Proc. Natl. Acad. Sd. USA Vol. 74, No. 8, pp. 3377-81, August 1977 Biophysics Quantum efficiency of photosynthetic energy conversion (photosynthesis/photophosphorylation/ferredoxin) RICHARD K. CHAIN AND DANIEL I. ARNON Department of Cell Physiology, University of California, Berkeley, Berkeley, California 94720 Contributed by Daniel I. Arnon, June 7,1977

ABSTRACT The quantum efficiency of photosynthetic with cyclic and noncyclic photophosphorylation and a "dark," energy conversion was investigated in isolated spinach chlo- enzymatic phase concerned with the assimilation of CO2 (8). roplasts by measurements of the quantum requirements of ATP has established that the formation by cyclic and noncyclic photophosphorylation cata- Fractionation of chloroplasts (9) light lyzed by ferredoxin. ATP formation had a requirement of about phase is localized in the membrane fraction (grana) that is 2 quanta per 1 ATP at 715 nm (corresponding to a requirement separable from the soluble stroma fraction which contains the of 1 quantum per electron) and a requirement of 4 quanta per enzymes of CO2 assimilation (10). Thus, in isolated and frac- ATP (corresponding to a requirement of 2 quanta per electron) tionated chloroplasts, investigations of photosynthetic quantum at 554 nm. When cyclic and noncyclic photophosphorylation efficiency can be focused solely on cyclic and noncyclic pho-. were operating concurrently at 554 nm, a total of about 12 account the conversion of quanta was required to generate the two NADPH and three ATP tophosphorylation, which jointly for needed for the assimilation of one CO2 to the level of glu- photon energy into chemical energy without the subsequent cose. or concurrent reactions of biosynthesis and respiration that cannot be avoided in whole cells. Few areas of photosynthesis have received more intensive The present investigation was undertaken not to reactivate theoretical and experimental study and generated more con- old and now dormant controversies but to relate overall pho- troversy than the efficiency with. which photosynthetic cells tosynthetic quantum efficiency to the quantum efficiency of convert the electromagnetic energy of light into chemical en- cyclic and noncyclic photophosphorylation, the two energy ergy (for review, see refs. 1 and 2). Two different concepts, conversion reactions in plant photosynthesis that, for reasons never reconciled during the lifetimes of their main protagonists, discussed elsewhere (8, 11), can be investigated in isolated emerged from the many investigations. One concept, espoused chloroplasts but not in whole cells. In cyclic photophosphor- by Warburg et al. (3), was that photosynthetic quantum con- ylation (12-14), ATP is the sole product and no 02 is produced version has an efficiency of about 90%-i.e., that energy (Eq. 1), whereas in noncyclic photophosphorylation (15, 16), equivalent to that of 3 einsteins of red quanta (42 kcal each) is 02 evolution is coupled with the reduction of ferredoxin and sufficient to liberate 1 mol of 02 (corresponding to 1/6 mol of the formation of ATP (Eq. 2). glucose, for which AGO' = 686/6 = 114 kcal). In contrast, hp Emerson (4) and his followers (5) concluded that photosynthetic ADP + Pi -. ATP [1] efficiency was much lower, of the order of 8 to 12 quanta per 02, a range that is widely accepted today even though values 4 Fdo. +2 H20 +2 ADP +2 Pi less than 8 have, at times, been obtained by investigators (6, 7) who did not share Warburg's conclusions. ','4Fdred+02+2ATP+4H+ [2] Most studies of photosynthetic quantum efficiency were based on measurements of light-induced production of 02 in which Fd0, is oxidized ferredoxin and Fdred is reduced fer- (corrected for concurrent respiration) during complete pho- redoxin. tosynthesis by whole cells, usually unicellular algae of the Reduced ferredoxin, an iron-sulfur protein electron carrier Chlorella type. Discordant results were attributed to experi- with a reducing power (Eo' = -420 mV) equal to that of mo- mental variables such as errors in methods (usually manometric) lecular