Planta

Plan ta (1989) 178: 545-552

9 Springer-Verlag 1989

Inhibition of photosynthesis by chilling in moderate light: a comparison of plants sensitive and insensitive to chilling Richard A.J. Hodgson 1. and John K. Raison 2 1 School of Biological Sciences, and 2 CSIRO Division of Food Processing and School of Biological Sciences, Macquarie University, N.S.W., 2109, Australia

Abstract. Photosynthetic activity, in leaf slices and

isolated thylakoids, was examined at 25 ~ C after preincubation of the slices at either 25 ~ C or 4 ~ C at a moderate photon flux density (PFD) of 450 g m o l . m - 2 . s -1, or at 4 ~ in the dark. The plants used were Spinacia oleracea L., Cucumis sativus L. and Nerium oleander L. which was acclimated to growth at 20~ or 45 ~ C. The plants were grown at a PFD of 550 g m o l . m -z. s-1. Photosynthesis, measured as CO2-dependent 02 evolution, was not inhibited in leaf slices from any plant after preincnbation at 25~ at a moderate PFD or at 4 ~ C in the dark. However, exposure to 4 ~ C at a moderate PFD induced an inhibition of CO2dependent 02 evolution within I h in C. sativus, a chilling-sensitive plant, and in 45 ~ C-grown N. oleander. The inhibition in these plants after 5 h reached 80% and 40%, respectively, and was independent of the CO2 concentration but was reduced at 02 concentrations of less than 3%. Methyl-viologen-dependent 02 exchange in leaf slices from these plants was not inhibited. There was no photoxidation of chlorophyll, in isolated thylakoids, or any inhibition of electron transport at photosystem (PS)II, PSI or through both photosystems which would account for the inhibition of photosynthesis. The conditions which inhibit photosynthesis in chilling-sensitive plants do not cause inhibition in S. oleracea, a chilling-insensitive plant, or in 20 ~ C-grown N. oleander. The CO2-dependent photosynthesis, measured at 5~ C, was re* Present address: Department of Biology, Washington University, Box 1137, St. Louis, MO 63130, USA Abbreviations: Chl=chlorophylt; D P I P H = r e d u c e d

form of 2,6-dichlorophenol-indophenol; D M Q = 2,5-dimethyl-p-benzoquinone ; M u = methyl viologen; 20~ = Nerium oleander grown at 20 ~ C; 45~ oleander grown at 45 ~ C; P F D = p h o t o n flux density (photon fluence rate); PSI and PSII = photosystem 1 and II, respectively

duced to about 3% of that recorded at 25~ in chilling-sensitive plants but only to about 30% in the chilling-insensitive plants. Methyl-viologen-dependent 02 exchange, measured at 5~ C, was greater than 25% of the activity at 25 ~ C in all the plants. The results indicate that the mechanism of the chilling-induced inhibition of photosynthesis does not involve damage to PSII. That inhibition of photosynthesis is observed only in the chillingsensitive plants indicates it is related, in some way, to the disproportionate decrease in photosynthetic activity in these plants at chilling temperatures. Key words: Chilling - Cueumis - N e r i u m

Photoinhibition of photosynthesis - Photosynthesis (active 02) - Spinacia - Temperature (chilling)

Introduction

Photosynthesis involves the interaction of a complex series of pathways and processes, some of which are light-dependent. If a pathway is for some reason severely restricted, such that dissipation of light energy is limited, then an inhibition of photosynthesis can develop (Osmond 1981). In a literal sense if the inhibition of photosynthesis is lightdependent the term photoinhibition (for a review, see Powles 1984) should be applied to describe the response. Experimentally, photoinhibition is most often induced by a high photon flux density (PFD) (i.e. above about 1000 p m o l . m - Z . s -1 or substantially greater than the irradiation experienced during growth), either alone or in combination with an additional stress (Powles 1984; Bj6rkman 1986). Under these conditions the inhibition is observed in a wide range of plants (Powles 1984; Bj6rkman 1986; Demmig and Bj6rkman 1987). The initial event(s) promoting photoinhibition are not related

