DECEMBER, 1949
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THE RELATIONSHIP OF PLANT PIGMENTS TO PHOTOSYNTHESIS JAMES H. C. SMITH Division of Plant Biology, Carnegie Institution of Washington, Stanford, California
I ) that only A s EARLY as 1779 it was proposed (6. the green parts of plants can dephlogisticate fixed air. In modern terms this means that only the green parts of plants are able to evolve oxygen when placed in an atmosphere containing carbon dioxide. This remained an almost unchallenged dictum until Englemann (1) a little over a hundred years later put it t o a rigorous test by means of his sensitive bacterial method. Englemann discovered that certain bacteria become immotile when placed in an atmosphere free of oxygen and regain their motility by introduction of the merest trace of oxygen. A trillionth (European units) of a milligram of oxygen, that is, about 20 oxygen molecules, can be detected by this method. By means of this fabulously sensitive test he examined colorless tissue from a large number of organisms, among which were the colorless tissue of the parenchyma cells of albino maple and ivy leaves, flower petals, and also the cells from many different kinds of animals. In no single case was it possible to detect the evolution of a trace of oxygen from the colorless protoplasm. I n contrast to this, when even the smallest chlorophyll body was illuminated, it evolved oxygen. From these experiments Englemann concluded that only the pigmentcontaining cells, and within these cells only the pigmented plasma particles, evolved oxygen in the light.
Englemann then proceeded to determine the effect of different wave lengths of light on the evolution of oxygen. He developed a micro technique ( 2 ) so that he could illuminate an organism or a piece of tissue with a spectrum of small dimeusions and determine t o which parts of the tissue the bacteria migrated. From the relative number of organisms surrounding the tissue illuminated with different wave lengths of light, he deduced the relative activities of the rays. He examined algae of different colors-green, blue-green, yellow-brown, and red-to determine whether their spectral sensitivity for the evolution of oxygen differed. The results he obtained are shown in Figure 1 (5). The solid line represents the photosynthetic activity and the broken line represents the relative light absorption of the organisms. The maximum assimilation in the green and yellow-brown algae occurs a t about 680 mp and corresponds to the red absorption band of chlorophyll. The yellow-brown algae have an almost equally high photosynthetic activity at about 520 mp, whereas the green algae have a second but much lower maximum of photosynthesis at around 490 mw. The red algae showed maximum photosynthesis in the middle portion of the spectrum a t about 570 mp, and this maximum photosynthesis is very much higher than that in the region vhere chlorophyll absorbs the most, at about 680 mp. The blue-green algae have a very
JOURX%L OF CHEMICAL EDUCATION
photosynthesis elicited th ,:?by. Figure 2 shows the results obtained by Emers~nand Lewis (4). Comparison of the total light absorbed (Figure 2A) as compared 60 with the amount of photosynthesis (active absorption) ,' shows that the photosynthesis rnns roughly parallel b -d 40.3 with the absorption. However, there is a deviation in YELLOW-BROWN 20 the short wave lengths. This can be accounted for by absorption of the carotenoids, which are known to $ the occur in Chlorella and to absorb in this region of the 80 spectrum. Based on the relative absorption of chlorophyll and carotenoids in the alcohol solution (Figure 2R) the carotenoids absorb as much as 75 per cent of the light in the region around 500 mu. There is oonsidera'ble discrepaniy, as Figure 2C shows, between the absorption of the extract and the absorption of the intact cells. Therefore, it is uncertain how quantitatively the absorption of the various pigments in the extract Wave Length in me Figure 1. Relation of Photoamthesis to Spectral Ahsovtion R o p r - can be assumed to represent their proportionate abtiesin D i f f e n t Cl-a of illme sorption in the living cells. The relatiw photmmthetia ability (*A)of different dames of algae at Perhaps the best test of what pigments are active in different wave lengths of light compared with their speotral absorption photosynthesis is to determine the quantum yield of - - 0)(Englemann (PI)). propertis (Cphotosynthesis a t different wave lengths of light and to broad band in the red end of the spectrum with a ma& compare this with the absorption of the pigments. mum photosynthetic activity a t about 620 mp. A The assumption is made that the effectiveness of light definite maximum of photosynthesis in blue light is ob- of different wave lengths depends only on the amount served only in the green algae. absorbed. The quantum yield for Chlorella is shown in The light absorption also differsin the different kinds Figure 20. Beyond 730 mp there is an exceedingly of algae, which indicates the presence of different pig- small amount of photosynthesis. The value rises ment complexes. From these experiments Englemann steadily to ahout 685 mp when it reaches a maximum of came to the conclusion that besides chloroph~ll other ahout 0.09. From '685 to 580 mp it remains at almost maximum value and then declines to about 0.065 a t pigments fundim in the assimilatory process. Englemann had a clear concept of the relation of 485 mp. The quantum yield then rises to about 0.08 photosynthesis to pigments in the living organism. at 420 mrr. The relationship of quantum yield to wave He realized that the action spectrum should correspond length reveals that in that region of the spectrum where to the