PhotosynthesisResearch 23: 195-203, 1990. © 1990KluwerAcademic Publishers.Printed in the Netherlands. Regular paper

Dynamics of Photosystem II heterogeneity in Dunaliella salina (green algae) Jeanne E. Guenther & Anastasios Melis Division of Molecular Plant Biology, 313 Hilgard Hall, University of California, Berkeley, CA 94720, USA Received6 March 1989; accepted 9 May 1989

Key words." photosystem II heterogeneity, QB-nonreducing centers, light-dependent activation, PS II repair cycle, thylakoid membrane Abstract

Based on the electron-transport properties on the reducing side of the reaction center, photosystem II (PS II) in green plants and algae occurs in two distinct forms. Centers with efficient electron-transport from QA to plastoquinone (QB-reducing) account for 75% of the total PS II in the thylakoid membrane. Centers that are photochemically competent but unable to transfer electrons from QA to QB (QB-nonreducing) account for the remaining 25% of total PS II and do not participate in plastoquinone reduction. In Dunaliella salina, the pool size of QB-nonreducing centers changes transiently when the light regime is perturbed during cell growth. In cells grown under moderate illumination intensity (500 #Em -2 s-l), dark incubation induces an increase (half-time 45 min) in the QB-nonreducing pool size from 25% to 35% of the total PS II. Subsequent illumination of these cells restores the steady-state concentration of QB-nonreducing centers to 25%. In cells grown under low illumination intensity (30#Em 2s J), dark incubation elicits no change in the relative concentration of QB-nonreducing centers. However, a transfer of low-light grown cells to moderate light induces a rapid (half-time 10 min) decrease in the QB-nonreducing pool size and a concomitant increase in the QB-reducing pool size. These and other results are explained in terms of a pool of QB-nonreducing centers existing in a steady-state relationship with QB-reducing centers and with a photochemically silent form of PS II in the thylakoid membrane ofD. salina. It is proposed that QB-nonreducing centers are an intermediate stage in the process of damage and repair of PS II. It is further proposed that cells regulate the inflow and outflow of centers from the QB-nonreducing pool to maintain a constant pool size of QB-nonreducing centers in the thylakoid membrane.

Abbreviations," Chl-chlorophyll, PS-photosystem, QA-primary quinone electron acceptor of PS II, QBsecondary quinone electron acceptor of PS II, LHC-light harvesting complex, F o- non-variable fluorescence yield, Fp~-intermediate fluorescence yield plateau level, Fmax-maximum fluorescence yield, Fiinitial fluorescence yield increase from Fo to Fpl (Fpl - F o), F v - total variable fluorescence yield (Fma x -- F o), DCMU - dichlorophenyl-dimethylurea

Introduction

Extensive research over the last several years has focused on the organization and structure of the photosynthetic complexes in the thylakoid membrane. In vascular plants and green algae, PS I is localized in stroma-exposed thylakoid membranes

(Andersson and Anderson 1980, Anderson and Haehnel 1982, Anderson and Melis 1983). It is composed of a chlorophyll a (Chl a) core complex and a Chl a-b accessory light-harvesting antenna (LHC I) containing a combined total of approximately 200Chl molecules (Melis and Anderson 1983). Unlike PS I centers, which form a uniform

