Cloning And Functional Analysis Of Wheat TaNAC29 And TaNAC2D In Response To Abiotic Stresses

Plants are frequently challenged by unfavorable environmental conditions, including high salinity, drought, and extreme temperatures, leading to severe negative effect on plant growth and development. Upon exposure to unfavorable environmental conditions, many related genes are induced. Transcription factors(TFs) are the one of such related gene family. Among TFs families, NAC(NAM, ATAF, and CUC) TFs and their corresponding cis-acting sequences act as molecular switches to regulate temporal and spatial gene expression. In recent years, an array of research on NACs involved in processes of growth and development, senescence, and in response to abiotic stress have been reported, while the functional analysis of NACs in the wheat crop is quite limited. In the present study, ten TaNAC genes were identified and cloned from bread wheat(Triticum aestivum L. cv. Chinese Spring). The biological functions of TaNAC29 and TaNAC2 D were further investigated. The main results are as follows:1) Through the wheat EST database searching, we succeeded in assembling and cloning of 10 wheat TaNAC genes, 9 of which were new TaNAC genes identified in wheat.2) The full-length cDNA of a novel NAC transcription factor gene, designated TaNAC29, was isolated from wheat. Sequence alignment suggested that TaNAC29 was located on chromosome 2BS. TaNAC29 was localized to the nucleus in wheat protoplasts, and proved to have transcriptional activation activities in yeast. Moreover, we demonstrated that L motif was sufficient for transactivation activity of TaNAC29. TaNAC29 was expressed at a higher level in the wheat leaves, and expression levels were much higher in mature senescing leaves than in young green leaves, indicating that TaNAC29 might be involved in the senescence process in wheat. The qRT-PCR analysis revealed that TaNAC29 transcripts were increased following treatments with salt, PEG6000, H2O2, and ABA in leaf and root of wheat. To examine TaNAC29 biological function, transgenic Arabidopsis plants overexpressing TaNAC29 were generated. Germination and root length assays of transgenic plants demonstrated that TaNAC29 overexpression plants had enhanced tolerances to high salinity and dehydration, and exhibited an ABA-hypersensitive response. When grown in the greenhouse, TaNAC29-overexpression plants showed the same tolerance response to salt and drought stresses at both the vegetative and reproductive growth period, and had delayed bolting and flowering in the reproductive growth period. Moreover, TaNAC29-overexpression plants accumulated lesser MDA, H2O2, while had higher SOD and CAT activities under high salinity and/or dehydration stress. Additionally, TaNAC29 significantly accelerated dark-induced leaf senescence in transgenic wheat, indicating that TaNAC29 positively regualted leaf senescence in plants.3) TaNAC2 D gene is located on chromosome 5BL. Transactivation activity assays also confirmed that TaNAC2 D was a transcriptional activator, and the C-terminal region(173-327) was enough to activate expression of reporter genes in yeast. TaNAC2D-GFP fusion protein was localized to the nucleus in wheat mesophyll protoplasts, which confirmed its function as a transcription regulator. Transcript levels of TaNAC2 D in the wheat leaf were higher than in other organs. Gene expression pattern analysis showed that TaNAC2 D was upregulated by high salinity, dehydration, ABA, and H2O2 treatments. To investigate biological function of TaNAC2 D, transgenic Arabidopsis overexpressing TaNAC2 D was generated. The 35-day-old transgenic Arabidopsis lines grown in the soil were treated with high salinity and drought stresses, respectively, TaNAC2 Doverexpression plants showed more sensitivity than that of WT plants. However, TaNAC2D-overexpression plants had enhanced tolerances to high salinity, dehydration and oxidative stress during seed germination and post-germination processes. To further understand these two different phenotypes, the expression levels of related marker genes were analyzed. The 14-day-old Arabidopsis seedlings grown on 1/2 MS medium were submerged in salt solution for 6 h or dehydrated for 1 h, respectively, the transcript levels of NCED3, RD29 A and RD29 B were all significantly up-regulated in TaNAC2D-overexpression plants than in WT plants. However, the 35-day-old plants grown in soil were incubated for 6 h in salt solution or dehydrated for 3 h, respectively, the expression levels of these marker genes were almost significantly down-regulated in transgnic plants compared to WT plants.In conclusion, TaNAC29-overexpression plants conferred salt and drought stresses tolerance during the whole plant growth period. TaNAC2D-overexpression plants also improved tolerances to mutiple abiotic stresses during seed germination and post-germination growth periods, while the 35-day-old transgenic plants grown in soil displayed more sensitivity to salt and drought stresses compared with WT plants. These findings shed some light on the complex mechanisms and role of NAC in a plants' response to environmental stresses.