Plant Molecular Biology 16: 71-79, 1991. © 1991 Kluwer Academic Publishers. Printed in Belgium.
71
Chloroplasts of Arabidopsis thaliana homozygous for the ch-1 locus lack chlorophyll b, lack stable LHCPII and have stacked thylakoids David L. Murray and Bruce D. Kohorn*
Botany Department, Duke University, Durham, NC 27706, USA (*author for correspondence) Received 11 July 1990; accepted in revised form 13 September 1990
Key words: LHCPII, chlorina, Arabidopsis
Abstract
We are interested in the mechanism of insertion of proteins into the chloroplast thylakoid membrane and the role that accessory pigments may play in this process. For this reason we have begun a molecular analysis of mutant plants deficient in pigments that associate with thylakoid membrane proteins. We have characterized plants that are homozygous for the previously isolated, recessive mutation chlorina-1 (ch-1) ofArabidopsis thaliana. Despite the lack of chlorophyll b and light-harvesting proteins of photosystem II (LHCPII) near normal levels of L H C P I I m R N A are found in the mutant, in contrast to L H C P I I m R N A levels in carotenoid-deficient mutants. The L H C P I I m R N A of chlorina-1 plants can be translated in vitro so it is likely that L H C P I I is not stable in ch- 1 plants. Moreover, the thylakoid membranes of ch- 1 plants remain appressed even though L H C P I I levels are drastically reduced.
Introduction
The means by which integral membrane proteins are inserted into lipid bilayers from an aqueous environment is poorly understood [37, 39]. It is clear, however, that not all proteins integrate into membranes co-translationally [37] and that specific regions of polypeptides mediate the integration process [39]. Recently a number of proteins have been implicated in aiding post-translational insertions [24]. To understand these processes we are studying the insertion, of the major light-harvesting chlorophyll a/b binding protein (LHCPII) into the thylakoid membrane. L H C P I I is synthesized as a precursor in the cytoplasm, is post-translationally imported into the chloroplast and then inserted into the thylakoid
membrane as a processed protein [22]. LHCPII ultimately becomes a member of the oligomeric chlorophyll-protein complex LHCII that channels light energy into photosystem II (PS II [35]). It has been suggested previously that chlorophyll b is required for the stability of L H C P I I in the thylakoid membrane [4], but whether chlorophyll b binding is required before, during, or after insertion of the protein into the membrane remains unclear. Both the site and pathway of chlorophyll b synthesis have yet to be discovered [6], and the existing nuclear mutations that affect chlorophyll b synthesis have been characterized in plants that have in the past not facilitated a molecular genetic analysis. We have begun a study of chlorophyll-deficient mutants of Arabidopsis in the hope that a biochemical characterization of
72 their chloroplasts will lead to a better understanding of chlorophyll b synthesis, and its role in L H C P integration. Here we describe a characterization of Arabidopsis that carries the recessive nuclear mutation chlorina-1 (ch-1). Chlorina-1 plants were originally isolated on the basis of their yellow green color and slow growth [13], and plants homozygous for ch-1 were shown to have reduced amounts of both chlorophyll b and stable L H C I I [ 13, 27, 31 ]. We are unable to detect chlorophyll b and show that of all the chlorophyll-protein complexes of the thylakoid, L H C I I is the most dramatically affected. In addition, stable L H C P I I is rare while L H C P I I m R N A is present at near normal levels in mutant plants. This m R N A can be translated in vitro suggesting that L H C P I I appears to be unstable in the absence of chlorophyll b. Moreover, this report shows that chloroplasts of ch-1 plants have appressed thylakoids despite the large deficiency in LHCPII.
Materials and methods
Arabidopsis thaliana Columbia, and theA. thaliana Columbia homozygous for ch-1 (obtained from Prof. Kranz, F R G ) were propagated on soil under 300 #mol photons m - 2s - 1 of light for four generations before analysis. Chlorophyll extractions and estimations were performed as described [26]. Extraction of thylakoid membranes from 4-week old plants, electrophoresis of denatured [ 16] and non-denatured proteins [14, 25], fluorography [17], and isolation [ 18] and analysis [34] of R N A were also carried out by published procedures. In vitro translations [ 14] and immuno-precipitations were performed as described using polyclonal sera prepared against Lemna gibba L H C P I I proteins [36]. Whole plants were radioactively labelled by first excising leaves under water, and then placing the stem in 1 ml of 40 mM H E P E S (pH 7.5) for 20 minutes. Radiolabelled 35S methionine (300/~Ci, Amersham) was added to each incuba-
1.2-
0-6°
"-~ 0
0
0
0
Fig. 1. Room temperature absorption spectra of acetonesoluble pigments from wild-type and ch-1 leaves. Pigments were extracted [26] from 0.5 g of fresh wild-type ( + ) and mutant (ch-1) leaves and the absorption spectra of equal amounts of extract are shown on the same relative scale. Horizontal scale shows wavelength in nanometers, vertical scale depicts relative absorption. tion, and the incorporation was terminated by the isolation of the thylakoids. Samples were prepared for electron microscopy by dissecting leaves directly into 5 ~o glutaraldehyde in phosphate-buffered saline (PBS), and shaken overnight at 4 °C. Leaves were then rinsed three times in PBS, placed in 2~o osmium tetroxide overnight, and then rinsed in water before a one-hour incubation in 1 ~o uranyl acetate. Samples were then dehydrated through an ethanol series, washed with propylene oxide, infiltrated with increasing proportions of Polybed resin in propylene oxide, and finally embedded in Polybed resin. Sections were cut on a Reichert-Jung ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a Zeiss EM 10 A electron microscope.
Results
Chlorophyll content of ch-1 plants is reduced Plants homozygous for the X-ray-induced ch-1 mutation [ 13] appear yellow-green under both
73
Fig. 2. Pigment-protein complexes of normal and ch-1 thylakoids. Isolated thylakoids were run in a non-denaturing gel that displays visible amounts of chlorophyll [14, 25] and that separates chlorophyll-protein complexes. + : wild type, ch-1: mutant thylakoids. Each lane contains 10/~g of chlorophyll. From top to bottom: photosystem I; core complex I; core complex II; light-harvesting complex Ilfl; core complex II~fl; light-harvesting complex II0tflz.
high and low light intensities and grow and senesce more slowly than wild type. Previous studies using thin-layer chromatography have failed to detect chlorophyll b, and we confirmed using a spectroscopic analysis that chlorophyll b levels are reduced [26]. Figure 1 shows the fluorescence scan of total extracted pigments. While wild-type plants have a calculated total chlorophyll content of 1.84 ktmol/g of fresh weight, ch-1 homozygotes have only 0.4 #mol/g. The calculated chlorophyll a/b ratio is 3.27 and 44.57 for wild-type and ch-1 plants, respectively. The absorption of other pigments make it difficult to determine whether chlorophyll b is totally absent, yet it is clear that it is significantly reduced in mutant plants.
