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I'HOTOSYNTHESIS1 WINSTON M. MANNING Wisconsin Geological and Natural History Survey, Madison, Wisconsin Received May 8, 1988 I. INTRODUCTION In this review major emphasis will be placed on the photochemical and kinetic aspects of photosynthesis. The influence of the various physiological factors is more fully discussed in other recent reviews of photosynthesis (23, 24, 58, 721. Except for studies of the physical and chemical properties of extracted plant pigments, investigations of photosynthesis are practically limited to a study of living plants. This limitation introduces complications not found in non-biological photochemical studies. In the first place, other chemical reactions, thermal as well as photochemical, undoubtedly occur during a period of photosynthesis. Unless proper allowance is made, these reactions may be of sufficient magnitude to obscure the significance of the photosynthesis measurements. In the second place, variations in factors influencing photosynthesis must be limited to those which will not kill or seriously injure the plant during the time when measurements are being made. In many cases, moreover, a variation in a particular environmental (external) factor may influence photosynthesis indirectly through its effect in the plant on internal factors other than those directly connected with the photosynthetic mechanism. This is particularly true for experiments with the higher plants. Emerson (23) has discussed more fully some of the limitations involved in photosynthesis research. II. THE MEASUREMENT OF PHOTOSYNTHESIS ' Only a brief outline of experimental procedure will be given here. More extensive descriptions of the earlier methods which have been em- ployed may be found in the monographs of Spoehr (70) and Stiles (741. Details of later procedures may be obtained from the original papers. Photosynthesis is usually measured by determining either the amount of carbon dioxide consumed or the amount of oxygen liberated, or both. ~ Contribution No. 11 to the Third Report of the Committee on Photochemistry, National Research Council. 117

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118 WINSTON M. MANNING Less direct methods may also be used, such as the measurement of the change in dry weight or sugar content of the plant material under investi- gation. However, results so obtained may involve factors other than the photosynthetic process. The measurement of photosynthesis in land plants With land plants photosynthesis may be measured by using either an entire plant or only part of the plant (often a single leaf). When a single leaf is studied, it is often detached from the rest of the plant, although many recent investigators have worked with attached leaves (41, 59, 60~. In either case the material to be investigated is usually enclosed in a light- transm~tting chamber.2 The change in carbon dioxide or oxygen com- position during a period of illumination or darkness may then be deter- mined. In practice a flow method is often employed in order to maintain a nearly constant carbon dioxide concentration in the reaction chamber. The change in gas composition during a period of darkness gives a measure of respiration. It is usually assumed that, for a given tempera- ture, this process proceeds at the same rate in light as in darkness. A correction for respiration is therefore made in calculating absolute photo- synthetic rates. The change in oxygen and carbon dioxide may be determined by gas analysis (17), but other methods, in which only carbon dioxide is deter- mined, are more frequently used. In the conductivity method (43, 59, 60, 71, 76), the carbon dioxide is absorbed by a solution of alkali at constant temperature (~ 0.01C.~. The conductivity of the hydroxide-carbonate mixture gives an accurate measure of the amount of carbon dioxide absorbed. With this method a continuous record of photosynthetic (or respiratory) rate may be obtained. Thomas and Hill (76) constructed a chamber large enough to enclose a field plot of wheat or alfalfa plants 6 feet square, and used the conducting ity method to obtain a continuous record of photosynthesis and respiration, the measurements sometimes extending over a period of several weeks. Instead of measuring the conductivity of the hydroxide-carbonate mixture, some investigators have titrated the mixture with standard acid (41~. This method requires less apparatus than the conductivity method, but it is not as suitable for continuous measurements. McAlister (53) has recently developed a very sensitive spectrometric method for determining carbon dioxide. This method is well adapted for continuous measurements of photosynthesis in land plants. A closed 2 In single leaf studies the chamber may be attached to the under side of the leaf, with the leaf forming part of the chamber wall (41, 60~. This permits nearly normal air circulation and transpiration.

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PHOTOSYNTHESIS 119 system is used, with the gas rapidly circulating through the plant chamber and through an optical absorption tube. The absorption by the 4.2~.3,u band of carbon dioxide is determined with a rock salt spectrograph and a vacuum thermocouple. A notable characteristic of this method is its rapid response to changes in photosynthetic rate. Photosynthesis measurements in algae and other aquatic plants Photosynthesis measurements in aquatic plants are usually carried out with the plant material suspended in water. However, both van den Honert (77) and van der Pasuw (78) have worked with moist films of Hormidium (a very small filamentous blue-green alga) attached to glass plates. The rate of oxygen evolution by the suspended plant material is usually used as the criterion of photosynthetic rate. The manometric method of Warburg (84) has been widely used for measuring changes in oxygen concentration in experiments with aquatic plants. In this method the plant material is suspended in a carbonate- bicarbonate buffer mixture (usually about 0.1 molar), and placed in a closed glass vessel connected to a manometer. The buffer concentration is high enough to maintain a practically constant carbon dioxide partial pressure in the gas phase, so that the increase in gas volume due to oxygen evolution can be read directly on the manometer. Vigorous shaking is necessary to maintain equilibrium between gas and liquid phases. With some modifications the manometric method can be applied to measure- ments carried out with an unbuffered nutrient solution (9, 85~. This modification requires the use of two vessels and depends upon the difler- ence in solubility of carbon dioxide and oxygen in the nutrient solution. With this differential type of manometer both oxygen and carbon dioxide changes can be measured. Another method frequently employed, especially in ecological studies, is the determination of dissolved oxygen in closed vessels by titration (Winkler method). This method avoids the use of the somewhat non- physiological buffer mixture and is much simpler than the differential manometric method. However, it does not permit a series of measure- ments on a single sample. Petering and Daniels (62) have recently applied the dropping-mercury electrode to a determination of dissolved oxygen changes in photosynthesis. As in the manometric method, repeated measurements may be made on a single sample of plant suspension. In at least two respects, this method is superior to the usual manometric method. It does not necessitate the use of buffer mixtures and it does not involve an equilibrium between gas and liquid phases. In speed of response it compares favorably with the spectrometric method of McAlister (53~.

