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Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 117
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 118
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 119
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 120
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
×
Page 121
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
×
Page 122
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
×
Page 123
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
×
Page 124
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
×
Page 125
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
×
Page 126
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
×
Page 127
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
×
Page 128
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
×
Page 129
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
×
Page 130
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
×
Page 131
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
×
Page 132
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 133
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 134
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 135
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 136
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 137
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 138
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 139
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 140
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 141
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 142
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 143
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 144
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 145
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 146
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 147
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 148
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 149
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 150
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 151
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 152
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 153
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 154
Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Suggested Citation:"Photosynthesis." National Research Council. 1938. Third Report of the Committee on Photochemistry: Reprint and Circular Series of the National Research Council. Washington, DC: The National Academies Press. doi: 10.17226/9565.
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Page 156

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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

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 (~ 0.01°C.~. 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.

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~.

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.

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

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-

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 10°C.; curve B is for a higher temperature, such as 20°C. 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.

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.

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. t°Aeeording 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. .,

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-

PHOTOSYNTHESIS 127 tom at 12° and 20°C., 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~.

128 WINSTON M. MANNING synthesis at saturating concentrations of carbon dioxide. They con- cluded, however, that in carbonate mixtures at a higher pH, other factors, probably carbonate and bicarbonate concentrations as well as free carbon dioxide concentration, were influencing the rate of photosynthesis. More- over, with the phosphate buyers, their measurements of photosynthesis in Chlorella as a function of carbon dioxide concentration more closely re- sembled van den Honert's results for Hormidium than Warburg's results for Chlorella in carbonate-bicarbonate buffers. On the basis of the results of Emerson and Green, it may be considered probable that van den Honert's curves (77), showing little or no temperature effect at low carbon dioxide concentration, are valid for Chlorella as well as for Hormid~um. Since a knowledge of the temperature effect at low carbon dioxide con- centrations is of importance in choosing a mechanism for the photosyn- thetic process (see page 841), it appears desirable that a further study should be made of this effect in both algae and higher plants. The inf hence of intermittent light on photosynthesis Warburg (82), Emerson and Arnold (25), and Pratt and Trelease (64) have studied the effect of intermittent light on photosynthesis in Chlorella. Warburg used a rotating sector which cut out half of the incident light. He found that at high light intensity and abundant carbon dioxide con- centration a given amount of light produced more photosynthesis when absorbed intermittently by the Chlorella than when it was absorbed continuously. Moreover, the improvement in yield depended upon the frequency of the flashing. At low light intensities, intermittent light produced no improvement in yield. The eject of intermittent light can be explained on the basis of the cyclic mechanism discussed above (page 823~. Thus, after a sufficiently long period of intense illumination, most of the chlorophyll molecules would be activated (or combined in an unstable compound) and waiting to undergo the so-called Blackman reaction. Photons absorbed by these molecules would presumably be without effect on the photosynthetic rate. But, if this period of intense illumination were followed by a sufficiently long period of darkness, the Blackman or thermal reaction should continue until completed. Then, at the beginning of the next light flash, all of the photons would presumably be absorbed by chlorophyll molecules ready to undergo the next photosynthetic cycle. By combining very short light flashes with long dark periods it should be possible, according to this picture, to have a plant utilize intermittent light of high intensity as efficiently as it can utilize continuous light of low intensity. Emerson and Arnold (25) have extended considerably the observations of Warburg. They used a neon tube as a light source, with an electrical circuit which gave very short light flashes. The circuit was constructed

PHOTOSYNTHESIS 129 so that the length of the dark periods could be varied without appreciable effect on the light flashes. The Chlorella was suspended in a buffer mix- ture and photosynthesis was measured manometrically. Figure 6 shows the results obtained by Emerson and Arnold in their study of the effect of dark time on the yield of photosynthesis per flash. The lower curve was obtained at 1.1°C., while the two upper series were obtained at 25°C. All of the measurements shown in figure 6 were made with Chlorella cells from the same culture. It appears that at 25°C. a dark period of 0.04 sec. is sufficient for practical completion of the thermal 5 4 to - .. on G) 3 2 8- o , ·' _ ~ 1 _ ~ 1 ., 1 2 Terms of a second FIG. 6. The effect of dark time on the yield of photosynthesis per flash of light. Open circles are points made at 25°C.; solid circles at 1.1°C. The crosses are a check made at 25°C. (Emerson and Arnold (25~. Or Blackman reaction, while at 1.1°C. a period of 0.2 or 0.3 sec. is required for practical completion. In these experiments of Emerson and Arnold, the duration of each light flash was less than 2 X 10-5 sec., a period which is short compared with the average duration of the thermal reaction, at least at 1.1°C. Figure 7 shows the results obtained with Chlorella at high light intensity and at 23.9°C. by Pratt and Trelease (64~. Using Chlorella cells suspended in heavy water as well as in ordinary water, they studied the effect of flashing light on the rate of photosynthesis. Each light flash in these