196 population with respect to antenna size and localization, PS II centers can be divided into two populations. The apparent heterogeneity in PS II has been examined by several investigators over the last ten years (Black et al. 1986, Melis et al. 1988). Two main aspects of PS II heterogeneity have prevailed in the literature. The PS II antenna size heterogeneity, also known as c~,/?heterogeneity, and the PS II reducing side heterogeneity. The antenna size heterogeneity refers to the occurrence of two distinct PS II populations with different light-harvesting Chl antenna sizes. The dominant form, PS II~, is localized in the grana partition regions (Anderson and Melis 1983) and is responsible for the majority of the water oxidation activity and plastoquinone reduction. In mature spinach chloroplasts, PS II~ centers represent 75% of the total PS II centers. These centers possess a Chl a core complex, an accessory Chl a-b lightharvesting inner antenna (LHC II-inner), and a peripheral antenna (LHC II-peripheral) containing a combined total of about 210-250 Chl a and Chl b molecules (Morrissey et al. 1989). In Dunaliella salina, PS II~ accounts for approximately 50% of the total PS II centers. The core and LHC II-inner are the same as in spinach, however, the LHC II-peri.pheral is considerably larger than in spinach resulting in a PS II~ antenna size with more than 250 Chl a and Chl b molecules (Guenther et al. 1988). PS II~ centers account for the remainder of the PS II centers in the thylakoid membrane (25% in spinach (Melis 1985) and 50% in D. salina (Guenther et al. 1988)) and are localized in the intergrana or stroma-exposed thylakoid membranes (Anderson and Melis 1983). They contain the Chl a core and the Chl a-b LHC II-inner components only and lack the LHC II-peripheral antenna (Melis 1985, Greene et al. 1988, Guenther et al. 1988). The Chl antenna size of PS II~ is estimated to be about 130 Chl (a + b) molecules (Morrissey et al., 1989). In addition to heterogeneity in the antenna size, PS II centers also display heterogeneity on the reducing side of QA with respect to electron flow to the plastoquinone pool. Several investigators have shown that a number of PS II centers, though photochemically competent, are unable to transfer electrons from QA to Q~ (Thielen and Van Gorkom 1981, Lavergne 1982, Melis 1985, Graan and Ort 1986, Greene et al. 1988, Guenther et al. 1988).

Using Lavergne's nomenclature (1982) these centers are termed PS II QB-nonreducing. There is overlap between the PS II~ and QBnonreducing centers. In spinach chloroplasts, the pools of PS II~ and QB-nonreducing centers are identical. This contention is supported by three independent lines of evidence (Melis 1985). In contrast, measurements in Dunaliella salina showed that the proportion of PS II centers with /%type antenna is 50%, while the proportion of QBnonreducing centers is 25% (Guenther et al. 1988). This suggested that, under physiological growth conditions, QB-nonreducing centers are a subpopulation of PS II~ centers in D. salina, and that there is a significant fraction of PS II~ centers which are active in the QA-QB electron-transport reaction and in the reduction of the plastoquinone pool. In this work we addressed the question of the PS II QB-nonreducing pool size and pool dynamics in D. salina cells grown under various illumination conditions. We found that the concentration of the QB-nonreducing centers changes transiently when the light environment is altered. These findings are discussed within the context of a recently proposed PS II repair cycle model (Guenther and Melis 1989). We suggest that QB-nonreducing centers may represent the first functional state in the repair of damaged PS II centers.

Materials and methods Dunaliella salina cultures were grown in media containing 2.0 M NaC1 as described (Pick et al. 1986). Ceils for quantitation of membrane components were obtained from cultures at the start of log growth. Optically thin cultures were maintained to avoid self-shading and illuminated by either 30/IEm-2s ~ (low-light) or 500#Em-2s (medium-light) of mixed fluorescent and incandescent light. Quantitation of PS II centers unable to transfer electrons to the plastoquinone pool (PS II QBnonreducing) was obtained with intact D. salina cells in vivo from the relative amplitude of the initial fluorescence yield increase (Fo to Fpl according to Forbush and Kok (1968)). Intact cells were suspended in their growth media either in the presence of 20 #M DCMU (to obtain Fmax)or in the absence of this inhibitor (to obtain Fv0. The percentage of