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120 WINSTON M. MANNING III. GENERAL DESCRIPTION OF THE PlIOTOSYNT1 IETIC PROCESS The equation for the reaction involved in photosynthesis in green plants is often written as follows: CO2 + H20 ~1/6 (C6H1206) + 02 i\H = 112,000 cal. (I) This equation, when reversed, becomes the one usually used to represent the normal respiratory process. It is usually assumed that formaldehyde is the first product of photo- synthesis, and that subsequent polymerization is responsible for the forma- tion of glucose or other carbohydrates. If formaldehyde is the first product, then HI for equation I becomes 134,000 cal. The subsequent polymerization reaction will then be exothermic. The necessary energy for photosynthesis is assumed to come from light absorbed by the two chlorophyll pigments in the plant, although it is possible that energy absorbed by other pigments, such as carotene and xanthophyll, may sometimes be utilized.3 It is rather remarkable that light of wave length as long as 7000 A. (see figure 2), for which Nhv is equal to 40,500 car., will bring about a reaction for which HI is 112,000 cal. Evidently photo- synthesis involves a more complex series of endothermic reactions than has been observed for any non-biological photoreaction. Photosynthesis at different wave lengths Figures 1 and 2 show a rather close correlation between relative rates of photosynthesis atdiBerent wave lengths (Hoover (42~) and the absorption spectra (in ether solution) of chlorophylls a and b (93~. Hoover's results were obtained with young wheat plants, using equal incident light intensi- ties (less than 300 foot-candles) at the various wave lengths. A wheat leaf contains enough chlorophyll to absorb a considerable fraction of the incident radiation, even in the region between 5000 and 6000 A.; this accounts for the relatively high minimum in the photosynthesis curve (figure 2~. Chlorophyll absorbs more strongly at 3660 A. than in the region between 5000 and 6000 A. (90~. Consequently the low rate of 3 Except in the Myxophyceae (blue-green algae) the chlorophyll pigments are found only in restricted regions of the plant cell, known as chloroplasts. These regions also contain the yellow pigments carotene and xanthophyll. Frequently a large number of the cells in a green leaf contain no chloroplasts, while in the remaining cells the number may vary from one to many. Consequently only a small fraction of the total leaf volume consists of chloroplasts and hence is able to carry on photosyn- thesis. In many of the algae, however, the chloroplasts constitute a much larger fraction of the total plant material. For example, the single chloroplast in the uni- cellular green alga Chlorella probably occupies half of the cell volume. This large proportion of photosynthetically active plant material constitutes one reason for the widespread use of Chlorella in photosynthesis investigations.

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PHOTOSYNTHESIS 121 photosynthesis at 3660 A. (figure 2) indicates a much lower quantum efficiency for the process than in the region frown 4000 to 7000 A. Below 3000 A. ultraviolet radiation is distinctly injurious (57, 4~. of _ 90 so 70 _ 60 a v ~ 50 A 40 3 2 IC I1 1 l , .: ? 1 l i -I! -' 1 ~ l ' o ~ I ~ ~ ~1 1 1 _ o 0 o ~ ~ ~ ~ ~ 8 ~ ~ ~ a ~ ~ 8 ~ ~ ~ ~ ~ n X ~ ~ ~ O WAVE LENGTH IN A. 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 o 0 = COuPONENT A ~~ = COUPONE NT B 11 l ,tl ill 1 i 1 1 ! _, ~ ~ ~ ~ I 1_ 1 1 1 1 1 100 90 So To 60 so 30 20 FIG. 1. The absorption spectra of chlorophylls a and b in ether (Zscheile (93~) The formaldehyde hypothesis The occurrence of formaldehyde as an intermediate product of photo- synthesis has been neither proved nor disproved (589. In any event its concentration must be very low, even during rapid photosynthesis, since

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122 WINSTON M. MANNING concentrations higher than a few hundredths of a per cent are distinctly toxic. The presence of small amounts of formaldehyde in green leaves has been reported by a number of investigators. Others have found that plants in the dark can utilize low concentrations of formaldehyde to form carbohydrates. These observations add plausibility to the hypothesis that formaldehyde is an intermediate product in the photosynthetic process, but do not constitute proof of the hypothesis, since formaldehyde 350 300 o a 150 ID if: o --1 ~ 1 ' 1 --- 1 '-'- 1 :~1 250 _ 200 _ 100 _ I' ~ r' 4\ x 1 1 1 3500 4500 5~0 WAVE LENGTH 6500 7500 FIG. 2. Rate of photosynthesis as a function of wave length (low light intensity). As, the corrected form of the curve obtained with large Christianson filters; B4, the corrected form of the curve obtained with small Christianson filters. Points marked X are the results obtained with line filters and quartz mercury arc. (Hoover (42~. may be produced by other metabolic processes. Also, formaldehyde is only one of several organic compounds which may be utilized by plants in the formation of carbohydrates (58~. The photosynthetic quotient The ratio of the number of moles of carbon dioxide absorbed to the number of moles of oxygen produced, /`CO2/AO~, iS called the photosyn-

