Chloroplast Membranes (Photosynthesis/Chloroplast Structure/Chloroplast Function/Reaction Center/Membrane Differentiation) JAN M

Home , Chloroplast membrane, Stroma (fluid), Thylakoid

Proc. Natl Acad. Sci. USA Vol. 80, pp. 745-749, February 1983 Biophysics

Localization of different photosystems in separate regions of chloroplast membranes (photosynthesis/chloroplast structure/chloroplast function/reaction center/membrane differentiation) JAN M. ANDERSON AND ANASTASIOS MELIS Division of Molecular Plant Biology, 313 Hilgard Hall, University of California, Berkeley, California 94720 Communicated by Daniel I. Amnon, November 4, 1982 ABSTRACT The stoichiometric amounts and the photoactiv- led to the hypothesis ofPSII heterogeneity in higherplant chlo- ity kinetics ofphotosystem I (PSI) and of the a and fl components roplasts (9). Analysis of the biphasic kinetics of PSII showed a of photosystem I (PSiH. and PSll^) were compared in spinach fast and nonexponential a component (PSIIa) and a slower ex- chloroplast membrane (thylakoid) fractions derived from ap- ponential (3 component (PSII,). The two types of PSII reaction pressed and nonappressed regions. Stroma-exposed thylakoid center complexes differ both in the effective light-harvesting fractions from the nonappressed regions were isolated by differ- Chl antenna size (10) and in the apparent midpoint redox po- ential centrifugation following a mechanical press treatment ofthe tential oftheir primary electron acceptor Q (11, 12). Mg2e ions chloroplasts. Thylakoid vesicles derived mainly from the ap- affect the organization of PSII. in the membrane but not that pressed membranes of grana were isolated by the aqueous poly- of PSII1l (13). Melis and Homann (13) proposed that PSII is mer two-phase partition method. Stroma-exposed thylakoids were located in grana partitions and PSII3 is located in stroma ex- foundtohaveachlorophylla/chlorophyllbratioof6.0andaPSII/ posed thylakoids. Further studies (14, 15) supported this con- PSI reaction center ratio of0.3. Kinetic analysis ofsystem II pho- cept, because the amounts of PSIIa and PSII, differed in mu- toactivity revealed the absence ofPSII, from stroma-exposed thy- tant tobacco compared with wild type, with PSII, increasing as lakoids. The photoactivity ofsystem Iin stroma-exposed thylakoids the amount of appressed relative to nonappressed membrane showed a single kinetic component identical to that ofunfraction- areaincreased. Hitherto, however, this hypothesis has not been ated chloroplasts, suggesting that PSI does not receive excitation the amounts ofthe two types ofPSII re- energy from the PSII-chlorophyll ab light-harvesting complex. tested by determining thylakoids are significantly enriched in both action center complexes in subthylakoid fractions. Thus, stroma-exposed In this paper, we report on the stoichiometric ratios and ki- PSI and PSp. Inside-out vesicles from the appressed membranes and PSI reaction ofgrana-partition regions had a chlorophyll a/chlorophyll b ratio netic properties of the photoactivity of PSII of2.0 and a PSI/PSI reaction center ratio of 10.0. The photoac- centers in thylakoid membrane fractions derived from grana tivity of system II showed the membranes of the grana-partition partitions and alsofrom stroma thylakoids. The results show that regions to be significantly enriched in PSI~,. We conclude that PSIIH is found exclusively in the grana-partition regions. In con- PSIIa is exclusively located in the membranes of the grana par- trast, stroma thylakoids are enriched in both PSI and PSII,,. titions while PSII, and PSI are located in stroma-exposed thylakoids. The low PSI reaction center (P700) content of vesicles de- MATERIALS AND METHODS ofthe PSI rived from grana partitions and the kinetic homogeneity Chloroplasts were isolated from leaves of spinach (Spinacia complex suggest total exclusion ofP700 as a functional component the Yeda grana-partition region. oleracea L.). The chloroplast isolation procedure, in the membrane of the press treatment, and the aqueous polymer two-phase partition mem- methods were similar to those described in ref. 5. Stoichio- The structural differentiation ofhigher plant chloroplast PSII