Planta (1990)182:161-168

P l a n t a 9 Springer-Verlag1990

The effects of photoinhibition on the photosynthetic light-response curve of green plant cells (Chlamydomonas reinhardtil) Jerry W. Leverenz, Stefan Falk, Carl-Magnus Pilstr~m, and G~ran Samuelsson Department of Plant Physiology, University of Umefi, S-901 87 Umefi, Sweden Received 2 August 1989; accepted 18 April 1990

Abstract. The photosynthetic response to light can be accurately defined in terms of (1) the initial slope (quantum yield); (2) the asymptote (light-saturated rate); (3) the convexity (rate of bending); and (4) the intercept (dark respiration). The effects of photoinhibition [which damages the reaction centre o f photosystem II (PSII)] on these four parameters were measured in optically thin cultures of green plant cells (Chlamydomonas reinhardtii). The convexity of the light-response curve decreased steadily from a value of 0.98 (indicating a sharply bending response) to zero (indicating Michaelis-Menten kinetics) in response to increasing photoinhibition. Photoinhibifion was quantified from the quantum yield o f inhibited cells relative to that o f control cells. The quantum yield was estimated by applying linear regression to low-light data or by fitting a non-rectangular hyperbola. Assuming the initial slope is linear allowed comparison with earlier work. However, as the convexity was lowered this assumption resulted in a significant underestimate of the true quantum yield. Thus, the apparent level of photoinhibition required for a zero convexity and the initial decrease in light-saturated photosynthesis depended upon how the quantum yield was estimated. If the initial slope o f the light response was assumed to be linear the critical level of inhibition was 60%. If the linear assumption was not made, the critical level was 40%. At the level of inhibition where the convexity reached zero, the light-saturated rate of photosynthesis also began to decrease, indicating that this level of inhibition caused photosynthesis to be limited at all light intensities by the rate of PSII electron transport. At this level of inhibition the Fm-F i signal (where Fm is maximal chlorophyll fluorescence and Fi is intermedi-

Abbreviations and symbols: Chl = chlorophyll content; DCMU = 3(3,4-dichlorophenyl)-l, 1-dimethylurea; Fo, Fi, Fm= initial, intermediate, and maximal Chl fluorescence of dark adapted cells; P = rate of net photosynthesis per unit chlorophyll (gmol-(mg Chl)-1. s-1); PSII = photosystem II; PQ = plastoquinone ; q~= initial slope to the light-response curve; 0=convexity (rate of bending) of the lightresponse curve of photosynthesis; Q=photosynthetically active photon flux density (400-700 nm, ~tmol.m-2. S-1)

ate chlorophyll fluorescence of dark adapted cells; Briantais etal. 1988) from the fluorescence induction curve was zero and the F r F o signal (where Fo is initial chlorophyll fluorescence of dark adapted cells) was 30% of the control, indicating dramatic reduction or complete elimination of one type o f PSII. These data do not contradict published mathematical models showing that the ratio of the maximum speed of electron transport in PSII relative to the maximum speed o f plastoquinone electron transport can determine the convexity of the photosynthetic response to light.

Key words: Chlamydomonas (photosynthesis) - Chlorophyta - Light-response curve (convexity) - Photoinhibition - Photosynthesis (light response curve)

Introduction Modeling the photosynthetic response to photon flux density (Q) with the following quadratic equation has become more and more c o m m o n because of the good empirical agreement between the model and experimental measurements (Prioul and Chartier 1977; Leverenz and Jarvis 1979; Marshall and Biscoe 1980; Terashima and Saeki 1985; Leverenz 1987, 1988; Zhang 1988; Johnson et al. 1989): oP 2 - ( ~ Q + Pma0P + r

= 0.0

Eq. (1)

The parameters of this equation are, the initial slope (~), the convexity (curvature, or rate of bending, 0) and the asymptote or light-saturated rate o f photosynthesis (Pro,x). A parameter for respiration (R) is usually added to give a correct intercept on the Y axis. In terms of describing the fundamental efficiency o f the photosynthetic apparatus, 0 is as important as ~. For example, when 0 for the elementary photosynthetic system is low the light-response curve immediately bends away from 9 at low Q and the quantum efficiency of

162

J.W. Leverenz et al. : Photoinhibition and the photosynthetic light-response curve 350 300 250

.E~20C =, 15C IX." 10C

5C

~o 0130

I

500

I

1000

80

15~00

2000

Q, )Jmot- m-2.s-1

Fig. l. Modelled photosynthetic response to light (using Eq. 1) where the initial slope is 1.835, the asymptote is 350 lxmol.(mg Chl) X.s 1, and the convexity is 0.976 (top curve), 0.500 (middle curve) or 0.000 (lower curve). Insert shows the response over a lower range of photon flux density for convexities of 0.976 (top curve) and 0.000 (bottom curve)