197 QB-nonreducing centers was determined from the ratio of the F i (Fo to Fpl) amplitude over the Fv (Fo to Fmax) fluorescence amplitude. Excitation was provided in the green region of the spectrum by a combination of CS 4-96 and CS 3-69 Corning filters. The actinic light intensity was 50 # E m -2 s -~. Cells were harvested by centrifugation at 1500 x g for 3 rain, resuspended in 50 m M Tricine (pH 7.8) containing 0.4M sucrose, 10raM NaC1 and 5raM MgC12, and then recentrifuged at 1500 x g for 3 rain. The pellet was resuspended in 50raM Tricine (pH 7.8) containing 10raM NaC1 and 5 m M MgC12 (hypotonic buffer) and passed twice through a Yeda press at a pressure of 13.7MPa. Centrifugation at 3000 x g for 5min pelleted unbroken cells and large cell fragments. The supernatant was then centrifuged at 45,000 x g for 10min. The pellet containing the thylakoid membranes was resuspended in fresh hypotonic buffer. Chlorophyll concentration was determined in 80% acetone (Arnon 1949, Melis et al. 1987). Samples were kept on ice until immediately prior to taking measurements when they were diluted to 100ffM Chl (a + b). Light-induced absorbance-difference measurements were made with a laboratory constructed split-beam spectrophotometer. The optical pathlength of the cuvette was 0.185 cm in the direction of the measuring beam. The concentration of the primary quinone electron-acceptor of PS II, QA, was determined from the amplitude of the light-minus-dark absorbance change at 320 nm in thylakoid membranes suspended in the presence of 20 #M D C M U and 2.0 mM potassium ferricyanide. The procedure of Pulles et al. (1976) was used to correct for particle flattening effects and a differential extinction coefficient of 13raM -~cm -1 was applied to the corrected absorbance difference at 320nm (Van G o r k o m 1974). Absorbance spectra of thylakoid membranes were measured using an Aminco DW2a spectrophotometer with a 3 nm slit width and opal quartz cuvettes mounted directly against the photomultiplier tube to minimize light scattering effects (Shibata 1958). The flattening correction factor at 320 nm was between 1.12 and 1.15 for D. salina thylakoids. The concentration of the reaction center of PS I, P700, was determined from the amplitude of the light-minus-dark absorbance change at 700nm of solubilized (0.02% SDS) chloroplast membranes

suspended in the presence of 2 mM sodium ascorbate and 200#M methyl viologen. An extinction coefficient of 64mM -~ cm ~ was applied for the calculation of P700 concentration (Hiyama and Ke 1971).

Results

The relative concentration of PS II Q~-nonreducing centers can be measured with leaf discs or algal cells in vivo (Melis 1985). Because QB-nonreducing centers are unable to transfer electrons from QA to QB, electrons accumulate on QA promptly upon illumination and this is reflected in the initial fluorescence yield increase from Fo to Fp~ (Fig. 1). Following a brief plateau (Fpl), the fluorescence yield increases further towards Fmax (Fig. 1). The lag in the fluorescence yield increase (Fp~) reflects the time needed for the reduction of the plastoquinone pool. Once the plastoquinone pool is reduced, electrons accumulate on QA with a concomitant fluorescence yield increase to Fmax. The fluorescence yield amplitude from F o to Fpl provides a relative measure of the pool size of PS II QBnonreducing centers in the thylakoid membrane. This assignment of the F o to Fpl transition to QBnonreducing centers is supported by the observation that even in the presence of artificial elec-

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tron-acceptors, such as potassium ferricyanide and/or dimethylbenzoquinone, the fluorescence yield increase from Fo to Fp~ still occurs, suggesting that a fraction of PS II centers are unable to transfer electrons from QA to QB (Melis 1985). The yield from F o to Fmax (i.e., Fv) gives a relative measure of the total pool ofPS II centers. Thus, the ratio of the amplitude of Fpl-F o over the amplitude of Fmax-Fo is a measure of the fraction of QB-nonreducing centers in the thylakoid membrane. The fluorescence induction curve in vivo shows many slower transients (Kautsky and Hirsch 1934), and therefore it is difficult to obtain the true FmaX with the approach shown in Fig. 1. To overcome this difficulty with D. salina cells in vivo, the amplitude of the Fmax was measured with cells suspended in the presence of D C M U . Figure 2 shows fluorescence induction traces in the presence and absence of D C M U obtained with intact D. salina cells. Figure 2 ( - D C M U ) shows the initial fluorescence yield increase (F o to Fp~) attributed to the photoactivity of QB-nonreducing centers. Figure 2 ( + D C M U ) establishes the maximum fluorescence yield (Fmax) when all PS II centers are in the reduced form. The results of Fig. 2 indicated that approximately 26% of all PS II centers were QB-nonreducing. In order to determine whether the pool size of PS II QB-nonreducing centers is constant or represents a steady-state value for cells grown under con-

