363
Planta 9 Springer-Verlag 1988
Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: Effect of light during growth on photoinhibition and recovery D.H. Greer and W.A. Laing Plant Physiology Division, D S I R , Private Bag, P a l m e r s t o n N o r t h , N e w Zealand
Abstract. Photoinhibition of photosynthesis was
induced in intact kiwifruit (Actinidia deliciosa (A. Chev,) C.F. Liang et A.R. Ferguson) leaves grown at two photon flux densities (PFDs) of 700 and 1 300 g m o l ' m - 2 . s 1 in a controlled environment, by exposing the leaves to PFD between 1 000 and 2000 gmol.m-2-s -1 at temperatures between 10 and 25 ~ C; recovery from photoinhibition was followed at the same range of temperatures and at a PFD between 0 and 500 gmol.m-2, s-1. In either case the time-courses of photoinhibition and recovery were followed by measuring chlorophyll fluorescence at 692 nm and 77K and by measuring the photon yield of photosynthetic 02 evolution. The initial rate of photoinhibition was lower in the high-light-grown plants but the long-term extent of photoinhibition was not different from that in low-light-grown plants. The rate constants for recovery after photoinhibition for the plants grown at 700 and 1 300 gmol. m - 2. s- 1 or for those grown in shade were similar, indicating that differences between sun and shade leaves in their susceptibility to photoinhibition could not be accounted for by differences in capacity for recovery during photoinhibition. Recovery following photoinhibition was increasingly suppressed by an increasing PFD above 20 gmol. m-2. s-1, indicating that recovery in photoinhibitory conditions would, in any case, be very slow. Differences in photosynthetic capacity and in the capacity for dissipation of non-radiative energy seemed more likely to contribute to Abbreviations and symbols: Fo, F~, F ~ = i n s t a n t a n e o u s . maximum, variable fluorescence; F~/F,. = fluorescence ratio; Fi = Fv at t = 0; Foo = Fv at t = oo ; KD = rate c o n s t a n t for non-radiative energy dissipation ( N R D ) ; Ke = rate constant for p h o t o c h e m istry; k ( F p ) = f i r s t - o r d e r rate constant for p h o t o i n h i b i t i o n ; k ( F r ) = f i r s t - o r d e r rate constant for recovery; P F D = p h o t o n flux density; PSII = p h o t o s y s t e m II; ~01p h o t o n yield o f 02 evolution (incident light)
differences in susceptibility to photoinhibition between sun and shade leaves of kiwifruit. Key words: Actinidia- Chlorophyll fluorescence - Light and growth - Light and photoinhibition
- Photoinhibition of photosynthesis (recovery) Photosynthesis (photon yield) - Temperature and photoinhibition.
Introduction
Leaves of many plant species grown in Shaded environments have been shown to be susceptible to photoinhibition of photosynthesis. In general, the leaves exhibit a reduction in light-limited and lightsaturated rates of photosynthesis when exposed to photon flux densities (PFD) in excess of that during their growth (see Powles 1984; Bj6rkman and Demmig 1987). However, the current understanding of photoinhibition has largely come from studies of shade-grown plants especially at low temperatures (Powles and Thorne 1981; Powles and Bj6rkman 1982; Greer et al. 1986, 1988). Intrinsic interspecific differences in susceptibility to photoinhibition have been established from a comparison of differences between shade-grown leaves of various species grown in similar conditions (Demmig and Bj6rkman 1987). Comprehensive investigations of recovery from photoinhibition have also been made on shadegrown plants. In Phaseolus vulgaris, it has been shown that recovery is temperature-dependent with rates of recovery increasing with increasing temperatures, and that recovery requires weak light for the maximum rates of recovery (Greer et al. 1986). In shade-grown kiwifruit (Actinidia deliciosa) leaves, recovery was shown to depend not
356
D.H. Greet and W.A. Laing: Effect of light on photoinhibition and recovery
only on temperature but also on the extent of photoinhibition, with the rate of recovery decreasing with increasing severity of photoinhibition (Greer and Laing 1988). To assess the impact of photoinhibition on field-crop growth, the effect of the light regime during growth needs to be understood. Demmig and Bj6rkman (1987) have shown for Gossypium hirsutum and Hedera canariensis that photoinhibition was more pronounced in leaves grown at a low PFD compared to those grown at a high PFD. Beyond this, however, little is known of the comparative effect of the incident PFD or temperature on the rate and extent of photoinhibition of plants grown at different PFDs. Demmig and Bj6rkman (1987) have also shown the rate of recovery to be higher in the high-light-grown plants. Again little is known of the comparative effect of temperature on this recovery. A regulatory mechanism to dissipate excess excitation energy during high-light exposure has been proposed to partially protect leaves from photoinhibition of primary photochemistry (Bj6rkman 1987a, b; Demmig and Bj6rkman 1987). This mechanism is the non-radiative (thermal) energy dissipation (NRD) of excess excitation energy and may involve the conversion of the xanthophyll pigment violaxanthin to zeaxanthin (Demmig et al. 1987). The relative rate constant for NRD can be estimated from fluorescence measurements at 77K since the quenching of fluorescence reflects increased non-radiative energy dissipation. Greer et al. (1988) have shown that shade-grown kiwifruit leaves under photoinhibitory conditions apparently have some capacity to dissipate excess energy by NRD but insufficient to prevent primary photochemical damage. For this and other species, an important question is the significance of NRD in protecting high-light-grown plants from photoinhibition. In this work, the effect of the PFD during the growth of kiwifruit leaves on their susceptibility to photoinhibition was studied. In particular, the effects of the incident PFD and the temperature during the exposure to high light on the kinetics of photoinhibition were examined. Similarly, the kinetics of recovery from photoinhibition were determined both with respect to temperature and the incident PFD. Materials and methods This study was carried out using the controlled-environment (C.E.) facilities at the Climate Laboratory, Plant Physiology Division, DSIR.
Plant material. Rooted cuttings of Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson were grown in 9-1 pots as described in Greer et al. (1988). For one set of plants, the vine was trained along a horizontal trellis 0.6 m above the pot, while for another set of plants, the vine was trained along a similar trellis 1.3 m above the pot. Both of the above sets of plants were exposed to day/night temperatures of 25/20+0.5 ~ C. The corresponding water-vapour pressure defcits were 0.63/0.47_+0.05 kPa. The plants with a 0.6-m trellis were grown in a C.E. room where the lighting for 11.5 h was provided by a water-screened array of four high-pressure discharge lamps (" Metalarc", 1 kW; G T E ; Sylvania, Drummondville, Que., Canada) and four quartz-iodide lamps ( " H a l o g e n " , 1 kW; Thorn, Enfield, U K ) giving a P F D of 700 ~tmol-m-2.s -1 at the leaf surface. The plants with the 1.3-m trellis were grown in a second C.E. room where the lighting for an 8-h photoperiod was provided by six high-pressure discharge lamps and six quartz-iodide lamps giving a P F D of 1300 ~tmol.m - 2 - s - 1 at the leaf surface. The photoperiod in both rooms was extended to 12.5 h with supplementary lighting from six tungsten-filament lamps (Par 38, 150 W ; G T E ; Sylvania, Winchester, Ky., USA). The leaves were maintained normal to the incident radiation and the plants were watered four times daily with a 0.5-strength Hoaglands nutrient solution (Brooking 1976). Photoinhibition and recovery. To induce photoinhibition, an intact leaf was sealed into a gas-exchange chamber and exposed to a P F D of 1000, 1500 or 2000/.tmol.m Z.s-1 from a highpressure discharge lamp as described earlier (Greer et al. 1988). The leaf temperature was held constant during the exposure to the light at 10, 20 or 25 ~ C. The CO2 partial pressure was monitored in an open gas-exchange system and was between 30 and 35 kPa. The 0 2 partial pressure and leaf-to-air watervapour difference were 21 kPa and approx. 1 kPa, respectively. The rest of the plant was at 25 ~ C and a P F D of approx. 100 lamol.m-2.s 1 To study recovery, the leaf was initially photoinhibited at 20~ and a P F D of 2000 g m o l . m - 2 - s 1 for 270 min. This pretreatment resulted in 64-68% photoinhibition. In standard recovery experiments, the P F D was reduced to 20 g m o l - m zs-1, provided by a 40-W incandescent lamp and the leaf temperature was held constant at 10, 20 or 25 ~ C. To evaluate the effect of P F D on recovery, additional experiments were carried out at PFDs of 0, 250 and 500 l.tmol-m-2-s- 1 at 25 ~ C. The high-pressure discharge lamp with appropriate neutral density screens was used to provide these PFDs. Further details are given in Greer et al. (1988). Photoinhibition and recovery assays. At intervals throughout each photoinhibition or recovery experiment, duplicate leaf discs (10 m m diameter) were punched from spaced locations on the leaf. These were kept in the dark for at least 20 min and then chlorophyll fluorescence at 692 nm and 77K measured as described in Greer et al. (1986). The instantaneous fluorescence (Fo) and the maximum fluorescence (Fro) were recorded and the variable fluorescence (F~ = F , , - Fo) and the fluorescence ratio (Fv/F,~) were calculated. All data were normalised to an initial level before photoinhibition of Fm= 100. At less frequent intervals, additional leaf discs (36 m m diameter) were punched and light-limited photosynthetic oxygen evolution was measured with a leaf-disc oxygen-electrode unit (LD2; Hansatech, Kings Lynn, Norfolk, U K ) and the p h o t o n yield on an incident-light basis, (~01) determined as described previously (Greer et al. 1986). Data analysis. Photoinhibition and recovery were measured by changes in F~ and ~01. Part of the change in Fv is reflected in
D.H. Greet and W.A. Laing: Effect of light on photo•177177
and recovery
changes in non-radiative energy dissipation and part in the inactivation of the primary photochemistry of photosystem II (PSII) (0gren and Oquist 1984; Bj6rkman 1987a, b). In shadegrown kiwifruit leaves, the decrease in F~ during exposure to high-light, was correlated directly with changes in the photon yield (Greer et al. 1988) and this relationship was confirmed in this study. Since decreases in F~ are indicative of changes in photon yield, a primary measure of photoinhibition (Bj6rkman 1987b), we have adopted F~ to measure photoinhibition. Data were fitted to an exponential equation using non-linear regression analysis described in Greer et al. (1986, 1988).
