Biosynthesis of P700-Chlorophyll a Protein Complex, Plastocyanin, And

Plant Physiol. (1986) 81, 60-66 0032-0889/86/81 /0060/07/$0 1.00/0

Biosynthesis of P700-Chlorophyll a Protein Complex, Plastocyanin, and Cytochrome b6/fComplex' Received for publication September 12, 1985 and in revised form December 18, 1985

TERUHIRO TAKABE*, TETSUKO TAKABE, AND TAKASHI AKAZAWA Department ofChemistry, Faculty ofScience and Technology, Meijo University, Tenpaku, Nagoya 468, Japan (Teruhiro Takabe); and Research Institutefor Biochemical Regulation, School ofAgriculture, Nagoya University, Chikusa, Nagoya 464, Japan (Tetsuko Takabe, T.A.)

ABSTRACT ofseveral subunit components (4, 16, 17, 23). The subunit I (60- 65 kD) is comprised of reaction center Chl (P700) and directly Changes in the amount of P700-chlorophyll a protein complex, plas- involved in the photochemical reaction (23). Although the func- tocyanin, and cytochrome b6/f complex during greening of pea (Pisum tion ofthe small subunits is largely obscure, it has been postulated sativum L.), wheat (Triticum aestivum L.), and barley (Hordeum vulgare that they have a role as electron acceptors of P700 and/or L.) leaves were analyzed by an immunochemical quantification method. structural components of the protein complex (4, 12). Recently, Neither subunit I nor II of P700-chlorophyll a protein complex could be Nechushtai and Nelson (18) have reported that a significant detected in the etiolated seedlings ofall three plants and the accumulation amount of subunit I is present in the etiolated seedlings of oat, of these subunits was shown to be light dependent. On the other hand, a bean, and spinach and its amount does not significantly change small amount of plastocyanin was present in the etiolated seedlings of all during greening, whereas the small subunits of P700-Chl a pro- three plants and its level increased about 30-fold during the subsequent tein complex accumulate only after those plants are illuminated. 72-hour greening period. Furthermore, cytochromef, cytochrome b6, and On the contrary, the absence ofsubunit I in the etiolated seedlings Rieske Fe-S center protein in cytochrome b6/fcomplex were also present of barley has been reported by Viering and Alberte (27). The in the etiolated seedings of all three plants. The level of each subunit effects of light on the biosynthesis of subunit I reported by these component increased differently during greening and their induction two groups are clearly different. Moreover, relatively few workers pattern differed from species to species. The accumulation of cytochrome have studied the synthesis of the small subunits of P700-Chl a bd/fcomplex was most profoundly affected by light in pea leaves, and the protein complex. We have studied the biosynthesis of large and levels of cytochrome Jf cytochrome b6, and Rieske Fe-S center protein small subunits of P700-Chl a protein complex during greening increased during greening about 10-, 20-, and more than 30-fold, respec- using other plant species. tively. In comparison to the case of pea seedlings, in wheat and barley Most biochemical studies on the accumulation of plastocy- leaves the level ofeach subunit component increased much less markedly. anin, Cytfl and Cyt b6 during greening have been performed by The results suggest that light regulates the accumulation of not only the spectroscopic methods (3, 7, 8, 10, 19). Previous results have chlorophyll protein complex but also the components of the electron shown that in the dark-grown bean leaves, only a very small transport systems. amount of Cyt fand b6 is present and a marked accumulation starts upon illumination (7). In contrast, dark-grown barley leaves contain a large amount of Cytfand b6 and there is little increase upon illumination (3, 8, 10, 19). In addition to Cyt f and b6, it is known that Cyt b6/fcomplex contains a Rieske Fe- S center protein and a 17 kD protein ofunknown function (13). Dark-grown higher plants are incapable of photosynthesis, The biosynthesis of Rieske Fe-S center protein and 17 kD primarily because they lack Chl and some polypeptides essential polypeptide during greening has not been studied. We have for the photosynthetic reactions (6). Upon illumination ofdark- measured the levels of above described proteins during chloro- grown plants, the synthesis of Chl and previously missing poly- plast development in pea, wheat, and barley by using an