hydrogen (17), serves directly as a reductant in some of 02 measurement, variations in the concurrent 02 con- reactions but for CO2 assimilation in plants the reductant is sumption by respiration, participation of respiratory interme- NADPH (E ' = -320 mV) which is formed enzymatically (18, diates in photosynthesis, need for supplementary (catalytic) 19) with no further input of photon energy: illumination, and nutritional history, age, and physiological status of the cells (1-5). Left unchallenged, however, was the 4 FdreM + 2 NADP+ + 4 H+ main (and, to us, dubious) premise underlying these studies with Fd-NADP whole cells-namely, that the photoproduction of 1 mol of 02 - 4Fdox +2NADPH+2H+ [3] always corresponds to the assimilation of 1 mol of CO2 to the reductase level of glucose and that, therefore, 02 evolution is a reliable ATP is a product of both cyclic and noncyclic photophos- measure of the total amount of chemical energy stored. phorylation (Eq. 1 and Eq. 2) which, according to recent evi- A different perspective and experimental approach to the dence, may proceed concurrently in isolated chloroplasts (20, question of photosynthetic quantum efficiency emerged from 21). Thus, the quantum efficiency of light-induced ATP for- studies of photosynthesis by isolated chloroplasts in which the mation by isolated chloroplasts provides a direct and reliable process was physically separated into a light phase concerned index of the efficiency with which photon energy is converted into chemical energy during photosynthesis. An measurements of The costs of publication of this article were defrayed in part by the argument against photosynthetic payment of page charges from funds made available to support the quantum efficiency in isolated chloroplasts is that isolation research which is the subject of the article. This article must therefore procedures may damage the photosynthetic apparatus and be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea.

3377 Downloaded by guest on September 24, 2021 3378 Biophysics: Chain and Arnon Froc. Natl. Acad. Sci. USA 74 (1977)

result in low efficiency. That these and other experimental was also confirmed through measurements of light intensity by hazards do exist is illustrated by the wide range of values for the chemical actinometry (at 420-nm) with the potassium fer- quantum efficiency of photosynthetic phosphorylation reported rioxalate actinometer of Hatchard and Parker (24). from different laboratories. The values (expressed henceforth The fraction of incident monochromatic illumination ab- as quantum requirements; minimal quantum requirements are sorbed by chloroplasts was measured in each quantum effi- equivalent to maximal quantum efficiencies) varied from 3 to ciency experiment with a large Ulbricht integrating sphere 200 quanta of absorbed light per molecule of ATP formed (see (50-cm diameter) in a manner similar to that described by review, ref. 22). Warburg and Krippahl (25). An RCA 6217 photomultiplier The present investigation was prompted by recent findings tube served as the light detector inside the sphere; the photo- (20, 21) that further documented the role of ferredoxin as the multiplier output was measured outside the sphere with a digital native catalyst of cyclic photophosphorylation (13, 14) and voltmeter (Hewlett-Packard model 3440A). Monochromatic characterized the optimal experimental conditions under which illumination to the sphere was provided by a light beam that high quantum efficiencies (low quantum requirements) of was isolated by using the same interference filters used in ferredoxin-catalyzed photophosphorylations would most likely providing actinic monochromatic illumination to the reaction be found. Low quantum requirements were indeed observed: mixtures. 