Ft= F~-- ( F ~ - Fi)e -kt where Ft is the variable fluorescence at any time, t, F~ is Fv at t=0, F~ is the steady-state variable fluorescence and k is the first-order rate constant. Relative values of the rate constant for non-radiative energy dissipation (NRD), KD, were determined according to Bj6rkman (1987b) and Greer et al. (1988). For comparison, data for shade-grown plants (i.e. where the PFD did not exceed 300 ~mol- m - 2. s- i) (Greer et al. 1988 ; Greet and Laing 1988) are included in the results.
Table 1. The control values of photon yield of 0 2 evolution
(tool 02" (mol photons)-1), fluorescence characteristics at 77K and 692 am, and the derived rate constant for non-radiative energy dissipation, for kiwifruit leaves grown at two PFDs. For comparison, data for leaves grown in 300 g m o l . m - 2 - s are included (Greer et al. 1988). The measurements of ~0~ and fluorescence were determined at the end of the 11.5-h dark period prior to any exposm'e to the light (mean • SE; n : 27) Characteristic
~i ~" ~ ~" ~/~ KDb
Growth PFD (Ixmol.m 2.s-1) 300
700
1300
0.0920• 4.8 • 41.1 • 36.2 • 0.882 • 8.4 •
0.0863• 4.2 • 23.0 • 18.8 • 0.815 • 14.9 •
0.0815 3.7 19.4 15.6 0.801 16.4
• • _+3.9 +3.6 _+0.030 +_3.3
These measurements have not been normalised to F,, = 100 b Calculated assuming the rate constant for photochemistry (Kp) = 70
357
Results
Comparison of fluorescence and photosynthetic characteristics. There was a general trend for fluorescence and the photon yield of non-photoinhibited control plants to decrease with increasing PFD during growth (Table 1). Fo, Fro, and F~ were typically 15-20% higher for leaves developed under a PFD of 700 ~tmol.m-2-s-1, than those leaves grown at 1300 g m o l ' m - Z ' s -1 while F~/Fm was only about 2% higher. Photon yield was about 6% higher in the 700 gmol. m - 2. s- 1_grown plants and this difference was significant (p=0.01). All of these characteristics were also higher in those leaves grown at 300 g m o l . m -2. s-1. ~0i and Fv/Fm were about 7% higher while Fv was nearly double that for the 700 g m o l . m - 2 , s-1-grown plants. As a consequence of these differences in Fo and F,,, the calculated initial relative value of the rate constant for N R D (KD) apparently increased with increasing PFD of growth. The light-saturated photosynthetic rate for those plants grown at 1300 gmol-m - 2 ' s -1 was 17.5 gmol ( C O z ) . m - 2 . s -1, about 22% higher than for the plants grown at 700 lamol'm-2, s-1, 1 4 . 3 ~ m o l ( C O z ) - m 2"s-1.
Daily changes in fluorescence characteristics and photon yield. For plants grown at 700 g m o l - m - 2 . s-1, there were no significant changes in any of the fluorescence characteristics through the day (Table 2). For the leaves grown at 1 300 g m o l . m - 2 -s-1, however, there was a significant decrease in F,,, Fv and Fv/Fm and a slight but non-significant increase in Fo. There was no evidence of a significant change in photon yield during the day for plants grown at either 700 or 1 300 ~tmol 9m - 2. s- 1.
Effect of PFD on photoinhibition. The time-courses of the decrease in the fluorescence emission and
Table 2. The effect of the PFD during growth on the photon yield (mol O2" (tool photons)- 1) and on the fluorescence characteristics for kiwifruit plants grown at two PFDs prior to and during exposure to the light (mean • SE, n = 14) Characteristic
Growth PFD (gmol " m - 2 .s -
1)
700
~Oi
Fo Fm F~ FvlF,.
1300
Before
After a
Before
After
0.0899• 3.1 • 18.0 • 14.9 • 0.827 •
0.0917-+0.0012 3.3 • 17.0 • 13.7 • 0.803 •
0.0750 • 0.0032 3.3 • 18.7 +1.2
0.0801• 3.9 • 14.3 • 10.3 • 0.725 •
" Measured as the mean value between 150 and 400 min exposure to the PFD
15.4
• 1.2
0.819 •
358
D.H. Greer and W.A. Laing: Effect of light on photoinhibition and recovery Table 3. The effect of the incident P F D at a temperature of 20 ~ C on the rate constant for photoinhibition, k(Fp), (mean _+ SE, n - 6) and on the extent o f photoinhibition, for intact kiwifruit leaves grown in three light regimes. The extent of photoinhibition was calculated from F~ at t = oo (F~) and expressed as 1 - F ~ / F ~ where Fi = Fv of the appropriate control leaves
100
so
80 .
40
Characteristic
F,
20
k(Fp)
16o
I
z00 Time
36o
I
500
(rain)
Growth P F D (gmolm-2.s-1)
300~
(• -3 9min a)
700 1300
Extent
300 a 700 1300
(%)
Exposure P F D (~mol, m - 2. S 1) 1000
-8.8+1.6 4.6_+2.3
1 500
2000
8.5 + 1.1
--
9.6_+2.4 5.5+1.8
10.3_+1.6 7.5_+1.4
-
92
--
59 50
62 64
81 74
100
a From Greer et al. (1988)
8o
B
60
40
9
o
9
20
o
10
~'
4
;
16o
2;o
36o
Time (rain)
~
4;o
soo
C~
0
x
2 0
6
16o
260
360
,60
500
Time (rain) Fig. 1A-C. The effect of an exposure at a P F D of either 1000 ( o - - o ) or 2 000 g m o l - m - 2 " S - 1 ( O - - O ) at 20 ~ C on the timecourse of (A) Fo and Fro, (B) Fv and (C) the photon yield ((00 for intact kiwifruit leaves grown at a P F D of 700 g m o l - m -2. s-1. Each line is the best fit to an equation described in the text and is the mean of three leaves
photon yield for leaves grown at a P F D of 700 ~tmol-m-2. s- 1 and exposed to a P F D of either 1 000 or 2000 g m o l . m - Z . s 1 are shown in Fig. 1. At both PFDs, Fo increased to the same extent whereas Fm decreased to a greater extent at the higher PFD. Fv therefore decreased exponentially
to approach a steady-state after about 400 rain with the initial decrease faster and ultimately greater at the higher PFD. Similar changes in photon yield were observed. The rate constant for photoinhibition, k(Fp), and the extent of photoinhibition for leaves exposed to a range of P F D s during the photoinhibitory treatment are shown in Table 3. For plants grown at both P F D s k(Fp) increased linearily with increasing exposure PFD, at least between 1 000 and 2 000 gmol. m - 2. s- 1. The severity of photoinhibition was also dependent on the P F D during the exposure for each treatment, increasing from about 50% at 1 000 ]amol -m - 2. s - 1 to 80% at 2 000 gmol. m - 2. s- 1. The susceptibility of kiwifruit leaves to photoinhibition was therefore dependent on the P F D during exposure. There was a higher initial rate of photoinhibition for the leaves grown at 700 compared with those at 1 300 ~mol. m - 2. s - 1 but there was apparently little difference in the extent of photoinhibition (Fig. 2). These results are similarly reflected in higher rate constants for photoinhibition (k(Fp)) at all photoinhibitory P F D s for those leaves grown at 700 g m o l - m - 2 , s-1 compared to those grown at 1 300 g m o l ' m -2" s - t (Table 3). However, k(Fp) for the 700 g m o l . m - 2 . s - < g r o w n plants was similar to that for plants grown in 300 ~mol- m - 2. s - t In contrast, the extent of photoinhibition in the plants from the two high-light regimes was similar at all P F D s but it increased markedly between plants grown in 300 g m o l - m - 2 . s - 1 and in either of the other two regimes. These results were confirmed by similar changes in the photon yield (not shown). Effect o f temperature on photoinhibition. The rate constant for photoinhibition, k(Fp), was only mar-
D.H. Greer and W.A. Laing: Effect of light on photoinhibition and recovery
A
1oo i,
E 8o
I Jr
6O
Q~
100
359
A ___,___--4,
c~ 8o o ~)
60
--~
40
0 i,
o 40 Fo
,-7" 2O
20
6
16o
260 Time
36o
46o
6
500
260
(min)
Time
360
I0
lOO
460
500
(min) l
T
B 80
~
60
-6 6 E ? 4
O
L,2 4O
0
vx 2 #.
