Junichi Obokata

Molecular Heterogeneity of Photosystem I (psaD, psaE, psaF, psaH, and psaL Are All Present in Isoforms in Nicotiana spp.)

The protein composition of photosystem I (PSI) was examined in Nicotiana spp. by high-resolution polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and immunoblot analysis. Five PSI proteins show polymorphism in an amphidiploid species, Nicotiana tabacum, but not in its ancestral diploid species, Nicotiana sylvestris and Nicotiana tomentosiformis. These Nicotiana spp. appear to have at least 18 PSI proteins per genome that range in molecular mass from 3 to 20 kD. They include the products of nuclear genes psaD, psaE, psaF, psaG, psaH, psaK, and psaL, the product of chloroplast gene psaC, N-terminally blocked proteins of 4.5 and 3.0 kD, and an unidentified protein of 12.5 kD. The psaD, psaF, psaH, and psaL products have two isoforms each that are distinguished by different mobilities in polyacrylamide gel electrophoresis, and the psaE product has four isoforms. The two isoforms of the psaD product have distinct amino acid sequences, indicating that they are encoded by different genes within the genome. Four isoforms of the psaE products can be classified into two groups by N-terminal amino acid sequence, indicating that at least two psaE genes are present in the genome. To examine whether the polymorphic nature of PSI is peculiar to Nicotiana spp., we carried out immunoblot analysis of the psaD and psaE products in isogenic lines of tomato (Lycopersicon esculentum), Arabidopsis thaliana, red bean (Vigna angularis), and corn (Zea mays). Two electrophoretically distinct isoforms were found for the psaD products of tomato, A. thaliana, and corn, and two isoforms of psaE products were detected in tomato, A. thaliana, and red bean. These results suggest that the nuclear-encoded subunits of PSI, except for the psaG and psaK products, generally have two isoforms.

Nucleotide sequence of a cDNA clone encoding a putative glycine-rich protein of 19.7 kDa in Nicotiana sylvestris

Glycine-rich proteins (GRPs) are a group of proteins characterized by a high content and repetitive sequences of glycine residues, and genes encoding them have been isolated from petunia [1, 5], bean [4], maize [2] and tobacco [8]. GRPs are considered to be the components of plant cell walls, because the cell walls of certain plant organs are rich in glycine [7]. A GRP of bean was indeed shown to be present in cell walls [4]. In Arabidopsis thaliana, five distinct cDNA clones encoding putative glycine-rich proteins were isolated, and they differ in both tissue-specific expression pattern and response to external stimuli [ 3 ]. Here we demonstrate the nucleotide sequence of Nicotiana sylvestris cDNA clone encoding a putative glycine-rich protein. Poly(A) + RNA was purified from whole plant of N. sylvestris, and cDNA library was constructed into 2gtl0. Screening of the library using a synthetic oligonucleotide probe (5GCIGA A / GGA A / GGCIGCIGCIGCIGCIACIAAA/GGAA/GGC-3) gave several positive clones, among which a cDNA clone designated ob7A1 was subjected toDNA sequencing (Fig. 1). The cDNA clone contains one large open reading frame, and the nucleotide sequence surrounding its initiation codon is G A G A A T G G C which matches the consensus sequence for the translation initiation in plant genes, AACAATGGC [ 6 ]. The open reading frame encodes a protein of 214 amino acids with a predicted molecular mass of 19.7 kDa and it contains much glycine (44.9~o). Computer analysis revealed that it corresponds to a putative glycine-rich protein, atGRP-2 ofA. thaliana [3], and hence we designate the protein encoded by ob7A1 as nsGRP-2. Genomic Southern hybridization probed with the insert DNA of ob7A1 showed that nsGRP-2 is encoded by a single-copy gene in N. sylvestris (data not shown), as is similar to the asGRP-2 in A. thaliana [3]. Comparison between the nsGRP-2 and atGRP-2 revealed the unique feature of these proteins (Fig. 2A). GRP-2 protein has two glycinerich domains (GRD 1 and GRD2) which are held between conserved domains of low glycine content (CD1, CD2, and CD3) as shown in Fig. 2B. The conserved domains, CD1, CD2 and CD3, are 81, 16 and 19 amino acid residues long, respectively, and their amino acid sequences are highly conserved between N. sylvestris and A. thaliana. On the other hand, the amino acid sequences in the glycine-rich domains are divergent between these plant species. The glycine-rich domain, GRD1, is 77 and 47 amino acid residues long in N. sylvestris and A. thaliana, respectively, and as for GRD2, it consists of 21 and 37 amino

Structure and expression of a nuclear gene for the PSI-D subunit of photosystem I inNicotiana sylvestris

