The Dependence of the Quantum Yield of Chlorella on Wave Lenghth of Light Author(s): Robert Emerson and Charlton M. Lewis Source: American Journal of Botany, Vol. 30, No. 3 (Mar., 1943), pp. 165-178 Published by: Botanical Society of America, Inc. Stable URL: http://www.jstor.org/stable/2437236 Accessed: 24-06-2016 13:38 UTC

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This content downloaded from 130.223.51.68 on Fri, 24 Jun 2016 13:38:58 UTC All use subject to http://about.jstor.org/terms THE DEPENDENCE OF THE QUANTUM YIELD OF CHLORELLA PHOTOSYNTHESIS ON WAVE LENGTH OF LIGHT 1

Robert Emerson and Charlton MI. Lewis

MlEASUREMENTS OF the efficiency of plhotosyntihe- ble of photosyn-thesis. But owiing to the complexity sis in different wave lengths of light have receiitly of the plant's assimilatory mechaniism, it seems ap- been used as an approach to the problem of the propriate to inquire to what extent the activity of physiological role of the pigments accompanving chloroplhyll bears out expectation. chlorophyll. Dutton and M'ianning (1941) concluded Many investigators have accepted Warburg and from their measurements with the diatom Nitzschia Negelein's (1923) measurements of the efficiency closterium that the light absorbed by fucoxanthin of Chlorella photosynthesis in different parts of the and other carotenoid pigments was available for spectrum as satisfactory proof of the constancv of assimilation. Emerson and Lewis the yield for light absorbed by chlorophyll. But (1942) made similar measurements with the blue these measurements were brought into question by green alga Chroococcus, and concluded that light the work of Manning, Stauffer, Duggar, and Dan- absorbed by the blue pigment phycocyanin gave a iels (1938) and Emerson and Lewis (1941) h-ave photosynthetic yield equal, within the experimental shown that Warburg and Negelein's results are error, to the yield for light absorbed by chlorophyll. subject to certain errors inherent in their method, They found, on the other hand, that the quantum and that their value for the quantum vield of photo- yield in the region of carotenoid absorption was synthesis should, therefore, be rejected. Since the much reduced, and regarded this as a clear indica- magnitude of the error arising from Warburg and tion that these pigments were not photosynthetic- Negelein's method may vary with wave length, their ally active with the full efficiency of chlorophyll and conclusions as to the dependence of quantum yield phycocyanin. However, when estimates of the pro- on wave length are also open to doubt. Further portion of light absorbed by the carotenoids were data on this point would have been desirable in any mnade from pigment extracts, and compared with case, because their conclusions were based only up- the dependence of quantum yield on wave length, on measurements in the yellow mercury line (578 evidence was obtained tlhat a fraction of the light m1u) and in a broad band of red light from 610 to absorbed by the carotenoids was probably available 690 m[u. Measurements were also made at 546 and for photosynthesis. The same technique has been 436 m[k, but at 436 the absorption was divided be- applied to the problem of carotenoid activity in the tween chlorophyll and carotenoid pigments, and at green alga , and the results 546 the measurements were regarded as somewhat are reported in the present paper. uncertain because of the very dense suspen- In the work with both Nitzschia and Chroococ- sions required to absorb all the incident light in this cus, the quantum yield for light absorbed bv chlo- region where chlorophyll absorption is low.2 rophyll was measured in a wave length region The above considerations make evident the de- where all or nearly all of the total light absorbed sirability of more precise information as to the de- was absorbed by chlorophyll. This yield for light pendence of quantum yield on wave length, particu- absorbed by chlorophyll was then compared with larly in the red region, where chlorophyll is the the yield for spectral regions where light absorp- principal light-absorbing pigment. The absorption tion was divided between chlorophyll and accessory of light by the accessory pigments of Chlorella is pigments, to show whether or not the energy ab- restricted to a narrower range of the visible spec- sorbed by the accessory pigments was being used trum than is the case for Nitzschia or Chrobcoccus, for photosynthesis. In making this comparison it leaving a broader region for study of photosynthe- was assumed that the yield for the fraction of light sis in light absorbed by chlorophyll alone. If the absorbed by chlorophyll did not vary with wave yield for light absorbed by chlorophyll, and the length, but remained the same even when a large dependence of this yield on wave length, cani be fraction of the light was absorbed by other pig- established by measurements in a spectral region ments. The Einstein photochemnical equivalent law where chlorophyll alone absorbs all the light, this predicts that the primary photochemical action may imiake possible a more satisfactory analysis of should be proportional to the number of absorbed measurements in other spectral regions, where ab- quanta, so it inight reasonably be expected that the sorption is divided between chlorophyll and other quantum yield for light absorbed by chlorophyll 2 The dense cell suspensions made the respiration read- should be independent of wave length, at least over ings unduly large in proportion to the photosyntlhesis. The apparent necessity of using such dense suspensions the spectral range within which chlorophyll is capa- to achieve total absorption of the incident light may have 1 Received for publication October 6, 1942. arisen from the error associated with Warburg and The experimental work reported in this communication Negelein's method of measurement. When this error was was carried out during the years 1938-40 at the Carnegie avoided, we found no indications of incomplete light Institution of Washington Laboratory of Plant , absorption, even with much thinner suspensions than they, , Calif. used (cf. Emerson and Lewis, 1941, p. 801-802).

[The Journal for Februarv (30: 83-163) was issued March 31, 1943.] AMERICAN JOURNAL OF BOTANY, VOL. 30, No. 3, MARCH, 1943.

165

This content downloaded from 130.223.51.68 on Fri, 24 Jun 2016 13:38:58 UTC All use subject to http://about.jstor.org/terms 166 AMERICAN JOURNAL OF BOTANY [Vol. 30, pigmiients of uncertain physiological activity. This compared with the absorption spectrum of the cells is the reason for the particular attention devoted to measured by direct methods. the red region in the present investigation. EXPERIMENTAL METHODS. MIost of tlhe teclh- In the course of the experimental work, two niques used have been fully described in earlier phenomena were encountered which raised special communications (Emerson and Lewis, 1939, 1941, problemiis in the measurement of the quantum yield 1942), and need only be mentioned briefly. Cells in certain portions of the spectrum. Exposure of were grown in the usual inorganic nutrient medium cells to the blue-green region sometimes caused a prepared in glass distilled . Manganese, considerable inierease in the apparent rate of res- boron, zinc, copper, and molybdenum were added piration. An unexpectedly sharp decline in the in concentrationis of 0.5, 0.25, 0.025, 0.005, and quantum yield was observed in the far red. Sinice 0.05 parts per millioii respectively. Cultures were these two observations are probably at least as sig- grown in continuous incandescent illumination at nificant as the original purposes of the work, they a temperature of about 20?C. An iiioculum of about are discussed more fully than might appear to be 1 cmm. cells in 200 ml. of medium produced a necessary in view of their apparent treatmeiit mere- growth of about 300 cmm. of cells in from five to ly as difficulties of an experimental character. seveii days. Cultures of about this density were har- The same general plan has been followed as in vested for the photosynthesis measurements. The the correspondinig work on Chroococcus (Emerson cells were centrifuged out of the culture medium, and Lewis, 1912). To obtain evidence concerning washed twice in a mixture of 85 parts M/10 sodium the activity of accessory pigments from measuire- bicarbonate and 15 parts M/10 potassiumi carbon- ate, suspended in the same mixture, and pipetted x I l l l lI / ~ into the manonieter vessel. Twenty-five ml. of fluid containing about 300 cmin. cells was the usual filling for the manometer vessel. This concentration of cells was usually sufficient for total absorption

