Tetsuko Takabe

Effects of hydrogen peroxide and nitric oxide on both salt and heat stress tolerance in rice

Abstract Higher plants growing in natural environments experience various abiotic stresses. H2O2 and nitric oxide (NO) free radicals are produced and cause oxidative damage to plants under various abiotic stress conditions. However, in the present study, we found that pretreating rice seedlings with low levels (

Enhanced tolerance to salt stress in transgenic rice that overexpresses chloroplast glutamine synthetase.

The potential role of photorespiration in the protection against salt stress was examined with transgenic rice plants. Oryza sativa L. cv. Kinuhikari was transformed with a chloroplastic glutamine synthetase (GS2) gene from rice. Each transgenic rice plant line showed a different accumulation level of GS2. A transgenic plant line, G39-2, which accumulated about 1.5-fold more GS2 than the control plant, had an increased photorespiration capacity. In another line, G241-12, GS2 was almost lost and photorespiration activity could not be detected. Fluorescence quenching analysis revealed that photorespiration could prevent the over-reduction of electron transport systems. When exposed to 150 mM NaCl for 2 weeks, the control rice plants completely lost photosystem II activity, but G39-2 plants retained more than 90% activity after the 2-week treatment, whereas G241-12 plants lost these activities within one week. In the presence of isonicotinic acid hydrazide, an inhibitor of photorespiration, G39-2 showed the same salt tolerance as the control plants. The intracellular contents of NH4+ and Na+ in the stressed plants correlated well with the levels of GS2. Thus, the enhancement of photorespiration conferred resistance to salt in rice plants. Preliminary results suggest chilling tolerance in the transformant.

EXPRESSION OF THE BETAINE ALDEHYDE DEHYDROGENASE GENE IN BARLEY IN RESPONSE TO OSMOTIC STRESS AND ABSCISIC ACID

When subjected to salt stress or drought, some vascular plants such as barley respond with an increased accumulation of the osmoprotectant glycine betaine (betaine), being the last step of betaine synthesis catalyzed by betaine aldehyde dehydrogenase (BADH). We report here cloning and characterization of BADH cDNA from barley, a monocot, and the expression pattern of a BADH transcript. An open reading frame of 1515 bp encoded a protein which showed high homology to BADH enzymes present in other plants (spinach and sugar-beet) and in Escherichia coli. Transgenic tobacco plants harboring the clone expressed high levels of both BADH protein and its enzymatic activity. Northern blot analyses indicated that BADH mRNA levels increased almost 8-fold and 2-fold, respectively, in leaves and roots of barley plants grown in high-salt conditions, and that these levels decreased upon release of the stress, whereas they did not decrease under continuous salt stress. BADH transcripts also accumulate in response to water stress or drought, indicating a common response of the plant to osmotic changes that affect its water status. The addition of abscisic acid (ABA) to plants during growth also increased the levels of BADH transcripts dramatically, although the response was delayed when compared to that found for salt-stressed plants. Removal of plant roots before transferring the plants to high-salt conditions reduced only slightly the accumulation of BADH transcripts in the leaves.

Salt tolerance of transgenic rice overexpressing yeast mitochondrial Mn-SOD in chloroplasts

Abstract The potential role of superoxide dismutase (SOD) in the protection against salt stress was examined using transgenic rice plants. The coding region of the yeast mitochondrial Mn-SOD gene was fused with the chloroplast targetting signal of glutamine synthetase gene and introduced into rice protoplasts by electroporation. Immunogold labeling experiments revealed that the yeast Mn-SOD was accumulated in the chloroplasts of transgenic rice. The total SOD activity in the control plant was mostly attributed to the activity of cytosolic and chloroplastic Cu/Zn-SOD. Total SOD activity in the transformant was about 1.7-fold that of the control under non-stressed conditions. The photosynthetic electron transport rates of control and transgenic rice were similar under non-stressed conditions. Upon salt stress (100 mM NaCl), the SOD activities decreased in both plants, but the decrease was faster in the control plant. The activities of overexpressed Mn-SOD and cytosolic Cu/Zn-SOD did not change upon salt stress in either the transgenic or control plants, whereas the chloroplastic Cu/Zn-SOD activity in control rice decreased significantly. At high salinity, the ascorbate peroxidase activity of the transformant was about 1.5-fold higher than that in the control. These results suggest that increased levels of ascorbate peroxidase and high levels of chloroplastic SOD in the transformant are important factors for salt resistance in rice.

Cloning of peroxisomal ascorbate peroxidase gene from barley and enhanced thermotolerance by overexpressing in Arabidopsis thaliana

A full-length cDNA clone (HvAPX1) encoding a peroxisomal type ascorbate peroxidase was isolated from barley (Hordeum vulgare cv. Haruna-nijyo) leaves by differential display. The deduced amino acid sequence of the HvAPX1 gene had 75.3% homology to that from the Gossypium hirsutum glyoxysomal APX gene and 72.1% homology to that from the Arabidopsis thaliana peroxisomal APX gene, APX3. Southern blot analysis indicated that a single-copy gene in the barley genome encoded HvAPX1. Northern blot analysis showed that the HvAPX1 transcript increased remarkably in response to heat, salt and abscisic acid treatment. Induction was not caused by treatment with hydrogen peroxide. The HvAPX1 gene was introduced into A. thaliana under control of the 35S RNA promoter of the cauliflower mosaic virus. The transgenic plants were significantly more tolerant to heat stress as compared with the wild-type.