Table 1. Maximum convexities (0) for the light-response curve of cells and leaves: only steady-state measurements of photosynthesis are considered. The 0 for gymnosperms is from Leverenz (1988), the value for C3 angiosperm leaves is from Prioul and Chartier (1977) and for isolated C3 cells from Terashima and Saeki (1985). The original light curve for Anacystis nidulans is from Samuelson et al. (1987) and for C4 angiosperm leaves are from Ludlow and Wilson (1971) and Robichaux and Pearcy (1980) Species or plant type

Maximum 0

Procaryote :

Anacystis nidulans

0.96

Eucaryotes:

Chlamydomonas reinhardtii Conifer leaves C3 angiosperm leaves C3 angiosperm cells C4 angiosperm leaves

0.98 0.97 0.97-0.98 0.965 0.97

photosynthesis decreases immediately and continuously as light is increased (Fig. 1). In addition, when 0 is low, Pmax is not reached even at full sunlight (2000 ~tmol. m-Z-s-1, Fig. 1). Thus a low 0 implies a reduced photosynthetic efficiency beginning at very low light and continuing on up to full sunlight. For individual photosynthetic cells to operate efficiently they must achieve both a high maximal quantum yield (high ~) and a high convexity (0 approaching 1.00, Fig. 1). Experimental evidence indicates that 0 of the unstressed C3-plant cell is typically close to 0.97 (Terashima and Saeki 1985) and is independent of temperature between 5 and 32~ C and C02 concentration between 35 and 200 Pa (Leverenz 1987, 1988). It was also shown that lower values of 0 for unstressed leaves are probably

the result of mutual shading, whereas the maximum 0 found for leaves is probably very close to that for individual, unstressed cells (Leverenz 1987, 1988). This information allows the analysis of published light-response curves for both cells and leaves to estimate 0 for cells. Such an analysis indicates that for many, oxygen-evolving, photosynthetic cells 0 may be between 0.97 and 0.98 (Table 1). However, at present we know of no systematic experiments on the effects of environmental stress on 0. In this paper we examine the effects of light stress (photoinhibition) on 0 using optically thin samples of unicellular algae to avoid complications as a result of mutual shading. Convexities above 0.90 have been modelled as the result of the diffusion resistance to CO2 being 10-50 times the "carboxylation resistance" (Prioul and Chartier 1977). However, experimental evidence does not support this model (Oya and Laisk 1976; Evans et al. 1986; Leverenz 1988; Zvalinskii and Litvin 1988). A sharp bending of the light-response curve has also been modelled to be the result of efficient sharing of energy between a number of connected photosystem II (PSII) reaction centres (Herron and Mauzerall 1972; Laisk and Niilsk 1981; Zvalinskii and Litvin 1984). Measurements typicall~ indicate that about 60% of the excitons arriving at a closed centre are transferred to a neighboring open centre (Briantais et al. 1988). By itself a transfer efficiency of 0.6 appears to be too small to give a 0 near 0.97 (Laisk and Niilsk 1981 ; Zvalinskii and Litvin 1984). A 0 of 0.97-0.98 can also be obtained by assuming the time it takes the PSII reaction centre to re-open after capturing an exciton (so it can capture another exciton) is 30-50 times faster than the turnover time of the plastoquinone (PQ) pool (Farquhar and Wong 1984) or other reactions outside the PSII complex (Zvalinskii and Litvin 1984, 1988). Such high relative speeds of reactions in the PSII complex have been confirmed in experiments (Forti 1987; Mathis and Rutherford 1987). Moreover, the ratio of PSII complexes to cytochrome b6f complexes is 0.9 or larger in most plants (Neale and Melis 1986; Ort and Baker 1989). Combined with the relatively high speed of PSII electron transport this could create the observed high 0 and make lightsaturated photosynthesis independent of substantial changes in PSII electron transport. The speed of reactions within the PSII complex remain high at temperatures down to about - 3 0 ~ (Mathis and Rutherford 1987). This is in agreement with the empirical evidence that 0 is independent of temperature between 5 and 32~ C (Leverenz 1988). Intuitively one can imagine that a high speed of turnover of the PSII reaction-centre complexes assures that over a significant range of photon flux densities the reaction centres can process excitons faster than they arrive and thus are open for nearly every newly arriving exciton. This gives the long quasi-linear initial slope to the light-response curve (Bj6rkman and Demmig 1987). However, at a critical photon flux density the PQ pool can no longer be reoxidized fast enough to keep up with the potential flow of electrons from Qa and the reaction centres can not re-open because of the lack of the Qb

