p California Association of Chemistry Teachers
I
Roderic 6. Park
Lawrence Radiation Laboratory and Botany Department university of California Berkeley
Advances in Photosynthesis
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Kind (I), Calvin (8), and Bassham (3) have reviewed advances in photosynthesis in THIS JOURNAL in recent years. Since the previous articles dealt primarily with the chemistry of photosynthesis, this article will emphasize the relation between photosynthetic chemistry and the molecular architecture of the photosynthetic center in plant cells. Photosynthesis is the biological conversion of eleotromagnetic energy into chemical energy. The chemical energy so obtained is then used by the organism for synthesis of intricate organic moleculcs from the chemical raw materials available in the external environment. Three examples will be given to illustrate this definition.
used directly to produce acetylphosphate which condenses to yield 0-keto acids which are eventually reduced to yield poly-b-hydroxybntyrate. The reducing power in this case is not supplied by light, but by acetate metabolized via the tricarboxylic acid cycle. In the first two examples of photosynthesis the carbon source was highly oxidized and the reducing agent was H,O or some inorganic compound such as H,S. In this case the carbon source for synthesis (acetate) is a t a more reduced level and serves not only as the carbon source but also as the reducing agent in place of H 2 0 or H2S. This reaction may be written as follows:
Types of Photosynthesis
The best known type of photosynthesis is that occurring in green plants. I n this type of photosynthesis CO* is the carbon source for the synthesis. The reduction of CO? to the level of sugar must be accompanied by an oxidation and in this case water is oxidiz~dto yield oxygen gas. In its simplest form this rcact,ion is written as follows: 2&0
light + COXchlorophyll CHzO + H,O + O1 --A
(1)
A second kind of photosynthesis is typified by the purple bacteria. Again Con is the carbon source for synthesis of the organism, but this time reduction of C o n is accompanied by the oxidation of one of a number of organic or inorganic compounds other than water. Oxidat,ion of HnS to yield free sulfur is a typical example. The general equation used to describe this reaction is: 2HsS
light + COI baeterlo-
CHIO
+ H1O + 25
(2)
ohlorophyll
A third kind of photosynthesis is carried out by some purple bacteria in which acetate rather than COz is the carbon source used for growth. The carbon source (acetate) is much more reduced than is C02. In this case chemical energy derived from sunlight is
Presented at the CACT Summer Conference, hilomar, California, August, 1961.
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light
acetate
organism
bacteriochlorophyll
+ CO1
In recent years it has become evident that these types of photosynthesis, though occurring in different organisms and superficially appearing different in terms of their synthetic chemistry, are actually closely related. This close relat,ionship extends not only into the biochemical mechanisms involved, but even into the molecular architecture of the photosynthetic apparatus. The following discussion will emphasize a few of the important experimental contribntions which have led to our present understanding of photosynthesis. Light and Dark Reactions
Over fifty years ago Blackman (4) pcrforrned experiments which showed that photosynt,hesis consisted of several broad classes of reactions. Blackman varied light intensity, C02 concentration, and temperature during the photosynthesis of green plants. He showed that the photosynt,hetic rate is temperatnre-independent when the photosynthetic rate is limited by low light intensity. Rlackman found, on the other hand, that the photosynthetic rate is temperat,uredependent when photosynt,hesis is limited by low Con concentrations. These data show that photosynthesis consists of two classes of reactions: temperatureindependent photochemical reactions which are responsible for conversion of electromagnetic energy to chemical potential and temperature-dependent enzyme reactions which utilize t,he energy from thc light reactions to fix CO1 into sugar.