546

R.A.J. Hodgson and J.K. Raison: Photoinhibition and chilling stress

to stomatal limitation of gas exchange or chlorophyll (Chl) photo-oxidation (Powles 1984). A consistent observation associated with photoinhibition induced by a high PFD is a quenching of the 77K fluorescence of both photosystem (PS)II and PSI, which can reflect damage to the reaction-center complex (Powles and Bj6rkman 1982; Powles 1984; Bj6rkman 1986). Simultaneous exposure to chilling (temperatures in the range 0 ~ C to about 15 ~ C) and a high PFD promotes a severe inhibition of photosynthesis in a broad selection of both chilling-sensitive and chilling-insensitive plants (Powles et al. 1983; Ogren etal. 1984; Yakir etal. 1985; Bongi and Long 1987; Hodgson et al. 1987; Wise and Naylor 1987). Photoinhibition has also been noted in a variety of both chilling-sensitive and chilling-insensitive plants after 20 h at 7~ C and a moderate PFD (Smillie et al. 1988). However, with chilling at a moderate PFD for only a few hours the inhibition of photosynthesis is evident only with chilling-sensitive plants (Kislyuk and Vas'kovskii 1971; Taylor and Rowley 1971 ; Wright and Simon 1973; Garber 1977; Lasley etal. 1979; Lindeman 1979; Mustfirdy et al. 1984; Moll and Steinback 1986; Hodgson et al. 1987; Peeler and Naylor 1988) and only when chilled below a particular critical temperature (Powles et al. 1983; Moll and Steinback 1986; Hodgson et al. 1987). The critical temperature for the initiation of photoinhibition under these conditions is coincident with that of a phase transition in the polar lipids of the thylakoids (Raison and Orr 1986; Hodgson et al. 1987) and thylakoid membranes (Havaux and Lannoye 1983). The phase transition in the thylakoid lipids adversely affects membrane-associated processes (Shneyour et al. 1973; Nobel 1974; Garber t977; Murata and Fork 1976) and is thought to be the basis for the dysfunction which inhibits photosynthesis at chilling temperatures (Hodgson et al. 1987). The aim of the experiments described here was to compare the effect of chilling at a moderate PFD for a few hours on photosynthesis in chillingsensitive and chilling-insensitive plants to gain some insight into how these conditions inhibit photosynthesis. The time of treatment was limited to a few hours as this would approximate the time tropical plants might experience chilling in the light in the cooler habitats of tropical latitudes after sunrise. The comparison included leaves from spinach (chilling-insensitive), cucumber (chilling-sensitive) and from an oleander clone, grown at either 20 ~ C (20~ or 45 ~ C (45~ Acclimation of oleander to different temperature re-

gimes alters the thermal response of the thylakoids and the polar lipids (Raison et al. 1982), the photosynthetic capacity and temperature optimum for photosynthesis (Badger et al. 1982) as well as the susceptibility of photosynthesis to inhibition by chilling (Hodgson et al. 1987). Thus, for oleander, growth at 20~ and 45~ produces plants which show a differential response to chilling and provides a model for comparative studies that obviate genetic differences.

Materials and methods Plant material. Seeds of Spinach (Spinacia oIeracea L. cv. Yates hybrid 107), cucumber (Cucumis sativus L. cv. Palomar) were obtained from New World Seeds Pry, Ltd., Galston, NSW., Australia. Oleander (Nerium oleander L.) was vegetatively propagated from a single bush. The plants were grown at 550 Ixmol. m - 2 - s -1 as described in Hodgson et al. (1987). The method for preparation and preincubation of the leaf slices was as described in Jones and Osmond (1973) and Hodgson et al. (1987). For the experiments examining the influence of bicarbonate (HCO~) concentration during the preincubation, 0.25 g fresh weight (FW) of leaf slices were ptaced in a glass jar to which was added 3 ml of N2-saturated buffer consisting of 0.1 M sorbitol, 0.5 mM CaSO4 and 50 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid (Hepes), pH 7.5, (Buffer A) and various concentrations of KHCO3. To examine the effect of 02 concentration during the preincubation, tubes with 0.25 g FW of leaf slices in 3 ml of Buffer A were either saturated with air, N2 or N2 plus 100 700 gM sodium dithionite, and sealed under

