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.

Growth regulation mechanisms in higher plants under microgravity conditions - changes in cell wall metabolism.

During Space Shuttle STS-95 mission, we cultivated seedlings of rice (Oryza sativa L. cv. Koshihikari and cv. Tan-ginbozu) and Arabidopsis (Arabidopsis thaliana L. cv. Columbia and cv. etr1-1) for 68.5, 91.5, and 136 hr on board, and then analyzed changes in the nature of their cell walls, growth, and morphogenesis under microgravity conditions. In space, elongation growth of both rice coleoptiles and Arabidopsis hypocotyls was stimulated. Also, the increase in the cell wall extensibility, especially that in the irreversible extensibility, was observed for such materials. The analyses of the amounts, the structure, and the physicochemical properties of the cell wall constituents indicated that the decreases in levels and molecular masses of cell wall polysaccharides were induced under microgravity conditions, which appeared to contribute to the increase in the wall extensibility. The activity of certain wall enzymes responsible for the metabolic turnover of the wall polysaccharides was increased in space. By the space flight, we also confirmed the occurrence of automorphogenesis of both seedlings under microgravity conditions; rice coleoptiles showed an adaxial bending, whereas Arabidopsis hypocotyls elongated in random directions. Furthermore, it was shown that spontaneous curvatures of rice coleoptiles in space were brought about uneven modifications of cell wall properties between the convex and the concave sides.

A cytokinin-repressed gene in cucumber for a bHLH protein homologue is regulated by light.

CRR12 was identified as a cytokinin-repressed gene encoding a cucumber homologue for a basic region/helix-loop-helix protein. The level of CRR12 mRNA decreased in response to either cytokinins or light in etiolated cotyledons. The level was low in cotyledons and leaves of light-grown plants, but it increased during dark incubation.

The nitrate reductase gene isolated from DNA of cultured spinach cells.

The gene encoding nitrate reductase was cloned from the DNA isolated from cultured spinach (Spinacia oleracea cv. Hoyo) cells and sequenced. The clone contains 7612 nucleotides of the gene which consists of four exons interrupted by three introns. The transcription start site was determined by primer extension analysis and located 193 bp upstream of the ATG translation initiation codon. The 5'-flanking region contains a TATA box and CAAT box.

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.

The level of mRNA transcribed from psaL, which encodes a subunit of photosystem I, is increased by cytokinin in darkness in etiolated cotyledons of cucumber.

A cDNA clone for an mRNA whose level increased within 2 h of the start of treatment with N6-benzyladenine in etiolated cotyledons of cucumber was isolated by differential hybridization. The cDNA was homologous to psaL, which encodes subunit XI (PSI-L) of photosystem I. The accumulation of psaL mRNA was specifically induced by cytokinins or light.

Cytokinin induces a rapid decrease in the levels of mRNAs for catalase, 3-hydroxy-3-methylglutaryl CoA reductase, lectin and other unidentified proteins in etiolated cotyledons of cucumber.

Large-scale differential hybridization was performed to examine rapid changes in gene expression caused by a phytohormone, cytokinin, in etiolated cotyledons of cucumber (Cucumis sativus L.). We isolated 86 cDNA clones for mRNAs whose levels decreased within 2 h of the start of treatment with N6-benzyladenine (BA). Partial nucleotide sequences showed that some of the cDNAs were homologous to those for catalase, 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) and a lectin. This is the first report that the levels of the mRNAs for those proteins are regulated by a cytokinin in darkness. Together with previous results [Teramoto et al. (1993) Physiol. Plant. 87: 584, (1994) Planta 193: 573, (1995) Planta 196: 387], the present study suggests that the cytokinin act to lower the levels of mRNAs transcribed from various genes in etiolated cotyledons.

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 and t-zeatin), but not by adenine or 2,4-dichlorophenoxyacetic acid (auxin). The results are discussed in relation to the primary action of cytokinin.

A Polypeptide That Induces Flowering in Lemna paucicostata at a Very Low Concentration.

A flower-inducing substance of high molecular mass, extracted from Lemna paucicostata, was purified to homogeneity. It had characteristics of a polypeptide, with an amino-terminal sequence of Leu-Val-Gly-Asn-Thr, and induced formation of flower buds of L. paucicostata 151 at a concentration of 10(-10) molar.

A flower-inducing substance of high molecular weight from higher plants.