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PHOTOSYNTHESIS 123 thetic quotient.4 If glucose or some other carbohydrate is the final prod- uct of photosynthesis, the photosynthetic quotient should have a value of 1, as in equation I. Maquenne and Demoussy (56) and Willstatter and Stoll (89) investigated a large number of different plants and found a quotient very close to unity. Apparent values markedly different from unity are sometimes obtained, but these may often be attributed to the effects of abnormal respiration or other processes. The value of the photosynthetic quotient constitutes the principal proof that a carbohydrate is the first product of photosynthesis. Many investi- gators have attempted to identify this carbohydrate product. From present evidence it may be considered probable that a hexose is the first product formed, though the prior formation of sucrose remains as a possibility.5 In diatoms (and also in some other plants) the products of photosyn- thesis are stored principally in the form of oils rather than as carbohydrates. Barker (10) has measured the photosynthetic quotient in two species of diatoms in an effort to determine whether the oils are formed as primary products of photosynthesis or whether they are secondary metabolic products. For complete conversion of the carbon dioxide to oils, the quotient should be approximately 0.70 instead of 1.00. For both species of diatoms Barker found a quotient close to 0.95, with a tendency for the value to increase slightly with increasing light intensity. He concluded that 10 per cent or less of the photosynthetic products appeared as stored fat and that this fat production was probably the result of a secondary reaction.6 The eject of light intensity and temperature on the rate of photosynthesis Figure 3 shows diagrammatically the relation between the rate of photosynthesis and light intensity at high carbon dioxide concentration. Curve A represents the behavior at a relatively low temperature, perhaps 10C.; curve B is for a higher temperature, such as 20C. While the type of behavior shown in figure 3 is characteristic of most 4 Some writers apply the term "photosynthetic quotient" to the reciprocal of this ratio, i.e., /~02/^CO2. 5 More complete discussions of this problem are given by Spoehr (reference 70, page 215) and Stiles (reference 74, page 151~. 6 When a simple alga, such as Chlorella, is grown under long-continued constant conditions, including constant illumination, it is probable that an equilibrium is reached between the rate (per unit of plant material) at which food is manufactured by photosynthesis and the rate at which it is used in growth. Under these conditions the apparent photosynthetic quotient (i.e., uncorrected for respiration) should give a measure of the average state of oxidation of the entire cell material. The oxygen eliminated during the reduction of nitrates to protein material should contribute noticeably to a lowering of the apparent quotient.

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124 WINSTON M. MANNING plants which have been studied, the exact numerical behavior may vary widely. Thus, in many land plants the maximum photosynthetic rate may not be reached until the light intensity approaches that of sunlight, while in other plants, particularly some of the algae, one-tenth of this intensity may produce the maximum rate.7 ma. . . fine snape of a rate-nght Intensity curve will depend on the fraction of light absorbed by the plant material under investigation. With a thick leaf containing an abundance of chlorophyll, or with a dense suspension of algae, different portions of the plant material will be exposed to widely ~0 - s I o IL o 350 / 1/ I LIGHT INT ENSITY _ FIG. 3 300 .5 ~ USA 200 t56 A' IN 5G 906 _ ~ ' ~ \ , _ ~ - / ~ 6 ,7 1 1 ~7 em,oerolure, C FIG. 4 FIG. 3. Rate of photosynthesis as a function of light intensity. A, low tempera- ture curve; B. high temperature curve. FIG. 4. Variation in rate of photosynthesis with temperature at high light inten- sities for N~tzschia closterium (O) and Niteschia palea (my. Photosynthesis is ex- pressed in cubic millimeters of evolved oxygen per hour per 10 cmm. of cells. (Bar- ker (10~. different light intensities. A very high incident intensity will then be necessary to produce a maximum photosynthetic rate in the chloroplasts farthest removed from the incident surface. As shown in figure 3, the rate of photosynthesis is nearly independent of temperature at low light intensities, but becomes temperature-depend- ent at higher intensities. Thus, for Chlorella Warburg (82) found a temperature coefficient (Q1O) of unity at approximately 6 X 103 ergs per 7 In some plants the rate of photosynthesis may increase to a maximum and then decrease again as the light intensity is increased (48, 54, 72). The reason for a de- crease in rate at high intensities is not definitely known. It may be caused by some form of injury, or it may be caused by the simultaneous occurrence of photooxidation processes.

