Nobuaki Hayashida

The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression

The complete nucleotide sequence (155 844 bp) of tobacco (Nicotiana tabacum var. Bright Yellow 4) chloroplast DNA has been determined. It contains two copies of an identical 25 339 bp inverted repeat, which are separated by a 86 684 bp and a 18 482 bp single‐copy region. The genes for 4 different rRNAs, 30 different tRNAs, 39 different proteins and 11 other predicted protein coding genes have been located. Among them, 15 genes contain introns. Blot hybridization revealed that all rRNA and tRNA genes and 27 protein genes so far analysed are transcribed in the chloroplast and that primary transcripts of the split genes hitherto examined are spliced. Five sequences coding for proteins homologous to components of the respiratory‐chain NADH dehydrogenase from human mitochondria have been found. The 30 tRNAs predicted from their genes are sufficient to read all codons if the ‘two out of three’ and ‘U:N wobble’ mechanisms operate in the chloroplast. Two sequences which autonomously replicate in yeast have also been mapped. The sequence and expression analyses indicate both prokaryotic and eukaryotic features of the chloroplast genes.

Two genes that encode Ca2+-dependent protein kinases are induced by drought and high-salt stresses in Arabidopsis thaliana

Two cDNA clones, AATCDPK1 and cATCDPK2, encoding Ca2+-dependent, calmodulin-independent protein kinases (CDPK) were cloned from Arabidopsis thaliana and their nucleotide sequences were determined. Northern blot analysis indicated that the mRNAs corresponding to the ATCDPK1 and ATCDPK2 genes are rapidly induced by drought and high-salt stress but not by low-temperature stress or heat stress. Treatment of Arabidopsis plants with exogenous abscisic acid (ABA) had no effect on the induction of ATCDPK1 or ATCDPK2. These findings suggest that a change in the osmotic potential of the environment can serve as a trigger for the induction of ATCDPK1 and ATCDPK2. Putative proteins encoded by ATCDPK1 and ATCDPK2 which contain open reading frames of 1479 and 1488 bp, respectively, are designated ATCDPK1 and ATCDPK2 and show 52% identity at the amino acid sequence level. ATCDPK1 and ATCDPK2 exhibit significant similarity to a soybean CDPK (51 % and 73%, respectively). Both proteins contain a catalytic domain that is typical of serine/threonine protein kinases and a regulatory domain that is homologous to the Ca2+-binding sites of calmodulin. Genomic Southern blot analysis suggests the existence of a few additional genes that are related to ATCDPK1 and ATCDPK2 in the Arabidopsis genome. The ATCDPK2 protein expressed in Escherichia coli was found to phosphorylate casein and myelin basic protein preferentially, relative to a histone substrate, and required Ca2+ for activation.

ATMPKs: a gene family of plant MAP kinases in Arabidopsis thaliana

We previously reported two cDNAs for MAP kinases (cATMPK1 and cATMPK2) from a dicot plant, Arabidopsis thaliana. We describe here the cloning and characterization of five additional cDNAs encoding novel MAP kinases in Arabidopsis, CATMPK3, cATMPK4, cATMPK5, cATMPK6, and cATMPK7. The amino acid residues corresponding to the sites of phosphorylation (Thr‐Glu‐Tyr) that are involved in the activation of animal MAP kinases are conserved in all the seven putative ATMPK proteins. Genes for MAP kinases in Arabidopsis constitute a family that contains more than seven members. Sequence analysis suggests that there are at least three subfamilies in the family of Arabidopsis genes for MAP kinases.

The gene for the 9 kd polypeptide, a possible apoprotein for the iron-sulfur centers A and B of the photosystem I complex, in tobacco chloroplast DNA

SummaryThe gene for the 9 kd polypeptide (a possible apoprotein for the iron-sulfur centers A and B) of photosystem I has been located in the small single-copy region of tobacco chloroplast DNA. This gene (psaC) was identified by comparing the N-terminal amino acid sequence of the spinach 9 kd polypeptide with the entire sequence of tobacco chloroplast genome. The gene organization is ndhE (101 codons) — 263 by spacer — psaC (S1 codons) — 94 by spacer - ndhD (509 codons). Northern blot hybridization revealed that psaC is transcribed in the chloroplasts. The deduced amino acid sequence and secondary structure are presented. The predicted polypeptide is rich in cysteine residues and contains a unique repeated sequence.