0 of the incident light, except for the green and in- frared regions of the spectrum, where denser sus- pensions were used. It was sometimes necessary to E 4 0 Red make a small correction for transmitted light, based C on visual estimation of the per cent transmitted. 0 Green The reliability of such estimates has been discussed A Blue in an earlier paper (1939, p. 819-20). Further ex- 2 / perience has shown that when the tranismitted light is less than 5 per cent, visual estimation introduces o no serious error. For measurements of the "active 0c0 I I I I I absorption," where it was desired to work with sus- 0 2 4 6 8 10 penisions absorbing only about half the incident Infensify in einsteins /cm2/ min. x 107 beam., about 40 cmin. cells were used in 25 ml. of Fig. 1. Photosyntlhesis as a function of intensity at carbonate mixture. The carbonate mixture is strong- three different wave lengths, 660 m,u (circles), 560 m, ly buffered for carbon dioxide and at 20? maintains (squares), and 460 m,u (triangles). The area of cross a constant concentration of about 0.2 millimoles per section of the light beam was 15.4 cm.2 at the vessel. The liter. Rates of photosynithesis were calculated from intensities that were ordinarily used for measuring quari- changes in oxygen pressure, measured manometric- tum yields varied from 0.1 to about 0.5 X 10-7 einsteins ally with a double cathetoineter reading in huii- per cm.2 per minute. dredths of a mnillimeter. The plhotosynthesis measureiiients were made at ments of the quantum yield of photosyiitlhesis at 200C., with light from a 1000-watt tungsten fila- different wave lengths, it was necessary to supple- ment lamp, passed through a large grating mono- ment the quantum yield measurements with esti- cliromator. The monochromator and lighting sys- mates of the proportions of light absorbed by the yellow and green pigments at each wave length. temr, constructed with the aid of a grant from the The quantum yield measurements have been made National Research Council, will be described in with dense suspensions of cells which absorbed all a separate publication (Lewis and Emerson, in the incident light. The proportions of light absorbed preparation for publication). Photosynthesis could by the yellow and green pigment components have be measured in the red region with band half- been estimated from absorption measurements on widths as inarrow as 50 A (width to half the extracted pigments. The conclusions drawn from a maximum intensity), and in blue with half widths comparison of quantum yields and light absorption of about 160 A. Scattered radiation outside the have been further supported by measurements with band being transmitted by the inonochromator was thin suspensions, which absorbed only about half reduced by means of colored glass filters trans- of the incident light. From measurements with thin mitting only in the region of the band being used. suspensions, the absorption spectrum of the photo- Scattered infrared radiation was reduced by a 1-cm. synthetically active pigments has been plotted and layer of a saturated solution of ferrous ammoniurn

This content downloaded from 130.223.51.68 on Fri, 24 Jun 2016 13:38:58 UTC All use subject to http://about.jstor.org/terms Mar., 1943] EMERSON AND LEWIS-CHLORELLA PHOTOSYNTHESIS 167

sulfate. By a simple change of a mirror and lens pendent of light intensity over a considerable range. in the light path, it was possible to project the en- Eichhoff (1939), however, has claimed that the tire light beam emitted by the monochromator shape of the curve for dependence of photosynthe- either into the vessel containing the assimilating sis on light intensity is different in white light and cells, or onto the receiving surface of a platinum in red light. Therefore, it seemed necessary to in- bolometer for measurement of radiation intensity. vestigate whether the measured quantum yield was We are again indebted to H. H. Strain for ex- independent of intensity over the intensity range traction of the cell pigments, separation of the yel- used for several wave lengths in the visible spec- low components by saponification and removal of trum. Figure 1 shows measureinents of rate of pho- the chlorophyll, and measurement of the absorp- tosynthesis plotted against light intensity in red, tion spectra both before and after separation. (For green, and blue light, up to the highest intensities a description of the technique of extraction and obtainable with the monochromator. Much more separation of the pigments, cf. Strain, 1938, p. 125- energy was available in the red region (points 132.) The absorption due to chlorophyll was com- plotted as open circles) than in the blue region puted as the difference between the absorption be- (triangle points). Measurements in green are fore and after removal of the chlorophyll. We plotted as squares, and run nearly up to the maxi- measured the absorption spectrum of live cells just mum intensity used in red. Since the purpose of this as in the work on Chroococcus, with a photronic experiment was only to show how nearly the rate of cell placed in contact with the window of an ab- sorption cell containing the algal suspension. photosynthesis was a linear function of light in- DEPENDENCE OF YIELD ON LIGHT INTENSITY.- tensity in the three different wave lengths, the three Before proceeding to a comparison of quantum sets of measurements were made with different cul- yields and absorption spectra, we must mention tures, and should not be used for comparing quan- certain experiments showing the dependence of the tum yields in differenit wave lengths. All the meas- quantum yield measurements on light intensity and urements fall pretty close to the same line. lending rate of respiration. The results of these experi- no support to Eiclilioff's suggestion that intensity ments lend support to the validity of the technique dependence is a function of wave length. The figure used for measuring quantum yields. shows that for the low intensities used for efficiency It has already been shown (Emerson and Lewis, measurements (about 5x 10-s einsteins/cm. 2/min. ), 1941, fig. 6) that the quantum yield for Chlorella the yield may be considered independent of inten- in sodium light (X = 589 m,u) is practicallv inde- sity in all three wave length regions.

c6) T Ti1 ~ I I I I -T

CTo

ha : a

:3-40 -0 (90 m 0.

m i minue r-60-

o 00

00 C-io 0

Fig. 2. Evidence for an effect of light on rate of oxygen consumption. The rate of pressure change (due to oxygen exchange) is based on readings of the manometer at one-minute intervals and is plotted in hundredths of a milli- meter of manometer fluid per minute. Vertical lines indicate the times at which the light was turned on or off. Light periods are indicated at the bottom of the graph by giving the wave length in m,t of the light used. The cells were in the dark for about 05 minutes preceding the beginning of observations. The vessel contained 260 cmm. of cells in 25 ml. of carbonate buffer. The vessel constant Ko was o 2.106. Intensities used were 8.0 X 10c einsteins per cm.2 per minute at 480 m,u; 4.1 X 10-8 at 435; and 7.3 X 10-8 at 560.

