We isolated two low temperature-inducible cDNAs designated as MLT5 and MLT31 from mungbean by subtractive suppression hybridization method. By rapid amplification of cDNA end technique, the full-length cDNAs designated MLT5 and MLT31 were obtained. The full-length cDNA of MLT5 contains an open reading frame of 324 nucleotides coding for 107 amino acids. The full-length cDNA of MLT31 contains an open reading frame of 444 nucleotides in length and capable of specifying a 16.5-kDa protein of 148 amino acids (aa) with an isoelectric point of 7.72. RNA expression of MLT5 was strongly induced by low temperature in the early time and also by wounding, NaCl and ABA. MLT31 mRNA was induced by NaCl and ABA but not by wounding and low temperature stress. MLT5 encodes a protein of unknown function, of which targeting site is predicted to be chloroplast membrane protein. To examine the localization of MLT5, MLT5-GFP was expressed in tobacco cells. It was shown that MLT5-GFP was localized to surface of chloroplast in tobacco cell. To examine the function of MLT31, MLT31 was expressed in Escherichia coli as His-fusion protein. Purified MLT31-His recombinant protein will be tested for ubiquitination activity in vitro. In addition, for the in vivo functional analysis of MLT31, MLT31 will be expressed in yeast ubc9 knock-out mutant. We propose that MLT5 and MLT31 play an important role in signal pathway of abiotic stress in plants.

We isolated wound-inducible genes using suppression subtractive hybridization (SSH) method and were able to obtain to clone W3 gene encoding dnaJ like protein. The full-length cDNA of W3 is 689 bp with an open reading frame (ORF) consisting of 163 amino acid (aa). Genomic southern blot confirmed that soybean genome has two copies of W3 gene. Northern blot analysis was also carried out for the gene expression during heat, NaCl, drought, wounding stresses. The expression of W3 gene specifically induced by heat, NaCl, wounding and drought stress. Using GFP fusion vector, W13-GFP was targeted both to nucleus. For the functional analysis of W3, His-tagged W3 recombinant protein was heterologously expressed in E. coli. The W3 recombinant cells showed enhanced heat tolerance compared to that of vector control cells. We suggest that dnaJ-like W3 protein function as molecular chaperone in the nucleus of the plant cell during various stresses.

Photorespiration reduces carbon fixation rate, but is essential process in plant. Photorespiration involves reactions in chloroplasts, peroxisomes, and mitochondria. In photorepiratory peroxisome, alanine glyoxylate aminotransferase (AGT) catalyzes alanine and glyoxylate into glycine and pyruvate. We isolated a low temperature-inducible cDNA encoding AGT from mungbean leaves. The full-length cDNA, designated as MLT9, contains an open reading frame of 1,203 nucleotides coding for a protein of 401 amino acids. Genomic DNA blotting showed that the mungbean genome has one copy of MLT9. MLT9 mRNA was induced not only by low temperature but also by drought stress, but ABA and NaCl did not induce RNA expression of MLT9. In mungbean, AGT activity was higher in the non-stressed leaves compared to the low-temperature treated leaves. Based on GFP/RFP targeting experiment, GFP-MLT9 fusion protein and SKL-RFP, a peroxisome marker, were colocalized to peroxisome in tobacco protoplasts. This suggests that peroxisomal MLT9 plays a role in photorespiratory metabolism in response to low temperature and drought stress.

We isolated low temperature inducible genes using suppression subtractive hybridization (SSH) method and were able to obtain to clone MLT7 gene encoding peroxiredoxin and aminotransferase. The full-length cDNA of MLT7 is 1,049 bp with an open reading frame (ORF) consisting of 261 amino acid (aa). Genomic southern blot confirmed that mungbean genome has two copies of MLT7 gene. Northern blot analysis was also carried out for the gene expression during ABA, NaCl, drought, wounding and H2O2 stresses. The expression of MLT7 gene significantly decreased by ABA, NaCl and drought stress, but wounding and H2O2 stress significantly induced MLT7 gene expression. Especially, H2O2 strongly induced the MLT7 gene expression. The expression of MLT7 gene during low temperature stress started to increase in 3 h after treatment, and than slightly decreased and again increased at 24 h. Using GFP fusion vector, GFP-MLT7 was targeted both to mitochondria and chloroplast. However, it was mostly targeted to mitochondria and partially targeted to chloroplast. For the functional analysis of MLT7, MLT7 recombinant protein was heterologously expressed in E. coli. The MLT7 recombinant cells showed enhanced antioxidant activity compared to that of vector control cells.

We isolated wound-inducible genes using suppression subtractive hybridization (SSH) method and were able to obtain to clone w123 gene encoding dnaJ like protein. The full-length cDNA of w123 is 689 bp with an open reading frame (ORF) consisting of 163 amino acid (aa). Genomic southern blot confirmed that soybean genome has two copies of w123 gene. Northern blot analysis was also carried out for the gene expression during heat, NaCl, drought, wounding stresses. The expression of w123 gene specifically induced by heat, NaCl, wounding and drought stress. Using GFP fusion vector, w123-smGFP was targeted both to nucleus. For the functional analysis of w123, His-tagged w123 recombinant protein was heterologously expressed in E. coli. The w123 recombinant cells showed enhanced heat tolerance compared to that of vector control cells. We suggest that dnaJ-like w123 protein function as molecular chaperone in the nucleus of the plant cell during various stresses.