130 WINSTON M. MANNING experiments lasted about 0.0045 sec. This period was much longer than that used by Emerson and Arnold. Consequently a considerable fraction of the chlorophyll molecules probably went through the reaction cycle more than once for each flash. The point corresponding to a dark period of 0.0122 sec. on the upper curve in figure 7 is apparently slightly lower than the maximum, suggest- ing that the Blackman reaction is not quite completed in 0.01 sec. at 24°C. This is in approximate accord with indirect calculations of Emerson and Arnold (26) which indicated an average time of 0.012 sec. for the com- pletion of a cycle (photochemical + Blackman reactions) in Chlorella at 25°C. The lower curve in figure 7 was obtained from experiments in which the algae were suspended in heavy water. Evidently deuterium oxide retards the Blackman reaction without appreciably affecting the photochemical reaction, since, with a long dark period, the amount of photosynthesis per flash is the same in heavy water as in ordinary water.~3 These observa- o 30 - 2s 2 0 ' 15 A ~ 10 on ~ s 1 ' I ' i ' I l H2~ ;/ 0.01 0.03 0.05 0.07 0.09 LO NGTH OF DARK PERIOD (SECONDS FIG. 7. Rate of photosynthesis in Chlorella vulgaris per minute of actual illumina- tion as a function of the length of the dark period. The values are proportional to the photosynthesis per flash. (Pratt and Trelease (64~. tions indicate that the specific effect of deuterium oxide is manifested in the Blackman reaction. Possibly the replacement of water by deute- rium oxide may reduce the rate of the Blackman reaction by affecting a specific enzyme system. Emerson and Arnold (25) have studied the effect of low concentrations of hydrogen cyanide on photosynthesis in Chlorella in flashing light. With a hydrogen cyanide concentration of 1.14 X 10-4 moles per liter, the effect was very similar to that of 99.9 per cent deuterium oxide, as reported by t3 with continuous light of high intensity, according to the measurements of Craig and Trelease (21), the rate of photosynthesis in D20 is about 40 per cent of the rate in H20, except at very high temperatures (see figure 5), where the rate in HERO falls more rapidly than the rate in D2O. At low light intensities the rates in D20 and H20 become nearly equal. These observations are in agreement with the conclusions indicated by the flashing light experiments. o

PHOTOSYNT1 IESIS 131 Pratt and Trelease (64) (see figure 7~. The Blackman reaction was re- tarded but the maximum yield per flash was unchanged. But, if the hydrogen cyanide was replaced by a low concentration of phenylurethan or thymol, the maximum yield of photosynthesis per flash was decreased, with only a slight retardation of the Blackman reaction.~4 Figure 8 shows the observations of Emerson and Arnold concerning the influence of carbon dioxide concentration on the yield of photosynthesis per flash. As before, the flash duration was less than 2 X 10-5 sec. They concluded from these experiments that carbon dioxide enters the process sj ~ 4 ~0 I_ ._ A: cot In ._ ,: 2 - >% o o Do, / / ,0 2 4 6 8 10 12 Dark time in hundredths of a second 1 - 1 FIG. S. The course of the dark reaction at two different concentrations of carbon dioxide. Open circles, carbon dioxide concentration = 71 X 10-6 moles per liter; solid circles, carbon dioxide concentration = 4.1 X 10-6 moles per liter. (Emerson and Arnold (25~. Of photosynthesis either before or (less likely) coincident with the photo- chemical reaction. If chlorophyll alone were involved in the light reac- tion, then, according to their argument, a lower carbon dioxide concentra- tion would not decrease the yield per flash but would necessitate longer dark periods for full utilization of the light. Since the opposite effect was actually found, Emerson and Arnold concluded that both chlorophyll and carbon dioxide (perhaps in combination) are required for the light reaction. ]4 In continuous light, narcotics such as phenylurethan and thymol inhibit photo- synthesis at both high and low light intensities, in contrast to hydrogen cyanide, which inhibits only at high intensities (83~.

132 WINSTON M. MANNING However, it appears that this conclusion is open to question from two points of view. In the first place, carbon dioxide concentration was varied by changing the proportion of carbonate to bicarbonate in the buffer mixtures. Later experiments of Emerson and Green (29) have thrown doubt on the validity of this method of obtaining low carbon dioxide con- centrations (see page 825~. Secondly, it appears that the argument of Emerson and Arnold (given in the preceding paragraph) would be strictly valid only if the average lifetime of an activated chlorophyll molecule were much longer than the average time required for the :Blackman reac- tion. Otherwise, the lower curve in figure 8 could be interpreted as the resultant of two curves, one a slowly rising curve, like the lower curve in figure 7, and the other a falling curve representing a decrease in concentra- tion of activated chlorophyll. Although the behavior illustrated in figure 8 may not give very definite evidence concerning the order in which certain steps in the reaction cycle may occur, it probably indicates, as Emerson has pointed out (reference 23, page 319), that carbon dioxide enters into a reaction in the photosyn- thetic cycle other than the Blackman reaction, since, if the latter reaction alone were affected by carbon dioxide concentration, the maximum yield of photosynthesis per flash should remain constant. However, this last conclusion is still subject to the uncertainty concerning the influence of carbonate-bicarbonate buffers. Other experiments involving the use of intermittent light will be dis- cussed below (see pages 831 and 845~. The induction period in photosynthesis A number of investigators have observed an induction period in photo- synthesis, but the characteristics of this period appear to vary widely in different plant species. In the marine alga Ulva, Osterhout and Haas (61) found that about 2 hr. were necessary for attainment of a steady rate at high light intensity. In the moss Mnium, Briggs (14) found an induc- tion period of nearly an hour. Emerson and Green (27) found a period of about 20 min. for the marine alga Gligartina. A period.of 3 min. or less has been found in Chlorella (83), Hormidium (78), Cabomba (69), and wheat (53~. After a 10-hr. period of darkness, however, McAlister found a longer induction period for wheat, about 12 min. Warburg found an induction period in Chlorella only at high light intensities, but for Cabomba, Smith observed an induction period at both high and low intensities. However, Warburg's measurements extended to lower intensity values than did those of Smith. This wide diversity of characteristics suggests that the induction period may be caused by different mechanisms in different plants. Smith (69) has derived an equation for the induction period, relating the relative rate

