PHOTOSYNTHESIS - Plant Physiology · Photosynthesis is sensitized, in algae of all divi-sions (and...

RED DROP AND ROLE OF AUXILIARY PIGMENTS IN PHOTOSYNTHESISR. EMERSON AND E. RABINOWITCH 1

PHOTOSYNTHESIS PROJECT, BOTANY DEPARTMENT, UNIVERSITY OF ILLINOIS

Note: When Robert Emerson was killed in aplane accident on February 4, 1959, much of the ex-perimental material accumulated in his two yearswork on the action spectrumn of photosynthesis in thefar-red region, remained uinpublished. He was topresent these resuilts to the Botanical Congress inMontreal in August, 1959. Instead, the followingpaper was presented, prepared on the basis of Emer-son's earlier talks and laboratory notes, and afterconsultation with his collaborators, R. V. Chalmersand C. Cederstrand. The theoretical discussion ofthe results is my own. The presentation of this paperwas intended as a memorial to Dr. Emerson; I wasurged to publish it to give all those zworking in thefield access to the results of Emerson's last, excitingresults. A paper presented by J. Myers (12) at thesame meeting provides both confirmation and inter-esting expansion of the phenomenon which is becom-ing known as the Emerson effect*. In particular, itshows that the so-called chromatic transient, as ob-served after the replacement of illumination with lightat 700 mA by illumination with a shorter-wave lightgiving the samne steady yield of photosynthesis, hasthe same action spectrum as the Emerson effect, i.e.,suggests a specific role of chlorophyll b.-E. RABINO-WITCH.

INTRODUCTION

From experiments on the action spectrum of photo-synthesis (largely by Emerson and co-workers), andof chlorophyll fluorescence (by French, and Duysens)performed up to 1957, an apparently consistent pic-ture emerged which could be summarized as follows.

Photosynthesis is sensitized, in algae of all divi-sions (and in the higher plants as well), most effec-tively by light quanta absorbed by chlorophyll a.Light absorbed by other, accessory pigments (chloro-phyll b and c, the phycobilins, and the carotenoids)can contribute to photosynthesis with an efficiencywhich is either lower, or, at best, equal to that of thelight absorbed by chlorophyll a. The efficiency ofaccessory pigments in sensitizing photosynthesisclosely parallels their efficiency in sensitizing the fluo-rescence of chlorophyll a. It is therefore plausible topostulate that the contribution of accessory pigmentsto photosynthesis is based on transfer of the lightenergy absorbed by them to chlorophyll a (where itcan be utilized to produce chlorophyll fluorescence,or to sensitize photosynthesis). A mechanism by

Received November 2, 1959.* Perhaps, it should be called the "second Emerson

Effect", the first being the carbon dioxide burst at thebeginning of the illumination period.

which this transfer is likely to occur has been demon-strated by physicists (Perrin, Vavilov, Forster, andothers), and is known as "resonance energy transfer"or "inductive resonance."

The efficiency of the energy transfer to chlorophyllfrom the several other pigments present in chloroplastsmust depend mainly on two factors: the intimacy oftheir association, which may involve both spatial close-ness and chemical attachment, and the overlapping ofthe fluorescence band of the accessory pigment andthe absorption band of chlorophyll a (which is themeasure of resonance between the two pigments).

If this picture is accepted, it can still be askedif the energy supply to chlorophyll a is the main pur-pose of the presence of the different accessory pig-ments in various photosynthetic organisms, or if someof them may have other physiological functions. Inthe case of the phycobilins, found mainly (but notonly) in deep-water red algae, the energy-collectingfunction appears plausible and may be vital, becauseof the predominance, deep under the sea, of green lightnot effectively absorbed by chlorophyll. This fact,and the high efficiency of energy transfer from thephycobilins to chlorophyll (80-95 %), observed inmeasurements of the action spectrum of chlorophyllfluorescence, supports the assumption that energysupply is in fact the main raison d'etre of red (andblue) pigments in Rhodophyceae. The case of fucox-anthol appears similar; this pigment, too, contributessignificantly to the absorption of submarine light bythe Phaeophyceae and Bacillariophyceae, and experi-ments show a high efficiency of energy transfer fromit to chlorophyll a. Other carotenoids, particularlythose found in green plants, contribute very little tothe total absorption of sunlight, and the efficiency ofenergy transfer from them to chlorophyll a is relative-ly low (20-50 %). It seems likely that the mainphysiological function of these pigments is different;the fact that they contribute excitation energy tochlorophyll a may well be merely a physically unavoid-able, but physiologically insignificant phenomenon.