J.W. Leverenz et al. : Photoinhibition and the photosynthetic light-response curve a c c e p t o r . A s a result a significant f r a c t i o n o f the excitons arrive w h e n the r e a c t i o n centre is closed, e x c i t o n s are lost to c o m p e t i n g p r o c e s s e s a n d a m e a s u r a b l e r e d u c t i o n in the slope o f the p h o t o s y n t h e t i c r e s p o n s e to light occurs. As a h i g h e r a n d h i g h e r f r a c t i o n o f the e x c i t o n s arrive w h e n the r e a c t i o n centre is c l o s e d the light-res p o n s e curve b e n d s f u r t h e r a n d f u r t h e r a w a y f r o m the l i n e a r initial slope until all a d d i t i o n a l excitons hit closed r e a c t i o n c e n t r e s a n d true light s a t u r a t i o n is achieved. T h e g r e a t e r the difference in speed b e t w e e n the t u r n o v e r o f P S I I relative to o t h e r r e a c t i o n s in the p h o t o s y n t h e t i c chain, the s h a r p e r is the t r a n s i t i o n b e t w e e n the initial slope a n d l i g h t - s a t u r a t e d p h o t o s y n t h e s i s ( F a r q u h a r a n d W o n g 1984; Z v a l i n s k i i a n d L i t v i n 1984, 1988). A test o f the a c c u r a c y o f the m o d e l s o f F a r q u h a r a n d W o n g (1984) a n d Z v a l i n s k i i a n d L i t v i n (1984, 1988) w o u l d be to use successively h i g h e r degrees o f p h o t o i n h i b i t i o n to cause a r e d u c t i o n in either the n u m b e r o r speed o f P S I I r e a c t i o n centres. T h e s e m o d e l s suggest t h a t as the p h o t o s y n t h e t i c system is f o r c e d to r u n with either fewer o r slower P S I I centres, 0 s h o u l d decrease. H o w ever, Pm,x s h o u l d n o t be affected b y p h o t o i n h i b i t i o n o f P S I I r e a c t i o n centres until p h o t o s y n t h e s i s is limited b y the c a p a c i t y for P S I I e l e c t r o n t r a n s p o r t at all light levels. A t this p o i n t the l i g h t - r e s p o n s e curve f o r p h o t o s y n t h e s i s s h o u l d be d e f i n e d b y the l i g h t - r e s p o n s e curve for P S I I e l e c t r o n t r a n s p o r t . Thus, w h e r e Pmax decreases 0 s h o u l d be zero, since P S I I e l e c t r o n t r a n s p o r t h a s M i c h a e l i s M e n t e n kinetics ( 0 = zero) ( O q u i s t et al. 1982; M~ienpfifi et al. 1987; A l b e r t s s o n a n d Yu 1988). P h o t o s y s t e m II r e a c t i o n centres a r e h e t e r o g e n e o u s (Black et al. 1986; Mfienp~i~i et al. 1987; B r i a n t a i s et al. 1988). O n e a s p e c t o f this h e t e r o g e n e i t y is t h a t 3 0 - 4 0 % o f the P S I I centres r e - o p e n m a n y times m o r e slowly t h a n the faster P S I I centres (ibid.). These slow centres a r e believed to cause the rise in c h l o r o p h y l l (Chl) fluorescence f r o m Fo to F1 in the curve for c h l o r o p h y l l fluorescence i n d u c t i o n ( B r i a n t a i s et al. 1988). T h e fastest P S I I centres r e - o p e n after c a p t u r i n g a n e x c i t o n with a halftime o f 0.4 m s ( E r i c k s o n et al. 1989 for e x a m p l e ) . T h e rise f r o m Fi to Fm in the f l u o r e s c e n c e - i n d u c t i o n curve is believed to arise f r o m fast centres ( B r i a n t a i s et al. 1988). It h a s b e e n r e p o r t e d t h a t fast P S I I r e a c t i o n centres are m o r e sensitive to p h o t o i n h i b i t i o n t h a n slow P S I I r e a c t i o n centres (M~ienpfi~i et al. 1987; B r i a n t a i s et al. 1988). I n this p a p e r we s h o w t h a t 0 is s t r o n g l y r e d u c e d b y p h o t o i n h i b i t i o n f r o m a v a l u e o f 0.98 in u n i n h i b i t e d cells to a v a l u e o f zero at 40 (or 6 0 ) % i n h i b i t i o n . We also s h o w t h a t the initial d e c r e a s e in Pm.x Occurs n e a r the p o i n t w h e r e 0 h a s d e c r e a s e d to zero. A t this level o f i n h i b i t i o n the fluorescence signal w h i c h is a s s o c i a t e d w i t h the fast P S I I centres (Fm-Fi) w a s n o l o n g e r m e a s u r able a n d o n l y 3 0 % o f the signal f r o m the slow c e n t r e s ( F i F o ) was left.