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Dark incubation, h Fig. 6. (A) The relative concentration of PS II QB-nonreducing centers and (B) the concentration of all photochemically competent PS II centers (Chl/QA) as a function of dark incubation oflowqight (30 #E m 2s l) grown D. salina cells. The cells were placed in the dark at time 0.

tinuous illumination, the system was perturbed in various ways. Figure 3 shows the fluorescence yield values of Fo, Fpl and Fma x for D. salina cells grown under 500 #E m 2s i light (0 h) and as a function of time during a dark incubation of the cell culture. F o and Fma x did not change significantly over a 6h dark incubation. However, F p increased substantially in the dark. From these data, the fraction of PS II QB-nonreducing centers was calculated (Fig. 4A). The fraction of QB-nonreducing centers increased from 24% to 35% of the total PS II centers. The half-time of the change was approximately 45 min. Figure 4B confirms that, over the same time period, the absolute number of PS II reaction centers that are photochemically competent (measured from the Chl/QA ratio) increased slightly. The data clearly show that, while the total population of PS II centers increased only slightly, the pool of QBnonreducing centers increased significantly when medium-light grown cells were incubated in the dark. To determine whether a similar change would occur in low-light grown cells, D. salina cells were grown at 30#Em 2s-I and the fraction of QBnonreducing centers was measured during dark incubation. Figure 5 shows the fluorescence yield values of Fo, Fpl and Fma x in the light (0h) and during the dark incubation. As opposed to medium-light grown cells, low-light grown cells did not demonstrate any significant changes in their fluorescence yield parameters. Therefore, no change is seen in the relative concentration of Qunonreducing centers (Fig. 6A) which remained constant at approximately 26% of total PS II centers. Low-light grown cells also showed no change in their absolute number of PS II centers (Chl/QA, Fig. 6B). The above results suggest that under continuous illumination conditions, the steady-state pool of PS I[ QB-nonreducing centers is approximately 25% of the total PS II concentration in the thylakoid membrane. However, perturbation of the growth conditions may lead to changes in the steady-state concentration of PS II QB-nonreducing centers. It would appear that low-light grown cells are not affected by dark incubation, however, mediumlight grown cells build up their reserve of QBnonreducing centers in excess of their normal steady-state amount when switched to the dark. To further understand the differential depen-

200 MEDIUM LIGHT

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Time, h Fig. 7. The relativeconcentration of PS II QB-nonreducingcenters as a function of time under differentillumination regimesin low-light (30#Em-2s-~)grownD. salinacells. Thecellsweretransferredtomedium-light(5OO#Em-Zs ~)at time 0. At 2 h they were transferred

to the dark. At 6 h (after 4 h of dark incubation) they were returned to low-light conditions. dence of the concentration of PS II QB-nonreducing centers on the growth light intensity, cells were grown in low-light (30 #E m 2 s-l), transferred to medium-light ( 5 0 0 # E m - 2 s -1) and then to the dark. Figure 7 shows the changes in the relative amount of QB-nonreducing centers that follow such transitions in light intensity. After being grown in low-light for several days the cells attained a steady-state PS II QB-nonreducing concentration of 26% of total PS II centers. At time 0 (Fig. 7) the cells were transferred to medium-light. Within 30 rain in medium-light the number of QBnonreducing centers decreased by about 50% from the initial concentration and stabilized at this level. After 2 h in medium-light, the cells were incubated in the dark. It was observed that, following a lag period of about 1 h, the number of QB-nonreducing centers began to increase in the dark. This increase Table 1. Photosynthetic apparatus characteristics in Dunaliella salina grown under low and medium-light intensities

Chl a/Chl b Chl/PS I Chl/PS II PS II~ PS II QB-nonreducing

Low-light

Medium-light

3.65 760 480 50% 26%

6.40 650 400 55% 24%

The relative concentration of Chl, PSI and PS II is given on a mol/mol basis. The fraction of PS II~and PS II QB-nonreducing centers is given as a percent of total PS II in the thylakoid membrane. The light intensities used were 30#Em-2s -l (low) and 500 #E m 2s 1 (medium).

