Planta (1996)198:300-309 P l a ~ n t a

O Springer-Verlag 1996

Photoinactivation of functional photosystem II and Dl-protein synthesis in vivo are independent of the modulation of the photosynthetic apparatus by growth irradiance Youn-II Park, Jan M. Anderson, Wah Soon Chow

CSIRO, Division of Plant Industry and Cooperative Research Centre for Plant Science, GPO Box 1600, Canberra, ACT 2601, Australia

Received: 25 May 1995/Accepted: 29 June 1995

Abstract. To investigate whether the in-vivo photoinhi- bition of photosystem II (PSII) function by excess light is an intrinsic property of PSII, the maximal photochemical efficiency of PSII (Fv/Fm) and the content of functional PSII (measured by repetitive flash yield of oxygen evolu- tion) were determined in leaves of pea (Pisum sativum L.), grown in 50 (low light), 250 (medium light), and 650 (high light) gmol photons.m-2-s -1. The modulation of PSII functionality in vivo was induced in 1.1% CO2 by varying either (i) the duration (0-2 h) of light treatment (fixed at 1800 gmol photons- m -2 "s-1) or (ii) irradiance (0-3200 gmol photons.m-2.s -1) at a fixed duration (1 h), after infiltration of leaves with water (control), lincomycin (an inhibitor of chloroplast-encoded protein synthesis), or a combination of lincomycin with nigericin (an uncoup- ler), through the cut petioles of leaves of 22-to 24-d-old plants. The reciprocity law of irradiance and duration of illumination for PSII function in vivo (Park et al. 1995, Planta 196: 401-411) holds in all differently light-grown peas, demonstrating that inactivation of functional PSII depends on photon exposure (mol photons.m-2), not on the rate of photon absorption. In vivo, PSII acts as an intrinsic "photon counter" and at higher photon expo- sures is inactivated following absorption of about 3 x 107 photons. There is a functional heterogeneity of PSII in vivo with 25 % less-stable PSIIs that are inactivated at low photon exposure, compared to 75% more-stable PSIIs regardless of modulation of the photosynthetic apparatus. We suggest that the less-stable PSIIs represent monomers located in the nonappressed granal margins, while the more-stable PSIIs are dimers located in the appressed grana membrane cores. The capacity for Dl-protein synthesis was the same in all the light-acclimated peas and saturated at low light, indicating that Dl-protein

Abbreviations: Dl-protein = psbA gene product; D2 protein = psbD gene product; Fo = chlorophyll fluorescence corresponding to open PSII reaction centres; Fv, Fm = variable and maximum flu- orescence after dark incubation, respectively; PS = photosystem; QB = secondary quinone electron acceptor Correspondence to: Dr Y.-I. Park; FAX: 61 (6) 246 5000; Tel: 61 (6) 246 5066; E-mail: park@pican.pi.csiro.au

repair is also an intrinsic property of PSII. This accounts for the low intensity required for recovery of photoin- hibition in sun and shade plants which is independent of light-harvesting antennae size or PSII/PSI stoichio- metries.

Key words: Dl-protein - Photoinhibition - Photon exposure - Photosystem II heterogeneity - Light acclima- tion - Pisum

Introduction

When plants are illuminated with excess light, loss of photosynthetic activity occurs. The primary site of photo- inactivation is the PSII complex; photoinhibition is observed as a decrease in the quantum yield of photosyn- thesis, followed by a decline of maximal photosynthetic capacity (Powles 1984). The Dl-protein, which together with D2-protein in the heterodimer of the PSII reaction- centre complex organizes the redox components of PSlI (Nanba and Satoh 1987), turns over 50-100 times faster than other thylakoid proteins in the light in vivo (Eaglesham and Ellis 1974) and is linked with photoin- hibition (Kyle et al. 1984). Net photoinhibition at high light occurs when the rate of Dl-protein degradation exceeds the capacity for Dl-protein synthesis (Greer et al. 1986). However, the complex phenomenon of photoin- hibition comprises both damaging and protective events, including down-regulation of PSII, reversible and irre- versible photoinactivation of PSII electron transport, as well as concomitant Dl-protein turnover (Prasil et al. 1992; Aro et al. 1993; Chow 1994; Ohad et al. 1994). Plants have also developed multiple protective and regulatory strategies that involve both short- and long-term re- sponses to reduce photoinhibition and optimize photo- synthetic performance (Chow 1994).

In this paper, we compare the susceptibility of PSII to photoinactivation and Dl-protein degradation in vivo with peas acclimated under different growth irradiances. It is well established that shade or low-light-grown plants are more susceptible to photoinhibition of photosynthesis

Y.-I. Park et al.: Photoinactivation of PSII and Dl-protein turnover in vivo 301

than are sun or high-l ight-grown plants (cf. Anderson and O s m o n d 1987; C h o w 1994). However , there is still debate on the cause of the differential susceptibility to photoinhibi t ion in vivo between differently light-ac- cl imated plants. The enhanced susceptibity of shade plants to excess light has been ascribed to various factors: a larger an tenna size of PSII , a lower rate of photo- synthesis (Powles 1984), a lower capaci ty for format ion of zeaxanthin via the xanthophyl l cycle (Demmig-Adams and Adams 1992; 1995), and /or a lower repair capaci ty of D l -p ro t e in (Oquist et al. 1992), p robab ly due to a slower degradat ion of phosphory la ted Dl -p ro t e in in higher plants (Aro et al. 1993; Rintam~iki et al. 1995).

