May 14, 1974 - Park and co-workers (Annu. Rev. ... In 1971 Sane and Park (37) studied a stroma lamella frac- ..... Photosynthetic action spectra of marine algae ...
Plant Physiol. (1974) 54, 386-391
Biosynthesis of Grana and Stroma Lamellae in Spinach1 Received for publication March 6, 1974 and in revised form May 14, 1974
FERNANDO HENRIQUES2 AND RODERIC PARK Department of Botany and Lasswr ence Berkeley Laboratory, University of California, Berkeley, California 94720 ABSTRACT Park and co-workers (Annu. Rev. Plant Physiol. 22: 395430) have suggested that stroma lamellae, which perform only photosystem I contain a developing photosystem II which becomes functional upon the folding of these membranes to form grana stacks. The present study was undertaken to test this hypothesis. The lipid and protein constituents of chloroplast membranes were pulse-labeled radioactively with 14C, and the specific activity of stroma and grana components were studied in a pulse-chase experiment. The components of both grana and stroma lamellae become labeled and decay at similar rates which suggests that the Park and Sane hypothesis is not correct. The results also show large differences in the turnover rates of some chloroplast membrane components supporting the multistep model for membrane synthesis and maintenance in this organelle.
Stroma and grana lamellae of higher plant chloroplasts constitute an elaborate complex of membranes where the conversion of light into chemical energy takes place. These two types of membranes have been separated physically and comparative studies of their structure, gross chemical composition, pattern of development, and enzymatic capabilities have been made (1, 5, 9, 15, 32-34, 36, 44). However, the biogenetic relationships between the two types of membranes, if any, are not established clearly. In 1971 Sane and Park (37) studied a stroma lamella fraction from spinach chloroplasts which contained only PSI3 and found a decline in the quantum yield for NADP photoreduction at wavelengths shorter than 700 nm. This result indicated the presence of a pool of Chl in stroma lamellae which was ineffective in the PSI reaction. Subsequent fluorescence studies of the stroma lamella preparations (30) and reports in the literature of similar observations on PSI fractions obtained by Triton (45) or digitonin (39, 47) treatments, provided evidence that the inactive Chl was not due to damaged PSII caused by the fractionation procedure. Support for the notion that the presence of uncoupled Chl was not a fractionation
1This project was supported by the Atomic Energy Commission and National Science Foundation Grant GB-25579X. 2 On leave from University of Luanda, Angola, Portugal. 'Abbreviations: PSI: photosystem I; PSII: photosystem II; PMS: phenazine methosulphate; DPC: 1, 5-diphenylcarbazide; DCIP: 2, 6-dichlorophenolindophenol; 10K: 10,000g; 160K: 160,000g; kd: kilodalton; SDS: sodium dodecyl sulfate.
artifact also came from the work of Homann and Schmid (18) on intact chloroplasts of the tobacco mutant NC95. Plastids from the yellow regions of the leaves of NC95 contain mainly stroma lamellae and perform primarily the PSI reaction. Homann and Schmid (18) showed, with intact chloroplasts from the yellow regions, a similar decline in quantum yield of PMS cyclic photophosphorylation at short wavelengths. All these results strongly suggest that the presence of photochemically inactive Chl is an inherent characteristic of stroma lamellae, and Sane and Park (30) proposed that this Chl might be related to sites of membrane biogenesis. Further characterization of the stroma lamellae indicated that some components of PSII are present in the unstacked membrane and the use of DPC as electron donor revealed incipient PSII activities in this fraction (29). Interestingly, this electron transport from DPC to DCIP showed characteristics very similar to those observed for PSII in developing bean chloroplasts (2), namely a large insensitivity to DCMU, and all these observations led Sane and Park to generalize their concept and hypothesize that the stroma lamellae contain a developing PSII which is completed upon the folding of these lamellae to form grana stacks (37). Such a scheme, although agreeing with the wealth of data on development of structure and function during the greening process of chloroplasts (10-12, 15, 16, 25, 31, 34, 49) and supporting the view that PSII activities are closely associated with grana appearance, is based only on circumstantial evidence and has received, so far, no direct experimental support. The present study was undertaken to test this