National Institute for Basic Biology



Associate Professor (adjunct):
Hirokazu Kobayashi
Research Associate:
Noritoshi Inagaki

The conversion of light energy into chemical energy used for CO2 assimilation is the process of photosynthesis, one of the most important biological events. The photosynthesis utilizing water as an electron donor occurs in cyanobacteria and in chloroplasts in all eukaryotic algae and plants. The architecture of machinery for photosynthesis is directed by both nuclear and plastid genes in plants. The extremely efficient energy conversion is ensured by the highly ordered organization of molecules in photochemical reaction centers. The projects in this division are aiming to elucidate the mechanisms underlying the biogenesis of the photosynthetic machinery and the dynamic organization of molecules in the photosystem II (PS II) reaction center.

I. Tissue-specific expression of plastid genes

(1) Mechanisms of suppressed levels of transcripts for plastid photosynthesis genes in nongreen tissues such as roots and calli were analyzed employing the higher plant Arabidopsis thaliana which is suited for further genetical dissection. A region encoding promoters of rbcL for the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) and atpB/E operon for b and e subunits of coupling factor one was cloned and sequenced. Transcripts both for rbcL and psbA (gene for D1 protein in PS II reaction center) were undetectable and 16S rRNA was detected at a low level in roots of A. thaliana. The run-on transcription experiment revealed that expression of rbcL and psbA was regulated at transcription or upper steps in gene expression. The copy number of plastid DNA in roots was one-fifth of that in green leaves on the basis of total cellular DNA. Digestion of DNA with methyl-sensitive and -insensitive isoschizomeric endonucleases and subsequent polymerase chain reaction (PCR), as well as in vitro run-off transcription, resulted in no evidence of the regulation by DNA modification. In spite of predominant suppression of expression of rbcL and psbA at transcription in roots and calli, 16S rRNA levels there decreased in the result of low RNA stability.

(2) Genes for s-like factors of bacterial-type RNA polymerase have not been characterized from any multicellular eukaryotes, although they likely play a crucial role in the expression of plastid photosynthesis genes. We have cloned three distinct cDNAs designated SIG1, SIG2, and SIG3 for polypeptides possessing amino acid sequences for domains conserved in s70 factors of bacterial RNA polymerases from A. thaliana. Each gene is present as one copy per haploid genome without any additional sequences hybridized in the genome. Transient expression assays using green fluorescent protein (GFP) demonstrated that N-terminal regions of the SIG2 and SIG3 open reading frames could function as transit peptides for import into chloroplasts. Transcripts for all three SIG genes were detected in leaves but not in roots, and induced in leaves of dark-adapted plants in rapid response to light illumination. Together with results of the analysis of tissue-specific regulation of transcription of plastid photosynthesis genes, expressed levels of the genes may influence transcription by regulating RNA polymerase activity in a green tissue-specific manner.

II. Dynamic aspects of molecular organization of PS II

We have focused on elucidation of molecular mechanism of the C-terminal processing of the D1 protein known to be an integral subunit of the PS II reaction center. D1 protein is synthesized as a precursor form furnished with a C-terminal extension. The specific excision of the C-terminus is well characterized to be an essential event for construction of the photosynthetic oxygen-evolving machinery. Therefore, the protease, CtpA, involved in this process was characterized in biochemical and molecular biological techniques. However, the catalytic mechanism of the protease has not yet been demonstrated. We have constructed a series of systematic mutants of the cyanobacterium Synechocystis sp. PCC 6803, in which one of the putative catalytic residues of the protease was substituted to alanine by conventional site-directed mutagenesis. Biological and biochemical analyses of these mutants suggest that essential residues for the protease reaction are Ser313 and Lys338 in the Synechocystis CtpA sequence. It is speculated that the residues constitute Ser-Lys dyad in the tertiary structure and the Ser residue of the dyad hydrolyze a peptide bond with a similar manner of b-lactamase, a Ser-Lys dyad type hydrolase for cleavage of the b-lactam ring of antibiotics.

Fig. 1.
Localization of GFPs fused to N-terminal regions of SIG2 and SIG3. GFP fusion constructs with the N-terminal regions of SIG2 ORF (SIG2-GFP, panels C and G) and SIG3 ORF (SIG3-GFP, panels D and H), and the transit peptide of small subunit of Rubisco (RBCS-GFP, panels B and F), as well as GFP alone (GFP, panels A and E), were introduced into tobacco leaves by particle bombardment. Guard cells were observed using MRC-1024 Confocal Imaging System (X 480) with excitation at 488 nm and emission at 520 nm (panels A-D), as well as excitation at 647 nm and emission at 666 nm (panels E-H). The same objects are shown in each pair of upper and lower panels.

Publication List:
Isono, K., Niwa, Y., Satoh, K. and Kobayashi, H. (1997) Evidence for transcriptional regulation of plastid photosynthesis genes in Arabidopsis thaliana roots. Plant Physiol., 114, 623-630.
Isono, K., Shimizu, M., Yoshimoto, K., Niwa, Y., Satoh, K., Yokota, A. and Kobayashi, H. (1997) Leaf-specifically expressed genes for polypeptides destined for chloroplasts with domains of s70 factors of bacterial RNA polymerases in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA, 94, 14948-14953.
Last Modified: 12:00, May 28, 1998