This content downloaded from 130.223.51.68 on Fri, 24 Jun 2016 13:38:58 UTC All use subject to http://about.jstor.org/terms 168 AMERICAN JOURNAL OF BOTANY [Vol. 30,

THE CORRECTION FOR RESPIRATION AND THE DE- in figure 2 shows responses to other treatments, the PENDENCE OF OXYGEN CONSUMPTION ON WAVE probable course of respiration during the light peri- LENGTH OF LIGHT.-A small effect of light on res- ods being drawn as a dotted line. The course of this piration is generally admitted to be a probable line is estimated from the drift in rate of pressure source of error in all measurements of photosynthe- change during the light exposures, on the assump- sis. According to Gessner (1938) the magnitude of tion that the rate of photosynthesis is constant, and the effect depends very much on previous dark that the changes are due to respiration. Exposures adaptation, but it does not seem to be directly de- to 435 or 560 my do not appear to interrupt the pendent on accumulation of the products of photo- slow decline in respiration which is evident during svnthesis, because ultraviolet irradiation, which the dark periods. Another exposure to 480 m,u just caused no photosynthesis, nevertheless resulted in before the change to 560 suggests another rise in small subsequent increases in respiration similar to respiration following the drop which had occurred those observed after exposure to visible light. during and after the exposure to 435 m,. This time Mothes, Baatz, and Sagromsky (1939) report that the rise is much smaller than it was with the first if infrared radiation is carefully filtered out. then exposure to 480. However, after a dark period of exposure to visible light has no effect on subsequent two and a half hours the effect is rather larger than respiration. Although these and similar observa- with the original exposure. It will be noted that all tions suggest only minor effects of light on respira- these changes in respiration are superimposed on tion, we have found that there is a fairly narrow a gradual downward trend which persists during range of wave lengths which may cause such large the whole course of the observations. increases in rate of oxygen consumption that they Other experiments were made to establish the easily become observable and constitute a serious approximate limits of the effect. Starting at the red obstacle to the correct measurement of photosyn- end of the spectrum, there was no clear influence thesis. In the following discussion we refer to these of light on respiration down to about 530 m,. A effects of light on oxygen consumption as effects on ten-minute exposure to this wave length caused "respiration," simply because the dark correction about a ten per cent increase in respiration, and applied to measurements of photosynthesis is con- shorter wave lengths gave increasing effects to ventionally designated as "respiration." We do not about 470. The effect was still prominent at 460, wish to imply that the physiological nature of the but by 435 my it was small or perhaps entirely ab- extra oxygen consumption is the same as ordinary sent. The maximum observed increase in respiration respiration, because we have no evidence whatever was from the last exposure shown in figure 2, which on this point. caused a rise of over 70 per cent. For such large Figure 2 shows the behavior of the rate of pres- effects, intensities considerably above those ordi- sure change when a suspension of cells was exposed narily used for the quantum yield measurements to light of 480 my, where the effect is about maxi- were required. mal. (The rates shown represent oxvgen ex- The rates of respiration of different samples of changed expressed in hundredths of a millimeter of cells were not equally sensitive to light of the region the manometer fluid per minute.) The observations near 480 m,u. Cells grown over neon tubes, instead begin after 75 minutes in darkness, when the rate of the usual incandescent lamps, showed particu- of respiration has dropped to a steady value of -85 larly large increases of respiration in response to per min. Exposure to light causes a change to -12 light in this region. The cells grown in neon light as a result of photosynthesis, but instead of main- developed a smaller amount of chlorophyll in pro- taining this new rate, as we should expect from portion to the yellow pigments, and were distinctlv measurements at other wave lengths (cf. Emerson yellow-green in color. With certain other samples and Lewis, 1941, fig. 5), the rate changes in the of cells, exposure to light of 480 m,a caused only course of a few minutes to -25 per minute. This negligible increases in respiration. In the experi- might be interpreted as a fall in rate of photosyn- ments with Chrobcoccus cells (Emerson and Lewis, thesis, but when the suspension is darkened after 1942) no specific effects of blue light on respira- 44 minutes of light exposure, the respiration is tion were observed. found to be much larger than it was before illu- The observations discussed above were made on mination, -112 instead of -85, suggesting strongly thick suspensions of cells. With thin suspensions that the alteration in rate of pressure change dur- the rate of respiration appeared to be altered to ing the early part of the light period was due to an approximately the same extent, but the effect was increase in respiration rather than a decrease in not ordinarily noticeable because of the much lower photosynthesis. By the end of the 15-minute dark rate of pressure change due to respiration. period the rate of respiration has dropped back to Since the effect is limited to wave lengths where about -100, and is still dropping rapidly. At this a considerable proportion of the absorbed light point, light of 480 m,u is given again, and this time must be absorbed by the carotenoids, a photochemi- a fairly steady rate of pressure change is attained cal acceleration of respiration by the yellow pig- almost at once, showing that a stationary state has ments is suggested. The large responses with the been reached, and the respiration neither rises nor yellow-green cells grown in neon light support this falls in response to the light. The rest of the curve suggestion. But a strictly photochemical effect of

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this kind would not be expected to carry over into our work on quantum yields, and indeed most quan- the ensuing dark period, nor should it be so slow titative work on photosynthesis, is based on the in starting as indicated by figure 2. assumed constancy of respiration during light ex- Various modifications of experimental procedure posure, one must keep in mind the possibility that were tried in order to avoid, as far as possible, the minior changes in respiration may bring about small errors in photosynthesis measurements which would and perhaps systematic errors in the computed rate arise from such large effects of light upon rate of of photosynthesis. Under the conditions of the quan- respiration. We found that with wave lengths pro- tuim yield measurements, the rate of respiration ducing such effects, a conditioning exposure of tends to decline slowly over a period of several about twenty minutes usually sufficed to bring the hours. We have found that some decline takes place respiration to a fairly steady rate. Subsequent meas- regardless of whether the cells are kept in light or urements of rate of photosynthesis by means of the darkness or whether they are exposed to the ten- usual alternating ten-minute periods of light and miniute alternations of light and darkness used for darkness then gave no indications of serious errors the photosynthetic measurements. The decline is due to changes in rate of respiration during illu- genierally slower when some illumination is given. mination. At first we thought it possible that much time could It may be that other wave lengths of light have be saved by measuring the rate of respiration only smaller effects on respiration which remain unde- at the beginning and end of a series of exposures tected because they do not persist long enough into to different wave lengths of light, and interpolating the subsequent dark periods to be evident. Since all values of respiration on the assumption that the

>1.2

m [ o Total Extracted Piqments

1.0 / Chlorophyll

/ine isofrtoaligmets;therk--o Carotenoids

0.8.

0.6.

0

000

0~~~~ /

0.2

0 400 440 480 520 560 600 640 680 720 Wa ve Le-nqth in m Flig. 3. Absorption spectra of ethanol solutions of pigments extracted quantitatively from Chiorella cells. The solid line is for total pigments; the broken line for the carotenoid fraction, after saponification and separation of the chlorophyll; and the dotted line for the chlorophyll fraction, obtained by subtracting the curve for carotenoids from that for total pigments. At wave lengths longer than 520 m,b the spectrum for chlorophyll coincides with that of the total extract. Complete absence of chlorophyll from the carotenoids after saponification is indicated by the four points of the carotenoid curve near 650 m,u.