hypothesis. Our approach was the following. Two spinach plants were allowed to photosynthesize with "4CO2 for 1 hr; immediately after this period, the stroma and grana lamellae of one plant were separated, and the specific radioactivity of their lipid and protein components was analyzed. The other plant was allowed to grow in the presence of atmospheric CO2 for 7 days more and after that subjected to identical treatment. The 7-day cold chase period was initially selected because the data of Shlyk, as reported by Kutiurin (21), showed that the half-life of Chl in tobacco leaves is 13.2 days. If the hypothesis is correct, one would expect to find a considerable amount of radioactivity in the stroma components immediately after the labeling period compared with the grana region. After 7 days, labeled stroma membranes would become folded into grana and if reformation of stroma membranes from cold precursors occurred, then, an increase in the relative amounts of radioactivity of the grana regions should accompany a decrease in radioactivity of the stroma. Though the results reported here do not support the sequential stroma lamellae -- grana lamellae biogenetic scheme hypothesized by Sane and Park (37), they do show that the protein components of the chloroplast membranes turn over with different rates, and this observation is discussed in relation to the assembly of the thylakoid membranes. 386
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nm using a Gilford gel-scanner accessory on a Beckmann DU spectrophotometer with 0.1 -mm slit. 14CO2 Assimilation. The "CO2 assimilation was conducted Molecular weights were determined from a standard plot in a closed system, described by Bassham and Kirk (6), in- using phosphorylase A (93,000 daltons), BSA (68,000 daltons), corporating an infrared gas analyzer, an ionization chamber ovalbumin (43,000 daltons), chymotrypsinogen (26,000 dalfor "CO2 measurements, and an oxygen analyzer, connected tons), myoglobin (17,000 daltons) and Cyt c (11,700 daltons) with a multipoint recorder. The illumination was provided as standard proteins. by two 250w photoflood lamps operated at 1 10 v and the light Counting Procedures. The radioactivity of the pigments was was passed through filters to prevent excessive heating and in- determined with a Geiger-Muller gas flow counter (Nuclear jury of plants. Chicago, Model 47D), the samples being counted for sufficient Spinach (Spinacia oleracea L.) plants approximately 1 time to accumulate 2500 counts. For measurements of radiomonth old with four fully expanded leaves were used in all activity in proteins, the gels were frozen in solid CO2 and experiments. The plants were allowed to photosynthesize for sliced into 1-mm pieces. The slices were incubated with 0.1 1 hr in the presence of 250 mCi of "CO2 and then either im- ml of 30% H20 for 8 hr at 60 C. After solubilization 12 ml mediately processed (1-hr plant) or grown for 7 days more of aquasol solution were added and the radioactivity was mea(7-day plant) in the presence of atmospheric CO2. Subsequent sured in a Packard Tri-Carb automatic liquid-scintillation treatment was identical in both cases. spectrometer, Model 3375. Isolation of Subchloroplast Fractions. Plants exposed to "CO. were diluted with market-purchased spinach leaves to give a RESULTS AND DISCUSSION final weight of 100 g, necessary for the subsequent fractionation procedure. Subchloroplast fractions were prepared as deLipids. The selective disruption of spinach chloroplasts by scribed by Sane and Park (37). Chloroplasts were suspended in the French pressure cell followed by fractional centrifugation 0.05 M potassium phosphate (pH 7.4)-0.15 M potassium chlo- permits the separation of grana stacks, pelleted at 10,000g, ride, passed through the French pressure cell, and the result- from the unstacked stroma lamellae pelleted at 160,000g. The ing brei was submitted to fractional centrifugation. The heavy biochemical characterization of these fractions showed that grana stacks were pelleted at 10,000g (hereafter called 10K the grana stacks were enriched in PSII and had a lower Chl fraction) and the lighter unappressed stroma lamellae were a/Chl b ratio than intact chloroplasts, whereas the stroma sedimented at 160,000g (160K fraction). The fractions were lamellae, containing only PSI, were characterized by a much washed with EDTA (1 mm, pH 8.0) for 30 min at 0 C. Total higher Chl a/Chl b ratio (36). The values of these ratios for Chl and the ratio of Chl a!Chl b were determined by Arnon's our preparations, obtained by the French pressure technique, method (4). are presented in