Chlorophyll-protein complexes are altered in ch-1
Previous findings show that ch-1 plants lack the chlorophyll-protein complex LHCII [ 13, 27]. We extend these results by analyzing thylakoid membranes in non-denaturing gels that maintain chlorophyl-protein complexes and produce little or no free chlorophyll [25]. We found that of all
Fig. 3. Protein composition of mutant and wild-type thylakoids. The entire lanes from the gel lanes shown in Fig. 2 (run in the direction of the horizontal arrows) were separated from each other and were placed on their side and subjected to electrophoresis in a denaturing gel in the direction of the vertical arrows. Lane a: thylakoids from ch-1 plants; lane d: normal thylakoids. Mutant (lane b) and normal (lane c) thylakoids were also fully denatured and run into the same denaturing gel, which was then stained with Coomassie blue. Also shown on the right are the sizes of molecular weight markers x 10 -3, Arrow and box indicate position of LHCP II. < < , PSI. < , CF1 ATPase. *, PSII polypeptides.
the thylakoid membrane proteins detected with Coomassie blue on denaturing polyacrylamide gels, only L H C P I I could not be detected in mutant plants. Bands corresponding to the core complexI and II (CCI and II in Fig. 2) are increased in abundance and this may reflect either the increased (or altered) accessibility of detergent to the thylakoid membrane in the absence of LHCII, or an increase in absolute protein levels. To determine the protein content of the various green bands the first-dimension gel lanes were separated from each other, and placed on their sides and run into a completely denaturing gel ([16], Fig. 3 lanes a and d). Thylakoids from
74
Fig. 4. Detection of LHCPII with antisera or 35S methionine. 35S labelled thylakoid membranes from wild-type ( + ) and mutant
(ch-1) plants were isolated, run in a denaturing gel, and transferred to nitrocellulose. Panel A: filter probed with anti-LHCPII antibody, and visualized as described [36]. Panel B: same filter subjected to autoradiography. Arrows point to position of LHCPII. Panel C: Total RNA from wild-type ( + ) or mutant (ch-1) plants was translated in vitroin the presence of 35S methionine, and the products were immunoprecipitated with anti-LHCPII antibody, run in a denaturing gel, and subjected to autoradiography. Numbers indicate Mr x 10 3.
mutant and wild-type plants were also denatured and run into this gel to serve as a reference (Fig. 3, lanes b and c respectively). Figure 3 shows such a gel stained with Coomassie blue, and d e m o n strates that the major polypeptides and thus complexes o f the wild-type thylakoids are present in ch-1 plants, with the exception o f L H C P I I (arrow and square). We do notice that other thylakoid polypeptides associated with L H C I I vary in abundance, especially those o f P S I I (*), but those o f P S I ( < < ) and CF1 A T P a s e ( < ) do not. Thus ch-1 thylakoids differ most dramatically from those o f the wild type in their lack o f chlorophyll b and L H C P I I polypeptides.
L H C P I I levels are reduced
Coomassie blue stain failed to detect L H P C I I in ch-1 thylakoids and so we used more sensitive methods o f detection: autoradiography o f in vivo labelled proteins, and immunoblotting. Wild-type and ch-1 plants were incubated with 35S methionine for three hours to label newly synthesized proteins, and the thylakoids were isolated, run into a denaturing gel, and transferred to nitrocellulose [34]. The nitrocellulose was then challenged and stained with a polyclonal serum that recognizes all L H C P I I variants [36], or subjected to autoradiography. Figure 4A shows the alkaline
75 phosphatase stain of the antibody-treated nitrocellulose and Fig. 4B is an autoradiograph of the same filter. In wild-type thylakoids abundant LHCPII can be detected with antibodies (Fig. 4A, lane + ) but only a faint signal heterogeneous in size is detected in thylakoids from ch-1 plants (lane ch-1). We have not determined which variant of LHCP is represented and we have been unable on numerous western blots with ch-1 thylakoids to detect stronger signals or bands that are more discrete. The autoradiogram of the labelled thylakoids detects a 28 kDa radioactive band in wild-type thylakoids (Fig. 4B, lane + ) but only a faint band is seen in thylakoids from ch- 1 plants. We also note that there are differences in the intensities between the labelled polypeptides for wild-type and mutant thylakoids, but these have not been investigated further.
Ch-1 L H C P I I m R N A can be translated
We next asked whether the absence of LHPCII was the consequence of a reduction in the levels of translatable LHCPII m R N A or to an increase
Fig. 5. mRNAs levels for LHCPII, nitrate reductase, and the large subunit of Rubisco (LSU). 2.5 #g of total RNA from wild-type (lanes a, c, e) and mutant (b, d, f) plants were run in 1.2% denaturing agarose gel, transferred to nitrocellulose, probed with 32p labelled coding sequences for LHCP (lanes a, b), nitrate reductase (lanes c, d), and LSU (lanes e, f), and subjected to autoradiography. Numbers on the right indicate size in kilobases.
in the rate of protein turnover. To address the former possibility equal amounts of mutant and wild-type total RNA were translated in the presence of 35S methionine in a wheat germ extract, and the products were immuno-precipitated with LHPCII antibody and analyzed by denaturing gel electrophoresis [ 16]. The results of this analysis are shown in Fig. 4C. Anti-LHCPII precipitates a 32 kDa radioactive protein from total translation products of both wild-type and mutant samples (Fig. 4C) and this protein corresponds in size to the precursor of LHCPII. Pre-immune serum does not precipitate any radioactive protein (data not shown). Thus LHPCII m R N A in ch-1 plants can be translated in vitro. We are therefore left with the second explanation that there is an increased turnover rate of LHCPII in ch- 1 plants. While equivalent amounts of LHCPII can be synthesized in vitro from isolated RNA, it was still unclear if the m R N A populations were affected. Therefore, equal amounts of total RNA from wild-type and ch-1 plants were run into a denaturing agarose gel, and the RNA was transferred to nitrocellulose [33]. Figure5 shows autoradiograms of these RNAs that have been probed with LHCPII coding sequence [8, 18]. We conclude that LHCPII m R N A levels in mutant tissue (lane b) are in fact increased relative to those of the wild type (lane a). (The larger size of the ch-1 m R N A is also reflected in the ethidium bromide stained ribosomal RNA and is a reproducible effect of the total RNA preparation, perhaps due to the different starch content of the mutant and wild-type plants.) We also probed these northern blots with another nuclear coding sequence nitrate reductase [7], and a chloroplast sequence the large subunit of ribulose 1,5-bisphosphate carboxylase (LSU [40]), to determine whether other cytoplasmic mRNAs and chloroplast mRNAs (respectively) were affected. Figure 5 also shows that in comparison to LHCP m R N A the chloroplast LSU m R N A level is not affected by the ch-1 mutation (lane e and f), and that cytoplasmic nitrate reductase mRNAs may be slightly reduced in abundance in ch-I plants (lane c and d), although accurate quantitation has not been carried out.
