Photosynthesis Research 14:259 267 (1987) © Martinus Nijhoff Publishers, Dordrecht - Printed in the Netherlands
Regular Paper
Reversal of heat-induced alterations in photochemical activities in wheat primary leaves N A R E N D R A N A T H MOHANTY, S.D.S. M U R T H Y & PRASANNA MOHANTY 1 School of Life Sciences, Jawaharlal Nehru University, NEW DELHI- 110 067, India; i Author for correspondence Received 12 May 1987; accepted in revised form 5 August 1987
Key words: high temperature-induced alterations, photochemical activities, post stress reversal, wheat Abstract. Chloroplasts isolated from elevated temperature treated 8-day-old continuouswhite-light-grown wheat primary leaves lost the ability to photo-oxidize water. Also, the ability of ascorbate to donate electrons to photosystem II declined. However, a significant increase in reduced dichlorophenolindophenol-supported photosystem-I-mediated methylviologen photo-reduction activity was observed. The plants stressed at 45 °C and 47 °C were subsequently grown at 25 °C and the partial photochemical activities were measured in chloroplasts isolated from the plants at 24-h intervals. The post stress alterations observed are (1) a significant restoration of water oxidation capacity in 45 °C and partial restoration in 47 °C-treated leaves. Ascorbate-supported photochemical activities recovered more or less in similar fashion; (2) reversal of enhanced photosystem I activitY in both 45 ° C and 47 °C treated leaves. These results suggest that the restoration in water oxidation capacity is possible in 45 °C treated leaves and is limited by the severity of heat stress in 47 °C-treated leaves. Restoration of water oxidation capacity vis-5.-vis to the reversal of heat-enhanced photosystern I activity also indicates the existence of possible endogenous control for repair of alterations during the post stress. Abbreviations: DCPIP-2,6, Dichlorophenol-indophenol, DCMU-3-(3,4-dichlorophenyl)-l, 1-dimethylurea, FeCN Ferricyanide, Hepe~N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid, PD-Phenylene diimine, MV-Methyl Viologen
Plants are often exposed to fluctuations in atmospheric temperature, which influences the functional activity of photosynthetic apparatus. At elevated temperature marked inhibition in photosynthetic efficiency occurs (Berry and Bjorkman 1980, Quinn and Williams 1985). The inhibition of whole-leaf photosynthesis by high temperature has been found to be due to the disruption of the functional integrity of the photosynthetic apparatus at the chloroplast level (Pearcy et al. 1977, Armond et al. 1978, Bauer and Senser 1979). The thylakoid membranes have been shown to be more heat-sensitive
260 than other biomembranes (Thebud and Santarius 1982). High-temperature treatment to whole plants, excised leaves or isolated chloroplasts results in significant or complete loss of photophosphorylation and photosystem (PS) II-mediated electron transport activity (Berry and Bjorkman 1980, Quinn and Williams 1985). In isolated chloroplasts subjected to elevated temperature, however, a stimulation in DCPIPH2-supported PS I activity is observed (Quinn and Williams 1985). Whole plants rather than the isolated chloroplasts should be preferred for studying the effect of heat stress on the functioning of the thylakoid membranes as chloroplast in vivo (i.e., inside the leaves) may exhibit differential heat tolerance compared to that of isolated chloroplasts. Furthermore, for a study on the post effect of high temperature stress, the attached leaves serve as the most suitable system as these provide the conducive environment for possible repair inside the chloroplast. A majority of the reports on the high temperature effects on photosynthesis have been confined to in vitro studies. Only a few reports on the post action of heat stress on photosynthetic activities such as, in ivy leaves (Bauer and Senser 1979) and in excised spinach leaves, (Yordanov et al. 1975) have been documented. Thus, a detailed study on the after-effect of heat stress on the various photochemical activities in chloroplasts will further our understanding of the repair of heat-damaged photosynthetic apparatus. In the present study, we have attempted to investigate the partial electron transport activities in chloroplast isolated from the intact wheat seedlings recovering at 25 °C after receiving the heat treatment at 45 °C and 47 °C for 10 min.
Materials and methods
Wheat (Triticum aestivum L. cv Kalyansona) seedlings were raised on Petri-plates under continuous white light (-~16Wm -2) at 25°C. Half strength Hoagland solution was supplied at 4-day intervals to the seedlings. Eight-day-old seedlings were exposed to various elevated temperatures (Fig. 1) for 10rain in low light ( 2 1Wm-2). The high-temperature (45 °C and 47 °C)-treated seedlings were further grown at 25 °C under continuous light (-~ 16Wm-2). Primary leaves of both control (untreated) and hightemperature (HT)-treated seedlings were sampled for chloroplast isolation and assay of photochemical activities at 24-h intervals. The chloroplast isolation and the polarographic measurement of the partial photochemical activities were done as described earlier (Sabat et al. 1986) with slight modifications. The leaves were homogenized in 25mM
261 T~ '2
H 2 0 ~ P D---,.Fe c N • ASC ~ M V
tJ E
E
200 16o
g u 120
Z
3 80
o E
0
L
~_
315
25 30 Temperature
410
1
45 50 t r e a t m e n t jC
Fig. 1. High-temperature (HT)-induced loss in water oxidation capacity and ascorbatesupported whole-chain electron transport activity. Chloroplasts were isolated from 8-d-old control (untreated) and HT-treated attached wheat primary leaves and subjected to various elevated temperatures for 10 min in light.
