Photosynthesis Research 10." 189-196 (1986) © Martinus N i j h o f f Publishers, Dordrecht - Printed in the Netherlands

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RELATIONSHIPS AMONG CELL CHLOROPHYLL CONTENT, PHOTOSYSTEM II LIGHT-HARVESTING AND THE QUANTUM YIELD FOR OXYGEN PRODUCTION IN CHLORELLA* ARTHUR C. LEY

i. ABSTRACT Cells of the green alga Chlorella vul~arls were grown under conditions where total Chl/cell varied by a factor of almost 80; from 0.02 fmol/cell to nearly 1.6 fmol/cell. The change in Chl/cell was accomplished by an approximately ll-fold increase in RCII/cell along with a 7-fold increase in Chl/RCII. The effective absorption cross section per RCII at 596 nm varied by a factor of 6, increasing with Chl/cell from a minimum of 20 A 2 to a maximum of 116 A 2. In contrast, over the same range of Chl/cell, the quantum requirement for 02 production remained relatively constant at I 0 . 4 ~ 1.8 quanta absorbed/O 2 evolved. The results are well described by a simple model in which changes in Chl/cell are produced by coordinated changes in reaction center and llght-harvesting complexes. The model predicts that between 20 and 40% of the light-harvesting chlorophyll-protein complexes commonly assigned to PSII, do not function as antenna for PSII. 2. INTRODUCTION Photosynthetic organisms often respond to changes in environmental conditions with changes in the composition of the photosynthetic apparatus. For example, both total environmental irradlance and its spectral distribution, have been shown to influence the composition of the photosynthetic apparatus (1-9). Organisms ranging from cyanobacterla (4,9) to unicellular eucaryotlc algae (2,3,7,8) to ferns (6) and higher plants (1,5) have all been shown to respond to changes in environmental light conditions. The responses which have been reported include changes in photosynthetic rates and turnover times (2,3,5,6), pigment content (I9), reaction center (RC) ratios (3-6) and photosystem antenna sizes (3,4,5-7). Advances in biochemical expertise have permitted the quantitative description of the pigment composition of plants in terms of specific plgment-protein complexes (10-20) of known compostion and function. Recent experiments have demonstrated that the relative amounts of the various pigment-protein complexes can also respond to changes in environmental conditions (5,6).

~This paper is dedicated to the memory of Warren L. Butler, a pioneer in the field of photobiology. His creativity and innovativeness as a scientist were matched only by his patience as a mentor.

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In a previous study (7) it was reported that culture conditions influenced the values of several PSll-related parameters in Chlorella including cell Chl content, absorption cross sections, and photosynthetic unit sizes. However, under the same conditions, the quantum requirement for 02 production was essentially unchanged. This report describes the results of a more extensive study of the relationships between cell Chl levels, PSII llght-harvesting properties, and the quantum requirement for 02 production. 3. MATERIALS AND METHODS Cells of the green alga Chlorella vulgaris were grown in batch cultures exposed to a wide range of irradian---------~esbetween about 106 erg/cm2-s and 102 erg/cm2-s as previously described (7). Cell cultures were grown with no additional aeration or CO 2 enrichment and it is likely that the cultures growing at the highest irradiances were CO2-1imited (21). In addition, due to their proximity to the metal arc lamp, the high irradiance cultures grew at somewhat higher temperature (25°C) than did the other cultures (20°C). Thus, several environmental factors probably influenced the properties of the cells reported here. Since the aim of this work is to investigate the more fundamental relationships between cell Chl levels and PSII properties, no effort has been to causally relate any cell property to any extrinsic environmental variable. Chl concentrations were determined from ethanol extracts of cells as described previously (7). When the ratio of Chl-a:Chl-b was greater than 5, a room temperature flourescence assay similar to that of Boardman and Thorne (22) was used. Calibrations using known mixtures of purified Chl-a and Chl-b in 95% ethanol indicate that this procedure is accurate for ratios of Chl-a:Chl-b up to 25. Cell counts were made in quadrupicate using a hemocytometer. Measurements of the photosynthetic unit size for 02 production (PSUo2) were determined as described previously (3) using flash rates of 5, I0, 15, and 20 Hz. As has been reported by Myers and Graham (23), the addition of a continuous far red (704 nm) irradiance had no effect on the magnitude of the 02 flash yields. The number of PSll reaction centers (RCII) per cell were calculated from PSUo2 and Chl/cell using the assumption of 4e" per 02 . Effective absorption cross sections per RCII (6-o~) were determined at 596 nm from laser flash energy saturation curves as described previously (7). The quantum requirement for 02 production was calculated from PSUo2, O--oz, and the in rive 596 nm absorption cross section of a Chl molecule,6-'~ as (15): Quantum Requirement = P5~o~ "~I (i) 4.RESULTS The culture growth conditions used in this study produced large changes in the pigment and PSll properties of Chlorella cells (Table I, Fig. IA-E). The data shown in Table I are arranged in order of decreasing Chl-(a+b)/cell, which correlates roughly with increasing growth irradiance. Cells containing more than 1.0 fmole Chl or less than 0.I fmole Chl grew at the lowest and highest irradiances, respectively. The values for Chl/cell shown in Table I vary by a factor of almost 80, from about 0.02 fmol/cell to about 1.6 fmol/cell. Over this range, Chl-a/cell increases by a factor of 60, while Chl-b/cell increases more than 800-fold. As a result, the ratio of Chl-a:Chl-b changes by at least an order of magnitude from _< 0.04 to about 0.4. The variation in Chl/cell is produced by changes in both RC/cell and