N2. Thylakoid isolation. Thylakoids were isolated by a procedure based on that of Anderson et al. (1971). Spinach and cucumber leaf slices (2 g FW) were chopped with a Moulinex (robot rnarinette) commercial tissue homogenizer, for 5-10 s in 75 ml of a buffer consisting of 0.33 M sorbitol, 1 m M MgClz, 1 mM MnC12, 1 mM ethylenediaminetetraacetic acid (EDTA), 30 mM 2-{[2-hydroxy-l,t-bis(hydroxymethyl)ethyl]amino}ethanesulphonic acid (Tes), pH 7.5, (Buffer B) with 5 mM dithiothreitol, 0.5% (w/v) bovine serum albumin and 1% (w/v) polyvinylpyrrolidone. Thylakoids were also isolated from oleander essentially as described above except the leaf slices were ground in a mortar and pestle. The homogenates were filtered through two layers of Miracloth (Calbiochem, Behring Diagnostics, La Jolla, Cal., USA) before centrifuging at 1000 .g for 10 min. The supernatant was discarded and the green pellet resuspended and washed in Buffer B, pH 7.5. The final chloroplast pellet was resuspended in a small volume of this buffer. All operations were carried out at 4 ~ C. Photosynthetic activity. Photosynthesis was measured as COzdependent 02 exchange as described in Jones and Osmond (1973) using a PFD of 1 100 ~mol-m-2. s-1. Electron transport was measured as methyl-viologen (MV)-dependent 02 exchange, in Buffer A with 2 m M MV. For the experiments described in Fig. 5, leaf slices were removed fi'om the preincubation conditions and incubated in the 02 electrode at 25 ~ C in the dark for a further 15 min with either KHCO3 or MV before measuring dark respiration, photosynthesis and electron transport. For the experiments described in Table 1 the leaf slices were not preincubated, but were assayed directly at 25 ~ C or

R.A.J. Hodgson and J.K. Raison : Photoinhibition and chilling stress 5~ C. All assays were completed within 30 min. Electron transport in thylakoids was measured in Buffer B, pH 8.0, using the equivalent of 30 gg of Chl. Whole-chain electron transport was measured following the addition of either 1 m M K3Fe(CN)6 or 1 m M MV; PSII activity was measured in the presence of 0.75 m M 2,5-dimethyl-p-benzoquinone (DMQ) and 1 m M K3Fe(CN)6; and PSI activity in the presence of 8 gM 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU), I m M ascorbate, I00 pM 2,6-dichlorophenolindophenol (DCPIP) and 1 m M MV. Electron transport was uncoupled with 2.5 m M NH4CI.

Estimation of chlorophyll and carotenoids. The pi~dnents were extracted from leaf slices or isolated thyalakoids with 80% (v/v) acetone-water_ The concentration of Chi and totai carotenoids was estimated as described by Arnon (1949) and Lichtenthaler et al. (1982), respectively.

Results The effect of chilling leaf slices in moderate light on the subsequent rate of both light-limited and light-saturated photosynthesis, measured at 25 ~ C, is shown in Fig. 1. Slices from both cucumber and 45~ show inhibition of light-limited and light-saturated photosynthesis following preincubation for 2.5 h at 4 ~ at a P F D of 450 gmol. m - z . s-~. The inhibition for cucumber is more severe than for 45~ There was no inhibition in either of these plants following preincubation at 25 ~ C at a moderate P F D or 4 ~ C in the dark.

