Abstract
Plant transcription factors involved in stress responses are generally classified by their involvement in either the abscisic acid (ABA)-dependent or the ABA-independent regulatory pathways. A stress-associated NAC gene from rose (Rosa hybrida), RhNAC3, was previously found to increase dehydration tolerance in both rose and Arabidopsis. However, the regulatory mechanism involved in RhNAC3 action is still not fully understood. In this study, we isolated and analyzed the upstream regulatory sequence of RhNAC3 and found many stress-related cis-elements to be present in the promoter, with five ABA-responsive element (ABRE) motifs being of particular interest. Characterization of Arabidopsis thaliana plants transformed with the putative RhNAC3 promoter sequence fused to the β-glucuronidase (GUS) reporter gene revealed that RhNAC3 is expressed at high basal levels in leaf guard cells and in vascular tissues. Moreover, the ABRE motifs in the RhNAC3 promoter were observed to have a cumulative effect on the transcriptional activity of this gene both in the presence and absence of exogenous ABA. Overexpression of RhNAC3 in A. thaliana resulted in ABA hypersensitivity during seed germination and promoted leaf closure after ABA or drought treatments. Additionally, the expression of 11 ABA-responsive genes was induced to a greater degree by dehydration in the transgenic plants overexpressing RhNAC3 than control lines transformed with the vector alone. Further analysis revealed that all these genes contain NAC binding cis-elements in their promoter regions, and RhNAC3 was found to partially bind to these putative NAC recognition sites. We further found that of 219 A. thaliana genes previously shown by microarray analysis to be regulated by heterologous overexpression RhNAC3, 85 are responsive to ABA. In rose, the expression of genes downstream of the ABA-signaling pathways was also repressed in RhNAC3-silenced petals. Taken together, we propose that the rose RhNAC3 protein could mediate ABA signaling both in rose and in A. thaliana.
Highlights
Drought, or dehydration, is one of the major limiting factors for plant growth, development, and productivity and plants have evolved a range of physiological, biochemical and molecular responses to promote drought stress tolerance [1]
Drought or dehydration-induced transcription factors (TFs) have been isolated from many plant species and demonstrated to be involved in drought tolerance, such as DREB2A, DREB2C, AREB1, and WRKY57 from A. thaliana [3,4], OsbZIP46 from Oryza sativa [5], TaMYB30-B from Triticum aestivum [6], RhNAC2 from Rosa hybrida [7], and ThbZIP1 from Tamarix hispida [8]
Subsequent sequence analysis of this region revealed a number of putative cis-elements, including elements associated with abscisic acid (ABA), cold, pathogen and wounding responses (Figure 1, Figure S1 and Table S2)
Summary
Dehydration, is one of the major limiting factors for plant growth, development, and productivity and plants have evolved a range of physiological, biochemical and molecular responses to promote drought stress tolerance [1]. TFs involved in stress responses are typically classified as being involved either in ABA-dependent or the ABA-independent regulatory pathways [9] and structure and sequence analyses of the promoters of ABA-dependent TF genes have identified many stress-related ciselements. These include the G-box (CACGTG, a MYC recognition site), the dehydration-responsive element/C-repeat (DRE/ CRT) and the ABA-responsive element [10,11]. Microarray analysis further revealed that constitutive overexpression of ABA-related TF genes, such as ABO3 and MYB96, generally increases the expression of downstream ABA-responsive genes, and results in drought tolerance [18,19]
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