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TABLE I. Pigment and photochemical characteristics of Chlorella cells h a v i n ~ different chlorophyll ~on~e~t, Chl I Chl a I Chl h I Chl b PSU022 RCII3 Cross 4 Chl Quantum 5 Cell Cell Cell Chl a Cell Section RCII Requirement 1.57 1.15 0.42 0.37 5480 6.9 113 377 14.5 1.32 0.98 0 34 0.35 4500 7.1 116 387 11.6 0.82 0.60 0 22 0.32 nd --95 317 .... 0.71 0.54 0 17 0.32 nd --96 320 .... 0.71 0.52 0 19 0.37 nd --98 327 .... 0.68 0.51 0 17 0.33 nd --90 300 .... 0.58 0.43 0 13 0.28 nd --85 283 .... 0.54 0.43 0 Ii 0.26 nd --74 247 .... 0.52 0.41 0 Ii 0.27 2800 4.5 nd ....... 0.51 0.39 0 12 0.31 nd --95 317 .... 0.50 0.39 0 Ii 0.30 2250 --80 267 8.4 0.38 0 12 0.50 0.33 3170 3.8 nd ....... 0.49 0.39 0 I0 0.27 2300 5.1 nd ....... 0.38 0 II 0.49 0.28 2150 5.5 nd ....... 0.48 0.37 0 ii 0.30 nd --95 317 .... 0.38 0 I0 0.48 0.27 2300 5.0 83 277 8.3 0.46 0.35 0 11 0.32 2910 3.8 82 273 10.7 0.44 0.35 0.094 0.27 3230 3.3 nd ....... 0.44 0.34 0.I0 0.30 nd --69 230 .... 0.43 0.34 0.089 0.26 nd --74 247 .... 0.43 0.33 0.096 0.29 1970 5.2 nd ....... 0.41 0.31 0.095 0.31 nd --82 273 .... 0.40 0.32 0.082 0.26 nd --71 237 .... 0.40 0.32 0,089 0.24 nd --78 260 .... 0.38 0.29 0.088 0.30 3000 3.0 nd ....... 0 37 0.30 0.075 0.25 2240 4.0 70 233 9.6 0 37 0.30 0.074 0.25 nd --65 217 .... 0 36 0.29 0.073 0.26 2590 3.4 83 277 9.4 0 36 0.29 0.073 0.26 2270 3.8 74 247 9.2 0 34 0.26 0.083 0.32 2930 2.8 nd ....... 0 34 0.26 0.78 0.30 2370 3.5 67 223 10.6 0 33 0.27 0.064 0.24 2180 3.6 77 257 8.5 0.25 0.065 0 31 0.26 2390 3.1 82 273 8.8 0 3O 0.25 0.065 0.26 2010 3.6 nd ....... 0 27 0.22 0.047 0.22 2600 2.5 nd ....... 0.22 0.049 0.23 2600 2.4 nd ....... 0 26 0 19 0.16 0.034 0.20 2090 2.2 nd ....... 0.22 2260 2.0 nd . . . . ~-0 19 0.16 0.037 0 17 0.15 0.021 0.14 1480 2.8 42 140 10.6 0 17 0.14 0.030 0.15 1540 2.6 43 143 10.8 0 16 0.14 0.022 0.15 1260 3.1 38 127 9.9 0.13 1150 2.7 nd ....... 0 13 0.ii 0.014 0.ii 0.010 0.09 1340 2.2 nd ....... 0 12 0.093 0.009 0.10 1300 1.9 nd ....... 0 I0 0.14 910 2.4 nd ....... 0.092 0.080 0.011 0.06 1030 2.0 22 73 14.1 0.085 0.080 0.005 0.04 850 1.4 21 70 12.1 0.051 0.002 0.053 0.035 0.004 0.10 760 1.2 nd ....... 0.039 0.04 670 1.0 22 73 9.2 0.028 0.027 0.001 0,04 960 0,6 nd ....... 0.021 0,001 0.022 2. mole Chl • flash/mole 02 3. 10b/cell 1. fmol/ce11 Units: 4. A 2 5. photons absorbed/O 2 p r o d u c e d