Twenty-five years later Emerson and Arnold (5) showed that the two classes of photosynthetic reactions, light reactions and dark reactions, can be separated in time. Algae illuminated with a bright flash of light followed by a suitable dark period carried out photosynthesis a t the same rate as algae receiving the same amount of light energy but under continuous illumination. Apparently the energy-rich chemical products resulting from the light flash were used in a subsequent dark period for the process of Con fixation. By adjusting the relative lengths of the light and dark periods, Emerson showed that threemillisecond flashes, followed by 20-millisecond dark periods, produced the same rate of photosynthesis as continuous light. Shorter dark periods reduced the photosynthetic yield. Emerson and Arnold (6) also showed that following a saturating light flash, approximately one COZmolecule was fixed per 2500 chlorophyll molecules. I t seemed that chlorophyll molecules operated in groups for absorption of light energy. The absorbed energy then migrated, possibly by resonance transfer, to an active site where the formation of the light reaction products took place. This group of chlorophylls acting together as a light gathering target was called the "photosynthetic unit." I n recent years improved experimental apparatus has revised the size of the photosynthetic unit down to about 300-500 chlorophyll molecules (7). The photosynthetic unit is an important factor in photosynthetic light reactions since only by an energy focusing mechanism can plants obtain enough energy a t one site within a time of milliseconds to fix COz into sugar. This mechanism becomes especially important a t low light intensities. Pathways of Electron Transport
The work of Blackman and of Emerson established that photosynthesis consists of light and dark reactions, but the mechanism of these reactions remained a subject for future exploration. Van Niel (8) in 1931, on the basis of comparisons between green plant photosynthesis, equation (I), and bacterial photosynthesis, equation ( Z ) , proposed the novel idea for that time that light energy was used to convert water rather than COZinto an oxidized and a reduced moiety. The reduced fraction would reduce C02and the oxidized fraction would lead to production of molecular oxygen. This prediction was beautifully demonstrated by Ruben and Kamen (9) in 1941 who showed, using lS0 tracer techniques, that the oxygen produced in green plant photosynthesis arises from the oxygen of water and not from the oxygen of COz. We would now combine the findings of thesc experiments in the following way. Absorption of light by the photosynthetic unit yields spatially separated oxidized and reduced sites resulting from electron transfer. This is the light reaction of photosynthesis. Subsequently, the oxidized moiety in green plant photosynthesis brings about oxidation of water to yield molecular oxygen. The reduced moiety eventually reduces the cofactor pyridine nucleotide (PN) to the reduced form PNH. The PNH so produced provides the reducing power necessary to reduce COI to the level of sugar. The photosynthetic unit and its associated electron transport pathways then consist of an electron pump
mechanism which uses the energy supplied by sunlight to remove electrons from water to produce oxygen. The e1ectror.s obtained from water are in turn raised to a sufficiently negative redox potential (Eof = -0.3 v) to reduce P N and subsequently to reduce
co,.
One other cofactor necessary for C02 fixation by plants is produced during the transport of electrons from water to PN. This cofactor is adenosine triphosphate (ATP). The synthesis of ATP from adenosine diphosphate (ADP) and inorganic phosphate (P,) in the photosynthetic apparatus of green plants was first demonstrated by Arnon in 1954 (10). This reaction uses energy which is available during the electron flow from water to PN. A similar coupling of ATP formation to electron flow in mitochondria accounts for the majority of energy available to aerobic organisms, such as man, for carrying out their life functions. The oxidation of water, production of PNH, and production of ATP are illustrated in Figure 1. PNH
H'
LIGHT system
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Figure 1. General concept of the light reactions and asrociated electron transport palhxays of green p l o d photosynthesis. There reactions are locolired within a chlorophyll containing porticle cdled the quantosome.
During the past ten years the electron path represented by a dashed line between the plus and minus signs in Figure 1 has been studied in great detail. This pathway is now known to involve one or more cytochromes (1I), possibly plastoquinone (12), and probably two pigment systems. One cytochrome in particular, cytochrome-f, is intimately involved in the primary act of photosynthesis. Illumination of chlorophyll-a brings about an immediate oxidation of cytochrome-f (1.3)'. Chance (14)has shown that this oxidation of cytochrome by chlorophyll can occur even a t liquid nitrogen temperatures. The fact that cytochrome oxidation can occur a t such a low temperature indicates that this is a solid state reaction, i.e., one not requiring participation of mobile chemical substances. Other information on the electron transport pathway comes again from the observations of Emerson who found that photosynthesis in green plants involves light absorption by more than one pigment system. One of the pigment systems involves chlorophyll-a and the other pigment system is an accessory pigment, for example chlorophyll-b or a phycohilliu. In the plant systems involving chlorophyll-a and an accessory pigment, Duysens (IS) has shown that illumination of chlorophyll-a brings about oxidation of cytochrome, whereas illumination of the accessory pigment brings about reduction of the cytochrome. Duysens found that inhibitors of oxygen evolution in photosynthesis also Volume 39, Number 8, August 1962
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inhibited reduction of cytochrome by the accessory pigment. Recently Arnon, et al. (16), reported experiments which support the experiments of Duysens. These experiments taken together lead to the formulation of the light reactions of green plant photosynthesis, shown in Figure 2. Light absorbed by the accessory pigment system, possibly by electron-hole separation as suggested by Calvin (16), brings about the oxidation of the water and reduction of some substance, possibly plastoquinone or cytochrome-b6. During the passage of the electrons through the cytochromes to a more positive potential, a portion of the
The external electron donor in this type of photosynthesis is the acetate itself rather than water or Has. The electron transport system of photosynthesis may he thought of as a light-driven electron pump. Light energy absorbed by chlorophyll leads to electron excitation. The energy of the excited electron is used for reduction of PN with concomitant oxidation of some electron donor and for production of ATP. In the special case of acetate assimilation in bacteria, only ATP is produced from the energy of the excited electron.
