Functional Characterization Of Three Stress-related NAC Transcription Factor Genes In Miscanthus Lutarioriparius

NAC transcription factors(TFs) play crucial roles in plant responses to abiotic and biotic stresses. Miscanthus lutarioriparius is one of Miscanthus species native to East Asia. It has attracted much attention as a potential lignocellulosic plant for bioenergy production; because of its high photosynthetic efficiency, superior biomass productivity as well as wide adaptability to various environments. However, little information of the functions of stress-related NAC TFs is available for M. lutarioriparius. In this study, a detailed functional characterization of three NAC TFs(Ml NAC4, Ml NAC5 and Ml NAC12) was carried out, the main results are as the followings:1. Phylogenetic analysis revealed that three NAC TFs(Ml NAC4, Ml NAC5 and Ml NAC12) were classified to ATAF subfamily of NAC proteins;2. The C-terminus YFP fusion constructs of three genes driven by Ca MV 35 S promoter were transiently expressed in tobacco epidermal cells, respectively. Subcellular localization of Ml NAC4/5/12-YFP fusion protein indicated that Ml NAC4/5/12 are nuclear proteins;3. The entire coding region, N-terminal domain and C-terminal domain of three genes were cloned into p GBKT7, respectively. The constructs and empty vector p GBKT7(negative control) were transformed into yeast strain AH109. Transactivation assay in yeast cells demonstrated that Ml NAC4/5/12 function as transcription activators and their activation domain are located in the C-terminus;4. A 1296 bp, 726 bp and 1465 bp DNA fragment of Ml NAC4/5/12 promoter were amplified by hi TAIL-PCR, respectively. Sequence analysis revealed that a number of stress-responsive related cis-acting elements were present in the Ml NAC4/5/12 promoter;5. Three promoter fragments were inserted upstream of GUS reporter gene in p CXGUS-P vector and transformed into Arabidopsis Col-0. The results indicated that p Ml NAC5::GUS was mainly expressed in the hypocotyl of 5-day-old seedlings, in the main veins of rosette leaves, at the junction of the stem and the petiole, in anthers and the abscission zone of mature siliques. In contrast, p Ml NAC12::GUS expression was relatively ubiquitous, which was detected in various tissues examined;6. Overexpression of Ml NAC5 and Ml NAC12 in Arabidopsis had impacts on a wide range of plant development processes including dwarfism, leaf senescence, leaf morphology, and late flowering under normal growth conditions;7. Ml NAC5 and Ml NAC12 overexpression both leads to ABA hypersensitivity in transgenic Arabidopsis during germination and the root elongation stage. In comparison to WT control, the transgenic lines exhibited much lower germination rates as well as much shorter root length under ABA treatment on 1/2 MS plate;8. Overexpression of Ml NAC5 in Arabidopsis significantly enhanced drought and cold tolerance. RT-q PCR analysis showed that some stress-responsive marker genes were significantly up-regulated in transgenic lines compared to WT.