The StEPEBP gene, which had previously been isolated from the potato plant, was introduced into potato plants under the control of the cauliflower mosaic virus (CaMV) 35S promoter for constitutive expression. The resulting transgenic plants showed visibly less cold damage compared with the empty vector transgenic control plants when both plants were stored in a -6degreesC freezer for 24 hours. In addition, this study attempted to isolate the StEPEBP regulated genes using a 3.1K cDNA chip spotted with the clones isolated from the cold-specific red pepper library. The microarray and Northern blot results revealed that 39 genes had a significantly differential expression level in the StEREBP transgenic plants. Of 39 genes, 34 and 5 genes were found to be up- and down-regulated, respectively. The genes for HSP90, a lichenase precursor, PR-1, a hexose transporter, dehydrin, peroxidase calreticulin, tyramine hydroxycinnamoyl transferase, the RD22 protein and the receptor protein kinase-related protein appeared to be up-regulated by StEREBP. However, the genes for the fruit ripening protein, the major-latex like protein and secretory peroxidase appeared to be down-regulated by StEREBP. In addition, the expression profiles of all 39 genes in response to cold, salt, ABA and drought stress were examined at the transcript level.

Changes of expression in cold-regulated mRNA levels in potato (Solanum tuberosum L. cv. Superior) were analyzed. A total of 12,000 cDNAs was subjected to reverse Northern blot analysis using ^(32)P-dCTP- labeled first strand cDNA generated from the total RNA isolated from 25℃- and 4℃-treated potato plants. A total of 245 cDNA clones were sequenced from a cDNA library constructed from the cold treatment. Based on the BLAST results, 103 cDNA clones were found to be redundant. The analyzed genes were classified into 12 groups according to their putative functions, where the 20.2 % of group I was associated with energy metabolism, 13.1％ of group XI with cell rescue and defense, 2.6％ of group Ⅷwith signal transduction. Among the cDNA clones up-regulated by cold treatment from the results of the reverse Northern blot analysis, 32 were used for the Northern blot analysis. Most of the cold-treated clones showed overexpression compared with the control, while some showed down-regulation. In general, it was found that cold stress related genes were overexpressed more than two-folds at 4℃ treatment.

Exploring the tolerant mechanism of crop plants to abiotic stresses causing decreased yield is one important research undertaking. The first step to achieve this goal is through isolation of genes that are regulated by such stresses. This study attempted to isolate genes that respond to cold temperature, one of the major abiotic stresses of crops. First, cDNA library with RNA extracted from vegetative leaf, stem, and root of potato plants treated at 4 degreesC and 25 degreesC for 8 hrs was constructed. To evaluate transcriptional regulation by abiotic stresses like cold (4 degreesC), salt (250 mM NaCl), drought (air drying, for 24 hrs), and pathogen treatment (zoospore treatment of Phytophthora infestans), 100 genes were spotted onto DNA chip. Results showed that seven clones of cold stress protein were up-regulated by cold treatment, while six clones of ADP-ribosylation factor-like protein were up-regulated by salt treatment. Pathogen up-regulated the expression of ubiquitin carrier and drought stress up-regulated the expressions of cysteine protease and photosystem II homologue. Expression of some clones showing up-regulation by cold treatment in cDNA microarray was compared with that of Northern blot analysis. Although the results of the microarray analysis were not completely consistent with that of the Northern blot analysis, there was a very strong relationship between the two analyses.

Complementary DNA for a gene encoding trehalose phosphorylase (TP) that reversibly catalyzes trehalose synthesis and degradation from alpha-glucose-1-phosphate (alpha-Glc-1-P) and glucose was cloned from Pleurotus sajor-caju. The cDNA of P. sajor-caju TP (designated PsTP, GenBank Accession No. AF149777) encodes a polypeptide of 751 amino acids with a deduced molecular mass of 83.7 kDa. The PsTP gene is expressed in mycelia, pilei, and stipes of fruiting bodies. Trehalose phosphorylase PsTP was purified from PsTP-transformed Escherichia coli. The enzyme catalyzes both the phosphorolysis of trehalose to produce alpha-Glc-1-P and glucose, and the synthesis of trehalose. The apparent K(m) values for trehalose and Pi in phosphorolytic reaction at pH 7.0 were 74.8 and 5.4 mM, respectively. The PsTP gene complemented Saccharomyces cerevisiae Deltatps1, Deltatps2 double-mutant cells, allowing their growth on glucose medium. Furthermore, yeast transformed with PsTP produced 2-2.5-fold more trehalose than non-transformants or cells transformed with empty vector only.

