A method for in vitro reconstitution of the chlorophyll a/b light-harvesting complex from LiDodSO4/ heat-denatured or acetone-extracted photosynthetic membranes has been developed. Characterization of the minimum components necessary for the functional organization of pigments in these membrane complexes reveals that xanthophylls are essential structural components. (12). Xanthophylls also protect thylakoid membranes from photosensitized oxidation (11, 13), and violaxanthin is involved in an epoxidation cycle that may remove excess reducing equivalents from the chloroplast (11). The parallel accumulation of thylakoid membranes and xanthophylls upon illumination ofdark-grown plants (14,15) and during the cell cycle (16) suggests these pigments are important in the biogenesis and/or stabilization of thylakoid membranes.We have devised a method for the in vitro reconstitution of LHCPII and show that the stable association of Chl-a and -b with apoproteins requires xanthophylls. Moreover, specific xanthophylls must be provided in the reconstitution mixtures to obtain LHCPII possessing spectral and excitation energy transfer characteristics comparable to those of native complexes. MATERIALS AND METHODSPreparation of Thylakoids and Thylakoid Polypeptides.Thylakoids from spinach were prepared by flotation in discontinuous sucrose gradients (17) (18). Xanthophylls of greater than 90% purity were obtained by preparative paper chromatography (Whatman 3MM) using 10% (vol/vol) acetone in petroleum ether (bp, 35-60°C) and elution with diethyl ether. The pigments were identified by their Rf value, absorption spectra, and acid-induced hypsochromic shift in ethanol (11,18,19). The xanthophylls were judged to be free of diacylglycerides by thin layer chromatography (8) and visualization with UV, with iodine vapors, or with charring. Xanthophyll concentrations were calculated as described (19). Purified Chl-a and -b (Sigma) were dissolved in diethyl ether; the absence of diacylglycerides was verified as above. Diacylglycerides (Sigma) were diluted as recommended by the supplier.Reconstitution of LHCPII from Thylakoid Membranes. Routinely, 15 ,ug of Chl-b is dried under vacuum at 25°C; subsequent steps are at 0°C. The residue is dissolved in 2 to 3 1,u of diethyl ether and then an equal volume of ethanol is added to promote pigment transfer into detergent solutions. Transfer is achieved most effectively by repeatedly adding 1-2 Al of the ethanol/ether solution to 20 ul of 30% (wt/vol) sucrose/5% (wt/vol) LiDodSO4 with mixing. Next, a 30-,ul aliquot of thylakoid membranes in gel sample buffer (see above) is added to the detergent mixture. Because complete denaturation of LHCPII polypeptides is necessary, the solution is heated at 100°C for 1 min. Reconstitution requires gradual freezing at -20°C and becomes progressively more complete as the mixture is subjected to three cycles of freezing (6-12 hr) and thawing (15 min at 20°C). Finally, the samples are subjected to LiDodSO4/PAGE at 4°C (20) using a 2.4-cm stacking gel and ...