Table I; the Chl a/Chl b ratio of the initial Extraction and Separation of the Pigments. The pigments chloroplasts was 2.95 for the 1-hr plant and 2.88 for the 7-day were extracted with 80% acetone and separated by two- plant, and the Chl a/Chl b ratios of the fractions fall within dimensional TLC on mannitol plates, according to Smith et the range expected for grana and stroma lamellae. al. (43). In these experiments 2% (v/v) methanol in petroleum Also presented in Table I are Chl a/Chl b ratios obtained ether (b.p. 30-60 C) in the first direction and 5% (v/v) acetone after separation of the different pigment components by TLC in petroleum naphtha (b.p. 60-70 C) in the second direction on mannitol plates. The differences found are minor, indicatwere used. The amounts of individual Chl a and b after separa- ing that an adequate separation of the Chl was obtained in the tion were calculated from the Mackinney coefficients (23) and chromatogram. In all the cases, distinct spots for carotenes, the ratio of Chl a! Chl b was used to assess the degree of resolu- lutein, violoxanthin, and neoxanthin were also visible, the tion obtained in the chromatogram. The recovery of total Chl carotenols being well resolved from the origin. from the plates averaged 96%. The data on 1"C incorporation into the Chl a and b of 10K Preparation and Determination of Protein. The membrane and 160K are reported in Table I. Although the individual protein fraction, recovered by centrifugation after lipid ex- carotenoids were analyzed, the amounts of radioactivity were traction in 80% acetone, was suspended at a concentration of too low for accurate measurement with the exception of /B1 mg of protein/ml in 10 mm sodium phosphate buffer (pH carotene. The carotenes accounted for a major portion of 7.0)-l % SDS-1 % /3-mercaptoethanol and was incubated at radioactivity of the pigment fraction in the 1-hr plant. During 45 C overnight. The suspension then was heated at 100 C the 7 days of chasing with atmospheric C02, the specific radiofor 20 min and dialyzed against 10 mm sodium phosphate activity of the carotenes decreased and was accompanied by an buffer, pH 7.0-0.1 % SDS-0.1 % ,B-mercaptoethanol for 6 hr. The amount of protein was estimated using the method of Table 1. Specific Radioactivity. of Cilorophylls a anidb after a 1-Hr Lowry et al. (22) with BSA suspended in the same buffer as a Labelinig Period anid after a 7-Day, Chase wit/i A tinospheric CO2 standard. Similar data were obtained from five additional experiments. Electrophoresis Technique. Acrylamide gel electrophoresis, in the presence of SDS, was carried out in cylindrical tubes Specific Radioactivity of b Ratio Chl a 'Chl b Ratio Chlorophylls (10 x 0.6 cm) containing 7% acrylamide and 0.23% methChl a'chl Fraction (af ter TLC) (pellet) ylene-bisacrylamide (24). The electrophoretic buffer consisted Chla Chlb of 0.05 M sodium phosphate (pH 7.0)-0.1% SDS-0.1% /Bmercaptoethanol (48). Approximately 70 jg of protein were cplig layered on the top of the gel and subjected to electrophoresis 1-Hr plant at 8 mamp/tube until the tracking dye (pyronine y) was about 2.37 403 2.52 104 1OK 1 cm from the end of the tube. Electrophoresis was completed 4.98 418 160K 119 5.16 in 5 to 6 hr. The gels were fixed and stained with 0.25% 7-Day plant Coomassie brilliant blue R in 7.5% acetic acid-50% methanol 2.53 287 2.48 154 IOK (48); the destaining was performed by several changes in Weber 5.51 305 160K 5.32 160 and Osborn solution. The destained gels were scanned at 560
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increase of that of the xanthophylls. These findings, previously reported by other workers for several different organisms, have been used to suggest that /3-carotene can act as a precursor for the more oxygenated forms of carotenoids (8, 19). Our observations are consistent with those of Roux and Husson (35) who found that /3-carotene has a much faster turnover rate than any other of the chloroplast pigments. The data presented in Table I for specific radioactivity of Chl a and Chl b agree quite well with published values (40). The higher specific radioactivity of Chl a compared with Chl b in the short term experiment and the decrease of this ratio during the 7 days support the generally accepted view that Chl b arises from Chl a, as first proposed by Shlyk et al. (40, 41). The 7-day cold chase in our experiments led to a 30% decline in specific radioactivity of Chl a, indicating a Chl turnover time not greatly different from that reported for tobacco (21). An important observation for our purpose is that the distribution of '4C into the Chl of grana and stroma is very similar, immediately after the labeling period. These results argue against the hypothesis of the stroma membrane as being more actively implicated in the biosynthesis of the Chl. The observation that in the 7-day plant the ratios specific radioactivity Chl alOK specific radioactivity Chl a6-K