The above-mentioned picture had one dark spot.Blinks, Haxo, and Yocum (9) had concluded, frommeasurements of the action spectra of photosynthesisin various species of red algae, that the photosyntheticefficiency of the light quanta absorbed by the phycobil-ins is generally higher; often much higher, than thatof the light quanta absorbed by chlorophyll a. Thissuggested direct sensitization of photosynthesis by thered pigment, without the intermediary of chlorophylla-in contradiction to the above-described picture.However, Duysens (3) found that a similar phenome-non can be observed also in the excitation of the fluo-rescence of chlorophyll a in these algae; it, too, isexcited more efficiently by light quanta absorbed bythe phycobilins than by those absorbed by chlorophylla itself. The hypothesis that the accessory pigments

477

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PLANT PHYSIOLOGY

contribute to photosynthesis by transferring their ex-

citation energy to chlorophyll a thus seemed to bevalid in this case, too; it was, however, paradoxicalthat light absorption by chlorophyll itself appearedto produce excited chlorophyll molecules less apt to

sensitize photosynthesis, or to emit fluorescence, thanthe excited chlorophyll molecules obtained by energy

transfer from the accessory pigments.

I. RED DROP. Emerson decided to investigatethis paradoxical situation in 1958. The detailed in-vestigation of the action spectrum of photosynthesisin Porphyridium, made in collaboration with M. Brody(1), changed the experimental picture substantially.The relative inefficiency of light absorbed by chloro-p4yfl1 a in red algae proved to be limited to the part

of the absorption spectrum above 650 mu; while at

644 mu (where the light absorption is divided aboutequally between chlorophyll and phycocyanin), theabsorbed quanta were found to be generally more ef-fective than the 546 mu quanta, absorbed entirely bythe phycobilins. In the extreme case (that of algaeadapted to green light, which is absorbed predomi-nantly by phycoerythrin), the two efficiencies were

equal (cf. fig 1). This suggests a situation similarto that found in other divisions of algae, where theefficiency of the accessory pigments can equal, butnot exceed, that of chlorophyll.

It remains to be seen whether a correction similarto that brought by M. Brody's measurements to theaction spectrum of photosynthesis of red algae, mustbe applied also to the above-mentioned conclusions ofDuysens et al, concerning the action spectrum ofchlorophyll a fluorescence in red algae; in other words,whether or not the quantum yield of directly excitedchlorophyll fluorescence also declines in these cellsonly above 650 my.

The puzzle of the action spectrum of photosynthesisin red algae was shifted by M. Brody's measurements

0.08~~~~~~~~~~~~~~~~~~~.

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0-00---400 450 500 550 600 650 700

WAVE LENGTH, mJU

FIG. 1. Action spectra of photosynthesis in Porphy-ridium pre-illuminated with green light (solid line) andblue light (dashed line). In the first case, the phycobilinsare about as efficient in sensitizing photosynthesis as

chlorophyll; in the second case they are only about halfas effective. There is no significant difference in the

pigment content of the two samples. [After M. Brody and

R. Emerson (1)].

-o

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680 700 720 740

Wave LenqthFIG. 2. Red end of the action spectrum of Chlorella.

Solid curve is extrapolation of results to complete absorp-tion. Width of spectral bands used is indicated. [AfterR. Emerson and C. Lewis (6)].

from the alleged low effectiveness of chlorophyll a as

such, to the low efficiency of light absorbed by chloro-phyll a > 650 mn'. It seemed significant that this isthe region where chlorophyll a becomes the only ab-sorbing pigment; one had to recall the observation,made by Emerson and Lewis (6) in 1943, that inChlorella, too, the quantum yield of photosynthesisdeclines in the extreme red part of the chlorophyllabsorption band, > 685 msl-the region where theabsorption by chlorophyll b is likely to be negligible,and practically all absorption must be due to chloro-phyll a, (cf. fig 2). Emerson therefore conceived a

working hypothesis, which seemingly ran contrary tothe above-outlined generally accepted concept. Hesuggested that in order for photosynthesis to proceedwith maximum efficiency, energy quanta must be con-

tributed by two pigments: by chlorophyll a, and byone of the auxiliary pigments-chlorophyll b in greenalgae, a phycobilin in red algae, etc.

