We isolated a DREB orthologue, MtDREB1C, from Medicago truncatula. Its deduced protein contains an AP2 domain of 57 amino acids. Yeast one-hybrid assay revealed that MtDREB1C specifically bound to the dehydration-responsive element (DRE) and activated the expression of HIS3 and LacZ reporter genes. In a transcriptional activation assay, coexpression of the MtDREB1C cDNA resulted in much higher (21.2 times) transactivation of the LacZ reporter gene than experiments performed without MtDREB1C. Transformation of Medicago revealed that overexpression of MtDREB1C suppressed shoot growth, and enhanced the freezing tolerance of M. truncatula. The MtDREB1C gene was transformed into China Rose (Rosa chinensis Jacq.) driven by Arabidopsis rd29A promoter. Southern-blot analysis showed that the target gene was integrated into the genome of a surviving transgenic rose plant. Northern-blot analysis illustrated that robust expression of MtDREB1C was only activated under stress conditions, and the expressed MtDREB1C mRNA reached maximum accumulation 10 h following freezing treatment. The performance of the transgenic line under freezing stress was superior to untransformed controls. This transgenic plant continued to grow, flowered under unstressed conditions, and was phenotypically normal. These facts indicate that the MtDREB1C gene, isolated from Medicago truncatula and driven by the Arabidopsis rd29A promoter, enhanced freezing tolerance in transgenic China Rose significantly without any obvious morphological or developmental abnormality.

Potato (Solanum tuberosum L.) is the fourth most important crop worldwide. Potato virus A (PVA) is one of the most harmful viruses infecting potatoes. However, the molecular mechanisms governing the responses to PVA infection in potato at the transcriptional and post-transcriptional levels are not well understood. In this study, we performed both mRNA and small RNA sequencing in potato leaves to identify the genes and miRNAs involved in the response to PVA infection. A total of 2,062 differentially expressed genes (DEGs) and 201 miRNAs (DEMs) were identified, respectively. Gene ontology (GO) and KEGG analysis revealed that these DEGs were involved in the transduction of pathogen signals, transcriptional reprogramming, induction of hormone signaling, activation of pathogenesis-related (PR) genes, and changes in secondary metabolism. Small RNA sequencing revealed 58 miRNA-mRNA interactions related to PVA infection. Some of the miRNAs (stu-miR482d-3p, stu-miR397-5p, etc) which target PR genes showed negative correlations between the DEMs and DEGs. Eight of the DEGs and three DEMs with their target genes were further validated by quantitative real time-PCR (qRT-PCR). Overall, this study provides a transcriptome-wide insight into the molecular basis of resistance to PVA infection in potato leaves and potenital candidate genes for improving resistance cultivars.

We isolated a DREB homologue gene, MtDREB2A, from Medicago truncatula. Its deduced protein contains an AP2 domain of 59 amino acids. The expression of MtDREB2A was significantly induced in roots by salt and drought treatments. Using megaprimer PCR, we deleted a Ser/ Thr-rich coding region between residues 142 and 190, and transformed MtDREB2A to a constitutive form, namely, MtDREB2a. Yeast one-hybrid assay revealed that both MtDREB2A and MtDREB2a specifically bound to the dehydration-responsive element (DRE) and activated the expression of the reporter genes of HIS3 and LacZ. Analysis of transcription activities of the proteins in yeast indicated that MtDREB2a could activate the expression of reporter gene, whereas MtDREB2A could not. Overexpression of MtDREB2a in transgenic M. truncatula resulted in significant dwarfed seedling. These results suggested that MtDREB2A functioned specially in response to salt and drought stresses in M. truncatula and that deletion of the Ser/Thr-rich region between residues 142 and 190 activated the transcriptional activation ability of MtDREB2A.