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Chl/RC. Values for PSU02 shown in Table I vary from about 800 Chl/O 2 to about 5500 Chl/O 2. Assuming that the PSUo2 measurements count all RCII (7,8,23,24), this variation in PSUo2 implies an ll-fold change in RCII/eelI, from 0.6x105 to 7xlO 5, and a 7-fold change in total cell Chl per RCII (-PSUo2--'. 4), from about 200 to about 1400. The effective absorption cross section per RCII (O--o~) provides a direct measure of the llght-harvesting power of the PSII antenna i_nnvivo. (For a more detailed discussion of the measurement and significance of O--oi see ref. 7 and the article by Mauzerall elsewhere in this volume.) Table I presents values for O-~L determined at 596 nm (the wavelength of the laser used for the measurements) in Chlorella cells having greatly different Chl content. Cross sections vary nearly 6-fold with increasing Chl/cell, from a minimum of 21 A 2 to a maximum of 116 A 2. For "bookkeeping" purposes it is useful to convert the measured in vivo cross section values into the specific number of Chl molecules acting as antenna for a RCII. The in rive absorption cross section for Chl at 596 nm, 0.3 A 2 (7), allows this conversion. The results of this calculation are shown in the eighth column of Table I. As total cell Chl levels change by a factor of 80, the specific size of the antenna available to an individual RCII changes by a factor of 6, from about 70 to nearly 400 Chl functioning as PSII antenna per RCII. The final column in Table I presents values for the quantum requirement for photosynthetic 02 production by cells having greatly different Chl content. Remarkably, despite the large variations in all the other parameters listed in Table I, the quantum requirement is essentially constant. The variation between the largest and smallest values shown in Table I is less than a factor of 2 and the average of all the measurements is 10.4 ~ 1.8. The quantum requirement of cells having very high or very low Chl/cell may be significantly greater than that of cells having intermediate levels of Chl/cell ( 1 2 Z 2 v s 9 . 6 ~ 0 . 9 5 , respectively). 5. DISCUSSION The values for the quantum requirement shown in Table I are larger than the theoretical minimum obtained by assuming that photosystems operate with no photochemical losses, that PSI is the sole oxidant of PSlI, and that cell Chl is equally divided between PSI and PSII. As shown by Eq. (i), the quantum requirement can be derived from three measured quantities: PSUo2,O-o~ I , and O-o~ It has previously been argued (7), that the measured values for O ~ are not greatly decreased by photochemical inefficiencies in PSII. The value for O-c~i is near the lower end of the range of molecular cross sections measured in vitro for Chl-a and Chl-b in various solvents (0.26 A 2 to 0.42 A 2, dep~ndlng on the Chl species and solvent, unpublished observations). Thus it appears that the quantum requirement is "too high" because, to a large extent, PSUo2 values are "too large". The observation, reported by Myers and Graham (23) and confirmed here, that a far red background irradianee does not increase 02 flash yields suggests that PSI is not limiting in the PSU02 measurement. If this is the case, the quantum requirement for 02 production is a direct reflection of the fraction of cell absorbance at 596 um that contributes to functional PSII llght-harvesting. The "elevated" values observed for the quantum requirement imply that this fraction is less than 0.5. Such a situation would arise if a significant fraction of the PSII antenna was associated with non-functlonal RCII (or reaction centers in which non-photochemical losses occur during the dark processes of 02 production), or if at 596 nm the total cell PSI absorption exceeds that of PSII by 20 to 30%.