This content downloaded from 130.223.51.68 on Fri, 24 Jun 2016 13:38:58 UTC All use subject to http://about.jstor.org/terms 170 AMERICAN JOURNAL OF BOTANY [Vol. 30, rate of decline had been linear. But measurements tum yield has beeni founid to be the saim1e at 00, 100, made in this way were unsatisfactory, because the and 200, altlhouighi the rate of respiration changed rate of decline was not the same during continuous by a factor of 5 over this range of teinperatures. light as witli alternating light and dark periods, anid The relative constancy of the quantum yield in a a series of exposures to different wave lengths nlumber of different organisms, as compared to wide seemed always to be accompanied by a certain num- variability in rate of respirationi, is further evidence ber of unpredictable small irregularities iu respira- that errors due to the method of estimating the res- tion. We, therefore, used alternating ten-minute piration correction must be snmall (cf. Emerson and periods of light and darkness for most of the meas- Lewis, 1941, tables 2 and 6). But in limiting cases, urements. It was often necessary to discard the such as at very low light intensities or toward the measurement during the first exposure to a new infrared where photosynthesis becomes very small, wave length or intensity, because such changes it may sometimes be impossible to distinguish be- seemed to be followed by small changes in respira- tween actual plhotosynthesis and small effects of tion, so that the rate of respiration before exposure light on respiration. to a new wave length was not strictly comparable COMPARISON OF ABSORPTION SPECTRA OF EX- with the rate after the exposure. TRACTED PIGMENTS AND INTACT CELLS.-Figure 3 There is good evidence that, with alternating ten- shows a plot of the absorption spectra of the pig- ininute periods of light and darkness, the dark read- ments extracted from Chlorella. The curve drawn ings ordinarily lead to a close estimate of the rate with a solid linie is for the alcoholic extract, con- of respiration duriiig the light exposures. Tlle quan- taining both chlorophyll and carotenoids. The

1.2 ~I_ _ _ _

Intact Cells

'-.~ Combined Ext ract s( C.0 x x Relative Absorption Lr 0 C by Carotenoids

0.6gWf 'I 60

0.4 40

- -I- -X-9

0.2 20

0 0 400 440 480 520 560 600 640 680 720 Wave Length in m, Fig. 4. Comparison of the absorption spectra of the extracted pigments and of intact Chlorellcl cells. The solid curve shows the absorption due to a suspension of intact cells 1.4 cm. thick and containing 0.96 cmm. of cells per ml. The spectrum of the extracted pigments, given by the broken line, was derived from figure 3 by combining the chlo- rophyll and carotenoid curves after shifting each one toward the red an amount intended to compensate for the wave length shifts accompanying extraction. All curves for extracted pigments represent a pigment concentration that corresponds quantitatively to the concentration of live cells used in obtaining the solid curve. (The two breaks in the latter curve are at points where the cell suspension was stirred and the filters on the monochromator changed.) The crosses show calculated values for the fraction of the total absorbed liglht that is absorbed by the carotenoids, based on the curves for the extracts after introduction of the wave length shifts. The ordinate scale for these points is at the right.

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broken curve slhows the absorption by carotenoids paring the absorption spectra, to avoid differeiices after saponification and removal of the chlorophyll. due merely to the shift of the bands which is knlowni A few measurements were made on the carotenoid to accompany extraction. For the Chilorella curves, solution at about 650 m,u, and the absence of sig- the red chlorophyll maximum has been shifted 15 nificant absorption in this region shows the effec- m[u toward longer wave lengths, anid the blue mllaxi- tiveness of the technique used for the removal of mum 6 muT, the chanige in amount of shift coml-ing the chlorophyll. The dotted curve from 400 to 520 at 540 inu. The carotenoid curve was shifted :1 mru shows the difference between the curve for the m[u toward the red. These adjustments were made carotenoids and the one for the total pigments, and before adding the curves to give the broken lie represents absorption due to chlorophyll. Beyond in figure 4. 520 practically all the absorption is due to cllo- In the case of the corresponding curves for rophyll. Chroococcus, whiclh were obtained by making simni- Figure 4 shows a comparison of the absorption lar adjustments (Emerson and Lewis, 1942, fig. 2), by the extracted pigments (broken curve) with the the absorption curve for the sum of the extracted absorption by a suspension of the living cells (solid pigmeints was sufficiently similar to the curve for curve). Both curves represent the same concentra- the intact cells to justify the conclusion that the tion of pigment per unit cross section of the light absorption characteristics of the individual conmpo- beam, and they were made with samples of cells nents had been altered but slightly by extraction, from the same suspension. The curves for the ex- and, hence, that the relative amounts of light ab- tracted pigments have been shifted to make the sorbed by the pigments in the living cells could be positions of the maxima coincide as nearly as pos- estimated from the relative amounts they absorbed sible with the maxima of the intact cells. It seems in the extracted condition. The saime cannot be said reasonable to make such adjustments before com- of the Chlorella curves. Absorption by the intact

0.10

x A~~~~~~~

E A :A A- Wave n In m, C A u~~~~~~~~~~~~~~~~~~~~~

I. x~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~N E F- A x V 0

c ~ ~ ~ ~ ~ A 0.9

0.05 400 440 A 480 520 560 600 640 68~~~~T u n 0 72

Fig. v>. The quantum yield of photosynthesis as a function of wave length for Chlorella. The points obt_ail." ved on

0.04 -

0.07d ymutpyn lltevleotieneahrnb A-to Symbol Facniy.Thefors sdaegvni

the figure. The band half widths that were commonly used in the various parts of the spectrum are indicated by hori- zontal lines of corresponding length.

