Arabidopsis atDjC53 and atDjC32 gene DnaJ-like protein homologous to DnaJ-like protein was characterized for the functional analysis of DnaJ-like protein. It was shown that atDjC53 and atDjC32 RNA expression is induced by heat shock stress and atDjC53- and atDjC32-GFP was targeted to the nucleus of protoplasts. The atDjC53 and atDjC32 promoter (1 kb) was isolated and fused to the GUS reporter gene to investigate gene regulation of atDjC53 and atDjC32 specific to heat shock stress or to developmental organ in the transgenic lines. RNAi and overexpression construct was employed to generate atDjC53 and atDjC32 knock-out plants for the study of their function. Molecular function of atDjC53 and atDjC32 is discussed in relation to heat shock and also developmental stages in Arabidopsis.

Heat shock transcription factors (HSFs) are the major heat shock factors regulating the heat stress response. They participate in regulating the expression of heat shock proteins (HSPs), which are critical in the protection against stress damage and many other important biological processes. In this study, a genome-wide analysis was carried out to identify all HSFs soybean genes. Twenty six nonredundant HSF genes (GmHsf) were identified in the latest soybean genome sequence. Chromosomal location, protein domain and motif organization of GmHsfs were analyzed in soybean genome. The phylogenetic relationships, gene duplications and expression profiles of GmHsf genes were also presented in this study. According to their structural features, the predicted members were divided into the previously defined classes A–C, as described in Arabidopsis. Using RT-PCR, the expression patterns of 26 GmHsf genes were investigated under heat stress. The data revealed that these genes presented different expression levels in response to heat stress conditions. Real-time (q)RT-PCR was performed to investigate transcript levels of five GmHsfs in response to multiple abiotic stresses. Differential expression of five GmHsfs implies their role during abiotic stresses. Subcellular localization using GFP-fusion protein demonstrated that GmHsf12 and GmHsf34 were restricted to the nucleus and GmHsf28 was localized in the nucleus and cytoplasm in plant. The results provide a fundamental clue for understanding of the complexity of the soybean HSF gene family and cloning specific function genes in further studies and applications.

Salinity stress severely affects plant growth and development causing crop loss worldwide. Suaeda asparagoides is a salt-marsh euhalophyte widely distributed in southwestern foreshore of Korea. To isolate salt tolerance genes from S. asparagoides, we constructed a cDNA library from leaf tissues of S. asparagoides that was treated with 200 mM NaCl. A total of 1,056 clones were randomly selected for EST sequencing, and 932 of them produced readable sequence. By sequence analysis, we identified 538 unigenes and registered each in National Center for Biotechnology Information. The 80 salt stress related genes were selected to study their differential expression. Reverse Transcriptase-PCR and Northern blot analysis revealed that 23 genes were differentially expressed under the high salinity stress conditions in S. asparagoides. They are functionally diverse including transport, signal transduction, transcription factor, metabolism and stress associated protein, and unknown function. Among them dehydrin (SaDhn) and RNA binding protein (SaRBP1) were examined for their abiotic stress tolerance in yeast (Saccharomyces cerevisiae). Yeast overexpressing SaDhn and SaRBP1 showed enhanced tolerance to osmotic, freezing and heat shock stresses. This study provides the evidence that SaRBP1 and SaDhn from S.asparagoides exert abiotic stress tolerance in yeast. Information of salt stress related genes from S. asparagoides will contribute for the accumulating genetic resources to improve osmotic tolerance in plants.

We have isolated wound-inducible genes from soybean using suppression subtractive hybridization (SSH) method and were able to obtain the full-length clone of GmDjp1 gene encoding DnaJ-like protein. The full-length cDNA of GmDjp1 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 GmDjp1 gene. Northern blot analysis showed that the RNA expression of GmDjp1 gene is specifically induced by heat, NaCl, wounding and drought stresses. It was demonstrated that GmDjp1-GFP was targeted to the nucleus in tobacco cell. GmDjp1 overexpression plants showed more susceptible to salt and heat stress compared to WT. RNA expression level of Hsp18.2 and Hsp25.3-P was lower than that of WT during recovery after heat hock in plants. This indicates that GmDjp1 may play a negative regulator to stress responses in plants.

A low temperature-inducible cDNA designated as VrUBC1 from mungbean (Vigna radiata) was isolated by subtractive hybridization method. By rapid amplification of cDNA end technique, the full-length cDNA of VrUBC1 was obtained. The full-length cDNA of VrUBC1 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. VrUBC1 mRNA was induced by NaCl and ABA, but not by wounding and low temperature stress. It was shown that VrUBC1-GFP was localized to the cytoplasm in tobacco cell. To examine the function of VrUBC1, VrUBC1 was expressed in Escherichia coli as His-fusion protein. Purified VrUBC1-His recombinant protein was shown to have ubiquitination activity in vitro. For the in vivo functional analysis of VrUBC1, VrUBC1 was expressed in yeast ubc4/5 double mutant. Stress tolerance was tested in the VrUBC1 overexpressing Arabidopsis transgenic plants. We propose that VrUBC1 play an important role in protein degradation processes during abiotic stress in plants.