Figure 2. The light reactions and associated electron transport pathwoyr of green plant photosyntherir. There reactions are localized wilhin o chlorophyll containing called the quantarome.
energy given up is used to form ATPfrom ADP and Pi. This phosphorylation is probably similar to the one observed in the respiratory system of mitochondria. Thus the terminal cytochrome, cytochrome-f, becomes reduced. Light absorbed by chlorophyll-a then brings about oxidat.ion of cytochrome-f and eventually reduction of PN to PNH. The reduction of PX to PNH requires two electrons and thus according to this scheme four quanta would be required to bring about PN reduction. Arnon and co-workers 115) ~, have shown that under certain circumstances in higher plant cells, electrons may follow a short-circuit pat,hway, utilizing energy absorbed by chlorophyll-a. This pathway is catalyzed by an added cofactor, phenazine methosulfate (PMS). The path followed by the electron in this pathway is illustrated by the dashed line shown in Figure 2. It, is seen that the electron, excited to a more negative potent,ial by the energy absorbed by chlorophyll-a, gives up its energy to form ATP in the passage back to cytochrome-f. This type of phosphorylation is called cyclic phosphorylation whereas the type associated with electron transport to PN is called noncyclic phosphorylation. The photosynthetic electron transport pathways for bacterial photosynthesis are in essence a simplificat,ion of the pathway found in grcen plants. The pathways for bacterial photosynthesis are presented in Figure 3. Bacterial photosynthesis, in which the carbon source is COa, differs from green plant photosynthesis in that the source of the electrons for Con reduction is not water, but some other reducing agent such as H,S. For this reason oxygen is never evolved in bacterial phot,osynthcsis. This pathway is shown in Figure 3a. A specialized case of bacterial photosynthesis is presented in Figure 36 in which the carbon source is acetat,e; see equation (3). 426
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Figure 3. (01 Light readion, and os9ociated electron transport pathways in bacterial photorynthesis in which COz server as the carbon source. There reoctionr ore localized within a chromotophore subunit described b y Frenkel (231. (b) Some as lo) except that acetate server or the carbon source.