This content downloaded from 130.223.51.68 on Fri, 24 Jun 2016 13:38:58 UTC All use subject to http://about.jstor.org/terms 172 AMERICAN JOURNAL OF BOTNNY [Vol. 30, cells is twice as higlh as absorption by the extracted cells can be much reduced by suspendinig the cells pigments in the green region, where absorption is in glycerine, and this results in a slight sharpening minimal, and the relationship is reversed in the red of the absorption maxima, but the changes are not and blue maxima, making an over-all difference of such as to bring the curve for the intact cells into about a factor of 4 between the two curves. One appreciably closer agreement with the curve f or must conclude that the absorption characteristics extracted pigments. This is an indication that our of the Chlorella pigments have been substantially photronic cell method of measuring absorption for altered by extraction, and that it may not be pos- the cell suspensions is fairly satisfactory from the sible to obtain a useful estimate of the proportions standpoint of integrating the scattered light. Fur- of light absorbed by the components in the cell ther evidence against the existence of serious errors from an examination of the absorption curves of the due to scattering is mentioned in connection with extracted components. Nevertheless we have in- figure 7. cluded in figure 4 a lightly drawn curve to show the The chief purpose of the absorption measure- percentage of total absorbed light which is ab- ments was to obtain an estimate of the distribution sorbed by the carotenoids. This curve is derived of light absorption between the yellow and green from the curves for the separated components after pigments, for comparisoni with the dependence of making the adjustments to compensate for shifts quantum yield on wave length. It is unfortunate in the absorption bands due to extraction, and rep- that in the case of Chlorella the absorption charac- resents the best available estimate of the light ab- teristics of the pigments are so much altered by ex- sorption due to carotenoids in the intact cells. traction, that the measurements on the extracted Since we are concerned here with the special pigments give only an uncertain picture of the pro- problem of establishing the distribution of light portions of light absorbed by the pigment compo- absorptioni between the pigment components in the nents in the living cell. But in order to obtain from living cell, no specific reference need be made to the quantum yield measurements evidence concern- the voluminous literature on the absorption spectra ing the possible participationi of the yellow pig- of extracted plant pigments. In an investigation, ments in photosynthesis, some estimationi of the the purposes of whiclh were similar to our own, proportion of light they absorb must be made. Since French (1937) used the absorption curves of pig- the curves plotted in figure 4 appear to be the best ment extracts to identify the absorption maxima of estimate available at present, we have used them, the red and green pigments in living Spirillum ru- in the following section, for comparison with the brum cells. dependence of quantum yield on wave length. Dutton and Manning (1941) in their work on QUANTUM YIELDS. Nineteen sets of measure- Nitzschia also based estimates of distribution of ments of the quantum yield of photosynthesis as a absorption in the intact cell upon measurements function of wave length have been plotted together made on pigment extracts, and emphasized the un- in figure 5. Each set of points, distinguished by a certainty inherent in the method. They made allow- different symbol, represents one series of measure- ance for a uniform shift in the absorption bands ments made with a freshly-harvested culture. The due to extraction, but made no quantitative compari- almost inevitable variability in the physiological son of absorption by extracts and by suspensions of activity of a series of cultures grown over a period live cells. of more than two months and the slight modifica- The unsatisfactory outcome of our comparison tions made in the technique of measurement as the between absorption by extracted pigments and ab- work progressed were held responsible for a slight sorption by intact cells in the case of Chlorella, as variability in the observed quantum yield at any compared to the relatively good agreement shown given wave length. But the dependence of yield on earlier by the curves for Chrobcoccus, is probably wave length appeared to be essentially the same in associated with the higher level of complexity of spite of this variability in the magnitude of the the Chlorella cells. The Chroococcus cells show no yield. In order to plot all the measurements in a internal structure whatever, the pigments appar- single figure without obscuring the dependence of ently being uniformly distributed throughout the yield on wave length because of the scatter of the protoplast. In Chlorella, on the other hand, the pig- points, it seemed necessary to make some adjust- ments are concentrated in the , a highly ment in those sets of points which fell uniformly specialized structure which occupies only a small above or below the level of the majority of meas- part of the total volume of the cell. This greater urements. The need for such an adjustment arises structural complexity is perhaps an indication of chiefly from the fact that some sets of measure- greater chemical complexity as well. ments covered nearly the entire spectral range un- The optically less homogeneous Chlorella cells der investigation, while others were limited to a probably also scatter light more strongly than the narrower region. Of the nineteen sets of measure- Chroococcus cells, but the differences between the ments, four were moved upward by a small factor solid and broken curves in figure 4 do not appear anid four were moved downward. In nlo case was to vary regularly with wave length, and, therefore, the adjustment larger than ten per cent. The other should not be attributed primarily to scattering. eleven sets have been plotted just as they were meas- The light-scattering by suspensions of Chlorella ured, without adjustment. The adjustments do not

This content downloaded from 130.223.51.68 on Fri, 24 Jun 2016 13:38:58 UTC All use subject to http://about.jstor.org/terms Malar.. 1943] EMERSON AND LEWIS CULORELLA PHOTOSYNTHESIS 173 appear to alter the conclusions to be drawn from TIrE LOW YIELD IN THE FAR RED.-The abrtupt the observations, and they bring out the dependence decline in yield in the far red starts at 685 m/A, of yield on wave length somewhat more clearly. whiere chlorophyll absorption is still strong, and Figure 5 shows that the yield is roughly constant where there are no known bands of pigments pres- from 580 to 685 m)u. From 685 toward the infrared, ent in sufficient concentration to compete seriously the yield drops sharply. This is discussed in greater with chlorophyll for the absorption of light. Be- detail in connection with figure 6. From 580 toward fore we consider possible interpretations of this de- the violet the yield declines to a minimum at about clinie in yield toward the inifrared, it seems neces- 490, and rises again toward 400 my. As in the case sary to consider certain potential sources of error of the Chroococcus measurements, the minimum peculiar to measurements in this region. As already near the maximum of the relative absorption by the stated in the section on methods, the quantum yield yellow pigments is qualitatively what would be ex- measurements are based on total absorption of the pected if the light absorbed by the yellow pigments incident light by the assimilating cells. Visual in- were not available for photosynthesis. The fact that spectioni has been found to be a sensitive test for the decline begins as near the red as 580 m[u would totality of absorption, but toward tile inifrared the indicate that carotenoid absorption is already be- sensitivity of the eye diminislhes to the vanishing coming appreciable. The extracted carotenoids, point, so transmission of the incident light can no however, show very slight absorption at wave longer be detected this way. Direct miieasurement lengths longer thani 520. The above interpretation of the absorption by the phiotronic cell method, of the decline in quantum yield would, therefore, which we have found so useful in the visible region, require that in the plant the carotenoid absorption is not feasible in the infrared, both because of the is not only shifted as a whole 14 m[u toward the red lower sensitivity of the cell, and because when the as comipared with the extract, but is also broadened absorption is small the error caused by scattered to such an extent that the tail of the band extends light becomes great. This is clearly shown in table an additional 45 m[u. This sort of broadening is to 1 which gives the percentage of the incident liglht be expected, but hardly to so marked a degree. TABLE 1. Scattered reflectioni front a cell suspension, an.d Figure 4 indicates that between 490 and 510 m,u fr-omi a suspensioni of India inlk. the yellow pigments absorb over 70 per cent of all the light absorbed, but the yield drops only from Per cent of lighlt scattered a maximum of about 0.090 in the red to a minimum Wave length India ink Cell suspension of 0.066, a decrease of less than 30 per cent. This decrease is too small to agree quantitatively with 450 0.76 0.62 the interpretation that the light absorbed bv the 500 0.64 0.79 carotenoids is inactive in photosynthesis. At 420 550 0.52 1.24 m,u the yellow pigments still appear to absorb about 600 0.49 0.75 650 0.48 0.61 30 per cent of all the light absorbed, but the yield 700 0.4 1.8 is only about 10 per cent below the value in red, 730 0.5 9.3 where there is Ino carotenoid absorption. These comparisons would be more significant if the curves for the extracted pigments agreed better which was scattered back toward the source of with the absorption of the intact cells. Since this illumination by a thick suspension of cells. The agreement is so poor it must be admitted in the first measurements were made with the photronic cell place that the dotted curve for the percentage of facing an illuminated suspension of cells, and placed absorption by carotenoids may be only a very rough just enough to one side to avoid light reflected from picture of the relationship in the living cells. Hence, the window of the vessel. For comparison, similar it is possible to do little more than speculate as to measurements with a suspension of india ink are the significance of the variations in quantum yield shown. In both cases readings are given as per cent with wave length. Possibly certain of the caro- of those obtained when a piece of white nmatte paper was placed over the window of the vessel. As al- tenoids are capable of playing a photochemical part ready noted by Warburg and Negelein (1922), the in carbon dioxide assimilation, and others are not. scattering of light from a cell suspension back Perhaps all are active, but with a relatively low toward the source of illumination is small with visi- efficiency. Another, though less probable, possi- ble light. At the red and blue absorption bands it bility is that the carotenoids are all photochemically is scarcely greater for the cells than for the ink. inactive in photosynthesis. In this case the ratio of In the green where absorption is less, the light carotenoid to chlorophyll absorption near 500 mn1t penetrates deeper into the suspension so that more would have to be only about one-third as great in cells contribute to the scattered light, and this is the intact cells as when measured in solution. Fuir- somewhat greater. Toward the infrared, as the ab- thier experimental work with cells containing dif- sorption falls to a very low value, scattering takes ferent proportions of carotenoid pigments and chlo- place from all the cells of the suspension, and rophyll might be expected to help in solving this amounts to almost ten per cent of that scattered problem. from white paper. At wave lengths toward the visi-