76
Fig. 6. Ultra-structure of normal and ch- 1 chloroplasts. Thin sections of wild-type (panels A and C) and mutant (panels B and D) leafs from tissue of equal age and light exposure. Bar represents 0.5 #m in panels A and B, and 0.1/~m in panels C and D.
Ch-1 thylakoids remain stacked L H C P I I harvests light energy for PSII, but there are indications that the complex also plays a structural role in maintaining the stacked arrangement of thylakoids [2, 32]. Since the L H C P I I levels are drastically lowered in ch-1 plants, we were interested to see if thylakoids from ch-1 plants had reduced amounts of stacked
thylakoids. Figure 6 shows representative electron micrographs of sectioned leaves from wildtype and ch-1 plants. The chloroplasts of the mutant are as numerous but generally more rounded and lighter in appearance than those of the wild type (panels B, D and A, C, respectively). It is not clear whether the lighter appearance is related to the lack of pigments and protein. Although the light appearance creates the impres-
77 sion that there is less stacking of mutant thylakoids, examination of multiple sections shows that thylakoids of ch-1 plants show significant stacking and the degree to which the thylakoids are stacked in the mutant is not directly related to the deficiency in LHCPII. Discussion
The absence of LHCPII in Arabidopsis homozygous for the recessive ch-1 mutation is not due to a lack of translatable LHCPII mRNA. Using antibodies and in vivo labelling LHCPII can only be detected at very reduced levels, yet in vitro translations and immuno-blots show the presence of abundant LHCPII mRNA. These results are consistent with those found for mutations that affect chlorophyll b synthesis in a number of other plant species [ 31 ]. LHCPII m R N A levels are not reduced in the absence of chlorophyll b and they in fact exhibit a slight increase in abundance. This increase is noticed in multiple RNA preparations and is limited to only certain messages as LSU m R N A within the chloroplast is not affected and the cytoplasmic m R N A for nitrate reductase may be slightly decreased in abundance. In vitro translation and immuno-precipitation experiments indicate that the LHCPII m R N A of ch-1 plants can be translated, leading to the conclusion that LHCPII is not stable in the absence of chlorophyll b. It is still possible that LHCPII m R N A can not be translated in ch-1 cytoplasm, but this would necessitate a cytoplasmic translation apparatus that is sensitive to pigment levels within the chloroplast. It is also unclear whether the increased LHCPII message level reflects an attempt to compensate for the increased turnover rate of the protein. The lack of an effect of a chlorophyll b reduction on nuclear m R N A levels is in strong contrast to that reported for carotenoid-deficient maize seedlings, where LHCP m R N A levels are greatly reduced [20]. This difference is logical if the differentiated state of the chloroplast and not the pigment levels themselves are tightly coupled to the nuclear control mechanisms.
Of additional significance is the condition of the thylakoid membranes in ch-1 plants. It has been suggested that LHCPII mediates thylakoid stacking as protease digestion of thylakoid membranes both removes the amino-A terminus of LHCPII, and unstacks the thylakoids [2, 32]. However, LHCPII levels are drastically reduced in ch-1 plants and the thylakoid membranes are still stacked. As many other thylakoid proteins are affected by protease treatment [2, 32] our results suggest that these proteins, and not LHCPII, may be more important in the stacking of thylakoids. The chlorina f2 mutant of barley also has reduced chlorophyll b [4], yet still has stacked membranes [3, 10]. However, stable LHCP varients are detected at levels sufficient to account for thylakoid stacking [28, 38]. The development of stacked thylakoid membranes is correlated with the onset of chlorophyll b synthesis in maize [ 1, 12], but these studies only correlate the presence of LHCPII with stacked thylakoids, and do not demonstrate a structural role for LHCPII in thylakoid architecture. It is apparent that there are several classes of mutations that affect chlorophyll b levels, and each are represented in several plant families [ 1, 3, 4, 5, 9, 11, 12, 19, 23, 29, 30]. The overriding conclusion from all of these studies is that there are several factors that lead to the elegant structure and physiology of the chloroplast. Our present study shows that in the absence of stable LHCPII, thylakoids can nevertheless be found in a stacked form. Thus the physical arrangement of the thylakoids is likely to be due to agents other than LHCPII. The site and pathway of chlorophyll b synthesis has not been resolved and therefore its role in stabilizing LHCPII is still unclear. While it is instructive to determine the regions of LHCPII that are required for chlorophyll b binding it is also important to characterize the biosynthesis of the pigment. This report is a required initial characterization of the ch-1 phenotype in Arabidopsis. The ch-1 locus has been mapped to chromosome 1 of Arabidopsis [13, 15] and proximal RFLP markers [21] will aid in the isolation
78 of the disrupted gene. With this approach we hope to eventually understand the role that chlorophyll b plays in allowing the stable insertion of LHCPII into thylakoid membranes.
Acknowledgements Support for this work was provided by National Institutes of Health grant GM39696-01 to B.D.K. and by a North Carolina Biotechnology Center Fellowship to D.L.M. This manuscript was written in partial fulfillment of a master's degree for D.L.M. We thank Andrea Auchincloss, Tracy Smith, Gerty Ward, Stephan Johnston, Jim Siedow, and John Boynton for their interest and comments, Shirley Casanas for the electron microscopy work, Nigel Crawford for the nitrate reductase cDNA, and Elaine Tobin for LHCP antiserum.