2 quanta per ATP under far-red monochromatic illumination Analytical Procedures and Reagents. Chlorophyll and (715 nm) that supported only cyclic photophosphorylation and NADPH were determined as described (23, 26). ATP was 4 quanta per ATP under short-wavelength monochromatic il- measured by the method of Hagihara and Lardy (27). Ferre- lumination (554 nm) that supported both cyclic and noncyclic doxin was isolated either from spinach leaves (28) or from the photophosphorylation. The theoretical implications of these blue-green alga Spirulina maxima (29). NADP+ and ADP were values and their relationship to measurements of quantum re- purchased from the Sigma Chemical Co. (St. Louis, MO). quirements in whole cells are discussed. RESULTS METHODS Quantum Requirements of Cyclic Photophosphorylation Chloroplasts. Chloroplasts were isolated from spinach leaves at 715 nm. Cyclic photophosphorylation in vivo occurs in (Spinacia oleracea var. High Pack) grown in a greenhouse in chloroplast lamellae that are in contact with the stroma fluid a nutrient solution culture (23) and freshly harvested before that contains dissolved 02 but in vitro the process traditionally each experiment. The chloroplast preparations were "broken" has been investigated under anaerobic conditions, for reasons chloroplasts, prepared as described (20) except that an addi- that are discussed elsewhere (8, 20). Recently (20), when fer- tional low-speed centrifugation step was added to remove any redoxin-catalyzed cyclic photophosphorylation by isolated residual large fragments. The broken chloroplasts used consisted chloroplasts was investigated under the more physiological of lamellar material depleted of soluble chloroplast compo- aerobic conditions, it was found to have several distinct features, nents. including a low ferredoxin requirement. Cyclic photophos- Aerobic Conditions. Unless otherwise indicated, the reac- phorylation was optimally catalyzed by the same low concen- tions were carried out in glass cuvettes (2-mm light path) filled trations of ferredoxin (10 ,gM) that are required for NADP+ with reaction mixture. Aerobic conditions, provided in all ex- reduction, concentrations much lower than those previously periments, mean here that the reaction mixtures were in used (100 ,M) for cyclic photophosphorylation under anaerobic equilibrium with air and contained dissolved oxygen (ca 0.25 conditions (30). mM) but were not otherwise deliberatedly aerated. Another feature of ferredoxin-catalyzed cyclic photophos- Illumination. Monochromatic illumination (715 or 554 nm) phorylation in the presence of 02 is its sensitivity to over- was provided by a light beam from a 250-W air-cooled tung- reduction by electrons released by photosystem II from water sten-halogen lamp (Type EHN, General Electric Co.); the beam (20). Experimentally, one way to poise the system and prevent was passed through interference filters (Baird-Atomic Co.) with overreduction is to use 715-nm illumination, a wavelength that 20-nm half-band width for 554-nm light and 10-nm half-band activates photosystem I but not photosystem II (14, 31, 32). Il- width for 715-nm light. lumination by 715-nm light generated only a "trickle" electron Actinic illumination by 554- and 715-nm light beams, neither flow from water, just adequate to maintain the proper poising of which is strongly absorbed by chloroplasts, ensured that the in the presence of 02(20). entire reaction mixture received uniform illumination despite As cyclic photophosphorylation is driven by photosystem I the fairly high chlorophyll concentration. Preliminary exper- (14, 32), the use of 715-nm illumination that is absorbed almost iments established that, within the range of illumination used, exclusively by photosystem I offered a great experimental ad- the rates of photochemical reactions were directly proportional vantage for obtaining the minimal quantum requirements for to light intensity. ATP formation by cyclic photophosphorylation. The fraction Light Measurements. Incident light intensity was measured of 715-nm light absorbed by photosystem II (and the resulting with a radiation meter (Radiometer model 65, Yellow Springs electron trickle) is so small that it can be disregarded (14, 20). Instrument Co.) calibrated against a National Bureau of Stan- By contrast, shorter wavelengths (<700 nm) are absorbed