547

Also, no inhibition was detected in spinach and 20~ following preincubation at either 4 ~ C or 25 ~ C at a moderate PFD. The rates of photosynthesis at light saturation (Fig. 1) are the same as the rates reported by Jones and Osmond (1973) for leaf slices. They are lower than the rates for leaf disks of sun plants but higher than those calculated for shade plants (Walker and Osmond 1986), assuming 510 mg Chl. m - 2. Since the rates of photosynthesis using leaf slices (Fig. 1) are within the range of values for leaf disks the use of slices in an aqueous environment was considered an appropriate technique for comparative studies of photosynthesis. The effect of increasing the time of preincubation on photosynthesis in leaf slices is shown in Fig. 2. Photosynthesis was not affected for at least 5 h when the preincubation was either at 4 ~ C in the dark or at 25 ~ C in a moderate PFD. Photosynthesis in cucumber leaf slices was inhibited by about 60% and in 45~ by about 20%, following preincubation at 4 ~ in a moderate P F D for 2.5 h. This increased to about 80% and 40% inhibition, respectively, when the preincubation was for 5 h. With spinach and 20~ exposed to the same conditions, no inhibition of photosynthesis was observed. The dark respiration (mean 02 uptake gmol. (rag Chl)- 1. h - 1 + SE, n >

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Photonflux density(pmol-rn-2-s -I) Fig. I, The response of photosynthesis to P F D in spinach, cucumber and 45 ~ and 20~ Photosynthesis was measured at 25 ~ C following preincubation for 2.5 h at either 25 ~ C (solid symbols) or 4 ~ C (open symbols) with 450 txmol, m 2. s-~ ( ) or in the dark ( ..... ). For spinach and 20~ the curves for preincubation in the light at 2 5 ~ and 4 ~ C are the same and only the curve for 4 ~ C in the light is shown. With cucumber and 45 ~ C-oleander the curves for preincubation at 25 ~ C in the light and 4 ~ in the dark are the same and only the curve for 4 ~ in the dark is shown. Each point represents an individual value

548

R.A.J. Hodgson and J.K. Raison : Photoinhibition and chilling stress

concentration had no adverse effect on photosynthesis. To assess the photosynthetic potential of leaf slices at chilling temperatures a direct comparison was made of photosynthetic activity at 25 ~ C and 5~ C. As shown in Table 1, both photosynthesis and electron transport are restricted at 5~ C in all plants. Photosynthesis, measured as CO2-dependent 02 exchange at 5~ C, was reduced to about 35% of the activity at 25 ~ C in spinach and 20 ~ oleander. For cucumber and 45~ photosynthesis was disproportionately reduced to about 3% of that observed at 25 ~ C. In comparison with photosynthesis there was little difference in the effect of temperature on the rate of electron transport, measured as MV-dependent 02 exchange, in the two groups of plants. As shown in Table 1, the capacity for electron transport at 5~ was about 25% of the activity observed at 25~ for all plants. Similarly, the rate of dark respiration at 5~ C was reduced, for all plants, to between 18% and 28% of that at 25 ~ C (Table 1). The Qlo for MV-dependent electron transport and dark respiration in leaf slices, calculated assuming a regular decline in rate with temperature, was about 2 for all the conditions and plants. The Q~o for photosynthesis however, was much higher for cucumber and 45~ reaching 5.1, whereas for spinach and 20~ it was less than half this value, 1.8 and 2.1, respectively. Shown in Fig. 3 a is the effect of varying the HCO~- concentration during preincubation on photosynthesis in spinach and cucumber. Photosynthesis in cucumber was little affected by lowering the H C O j concentration, whereas with spinach it was inhibited up to 20% as the concentration of HCO~- was reduced below about 60 mM. The results of a similar study of the effect of 02 concentration during the preincubation on the inhibition of photosynthesis is shown in Fig. 3 b. For cucumber the inhibition increased with increasing 02,