Al though it was recognised that D l -p ro t e in degrada- t ion occurs at low light and increases with increasing irradiance (Mat too et al. 1984), photo inhib i tory studies bo th in vitro and in vivo have mainly concentra ted on high light stress (Chow 1994; O h a d et al. 1994). Little at tent ion has been given to exploring D l -p ro t e in turnover in vivo under low light intensity, yet it is only at very low intensity (up to an irradiance of 50 lmaol p h o t o n s - m - 2 , s-1) that light is limiting for PSI I function. Recently, in an integrative approach to study photoinhibi t ion in vivo, we have explored photoinhibi t ion in pea leaves f rom plants acclimated to medium growth irradiance, as well as some of the photoprotect ive strategies which enable PSI I to remain functional, not only under high light, but also and equally impor tant ly under normal light (Park et al. 1995). By extending this study to peas g rown under low, medium and high irradiances, we demonst ra te that the photoinac- t ivation of functional PSI I is intrinsic and independent of l ight-harvesting antennae size or P S I I / P S I stoichiometry. Fur the rmore D l -p ro t e in synthesis is also an intrinsic function of PSII , independent of maximal photosythet ic capaci ty and acclimation of the photosynthet ic appara tus to light. We also demonst ra te a functional heterogeneity of PSI I in vivo.

Materials and methods

Plant material. Pea (Pisum sativum L. cv. Greenfeast) was grown from seed in a compost/perlite mixture (1:1) and watered every second day with a water-soluble fertilizer, 'Aquasol' (Hortico Ltd., Revesby, Sydney, Australia) (for low- and medium-light peas) or half-strength Hoagland nutrient solution (high-light peas). Low- (50 lamol photons'm- 2. s- 1) and medium-light (250 larnol photons" m-2.s-1) plants were grown in growth chambers (12 h light/22~ 12 h dark/18~ Illumination was provided by fluorescent tubes (Philips TLD 58 W/86). High-light (650 lamol photons-m-2"s -I) peas were grown in growth cabinets (12 h light/26~ 12 h dark/22~ under Siemens (Wotan) Power Star HQ1T WD 400 lamps. To ensure uniformity of leaves, the fourth leaf pair, represent- ing the youngest fully-expanded pair of leaves when the plants were 22-24 d old, was collected in the morning at about 1-2 h into the photoperiod.

Inhibitor treatments. Leaf petioles were cut under water and trans- ferred to small Eppendorf tubes containing water (untreated) or 0.6 mM lincomycin with or without nigericin (1 IxM) (Sigma, St. Louis, Mo, USA), and then allowed to transpire for 2 h in a fumehood with dim light (20 ~unol photons'm-2"s - 1). The con- centrations of inhibitors in the bulk leaf tissue, estimated according

to Bilger and Bj6rkman (1994), were 1.2 mM lincomycin and 1.5 ~tM nigericin.

Light treatments. To measure both functional PSII reaction centres and fluorescence parameters as a function of photon exposure (mol photons-m-2), control and inhibitor-treated leaf discs (3.64 cm 2) were exposed to various irradiances (0-32001amol photons" m-2"s -1) for 1 h or to 1800 ktmol photons'm-2"s -~ for various durations (0-2 h) in the chamber of a leaf-disc oxygen electrode (Hansatech, King's Lynn, Norfolk, UK), through which humidified air containing 1.1% CO2 at 25 ~ was passed. The treatment light was provided by a slide projector.

Determination of functional PSII and PSI reaction centres. The num- ber of functional PSII reaction centres was determined according to Chow et al. (1989, 1991) using a leaf-disc oxygen electrode system (Hansatech). Following an initial dark equilibration of the leaf disc for about 10 min, repetitive single-turnover xenon flashes (10 Hz, 2.5 I~s full width at half peak intensity; type FX200; EG & G Electro Optics, Salem, Mass., USA) of a saturating intensity were applied for 4 min, followed by 4 min of darkness. This was followed by a second cycle of flashes and darkness. The slight heating artefact due to the flashes was taken into account and any limitation of electron trans- port by PSI was avoided by the use of background far-red light (Chow et al. 1991). The amount of functional PSII reaction centres was expressed in mmol PSII-mol Chl- 1. The number of PSI reac- tion centres was determined from the absorbance change at 703 nm induced by blue-green light (Coming 4-72, 500 ~tmol photons- m-2.s-1) after correcting for chlorophyll fluorescence (Chow and Hope 1987). The chlorophyll in leaf discs was determined from aqueous buffered 80% acetone extracts (25 mM Hepes, pH 7.5), using the extinction coefficients and wavelengths of Porra et al. (1989). The content of functional PSII reaction centres after a light treatment was calculated as a percentage of the initial value obtained before the light treatment.

Determination of chlorophyll (Chl ) fluorescence parameters at room temperature. Fluorescence parameters (Fo, chlorophyll fluorescence corresponding to open PSII reaction centres; Fv and Fm, variable and maximum fluorescence after dark incubation; Fv/Fm) were measured at room temperature after measurement of functional PSII reaction centres (either following exposure to 18001amol photons-m-E.s - ~ for different durations or to varying irradiances for 1 h). After light treatments, leaf discs were dark-treated for 30 min in leaf clips of a Plant Efficiency Analyser (Hansatech). Excitation light for fluorescence was given at 80% of maximum for 5 s. To allow for variation in chlorophyll content among leaves, all fluorescence signals were normalized to Fo values in dark-treated controls prior to light treatment.

