Toshisuke Iwasaki

Molecular Cloning of a Novel Importin a Homologue from Rice, by Which Constitutive Photomorphogenic 1 (COP1) Nuclear Localization Signal (NLS)-Protein Is Preferentially Nuclear Imported*

Nuclear import of proteins that contain classical nuclear localization signals (NLS) is initiated by importin α, a protein that recognizes and binds to the NLS in the cytoplasm. In this paper, we have cloned a cDNA for a novel importin α homologue from rice which is in addition to our previously isolated rice importin α1a and α2, and we have named it rice importin α1b. In vitro binding and nuclear import assays using recombinant importin α1b protein demonstrate that rice importin α1b functions as a component of the NLS-receptor in plant cells. Analysis of the transcript levels for all three rice importin α genes revealed that the genes were not only differentially expressed but that they also responded to dark-adaptation in green leaves. Furthermore, we also show that the COP1 protein bears a bipartite-type NLS and its nuclear import is mediated preferentially by the rice importin α1b. These data suggest that each of the different rice importin α proteins carry distinct groups of nuclear proteins, such that multiple isoforms of importin α contribute to the regulation of plant nuclear protein transport.

Functional Analysis and Intracellular Localization of Rice Cryptochromes

Blue-light-receptor cryptochrome (CRY), which mediates cotyledon expansion, increased accumulation of anthocyanin, and inhibition of hypocotyl elongation, was first identified in Arabidopsis. Two Arabidopsis cryptochromes (AtCRY1 and AtCRY2) have been reported to be localized to the nucleus. However, there is no information on the cryptochromes in monocotyledons. In this study, we isolated two cryptochrome cDNAs, OsCRY1 and OsCRY2, from rice (Oryza sativa) plants. The deduced amino acid sequences of OsCRY1 and OsCRY2 have a photolyase-like domain in their N termini and are homologous to AtCRY1. To investigate the function of OsCRY1, we overexpressed a green fluorescence protein-OsCRY1 fusion gene in Arabidopsis and assessed the phenotypes of the resulting transgenic plants. When the seedlings were germinated in the dark, no discernible effect was observed. However, light-germinated seedlings showed pronounced inhibition of hypocotyl elongation and increased accumulation of anthocyanin. These phenotypes were induced in a blue-light-dependent manner, indicating that OsCRY1 functions as a blue-light-receptor cryptochrome. We also examined the intracellular localization of green fluorescence protein-OsCRY1 in the transgenic plants. It was localized to both the nucleus and the cytoplasm. We identified two nuclear localization domains in the primary structure of OsCRY1. We discuss the relationship between the function and intracellular localization of rice cryptochromes by using additional data obtained with OsCRY2.

Cloning of a cDNA that encodes farnesyl diphosphate synthase and the blue-light-induced expression of the corresponding gene in the leaves of rice plants☆

A cDNA encoding farnesyl diphosphate synthase (FPPS), a key enzyme in isoprenoid biosynthesis, was isolated from a cDNA library constructed from mRNA that had been prepared from etiolated rice (Oriza sativa L. variety Nipponbare) seedlings after three hours of illumination by a subtraction method. The putative polypeptide deduced from the 1289 bp nucleotide sequence consisted of 353 amino acids and had a molecular mass of 40 676 Da. The predicted amino acid sequence exhibited high homology to those of FPPS from Arabidopsis (73% to type 1, 72% to type 2) and white lupin (74%). Southern blot analysis showed that the rice genome might contain only one gene for FPPS. The highest level of expression of the gene was demonstrated in leaves by RNA blot analysis. Moreover, light, in particular blue light, effectively enhanced expression of the gene.

Cloning of cDNA encoding the rice 22 kDa protein of Photosystem II (PSII-S) and analysis of light-induced expression of the gene

Cloning of rice cDNA encoding the chlorophyll-binding 22 kDa protein of Photosystem II (PSII-S) and the light-induced expression of the gene are reported. One of the light-responsive cDNA clones, isolated by screening with a light-specific subtracted cDNA probe, was shown to encode PSII-S of rice. Genomic Southern analysis suggested that the PSII-S gene, psbS, is a single-copy gene in rice. A brief exposure to red light induced a severalfold increase in the steady state level of PSII-S transcripts in etiolated seedlings. The red light effect was reversed by far-red light, suggesting involvement of phytochrome in the PSII-S gene regulation. Prolonged exposure (3 h) to blue light, however, revealed a much stronger effect than red light on the accumulation of PSII-S transcripts in the etiolated seedlings. In dark-adapted green plants, prolonged exposure to blue light induced re-accumulation of transcripts encoding PSII-S, whereas red light had little effect.

Evolutionary origin of two genes for chloroplast small heat shock protein of tobacco

Two different cDNA clones for the chloroplast small heat shock protein (smHSP) were isolated from tobacco (Nicotiana tabacum cv. Petit Havana SR1). One of the cDNAs (type I) has a full-length open reading frame (ORF) of the smHSP of 26.6 kDa. By contrast, the other one (type II) contains an additional nucleotide that causes the frame shift inside a putative ORF for the smHSP. If this nucleotide is neglected, type II cDNA encodes the smHSP that is 89% identical to that encoded by type I cDNA. Southern blot and polymerase chain reaction (PCR) analyses with genomic DNA indicated that tobacco has two different smHSP genes while two ancestors of tobacco, N. sylvestris and N. tomentosiformis, have a single gene that each corresponds to one of the two genes of tobacco. It was also found that one of the tobacco genes has an ORF for the smHSP disrupted by nucleotide insertion in the same way as type II cDNA, while both ancestor genes have a functional ORF. These results suggest that the two smHSP genes of tobacco had been derived from the two ancestor species, and that one of the two genes had been disrupted by nucleotide insertion during the course of the evolution of tobacco. Northern blot and reverse transcription (RT)-PCR analyses demonstrated that both the tobacco genes are expressed upon heat stress, exhibiting different dependences on temperature.