and PSI reaction centers were branes into grana stacks and stroma-exposed thylakoids is par- metric measurements of with stroma-exposed thy- taken with a sensitive optical difference spectrophotometer (8). alleled by a functional differentiation, The geometry ofthe apparatus and the experimental conditions lakoids having mainly photosystem (PS) I and grana stacks being have been reported (16, 17). For enriched in PSII (1). The introduction ofaqueous polymer two- for the kinetic measurements inside- determination of the primary photoactivity kinetics of the PSI phase partition by Albertsson (2) allowed the isolation of chloroplasts were first treated with cy- out vesicles derived mainly from grana partitions-i.e., the ap- reaction center (P700), even more anide for 60 min to inhibit plastocyanin (18). The Chl concen- pressed membranes of the grana stacks-that were Chl b ratios were determined according enriched in PSII (3, 4). Andersson and Anderson (5) demon- trations and the a/Chl strated a marked lateral heterogeneity in the distribution ofthe to ref. 19. chlorophyll (Chl)-protein complexes along the thylakoid membrane of spinach chloroplasts. Thylakoid membrane vesicles RESULTS derived from the granapartitions showed an enrichment in PSII To avoid the use of harsh detergents, which might disturb the Chl-protein complex and its associated Chl ab-protein of the in vivo organization ofthe supramolecular complexes ofthe thy- light-harvesting complex (LHC) and a substantial depletion in lakoids, the chloroplasts were fractionated mechanically by the PSI Chl-protein complex. These results led to the hypothesis Yeda press treatment. The Yeda press fractionation ofthe spin- that PSI is excluded from the grana-partition regions (5, 6). In ach thylakoids was followed by separation of the grana stacks contrast, stroma-exposed thylakoids were enriched in PSI but (Y-40) from the stroma-exposed thylakoids (Y-100) by differ- they always contained a small complement of PSII (5-8). ential centrifugation (5). We then used the aqueous polymer Recognition of the biphasic nature of PSII photoactivity has two-phase partition (2) to separate the right-side-out vesicles Abbreviations: Chl, chlorophyll; PS, photosystem; LHC, light-har- The publication costs ofthis article were defrayed in part by page charge primary electron ac- payment. This article must therefore be hereby marked "advertise- vesting complex; P700, reaction center of PSI; Q, ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. ceptor of PSII. 745 Downloaded by guest on September 30, 2021 746 Biophysics: Anderson and Melis Proc. Natl. Acad. Sci. USA 80 (1983)

(fraction T-2), with both PSI and PSII characteristics, from the Time, s inside-out vesicles (fraction B-3), which are highly enriched in 0 0.2 0.4 PSII and are derived mainly from the granapartitions (3-5). The -. amounts of PSI and PSII reaction centers were measured directly from the light-induced oxidation of the P700 (PSI) and A from the light-induced reduction of Q, the primary electron acceptor of PSII. I I The Chl a/Chl b ratios and the Q and P700 contents of un- 0ID~~~-d fractionated thylakoids and various subchloroplast fractions derived from appressed and nonappressed membrane regions are compared in Table 1. In mature unfractionated chloroplasts, the Chl a/Chl b ratio was about 2.8 and the ratios Chl/P700 = 580 and Chl/Q = 297 were comparable with those reported (20) for a variety ofhigher plant chloroplasts. Thus, spinach thylakoids appeared to have almost twice as much PSII reaction center as PSI reaction center (Q/P700 = 1.95). The stroma-exposed thylakoid membranes (fraction Y-100) FIG. 1. Kinetic traces: time course of P700 photooxidation. (A) derived from the Yeda press treatment (5) had a Chl a/Chl b Unfractionated chloroplasts at 238 ,uM Chl (a+b) in the presence of ratio ofabout 6 and were significantly enriched in PSI. The Q/ 200 puM methylviologen. Chl a/Chl b = 2.79; result presented is the P700 ratio was 0.3 in agreement mean of 16 individual measurements; AA700 = lo-3 (B) Isolated with a previous study (8). In stromathylakoidsat 23 ,IMChl (a+b) inthe presence of 200 p.M meth- contrast, the inside-out thylakoid