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PHOTOSYNTHESIS 125 cm.2 per second,8 but a coefficient of from 2 to 5 (depending upon the temperature) at an intensity of approximately 100 X 103 ergs per cm.2 per second. From the type of behavior shown in figure 3, as well as from other evi- dence to be discussed below, it must be concluded that photosynthesis is a complex cyclic reaction, involving at least one thermal reaction, usually known as the Blackman reaction,9 in addition to whatever photoreactions are necessary. According to this view, at low light intensities, where the rate of photosynthesis is approximately proportional to the intensity, the time between the absorption of successive photons, by that portion of the mechanism involved in the reduction of a single carbon dioxide molecule, will be sufficient to permit the Blackman reaction to be completed. Light is then said to be the limiting factor. But at high intensities, where the rate has practically reached its maximum value (so-called light satura- tion), the rate will presumably be limited only by the rate of the Black- man reaction.~ This implies that photons are absorbed so rapidly by the mechanism that the average time between the completion of the Black- man reaction and the next light absorption will be very short compared with the average time required for the Blackman reaction. An increase in temperature will increase the rate of the Blackman reaction without appreciably altering the rate of the light reaction. s In this research Warburg made only approximate estimates of absolute inten- . . sties. 9 The term "Blackman reaction" as used in this paper refers to any reaction or reactions which contribute to the temperature dependence of the photosynthetic process. It is named for F. F. Blaekman, who, in 1905 (12), formulated his principle of limiting factors in the following words: "When a process is conditioned as to its rapidity by a number of separate factors, the rate of the process is limited by the pace of the 'slowest' factor." Application of this principle did much to clarify the eon- tradietory results of earlier research on photosynthesis. Many subsequent investigators appear to have interpreted this principle too literally, i.e., they have assumed that for measurements under ideal conditions only a single factor could influence the rate of photosynthesis at any one time. According to this view, an increase in the intensity of a particular variable should result in an abrupt transition from direct dependence of the rate on this variable to complete independence. Such a transition is contrary to any reasonable kinetic formulation of the process. tAeeording to a recent mechanism of Franck and Herzfeld (34), the rate of the Blaekman reaction is not limiting at high light intensity (see page 850~. ~i Warburg and Uyesugi (87) and Yabusoe (92) found some similarities between the Blackman reaction and the rate of decomposition of hydrogen peroxide by the en- zyme eatalase. There has therefore been a widespread belief that the Blaekman reaction consists of a reaction between eatalase and a peroxide. Hence many pos- tulated mechanisms have included hydrogen peroxide or organic peroxides as inter- mediate products in photosynthesis. However, Emerson and Green (28) have re- eently made further comparisons of the two reactions without finding any significant similarity. .,

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126 WINSTON M. MANNING \ Figures 4 and 5 illustrate the effect of temperature on the rate of photo- synthesis at high light intensity and high carbon dioxide concentration. Figure 4 gives the results obtained by Barker (10) with two species of diatoms; figure 5 gives the results obtained by Craig and Trelease (21) with Chlorella vulgaris. The lower curves in figure 5 are for rate measure- ments in 99.9 per cent heavy water. (The heavy water experiments will be discussed below, page 828.) The decrease in photosynthetic rate at high temperatures is probably due to injury to the plant material. Fur- ther evidence of injury is found in the fact that the photosynthetic rate at high temperatures is not constant, but decreases with time. Injury may be due to deactivation of enzymes (or of an enzyme-producing mechanism) connected with the Blackman reaction, or it may be due to a change in the physical condition of the chloroplasts. The Arrhenius equation may be used to calculate apparent activation energies for the temperature-sensitive reaction in photosynthesis. How- G O O4-~;r T~ o ~ ! 1 1 1 1 1 26 34 42 30 38 46 30 38 'C FIG. 5. Rate of photosynthesis as a function of temperature. For H20 shown by circles with vertical bars, and for D20 shown by circles with horizontal bars. (Craig and Trelease (21~. ever, in view of the complex system involved, the significance of the figures so obtained is doubtful. The apparent activation energy varies for difler- ent species and decreases with increasing temperature. The low tem- perature value for Chlorella pyrenoidosa and Hormidium flaccidum has been reported as being approximately 20,000 cal. (27, 78~. Barker (10) found a value of about 30,000 cal. for the diatoms Nitzschia closterium and Nitzschia palea (see figure 4~. Emerson and Green (27) found a value of approximately 50,000 cal. for Chlorella vulgaris and for the marine alga Gigartina harveyana. The effect of carbon dioxide concentration on the rate of photosynthesis Variation in carbon dioxide concentration apparently affects the rate of photosynthesis in a manner similar to variation in light intensity. Van den Honert (reference 77, page 225), working with moist films of Hormid-

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PHOTOSYNTHESIS 127 tom at 12 and 20C., and using high light intensity, obtained curves essentially like those of figure 3 (with light intensity replaced by carbon dioxide concentration). In both curves the rate of photosynthesis reached its maximum value at a carbon dioxide concentration of about 0.04 per cent by volume. (Normal carbon dioxide concentration in air is approxi- mately 0.03 per cent.) For young wheat plants Hoover, Johnston, and Brackett (43) found that a higher carbon dioxide concentration, approxi- mately 0.15 per cent, was necessary for a maximum rate of photosynthesis (light intensity one-fourth of that of sunlight). Light saturation was not reached in this work; at higher light intensities still higher carbon dioxide concentrations would have been required. In general, the dif- fusion of carbon dioxide into a chloroplast will encounter more resistance in a leaf, where most of the chloroplast-containing cells are usually some distance from the leaf surface, than in a small unicellular or filamentous alga, where every vegetative cell is photosynthetically active and not surrounded by other cells. A higher carbon dioxide concentration will then be necessary for leaves in order to overcome this diffusion resist- ance.~2 For Chl~rella in liquid suspension Emerson and Green (29) found that a carbon dioxide concentration of 10 or 15 X 10-6 moles per liter was sufficient for maximum photosynthesis. Smith (68), using the water plant Cabomba, has studied the effect of light intensity and carbon dioxide concentration on the photosynthetic rate. He found that both variables affected the rate in the same way. However, there is disagreement concerning the effect of temperature at low carbon dioxide concentrations. According to van den Honert's curves (reference 77, page 225), temperature is without effect on the rate at low concentrations, but Warburg (82) and Emerson (23) found a high temperature coefficient for Chlorella at low carbon dioxide concentrations. Both Warburg and Emerson used the manometric method in their measure- ments; the Chlorella was suspended in carbonate-bicarbonate buffer mixtures and the concentration of free carbon dioxide was varied by vary- ing the ratio of carbonate to bicarbonate. At least three possible objec- tions may be raised to the use of these buff ers for regulating carbon dioxide concentration: (~) In varying the carbon dioxide concentration, the pH is also varied. The high pH necessary to obtain limiting carbon dioxide concentrations may influence the photosynthetic rate. (2) A high con- centration of carbonate or bicarbonate ions may inhibit photosynthesis. (~) It is possible that water plants may be able to use carbonate or bicar- bonate in the photosynthetic process (3~. Emerson and Green (29), work- ing with phosphate buffers, found that within the range from pH 4.6 to 8.9, neither bicarbonate nor hydrogen ion influenced the rate of photo- 72 The problem of diffusion resistance has been discussed by James (47~.