The PSI-D subunit is the ferredoxin-binding site of photosystem I, and is encoded by the nuclear genepsaD. We isolated apsaD genomic clone fromNicotiana sylvestris, by screening a genomic library with apsaD cDNA which we previously cloned fromN. sylvestris (Yamamotoet al., Plant Mol Biol 17: 1251, 1991). Nucleotide sequence analysis revealed that this genomic clone contains apsaD gene, which does not correspond to thepsaD cDNA, so we designated these genespsaDb andpsaDa, respectively. ThepsaDb clone encodes a protein of 214 amino acids uninterrupted by introns. The N-terminal sequence determined for theN. sylvestris PSI-D protein encoded bypsaDb begins at the 49th residue. The products ofpsaDa andpsaDb share 82.7% and 79.5% identity at the amino acid and nucleotide levels, respectively. Genomic Southern analysis showed that two copies ofpsaD are present in theN. sylvestris genome. Ribonuclease protection assays and immunoblot analysis inN. sylvestris indicate that both genes are expressed in leaves, stems and flower buds, but neither is expressed in roots. During leaf development, the ratio ofpsaDb topsaDa mRNA increases from 0.12 in leaf buds to 0.36 in mature leaves. The relative abundance of the corresponding proteins decreased over the same developmental period. These results indicate that differential regulation mechanisms controlpsaDa andpsaDb expression at both the mRNA and protein levels during leaf development.

Nucleotide sequence of cDNA clones encoding PSI-D2 protein of photosystem I in Nicotiana sylvestris

Yoshiharu Yamamoto 1,2, Hideo Tsuji 1 Nobuaki Hayashida 3, Kazuhito Inoue 4 and Junichi Obokata 2. 1Department of Botany, Faculty of Science, Kyoto University, Kyoto 606, Japan; 2Department of Botany, Faculty of Science, Hokkaido University, Sapporo 060, Japan (* author for correspondence); 3 RIKEN Institute, Tsukuba 305, Japan; 4Department of Biological Sciences, School of Science, Kanagawa University, Hiratsuka, Kanagawa 259-12, Japan

Cloning of a nuclear-encoded photosystem I gene, psaEb, in Nicotiana sylvestris

PSI is a multiprotein pigment complex in thylakoid membranes and mediates light-driven electron transport from plastocyanin to ferredoxin. PSI consists of at least 13 subunits, designated PSI-A through PSI-L and PSI-N, which are encoded in the nuclear or plastid genome (Bryant, 1992). The gene for the PSI-E subunit is designated as psaE and is located in the nuclear genome in higher plants and green algae (Bryant, 1992) and in the plastid genome in red algae (Reith, 1992). psaE cDNAs have been isolated from several plant species (Bryant, 1992), whereas genomic clones have not been isolated as yet. In this study, we isolated what to our knowledge is the first nuclear-encoded psaE gene. We screened a Nicotiana sylvestris genomic library in ADASH vector using psaE cDNA clones (Obokata et al., 1994) as probes and isolated a genomic clone named kuEG3. This clone has a 14.7-kb insert, and a 2.2-kb region containing a psaE gene was sequenced (Table I). Comparison of the nucleotide sequences of kuEG3 with the psaE cDNA clones revealed that this genomic clone contains the psaEb gene. The protein-coding region of psaEb is interrupted by two introns of 456 and 198 bp. These introns have the consensus dinucleotides, GT and AG (Hanley and Schuler, 1988), at their 5 and 3 borders, respectively. According to the exon-shuffling hypothesis, introns were present in the most ancient genes (Gilbert et al., 1986). If an ancient psaE gene originally located in the plastid genome were transferred to the nuclear genome during plant evolution, as for the genes rp122 (Gantt et al., 1991) and tufA (Baldauf and Palmer, 1990), these introns would have been maintained in the nuclear genome of land plants but lost during the subsequent evolution of red algae genomes. The psaEb gene isolated here has several sequence elements homologous to well-defined cis-elements of other photoregulated genes, such as the GT-1 box of rbcS (Green et al., 1988) and the GATA motif of Lkca and Lkcb genes (Castresana et al., 1987). In addition, the R3 and R5 motifs previously found in the tobacco genes psaD (Yamamoto et

Nuclear-Coded Subunits of PSI in Nicotiana

The PSI complex in thylakoid membranes contains subunits encoded each by the nuclear and chloroplast genomes. A few years ago we initiated research on PSI biogenesis with a special emphasis on the contribution of the nuclear genome. In this article we would like to summarize our recent work briefly, and then discuss the role of the nuclear genome in relation to the evolution of photosynthetic apparatus. The details of our recent work will be described elsewhere.