Although the red drop in the quantum yield ofphotosynthesis of green algae above 680 mit was ratherconclusively established by the 1943 study, the rapidlydeclining absorptive capacity of the cells in this regionand their capacity for selective scattering of light on

the long-wave side of the absorption peak (discoveredby Latimer (10), cf. fig 3) permitted some doubt as

to the reality of the drop; it could conceivably have

been caused by overestimation of the number of quantaactually absorbed by chlorophyll in this region. A

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478



479EMERSON AND RABINOWITCH-RED DROP AND AUXILIARY PIGMENTS

more precise study of the red drop was therefore un-dertaken by Emerson and Chalmers. Preliminaryresults have been presented in two communicationsto the National Academy of Sciences (5). Somespeculations on the implications of the findings werediscussed in a talk (4) before the Phycological So-ciety. The main body of results remained unpublishedat the time of Emerson's death.

II. ENHANCING EFFECT OF AUXILIARY LIGHT.The far-red quantum drop made many people thinkthat perhaps in this part of the spectrum, energyquanta may be too small to sustain photosynthesis, orat least to do so with a good yield. This seemed anattractive explanation, despite the fact that spectro-scopic theory suggests that within a given electronicabsorption band, the electronically excited molecules,after thermal equilibration with the medium (whichis likely to be completed, in a condensed medium, be-fore the photochemical reaction takes place), musthave the same vibrational energy distribution, and thusalso identical reactivity, whatever the specific size ofthe absorbed quantum.

If insufficient vibrational energy of the electronic-ally excited chlorophyll molecules had something todo with the red drop, one could expect the quantumyield in this spectral region to improve with increas-ing temperature. The first experiments of Emersonet al (5) were therefore concerned with the effectof temperature on the quantum yield in the far red.A temperature effect was in fact found, but it had theopposite sign from that expected; the far red light wasused more effectively at 50 C than at 200 C! (fig 4).

This finding, which in any case did not supportthe suggested interpretation of the red drop, was notfurther pursued, since another one, obtained at aboutthe same time, appeared more exciting, supporting,as it did, the interpretation of the drop as indicativeof the importance, for photosynthesis, of quanta ab-sorbed by accessory pigments. This was the find-ing (figs 4, 5) that the quantum yield of photosyn-thesis in far red light can be substantially improvedby auxiliary illumination with light of shorter wavelength. The average quantum yield obtained in twosuperimposed beams proved to be substantially higherthan the average of the quantum yields obtained byusing the two light beams separately. Since in theauxiliary light per se, the yield was the usual one(8-12 quanta per molecule of 0), while in the farred beam per se it was substandard (20-50 quantaper molecule of 02), it was natural to ascribe theeffect of the mixing of two lights to the improvementof the photochemical effectiveness of the quanta of farred light by the auxiliary light. (The average quan-tum requirement of two combined beams never wasbelow the familiar minimum of 8 to 12 quanta.

The next question was: what was important, thesize of the quanta of the auxiliary light, or the pigmentthat absorbed them? This required a determinationof the action spectrum of the auxiliary light, a taskthat proved extremely laborious and time-consuming.The method used was to combine far red illumination

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FIG. 3. Selective scattering of light by Chlorella cellsuspension. [After P. Latimer and E. Rabinowitch(10) ].

FIG. 4. Red drop of quantum yield of photosynthesisin Chlorella. Solid line, 50 C; dashed line 20° C; dottedline, 20° C with supplementary light of shorter wave-length. [After R. Emerson, R. V. Chalmers, and C.Cederstrand (5) ].

FIG. 5. Proportionality of the rate of photosynthesisto light intensity in far-red and auxiliary light. [AfterR. Emerson, R. V. Chalmers, and C. Cederstrand (5)].

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PLANT PHYSIOLOGY

YIELD WITH SUPPLEMENTAR' Y LIGHT

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FIG. 6. Effect of monochromatic supplementary light on the yield of photosynthesis in far red light for theblue-green alga Anacystis, compared to the fraction of light absorbed by phycocyanin. [After R. Emerson, R. V.Chalmers, and C. Cederstrand, unpubl.].

FIG. 7. Effect of monochromatic supplementary light on the yield of photosynthesis in far-red light for thegreen alga Chlorella, compared to the fraction of light absorbed in chlorophyll b. [after R. Emerson, R. V. Chal-mers, and C. Cederstrand, unpubl.].