Exogenous Glycinebetaine Accumulation and Increased Salt-tolerance in Rice Seedlings

Exposure of 28-day-old rice seedlings for 6 days to 150 mm NaCl was found to induce drastic decreases in relative water contents, chlorophyll, and proteins in the leaves. This effect was largely prevened when before the exposure to NaCl the seedlings were treated for 4 days with 15 mm glycinebetaine. Although rice plants do not accumulate glycinebetaine endogenously, added glycinebetaine was found to be taken up by the roots and to accumulate in the leaves to reach a concentration of up to 5.0 μmol per gram fresh weight. The level is comparable with those of barley and wheat, which are well known glycinebetaine accumulators, under salt stress. The quantum yield of PSII was decreased by 27% under salt stress. This decrease was also largely prevented by glycinebetaine application.

Molecular cloning and functional characterization of two kinds of betaine-aldehyde dehydrogenase in betaine-accumulating mangrove Avicennia marina (Forsk.) Vierh.

Glycinebetaine is an important osmoprotectant in bacteria, plants, and animals, but only little information is available on the synthesis of glycinebetaine in tree plants. Among four mangrove species, glycinebetaine could be detected only in Avicennia marina. Pinitol was the main osmoprotectant in the other three species. The level of glycinebetaine in A. marina increased under high salinity. Betaine-aldehyde dehydrogenase (BADH) was detected in all four species, but choline monooxygenase could not be detected. A cDNA library was constructed from the leaves of A. marina. Two kinds of BADH cDNAs were isolated, one homologous to the spinach chloroplast BADH, and the other with unique residues SKL at the end of C-terminus. The BADH transcription levels of the former were higher than those of the latter. The levels of the former BADH increased at high salinity whereas those of the latter were independent of salinity. BADHs were expressed in Escherichia coli and purified. Two kinds of A. marina BADHs exhibited similar kinetic and stability properties, but were significantly different from those of spinach BADH. A. marina BADHs efficiently catalyzed the oxidation of betainealdehyde, but not the oxidation of ω-aminoaldehydes and were more stable at high temperature than the spinach BADH.

Overexpression of DnaK from a halotolerant cyanobacterium Aphanothece halophytica acquires resistance to salt stress in transgenic tobacco plants

Abstract To test the role of the heat shock protein DnaK/Hsp70 in salt tolerance, transgenic plants of Nicotina tabacum cv Petit Havana SR1 were made with DnaK1 from a halotolerant cyanobacterium Aphanothece halophytica ( A. halophytica ) overexpressed in the cytosol. The growth rate and photosynthetic activities of the transgenic and control tobacco plants were similar under non-stressed conditions. The CO 2 assimilation rate of the control plants decreased with increasing concentration of NaCl. After 3 days of treatment with 0.6 M NaCl, the CO 2 fixation rate decreased to 40% of that in the non-stressed plants whereas its activity in the transgenic plants was about 85% of that in the non-stressed plants. Similar results were observed for the stomatal transpiration. The sodium contents in leaves of the control plants were significantly increased by salt stress whereas those in the transgenic plants remained at levels similar to those in the non-stressed plants. Total protein contents and ribulose 1,5-bis phosphate carboxygenase and oxygenase (RuBisCO) levels were decreased by salt stress in both the transgenic and control plants but the decrease was slight in the transgenic tobacco. All these data clearly indicate that the expression of DnaK1 from a halotolerant cyanobacterium A. halophytica improved the salt tolerance of the tobacco plant.

Enhanced expression of a nuclease gene in leaves of barley plants under salt stress

We isolated a cDNA clone, Bnuc1, encoding a nuclease I from leaves of salt-stressed barley (Hordeum vulgare L. cv. Haruna-nijyo) by the differential display method. Northern blot analysis revealed that the transcript of Bnuc1 gene was increased dramatically in barley leaves under salt stress. The expression of Bnuc1 gene was also increased by exogenously applied abscisic acid (ABA) in leaves, but not by gibberellic acid (GA) during seed germination. Furthermore, Bnuc1 gene was expressed more in old leaves than in young leaves during both salt stress and natural senescence. Salt-inducible nuclease activity possibly corresponding to the Bnuc1 gene was detected, and was much higher in old leaves than in young leaves under salt stress.

Analysis of salt-inducible genes in barley roots by differential display.

Abstract To obtain insight into the comprehensive molecular characteristics related to the mechanisms of salt tolerance, we performed a large-scale screening of salt-inducible genes in barley roots by differential display. A comparative analysis of gene expression between control and salt-stressed conditions led to the detection of 218 cDNA clones induced by salt. Sequence analysis and database searching revealed that 133 cDNA clones have homology to known proteins. Twenty-four salt-inducible clones were identified as genes for signal transduction (e.g., phosphatidylinositol-4-phosphate-5-kinase, mitogen-activated protein kinase, transcription factor, receptor protein kinase, and protein phosphatase 2A). We also detected clones encoding glutathione reductase, thioredoxin-like protein, trehalose-6-phosphate synthetase, and heat shock proteins in the category of typical stress tolerance. Furthermore, we have obtained genes encoding membrane transporters, members of the P450 family, enzymes involved in RNA metabolism or function, and enzymes of sugar or amino acid metabolism. It must be noted that most genes were expressed strongly in roots, but only rarely or weakly in leaves. In addition, some clones were newly found as salt-inducible genes encoding SCARECROW, splicing factor and apoptosis protein. In this research, it was shown that differential display is a powerful tool for a large-scale cloning of cDNAs induced by salt and these results are very useful for understanding the mechanisms of plant salt tolerance.