by dards (Washington, DC) radiation standard. Selected confir- photosystems II and I and generate, in addition to ATP, re- matory measurements of incident light intensity were made ducing power (and 02) and would not be expected to give the with a calibrated Eppley quartz-window surface-type linear same low quantum requirements for ATP formation as were thermopile. Incident illumination remained constant, as indi- observed under long-wavelength illumination. cated by concordant measurements before and after each ex- These considerations are supported by the results shown in periment and by little or no variation from day to day. Table 1. At 715 nm, aerobic, ferredoxin-catalyzed cyclic pho- Incident light intensity was measured by placing the mea- tophosphorylation required only about 2 quanta per ATP, suring device where the sample cuvette was normally illumi- whereas at 554 nm the quantum requirement increased sev- nated in the apparatus. Corrections were applied for measured eralfold. reflection losses (7.5%) at the incident-to-cuvette side. We reported elsewhere (21) that chloroplasts isolated from The reliability of the incident light intensity measurements young plants had considerably higher rates of ATP formation Downloaded by guest on September 24, 2021 Biophysics: Chain and Amon Proc. Natl. Acad. Sci. USA 74 (1977) 3379

Table 1. Quantum requirements of ferredoxin-catalyzed cyclic photophosphorylation at 554- and 715-nm monochromatic illumination Illumination Light Quantum Time, absorbed, ATP formed, requirement, X, (nm) min ME Mmol ME/Mmol ATP Z Fd +NADPH E 554 2.5 1.01 0.134 7.5 :L 3.0 - 5.0 2.02 0.205 9.9 0 10.0 4.04 0.366 11.0 2.5 00 715 2.5 0.42 0.187 2.2 0 5.0 0.84 0.340 2.5 4I- 10.0 1.68 0.787 2.1 1.5 The reaction mixture (0.65 ml) contained broken chloroplasts (see Methods) (equivalent to 332 Mg of chlorophyll) and the following 1.04 (mM): Tricine-KOH buffer (pH 8.2), 200; MgCl2, 5.0; ADP, 7.5; K2H32PO4, 7.5; and spinach ferredoxin, 0.01. Temperature, 15°-17°; 0.5- age of plants, 25-29 days. Incident light intensity at each wavelength was 1 X 104 ergs-cmn2-sec-1, corresponding to 0.77 microeinstein (JE) 0.2 0.4 0.6 0.8 1.0 1.2 1.4 per min at 554 nm and 0.99 ME per min at 715 nm. DCMU, MM FIG. 1. Experimental conditions as in Table 1 except that 2.5mM than did chloroplasts from old plants. The effect of age was also NADPH and DCMU were added as indicated. The cuvettes were il- reflected in the quantum requirements of cyclic photophos- luminated by 554-nm light for 30 min. Incident light intensity, 2 phorylation at 715 nm (Table 2). Chloroplasts from young X 104 ergs-cm-2-sec-1; age of plants, 25-29 days. Fd = ferredoxin. plants again exhibited a requirement of about 2 quanta per ATP. The requirement increased substantially in chloroplasts 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). The effect isolated from fully mature leaves. of NADPH was unexpectedly found to be similar to that of The requirement of 2 quanta per ATP was obtained by actual DCMU in poising the system by restricting electron flow from measurements of incident light intensity (and the fraction ab- water. The mechanism of NADPH action was found to be a sorbed) without extrapolation to zero light intensity (cf. 33). No reduction of C-550 (20), a chloroplast component (41) that, from problems caused by a "low-light-intensity lag" (22) were en- an operational point of view, acts as an electron acceptor in the countered in the ferredoxin-catalyzed cyclic photophosphor- reaction center of photosystem 11 (42). ylation. We consider this back electron flow from NADPH via fer- Evidence from this laboratory (ref. 31 and figure 8 in ref. 32) redoxin to (>550 to be the physiological poising mechanism that and from other laboratories (33-40) indicates that the quantum triggers the operation of cyclic photophosphorylation alone in requirements for electron transport are 1 quantum per electron situations when excess ATP (but not reducing power) is needed; in far-red light (e.g., 715 nm) that activates photosystem I and NADPH would then