This content downloaded from 130.223.51.68 on Fri, 24 Jun 2016 13:38:58 UTC All use subject to http://about.jstor.org/terms 174 AMERICAN JOURNAL OF BOTANY [Vol. 30, ble from 700 inju, where absorption is higlh and scat- 683 anld 698 m[u the thinnlest suspension- gave a vield tering low, the photocell appears to give quite satis- practically equal to the yield for the thickest sus- factory measurements of absorption. But toward the pension. But beyond 698 the yield for the thinniest infrared from 700, accurate measurements of ab- suspension falls more and more below that for the sorption would clearly require better integration of others, a clear indication of incomplete absorption the scattered light than can be expected of such a of the inicident light by the thin suspensioni. At 728 simple arrangement as we have used. mnk, the measureinents show that even- the thickest Without measurement of the scattered light, it suspension could not be depenided upon to absorb is nevertheless possible to test whether a given cell all the incident radiation. The lower cuLrve drawni suspension transmits an appreciable amount of the in figure 6 shows how the yield might have been incident beam. The yield of photosynthesis per unit thouglht to depen-d on wave length if the measure- of incident light may be measured with different ments had been made only with the 246 cmm. sus- pension, which had been adequate for total absorp- tion at 690 m[u. The upper curve shows the esti- mated actual yield, making a generous allowanice 0.08 for the incomplete absorption as revealed by the IL 246 cmm. Cells observed dependenice of yield on suspen-sion densitv. These measurements show beyond doubt that both 0 493 ii " the absorption of light and the quantum yield drop 0 986 verv rapidly between 685 and 730 m[u. Probablv 0.06 there is a similar decline in the yield for ChroocoC- clls (Emerson and Lewis, 1942, fig. 4) in this part E of the spectrum, but measurements with different suspension densities were not made, so the observed decline may be due in large part to incomplete absorption. The most plausible interpretatioii of the sharp drop in quantum yield of photosynthesis toward the infrared seems to be that the light quanta of this spectral region no longer provide sufficient 0.02 energy for the photochemical primary process. This seems reasonable if, as has sometimes been sug- 0 gested, the capacity of chloroplhyll to effect photo- synthesis is linked with the energy level required for the emission of fluorescence (cf. Franck and 0- Herzfeld, 1937, p. 251; also Franck, French anid 680 700 720 740 Puck, 1 941). Experiments made by Vermeulen, Wave Length Wassink and Reman (1937) give considerable sup- port to this suggestion. They measured the fluores- Fig. 6. Decline in quantum yield in the far red. The cence spectrum of chlorophyll, and found that with points slhow the apparent quantum yield witlh tlhree dif- three widely separated wave lengtlhs of excitinig ferent suspension densities, calculated on the basis of incident light. The solid curve shows an estimate of the light, the fluorescence band was alwavs the same. true yield, based on the assumption that differences be- They found the quantum vield of fluorescence was tween values obtained with different suspension denisities constant with increasing wave lengths up to 6241 are due to differences in absorption. The volume of cells m,u. The maximum of the principal fluLorescence used in eaclh case (in 25 ml. of fluid) is given in the figure. band is generally given as about 685 mp, for live cells (cf. Dhere, 1937), and measurements with ex- suspension densities. At low light intensities the citing light closer to this than was used by Vermeu- yield is independent of suspension density, apart len et al. would be unsatisfactory because of the from dependence of absorption on density. If a increasing difficulty of distinguishing between the thick suspension gives a higher apparent yield than fluorescent light and scattered light from the ex- a thinner one, then absorption of the incident beam citing source. It is, therefore, impossible to say how by the thinner one must be incomplete. If the thin loiig the wave length of the exciting light may be suspension gives a yield equal to that of the thicker and still raise chlorophyll to the energy level nieces- one, then presumably absorption is equally good in sary for the emission of fluorescence. But if the both cases. Figure 6 shows measurements of the fluorescence maximum is at 685 m[k, one would ex- quantum yield with three different suspension densi- pect that wave lengths longer than this would ex- ties, covering the region of the steep decline shown cite fluorescence of this band with rapidly decreas- in figure 5. The wave length scale has been ex- ing efficiency, and that this might be reflected in panded, so the slope appears less steep. The results a decreased quantum yield of photosynthesis. The with the three suspension densities are plotted with wave length at which the photosynthetic yield be- distinguishing- symbols. The figure shows that at gins to drop is very close to 685 m,u.

This content downloaded from 130.223.51.68 on Fri, 24 Jun 2016 13:38:58 UTC All use subject to http://about.jstor.org/terms Mar., 1943] EMERSON AND LEWIS CHLORELLA PHOTOSYNTIHESIS 175