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146 WINSTON M. MANNING ing of fluorescence by oxygen to the utilization of chlorophyll excitation energy in the process of photosynthesis. Kautsky and Hormuth (49) suggested that oxygen forms an addition compound with some substance in the chloroplast, and that this compound serves to transfer the chloro- phyll excitation energy to carbonic acid, which is thereupon reduced to formaldehyde or carbohydrate. Kautsky's interpretation is open to question from several points of view. In the first place, recent experiments by Gaffron (39), with Chlorella, indicate that oxygen is not essential for photosynthesis. Gaf- fron observed that when Chlorella was illuminated following 15 hr. in darkness in a nitrogen atmosphere, carbon dioxide consumption proceeded normally. However, oxygen production did not occur for some time, presumably because of oxidation of excess intracellular fermentation products. Secondly, Kautsky used the 3660 A. line of mercury to excite fluores- cence. This wave length is relatively inefficient for photosynthesis (see page 818~.. Thirdly, as Emerson (23) has pointed out, a plant exposed to high light intensity is using only a small fraction of the absorbed energy in the photosynthetic process. Consequently, changes in fluorescent intensity might occur quite independently of the photosynthetic process. Franck and Wood (36), while not considering oxygen as necessary for photosynthesis, concluded that there is a close connection between the processes of photosynthesis and fluorescence. They interpreted the vari- ation in fluorescent intensity as being due to the formation and disap- pearance of a strongly fluorescent radical. According to the mechanism of Kautsky and Hormuth (49), it would appear that, at low light intensities, the percentage yield of fluorescence should increase with increasing intensity of the incident light, approach- ing a maximum value after the attainment of a maximum photosynthetic rate. If this type of relation could actually be established, it would constitute strong evidence for a connection between photosynthesis and fluorescence. The relation between chlorophyll concentration and the maximum rate of photosynthesis Emerson (22) and Fleischer (33) have studied the effect of varying chlorophyll concentration on the rate of photosynthesis in Chlorella. Emerson grew Chlorella in a nutrient solution containing glucose, and varied the chlorophyll concentration by varying the concentration of iron in the nutrient solution. Fleischer varied the chlorophyll concentra- tion in three different ways. In some of his series the iron concentration was varied, while in other series the magnesium or nitrogen concen-

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PHOTOSYNTHESIS 147 "rations were varied. Except for samples deficient in magnesium, Emer- son and Fleischer both found that, at high light intensity and abundant carbon dioxide concentration, the rate of photosynthesis per unit volume of cells was approximately proportional to the amount of chlorophyll. When chlorophyll deficiency was caused by insufficient magnesium, Fleischer found an abnormally low rate of photosynthesis, and conse- quently concluded that magnesium influences the process of photosyn- thesis in some way other than by its influence on chlorophyll production. Emerson and Arnold (26) have studied the relation in Chlorella between chlorophyll content and the rate of photosynthesis in intermittent light of high intensity. In these experiments the chlorophyll content was varied by varying the light conditions under which the Chlorella was cul- tured. As in previous work (25), they used light flashes which lasted only about 10-5 sec., a period which is short compared with the duration of the dark or Blackman reaction (see page 827~. Sufficient time was allowed between flashes for completion of the dark reaction, so that light saturation in their intermittent light experiments implied, not that the rate of the dark reaction was limiting, as it is in the case of saturation with continuous light, but that the photochemical mechanism was saturated. Under these conditions the maximum rate was again found to be approximately proportional to the chlorophyll concentration. Emerson and Arnold calculated the ratio between the total number of chlorophyll molecules present in the reaction vessel and the number of carbon dioxide molecules reduced per single light flash. Instead of finding a value of 1 (or of 3 or 4 or 5, as would be expected in case a series of three or four or five al- ternate photochemical and thermal reactions were required for the photo- synthetic cycle), they found a value of approximately 2500. This figure represented the average for all chlorophyll concentrations; its value was apparently independent of chlorophyll concentration. In similar ex- , periments Kohn (52) found that light saturation was reached when only about one chlorophyll molecule in a hundred absorbed one or more quanta in a single flash. Arnold and Kohn (6) have determined the minimum value of the ratio of chlorophyll molecules to carbon dioxide molecules reduced per flash for several different species in different divisions of the plant kingdom. They found the value to lie between 2000 and 4000 in all cases. Emerson (23, 24) has suggested that carbon dioxide probably does not combine with chlorophyll prior to the photoreaction, and that the ratio of about 2500 probably represents the ratio between chlorophyll and some other internal factor, perhaps the substance which combines with carbon dioxide. However, according to this mechanism, one would expect a very low quantum efficiency, even with low intensity continuous light. The high efficiency values reported by Warburg and Negelein (86), or