PHOTOSYNTHESIS 133 of photosynthesis to the duration of illumination, which satisfactorily describes his own data and also data obtained by several other investi- gators. However, the application of this equation to a particular series of data depends upon the evaluation of a constant which determines the time scale. Most methods for measuring photosynthesis have too great a time lag to permit a satisfactory direct study of an induction period as short as 3 min. Warburg, van der Paau~, and Smith measured photosynthesis manometrically in their experiments, and resorted to an indirect integra- tion method for studying the short induction periods which they encoun- tered. Smith, for example, determined respiration for a 30-mint period and then exposed the plant (Cabomba) to light for 1 min. The manometer was not read until after a further 5- or 10-mint dark period. The rate so determined was, in effect, the resultant of 1 min. of photosynthesis super- imposed on a 6- or 11-mint period of respiration. The procedure was then repeated for the same or for successively longer light exposures. The methods of Warburg and van der Paauw were similar to this. A potential source of error in this type of procedure is the possible stimulation of respiration by light. With a short light period such as 1 min., even a slight increase in respiratory rate during the subsequent 5- or 10-mint dark period would greatly reduce the apparent yield of photosynthesis. Because of the very small time lag in his apparatus (see page 816), McAlis- ter (53) was able to determine the induction period in wheat from con- tinuous measurements of the photosynthetic rate. Considering the amount of carbon dioxide lost to photosynthesis as a measure of the induction period, he found that, in wheat, this quantity decreased with decreasing light intensity, apparently approaching zero at zero intensity. This result is in qualitative agreement with the behavior in Chlorella (83) and in Cabomba (69~. McAlister also found the induction period in wheat to be nearly twice as long at 12°C. as at 31°C. Van der Pasuw (78) observed a similar temperature effect in Hormidi?~m. In intermittent light, with equal light and dark periods, the rate of photosynthesis in wheat passes through a minimum for light and dark periods of about 1 min. (53~. With longer intervals a smaller percentage of total illumination time is taken up by the induction periods, while at shorter intervals it is probable that two factors contribute to an increase in rate. One is the occurrence of the Blackman reaction during the dark periods (see page 8261; the other is the probability that, after a short dark interval, the induction period is less pronounced than after a longer interval. No very convincing mechanism has so far been advanced for the induc- tion process in photosynthesis. Since three or more quanta of red light are necessary to supply energy for the reduction of one molecule of carbon

134 WINSTON M. MANNING dioxide to carbohydrate, it is often assumed that the process occurs through a series of three or more intermediate photochemical reactions (however, see page 851~. On this basis it would appear probable that, after a long dark period, each reaction unit in the chloroplast would have to undergo some or all of this series of reactions before evolving oxygen or absorbing additional carbon dioxide. This should produce an induction period last- ing until the establishment of equilibrium between the concentration of the various intermediates. Furthermore, the magnitude of the induction effect after a dark interval should give a measure of the extent to which the various intermediate compounds were decomposed during the dark interval. According to this picture the induction period should last much longer at low light intensities than at high intensities. This is contrary to the observed facts. Consequently, it is evident that processes other than those considered in the preceding paragraph must play a part in the induc- tion period. According to the mechanism of Franck and Herzfeld (34), oxygen is evolved after the first photochemical step in the series of reduction reac- tions. They suggested that the induction period is the result of photo- oxidation processes (see page 850~. It appears that further data concerning the influence on the induction period of various external and internal factors are greatly to be desired. Quantum e;~c~ency of photosynthesis A knowledge of the quantum efficiency of the photochemical process in photosynthesis is essential to any detailed consideration of the kinetics of the process. Unfortunately, the actual measurement of this quantity is accompanied by difficulties not encountered in quantum efficiency measurements carried out in non-living systems. Some of these difficulties will be considered in the following paragraphs. Because of the complexity of the photosynthetic process, many factors other than the efficiency of the photoprocess may influence quantum effi- ciency values determined from a measurement of the overall reaction. To a certain extent, the influence of these other factors may be minimized by using low light intensities, high carbon dioxide concentration, and a relatively high temperature. In this range, since the rate of photosyn- thesis is nearly proportional to light intensity, the quantum efficiency should be nearly constant and relatively independent of other external variables. Under these conditions, however, the necessary correction for respiration becomes relatively very large. If it were definitely established that the respiratory and photosynthetic processes are independent and unaffected by each other, and that the rate of respiration is not influenced by light, then respiration corrections, even when large, would not seriously