tend to accumulate (20, 21). Such a sit- 2 quanta per electron under short-wavelength illumination (e.g., uation would arise, for example, during protein synthesis, when 554 nm) that activates photosystems I and II. The observed ATP is required for the activation of amino acids by amino acyl requirement of two 715-nm quanta per ATP is consistent with synthetases to form amino acid adenylates. the concept that the formation of one ATP involves a light- Experimentally, the poising effect of NADPH is replace- induced transfer of two electrons via photosystem I and that able by DCMU or, as already discussed, far-red monochromatic cyclic photophosphorylation has only one site of ATP forma- light. A comparison of the stimulatory effects of NADPH and tion. DCMU on cyclic photophosphorylation is given in Fig. 1. The Quantum Requirements of Cyclic Photophosphorylation action of NADPH was in the main similar and additive to the at 554 nm. We have recently reported (20) that under short- action of DCMU. In the absence of DCMU, the addition of wavelength monochromatic illumination (equivalent to white NADPH more than doubled the rate of cyclic photophospho- light as far as the photooxidation of water is concerned) the rylation. At lower concentrations of DCMU, the addition of operation of ferredoxin-catalyzed cyclic photophosphorylation NADPH gave a further substantial increase in photophospho- was greatly stimulated either by the addition of NADPH or by rylation but at higher concentrations, DCMU alone was more the addition of the well-known inhibitor of photosystem II, effective than the combination of DCMU and NADPH. When the system was optimally poised by DCMU alone, the addition of NADPH was inhibitory. Table 2. Effect of age of plants on quantum requirements of The beneficial poising effect of NADPH and DCMU on ferredoxin-catalyzed cyclic photophosphorylation at 715 nm cyclic photophosphorylation was reflected in lower quantum Old plants (42 days) Young plants (27 days) requirements. About 10 quanta per ATP were required in the Quantum Quantum unpoised system, 6 quanta in the presence of NADPH, and 4 Light require- Light require- quanta in in the presence of DCMU (Table 3). In these exper- ab- ATP ment, ab- ATP ment, iments, optimal poising and the lowest quantum requirement Time, sorbed, formed, ME/zmol sorbed, formed, ME/,Mmol were obtained with the concentration of DCMU used. The min ME M4mol ATP (,ME) Mmol ATP concentrations of NADPH and DCMU needed for optimal poising varied seasonally, depending on the relative activity of 2.5 0.58 0.106 5.5 0.50 0.218 2.3 photosystem II. 5.0 1.16 0.164 7.1 1.00 0.378 2.6 When cyclic photophosphorylation was optimally poised, 10.0 2.32 0.305 7.6 2.00 0.805 2.5 4 quanta per ATP represented the lowest quantum requirement Experimental conditions not otherwise specified were as in for cyclic photophosphorylation at 554 nm. Based on the Table 1. premise that the formation of one ATP involves the transfer of Downloaded by guest on September 24, 2021 3380 Biophysics: Chain and Arnon Proc. Nati. Acad. Sci. USA 74 (1977)

Table 3. Effect of NADPH and 3-(3,4-dichlorophenyl)-1,1- Table 4. Quantum requirements of NADP+ reduction and dimethylurea (DCMU) on quantum requirements of cyclic concurrent noncyclic and cyclic photophosphorylations at 554 nm photophosphorylation at 554 nm as influenced by the age of plants Illumination ATP Quantum Quantum Time, Absorbed formed, requirement, requirements, min AE '4mol ME/,gmol ATP Illumination Light ATP NADPH uE/,umol time, absorbed, formed, formed, ATP NADPH Control min ,E ,umol ,umol formed formed 2.5 1.01 0.134 7.5 5.0 2.02 0.205 9.9 Old plants (42 days) 10.0 4.04 0.366 11.0 2.5 1.13 0.173 0.152 6.5 7.4 +DCMU 5.0 2.26 0.324 0.268 7.0 _ 8.4 10.0 4.52 0.562 0.528 8.0 8.6 2.5 1.01 0.252 4.0 5.0 2.02 0.469 4.3 Young plants (27 days) 10.0 4.04 0.979 4.1 2.5 1.05 0.277 0.165 3.8 6.4 +NADPH 5.0 2.10 0.522 0.360 4.0 5.8 10.0 4.20 1.025 0.716 4.1 5.9 2.5 1.03 0.190 5.4 5.0 2.06 0.338 6.1 NADP+ (2.5 mM) was added where indicated. Experimental con- 10.1 4.12 0.621 