FIG. 8. Effect of monochromatic supplementary light on the yield of photosynthesis in far-red light for thediatom Navicula, compared to the absorption spectrum of fucoxanthol. [after R. Emerson, R. V. Chalmers, andC. Cederstrand, unpubl.].

FIG. 9. Effect of monochromatic supplementary light on the yield of photosynthesis for the red alga Porphyridi-um, compared to the absorption spectrum of phycoerythrin. [after R. Emerson, R. V. Chalmers, and C. Cederstrand,unpubl.].

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EMERSON AND RABINOWITCH-RED DROP AND AUXILIARY PIGMENTS

of certain constant intensity with the illumination byauxiliary light of an intensity giving, by itself, ap-proximately the same amount of photosynthesis. Thecurve showing the yield of combined illumination,minus the yield caused by the auxiliary light alone,was plotted as function of wave length of the auxiliarylight, and compared with the curve showing the rela-tive proportion of light absorbed, at the same wave-lengths, by the auxiliary pigment (or simply with theabsolute absorption curve of this pigment).

Figures 6 to 9 show this comparison for algae offour divisions, containing phycocyanin, chlorophyllb, fucoxanthol, and phycoerythrin as main auxiliarypigment. In every case, the action spectrum of sup-plementary light is strikingly similar to the (relativeor absolute) absorption spectrum of the auxiliarypigment.

The chosen method of comparison implied thatthe stimulating effect of auxiliary light on the far-red yield is proportional to (or at least, rises mo-notonously with) the intensity of the auxiliary light.This was checked by experiments with constant in-tensity of far-red, and varying intensity of auxiliarylight. It clearly shows that the effect of auxiliarylight is by no means a catalytic one [as suggested byWarburg (14) for the special case of blue light addedto light of greater wave lengths], but rises with itsintensity until it begins to show saturation, in thesame intensity region where photosynthesis becomeslight-saturated.

Considering the uncertainties involved in the cal-culation, the parallelism of the curve pairs in figures6 to 9 appears striking. It justifies the conclusionthat the enhancing effect of light of a certain wave-length on the yield in the far red parallels the propor-tion of light absorbed, at this wavelength, by the mainauxiliary pigment-chlorophyll b in Chlorella, phyco-cyanin in Anacystis, fucoxanthol in Navicula, andphycoerythrin in Porphyridium. It seems that thefar-red quanta, absorbed by chlorophyll a, are mosteffectively supplemented by quanta absorbed by anauxiliary pigment, and not by other quanta absorbedby chlorophyll a itself.

Since in an average higher plant a broad band ofred and orange light, which is absorbed to about %by chlorophyll b and about 34 by chlorophyll a, canproduce photosynthesis with full efficiency, it seemsthat quanta absorbed by chlorophyll a at the shorterwavelengths do not need supplementation by quantaabsorbed by other pigments.

One matter that needs further attention is the ap-pearance on figures 6 to 9, of negative effects. Ifconfirmed by further experiments, they may meanthat under certain conditions, combinations of far-redlight with light of shorter wave lengths may decreaserather than increase the total yield (even though theexperiments were carried out at light intensities farbelow saturation). In this case, it may be that theeffect is due to decline in the efficiency of the auxiliaryshort-wave light to below the usual value of 8 to 10quanta per 02 molecule, rather than to a further de-

cline in the efficiency of the far-red light. It is verylikely that the need for balancing the excitation ofthe several pigments present in chloroplasts manifestsitself in various ways, and not only in the inefficiencyof far-red light absorbed only by chlorophyll a.

III. EXPERIMENTS ON XANTHOPHYCEAE (HETER-OKONTAE). According to data in the literature (13)Xanthophyceae contain no chlorophyll b. If Emer-son's hypothesis is correct, they should show no effectof supplementary light on the yield in far-red liglht.This was in fact confirmed by experiments withPolyedriella. However, measurements of actionspectra showed that this difference was due to theextension of high quantum yields out to 690 me', andnot (as was expected) to the drop of the yield > 680my being insensitive to auxiliary light! Since thesealgae were grown in a medium containing organic nu-trients, it was suspected that the presence of such nu-trients may permit full photosynthetic rate at 690 mys,without the aid of supplementary light. Preliminaryexperiments by R. Govindjee suggested that if Polye-driella is precipitated from the organic and transfer-red into the inorganic medium, and the transfer is re-peated several times, the red drop does appear afterabout five transfers. This suggests that Polyedriellagrown in organic medium may contain a substrate(or a catalyst) which permits the cell to utilize lightat 680 to 690 my with full efficiency, without the helpof light of shorter wavelengths.