In many organisms, trehalose protects against several environmental stresses, such as heat, desiccation, and salt, probably by stabilizing protein structures and lipid membranes. Trehalose synthesis in yeast is mediated by a complex of trehalose-6-phosphate synthase (TPS1) and trehalose-6-phosphate phosphatase (TPS2). In this study, genes encoding TPS1 and TPS2 were isolated from Zygosaccharomyces rouxii (designated ZrTPS1 and ZrTPS2, respectively). They were functionally identified by their complementation of the tps1 and tps2 yeast deletion mutants, which are unable to grow on glucose medium and with heat, respectively. Full-length ZrTPS1 cDNA is composed of 1476 nucleotides encoding a protein of 492 amino acids with a molecular mass of 56 kDa. ZrTPS2 cDNA consists of 2843 nucleotides with an open reading frame of 2700 bp, which encodes a polypeptide of 900 amino acids with a molecular mass of 104 kDa. The amino acid sequence encoded by ZrTPS1 has relatively high homology with TPS1 of Saccharomyces cerevisiae and Schizosaccharomyces pombe, compared with TPS2. Western blot analysis showed that the antibody against S. cerevisiae TPS1 recognizes ZrTPS1. Under normal growth conditions, ZrTPS1 and ZrTPS2 were highly and constitutively expressed, unlike S. cerevisiae TPS1 and TPS2. Salt stress and heat stress reduced the expression of the ZrTPS1 and ZrTPS2 genes, respectively.

A 1.2-kb full-length cDNA sequence of a glyceraldehyde-3-phosphate dehydrogenase (GPD) gene was isolated from the mushroom, Pleurotus sajor-caju. The full-length cDNA of the GPD gene consists of 1248 nucleotides, predicted to encode a 36-kDa polypeptide consisting of 335 amino acid residues. Sequence analysis revealed that the GPD gene has more than 72-78% amino acid sequence homology with those of other Basidiomycetes. Expression of the GPD gene increased when P. sajor-caju was treated with various abiotic stresses, such as salt, cold, heat, and drought. There was an eightfold induction by drought treatment. Salt and cold stress induced four- and twofold induction of GPD gene expression, respectively. There was also a fivefold induction by heat stress. The GPD gene exhibits different expression patterns under different stress conditions. It reached its maximum expression level within two hours under cold or heat treatment. The mRNA levels of this gene increased proportionally to increasing treatment time under salt or dry conditions. Because the expression of GPD was significantly increased, we tested whether GPD could confer abiotic stress resistance when it was introduced into yeast cells. For this, a transgenic yeast harboring P. sajor-caju GPD was generated under the control of a constitutively expressed GAL promoter. The results from biofunctional analyses with GPD yeast transformants showed that GPD yeast transformants had significantly higher resistance to cold, salt, heat, and drought stresses.

In yeast, trehalose-6-phosphate synthase is a key enzyme for trehalose biosynthesis, encoded by the structural gene TPS1. Trehalose affects sugar metabolism as well as osmoprotection against several environmental stresses, such as heat and desiccation. The TPS1 gene of Saccharomyces cerevisiae was engineered under the control of the CaMV 35S promoter for constitutive expression in transgenic potato plants by Ti-plasmid of Agrobacterium-mediated transformation. The resulting TPS1 transgenic potato plants exhibited various morphological phenotypes in culture tubes, ranging from normal to severely retarded growth, including dwarfish growth, yellowish lancet-shaped leaves, and aberrant root development. However, the plants recovered from these negative growth effects when grown in a soil mixture. The TPS1 transgenic potato plants showed significantly increased drought resistance. These results suggest that the production of trehalose not only affects plant development but also improves drought tolerance.