Hepes isolation buffer (pH 7.5) containing 400 mM sucrose, 10 mM MgC12 and 5 mM KCI. Chlorophyll (Chl) was estimated according to Arnon (1949). The assay mixture for the measurement of PS-II-mediated oxygen evolution activity contained 0.3 mM FeCN and 0.2 mM PD in three ml of the 25 mM Hepes reaction buffer (pH7.8) containing 100mM sucrose, 10mM MgC12 and 5mM KC1. For H20 to methylviologen electron transport assay, 2 m M azide and 1 mM MV were used. Ascorbate-supported whole-chain electron transport activity was measured by using 5 mM ascorbate, 2 mM azide and I mM MV. P S I assay mixture contained 0.1 mM DCPIP, 5 mM ascorbate, 2 mM azide, 0.005 m M D C M U and 1 mM MV. In all the assays chloroplasts equivalent to 30 ~tg Chl was used. The assays were conducted at 25 °C under saturating light intensity (-~ 400 Wm 2).
Results and discussion
PS-II-mediated 02 evolution activity measured polarographically as electron transport activity (H20 ---, PD ~ FeCN) in chloroPlasts isolated from 8-day-old wheat primary leaves treated at various elevated temperatures declined with increase in temperature above 37.5 °C (Fig. 1). No measurable 02 evolution activity was detected in chloroplasts isolated from 47 °Ctreated plants indicating the complete loss of water oxidation capacity. The sensitiveness of water oxidation complex to high temperature is also well documented (Katoh and San-Pietro 1967, Yamashita and Butler 1968, Santarius 1975, Bauer and Senser 1979, Krishnan and Mohanty 1984).
262 High-temperature-stressed chloroplasts with impaired water oxidation system could photo-oxidize artificial electron donors like hydroxylamine (Bennoun and Joliot 1969), 1,5-diphenyl carbazide (Vernon and Shaw 1969) or ascorbate (Ben-Hayyim and Avron 1970) in the presence of a Hill oxidant. Ascorbate is known electron donor of PS II (Katoh and San-Peitro 1967, Ben-Hayyim and Avron 1970). Ascorbate electron donation depends upon the integrity of chloroplasts (Trebst 1974). We have investigated the ascorbate-supported whole-chain electron transport activity in chloroplasts isolated from control and HT-treated leaves. The ascorbate-mediated electron transport activity is reported to be DCMU-sensitive. However, in our assay conditions only 75-80% of ascorbate-supported electron transport activity was found to be DCMUsensitive. The ability of ascorbate to support PS-II-mediated electron transport activity in chloroplasts isolated from HT-treated leaves declined with the increase in temperature (Fig. 1). The residual activity obtained above 45 °C was found to be mostly DCMU-insensitive. Failure of ascorbate to donate electrons appreciably to PS II above 45°C is suggestive of heatinduced alteration at PS II level over and above the impairment at the oxygen-evolving complex (OEC). Similar conclusions have been drawn from studies on diphenyl carbazide supported PS II photochemical activity (Yamashita and Butler 1968, Santarius 1975, Pearcy et al. 1977, Gounaris et al. 1983) and Chl a fluorescence (Pearcy et al. 1977, Armond et al. 1978) in high-temperature-stressed chloroplasts. Figure 2 reveals the effect of elevated temperature on the reduced DCPIPsupported PS-I-mediated MV photoreduction activity. In chloroplasts isolated from HT-treated leaves, a significant enhancement in P S I activity
_654Asc,ocp,~550 E 500l-
~: ~so E~_35
J I I I I I 25 30 35 40 45 50 Tempera|ure lreclt men I~C
Fig. 2. High-temperature-induced enhance in reduced DCPIP-supported P S I activity. Reduced DCPIP-supported PS-I-mediated MV photoreduction activity in chloroplasts isolated from 8-d-old control and HT-treated attached wheat primary leaves is shown as the function of temperature treatment.
