Planta (1989)180:32-39
P l a n t ~ 9 Springer-Verlag1989
Photoinhibition of photosynthesis in intact kiwifruit ( A ctinidin deliciosa ) leaves: effect of growth temperature on photoinhibition and recovery D.H. Greer and W.A. Laing Plant Physiology Division, DSIR, Private Bag, Palmerston North, New Zealand
Abstract. Intact leaves of kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson) from plants grown in a range of controlled temperatures from 15/10 to 30/25~ C were exposed to a photon flux density (PFD) of 1 500 gmol. m - 2. s - 1 at leaf temperatures between 10 and 25 ~ C. Photoinhibition and recovery were followed at the same temperatures and at a PFD of 20 gmol. m - 2 . s 1 by measuring chlorophyll fluorescence at 77 K and 692 nm, by measuring the photon yield of photosynthetic 02 evolution and light-saturated net photosynthetic CO2 uptake. The growth of plants at low temperatures resulted in chronic photoinhibition as evident from reduced fluorescence and photon yields. However, low-temperaturegrown plants apparently had a higher capacity to dissipate excess excitation energy than leaves from plants grown at high temperatures. Induced photoinhibition, from exposure to a PFD above that during growth, was less severe in low-temperaturegrown plants, particularly at high exposure temperatures. Net changes in the instantaneous fluorescence, Fo, indicated that little or no photoinhibition occurred when low-temperature-grown plants were exposed to high-light at high temperatures. In contrast, high-temperature-grown plants were highly susceptible to photoinhibitory damage at all exposure temperatures. These data indicate acclimation in photosynthesis and changes in the Abbreviations and symbols: Fo, Fro, F~=instantaneous, maximum, variable fluorescence; Fv/Fm = fluorescence ratio; Fi = Fv at t = 0 ; Fc=Fv of control leaves; Fo~ =Fv at t = or; Ko=rate constant for non-radiative energy dissipation; Kp=rate constant for photochemistry; k(Fp)=first-order rate constant for photoinhibition; k(Fr)= first-order rate constant for recovery; PFD=photon flux density; P~=light-saturated net photosynthesis; cpi = photon yield of 02 evolution (incident light)
capacity to dissipate excess excitation energy occurred in kiwifruit leaves with changes in growth temperature. Both processes contributed to changes in susceptibility to photoinhibition at the different growth temperatures. However, growth temperature also affected the capacity for recovery, with leaves from plants grown at low temperatures having moderate rates of recovery at low temperatures compared with leaves from plants grown at high temperatures which had negligible recovery. This also contributed to the reduced susceptibility to photoinhibition in low-temperature-grown plants. However, extreme photoinhibition resulted in severe reductions in the efficiency and capacity for photosynthesis. Key words: Actinidia - Chlorophyll fluorescence - Growth temperature - Photoinhibition of photosynthesis (recovery) - Temperature and photoinhibition
Introduction Kiwifruit (Actinidia deliciosa) plants are highly susceptible to photoinhibition of photosynthesis (i.e. to reductions in both the photon yield and the rate of light-saturated photosynthesis), especially when grown at low photosynthetic photon flux densities (PFD) (Greet et al. 1988; Greer and Laing 1988a, b). In addition, high PFD during growth induces chronic photoinhibition, i.e. a more or less permanent reduction in the photon yield of photosynthesis. These results are consistent with other studies that have compared the susceptibility to photoinhibition between sun and shade leaves of other species (Powles and Critchley
D.H. Greer and W.A. Laing: G r o w t h temperature effects on photoinhibition
1980; Demmig and Bj6rkman 1987; Seemann et al. 1987). Although the effect of PFD during growth on susceptibility to photoinhibition is well documented, especially for sun and shade grown leaves, the effect of temperature during growth is not. In a comprehensive review of photoinhibition, Powles (1984) made no mention of how changes in growth temperature would alter the predisposition of leaves to high-light stress. Subsequently, Moll and Steinback (1986) have shown that barley leaves grown at 10~ C were more able to resist photoinhibition than those grown at 25/20 ~ C. Similarly, maize grown at the low temperature was less susceptible to photoinhibition compared with those grown at a higher temperature (Greer and Hardacre 1989). Krause and Somersalo (1988) showed low-temperature hardening significantly reduced the susceptibility of spinach to photoinhibition in comparison with unhardened plants. In other studies the effects of growth temperature and growth PFD have been compounded, making interpretation difficult (Seemann et al. 1987; Smillie et al.
1988). Photoinhibition of photosynthesis in kiwifruit leaves is highly dependent on the exposure temperature (Greet et al. 1988; Greer and Laing 1988b) and is most probably related to the temperature-dependency of photosynthesis (()quist et al. 1987), but it is unknown whether temperature during growth affects the susceptibility of photosynthesis in kiwifruit leaves to photoinhibition. Kiwifruit leaves grown at a range of temperatures have a considerable capacity to adjust their temperature optimum for photosynthesis (0.6 ~ C per 1~ C shift in growth temperature; Laing 1985) and develop photosynthetic rates at about 80% of that for the optimum growth temperature when grown at temperatures as low as 15 ~ C. This great ability of the photosynthetic apparatus of kiwifruit leaves to acclimate to growth temperature indicates that they have some capability, perhaps by being able to effectively dissipate excess excitation energy, to resist photoinhibition when grown at low temperatures. Our objectives were to examine the effects of growth temperature on the susceptibility of kiwifruit leaves to photoinhibition, and to post-photoinhibition recovery. In keeping with the recent findings that photoinhibition, in part, involves an increase in non-radiative energy dissipation and/or inactivation of the primary photochemistry (Bj6rkman 1987; Demmig et al. 1987, 1988; Greer 1988), the relative contribution of these two processes to photoinhibition in kiwifi'uit leaves was considered.
33
Material and methods Plant material. Rooted cuttings of Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson were grown in the controlledenvironment (C.E.) facilities in the D S I R Climate Laboratory, in 9-1 pots, with the vine trained along a horizontal trellis. The plants were grown at day/night temperatures of 30/25, 25/ 20, 20/15, and 15/10 (+_0.5 ~ C) at a P F D of 700 ~ m o l - m -2 .s -1 for 11.5 h with an additional l-h daylength extension o f photoperiod lights (see Greer and Laing 1988b for further details). New plants were used at each change in temperature and sufficient time was allowed for full development of new leaves at each temperature (three weeks at 30/25 ~ C increasing to eight weeks at 15/10 ~ C). At the lowest temperature regime, bud growth was severely inhibited but leaf primordia were still able to expand. There was no evidence of photobleaching of chlorophyll at the lower growth temperatures, and Morgan et al. (1985) found similar leaf chlorophyll content in kiwifruit leaves over the same range of temperatures. Photoinhibition and recovery treatments. To induce photoinhibition, an attached intact leaf was sealed into a gas-exchange chamber and exposed to a P F D of 1 500 lamol, m - 2. s - 1 from a high-pressure discharge lamp (Greer et al. 1988). The leaf temperature was held constant during the high-light exposure at 10, 20 or 25 ~ C. For those plants grown at 25/20 ~ C, photoinhibition was only assessed at an exposure temperature of 20 ~ C and the comparable data from Greer and Laing (1988b) utilised for the other exposure temperatures. For the plants grown at 30/25 ~ C, photoinhibition was additionally measured at an exposure temperature of 30 ~ C. The CO2 partial pressure was monitored in an open gas-exchange system and was between 30 and 35 Pa. The 02 partial pressure and leaf-to-air watervapour difference were 21 kPa and 1 kPa, respectively. To study recovery, the leaf was initially photoinhibited at 20 ~ C and at a P F D of 1 500 gmol. m - 2. s - a for 300 min, resulting in about 6(~70% photoinhibition. The P F D was then reduced to 20 g m o l . m 2 - s - t , provided by a 40-W incandescent lamp, and the leaf temperature was held constant at 10, 20 or 25 ~ C. For plants grown at 30/25 ~ C, recovery was also measured at a leaf