References 1. Allen KD, Duysen ME, Staehelin LA: Biogenesis of thylakoid membranes is controlled by light intensity in the conditional chlorophyll b-deficient CD3 mutant of wheat. J Cell Biol 107:907-919 (1988). 2. Anderson JM: Photoregulation of the composition, function, and structure of thylakoid membranes. Ann Rev Plant Physiol 37:93-136 (1986). 3. Bassi R, Hinz U, Barbato R: The role of the light harvesting complex and photosystem II in thylakoid stacking in the chlorina-f2 barley mutant. Carlsberg Res Commun 50:347-367 (1985). 4. Bellemare G, Bartlett SG, Chua NH: Biosynthesis of chlorophyll a/b-binding polypeptides in wild type and the chlorina-fz mutant of barley. J Biol Chem 257: 7762-7767 (1982). 5. Boardman NK, Highkin HR: Studies on a barley mutant lacking chlorophyll b. I. Photochemical activity of isolated chloroplasts. Biochim Biophys Acta 126:189-199 (1966). 6. Castelfranco PA, Beale SI: Chlorophyll biosynthesis: recent advances and areas of current interest. Annu Rev Plant Physiol 34:241-278 (1983). 7. Crawford N, Smith M, Bellissimo D, Davis R: Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains. Proc Natl Acad Sci USA 85:5006-5010 (1988).
8. Feinberg AP, Vogelstein B: A technique for radio-labeling DNA restriction fragments to high specific activity. Anal Biochem 132:6-13 (1983). 9. Freeman TP, Duysen ME, Olson NH, Williams ND: Electron transport and chloroplast ultrastructure of a chlorophyll-deficient mutant of wheat. Photosyn Res 3: 179-189 (1982). 10. Goodchild DJ, Highkin HR, Boardman NK: The fine structure of chloroplasts in a barley mutant lacking chlorophyll b. Exp Cell Res 43:684-688 (1966). 11. Greene B, Allred DR, Morishige DT, Staehelin LA: Hierarchical response of light harvesting chlorophyll proteins in a light-sensitive chlorophyll b-deficient mutant of maize. Plant Physiol 87:357-364 (1988). 12. Henningsen KW, Nielsen NC, Smillie RM: The effect of nuclear mutations on the assembly of photosynthetic membranes in barley. Port Acta Biol ser A 14:323-344 (1974). 13. Hirono Y, Redei GP: Multiple allelic control of chlorophyll b level in Arabidopsis thaliana. Nature 197: 1324-1325 (1963). 14. Kohorn BD, Harel E, Chitnis PR, Thornber JP, Freddie B, Tobin EM: Functional and mutational analysis of the light harvesting chlorophyll a/b binding protein of thylakoid membranes. J Cell Biol 102:972-981 (1986). 15. Koornneef M, van Eden J, Hanhart CJ, Stam P, Braaksma FJ, Feenstra WJ: Linkage map of Arabidopsis thaliana. J Hered 74:265-272 (1983). 16. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 (1970). 17. Laskey RA, Mills AD: Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem 56:335-341 (1975). 18. Leutweiler LS, Meyerowitz EM, Tobin EM: Structure and expression of three light-harvesting chlorophyll ab-binding protein genes in Arabidopsis thaliana. Nucl Acids Res 14:4051-4064 (1986). 19. Markwell JP, Webber AN, Lake B: Mutant of sweet clover (Melilotus alba) lacking chlorophyll b. Plant Physiol 77:948-951 (t985). 20. Mayfield SP, Taylor WC: Carotenoid-deficient maize seedlings fail to accumulate light-harvesting chlorophyll a/b-binding protein (LHPC) mRNA. Eur J Biochem 144: 79-84 (1984). 21. Meyerowitz EM: Arabidopsis thaliana. Ann Rev Genet 21:93-111 (1987). 22. Murphy DJ: The molecular organization of the photosynthetic membranes of higher plants. Biochim Biophys Acta 864:33-94 (1986). 23. Nakatani HY, Baliga V: A clover mutant lacking the chlorophyll a and b containing protein antenna complexes. Biochem Biophys Res Commun 131:182-189 (1985). 24. Pellham H: Coming in from the cold. Nature 332: 776-777 (1988).
79 25. Peter GF, Thornber JP: In: Rogers LJ (ed) Methods in Plant Biochemistry Vol. II. Amino acids, protein, and nucleic acids. Academic Press, in press. 26. Porra RJ, Thompson WA, Kreidemann PE: Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b with NN-dimethylformamide, methanol, buffered aqueous acetone or alkaline pyridine. Biochim Biophys Acta 975: 384-394. 27. Ruhle W, Reilander H, Otto K-D, Wild A: Chlorophyllprotein-complexes of thylakoids of wild type and chlorophyll b mutant ofArabidopsis thaliana. Photosyn Res 4: 301-305 (1983). 28. Ryrie JJ: Immunological evidence for the apoprotein of the light-harvesting chlorophyll protein complex in a mutant of barley lacking chlorophyll b. Eur J Biochem 121:149-155 (1983). 29. Schwartz HP, Kloppstech K: Effects of nuclear gene mutations on the structure and function of plastids in pea. The light-harvesting chlorophyll a/b protein. Planta 155:116-123 (1982). 30. Simpson DJ, von Wettstein D: Macromolecular physiology of plastids. XIV. Viridis mutants in barley; genetic, fluoroscopic and ultrastructural characterization. Carlsberg Res Commun 45:283-314 (1980). 31. Somerville CR: Analysis of photosynthesis with mutants of higher plants and algae. Annu Rev Plant Physiol 37: 467-507 (1986). 32. Steinback KE, Burke JJ, Arntzen CJ: Evidence for the role of the surface exposed segments of the lightharvesting complex in cation-mediated control of chloroplast structure and function. Arch Biochem Biophys 195: 546-557 (1979).
33. Thomas PS: Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201-5205 (1980). 34. Towbin T, Staehelin T, Gordon J: Electrophoretic transfer of protein from polyacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 80:4350-4354 (1979). 35. Thornber JP: Biochemical characterization and structure of pigment-proteins of photosynthetic organisms. In: Staehelin LA, Arntzen CJ (eds) Encyclopedia of Plant Physiology. Vol. 19. Photosynthesis III. pp. 98-142. Springer-Verlag, Berlin, Heidelberg (1986). 36. Tobin EM, Turkaly E: Kinetin affects rates of degradation ofmRNAs encoding two major chloroplast proteins in Lemna gibba L. G-3. J Plant Growth Regul 1:3-13 (1982). 37. Verner K, Schatz G: Protein translocation across membranes. Science 241:1307-1313 (1988). 38. White MJ, Green BR: Polypeptides belonging to each of the three major chlorophyll a + b protein complexes are present in a chlorophyll-b-less barley mutant. Eur J Biochem 165:531-535 (1987). 39. Wickner W: Mechanisms of membrane assembly: general lessons from the study of M13 coat protein and Escherichia eoli leader peptidase. Biochemistry 27: 1081-1086 (1988). 40. Zurawski G, Whitfield PR, Bottomly W: Sequence for the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase from pea chloroplasts. Nucl Acids Res 14:3975 (1986).