vesicles, fraction B-3 derived ylviologen. Chl a/Chl b = 6.0; result presented is the mean of 32 in- mainly from the chloroplast partition regions, had a low Chl a/ dividual measurements; AA700 = 5 x 10'. Before fractionation, the Chl b ratio (about 2.1) and were markedly enriched in PSII (Q/ isolated chloroplasts were treated with cyanide to inhibit plastocyanin P700 = 9.6). As expected from theirknown photochemical char- (18). acteristics (4), the right-side-out vesicles (fraction T-2) from the grana, were slightly depleted in Q relative to chloroplasts (Table functionally homogeneous, in contrast to the observed heter- 1). ogeneity of PSII in higher plant thylakoids (9-15). Hence, a Comparison of the amount of P700 in stroma-exposed thy- careful comparison of the kinetics of the PSI photoactivity was lakoids (Y-100) with that in the grana partitions (B-3) shows a undertaken for unfractionated chloroplasts and the stroma thy- 10-fold depletion ofP700 in fraction B-3 relative to Y-100 (Table lakoid fraction (Y-100). 1). This is similar to the depletion of the PSI complex assayed The photooxidation kinetics of P700 induced by weak con- previously by NaDodSO4/polyacrylamide gel electrophoresis tinuous illumination is shown in Fig. 1. In this approach, the (5). Since fraction B-3 is known to be contaminated with right- rate of P700 photooxidation is limited by the rate of light ab- side-out vesicles (4), the depletion of P700 from the membrane sorption by PSI. The rate of light absorption by each PSI unit ofthe partition region must be greater in vivo than that detected depends directly on the antenna size of the light-harvesting in this study. These results support the concept of the lateral pigments associated with PSI. In principle then, one can obtain separation ofthe two photosystems in the thylakoid membrane an accurate estimate of the antenna size of the light-harvesting ofhigher plant chloroplasts (see Discussion). The results are in pigments of PSI from measurement ofthe kinetics of P700 pho- agreement with those of a previous study (8), which showed tooxidation (16). Fig. 1A shows the P700 photooxidation kinetics grana stacks (Chl a/Chl b, 2.3) to have a Q/P700 ratio of 2.5. of unfractionated chloroplasts while Fig. 1B presents the P700 They disagree with a third study (21), which showed equal kinetics from stroma thylakoids. The semilogarithmic plot ofthe amounts of Q and P700 and invariable Chl/Q and Chl/P700 kinetic data of Fig. 1 is a straight line that has the same slope ratios in chloroplasts and inside-out vesicles. for both samples (Fig. 2). Thus, it is verified that the P700 pho- In view of the extreme depletion of the PSI complex in the tooxidation is a monophasic first-order function of time occur- grana partitions, it was suggested that perhaps all of the PSI ring with the same rate in both unfractionated chloroplasts and complex is excluded from the partition region in vivo (5, 6, 22). the isolated stroma thylakoid fraction. Physical separation ofthe Ifthis were so, then the PSI complex might be structurally and PSI complex in the stroma thylakoid fraction from the PSII-Chl

Table 1. Chl a/Chl b ratios and Q and P700 contents in chloroplast membrane fractions 0 Chla/Chl b Chl/P700 Chl/Q Q/P700 Chloroplasts 2.78 580 297 1.95 Stroma thylakoids (Y-100) 5.9 262 875 0.30 -2 Inside-out grana partitions (B-3) 2.07 2,642 273 9.6 - Right-side-out grana 1 -3 (T-2) 2.81 534 316 1.7 Unfractionated chloroplasts, stroma thylakoids (Y-100), and grana -4F partitions (B-3) were obtained asdescribed inref 5. Chl/P700 andChl/ Q ratios are based on total Chl (a+b) in the sample. Q and P700 con- 0 0.2 0.4 centrations were measured as described in ref. 8. Reproducibility ofthe Time, s results forunfractionated chloroplasts, isolated stroma thylakoids, and the right-side-out vesicles (T-2) was 10-20%. Different inside-out FIG. 2. First-order kinetic analysis of the traces shown in Fig. 1. grana-partition (B-3) preparations showed greater variability (i.e., Q/ Unfractionated chloroplasts (o) and isolated stroma thylakoids (o) P700 ratio variations were 6-10). gave identical rate constants as shown by the semilogarithmic slopes. Downloaded by guest on