immu- peptides occurs together with the induction of photosynthetic nochemical quantification method (25). For this study, specific activities. antibodies raised against subunits I and II ofP700-Chl a protein The expression of both nuclear and chloroplastic genomes is complex, plastocyanin, Cyt f Cyt b6, and Rieske Fe-S center required to achieve light-regulated chloroplast development. The protein were used throughout. biosynthesis of light-harvesting Chl a/b protein associated with PSII and ribulose 1,5-bisphosphate carboxylase has been exten- MATERIALS AND sively studied (2, 5, 20, 26). The increased formation of these METHODS proteins occurring upon illumination of dark-grown plants is Plant Materials. The pea (Pisum sativum L.), wheat (Triticum partly due to the increase in levels ofthe corresponding mRNAs aestivum L.), and barley (Hordeum vulgare L.) seeds were soaked (2, 5, 20, 26). In contrast, there is a paucity of information in tap water overnight and planted in vermiculite. Pea seedlings concerning the biosynthesis of P700-Chl a2 protein complex, were grown in darkness at 20°C, and both wheat and barley plastocyanin, and Cyt b6/fcomplex. seedlings were grown at 28°C. The period of etiolation was 12 d The P700-Chl a protein complex in higher plants is composed for pea, 10 d for wheat, and 8 d for barley. At the end of these periods, the plants were illuminated with tungsten lamps provid- ' Supported in part by a Grant-in-Aid for Scientific Research from the ing 10 W/m2. After exposure to light for various times, apical Ministry of Education, Science and Culture ofJapan. buds approximately 2 cm long of pea and the upper 4 to 5 cm 2 Abbreviation: P700, reaction center chlorophyll of photosystem I. of primary leaves of wheat and barley were harvested and used 60 BIOSYNTHESIS OF CHLOROPLAST PROTEINS 61 for protein extraction. Electron paramagnetic resonance spectra were made using a Protein Extraction. Seedlings (2 g) were frozen in liquid N2, JEOR JES-FE 1 XG X band spectrometer with an Oxford Instru- powdered by means of pestle and mortar, and further homoge- ment liquid helium cryostat and variable temperature system. nized using 2 ml of 50 mM Tris-HCl (pH 8.0) buffer containing Microwave power and temperature for electron paramagnetic 0.2 M sucrose, 10 mM NaCl, 0.1% (w/v) NaN3, and 1 mM resonance spectra were 5 mW and 13 K, respectively. Spectro- phenylmethylsulfonyl fluoride. The homogenate was filtered scopic method to estimate the contents of Cyt f and b6 in the through two layers of Miracloth (Calbiochem) and used as the membranes was carried out using a Hitachi 557 double beam total protein fraction of leaves. The membrane and soluble spectrophotometer according to the procedure ofLeto and Miles protein fractions of leaves were prepared by centrifugation ofthe (15). total fraction for 5 min at 15,800g. Each fraction was frozen and Electrophoresis was carried out usually on 13% (w/v) acryl- stored at -80°C until analyzed. amide-SDS gels according to Laemmli (14). Proteins and hemes Protein Purification and Antibody Preparation. The P700-Chl were stained with Coomassie brilliant blue R-250 and 3,3',5,5'- a protein complex from spinach chloroplasts was purified as tetramethylbenzidine/hydrogen peroxidase (24), respectively. previously described (23). The photochemically active reaction Protein was determined according to the method of Lowry and center protein containing only subunit I was prepared from the Chl was determined according to Arnon as previously described. purified P700-Chl a protein complex as previously described (23). The polypeptides of subunits I and II of P700-Chl a protein RESULTS complex were prepared by electroelution from polyacrylamide gels after SDS-electrophoresis of the purified reaction center Protein Isolation and Antibody Preparation. Polypeptides of protein and P700-Chl a protein complex, respectively. Cyt b6 and Rieske Fe-S center protein were isolated from the preparation of native Cyt b6/fcomplex as described under "Ma- Cyt b6/fprotein complex from spinach was purified according terials and Methods." The absorption spectrum of the Cyt b6/f to the method of Hurt and Hauska (13) after a minor modifica- complex preparation was essentially the same as that reported by tion. After fractionation by (NH4)2SO4, Cyt b6/f complex was Hurt and Hauska (13) (data not shown). The specific activity of applied to a Sephacryl S-300 column (90 