71
Chloroplasts of Arabidopsis thaliana homozygous for the ch-1 locus lack chlorophyll b, lack stable LHCPII and have stacked thylakoids David L. Murray and Bruce D. Kohorn*
Botany Department, Duke University, Durham, NC 27706, USA (*author for correspondence) Received 11 July 1990; accepted in revised form 13 September 1990
Key words: LHCPII, chlorina, Arabidopsis
Abstract
We are interested in the mechanism of insertion of proteins into the chloroplast thylakoid membrane and the role that accessory pigments may play in this process. For this reason we have begun a molecular analysis of mutant plants deficient in pigments that associate with thylakoid membrane proteins. We have characterized plants that are homozygous for the previously isolated, recessive mutation chlorina-1 (ch-1) ofArabidopsis thaliana. Despite the lack of chlorophyll b and light-harvesting proteins of photosystem II (LHCPII) near normal levels of L H C P I I m R N A are found in the mutant, in contrast to L H C P I I m R N A levels in carotenoid-deficient mutants. The L H C P I I m R N A of chlorina-1 plants can be translated in vitro so it is likely that L H C P I I is not stable in ch- 1 plants. Moreover, the thylakoid membranes of ch- 1 plants remain appressed even though L H C P I I levels are drastically reduced.
Introduction
The means by which integral membrane proteins are inserted into lipid bilayers from an aqueous environment is poorly understood [37, 39]. It is clear, however, that not all proteins integrate into membranes co-translationally [37] and that specific regions of polypeptides mediate the integration process [39]. Recently a number of proteins have been implicated in aiding post-translational insertions [24]. To understand these processes we are studying the insertion, of the major light-harvesting chlorophyll a/b binding protein (LHCPII) into the thylakoid membrane. L H C P I I is synthesized as a precursor in the cytoplasm, is post-translationally imported into the chloroplast and then inserted into the thylakoid
membrane as a processed protein [22]. LHCPII ultimately becomes a member of the oligomeric chlorophyll-protein complex LHCII that channels light energy into photosystem II (PS II [35]). It has been suggested previously that chlorophyll b is required for the stability of L H C P I I in the thylakoid membrane [4], but whether chlorophyll b binding is required before, during, or after insertion of the protein into the membrane remains unclear. Both the site and pathway of chlorophyll b synthesis have yet to be discovered [6], and the existing nuclear mutations that affect chlorophyll b synthesis have been characterized in plants that have in the past not facilitated a molecular genetic analysis. We have begun a study of chlorophyll-deficient mutants of Arabidopsis in the hope that a biochemical characterization of
72 their chloroplasts will lead to a better understanding of chlorophyll b synthesis, and its role in L H C P integration. Here we describe a characterization of Arabidopsis that carries the recessive nuclear mutation chlorina-1 (ch-1). Chlorina-1 plants were originally isolated on the basis of their yellow green color and slow growth [13], and plants homozygous for ch-1 were shown to have reduced amounts of both chlorophyll b and stable L H C I I [ 13, 27, 31 ]. We are unable to detect chlorophyll b and show that of all the chlorophyll-protein complexes of the thylakoid, L H C I I is the most dramatically affected. In addition, stable L H C P I I is rare while L H C P I I m R N A is present at near normal levels in mutant plants. This m R N A can be translated in vitro suggesting that L H C P I I appears to be unstable in the absence of chlorophyll b. Moreover, this report shows that chloroplasts of ch-1 plants have appressed thylakoids despite the large deficiency in LHCPII.
Materials and methods
Arabidopsis thaliana Columbia, and theA. thaliana Columbia homozygous for ch-1 (obtained from Prof. Kranz, F R G ) were propagated on soil under 300 #mol photons m - 2s - 1 of light for four generations before analysis. Chlorophyll extractions and estimations were performed as described [26]. Extraction of thylakoid membranes from 4-week old plants, electrophoresis of denatured [ 16] and non-denatured proteins [14, 25], fluorography [17], and isolation [ 18] and analysis [34] of R N A were also carried out by published procedures. In vitro translations [ 14] and immuno-precipitations were performed as described using polyclonal sera prepared against Lemna gibba L H C P I I proteins [36]. Whole plants were radioactively labelled by first excising leaves under water, and then placing the stem in 1 ml of 40 mM H E P E S (pH 7.5) for 20 minutes. Radiolabelled 35S methionine (300/~Ci, Amersham) was added to each incuba-
1.2-
0-6°
"-~ 0
0
0
0
Fig. 1. Room temperature absorption spectra of acetonesoluble pigments from wild-type and ch-1 leaves. Pigments were extracted [26] from 0.5 g of fresh wild-type ( + ) and mutant (ch-1) leaves and the absorption spectra of equal amounts of extract are shown on the same relative scale. Horizontal scale shows wavelength in nanometers, vertical scale depicts relative absorption. tion, and the incorporation was terminated by the isolation of the thylakoids. Samples were prepared for electron microscopy by dissecting leaves directly into 5 ~o glutaraldehyde in phosphate-buffered saline (PBS), and shaken overnight at 4 °C. Leaves were then rinsed three times in PBS, placed in 2~o osmium tetroxide overnight, and then rinsed in water before a one-hour incubation in 1 ~o uranyl acetate. Samples were then dehydrated through an ethanol series, washed with propylene oxide, infiltrated with increasing proportions of Polybed resin in propylene oxide, and finally embedded in Polybed resin. Sections were cut on a Reichert-Jung ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a Zeiss EM 10 A electron microscope.
Results
Chlorophyll content of ch-1 plants is reduced Plants homozygous for the X-ray-induced ch-1 mutation [ 13] appear yellow-green under both
73
Fig. 2. Pigment-protein complexes of normal and ch-1 thylakoids. Isolated thylakoids were run in a non-denaturing gel that displays visible amounts of chlorophyll [14, 25] and that separates chlorophyll-protein complexes. + : wild type, ch-1: mutant thylakoids. Each lane contains 10/~g of chlorophyll. From top to bottom: photosystem I; core complex I; core complex II; light-harvesting complex Ilfl; core complex II~fl; light-harvesting complex II0tflz.
high and low light intensities and grow and senesce more slowly than wild type. Previous studies using thin-layer chromatography have failed to detect chlorophyll b, and we confirmed using a spectroscopic analysis that chlorophyll b levels are reduced [26]. Figure 1 shows the fluorescence scan of total extracted pigments. While wild-type plants have a calculated total chlorophyll content of 1.84 ktmol/g of fresh weight, ch-1 homozygotes have only 0.4 #mol/g. The calculated chlorophyll a/b ratio is 3.27 and 44.57 for wild-type and ch-1 plants, respectively. The absorption of other pigments make it difficult to determine whether chlorophyll b is totally absent, yet it is clear that it is significantly reduced in mutant plants.