temperature of 30~ while for those grown at 25/20 ~ C recovery was only measured at a leaf temperature of 25 ~ C, and data for other leaf temperatures were obtained from Greer and Laing (1988b). Photoinhibition and recovery assays. At intervals, duplicate leaf discs (10 m m diameter) were punched from spaced locations on the leaf. Chlorophyll fluorescence at 692 n m and 77 K was measured on these samples as previously described (Greer et al. 1986, 1988). The instantaneous fluorescence (Fo) and the maxim u m fluorescence (fm) were recorded and the variable fluorescence (Fv=Fm--Fo) and the fluorescence ratio (Fv/Fm) were calculated. D a t a were normalised to an initial pre-photoinhibition level of Fm= 100, except where indicated. At less-frequent intervals, additional leaf discs (36 mm diameter) were punched and light-limited photosynthetic oxygen evolution was measured as described by Greer et al. (1986) and the p h o t o n yield on an incident-light basis, (Pi determined. In addition, the net photosynthetic rate during photoinhibition was monitored. Data analysis. Photoinhibition and recovery were measured by changes in Fv and ~0i. In kiwifruit leaves, these two parameters were linearly related in contrast to the relationship between Fv/Fm and q~i which was curvilinear (Fig. l). Fluorescence and photon-yield data were fitted to the following exponential equation using non-linear regression analysis, Ft=F~ --(F~ - F O e -k'
(1)
34
D.H. Greet and W.A. Laing: Growth temperature effects on photoinhibition 80
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.2 00 ~, (x 10 -2 m o l . m o l - ' ) Fig. 1 A, B. The relationship between the photon yield of oxygen evolution ~0~, tool 02" (photons moo - ~, and A the variable fluorescence (F~) and B the fluorescence ratio (Fv/F~) during the exposure of kiwifruit leaves to photoinhibition at 20 ~ C and a P F D of 1500 p m o l . m - 2. s- 1 ( e - - e ) or during the subsequent recovery from photoinhibition ( I - - I ) . The lines drawn are the least-squares fit to the photoinhibition data only
where Ft is the variable fluorescence or photon yield at any time, t; Fi is Fv or q~i at t = 0 ; F ~ is Fv or cp~ at t----m, and k is the first-order rate constant. The relative value of the rate constant for non-radiative energy dissipation, K , was determined according to Greer
(1988). Results
Comparison of fluorescence and photosynthetic characteristics in control leaves. There were signifi-
cant differences in the fluorescence and photosynthetic characteristics between control leaves from plants grown at the four temperatures (Table 1). The photon yield was highest in leaves from plants grown at 25/20 ~ C and it decreased to about 80% of maximum at the lowest growth temperature. Light-saturated net photosynthesis was highest in leaves from plants grown at 20/15 ~ C and this decreased by up to 35% at the other growth temperatures. By contrast, the fluorescence characteristics were nearly all maximal for plants grown at 30/ 25 ~ C and decreased at lower growth temperatures. In particular, Fv was depressed to 20% of maxim u m at 15/10 ~ C and Fv/Fm to 65% of the maximum. As a consequence of the differences in Fo and Fm, the relative value of the rate constant for non-radiative energy dissipation (KD) apparently increased fourfold from the 30/25~ C to the 15/ 10 ~ C growth temperature. All these measurements show that chronic photoinhibition occurred in kiwifruit leaves, especially during growth at low temperatures. Removing plants from darkness and transferring them to weak light ( 1 0 g m o l . m - 2 . s -1) at 250C for 180-200rain resulted in significant changes in each of these characteristics, especially in leaves from plants grown at lower temperatures (Table 2). In all but the lowest growth temperature, Fo decreased by about 7% whereas at 15/10 ~ C, Fo increased by a similar amount. Fro, Fv and Fv/ Fm each increased in leaves of plants from all growth temperatures although the values still r e mained significantly lower than those in leaves from plants grown at 30/25 ~ C. There were also increases in ~0i of between 3 and 10% following the transfer. The ability of leaves from plants grown at 15/ 10 ~ C to recover from chronic photoinhibition was
Table 1. The control values of photon yield of oxygen evolution ((p i), light-saturated photosynthesis (P s), fluorescence characteristics at 77 K and 692 nm, and the derived rate constant for nonradiative energy dissipation (KD) for kiwifruit leaves grown at four temperatures. The measurements of ~01 and of fluorescence were determined at the end of the 12.5-h dark period prior to any exposure to the light (mean_+ SE, n--=10) Characteristic
(p ia Ps b
Fo F,, Fv Fv/Fm KD
Growth temperature (~ C) 15/10
20/15
25/20
30/25
0.0612 -+ 0.0035 12.4-+0.1 4,3-+0.2 8.9_+0.7 4.7 + 0.6 0.494 _ 0.031 63.1
0.0682 • 0.0014 19.3 -t-0.8 5.6_+0.2 18.0___0.9 12.3 _+0.8 0.687 4- 0.014 30.1
0.0754 4- 0.0011 14.3 ___0.3 5.5_+0.1 22.9-+0.7 17.4 _+0.7 0.757 _+0.008 20.8
0.0695 4-_0.0014 13.5 +0.05 5.2-+0.1 27.2_+0.8 21.8 _+0.7 0.809 • 0.003 15.7
" mol 02" (mol photons)-1 measured at 25 ~ C b gmol C O 2 - m - Z ' s -~ measured at 20 ~ C and 1 500 g m o l - m - 2 " s -1
D.H. Greer and W.A. Laing: G r o w t h temperature effects on photoinhibition
35
Table 2. The control values of p h o t o n yield of oxygen evolution and fluorescence characteristics at 77 K and 692 n m in kiwifruit leaves grown at four temperatures. The measurements were determined after the plants had been left for 180-200 min at a P F D of 10 g m o l - m - z . s - 1 and 25 ~ C. The percentage change indicated is the difference between these measurements and those for leaves measured immediately from the dark (Table I) (mean • SE, n = 10) Characteristic
(~0i a % Fo % Fm % F~ %
Fv/Fm %
G r o w t h temperature (~ C) 15/10
20/15
25/20
30/25
0.0670 • 0.0020 +10 4.6-t-0.2 +8 14.2 + 1.3 +60 9 . 6 _ 1.2 +106 0.659 _+0.023 +34
0.0706 _ 0.0016 +4 5.2_+0.2 -7 22.1 + 1.5 +24 17.1 _+1.4 +39 0.762-+0.001 +12
0.0780 ___0.0008 +3 5.1 +_0.1 -8 23.2 _+0.5 +1 18.1 _+0.5 +5 0.781 _+0.004 +3
0.0740 4- 0.0014 +6 4.8___0.3 -6 29.0 _+1.9 +7 24.2 + 1.7 +11 0.833 4-0.0 +3
" mol O2' (mol p h o t o n s ) - 1 measured at 25 ~ C
further assessed at a recovery temperature of 25 ~ C and at a P F D of 20 g m o l . m - 2 . s - 1 (Fig. 2). The recovery of Fv was slow and only 50% complete after 1 800 min. Full recovery to the level of the plants grown at 25/20 ~ C would be predicted from eqn. 1 to take up to 20 d. Effect o f growth temperature on photoinhibition. When leaves from plants grown at 25/20 ~ C were exposed to a high P F D , there was an initial rapid decrease in both Fv and Fv/F m that declined exponentially to approach a steady-state in about 450 rain (Fig. 3). In contrast, Fo increased at a more or less linear rate by 1.3-fold whereas Fv de-
creased by 76% and Fv/F m by 44%. Over the same time the photon yield was reduced by 40% and light-saturated net photosynthesis by 20%. Comparable results were observed in leaves from plants grown at other temperatures (not shown). Growth temperature, however, had little effect on the first-order rate constant for photoinhibition, k(Fp), at any exposure temperature (Table 3).
80
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Fig. 2. Recovery from chronic photoinhibition at 25~ and a P F D of 20 g m o l - m - Z - s -1 of the instantaneous (Fo, o - - 9 and variable (Fv, e - - o ) fluorescence and the p h o t o n yield ((Pi, m - - m ) in kiwifruit leaves from plants grown at 15/10 ~ C and a P F D of 700 g m o l . m -z .s -a. Each line is a least-squared exponential regression fit and the data are the means of three leaves (data not normalized)
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Time ( m i n ) Fig. 3A, B. The time course of fluorescence changes in A Fo, 9 Fv, $ - - @ ; Fv/Fm, rn--D and B p h o t o n yield ((p i) and light-saturated net photosynthesis (Ps) in kiwifruit leaves from plants grown at 25/20 ~ C and exposed to a P F D of 1500 gmol. m - 2. s - 1 at 20 ~ C. Each line, except photosynthesis, is the leastsquared exponential regression fit and the data are the mean of two leaves
36
D.H. Greer and W.A. Laing: Growth temperature effects on photoinhibition
Table 3. The effect of temperature during exposure to a PFD of 1 500 gmol-m-2.s -1 on the rate constant for photoinhibition, k(Fp) in intact kiwifruit leaves grown in four temperature regimes. Data are mean k(Fp) x 10 -3.rain 1 • n=6 Exposure temperature (~ (2)
Growth temperature (~ C)
30 25 20 10
12.3,+5.1 11.0_+3.6 11.0• 12.6+_4.0 14.2,+2.3 9.4• 1.2 6.2• 7.7• 9.0,+1.0"