September 30, 2021 Biophysics:- Anderson and Melis Proc. Natd Acad. Sci. USA 80 (1983) 747 ab LHC in the grana did not result in the loss of any portion of the functional light-harvesting antenna of PSI. The result shows that the antenna unit size of PSI in stroma thylakoids is identical to that ofintact chloroplasts. The finding that the antenna unit size ofPSI in unfractionated chloroplasts is the same as that in isolated stroma thylakoids a -2 excludes the possibility that PSI was also located in the membranes of the grana-partition regions where it would have in- -3 _ teracted with the PSII LHC. The evidence suggests thatin vivo the PSI complex is exclusively located in stroma-exposed thylakoids. -4 Unlike those of PSI, the primary photoactivity kinetics of 0 0.2 0.4 0.6 PSII are complex (9, 10). In view ofthe PSII differentiation into Time, s PSIIH and PSII in higher plant chloroplasts, we undertook to test the hypothesis (13, 14) that membranes of the grana-par- FIG. 4. First-order kinetic analysis of fluorescence traces shown tition regions and stroma-exposed thylakoids are enriched in in Fig. 3;The relative PSII concentration in different unfractionated PSIIa and PSII,, respectively. We did so by measuring the Chl chloroplast preparations (o) was 20-35% of the total PSII; in the iso- a fluorescence induction kinetics ofthe various thylakoid prep- lated stroma thylakoids (e), it was 85-99% of total PS11. arations in the presence of the electron transport inhibitor 3- mea- In this approach, the rescence induction in Fig. 5B. From the results ofseveral (3',4'-dichlorophenyl)-1, 1-dimethylurea. surements, we have determined that, in the grana partitions, area over the fluorescence induction curve is directly propor- of. 23). Comparison the relative concentration of PSII, was reduced to 25-35% tional to the photoreduced fraction of Q (10, that found in the control chloroplasts. The result provides fur- of the fluorescence induction curves of unfractionated chloro- that PSII,3 is excluded from the plasts and stroma-exposed thylakoids shows that the slow P. ther support for the hypothesis component accounts for a.small portion ofthe variable fluores- partition region of grana thylakoids. cence in,the unfractionated chloroplasts but for a major portion DISCUSSION 3). of the variable fluorescence from stroma thylakoids (Fig. The results presented in Table 1 for the amounts ofQ and P700 A quantitative presentation ofthe same results (Fig. 4) shows show an enrichment of Q and a significant depletion of P700 in. the semilogarithmic plots oftheareas over the two fluorescence the membranes of the grana partitions and an enrichment of kinetic curves (13, 23). From the intercepts ofthe linear phases P700, hence ofPSI, in the stroma thylakoid region. They agree with the ordinate at zero time, in several different preparations,- with the previous study (5), which compared the relative dis- we determined that PSII.3 accounts for 20-35% ofthetotal PSII in the chloroplasts but for 85-99%ofthe total PSII in the stroma thylakoid fraction. This observation directly supports the hypothesis that ( centers are located in stroma-exposed thylakoids 0.8 - A (13, 14). Recently (17), we presented evidence based on kinetic ==j 06 B- = analysis suggesting that the fluorescence yield ofPSIIa, but not that ofPSII,,, strongly depends on the Mg-' content ofthe chlo- 0.6- roplast suspension medium. In this work, we verified directly 10.02 . that the fluorescence yield from stroma thylakoids is largely 0.4- independent of Mg2" ions (data not shown). The fluorescence induction kinetics of unfractionated chlo- ~0 roplasts (C) and inside-out vesicles derived from the chloroplast partition region (B-3) are compared-in Fig. 5.. In contrast to the 0 0.2 0.4 results in Fig. 3, the fluorescence kinetics ofthe thylakoids from the grana-partition region are-considerably faster than those of the control chloroplasts, suggesting selective elimination ofthe slow (3 phase from the appressed membrane fraction. This is -1 evidenced in the semilogarithmic plots of the areas over fluo-