Results

The Chl a/b ratios of the variously acclimated pea leaves, a sensitive index of bo th accl imation to growth irradiance and susceptibility to photoinhibi t ion of PSI I (Aro et al. 1993), were 3.17, 3.32 and 3.88, respectively, in peas g rown in low (50 ~tmol p h o t o n s . m - 2 . s - 1 ) , medium (2501~mol p h o t o n s - m - 2 - s -1) and high (650~tmol p h o t o n s ' m -2 �9 s-1) irradiance (Table 1). The decrease in Chl a/b ratios indicates that the PSIIs of low-light peas have the largest PSI I l ight-harvesting antennae. There is also an alter- at ion in the amounts of functional PSII , but not that of PSI on a chlorophyll basis with accl imation (Table 1): thus the P S I I / P S I stoichiometries also differ with acclima- tion. Figure 1, where the photosynthet ic rate is ex- pressed as utilized pho tons by assuming that ten pho tons

302 Y.-I. Park et al.: Photoinactivation of PSII and Dl-protein turnover in vivo

Table 1. Comparison of Chl a/b ratios, amounts of functional PSII and PSI reaction centres (mmol.mol Chl- 1), and maximal photosynthetic capacity (P,,,x; gmol 02"m-2"s - 1) of leaves of peas grown in low, medium, and high light. Values are mean _ SE (n = 4-10)

Peas Chl a/b PSII PSI P,,ax PSII/PSI

Low-light pea 3.17 _+ 0.01 2.30 _+ 0.01 1.55 _+ 0.06 13.9 _+ 0.46 1.48 Medium-light pea 3.32 _ 0.05 2.66 _+ 0.02 1.57 • 0.02 21.6 _+ 1.45 1.69 High-light pea 3.88 _+ 0.04 2.98 -I- 0.08 1.50 _+ 0.01 26.9 -4- 0.09 1.99

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Fig. 1. Light response curves of photosynthetic 02 evolution of pea leaves acclimated to low (50 gmol photons.m-2-s -1, O), medium (250gmol photons-m-2.s -1, A), and high (650gmol photons .m-2-s- 1, 0) growth irradiance, expressed as utilized irra- diance assuming that 10 photons are utilized for the evolution of one 02 molecule. The absorbed irradiance is also shown to demonstrate the excess of absorbed over utilized irradiance

are needed for the evolution of one oxygen molecule (BjSrkman and Demmig 1987), shows the photosynthetic 02 evolution in pea leaves grown under various light irradiances. Photosynthetic rates were saturated at about 200 gmol pho tons .m- a.s- 1 in low-light-acclimated pea leaves and 450 gmol photons, m - 2. s- 1 in both medium- and high-light peas. Figure 1 also shows the absorbed irradiance (calculated as the product of incident irra- diance and a factor of 0.9, the measured absorptance of pea leaves), greatly exceeds the utilized irradiance. Excess light, the gap between the absorbed photons and the photosynthetic utilization of absorbed light, increases drastically with increasing irradiance, particularly above the irradiance where photosynthesis is saturated. Hence low-light peas are exposed to more excess light than are the medium- and high-light peas at above-saturating irradiance.

Low-light peas are the most susceptible to photon expo- sure. The maximum efficiency of PSII assayed by the chlorophyll fluorescence ratio, Fv/Fm, is often used as a simple measure of PSII function in photoinhibited leaves because of an approximately linear correlation with the quantum yield of light-limited 02 evolution (Demmig and Bj6rkman 1987) and the number of functional PSII reaction centres (Oquist et al. 1992; Russell et al. 1995). To avoid any limitation of utilization of absorbed light by photosynthesis during the photoinhibitory light treat- ment, either control or inhibitor-treated pea leaf discs

were illuminated in the presence of 1.1% C O 2 (Park et al. 1995), rather than floating leaves or discs on water under atmospheric CO2 as used in previous studies (Oquist- et al. 1992; Aro et al. 1993). We determined the photo- inactivation of PSII as a function of photon exposure (mol photons.m-2, Bell and Rose 1981), which being the product of irradiance and duration of light treatment takes both parameters into cognisance.

Figure 2a shows changes of Fv/Fm as a function of photon exposure (mol photons.m-Z) in peas acclimated to various growth light. With increasing photon exposure, Fv/Fm ratios in control leaves of medium-light (closed triangles) and high-light (closed diamonds) peas remained nearly unchanged up to 7 mol.m -2, and then decreased slightly, in contrast to low-light peas (closed circles) which showed a decline in Fv/Fm even at low photon exposures. Under conditions of low photon exposure where there is no net loss of PSII function or Dl-protein content, the rate of D 1-protein degradation can only be revealed when the rate of de novo synthesis of Dl-protein is blocked. Lincomycin, which is readily taken up in the transpiration stream, was used to inhibit chloroplast-encoded protein synthesis. It is known that lincomycin exacerbates photo- inhibition without causing any other effects on chloro- plast-encoded proteins other than the Dl-protein or photosynthesis (Tyystjiirvi et al. 1992; Aro et al. 1993). In all lincomycin-treated leaves (Fig. 2a; open symbols), it is clearly seen that the Fv/Fm ratio in low-light peas de- clined biphasically, compared with the pseudolinear de- crease in medium- and high-light peas. Taken together, low-light peas are more sensitive to photoinhibitory light treatment than medium- and high-light peas, especially under low photon exposure, confirming earlier results (Aro et al. 1993).