20 0
6
,60
26o Time
36o
46o
soo
(min)
Fig. 2A, B. The effect of exposing intact kiwifruit leaves grown at a P F D of either 700 ( o - - o ) or 1300 gmol- m - 2. s - 1 ( e - - e ) to a P F D of 1 500 g m o l . m - 2 - s - ~ at 20 ~ C on the change in
(A) Fo and F,,, and (B) the change in F,. Each line is the mean of three leaves
Table 4. The effect of temperature during exposure to a P F D of 1 500 Ixmol-m -2-s -1 on the rate constant for photoinhibition, k(Fp) (mean_+ SE, n = 6) and on the extent of photoinhibition, for kiwifruit leaves grown in three light regimes Characteristic
k(Fp) ( x l 0 -3 9 min -1)
Extent (%)
Growth P F D (~tmolm - 2 . S -1) 300 ~ 700 1300
300a 700 1300
Temperature (~ C) 10 9.0_+0.6 9.0_+1.0 4.7_+2.0
98 84 94
20 8.5+_1.1 9.6_+2.4 5.5_+1.8
92 62 64
0
6
,6o
260
360
,60
500
T i m e (min) Fig. 3A, B. The time-course of recovery at a PFD of 20 gmolm 2.s-1 and at 25~ of (A) ( e - - e ) Fo, (e--i) Fm and ( o - - o ) F, and (B) photon yield (~00 of intact kiwifruit leaves grown at a PFD of 700 lamol'm-Z-s-1 after a 270-min exposure to a PFD of 2000 gmol-m 2.s-~ at 20~ Each line is the mean of three leaves 700 a n d 1 300 g m o l . m - 2 - s 1 T h e extent o f p h o toinhibition was m o r e strongly t e m p e r a t u r e - d e p e n d e n t with p h o t o i n h i b i t i o n m u c h m o r e severe at the lowest t e m p e r a t u r e . Plants g r o w n in 300 g m o l . m - 2. s - J were the m o s t susceptible to p h o t o i n h i b i tion at all t e m p e r a t u r e s .
25 10.5_+1.3 11.0_+3.0 _b
81 39 49c
From Greer et al. (1988) b Kinetics of photoinhibition were linear rather than exponential (F~= 85.4~0.084 t) c Calculated at t = 500 min ginally d e p e n d e n t o n t e m p e r a t u r e (Table 4). It was a g a i n evident that k(Fp) did not v a r y between plants g r o w n in 300 or at 700 ~ t m o l ' m -2" s - 1 b u t did decrease significantly b e t w e e n plants g r o w n at
Time-course and effect of temperature on recovery. The time-courses o f the recovery o f Fo, Fm a n d Fv a n d ~0~at 25 ~ C a n d 20 g m o l . m - 2. s - 1 following p h o t o i n h i b i t i o n o f leaves g r o w n at a P F D o f 700 ~ m o l . m - 2 . s - 1 are s h o w n in Fig. 3. R e c o v e r y was c o n f i r m e d b y an increase in Fm with a c o n c o m i t a n t decrease in Fo. T h e increase in F~ was closely p a r a l leled b y an increase in qh. R e c o v e r y o f Fv was strongly t e m p e r a t u r e - d e p e n d e n t with negligible r e c o v e r y at 1 0 ~ a n d an increasing rate f r o m 20 to 25 ~ C (Table 5). H o w ever, there was little or n o difference b e t w e e n plants g r o w n at the v a r i o u s light regimes in the kinetics o f r e c o v e r y at these t e m p e r a t u r e s . Similar results were o b s e r v e d for r e c o v e r y o f ~0~ (not shown).
360
D . H . G r e e r a n d W . A . L a i n g : Effect o f light on p h o t o i n h i b i t i o n a n d recovery
Table 5. T h e effect o f t e m p e r a t u r e at a P F D o f 20 p . m o l . m -z" s - 1 o n the rate c o n s t a n t for recovery f r o m p h o t o i n h i b i t i o n , k(F~), following p h o t o i n h i b i t i o n at 20 ~ C a n d a P F D o f 2000 p m o l . m 2. s - ~ o f kiwifruit leaves g r o w n in three light regimes (mean_+ SE, n = 6). T h e extent o f p h o t o i n h i b i t i o n was between 64 a n d 6 8 % o f the F~ o f control leaves in all cases Characteristic
k(Fr) ( x 1 0 - 3 " m i n -1)
Growth PFD (btmol . m 2 - s - 1 )
300 b 700 1300
Temperature a
(o C)
20
25
1.6__+0.2 2.0_+0.3 2.4_+0.3
4.7_+0.4 5.5_+0.5 4.0_+0.3
N o recovery occurred at 10 ~ C b F r o m G r e e r a n d L a i n g (1988)
100
8O o
a.~
9
9
Effect of PFD on recovery. In Fig. 4 is shown a comparison of the time-courses of recovery at P F D s ranging from 0 to 250 g m o l - m - 2 - s -1 of leaves grown at a P F D of 1300 ~tmol.m-Z.s -1 Recovery in the dark was exceedingly slow and only 21% complete in 450 min. Recovery occurred at both 20 and 250 Ixmol" m - 2 . s-1, but was slow at the higher PFD, and only 51% complete in 450 min compared with about 90% at 20 gmol . m - 2 9s- 1. Recovery at a P F D of 500 gmol. m - 2. s- 1 (not shown) was even slower than that at 250 gmol. m - 2. s- 1 and was only about 40% complete in the same time. Thus, recovery was maximal at the lowest light level. A similar response occurred in the 700 g m o l . m - Z . s - l - g r o w n plants (not shown). The rate constant for recovery, (k(Fr)), as a function of the P F D is shown in Fig. 5. This shows that k(Fr) increased dramatically, about seven-fold, from the dark with the addition of weak light but then declined with further increases in PFD. This would indicate that recovery at the P F D s at which the plants were grown would not be much higher than in the dark.
4O
Discussion
;
1;0
2;0
3;0
4;0
soo
Time (rain) Fig. 4. T h e t i m e - c o u r s e o f recovery o f Fv at 2 5 ~ a n d at a P F D o f 0 ( n - - n ) , 20 ( o - - o ) or 250 b t m o l - m - 2 - s i ( e - - e ) for intact kiwifruit leaves g r o w n at a P F D o f 1300 g m o l . m 2. s -1 following a 2 7 0 - m i n e x p o s u r e to a P F D o f 2000 g m o l . m 2. s - a at 20 ~ C. E a c h line is the m e a n o f three leaves
_t.,, 4 I r ~
E ?
5
O ,r
x
2
L~ v
;
1 ,o PFD
3;0
(/~mol.m
4;0 -z.
s-')
Fv, k(F~), at 2 5 ~ as a f u n c t i o n o f P F D d u r i n g the recovery period for intact kiwifruit leaves g r o w n at a P F D o f 1 300 g m o l . m - 2 s - 1 following p h o t o i n h i b i t i o n at a P F D o f 2000 g m o l . m - 2 - s - x Fig. 5. T h e rate c o n s t a n t for recovery o f
When kiwifruit leaves were grown in high-light there were marked changes in fluorescence emission at 77K, notably F~/Fm, compared with those leaves grown at low-light. Fluorescence changes in a given leaf are indicative of a redistribution of energy within PSII, with the quenching of fluorescence reflecting an increase in the rate constant for non-radiative energy dissipation, (Ko) while a rise in Fo reflects a decrease in the rate constant for photochemistry in PSII, (Kv) (0gren and Oquist 1984, Bj6rkman 1987b). Assuming that Ke was constant between leaves from the three light regimes, it appears that the relative value for KD increased with increasing P F D of growth 9 This might indicate there was an increased capacity to protect the photosynthetic apparatus from highlight stress when grown at high-light. This however, needs biochemical confirmation of, for example, differences in zeaxanthin formation (Demmig et al. 1987). Correlated with the changes in fluorescence were changes in the photon yield9 This indicates the kiwifruit leaves have some propensity to become photoinhibited during the growth conditions, the more so the higher the PFD. This chronic photoinhibition was, however, relatively minor since a four-fold increase in P F D resulted in no more than an 11% decrease in photon yield. In spite of this, the leaves also exhibited a capacity to accli-
D.H. Greer and W.A. Laing: Effect of light on photoinhibition and recovery
mate to the higher PFDs by increasing the maximum photosynthetic rates (Laing 1985; this study). Similar divergent responses to an increase in growth PFD by this shade-tolerant canopy vine (Ferguson 1988) have been reported in other shade-tolerant species (see Anderson and Osmond 1987). No additional diurnal changes in fluorescence or photon yield were detected for kiwifruit leaves grown and exposed to 700 gmol.m-2-s -1. For those plants grown at 1 300 btmol-m-2, s-1, however, there was a marked diurnal reduction in Fro, F~ and Fv/Fm, and a slight increase in Fo. Thus the chronic photoinhibition was exacerbated through the day although only to a minor extent since there were no detectable changes in photon yield. Ben et al. (1987) also observed a decrease in F~/F,, in Helianthus annuus leaves exposed to the sun during the day, again without concomitant changes in the photon yield. Bj6rkman and Powles (1984) similarly observed a diurnal reduction in F,, when Nerium oleander was exposed to the full midsummer PFD. These fluorescence changes probably reflect changes in Kp and Ko in the upper surface of leaves only, since they apparently have little effect on the well-being of the leaf as a whole. Severe photoinhibitory damage to PSII resulted when kiwifruit leaves from all light regimes were exposed to PFDs substantially above that in which they were grown. This was manifested by a decrease in the photon yield and by a concomitant decrease in F,, and an increase in Fo, indicating that direct damage at or near the reaction centres was occurring (CMand et al. 1986). The extent of the photoinhibition was highly dependent on temperature, increasing in severity with decreasing temperatures but the kinetics of the process were essentially independent of temperature. Similar effects of temperature were observed in shade-grown kiwifruit leaves (Greer et al. 1988) indicating that the temperature-dependency of photoinhibition is not affected by the PFD during growth as {)quist et al. (1987) had proposed. The extent of photoinhibition also depended on the PFD during the highqight exposure, increasing as the PFD increased. Similar results have been observed in lowlight-grown Phaseolus vulgar& (Powles and Thorne 1981), Nerium oleander (Powles and Bj6rkman 1982) and Lemna gibba ({)gren et al. 1984). The exacerbation of photoinhibition by increasing PFD is expected since the absorption of excitation energy would increasingly exceed its rate of utilisation by photochemistry and rate of non-radiative dissipation (Bj6rkman 1987 a). In contrast to temperature, the kinetics of pho-
361