cm x 3 cm2) equili- plastoquinol-plastocyanin oxidoreductase was high (about 10 brated with 10 mm Tris-HCl (pH 8.0) and 0.05% (v/v) Triton .umol-nmol-'.h'). The electron paramagnetic resonance spec- X-100. Fractions rich in Cyt b6/fcomplex were concentrated by trum of the purified Cyt b6/fcomplex showed the characteristic ultrafiltration (Millipore Immersible CX-10) and applied to the of Rieske center same column once again. Cyt b6 and Rieske Fe-S center proteins signal (g= 1.89) Fe-S protein (data not shown). were prepared by the electroelution of a polyacrylamide gel after The polypeptide composition of the purified Cyt b6/f complex tested by SDS-PAGE is presented in Figure 1 track 1. The four SDS-electrophoresis of the purified Cyt b6/fcomplex. Cyt fand main bands of 33, 23, 20, and 17 kD can be seen by Coomassie plastocyanin were purified from Brassica komatsuna and spin- blue staining. Two heme-associated bands of 33 and 23 kD were ach, respectively, as previously described (21, 22). identified as Cyt fand b6, respectively (Fig. 1, track 2). Rieske Purified specimens of each individual protein or subunit (0.5 Fe-S center protein could be separated from the Cyt b6/fcomplex mg) were mixed with an equal volume of Freund's complete by hydroxyapatite column chromatography. The bands for adjuvant and injected subcutaneously into white female rabbits. Rieske Fe-S center protein and its depleted Cyt b6/fcomplex are Then 0.5 mg proteins mixed with Freund's incomplete adjuvant shown in Figure 1, tracks 3 and 4, respectively. Identification of was injected twice at 2-week intervals. Rabbits were bled 2 to 3 the 20 kD polypeptide as the Rieske Fe-S center protein was weeks after the last booster injection, and specificity of the confirmed by its electron paramagnetic resonance spectra (data antibodies obtained was examined by an immunodecoration not shown). analysis (see below). Immunological specificity was examined for the quantitative Detection of the Relative Amounts of Polypeptides in the analysis of the developmental formation of P700-Chl a protein Seedlings. To measure the relative amounts of polypeptides in complex, plastocyanin, and Cyt b6/fcomplex. It was of prime the leaves, samples were electrophoresed on 13% (w/v) acryl- importance to obtain specific antisera. The antiserum raised amide-SDS gels according to the methods of Laemmli (14). The against CytfJ Cyt b6, and Rieske Fe-S center protein only cross- proteins separated were electrophoretically transferred to the reacted with 33 (Fig. 1, track 5), 23 (track 6), and 20 kD (track nitrocellulose filters by using a Marysol (Tokyo, Japan) Trans 7) of the purified Cyt b6/fcomplex, respectively. Specificity of Blot apparatus. The nitrocellulose filters were presoaked for 1 h the antibodies against subunits I and II of P700-Chl a protein in 10 mm phosphate buffer (pH 7.5) containing 150 mM NaCl, complex from spinach is also shown in Figure 1. As can be seen 0.1% (v/v) Tween 20, 0.1% (w/v) NaN3, and 5% (w/v) skim in track 8, P700-Chl a protein complex contains five polypeptides milk (PBS-Tween-Milk solution), and then treated with the (60-65, 23, 20, 18, and 12 kD). The antibodies against subunits specific antibodies for 1 h at room temperature. After washing I and II ofP700-Chl a protein complex reacted only with a single with PBS-Tween-Milk solution for 1 h, alkaline phosphatase- antigenic protein in the complex (Fig. 1, tracks 9 and 10). conjugated goat anti-rabbit antibody was incubated with the Immunological cross-reactivities of each antibodies against the filter papers for 50 min. The antigen-antibody complex was then spinach polypeptides such as subunits I and II of P700-Chl a detected by immersing the blot sections in 0.15 M barbital sodium protein complex, plastocyanin, Cyt b6, and Rieske Fe-S center (pH 9.0), 4 mM MgCl2, 0.01% (w/v) Nitroblue tetrazolium, and protein with the corresponding proteins ofpea, wheat, and barley 0.005% (w/v) 5-bromo-4-chloro-3-indolyl-phosphate. The re- was observed. It was also confirmed that antibody against Cyt f sulting purple bands were scanned by a densitometer (Shimadzu from Brassica komatsuna cross-reacted with Cytffrom all three Dual-Wavelength TLC Scanner CS-910). species. Linearity of staining intensity and the antigen amount Assay Methods. Activity of Cyt b6/f complex was assayed was observed. using a Hitachi 557 spectrophotometer by measuring the activity Formation