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Fig. 2. The effect of preincubating leaf slices of spinach, cucumber, 45 ~ and 20~ at either 25~ (solid symbols) or 4~ (open symbols) in the light ( ) and dark (..... ) on CO2-dependent 02 evolution at 25 ~ C. Control rates of photosynthesis (mean ~tmol Oz' (mg Chl)- 1 h 1 + SE, n > 6) for spinach, cucumber, 20~ and 45~ were, in the sequence given, 77+10, 108+14, 108+14 and 8 7 + 9

7) in cucumber (13 + 4) and 45~ leaf slices (11 +2) was inhibited by about 25% and 7%, respectively, following 5 h at 4 ~ at a moderate PFD. Spinach (22_+1) and 20~ (22+4) leaf slices showed no inhibition of dark respiration. The HCO~ concentration in Buffer A (see Materials and methods) during preincubation was maintained at 75 m M to ensure adequate CO2 for all the tissue over the relatively long preincubation period. The observation that slices assayed before and after 5 h of preincubation, under control conditions of 25 ~ C, a moderate PFD, and at the high HCO~ concentration, had equivalent photosynthetic activity (Fig. 3) shows that the high HCO 3

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Fig. 3a, b. The effect of HCO;- Ca) and O2 (b) concentration during preincubation of Leaf slices of spinach, cucumber, 45 ~ and 20~ at 4 ~ C in the light for 2.5 h on COz-dependent 02 evolution at 25~ C. Data are individual values from several experiments

R.A.J. Hodgson and J.K. Raison: Photoinhibition and chilling stress

549

Table 1. The effect of preincubation temperature and P F D on COz-dependent photosynthesis, MV-dependent electron transport and dark respiration. The rates were measured using leaf slices and are the mean o f three experiments Plant and parameter

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151 105 4 4 5.1

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20 14 3 24 2.1

18 13 2 18 2.4

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124 91 21 24 2.1

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Cucumber gmol .(g FW) -1 -h -1, 25 ~ C ~mol. (rag Chl) - i. h - i. 25 ~ C gmol- (rag Chl) - 1. h - 1, 5~ C % of 25 ~ value Qa0 5~ C

20~ gmol-(g FW) -1 .b -1, 25 ~ C lamol-(mg Chl)- a. h - 1, 25 ~ C g m o l - ( m g C h l ) - l . h 1, 5o C % of 25 ~ value Qlo 5~176 C

45~ gmol- (g F W ) - 1. h - 1, 25 ~ C gmol. (rag Chl) - t. h - 1, 5o C g m o l - ( m g C h l ) - I h 1, 5o C % of 25 ~ value Qlo 5~176 C

" Oz evolution after accounting for dark respiration u 02 uptake accounting for dark respiration M and H refer to moderate (350 p~mol,m - 2 . s-1) and high (980 gmol-m z. s-1) P F D d Qlo calculated on the difference between rates at 25 ~ C and 5~ C

reaching a maximum at about 8%. Beyond this level increasing the O2 concentration did not increase the severity of the inhibition. For spinach, photosynthesis was not inhibited by the 02 concentration present when chilled during preincubation. The effect of chilling leaf slices at a moderate PFD on the activity of electron transport was investigated using thylakoids isolated from the slices after preincubation. As shown in Fig. 4, preincubation did not reduce the capacity of thylakoids for either whole-chain electron transport or for PSII activity in particular. Similarly, preincubation did not affect PSI except in thylakoids from cucumber, where it was inhibited by about 16%. This inhibition was, however, not observed as a decrease in whole-chain electron transport. To determine if the inhibition of PSI in cucumber could be attributed to a disruption of membrane integrity during isola-

tion of the thylakoids, the rate of electron transport in intact slices was determined by measuring MV-dependent O2 exchange. The results, shown in Fig. 5, indicate electron transport to PSI was not inhibited in cucumber when photosynthesis was substantially inhibited. Similarly there was no inhibition of electron transport in the slices from spinach. Chilling for up to 5 h at 4 ~ at a moderate PFD caused no significant loss of either total Chl or carotenoids in any of the plants. Values for cucumber + S E , n > 1 7 , before and after the preincubation, were: for Chl 1.27_+0.13 and 1.25_+0.14 gg. (g FW) - t , for the Chl a/b ratio 3.1 +0.1 and 3.2___ 0.2, and for total carotenoid 202 • 20 and 198 • gg. (g F W ) - 1. There was also no effect of preincubation at 4 ~ C in the dark on pigment concentrations in any of the plants.