Fixation of Carbon Dioxide
A major achievement in photosynthesis research was the elucidation of the reactions by which Con is accepted into an organic compound and is subsequently converted into sugars with regeneration of the COz acceptor. These dark reactions were worked out primarily by Calvin and co-workers (17). This pathway as it is now formulated is presented in Figure 4. The Cop acceptor is a five-carbon sugar ribulose diphosphate (RuDP). The unstable six-carbon intermediate which is formed from C02 and RuDP breaks down into two 3-phosphoglyceric acid (PGA) molecules. This reaction is catalyzed by the enzyme carboxydismutase and is not an energy requiring reaction. PGA is the first stable product of CO1 fixation during green plant photosynthesis that one can detect using carbon-14 tracer techniques. The carbon-14 label subsequently appears in the other components of the photosynthetic carbon cycle shown in Figure 4. Eventually more CO1 acceptor (RuDP) is formed which allows contin~at~ionof the photosynthetic cycle. Bassham has described these reactions in some detail in THIS JOURNAL (3). It is shown in Figure 4 that energy supplied by thc photosynthet,ic unit and its associated
electron transport pathways must be introduced into the carbon cycle a t two places. Energy is first introduced during the reduction of the first product of photosynthesis (PGA) to the level of sugar (triosphosphate). This reaction consumes one molecule of ATP and one molecule of PNH. Since for each carbon dioxide molecule fixed there are two PGA molecules produced, the total ATP and PNH requirement for this part of the cycle will be two molecules of ATP and two molecules of PNH. The other point in the cycle where energy is required is during the conversion of ribulose 5-phosphate into RnDP. This reaction consumes one additional molecule of ATP. Thus the total energy required for fixing one molecule of COzinto sugar as outlined in Figure 4 is two molecules of PNH and 3 molecules of ATP. The role of the light and electron transport reactions of photosynthesis is to provide the ATP and PNH molecules consumed during COzfixation. The discussion of the light reactions of photosynthesis has been made possible largely through the use of broken cell systems. I n the case of the dark reactions in which COXis fixed into sugars, it was possible to feed the cells with carbon-14 labeled hicarbonate and then after varying lengths of time to determine into which compounds the bicarbonate had been assimilated. Plant cells, in particular algal cells, readily assimilate bicarbonate ions through the cell membrane. On the other hand, many of the compounds that one would like to feed the cell in order to elucidate the electron transport pathway of the light reactions will not penetrate the cell membrane. In general phosphorylated substances such as ATP, ADP, PN, or PNH will not penetrate cells when fed externally. Thus, before investigators could find the role of these compounds in photosynthesis, it was
necessary to break the cell in such a way that these compounds could have access to the photosynthetic sites within the cell. It was shown over 20 years ago by Hill (18) that a t least some of the light reactions associated with photosynthesis are localized in chloroplasts-the chlorophyll containing bodies of plant cells. Hill showed that when chloroplasts were isolated from the cell in suitable media they would carry out a lightdependent evolution of oxygen, if a suitable oxidant such as iron (1II)ion was supplied. Electron transport from water resulted in oxygen evolution and reduction of iron(II1) to iron(I1) ion. Hill tried to find whether carbon dioxide could also serve as an oxidant for photosynthesis and thus he reduced to the level of sugar. His experiments were unsuccessful and, though he had shown that the light reactions of photosynthesis were associated with the chloroplasts of higher plant cells, his efforts to demonstrate COz fixation by the chloroplasts remain inconclusive. I t was not until 1954 that Arnon (10) demonstrated that isolated chloroplasts could also carry out fixation of C02 by a pathway similar to that observed in intact plant cells by Calvin. Thus, from the work of Hill and Arnon, it is known that both the light reactions and dark reactions of photosynthesis occur within the chloroplast of the plant cell. Chloroplast Structure
What is the architecture of this body, the chloroplast, which allows such an amazing diversity of reactions to go on within it? The answer to this question has come from structural information obtained with the electron microscope, and also from separation of the isolated chloroplast into fractions which will individually carry out either the light and electron transport reactions of photosynthesis or the dark reactions of
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photosynthesis. Recombination of these fractions is necessary in order to reconstitute the entire photosynthetic process. Figure 5 is an electron micrograph of a thin section of a spinach chloroplast. I t is seen that the chloroplast is bound by a membrane, and within this membrane there is a highly stained lamellar strncture. This lamellar structure lies embedded in a granular material called the stroma. Recently in our
....,.,..
This fraction carried out the light reactions of photosynthesis, oxygen evolution, and photosynthetic phosphorylation. The supernatant on the other hand contained no chlorophyll, hut contained the enzymes necessary for carrying out t,he COs fixation reactions of photosynthesis. The question arose as to how small a fragment of the particulate structures in the green precipitate is sufficient to support C02 fixation in the supernatant fraction. Lamellar fragments as small as 800 A across and 100 A in thickness, containing several thousand chlorophyll molecules, retain full activity. Improved techniques will probably allow us to go to considerably smaller chlorophyllcontaining particles than this. The particulate nature of the spinach lamellar structures is especially evident in frozen dried and shadowed material. Recently, however, a number of micrographs of thin sections of chloroplasts have appeared in the literat,ure (to),in which the granular appearance of the lamellar structures is evident,. The granular subpnits are oblate spheres 200 A in diameter, are 100 A thick, and are osmiophilic over one surface. We have chosen the term quantasome to describe this particle. Our reasons for thinking that the quantasome is a particle of fundamental importance to photosynthesis are outlined below. The architecture of the chlorophyll-containing lamellar stmctures found in various members of the plant kingdom is highly varied (80-22). However, they can all be constructed from the quantasome particle.