15/10
20/15
25/20
Table 4. The effect of growth and exposure temperatures on the net changes in Fo in kiwifruit leaves before and after their exposure to a PFD of 1 500 gmol-m-2.s -1 (mean,+SE, n=6) Growth temperature (o c)
Exposure temperature (o c)
Exposure time (min) Change (%) 0 380-
30/25
30 25 20 10
25/20
258
5.0-+0.3 5.7-+0.4 6.0-+0.4 5.1,+0.2 4.2-+0.3 5.5-+0.3 4.9-+0.2 6.2• 5.3• 6.1• 4.0,+0.1 4.3• 4.7•
30/25 13.1 ,+ 3.3 15.5+1.7 16.0• 1.8 8.5•
a Data from Greer and Laing (1988b) 20/15
Nevertheless, there was some tendency for photoinhibition to be faster in leaves from plants grown at 30/25~ than in leaves from plants grown at the other temperatures. There was a marked decline in k(Fp) at an exposure temperature o f 10 ~ C in leaves f r o m plants f r o m all g r o w t h t e m p e r a t u r e s c o m p a r e d to the higher exposure temperatures - that is, p h o t o i n h i b i t i o n t o o k a b o u t twice as long to reach a steady-state at 10 ~ C t h a n at 20 or 25 ~ C. The extent o f p h o t o i n h i b i t i o n was d e p e n d e n t o n b o t h the g r o w t h a n d exposure temperatures (Fig. 4). It decreased with decreasing t e m p e r a t u r e in which the plants were g r o w n with a b o u t 5 15% less p h o t o i n h i b i t i o n in leaves f r o m plants g r o w n at 1 5 / 1 0 ~ c o m p a r e d with those leaves f r o m plants g r o w n at 30/25 ~ C. However, this effect o f
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Fig. 4, The effect of growth temperature on the extent of photoinhibition (mean• n= 6) in kiwifruit leaves exposed to a PFD of 1500 gmol.m-Z.s -1 at a range of temperatures: 10~ C, o - - o ; 20~ C, e - - e ; 25~ C, n - - t 3 ; 30~ C, =. The extent was calculated from F~ at t= ~ (F~) and expressed as (1-Fo~/Fc) x 100 where Fc=F,, of the appropriate controls. With the exception of those leaves exposed at 10~ C, there were significant (P=0.05) linear regressions between the extent of photoinhibition and growth temperature at each exposure temperature
15/10
20 l0 b 25 20 10 25 20 10
6.2-+0.2 7.3,+0.1 7.9-+0.4 7.4-+0.4 4.6-+0.3 6.8,+0.1 8.1,+0.4 5.9• 6.2-+0.2 7.1• 3.2-+0.2 4.5-+0.5 5.5-+0.6
+24 +28 +32 +45 +10 +24 +65 --5 +17 +16 --21 + 4 +17
a Averaged between 300 and 440 min b Greer and Laing (1988b) g r o w t h t e m p e r a t u r e was m o s t m a r k e d at higher exposure temperatures. A t 10 ~ C, p h o t o i n h i b i t i o n was severe, irrespective o f the t e m p e r a t u r e in which the plants were grown. Similar results were evident f r o m m e a s u r e m e n t s o f the p h o t o n yield a n d lightsaturated net p h o t o s y n t h e s i s (not shown), q~ decreased b y 15 30%, 3 0 - 5 5 % a n d 6 5 - 8 5 % at exposure temperatures o f 25, 20 a n d 10 ~ C, with the m i n i m u m c h a n g e tending to o c c u r in the leaves f r o m plants g r o w n at 20/15 ~ C. C o m p a r a b l e decreases in net p h o t o s y n t h e s i s at these temperatures were 1 5 - 2 0 % , 2 0 - 3 5 % a n d 50 65%. There were m a r k e d differences in the response o f Fo to high-light exposure in leaves f r o m plants g r o w n at the various temperatures (Table 4). In leaves f r o m plants g r o w n at 30/25 a n d 25/20 ~ C, F0 increased at all exposure temperatures, t h o u g h m o r e so at the low t h a n at the high exposure temperatures. A similar effect o f exposure t e m p e r a t u r e on changes in Fo o c c u r r e d in leaves f r o m plants g r o w n at 20/15 a n d 15/10 ~ C. However, at an exposure t e m p e r a t u r e o f 25 ~ C, Fo decreased slightly in leaves f r o m plants g r o w n at 20/15 ~ C and m o r e m a r k e d l y in leaves f r o m plants g r o w n at 15/10 ~ C. Relative changes in F m (riot shown) were m o r e conservative t h a n in Fo a n d were n o t d e p e n d e n t on g r o w t h temperature. A t exposure temperatures o f 25, 20 and 10 ~ C, Fm decreased, in the sequence given, by 35%, 4 5 % a n d 55%.
Effect of growth temperature on recovery. G r o w t h t e m p e r a t u r e h a d a significant effect o n the rate c o n s t a n t for recovery (Fig. 5). F o r each recovery
D.H. Greer and W.A. Laing: G r o w t h temperature effects on p h o t o i n h i b i t i o n T
T
r
T
A 25oc
200 09 > O r
1 50 1 O0
~6
2~176 lOOC
E
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x
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7
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5
0
20oC
X
10=C
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Growfh temperafure (~ Fig. 5A, B. The effect of growth temperature on A the extent
of recovery (mean, n = 6) in intact kiwifruit leaves at a range of temperatures: 10~ C, o o; 20~ C, e - - e ; 25~ C, n - - n ; 30~ C, =, and B the rate constant for recovery, k(Fr) (mean+ SE, n=6) followingphotoinhibition at 20~ and a PFD of 1500 gmol.m 2. s- x. The extent of recovery was determined as (F| where Fc is the control Fv, Fi the Fv at the start of the recovery phase and F~ the Fv at the end of the recoveryphase (i.e. 440 min)
temperature, k(Fr) was always highest in leaves from plants grown at 15/10 ~ C. At a recovery temperature of 10 ~ C, only those plants grown at 15/ 10~ C recovered significantly. Similar trends were evident with the extent of recovery. However, the 15/10 ~ C-grown plants recovered to a level above their initial control values of F~, but this can be accounted for by some recovery of the chronic photoinhibition in addition to the recovery of the induced photoinhibition. In absolute terms, however, recovery in the leaves from plants grown at 15/10~ C was still incomplete compared with that in leaves from plants grown at the higher temperatures. Discussion
Kiwifruit leaves suffered chronic or semi-permanent photoinhibition at a moderate P F D of 700 gmol. m - 2. s- 1 when grown at low temperatures. This was apparent from a sustained quenching of fluorescence emission at 77 K, and from reductions of both Fv/Fm and the photon yield of O2 evolution. The values reported here were significantly lower than those for kiwifruit leaves grown
37
in shade (Greer et al. 1988) and show a similar reduction to that occurring when kiwifruit leaves were grown at a high P F D at 25/20 ~ C (Greer and Laing 1988 b). The severity of the chronic photoinhibition was, however, directly dependent on growth temperature and strongly increased when the growth temperature declined. These results conform with the effects of temperature on vegetative growth of kiwifruit (Morgan et al. 1985) who showed that a high PFD inhibited growth at low temperatures, indicative of photoinhibition. Chronic photoinhibition, consistent with differences in shoot growth rates, was similarly observed in two maize hybrids grown at low temperatures (Greer and Hardacre 1989) and in spinach grown at low compared with high temperatures (Krause and Somersalo 1988). There was some capacity of the leaves from plants grown at 15/10~ C to repair chronic photoinhibitory damage. However, this recovery was estimated to be several orders of magnitude slower than recovery from induced photoinhibition. The slow rate may result because resynthesis of part or even the entire chloroplast is required (Anderson and Andersson 1988) as opposed to resynthesis of the few thylakoid proteins considered to be necessary for recovery from induced photoinhibition (Ohad et al. 1984; Cleland et al. 1986; Greer et al. i986). Concomitant decreases in fluorescence, photon yield and light-saturated net photosynthesis occurred when kiwifruit leaves were exposed to a P F D in excess of that received by leaves during growth. A typical level of photoinhibition at the upper leaf surface, that is, a 65% decrease in Fv, corresponded to a 45% decrease in photon yield and a 25% decrease in light-saturated net photosynthesis. Similar changes in these same parameters were observed in Nerium oleander (Powles and Bj6rkman 1982) and for photon yield and photosynthesis in Phaseolus vulgaris (Powles and Thorne 1981). These differences in extents of photoinhibition occur because of light attenuation within the leaf (Knapp et al. 1988) and the resultant gradient in photoinhibition (Powles and Bj6rkman 1982; Krause and Somersalo 1988). Reductions in Fv in kiwifruit leaves occurred more rapidly then did changes in photon yield (Fig. 3 ; see also Bj6rkman and Demmig 1987) and, as a consequence, extreme photoinhibition at the upper leaf surface (90% decrease in Fv) resulted in an 80% decrease in photon yield and a 65% loss of light-saturated net photosynthesis. Such a severe reduction would have serious consequences for the productivity of such leaves.