550

R.A.J. Hodgson and J.K. Raison: Photoinhibition and chilling stress

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Fig. 5. The photosynthetic 02 exchange in leaf slices of spinach, cucumber, 45 ~ and 20~ after a preincubation at 4 ~ C in the light for 2.5 h. The electron acceptors were HCO~ or MV. Control rates of photosynthesis (mean gmol 0 2 ' ( m g C h l ) - I . h - I _ + S E , n > 3 ) were 774-10 and 13.54-1.1 (spinach) and 1084-14 and 15.04-1.3 (cucumber), using HCO~ and MV, respectively

In quantitative terms the inhibition of photosynthesis exhibited by cucumber and 45~ under conditions of a moderate PFD and chilling (Fig. 1), is, in general, similar to the photoinhibition induced by a high PFD (Powles et al. 1983). However, there are marked qualitative differences. The most obvious one is that the expression of photoinhibition in leaf slices, induced by chilling at a moderate PFD, does not appear to involve an inhibition of PSII activity. It is dependent on oxygen (Fig. 3), light (Fig. 1) and on a simultaneous chilling stress, and is thus evident only in plants sensitive to chilling (Fig. 1 and Hodgson et al. 1987). In recognition of these characteristics the inhibition described here is referred to as chilltemperature photoinhibition to distinguish it from the photoinhibition induced by a high PFD, or a high PFD in combination with temperature extremes, limiting nutrients or low levels of CO2 (Ogren et al. 1984; Powles 1984; Bj6rkman 1986; Moll and Steinback 1986; Bongi and Long 1987). Chill-temperature photoinhibition is evident only in cucumber and 45~ (Fig. 1) and in these plants the polar lipids from the leaves of both these plants show phase transitions at about 12~ and 7~ C, respectively (Hodgson et al. 1987), typical of plants sensitive to chilling. This relationship between susceptibility of the plant to chilltemperature photoinhibition and sensitivity to chilling is in marked contrast with the photoinhibition induced by a high PFD (Powles et al. 1983; Bj6rkman 1986; Demmig and Bj6rkman 1987) for which all plants tested appear susceptible to this treatment. Chilling at a moderate PFD inhibited photosynthesis by up to 80%, but did not cause a loss of chloroplast pigments in any of the plants. Other studies (Garber 1977; Taylor and Rowley 1971) also show there is considerable inhibition of photosynthesis before there is a measurable photo-oxidation of Chl, even under conditions of a high PFD (Powles 1984). Thus, it is unlikely that photo-oxidation of Chl plays a role during the relatively short period in which chill-temperature photoinhibition develops. A blockage or restriction in the electron-transport chain has the potential to inhibit the nondestructive dissipation of energy from the thylakoids and therefore promote chill-temperature photoinhibition. In thylakoids isolated from plants chilled at a moderate PFD for 5 h there was no major disruption in electron transport at either PSI or PSII or in whole-chain electron flow to MV