ahsorption of the pigments which are active in photosynthesis in the organism. In order to make a rigorous determination of mhat pigments are active in photosynthesis and to what extent, it would be necessary to determine quantitatively the absorption of each pigment in the organism a t each wave length and compare this with the amount of photosynthesis produced. Still, a t the present time, this procedure is impossible because t.here is no way to determine the absorption of each pigment individually in the living organism. As an alternative procedure, determination of the absorption of the extracted pigments has been resorted to. But the absorption spectra in the extracts differ from t,hose in the living organisms and the magnitudes of the differences vary from organism to organism. This makes it impossible to compare quantitatively the absorption of the pigments in the extract with the absorption of the pigment in the organism. The best that can he done is to make an approximation of the Figure 2. Rektion of Photosynthesis to Spactrnl Abmorption P r o ~ r ties in Chlorellowemoidos. absorption of the individual pigments in the living (A) Total abwrption of the organiam compared with the "active" ahorganism from their absorption in the extract and comi. r,"theperosntofinddentlightwbichrasultad in photosynthesis." pare this with the photosynthesis a t various wave amption, ( B ) Estimated percentage of the light absorbed by the aamtenoids. leneths. (C) Compmiaon of the optical densities of a suspension of intact celh i s an example, let us first look a t the green alga, and of an extract of the same quantity of oells sdiuated for wavclength ChImeZla. The absorption of light by the living cell s b i ~ ~ ~yields at difi-t lenstb. of light ~ of u ~ ~ ; has been measured and compared with the amount of (Emerson sod Lewis (4)). so
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DECEMBER, 1949
the carotenoids absorb light the quantum yield decreases conspicuously. The decrease is not so great as the absorption by the carotenoids in extracts of pigments might indicate, for whereas the quantum yield decreases about 30 per cent there is indication that the relative amount of light absorbed by the carotenoids increases to about 70 or 75 per cent. The decrease in quantum yield in this region is evidence that the carotenoids as a whole are not as photosynthetically active as the chlorophylls. But the quantitative relations may be interpreted to mean that the carotenoids are a t least partially active. Whether the carotenoid pigments are all less active or whether some are completely inactive and others fully active still remains in doubt. Both chlorophylls a and b appear to be active. Whether they are active to the same extent is not yet clear. At the wave lengths near the maximum absorption of chlorophyll b there appears to be a slight diminution in the quantum yield, the reality of which is questionable. From this discussion it appears justifiable to conclude that in the ereen alea. Chlwella. the ~iements chiefly active in p~otosyn&&is are the chldrGhylls. If the carotenoids are active, they are, as a whole, considerably less efficientthan the chlorophylls. The blue-green alga, Chroococcus, has also been examined by Emerson and Lewis (5) relative to the total absorption and active absorption. In Figure 3A it is seen that the active absorption, shown by the broken line, parallels the total absorption a t the longer wave lengths, but falls far short of the total absorption in the middle portion of the spectrum. This is the region of the spectrum where the carotenoids are chiefly responsible for the absorption, as the curves in Figure 3B show. The peak around 680 mp is due to the absorption of chlorophyll. The peak at 620 mp is due to the absorption by phycocyanin. In this organism the absorption of the intact cells and of the combined extracts very nearly parallel each other (Figure 3C) and for this reason it may be assumed that the absorption of the pigments in solution bears a direct relationship to the absorption of the pigments in the living cells. Calculated on this basis, the relative absorption by the different pigments is shown in Figure 3B. When the quantum yield (Figure 3 0 ) is compared to the absorption of the different pigments it is clear that in the region where both chlorophyll and phycocyanin absorb the quantum efficiency is nearly constant. It is in the region where the carotenoids absorb most strongly that the quantum yield falls off decidedly. Although the quantum yield falls off sharply in this region, the decrease is less than what would be expected from the absorption by carotenoids in extracts of the alga (cf. broken line, Figure 3D). A review of the facts indicates that light absorbed by chlorophyll and phycocyanin is photosynthetically active and to nearly the same extent. Light absorbed by the carotenoids is used to only a slight extent if a t all. The yellow-brown alga, Nitzschia closterium, has been
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3. Relation of Photoaynthds to Spectre1 Ab.ozption Rope. tie. in Chrooeoccu.
( A ) Tots1 absorption of the organism oornpmed with "active" absorption. ( B ) Estimated percentwe of the light absorbed by the vatioue dassea of pigments. (C) Comparison of the absorption speotra of intact oella and extracts of the same quantity of cells adjuted for wave-length ahift (aaa text). ( D ) Quantum yields of photoavnthesis at ditlerent wave lendhs ~ht. . of l i. Ths solid line repreaentn the satual obaervationa. The broken line represeots valves calculated on the assumption that the yield is 0.08 for light absorbed by chlorophyll and phycocusnin and zero far Light absorbed by carotenoids (Emerson and Lewis ( 5 ) ) .
