Functional Analysis of Two NAC Transcription Factors and Transcriptome Analysis under Drought in Citrullus Colocynthis

Abstract

Citrullus colocynthis (L.) Schrad, closely related to watermelon (Citrullus lanatus L.), belongs to the Cucurbitaceae family. It can survive arid environments by maintaining its water content without wilting of the leaves or desiccation under severe stress conditions. NAC (NAM, ATAF1,2, CUC2) transcription factors play important roles in plant growth, development, and responses to abiotic and biotic stress. Two novel NAC transcription factors (CcNAC1 and CcNAC2) were isolated from Citrullus colocynthis. The characterization of the two genes was studied in order to gain an understanding of their function under abiotic stress conditions. Manipulation of transcription factors represents a potential strategy for development of transgenic stress tolerant plants. Promoter regions, transgentic GUS assays etc. studies results indicated that CcNAC1 and CcNAC2 may have multiple functions to regulate the plant’s defense responses to abiotic stress. Further studies on the functional role of these genes to different qualities of light and auxin were based on the in silico analysis of the CcNAC1 and CcNAC2 promoter regions, which revealed the presence of several light-associated motifs. The impact of both light and auxin on CcNAC1 and CcNAC2 gene expression was examined in C. colocynthis leaves, and using reporter (pCcNAC1, 2::GUS) lines in Arabidopsis. Furthermore the effects of constitutive overexpression (OE-CcNAC1, 2) lines in Arabidopsis were also examined under a range of conditions to confirm reporter line linkages. White, blue, red, and far red light treatments resulted in similar patterns of quantitative changes in CcNAC1and CcNAC2 expression in both species, with the highest transcript increases associated with red light. Photomorphogenic changes in Arabidopsis hypocotyls were correlated with changes in gene transcript levels. In the absence of light hypocotyls of OE-CcNAC1 and OE-CCNAC2 lines were significantly longer as compared to hypocotyls of wild type seedlings. The addition of exogenous auxin (+IAA) to growth medium also resulted in changes to the hypocotyl lengths of overexpression lines and spatiotemporal reporter line changes in Arabidopsis seedlings. Our data suggest that CcNAC1, 2 might be functionally important in the light signaling pathway, and appear connected to the phytohormone auxin. Different light and dark treatments resulted in quantitative and apatiotemporal changes in CcNAC1 and CcNAC2 expression patterns. This study points to the relationship between the auxin, light and NAC TFs. We also used high throughput mRNA Illumina sequencing technology and bioinformatic strategies to analyze the leaf transcriptome of C. colocynthis under drought treatment. Leaf samples following 4 days of water deficit treatment were used for RNA extraction for library construction and Illumina sequencing. qRT-PCR analysis of drought induced genes was performed to confirm the accuracy of RNA sequencing. More than 5038 whole cDNAs were identified and 2545 genes showed significantly changes during drought as compared with Day 1. Principle component analysis showed that drought was the major factor in regulation of the transcriptome changes and many candidate drought stress related genes were detected. Our data imply that there are transcriptional changes in C.colocynthis under drought stress, providing the understanding of the molecular regulation mechanism of plant drought resistance.