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Fig. IA shows that Chl-b/cell is a remarkabl~ linear function of Chl-a/cell. A llnear regression on the data (r~-0.985) has a slope of 0.38 (corresponding to an integral ratio Chl-a:Chl-b of 8:3) and an x-axis intercept of 0.08 fmol Chl-a/cell. Cells having less than about 0.08 fmole Chl-a contain essentially no Chl-b. Since Chl-b is thought to be contained only in the light-harvesting complexes of PSI and PSII (i0,Ii,14,16,18,20), it may be that these cells contain only RC complexes. Two aspects of the data shown in Table I support this possibllty. The first is that at low Chl/cell, PSUo2 values are similar to those measured in organisms (cyanobacteria and red algae) which lack Chl-b and LHCP (4,9). The second is that ~-5~ is constant at 21 A 2 for low values of Chl/cell. Thus for cells containing less than 0.08 fmol Chl-a, changes in Chl/cell would be accomplished solely by changes in RC/cell. 21 A 2 corresponds to an l n v l v o RCII complex antenna size of about 75 Chl-a. This is larger than the value of 50 Chl-a/RCII observed with isolated complexes (12,19). It may be that the in vlvo complex is larger than the isolated particle, or that the value for o-c~ in the complex is larger than the overall average for Chl in the cells. At low Chl/cell, the ratio of total cell Chl to RCII is about 220:1. Since 75 Chl-a act as antenna per RCII, about 145 Chl-a are associated with PSI per RCII. Furthermore, since both Chl/cell:RCII and O-o~ are constant at low Chl/cell, it seems likely that the ratio RCI:RCII is also constant in this range. The ratio RCII:RCI in Chlorella has been reported to be about 1.4:1 (23). If this is the case at low Chl/cell, the RCI complex contains about 200 Chl-a as antenna. If, on the other hand, the RCI complex is similar to that deduced for cyanobacteria (9) or the PSI "core" particle of higher plants (17,18) and contains about 120 Chl-a, then the ratio RCII:RCI at low Chl/cell is about 0.8. For values of Chl-a/cell greater than about 0.08 fmole, Chl-b/cell is almost a linear function of Chl-a/cell. This observation implies that increases in cell Chl are achieved by the addition of Chl-a and Chl-b in a fixed molar ratio. Correction of the data shown in Fig. IA for the measured increase in RCll/cell (with the associated increase in Chl-a only) gives the ratio with which Chl-a and Chl-b are added in light-harvestlng complexes: Chl-a:Chl-b - 2.1:1. The data in Table I can be used to calculate that for each increment of 2xlO 5 RCII/cell, total cell Chl/RCII also increases by about 210 Chl. Of these 210 Chl, 140 are Chl-a and 70 are Chl-b. At present, two general classes of llght-harvesting plgment-proteln complexes can be distinguished: LHCP and LHCI (5,6,10,16,20). LHCPs are characterized by relatively low ratios of Chl-a:Chl-b (between i:I and 2:1) and are usually considered to function only as antenna for RCII (5,6,10,11). LHCI (or CP O) functions in the PSI antenna and has relatively high ratios of Chl-a:Chl-b; 4:1 for LHCI (13,14,16) and about 6:1 for CP O (15,20). The results described above suggest a simple model relating relating cell Chl content, numbers of RCII and their antenna sizes, and quantum requirements. Cells can contain RC complexes (RCI and RCII) and light-harvesting pigment-proteln complexes (LHCP and LHCI). The RCII complex contains 75 Chl-a acting as antenna per photochemically active site. An additional 145 chl-a per RCII are associated with PSI (with an unspecified number of RCI). When cells contain less than about 2xlO 5 RCII, they contain no LHCP or LHCI and changes in Chl/cell result solely from changes in RC/cell. Cells having more than 2xlO 5 RCII/cell contain both reaction center and light-harvesting complexes. Changes in Chl/cell

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FIGURE LEGEND: Figure I. Relationships between cell Chlorophyll-a levels and other physiologically important parameters in Chlorella. A.) Chlob/cell B.) FSUo2, C.) RCII/cell, D.) ~-oz , and E.) Quantum Requirement for 0 2 production. The curves drawn t h r o u g h t h e data were calculated as described in the text.