of P700-Chl a Protein Complex in Pea, Wheat, and of plastoquinol-plastocyanin oxidoreductase. The wavelength Barley Seedlings. In pea, there was a prominent lag of Chl pairs 597 to 540 nm was used. The assay mixture contained in formation about 24 h after exposure of the plant to light. On the a total volume of 1 ml: Cyt b6/f complex 10 nm, oxidized other hand, in both wheat and barley Chl formation started plastocyanin 2 ,M, 10 mm Tris-HCl (pH 8.0), and 0.05% (v/v) instantaneously upon illumination. To measure changes ofpoly- Triton X-100. The reaction was started by the addition ofspinach peptide abundance in leaves during chloroplast development, an plastoquinone (13). immunological quantification method was employed. It was 62 TAKABE ET AL. Plant Physiol. Vol. 81, 1986

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FIG. 1. Identification ofpolypeptides and cross-reactivity ofantibodies. (1), Protein stain ofCyt b6/fcomplex; (2), heme stain ofCyt b6/fcomplex; (3), protein stain of Rieske Fe-S center protein; (4), protein stain of Rieske Fe-S center protein depleted Cyt b6/f complex; (5), immunological blotting of Cytf; (6), immunological blotting of Cyt b6; (7), immunological blotting of Rieske Fe-S center protein; (8), protein staining of P700-Chl a protein complex; (9), immunological blotting of subunit I of P700-Chl a protein complex; (10), immunological blotting of subunit II of P700-Chl a protein complex. found that the subunits I and II of P700-Chl a protein complex etiolated seedlings of barley was almost the same as that of are not detectable in the dark-grown seedlings of pea, wheat, and greened one (Fig. 5). These results were also confirmed by the barley. Figure 2 shows that subunit I of P700-Chl a protein heme-staining and the spectroscopic assay (data not shown). We complex is undetectable in the etiolated seedlings of pea (Fig. also found that Cyt b6 was present in the etiolated seedlings of 2A) and wheat (Fig. 2B), and its accumulation is light dependent. all three plants (Figs. 3 and 5). The presence of large amount of Moreover, it took 24 h for pea and 12 h for wheat for the Cyt fand b6 in the etiolated barley seedlings has been reported appearance of the subunit I after exposure of the plant to light. by heme-staining and incorporation of ['4CJ6-aminolevulinic A similar pattern was observed with the light-induced formation acid (I1), which is consistent with the present result (Fig. 5). of subunit II of P700-Chl a protein complex in the pea (Fig. 2C) Only a small amount of Rieske Fe-S center protein was de- and wheat (Fig. 2D) leaves. The pattern of accumulation of tected in the etiolated pea leaves and the level increased more subunits I and II in barley leaves is presented in Figure 5. than 30-fold during greening (Fig. 3E). In the etiolated seedlings Appearance of Cyt btf Complex and Plastocyanin. The Cyt of wheat and barley, a relatively higher amount of the protein b6/fcomplex contains four main polypeptides, of which only a existed and the level increased about 5-fold and 1.7-fold, respec- Rieske Fe-S center protein is encoded by the nuclear DNA (1). tively (Figs. 3F and 5). We attempted to analyze the amount ofCytf, Cyt b6, and Rieske The formation of plastocyanin was analyzed by using the Fe-S center protein by the same immunochemical technique as antibody directed against spinach plastocyanin. This protein has described above. We have found that Cyt f was present in the been reported to be encoded by the nuclear DNA (6). Since it is etiolated seedlings of pea, wheat, and barley, but the relative known that plastocyanin is easily released from thylakoid mem- amounts differed from one species to the other. Etiolated seed- branes, the total protein fraction from seedlings at various stages lings of pea contain a small amount of Cyt f and its amount were electrophoresed and subjected to the immunoblotting. As increased about 10-fold upon 72 h illumination as shown in shown in Figures 4 and 5, only a small amount of plastocyanin Figure 3A. In contrast, etiolated wheat seedlings contained a was present in the etiolated seedlings, and its level increased significant amount of Cytfand its amount increased only about about 30-fold during the 72 h greening period in all three plant 2-fold upon illumination (Fig. 3B). The amount of Cyt fin the species tested. BIOSYNTHESIS OF CHLOROPLAST PROTEINS 63 A pea B wheat 1 2 3 4 5 1 2 3 4 5

94.