Material and methods Cultivation of cells. Chlamydomonas reinhardtii cells (wild-type strain c 137 mt § obtained from Dr. D.D. Kaska, see Kaska et al.

163

1987) were grown photoautotrophically at 27~ in axenic constant-density cultures (ACC 400; Techtum Instruments, Ume~i, Sweden) with air bubbling through the cultures. A set of fluorescent tubes (Type TL 20W/55; Phillips F6rs~iljning, Stockholm, Sweden) was used to give a Q of 100-125 I.tmol.m-2.s -1 at the centre of the culture tubes. The growth medium was as described by Lidholm et al. (1987) and Falk et al. (1990); however, the rate of bubbling of air in the culture media was higher than that used previously. In addition, algal density was kept slightly lower, between 2.5 and 3 lag Chl-ml-1. This resulted in cells with more consistent and higher photosynthetic capacities. Photoinhibition treatment. Subsamples from the cell cultures were photoinhibited in glass tubes (1.5 cm diameter) immersed in a temperature-controlled water bath. The growth medium was supplemented with 1 mM NaHCO3 at the beginning of the photoinhibitory treatment. Air was bubbled through the media to provide stirring. Metal-halogen lamps (Type HQI-TS 400W; Osram, Stockholm, Sweden), mounted in reflectors facing one glass wall of the water bath, were used to illuminate the samples. The desired photoinhibitory light was obtained by altering the distance between the light and the water bath. The photoinhibitory treatment was performed for 60 min at 27~ at Q of 1050, 1400, 1600, 1800, 2050, and 2200 lamol, m - 2 . s - 1 . To calculate the level of inhibition the initial slope for oxygen evolution was estimated by linear regression with Q and compared with that for uninhibited control cells. A second method to estimate the level of inhibition was to use Eq. (1) to estimate the maximum quantum yield. This second method, in theory, eliminates changes in 0 as a factor affecting the estimate of the maximum quantum yield of photosynthesis (Leverenz 1987). To assure the same degree of photoinhibition for all measured data points in each individual light-response curve, repair was prevented by the addition of chloramphenicol (Lidholm et al. 1987) to the oxygen-electrode cell for both control and inhibited cells. Measurement of photosynthesis. Rates of photosynthesis and respiration were measured using a temperature-controlled Clark-type oxygen electrode (Hansatech, King's Lynn, Norfolk, UK). Samples were flushed with N2 before measurement to reduce the oxygen tension in the electrode to 15 _+2% and temperature was kept at 27 + 1~ C. Prior to the measurements, 4 mM NaHCO3 was added to each sample and chloramphenicol was added to a concentration of 100 lag-ml- 1 from a stock solution of 33 mg-ml- 1 ethanol. The rate of oxygen evolution or uptake was recorded on a strip-chart recorder (Sekonic, Tokyo, Japan; SS-250F) and was calculated from the slope of the curve as soon as it stabilized. The maximum slope within 15 min was used. If the slope decreased steadily, however, indicating the onset of additional photoinhibition, the data were not used. This additional photoinhibition occurred during the measurements of 02 evolution at higher photon flux densities, limiting the maximum light that could be used to measure photosynthesis. This was more noticeable in the more highly inhibited samples. Chlorophyll. Following a measurement of photosynthesis each sample of cells was frozen. After a number of samples were obtained they were each centrifuged down and resuspended in 85% acetone. Chlorophyll content was estimated using the equations of Arnon (1949). Absorptance was measured using a Shimadzu " M P S " spectrophotometer with a half-band width of 2 nm (Shimadzu, Kyoto, Japan). Measurement of PSIIfluorescence. Fluorescence was measured at room temperature. The fluorescence signal was generated with a Plant Stress Meter (BioMonitor; S.C.I., Ume/t, Sweden) with an actinic light of 400 lamol.m-2-s-1. This signal was recorded and stored on a computer hard disk using an oscilloscope card (PCSCOPE T12840; Intelligent Messtechnik, Backning, F R G ) w i t h a sampling frequency of 10 kHz. The stored fluorescence signal was analysed for values of Fr,, Fi, and Fo; Fo was defined as the