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and specific radioactivity Chl bloK specific radioactivity Chl b1s,,1
remain constant, supports the "1-hr" data and definitely rules out the hypothesis of preferential localization of chlorophyll biosynthesis centers in the stroma lamellae with later migration of the new pigment molecules to the grana region by stacking of the single membranes. Contrarily, it suggests that both stroma lamellae and grana stacks have their own centers for the formation of pigments and these incorporate '4C at about equal rates. These results are in apparent disagreement with the extensive work done by Shlyk and co-workers (42) on centers of Chi biosynthesis. These authors repeatedly reported the existence of "light particles" in chloroplasts where Chl synthesis preferentially takes place. However, by reasons of differences in the procedures followed and by an absence of a correspondence between the light particle and its counterpart in intact chloroplasts, it is not easy to compare their results with ours, and we will not attempt it here. The results of Goldberg and Ohad (14) with the y-l mutant of the green algae Chlamydomnonas reinhardi are of comparative interest. These authors investigated the distribution of labeled lipids between the stacked and unpaired photosynthetic membranes of this organism and found that the radioactive pattern obtained after a short pulse with acetate-H3 is not changed by a chase period with cold acetate, suggesting that no migration of membrane material occurs in the mutant chloroplast, in agreement with our conclusions. The observation, also made by the authors, that unpaired membranes incorporate acetate-H; twice as fast as the stacked membranes, apparently in contradiction with our results, may depend on the fact that the paired membranes of Chlamnydomonas are relatively isolated from the chloroplast matrix. Peptides. The proteins of the photosynthetic membranes, solubilized by SDS as described under "Material and Methods," show a complex electrophoretic pattern discussed in detail elsewhere (26). Comparison of their electrophoretic mobilities with those of known proteins gives the mol wt estimates in kilodaltons shown in Figures 1 and 2; no mol wt are assigned to peaks below 18 kd since the plot relating mol wt with relative mobilities is not linear at these low values. A comparison of the densitometric tracings of the 10K and 160K fractions shows a great similarity in the polypeptide composition of both fractions; however, minor qualitative differences can be seen. Prominent differences are the absence of the 36 kd peak in the 10K fraction and the presence of a 39 kd peak in this fraction which is lacking in the 160K. The peak with mol wt 54 kd in the stroma-membrane preparation represents two unresolved peaks with 56 kd and 54 kd (shown by the use of im-
proved electrophoretic technique). Quantitatively, the stroma and grana lamellae show im-
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portant differences. The 23 kd peak, representing the major polypeptide component of the 10K fraction and accounting for approximately 30% of the total membrane protein in the grana, is much smaller in the 160K fraction where the two high mol wt components, 60 kd and 54 kd, clearly predominate. The 160K fraction is comparatively enriched in the 19 kd and 21 kd components, whereas the 10K fraction, on the other hand, contains more of the 30 kd peak. Some of these peaks have been tentatively identified with known components of the photosynthetic membranes (26, 46). However, no reliable identification has been presented so far with possible exception for the peaks 60 kd and 56 kd, presumed to be two subunits of the coupling factor. The pattern of '4C incorporation immediately following the