263 was observed. Thermal-induced stimulation in P S I activity has been also reported under in vitro conditions (Armond et al. 1978, Stidham et al. 1982, Gounaris et al. 1983). In in vitro conditions, the heat-induced stimulation in P S I activity was observed even in the presence of uncouplers which led to the suggestion that the enhancement in P S I activity was not reflection of release from photosynthetic control on thermal uncoupling (Thomas et al. 1986). Heat-induced structural alterations at the oxidizing side of P S I have been suggested (Sabat et al. 1986, Thomas et al. 1986). The mechanism of heat-induced stimulation in PSI activity in vivo is not clear and its physiological role is yet to be understood. Plants upon return to low temperature after brief exposure to high temperature try to withstand the heat injury. The post heat-stress repair of thylakoid photochemical activity in intact plants is little understood and such a study will be of importance as it may provide insight into the possible pattern of repair to heat damage in the attached leaves of high-temperaturesusceptible crop like wheat. Wheat seedlings stressed at 45 °C and 47 °C are selected for the analysis of post heat effects as the former with marginal water oxidation capacity and latter with complete impaired water oxidation capacity (see Fig. 1) represent two different stages for possible repair during post stress. Total Chl content estimated, revealed significant loss in Chl amount after 72 h in 47 °C-treated leaves (data not shown). Thus, the experiments were restricted to 72 h in 47 °C-treated leaves in order to exclude the effects of aging induced by HT-treatment. A differential pattern of restoration of water oxidation capacity in chloroplasts isolated from 45 °C and 47 °C treated primary leaves were observed (Fig. 3). In 45 °C-treated leaves a significant restoration in water oxidation capacity was observed indicating the possibility of total restoration during post stress. However, 47 °C-treated leaves with no measurable water oxidation activity at 0 h recovered partially to 20% of control value. This suggests that the restoration in water oxidation capacity in HT-treated leaves is dependent on the severity of heat stress. Heat-induced impairment of water oxidation capacity was attributed to the loss of M n +2 (Cheniae and Martin 1970) and also to the loss of extrinsic polypeptides from OEC (Volger and Santarius 1981, Nash et al. 1985). Recovery of water oxidation capacity could be due to the reorganization at the OEC site during post stress. Our results are in agreement with the findings of Bauer and Senser (1979) that the repair in water oxidation capacity is possible as long as heat stress is not severe to cause necrosis in ivy leaves. Failure of ascorbate to donate electrons appreciably to PS II in chloroplasts isolated from heat-stressed leaves (Fig. 1) indicates the existence of second heat-sensitive site. We have attempted to analyze the pattern of
264 --45
~, 6O o~ o
*C
j,~
"650
,2
°20 c
> o u ~
j/¢/ ,4
0
2J4 -- ~8 7'2916 Time after temperolure treatment,h
~-
Fig. 3. Percentage of recovery in water oxidation capacity in chloroplasts isolated from HT-treated attached primary leaves during post stress. Whole-chain electron transport activity (H20 ~ MV) in closed circles (e) and PS-II-mediated 02 evolution activity (H20 ~ PD FeCN) in open circles (O) for 45°C - and 47°C-treated plants. The corresponding control (100%) values for H 2 0 ~ MV assay are 88, 82, 77, 79 and 87 tt mole 02 consumed mg Chl Ihand for H 2 0 ~ P D - - * F e C N assay 172, 183, 167, 175 and 180/t mole 02 evolved mg Chl ~h -~ at 0, 24, 48, 72 and 96 h, respectively, of post stress. The SD is not more than 5%.
repair possible at this heat-sensitive site during post stress using ascorbatesupported whole-chain electron transport assay. Ascorbate-supported whole-chain electron transport activity in chloroplasts isolated from HTtreated leaves restored significantly in 45 °C-treated plants. However, marginal restoration was noticed in 47 °C-treated plants during post stress (Fig. 4). Our observations suggest that both the impairments at OEC site and PS II site could be repaired during post stress at least in 45 °C-treated plants. ~8o
ASC'-)'MV
E
....
45"C o
70 >,60 > ou 5 0
.£ b40
I
I
712 96 Time after temperature trealment, h ;
24
/-.8
Fig. 4. Percentage recovery in ascorbate-supported whole-chain-mediated MV photoreduction activity in chloroplasts isolated at 24h intervals from HT-treated attached wheat primary leaves. The corresponding control (100%) values are, 160, 170, 172, 178 and 167# mole 02 consumed mg Chl-th -~ at 0, 24, 48, 72 and 96 h, respectively, of post stress. The SD is not more than 5%.