reached a new steady-state value after about 2h. When the cells were returned to low-light conditions (6 h) the number of Q~-nonreducing centers was lowered rapidly, followed by a slow increase. This slow increase in the concentration of PS II QB-nonreducing centers continued for several hours until gradually the cells reached their original steady-state concentration of 26%. The above results suggest that light plays a role in the PS I! QB-nonreducing pool dynamics, possibly by mediating "activation" of the QA-QB interaction and conversion of the QB-nonreducing centers to Q~-reducing centers (Guenther and Melis 1989). To gain a better understanding of the mechanism involved in the lowering of PS II QBnonreducing center concentration upon illumination of dark-adapted cells, a number of experiments tested the effectiveness of different conditions in bringing about this phenomenon. It was found that the amplitude and rate of decrease in QB-nonreducing centers was the same for light intensities between 10 and 500 #E m - 2 s- ~. Moreover, the decrease was not observed if the experiment was carried out at 0 °C, suggesting the participation of an enzymatic reaction or membrane conformational change in the light-induced lowering of the concentration of PS II QB-nonreducing centers. For comparison purposes, Table 1 shows a quantitation of the components of the photochemical apparatus in cells grown under the two light regimes employed in this work. The only major difference between the two groups of cells is the

201

repair Photochemically silent stoge

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Fig. 8. Schematic describing the dynamics of the PS II QBnonreducing center pool size. It is postulated that QB'nonreducing centers are converted to a Qn-reducing form in a lightdependent reaction. A photochemically silent "transient stage" of PS II also participates in a damage-and-repair cycle in the thylakoid membrane.

lower Chl a/Chl b ratio and the higher chlorophyll content per reaction center for the low-light grown cells, presumably due to a larger LHC II-peripheral antenna.

Discussion

In earlier work the concentration of PS II QBnonreducing centers in D. salina grown under continuous 80 #E m -2 s 1 illumination was measured and found to be approximately 25% of the total PS II in the thylakoid membrane (Guenther et al. 1988). In this work the same estimate was obtained from cells grown under 30 and 500#Em-2s -1 of continuous illumination. This suggests that under different continuous illumination regimes, the steady-state concentration of PS II QB-nonreducing centers is constant and equal to approximately one-fourth of the total PS II centers in D. salina. To determine whether the pool size of QB-nonreducing centers is static or dynamic, the growth conditions were perturbed and the effect on the steady-state concentration of QB-nonreducing centers was measured. Perturbation of the light regime during cell growth resulted in transient changes in the relative PS II QB-nonreducing concentration followed by a return to the 25% steady-state value. The results can best be explained in terms of a pool of PS II

Q~-nonreducing centers existing in a steady-state relationship with the rest of PS II in the thylakoid membrane of D. salina. When cells grown under medium-light (500#Em-2s -1) were incubated in the dark, the QB-nonreducing pool size increased to about 35% of the total PS II centers (Fig. 4A). There was no lag period in the increase of this pool size upon dark incubation of the cells, as would be expected if de novo biosynthesis of PS II proteins were involved. Rather, we propose that there are PS II centers in the thylakoid membrane which are photochemically silent. These centers cannot be detected either by fluorescence induction kinetics or by photoreduction of QA or pheophytin (Fig. 8). This photochemically silent state of PS II probably results from damage to the reaction center and represents centers in the process of repair. Upon repair, these centers become photochemically competent but remain impaired in the QA-QB electrontransfer reaction (PS II QB-nonreducing centers). The QB-nonreducing centers are later converted to a QB-reducing form and become fully active in the process of plastoquinone reduction. The cells regulate the inflow and outflow of centers from the QB-nonreducing pool in an attempt to maintain a constant pool size of PS II QB-nonreducing centers in the thylakoid membrane. To explain the increase in the QB-nonreducing pool size in the dark, we postulate that outflow of centers from the QBnonreducing pool is light-dependent and ceases immediately upon dark incubation of the cells. Meanwhile, the process of PS II addition to the QB-nonreducing pool continues in the dark until all PS II centers in the photochemically silent stage have become photochemically competent. This explanation of the data is consistent with the observation of a slight increase in the total number of photochemically competent PS II centers during this period of time (Fig. 4B), adequate to account for the increase in the number of QB-nonreducing centers. Low-light grown cells transferred to mediumlight conditions showed a rapid decrease in the pool size of QB-nonreducing centers (Fig. 7). This result implies that transient exposure of cells to higher light intensity conditions accelerates the outflow of PS II centers from the Qa-nonreducing pool. During this period there was no net change in the total number of photochemically competent PS II centers in the thylakoid membrane suggesting that

