented). The pH response of DCPIP reduction seem to be altered by water stress. In the control, maximum reduction is found at pH 6.0 and 6.5 which shifts to pH 7.0 due to water stress. Ratio of uncoupled to coupled rates showed higher degree of uncoupling in water stressed plants. Uncoupling of photophosphorylation and pH shift have been also observed due to fatty acid accelerated ageing of isolated chloroplasts (SIEGENTHALER and RAWLER 1977). Thus, it appears that there is a good deal of parallelism with respect to changes in photochemical activities accompanying water stress and ageing. Acknowledgements Thanks are due to the Dean, School of Life Science for providing laboratory facilities. The help rendered by Drs. K. M. SRIVASTAVA and V. K. KOCHTIAR in preparing the ma.nuscript is thankfully acknowledged.
412
N. KUMAR and P. MOHANTY, Effect of Water Stress on the Photosynthetic Activity
References ARNON, D. 1.: Copper enzymes in isolated chloroplasts: Polyphcnol oxidase in Beta vulgaris. Plant Physiol. 24, 1- 5 (1949). BARRS, H. D.: Determination of water deficit in plant tissues. In: T. T. KOZLOWSKI ed. Water Deficit and Plant Growth, Vol. 1. Development control and measurement, pp.239- 368. Academic Press, New York 1968. BHARDWAJ, R., and SINGHAL, G. S.: Effect of water stress on photochemical activity of chloroplasts during greening of etiolated barley seedlings. Plant Cen Physiol. 22, 155- 162 (1981). BOARDMAN, N. K: Development of Chloroplast. Structure and Function. In: A. TREBST and M. AvRON (cds.). Encyclopedia of plant physiology, New Series Vol. V, pp. 583- 600, Springer 1977. FELLOWS, R. J., and BOYER, J. S.: Structure and activity of chloroplasts of sun-flower leaves having various water potentials. Planta 132, 229- 239 (1976). HSIAO, T.: Plant responses -to water stress. Ann. Rev. Plant Physiol. 24, 519- 570 (1973). KECK, R. W., and BOYER, J. S.: Chloroplast response of low leaf water potentials. III Differing inhibition of electron transport and photophosphorylation. Plant Physiol. 63, 474- 479 (1974). KUMAR, N., and MOIiANTY, P.: Effect of 6-Benzyl Aminopurine on the Stabilization of Absorption Spectrum and Hill Activity of Isolated Chloroplasts. Biochem. Physiol. Pflanzen 117, 137- 142 (1982). MOIIANTY, P., and BOYER, J. S.: Chloroplast response to low leaf water potentials. IV. Quantum yield is reduced. Plant Physiol. 61 704- 709 (1976). MULLER, M., and SANTARlUS, K. A.: Changes in chloroplast membrane lipids during adaptation of barley to extreme salinity. Plant Physiol. 62, 326-329 (1978). OKAMOTO, T., KOTOH, S., and MURAKAMI, S.: Linolenic acid binding by chloroplasts. Plant Cell Physiol. 18, 551-560 (1977). POLJAKOFF-MAYBER, P.: Ultrastructural consequences of drought: In: L. L. PALEG and D. ASPIN"HL (eds.). The Physiology and Biochemistry of Drought Resistance in Plants. pp.389- 401, Academic Press 1981. POTTER, J. R., and BOYER, J. S.: Chloroplast response to low water potentials. II Role of osmotic potential. Plant Physiol. 51, 993-997 (1973). SIEGENTHALER, P. A., and RAWLER, A.: Ageing of the photosynthetic appa.ratus V. Change in pH dependence of electron transport and relationships to endogenous free fatty acids. Plant Sci. Letters 9, 265- 273 (1977). VIRGIN, H. I.: Chlorophyll formation and water deficit. Physiol. Plant 18, 998- 1000 (1965).
Received October 25, 1982 ; accepted December 28, 1982 Authors' address: NIKlIlL KU1tfAR and PR ASANNA MOHAN'I'Y, School of Life Science, J.N.U., INDIA -110067 New Delhi.
