In Arabidopsis thaliana, we identified a novel gene of a NADPH-protochlorophyllide oxidoreductase (POR) isoform, which catalyzes the light-dependent protochlorophyllide a reduction in the chlorophyll (Chl) biosynthetic pathway. The deduced amino acid sequence of the novel POR isoform (PORC) showed significant identities (V V75%) with the previously isolated two POR isoforms of A. thaliana. Contrasting with these POR isoforms, the PORC transcript increased in etiolated seedlings by illumination, and was dominantly expressed in immature and mature tissues. The present results demonstrated that Chl biosynthesis and chloroplast biogenesis in A. thaliana are controlled by three POR isoforms, which are differentially controlled by light and development.

; NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide. To elucidate the physiological function of three differentially regulated POR isoforms (PORA, PORB and PORC) in Arabidopsis thaliana, we isolated T-DNA tagged null mutants of porB and porC. The mature seedlings of the mutants had normal photosynthetic competencies, showing that PORB and PORC are interchangeable and functionally redundant in developed plants. In etiolated seedlings, only porB showed a reduction in the photoactive protochlorophyllide and the size of prolamellar bodies (PLBs), indicating that PORB, as well as PORA, functioned in PLB assembly and photoactive protochlorophyllide formation in etiolated seedlings. When illuminated, the etiolated porB seedling was able to green to a similar extent as the wild type, whereas the greening was significantly reduced under low light conditions. During greening, high light irradiation increased the level of PORC protein, and the greening of porC was repressed under high light conditions. The porB, but not porC, etiolated seedling was more sensitive to the far-red block of greening than the wild type, which is caused by depletion of endogenous POR proteins resulting in photo-oxidative damage. These results suggest that, at the onset of greening, PLBs are important for efficient capture of light energy for photoconversion under various light conditions, and PORC, which is induced by high light irradiation, contributes to photoprotection during greening of the etiolated seedlings.

Cytosolic malate dehydrogenase (cytMDH) was captured by thioredoxin affinity chromatography as a possible target protein of cytosolic thioredoxin (Yamazaki, D., Motohashi, K., Kasama, T., Hara, Y., and Hisabori, T. (2004) Plant Cell Physiol. 45, 18 -27). To further dissect this interaction, we aimed to determine whether cytMDH can interact with the cytosolic thioredoxin and whether its activity is redox-regulated. We obtained the active recombinant cytMDH that could be oxidized and rendered inactive. Inactivation was reversed by incubation with low concentrations of dithiothreitol in the presence of recombinant Arabidopsis thaliana thioredoxin-h1. Inactivation of cytMDH was found to result from formation of a homodimer. By cysteine mutant analysis and peptide mapping analysis, we were able to determine that the cytMDH homodimer occurs by formation of a disulfide bond via the Cys 330 residue. Moreover, we found this bond to be efficiently reduced by the reduced form of thioredoxin-h1. These results demonstrate that the oxidized form cytMDH dimer is a preferable target protein of the reduced form thioredoxin-h1 as suggested by thioredoxin affinity chromatography. Malate dehydrogenase (MDH)3 catalyzes the reversible reaction of oxaloacetate to malate utilizing the NAD ϩ /NADH or NADP ϩ /NADPH cofactor system and is known to occur ubiquitously in mammals, plants, and most prokaryotes. NAD-dependent MDHs (EC 1.1.1.37) generally exist as a homodimer with a subunit of 32-37 kDa (1, 2), whereas NADP-dependent MDH (EC 1.1.1.82) possesses a larger subunit of ϳ42 kDa (3, 4). In plants, MDH isoforms have been reported in the cytosol and in organelles and have been found to carry out important roles in a number of metabolic pathways (5). In general, MDH proteins are grouped into five classes according to their location within the cell; (i) cytosolic NAD-dependent MDH (cytMDH); (ii) mitochondrial NAD-dependent MDH, which is part of the tricarboxylic acid cycle; (iii) glyoxisomal and peroxisomal NAD-dependent MDHs, which are involved in photorespiration; (iv) chloroplast NADPdependent MDH, which is required for the transfer of reducing equivalents from chloroplast stroma to cytosol; and (v) chloroplast NAD-dependent MDH (6).Among the classes listed, chloroplast NADP-dependent MDH is a thiol enzyme that has been subject of intense study (4,(7)(8)(9)(10)(11)(12) and that uses NADP ϩ for catalysis. Chloroplast NADPdependent MDH possesses both N-and C-terminal extensions, both of which harbor a redox-sensitive cysteine pair (4,(12)(13)(14). Central to the regulation of chloroplast MDH, ferredoxin-thioredoxin reductase sources electrons from photosynthesis to reduce chloroplast thioredoxins (Trx) (8). Trxs are an important class of regulatory proteins that modulate the activity of a wide variety of thiol-containing chloroplast enzymes by the reduction of specific disulfide bonds (15). In the light, excess NADPH (produced by photosynthesis but surplus to CO 2 fixation requirements) induces the conversion of oxaloacetate to malate, r...

Actinophage TG1 forms stable lysogens by integrating at a unique site on chromosomes of Streptomyces strains. The phage (attP(TG1)) and bacterial (attB(TG1)) attachment sites for TG1 were deduced from comparative genomic studies on the TG1-lysogen and nonlysogen of Streptomyces avermitilis. The attB(TG1) was located within the 46-bp region in the dapC gene (SAV4517) encoding the putative N-succinyldiaminopimelate aminotransferase. TG1-lysogens of S. avermitilis, however, did not demand either lysine or diaminopimelate for growth, indicating that the dapC annotation of S. avermitilis requires reconsideration. A bioinformatic survey of DNA databases using the fasta program for the attB(TG1) sequence extracted possible integration sites from varied streptomycete genomes, including Streptomyces coelicolor A3(2) and Streptomyces griseus. The gene encoding the putative TG1 integrase (int(TG1)) was located adjacent to the attP(TG1) site. TG1 integrase deduced from the int(TG1) gene was a protein of 619 amino acids having a high sequence similarity to phiC31 integrase, especially at the N-terminal catalytic region. By contrast, sequence similarities at the C-terminal regions crucial for the recognition of attachment sites were moderate or low. The site-specific recombination systems based on TG1 integrase were shown to work efficiently not only in Streptomyces strains but also in heterologous Escherichia coli.