Chlorophyll-protein complexes are altered in ch-1
Previous findings show that ch-1 plants lack the chlorophyll-protein complex LHCII [ 13, 27]. We extend these results by analyzing thylakoid membranes in non-denaturing gels that maintain chlorophyl-protein complexes and produce little or no free chlorophyll [25]. We found that of all
Fig. 3. Protein composition of mutant and wild-type thylakoids. The entire lanes from the gel lanes shown in Fig. 2 (run in the direction of the horizontal arrows) were separated from each other and were placed on their side and subjected to electrophoresis in a denaturing gel in the direction of the vertical arrows. Lane a: thylakoids from ch-1 plants; lane d: normal thylakoids. Mutant (lane b) and normal (lane c) thylakoids were also fully denatured and run into the same denaturing gel, which was then stained with Coomassie blue. Also shown on the right are the sizes of molecular weight markers x 10 -3, Arrow and box indicate position of LHCP II. < < , PSI. < , CF1 ATPase. *, PSII polypeptides.
the thylakoid membrane proteins detected with Coomassie blue on denaturing polyacrylamide gels, only L H C P I I could not be detected in mutant plants. Bands corresponding to the core complexI and II (CCI and II in Fig. 2) are increased in abundance and this may reflect either the increased (or altered) accessibility of detergent to the thylakoid membrane in the absence of LHCII, or an increase in absolute protein levels. To determine the protein content of the various green bands the first-dimension gel lanes were separated from each other, and placed on their sides and run into a completely denaturing gel ([16], Fig. 3 lanes a and d). Thylakoids from
74
Fig. 4. Detection of LHCPII with antisera or 35S methionine. 35S labelled thylakoid membranes from wild-type ( + ) and mutant
(ch-1) plants were isolated, run in a denaturing gel, and transferred to nitrocellulose. Panel A: filter probed with anti-LHCPII antibody, and visualized as described [36]. Panel B: same filter subjected to autoradiography. Arrows point to position of LHCPII. Panel C: Total RNA from wild-type ( + ) or mutant (ch-1) plants was translated in vitroin the presence of 35S methionine, and the products were immunoprecipitated with anti-LHCPII antibody, run in a denaturing gel, and subjected to autoradiography. Numbers indicate Mr x 10 3.
mutant and wild-type plants were also denatured and run into this gel to serve as a reference (Fig. 3, lanes b and c respectively). Figure 3 shows such a gel stained with Coomassie blue, and d e m o n strates that the major polypeptides and thus complexes o f the wild-type thylakoids are present in ch-1 plants, with the exception o f L H C P I I (arrow and square). We do notice that other thylakoid polypeptides associated with L H C I I vary in abundance, especially those o f P S I I (*), but those o f P S I ( < < ) and CF1 A T P a s e ( < ) do not. Thus ch-1 thylakoids differ most dramatically from those o f the wild type in their lack o f chlorophyll b and L H C P I I polypeptides.
L H C P I I levels are reduced
Coomassie blue stain failed to detect L H P C I I in ch-1 thylakoids and so we used more sensitive methods o f detection: autoradiography o f in vivo labelled proteins, and immunoblotting. Wild-type and ch-1 plants were incubated with 35S methionine for three hours to label newly synthesized proteins, and the thylakoids were isolated, run into a denaturing gel, and transferred to nitrocellulose [34]. The nitrocellulose was then challenged and stained with a polyclonal serum that recognizes all L H C P I I variants [36], or subjected to autoradiography. Figure 4A shows the alkaline
75 phosphatase stain of the antibody-treated nitrocellulose and Fig. 4B is an autoradiograph of the same filter. In wild-type thylakoids abundant LHCPII can be detected with antibodies (Fig. 4A, lane + ) but only a faint signal heterogeneous in size is detected in thylakoids from ch-1 plants (lane ch-1). We have not determined which variant of LHCP is represented and we have been unable on numerous western blots with ch-1 thylakoids to detect stronger signals or bands that are more discrete. The autoradiogram of the labelled thylakoids detects a 28 kDa radioactive band in wild-type thylakoids (Fig. 4B, lane + ) but only a faint band is seen in thylakoids from ch- 1 plants. We also note that there are differences in the intensities between the labelled polypeptides for wild-type and mutant thylakoids, but these have not been investigated further.
Ch-1 L H C P I I m R N A can be translated
We next asked whether the absence of LHPCII was the consequence of a reduction in the levels of translatable LHCPII m R N A or to an increase
Fig. 5. mRNAs levels for LHCPII, nitrate reductase, and the large subunit of Rubisco (LSU). 2.5 #g of total RNA from wild-type (lanes a, c, e) and mutant (b, d, f) plants were run in 1.2% denaturing agarose gel, transferred to nitrocellulose, probed with 32p labelled coding sequences for LHCP (lanes a, b), nitrate reductase (lanes c, d), and LSU (lanes e, f), and subjected to autoradiography. Numbers on the right indicate size in kilobases.
in the rate of protein turnover. To address the former possibility equal amounts of mutant and wild-type total RNA were translated in the presence of 35S methionine in a wheat germ extract, and the products were immuno-precipitated with LHPCII antibody and analyzed by denaturing gel electrophoresis [ 16]. The results of this analysis are shown in Fig. 4C. Anti-LHCPII precipitates a 32 kDa radioactive protein from total translation products of both wild-type and mutant samples (Fig. 4C) and this protein corresponds in size to the precursor of LHCPII. Pre-immune serum does not precipitate any radioactive protein (data not shown). Thus LHPCII m R N A in ch-1 plants can be translated in vitro. We are therefore left with the second explanation that there is an increased turnover rate of LHCPII in ch- 1 plants. While equivalent amounts of LHCPII can be synthesized in vitro from isolated RNA, it was still unclear if the m R N A populations were affected. Therefore, equal amounts of total RNA from wild-type and ch-1 plants were run into a denaturing agarose gel, and the RNA was transferred to nitrocellulose [33]. Figure5 shows autoradiograms of these RNAs that have been probed with LHCPII coding sequence [8, 18]. We conclude that LHCPII m R N A levels in mutant tissue (lane b) are in fact increased relative to those of the wild type (lane a). (The larger size of the ch-1 m R N A is also reflected in the ethidium bromide stained ribosomal RNA and is a reproducible effect of the total RNA preparation, perhaps due to the different starch content of the mutant and wild-type plants.) We also probed these northern blots with another nuclear coding sequence nitrate reductase [7], and a chloroplast sequence the large subunit of ribulose 1,5-bisphosphate carboxylase (LSU [40]), to determine whether other cytoplasmic mRNAs and chloroplast mRNAs (respectively) were affected. Figure 5 also shows that in comparison to LHCP m R N A the chloroplast LSU m R N A level is not affected by the ch-1 mutation (lane e and f), and that cytoplasmic nitrate reductase mRNAs may be slightly reduced in abundance in ch-I plants (lane c and d), although accurate quantitation has not been carried out.