Short Communication
Effect of Water Stress on the Photosynthetic Activity of Greening Wheat Seedlings NIKHIL KUMAR') and PRASANNA MOHANTY School of Life Science, J.N.U., New Delhi, India Key T er mIn d ex : water stress, photochemical activity, uncoupling, ageing; Triticum aestivum
Summary RWC, total chrolorophyll and the activities of PS II and PS I was inhibited by water stress, the effect being morc in PS II (H 2 0 -+ FeCN, 0 2 1) as compared to PS I (DCPIPH2' -+ MV, 02~)' pH response of PS II (H2 0 -+ DCPIP) reaction showed the maximum activity around pH 6.0 to 6.5 in control which was shifted to pH 7.0 due to water stress. Th e extent of uncoupling was also higher in plants subjected to water stress.
The development of photochemical activies during greening of etiolated plants is inhibited due to water stress (VIRGIN 1966). In a fully grown leaf, water stress inhibit various partial reactions of photosynthesis and net photosynthesis (KECK and BOYER 1974; MOHANTY and BOYER 1976). Prolonged high water stress is known to cause certain structural changes in the chloroplasts which are found to be correlated with the inhibition in PS II activity (FELLOWS and BOYER 1976). Accumulation of osmophilic granules in the chloroplasts due to water stress has been documented in several cases (OKAMOTO and KAToH 1977; POLJAKOFF-MAYBER 1981). The effect of water stress on different photochcmical activities has been well worked out, howcver, there appears to bc lack of information regarding the pH characteristics of these reactions. In this report we communicate our findings on the development of photochemical activities especially the pH characteristics in etiolated wheat seedling subjected to water stress. Wheat seedlings (Triticum aestivum C.V.) were grown in dark at 28 °0 for 7 d after soaking. On the 7th d roots were flooded with PEG solution (30 g PEG/1OO ml H,O) and the seedlings kept in the dark for another 24 h. Greening was carried out in white light for 12 and 24 h. Relative water content has been calculated according to BARRS (1968). Chloroplasts were isolated at 0- 4"C in the medium containing 0.331\1 sucrose, 10 mM NaCI, 20 mM HEPES·KOH (pH 7.8) and 0.2 % BSA (w/v) by homogenising the leaves in a mortar pestle. The homogenate was passed through double layer of mira cloth. The pellets containing chloroplasts, obtained after centrifugation at 5,000 g for 10 min, suspended in the isolation buffer to give ca. 1 rug Chl/ml. Chlorophyll estima-
1) Present address: National Botanical Resea.rch Institute, Lucknow - 226001, V.P., India. Abbreviations: RWC - relative water content, FeCN - ferrieyanide, DCPIP - 2, 6 dichloro~ phenol indophenol, MV - methyl viologen, PS I - photosystem It PS II - photosystem II
410
N.
KUMAR
and P.
MOHANTY
Table 1. RWC, Total chlorophyll and photochemical activities of isolated chloroplasts of greening wheat seedlitlgs under water stress, The reaction mixture for oxygen evolution contained 0.15 M sucrose, 10 mM NaCl 5 rnM MgC1 2 • 20 mM HEPES·KOH (pH 7.0), 10 roM K, Fe(CN),. For uptake assay, the reaction mixture con· sisted 01 0.15 M sucrose, 10 mM NaCl, 5 mM MgCl 2, 20 mM HEPES·KOH (pH 8.0), 0.3 mM DCMU, 1 mM DCPIP, 0.2 M Sodium ascorbate, 0.5 mM Sodium azide. Total reaction volume was 3 ml with chloroplast equivalent to 5- 10ttg Chi/ml. Reaction mixtures were sparged with N2 or O2 for monitoring oxygen evolution and uptake, respectively
Hours of greening
12
RWC (%) Total chlorophyll mg!g d.w. PS II (H2 0 ~ FeCN) pmoles O2 evolved/rug Chl!h PS I (DCPIPH2 ~ MV) ,umoles O2 uptake/ mg Chl!h
24
Control 94 2.56
Stress 84 1.74
Control 92 6.34
Stress 73 4.10
67.90
39.60
80.89
50.13
476.30
390.10
706.00
560.21
tion was done according to ARNON (1949). Oxygen evolution and uptake were measured polarographically using YS I Oxygen electrode. DCPIP reduction was measured at 590 nrn as described by K UMA R and 1\{OIiANTY (1982).