PHOTOSYNTHESIS 135 reduce the accuracy of photosynthesis measurements. However, van der Paauw (78) found that, in Hormidium, respiration was more than twice as rapid immediately after an hour of strong illumination as it had been before the photosynthetic period. Two hours after exposure, the respira- tion had dropped to its normal rate. In an atmosphere devoid of carbon dioxide, van der Paauw found that the respiratory rate was actually in- creased during illumination. However, it is possible that, in the absence of carbon dioxide, light may have produced an abnormal efl ect. Petering and Daniels (62) have observed a temporary increase in the rate of respiration for Chlorella after exposure to light, the extent of the increase depending on the previous treatment. On the other hand, McAlister (53) failed to find any stimulation in wheat plants after illumination at high light intensities, despite the fact that a very sensitive method was used. The difference between the response of wheat on the one hand, and Hormidium and Chlorella on the other, may be partially due to the much larger fraction of total cell volume occupied by the chloroplasts in the case of the algae. The measurement of light absorption in plant material is difficult. Pigments are irregularly distributed and much light is lost by refraction and scattering. From this point of view, a small unicellular alga, such as Chlorella, is more satisfactory than a leaf of a higher plant. Chlorella in suspension settles only slowly, making it easy to maintain even distribu- tion. Moreover, by varying the concentration of the suspension, the fraction of incident light absorbed can be varied at will. Not all of the absorbed light is absorbed by chlorophyll. The amount absorbed by other substances can be estimated from measurements made with extracted pigments, but such an estimate is uncertain, since the opti- cal properties and distribution of the various pigments are altered by the extraction process. Moreover, it is not known whether light absorbed by chloroplast pigments other than chlorophyll can contribute to photo- synthesis. At wave lengths longer than 5500 A, the uncertainty due to these other pigments is minimized, since their absorption is very slight at longer wave lengths. Another difficulty is found in the fact that, in some plant material, a large part of the chlorophyll may be inactive so far as ability to produce photosynthesis is concerned. After reaching maturity a leaf usually decreases in photosynthetic activity, even though the amount of cliloro- phyll may remain constant or increase. Because of this, it is advisable to use only young or actively growing material for quantum efficiency measurements. Finally, there is a possibility that the mechanism of photosynthesis is not the same for all plants. In this case the overall efficiency would prob- ably vary in different plants, even if all measurements were made under

136 WINSTON M. MANNING ideal conditions. However, the apparently identical nature of the pig- ments in many different plant species, as well as other similarities in the photosynthetic process, suggest that the photochemical reactions are the same, at least throughout the green algae (~Chlorophyceae) and the higher divisions of the plant kingdom (mosses, ferns, and seed plants). The first measurements of the quantum efficiency of photosynthesis were made by Warburg and Negelein (85, 86), with the alga Chlorella as plant material. A differential manometer was used for the photosynthesis measurements. A mercury arc with filters served as a source of mono- chromatic light except in the red, where a filament lamp with filters was used to obtain the region 6100-6900 A. To avoid the necessity of measur- ing transmitted light, Warburg and Negelein used very heavy suspensions of the algae. Except for a small amount of reflection, practically all of the incident light was absorbed. At any one time most of the Chlorella cells were thus receiving only a small fraction of the incident light intensity. To reduce the value of the respiration correction, these investigators carried out their measurements at a temperature of 10°C., where respira- tion is much slower than at 20° or 25°C. At wave lengths of 6600, 5780, and 5461 it. Warburg and Negelein found a quantum efficiency of approximately 0.25 molecule of carbon dioxide per quantum. The observed value at 4360 A. was lower, about 0.20, but an approximate correction for light absorbed by the carotene and xanthophyll pigments increased the 4360 A. value to 0.25 or slightly higher. The incident light intensity in these experiments varied from 575 to 3500 ergs per cm.2 per second. Within this range the quantum efficiency appeared to be independent of light intensity. At 6600 A. Nhv = 43,000 car., so that a quantum efficiency of 0.25 corresponds to an energy efficiency of 65 per cent on the basis of equation I. If formaldehyde is the first product of photosynthesis, then AH for equation I becomes 134,000 cal. and the corresponding energy efficiency is 78 per cent. In either case the energy efficiency is surprisingly large, particularly since one or more of the intermediate reactions in photo- synthesis is probably exothermic. The quantum efficiency of photosynthesis in Chlorella has recently been investigated by Manning, Stauffer, Duggar, and Daniels (55~. These investigators found quantum efficiencies much lower than those reported by Warburg and Negelein. Most of the experiments were carried out at 25°C., with high carbon dioxide concentrations. Light intensities varied from 830 to 24,000 ergs per cm.2 per second. A mercury arc was used with a monochromator to give monochromatic light of wave lengths 5461 and 4360 A. Other measurements were made with polychromatic light from the mercury arc, and still others with light from a tungsten filament lamp.