R.A.J. Hodgson and J.K. Raison: Photoinhibition and chilling stress

(Fig. 4). The small inhibition of PSI activity (16%) displayed by thylakoids from cucumber could not account for the 80% inhibition of photosynthesis. In addition, the importance of this inhibition in PSI activity is perplexing. Others (Kislyuk and Vas'kovskii 1972; Garber 1977; Lindeman 1979) have noted a small inhibition in PSI activity in the first 20 h of chilling but this would not account for the severe inhibition noted in chilling-sensitive plants after only 5 h of chilling (Fig. 2). Another feature which distinguishes the chilltemperature photoinhibition from the inhibition produced by a high PFD, is that in the former PSII activity is not inhibited (Fig. 4). This result is consistent with those of Kislyuk and Vas'kovskii (1972) and Linderman (1979) who show that chilling at a moderate PFD does not inhibit PSII activity for at least 20 h, and those of Mfienp/ifi et al. (1988) who show only as little as 9% inhibition of PSII activity in chilling-sensitive pumpkin after 5 d of chilling at a moderate PFD. The observation that MV-dependent 02 exchange is not inhibited by chilling at a moderate PFD is consistent with the lack of inhibition in PSII activity. Photosystem II is damaged and MV-dependent 02 exchange is inhibited by a high PFD (Whitelam and Codd 1983). This further supports the view that different mechanisms are involved. Some indication of the site of chill-temperature photoinhibition can be obtained from the different effect of the treatment on CO2-dependent and MV-dependent 02 exchange. The former involves both electron transport through the photosystems as well as carbon fixation, while the latter depends only on the rate of electron transport to PSI. Thus inhibition of CO2-dependent O2 exchange without an inhibition of MV-dependent 02 exchange (Fig. 5) indicates that a site of dysfunction in chilltemperature photoinhibition is located somewhere on the reducing side of PSI. This could be at the terminal components of thylakoid electron transport (ferredoxin-FeS complex, ferredoxin-NADP reductase) and-or in reactions of the Calvin cycle. In a further examination of photosynthesis in leaf slices, undertaken to test the view that chilltemperature photoinhibition results from damage on the reducing side of PSI, photosynthesis and electron transport were assayed at 5~ C instead of 25 ~ C (Table 1). For CO2-dependent 02 exchange, chilling-sensitive and chilling-insensitive plants are differentially affected but for MV-dependent 02 exchange there is no distinction between the two plant groups. Because CO2 and M u act as acceptors at different locations in the chloroplast, these data (Table 1) are taken as support for the assump-

551

tion (derived from Figs. 4, 5) that the site of damage in chill-temperature photoinhibition is not in PSII but is either in the terminal components of electron transport and-or in the Calvin cycle. The ability of chilling-insensitive plants to tolerate or avoid the disruption(s) produced by chilling at a moderate PFD may be related to their capacity to fix carbon at chilling temperatures (Table 1). This suggestion is derived from the proposal that susceptibility to photoinhibition is regulated, under the particular stress conditions, by the rate of carbon metabolism in the tissue (Osmond 1981). Thus, decreasing CO2 fixation, by decreasing COz concentration for exampl e, can exacerbate photoinhibition at 25 ~ C (Powles and Critchley 1980). This effect of COz concentration is not seen for chilling-induced photoinhibition in cucumber (Fig. 3 a) or Phaseolus vulgaris (Powles et al. 1983). However, for spinach, limited chill-temperature photoinhibition develops when carbon fixation is reduced by lowering the CO/ concentration (Fig. 3 a). This result indicates that spinach maintains an adequate rate of carbon fixation at chilling temperatures in the light and thus avoids the conditions that lead to photoinhibition. Taken together our results indicate that the lesion associated with chill-temperature photoinhibition is not associated with photo-oxidation of pigments or damage to PSII. It is more likely that it involves some components on the reducing side of PSI. The dependence of chill-temperature photoinhibition on 02 (Fig. 3 b) indicates that the inactivation of the photosynthetic process is mediated through the effects of an active 02 species, such as superoxide, hydrogen peroxide and-or singlet oxygen (for reviews, see Elstner 1982; Halliwell 1984) and this aspect is being investigated. This work was supported in part by a Macquarie University Research Grant.