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are accomplished by coordinated changes in the numbers of both RC and light-harvesting complexes. Each addition of 105 RCII is accompanied by an increase in the ratio of total cell Chl to RCII by the addition of 208 Chl/RCII. 121 of the 208 Chl (67 Chl-a and 54 Chl-b, Chl-a:Chl-b-l.2:l) are associated with LHCP. The remaining 87 Chl (70 Chl-a and 17 Chl-b; Chl-a:Chl-b-4.1:l) function in LHCI. This model was used to calculate the curves shown in Fig. IA-E. Considering the oversimplifications inherent in the model, the calculated curves fit the data quite well. For simple bulk properties of the cells (Chl-b v__ssChl-a, PSU02 v__ssChl-a, and RCII v s Chl-a), the model is adequate as described above. However, calculations o f PSII-specific properties such as cross sections or quantum yields require that the distribution of light-harvesting complexes be specified. In Fig. ID,E, three curves are plotted. The solid curves were obtained by assuming that LHCP associates exclusively with RCII and that LHCI functions solely in PSI. The two other curves in panels D and E were obtained by assuming that some fraction, designated "f", of LHCP did not function as antenna for RCII. In both panels, the dashed and dotted curves were obtained using f-0.2 and f-0.3, respectively. It is clear from the plots shown in Fig. 1 that f-0 does not fit the data. It is possible to generate fits to the data similar to those shown in Fig. 1 by assuming that LHCI has ratios Chl-a:Chl-b which are greater than 4 (and thus is more similar to CP O). In these cases, even larger values for f are required (up to 0.4 for Chl-a:Chl-b - 6) to provide fits to the data comparable to those shown in Fig. ID,E. Finally, it appears that the data shown in Fig. 1 might be best fit if f is allowed to increase with increasing Chl/cell; i.e., as Chl/cell increases, the fraction of LHCP which functions in the PSII antenna decreases. It is clear from plots such as those shown in Fig. I D , E that a significant fraction (between ~20 and 40%, depending on assumptions concerning the composition of LHC!) of LHCP does not function as light-harvesting antenna for PSII. Presumably, this fraction is available for use by PSI. In this case, the magnitude of f might be controled via the operation of a protein kinase/phosphotase system similar to that described in higher plants (25,26). 6. ACKNOWLEDGEMENTS This research was supported in part by the United States Department of Agriculture (Contract # 5901941 9-03289) and the National Science Foundation (Grant # PCM 8316373). I thank Dr. David Mauzerall for many helpful discussions and suggestions throughout the course of this work. 7. REFERENCES i. 2. 3. 4. 5. 6. 7. 8. 9.

Boardman NK (1977) Ann. Rev. Plant Physiol. 2 8 : 3 5 5 - 3 6 3 Falkowskl PG and Owens TG (1980) Plant Physiol. 6 6 : 6 3 2 - 6 3 5 Falkowski PG, Owens TG, Ley AC and Mauzerall DC (1981) Plant Physiol. 6 8 : 9 6 9 - 9 7 3 Kawamura M, Mimuro M and Fujita Y (1979) Plant Ceil Physiol. 20: 697-705 Leong T-Y and Anderson JM (1984) Photosyn. Res. 5 : 1 0 5 - 1 1 5 Leong T-Y, Goodchild DJ and Anderson JM (1985) Plant Physiol. 78: 561-567 Ley AC and Mauzerall DG (1982) Biochlm. Biophys. Acta 6 8 0 : 9 5 - 1 0 6 Myers J and Graham J-R (1971) Plant Physiol. 4 8 : 2 8 2 - 2 8 6 Myers J, Graham J-R and Wang RT (1980) Plant Physiol. 6 6 : 1 1 4 4 - 1 1 4 9

196 wl0. Anderson JH (1980) FEBS Left. 117:327-331 Ii. Anderson JM, Waldron JC and Thorne SW (1978) FEBS Lett. 92:27-233 12. Diner BA and Wollman F-A (1980) Eur. J. Biochem. Ii0:521-527 13. Dunahay TG and Staehelin LA (1985) Plant Physiol. 78:606-613 14. Haworth P, Watson JL and Arntzen CJ (1983) Bioohlm. Biophys. Aeta 724: 151-158 15. Ish-Shalon D and Ohad I (1983) Biochlm. Biophys. Acta 722:498-507 16. Lam E, Oritz W, Hayfield S and R. Malkln (1984) Plant Physiol. 74: 650-655 17. Mullet JE, Burke JJ and Arntzen CJ (1980) Plant Physiol. 65:814-822 18. Mullet JE, Burke JJ and Arntzen CJ (1980) Plant Physiol. 65:823-827 19. Satoh K and Butler WL (1978) Plant Physiol. 61:373-378 20. Wollman F-A and Bennoun P (1982) Biochlm. Biophys. Aeta 680:352-360 21. Myers J (1951) Ann. Rev. Hicrobiol. 5 : 1 5 7 22. Boardman NK and Thorne SW (1971) Biochim. Biophys. Acta 253:221-231 23. Myers J and Graham J-R (1983) Plant Physiol. 73:440-442 24. Myers J, Graham J-R and Wang RT (1983) Biochim. Biophys. Aeta 722: 282-290 25. Bennet J (1983) Phil. Trans. R. Soc. Lon. B302:113-125 26. Horton P (1983) FEBS Lett. 152:47-52

Author's address: Department of Cellular and Developmental Biology, Harvard University, 16 Divinity Avenue, Cambridge, Massachusetts 02138 (U.S.A.)

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