6.6 ditions not otherwise specified were as in Table 1. Experimental conditions as in Table 1 (554-nm illumination) ex- two NADPH and the three ATP that are consumed in the cept that 2.5 mM NADPH and 13 IAM DCMU were added where in- assimilation of one CO2 to the level of glucose (43). It is of in- dicated. terest that Egneus et al. (44), who measured the quantum re- two electrons, the results signify a requirement of 2 quanta per quirement for CO2 assimilation by isolated spinach chloroplasts, electron under short-wavelength illumination, in contrast to found a requirement of about 12 quanta per CO2 at 674 nm. a requirement of 1 quantum per electron under long-wave- length illumination (Table 1). DISCUSSION Quantum Requirements in Concurrent Cyclic and Non- Past investigations of the efficiency with which photosynthetic cyclic Photophosphorylations. In the presence of substrate cells convert light into chemical energy have usually relied on amounts of NADP+, 554-nm illumination activated simulta- measurements of photoproduction of oxygen, in the belief that neously both cyclic and noncyclic photophosphorylations, a fact they signified the production of sugars as end-products in which reflected in the excess of ATP formed over NADP+ reduced the chemical energy is stored. This belief derives its justification and in ATP:NADPH ratios greater than 1.0 (20, 21). Here, the from the photosynthetic quotient (02 evolved:CO2 fixed) being monochromatic illumination, which was well below saturation close to 1.0. But it has long been recognized that so small a (as required for measurements of quantum requirements), was variation from unity as 3%, which is well within the observed partitioned between cyclic and noncyclic photophosphoryla- limits of reported measurements (cf. refs. 1 and 2), might in- *tion, each of which contributed a portion of the total ATP dicate the formation of as much as 15% protein (45), the syn- formed. By taking into account the total number of quanta thesis of which requires much more ATP than does that of absorbed by both systems and the total amount of ATP formed sugars. The extra ATP needed for the synthesis of proteins and by them, we obtained a requirement of 4 quanta per ATP with other biopolymers (46) can be provided by regulation (20, 21) chloroplasts isolated from young plants; the quantum re- of ferredoxin-catalyzed cyclic photophosphorylation, a reaction quirement per ATP was almost twice as high with chloroplasts that does not produce oxygen. from old plants (Table 4). Oxygen evolution by whole cells is not a reliable measure of Thus, at 554 nm, the same requirement of 4 quanta per ATP the total chemical energy derived from the photosynthetic (equal to 2 quanta per electron) was obtained, regardless quantum conversion process for at least two reasons. First, as whether the ATP was formed by cyclic photophosphorylation just stated, 02 evolution does not reflect the photoproduction alone or by cyclic and noncyclic photophosphorylation oper- of ATP by cyclic photophosphorylation. Second, 02 evolution ating concurrently. When the two systems operated concur- reflects the photoproduction of reducing power but pho- rently, the fraction of absorbed quanta used for noncyclic tosynthetic cells may use the reducing power for purposes other photophosphorylation also generated NADPH in addition to than CO2 assimilation. For example, reduced ferredoxin and 02 (see Table 4 and Eqs. 1-3). When cyclic photophosphor- pyridine nucleotides reduce sulfite to sulfide (47) and nitrate ylation operated alone, the only product was ATP and the to ammonia (48-50). Because Chlorella cells contain (on a surplus photon energy that could have been used at shorter dry-weight basis) about 50% C and 10% N (51), just the re- wavelengths to generate NADPH was lost. duction of nitrate to ammonia (a reaction that requires eight An accurate measurement of the quantum requirement for electrons as contrasted with four electrons for the reduction of NADP reduction was not possible because there was no direct C02) could account for