,~... ,.
Figure 5. Electron micrograph d on O,Opstained thin section of a spinach chloroplo~t.
laboratory we subjected whole isolated chloroplasts to fragmentation by sound waves (19). The fragmented pieces were then separated from one another by fractional centrifugation. One ohtained, after centrifugation, three layers in a centrifuge tube. There was a green precipitate at the bottom of the tube, a slightly yellow protein supernatant overlying the green precipitate, and a yellow lipid material which rose to the surface of the tube. Electron micrographs of the first two fractions are shown in Figure 6. The green precipitate was shown to correspond to the dark lamellar strncture contained within the chloroplast membrane. The green precipitate is composed of doublc layered structures made up of small particles.
Figure 6.
la)
Eiectrcn micrograph of chromium rhodowed green precipi-
tote. 8 8 0 A paiyrtyrene latex present. lbl Electron microgroph of chromium shadowed rupernotont. 1 8 8 0 A polyrtyrene lotex present.
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Figure 7. Models for chioroplort lornellor rtructurer which ore conrtrv~ted from the quontasome particle.
Figure 7 shows how the qnantasome may be used to construct three of the principal types of chlorophyllcontaining lamellar structures thus far described. The upper lamellar structure in Figure 7 is the type found in the chloroplasts of blue green algae (21). The middle lamellar structure with the two thicken~d center lines is the type found in Euglena ($8). The lower structure is the type found in spinach and tobacco chloroplasts (19, $0). When the quantasomes are placed back to back an osmiophilic line of double thickness is obtained. In chloroplasts of the spinach and tobacco type, these thickened areas are called the grana areas. Observations by fluorescence microscopy indicate that chlorophyll is more concentrated in the grana than in other areas of the chloroplast. I t has
often been maintained that grana are the only portion of the chloroplast lamellar structure which contains chlorophyll. Evidence obtained by Park and Pan (19) would dispute this idea and suggest that chlorophyll is distributed uniformly throughout the lamellar structure of the chloroplast. Chlorophyll appears more evident in the grana areas because of the greater number of lamellar structures stacked up in these areas, as shown in Figure 7. The stroma and grana lamellae supported photosynthesis equally well. Thus the quantasome, whether located in the grana lamellae or the stroma lamellae, probably contains chlorophyll and will support COzfixation. The number of chlorophyll molecules which could be stacked on one surface of a 200 A quantasome can he estimated. The chromophore gortion of the chlorophyll molecule is approximately 15 A on a side. Thus if the chlorophyll molecules are stacked together at a 45 degree angle to the plane of the surface, it would be possible to place several hundred chlorophyll molecules on the surface of one of the lamellar subunits. This number of chlorophyll molecules is surprisingly close to the size of the photosynthetic unit as initially described by Emerson and Arnold and more recently by Kok (7). I t may be that the smallest lamellar particle which carries out all the light and electron transport reactions of photosynthesis is the 200 A diameter quantasome which we can see with the electron microscope. Frenkel (BS) has shown that a fully active subunit similar in size to the quantasome exists in the bacterial photosynthetic apparatus (the chromatophore). The protein supernatant overlying the green precipitate after the chloroplast fractionation experiment was responsible for the dark C 0 2 fixation reactions of photosynthesis. This fraction apparently corresponds to the stroma material of the intact chloroplast. Stroma is the granular material shown in Figure 5, in which the chlorophyll-containing lamellar structure of the chloroplast is embedded. Thus there is actually a morphological basis for the two types of photosynthetic reactions: light reactions, with their associated electron transport pathways, and dark reactions, which were initially described from a purely physiological point of view. The light reactions occur in the lamellar structure of the chloroplast and the dark reactions occur in the stroma of the chloroplast. Both these fractions are necessary to carry out photosynthesis. This is shown in Table 1 in which the COz fixation capacity of the green precipitate alone (the lamellar structures) and of the protein supernatant alone (the stroma) are given. When the two fractions are mixed together, there is a synergistic effect on carbon dioxide fixation. The fixation capacity of the mixed fractions is some 65 times greater than that of the two fractions taken separately. This is to be expected from our model, since the light reactions of the lamellar structures produce the energetic cofactors required to drive C 0 2fixation in the stroma. Thus we see the chloroplasts as a very remarkable factory for carrying out a complicated synthetic process involving conversion of electromagnetic energy into chemical energy. Much effort in research on photosynthesis is now being directed toward understanding the mechanisms
Table 1.