PHOTOSYNTHESIS 137 In some experiments a gas stream was bubbled through the algal suspen- sion and photosynthesis measured by gas analysis, while in other experi- ments a closed system was used, oxygen being determined by the Winkler method. In all cases the algal suspensions were less concentrated than those of Warburg and Negelein, since from 10 to 50 per cent of the incident light was transmitted through the back of the reaction vessel. The quantum efficiency values obtained by Manning, Stauffer, Duggar, and Daniels were quite variable, but the maximum value of approxi- mately 0.06 is so far below the figure obtained by Warburg and Negelein that it is difficult to attribute the discrepancy to differences in experi- mental procedure.~5 Different strains of Chlorella (possibly difl Brent species) may have been used in the two investigations (reference 55, page 272), but it is unlikely that such minor differences would cause any funda- mental difference in the photosynthetic mechanism. Quantum efficiency measurements, in Chlorella exposed to various intensities of sunlight below the surface of a lake, have been made by Manning, Juday, and Wolf (54~. The maximum value approached at low light intensities was approximately 0.05. Burns (18) has calculated quantum efficiencies from measurements of photosynthesis in white pine trees. In his experiments the top of a young tree was enclosed in a bell jar containing 1 per cent of carbon dioxide. The decrease in carbon dioxide concentration after 2 hr. of illumination was determined by gas analysis. Light sources were the 5890 A. line of sodium, the 5780 A. line of mercury, and polychromatic light with filters. The quantum efficiency for the yellow lines of sodium and mercury was approximately 0.13; for the polychromatic light the value was approxi- mately 0.11. The light intensities used were apparently greater than 10,000 ergs per cm.2 per second. In Burns' experiments it was necessary to estimate the amount of absorbed light by determining the absorption of an acetone solution of the extracted pigments. This procedure is subject to some uncertainty, since the pigment distribution is of course very different in the two cases. Such an approximation is apparently unavoidable when working with a light-absorbing system as complex as this. lain both investigations it was evident that the previous conditions of growth played an important part in determining quantum efficiency values. Warburg and Negelein found that a week of growth at high light intensity, followed by a week of growth at low light intensity, gave the most favorable results for their strain of Chlorella. It is especially necessary to avoid using old cultures which have passed the period of most active growth. Chlorella cells from such cultures often contain a high chlorophyll concentration, but nevertheless show a low photosynthetic ef- ficiency. It is possible that much of the chlorophyll in such cells is unable to trans- fer absorbed energy to the photosynthetic mechanism.

138 WINSTON M. MANNING Briggs (13) has determined the energy utilization for photosynthesis in the leaves of the bean (Phaseolus vulgaris), the elder (Sambucus nigra), and the elm. Most of the light intensities were in the neighborhood of 5000 ergs per cm.2 per second. The higher quantum efficiencies estimated from Briggs' data are in approximate agreement with those determined by Burns. The present data concerning the problem of the quantum efficiency of photosynthesis are in serious disagreement. Evidently a further study of the problem is greatly to be desired. It appears probable that accurate and extensive data on the variation of the quantum efficiency as a func- tion of wave length in different species may help to dispel the uncertainty concerning the photosynthetic activity of pigments other than chlorophyll, and also serve to provide a more secure basis for the postulation of chemi- cal and kinetic mechanisms for the process. Photosynthesis in bacteria The problem of photosynthesis in bacteria is a very interesting one, and may have an important bearing on the problem of photosynthesis in green plants. An adequate discussion of bacterial photosynthesis would require many pages; only a few aspects of the problem will be considered here. More complete information may be obtained from recent publications of van Niel (79, 80), whose researches have contributed a large portion of our present knowledge concerning bacterial photosynthesis. In the presence of light, hydrogen sulfide, and carbon dioxide, the green and purple sulfur bacteria are able to develop in entirely inorganic media. These bacteria grow only under anaerobic conditions, and no oxygen is given on during their development. Instead, sulfur or sulfuric acid is produced. Assuming that formaldehyde is the first product, the equation for the nr~r~.~.~ in t.h~ Ron ~~,lf,~r bacteria may be written: ,~ ~ ~ v ~~ ~ ~ ~ v a_ ~ e) ~ — _ light CO2 + 2H2S ) (CH20) + H20 + 2S AH and in the purple bacteria: light 2CO2 + H2S + 2H20 ) 2(CH20) + H2SO4 AH 22,000 cal. (II) 72,000 cal. (III) Apparently the green sulfur bacteria can utilize only hydrogen sulfide as a hydrogen donor for the reduction of carbon dioxide, but the purple sulfur bacteria can carry on photosynthesis in the presence of a number of substances other than hydrogen sulfide. Among these are sodium sul- fite, sodium thiosulfate, sulfur, hydrogen, and various organic substances