C pea Dsoar wheat 1 2 3 4 5 1 2 3 4 5

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FIG. 2. Immunological detection of subunits I and II of P700-Chl a protein complex during greening of etiolated pea and wheat seedlings. For experimental details see text. In each case 20 gg leaf membrane proteins were electrophoresed. A, Subunit I of pea seedlings; B, subunit I of wheat seedlings; C, subunit II of pea seedlings; D, subunit II of wheat seedlings. Exposure to light for dark-grown leaves was: 0 h (lane 1), 12 h (lane 2), 24 h (lane 3), 48 h (lane 4), and 72 h (lane 5). 64 TAKABE ET AL. Plant Physiol. Vol. 81, 1986

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FIG. 3. Immunological detection of Cyt f Cyt b6, and Rieske Fe-S center protein during greening of etiolated pea and wheat seedlings. The experimental conditions were the same as those of Figure 2. A, Cyt fof pea seedlings; B, Cytfof wheat seedlings; C, Cyt b6 of pea seedlings; D, Cyt b6 of wheat seedlings; E, Rieske Fe-S protein of pea seedlings; F, Rieske Fe-S protein of wheat seedlings. The exposure of light was: 0 h (lane 1), 12 h (lane 2), 24 h (lane 3), 48 h (lane 4), and 72 h (lane 5). DISCUSSION from several higher plant species (4, 16, 17, 23) and its biosynthesis has been studied (12, 18, 27). Vierling and Alberte (27) Numerous investigations have given evidences that etioplasts have reported that little or no subunit I accumulates in the contain both soluble and insoluble membranous polypeptide etiolated barley seedlings, whereas the accumulation of a signif- components required for photosynthesis, but they lack Chl and icant amount of subunit I in the etiolated oat, bean, and spinach organized structure of photosynthetic membrane system (6). leaves has been reported by Nechushtai and Nelson (18). Heij et Extensive studies have been carried out on the biosynthesis of al. (9) have also detected subunit I in the dark-grown thylakoid light-harvesting...Chl a/b protein (2, 5). It has been hypothesized ....~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. membranes of Spirodela oligorhiza using antiserum directed that synthesis of this protein is regulated by light and the stabi- against Pisum subunit I. Our data are consistent with the previous lization of the mRNA and the accumulation of this protein finding by Viering and Alberte of the light-dependent biosyn- depends on the availability of Chl (2, 5). thesis ofsubunit I in barley leaves (27). Since we could not detect Recently, P700-Chl a protein complex has been highly purified the subunits I and II of P700-Chl a protein in the dark-grown BIOSYNTHESIS OF CHLOROPLAST PROTEINS 65

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FIG. 4. Immunological detection of plastocyanin during greening of etiolated (A) pea and (B) wheat seedlings. The proteins of total fraction (40 gg) were electrophoresed. The exposure of light was: 0 h (lane 1), 12 h (lane 2), 24 h (lane 3), 48 h (lane 4), and 72 h (lane 5). seedlings, we conclude that these subunits are absent or present abundance of plastocyanin in barley leaves does not change in a very low amount in the etiolated seedlings of pea, wheat, during greening. Results of our investigation are consistent with and barley. Therefore, it is important to elucidate the mecha- those previous investigations showing its accumulation in the nisms for the coordinate synthesis ofChl and subunit I compared etiolated seedlings. However, we have found that the level ofthe with the light-harvesting Chl a/b protein in the plant species. protein increased about 30-fold during greening of pea, wheat, Subunit II of P700-Chl a protein complex was also not detect- and barley leaves. In pea leaves, light-regulated biosynthesis of able in the etiolated pea, wheat, and barley seedlings, and a ribulose 1,5-bisphosphate carboxylase has