To test on Polyedriella the theory of chlorophyll babsorption as source of the auxiliary light effect onthe yield in the far red, one would have to make theexperiment on Polyedriella cultures repeatedly trans-ferred into an inorganic medium; and this experi-ment has not yet been carried out.

IV. EFFECTS OF ORGANIC NUTRITION ON REDDROP IN CHLORELLA AND PORPHYRIDIUMi. The ob-servations on Polyedriella raised the questionwhether the red drop in other algae also could be af-fected by organic nutrition. Experiments withChlorella grown in glucose solution showed nochange, but algae grown in the presence of beef brothor earth extract did show a shift of the red drop tolong wave lengths, (fig 10).

Because of Myers' findings in experiments onalgal growth, it was suspected that vitamin K may bea possible active ingredient of broth or earth extract;experiments with the addition of this (and other) pureorganic micronutrients are planned.

Even considerable alterations in the composition ofthe inorganic growth medium had no effect on thered drop in Chlorella.

V. EFFECT OF TIME INTERVAL BETWEEN ABSORP-TION OF FAR-RED LIGHT AND SUPPLEMENTARY LIGHT.In the experiments described above, the experimentalarrangement was that shown in figure 11. The far-red light, isolated by glass filters, entered the mano-meter vessel from above, while the auxiliary lightcame from below, usually through a monochromator(except in blue-violet light, where filters had to be

481



PLANT PHYSIOLOGY

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FIG. 10. Effect of earth extract on the red drop inChlorella. [after R. Emerson and R. Govindjee, unpubl.].

used). In the majority of the experiments, bothbeams were practically completely absorbed. In apreliminary experiment, the auxiliary beam was sentfrom below and the far-red light from the side, andthe enhancing effect was found to be considerablysmaller. Since in the second arrangement, a cellneeds less time to get from the bottom layer of thevessel, (where it has the greatest chance to absorbauxiliary light) into the layer where its chance toabsorb far-red light is highest, this observation raisedthe possibility that a certain time interval betweenthe two absorption acts may favor the effect. Thissuggests experiments in which the two beams wouldbe alternated at variable time intervals.

VI. INTERPRETATION OF RED DROP. Emerson'sconclusion that photosynthesis required (or, at least,is enhanced by) energy absorption in an auxiliarypigment, permits of several hypothetical interpreta-tions. The simplest one is to postulate that two (ormore) different primary photochemical processes areinvolved in photosynthesis, and are preferentially sen-sitized by the several pigments. For example, onecould imagine that photosynthesis requires bothphotoreduction (of CO2 or of a substrate derivedfrom it, such as a carboxylic acid), and photoxidation(of water or a substrate derived from it). One coulddescribe essentially the same concept by saying thateach hydrogen atom (or electron) must be photo-activated twice on its path from the ultimate donor,water, to the ultimate acceptor, carbon dioxide, andthat each of these two steps is preferentially sensitizedby one or the other of the pigments. To use a con-crete example, one could imagine a mechanism of thefollowing type:

hvI. Chl a + H20 02 + rChl a

hv

II. Chl b + CO -{ CH20 + o Chl b

III. o Chl b + r Chl a- Chl b + Chl a

-chlorophyll a serving preferentially as photoxidantand chlorophyll b (or another auxiliary pigment)preferentially as photoreductant (or vice versa), anda dark reaction, III, between the reduced and theoxidized pigment restoring the original system. (Isay "preferentially" because in the steady state, re-actions I and II must proceed at the same rate; if onlyChl b could sensitize reaction II, the maximum yieldcould be obtained only in light absorbed to one-halfby chlorophyll b, and one-half by chlorophyll a, andnot in a broad red-orange band absorbed to only one-fourth by the b component.)

The above interpretation of Emerson's findingsis made unlikely, if not impossible, by the evidence,derived from fluorescence experiments, that of thequanta absorbed by chlorophyll b (as well as byphycobilins, or fucoxanthol), a very large fraction istransferred to chlorophyll a by resonance. This evi-dence is not as precise as could be desired, and therelevant experiments need to be repeated with im-proved techniques; but on the whole, they seem con-vincing.