265 Repair in 47 °C-treated plants are marginal, possibly because of the severity of heat stress. Further repair in 47 °C-treated plants may take long time which could not be analyzed because of complexity arising due to the induction of aging process. Recently Weis et al. (1986) have reported that the period necessary for restoration of photosynthesis after mild heat stress depended on the extent of heat damage in Liverwort thalli. Figure 5 reveals the reversal of heat-induced enhancement in PSI activity in chloroplasts isolated from both 45 °C- and 47 °C-treated plants. The heat-induced stimulation in DCPIPH2-supported PS I activity observed at 0 h in chloroplasts isolated from 45 °C- and 47 °C-treated primary leaves disappeared during 48 h after the heat treatment. Our result suggests that heat-induced alterations responsible for enhancement in PS I activity are repairable during the post stress. We report that the high-temperature-induced alterations in terms of impairments at OEC site and PS II site and alteration at PSI site responsible for enhancement in DCPIPHz-supported PSI activity are repairable during post stress. Restoration of water oxidation capacity is dependent on the severity of heat stress as evidence in 47°C-treated leaves. The reversal of heat-enhanced PSI activity simultaneous to the restoration in water oxidation capacity and PS II activity indicates the existence of control to reverse the HT-induced alterations of the photosynthetic apparatus. Further studies are needed to ascertain the physiological significance of stimulation in PSI activity, if any, and to underline the mechanism of repair during post HT-stress. ~160-- { ~ "c
--45°C . . . . 47 °C
a-140 ~ " ~
\ x\ \
>--12o
m °80
x_
a_
~
I
I
I
I
0
24
48
72
X,m~: aft~?r temperature: treatmentjh
Fig. 5. Post effect of HT-treatment on the reduced DCPIP-supported P S I activity. Reduced DCPIP-supported PS-I-mediated MV photoreduction activity of chloroplasts isolated from HT-treated attached primary leaves measured at 24-h intervals shown in percentage of corresponding control values. Respective control (100%) values at 0, 24, 48 and 72 h of post stress are 380, 420 408 and 455 # mole of O2 consumed mg Chl ~h ~. The SD is not more than 50/0.
266 Acknowledgement Supported by grant No. FG-ln-575 (IN-SEA-170) to PM. NM is thankful to University Grants Commission for fellowship.
References Armond PA, Schreiber U and Bjorkman O (1978) Photosynthetic acclimation to temperature in the desert shrub Larrea divericata. II. Light harvesting efficiency and electron transport. Plant Physiol 61:411-415 Arnon DI (1949) Copper enzymes in isolated chloroplasts Polyphenol oxidase in Beta vulgaris. Plant Physiol 24:1-15 Bauer H and Senser M (1979) Photosynthesis of Ivy leaves (Hedera helix L.) after heat stress. II. Activity of ribulose bisphosphate carboxylase, Hill reaction and chloroplast ultrastructure. Z Pflanzenphysiol Bd 91.S: 35%369 Ben-Hayyim G and Avron M (1970) Involvement of photosystem two in nonoxygen evolving non-cyclic and in cyclic electron flow processes in chloroplasts. Eur J Biochem 15:155 160 Bennoun P and Joliot A (1969) Etude de La Photooxidation de L'Hydroxylamine Par les chloroplasts D'epinards Biochim. Biophys Acta 189:85-94 Berry J and Bjorkman O (1980) Photosynthetic response and adaptation to temperature in higher plants. Ann Rev Plant Physiol 31:491-543 Cheniae GM and Martin IF (1970) Site of manganese within photosystem II. Roles in 02 evolution and system II. Biochim Biophys Acta 197:219-239 Gounaris K, Brain APR, Quinn PJ and Williams WP (1983) Structural and functional changes associated with heat-induced phase separations of non-bilayer lipids in chloroplast thylakoid membranes. FEBS Lett 153:47 52 Katoh S and San-Pietro A (1967) Ascorbate-supported NADP photoreduction by heated Euglena chloroplasts. Arch Biochem Biophys 122:144-152 Krishnan M and Mohanty P (1984) Reactivation by chloride of Hill-activity in heat- and tris-heated thylakoid membranes from Beta vulgaris. Photosynth Res 5:185-198 Nash D, Miyao M and Murata N (1985) Heat inactivation of oxygen evolution in photosystem II particles and its acceleration by chloride depletion and exogenous manganese. Biochim Biophys Acta 807:127-133 Pearcy RW, Berry JA and Ford DC (1977) Effects of growth temperature on the thermal stability of the photosynthetic apparatus of Atriplex lentiformis (Torr.) Wats. Plant Physiol 59:873-878 Quinn PJ and Williams WP (1985) Environmentally induced changes in chloroplast membranes and their effects on photosynthetic function. In: Barber J and Baker NR (eds) Photosynthetic Mechanisms and the Environment. pp 1-47. Amsterdam: Elsevier Science Publishers, Biomedical Division. Sabat SC, Mohanty N and Mohanty P (1986) Heat-induced alteration in electron donation sites of ascorbate and ascorbate-reduced catechol in the electron transport chain of Amaranthus chloroplasts. Ind J Biochem Biophys 23:266-269 Santarius KA (1975) Sites of heat sensitivity in chloroplasts and differential inactivation of cyclic and non-cyclic photophsphorylation by heating. J Therm Biol 1:101-107 Stidham MA, Uribe EG and Williams GJ III (1982) temperature dependence of photosynthesis in Agropyron smithii Rydb. II. Contribution from electron transport and photophosphorylation. Plant Physiol 69:929-934