toinhibition depended on the PFD during the highlight exposure, with the rate constant for photoinhibition, k(Fp) increasing linearly with increasing PFD, at least between 1000 and 2 000 btmol-m-2 s- 1 for plants grown at both 700 and 1 300 gmolm - 2. s- 1. A similar linear increase in the rate constant for photoinhibition was observed in shadegrown Monstera deliciosa by Demmig and Bj6rkman (1987). These studies on higher plants are thus consistent with the effect of PFD on photoinhibition in unicellular algae (Kok 1956; Neale 1987). It was evident, however, that the rate constant for photoinhibition in kiwifruit leaves grown at 1 300 gmol-l-n -2. S-1 was lower than in plants grown at 700 Ixmol-m 2"s-1 (Fig. 2, Table 3). k(Fp) was from 1.3- to 1.9-fold higher in those plants grown at 700 g m o l ' m - 2 , s-1 and this relative difference was maintained over all temperatures and exposure PFDs. The extent of photoinhibition was, however, not different after long exposures, indicating that the leaves were about equally susceptible. Leaves grown at 300 btmol" m - 2 ' s - 1 , by contrast, were much more susceptible than leaves grown at the two higher PFDs though there was little difference in k(Fp) from that in the leaves from 700 gmol. m-2. s-1. Seeman et al. (1987) also observed a 1.5- to 2-fold difference in the rate constant for Phaseolus vulgaris leaves grown at about 550 and 1 100 btmol- m - 2. s - 1 whereas Powles and Critchley (1981) noted that the susceptibility to photoinhibition in the same species increased significantly when the PFD during growth decreased from 25 to 6% of full sunlight. The response to the growth PFD was therefore similar to that observed here. Demmig and Bj6rkman (1987) observed that the increased susceptibility to photoinhibition between high- and low-light-grown plants persisted even when dissipation of energy was prevented by inhibiting photosynthesis. Additionally, recovery from photoinhibition was faster in high- compared with low-light-grown plants. A similar result was observed in Lemna gibba by Skogen et al. (1986). From their results, Demmig and Bj6rkman (1987) proposed that differences in capacity for recovery contributed to the differences in susceptibility to photoinhibition between sun and shade leaves. In kiwifruit leaves, however, the rates of post-photoinhibition recovery were similar for leaves grown over a four-fold range in PFD (Table 5), in spite of inherent differences in both the kinetics and extent of photoinhibition. Greer and Laing (1988) have shown previously in kiwifruit leaves that the rate constant for recovery, k(Fr), was highly dependent on the extent of photoinhibition. This has also been recognised by Demmig and Bj6rkman
362
D.H. Greer and W.A. Laing: Effect of light on photoinhibition and recovery
(1987). In their comparison of the differences in recovery rate constants between sun and shade leaves of Gossypium and Hedera, there were great differences in the initial level of photoinhibition. Differences in recovery, therefore, could have reflected differences in the extent of photoinhibition. It is axiomatic that it is the recovery occurring during photoinhibition rather than following photoinhibition that would be expected to affect the susceptibility to photoinhibition. However, the recovery of kiwifruit leaves following photoinhibition was substantially inhibited by increases in the PFD between 20 and 500 g m o l - m - 2 " s - 1 . Results consistent with this were also observed by Greer et al. (1986) with Phaseolus vulgaris and by Skogen et al. (1986) with Lemna gibba. By extrapolating the effect of PFD on the recovery rate constant (Fig. 5) to those PFDs where photoinhibition was determined, it would appear that recovery occurring during the high-light exposure was very slow. Thus it seems unlikely that recovery occurring during photoinhibition could account for the differences in susceptibility to photoinhibition between sun and shade leaves of kiwifruit. Differences in photosynthetic capacity between these kiwifruit plants, instead, are more likely to conform with high-light-grown leaves being less susceptible to photoinhibition (Bj6rkman et al. 1972; Anderson and Osmond 1987). In summary, the results of this study have confirmed that kiwifruit leaves are susceptible to photoinhibition. Although growth at high PFDs tends to ameliorate the susceptibility to photoinhibition, a chronic but low level of photoinhibition occurs in response to the high light. However, the incident PFD and the temperature are the most important factors, affecting both photoinhibition and recovery, and the balance between these two processes determines the extent of damage to the primary photochemical apparatus. References Anderson, J.M., Osmond, C.B. (1987) Sun-shade responses: compromises between acclimation and photoinhibition. In: Topics in photosynthesis, vol. 9: Photoinhibition, p 1 38, Kyle, D.J., Osmond, C.B., Arntzen, C.J., eds. Elsevier, Amsterdam Ben, G-Y., Osmond, C.B., Sharkey, T.D. (1987) Comparisons of photosynthetic responses of Xanthium strumarium and Helianthus annuus to chronic and acute water stress in sun and shade. Plant Physiol. 84, 476-482 Bj6rkman, O. (1987a) High irradiance stress in higher plants and interaction with other stress factors. Progr. Photosynth. Res. 4, 11-18 Bj6rkman, O. (1987b) Low-temperature chlorophyll fluorescence in leaves and its relationship to photon yield of photo-
synthesis 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., Boardman, N.K., Anderson, J.M., Thorne, S.W., Goodchild, D.J., Pylotis, N.Z. (1972) Effect of light intensity during growth of Atriplex patula on the capacity of photosynthetic reactions, chloroplast components and structure. Carnegie Inst. Wash. Yearb. 71, 115135 Bj6rkman, O., Demmig, B. (1987) Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins. Planta 170, 48%504 Bj6rkman, O., Powles, S.B. (1984) Inhibition of photosynthetic reactions under water stress: interaction with light level. Planta 161,490-504 Brooking, I.R. (1976) Soilless potting media for controlled-environment facilities. N.Z.J. Exp. Agric. 4, 203-208 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 (77K) and photon yield of Oz evolution in leaves of higher plants. Planta 171, 171-184 Demmig, B., Winter, K., Krtiger, 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 Ferguson, A.R. (1988) Botanical Nomenclature: Actinidia chinensis, A ctinidia deliciosa, and Actinidia setosa. In: Kiwifruit: Science and Management, in press, Warrington, I.J., Weston, G.C., eds. N.Z. Soc. for Horticultural Science in conjunction with Ray Richards Publisher, Auckland, N.Z. 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., Laing, W.A. (1988) Photoinhibition of photosynthesis in intact kiwifruit (Actinidia delieiosa) leaves: Recovery and its dependence on temperature. Planta 174, 159-165 Greer, D.H., Laing, W.A., Kipnis, T. (1988) Photoinhibition of photosynthesis in intact kiwifruit (Actinidia delieiosa) leaves: Effect of temperature. Planta 174, 152-158 Kok, B. (1956) On the inhibition of photosynthesis by intense light. Biochim. Biophys. Acta 21,234-244 Laing, W.A. (1985) Temperature and light response curves for photosynthesis in kiwifruit (Aetinidia chinensis) cv. Hayward. N.Z.J. Agric. Res. 28, 117-124 Neale, P.J. (1987) Algal photoinhibition and photosynthesis in the aquatic environment. In: Topics in photosynthesis, vol. 9: Photoinhibition, pp. 3%68, Kyle, D.J., Osmond, C.B., Arntzen, C.J., eds. Elsevier, Amsterdam Ogren, E., 13quist, G. (1984) Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. III. Chlorophyll fluorescence at 77K. Physiol. Plant. 62, 193-200 ()gren, E., Oquist, G., Hallgren, J.-E. (1984) Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. I. Photosynthesis in vivo. Physiol. Plant. 62, 181-186 Oquist, G., Greer, D.H., Ogren, E. (1987) Light stress at low temperature. In: Topics in photosynthesis, vol. 9: Photoinhibition, pp. 6%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, 15~44 Powles, S.B., Bj6rkman, O. (1982) Photoinhibition of photo-
D.H. Greer and W.A. Laing: Effect of light on photoinhibition and recovery synthesis: 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 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 mierosora. Planta 152, 471 477 Seeman, J.R., Sharkey, T.D., Wang, J., Osmond, C.B. (1987)
363
Environmental effects on photosynthesis, nitrogen-use efficiency, and metabolite pools in leaves of sun and shade plants. Plant Physiol. 84, 796-802 Skogen, D., Chaturvedi, R., Weidemann, F., Nilsen, S. (1986) Photoinhibition of photosynthesis: Effect of light quality and quantity on recovery from photoinhibition in Lemna gibba. J. Plant Physiol. 126, 195-205
Received 7 December 1987; accepted 25 March 1988
Planta 9 Springer-Verlag 1988
Photoinhibition of photosynthesis in intact kiwifruit (Actinidia deliciosa) leaves: Effect of light during growth on photoinhibition and recovery D.H. Greer and W.A. Laing Plant Physiology Division, D S I R , Private Bag, P a l m e r s t o n N o r t h , N e w Zealand
Abstract. Photoinhibition of photosynthesis was