We also determined the number of functional PSII centres in pea leaves by 02 evolution from repeti- tive single-turnover saturating flashes (Chow et al. 1991). This method gives a sensitive assay of the total number of functional PSIIs in vivo. Figure 2b shows the change in the number of functional PSIIs (plotted as a percentage of the initial values) as a function of photon exposure in various light-acclimated peas. Loss of PSII functional- ity in control leaves with respect to photon exposure (Fig. 2b; closed symbols) clearly demonstrated that low-light leaves began to be inactivated at a lower photon exposure than medium leaves, and in turn than high-light leaves, also showing that high-light-accli- mated peas were most resistant to a given light stress. In all cases, this loss of PSII functionality (Fig. 2b) began to decline almost linearly above a photon exposure corresponding to 1 h illumination at 2-3 times growth irradiance.

Y.-I. Park et al.: Photoinactivation of PSII and Dl-protein turnover in vivo 303

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Fig. 2a, h. Change of the estimated maximal photochemical effi- ciency of PSII (Fv/Fm) (a), and functional PSII (expressed as the percentage of initial value) (b), as a function of photon exposure in pea leaf discs grown at low, medium, and high light. Pea leaf discs (3.64 cm 2) from control and lincomycin-treated leaves were exposed to (i) 0-3000 larnol photons'm-2-s -1 for 1 h, or (ii) 1800p.mol photons-m-E's -1 for various durations ((~2h) in an Hansatech leaf-disc electrode system supplied with humidified air (1.1% CO2). The fraction of functional PSII expressed as mmol PSII-mol Chl-~, and measured by repetitive flash-induced 02 evolution according to Chow et al. (1991), corresponding to 100% in low-, medium-, and high-light peas was 2.30 -t- 0.08, 2.66 _+ 0.02, 2.98 -t- 0.08 (n = 4~10), respectively. O, low-light pea, control; (3, low-light pea, lincomycin- treated; &, medium-light pea, control; /x, medium-light pea, lin- comycin-treated; O, high-light pea, control; <>, high-light pea, lin- comycin-treated. Mean values ( -t- SE) for 3-4 leaf disks are shown

Reciprocity between irradiance and duration of light treat- ment in differently acclimated peas. In 1966, Jones and Kok showed that inactivation of PSII electron transport with isolated spinach chloroplasts or Chlorella cells fol- lowed the general exponential at tenuation law, Rt =Ro e - ct, where Ro is the initial rate of PSII electron transport, Rt is the rate after exposure to photoinhibitory light dur- ing time t, and c is a rate constant related to irradiance. We reasoned that if this were so for leaves, there should be reciprocity of time and irradiance, the product of which is photon exposure (Bell and Rose 1981), at least under conditions of high photon exposure where the photo- protective strategies might be overwhelmed. For Fig. 6, we determined the content of functional PSII in pea leaves over a very wide range of irradiance and duration. Photon exposure was obtained in two ways: (i) light treatment for l h at 0 -3000pmol pho tons .m-2 . s -1 ; (ii)fixed irra- diance of 1800 pmol pho t ons .m -2 . s -1 for 2min to 2 h

(Fig. 2). Exactly the same pattern of PSII inactivation was obtained with either mode of exposure to light, for control or lincomycin-treated leaves of medium-light peas at each photon exposure (Park et al. 1995). Further, for both low-light leaves or high-light leaves it did not matter whether the photon exposure was obtained by fixed irradiance with varying time, or vice versa (data not shown). This convincingly demonstrates a reciprocity be- tween irradiance and duration of illumination in vivo over the entire physiological range of photon exposure.

Degradation of Dl-protein in vivo. In the presence of lincomycin, declines of PSII functionality in vivo showed profiles very different from those of control leaves (Fig. 2b; open symbols): contrary to a multiphasic decline in medium-light peas, the fraction of functional PSIIs of the low- and high-light peas in the presence of lincomycin decreased rather biphasically. An estimate of the effect of particular photoprotective strategies in differently light- acclimated pea leaves, can be ascertained by comparison of the increase in the number of nonfunctional PSII in inhibitor-treated leaves at a given photon exposure, with those of untreated leaves. To compare then the relative activity of the Dl-prote in repair system, the increase in the number of nonfunctional PSIIs (calculated from Fig. 2b by subtracting the number of nonfunctional PSIIs in con- trol from that in lincomycin-treated leaves at any given photon exposure), was plotted against photon exposure (Fig. 3). Significantly and unexpectedly, there is no clear difference in the number of nonfunctional PSII complexes between the differently light-acclimated pea leaves over the entire range of photon exposure: the scatter of the data for each growth irradiance overlaps with the means (open diamonds) for the three growth irradiances. These results indicate a constant capacity of Dl-prote in synthesis in all light-acclimated peas.