76
Fig. 6. Ultra-structure of normal and ch- 1 chloroplasts. Thin sections of wild-type (panels A and C) and mutant (panels B and D) leafs from tissue of equal age and light exposure. Bar represents 0.5 #m in panels A and B, and 0.1/~m in panels C and D.
Ch-1 thylakoids remain stacked L H C P I I harvests light energy for PSII, but there are indications that the complex also plays a structural role in maintaining the stacked arrangement of thylakoids [2, 32]. Since the L H C P I I levels are drastically lowered in ch-1 plants, we were interested to see if thylakoids from ch-1 plants had reduced amounts of stacked
thylakoids. Figure 6 shows representative electron micrographs of sectioned leaves from wildtype and ch-1 plants. The chloroplasts of the mutant are as numerous but generally more rounded and lighter in appearance than those of the wild type (panels B, D and A, C, respectively). It is not clear whether the lighter appearance is related to the lack of pigments and protein. Although the light appearance creates the impres-
77 sion that there is less stacking of mutant thylakoids, examination of multiple sections shows that thylakoids of ch-1 plants show significant stacking and the degree to which the thylakoids are stacked in the mutant is not directly related to the deficiency in LHCPII. Discussion
The absence of LHCPII in Arabidopsis homozygous for the recessive ch-1 mutation is not due to a lack of translatable LHCPII mRNA. Using antibodies and in vivo labelling LHCPII can only be detected at very reduced levels, yet in vitro translations and immuno-blots show the presence of abundant LHCPII mRNA. These results are consistent with those found for mutations that affect chlorophyll b synthesis in a number of other plant species [ 31 ]. LHCPII m R N A levels are not reduced in the absence of chlorophyll b and they in fact exhibit a slight increase in abundance. This increase is noticed in multiple RNA preparations and is limited to only certain messages as LSU m R N A within the chloroplast is not affected and the cytoplasmic m R N A for nitrate reductase may be slightly decreased in abundance. In vitro translation and immuno-precipitation experiments indicate that the LHCPII m R N A of ch-1 plants can be translated, leading to the conclusion that LHCPII is not stable in the absence of chlorophyll b. It is still possible that LHCPII m R N A can not be translated in ch-1 cytoplasm, but this would necessitate a cytoplasmic translation apparatus that is sensitive to pigment levels within the chloroplast. It is also unclear whether the increased LHCPII message level reflects an attempt to compensate for the increased turnover rate of the protein. The lack of an effect of a chlorophyll b reduction on nuclear m R N A levels is in strong contrast to that reported for carotenoid-deficient maize seedlings, where LHCP m R N A levels are greatly reduced [20]. This difference is logical if the differentiated state of the chloroplast and not the pigment levels themselves are tightly coupled to the nuclear control mechanisms.
Of additional significance is the condition of the thylakoid membranes in ch-1 plants. It has been suggested that LHCPII mediates thylakoid stacking as protease digestion of thylakoid membranes both removes the amino-A terminus of LHCPII, and unstacks the thylakoids [2, 32]. However, LHCPII levels are drastically reduced in ch-1 plants and the thylakoid membranes are still stacked. As many other thylakoid proteins are affected by protease treatment [2, 32] our results suggest that these proteins, and not LHCPII, may be more important in the stacking of thylakoids. The chlorina f2 mutant of barley also has reduced chlorophyll b [4], yet still has stacked membranes [3, 10]. However, stable LHCP varients are detected at levels sufficient to account for thylakoid stacking [28, 38]. The development of stacked thylakoid membranes is correlated with the onset of chlorophyll b synthesis in maize [ 1, 12], but these studies only correlate the presence of LHCPII with stacked thylakoids, and do not demonstrate a structural role for LHCPII in thylakoid architecture. It is apparent that there are several classes of mutations that affect chlorophyll b levels, and each are represented in several plant families [ 1, 3, 4, 5, 9, 11, 12, 19, 23, 29, 30]. The overriding conclusion from all of these studies is that there are several factors that lead to the elegant structure and physiology of the chloroplast. Our present study shows that in the absence of stable LHCPII, thylakoids can nevertheless be found in a stacked form. Thus the physical arrangement of the thylakoids is likely to be due to agents other than LHCPII. The site and pathway of chlorophyll b synthesis has not been resolved and therefore its role in stabilizing LHCPII is still unclear. While it is instructive to determine the regions of LHCPII that are required for chlorophyll b binding it is also important to characterize the biosynthesis of the pigment. This report is a required initial characterization of the ch-1 phenotype in Arabidopsis. The ch-1 locus has been mapped to chromosome 1 of Arabidopsis [13, 15] and proximal RFLP markers [21] will aid in the isolation
78 of the disrupted gene. With this approach we hope to eventually understand the role that chlorophyll b plays in allowing the stable insertion of LHCPII into thylakoid membranes.
Acknowledgements Support for this work was provided by National Institutes of Health grant GM39696-01 to B.D.K. and by a North Carolina Biotechnology Center Fellowship to D.L.M. This manuscript was written in partial fulfillment of a master's degree for D.L.M. We thank Andrea Auchincloss, Tracy Smith, Gerty Ward, Stephan Johnston, Jim Siedow, and John Boynton for their interest and comments, Shirley Casanas for the electron microscopy work, Nigel Crawford for the nitrate reductase cDNA, and Elaine Tobin for LHCP antiserum.