Effects of methyl jasmonate and elicitor on the activation of phenylalanine ammonia-lyase and the accumulation of scopoletin and scopolin in tobacco cell cultures

Abstract The effects of methyl jasmonate (MJ) and elicitor on the phenylpropanoid metabolism in tobacco was studied. It was found that suspended cells of tobacco, responded to MJ and elicitor from the plant pathogenic fungus Fusarium solani with differential formation of phenolic compounds. Both MJ and elicitor induced phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity to comparable levels. Northern hybridization showed that the increase in PAL activity was preceded by PAL mRNA accumulation. PAL was induced transcriptionally and enzymatically faster in elicitor-treated cells as compared to MJ-treated cells. HPLC of extracts from tobacco cells and their culture filtrate treated with MJ, showed an induction of the syntheses of coumarin-scopoletin and scopolin. This effect was not seen in elicitor-treated or control cell cultures. Scopolin was mainly found within the cell (99%), whereas scopoletin accumulated in the culture filtrate (90%). We conclude that though MJ mimics stress, indicating its possible role in the plant defense metabolism, responses of tobacco cells to MJ and elicitor are distinct.

The gene encoding a calcium-dependent protein kinase located near the sbe1 gene encoding starch branching enzyme I is specifically expressed in developing rice seeds

A gene (spk) encoding a Ca(2+)-dependent protein kinase (SPK) is located in the region immediately upstream of the sbe1 gene encoding a starch branching enzyme. The spk gene is specifically expressed in developing seeds and its expression pattern is very similar to those of genes encoding starch-synthesizing enzymes such as sbe1 and waxy, seed lipid-synthesizing enzymes, as well as genes encoding seed storage proteins. A full-length spk cDNA was isolated from a cDNA library constructed from developing seeds. The deduced amino acid sequence showed that SPK has a high degree of homology to soybean and carrot Ca(2+)-dependent protein kinase, both of which contain calmodulin domains. The calmodulin domain, as well as the catalytic subdomain consensus regions of protein kinases are highly conserved in SPK. These results suggest that a tissue- and stage-specific protein kinase, SPK, is involved in the synthesis of seed storage compounds during seed development. They also strongly suggest that Ca2+ is required for seed development.

Identification of a new gene in the chloroplast genome encoding a low-molecular-mass polypeptide of photosystem-II complex

Low‐molecular‐mass polypeptides in spinach photosystem II membranes were separated by SDS‐polyacrylamide gel electrophoresis. The partial amino acid sequence of one of the polypeptides was determined. Comparison of this sequence with the entire nucleotide sequence of the tobacco chloroplast genome revealed that this polypeptide is encoded in the chloroplast genome. The gene for the polypeptide, designated as psbK, is located between the genes for tRNASer and tRNAGln in the large single‐copy region and oriented in the direction opposite to the tRNA genes. The amino acid sequence deduced from the gene indicates that the translation product consists of 98 amino acid residues and its 62nd residue corresponds to the amino‐terminus of the mature polypeptide. The putative polypeptide consists of 37 amino acid residues with a molecular mass of 4285 Da and has a single membrane‐spanning segment. Northern blot hybridization analysis revealed that psbK was transcribed in the chloroplast.

TWO GENES THAT ENCODE RIBOSOMAL-PROTEIN S6 KINASE HOMOLOGS ARE INDUCED BY COLD OR SALINITY STRESS IN ARABIDOPSIS THALIANA

We have isolated two closely related cDNA clones (cATPK19 and cATPK6) with homology to protein‐serine/threonine kinases from Arabidopsis thaliana using the polymerase chain reaction (PCR). The deduced amino acid sequences of the ATPK19 and ATPK6 contain all 11 conserved regions of the catalytic domain of protein kinases, and have homology to p70 ribosomal S6 kinases (52%). ATPK19 and ATPK6 have putative PEST regions in their N‐ and C‐terminal regions, and these regions also contain putative phosphorylation sites that are recognized by casein kinases or proline‐directed protein kinases such as cdc2, MAP kinase, and p54 MAP‐2 kinase (SAPK). The transcription levels of the ATPK19 and ATPK6 genes rapidly and markedly increased when plants were subjected to cold or high‐salt stresses. These observations suggest that ATPK19 and ATPK6 may function in the adaptation of plant cells to cold or high‐salt conditions, providing an understanding of the role of protein phosphorylation in plant responses to environmental stresses.

Optical properties of diatom silica frustule with special reference to blue light

Using fresh water diatom, identified as Melosira variance which is growing faster than other algae in the ponds, we characterized the ultrastructure of its silica frustule by electron microscopic observations. On hypothesizing that the ultrastructure has some meanings in photosynthesis, behaviors of a single frustule toward light irradiation were analyzed using an optical fiber. We found that the frustule absorbed light mainly in the blue wavelength region. The theoretical analyses of optical properties supported experimental results and suggested a strong interaction between blue light and inner silica materials. It is speculated that the interaction served for partial extinction of excessive irradiation of blue light and for enhancing photosynthesis of the diatom.

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.