38
D.H. Greer and W.A. Laing: Growth temperature effects on photoinhibition
Growing kiwifruit plants at different temperatures had a significant effect on their susceptibility to photoinhibition. Differences in susceptibility between plants grown at the different temperatures were manifested more particularly at high exposure temperatures where leaves from plants grown at 15/10~ C were about 15-20% less photoinhibited than were those leaves from plants grown at 30/ 25 ~ C (Fig. 4). However, leaves from plants grown at low temperatures were also chronically photoinhibited, hence the total extent of photoinhibition must have included some degree of chronic photoinhibition. Therefore, leaves of kiwifruit plants grown at low temperatures were less susceptible to photoinhibition, particularly at high exposure temperatures, than were leaves from plants grown at high temperatures. The capacity of kiwifruit leaves to photosynthetically acclimate to low temperatures (Laing 1985) could account, in part, for the reduced susceptibility to photoinhibition of the low-temperature-grown plants. However, it is apparent that these low-temperature-grown kiwifruit plants also have an increased capacity to dissipate excess energy non-radiatively, and this must contribute to the resistance. As the growth temperature declined there was a marked shift from a rising to a falling Fo during photoinhibition. Notably, at an exposure temperature of 25 ~ C, the change in Fo shifted from a 28% increase in plants grown at 30/25 ~ C to a 21% decrease in plants grown at 15/10 ~ C. The net change in Fo is indicative of relative changes in the rate constants for both non-radiative energy dissipation and for the photochemistry of photosystem II, with a rise reflecting a decrease in the rate constant for photochemistry, Kp and a fall reflecting an increase in the rate constant for non-radiative energy dissipation, KD (Bj6rkman 1987; Demmig et al. 1987, 1988). Thus the relative dominance of these two processes in kiwifruit leaves apparently shifted with changes in growth temperature notably at the higher exposure temperatures. In plants grown at high temperatures, changes in Kp during photoinhibition dominated the decline in Fv while in plants grown at low temperatures, changes in KD would have contributed relatively more. There is, therefore, an apparently greater capacity to dissipate excess excitation energy, through either the violaxanthin-zeaxanthin cycle (Demmig et al. 1987, 1988) or the cytochrome b-559 cycle (Thompson and Brudvig 1988), in kiwifruit plants grown at low compared with high temperatures. Under the range of conditions that kiwifruit plants have been grown (Greer et al. 1988; Greer
and Laing 1988b), growth P F D affected the susceptibility of leaves to photoinhibition more so than growth temperature. Changes in the growth P F D from 1 300 to 300 gmol. m - 2 s - 1 resulted in a 1.7-fold increase in the susceptibility to photoinhibition while increasing the growth temperature from 15/10~ C to 30/25~ C resulted in a 1.2-fold increase in susceptibility to photoinhibition. However, whether grown at different P F D s or at different temperatures, photoinhibition in kiwifruit leaves was mostly dependent on the exposure temperature. Recovery from photoinhibition following the high-light exposure, was also strongly temperature-dependent. This is consistent with earlier results (Greer and Laing 1988 a, b). However, growth temperature also had a significant affect on the first-order rate constant, k (Fr) with recovery tending to decline as growth temperatures increased such that leaves from plants grown at 15/10~ C were able to recover at a moderate rate whereas leaves from plants grown at 25/20 ~ C and 30/25 ~ C recovered only negligibly at 10 ~ C. Thus, while recovery was generally fastest in plants grown at, and exposed to high temperatures, nevertheless it was apparent that significant recovery could occur at low temperatures in plants grown at low temperatures. This undoubtably contributes to the reduced susceptibility to photoinhibition of the lowtemperature-grown plants since the extent of damage is directly ameliorated by the capacity of the leaves to recover (Ohad et al. 1984; Greer and Laing 1988a, b). Growth of kiwifruit plants at low temperatures increases the capacity for recovery at low leaf temperatures without, however, reducing the capacity for recovery at high leaf temperatures. Thus, there in some acclimation potential in the recovery process, though it remains to determine how this is manifested biochemically. These results, however, support the proposal of Krause and Somersalo (1988) that long-term growth at low temperatures leads to an acclimation of the recovery process. However, in kiwifruit leaves, the temperature experienced during recovery has the greatest impact in determining the rate of recovery from photoinhibition. In summary, growth temperature has an important effect in predisposing kiwifruit leaves to photoinhibition. On one hand, plants grown at low temperatures are significantly affected by a chronic damage but on the other, have a greater capacity to resist induced photoinhibition from high-light exposures than plants grown at high temperature. The latter
D.H. Greer and W.A. Laing: Growth temperature effects on photoinhibition
feature apparently arises from both an increased capacity to dissipate excess energy and photosynthetic acclimation at low growth temperature. In addition, an increased capacity for recovery further reduces photoinhibition. Additional studies are required to elucidate the biochemical mechanisms that are associated with this increased resistance. However, the efficiency and capacity for photosynthesis is considerably depressed by the effects of high-light and it is now important to determine what impact this has on crop growth in field situations. We would like to thank Robert Southward and the Technical Services Group of the Climate Laboratory for technical assistance.
References Anderson, J.M., Andersson, B. (1988) The dynamic photosynthetic membrane and regulation of solar energy conversion. Trends Biochem. Sci. 13, 351-355 Bj6rkman, O. (1987) Low-temperature chlorophyll fluorescence in leaves and its relationship to photon yield of photosynthesis in photoinhibition. In: Topics in photosynthesis, vol. 9: Photoinhibition, pp. 123-144, Kyle, D.J., Osmond, C.B., Arntzen, C.J., eds. Elsevier, Amsterdam Bj6rkman, O., Demmig, B. (1987) Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta 170, 489 504 Cleland, R.E., Melis, A., Neale, P.J. (1986) Mechanism of photoinhibition: photochemical reaction centre inactivation in system II of chloroplasts. Photosynth. Res. 9, 79 88 Demmig, B., Bj6rkman, O. (1987) Comparison of the effect of excessive light on chlorophyll fluorescence (77 K) and photon yield of 02 evolution in leaves of higher plants. Planta 171, 171 184 Demmig, B., Winter, K., Kriiger, A., Czygan, F.C. (1987) Photoinhibition and zeaxanthin formation in intact leaves. A possible role of the xanthophyll cycle in the dissipation of excess light energy. Plant Physiol. 84, 218-224 Demmig, B., Winter, K., Krfiger, A., Czygan, F.-C. (1988) Zeaxanthin and the heat dissipation of excess light energy in Nerium oleander exposed to a combination of high light and water stress. Plant Physiol. 87, 17 24 Greer, D.H. (1988) Effect of temperature on photoinhibition and recovery in Actinidia deliciosa. Aust. J. Plant Physiol. 15, 195-205 Greer, D.H., Berry, J.A., Bj6rkman, O. (1986) Photoinhibition of photosynthesis in intact bean leaves: role of light and temperature, and requirement for chloroplast-protein synthesis during recovery. Planta 168, 253-260 Greer, D.H., Hardacre, A.K. (1989) Photoinhibition of photosynthesis and its recovery in two maize hybrids varying in low temperature tolerance. Aust. J. Plant Physiol. 16, 189-198 Greer, D.H., Laing, W.A. (1988a) Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: re-
39
