Go Takeba

Three cDNA sequences coding for glutamine synthetase polypeptides in Oryza sativa L.

Atsushi Sakamoto i, Masahiro Ogawa 3, Takehiro Masumura l, Daisuke Shibata 1, Go Takeba 2, Kunisuke Tanaka 1 and Shoji Fujii 1 1Laboratory of Biochemistry, Faculty of Agriculture, Kyoto Prefectural University, Shimogamo, Kyoto 606, Japan; 2Laboratory of Applied Biology, Faculty of Living Science, Kyoto Prefectural University, Shimogamo, Kyoto 606, Japan; 3Laboratory of Food Production, The Research Institute for Food Science, Kyoto University, Ufi, Kyoto 611, Japan

cDNA cloning of an mRNA encoding a sulfur-rich 10 kDa prolamin polypeptide in rice seeds

Using a rice maturing seed pUC9 expression library, we isolated a cDNA clone corresponding to 10 kDa sulfurrich prolamin by immunoscreening. A longer cDNA clone was obtained from a λgtll library by plaque hybridization using this 32P-labeled cDNA as a probe. A polypeptide sequence composed of 134 amino acids was deduced from the nucleotide sequence. A 24 amino acid signal peptide was assigned by computer calculation for the membrane spanning region and Edman sequencing of the purified mature polypeptide. Remarkably, 20% of methionine and 10% of cysteine were found in the mature polypeptide as well as high contents of glutamine, and hydrophobic amino acids. Part of the amino acid sequence was homologous with a conserved cysteine-rich region found in other plant prolamins. Two repeats of amino acid sequence were found in the polypeptide.

Noncoding RNA for CR20, a cytokinin-repressed gene of cucumber

The CR20 gene was identified as a cytokinin-repressed gene in excised cotyledons of cucumber. We determined the sequences of some CR20 cDNAs with different structures and sequenced genomic clones for CR20. This gene consisted of three exons, and there were at least three types of transcript, which seemed to be generated by alternative splicing of the second intron. None of the CR20 transcripts included a long open reading frame (ORF). We isolated a cDNA of Arabidopsis thaliana with cucumber CR20 cDNA as a probe. This cDNA for a gene designated AtCR20-1 also lacked a long ORF. A region of 180 nucleotides was conserved in the CR20 RNA of cucumber and the AtCR20-1 RNA of Arabidopsis, although the homology was relatively low when the entire sequences were compared. Each conserved region consisted of seven elements, and seems to form stable secondary structure. These suggest that CR20 RNA may function as an RNA that is not translated into a protein.

Phytochrome-mediated activation of the gene for cytosolic glutamine-synthetase (GS1) during imbibition of photosensitive lettuce seeds

A full-length cDNA encoding glutamine synthetase was isolated from a λgt11 library constructed from the poly(A)+ RNA isolated from lettuce seeds incubated under red light. The nucleotide sequence of the cDNA and the deduced sequence of amino acids showed a high degree of homology to those of the cytosol-type glutamine synthetase from other plants. Northern and dot-blot analyses of poly(A)+ RNA extracted from the seeds incubated under various light conditions showed that the activation of the gene for cytosolic glutamine-synthetase during imbibition of lettuce seeds is directly or indirectly regulated by phytochrome.

Hypergravity-induced changes in gene expression in Arabidopsis hypocotyls

Under hypergravity conditions, the cell wall of stem organs becomes mechanically rigid and elongation growth is suppressed, which can be recognized as the mechanism for plants to resist gravitational force. The changes in gene expression by hypergravity treatment were analyzed in Arabidopsis hypocotyls by the differential display method, for identifying genes involved in hypergravity-induced growth suppression. Sixty-two cDNA clones were expressed differentially between the control and 300 g conditions: the expression levels of 39 clones increased, whereas those of 23 clones decreased under hypergravity conditions. Sequence analysis and database searching revealed that 12 clones, 9 up-regulated and 3 down-regulated, have homology to known proteins. The expression of these genes was further analyzed using RT-PCR. Finally, six genes were confirmed to be up-regulated by hypergravity. One of such genes encoded 3-hydroxy-3-methylglutaryl-Coenzyme A reductase (HMGR), which catalyzes a reaction producing mevalonic acid, a key precursor of terpenoids such as membrane sterols and several types of hormones. The expression of HMGR gene increased within several hours after hypergravity treatment. Also, compactin, an inhibitor of HMGR, prevented hypergravity-induced growth suppression, suggesting that HMGR is involved in suppression of Arabidopsis hypocotyl growth by hypergravity. In addition, hypergravity increased the expression levels of genes encoding CCR1 and ERD15, which were shown to take part in the signaling pathway of environmental stimuli such as temperature and water, and those of the alpha-tubulin gene. These genes may be involved in a series of cellular events leading to growth suppression of stem organs under hypergravity conditions.