In Table 2, we compare the content of functional PSII in lincomycin-treated leaves with the remaining content of

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Fig. 3. The increase of nonfunctional PSII of lincomycin-treated leaves from variously light-acclimated peas as a function of photon exposure. The fraction of nonfunctional PSIIs was calculated from Fig. 2b by subtracting the percentage of nonfunctional PSII in lincomycin-treated leaves from that in water-treated leaves given the same photon exposure. O, low-light pea; A, medium-light pea; 0, high-light pea; ~, mean value for the three growth irradiances

304 Y.-I. Park et al.: Photoinactivation of PSII and Dl-protein turnover in vivo

Table 2. Comparison of fractions of functional PSII with the re- maining content of labelled Dl-protein after 1 h exposure of low-, medium-, and high-light pea leaves to given irradiances. The remain- ing fraction of functional PSII [PSIIF] in lincomycin-treated leaves was obtained from Fig. 2b. The labelled Dl-protein content [DI] was determined from previous [-S35]methionine pulse-chase experi- ments (Aro et al. 1993) using the equation n/no = exp[ - (in 2)t/ tl/2], where n/no is the fraction of remaining content at given time (t), n is the quantity at given time, no is the initial quantity, and tl/2 is the half-life

Photon exposure Peas (mol photons-m- z)

0.18 1.44 5.76 10.1

Low-light pea [PSIIF] 0.89 0.69 0.53 - [D1] 0.86 0.76 0.58 -

Medium-light pea [PSIIv] 0.86 0.76 0.66 - [D1] 0.90 0.78 0.65

High-light pea [PSIIF] 0.89 0.78 0.62 0.46 I-D1] 0.90 0.76 0.54 0.45

Dl-protein (calculated from the rate constants of D1- protein degradation at different irradiances, previously determined in this laboratory by pulse-chase experiments using [3SS]methionine (Aro et al. 1993)). There is excellent agreement between the two parameters at several photon exposures for low-, medium- and high-light-acclimated peas (Table 2). This clearly demonstrates that the loss of functional PSII reaction centres in the presence of linco- mycin was comparable to gross degradation of Dl-pro- tein in the absence of the inhibitor for all acclimated peas. Hence in ou r experiments, since the net loss of functional PSII in lincomycin-treated leaves was associated with gross loss of Dl-protein in untreated leaves (Figs. 2b, 3), it allows us to equate PSII photoinactivation with the degradation of Dl-protein. Thus, our results not only demonstrate that the relative activities of Dl-protein degradation are similar in the differently light-acclimated peas, but also show that there is a balance between photo- inactivation of PSII and its subsequent recovery, via D1- protein degradation, de novo synthesis of Dl-protein and reassembly of PSII in all cases.

tional heterogeneity in vivo (Park et al. 1995): about 25% of the total functional PSII complexes were inactivated under very low photon exposure, with no concomitant decline in Fv/Fm ratios. With increasing photon exposure there was a linear decline in both PSII functionality and Fv/Fm ratios (Fig. 4; Park et al. 1995). To explore further this functional heterogeneity of PSII in vivo, the relation- ship between Fv/Fm ratios and the content of functional PSII was also investigated in low- and high-light grown peas for control, lincomycin-treated and lincomycin and nigericin-treated leaves (Fig. 4). Uncouplers such as nigericin exacerbate photoinhibition in chloroplasts (Krause and Behrend 1986) and leaves (Ogren 1991), due to the inhibition of generation of the transthylakoid pH- mediated gradient (Park et al. 1995). Significantly, the three profiles (Fig. 4) are very similar with each of the light-acclimated peas, with about 25% of the functional PSIIs being inactivated under low photon exposure, with- out appreciable decrease of the Fv/Fm ratio. With in- creasing photon exposure, however, a linear relationship between the two parameters was then established, regard- less of light acclimation.

The fluorescence parameter, 1/Fm is, to a first approx- imation, a measure of non-photochemical loss of excita- tion energy from light-harvesting antennae (Havaux et al. 1991; Walters and Horton 1993). Figure 5 shows the behavior of 1/Fm with respect to the fraction of functional PSII centres in the various light-grown pea leaves, treated with or without inhibitors. There was an apparent bi- phasic increase of 1/Fm as functional PSII centres de- clined in all pea leaves with increasing photon exposure. Note that again about 25% of the functional PSII com- plexes were inactivated in all pea leaves, before there was any substantial increase in 1/Fm (Fig. 5). As further loss of functional PSII occurred, 1/Fm values in the presence of lincomycin alone were greater than those in the presence of lincomycin plus nigericin (inverted open triangles) for both high- and medium-light leaves, perhaps more so in high- than medium-light leaves. However, in low-light peas, treatment with lincomycin had the same effect on 1/Fm, regardless of the presence or absence of nigericin.

Discussion

Synthesis of Dl-protein in vivo. Examination of the increase in the fraction of nonfunctional PSIIs in lin- comycin-treated pea leaves as a function of photon expo- sure (Fig. 3) shows a threshold effect. This means that Dl-protein synthesis in vivo saturates already at about 70 gmol photons- m - 2. s- 1, as deduced from the maximal increase in nonfunctional PSII in all lincomycin-treated leaves from peas acclimated to low, medium and high growth irradiance (Fig. 3).

Functional heterogeneity of PSII in vivo in light,acclimated peas. There is a marked discrepancy between estimated PSII efficiency, Fv/Fm (Fig. 2a) and the fraction of func- tional PSII centres (Fig. 2b)as a function of photon expo- sure. Previously, we demonstrated from the relationship between Fv/Fm ratio and the number of functional PSII complexes in medium-light peas, that PSII shows a func-

Photosystem II is an intrinsic "photon counter". Light in- activation of PSII function in vitro (Jones and Kok 1966; Bj6rkman et al. 1972) and in vivo (Park et al. 1995) is determined by photon exposure, the product of irradiance and duration of illumination. To explore further this property in vivo, we used differently light-acclimated pea leaves, with or without lincomycin (inhibitor of chloro- plast-encoded proteintranslation) to interfere with turn- over of the Dl-protein, the main photoprotective strategy adopted by medium-light peas (Park et al. 1995), and low- and high-light peas (data not shown). The dependency of PSII inactivation on photon exposure is deafly seen in the differently light-acclimated pea leaves, in terms of both Fv/Fm ratios and the fraction of functional PSIIs against photon exposure (Fig. 2). Reciprocity of intensity and time of illumination for the different modes of photon exposure was obtained for all of the variously acclimated