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titative relationship is difficult because the amounts of different polypeptides present in the fractions are not easily measured, the specific activities of the different polypeptides in both fractions are similar after this short time of "4C incorporation. For example, in the 30 kd component of the 10K fraction, the absorbance is about 1 and the radioactivity is about 1500. In the 160K fraction, this same component has an absorbance of 0.6 and a radioactivity of about 800. The 7-day plant shows a parallel decrease in radioactivity in both fractions (Fig. 3 and 4), contrary to what would be expected if a precursor-product relationship existed between the stroma and grana lamellae. These data would appear to rule out the initial hypothesis that stroma contains some incomplete PSII components that are assembled into a functional PSII unit upon stacking of the single stroma lamellae. A comparison of Figure 1 with Figure 3, and Figure 2 with Figure 4, indicates a heterogeneity in the rate of loss of radioactivity among the different membrane proteins. For example, the 30 kd component not only becomes labeled more rapidly, but decays much faster than other components whereas the 23 kd and the two high mol wt components, 60 kd and 56 kd, have slow decays. However, one has to be cautious in the interpretation of decay kinetics because of possible reutilization of some labeled material. We interpret these data as indicating a heterogeneity in the turnover rates of the different membrane polypeptides and this observation has a bearing on the concepts
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assimilation period into the protein of stroma and grana lamellae is also shown in Figures and 2. The radioactivity measurements, in a qualitative way, closely match the densitometric tracings of the membrane polypeptides. It is interesting to note that the radioactivity measurements appear to resolve the smooth peak in the lowest portion of the gel into a number of different peaks. This indication of several components in this region of the gel was subsequently demonstrated by increasing the acrylamide concentration to 10% which resolved distinct peaks. Though there is a qualitative match between radioactivity and densitometric tracings in Figures and 2, the amount of radioactivity does not parallel the density. In the 10K fraction, although the 23 kd polypeptide is the most radioactive peak, it is clear that the 30 kd and 25 kd peaks are relatively more labeled. Also, in the 160K fraction, more radioactivity is proportionally incorporated in the 30 kd and 24 kd peaks than in the 60 kd and 54 kd components. In both membrane fractions the two high mol wt components show a slow rate of '4C incorporation suggesting a lower turnover of these components. The Sane and Park hypothesis suggested that components of stroma lamellae might become labeled more rapidly than those of grana lamellae; the data (Fig. 1 and 2) fail to show any preferential incorporation of the label into the stroma lamellae polypeptides. Although the establishment of a quan-
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PSII which becomes completed upon the folding of these membranes to form grana stacks. The lipid and protein components of stroma and grana have been labeled with '4C and the specific radioactivity of these components was analyzed immediately following the labeling period and subsequent to a 7-day chase with atmospheric CO2. According to the hypothesis, it was anticipated that a preferential incorporation of label into the stroma components compared with grana should occur in the short term experiment, and an inversion of this situation would be found 7 days later as a result of stacking of labeled stroma lamellae to form the grana and reformation of the former from cold precursors. The data reported in this paper are not consistent with that biogenetic model for chloroplast membranes and favor the view that stroma and grana membranes have distinctive centers of biosynthesis, working independently and apparently at similar rates. We are, therefore, left with the intriguing problem of the presence of a large pool of photochemically inactive Chl and protein components in the stroma lamellae which apparently are not transformed into PSII functional elements. More work is necessary to clarify the significance of this "inactive Chl pool." When this problem is explored, it should not be forgotten that previous references to inactive Chl (17) were merely an expression of our ignorance of its true function. Our results are also of interest regarding the proposed models for thylakoid assembly. A stepwise assembly has been generally accepted, based mainly on the time-course appearance of the different photochemical activities. The heterogeneity found in our work on the turnover rates of the chloroplast membrane polypeptides favors this multistep model and reinforces the concept of the membrane as a very dynamic structure in continuous exchange with the medium through breakdown and resynthesis of its components.