covery and its dependence on temperature. Planta 174, 159165 Greer, D.H., Laing, W.A. (1988b) Photoinhibition of photosynthesis in intact kiwifruit (Actinidia delieiosa) leaves: effect of light during growth on photoinhibition and recovery. Planta 175, 355-363 Greer, D.H., Laing, W.A., Kipnis, T. (1988) Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: effect of temperature. Planta 174, 152-158 Knapp, A.K., Vogelmann, T.C., McClean, T.M., Smith, W.K. (1988) Light and chlorophyll gradients within Cucurbita cotyledons. Plant Cell Environ. 11, 257-263 Krause, G.H., Somersalo, S. (1988) Fluorescence as a tool in photosynthesis research; application in studies of photoinhibition, cold acclimation and freezing stress. Philos. Trans. R. Soc. Lond. [Biol] 323, 281-294 Laing, W.A. (1985) Temperature and light response curves for photosynthesis in kiwifruit (Actinidia chinensis) cv. Hayward. N.Z.J. Agric. Res. 28, 117-124 Moll, B.A., Steinback, K.E. (1986) Chilling sensitivity in Oryza sativa: The role of protein phosphorylation in protection against photoinhibition. Plant Physiol. 80, 420423 Morgan, D.C., Warrington, I.J., Halligan, E.A. (1985) Effect of temperature and photosynthetic photon flux density on vegetative growth of kiwifruit (Aetinidia chinensis) N.Z.J. Agric. Res. 28, 109-116 Ohad, I., Kyle, D.J. Arntzen, C.J. (1984) Membrane protein damage and repair: removal and replacement of inactivated 32-kilodalton polypeptides in chloroplast membranes. J. Cell Biol. 99, 481485 Oquist, G., Greer, D.H., Ogren, E. (1987) Light stress at low temperature. In: Topics in photosynthesis, vol. 9: Photoinhibition, pp. 67-87, Kyle, D.J., Osmond, C.B., Arntzen, C.J., eds. Elsevier, Amsterdam Powles, S.B. (1984) Photoinhibition of photosynthesis induced by visible light. Annu. Rev. Plant Physiol. 35, 1544 Powles, S.B., Bj6rkman, O. (1982) Photoinhibition of photosynthesis: Effect on chlorophyll fluorescence at 77 K in intact leaves and in chloroplast membranes of Nerium oleander. Planta 156, 92107 Powles, S.B., Critchley, C. (1980) Effect of light intensity during growth on photoinhibition of attached intact bean leaflets. Plant Physiol. 65, 1181 1187 Powles, S.B., Thorne, S.W. (1981) Effect of high-light treatments in inducing photoinhibition of photosynthesis in intact leaves of low-light grown Phaseolus vulgaris and Lastreopsis microsora. Planta 152, 471477 Seemann, J.R., Sharkey, T.D., Wang, J., Osmond, C.B. (1987) Environmental effects on photosynthesis, nitrogen-use efficiency, and metabolite pools in leaves of sun and shade plants. Plant Physiol. 84, 796~802 Smillie, R.M., Hetherington, S.E., He, J., Nott, R. (1988) Photoinhibition at chilling temperatures. Aust. J. Plant Physiol. 15, 207-222 Thompson, L.K., Brudvig, G.W. (1988) Cytochrome b-559 may function to protect photosystem II from photoinhibition. Biochemistry 27, 6653 6658
Received 17 March; accepted 26 July 1989
P l a n t ~ 9 Springer-Verlag1989
Photoinhibition of photosynthesis in intact kiwifruit ( A ctinidin deliciosa ) leaves: effect of growth temperature on photoinhibition and recovery D.H. Greer and W.A. Laing Plant Physiology Division, DSIR, Private Bag, Palmerston North, New Zealand
Abstract. Intact leaves of kiwifruit (Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson) from plants grown in a range of controlled temperatures from 15/10 to 30/25~ C were exposed to a photon flux density (PFD) of 1 500 gmol. m - 2. s - 1 at leaf temperatures between 10 and 25 ~ C. Photoinhibition and recovery were followed at the same temperatures and at a PFD of 20 gmol. m - 2 . s 1 by measuring chlorophyll fluorescence at 77 K and 692 nm, by measuring the photon yield of photosynthetic 02 evolution and light-saturated net photosynthetic CO2 uptake. The growth of plants at low temperatures resulted in chronic photoinhibition as evident from reduced fluorescence and photon yields. However, low-temperaturegrown plants apparently had a higher capacity to dissipate excess excitation energy than leaves from plants grown at high temperatures. Induced photoinhibition, from exposure to a PFD above that during growth, was less severe in low-temperaturegrown plants, particularly at high exposure temperatures. Net changes in the instantaneous fluorescence, Fo, indicated that little or no photoinhibition occurred when low-temperature-grown plants were exposed to high-light at high temperatures. In contrast, high-temperature-grown plants were highly susceptible to photoinhibitory damage at all exposure temperatures. These data indicate acclimation in photosynthesis and changes in the Abbreviations and symbols: Fo, Fro, F~=instantaneous, maximum, variable fluorescence; Fv/Fm = fluorescence ratio; Fi = Fv at t = 0 ; Fc=Fv of control leaves; Fo~ =Fv at t = or; Ko=rate constant for non-radiative energy dissipation; Kp=rate constant for photochemistry; k(Fp)=first-order rate constant for photoinhibition; k(Fr)= first-order rate constant for recovery; PFD=photon flux density; P~=light-saturated net photosynthesis; cpi = photon yield of 02 evolution (incident light)
capacity to dissipate excess excitation energy occurred in kiwifruit leaves with changes in growth temperature. Both processes contributed to changes in susceptibility to photoinhibition at the different growth temperatures. However, growth temperature also affected the capacity for recovery, with leaves from plants grown at low temperatures having moderate rates of recovery at low temperatures compared with leaves from plants grown at high temperatures which had negligible recovery. This also contributed to the reduced susceptibility to photoinhibition in low-temperature-grown plants. However, extreme photoinhibition resulted in severe reductions in the efficiency and capacity for photosynthesis. Key words: Actinidia - Chlorophyll fluorescence - Growth temperature - Photoinhibition of photosynthesis (recovery) - Temperature and photoinhibition
Introduction Kiwifruit (Actinidia deliciosa) plants are highly susceptible to photoinhibition of photosynthesis (i.e. to reductions in both the photon yield and the rate of light-saturated photosynthesis), especially when grown at low photosynthetic photon flux densities (PFD) (Greet et al. 1988; Greer and Laing 1988a, b). In addition, high PFD during growth induces chronic photoinhibition, i.e. a more or less permanent reduction in the photon yield of photosynthesis. These results are consistent with other studies that have compared the susceptibility to photoinhibition between sun and shade leaves of other species (Powles and Critchley
D.H. Greer and W.A. Laing: G r o w t h temperature effects on photoinhibition
1980; Demmig and Bj6rkman 1987; Seemann et al. 1987). Although the effect of PFD during growth on susceptibility to photoinhibition is well documented, especially for sun and shade grown leaves, the effect of temperature during growth is not. In a comprehensive review of photoinhibition, Powles (1984) made no mention of how changes in growth temperature would alter the predisposition of leaves to high-light stress. Subsequently, Moll and Steinback (1986) have shown that barley leaves grown at 10~ C were more able to resist photoinhibition than those grown at 25/20 ~ C. Similarly, maize grown at the low temperature was less susceptible to photoinhibition compared with those grown at a higher temperature (Greer and Hardacre 1989). Krause and Somersalo (1988) showed low-temperature hardening significantly reduced the susceptibility of spinach to photoinhibition in comparison with unhardened plants. In other studies the effects of growth temperature and growth PFD have been compounded, making interpretation difficult (Seemann et al. 1987; Smillie et al.
1988). Photoinhibition of photosynthesis in kiwifruit leaves is highly dependent on the exposure temperature (Greet et al. 1988; Greer and Laing 1988b) and is most probably related to the temperature-dependency of photosynthesis (()quist et al. 1987), but it is unknown whether temperature during growth affects the susceptibility of photosynthesis in kiwifruit leaves to photoinhibition. Kiwifruit leaves grown at a range of temperatures have a considerable capacity to adjust their temperature optimum for photosynthesis (0.6 ~ C per 1~ C shift in growth temperature; Laing 1985) and develop photosynthetic rates at about 80% of that for the optimum growth temperature when grown at temperatures as low as 15 ~ C. This great ability of the photosynthetic apparatus of kiwifruit leaves to acclimate to growth temperature indicates that they have some capability, perhaps by being able to effectively dissipate excess excitation energy, to resist photoinhibition when grown at low temperatures. Our objectives were to examine the effects of growth temperature on the susceptibility of kiwifruit leaves to photoinhibition, and to post-photoinhibition recovery. In keeping with the recent findings that photoinhibition, in part, involves an increase in non-radiative energy dissipation and/or inactivation of the primary photochemistry (Bj6rkman 1987; Demmig et al. 1987, 1988; Greer 1988), the relative contribution of these two processes to photoinhibition in kiwifi'uit leaves was considered.
33