References Anderson, J.M., Boardman, N.K., Spencer, D. (1971) Phosphorylation by intact chloroplasts from maize bundle sheath. Biochim. Biophys. Acta 245, 253-258 Arnon, D,I. (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris L. Plant Physiol. 41, I 15 Badger, M.R., Bj6rkman, O., Armond, P.A. (1982) An analysis of photosynthetic response and adaption to temperature in the higher plants: temperature acclimation in the desert evergreen Neriurn oleander L. Plant Cell Environ. 5, 85-99 Bj6rkman, O. (1986) High-irradiance stress in higher plants and interaction with other stress factors. In: Progress in photosynthesis research, vol. IV, pp. 1.1-16, Biggins, J., ed. Martinus Nijhoff, Dordrecht, The Netherlands

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R.A.J. Hodgson and J.K. Raison: Photoinhibition and chilling stress

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tion between light and temperature. I. Photosynthesis in vivo. Physiol. Plant. 62, 181 186 Osmond, C.B. (1981) Photorespiration and photoinhibition. Some implications for the energetics of photosynthesis. Biochim. Biophys. Acta 639, 77-98 Peeler, T.C., Naylor, A.W. (1988) A comparison of the effects of chilling on leaf gas exchange in pea (Pisum sativum L.) and cucumber (Cucumis sativus L.). Plant Physiol. 86, 143146 Powles, S.B. (1984) Photoinhibition of photosynthesis induced by visible light. Annu. Rev. Plant Physiol. 35, 15~44 Powles, S.B., Berry, J.A., Bj6rkman, O. (1983) Interaction between light and chilling temperature on the inhibition of photosynthesis in chilling-sensitive plants. Plant Cell Environ. 6, 117-123 Powles, S.B., Bj6rkman, O. (1982) Photoinhibition of photosynthesis: effect on chlorophyll fluorescence at 77K in intact leaves and in chloroplast membranes of Nerium oleander. Planta 156, 97-107 Powles, S.B., Critchley, C. (1980) Effect of light intensity during growth in photoinhibition of intact attached bean leaflets. Plant Physiol. 65, 1181-1187 Raison, J.K., Pike, C.S., Berry, J.A. (1982) Growth temperature-induced alterations in the thermotropic properties of N. oleander membrane lipids. Plant Physiol. 70, 215-218 Raison, J.K., Orr, G.R. (1986) Phase transitions in thylakoid polar lipids of chilling-sensitive plants: a comparison of detection methods. Plant Physiol. 80, 638 645 Shneyour, A., Raison, J.K., Smillie, R.M. (1973) The effect of temperature of the rate of photosynthetic electron transfer in chloroplasts of chilling-sensitive and chilling-resistant plants. Biochim. Biophys. Acta 292, 152-161 Smillie, R.M., Hetherington, S.E, He, J., Nott, R. (1988) Photoinhibition at chilling temperatures. Aust. J. Plant Physiol. 15, 207-22 Taylor, A.O., Rowley, J.A. (1971) Plants under climatic stress. I. Low temperature, high light effects on photosynthesis. Plant Physiol. 47, 713-718 Walker, D.A., Osmond, C.B. (1986) Measurement of photosynthesis in vivo with a leaf disc electrode : correlation between light dependence of steady-state photosynthetic 02 evolution and chlorophyll a fluorescence transients. Proc. R. Soc. London Ser. B 227, 267-280 Whitelam, G.C., Codd, G.A. (1983)Photoinhibition of photosynthesis in the cyanobacterium Microcystis aeruginosa. Planta 157, 561-566 Wise, R.R., Naylor, A.W. (1987) Chilling-enhanced photooxidation. The peroxidative destruction of lipids during chilling injury to photosynthesis and ultrastructure. Plant Physiol. 83, 272-277 Wright, M., Simon, E.W. (1973) Chilling injury in cucumber leaves. J. Exp. Bot. 24, 400411 Yakir, D., Rudich, J., Bravdo, B.A. (1985) Photoacoustic and fluorescence measurements of the chilling response and their relationship to carbon dioxide uptake in tomato plants. Planta 164, 345-353 Received 9 December 1988; accepted 14 March 1988