PHOTOSYNTHESIS 139 (79~.~6 For the reaction with hydrogen (37), the probable equation is light CO2 + 2H2 - ) (CH20) + H20 /\H _ 2000 cal. (IV) As Spoehr and Smith have pointed out (72), it is questionable whether the processes represented by equations II and IV can, strictly speaking, be called photosynthesis, at least if the word is used to denote an accumu- lation of light energy in the form of chemical energy. As in the higher plants, the formaldehyde or other intermediate product is presumably polymerized almost immediately to carbohydrates. If equations II and IV are written with glucose instead of formaldehyde as the reduction product, AH becomes approximately zero for equation II and negative for equation IV. Bacterio-chlorophyll, a pigment closely related to the chlorophyll found in green plants (32), occurs in both of these groups of bacteria. The purple bacteria also contain one or more red pigments, probably related to the carotenoid pigments of green plants. Bacterio-chlorophyll shows a strong absorption in the region between 8000 and 90ao A., in addition to its visible absorption bands. Consequently the sulfur bacteria are able to utilize infrared radiation in the reduction of carbon dioxide. French (38) has studied the effect of different wave lengths on the rate of carbon dioxide assimilation by one of the purple bacteria (Spirillum rnbrum). Using very thin suspensions of the bacteria, he found that the reaction rate corresponded closely to the absorption spectrum of the green pigment, even in regions where the red pigment absorbed strongly. Con- sequently he concluded that the red pigment is photoche~nically inactive in photosynthesis. Van Niel has suggested that the various equations for photosynthesis (I to IV) should be considered as special cases of a general type reaction: CO2 + 2H2A + nhv > (CH20) + H20 + 2A (V) where H2A represents a substance able to furnish hydrogen for the reduc- tion of carbon dioxide. Quantum efficiency of photosynthesis in bacteria Roelefson (66) has made some measurements of the quantum efficiency of photosynthesis in the purple sulfur bacteria. After making corrections for light absorbed by red pigments, he concluded that the maximum quantum efficiency is approximately 0.25 molecule of carbon dioxide per t6 The purple bacteria referred to here are members of the Thiorhodaceae. Another group of photosynthetically active purple bacteria, the Athiorhodaceae, apparently require organic compounds instead of sulfur compounds as reducing agents.

140 WINSTON M. MANNING quantum absorbed, in agreement with the measurements of Warburg and Negelein (86) for the alga Chlorella. French (37), using the purple bacterium Streptococcus varians, has made quantum efficiency measurements in which infrared radiation (wave lengths 8520 + 8940 A., from a cesium lamp) was used as the energy source. The red pigments do not absorb appreciably in this wave length region. The bacterial suspension was in equilibrium with an atmosphere of 5 per cent carbon dioxide and 95 per cent hydrogen. Consequently the overall reaction presumably corresponded to that of equation IV. French's measurements were complicated by the fact that he found a sigwoid curve for the variation in reaction rate as a function of light intensity. Thus, at low intensities the rate was proportional to a higher power of intensity than the first power. Quantum efficiencies were cal- culated from the maximum slopes observed for his rate versus light intensity curves. The maximum slope occurred at various light intensi- ties, depending upon the type of pretreatment of the bacteria. By extrapolation French found that as the position of the maximum slope approached zero light intensity, the quantum efficiency appeared to approach a value of 0.25 (or 0.5 on the basis of hydrogen), in agreement with the estimates of Roelefson. As Emerson has pointed out (24), there is some doubt concerning the validity of this indirect method of calcu- lating quantum efficiencies, particularly since it yields higher efficiency values than those actually prevailing in French's experiments. French agrees with van Niel (79) in believing that the agreement be- tween the quantum efficiencies observed for purple bacteria and those observed for Chlorella by Warburg and Negelein (86) indicate that a simi- lar sequence of ~hotoreactions is involved, despite the great difference in However. recent experiments with Chlorella (54, energy requirements. 55) have created some doubt concerning the correctness of the quantum efficiency values obtained by Warburg and Negelein (see page 835~. IV. THE ROLE OF CHLOROPHYLL IN THE PHOTOSYNTHETIC PROCESS Chlorophyll production The manner in which chlorophyll is manufactured by green plants is a problem of fundamental physiological importance. It will be men- tioned only briefly in this review, since it probably is not a direct part of the problem of photosynthesis. Further information on chlorophyll production may be obtained from articles by Spoehr and Smith (72), Inman, Rothemund, and Kettering (46), and Rothemund (67~. In most of the higher plants light is necessary for chlorophyll produc- tion. However, other plants, particularly some of the algae, can appar- ently produce chlorophyll in the dark. Etiolated plants, although usually

PHOTOSYNTHESIS 141 containing no chlorophyll, have been found to contain a very small amount of another green pigment which is usually called protochlorophyll. It differs spectroscopically from chlorophyll. When an etiolated plant is exposed to light, most of this green pigment disappears and is perhaps transformed into chlorophyll (67~. Most plants develop a higher chlorophyll concentration when grown in light of relatively low intensity than when grown in very bright light. Consequently several investigators have suggested that, in addition to its role in chlorophyll formation, light may also produce chlorophyll de- composition. According to this view, the chlorophyll concentration in a plant under given conditions can be regarded as a steady state concen- tration, with the rate of chlorophyll formation being approximately bal- anced by its rate of decomposition. The production of chlorophyll is, of course, also dependent upon the proper supply of mineral nutrients; perhaps the most conspicuous of these are magnesium, which enters into the chlorophyll molecule, and iron, which apparently acts as a catalyst in some step in the formation process. Chemical properties of chlorophyll The structure and chemical properties of extracted chlorophyll are now known rather completely, largely as a result of the investigations of Will- statter, Stoll, Fischer, and Conant. The subject has recently been re- viewed by Fischer (30) and by Steele (73~. Figure 9 shows the probable formula for chlorophyll a (30~. Fischer's formula for chlorophyll b is identical with the formula for a, except that the methyl group in the 3- position is replaced by a formyl group. Willstatter and Stoll (89) have found that the a:b ratio for chlorophyll in leaves is approximately 3. They found that this ratio was practically constant for many different species and for different environmental condi- tions. However, other investigators have found evidence that the ratio depends somewhat upon external conditions. Working with etiolated corn seedlings, Inman (45) found that, shortly after exposure to light, the chlorophyll a:b ratio was very high, approxi- mately 22:1. After an additional 90 min. of light exposure the ratio dropped to 17:1. Similar behavior has been observed in oat seedlings by Burr and Miller (19~. The simplest explanation for this behavior would be that chlorophyll a is produced first, and that chlorophyll b is an oxida- tion product of chlorophyll a. However, this type of oxidation would be very difficult to bring about in vitro. In some of the earlier theories concerning the photosynthetic mecha- nism, it was assumed that the a and b components formed a photo-acti- vated oxidation-reduction system, so that the cooperation of both components would be necessary to carry on photosynthesis. Aside from .