Material and methods Plant material. Rooted cuttings of Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson were grown in the controlledenvironment (C.E.) facilities in the D S I R Climate Laboratory, in 9-1 pots, with the vine trained along a horizontal trellis. The plants were grown at day/night temperatures of 30/25, 25/ 20, 20/15, and 15/10 (+_0.5 ~ C) at a P F D of 700 ~ m o l - m -2 .s -1 for 11.5 h with an additional l-h daylength extension o f photoperiod lights (see Greer and Laing 1988b for further details). New plants were used at each change in temperature and sufficient time was allowed for full development of new leaves at each temperature (three weeks at 30/25 ~ C increasing to eight weeks at 15/10 ~ C). At the lowest temperature regime, bud growth was severely inhibited but leaf primordia were still able to expand. There was no evidence of photobleaching of chlorophyll at the lower growth temperatures, and Morgan et al. (1985) found similar leaf chlorophyll content in kiwifruit leaves over the same range of temperatures. Photoinhibition and recovery treatments. To induce photoinhibition, an attached intact leaf was sealed into a gas-exchange chamber and exposed to a P F D of 1 500 lamol, m - 2. s - 1 from a high-pressure discharge lamp (Greer et al. 1988). The leaf temperature was held constant during the high-light exposure at 10, 20 or 25 ~ C. For those plants grown at 25/20 ~ C, photoinhibition was only assessed at an exposure temperature of 20 ~ C and the comparable data from Greer and Laing (1988b) utilised for the other exposure temperatures. For the plants grown at 30/25 ~ C, photoinhibition was additionally measured at an exposure temperature of 30 ~ C. The CO2 partial pressure was monitored in an open gas-exchange system and was between 30 and 35 Pa. The 02 partial pressure and leaf-to-air watervapour difference were 21 kPa and 1 kPa, respectively. To study recovery, the leaf was initially photoinhibited at 20 ~ C and at a P F D of 1 500 gmol. m - 2. s - a for 300 min, resulting in about 6(~70% photoinhibition. The P F D was then reduced to 20 g m o l . m 2 - s - t , provided by a 40-W incandescent lamp, and the leaf temperature was held constant at 10, 20 or 25 ~ C. For plants grown at 30/25 ~ C, recovery was also measured at a leaf temperature of 30~ while for those grown at 25/20 ~ C recovery was only measured at a leaf temperature of 25 ~ C, and data for other leaf temperatures were obtained from Greer and Laing (1988b). Photoinhibition and recovery assays. At intervals, duplicate leaf discs (10 m m diameter) were punched from spaced locations on the leaf. Chlorophyll fluorescence at 692 n m and 77 K was measured on these samples as previously described (Greer et al. 1986, 1988). The instantaneous fluorescence (Fo) and the maxim u m fluorescence (fm) were recorded and the variable fluorescence (Fv=Fm--Fo) and the fluorescence ratio (Fv/Fm) were calculated. D a t a were normalised to an initial pre-photoinhibition level of Fm= 100, except where indicated. At less-frequent intervals, additional leaf discs (36 mm diameter) were punched and light-limited photosynthetic oxygen evolution was measured as described by Greer et al. (1986) and the p h o t o n yield on an incident-light basis, (Pi determined. In addition, the net photosynthetic rate during photoinhibition was monitored. Data analysis. Photoinhibition and recovery were measured by changes in Fv and ~0i. In kiwifruit leaves, these two parameters were linearly related in contrast to the relationship between Fv/Fm and q~i which was curvilinear (Fig. l). Fluorescence and photon-yield data were fitted to the following exponential equation using non-linear regression analysis, Ft=F~ --(F~ - F O e -k'
(1)
34
D.H. Greet and W.A. Laing: Growth temperature effects on photoinhibition 80
".f
60 40 20 0
I
'
I
'
I
'
.8
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,6 .4
.2 00 ~, (x 10 -2 m o l . m o l - ' ) Fig. 1 A, B. The relationship between the photon yield of oxygen evolution ~0~, tool 02" (photons moo - ~, and A the variable fluorescence (F~) and B the fluorescence ratio (Fv/F~) during the exposure of kiwifruit leaves to photoinhibition at 20 ~ C and a P F D of 1500 p m o l . m - 2. s- 1 ( e - - e ) or during the subsequent recovery from photoinhibition ( I - - I ) . The lines drawn are the least-squares fit to the photoinhibition data only
where Ft is the variable fluorescence or photon yield at any time, t; Fi is Fv or q~i at t = 0 ; F ~ is Fv or cp~ at t----m, and k is the first-order rate constant. The relative value of the rate constant for non-radiative energy dissipation, K , was determined according to Greer
(1988). Results
Comparison of fluorescence and photosynthetic characteristics in control leaves. There were signifi-
cant differences in the fluorescence and photosynthetic characteristics between control leaves from plants grown at the four temperatures (Table 1). The photon yield was highest in leaves from plants grown at 25/20 ~ C and it decreased to about 80% of maximum at the lowest growth temperature. Light-saturated net photosynthesis was highest in leaves from plants grown at 20/15 ~ C and this decreased by up to 35% at the other growth temperatures. By contrast, the fluorescence characteristics were nearly all maximal for plants grown at 30/ 25 ~ C and decreased at lower growth temperatures. In particular, Fv was depressed to 20% of maxim u m at 15/10 ~ C and Fv/Fm to 65% of the maximum. As a consequence of the differences in Fo and Fm, the relative value of the rate constant for non-radiative energy dissipation (KD) apparently increased fourfold from the 30/25~ C to the 15/ 10 ~ C growth temperature. All these measurements show that chronic photoinhibition occurred in kiwifruit leaves, especially during growth at low temperatures. Removing plants from darkness and transferring them to weak light ( 1 0 g m o l . m - 2 . s -1) at 250C for 180-200rain resulted in significant changes in each of these characteristics, especially in leaves from plants grown at lower temperatures (Table 2). In all but the lowest growth temperature, Fo decreased by about 7% whereas at 15/10 ~ C, Fo increased by a similar amount. Fro, Fv and Fv/ Fm each increased in leaves of plants from all growth temperatures although the values still r e mained significantly lower than those in leaves from plants grown at 30/25 ~ C. There were also increases in ~0i of between 3 and 10% following the transfer. The ability of leaves from plants grown at 15/ 10 ~ C to recover from chronic photoinhibition was
Table 1. The control values of photon yield of oxygen evolution ((p i), light-saturated photosynthesis (P s), fluorescence characteristics at 77 K and 692 nm, and the derived rate constant for nonradiative energy dissipation (KD) for kiwifruit leaves grown at four temperatures. The measurements of ~01 and of fluorescence were determined at the end of the 12.5-h dark period prior to any exposure to the light (mean_+ SE, n--=10) Characteristic
(p ia Ps b
Fo F,, Fv Fv/Fm KD
Growth temperature (~ C) 15/10
20/15
25/20
30/25
0.0612 -+ 0.0035 12.4-+0.1 4,3-+0.2 8.9_+0.7 4.7 + 0.6 0.494 _ 0.031 63.1
0.0682 • 0.0014 19.3 -t-0.8 5.6_+0.2 18.0___0.9 12.3 _+0.8 0.687 4- 0.014 30.1
0.0754 4- 0.0011 14.3 ___0.3 5.5_+0.1 22.9-+0.7 17.4 _+0.7 0.757 _+0.008 20.8
0.0695 4-_0.0014 13.5 +0.05 5.2-+0.1 27.2_+0.8 21.8 _+0.7 0.809 • 0.003 15.7
" mol 02" (mol photons)-1 measured at 25 ~ C b gmol C O 2 - m - Z ' s -~ measured at 20 ~ C and 1 500 g m o l - m - 2 " s -1
D.H. Greer and W.A. Laing: G r o w t h temperature effects on photoinhibition
35
Table 2. The control values of p h o t o n yield of oxygen evolution and fluorescence characteristics at 77 K and 692 n m in kiwifruit leaves grown at four temperatures. The measurements were determined after the plants had been left for 180-200 min at a P F D of 10 g m o l - m - z . s - 1 and 25 ~ C. The percentage change indicated is the difference between these measurements and those for leaves measured immediately from the dark (Table I) (mean • SE, n = 10) Characteristic
(~0i a % Fo % Fm % F~ %
Fv/Fm %
G r o w t h temperature (~ C) 15/10
20/15
25/20
30/25
0.0670 • 0.0020 +10 4.6-t-0.2 +8 14.2 + 1.3 +60 9 . 6 _ 1.2 +106 0.659 _+0.023 +34
0.0706 _ 0.0016 +4 5.2_+0.2 -7 22.1 + 1.5 +24 17.1 _+1.4 +39 0.762-+0.001 +12
0.0780 ___0.0008 +3 5.1 +_0.1 -8 23.2 _+0.5 +1 18.1 _+0.5 +5 0.781 _+0.004 +3
0.0740 4- 0.0014 +6 4.8___0.3 -6 29.0 _+1.9 +7 24.2 + 1.7 +11 0.833 4-0.0 +3
" mol O2' (mol p h o t o n s ) - 1 measured at 25 ~ C
further assessed at a recovery temperature of 25 ~ C and at a P F D of 20 g m o l . m - 2 . s - 1 (Fig. 2). The recovery of Fv was slow and only 50% complete after 1 800 min. Full recovery to the level of the plants grown at 25/20 ~ C would be predicted from eqn. 1 to take up to 20 d. Effect o f growth temperature on photoinhibition. When leaves from plants grown at 25/20 ~ C were exposed to a high P F D , there was an initial rapid decrease in both Fv and Fv/F m that declined exponentially to approach a steady-state in about 450 rain (Fig. 3). In contrast, Fo increased at a more or less linear rate by 1.3-fold whereas Fv de-
creased by 76% and Fv/F m by 44%. Over the same time the photon yield was reduced by 40% and light-saturated net photosynthesis by 20%. Comparable results were observed in leaves from plants grown at other temperatures (not shown). Growth temperature, however, had little effect on the first-order rate constant for photoinhibition, k(Fp), at any exposure temperature (Table 3).
80
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Fig. 2. Recovery from chronic photoinhibition at 25~ and a P F D of 20 g m o l - m - Z - s -1 of the instantaneous (Fo, o - - 9 and variable (Fv, e - - o ) fluorescence and the p h o t o n yield ((Pi, m - - m ) in kiwifruit leaves from plants grown at 15/10 ~ C and a P F D of 700 g m o l . m -z .s -a. Each line is a least-squared exponential regression fit and the data are the means of three leaves (data not normalized)