Y.-I. Park et al.: Photoinactivation of PSII and Dl-protein turnover in vivo 305

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;0 ;0 100 120 PSII (% of Initial)

Fig. 4. Relationship between the maximal photochemical efficiency of PSII (Fv/Fm) and the fraction of functional PSII (expressed as a percentage of initial) in variously light-grown pea leaf discs, untreated and treated with lincomycin and a combination of lincomycin and nigericin. Leaf discs were pre-exposed to varying irradiance for 1 h, or at 1800 ~mol photons'm-2.s -1 for varying duration, in a Hansatech leaf-disc electrode system with humidified air containing 1.1% CO2, and then functional PSIIs were measured in same leaf-disc eletrode system containing 21% 02 and 1% CO2 at 25 ~ Fluorescence parameters of the same leaf disks were measured with a Plant Efficiency Analayzer (Hansatech) after 30 min-dark incubation. O, low-light pea, control; �9 low-light pea, lincomycin- treated; &, medium-light pea; A, medium-light pea, lincomycin- treated; O, high-light pea, control; ~, high-light pea, lincomycin- treated; V, low-, medium-, and high-light pea treated with lincomycin in the presence of nigericin

peas (Fig. 2; Pa rk et al. 1995). This convincingly demon- strates that PSI I is inactivated in a manner depending only on the number of absorbed photons , and not the rate of the absorpt ion, regardless of the growth irradiance.

0 .7

0 .6

0 .5

E 0.4

~ 0 . 3

0.2

0.1

0.0 0.7

0 . 6

0.5

~ 0.4 u_ ~ 0 . 3

0.2

0.1

0.0 0.7

0 . 6

0 . 5

~ 0.4

0.2

0.1

0.0 0

. , . , . , . , . , ,

Low pea

o

o �9

i i I i i i

�9 , . , , , . , . , ,

~ ^ Med. pea

v ~7~7 zx

f i i i i

�9 , , , . , , , , , ,

High pea

'7 O

i r I , I I t

2 0 4 0 6 0 ' 8 0 ' 1 0 0 ' 1 2 0

Functional PSII (% of Initial)

Fig. 5. Relationship between l/Fm and the number of functional PSIIs (expressed as a percentage of initial) in variously light-grown pea leaf discs, untreated and treated with lincomycin and a combina- tion of lincomycin and nigericin. See Fig. 4, for experimental condi- tions and symbols

This reciprocity is not influenced b y maximal pho tosyn- thesis with high- and medium-ligh t peas having m a x i m u m pho ton utilization rates of about 450 pmol p h o t o n s - m - z �9 s - 1 compared to 200 pmol p h o t o n s . m - z. s - 1 with low- light peas (Fig. 1). Al though the ability to utilize absorbed quan ta by photosynthesis is one of the front-line stategies for coping with excess light (Chow 1994), the property of reciprocity for the photoinactivation of PSII is independent of maximal photosynthetic rates (Fig. 1), Accl imation also markedly influences the compos i t ion of the pho tosyn- thetic apparatus: sun and high-light plants have more PSI I complexes, each with smaller l ight-harvesting antennae relative to PSI, while shade and low-light plants

306 Y.-I. Park et al.: Photoinactivation of PSII and Dl-protein turnover in vivo

have fewer PSII complexes, each with larger light-harvest- ing antennae relative to PSI (Anderson 1986; Anderson and Osmond 1987). However, our results with the ac- climated peas clearly show that differences in light-harvest- ing antennae and the PSII /PSI stoichiometries (Table 1) do not affect the reciprocity between irradiance and duration of light treatment.

Assuming that reciprocity of irradiance and duration of illumination in vivo holds generally for higher-plant PSII, we hypothesize that the photoinactivation of PSII is due only to its ability to sum up the photons absorbed, and is independent of the rate of absorption. When a cer- tain number of photons have been counted in control leaves under low photon exposure, Dl-protein degrada- tion and synthesis are needed for repair to occur in order to avoid net loss of functional PSII under normal light. This form of "wear and tear" of PSII depends only on the number of photons absorbed, but not on the rate of photon exposure. We suggest that the "photon-counting" capability presumably arises from the finite probability of toxic products or unstable states being generated after the absorption of a certain number of photons. Above 2-3 times the growth irradiance, which is about equiv- alent to the saturating irradiance for photosynthesis in all acclimated peas, approximately one PSII was inac- tivated after absorption of 3.3 x 107, 3.0x 107, and 3.7 x 10 v photons in low-, medium- and high-light peas, respectively (calculated from the slopes of PSII photoinac- tivation at medium to high photon exposure in Fig. 2b, knowing that 100% of initial functional PSII is equivalent to ca. 1 ~tmol PSII.m-2). This rather constant quantum yield of PSII photoinactivation suggests that the photon- counting efficiency of PSII is unique and an intrinsic property of higher plant PSII. But the extent of PSII photoinactivation at given photon exposure is variable due to the photoprotective strategies in operation in particular leaves as shown by biphasic or multiphasic declines of the fraction of functional PSII in lincomycin- treated leaves (Fig. 2b).