induced in intact kiwifruit (Actinidia deliciosa (A. Chev,) C.F. Liang et A.R. Ferguson) leaves grown at two photon flux densities (PFDs) of 700 and 1 300 g m o l ' m - 2 . s 1 in a controlled environment, by exposing the leaves to PFD between 1 000 and 2000 gmol.m-2-s -1 at temperatures between 10 and 25 ~ C; recovery from photoinhibition was followed at the same range of temperatures and at a PFD between 0 and 500 gmol.m-2, s-1. In either case the time-courses of photoinhibition and recovery were followed by measuring chlorophyll fluorescence at 692 nm and 77K and by measuring the photon yield of photosynthetic 02 evolution. The initial rate of photoinhibition was lower in the high-light-grown plants but the long-term extent of photoinhibition was not different from that in low-light-grown plants. The rate constants for recovery after photoinhibition for the plants grown at 700 and 1 300 gmol. m - 2. s- 1 or for those grown in shade were similar, indicating that differences between sun and shade leaves in their susceptibility to photoinhibition could not be accounted for by differences in capacity for recovery during photoinhibition. Recovery following photoinhibition was increasingly suppressed by an increasing PFD above 20 gmol. m-2. s-1, indicating that recovery in photoinhibitory conditions would, in any case, be very slow. Differences in photosynthetic capacity and in the capacity for dissipation of non-radiative energy seemed more likely to contribute to Abbreviations and symbols: Fo, F~, F ~ = i n s t a n t a n e o u s . maximum, variable fluorescence; F~/F,. = fluorescence ratio; Fi = Fv at t = 0; Foo = Fv at t = oo ; KD = rate c o n s t a n t for non-radiative energy dissipation ( N R D ) ; Ke = rate constant for p h o t o c h e m istry; k ( F p ) = f i r s t - o r d e r rate constant for p h o t o i n h i b i t i o n ; k ( F r ) = f i r s t - o r d e r rate constant for recovery; P F D = p h o t o n flux density; PSII = p h o t o s y s t e m II; ~01p h o t o n yield o f 02 evolution (incident light)
differences in susceptibility to photoinhibition between sun and shade leaves of kiwifruit. Key words: Actinidia- Chlorophyll fluorescence - Light and growth - Light and photoinhibition
- Photoinhibition of photosynthesis (recovery) Photosynthesis (photon yield) - Temperature and photoinhibition.
Introduction
Leaves of many plant species grown in Shaded environments have been shown to be susceptible to photoinhibition of photosynthesis. In general, the leaves exhibit a reduction in light-limited and lightsaturated rates of photosynthesis when exposed to photon flux densities (PFD) in excess of that during their growth (see Powles 1984; Bj6rkman and Demmig 1987). However, the current understanding of photoinhibition has largely come from studies of shade-grown plants especially at low temperatures (Powles and Thorne 1981; Powles and Bj6rkman 1982; Greer et al. 1986, 1988). Intrinsic interspecific differences in susceptibility to photoinhibition have been established from a comparison of differences between shade-grown leaves of various species grown in similar conditions (Demmig and Bj6rkman 1987). Comprehensive investigations of recovery from photoinhibition have also been made on shadegrown plants. In Phaseolus vulgaris, it has been shown that recovery is temperature-dependent with rates of recovery increasing with increasing temperatures, and that recovery requires weak light for the maximum rates of recovery (Greer et al. 1986). In shade-grown kiwifruit (Actinidia deliciosa) leaves, recovery was shown to depend not
356
D.H. Greet and W.A. Laing: Effect of light on photoinhibition and recovery
only on temperature but also on the extent of photoinhibition, with the rate of recovery decreasing with increasing severity of photoinhibition (Greer and Laing 1988). To assess the impact of photoinhibition on field-crop growth, the effect of the light regime during growth needs to be understood. Demmig and Bj6rkman (1987) have shown for Gossypium hirsutum and Hedera canariensis that photoinhibition was more pronounced in leaves grown at a low PFD compared to those grown at a high PFD. Beyond this, however, little is known of the comparative effect of the incident PFD or temperature on the rate and extent of photoinhibition of plants grown at different PFDs. Demmig and Bj6rkman (1987) have also shown the rate of recovery to be higher in the high-light-grown plants. Again little is known of the comparative effect of temperature on this recovery. A regulatory mechanism to dissipate excess excitation energy during high-light exposure has been proposed to partially protect leaves from photoinhibition of primary photochemistry (Bj6rkman 1987a, b; Demmig and Bj6rkman 1987). This mechanism is the non-radiative (thermal) energy dissipation (NRD) of excess excitation energy and may involve the conversion of the xanthophyll pigment violaxanthin to zeaxanthin (Demmig et al. 1987). The relative rate constant for NRD can be estimated from fluorescence measurements at 77K since the quenching of fluorescence reflects increased non-radiative energy dissipation. Greer et al. (1988) have shown that shade-grown kiwifruit leaves under photoinhibitory conditions apparently have some capacity to dissipate excess energy by NRD but insufficient to prevent primary photochemical damage. For this and other species, an important question is the significance of NRD in protecting high-light-grown plants from photoinhibition. In this work, the effect of the PFD during the growth of kiwifruit leaves on their susceptibility to photoinhibition was studied. In particular, the effects of the incident PFD and the temperature during the exposure to high light on the kinetics of photoinhibition were examined. Similarly, the kinetics of recovery from photoinhibition were determined both with respect to temperature and the incident PFD. Materials and methods This study was carried out using the controlled-environment (C.E.) facilities at the Climate Laboratory, Plant Physiology Division, DSIR.
Plant material. Rooted cuttings of Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson were grown in 9-1 pots as described in Greer et al. (1988). For one set of plants, the vine was trained along a horizontal trellis 0.6 m above the pot, while for another set of plants, the vine was trained along a similar trellis 1.3 m above the pot. Both of the above sets of plants were exposed to day/night temperatures of 25/20+0.5 ~ C. The corresponding water-vapour pressure defcits were 0.63/0.47_+0.05 kPa. The plants with a 0.6-m trellis were grown in a C.E. room where the lighting for 11.5 h was provided by a water-screened array of four high-pressure discharge lamps (" Metalarc", 1 kW; G T E ; Sylvania, Drummondville, Que., Canada) and four quartz-iodide lamps ( " H a l o g e n " , 1 kW; Thorn, Enfield, U K ) giving a P F D of 700 ~tmol-m-2.s -1 at the leaf surface. The plants with the 1.3-m trellis were grown in a second C.E. room where the lighting for an 8-h photoperiod was provided by six high-pressure discharge lamps and six quartz-iodide lamps giving a P F D of 1300 ~tmol.m - 2 - s - 1 at the leaf surface. The photoperiod in both rooms was extended to 12.5 h with supplementary lighting from six tungsten-filament lamps (Par 38, 150 W ; G T E ; Sylvania, Winchester, Ky., USA). The leaves were maintained normal to the incident radiation and the plants were watered four times daily with a 0.5-strength Hoaglands nutrient solution (Brooking 1976). Photoinhibition and recovery. To induce photoinhibition, an intact leaf was sealed into a gas-exchange chamber and exposed to a P F D of 1000, 1500 or 2000/.tmol.m Z.s-1 from a highpressure discharge lamp as described earlier (Greer et al. 1988). The leaf temperature was held constant during the exposure to the light at 10, 20 or 25 ~ C. The CO2 partial pressure was monitored in an open gas-exchange system and was between 30 and 35 kPa. The 0 2 partial pressure and leaf-to-air watervapour difference were 21 kPa and approx. 1 kPa, respectively. The rest of the plant was at 25 ~ C and a P F D of approx. 100 lamol.m-2.s 1 To study recovery, the leaf was initially photoinhibited at 20~ and a P F D of 2000 g m o l . m - 2 - s 1 for 270 min. This pretreatment resulted in 64-68% photoinhibition. In standard recovery experiments, the P F D was reduced to 20 g m o l - m zs-1, provided by a 40-W incandescent lamp and the leaf temperature was held constant at 10, 20 or 25 ~ C. To evaluate the effect of P F D on recovery, additional experiments were carried out at PFDs of 0, 250 and 500 l.tmol-m-2-s- 1 at 25 ~ C. The high-pressure discharge lamp with appropriate neutral density screens was used to provide these PFDs. Further details are given in Greer et al. (1988). Photoinhibition and recovery assays. At intervals throughout each photoinhibition or recovery experiment, duplicate leaf discs (10 m m diameter) were punched from spaced locations on the leaf. These were kept in the dark for at least 20 min and then chlorophyll fluorescence at 692 nm and 77K measured as described in Greer et al. (1986). The instantaneous fluorescence (Fo) and the maximum fluorescence (Fro) were recorded and the variable fluorescence (F~ = F , , - Fo) and the fluorescence ratio (Fv/F,~) were calculated. All data were normalised to an initial level before photoinhibition of Fm= 100. At less frequent intervals, additional leaf discs (36 m m diameter) were punched and light-limited photosynthetic oxygen evolution was measured with a leaf-disc oxygen-electrode unit (LD2; Hansatech, Kings Lynn, Norfolk, U K ) and the p h o t o n yield on an incident-light basis, (~01) determined as described previously (Greer et al. 1986). Data analysis. Photoinhibition and recovery were measured by changes in F~ and ~01. Part of the change in Fv is reflected in
D.H. Greet and W.A. Laing: Effect of light on photo•177177
and recovery
changes in non-radiative energy dissipation and part in the inactivation of the primary photochemistry of photosystem II (PSII) (0gren and Oquist 1984; Bj6rkman 1987a, b). In shadegrown kiwifruit leaves, the decrease in F~ during exposure to high-light, was correlated directly with changes in the photon yield (Greer et al. 1988) and this relationship was confirmed in this study. Since decreases in F~ are indicative of changes in photon yield, a primary measure of photoinhibition (Bj6rkman 1987b), we have adopted F~ to measure photoinhibition. Data were fitted to an exponential equation using non-linear regression analysis described in Greer et al. (1986, 1988).