It would be very difficult to explain the approxi-mate equality of the quantum yields of fluorescence ofchlorophyll a when excited by light absorbed by thispigment itself and when excited by light absorbed bychlorophyll b (or fucoxanthol) without assumingthat most of the quanta absorbed by these auxiliarypigments are transferred to chlorophyll a. (True, theactual fluorescence yield is only, < 3 %, but it is areliable index of what happens to the totality of theabsorbed quanta).

An alternative interpretation of Emerson's resultsis to begin with the fact that most of the quanta ab-sorbed by the auxiliary pigments are transferred tochlorophyll a, and thus cannot produce directly anyspecific photochemical effects. Instead, chlorophyll

LIGHT BEAM OF FIXED WAVE LENGTH-A:---::-: :--- -:- - -- I SIOURTCEMIRROR LNT IH

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REACTIONVESSEL

LIGHT BEAM OF VARIABLE WAVE LENGTH

MIRROR

FIG. 11. Arrangement of two light beams in experi-ments with antiparallel beams. [after R. Emerson,unpubl.].

482




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630 640 650 660 670

FIG. 12. Order of energy levelsnon-fluorescent forms of chlorophyll.S, higher 1 7T7 level, S; -3 T7T level

FIG. 13. Possible shapes of the achlorophyll in the fluorescent state (cthe non-fluorescent state (solid line).the n g band lies on the short-wave siband; in the second case, it lies onproviding a possible explanation of th

a, carrying the transferred quanta,to bring about preferentially a primprocess different from the one causeby the lone-wave auanta absorbed

process is initiated by long-lived molecules in the| triplet state; this explains why it often does not com-.-_-}i-- pete with fluorescence, i.e., why the substrate of the

photochemical reaction often does not quench fluores-cence. (This does not mean that photochemicalprocesses cannot be initiated by the S*-statein whichcase the substrate will quench the fluorescence).

The result of the competition between the processesS* - S and S - T in figure 12 can be strongly af-fected by the presence of a third, (so-called n s)excited state. If this state is located between the

fluorescont (-H20) S*-and the T-state, its presence catalyzes the conver-rophyll sion to triplet, and thus reduces or altogether quenches

fluorescence. If the n ir-state is located above theS*-state, it has no such effect. This is the presentlyaccepted explanation of the enhancing effect of polarmolecules on the fluorescence of chlorophyll: theproximity of polar molecules shifts the n it-state up-wards and the S*-state downwards, thus favoring

/ \ | fluorescence. In the hypothesis we are now consider-/ \ z ing, the two forms of chlorophyll complexes must both

\I belong to the same, fluorescent type.Franck (7) suggested a different interpretation.

In it, the two kinds of chlorophyll involved in photo-synthesis are a non-fluorescent form, in which theexcited molecules in the S*-state are instantaneously

660 690 7 00 converted, via the n 7r-state, into metastable moleculesmj T; and a fluorescent kind, which permits the S*-state

in fluorescent and to survive long enough either to fluoresce, or to mi-(Lowest 1 ff level, grate, by resonance, through a sequence of chloro-is T.) phyll molecules, until transferred to a molecule already

bsorption curves of in the T-state, raising it into an excited triplet state,lashed line) and in T*. The latter has an energy content so high as to

In the first case, permit direct sensitization of an electron (or hydro-ide of the main sf gen) transfer from H2O to RCOOH (or their equiv-its long-wave side, alents on the energy scale).e red drop. Absorption on the long-wave side of the main ab-

sorption peak, > 690 mnL, is suggested, in Franck'stheory, to be due predominantly to n 7r transitions in

must be postulated the non-fluorescent state (fig 13) it therefore pro-iary photochemical duces predominantly T-molecules, which alone cannotd (preferentially) bring about photosynthesis. Absorption in the mainLbv chloroDhvll a band, 650 to 690 mA, on the other hand, excites both

itself. To make this plausible, one could postulate,for example, the existence of two types of chloro-phyll a complexes in the cell, e.g., such associatedwith a reductant and such associated with an oxidant,and assume that one of these two types is closer to theauxiliary pigment than the other.

Somewhat awkward is the necessity to postulateequal fluorescence yield of both types. Livingston(11) has shown how different is, for example, thefluorescence of chlorophyll associated with polarmolecules (water, alcohol, amines) from that ofchlorophyll surrounded exclusively by non-polar lipoidmolecules. The fluorescence efficiency is generallydetermined by competition between the radiativetransfer from the fluorescent (excited singlet) stateto the ground singlet state, and the non-radiativetransfer to the metastable triplet state (fig 12). Ithas been often postulated that the photochemical

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FIG. 14. Differential spectra of young and old cul-tures of Euglena suggesting the existence in vivo of achlorophyll form with absorption peak at 695 my [afterC. S. French (8) ].