-3-3 8a) -4- 0Go a) -5. ~0 0 0.2 0.4 a) 5.4 Time, s 0 0.2 0.4 0.6 0.8- FIG. 5. (A) Kinetics of the variable Chl a fluorescence of unfrac- = Time, s tionated chloroplasts (curve C; Chl a/Chl b 2.9) and inside-out thylakoids derived from the 'grana-partition regions (B-3, Chl a/Chl b of a of unfraction- = 2.2). Conditions are as in Fig. 3. (B) First-order kinetic analysis FIG. 3. Kinetics of the variable Chl fluorescence concentration = isolated stroma the fluorescence traces shown in A. The relative PSI[[ ated chloroplasts (Chl a/Chl b 2.65; upper curve) and 25% of the = The reaction mixtures in the unfractionated chloroplasts (o) was approximately thylakoids (Chl a/Chl b 5.7; lower curve). the contained 30 ,uM Chl (a+b)/50 PM 3-(3',4'-dichlorophenyl)-1,1-di- total PS1; in the inside-out thylakoids derived from grana parti- methylurea/2 mM hydroxylamine. tions (e), it was5-10% of total PS11. Downloaded by guest on September 30, 2021 748 Biophysics: Anderson and Melis Proc. Natl. Acad. Sci. USA 80 (1983) tribution of Chl-protein complexes in the two membrane regions. Here, we found only 20% of the total P700 in the B-3 fraction compared with that ofchloroplasts (Table 1). This par- allels the depletion of the coupling factor (CF1) in the B-3 fraction (24). Because CF1 is likely to be restricted to nonappressed membranes only (25), its presence in the B-3 fraction could be attributed to contamination (24), as is the case with P700. Thus, one may argue that the low P700 content ofthe grana-partition DP pi vesicles (B-3) represents contamination from stroma-exposed CFE 1- ~ SUEM thylakoids containing CF1 (4, 24) and perhaps less than perfect CFI ( Grno prtition ) } separation ofthe partition region from the adjacent stroma thylakoid region. Therefore, it is highly likely that the chloroplast FIG. 6. Schematic showing the large membrane area where adja- grana-partition region in vivo has a Q/P7-00 ratio much higher cent grana thylakoids are appressed at the partition region and the than the detected ratio of9.6, probably exluding P700 as a func- much smaller stroma-exposed grana margins. PSll. is exclusively lo- tional component ofthis appressed membrane region altogether cated in the grana-partition membrane region along with most of the (5, 6). plastoquinone pool (8). The electrochemical proton gradient generated The kinetic analysis ofsystem II photoactivity (Fig. 5) showed specifically by the turnover of PSI1, is dissipated via the coupling fac- -contain tor (CF1), driving ATP synthesis from ADP and inorganic phosphate. that grana-partition regions primarily PSIIa and are The photolysis of H20 occurs in the hydrophobic region of the mem- depleted ofPSII1. The one-third to one-fourth ofPSII, detected brane, and the resulting electrons are processed by PSH1, (data not in fraction B-3 can be attributed to the known contamination shown). ofthis fraction with about 25% right-side out vesicles from nonappressed membranes (4). The overall structural-functional profile of the grana-partition region, therefore, is one of high located together with PSI. Thus, PSIIa and PSII, differ in their PSII, density and a lack ofboth PSII, and PSI reaction center -structural and functional arrangements [photochemical antenna complexes. We propose that such a structural arrangement in size difference = 2-2.5 (10), apparent midpoint redox potential the grana partitions favors the build-up of an electrochemical difference for their primary electron acceptor Q (11, 12), and proton gradient resulting from H+ release specifically by the lateral separation along the chloroplast thylakoid membrane photooxidation ofH20. This hypothesis is supported by the fol- (13)]. lowing concurring conditions unique for grana-containing chlo- The functional significance ofthe differentiation ofPSII. and roplasts. (i) The small lumen volume of grana thylakoids mini- PSII, in mature higher plant chloroplasts is unknown. The mizes the space within which an electrochemical proton major PSIIa with its larger antenna size and enhanced Chl b gradient is formed. (ii) The high density ofwater-splitting and content will be the predominant source of electrons for linear proton-evolving PSII, in the partition region ofgrana thylakoids electron transport and noncyclic photophosphorylation. The provides the bulk