164

J.W. Leverenz et al. : Photoinhibition and the photosynthetic light-response curve

signal-height 4.0 ms after the beginning of shutter-opening (fullopen time was 3 ms). The sample was dark-adapted for 10 min before measurement with chloramphenicol (100 ~tg.m1-1) and NaHCO3 (4 mM) added. Values of Fo, Fi, and Fm were used to calculate Fm-Fi and Fi-Fo. Results are presented as the mean value from four measurements at each level of inhibition. To check if the actinic light was of sufficient strength to give a true Fro, fluorescence was measured from 3-(3,4-dichlorphenyl)-l,l-dimethylurea (DCMU)-treated samples (10 -5 M) at the same occasion as the normal room-temperature fluorescence values. Modelling the light-response curve. The light-response curve was modelled as described previously (Leverenz 1987, 1988) with the alterations noted below. The symbol 0 is used for convexity in this paper which is equivalent to M used previously (Leverenz 1987, 1988). The model was fitted using a SPSS least-squares statistical package to all data in each data set from dark to the point where incipient photoinhibition was detected, unless a definite Kok effect (Sharp et al. 1984) was observed. Because of the formulation of the equation in the computer program the value of 0 could not be allowed to go to zero. Instead it was allowed to go to 1.10 -1~ When 0 was below 1 "10 -9 it was found that fitting the data to a rectangular hyperbola (0=zero) resulted in a smaller root mean squared of the residuals (more precision) and a more even distribution of modelled minus observed values with respect to Q (an unbiased fit). In contrast to previous experience with light-response curves of conifer foliage (Leverenz 1987, 1988), altering the estimated initial slope (within limits dictated by the noisy data) had no significant effect on 0 but was reflected in an altered estimated rate of dark respiration. This gave us confidence that the values of 0 are reliable.

Table 3. Top: The effect of photoinhibition at different photon flux densities for 1 h at 27~ C on modelled parameters of the photosynthetic light-response curve. Data from Falk et al. (1990) are indicated by a. Bottom: ratios of the quantum yields estimated from a linear model to those estimated from Eq. 1 Inhibition light (~tmol-m-2.s -1)

Parameters

Sample size

4~

0

Pmax

R

0a 1600 a 0 1050 1400 1800 2050 2200

1.049 0.687 1.835 1.642 1.428 1.125 0.682 0.188

0.976 0.198 0.978 0.802 0.263 0.000 0.000 0.000

209 216 300 404 278 197 77 35

3 28 28 54 79 51 51 52

10 10 15 15 13 13 13 15

0

Linear model/non-linear model

O.976a 0.978 0.802 0.263 0.198" 0.000 0.000 0.000

1.00 1.02 0.95 0.86 0.77 0.67 0.73 0.70

Results Because we c o u l d n o t m e a s u r e true l i g h t - s a t u r a t e d photosynthesis for m o s t levels o f p h o t o i n h i b i t i o n , we wished to k n o w h o w m u c h o f a light-response curve was req u i r e d to o b t a i n a reliable estimate o f 0. A low 0 implies that the light curve is b e n d i n g even at very low p h o t o n flux densities (Fig. 1). T h u s even low-light d a t a should c o n t a i n i n f o r m a t i o n o n 0 a n d it s h o u l d n o t be necessary to sample the entire light-response curve to o b t a i n a n a c c u r a t e ( u n b i a s e d ) estimate o f 0. However, noise in the d a t a m a y result in u n a c c e p t a b l y low precision in the estimate o f 0. To test this the initial d a t a sets for c o n t r o l a n d i n h i b i t e d cells were r e d u c e d to a smaller a n d smaller r a n g e in Q by s e q u e n t i a l l y e l i m i n a t i n g the m e a s u r e m e n t at the highest Q. T h e m o d e l was t h e n re-fitted to each Table 2. The effect of fitting Eq. (1) to smaller and smaller subsampies of complete data sets (Fig. 3, left) on the estimate of convexity (0). Shown are the maximum photon flux densities (Q) of each subsample, the sample size of each subsample, and the estimated convexity (0). Data from uninhibited cells and cells inhibited at 1400 ~tmol.m-2-s -I for 1 h (Fig. 2, left) Maximum Q (~tmol.m- 2. s - 1)