142 WINSTON M. MAxNING the difficulty involved in the methyl-formyl oxidation, further evidence that the a and b components do not function as an oxidation-reduction system is found in the observation of Fischer and Breitner (31) that chloro- phyll b does not occur in several of the red algae (Porphyra tenera, Bangia fuscopurpurea, and Polysiphonia nigrescens). On the other hand, Beber and Burr (11) observed no photosynthesis in etiolated oat seed- lings until long after a perceptible amount of chlorophyll a was formed. Their experiments suggested that chlorophyll b is necessary for photosyn- thesis in oat seedlings. Further evidence concerning the degree of inter- dependence of the two chlorophylls might be obtained from quantum H3C, CH CH2 I I H3C,I ~ ,Ic2H6 //~N; >N/ \ HO/ Mg ., :N~C l/iIII:/ H3C=CH2 HC10 bH2 \C/' boom ll COOCH3 FIG. 9. Fischer's formula for chlorophyll a (Steele (73)) AH HCH3 CH efficiency measurements with wave lengths chosen to correspond to the respective absorption maxima of the two pigments. Stoll (75), on the basis of his own and other investigations, has con- cluded that the action of both chlorophylls in the photosynthetic process is in large part due to the labile hydrogen atom attached to the carbon atom in the 10-position (figure 9~. According to his point of view, the existence of two chlorophyll components may be advantageous to a plant because of the increased range of wave lengths which can thus be absorbed (see figure 1~. This might be particularly useful to a plant growing in dim light. In the chloroplast the position of the absorption maxima for the two

PHOTOSYNTHESIS 143 chlorophylls is shifted approximately 200 A. to the red as compared to the position for the extracted pigments in ether solution. This shift is usually ascribed to differences in the optical- properties of the solvents. Albers and Knorr (2) have recently studied the absorption spectra of single chloroplasts in the region from 6640 to 7040 A. They found evi- dence for several maxima in the absorption band usually attributed to chlorophyll a. They suggested that the several maxima may be due to the presence of compounds between chlorophyll a and various inter- mediates in the photosynthetic process. The phytyl group in the chlorophyll molecule has little influence on the optical properties. Replacement of the phytyl group by a methyl group produces only a slight change in the chlorophyll absorption spectrum. The optical properties are due principally to the ring of conjugated double bonds surrounding the central magnesium atom. The phytyl group, according to Stoll (75), may serve to make chlorophyll lipoid-soluble, thus permitting proper distribution in or on the plastic. Stoll suggests that, in addition to maintaining the conjugated bond system, magnesium is important in maintaining the proper degree of reac- tivity with carbonic acid. Chlorophyll forms a complex with carbonic acid in vitro, and Stoll, as well as many other investigators, suggests that a similar complex is formed in the plant. As mentioned above (page 825), earlier evidence indicated that the rate of photosynthesis is temperature- dependent even at very low carbon dioxide concentrations. This was regarded as additional evidence for the formation of a carbonic acid (or carbon dioxide) complex with chlorophyll during the photosynthetic proc- ess. But the experiments of van den Honert (77) and of Emerson and Green (29) have indicated that the temperature dependence at low carbon dioxide concentrations may be only apparent, and due to complications resulting from the use of buffer mixtures. These observations weaken somewhat the evidence for participation of a carbonic acid-chlorophyll complex in photosynthesis. It has frequently been observed that chlorophyll in solution is decom- posed by the action of light. Albers and Knorr (1, 51) have followed this decomposition in the presence of various gases, by observing changes in the fluorescence spectra of chlorophylls a and b. Porret and Rabino- witch (63) have studied the bleaching of chlorophyll dissolved in methanol. In the presence of oxygen the quantum yield was approximately 10-6. This value was independent of oxygen concentration over a wide range, but the bleaching disappeared on complete removal of oxygen. In the absence of oxygen Porret and Rabinowitch (63) found that in- tense light caused a reversible bleaching of the red absorption band of chlorophyll. Using a 10-6 molar solution of chlorophyll in methanol, and a light intensity such that each chlorophyll molecule absorbed about 10 quanta per second, the bleaching of the red band amounted to about I

