Overexpression of TaMYB4 Confers Freezing Tolerance in Arabidopsis thaliana.

Freezing stress is one of the main factors limiting the growth and yield of wheat. In this study, we found that TaMYB4 expression was significantly upregulated in the tillering nodes of the strong cold-resistant winter wheat variety Dongnongdongmai1 (Dn1) under freezing stress. Weighted gene co-expression network analysis, qRT-PCR and protein-DNA interaction experiments demonstrated that monodehydroascorbate reductase (TaMDHAR) is a direct target of TaMYB4. The results showed that overexpression of TaMYB4 enhanced the freezing tolerance of transgenic Arabidopsis. In TaMYB4 overexpression lines (OE-TaMYB4), AtMDHAR2 expression was upregulated and ascorbate-glutathione (AsA-GSH) cycle operation was enhanced. In addition, the expression of cold stress marker genes such as AtCBF1, AtCBF2, AtCBF3, AtCOR15A, AtCOR47, AtKIN1 and AtRD29A in OE-TaMYB4 lines was significantly upregulated. Therefore, TaMYB4 may increase freezing tolerance as a transcription factor (TF) in Arabidopsis through the AsA-GSH cycle and DREB/CBF signaling pathway. This study provides a potential gene for molecular breeding against freezing stress.

Transcriptome analysis of the winter wheat Dn1 in response to cold stress.

BACKGROUND: Heilongjiang Province has a long and cold winter season (the minimum temperature can reach -30 ℃), and few winter wheat varieties can safely overwinter. Dongnongdongmai1 (Dn1) is the first winter wheat variety that can safely overwinter in Heilongjiang Province. This variety fills the gap for winter wheat cultivation in the frigid region of China and greatly increases the land utilization rate. To understand the molecular mechanism of the cold response, we conducted RNA-sequencing analysis of Dn1 under cold stress. RESULTS: Approximately 120,000 genes were detected in Dn1 under cold stress. The numbers of differentially expressed genes (DEGs) in the six comparison groups (0 ℃ vs. 5 ℃, -5 ℃ vs. 5 ℃, -10 ℃ vs. 5 ℃, -15 ℃ vs. 5 ℃, -20 ℃ vs. 5 ℃ and -25 ℃ vs. 5 ℃) were 11,313, 8313, 15,636, 13,671, 14,294 and 13,979, respectively. Gene Ontology functional annotation suggested that the DEGs under cold stress mainly had "binding", "protein kinase" and "catalytic" activities and were involved in "oxidation-reduction", "protein phosphorylation" and "carbohydrate metabolic" processes. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that the DEGs performed important functions in cold signal transduction and carbohydrate metabolism. In addition, major transcription factors (AP2/ERF, bZIP, NAC, WRKY, bHLH and MYB) participating in the Dn1 cold stress response were activated by low temperature. CONCLUSION: This is the first study to explore the Dn1 transcriptome under cold stress. Our study comprehensively analysed the key genes involved in cold signal transduction and carbohydrate metabolism in Dn1 under cold stress. The results obtained by transcriptome analysis could help to further explore the cold resistance mechanism of Dn1 and provide basis for breeding of cold-resistant crops.

Glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase genes of winter wheat enhance the cold tolerance of transgenic Arabidopsis.

In this study, winter wheat G6PDH (TaG6PDH) and 6PGDH (Ta6PGDH) were investigated. Both their expression and their activity were upregulated under cold stress, suggesting that TaG6PDH and Ta6PGDH positively respond to cold stress in winter wheat. Exogenous abscisic acid (ABA) treatment markedly increased the expression and activity levels of TaG6PDH and Ta6PGDH in winter wheat under cold stress. Subsequently, TaG6PDH-and Ta6PGDH were overexpressed in Arabidopsis, and showed stronger reactive oxygen species (ROS)-scavenging ability and higher survival rate compared with wild-type (WT) plants under cold stress. In addition, we found that TaG6PDH and Ta6PGDH overexpression can promote the oxidative pentose phosphate pathway (OPPP) in the cytoplasm and peroxisomes of Arabidopsis. In summary, Arabidopsis overexpressing TaG6PDH and Ta6PGDH showed improved cold tolerance.

tae-miR399-UBC24 Module Enhances Freezing Tolerance in Winter Wheat via a CBF Signaling Pathway.

Although the regulation of Pi homeostasis by miR399 has been studied in various plant species, its underlying molecular mechanism in response to freezing stress is still poorly understood. In this work, we found that the expression of tae-miR399 and its target gene TaUBC24 in the tillering nodes of the strong cold-resistant winter wheat cultivar Dongnongdongmai1 (Dn1) was not only significantly altered after severe winters but also responsive to short-term freezing stress. TaUBC24 physically interacted with TaICE1. Enhanced freezing tolerance was observed for tae-miR399-overexpressing Arabidopsis lines. Under freezing stress, overexpression of tae-miR399 ultimately decreased the expression of AtUBC24, inhibiting the degradation of AtICE1, which increased the expression of genes involved in the CBF signaling pathway and starch metabolism and promoted the activities of antioxidant enzymes. These results will improve our understanding of the molecular mechanism through which the miR399-UBC24 module plays a cardinal role in regulating plant freezing stress tolerance through mediation of downstream pathways.