Nitrogen-limited Chlamydomonas reinhardtii is chlorotic and very deficient in chlorophyll a/b lightharvesting complexes (LHC). Rates of synthesis of photosynthetic proteins, but especially the LHC apoproteins, are reduced 10-to 40-fold. Moderately high levels of chloroplast transcripts accumulate in nitrogen-limited cells, and there is a correlation between chloroplast DNA levels and chloroplast mRNA abundance. In contrast, nuclear transcripts encoding LHCII and ribulose 1,5-bisphosphate carboxylase small subunits are markedly reduced. Thus, nitrogen availability affects chloroplast protein synthesis by inhibition of translation and, to a lesser extent, chloroplast DNA amplification. Regulation of nuclear-encoded photosynthetic proteins by nitrogen is achieved through mechanisms affecting transcription and/or mRNA stability.Nitrogen (N) assimilation, photosynthetic electron transport, and CO2 fixation are interwoven processes limiting plant growth. Reducing equivalents generated from photosynthetic electron transport are required for nitrate reduction, and ATP-dependent NH' assimilation into amino acids occurs primarily within plastids (1). In turn, amino acid recycling is integral to photorespiration (2). N availability not only restricts the photosynthetic efficiency of plants but also it appears to regulate photosynthate utilization. Typically, products such as starch, lipid, and carotenoids accumulate in N-deficient plants (3-7). Despite major effects of N on photosynthetic pigments, information on its role in the regulation of photosynthetic gene expression and the synthesis of photosynthetic proteins is meager (8-10). 2678 branes were prewashed [1 hr at 650C in 0.2x SSPE (lx SSPE = 0.15 M NaCl/50 mM Na2HPO4, pH 7.4/5 mM EDTA) containing 0.5% NaDodSO4], prehybridized (4 hr at 420C), and hybridized (24-48 hr at 420C) as described (14). Blots were washed twice for 30 min at 260C in 2x SSPE/0.1% NaDodSO4 and twice for 15 min at 50'C in 0.1x SSPE/0.1% NaDodSO4 and then were exposed to Kodak XAR film at -70'C without intensifying screens. Dextran sulfate was used with pHS16 (see below) (15). For dot blots, 50 /.d of equal amounts of RNA (adjusted with wheat-germ tRNA) were spotted onto Zeta Probe under low-vacuum conditions and processed as described above. MATERIALS AND METHODSSouthern Blot Analysis. DNA in the LiCI-soluble fraction of nucleic acid extracts was precipitated with ethanol and resuspended in water. DNA was digested with restriction enzymes (see the legend to Fig. 7), treated for 30 min at 37°C with boiled RNase at 30 ,g/ml, and resolved on agarose gels. Before transfer to GeneScreenPlus (DuPont), DNA was depurinated (0.2 M HCO for 5 min), denatured (1.5 M NaCl/0.5 M NaOH), and neutralized (3 M NaCl/0.5 M Tris, pH 7.0). Prehybridization and hybridization solutions were as above but with NaDodSO4 at 0.5%. For dot blots, equal amounts of DNA (adjusted with herring sperm DNA) were denatured (0.25 M NaOH for 10 min), neutralized, and applied to GeneScreenPlus (see above).DNA Probes. pHS16 recog...

Distinctive properties are identified in the molecular structure of ribulose, 1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in chlorophyll c-containing alpe (i.e., chromophytes). Using purified enzyme from Cryptomonas sp., Coccolithophora sp., and Cylindrothecafusiformis, we have determined that the RuBPCase holoenzyme of each species has a molecular weight, subunit composition, and isoelectric points of its subunits similar to the purified enzymes from pea and Chlamydomonas reinhardtii. The large subunits from chromophytes exhibit microheterogeneity in their isoelectric points, whereas two to four well-resolved isoelectric variants of the small subunit were observed in each RuBPCase preparation. In spite of the high degree of similarity in terms of physical properties, both the small and large RuBPCase subunits of the chromophytes are structurally different from those of chlorophytes; immunological studies demonstrate that RuBPCase subunits of these two groups have few antigenic determinants in common.Ribulose 1,5-bisphosphate carboxylase/oxygenase is a bifunctional enzyme which initiates the photosynthetic reduction of CO2 and the first step of the photorespiratory pathway. Present in all autotrophs, RuBPCase3 is probably the most abundant enzyme in nature. In vascular plants, green algae, and most bacteria, the RuBPCase holoenzyme is a high mol wt complex (-550 kD) composed of eight copies each of large (-55 kD) and small (-15 kD)

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ABSTRACIPhosphorylated thylakoid proteins of spinach (Spinacia okracea L.) and pea (Pisum sativum L.) were solubilized, fractionated by sucrose density gradient centrifugation, and analyzed by gel electrophoresis and crossed immunoelectrophoresis to identify the phosphoproteins. It was found that in addition to intense phosphorylation of light-harvesting chlorophyll complex II, four photosystem II components, CP43 apoprotein, Dl, D2, and a 10 to 11 kilodalton protein, are substantially phosphorylated in the light. Furthermore, the CP43 apoprotein, Dl and D2 can be resolved into two electrophoretic subspecies, only one of which is phosphorylated. This indicates that only a fraction of the PSII polypeptides is phosphorylated. Finally, analysis of detergent procedures suggests that the 10 to 11 kilodalton phosphoprotein is a peripheral component of the Orevolving PSII reaction center complex.In studies of the substrates of thylakoid protein kinases, Steinback et aL (22) identified (7, 14).