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483



PLANT PHYSIOLOGY

types of chlorophyll. Depending on their ratio, itcan either produce about equal numbers of S* and T-molecules, and thus ultimately, a large number ofT*-molecules and a high yield of photosynthesis (asin Chlorella), or predominantly T-molecules, andthus a low yield of chlorophyll fluorescence and photo-synthesis (as in Porphyridium). Finally, (develop-ing Franck's interpretation so as to fit M. Brody'sfindings), one can suggest that absorption at 640 to650 my could excite predominantly the fluorescentspecies (in which the absorption on the short-waveside of the main peak is reinforced by the n ir-transi-tion!), and thus give, in all cells, a high yield offluorescence and of photosynthesis.

It is to be noted that under no condition is therea danger of shortage of molecules in the T-state, sinceeven in the extreme case of exclusive excitation offluorescent molecules, the transition S* - T is themost likely fate of each S*-molecule, unless enoughT-molecules have accumulated for the transition S*+ T - S + T* to become equally likely.

Another note: as Franck pointed out, similar ki-netic relationships can be derived also by considering,

zwI-z

ziA

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instead of a double excitation leading to the T*-state,two separate primary photochemical processes, onesensitized by molecules in the S*-state and one sensi-tized by molecules in the T-state, with the full quan-tum yield being only possible if the rates of the twoprocesses are equal.

The assumption that there are (at least) two dif-ferent kinds of chlorophyll in the chloroplasts imposesitself quite independently from Franck's ingenioushypothesis. The width of the red band in vivo alonesuggests it (Duysens, personal communication).The analysis of the shape of the absorption band byFrench (8) provides quantitative evidence that thered band has a complex nature, and that its composi-tion varies from species to species. One minor band,found in the absorption spectrum of some cells byFrench, has a peak at about 695 mi', and could thusqualify as the band responsible for the drop in quan-tum yield of photosynthesis in Chlorella > 685 mA(fig 14); it may be the n r band of the non-fluores-cent chlorophyll component postulated by Franck.

However, French also noted (in Euglena cells) afluorescence band at 705 my, which he associated

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0-iU-

600 700mP,lJm.u

FIG. 15. Fluorescence spectrum of Chlorella at room temperature (dashed line) and at -193° C. (solid line).[after S. S. Brody (2)].

FIG. 16. Fluorescence spectra of chlorophyll a solutions in ethanol. Dots: concentrated solution at -193°C;triangles, same solution at room temperature; circles, dilute solution at -193° C; crosses, same solution at roomtemperature. [after S. S. Brody (2)].

484




with the 695 mi absorption band. S. Brody (2)found a fluorescence band in the same position inlow-temperature (liquid air) fluorescence spectrumsof Chlorella (fig 15), and of concentrated (but notof diluted!) chlorophyll solutions (fig 16), and at-

tributed it to dimeric (or higher polymeric) chloro-phyll molecules. There is no doubt that chlorophyll,similarly to other dyes, does dimerize in concentratedsolutions. From concentration quenching data,Weber (personal communication) calculated a di-merization constant of 4.5 for chlorophyll a in ether,which implies about 3 % dimerization at 10-2 m/l.The proportion of dimers may increase considerablyat low temperature, and it is not implausible thatdimeric molecules, non-fluorescent at room tempera-ture, may become fluorescent at -190° C. To whatextent dimerization can be expected to occur inchlorophyll in vivo, which is probably adsorbed on

protein as a monomolecular layer, is uncertain. Brodysuggested that the red drop in the action spectrum ofphotosynthesis is caused by dimers (or higher poly-mers) with an absorption band at 695 mA; and thatthe much earlier drop in red algae could be explainedby the same dimers being present in a much higherproportion.

Whatever specific hypothesis would be supportedby future studies, it is highly plausible that the reddrop is associated with the existence of several formsof chlorophyll a in vivo, and with the incapacity ofone of these forms (or, to be more cautious, of one

absorption band of this form) to sensitize photo-synthesis with full maximum quantum yield; the en-

hancing effect of light absorbed by other pigments ismost probably due to the preferential transfer ofexcitation energy from these pigments to anothermore active form of chlorophyll a.