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148 WINSTON M. MANNING even those found by Manning, Staufler, Duggar, and Daniels (55), would thus be very difficult to explain. The photosynthetic Unit One way of reconciling the small yield of carbon dioxide reduced per flash with the relatively high quantum efficiencies observed at low light intensities is to assume that light absorbed by a large number of chloro- phyll molecules can be made available to a single carbon dioxide molecule. This assumption implies (unless one postulates an extremely long life for excited chlorophyll) that, in the chloroplast, the chlorophyll molecules are distributed in the form of groups or units, each containing a large num- ber of individual molecules. Arnold and Kohn (6), Gaffron and Wohl (40), and Weiss (88) have favored this interpretation. The postulated size of such a unit would depend upon the number of successive photo- chemical reactions which are required to reduce one carbon dioxide molecule, and also on the number of carbon dioxide molecules associated with each group. Weiss (88) has suggested that a single unit may have carbon dioxide molecules adsorbed on the surface, with one carbon dioxide molecule to each surface molecule of chlorophyll. Assuming four succes- sive photoreactions, it would thus be necessary to have 500 or 600 mole- cules in the interior for each surface molecule of chlorophyll. Weiss calculated that, if such a unit were spherical, its radius should be of the order of 0.4~. Globules of this general size have been observed in photo- graphs of chloroplasts (44~. However, it is difficult to conceive of energy transmission through a unit of this magnitude without enormous losses in the form of heat. Gaflron and Wohl (40) obtained additional support for the existence of of a photosynthetic unit from approximate calculations in which the data of numerous investigators were used. They found that the maximum photosynthetic rate (light saturation) corresponded to the reduction of one carbon dioxide molecule by each chlorophyll molecule every 10 or 20 sec. This period is approximately 1000 times as long as the average time for the dark or Blackman reaction (25, 26~. Assuming a single dark reaction in each cycle, this would indicate a unit of about 1000 chlorophyll molecules for each carbon dioxide molecule. It has also been suggested (34, 91) that the existence of a photosyn- thetic unit would account for the absence of a long induction period at low light intensities. If a single chlorophyll molecule were obliged to absorb four or more quanta before oxygen could be liberated, a long induction period would be required at low light intensities. But, if a unit of 500 or 1000 chlorophyll molecules were available for the reduction of each carbon dioxide molecule, the necessary quanta would be absorbed

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PHOTOSYNTHESIS 149 within a second or two, even at the lowest intensities that have been used in studies of photosynthesis. If the process of photosynthesis involves a series of photochemical reactions, any thermal decomposition of intermediate products would result in a lowering of the overall efficiency of the process. If one or more of t.~.~n intermediate products were sufficiently unstable to decompose 1 ~ 1 1 _ ~ _1 _~ ~ ~ AL ~~ 1~~ d CAN ~ ~1~ ~ Am ~~ appreciably wlt~m a period of a minute or less, men a plant SIlOUl~ O~ greatly benefited at low light intensities by the cooperation of more than one chlorophyll molecule in the reduction of a carbon dioxide molecule, - since the time between successive photoreactions would be shortened in proportion to the number of chlorophyll molecules per unit. As Gaflron and Wohl (40) have pointed out, a variation in the size of the photosynthetic unit might account for the phenomenon of light adaptation, by whICh plants of the same species may be conditioned to efficient use of either high or low light intensities. A plant accustomed to high intensities will often photosynthesize more rapidly in bright light than will one accustomed to low intensities, and vice versa. =7 ~ A large num- ber of small units would permit more rapid photosynthesis in bright light, while, with weak light, large units would serve to minimize the possible losses due to decomposition of intermediates. Thermal decomposition of intermediate products might account for the type of behavior shown at low light intensities by the species of purple bacteria which French (37) used in his measurements of quantum e~- ciency (see page 838~. At low light intensities French's measurements indicated a decreasing efficiency as intensity decreased. If thermal de- composition of intermediates was responsible for this behavior, it would be expected that an increase in temperature should increase the rate of decomposition, and thus cause the diminished efficiency to occur at higher light intensities. This effect was actually observed by French (reference 37, figure 5~. However, it should be remembered that the change in AH is small for the reaction studied by French. The only reason for assum- ing a series of photoreactions for the process is its apparent similarity to green plant photosynthesis. Perhaps the strongest argument against the existence of a photosyn- thetic unit is the difficulty involved in picturing a model which would permit an efficient transfer of energy from every region where visible light could be absorbed to the point where it could be utilized for carbon dioxide reduction. Franck and Herzfeld (34) considered that the fluorescence of chloro- phyll in living plants is an indication of unimolecular dispersion. Since colloidal chlorophyll does not fluoresce in vitro, they argue that chloro- phyll in units of 500 or more should not fluoresce in the chloroplast.