Low-light peas are more susceptible to photoinhibi- tion than medium and high-light peas (Fig. 2), confirm- ing earlier results with peas (Oquist et al. 1992; Aro et al. 1993). The dependence of the fluorescence para- meter, 1/Fm (a qualitative indicator of the loss of excita- tion energy by the light-harvesting antennae (Havaux et al. 1991)) showed that low-light peas have the least capability to avoid over-excitation of PSII reaction centres by means of the transthylakoid pH-gradient- mediated thermal dissipation of excitation energy (Fig. 5). Thus we conclude that low-light peas are more prone to photoinhibition than medium- or high-light peas, due not only to their lower utilization efficiency of absorbed light by photosynthesis (Fig. 1) but also their lower capacity for dissipation of excess light via a trans- thylakoid pH-gradient.

Functional heterogeneity of PSII in vivo is an intrinsic property of PSII. Low-light-acclimated leaves have rela- tively more stacked thylakoid membranes compared with high-light leaves (Anderson 1986), perhaps implying the possibility of a difference in the proportion of stable PSII to less-stable PSII reaction centres between differently

light-acclimated peas. To check this hypothesis, the rela- tionship between Fv/Fm and the number of functional PSII was investigated in differently light-acclimated pea leaves (Fig. 4). In all leaves, surprisingly, the curvilinear relationship holds, showing that about 25% of the frac- tion of functional PSII could be decreased without a concomitant loss of maximal photochemical efficiency of PSII, at low photon exposure. Therefore, the fraction of less-stable PSII is constant regardless of acclimation to light intensity.

Antenna size heterogeneity of PSII has been observed empirically from the biphasic kinetics of chlorophyll flu- orescence induction (Melis 1991). The PSII~s with larger light-harvesting antennae are located in appressed granal membranes and PSII~s with smaller light-harvesting an- tennae occur in stroma-exposed thylakoids (Anderson and Melis 1983; Melis and Anderson 1983). Functional heterogeneity of PSII has also been observed in vitro with QB-reducing and QB-nonreducing centres (cf. Melis 1991). In this paper we demonstrate functional PSII heterogen- eity in vivo with respect to oxygen evolution (Fig. 4), with 25% of PSIIs being easily inactivated at very low photon exposure, compared to 75% more-stable PSII centres for all acclimated peas. The less-stable PSII centres cannot be equated with QB-nonreducing PSII centres, however, since they are initially functional under repetitive saturat- ing flashes. As these 25 % less-stable PSII centres in each of the acclimated peas also register no Fv/Fm changes with respect to decline in the content of functional PSII at low photon exposure (Fig. 4), we suggested that they must undergo maximum spillover of excess excitation energy to PSI (Park et al. 1995). Consistent with this idea, is our finding that despite the inactivation of this sub-population under low photon exposure (Fig. 5; Park et al. 1995) the fluorescence parameter, 1/Fm, indicative of energy dissi- pation from the light-harvesting antenna (Havaux et al. 1991), is also unaltered.

Several further lines of evidence suggest that the low- light-inactivated PSIIs correspond to PSIIs located in nonappressed granal margin domains (Fig. 6) (Park et al. 1995). First, they have the same properties as PSII com- plexes in the granal margins; these PSIIs are of the PSII~ subtype with respect to antenna size, but are QB-reducing with respect to electron transport (Wollenberger et al. 1995). Second, although the fluorescence properties of granal margin PSIIs have not yet been examined, granal margins (which also possess PSI) have a higher chloro- phyll content compared to appressed granal cores or stromal thylakoids (Wollenberger et al. 1995), so spillover of excitation energy should be feasible. Third, there is mounting evidence in favour of PSIIa of appressed grana membrane cores existing as dimers in vivo (Santini et al. 1994; Boekema et al. 1995). During Dl-protein turnover, Barbato et al. (1992) indicate that it is monomeric PSIIs which laterally migrate out from appressed granal domains to the stroma-exposed thylakoids, where D1- protein replacement occurs (cf. Prasil et al. 1992). We conclude that the less-stable PSIIs which are inactivated at low light, represent monomeric PSIIs in the grana margins, en route back to granal domains after being repaired, while the more-stable PSIIs are those of ap- pressed grana cores (Fig. 6) (Park et al. 1995).

Y.-I. Park et al.: Photoinactivation of PSII and Dl-protein turnover in vivo

PSII Monomer

Fig. 6. Schema showing more-stable functional PSII dimers located in the appressed granal membrane cores, less-stable functional PSII monomers located in the non-appressed grana margins, and a ribo- some with psbA mRNA attached to a Dl-protein-depleted PSII located in non-appressed stromal thylakoids

Synthesis of Dl-protein in vivo. The increase in the fraction of nonfunctional PSII reaction centres in lincomycin- treated leaves compared to control leaves with respect to photon exposure was not only the same in the differently light-acclimated peas (20-30%), but also was constant over the entire range of irradiance (Fig. 3), indicating a constant capacity for Dl-protein synthesis. Further- more, there is excellent agreement between the net loss of functional PSII in lincomycin-treated leaves and the gross loss of Dl-protein in the absence of the inhibitor (Table 2). Hence in our experiments, we can equate the photoinac- tivation of functional PSII with the degradation of D1- protein. Regardless of light acclimation, the capacity of Dl-protein synthesis is the same in all peas (Fig. 2b). This confirms previous results demonstrating that the rates of Dl-protein degradation in light-acclimated peas illumin- ated with medium light (400~molphotons-m-2.s -1) were identical, and equivalent to rates of decline of Fv/Fm ratios in lincomycin-treated leaves (Aro et al. 1993). More- over, since our results show that there is a balance between photoinactivation of PSII, and subsequent recovery, via Dl-protein degradation, de novo synthesis of Dl-protein and reassembly of PSII for all pea leaves, Dl-protein synthesis is an intrinsic property of PSIl.