267 Thebud R and Santarius KA (1982) Effects of high-temperature stress on various biomembranes of leaf cells in situ and in vitro. Plant Physiol 70: 200-205. Thomas PG, Quinn PJ and Williams WP (1986) The origin of photosystem I mediated electron transport stimulation in heat-stressed chloroplasts. Planta 167:133-139 Trebst A (1974) Energy conservation in photosynthetic electron transport of chloroplasts. Ann Rev Plant Physiol 25:423-458 Vernon LP and Shaw ER (1969) Photoreduction of 2,6-dichlorophenolindophenolby diphenylcarbazide: A photosystem 2 reaction catalyzed by Tris-washed chloroplasts and subchloroplasts fragments. Plant Physiol 44:1645 1649 Volger H and Santarius KA (1981) Release of membrane proteins in relation to heat injury of spinach chloroplsts. Physiol Plant 51:195-200 Weis E, Wamper D and Santarius KA (1986) Heat sensitivity and thermal adaptation of photosynthesis in Liverwort thalli. Oecologia 69:134~139 Yamashita T and Butler WL (1968) Inhibition of chloroplast UV-irradiation and heat-treatment. Plant Physiol 43:2037-2040 Yordanov I, Zeinalov Y and Stamenova M (1975) Influence of post-action of high temperatures on photosynthetic activity, composition of lamellar proteins and spectral characteristics of pigment system I and II. Biochem Physiol Pflanzen 168:567-573
Regular Paper
Reversal of heat-induced alterations in photochemical activities in wheat primary leaves N A R E N D R A N A T H MOHANTY, S.D.S. M U R T H Y & PRASANNA MOHANTY 1 School of Life Sciences, Jawaharlal Nehru University, NEW DELHI- 110 067, India; i Author for correspondence Received 12 May 1987; accepted in revised form 5 August 1987
Key words: high temperature-induced alterations, photochemical activities, post stress reversal, wheat Abstract. Chloroplasts isolated from elevated temperature treated 8-day-old continuouswhite-light-grown wheat primary leaves lost the ability to photo-oxidize water. Also, the ability of ascorbate to donate electrons to photosystem II declined. However, a significant increase in reduced dichlorophenolindophenol-supported photosystem-I-mediated methylviologen photo-reduction activity was observed. The plants stressed at 45 °C and 47 °C were subsequently grown at 25 °C and the partial photochemical activities were measured in chloroplasts isolated from the plants at 24-h intervals. The post stress alterations observed are (1) a significant restoration of water oxidation capacity in 45 °C and partial restoration in 47 °C-treated leaves. Ascorbate-supported photochemical activities recovered more or less in similar fashion; (2) reversal of enhanced photosystem I activitY in both 45 ° C and 47 °C treated leaves. These results suggest that the restoration in water oxidation capacity is possible in 45 °C treated leaves and is limited by the severity of heat stress in 47 °C-treated leaves. Restoration of water oxidation capacity vis-5.-vis to the reversal of heat-enhanced photosystern I activity also indicates the existence of possible endogenous control for repair of alterations during the post stress. Abbreviations: DCPIP-2,6, Dichlorophenol-indophenol, DCMU-3-(3,4-dichlorophenyl)-l, 1-dimethylurea, FeCN Ferricyanide, Hepe~N-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid, PD-Phenylene diimine, MV-Methyl Viologen
Plants are often exposed to fluctuations in atmospheric temperature, which influences the functional activity of photosynthetic apparatus. At elevated temperature marked inhibition in photosynthetic efficiency occurs (Berry and Bjorkman 1980, Quinn and Williams 1985). The inhibition of whole-leaf photosynthesis by high temperature has been found to be due to the disruption of the functional integrity of the photosynthetic apparatus at the chloroplast level (Pearcy et al. 1977, Armond et al. 1978, Bauer and Senser 1979). The thylakoid membranes have been shown to be more heat-sensitive
260 than other biomembranes (Thebud and Santarius 1982). High-temperature treatment to whole plants, excised leaves or isolated chloroplasts results in significant or complete loss of photophosphorylation and photosystem (PS) II-mediated electron transport activity (Berry and Bjorkman 1980, Quinn and Williams 1985). In isolated chloroplasts subjected to elevated temperature, however, a stimulation in DCPIPH2-supported PS I activity is observed (Quinn and Williams 1985). Whole plants rather than the isolated chloroplasts should be preferred for studying the effect of heat stress on the functioning of the thylakoid membranes as chloroplast in vivo (i.e., inside the leaves) may exhibit differential heat tolerance compared to that of isolated chloroplasts. Furthermore, for a study on the post effect of high temperature stress, the attached leaves serve as the most suitable system as these provide the conducive environment for possible repair inside the chloroplast. A majority of the reports on the high temperature effects on photosynthesis have been confined to in vitro studies. Only a few reports on the post action of heat stress on photosynthetic activities such as, in ivy leaves (Bauer and Senser 1979) and in excised spinach leaves, (Yordanov et al. 1975) have been documented. Thus, a detailed study on the after-effect of heat stress on the various photochemical activities in chloroplasts will further our understanding of the repair of heat-damaged photosynthetic apparatus. In the present study, we have attempted to investigate the partial electron transport activities in chloroplast isolated from the intact wheat seedlings recovering at 25 °C after receiving the heat treatment at 45 °C and 47 °C for 10 min.