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of thylakoid assembly. Kirk (20) has recently presented a critical review on the process of thylakoid assembly. Based on the observation of a time-course acquisition of the different photosynthetic activities, it seems that "there is a stepwise assembly process, by which first a membrane is built up containing a certain minimum number of components, with other constituents being added later.. ." (20). Indeed, extensive work done during the last few years on chloroplast development has provided much evidence for a gradual acquisition of the different photochemical capabilities throughout the greening process of this organelle. Such evidence favors a stepwise model for thylakoid membranes assembly of several higher plant chloroplasts and is supported by our observations of a marked asynchrony in the turnover rates of the different spinach chloroplast membrane polypeptides. A stepwise membrane-assembly model has also been postulated for the photosynthetic lamellae of the alga Chlamydomonas (13, 27, 38) and other membrane systems of eucaryotic cells, namely mitochondria (7) and endoplasmic reticulum (3, 28). CONCLUSION In this study we attempted to test the hypothesis proposed by Sane and Park that stroma lamellae contain a developing
ALLEN-, C. F., P. GOOD, T. TROSPER, AND R. B. PARK. 1972. Chlorophyll, glycerolipid and protein ratios in spinach chloroplast grana and stroma lamellae. Biochem. Biophys. Res. Commoin. 48: 907-913. 2. ANDERSON, J. MI. AND N. K. BOARDMAN. 1964. Studies on the greening of darkI,grown bean plants. II. Dev elopment of photochemical activity. .Aust. J. 1.
Biol. Sci. 17: 93-101. 3. ARIAS, I., D. DOYLE, AND R. SCHIMKE. 1969. Studies on the synthesis and degradation of proteins of the endoplasmic reticulum of rat liver. J. Biol. Chem. 244: 3303-3315. 4. ARNON, D. I. 1949. Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24: 1-13. 5. ARNTZEN, C. J., R. A. DILLEY, AND F. L. CRANE. 1969. A comparison of chloroplast membrane surfaces visualized by freeze-etch and negative staining techniques, and ultrastructural characterization of membrane fractions obtained from digitonin-treated spinach chloroplasts. J. Cell Biol. 43: 16-31. 6. BASSHANI, J. A. AND Al. KIRK. 1966. Rapid inhibitor-induced changes in the 14C-labeled compounds formed by the photosynthetic carbon-reduction cycle. Advan. Tracer Methodol. 3: 211-221. 7. BEATTIE, D., R. E. BASFOIRD, AN-D S. KORITZ. 1967. The tturnov-er of protein components of mitochondria fr-om rat liver, kidney ancl brain. J. Biol. Chem. 242: 4584-4586. 8. BLASS, V., J. NI. ANDERSON, AND M1. CALVIN. 1939. Biosyntlhesis and possible functional relationships among the carotenoids, and between chlorophyll a and chlorophyll b. Plant Physiol. 34: 329-333. 9. BOARDMAN, N. K. AND J. M. ANDERSON-. 1964. Studies on the greening of dark-grown bean plants. I. Formation of chloroplasts from proplastids. Aust. J. Biol. Sci. 17: 86-92. 10. BOURDU, R., Y. MATHIEIJ, M. MIGINIAC-MASLOW, R. REMY, AND A. MOYSE. 1968. Structure granaire reduction du NADP et photophosphorylation des
chloroplastes isol6sfeuilles d'orge. Planta 80: 191-210. 11. DOWNTON, W. J., J. A. BERRYAND, AND E. B. TREGUNNA. 1970. C4-photosynthesis: noncyclic electron flow and grana development on bundle-sheath chloroplasts. Z. Pflanzenphysiol. 63: 194-198. 12. DOWNTON, W. J. AND N. A. PYLIOTIS. 1971. Loss of photosystem II (luring ontogeny of sorghum-bundle sheath chloroplasts. Can. J. Bot. 49: 179-180. 13. EYTAN, G. AN-D I. OHAD. 1972. Biogenesis of chloroplast membranes. VII. The preservation of membrane homogeneity during de-elopment of the photo-
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synthetic lamellar system in an algal mutant (Chlamydomonas reinhardi
y-1). J. Biol. Chem. 247: 112-121. 14. GOLDBERG, I. AND I. OHAD. 1970. Biogenesis of chloroplast membranes. V. A radioautographic study of membrane growth in a mutant of Chlamydomonas reinhardi y-1. J. Cell Biol. 44: 572-591.