References 1. Allen KD, Duysen ME, Staehelin LA: Biogenesis of thylakoid membranes is controlled by light intensity in the conditional chlorophyll b-deficient CD3 mutant of wheat. J Cell Biol 107:907-919 (1988). 2. Anderson JM: Photoregulation of the composition, function, and structure of thylakoid membranes. Ann Rev Plant Physiol 37:93-136 (1986). 3. Bassi R, Hinz U, Barbato R: The role of the light harvesting complex and photosystem II in thylakoid stacking in the chlorina-f2 barley mutant. Carlsberg Res Commun 50:347-367 (1985). 4. Bellemare G, Bartlett SG, Chua NH: Biosynthesis of chlorophyll a/b-binding polypeptides in wild type and the chlorina-fz mutant of barley. J Biol Chem 257: 7762-7767 (1982). 5. Boardman NK, Highkin HR: Studies on a barley mutant lacking chlorophyll b. I. Photochemical activity of isolated chloroplasts. Biochim Biophys Acta 126:189-199 (1966). 6. Castelfranco PA, Beale SI: Chlorophyll biosynthesis: recent advances and areas of current interest. Annu Rev Plant Physiol 34:241-278 (1983). 7. Crawford N, Smith M, Bellissimo D, Davis R: Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains. Proc Natl Acad Sci USA 85:5006-5010 (1988).
8. Feinberg AP, Vogelstein B: A technique for radio-labeling DNA restriction fragments to high specific activity. Anal Biochem 132:6-13 (1983). 9. Freeman TP, Duysen ME, Olson NH, Williams ND: Electron transport and chloroplast ultrastructure of a chlorophyll-deficient mutant of wheat. Photosyn Res 3: 179-189 (1982). 10. Goodchild DJ, Highkin HR, Boardman NK: The fine structure of chloroplasts in a barley mutant lacking chlorophyll b. Exp Cell Res 43:684-688 (1966). 11. Greene B, Allred DR, Morishige DT, Staehelin LA: Hierarchical response of light harvesting chlorophyll proteins in a light-sensitive chlorophyll b-deficient mutant of maize. Plant Physiol 87:357-364 (1988). 12. Henningsen KW, Nielsen NC, Smillie RM: The effect of nuclear mutations on the assembly of photosynthetic membranes in barley. Port Acta Biol ser A 14:323-344 (1974). 13. Hirono Y, Redei GP: Multiple allelic control of chlorophyll b level in Arabidopsis thaliana. Nature 197: 1324-1325 (1963). 14. Kohorn BD, Harel E, Chitnis PR, Thornber JP, Freddie B, Tobin EM: Functional and mutational analysis of the light harvesting chlorophyll a/b binding protein of thylakoid membranes. J Cell Biol 102:972-981 (1986). 15. Koornneef M, van Eden J, Hanhart CJ, Stam P, Braaksma FJ, Feenstra WJ: Linkage map of Arabidopsis thaliana. J Hered 74:265-272 (1983). 16. Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 (1970). 17. Laskey RA, Mills AD: Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem 56:335-341 (1975). 18. Leutweiler LS, Meyerowitz EM, Tobin EM: Structure and expression of three light-harvesting chlorophyll ab-binding protein genes in Arabidopsis thaliana. Nucl Acids Res 14:4051-4064 (1986). 19. Markwell JP, Webber AN, Lake B: Mutant of sweet clover (Melilotus alba) lacking chlorophyll b. Plant Physiol 77:948-951 (t985). 20. Mayfield SP, Taylor WC: Carotenoid-deficient maize seedlings fail to accumulate light-harvesting chlorophyll a/b-binding protein (LHPC) mRNA. Eur J Biochem 144: 79-84 (1984). 21. Meyerowitz EM: Arabidopsis thaliana. Ann Rev Genet 21:93-111 (1987). 22. Murphy DJ: The molecular organization of the photosynthetic membranes of higher plants. Biochim Biophys Acta 864:33-94 (1986). 23. Nakatani HY, Baliga V: A clover mutant lacking the chlorophyll a and b containing protein antenna complexes. Biochem Biophys Res Commun 131:182-189 (1985). 24. Pellham H: Coming in from the cold. Nature 332: 776-777 (1988).
79 25. Peter GF, Thornber JP: In: Rogers LJ (ed) Methods in Plant Biochemistry Vol. II. Amino acids, protein, and nucleic acids. Academic Press, in press. 26. Porra RJ, Thompson WA, Kreidemann PE: Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b with NN-dimethylformamide, methanol, buffered aqueous acetone or alkaline pyridine. Biochim Biophys Acta 975: 384-394. 27. Ruhle W, Reilander H, Otto K-D, Wild A: Chlorophyllprotein-complexes of thylakoids of wild type and chlorophyll b mutant ofArabidopsis thaliana. Photosyn Res 4: 301-305 (1983). 28. Ryrie JJ: Immunological evidence for the apoprotein of the light-harvesting chlorophyll protein complex in a mutant of barley lacking chlorophyll b. Eur J Biochem 121:149-155 (1983). 29. Schwartz HP, Kloppstech K: Effects of nuclear gene mutations on the structure and function of plastids in pea. The light-harvesting chlorophyll a/b protein. Planta 155:116-123 (1982). 30. Simpson DJ, von Wettstein D: Macromolecular physiology of plastids. XIV. Viridis mutants in barley; genetic, fluoroscopic and ultrastructural characterization. Carlsberg Res Commun 45:283-314 (1980). 31. Somerville CR: Analysis of photosynthesis with mutants of higher plants and algae. Annu Rev Plant Physiol 37: 467-507 (1986). 32. Steinback KE, Burke JJ, Arntzen CJ: Evidence for the role of the surface exposed segments of the lightharvesting complex in cation-mediated control of chloroplast structure and function. Arch Biochem Biophys 195: 546-557 (1979).
33. Thomas PS: Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201-5205 (1980). 34. Towbin T, Staehelin T, Gordon J: Electrophoretic transfer of protein from polyacrylamide gel to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 80:4350-4354 (1979). 35. Thornber JP: Biochemical characterization and structure of pigment-proteins of photosynthetic organisms. In: Staehelin LA, Arntzen CJ (eds) Encyclopedia of Plant Physiology. Vol. 19. Photosynthesis III. pp. 98-142. Springer-Verlag, Berlin, Heidelberg (1986). 36. Tobin EM, Turkaly E: Kinetin affects rates of degradation ofmRNAs encoding two major chloroplast proteins in Lemna gibba L. G-3. J Plant Growth Regul 1:3-13 (1982). 37. Verner K, Schatz G: Protein translocation across membranes. Science 241:1307-1313 (1988). 38. White MJ, Green BR: Polypeptides belonging to each of the three major chlorophyll a + b protein complexes are present in a chlorophyll-b-less barley mutant. Eur J Biochem 165:531-535 (1987). 39. Wickner W: Mechanisms of membrane assembly: general lessons from the study of M13 coat protein and Escherichia eoli leader peptidase. Biochemistry 27: 1081-1086 (1988). 40. Zurawski G, Whitfield PR, Bottomly W: Sequence for the gene for the large subunit of ribulose 1,5-bisphosphate carboxylase from pea chloroplasts. Nucl Acids Res 14:3975 (1986).