Isolation of a cDNA clone for a cytokinin-repressed gene in excised cucumber cotyledons.

Rapid changes in gene expression were studied during incubation of cucumber (Cucumis sativus L.) cotyledons with cytokinins in darkness. Complementary-DNA clones for mRNAs whose levels decreased within 4 h of treatment with N6-benzyladenine (BA) were isolated by differential hybridization. One of them (CR9) was sequenced. It is 588 bp long, and would encode a protein consisting of 137 amino-acid residues and having a molecular mass of 15 kDa. The sequence shows a high homology with a light-induced gene from rice. Northern blot analysis of the CR9 transcript showed the level of the mRNA (0.7 kb) to decrease tenfold within 4 h of BA treatment, i.e. well before BA-induced cotyledon expansion was observed. The repression became greater with increasing concentration of BA (10−8–10−5 M). The expression of the CR9 gene was repressed specifically by cytokinins (BA, isopentenyladenine andt-zeatin), but not by adenine or 2,4-dichlorophenoxyacetic acid (auxin). The results are discussed in relation to the primary action of cytokinin.

Expression Pattern and Gene Structure of Phenylalanine Ammonia-Lyase in Pharbitis nil

PAL gene expression were examined in flower buds and irradiated hypocotyls in Pharbitis nil. PAL activity and transcript levels were correlated with the accumulation of anthocyanin. Both in flower buds and hypocotyls, transcript levels, PAL activity, and then the amount of anthocyanin, increased. The PAL transcript was abundant in flower buds for a few days before flower opening. But the increase in PAL transcript induced by irradiation was temporal in hypocotyls. Phytochrome was shown to be involved in inducing the accumulation of anthocyanin in hypocotyls. To examine the mechanism regulating the expression of the PAL gene, the gene was cloned and sequenced, and the promoter region was compared with that of other PALs. The gene had two exons separated by an intron of 989 bp with consensus sequences at the intron/exon border. The predicted primary structure of the PAL protein consists of 711 amino acids. The promoter region was AT-rich and there were sequences similar to box 1, box 2, an AT-1 binding site and a G box. The role of PAL in the accumulation of anthocyanin is discussed.

Changes in expression of two cytokinin-repressed genes, CR9 and CR20, in relation to aging, greening and wounding in cucumber

The expression of two cytokinin-repressed genes, CR9 and CR20, was investigated in cucumber (Cucumis sativus L.). In excised cotyledons, the level of the CR20 transcript markedly decreased in the early periods of treatment with N6-benzyladenine (BA), i.e. well before BA-induced expansion of cotyledons was observed. The repression of CR20 was BA-dose dependent and highly specific for cytokinins. These features were similar to those of CR9 reported previously (H. Teramoto et al., 1994, Planta 193, 573–579). Furthermore, levels of the CR9 and CR20 transcripts decreased during the early phase of greening and soon after wounding of cotyledons. The levels were much lower in young leaves than in mature or senescent leaves. Because cytokinins are thought to control greening and aging of leaves, and to mediate response to wounding, the expression of these two genes could be closely related to the action of such hormones. However, there were some differences between the expression of CR9 and CR20, i.e. the pattern of diurnal changes and the transcript levels in roots. Therefore, some other factors in addition to cytokinins appear to differentially affect the expression of these two genes. Several transcripts of CR20 of different lengths (0.8–2.3 kb) were detected by Northern blot analysis. No long open reading frames could be detected in two CR20 cDNAs with different structures.

Phytochrome-mediated accumulation of free amino acids in embryonic axes of New York lettuce seeds.

Red light given to New York lettuce seeds incubated on a 0.3 m mannitol solution increased the amount of free amino acids in the embryonic axes, far-red light reversed this effect. Germination in response to red light given at various times during imbibition was closely related to the amount of free amino acids accumulated in the axes in response to the red light. The analyses of the free amino acids that accumulated in the axes of seeds incubated on 0.3 m mannitol indicated that the free amino acids that accumulated on red-light irradiation were produced by the degradation of storage protein. This is evidence that red-light-increased germination may be caused by the accumulation of free amino acids in the embryonic axes of New York lettuce seeds.

Accumulation of free amino acids in the tips of non-thermodormant embryonic axes accounts for the increase in the growth potential of New York lettuce seeds

Accumulation of free amino acids took place only in growing axes during the first 24 hr of imbibition at 18°C. A ninhydrin-positive section was shown to be located at the tips of the axes by a histochemical technique. The amount of free amino acids accumulated just at the breaking of the seed coats in the non-dormant axes was great enough to account for the increase in the growth potential, i.e., the force to rupture the testa. About 72% of the accumulated free amino acids in the non-thermodormant embryonic axes was comprised of glutamine and glutamate.