We have obtained no clear evidence of photosyn- process. At shorter wave lengths than 685 imn, it thesis at wave lengths loniger than the series in is fair to expect that there will always be sufficient figure 6. Measurements also were made at 760 and energy for the photochemical primary process. 800 m,u, with very dense suspensions of cells and Therefore, it is between 685 m1, and the region high intensities of radiation. There were consistenlt where carotenoid absorption begins, that we slhould differences in rate of pressure change between look for conistancy of the quantum yield in order to "dark" periods and periods of exposure to radia- support our assumption that the yield for light ab- tion. These differences amounited to about one per sorbed by chloroplhyll is constant in regions wlhere cent of the respiration rate and were too small to part of the light is absorbed by the yellow pigments. justify the conclusion that the infrared radiation THE YIELD FOR LIGHT ABSORBED BY CHLOROPHYLL. had caused photosynthesis. The pressure differences -The points in figure 5 leave room for doubt as to could have been accouinted for either by a small the conistanicy of the quantum yield in this region, inhibitory effect of infrared radiation oni respira- although from about 580 to 685 there are only minor tion or by photosynthesis due to the small amount variations. The principal departure from constanev of scattered visible liglht coming from the optical is the small minimum at about 660 m[u. The appar- surfaces of the monochromator. In some instances ent deviation in this region appears to be outside a filter of 0.5 per cent iodine in carbon tetraclhlo- the limits of error from instrumental sources, so ride was used to absorb scattered visible radiation, there is little doubt of its physiological origin. All but a trace of deep red was still transmitted. This attempts to obtain a uniform quantumi yield through impurity was not measured, but had it conitainied as the red region by modifying the method of making much as 1.5 per cent of the total energy of the beam, the respiration correction, changing the sequence which seems quite probable, it would account for the in which the different wave lengths were studied, observed effect. The lack of clearly demonistrable etc., were unsuccessful. Therefore, although the ap- photosynthesis at 760 and 800 mni does not nieces- parent variations in yield are admittedly no greater sarily indicate that the quantum yield has de- than might arise from such errors as small effects creased to zero, since the absorption by chlorophyll of light on respiration, nevertheless it seems neces- may be vanishingly small. Extracted chlorophyll sary to consider the possibility that the quantum in most solvents shows no appreciable absorption yield may vary slightly in the red region. Either the beyond 700 m/u. Van Gulik (1915), however, re- yield for light absorbed by chlorophyll may niot be ports that chlorophyll in carbon bisulfide shows constant or there may be anl inactive pigment pres- quite appreciable absorption in this part of the in- ent which absorbs an appreciable fraction of the frared. Possibly chlorophyll in carbon bisulfide light in the region around 660 my. gives a closer approximation to the absorption by Chlorophyll b may be expected to absorb a suffi- chlorophvll in live cells, in which case the quantum cient proportion of the light in the neighborhood of yield in the infrared must be very small. 660 mIu, so that if it were inactive it could account Eichhoff (1939) reported no diminution in the for the observed dip in the quantum yield in this yield of photosynthesis in infrared out to 800 mnj. region. The absorption curve for live cells (fig. 4) Our observations afford no support for his conclu- shows a definite shoulder at 660 mnt, presumably sion, and we wonder if his results may not have due to the red maximum for the b component. The been due to scattered visible radiation. Though he asymmetry in the curve for the extracted pigments measured the energy distribution of the radiation is by contrast extremely slight, and this may be be- photographically, it is not clear whether he made cause the absorption maxima for the two compo- sufficient allowance for the fact that an appreciable nents are much closer together in methanol than in minimum exposure is necessary to produce measur- the live cells. Extraction seems to cause a greater able darkening of an emulsion. shift in the maximum of the a component, so that In using the dependence of quantum vield on the b maximum becomes obscured. The same con- wave length (fig. 5) as evidence concerning photo- clusion may be drawn from curves published by chemical activity on the part of the carotenoids, we Katz and Wassink (1939, p. 100, fig. le). In recen;t assumed that the yield for the fraction of the light papers on the absorption spectra of the two chloro- absorbed by chlorophyll did not vary with wave phyll components there appears to be no general length. Constancy of the observed quantum yield statement to the effect that the maximum for the a in spectral regions where all the absorbed radiation component is more subj ect to change than that of is absorbed by chlorophyll would support this as- the b component as a result of extraction or trans- sumption. However, the decline in yield beyond fer from one solvent to another. Egle (1939), how- 685 m/A (fig. 6) should not be regarded as contrary ever, gives a table showing the positions of the red evidence, even though in this region no other pig- bands for the two chlorophyll components for a ments compete appreciably with chlorophyll for series of solvents, and a number of pairs of solvenits absorption of the light. In our opinion the decline can be selected which show greater shift of the banid toward the infrared must be due to failure of the for the a component (cf. especially alcohol and car- quanta of lower frequency to provide sufficient en- bon bisulfide). It seems probable on the basis of ergy to raise chlorophyll to the excited state re- these considerations that the red absorption maxi- quired for carrying out the photochemical primary ma for the two chlorophyll components, though onlv

This content downloaded from 130.223.51.68 on Fri, 24 Jun 2016 13:38:58 UTC All use subject to http://about.jstor.org/terms 176 AMERICAN JOURNAL OF BOTANY [Vol. 30, about 15 mn/ apart in iiiethlanol, are separated by mjt. The dip in the curve at 660 mi is about as large more nearly 30 m/i in the live cells. This would as might be expected oni the basis of this interpreta- mean that even partial inactivity of chlorophyll b tioin. If Chlorella is typical of otlier green plants, should produce a noticeable dip in the quantum it probably has two to three times as much of the a yield curve near 660 ini. component as of the b, anid the specific absorption If the long wave limit of full photosynthletic effi- coefficient for a is considerably higher. From 660 ciency is related to the maximum of the fluorescence to 685 the proportion of light absorbed by the b band in the way that has been indicated above, then component must decrease rapidly, and this would one might expect that the efficiency for light ab- account for the rise in yield toward 685. sorbed by each chlorophiyll component would have It has already been meentioned that the experi- its own limit, determined by the position of the menetal evidence for the minimum drawni in the fluorescence band for that component. According quantum yield curve at 660 mjt is not thoroughly to this reasoning, the phlotosynitlhetic efficiency for satisfactory. The above interpretation is put for- light absorbed by chlorophyll b might be expected ward as a suggestion whiclh appears to us promis- to show a sudden drop beginning near the priniciple ing, and not as an established fact. But we feel that fluorescence maximum for this component. The the experimental evidence justifies the hope that fluorescence bands for the two chlorophyll compo- further attention to the behavior of the quanitum nents are close to the respective absorption maxi- yield in the red region, possibly witlh even nar- ma, and like the absorption maxima they are more rower wave length bands, will lead to useful cor- widely separated in the living cell than in ordinary relations between the absorption and fluorescence solvenits. Dliere (1937) reports that for Ulva fronds of the chlorophyll components, and their activity in the fluorescence maximum for chlorophyll b is at assiinilation. 655.5, and for the a component at 684.7. These From the evidence available, it seems reasonable values agree well with the regionis where the quani- to regard the yield for light absorbed by chloro- tum yield of photosynthesis shows a decline-tlie pliyll as approximately constant where there is no first at about 660, and the second just beyond 685 competition from other pigmenits. The extension of

bc

(3 0~~~~~~~~. 0 0oo 0^? w -0 Total Absorption 80~~~~~~~~~~~~~~~~~~~~~~~ c - ... Active Absorpfion

0 ~ ~ ~ ~ ~~~

0 A 'IO " A

40~~~~~~~~~~~~~~~~~~~~~~~~ 20~~~~~~~~~~~~~~~~~~~~~~~~~

42 c mm. 40 cmm. 39 cmm.\ 20

400 440 480 520 560 600 640 680 720 Wave Length i n m,u

Fig. 7. Comparison of the absorption by the cells and that part of the absorption which is active in photosynthesis. These measurements are made with thin suspensions, giving inicomplete absorption. The "total" absorption is measured directly. The "active" absorption is calculated from the measured photosvnthesis on the assumption that all light used for photosynthesis gives a quantum yield of 0.084. This value is chosen to give agreement between the two curves in the red, where all absorption is assumed to be active. The quantity of cells used in each run, and the half widths of the bands of radiation are shown on the figure. The cells used in the run covering the red pIalt of the spectrum were from a separate culture from the others.