Ft= F~-- ( F ~ - Fi)e -kt where Ft is the variable fluorescence at any time, t, F~ is Fv at t=0, F~ is the steady-state variable fluorescence and k is the first-order rate constant. Relative values of the rate constant for non-radiative energy dissipation (NRD), KD, were determined according to Bj6rkman (1987b) and Greer et al. (1988). For comparison, data for shade-grown plants (i.e. where the PFD did not exceed 300 ~mol- m - 2. s- i) (Greer et al. 1988 ; Greet and Laing 1988) are included in the results.
Table 1. The control values of photon yield of 0 2 evolution
(tool 02" (mol photons)-1), fluorescence characteristics at 77K and 692 am, and the derived rate constant for non-radiative energy dissipation, for kiwifruit leaves grown at two PFDs. For comparison, data for leaves grown in 300 g m o l . m - 2 - s are included (Greer et al. 1988). The measurements of ~0~ and fluorescence were determined at the end of the 11.5-h dark period prior to any exposm'e to the light (mean • SE; n : 27) Characteristic
~i ~" ~ ~" ~/~ KDb
Growth PFD (Ixmol.m 2.s-1) 300
700
1300
0.0920• 4.8 • 41.1 • 36.2 • 0.882 • 8.4 •
0.0863• 4.2 • 23.0 • 18.8 • 0.815 • 14.9 •
0.0815 3.7 19.4 15.6 0.801 16.4
• • _+3.9 +3.6 _+0.030 +_3.3
These measurements have not been normalised to F,, = 100 b Calculated assuming the rate constant for photochemistry (Kp) = 70
357
Results
Comparison of fluorescence and photosynthetic characteristics. There was a general trend for fluorescence and the photon yield of non-photoinhibited control plants to decrease with increasing PFD during growth (Table 1). Fo, Fro, and F~ were typically 15-20% higher for leaves developed under a PFD of 700 ~tmol.m-2-s-1, than those leaves grown at 1300 g m o l ' m - Z ' s -1 while F~/Fm was only about 2% higher. Photon yield was about 6% higher in the 700 gmol. m - 2. s- 1_grown plants and this difference was significant (p=0.01). All of these characteristics were also higher in those leaves grown at 300 g m o l . m -2. s-1. ~0i and Fv/Fm were about 7% higher while Fv was nearly double that for the 700 g m o l . m - 2 , s-1-grown plants. As a consequence of these differences in Fo and F,,, the calculated initial relative value of the rate constant for N R D (KD) apparently increased with increasing PFD of growth. The light-saturated photosynthetic rate for those plants grown at 1300 gmol-m - 2 ' s -1 was 17.5 gmol ( C O z ) . m - 2 . s -1, about 22% higher than for the plants grown at 700 lamol'm-2, s-1, 1 4 . 3 ~ m o l ( C O z ) - m 2"s-1.
Daily changes in fluorescence characteristics and photon yield. For plants grown at 700 g m o l - m - 2 . s-1, there were no significant changes in any of the fluorescence characteristics through the day (Table 2). For the leaves grown at 1 300 g m o l . m - 2 -s-1, however, there was a significant decrease in F,,, Fv and Fv/Fm and a slight but non-significant increase in Fo. There was no evidence of a significant change in photon yield during the day for plants grown at either 700 or 1 300 ~tmol 9m - 2. s- 1.
Effect of PFD on photoinhibition. The time-courses of the decrease in the fluorescence emission and
Table 2. The effect of the PFD during growth on the photon yield (mol O2" (tool photons)- 1) and on the fluorescence characteristics for kiwifruit plants grown at two PFDs prior to and during exposure to the light (mean • SE, n = 14) Characteristic
Growth PFD (gmol " m - 2 .s -
1)
700
~Oi
Fo Fm F~ FvlF,.
1300
Before
After a
Before
After
0.0899• 3.1 • 18.0 • 14.9 • 0.827 •
0.0917-+0.0012 3.3 • 17.0 • 13.7 • 0.803 •
0.0750 • 0.0032 3.3 • 18.7 +1.2
0.0801• 3.9 • 14.3 • 10.3 • 0.725 •
" Measured as the mean value between 150 and 400 min exposure to the PFD
15.4
• 1.2
0.819 •
358
D.H. Greer and W.A. Laing: Effect of light on photoinhibition and recovery Table 3. The effect of the incident P F D at a temperature of 20 ~ C on the rate constant for photoinhibition, k(Fp), (mean _+ SE, n - 6) and on the extent o f photoinhibition, for intact kiwifruit leaves grown in three light regimes. The extent of photoinhibition was calculated from F~ at t = oo (F~) and expressed as 1 - F ~ / F ~ where Fi = Fv of the appropriate control leaves
100
so
80 .
40
Characteristic
F,
20
k(Fp)
16o
I
z00 Time
36o
I
500
(rain)
Growth P F D (gmolm-2.s-1)
300~
(• -3 9min a)
700 1300
Extent
300 a 700 1300
(%)
Exposure P F D (~mol, m - 2. S 1) 1000
-8.8+1.6 4.6_+2.3
1 500
2000
8.5 + 1.1
--
9.6_+2.4 5.5+1.8
10.3_+1.6 7.5_+1.4
-
92
--
59 50
62 64
81 74
100
a From Greer et al. (1988)
8o
B
60
40
9
o
9
20
o
10
~'
4
;
16o
2;o
36o
Time (rain)
~
4;o
soo
C~
0
x
2 0
6
16o
260
360
,60
500
Time (rain) Fig. 1A-C. The effect of an exposure at a P F D of either 1000 ( o - - o ) or 2 000 g m o l - m - 2 " S - 1 ( O - - O ) at 20 ~ C on the timecourse of (A) Fo and Fro, (B) Fv and (C) the photon yield ((00 for intact kiwifruit leaves grown at a P F D of 700 g m o l - m -2. s-1. Each line is the best fit to an equation described in the text and is the mean of three leaves
photon yield for leaves grown at a P F D of 700 ~tmol-m-2. s- 1 and exposed to a P F D of either 1 000 or 2000 g m o l . m - Z . s 1 are shown in Fig. 1. At both PFDs, Fo increased to the same extent whereas Fm decreased to a greater extent at the higher PFD. Fv therefore decreased exponentially
to approach a steady-state after about 400 rain with the initial decrease faster and ultimately greater at the higher PFD. Similar changes in photon yield were observed. The rate constant for photoinhibition, k(Fp), and the extent of photoinhibition for leaves exposed to a range of P F D s during the photoinhibitory treatment are shown in Table 3. For plants grown at both P F D s k(Fp) increased linearily with increasing exposure PFD, at least between 1 000 and 2 000 gmol. m - 2. s- 1. The severity of photoinhibition was also dependent on the P F D during the exposure for each treatment, increasing from about 50% at 1 000 ]amol -m - 2. s - 1 to 80% at 2 000 gmol. m - 2. s- 1. The susceptibility of kiwifruit leaves to photoinhibition was therefore dependent on the P F D during exposure. There was a higher initial rate of photoinhibition for the leaves grown at 700 compared with those at 1 300 ~mol. m - 2. s - 1 but there was apparently little difference in the extent of photoinhibition (Fig. 2). These results are similarly reflected in higher rate constants for photoinhibition (k(Fp)) at all photoinhibitory P F D s for those leaves grown at 700 g m o l - m - 2 , s-1 compared to those grown at 1 300 g m o l ' m -2" s - t (Table 3). However, k(Fp) for the 700 g m o l . m - 2 . s - < g r o w n plants was similar to that for plants grown in 300 ~mol- m - 2. s - t In contrast, the extent of photoinhibition in the plants from the two high-light regimes was similar at all P F D s but it increased markedly between plants grown in 300 g m o l - m - 2 . s - 1 and in either of the other two regimes. These results were confirmed by similar changes in the photon yield (not shown). Effect o f temperature on photoinhibition. The rate constant for photoinhibition, k(Fp), was only mar-
D.H. Greer and W.A. Laing: Effect of light on photoinhibition and recovery
A
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Fig. 2A, B. The effect of exposing intact kiwifruit leaves grown at a P F D of either 700 ( o - - o ) or 1300 gmol- m - 2. s - 1 ( e - - e ) to a P F D of 1 500 g m o l . m - 2 - s - ~ at 20 ~ C on the change in
(A) Fo and F,,, and (B) the change in F,. Each line is the mean of three leaves
Table 4. The effect of temperature during exposure to a P F D of 1 500 Ixmol-m -2-s -1 on the rate constant for photoinhibition, k(Fp) (mean_+ SE, n = 6) and on the extent of photoinhibition, for kiwifruit leaves grown in three light regimes Characteristic
k(Fp) ( x l 0 -3 9 min -1)
Extent (%)
Growth P F D (~tmolm - 2 . S -1) 300 ~ 700 1300
300a 700 1300
Temperature (~ C) 10 9.0_+0.6 9.0_+1.0 4.7_+2.0
98 84 94
20 8.5+_1.1 9.6_+2.4 5.5_+1.8
92 62 64
0
6
,6o
260
360
,60
500
T i m e (min) Fig. 3A, B. The time-course of recovery at a PFD of 20 gmolm 2.s-1 and at 25~ of (A) ( e - - e ) Fo, (e--i) Fm and ( o - - o ) F, and (B) photon yield (~00 of intact kiwifruit leaves grown at a PFD of 700 lamol'm-Z-s-1 after a 270-min exposure to a PFD of 2000 gmol-m 2.s-~ at 20~ Each line is the mean of three leaves 700 a n d 1 300 g m o l . m - 2 - s 1 T h e extent o f p h o toinhibition was m o r e strongly t e m p e r a t u r e - d e p e n d e n t with p h o t o i n h i b i t i o n m u c h m o r e severe at the lowest t e m p e r a t u r e . Plants g r o w n in 300 g m o l . m - 2. s - J were the m o s t susceptible to p h o t o i n h i b i tion at all t e m p e r a t u r e s .