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Time ( m i n ) Fig. 3A, B. The time course of fluorescence changes in A Fo, 9 Fv, $ - - @ ; Fv/Fm, rn--D and B p h o t o n yield ((p i) and light-saturated net photosynthesis (Ps) in kiwifruit leaves from plants grown at 25/20 ~ C and exposed to a P F D of 1500 gmol. m - 2. s - 1 at 20 ~ C. Each line, except photosynthesis, is the leastsquared exponential regression fit and the data are the mean of two leaves
36
D.H. Greer and W.A. Laing: Growth temperature effects on photoinhibition
Table 3. The effect of temperature during exposure to a PFD of 1 500 gmol-m-2.s -1 on the rate constant for photoinhibition, k(Fp) in intact kiwifruit leaves grown in four temperature regimes. Data are mean k(Fp) x 10 -3.rain 1 • n=6 Exposure temperature (~ (2)
Growth temperature (~ C)
30 25 20 10
12.3,+5.1 11.0_+3.6 11.0• 12.6+_4.0 14.2,+2.3 9.4• 1.2 6.2• 7.7• 9.0,+1.0"
15/10
20/15
25/20
Table 4. The effect of growth and exposure temperatures on the net changes in Fo in kiwifruit leaves before and after their exposure to a PFD of 1 500 gmol-m-2.s -1 (mean,+SE, n=6) Growth temperature (o c)
Exposure temperature (o c)
Exposure time (min) Change (%) 0 380-
30/25
30 25 20 10
25/20
258
5.0-+0.3 5.7-+0.4 6.0-+0.4 5.1,+0.2 4.2-+0.3 5.5-+0.3 4.9-+0.2 6.2• 5.3• 6.1• 4.0,+0.1 4.3• 4.7•
30/25 13.1 ,+ 3.3 15.5+1.7 16.0• 1.8 8.5•
a Data from Greer and Laing (1988b) 20/15
Nevertheless, there was some tendency for photoinhibition to be faster in leaves from plants grown at 30/25~ than in leaves from plants grown at the other temperatures. There was a marked decline in k(Fp) at an exposure temperature o f 10 ~ C in leaves f r o m plants f r o m all g r o w t h t e m p e r a t u r e s c o m p a r e d to the higher exposure temperatures - that is, p h o t o i n h i b i t i o n t o o k a b o u t twice as long to reach a steady-state at 10 ~ C t h a n at 20 or 25 ~ C. The extent o f p h o t o i n h i b i t i o n was d e p e n d e n t o n b o t h the g r o w t h a n d exposure temperatures (Fig. 4). It decreased with decreasing t e m p e r a t u r e in which the plants were g r o w n with a b o u t 5 15% less p h o t o i n h i b i t i o n in leaves f r o m plants g r o w n at 1 5 / 1 0 ~ c o m p a r e d with those leaves f r o m plants g r o w n at 30/25 ~ C. However, this effect o f
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femperafure
3'0 (~
Fig. 4, The effect of growth temperature on the extent of photoinhibition (mean• n= 6) in kiwifruit leaves exposed to a PFD of 1500 gmol.m-Z.s -1 at a range of temperatures: 10~ C, o - - o ; 20~ C, e - - e ; 25~ C, n - - t 3 ; 30~ C, =. The extent was calculated from F~ at t= ~ (F~) and expressed as (1-Fo~/Fc) x 100 where Fc=F,, of the appropriate controls. With the exception of those leaves exposed at 10~ C, there were significant (P=0.05) linear regressions between the extent of photoinhibition and growth temperature at each exposure temperature
15/10
20 l0 b 25 20 10 25 20 10
6.2-+0.2 7.3,+0.1 7.9-+0.4 7.4-+0.4 4.6-+0.3 6.8,+0.1 8.1,+0.4 5.9• 6.2-+0.2 7.1• 3.2-+0.2 4.5-+0.5 5.5-+0.6
+24 +28 +32 +45 +10 +24 +65 --5 +17 +16 --21 + 4 +17
a Averaged between 300 and 440 min b Greer and Laing (1988b) g r o w t h t e m p e r a t u r e was m o s t m a r k e d at higher exposure temperatures. A t 10 ~ C, p h o t o i n h i b i t i o n was severe, irrespective o f the t e m p e r a t u r e in which the plants were grown. Similar results were evident f r o m m e a s u r e m e n t s o f the p h o t o n yield a n d lightsaturated net p h o t o s y n t h e s i s (not shown), q~ decreased b y 15 30%, 3 0 - 5 5 % a n d 6 5 - 8 5 % at exposure temperatures o f 25, 20 a n d 10 ~ C, with the m i n i m u m c h a n g e tending to o c c u r in the leaves f r o m plants g r o w n at 20/15 ~ C. C o m p a r a b l e decreases in net p h o t o s y n t h e s i s at these temperatures were 1 5 - 2 0 % , 2 0 - 3 5 % a n d 50 65%. There were m a r k e d differences in the response o f Fo to high-light exposure in leaves f r o m plants g r o w n at the various temperatures (Table 4). In leaves f r o m plants g r o w n at 30/25 a n d 25/20 ~ C, F0 increased at all exposure temperatures, t h o u g h m o r e so at the low t h a n at the high exposure temperatures. A similar effect o f exposure t e m p e r a t u r e on changes in Fo o c c u r r e d in leaves f r o m plants g r o w n at 20/15 a n d 15/10 ~ C. However, at an exposure t e m p e r a t u r e o f 25 ~ C, Fo decreased slightly in leaves f r o m plants g r o w n at 20/15 ~ C and m o r e m a r k e d l y in leaves f r o m plants g r o w n at 15/10 ~ C. Relative changes in F m (riot shown) were m o r e conservative t h a n in Fo a n d were n o t d e p e n d e n t on g r o w t h temperature. A t exposure temperatures o f 25, 20 and 10 ~ C, Fm decreased, in the sequence given, by 35%, 4 5 % a n d 55%.
Effect of growth temperature on recovery. G r o w t h t e m p e r a t u r e h a d a significant effect o n the rate c o n s t a n t for recovery (Fig. 5). F o r each recovery
D.H. Greer and W.A. Laing: G r o w t h temperature effects on p h o t o i n h i b i t i o n T
T
r
T
A 25oc
200 09 > O r
1 50 1 O0
~6
2~176 lOOC
E
50
O
x
0
Ld
B T
.E E
7
25~
5
0
20oC
X
10=C
LI.-
0
Growfh temperafure (~ Fig. 5A, B. The effect of growth temperature on A the extent
of recovery (mean, n = 6) in intact kiwifruit leaves at a range of temperatures: 10~ C, o o; 20~ C, e - - e ; 25~ C, n - - n ; 30~ C, =, and B the rate constant for recovery, k(Fr) (mean+ SE, n=6) followingphotoinhibition at 20~ and a PFD of 1500 gmol.m 2. s- x. The extent of recovery was determined as (F| where Fc is the control Fv, Fi the Fv at the start of the recovery phase and F~ the Fv at the end of the recoveryphase (i.e. 440 min)
temperature, k(Fr) was always highest in leaves from plants grown at 15/10 ~ C. At a recovery temperature of 10 ~ C, only those plants grown at 15/ 10~ C recovered significantly. Similar trends were evident with the extent of recovery. However, the 15/10 ~ C-grown plants recovered to a level above their initial control values of F~, but this can be accounted for by some recovery of the chronic photoinhibition in addition to the recovery of the induced photoinhibition. In absolute terms, however, recovery in the leaves from plants grown at 15/10~ C was still incomplete compared with that in leaves from plants grown at the higher temperatures. Discussion
Kiwifruit leaves suffered chronic or semi-permanent photoinhibition at a moderate P F D of 700 gmol. m - 2. s- 1 when grown at low temperatures. This was apparent from a sustained quenching of fluorescence emission at 77 K, and from reductions of both Fv/Fm and the photon yield of O2 evolution. The values reported here were significantly lower than those for kiwifruit leaves grown
37
in shade (Greer et al. 1988) and show a similar reduction to that occurring when kiwifruit leaves were grown at a high P F D at 25/20 ~ C (Greer and Laing 1988 b). The severity of the chronic photoinhibition was, however, directly dependent on growth temperature and strongly increased when the growth temperature declined. These results conform with the effects of temperature on vegetative growth of kiwifruit (Morgan et al. 1985) who showed that a high PFD inhibited growth at low temperatures, indicative of photoinhibition. Chronic photoinhibition, consistent with differences in shoot growth rates, was similarly observed in two maize hybrids grown at low temperatures (Greer and Hardacre 1989) and in spinach grown at low compared with high temperatures (Krause and Somersalo 1988). There was some capacity of the leaves from plants grown at 15/10~ C to repair chronic photoinhibitory damage. However, this recovery was estimated to be several orders of magnitude slower than recovery from induced photoinhibition. The slow rate may result because resynthesis of part or even the entire chloroplast is required (Anderson and Andersson 1988) as opposed to resynthesis of the few thylakoid proteins considered to be necessary for recovery from induced photoinhibition (Ohad et al. 1984; Cleland et al. 1986; Greer et al. i986). Concomitant decreases in fluorescence, photon yield and light-saturated net photosynthesis occurred when kiwifruit leaves were exposed to a P F D in excess of that received by leaves during growth. A typical level of photoinhibition at the upper leaf surface, that is, a 65% decrease in Fv, corresponded to a 45% decrease in photon yield and a 25% decrease in light-saturated net photosynthesis. Similar changes in these same parameters were observed in Nerium oleander (Powles and Bj6rkman 1982) and for photon yield and photosynthesis in Phaseolus vulgaris (Powles and Thorne 1981). These differences in extents of photoinhibition occur because of light attenuation within the leaf (Knapp et al. 1988) and the resultant gradient in photoinhibition (Powles and Bj6rkman 1982; Krause and Somersalo 1988). Reductions in Fv in kiwifruit leaves occurred more rapidly then did changes in photon yield (Fig. 3 ; see also Bj6rkman and Demmig 1987) and, as a consequence, extreme photoinhibition at the upper leaf surface (90% decrease in Fv) resulted in an 80% decrease in photon yield and a 65% loss of light-saturated net photosynthesis. Such a severe reduction would have serious consequences for the productivity of such leaves.