Materials and methods
Wheat (Triticum aestivum L. cv Kalyansona) seedlings were raised on Petri-plates under continuous white light (-~16Wm -2) at 25°C. Half strength Hoagland solution was supplied at 4-day intervals to the seedlings. Eight-day-old seedlings were exposed to various elevated temperatures (Fig. 1) for 10rain in low light ( 2 1Wm-2). The high-temperature (45 °C and 47 °C)-treated seedlings were further grown at 25 °C under continuous light (-~ 16Wm-2). Primary leaves of both control (untreated) and hightemperature (HT)-treated seedlings were sampled for chloroplast isolation and assay of photochemical activities at 24-h intervals. The chloroplast isolation and the polarographic measurement of the partial photochemical activities were done as described earlier (Sabat et al. 1986) with slight modifications. The leaves were homogenized in 25mM
261 T~ '2
H 2 0 ~ P D---,.Fe c N • ASC ~ M V
tJ E
E
200 16o
g u 120
Z
3 80
o E
0
L
~_
315
25 30 Temperature
410
1
45 50 t r e a t m e n t jC
Fig. 1. High-temperature (HT)-induced loss in water oxidation capacity and ascorbatesupported whole-chain electron transport activity. Chloroplasts were isolated from 8-d-old control (untreated) and HT-treated attached wheat primary leaves and subjected to various elevated temperatures for 10 min in light.
Hepes isolation buffer (pH 7.5) containing 400 mM sucrose, 10 mM MgC12 and 5 mM KCI. Chlorophyll (Chl) was estimated according to Arnon (1949). The assay mixture for the measurement of PS-II-mediated oxygen evolution activity contained 0.3 mM FeCN and 0.2 mM PD in three ml of the 25 mM Hepes reaction buffer (pH7.8) containing 100mM sucrose, 10mM MgC12 and 5mM KC1. For H20 to methylviologen electron transport assay, 2 m M azide and 1 mM MV were used. Ascorbate-supported whole-chain electron transport activity was measured by using 5 mM ascorbate, 2 mM azide and I mM MV. P S I assay mixture contained 0.1 mM DCPIP, 5 mM ascorbate, 2 mM azide, 0.005 m M D C M U and 1 mM MV. In all the assays chloroplasts equivalent to 30 ~tg Chl was used. The assays were conducted at 25 °C under saturating light intensity (-~ 400 Wm 2).
Results and discussion
PS-II-mediated 02 evolution activity measured polarographically as electron transport activity (H20 ---, PD ~ FeCN) in chloroPlasts isolated from 8-day-old wheat primary leaves treated at various elevated temperatures declined with increase in temperature above 37.5 °C (Fig. 1). No measurable 02 evolution activity was detected in chloroplasts isolated from 47 °Ctreated plants indicating the complete loss of water oxidation capacity. The sensitiveness of water oxidation complex to high temperature is also well documented (Katoh and San-Pietro 1967, Yamashita and Butler 1968, Santarius 1975, Bauer and Senser 1979, Krishnan and Mohanty 1984).