25 10.5_+1.3 11.0_+3.0 _b
81 39 49c
From Greer et al. (1988) b Kinetics of photoinhibition were linear rather than exponential (F~= 85.4~0.084 t) c Calculated at t = 500 min ginally d e p e n d e n t o n t e m p e r a t u r e (Table 4). It was a g a i n evident that k(Fp) did not v a r y between plants g r o w n in 300 or at 700 ~ t m o l ' m -2" s - 1 b u t did decrease significantly b e t w e e n plants g r o w n at
Time-course and effect of temperature on recovery. The time-courses o f the recovery o f Fo, Fm a n d Fv a n d ~0~at 25 ~ C a n d 20 g m o l . m - 2. s - 1 following p h o t o i n h i b i t i o n o f leaves g r o w n at a P F D o f 700 ~ m o l . m - 2 . s - 1 are s h o w n in Fig. 3. R e c o v e r y was c o n f i r m e d b y an increase in Fm with a c o n c o m i t a n t decrease in Fo. T h e increase in F~ was closely p a r a l leled b y an increase in qh. R e c o v e r y o f Fv was strongly t e m p e r a t u r e - d e p e n d e n t with negligible r e c o v e r y at 1 0 ~ a n d an increasing rate f r o m 20 to 25 ~ C (Table 5). H o w ever, there was little or n o difference b e t w e e n plants g r o w n at the v a r i o u s light regimes in the kinetics o f r e c o v e r y at these t e m p e r a t u r e s . Similar results were o b s e r v e d for r e c o v e r y o f ~0~ (not shown).
360
D . H . G r e e r a n d W . A . L a i n g : Effect o f light on p h o t o i n h i b i t i o n a n d recovery
Table 5. T h e effect o f t e m p e r a t u r e at a P F D o f 20 p . m o l . m -z" s - 1 o n the rate c o n s t a n t for recovery f r o m p h o t o i n h i b i t i o n , k(F~), following p h o t o i n h i b i t i o n at 20 ~ C a n d a P F D o f 2000 p m o l . m 2. s - ~ o f kiwifruit leaves g r o w n in three light regimes (mean_+ SE, n = 6). T h e extent o f p h o t o i n h i b i t i o n was between 64 a n d 6 8 % o f the F~ o f control leaves in all cases Characteristic
k(Fr) ( x 1 0 - 3 " m i n -1)
Growth PFD (btmol . m 2 - s - 1 )
300 b 700 1300
Temperature a
(o C)
20
25
1.6__+0.2 2.0_+0.3 2.4_+0.3
4.7_+0.4 5.5_+0.5 4.0_+0.3
N o recovery occurred at 10 ~ C b F r o m G r e e r a n d L a i n g (1988)
100
8O o
a.~
9
9
Effect of PFD on recovery. In Fig. 4 is shown a comparison of the time-courses of recovery at P F D s ranging from 0 to 250 g m o l - m - 2 - s -1 of leaves grown at a P F D of 1300 ~tmol.m-Z.s -1 Recovery in the dark was exceedingly slow and only 21% complete in 450 min. Recovery occurred at both 20 and 250 Ixmol" m - 2 . s-1, but was slow at the higher PFD, and only 51% complete in 450 min compared with about 90% at 20 gmol . m - 2 9s- 1. Recovery at a P F D of 500 gmol. m - 2. s- 1 (not shown) was even slower than that at 250 gmol. m - 2. s- 1 and was only about 40% complete in the same time. Thus, recovery was maximal at the lowest light level. A similar response occurred in the 700 g m o l . m - Z . s - l - g r o w n plants (not shown). The rate constant for recovery, (k(Fr)), as a function of the P F D is shown in Fig. 5. This shows that k(Fr) increased dramatically, about seven-fold, from the dark with the addition of weak light but then declined with further increases in PFD. This would indicate that recovery at the P F D s at which the plants were grown would not be much higher than in the dark.
4O
Discussion
;
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Time (rain) Fig. 4. T h e t i m e - c o u r s e o f recovery o f Fv at 2 5 ~ a n d at a P F D o f 0 ( n - - n ) , 20 ( o - - o ) or 250 b t m o l - m - 2 - s i ( e - - e ) for intact kiwifruit leaves g r o w n at a P F D o f 1300 g m o l . m 2. s -1 following a 2 7 0 - m i n e x p o s u r e to a P F D o f 2000 g m o l . m 2. s - a at 20 ~ C. E a c h line is the m e a n o f three leaves
_t.,, 4 I r ~
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Fv, k(F~), at 2 5 ~ as a f u n c t i o n o f P F D d u r i n g the recovery period for intact kiwifruit leaves g r o w n at a P F D o f 1 300 g m o l . m - 2 s - 1 following p h o t o i n h i b i t i o n at a P F D o f 2000 g m o l . m - 2 - s - x Fig. 5. T h e rate c o n s t a n t for recovery o f
When kiwifruit leaves were grown in high-light there were marked changes in fluorescence emission at 77K, notably F~/Fm, compared with those leaves grown at low-light. Fluorescence changes in a given leaf are indicative of a redistribution of energy within PSII, with the quenching of fluorescence reflecting an increase in the rate constant for non-radiative energy dissipation, (Ko) while a rise in Fo reflects a decrease in the rate constant for photochemistry in PSII, (Kv) (0gren and Oquist 1984, Bj6rkman 1987b). Assuming that Ke was constant between leaves from the three light regimes, it appears that the relative value for KD increased with increasing P F D of growth 9 This might indicate there was an increased capacity to protect the photosynthetic apparatus from highlight stress when grown at high-light. This however, needs biochemical confirmation of, for example, differences in zeaxanthin formation (Demmig et al. 1987). Correlated with the changes in fluorescence were changes in the photon yield9 This indicates the kiwifruit leaves have some propensity to become photoinhibited during the growth conditions, the more so the higher the PFD. This chronic photoinhibition was, however, relatively minor since a four-fold increase in P F D resulted in no more than an 11% decrease in photon yield. In spite of this, the leaves also exhibited a capacity to accli-
D.H. Greer and W.A. Laing: Effect of light on photoinhibition and recovery
mate to the higher PFDs by increasing the maximum photosynthetic rates (Laing 1985; this study). Similar divergent responses to an increase in growth PFD by this shade-tolerant canopy vine (Ferguson 1988) have been reported in other shade-tolerant species (see Anderson and Osmond 1987). No additional diurnal changes in fluorescence or photon yield were detected for kiwifruit leaves grown and exposed to 700 gmol.m-2-s -1. For those plants grown at 1 300 btmol-m-2, s-1, however, there was a marked diurnal reduction in Fro, F~ and Fv/Fm, and a slight increase in Fo. Thus the chronic photoinhibition was exacerbated through the day although only to a minor extent since there were no detectable changes in photon yield. Ben et al. (1987) also observed a decrease in F~/F,, in Helianthus annuus leaves exposed to the sun during the day, again without concomitant changes in the photon yield. Bj6rkman and Powles (1984) similarly observed a diurnal reduction in F,, when Nerium oleander was exposed to the full midsummer PFD. These fluorescence changes probably reflect changes in Kp and Ko in the upper surface of leaves only, since they apparently have little effect on the well-being of the leaf as a whole. Severe photoinhibitory damage to PSII resulted when kiwifruit leaves from all light regimes were exposed to PFDs substantially above that in which they were grown. This was manifested by a decrease in the photon yield and by a concomitant decrease in F,, and an increase in Fo, indicating that direct damage at or near the reaction centres was occurring (CMand et al. 1986). The extent of the photoinhibition was highly dependent on temperature, increasing in severity with decreasing temperatures but the kinetics of the process were essentially independent of temperature. Similar effects of temperature were observed in shade-grown kiwifruit leaves (Greer et al. 1988) indicating that the temperature-dependency of photoinhibition is not affected by the PFD during growth as {)quist et al. (1987) had proposed. The extent of photoinhibition also depended on the PFD during the highqight exposure, increasing as the PFD increased. Similar results have been observed in lowlight-grown Phaseolus vulgar& (Powles and Thorne 1981), Nerium oleander (Powles and Bj6rkman 1982) and Lemna gibba ({)gren et al. 1984). The exacerbation of photoinhibition by increasing PFD is expected since the absorption of excitation energy would increasingly exceed its rate of utilisation by photochemistry and rate of non-radiative dissipation (Bj6rkman 1987 a). In contrast to temperature, the kinetics of pho-
361