1454 1317 1057 618 440 288 209

Sample size

15 14 13 12 11 10 9

Estimated 0 Control

Inhibited

0.978 0.979 0.988 0.975 0.996 1.000 0.933

---0.263 0.513 0.553 0.000

o f these s u b s a m p l e s o f the entire d a t a set. This analysis indicated that it was only necessary to m e a s u r e the lightresponse curve u p to a Q o f 209 ktmol, m - 2. s - 1 to k n o w that 0 for the c o n t r o l cells was above 0.93 a n d that 0 for the inhibited cells was below 0.55 (Table 2). T h u s while s u b s t a n t i a l v a r i a t i o n s in estimated 0 occurred, p r o b a b l y as a result o f noise in the data, a complete light response curve was n o t necessary to show that 0 was d r a m a t i c a l l y decreased in i n h i b i t e d cells (Table 2). As a result of this test, we increased the n u m b e r o f samples below 600 I x m o l . m - z. s - 1 in s u b s e q u e n t experim e n t s (Table 3). This increased the precision o f the estimates o f 0 ( D r a p e r a n d Smith 1966). Sample light-response curves in Fig. 2 show the r a n g e in q u a l i t y o f d a t a a n d the fit o f the m o d e l l e d curve. Each d a t u m p o i n t represents a m e a n o f three to five m e a s u r e m e n t s . To further illustrate the c h a n g e in 0 we show in Fig. 3 the best fit o f the m o d e l w h e n 0 is set to zero for c o n t r o l cells or to 0.976 for i n h i b i t e d cells. C o m p a r i s o n o f o u r c o n t r o l light curve (Fig. 2) with the c o n t r o l light curve m e a s u r e d by Falk et al. (1990) (Fig. 3) indicates a higher ~ a n d a higher P . . . . b u t identical 0 (Table 3). A c o m p a r i s o n o f two electrodes m a d e by two people t a k i n g s i m u l t a n e o u s samples o f algae, showed that the two electrodes gave equal rates o f photosynthesis which varied over time. This indicated that s u b s t a n t i a l v a r i a t i o n in Pmax was occurring. As a result o f this t e m p o r a l v a r i a t i o n it was necessary to m e a s u r e Pm.x for a c o n t r o l sample j u s t prior to each p h o t o i n h i b i tory t r e a t m e n t to o b t a i n a n accurate m e a s u r e o f the effect o f i n h i b i t i o n o n Pmax. F o r the c o n t r o l s Pmax varied

J.W. Leverenz et al. : Photoinhibition and the photosynthetic light-response curve

165

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150

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Fig. 2. Photosynthetic responses to light in uninhibited and in photoinhibited C. reinhardtii cells. Solid lines show the modelled curves (Eq. 1, parameters in Table 3) and the symbols show measured rates of photosynthesis. The top figure shows data from control (upper curve) and cells inhibited at 1400 ~tmol- m - 2. s - 1. The bottom figure shows data from cells inhibited at 1800 (upper curve) and 2050 txmol-m-2.s-1. Note change in scale. Error bars show 95% confidence limits at the highest light level

,

2 0

400 600 Incident Q ;Jmol 9m-2. s-1

800

'o

10 0

Fig. 3. Sample light-response curves showing the fit of Eq. 1 to the data for control (top figure) and inhibited cells (bottom figure, redrawn from Falk et al. 1990). Solid lines show the fit which minimized the residual errors (0 was 0.976 for control and 0.198 for inhibited cells). The best fits where 0 was pre-determined are shown by the dashed lines. For uninhibited cells 0 was fixed at zero (top figure) whereas for inhibited cells it was fixed to 0.976 (bottom figure)

between 279 and 385 ~tmol-(mg C h l ) - 1 - h - t . The absolute rates of photosynthesis do not affect 0 (Leverenz 1987) so control estimates of 0 were not made for every photoinhibitory treatment. Modelled parameters of the light-response curves for each level of inhibition are given in Table 3. Figure 4 shows the relationship of the modelled parameters, 0 and Pmax to photoinhibition. In this figure,

166

J.W. Leverenz et al. : Photoinhibition and the photosynthetic light-response curve

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0 i--:

-

photoinhibition was measured as a decrease in the maxim u m q u a n t u m yield as estimated by linear regression. This is consistent with using the rate of photosynthesis measured at one standard low light level to estimate quantum yield as done by Kok (1956). From zero to 50% inhibition Pmax was constant. As noted by Kok (1956) a transition occurred at 60% photoinhibition where P~na~ became limited by the degree o f photoinhibition (Fig. 4). With greater than 60% inhibition Pmax decreased towards a value of zero at 100% inhibition (Fig. 4). In contrast to Pmax, 0 decreased steadily towards a value o f zero at 60% inhibition. Importantly, 0 reached a value o f zero where Pmax began to drop. A linear regression can be used to describe this decrease where 0 = - 0 . 0 1 6 8 . I + 0 . 9 8 3 , r2=0.963. Above 60% inhibition 0 remained at zero. The b o t t o m o f Table 3 shows the ratio of 9 estimated by linear regression versus 4~ estimated by Eq. 1. This ratio decreased with 0 from a value close to 1.00. When 0 was zero, the estimate o f 4~ based on linear regression was 3 0 + 3% lower. Thus, although the data were noisy (Fig. 2), the differences in the estimates of 4~ were as predicted by an earlier numerical analysis (Leverenz 1987). Based on 4~ obtained by fitting Eq. I (Table 3) to estimate the degree o f photoinhibition, the same patterns o f response of Pmax and 0 to inhibition occurred (Fig. 5) but the level o f inhibition at which Pro,, dropped and at which 0 went to zero was 40% instead of 60%. Figure 6 shows the relationship between chlorophyll fluorescence and the level of photoinhibition of ~b. Measurements on D C M U - t r e a t e d cells gave the same values of Fm as measurements on cells without D C M U (data not shown). This indicates that the decrease in Fm-Fi was not a result o f insufficient actinic light to obtain a true Fm. A 95% decrease in Fm-Fi occurred at the lowest level of inhibition (11%). It was no longer possible to measure Fm-Fi at 34% inhibition. The Fi-Fo signal was less sensitive to inhibition. Fi-Fo was 30% of the control at the point (39% inhibition) were Pmax de-

+.

0.(]

i

4'0 6'0 8'0 100 % Inhibition Fig. 4. The effect of photoinhibition on the light-saturated rate of photosynthesis relative to that measured for control cells (o), and on the convexity of the light-response curve (+). Data for 50% inhibition are from Falk et al. (1990). The initial slope was assumed to be linear to calculate the degree of inhibition

0~ o

0.2

m

0

i

2'0

i

4'0 6'0 % Inhibition

+- -'-~ i

i

8'0

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Fig. 5. The effects of photoinhibition on the light-saturated rate of photosynthesis relative to control cells (o) and on the convexity of the light-response curve (+). Data for 50% inhibition are from Falk et al. (1990). Equation (1) was used to estimate the initial slope in order to calculate the degree of inhibition

i

i

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100 80

w 6c "6 4C |

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0

I

2'0

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I

60

80

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Fig. 6, The effects of photoinhibition on PSII fluorescence relative to control cells. Equation 1 was used to estimate the initial slope in order to calculate the degree of inhibition. Solid symbols show the change in Fi-Fo and the open symbols show the change in Fm-Fi. Error bars show the maximum 95% confidence intervals when it exceeds the symbol size creased and 0 reached zero. These patterns of decrease agree with the higher sensitivity of PSII~ (or fast PSII) to photoinhibition than PSII/~ (or slow PSII) (Mfiempfi/i et al. 1987; Briantais et al. 1989). Discussion

In spite of the variation in the data, this study showed a clear decrease in 0 with photoinhibition (Tables 2, 3, Figs. 2, 3). This decrease in 0 has implications for making unbiased estimates of ~. Two methods have been used to estimate 4~ in this study. The first was to assume a linear initial slope and use linear regression to be consistent with the method used by Kok (1956, Fig. 10). The second was to fit Eq. (J) to the data. The two methods give the same result ( + 2 % ) when 0 was 0.98 (Table 3), but using the linear assumption apparently resulted in an increasing bias in the estimate of the true maximum quantum yield as 0 decreased. When 0 was