38
D.H. Greer and W.A. Laing: Growth temperature effects on photoinhibition
Growing kiwifruit plants at different temperatures had a significant effect on their susceptibility to photoinhibition. Differences in susceptibility between plants grown at the different temperatures were manifested more particularly at high exposure temperatures where leaves from plants grown at 15/10~ C were about 15-20% less photoinhibited than were those leaves from plants grown at 30/ 25 ~ C (Fig. 4). However, leaves from plants grown at low temperatures were also chronically photoinhibited, hence the total extent of photoinhibition must have included some degree of chronic photoinhibition. Therefore, leaves of kiwifruit plants grown at low temperatures were less susceptible to photoinhibition, particularly at high exposure temperatures, than were leaves from plants grown at high temperatures. The capacity of kiwifruit leaves to photosynthetically acclimate to low temperatures (Laing 1985) could account, in part, for the reduced susceptibility to photoinhibition of the low-temperature-grown plants. However, it is apparent that these low-temperature-grown kiwifruit plants also have an increased capacity to dissipate excess energy non-radiatively, and this must contribute to the resistance. As the growth temperature declined there was a marked shift from a rising to a falling Fo during photoinhibition. Notably, at an exposure temperature of 25 ~ C, the change in Fo shifted from a 28% increase in plants grown at 30/25 ~ C to a 21% decrease in plants grown at 15/10 ~ C. The net change in Fo is indicative of relative changes in the rate constants for both non-radiative energy dissipation and for the photochemistry of photosystem II, with a rise reflecting a decrease in the rate constant for photochemistry, Kp and a fall reflecting an increase in the rate constant for non-radiative energy dissipation, KD (Bj6rkman 1987; Demmig et al. 1987, 1988). Thus the relative dominance of these two processes in kiwifruit leaves apparently shifted with changes in growth temperature notably at the higher exposure temperatures. In plants grown at high temperatures, changes in Kp during photoinhibition dominated the decline in Fv while in plants grown at low temperatures, changes in KD would have contributed relatively more. There is, therefore, an apparently greater capacity to dissipate excess excitation energy, through either the violaxanthin-zeaxanthin cycle (Demmig et al. 1987, 1988) or the cytochrome b-559 cycle (Thompson and Brudvig 1988), in kiwifruit plants grown at low compared with high temperatures. Under the range of conditions that kiwifruit plants have been grown (Greer et al. 1988; Greer
and Laing 1988b), growth P F D affected the susceptibility of leaves to photoinhibition more so than growth temperature. Changes in the growth P F D from 1 300 to 300 gmol. m - 2 s - 1 resulted in a 1.7-fold increase in the susceptibility to photoinhibition while increasing the growth temperature from 15/10~ C to 30/25~ C resulted in a 1.2-fold increase in susceptibility to photoinhibition. However, whether grown at different P F D s or at different temperatures, photoinhibition in kiwifruit leaves was mostly dependent on the exposure temperature. Recovery from photoinhibition following the high-light exposure, was also strongly temperature-dependent. This is consistent with earlier results (Greer and Laing 1988 a, b). However, growth temperature also had a significant affect on the first-order rate constant, k (Fr) with recovery tending to decline as growth temperatures increased such that leaves from plants grown at 15/10~ C were able to recover at a moderate rate whereas leaves from plants grown at 25/20 ~ C and 30/25 ~ C recovered only negligibly at 10 ~ C. Thus, while recovery was generally fastest in plants grown at, and exposed to high temperatures, nevertheless it was apparent that significant recovery could occur at low temperatures in plants grown at low temperatures. This undoubtably contributes to the reduced susceptibility to photoinhibition of the lowtemperature-grown plants since the extent of damage is directly ameliorated by the capacity of the leaves to recover (Ohad et al. 1984; Greer and Laing 1988a, b). Growth of kiwifruit plants at low temperatures increases the capacity for recovery at low leaf temperatures without, however, reducing the capacity for recovery at high leaf temperatures. Thus, there in some acclimation potential in the recovery process, though it remains to determine how this is manifested biochemically. These results, however, support the proposal of Krause and Somersalo (1988) that long-term growth at low temperatures leads to an acclimation of the recovery process. However, in kiwifruit leaves, the temperature experienced during recovery has the greatest impact in determining the rate of recovery from photoinhibition. In summary, growth temperature has an important effect in predisposing kiwifruit leaves to photoinhibition. On one hand, plants grown at low temperatures are significantly affected by a chronic damage but on the other, have a greater capacity to resist induced photoinhibition from high-light exposures than plants grown at high temperature. The latter
D.H. Greer and W.A. Laing: Growth temperature effects on photoinhibition
feature apparently arises from both an increased capacity to dissipate excess energy and photosynthetic acclimation at low growth temperature. In addition, an increased capacity for recovery further reduces photoinhibition. Additional studies are required to elucidate the biochemical mechanisms that are associated with this increased resistance. However, the efficiency and capacity for photosynthesis is considerably depressed by the effects of high-light and it is now important to determine what impact this has on crop growth in field situations. We would like to thank Robert Southward and the Technical Services Group of the Climate Laboratory for technical assistance.
References Anderson, J.M., Andersson, B. (1988) The dynamic photosynthetic membrane and regulation of solar energy conversion. Trends Biochem. Sci. 13, 351-355 Bj6rkman, O. (1987) Low-temperature chlorophyll fluorescence in leaves and its relationship to photon yield of photosynthesis in photoinhibition. In: Topics in photosynthesis, vol. 9: Photoinhibition, pp. 123-144, Kyle, D.J., Osmond, C.B., Arntzen, C.J., eds. Elsevier, Amsterdam Bj6rkman, O., Demmig, B. (1987) Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta 170, 489 504 Cleland, R.E., Melis, A., Neale, P.J. (1986) Mechanism of photoinhibition: photochemical reaction centre inactivation in system II of chloroplasts. Photosynth. Res. 9, 79 88 Demmig, B., Bj6rkman, O. (1987) Comparison of the effect of excessive light on chlorophyll fluorescence (77 K) and photon yield of 02 evolution in leaves of higher plants. Planta 171, 171 184 Demmig, B., Winter, K., Kriiger, A., Czygan, F.C. (1987) Photoinhibition and zeaxanthin formation in intact leaves. A possible role of the xanthophyll cycle in the dissipation of excess light energy. Plant Physiol. 84, 218-224 Demmig, B., Winter, K., Krfiger, A., Czygan, F.-C. (1988) Zeaxanthin and the heat dissipation of excess light energy in Nerium oleander exposed to a combination of high light and water stress. Plant Physiol. 87, 17 24 Greer, D.H. (1988) Effect of temperature on photoinhibition and recovery in Actinidia deliciosa. Aust. J. Plant Physiol. 15, 195-205 Greer, D.H., Berry, J.A., Bj6rkman, O. (1986) Photoinhibition of photosynthesis in intact bean leaves: role of light and temperature, and requirement for chloroplast-protein synthesis during recovery. Planta 168, 253-260 Greer, D.H., Hardacre, A.K. (1989) Photoinhibition of photosynthesis and its recovery in two maize hybrids varying in low temperature tolerance. Aust. J. Plant Physiol. 16, 189-198 Greer, D.H., Laing, W.A. (1988a) Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: re-
39
covery and its dependence on temperature. Planta 174, 159165 Greer, D.H., Laing, W.A. (1988b) Photoinhibition of photosynthesis in intact kiwifruit (Actinidia delieiosa) leaves: effect of light during growth on photoinhibition and recovery. Planta 175, 355-363 Greer, D.H., Laing, W.A., Kipnis, T. (1988) Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: effect of temperature. Planta 174, 152-158 Knapp, A.K., Vogelmann, T.C., McClean, T.M., Smith, W.K. (1988) Light and chlorophyll gradients within Cucurbita cotyledons. Plant Cell Environ. 11, 257-263 Krause, G.H., Somersalo, S. (1988) Fluorescence as a tool in photosynthesis research; application in studies of photoinhibition, cold acclimation and freezing stress. Philos. Trans. R. Soc. Lond. [Biol] 323, 281-294 Laing, W.A. (1985) Temperature and light response curves for photosynthesis in kiwifruit (Actinidia chinensis) cv. Hayward. N.Z.J. Agric. Res. 28, 117-124 Moll, B.A., Steinback, K.E. (1986) Chilling sensitivity in Oryza sativa: The role of protein phosphorylation in protection against photoinhibition. Plant Physiol. 80, 420423 Morgan, D.C., Warrington, I.J., Halligan, E.A. (1985) Effect of temperature and photosynthetic photon flux density on vegetative growth of kiwifruit (Aetinidia chinensis) N.Z.J. Agric. Res. 28, 109-116 Ohad, I., Kyle, D.J. Arntzen, C.J. (1984) Membrane protein damage and repair: removal and replacement of inactivated 32-kilodalton polypeptides in chloroplast membranes. J. Cell Biol. 99, 481485 Oquist, G., Greer, D.H., Ogren, E. (1987) Light stress at low temperature. In: Topics in photosynthesis, vol. 9: Photoinhibition, pp. 67-87, Kyle, D.J., Osmond, C.B., Arntzen, C.J., eds. Elsevier, Amsterdam Powles, S.B. (1984) Photoinhibition of photosynthesis induced by visible light. Annu. Rev. Plant Physiol. 35, 1544 Powles, S.B., Bj6rkman, O. (1982) Photoinhibition of photosynthesis: Effect on chlorophyll fluorescence at 77 K in intact leaves and in chloroplast membranes of Nerium oleander. Planta 156, 92107 Powles, S.B., Critchley, C. (1980) Effect of light intensity during growth on photoinhibition of attached intact bean leaflets. Plant Physiol. 65, 1181 1187 Powles, S.B., Thorne, S.W. (1981) Effect of high-light treatments in inducing photoinhibition of photosynthesis in intact leaves of low-light grown Phaseolus vulgaris and Lastreopsis microsora. Planta 152, 471477 Seemann, J.R., Sharkey, T.D., Wang, J., Osmond, C.B. (1987) Environmental effects on photosynthesis, nitrogen-use efficiency, and metabolite pools in leaves of sun and shade plants. Plant Physiol. 84, 796~802 Smillie, R.M., Hetherington, S.E., He, J., Nott, R. (1988) Photoinhibition at chilling temperatures. Aust. J. Plant Physiol. 15, 207-222 Thompson, L.K., Brudvig, G.W. (1988) Cytochrome b-559 may function to protect photosystem II from photoinhibition. Biochemistry 27, 6653 6658
Received 17 March; accepted 26 July 1989