This content downloaded from 130.223.51.68 on Fri, 24 Jun 2016 13:38:58 UTC All use subject to http://about.jstor.org/terms Mar., 1943] EMERSON AND LEWVIS--CHLORELLA PHOTOSYNTHESIS 177 this conclusion to the blue and green region, where pensions. If the light absorption were seriously in absorption is divided between chlorophyll and the error due to incorrect measurem-ient of the scattered carotenoids, must be regarded as only a working light, one would hardly expect such good agreem-ient hypothesis the purpose of which is to provide a between the active absorptioni anid the directly logical basis for consideration of the evidence con- measured absorptioni in the red regioin. cerning photochemical activity on the part of the Comparison of figure 7 with the correspondinig carotenoids. curves for Chroococcus (Emiersoni and L ewis, 1942, COMPARISON OF ACTIVE ABSORPTION AND TOTAL fig. 5) brings out several interesting points. The ABSORPTION. -The quantum yield mieasurements Chlorella curve shows no maxiiimumii at 620 iily, with Chroococcus (Emerson and Lewis, 1942) were wlhere the absorption due to phycocyaniii in Chro- supplemented with measurements on thin suspen- ococcus showed a maximum in both the active ab- sions, where only about half of the incident light sorption and the total absorption. The rise in the was absorbed. To find the "active absorption," or active absorption from green toward violet begins absorption spectrum of the pigments photochemi- for Chlorella at about 540 nifk, while for Chroococ- cally active in photosynthesis, the per cent of inci- cus it is not evident until 480. This difference is dent light which resulted in photosyinthesis was presumably due to the absence of chloroplhyll b in plotted at each wave length, on the assumption that Chro6coccus. It miiay also be associated with differ- all the light utilized for photosynthesis acted witlh ences in the absorption spectra of the carotenoid the same quantum yield. The active absorption pigments of these two organisms (cf. Emerson and measured in this way was compared with the ab- Lewis, 1942, p. 582). The absorption curves for the sorption measured directly with the photronic cell, extracted pigments indicate that the yellow pig- using the same suspension of cells and the same ments of Chrobcoccus absorb further toward the red slit widths as for measuring the active absorp- than do the carotenoids of Chlorella. This is in tion. Similar measurements have been made for accord with the differences in the quantuLim yield Chlorella, and the results are plotted in figure 7. curves for the two organisms, if we accept the evi- The solid curve shows the total absorption, and dence that light absorbed by the yellow pigments is the broken curve shows the active absorption. These inactive or only partially active in photosvnthesis. curves offer independent confirmation of the low The yield for Chroococcus, which showed stroniger yield between 690 and 710 mnyj, where the active absorption by carotenoids, drops more steeply, go- absorption falls below the measured absorption. ing from red toward green oii the wave length scale, The two curves run close together from 690 to 570, and goes to a lower level than the yield for indicating that all the absorbed light is used for Chlorella. photosynthesis with the maximum yield. The strong The combined evidence from the quantuiii vield asymmetry of the red absorption band is presum- measurements with dense suspensions and the meas- ably due to chlorophyll b, and the action spectrum urements of active absorption discussed above indi- repeats this asymmetry. Evidence of reduced ac- cates the probability that part of the energy ab- tivity of the b component at 660 myp is lacking in sorbed by the carotenoids must be available for figure 7, but the expected effect would be small, and photosynthesis. The net yield for the enitire fraction greater refinement of the technique, as well as more of light absorbed by the yellow pigimients must be observations, might be required to bring it out in considerably lower than the yield for the fraction an experiment of this kind. From 560 my on into absorbed by chlorophyll. the violet, the action spectrum falls well below the absorption curve, presumably because part of the SUMMARY absorption is due to pigments photochemically in- Measurements of the quantum yield of Chlorella active in photosynthesis. But again the interpreta- photosynthesis have beeii made at different wave tion is quantitatively unsatisfactory because, if all lengths of light, and compared with the estimated the yellow pigments are inactive, figure 4 indicates that there should be much more divergence at 490 distribution of light absorption between the yellow mni. than is shown in figure 7, and the divergence and greeni pigment components. should not start as early as it does in the grEen. All The estinlation of light absorption by the pig- through the blue one would expect to find n1ore dif- ments in the living cell was based oil absorption ference between the active absorption and the total curves for the extracted and separated components. absorption, if the light absorbed by the carotenoids The absorption characteristics of the pigments were were inactive, and if the yield for chlorophyll were found to be sufficiently altered by extraction so that constant. the conclusions drawn from comparison of absorp- Figure 7 offers general confirmation of the con- tion and photosynthetic yield could lead to only clusions drawn from the direct quantum yield meas- tentative conclusions as to the activity of accessory urements with totally absorbing suspensions (fig. pigments. 5). It also supports our opinioni that the difference The quantum yields were measured manometric- between the absorption curves for intact cells and ally, on the basis of oxygen exclhange. Preliminary extracted pigments (fig. 4) is not due primarilv to measurenients established that the depenidence of errors arising from light scattering by the cell sus- yield on light intensity was the same for the red,

This content downloaded from 130.223.51.68 on Fri, 24 Jun 2016 13:38:58 UTC All use subject to http://about.jstor.org/terms 178 AMERICAN JOURNAL OF BOTANY [Vol. 30, green, and blue regions of the spectrum, at low excited state required for the photochemical pri- light intensities. mary process. The minor variations in yield from Wave lengths of light in the neighborhood of 480 685 to 590 mpt seemed to be connected with differ- m,L were found under certain conditions to cause ences in the minimum energy required for excita- large temporary changes in rate of oxygen con- tion of the a and b components of chlorophyll. At sumption. Measurements in this region required frequencies higher than the required minimum, the modification of the usual technique, in order to results indicated that the yield for light absorbed avoid serious errors in estimating the correction to by chlorophyll was essentially constant, at least in be applied for respiration during light exposures. the region where there was no appreciable absorp- The quantum yield was found to be vanishingly tion by the yellow pigments. When part of the light small in the far red beyond 730 m,u. From 730 to was absorbed by the yellow pigments, the evidence 685, it rose steeply to a value of about 0.09. Apart suggested some photochemical activity on the part from minor variations, it maintained a constant of the.yellow pigments, but indicated that the net level to about 580 m/A, then declined to a minimum quantum yield for all the light absorbed by these of about 0.065 at 485 mj,v, and rose again to nearly pigments must be considerably smaller than the 0.08 at 420 m,u. The source of monochromatic light yield for light absorbed by chlorophyll. provided insufficient intensity for satisfactory meas- WILLIAM G. KERCKIIOFF LABORATORIES OF THE BIOLOGI- urement further toward the ultraviolet. CAL SCIENCES, The significance of the quantum yield measure- CALIFORNIA INSTITUTE OF TECHNOLOGY, ments was discussed. The low yield at wave lengths PASADENA, CALIFORNIA, AND longer than 685 ml. was attributed to failure of the MIOUNT WILSON OBSERVATORY, lower frequency quanta to raise chlorophyll to the PASADENA, CALIFORNIA

LITERATURE CITED

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