202 overall, PS II centers were neither degraded nor synthesized. Rather, the result suggests that QBnonreducing centers were being converted (or activated) to a QB-reducing form. When dark-adapted cells were placed in the light there was an immediate decrease in the concentration of QB-nonreducing centers (Fig. 7). This finding implied that the activation of the QBnonreducing centers is light dependent. Further experiments revealed that this activation will occur in weak light, i.e., in less than 10#Era 2s ~. Activation did not occur if cells were placed on ice (0 °C) implying that a conformational change or enzymatic reaction was involved. However, dark incubation of low-light grown cells did not result in a transient increase in the QB-nonreducing pool size in D. salina (Fig. 6A). This may be attributed to a slow rate of damage and repair of PS II centers in low-light resulting in a negligibly small concentration of photochemically silent centers. The question arises as to the size of the population of PS II occurring in the photochemically silent stage under physiological conditions. With cells grown under moderate light intensities (500/~E m-2 s- ~) the steady-state concentration of these silent centers represents approximately 10% of the total PS II centers (Fig. 4A). On the basis of photosystem stoichiometry (Table 1) and Chl antenna sizes in D. salina (Guenther et al. 1988), this would account for less than 3% of the total chlorophyll in the thylakoid membrane. Even if this population of silent PS II were larger at higher light intensities, it would need to exceed 35% of the total PS II centers before accounting for 10% of the total chlorophyll. Thus, under physiological conditions, the steady-state concentration of chlorophyll associated with PS II centers in the photochemically silent stage is low. We interpreted the fluorescence yield increase from F o t o Fpl to represent the accumulation of electrons on QA in PS II centers unable to transfer electrons from Q2 to QB. This differs from the interpretation of the initial fluorescence yield increase given by Schreiber and Neubauer (1987). We believe our approaches address altogether different phenomena, They measured changes in fluorescence yield upon illumination of intact chloroplasts with strong white actinic light (up to 26,00 #E m -2 s -1) which results in the reduction of the plastoquinone pool within a few millseconds.

Under these conditions they found the initial fluorescence yield to be modulated by the S-state of the Mn cluster in the water splitting complex. In contrast, our measurements were made with weak, nonsaturating green light (50 #Em 2s- ~) in which the rate of electron-transport to the plastoquinone pool was light-limited. In summary, the results from our work indicate that the relative concentration of PS II QBnonreducing centers in the thylakoid membrane is dynamic. Under continuous illumination the plant maintains a steady-state pool size of QB-nonreducing centers. A change in environmental light conditions creates transient changes in the pool size of the QB-nonreducing centers. Light is required for the conversion of QB-nonreducing to QB-reducing centers. The results are consistent with the proposed operation of a PS II repair cycle in the chloroplast (Baker and Webber 1988, Guenther and Melis 1989) in which, under physiological growth conditions, damage and repair to the PS II reaction centers occurs continuously and involves the PS II heterogeneity phenomenon. The rate of damage varies with the light intensity resulting in different rates of QB-nonreducing center accumulation and conversion. The cells regulate these rates, within limits, so that a steady-state concentration of PS II QB-nonreducing centers is maintained. The phenomenon discussed in this work may have significant implications for photoinhibition. Inhibition of photosynthesis occurs when the rate of light-absorption at PS II by far exceeds the rate of light utilization at the reaction center resulting in damage to the center (Powles 1984). According to the repair cycle model, chloroplasts would rapidly deplete their reserve of QB-nonreducing centers upon high light exposure. Photoinhibition would then manifest itself, when the rate of damage exceeded the rate of repair (Greer et al. 1986) such that the concentration of photochemically competent PS II centers in the thylakoid membrane was lowered significantly.

Acknowledgement The work was supported by NSF DCB-8815977 grant.

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