Results presented in the Table 1 show that water stress leads to water loss by the tissue as evident by the decline in RWC. This is also accompanied by the inhibition in the accumulation of chlorophyll in the seedlings. Water stress is known to inhibit some of the key enzymes of chlorophyll bio·synthesis (VIRGIN 1966). Therefore, the low ac· cumulation of chlorophyll might be due to the inhibition in biosynthesis. However, it should be mentioned that activity of many hydrolases is increased due to water stress (HSIAO 1973), which may also lead to the break·down of the pigment in the greening leaves. Data in the Table 1 show that PS I and PS II rates are lowered due to water stress. The extent of inhibition is more for PS II than PS I showing that the organisation of PS II is more labile than PS 1. FELLOWS and BOYER (1976) have shown that pro· longed severe water stress leads to conformational (lamellar thickness) and configura· tional changes (intra thylakoid spacing) in thylakoids in situ but in the isolated chloro· plasts, only conformational changes persist. They could find a correlation between the decrease in lamellar thickness and loss in PS II activity. Accumulation of osmophilic granules in the chloroplast due to water stress has been demonstrated in many cases (FELLOWS and BOYER 1976; POLJAKOFF-MAYBER 1981)
Effect of Water Stress on the Photosynthetic Activity
411
Table 2. pH profile of the dye reduction by isolated chloroplasts at 12 h of greening under water stress. Reaction mixture: 0.15 M sucrose. 10 mM Nacl. 5 mM Mgcl 2 • 20 mM MES-KOH (pH 6.0 and 6.5), HEPES-KOH (pH 7.0 and 8.0) and chloroplast equivalent 5- 10l'g Chl/ml and with or without
20 mM NH,CI) pH
I'moles DCPIP reduced' mg Chi· h Water stress
Control
6.0 6.5 7.0 8.0
NH,CI
138.00 ± 10.5 136.00 ± 17.7 112.67 ± 15.87 99.76 ± 10.85
+ NH,CI
- NH,CI
+ NH,CI
95.00
150.72 ± 22.00 160.10 ± 28.00 147.44 ± 18.00
± 14.86
99.27 17.43 106.00 ± 13.90 91.55 ± 12.10
±
109.60 ± 14.90 114.94 ± 10.26 115.5 ± 19.00
which may be a source for the release of free fatty acids. Inhibition of various photochemical activities due to ageing of isolated chloroplasts and fatty acid induced ageing is well known (OKAMOTO and KATOH 1977; SIEGENTHALER and RAWLER 1977). Unsaturated fatty acids like linolinic acid have been shown to cause a loss in Mn, thus, inactivating PS II. BHARDWAJ and SINGHAL (1981), by using artificial elcctron donors of PS II, have shown that the oxidising side of PS II near oxygen evolving site is most affected due to water stress. It is, therefore, quite possible that a part of the inhibition in photochemical activities (especially PS II) may be due to the release of fatty acids from the lipid droplets formed during water stress. It is worthy of note that fatty acid inhibition is also more pronounced for PS II and PS I, as is the case with water stress_ Uncoupling of photophosphorylation due to water stress has been reported by KEK and BOYER (1974) in sun-flower leaves. Table 2 shows coupled and uncoupled rates of DCPIP reduction by the chloroplasts after 12 h of greening. At this stage, photosynthetic units are small as evident by light saturation curve (data not presented). The pH response of DCPIP reduction seem to be altered by water stress. In the control, maximum reduction is found at pH 6.0 and 6.5 which shifts to pH 7.0 due to water stress. Ratio of uncoupled to coupled rates showed higher degree of uncoupling in water stressed plants. Uncoupling of photophosphorylation and pH shift have been also observed due to fatty acid accelerated ageing of isolated chloroplasts (SIEGENTHALER and RAWLER 1977). Thus, it appears that there is a good deal of parallelism with respect to changes in photochemical activities accompanying water stress and ageing. Acknowledgements Thanks are due to the Dean, School of Life Science for providing laboratory facilities. The help rendered by Drs. K. M. SRIVASTAVA and V. K. KOCHTIAR in preparing the ma.nuscript is thankfully acknowledged.