144 WINSTON M. MANNING 1 per cent. Therefore, assuming the quantum efficiency of the bleaching process to be 1, they estimated an average lifetime for the bleached state of about 10-3 sec. The degree of bleaching was approximately propor- tional to the square root of light intensity. Porret and Rabinowitch suggested that the bleaching was probably due to a dissociation into "dehydrochlorophyll" and a hydrogen atom (perhaps from carbon atom No. 10; see figure 9~. Ifs the presence of formic acid the reversible bleach- ing was increased to 10 per cent. With a dissolved oxygen concentration of 10-5 molar, the bleaching with formic acid was reduced about 50 per cent. Apparently the oxygen either formed a complex with chlorophyll or removed the chlorophyll excitation before dissociation could occur. Rabinowitch and Weiss (65) have found that chlorophyll in methyl alcohol solution is transformed by certain oxidizing agents, particularly ferric chloride, into a yellow product. The chlorophyll could be restored to its original condition by the action of ferrous chloride. The oxidation process was accelerated by light. In the yellow oxidation product the red absorption bands were virtually eliminated, and the blue bands shifted toward shorter wave lengths. A rather peculiar feature of the oxidation process was that the blue band of chlorophyll b was changed much more slowly than the red band of b, and also more slowly than either the red or blue bands of the a component. This suggests that the blue absorp- tion band of b may be produced by a chromophoric group different from those responsible for the other absorption bands of chlorophyll. Rabino- witch and Weiss consider it probable that the oxidation of a chlorophyll molecule results in the formation of a positively charged chlorophyll ion, which perhaps then breaks down into a hydrogen ion and dehydro- chl~rophyll. It is probable that, in the process of extraction from plant cells, the chlorophyll pigments undergo some sort of chemical change. Some in- vestigators (e.g., Stoll (75) and Franck and Herzfeld (34~) have suggested that, in the chloroplast, chlorophyll may be combined with a protein. Its specific effectiveness in photosynthesis would thus depend upon its attachment to a particular colloidal carrier. In any event, attempts to bring about carbon dioxide reduction by extracted chlorophyll have thus far been unsuccessful.~7 Whether this failure is due principally to changes t7 Baly, Stephen, and Hood (8) have reported the formation of carbohydrates from carbon dioxide and water, with visible light as the energy source. The light-absorb- ing agent in their experiments was not chlorophyll, but an aqueous suspension of nickel or cobalt carbonate. However, a number of other investigators have tried, without sucess, to repeat the experiment of Baly, Stephen, and Hood. It may be that these other investigators have failed to duplicate with sufficient exactness the procedure described by Baly et al., but, as Emerson (23) has suggested, it seems rea- sonable to require that the production of photosynthesis in vitro should be capable of repetition before it can be regarded as an established fact.

PHOTOSYNTHESIS 145 in the chlorophyll, or to changes in the environment, or both, is as yet an unanswered problem. Chlorophyll fluorescence Solutions of chlorophyll emit a bright red fluorescence when illuminated with visible or near ultraviolet radiation. It has been estimated that, under favorable conditions, about 10 per cent of the absorbed radiation may be recovered as fluorescence (36~. Chlorophyll in living cells also fluoresces, but much less strongly than solutions of the extracted pig- ment. The maximum yield for chlorophyll fluorescence in living material is of the order of 0.01 per cent (81~. Colloidal solutions of chlorophyll do not fluoresce. The fact that fluorescence occurs in the living cell is often regarded as an indication that chlorophyll is in a dissolved state in the chloroplast. However, the very low intensity of fluorescence in the chloroplast lessens the force of this argument. Zscheile (94) has determined the fluorescence spectra of chlorophylls a and b in ether solution. The exciting radiation was supplied by a tung- sten filament lamp. The a component showed two bands and the b component three bands in the region 6300-8200 A. No observations were made at wave lengths below 6300 A. Kautsky and coworkers (49, 50) and Franck and Wood (36) have stud- ied the eject of illumination time on the intensity of fluorescence in living leaves. In Kautsky's experiments the 3660 A. line of mercury was used as the exciting source, while, in the experiments of Franck and Wood, a tungsten filament lamp with a blue filter was used to produce fluores- cence. The results of these two series of investigations, while not identi- cal, showed the same general type of behavior. Following a long period of darkness, the fluorescent intensity from an illuminated leaf is at first very low, but increases rapidly to a maximum after 2 to 5 sec. of illumi- nation. The intensity then diminishes until a constant level is reached after about a minute. At a temperature of 35°C., Kautsky and Marx (50) found that this constant intensity was approximately two-thirds of the temporary maximum value, but at 0.1°C. the intensity remained con- stant at nearly the maximum value. In the presence of oxygen, the fluorescent intensity was reduced to about half the value observed in the absence of oxygen (49~. Franck and Levi (35) found that weak alcoholic or acetonic extracts of leaves, when irradiated in the presence of oxygen, showed a varia- tion in fluorescent intensity similar to that found in leaves. Kautsky concluded from his investigations that there is an intimate connection between the process of photosynthesis and the process of fluorescence. Earlier investigations (89) indicated that oxygen is neces- sary for photosynthesis. Consequently, Kautsky attributed the quench-

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-

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

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

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.

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.

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)

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

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.

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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