Overexpressing hemp salt stress induced transcription factor genes enhances tobacco salt tolerance

Although NAM ‘no apical meristem’-ATAF1/2 ‘arabidopsis transcription activation factor 1/2’-CUC2 ‘cup-shaped cotyledon 2’ (NAC) transcription factors (TFs) have been recognized to play important roles in plant responses to various abiotic stresses, the possible functions and potential mechanism of NAC TF genes towards salt resistance in hemp (Cannabis sativa L., Cs) is poorly understood. Therefore, three novel hemp NAC genes (CsNAC1, CsNAC2, and CsNAC3) up-regulated significantly under salt stress were cloned, and their functional characterization in salt tolerance were investigated in tobacco (Nicotiana tabacum L., Nt) for the first time. The proteins CsNAC1, CsNAC2, and CsNAC3 were predicted to be localized in nucleus when transiently expressed in hemp stem derived protoplasts. The proteins CsNAC1 and CsNAC2 were revealed as putative members of the ATAF subfamily sharing high similarities in tertiary structure, while the protein CsNAC3 was a homolog of the AtNAC3 subfamily in Arabidopsis [Arabidopsis thaliana (L.) Heynh., At]. Overexpression vectors of these three genes were constructed driven by CaMV35S and successfully transferred into tobacco plants (cultivar K326). The physiological and metabolic response of CsNAC2-overexpressing transgenic tobacco to salt (NaCl) stress was quite similar to those of the wild type (WT). In contrast, in CsNAC1-and CsNAC3-overexpressing transgenic tobacco, the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and the contents of chlorophyll, soluble protein and soluble sugar increased significantly, accompanied by a remarkable decrease in the malonaldehyde (MDA) content. Furthermore, all the six key stress-responsive genes were remarkably up-regulated when exposed to salt stress. The improved growth phenotype and physiological regulation ability of CsNAC1- and CsNAC3-overexpressing transgenic tobacco indicate the genes CsNAC1 and CsNAC3 may function as positive regulators in plant response to salt stress and have potential value in transgenic breeding to improve the salt tolerance in hemp.