412
N. KUMAR and P. MOHANTY, Effect of Water Stress on the Photosynthetic Activity
References ARNON, D. 1.: Copper enzymes in isolated chloroplasts: Polyphcnol oxidase in Beta vulgaris. Plant Physiol. 24, 1- 5 (1949). BARRS, H. D.: Determination of water deficit in plant tissues. In: T. T. KOZLOWSKI ed. Water Deficit and Plant Growth, Vol. 1. Development control and measurement, pp.239- 368. Academic Press, New York 1968. BHARDWAJ, R., and SINGHAL, G. S.: Effect of water stress on photochemical activity of chloroplasts during greening of etiolated barley seedlings. Plant Cen Physiol. 22, 155- 162 (1981). BOARDMAN, N. K: Development of Chloroplast. Structure and Function. In: A. TREBST and M. AvRON (cds.). Encyclopedia of plant physiology, New Series Vol. V, pp. 583- 600, Springer 1977. FELLOWS, R. J., and BOYER, J. S.: Structure and activity of chloroplasts of sun-flower leaves having various water potentials. Planta 132, 229- 239 (1976). HSIAO, T.: Plant responses -to water stress. Ann. Rev. Plant Physiol. 24, 519- 570 (1973). KECK, R. W., and BOYER, J. S.: Chloroplast response of low leaf water potentials. III Differing inhibition of electron transport and photophosphorylation. Plant Physiol. 63, 474- 479 (1974). KUMAR, N., and MOIiANTY, P.: Effect of 6-Benzyl Aminopurine on the Stabilization of Absorption Spectrum and Hill Activity of Isolated Chloroplasts. Biochem. Physiol. Pflanzen 117, 137- 142 (1982). MOIIANTY, P., and BOYER, J. S.: Chloroplast response to low leaf water potentials. IV. Quantum yield is reduced. Plant Physiol. 61 704- 709 (1976). MULLER, M., and SANTARlUS, K. A.: Changes in chloroplast membrane lipids during adaptation of barley to extreme salinity. Plant Physiol. 62, 326-329 (1978). OKAMOTO, T., KOTOH, S., and MURAKAMI, S.: Linolenic acid binding by chloroplasts. Plant Cell Physiol. 18, 551-560 (1977). POLJAKOFF-MAYBER, P.: Ultrastructural consequences of drought: In: L. L. PALEG and D. ASPIN"HL (eds.). The Physiology and Biochemistry of Drought Resistance in Plants. pp.389- 401, Academic Press 1981. POTTER, J. R., and BOYER, J. S.: Chloroplast response to low water potentials. II Role of osmotic potential. Plant Physiol. 51, 993-997 (1973). SIEGENTHALER, P. A., and RAWLER, A.: Ageing of the photosynthetic appa.ratus V. Change in pH dependence of electron transport and relationships to endogenous free fatty acids. Plant Sci. Letters 9, 265- 273 (1977). VIRGIN, H. I.: Chlorophyll formation and water deficit. Physiol. Plant 18, 998- 1000 (1965).
Received October 25, 1982 ; accepted December 28, 1982 Authors' address: NIKlIlL KU1tfAR and PR ASANNA MOHAN'I'Y, School of Life Science, J.N.U., INDIA -110067 New Delhi.