Fixation of K O 1b y Various Fractions from Sonicallv Fragmented Chloro~lasts
chlorophyll' Light Dark
Fraction Total sonicate 0 to 14,000 X g precipitate 0 to 14,000 X a du.5 supernatant - 14,000 to 110,000 X g precipitate 14.00 to 110.000 X e olus
Supernatant refers to the supernatant liquid obtained from 145,000 X g centrifugation. A fixation rate of 3 X lO%ounts/min/me chloro~h~ll/30 min is eouwalent to 1 m~cromoleof COI
by which light reactions within the lamellar structure occur. It is appealing to think that the 200 A quantasome is perhaps a morphological expression of the photosynthetic unit of Emerson and Arnold. This small particle holds the answers to much of what we do not know about photosynthesis. Experiments are presently being directed toward iinding the pigment orientations and associations within these particles and toward elucidation of the electron transport pathways which lead to oxidation of water and to reduction of PN. It is hoped that these studies may eventually lead us to the exact mechanism by which the electronic excitation of chlorophyll is converted into the chemical potential usable for synthetic reactions of photosynthesis. Literature Cited
( 1 ) KIND,C. A., J. CHEM. EDUC.,33,530 (1956). M.. J. CHEM. EDUC.. 35.428119581. 121 CALVIN. J:A., J. CHEM. EDUC.,'~~, i ~ (1961). i i3j BASSHAM, F. F., Ann. Bol., 19,281 (1905). ( 4 ) BLACKMAN, R., AND ARNOLD, A., J . Gen. Physiol., 15, 391 ( 5 ) EMERSON,
- - -- .
llYR2) , ,
( 6 ) EMERSON, R., AND ARNOLD, A,, J . G a . Physiol., 16, 191 11097\ ,A""-,. ( 7 ) KOK, B., AND BUSINQER, J. A,, i n "Research in Photosynthesis," edited by H. GAFFRON, Interscience Publishers. Ine.. New York. 1957. D. 357. (8) TAN N I E C. ~ B.,A ~ e h~ikrobiol.; . 3,1(1931). ( 9 ) RUBEN,S., ETAL.,J . A m . Chern. Sac., 63,877(1941). D. I., ALLEN,M. B., AND WHATLEY, F. R., Nature, (10) ARNON, 174,394 (1954). M . D., in "Research in Photosynthesis," edited by (11) KAMEN, H. GAFFRON, Interscience Publishers, h e . , 1957, p. 149. N. I . , Prm. Nat.Acad. Ski., 45,1696 (1959). (12) BISHOP, L. N. M.. AMESZ. J.. AND KAMP.B. M.. Nature. 1131 , , DUYSENS. 190,5i0(1961). ' (14) CHANCE, B., AND NISHIMWA, M., Pmc. Nat. Acad. Sci., 46, 19~19ROI -( 1 5 ) ARNON, D. I., ETAL., Prac. Nat.Aead. Sci.,47,1314(1961). (16) CAMIN.M., J . l'heowt. B i d , 2,258 (1961). J. A,, AND CALVIN, M., "The Path of Carbon in (17) BASSHAM, Photosvnthesis." Prentiee-Hall. h e . .. Englewood Cliffs. New Jersey, 1957. (181 HILL.R.. Pmc. Rou. Sac. (London1.B 127.192 (19391. i i g j PA&, R:B., AND +OX,N: G., J . M O LB G . , 3 , i ( i ~ t i i ) . (20) WEIER,T . E., Amer. J m r . Bot., 48,615 (1961). (21) RIS, H., AND slNcH, R.N., J . Biophys. Biocha. Cytot., 9 , 63 (1961). P., J . Ult~(L91mtur(: Research, 4,127 (1960). (22) GIBES,SILRAH W., AND HICKMAN, D. D., J. Biophys. Riocha. (23) FREXKEL, Cytol., 6,285 (1959).
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