262 High-temperature-stressed chloroplasts with impaired water oxidation system could photo-oxidize artificial electron donors like hydroxylamine (Bennoun and Joliot 1969), 1,5-diphenyl carbazide (Vernon and Shaw 1969) or ascorbate (Ben-Hayyim and Avron 1970) in the presence of a Hill oxidant. Ascorbate is known electron donor of PS II (Katoh and San-Peitro 1967, Ben-Hayyim and Avron 1970). Ascorbate electron donation depends upon the integrity of chloroplasts (Trebst 1974). We have investigated the ascorbate-supported whole-chain electron transport activity in chloroplasts isolated from control and HT-treated leaves. The ascorbate-mediated electron transport activity is reported to be DCMU-sensitive. However, in our assay conditions only 75-80% of ascorbate-supported electron transport activity was found to be DCMUsensitive. The ability of ascorbate to support PS-II-mediated electron transport activity in chloroplasts isolated from HT-treated leaves declined with the increase in temperature (Fig. 1). The residual activity obtained above 45 °C was found to be mostly DCMU-insensitive. Failure of ascorbate to donate electrons appreciably to PS II above 45°C is suggestive of heatinduced alteration at PS II level over and above the impairment at the oxygen-evolving complex (OEC). Similar conclusions have been drawn from studies on diphenyl carbazide supported PS II photochemical activity (Yamashita and Butler 1968, Santarius 1975, Pearcy et al. 1977, Gounaris et al. 1983) and Chl a fluorescence (Pearcy et al. 1977, Armond et al. 1978) in high-temperature-stressed chloroplasts. Figure 2 reveals the effect of elevated temperature on the reduced DCPIPsupported PS-I-mediated MV photoreduction activity. In chloroplasts isolated from HT-treated leaves, a significant enhancement in P S I activity
_654Asc,ocp,~550 E 500l-
~: ~so E~_35
J I I I I I 25 30 35 40 45 50 Tempera|ure lreclt men I~C
Fig. 2. High-temperature-induced enhance in reduced DCPIP-supported P S I activity. Reduced DCPIP-supported PS-I-mediated MV photoreduction activity in chloroplasts isolated from 8-d-old control and HT-treated attached wheat primary leaves is shown as the function of temperature treatment.
263 was observed. Thermal-induced stimulation in P S I activity has been also reported under in vitro conditions (Armond et al. 1978, Stidham et al. 1982, Gounaris et al. 1983). In in vitro conditions, the heat-induced stimulation in P S I activity was observed even in the presence of uncouplers which led to the suggestion that the enhancement in P S I activity was not reflection of release from photosynthetic control on thermal uncoupling (Thomas et al. 1986). Heat-induced structural alterations at the oxidizing side of P S I have been suggested (Sabat et al. 1986, Thomas et al. 1986). The mechanism of heat-induced stimulation in PSI activity in vivo is not clear and its physiological role is yet to be understood. Plants upon return to low temperature after brief exposure to high temperature try to withstand the heat injury. The post heat-stress repair of thylakoid photochemical activity in intact plants is little understood and such a study will be of importance as it may provide insight into the possible pattern of repair to heat damage in the attached leaves of high-temperaturesusceptible crop like wheat. Wheat seedlings stressed at 45 °C and 47 °C are selected for the analysis of post heat effects as the former with marginal water oxidation capacity and latter with complete impaired water oxidation capacity (see Fig. 1) represent two different stages for possible repair during post stress. Total Chl content estimated, revealed significant loss in Chl amount after 72 h in 47 °C-treated leaves (data not shown). Thus, the experiments were restricted to 72 h in 47 °C-treated leaves in order to exclude the effects of aging induced by HT-treatment. A differential pattern of restoration of water oxidation capacity in chloroplasts isolated from 45 °C and 47 °C treated primary leaves were observed (Fig. 3). In 45 °C-treated leaves a significant restoration in water oxidation capacity was observed indicating the possibility of total restoration during post stress. However, 47 °C-treated leaves with no measurable water oxidation activity at 0 h recovered partially to 20% of control value. This suggests that the restoration in water oxidation capacity in HT-treated leaves is dependent on the severity of heat stress. Heat-induced impairment of water oxidation capacity was attributed to the loss of M n +2 (Cheniae and Martin 1970) and also to the loss of extrinsic polypeptides from OEC (Volger and Santarius 1981, Nash et al. 1985). Recovery of water oxidation capacity could be due to the reorganization at the OEC site during post stress. Our results are in agreement with the findings of Bauer and Senser (1979) that the repair in water oxidation capacity is possible as long as heat stress is not severe to cause necrosis in ivy leaves. Failure of ascorbate to donate electrons appreciably to PS II in chloroplasts isolated from heat-stressed leaves (Fig. 1) indicates the existence of second heat-sensitive site. We have attempted to analyze the pattern of
264 --45
~, 6O o~ o
*C
j,~
"650
,2
°20 c
> o u ~
j/¢/ ,4
0
2J4 -- ~8 7'2916 Time after temperolure treatment,h
~-
Fig. 3. Percentage of recovery in water oxidation capacity in chloroplasts isolated from HT-treated attached primary leaves during post stress. Whole-chain electron transport activity (H20 ~ MV) in closed circles (e) and PS-II-mediated 02 evolution activity (H20 ~ PD FeCN) in open circles (O) for 45°C - and 47°C-treated plants. The corresponding control (100%) values for H 2 0 ~ MV assay are 88, 82, 77, 79 and 87 tt mole 02 consumed mg Chl Ihand for H 2 0 ~ P D - - * F e C N assay 172, 183, 167, 175 and 180/t mole 02 evolved mg Chl ~h -~ at 0, 24, 48, 72 and 96 h, respectively, of post stress. The SD is not more than 5%.
repair possible at this heat-sensitive site during post stress using ascorbatesupported whole-chain electron transport assay. Ascorbate-supported whole-chain electron transport activity in chloroplasts isolated from HTtreated leaves restored significantly in 45 °C-treated plants. However, marginal restoration was noticed in 47 °C-treated plants during post stress (Fig. 4). Our observations suggest that both the impairments at OEC site and PS II site could be repaired during post stress at least in 45 °C-treated plants. ~8o
ASC'-)'MV
E
....