toinhibition depended on the PFD during the highlight exposure, with the rate constant for photoinhibition, k(Fp) increasing linearly with increasing PFD, at least between 1000 and 2 000 btmol-m-2 s- 1 for plants grown at both 700 and 1 300 gmolm - 2. s- 1. A similar linear increase in the rate constant for photoinhibition was observed in shadegrown Monstera deliciosa by Demmig and Bj6rkman (1987). These studies on higher plants are thus consistent with the effect of PFD on photoinhibition in unicellular algae (Kok 1956; Neale 1987). It was evident, however, that the rate constant for photoinhibition in kiwifruit leaves grown at 1 300 gmol-l-n -2. S-1 was lower than in plants grown at 700 Ixmol-m 2"s-1 (Fig. 2, Table 3). k(Fp) was from 1.3- to 1.9-fold higher in those plants grown at 700 g m o l ' m - 2 , s-1 and this relative difference was maintained over all temperatures and exposure PFDs. The extent of photoinhibition was, however, not different after long exposures, indicating that the leaves were about equally susceptible. Leaves grown at 300 btmol" m - 2 ' s - 1 , by contrast, were much more susceptible than leaves grown at the two higher PFDs though there was little difference in k(Fp) from that in the leaves from 700 gmol. m-2. s-1. Seeman et al. (1987) also observed a 1.5- to 2-fold difference in the rate constant for Phaseolus vulgaris leaves grown at about 550 and 1 100 btmol- m - 2. s - 1 whereas Powles and Critchley (1981) noted that the susceptibility to photoinhibition in the same species increased significantly when the PFD during growth decreased from 25 to 6% of full sunlight. The response to the growth PFD was therefore similar to that observed here. Demmig and Bj6rkman (1987) observed that the increased susceptibility to photoinhibition between high- and low-light-grown plants persisted even when dissipation of energy was prevented by inhibiting photosynthesis. Additionally, recovery from photoinhibition was faster in high- compared with low-light-grown plants. A similar result was observed in Lemna gibba by Skogen et al. (1986). From their results, Demmig and Bj6rkman (1987) proposed that differences in capacity for recovery contributed to the differences in susceptibility to photoinhibition between sun and shade leaves. In kiwifruit leaves, however, the rates of post-photoinhibition recovery were similar for leaves grown over a four-fold range in PFD (Table 5), in spite of inherent differences in both the kinetics and extent of photoinhibition. Greer and Laing (1988) have shown previously in kiwifruit leaves that the rate constant for recovery, k(Fr), was highly dependent on the extent of photoinhibition. This has also been recognised by Demmig and Bj6rkman
362
D.H. Greer and W.A. Laing: Effect of light on photoinhibition and recovery
(1987). In their comparison of the differences in recovery rate constants between sun and shade leaves of Gossypium and Hedera, there were great differences in the initial level of photoinhibition. Differences in recovery, therefore, could have reflected differences in the extent of photoinhibition. It is axiomatic that it is the recovery occurring during photoinhibition rather than following photoinhibition that would be expected to affect the susceptibility to photoinhibition. However, the recovery of kiwifruit leaves following photoinhibition was substantially inhibited by increases in the PFD between 20 and 500 g m o l - m - 2 " s - 1 . Results consistent with this were also observed by Greer et al. (1986) with Phaseolus vulgaris and by Skogen et al. (1986) with Lemna gibba. By extrapolating the effect of PFD on the recovery rate constant (Fig. 5) to those PFDs where photoinhibition was determined, it would appear that recovery occurring during the high-light exposure was very slow. Thus it seems unlikely that recovery occurring during photoinhibition could account for the differences in susceptibility to photoinhibition between sun and shade leaves of kiwifruit. Differences in photosynthetic capacity between these kiwifruit plants, instead, are more likely to conform with high-light-grown leaves being less susceptible to photoinhibition (Bj6rkman et al. 1972; Anderson and Osmond 1987). In summary, the results of this study have confirmed that kiwifruit leaves are susceptible to photoinhibition. Although growth at high PFDs tends to ameliorate the susceptibility to photoinhibition, a chronic but low level of photoinhibition occurs in response to the high light. However, the incident PFD and the temperature are the most important factors, affecting both photoinhibition and recovery, and the balance between these two processes determines the extent of damage to the primary photochemical apparatus. References Anderson, J.M., Osmond, C.B. (1987) Sun-shade responses: compromises between acclimation and photoinhibition. In: Topics in photosynthesis, vol. 9: Photoinhibition, p 1 38, Kyle, D.J., Osmond, C.B., Arntzen, C.J., eds. Elsevier, Amsterdam Ben, G-Y., Osmond, C.B., Sharkey, T.D. (1987) Comparisons of photosynthetic responses of Xanthium strumarium and Helianthus annuus to chronic and acute water stress in sun and shade. Plant Physiol. 84, 476-482 Bj6rkman, O. (1987a) High irradiance stress in higher plants and interaction with other stress factors. Progr. Photosynth. Res. 4, 11-18 Bj6rkman, O. (1987b) Low-temperature chlorophyll fluorescence in leaves and its relationship to photon yield of photo-
synthesis 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., Boardman, N.K., Anderson, J.M., Thorne, S.W., Goodchild, D.J., Pylotis, N.Z. (1972) Effect of light intensity during growth of Atriplex patula on the capacity of photosynthetic reactions, chloroplast components and structure. Carnegie Inst. Wash. Yearb. 71, 115135 Bj6rkman, O., Demmig, B. (1987) Photon yield of O2 evolution and chlorophyll fluorescence characteristics at 77K among vascular plants of diverse origins. Planta 170, 48%504 Bj6rkman, O., Powles, S.B. (1984) Inhibition of photosynthetic reactions under water stress: interaction with light level. Planta 161,490-504 Brooking, I.R. (1976) Soilless potting media for controlled-environment facilities. N.Z.J. Exp. Agric. 4, 203-208 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 (77K) and photon yield of Oz evolution in leaves of higher plants. Planta 171, 171-184 Demmig, B., Winter, K., Krtiger, 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 Ferguson, A.R. (1988) Botanical Nomenclature: Actinidia chinensis, A ctinidia deliciosa, and Actinidia setosa. In: Kiwifruit: Science and Management, in press, Warrington, I.J., Weston, G.C., eds. N.Z. Soc. for Horticultural Science in conjunction with Ray Richards Publisher, Auckland, N.Z. 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., Laing, W.A. (1988) Photoinhibition of photosynthesis in intact kiwifruit (Actinidia delieiosa) leaves: Recovery and its dependence on temperature. Planta 174, 159-165 Greer, D.H., Laing, W.A., Kipnis, T. (1988) Photoinhibition of photosynthesis in intact kiwifruit (Actinidia delieiosa) leaves: Effect of temperature. Planta 174, 152-158 Kok, B. (1956) On the inhibition of photosynthesis by intense light. Biochim. Biophys. Acta 21,234-244 Laing, W.A. (1985) Temperature and light response curves for photosynthesis in kiwifruit (Aetinidia chinensis) cv. Hayward. N.Z.J. Agric. Res. 28, 117-124 Neale, P.J. (1987) Algal photoinhibition and photosynthesis in the aquatic environment. In: Topics in photosynthesis, vol. 9: Photoinhibition, pp. 3%68, Kyle, D.J., Osmond, C.B., Arntzen, C.J., eds. Elsevier, Amsterdam Ogren, E., 13quist, G. (1984) Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. III. Chlorophyll fluorescence at 77K. Physiol. Plant. 62, 193-200 ()gren, E., Oquist, G., Hallgren, J.-E. (1984) Photoinhibition of photosynthesis in Lemna gibba as induced by the interaction between light and temperature. I. Photosynthesis in vivo. Physiol. Plant. 62, 181-186 Oquist, G., Greer, D.H., Ogren, E. (1987) Light stress at low temperature. In: Topics in photosynthesis, vol. 9: Photoinhibition, pp. 6%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, 15~44 Powles, S.B., Bj6rkman, O. (1982) Photoinhibition of photo-
D.H. Greer and W.A. Laing: Effect of light on photoinhibition and recovery synthesis: 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 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 mierosora. Planta 152, 471 477 Seeman, J.R., Sharkey, T.D., Wang, J., Osmond, C.B. (1987)
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Environmental effects on photosynthesis, nitrogen-use efficiency, and metabolite pools in leaves of sun and shade plants. Plant Physiol. 84, 796-802 Skogen, D., Chaturvedi, R., Weidemann, F., Nilsen, S. (1986) Photoinhibition of photosynthesis: Effect of light quality and quantity on recovery from photoinhibition in Lemna gibba. J. Plant Physiol. 126, 195-205
Received 7 December 1987; accepted 25 March 1988