33.
15. GOODCHILD, D. J. AND R. B. PARE. 1971. Further evidence for stroma lamellae
34.
as a source of photosystem I fractions from spinach chloroplasts. Biochim. Biophys. Acta 226: 393-399.
35.
15. GYLDEN-HOLM, A. 0. AND F. R. WHATLEY. 1968. The onset of photophosphoryla17. 18. 19.
20.
21.
22. 23. 24.
25. 26. 27.
28. 29.
30.
31.
32.
tion in chloroplasts isolated from developing bean leaves. New Phytol. 67: 461-468. HANO, F. T. AND L. R. BLI-NKS. 1950. Photosynthetic action spectra of marine algae. J. Gen. Physiol. 33: 389-422. HO.MAN-, P. H. AND G. H. SCHMID. 1967. Photosynthetic reactions of chloroplasts with unusual characteristics. Plant Physiol. 42: 16191632. JENSENT, S. L., G. COHEN-BAZIRE, T. 0. NAKAYAMA. AN-D R. Y. STANIER. 1958. The path of carotenoid synthesis in a photosynthetic bacterium. Biochim. Biophys. Acta 29: 477-498. KIRK, J. T. 0. 1971. Chloroplast structure an(d bigenesis. Annu. Rev. Biochem. 40: 161-196. KUTIURIN, V. Ml. 1956. Chlorophyll turnover in plants. Dokl. Akad. Nauk SSSR 106: 355-357. LOWRY, 0. H., N. J. RosEaRoUGH, S. L. FARR, A'ND R. J. RANDALL. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275. MACKINNEY, G. 1941. Absorption of light by chlorophyll solutions. J. Biol. Chem. 140: 315-322. !MCDONAGH, J., H. MESSEL, R. P. NICDON-AGH, JR., G. MURANO, AND B. BLOMBACK. 1972. 'Molecular weight analysis of fibrinogen and fibrin chains by an improved sodium dodecyl sulfate gel electrophoresis method. Biochim. Biophys. Acta 257: 135-142. MILLER, 'M. AND P. S. NOBEL. 1972. Liglit-indluced changes in the ultrastructure of pea chloroplasts in vivo. Relationship to development and photosynthesis. Plant Physiol. 49: 535-541. ,NOLA-N, WILLIAM. 1973. Comparative studies on chloroplast grana and stroma lamellae. Ph.D. thesis. University of California, Berkeley. OHAD, I., P. SIEKEVITZ, AND G. E. PALADE. 1967. Biogenesis of chloroplast membranes. I. Plastid differentiation in a dark-grown algal mutant (Chlamydomnonas reinhardi). J. Cell Biol. 35: 521-552. OIMURA, T., P. SIEKEVITZ, AND G. E. PALADE. 1967. Turnover of constituents of the endoplasmic reticulum membranes of rat hepatocytes. J. Biol. Chem. 242: 2389-2396. PARK, R. B. AND R. V. SANE. 1971. Distribution of function and structure in chloroplast lamellae. Annu. Rev. Plant Physiol. 22: 395-430. PARK, R. B., K. E. STEINBACK, AND P. V. SANE. 1971. Distribution of variable fluorescence among subchloroplast fractions. Biochim. Biophys. Acta 253: 204-207. PHUNG-NHU-HLNG, S., A. HOARAU, AND A. MOYSE. 1970. Etude de l'evolution en chloroplastes des plastes etioles d'orge. II. Photophosphorylation et photor6duction du NADP, formation de ferr6doxin, en eclairement continu et par l'action d'eclairs. Z. Pflanzenphysiol. 62: 245-258. PHUNG-NHTJ-HUNG, S., A. LACOURLY, AND C. SARDA. 1970. Etude de 1'evolu-
36.