SUMMARY

The enhancement of the quantum yield of photo-synthesis in far red light (>680 mA) by auxiliarylight of shorter wavelengths is measured as a functionof the wavelength of auxiliary light, for green

(Chlorella), brown (Navicula), red (Porphyridium),and blue-green algae (Anacystis). The resulting"action spectra of the Emerson effect" show sharppeaks in the regions where the contribution of auxilia-ry pigments (chlorophyll b in Chlorella, chlorophyll c,

and fucoxanthol in Navicula, phycoerythrin in Por-phyridium, phycocyanin in Anacystis) to total lightabsorption is highest. This appears offhand as evi-dence that excitation of chlorophyll a alone is insuffi-cient to produce photosynthesis (at least, not with a

high yield), and simultaneous excitation of one of theauxiliary pigments is needed. However, the highyield of chlorophyll fluorescence in vivo sensitizedby auxiliary pigments contradicts this interpretation,since it suggests highly efficient transfer of excita-tion energy from auxiliary pigments to chlorophyll a.

A solution can be sought in the assumption of (at

least) two different forms of chlorophyll a, whichneed to be excited to produce photosynthesis. Onlyone is excited by far red light, the other can be excitedeither directly, e.g., by near red light, or indirectly,by energy transfer from the auxiliary pigments.

LITERATURE CITED1. BRODY, M. 1958. Thesis, Univ. of Illinois. Brody,

M. and R. Emerson. 1959. The quantum yieldof photosynthesis in Porphyridium cruentum. Jour.Gen. Physiol. 43: 251.

2. BRODY, S. S. 1958. A new excited state of chloro-phyll. Science 128: 838-839.

3. DUYSENs, L. N. M. 1952. Transfer of ExcitationEnergy in Photosynthesis. Thesis, Univ. ofUtrecht.

4. EMERSON, R. and R. V. CHALMERS. 1958. Specu-lations concerning the function and phylogeneticsignificance of the accessory pigments in algae.Phycol. Soc. Amer. News Bull. 11: 51-56.

5. EMERSON, R., R. V. CHALMERS, and C. CEDERSTRAND.1957. Some factors influencing the long-wave limitof photosynthesis. Proc. Nat. Acad. Sci., U.S. 43:133-143. Abstract: Dependence of yield of photo-synthesis in long-wave red on wavelength and in-tensity of supplementary light. Science 125: 746.Also, Yield of photosynthesis from simultaneousillumination with pairs of wavelengths. Science127: 3305.

6. EMERSON, R. and C. LEWIS. 1943. The dependenceof the quantum yield of Chlorella photosynthesis onwave length of light. Amer. Jour. Bot. 30: 165-178.

7. FRANCK, J. 1958. Remarks on the long-wave-length limits of photosynthesis and chlorophyllfluorescence. Proc. Nat. Acad. Sci., U.S. 44: 941-948.

8. FRENCH, C. S. 1958. Various forms of chlorophylla in plants. Brookhaven Symposia in Biology 11:65-73.

9. HAXO, F. T. and L. R. BLINKS. 1950. Photosyn-thetic action spectra of marine algae. Jour. Gen.Phys. 33: 389-422.

10. LATIMER, P. and E. RABINIOWITCH. 1956. Selectivescattering of light by pigment-containing plant cells.Jour. Chem. Phys. 24: 480. 1959. Selectivescattering of light by pigments in vivo. Archiv.Biochem. Biophys. 84: 428-441.

11. LIVINGSTONr, R., W. F. WATSON, and J. MCARDLE.1949. Activation of the fluorescence of chlorophyllsolutions. Jour. Amer. Chem. Soc. 71: 1542-1550. Livingston, R. and S. Weil. 1952. Activa-tion of the fluorescence of chlorophyll solutions.Nature 170: 750-752. Cf. also, Evstigneev, Vr. B.,V. A. Gavrilova, and A. A. Krasnovsky. 1949.Doklady Acad. Sci., USSR 66: 1133, 70: 261.

12. MYERS, J. and C. S. FRENCH. 1960. Relationshipbetween time course, chromatic transient, and en-

hancement phenomena of photosynthesis. PlantPhysiol. (in process)

13. STRAIN, H. H. 1951. The Pigments of Algae. In:Manual of Phycology G. M. Smith, ed. ChronicaBotanica Co., Waltham, Pp. 243-262.

14. WARBURG, O., G. KRIPPAHL, W. SCHROEDER. W.

BUCHHOLZ, and E. THEEL. 1954. Zeits. Natur-forsch. 8b: 164.

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