well over a third of the total pho- way to segregate the quanta that were used solely in noncyclic tosynthetically generated reducing power and a corresponding photophosphorylation, in which NADPH is formed. Using the evolution of 02 that has no direct connection with CO2 assim- total number of quanta absorbed by both cyclic and noncyclic ilation. photophosphorylation, we calculated a requirement of about The photoproduction of extra ATP by cyclic photophos- 6 quanta per NADPH for chloroplasts from young plants and phorylation and the diversion of photosynthetically generated about 8 quanta for chloroplasts from old plants (Table 3). reducing power for purposes other than CO2 assimilation may With chloroplasts from young plants, these quantum re- have occurred to a varying degree (and without detection) in quirements for a concurrent formation of ATP and NADPH whole cells, thereby accounting, at least in part, for the vari- add up to a requirement of 12 quanta for the generation of the ability of results in past investigations of photosynthetic Downloaded by guest on September 24, 2021 Biophysics: Chain and At-non Proc. Nat!. Acad. Sci. USA 74 (1977) 3381 quantum efficiency that relied on measurements of 02 evolu- 17. Tagawa, K. & Amnon, D. I. (1962) Nature 195,537-543. tion by algae. Particularly suspect in this regard would be 18. Shin, M., Tagawa, K. & Anion, D. L. (1963) Biochem. Z. 338, long-term experiments that were used to bolster the validity of 84-96. opposing views on the efficiency of photosynthetic conversion 19. Shin, M. & Anion, D. I. (1965) J. Biol. Chem. 240,1405-1411. 20. Anion, D. I. & Chain, R. K. (1975) Proc. Nat!. Acad. Sci. USA of energy. The long duration (up to 8 hr) of such experiments 72,4961-4965. with unicellular algae (5, 52, 53) is equal to, or greater than, the 21. Amnon, D. I., & Chain, R. K. (1977) in Photosynthetic Organelles, generation time of these cells (54) and must have encompassed Plant & Cell Physiol. Special Issue No. 3, 129-147. a wide range of biosynthetic reactions (other than carbohydrate 22. Avron, M. & Neumann, J. (1968) Annu. Rev. Plant Physiol. 19, synthesis) that used ATP and reducing power in varying pro- 137-166. portions.; 23. Anion, D. I. (1949) Plant Physiol. 24, 1-15. Photosynthetic quantum efficiency can be estimated reliably 24. Hatchard, C. G. & Parker, C. A. (1956) Proc. R. Soc. London Ser. only whenhe identity of the photosynthetic products and the A 235,518-568. energy requirements for their synthesis are known. Few, if any, 25. Warburg, 0. & Krippahl, G. (1954) Z. Naturforsch., Tell B 9, past investigations of quantum efficiency in whole cells iden- 181-182. 26. Del Campo, F. F., Ramirez, J. M. & Arnon, D. I. (1968) J. Blol. tified the photosynthetic products formed (other than 02) at Chem. 243,2805-2809. the expense of light energy. These considerations argue in favor 27. Hagihara, B. & Lardy, H. A. (1960) J. Biol. Chem. 235, 889- of isolated chloroplasts over whole cells for investigations of 894. photosynthetic quantum efficiency. With isolated chloroplasts 28. Losada, M. & Amnon, D. I. (1964) in Modern Methods ofPlant it is possible to determine the quantum requirements of ATP Analysis, eds. Linskens, H. F., Sanwal, B. D. & Tracey, M. V. and NADPH formation regardless of how these carriers of (Springer-Verlag, Berlin), Vol. 7, pp. 569-615. chemical energy and reducing power (formed by cyclic and 29. Hall, D. O., Rao, K. K. & Catmnack, R. (1972) Biochem. Blophys. noncyclic photophosphorylation) are later used in biosynthetic Res. Commun. 47,798-803. reactions. Concerns over to the 30. Anion, D. I., Tsujimoto, H. Y. & McSwain, B. D. (1967) Nature possible damage photosynthetic 214,562-566. apparatus during isolation of chloroplasts and a resultant low 31. McSwain, B. D. & Anion, D. I. (1968) Proc. Natl. Acad. Sci. USA efficiency of energy conversion are not warranted by the results 61,989-996. of this study, in which quantum requirements in agreement 32. Arnon, D. I. (1971) Proc. Nat!. Acad. Sct. 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