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150 WINSTON M. MANNING However, the very low intensity of fluorescence in vivo weakens the argument. Rabinowitchi~ has suggested that the weak chlorophyll fluorescence observed in living material may be emitted by a few "mis- placed" chlorophyll molecules which may play no part in photosynthesis. Wohl (91) has discussed in detail various arguments favoring the existence of a photosynthetic unit. He also has suggested several ex- periments with intermittent light which might add to our knowledge of the photoprocess involved in photosynthesis. The existence of a photosynthetic unit has thus far been neither proved nor disproved. Its existence would offer an explanation for several difler- ent groups of experiments, but on the other hand, various arguments, largely based on physical grounds, can be offered against it. The mechanism of photosynthesis Several recent investigators have proposed kinetic mechanisms for the process of photosynthesis, e.g., Arnold (5), Baly (7), Briggs (15), Burk and Lineweaver (16), Franck and Herzfeld (34), and Smith (68~. Of these, the mechanism of Franck and Herzfeld is the only one which in- cludes a detailed consideration of the photochemical reactions which may be involved. Other investigators have suggested a variety of chemical mechanisms without going into detail regarding the kinetics of the proc- ess ((:onant, Dietz, and Kamerling (20), Gaflron and Wohl (40), James (47), Kautsky and Hormuth (49), Stoll (75), van Niel (79~. In most of these mechanisms it is assumed that the quantum efficiency for the photochem~cal reaction is 0.25, in accord with the observations of Warburg and Negelein (86~. However, the low efficiency values found by recent investigators (see page 835) suggest that the value 0.25 may be too high. It has also been generally assumed that the photosynthetic process includes ~ series of four successive photochemical steps, AH for a single step being limited to the energy supplied by a single quantum. In van Niel's mechanism, however, the four photochemical steps are assumed to be identical, resulting in each case in the formation of a hydrogen atom; the four hydrogen atoms then presumably bring about the reduction of carbon dioxide to formaldehyde. Van Niel's mechanism is represented by the following equations: 4EChlorophyll.H20 + he > Chlorophyll.OH + Hi (1) CO2 + 4H ~ (CH20) + H20 (2) 2~2Chlorophyll.OH + H2A > 2Chlorophyll.H20 + A] (3) . . _ (V) CO2 + 2H2A + 4hY > (CH20) + H20 + 2A 18 Private communication.

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PHOTOSYNTHESIS 151 For reaction 1 to be energetically possible, the chlorophyll H2O com- pound would have to be a more intimate compound of chlorophyll and water than in the case of an ordinary hydrate. An alternative way of writing reaction 1 would be 4[Chlorophyll.H20 + he ~ Dehydrochlorophyll H2O + Hi the hydrogen atom thus being split from the chlorophyll portion of the hydrated molecule. In reaction 3, H2A would be water in the case of algae and higher plants, and hydrogen sulfide in the case of the green sulfur bacteria. Without modification this series of reactions could not take place (ex- cept perhaps in bacteria) with a quantum efficiency as high as 0.25, since reaction 2 is exothermic to the extent of about 150,000 cal. The energy thus wasted would be nearly equivalent to the total energy supplied by four photons at 7000 A. For HERA = H20, reaction 3 would be endo- thermic. The necessary energy would presumably have to be supplied by additional photons, unless reactions 2 and 3 were considered to be coupled in some manner so that the excess energy from reaction 2 could be utilized for reaction 3. The mechanism of Franck and Herzfeld (34) was designed to avoid the necessity of assuming a photosynthetic unit. They assumed, as did Stoll (75), that carbonic acid forms a complex with chlorophyll. This complex, in turn, was assumed to be combined with an organic molecule ROH, present in abundance. Franck and Herzfeld suggested that ROH may be a protein which forms the main body of the chloroplasts. The fol- lowing equations represent the forward reactions postulated by Franck and Herzfeld. They state that the equations are suggested as a working hypothesis, not as a final solution. HOE HO R Chl >C=O,ROH + he - ~ Chl >C/O OH (1) HO HO Carbonic acid HO\ /R Peracid HO ~ \ Chl >COO OH + enzyme ~ Chl + >C O + 202 + H20 (2) HO R Peracid H/ Formic acid HOE R acid HOE Chl >C=0 + H20 + he ~ Chl >C=O,ROH (3) R R acid HO\ H' Formic acid HO\ OR Chl ~C=O,ROH + he ~ Chl H Peraldehyde NC' O OH (4)

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152 WINSTON M. MANNING HO R Chl ECHO OH + enzyme > Chl + H' Peraldehyde HO R Chl COHN + he ~ Chl + H/ R aldehyde HO R \C~OH + 2O2 (5) H/ R aldehyde H\ TIC O + ROH (6) H' Formaldehyde The two enzyme reactions, in which oxygen is evolved, constitute the temperature-sensitive or Blackman reaction. According to Franck and Herzfeld, the calculated binding energy of OH to O in the two peroxides is so weak that the OH radical will be split off whenever the peracid or peraldehyde complexes absorb a photon. At low light intensities the probability of such an absorption would be negligible, because of the speed of the enzyme reactions. But, at intensities corresponding to light saturation in most leaves, they estimated this probability to be about TOO- According to their postulation, the photodecomposition of the permolecules into two radicals initiates chain reactions (back reactions in this case), each chain resulting in the decomposition of a large number of permolecules. With these chains light saturation is thus reached at a much lower intensity than would be required if the rate were limited by the Black- man reactions. By assuming a sufficient chain length (~103 at light satu- ration), saturation, both in flashing and in continuous light, can be ex- plained without the assumption of a photosynthetic unit. Franck and Herzfeld suggested that, after a long dark period, much of the chlorophyll may be attached to intermediate respiratory products, probably plant acids. Illumination could then cause photosynthesis, as in the case of chlorophyll-carbonic acid or chlorophyll-formic acid complexes. However, according to Franck and Herzfeld, photooxida- tion of these plant acids, probably by a chain mechanism, should also take place to a large extent. Until the accumulated respiratory products were exhausted, this oxygen consumption would largely counterbalance the photosynthetic oxygen production, thus producing an induction period. Increased probability of oxidation at high light intensities would cause a more noticeable underproduction of oxygen at high intensities than at low intensities, in agreement with experimental results (53~. The kinetic equations derived by Franck and Herzfeld are in reason- able agreement with experimental facts. However, this cannot be con- sidered as a criterion for the correctness of the mechanism, since equa- tions derived by other investigators on the basis of various mechanisms are also in fairly good agreement with experimental results. The reverse chain reactions of Franck and Herzfeld have been subjected