37. 38.
39. 40.
391
tion en chloroplastes des plastes etioles d'orge. I. Structure examin6e par cryodecopage. Z. Pflanzenphysiol. 62: 1-16. REMY, R. 1969. Etude de la structure des lamelles chloroplastiques de vicia faba L. par la technique du cryod6capage. Compt. Rend. Acad. Sci. Paris 268: 3057-3060. RHODES, M. J. AND E. W. YEMM. 1966. The development of chloroplasts and photosynthetic activities in young barley leaves. New Phytol. 65: 331-342. RoCx, E. AND C. HussoN. 1952. Pigments des chloroplastes et photosynth6se. Compt. Rend. Acad. Sci. Paris 235: 1154-1155. SANE, P. V., D. J. GOODCHILD, AND R. B. PARK. 1970. Characterization of chloroplast photosystems 1 and 2 separated by a nondetergent method. Biochim. Biophys. Acta 216: 162-178. SANE, P. V. AND R. B. PARK. 1971. Action spectra of photosystem I and photosystem II in spinach chloroplast grana and stroma lamellae. Biochim. Biophys. Acta 253: 208-212. SCHULDINER, S. AND I. OHAD. 1969. Biogenesis of chloroplast membranes. III. Light-dependent induction of proton-pump activity in whole cells and its correlation to cytochrome f photo-oxidation during greening of Chlamydomonas reinhardi mutant (y-1). Biochim. Biophys. Acta 180: 165-177. SCHWARTZ, M. 1967. Quantum yield of ferrocytochrome c photo-oxidation in chloroplast particles. Nature 213: 1187-1188. SHLYK, A. A. 1971. Biosynthesis of chlorophyll b. Annu. Rev. Plant Physiol.
22: 169-184. 41. SHLYE, A. A., T. N. GODNEv, R. M. ROTFARB, AND YA. P. LYAKHOVICH. 1957. The peculiarities of the two chlorophyll components of biosynthesis in the process of rejuvenation. Proc. Acad. Sci. USSR. Biochemistry Section (English translation). 113: 103-106. 42. SHLYK, A. A., I. V. PRD-ONIKOVA, L. I. FRADKIN, G. N. NIKOLAYEVA, AND G. E. SAVCHENKO. 1969. Relationship between chlorophyll metabolism and heterogeneity of pigment apparatus. In: Progress in Photosynthesis Research. V'ol. II. pp. 572-591. 43. SMITH, L. W., R. W. BREIDENBACH, AND D. RUBENSTEIN. 1965. Thin-layer chromatography of plant pigments on mannitol or sucrose. Science 148: 508-509. 44. TROSPER, T. AND C. ALLEN. 1973. Carotenoid composition of spinach chloroplast grana and stroma lamellae. Plant Physiol. 51: 584-585. 45. VERNON, LEO, B. KE, AND E. R. SHAW. 1967. Relationship of PIoe, electronspin resonance signal, and photochemical activity of a small chloroplast particle obtained by the action of Triton X-100. Biochemistry 6: 2210-2220. 46. VERNON, LEO AND S. KLEIN. 1974. Protein composition of spinach chloroplasts and their photosystem I and photosystem II subfragments. Photochem. Photobiol. 19: 43-49. 47. VREDENBERG, W. J. AND L. SLOOTEN. 1967. Chlorophyll a fluorescence and photochemical activities of chloroplast fragments. Biochim. Biophys. Acta 143: 583-594. 48. WEBER, K. AND M. OSBORN. 1969. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244: 4406-4412. 49. Woo, K. C., J. M. ANDERSON, N. K. BOARDOMAN, W. J. DOWNTON, AND C. B. OSMOND. 1970. Deficient photosystem II in agranal bundle-sheath chloroplasts of C-4 plants. Proc. Nat. Acad. Sci. U.S.A. 67: 18-25.