45"C o
70 >,60 > ou 5 0
.£ b40
I
I
712 96 Time after temperature trealment, h ;
24
/-.8
Fig. 4. Percentage recovery in ascorbate-supported whole-chain-mediated MV photoreduction activity in chloroplasts isolated at 24h intervals from HT-treated attached wheat primary leaves. The corresponding control (100%) values are, 160, 170, 172, 178 and 167# mole 02 consumed mg Chl-th -~ at 0, 24, 48, 72 and 96 h, respectively, of post stress. The SD is not more than 5%.
265 Repair in 47 °C-treated plants are marginal, possibly because of the severity of heat stress. Further repair in 47 °C-treated plants may take long time which could not be analyzed because of complexity arising due to the induction of aging process. Recently Weis et al. (1986) have reported that the period necessary for restoration of photosynthesis after mild heat stress depended on the extent of heat damage in Liverwort thalli. Figure 5 reveals the reversal of heat-induced enhancement in PSI activity in chloroplasts isolated from both 45 °C- and 47 °C-treated plants. The heat-induced stimulation in DCPIPH2-supported PS I activity observed at 0 h in chloroplasts isolated from 45 °C- and 47 °C-treated primary leaves disappeared during 48 h after the heat treatment. Our result suggests that heat-induced alterations responsible for enhancement in PS I activity are repairable during the post stress. We report that the high-temperature-induced alterations in terms of impairments at OEC site and PS II site and alteration at PSI site responsible for enhancement in DCPIPHz-supported PSI activity are repairable during post stress. Restoration of water oxidation capacity is dependent on the severity of heat stress as evidence in 47°C-treated leaves. The reversal of heat-enhanced PSI activity simultaneous to the restoration in water oxidation capacity and PS II activity indicates the existence of control to reverse the HT-induced alterations of the photosynthetic apparatus. Further studies are needed to ascertain the physiological significance of stimulation in PSI activity, if any, and to underline the mechanism of repair during post HT-stress. ~160-- { ~ "c
--45°C . . . . 47 °C
a-140 ~ " ~
\ x\ \
>--12o
m °80
x_
a_
~
I
I
I
I
0
24
48
72
X,m~: aft~?r temperature: treatmentjh
Fig. 5. Post effect of HT-treatment on the reduced DCPIP-supported P S I activity. Reduced DCPIP-supported PS-I-mediated MV photoreduction activity of chloroplasts isolated from HT-treated attached primary leaves measured at 24-h intervals shown in percentage of corresponding control values. Respective control (100%) values at 0, 24, 48 and 72 h of post stress are 380, 420 408 and 455 # mole of O2 consumed mg Chl ~h ~. The SD is not more than 50/0.
266 Acknowledgement Supported by grant No. FG-ln-575 (IN-SEA-170) to PM. NM is thankful to University Grants Commission for fellowship.
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267 Thebud R and Santarius KA (1982) Effects of high-temperature stress on various biomembranes of leaf cells in situ and in vitro. Plant Physiol 70: 200-205. Thomas PG, Quinn PJ and Williams WP (1986) The origin of photosystem I mediated electron transport stimulation in heat-stressed chloroplasts. Planta 167:133-139 Trebst A (1974) Energy conservation in photosynthetic electron transport of chloroplasts. Ann Rev Plant Physiol 25:423-458 Vernon LP and Shaw ER (1969) Photoreduction of 2,6-dichlorophenolindophenolby diphenylcarbazide: A photosystem 2 reaction catalyzed by Tris-washed chloroplasts and subchloroplasts fragments. Plant Physiol 44:1645 1649 Volger H and Santarius KA (1981) Release of membrane proteins in relation to heat injury of spinach chloroplsts. Physiol Plant 51:195-200 Weis E, Wamper D and Santarius KA (1986) Heat sensitivity and thermal adaptation of photosynthesis in Liverwort thalli. Oecologia 69:134~139 Yamashita T and Butler WL (1968) Inhibition of chloroplast UV-irradiation and heat-treatment. Plant Physiol 43:2037-2040 Yordanov I, Zeinalov Y and Stamenova M (1975) Influence of post-action of high temperatures on photosynthetic activity, composition of lamellar proteins and spectral characteristics of pigment system I and II. Biochem Physiol Pflanzen 168:567-573