of protons released during linear electron location of PSII, in appressed membranes ensures maximum transport. These protons are necessary for build-up ofthe elec- trapping of PSII light and hence ensures that the pool ofplas- trochemical proton gradient in the small space ofthe grana thy- toquinone will be mainly reduced so that PSI, which governs lakoid lumen, important for the efficient generation of ATP. overall photosynthetic rate, can function effectively (22). The (iii) The hydrophobic nature of the grana-partition region may minor PSII, with its smaller antenna size and relatively less minimize furtherproton loss (leakage) across most ofthe granum chlorophyll b may be required in stroma-exposed thylakoids to thylakoid membrane. The electrochemical proton gradient re- ensure a slow electron flow, enough for regulatory functions and sulting from the photochemical activity of PSIIa would be con- to poise the cyclic photophosphorylation (28). sumed by ATP formation by the coupling factor bound nearby In contrast to the observed functional heterogeneity of PSII on the stroma-exposed margin area of each granum thylakoid in higher plant chloroplasts, there is no indication of hetero- (Fig. 6). If this were the case, one would expect the grana thy- geneity in PSI (16, 29). The combination ofthe.Yeda press and lakoids to be a PS11-specific domain for protons released by the the aqueous two-phase partition treatment of the thylakoids oxidation ofwater (26, 27). Although the thylakoid lumen ofthe resulted in significant separation of PSII,, from PSI. Thus, the grana is known to be continuous with that ofstroma thylakoids, PSI complex was largely separated from the main Chl ab LHC it is conceivable that there exists a mechanism preventing pro- proteins contained in the.grana partition. The identical mono- tons released in the grana thylakoids from migrating into the phasic first-order P700 photooxidation kinetics in unfraction- lumen of stroma thylakoids and vice versa (27). Thus, it appears ated chloroplasts (Fig. 1A) and in isolated stroma thylakoids that the structural differentiation of membranes into (Fig. 1B) argue that the physical separation of PSI from the Chl chloroplast ab LHC occurred without a loss in light-harvesting pigment for grana and stroma thylakoids facilitates both a lateral separation PSI. The of the various electron transport components and a differentia- only interpretation of this result is that, in vivo, PSI tion of the electrochemical proton-gradient-formation process cannot receive excitation energy from the PSII-Chl ab LHC; at the two sites. i.e., PSI occurs as a separate entity in the stroma-exposed thy- In addition to PSI, stroma-exposed thylakoids possess some lakoid membranes. Hence, the continuous array models show- PSII activities (7), about 10-12% ofthe total PSII-associated Chl ing that the main Chl ab LHC contacts both PSI and PSII re- ab LHC (5) and significant amounts of Q (8), having a Q/P700 action center complexes (30) are not tenable. This has been ratio of0.3 (Table 1). The kinetic analysis ofsystem II photoac- suggested recently (16, 17) in direct measurements of the con- tivity (Figs. 3 and 4) showed stroma-exposed thylakoids to control exerted by Mg2+ on the purported distribution of excitation tain PSII, and lack PSII,. Because the stroma thylakoids are energy from the Chl ab LHC to PSII and PSI: no appreciable unlikely to be contaminated with grana-partition fragments, due effect of Mg2+ could be detected on the rate of-light absorption to the gentle fractionation by the Yeda press, the result suggests by PSII and PSI. The important implication of our findings is that PSII, is normally located only in stroma-exposed thyla- that interpretations on the phenomena of "spillover" and thy- koids. Such a locus would be found either in the end thylakoids lakoid unstacking (31), membranephosphorylation (32, 33), and of the grana stacks or in the stroma thylakoids so that PSII,3 is the state I-state II transition (34) may have to be reassessed. Downloaded by guest on September 30, 2021 Biophysics: Anderson and Melis Proc. NatL Acad. Sci. USA 80 (1983) 749

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