been extensively stud- measurable accumulation of the polypeptide became prominent ied (20, 26). This protein is present in the etiolated pea leaves at almost the same time as that of subunit I, supporting a and the level increases 15-fold during 72 h greening period (20). coordinated synthesis of the subunits I and II in the P700-Chl a In comparison to this case, the light-dependent accumulation of protein complex. Recently, the absence of two small subunits in plastocyanin seems quite significant. the etiolated barley seedlings has also been reported (12). Thus, We have also found that in pea leaves the levels of Cyt f Cyt the synthesis of small subunits of P700-Chl a protein complex is b6, and Rieske Fe-S center protein are regulated by light in much induced by light in all plant species tested hitherto (12, 18). the same way as that of plastocyanin. The light effect on the There have been two reports concerning the biosynthesis of biosynthesis ofthese polypeptides is less significant in wheat and plastocyanin during greening. Haslett and Cammack (8) have barley leaves. In all three plant species, the levels of Cyt f, Cyt reported that plastocyanin was detectable in the etiolated leaves b6, and Rieske Fe-S center protein increased in a time sequential of Phaseolus vulgaris by electron paramagnetic resonance spec- manner. The pattern of induction of Cyt fand b6 as presented troscopy and the level increased about 8-fold 120 h after illumi- in Figures 3 is somewhat different even though it is known that nation. They have also suggested that phytochrome is involved both genes are located on the chloroplast genome. Therefore, it in the accumulation of the polypeptide in bean leaves. On the is worthwhile to elucidate the differential light effect on the other hand, Plesnicar and Bendall (19) have reported that the biosynthesis of both cytochromes. It must be also noted that the 66 TAKABE ET AL. Plant Physiol. Vol. 81, 1986 a/b protein. Control of messenger RNA activity by light. Eur J Biochem 118: 71-80 6. ELLIs RJ 1981 Chloroplast proteins: synthesis, transport, and assembly. Annu Rev Plant Physiol 32: 111-137 7. GREGORY P, JW BRADBEER 1973 Plastid development in primary leaves of Phaseolus vulgaris. The light-induced development of the chloroplast cytochromes. Planta 109: 317-326 8. HASLETT BG, R CAMMACK 1974 The development of plastocyanin in greening bean leaves. Biochem J 144: 567-572 9. HEIJ HT, AG JOCHEMSEN, PTJ WILLEMSEN, GSP GROOT 1984 Protein synthesis during chloroplast development in Spirodela oligorhiza. Coordinated synthesis of chloroplast-encoded and nuclear-encoded subunits of ATPase and ribulose-1,5-bisphosphatase carboxylase. Eur J Biochem 138: 161-168 10. HENNINGS KW, NK BOARDMAN 1973 Development ofphotochemical activity and the appearance of the high potential form of cytochrome b-559 in greening barley seedlings. Plant Physiol 51: 1117-1126 1 1. H0YER-HANsEN G 1980 Identification of haem-proteins in thylakoid polypeptide patterns of barley. Carlsberg Res Commun 45: 167-176 12. HOYER-HANSEN G, LS H0NBERG, DJ SIMPSON 1985 Monoclonal antibodies used for the characterization ofthe two putative iron-sulphur center proteins associated with photosystem I. Carlsberg Res Commun 50: 23-35 13. HURT E, G HAUSKA 1981 A cytochrome f/b6 complex of five polypeptides with plastoquinol-plastocyanin oxidoreductase activity from spinach chloroplasts. Eur J Biochem 117: 591-599 14. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of the head ofbacteriophage T4. Nature 277: 680-685 FIG. 5. Pattern of the accumulation of chloroplast proteins during 15. LETO KJ, D MILES 1980 Characterization ofthree photosystem II mutants in greening ofetiolated barley seedlings. Basic experimental conditions were Zea mays L. lacking a 32,000 dalton lamellar polypeptide. Plant Physiol 66: the same as those of pea and wheat leaves. A, (0), subunit I of P700-Chl 18-24 16. M0LLER BL, G H0YER-HANSEN, RG HILLER 1981 Functional identification a protein complex; (A), subunit II of P700-Chl a protein complex; (U), ofbarley thylakoid polypeptides resolved by SDS-polyacrylamide gel electro- plastocyanin. B, (0), Cytf (A), Cyt b6; (U), Rieske Fe-S center protein. phoresis. InG Akoyunoglou, ed, Photosynthesis. III. Structure and Molecular Organization ofthe Photosynthetic Apparatus. Balaban Int. Science Service, level of Rieske Fe-S center protein which is encoded in the Philadelphia, pp 245-256 nuclear increased the latest. One that 17. MULLETJE, JE BURKE,CJ ARNTZEN 1980 Chlorophyll proteins ofphotosystem genome might postulate I. Plant Physiol 65: 814-822 the formation of this protein is a limiting element for the expres- 18. NECHUSHTAI R, N NELSON 1985 Biogenesis of photosystem I reaction center sion of Cyt b6/fcomplex activity during the greening of etiolated during greening ofoat, bean and spinach leaves. Plant Mol Biol 4: 377-384 plants. However, further study on the relationship between bio- 19. PLESNICAR M, DS BENDALL 1973 The photochemical activities and electron of and heme or iron-sulfur in carriers of developing barley leaves. Biochem J 136: 803-812 synthesis apo-protein centers Cyt 20. SASAKI Y, M ISHIYE, T SAKIHAMA, T KAMIKUBO 1981 Light-induced increase b6ffcomplex is needed to establish this. Our data suggest that of mRNA activity coding for the small subunit of ribulose 1,5-bisphosphate light regulates biosynthesis of not only Chl protein complex but carboxylase. J Biol Chem 256: 2315-2320 also electron transport carriers in chloroplasts. Information con- 21. TAKABE T, S NIWA, H ISHIKAWA, M MIYAKAWA 1980 Ionic strength and pH effects on the rate of reduction of spinach plastocyanin by ascorbate. J cerning the levels of the corresponding mRNAs is needed to Biochem 87: 111-115 better understand the biosynthetic mechanisms of those chloro- 22. TAKABE T, S NIWA, H ISHIKAWA, K TAKENAKA 1980 Electron transfer plast proteins. reactions of cytochromeffrom Brassica komatsuna with hexacyanofferate. J Biochem 88: 1167-1176 23. TAKABE T, H ISHIKAWA, S NIWA, S ITOH 1983 Electron transfer between LITERATURE CITED plastocyanin and P700 in highly-purified photosystem I reaction center complex. Effects ofpH, cations, and subunit peptide composition. J Biochem 1. ALT J, P WESTHOFF, BB SEARS, N NELSON, E HURT, G HAUSKA, RG HERRM- 94:1901-1911 ANN 1983 Genes and transcripts for the polypeptides of the cytochrome b6/ 24. THOMAS PE, D RYAN, W LEVIN 1976 An improved stainning procedure for fcomplex from spinach thylakoid membranes. EMBO J 2: 979-986 the detection of the peroxidase activity of cytochrome P450 on sodium 2. APEL K, K KLOPPSTECH 1978 The plastid membranes of barley (Hordeum dodecyl sulfate polyacrylamide gels. Anal Biochem 75: 168-176 vulgare). Light-induced appearance of mRNA coding for the apoprotein of 25. TOBIN H, T STAEHELIN, J GORDON 1979 Electrophoretic transfer of proteins the light-harvesting chlorophyll a/b protein. Eur J Biochem 85: 581-588 from polyacrylamide gels to nitrocellulose sheets: Procedure and some 3. BALTIMORE BG, R MALKIN 1977 Appearance of membrane-bound iron-sulfur applications. Proc Natl Acad Sci USA 76: 4350-4354 centers and the photosystem I reaction center during greening of barley. 26. TOBIN EM, JL SuTTIF 1980 Light effects on the synthesis of ribulose 1,5- Plant Physiol 60: 76-80 bisphosphate carboxylase in Lemna gibba L. G-3. Plant Physiol 65: 641- 4. BENGIS C, N NELSON 1977 Subunit structure of chloroplast photosystem I 647 reaction center. J Biol Chem 252: 4564-4569 27. VIERING E, RS ALBERTE 1983 Regulation of synthesis of the photosystem I 5. CUMING AC, J BENNETT 1981 Biosynthesis of the light-harvesting chlorophyll reaction center. J Cell Biol 97: 1806-1814