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PHOTOSYNTHESIS 153 to some criticism (91~. Aside from doubts concerning this part of their mechanism, it is evident that to account for the high efficiencies found by Warburg and Negelein, the various forward reactions in the mechanism of Franck and Herzfeld would have to proceed with remarkable efficiency. Not only would the quantum efficiencies for each photoreaction have to be nearly unity, but the intermediate products would have to remain practically unchanged during the periods between the absorption of successive quanta by a single chlorophyll molecule. This period, accord- ing to calculations of Gafiron and Wohl (40), was about 10 min. in many of the experiments of Warburg and Negelein. But Franck and Herzfeld explained the induction period by assuming that, in the dark, a consider- able fraction of the chlorophyll molecules become attached to interme- diate respiratory products. The experiments of McAlister with wheat (53) indicated that 10 min. in the dark is sufficient to produce a notice- able induction period. Thus, in its present form, the mechanism of Franck and Herzfeld can hardly be reconciled with the results of Warburg and Negelein. It could much more easily account for the efficiencies found by Manning, Staufler, Duggar, and Daniels (55~. But, if the maximum quantum efficiency for photosynthesis is actually only 0.06 instead of 0.25, then there is no very good reason for choosing a mechanism in- volving four photoreactions, rather than one involving five or possibly more reactions. In some respects it may be more desirable to postulate a mechanism involving only a single photoreaction, as in van Niel's mechanism (79~. This result could also be obtained in a manner quite different from that suggested by van Niel. The following scheme indicates a possible mecha- nism of this type. In the series of substances A:, As, An_l, A=, carbon dioxide or car- bonic acid is represented by A: and formaldehyde or other product is represented by An, with the other symbols representing intermediate substances. Assuming for simplicity that there are three intermediates, the following series of changes might conceivably take place: Al + As ~ 2A3 2tA3 + As > 2A4] 4tA4+hv~A5] (1) (2) (3) The overall equation would then be Al + 4hv > As. Water, oxygen, and other possible reactants are ignored in the above equations. Reactions 1 and 2 would presumably be enzyme reactions. The Blackman reaction might consist of one or more reactions like reaction 1 or 2, or it might be due to intermediate reactions not included in this or the following scheme.

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154 WINSTON M. MANNING To avoid postulating an enzyme reaction involving carbonic acid, one might use the following type of scheme: 4[A1 + he ~ As] 2[2A2 ~ A1 + As] 2A3~A1 + A5 (1') (2') (3') The sum of these reactions again gives the equation Al + 4hv ~ As. Disproportionation reactions similar in principle to 2' and 3' are probably necessary in the production, by a plant, of such compounds as fats and oils, where presumably much of the energy comes from the oxidation of glucose. If the maximum quantum efficiency is much lower than 0.25, many mechanisms of this type are possible, particularly ones involving more than three intermediate products. With a mechanism of the type just proposed, a relatively high quantum efficiency could be maintained at very low light intensities without neces- sitating stable intermediate substances, such as would be necessary with a mechanism like that of Franck and Herzfeld. Perhaps the principal value of this discussion has been to give an indication of how little actually is known concerning the chemistry of the photosynthetic process. When such entirely different mechanisms can possess even a small degree of plausibility, the need for further definite information becomes evident. REFERENCES (1) ALBERS, v. M., AND KNORR, H. v.: Cold Spring Harbor Symposia Quant. Biol. 3, 87 (1935~. (2) (3) ARENS, K. : Jahrb. wiss. Botan. 83,561 (1936~. (4) ARNOLD, w. : J. Gen. Physiol. 17, 135 (1933~. (5) ARNOLD, w.: Cold Spring Harbor Symposia Quant. Biol. 3, 124 (1935~. `6> . --~ (7) (8) ALBERS, v. M., AND KNORR, H. v.: Plant Physiol. 12, 833 (1937~. ARNOLD, w.' AND KOHN, H. I. : J. Gen. Physiol. 18, 109 (1934~. BALY, E. C. C.: Proc. Roy. Soc. (London) B117, 218 (1935~. BALY, E. C. C., STEPHEN, w. E., AND HOOD, N. R. : Proc. Roy. Soc. (London) A116, 212 (1927~. (9) BARCROFT, J. : J. Physiol.37,12 (1908~. (1O) BARKER, H. A. : Archiv Mikrobiol. 6, 141 (1935~. (1l) BEBER, A. J., AND BITER' G. O.: Paper read at Indianapolis meeting of Ameri- can Society of Plant Physiologists (1937). (12) BLACKMAN, F. F.: Ann. Botany 19, 281 (1905). (13) BRIGGS, G. E.: Proc. Roy. Soc. (London) B106, 1 (1929~. (14) BRIGGS, G. E.: Proc. Roy. Soc. (London) B113,1 (1933~. (15) BRIGGS, G. E. : Biol. Rev. Cambridge Phil. Soc.10,460 (1935~. (16) BURE, D., AND LINEWEAVER, H.: Cold Spring Harbor Symposia Quant. Biol. 3, 165 (1935~.

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