J.W. Leverenz et al. : Photoinhibition and the photosynthetic light-response curve zero the difference in estimated 4 was 30_+ 3%. With these data one can not distinguish which model gives a smaller residual error at low light. This is in agreement with the high R 2 statistic (0.993) found by Leverenz (1987) even when 4 is underestimated by 30%. However, except when 0 is 1.00 there is no truely linear initial slope to a response curve. Thus it appears to be a good hypothesis that Eq. (1) should provide a more accurate (unbiased) estimate of the maximum quantum yield than linear regression when applied to optically thin solutions of photosynthetic cells. Reducing the range of light over which the linear model is fitted will decrease the bias in the estimate of 4. However, with noisy data, this will reduce the precision (increase r a n d o m variation) of the estimate o f 4. Moreover, when 0 is zero it is often difficult to decide whether a K o k inflection has occurred. This makes selection of the proper " l i n e a r " initial slope subjective. We conclude that Eq. (1) will give the most precise or most accurate estimates of 4 when fitted to light-response curves for optically thin solutions of cells. The response o f Pmax to photoinhibition, estimated in this study by assuming a linear initial slope (Fig. 4), is in agreement with the experimental observations of Kok (1956) showing a 60% decrease in 4 (estimated by linear regression) was required before a decrease in Pm~x is observed. Thus fitting Eq. (1) appears to provide accurate estimates of Pm,* at least up to 60% photoinhibition, eventhough Pro,, was not experimentally observed (except for control cells). It is also in agreement with experiments made by Heber et al. (1988) where D C M U (instead o f photoinhibition) was used to titrate away PSII reaction centres, allowing for the fact that photoinhibition may reduce 4 less than it reduces the number of PSII reaction centres (Raven and Samuelsson 1986.). Ogren etal. (1984) found that P measured at 1000 g m o l . m - 2 , s-1 decreased at lower levels o f inhibition than observed for Pma~ in this study. This is an expected result if a decrease in 0 occurred (Figs. 1, 2). Ogren et al. (1984) considered P measured at 1000 gmol. m - 2. s - 1 to be "light-saturated" ; however, the analysis of this paper indicates that we have to use the term "light-saturated photosynthesis" in a more strict sence. We propose that the term "light-saturated photosynthesis" only be applied to the asymptote of the photosynthetic light-response curve. When 0 is low the least biased way to estimate the rate of light-saturated photosynthesis may be through use of Eq. (1). It is often observed that photoinhibition results in a much larger decrease in the quantum yield than in the light-saturated rate of photosynthesis, even in intact leaves (Bj6rkman etal. 1988; Oquist and Malmberg 1989). This indicates similar responses in unicellular algae and the cells of higher-plant leaves. The decrease in Pmax at 40 (or 60)% photoinhibition (Figs. 4, 5; Kok 1956) may be related to the reported heterogeneity in PSII. Assuming no limitation caused by longer diffusion distances for PQ when many PSII centres are photoinhibition, one would predict Pmaxto drop when all but 2 - 3 % of the fast PSII centres were destroyed or altered since they have 30-50 times faster

167

electron transport than through the PQ pool. Since the Fm-Fi rise in fluorescence is reported to come from these fast centres (Briantais et al. 1988), the Fm-Fi signal should disappear near the point where Pmax decreased. We found the Fm-Fi signal to disappear at 34% inhibition (Fig. 6) whereas Pmax decreased at 39% inhibition (Fig. 5). This indicates that either Pma* is not limited by the capacity of the slow PSII reaction centres or that these fluorescence signals cannot be used to quantify the number of fast and slow PSII reaction centres (Raven and Samuelsson 1986; Krause 1988). Three mechanisms have been modelled to control 0: (1) Efficient exciton transfer between PSII centres. (2) The high relative speed of the reactions within the PSII complex, and (3) both these mechanisms acting together (Zvalinskii and Litvin 1984). Photoinhibition is associated with the rapid disappearance of Fm-Fi (Fig. 6; Briantais et al. 1988) which apparently arises from fast PSIIa (Melis 1985). Since PSII~ is associated with PSII to PSII exciton transfer, photoinhibition reduced both the probability of exciton transfer between PSII centres and the number of fast centres. Whether hypothesis 1, 2 or 3 above is true cannot be determined by photoinhibition alone. The steady decrease in 0 to zero at 40% photoinhibition and the simultaneous decrease in Pmax at 40% inhibition does not contradict any of the above three hypotheses. If photoinhibition had no effect on 0 there would be strong reason to reject these three hypotheses. Zvalinskii and Litvin (1988) showed that if the maximum speed of PSII is sufficiently high relative to only one other process (for example PQ turnover), 0 remains high and virtually independent o f the speed of other reactions in the system. This agrees with the analysis of Prioul and Chartier (1977) showing that when 0 is high, diffusion resistance has no significant effect on 0. Moreover, the speed of turnover of the Calvin cycle or of the phosphate cycle (Sharkey 1985) are probably of secondary importance once a photosynthetic system is built with a high speed of PSII re-opening relative to the recycling of PQ (Zvalinskii and Litvin 1988). In agreement, no effect of CO2 partial pressure or of low temperature on 0 has been found (Leverenz 1988) and both C3 and C , plants can achieve a high 0 (Table 1). The present investigation was supported by the Swedish Council for Forestry and Agricultural Research, the Swedish Environmental Protection Board, and the Swedish Natural Science Research Council. We thank Dr. Deborah D. Kaska (Department of Biological Sciences, University of California, Santa Barbara, Calif., .USA) for giving us Chlamydomonasalgae. We thank Professor G. Oquist (Department of Plant Physiology, University of Ume/t, Ume/t, Sweden) for his encouragement, valuable comments and discussion. References

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J.W. Leverenz et al. : Photoinhibition and the photosynthetic light-response curve

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