The flower-inducing activities of aqueous extracts of several plants were fractionated by gel filtration. Three major peaks, corresponding to molecular weights of about 120, 20 to 30, and 5 to 10 kilodaltons, were detected in extracts of Lemna, Pharbitis, and Brassica. The latter two peaks may be degradation products generated during the extraction procedure. In extracts of soybean seeds, only the peak of material of 120 kilodaltons was detected. This is the first published report of a high molecular mass substance with florigenic activity in Lemna plants. The florigenic substance had some properties associated with proteins (or polypeptides), but the activity was unaffected by treatment with proteinase K.

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 lambda gt11 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.

Molecular Cloning and Ethylene Induction of mRNA Encoding a Phytoalexin Elicitor-Releasing Factor, beta-1,3-Endoglucanase, in Soybean.

Soybean (Glycine max) beta-1,3-endoglucanase (EC 3.2. 1.39) is involved in one of the earliest plant-pathogen interactions that may lead to active disease resistance by releasing elicitor-active carbohydrates from the cell walls of fungal pathogens. Ethylene induced beta-1,3-endoglucanase activity to 2- to 3-fold higher levels in cotyledons of soybean seedlings. A specific polyclonal antiserum raised against purified soybean beta-1,3-endoglucanase was used to immunoprecipitate in vitro translation products, demonstrating that ethylene induction increased translatable beta-1,3-endoglucanase mRNA. Several cDNA clones for the endoglucanase gene were obtained by antibody screening of a lambda-gt11 expression library prepared from soybean cotyledons. Hybrid-select translation experiments indicated that the cloned cDNA encoded a 36-kilodalton precursor protein product that was specifically immunoprecipitated with beta-1,3-endoglucanase antiserum. Escherichia coli cells expressing the cloned cDNA also synthesized an immunologically positive protein. Nucleotide sequence of three independent clones revealed a single uninterrupted open reading frame of 1041 nucleotides, corresponding to a polypeptide of 347 residue long. The primary amino acid sequence of beta-1,3-endoglucanase as deduced from the nucleotide sequence was confirmed by direct amino acid sequencing of trypsin digests of the glucanase. The soybean beta-1,3-endoglucanase exhibited 53% amino acid homology to a beta-1,3-glucanase cloned from cultured tobacco cells and 48% homology to a beta-(1,3-1,4)-glucanase from barley. Utilizing the largest cloned cDNA (pEG488) as a hybridization probe, it was found that the increase in translatable beta-1,3-endoglucanase mRNA seen upon ethylene treatment of soybean seedlings was due to 50- to 100-fold increase in steady state mRNA levels, indicating that ethylene regulates gene expression of this enzyme important in disease resistance at the level of gene transcription.

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 (32)P-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.

Changes revealed by a tracer technique in the amino acid metabolism of thermodormant and non-dormant New York lettuce seeds.

The metabolism of glycerol-(14)C(U) in non-thermodormant New York lettuce seeds was compared with that in thermodormant ones in order to find metabolic changes and a possible osmotic substance(s) in the non-dormant seeds. The higher incorporation of radioactivity into cationic substances in the non-dormant seeds revealed rapid synthesis and large pool sizes for some amino acids, especially Gln and Glu, before radicle protrusion. The enormous accumulation of Gln and Glu in germinating seeds indicates that these amino acids may act as osmotic substances in the germination of New York lettuce seeds. There were no indications that an osmotic substance(s) is included in the neutral and acidic fractions of the 80%-ethanol extracts. Time course analyses of free amino acids showed changes in the metabolism of free amino acids, especially Arg and Asn, at the early stage (4 to 8 hr) of imbibition both in thermodormant and in non-dormant seeds. These data are evidence that the metabolism of amino acids in New York lettuce seeds was affected by temperature treatment from the early stage of imbibition.

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.

Effects of temperature, red light and hormones on the accumulation of free amino acids in osmotically growth-inhibited embryonic axes of New York lettuce seeds.

Free amino acids accumulated in non-thermodormant embryonic axes of New York lettuce seeds from the 16th to the 24th hour of imbibition, even when germination was prevented with 0.3 m mannitol. The accumulation of free amino acids in the embryonic axes was affected by temperature, red light and hormones (gibberellic acid, kinetin and abscisic acid), and was correlated with the growth potential of the axes. These data are evidence that the accumulation of free amino acids in the embryonic axes is not the result of axes-growth, but one of its causes.