examined by Dutton and Manning (6). In Figure 4A are shown combined the relative amounts of light absorbed by the different pigments in an extract of Nitzschia closterium. The percentage of light absorbed by each of the pigments is shown in Figure 4B. This is compared with the relative quantum yields of photosynthesis obtained a t different wave lengths (crosses) in percentage of the quantum yield a t 665 mp. From the results obtained it is clear that where the carotenoids absorb the most, there the relative quantum yield is depressed the most. However, at 436 mp, where about 50 per cent of the light is absorbed by the carotenoid fucoxanthin, the relative quantum yield is reduced very little. It may be that this carotenoid is particularly active in photosynthesis. The fact that the quantum yield is lowered to only about 70 per cent of that when all the light is absorbed by chlorophyll, although 93 per cent of the light is absorbed by the carotenoids is strong indication that the carotenoids are partially responsible for the light absorbed and used in photosynthesis. Besides chlorophyll a this organism has been shown to contain chlorophyll c by Strain, Manning, and Hardin (7). What its contribution to photosynthesis is remains to be determined. The red algae.present an interesting situation. As was shown in the action spectrum curves obtained by Englemann, reproduced in Figure 1 (RED), the oxygen production was highest in green light at about 560 mp where phycobilins absorb strongly. This gave indication of the activity of the phycobilins. In the regions where chlorophyll shows its maximum absorption, in red and in blue light, the assimilation was only about 20
JOURNAL OF CHEMICAL EDUCATION
634
Figvrr 4.
Rolation of Photosynthesis to Sp-ctrd Abaor~tionprop.^t i u in NiL..~hi. closte,i"m
(A) A summation of the relative absorption ooeffioients (in acetone) at different wave lengths for the component pigment fractions from A'. cloatcrium.
( B ) The peroentase of the total absorption ss&ibsble to ohlorophyll o to fucorhnthin - - , and to total earotenoids - - - compared with the relative suantum yields of photosynthesis (crosses) a t different wave lengths (Dutton and Manning ( 8 ) ) .
-,
per cent of that in green light. And even though chlorophyll obviously is absorbing in the red region of the spectrum, its photosynthetic activity seems to he much depressed as compared to the phycobilins. The photosynthetic activity of red algae has been investigated by Blinks and his co-workers during the past few years by the use of modern techniques. The data published by Haxo and Blinks (8) are quoted here. A red alga, Schizymenia, and a green alga, Ulca, are compared as to their rates of oxygen evolution when illuminated with light of different wave lengths. The rates for both algae are arbitrarily fixed at 100 for red light (620-650 mp) and given the appropriate relative values at the other wave lengths. 435.8 rnN (blue) Schizymenia (red alga) 48 to 53 Ulva (grecn alga) 94
546 mrr (green) 288 to 340 46
sorbs further to the red than chlorophyll a--chlorophyll d maximum 696 mp, chlorophyll a maximum 665 mp in methyl alcohol. The effectiveness of this pigment in photosynthesis has not been determined. Certain bacteria carry on another type of photosynthesis--carbon dioxide is taken up but no oxygen is evolved when the organisms are illuminated. The hydrogen donors are substances other than water. Among these bacteria is the purple bacterium, Spirillum rubrum. French (10) has measured the action curve of this organism for the assimilation of carbon dioxide with butyrate as hydrogen donor and compared it with the absorption spectrum of the organism. In Figure 5A these two spectra are shown. I t is clear that the two do not coincide throughout. Where the carotenoids ahsorh most, there is a great diminution of photoassimilation. There is a coincidence at about 600 mp of an absorption band of the green pigment, bacteriochlorophyll, and a maximum in the action curve (Figure 5C) which supports the supposition that bacteriochlorophyll is responsible for the absorption of light active in the photoassimilation. This coincidence is evident in both the living cells and the methanol extract of the cells. From these measurements, French has concluded that the yellow pigments are inactive. The absorption spectra of the live cells and of the pigment extracts from S. rubrum differto an extraordinary degree (Figure 5B). Another means for determining the relationship of pigments to photosynthetic activity is to follow the genesis of photosynthetic activity simultaneously with the formation of pigments. Seedlings which are germinated in the dark produce leaves which contain no chlorophyll and have little or no photosynthetic activity. When such leaves are placed in the light they form chlorophyll and develop photosynthetic activity. Is this activity conditioned by the amount of chlorophyll which is formed?
6t0450 mu (red) 100 100
The green alga has its least activity where the red alga has its greatest activity; and the green alga has its greatest activity in the spectral regions where chlorophyll absorbs the most, whereas, the red alga has its greatest activity where the phycohiliu, phycoerythrin, absorbs the most. Various red algae differsomewhat in the wave lengths at which they show maxima of photosynthetic activity. This may possibly be ac'counted for by variation in the spectral absorption of the different algae caused by the various phycohilins they contain. The results demonstrate that in such organisms as Schizymenia the light absorbed by pigments other than chlorophyll is more effective than light absorbed by chlorophyll. Manning and Strain (9) have reported the presence of chlorophyll d in many red algae. This pigment ab-
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Figure 5.
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Relotion of Phetosynthesis to Spectral Absorption Properties in Spirillum r v b r u m
(A) Comwrison of the speotral absorption of living cells with the "aoLive" hbsorption. ( B ) Comparison of the spectral absorption of the living cells and of a methanol extract of the cells. (C) The "active" absorption compared with the spectral absorption of the methanol extract of oells (French (lo)).
DECEMBER, 1949
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