Synthesis of Dl-protein in vivo. Regardless of maximal photosynthetic capacity, the threshold for light saturation required for Dl-protein synthesis was the same in all the variously light-acclimated peas (Fig. 3). Moreover, D1- protein synthesis was saturated under low light compara- ble to about 70 ~mol photons 'm-2"s- x. We conclude also that higher plants, regardless of acclimation, possess the same intrinsic capacity for Dl-protein synthesis in order for PSII to remain functional under normal irradiance. Synthesis of D 1-protein in vivo by psbA mRNA is transla- tionally regulated in higher plants with the accumulation or stabilization of Dl-protein being strictly light-depen- dent (Danon and Mayfield 1991; Taniguchi et al. 1993). Given that Dl-protein degradation occurs at very low photon exposure, it is indeed necessary that Dl-protein

307

synthesis should also saturate at low light, since only if photoinactivated Dl-protein is rapidly replaced by new Dl-protein will there be no loss of PSII activity. With isolated pea chloroplasts, Taniguchi et al. (1993) showed that Dl-protein synthesis and the level of stromal ATP saturated at the same low intensity; even lower light inten- sities would be required in vivo. Additionally, Chow (1994) postulated that the low threshold of light required for optimal recovery from photoinhibition is due to the need to maintain an optimal stromal pH for protein syn- thesis, known also to be saturated at low light. Further, light modulates the translation of psbA mRNA by chang- ing the redox state of thioredoxin via reduced ferredoxin (Danon and Mayfield 1994).

Turnover of Dl-protein in vivo. Unexpectedly, we also find that peas acclimated to varying growth irradiances all possess the same fraction of less-stable PSII complexes (Fig. 4). The amount of grana stacks, determined as the granal cross-sectional area per chloroplast, is doubled in low-light compared to high-light pea chloroplasts (Ander- son and Aro 1994). However, the relative area of granal margins will remain much the same with small or large granal stacks. If it is true that the less-stable PSII mono- mers in the nonappressed granal margins are indeed,"in- termediates", in the Dl-protein turnover cycle, then two important corollaries must follow. First, the monomeric PSIIs should comprise the same fraction of total PSIIs, regardless of thylakoid membrane stacking; this is indeed so (Fig. 3). Second, the rate of Dl-protein synthesis under low to high irradiance should not be affected by maximum photosynthetic capacity: this is also evident in Fig. 3. Hence, our finding of a constant fraction of monomeric PSIIs strengthens our conclusion that higher plants, re- gardless of light acclimation, have an intrinsic capacity for both Dl-protein degradation and Dl-protein synthesis in vivo, and hence for Dl-protein turnover.

Recovery from photoinhibition requires only low light inten- sity. Clearly Dl-protein turnover is essential for PSII to remain functional in low light (Greenberg et al. 1987; Keren et al. 1995; Park et al. 1995). Thus, photoinhibition is not confined to high light, but occurs also under normal light (Figs. 2, 3). This finding sheds light on the recovery from photoinhibition in vivo after sustained high irra- diance which requires only very low irradiance (5-50 gmol photons.m-Z.s-1), a notable if hitherto puzzling feature of recovery (Aro et al. 1994). For example, peas acclimated to low, medium or high growth irradiances with very different maximal photosynthetic capacities, showed opti- mal recovery at the same low light, although recovery was almost as good at higher irradiance for the high-light plants (Aro et al. 1994). Hence the low-threshold irra- diance for recovery, obtained with either sun or shade plants, demonstrates that recovery from photoinhibition is also an intrinsic property of PSII that saturates at low photon exposure.

Physiological implications of an intrinsic capacity for D1- protein turnover in higher plants. The universal necessity for a similar intrinsic capacity for Dl-protein turnover to maintain PSII functionality in vivo (demonstrated by our

308 Y.-I. Park et al.: Photoinactivation of PSII and Dl-protein turnover in vivo

results discussed above) under low to sa tura t ing i r ra- d iance is ex t remely i m p o r t a n t for all plants , regardless of acc l imat ion . The a t t enua t i on of l ight th rough c rop and forest canopies is very marked , and even more severe with increase in water depth. Indeed, mos t terres t r ia l and aqua- tic ch lo rop las t s are exposed to only subsa tu ra t ing l ight for mos t of the day. F o r example , pho tosyn the t i c efficiency at low light, ra ther than s t au ra t ing light, is the ma jo r deter- m i n a n t of c rop pho tosyn the t i c pe r fo rmance (Ort and Baker 1988). If shade ch lo rop las t s of p lants were unable to ca r ry out D l - p r o t e i n repa i r with ease at low light, they wou ld be at a d i sadvantage ; however , with PSI I ac t ing as an intr insic " p h o t o n counter" , the t r igger ing of D l - p r o - tein for degrada t ion , de novo synthesis of D l - p r o t e i n and the reassembly of PSI I depends only on the to ta l number of p h o t o n s received, and is i ndependen t of the pa r t i cu la r l ight env i ronment , be it f luctuat ing or s ta t ionary .

Financial support for this research by the Department of Em- ployment, Education and Training/Australian Research Council International Research Fellowships Program (Korea) is gratefully acknowledged.

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