Two NAC transcription factors from Citrullus colocynthis, CcNAC1, CcNAC2 implicated in multiple stress responses

NAC (no apical meristem, Arabidopsis transcription activation factor 1 and 2, cup-shaped cotyledon 2) transcription factors (TFs) play important roles in plant growth, development, and responses to abiotic and biotic stress. Two novel NAC TFs were isolated from Citrullus colocynthis, a highly drought-tolerant cucurbit species: CcNAC1 and CcNAC2 each with conserved A-E NAC domains. Subcellular location of CcNAC1 and CcNAC2 investigated via transient expression of 35S::CcNAC1::GFP and 35S::CcNAC2::GFP fusion constructs in Arabidopsis protoplasts, revealed nuclear localization. The transactivation ability of CcNACs was examined in the GAL4 yeast assay system, and showed that only the C-terminal domain of CcNAC1 has the ability to activate reporter genes LacZ and His3. The CcNAC genes accumulated in a tissue-specific manner with expression levels in male flowers of C. colocynthis higher than leaves, hypocotyls or roots. Genome walking was used to isolate the CcNAC1 and CcNAC2-promoter regions. A high number of stress-related sequence motifs were detected, especially in the CcNAC1 promoter. C. colocynthis seedlings were treated with PEG, abscisic acid, salicylic acid (SA), jasmonic acid (JA), H2O2, ethylene, gibberellic acid (GA), wounding or salt. High CcNAC1 expression levels were detected following JA application, and wounding, while high CcNAC2 levels followed treatment with GA, JA, SA, and wounding, indicative of differential regulation of these stress responsive TFs in this cucurbit species.