Role of lncRNAs in cis- and trans-regulatory responses to salt in Populus trichocarpa.

Long non-coding RNAs (lncRNAs) are emerging as versatile regulators in diverse biological processes. However, little is known about their cis- and trans-regulatory contributions in gene expression under salt stress. Using 27 RNA-seq data sets from Populus trichocarpa leaves, stems and roots, we identified 2988 high-confidence lncRNAs, including 1183 salt-induced differentially expressed lncRNAs. Among them, 301 lncRNAs have potential for positively affecting their neighboring genes, predominantly in a cis-regulatory manner rather than by co-transcription. Additionally, a co-expression network identified six striking salt-associated modules with a total of 5639 genes, including 426 lncRNAs, and in these lncRNA sequences, the DNA/RNA binding motifs are enriched. This suggests that lncRNAs might contribute to distant gene expression of the salt-associated modules in a trans-regulatory manner. Moreover, we found 30 lncRNAs that have potential to simultaneously cis- and trans-regulate salt-responsive homologous genes, and Ptlinc-NAC72, significantly induced under long-term salt stress, was selected for validating its regulation of the expression and functional roles of the homologs PtNAC72.A and PtNAC72.B (PtNAC72.A/B). The transient transformation of Ptlinc-NAC72 and a dual-luciferase assay of Ptlinc-NAC72 and PtNAC72.A/B promoters confirmed that Ptlinc-NAC72 can directly upregulate PtNAC72.A/B expression, and a presence/absence assay was further conducted to show that the regulation is probably mediated by Ptlinc-NAC72 recognizing the tandem elements (GAAAAA) in the PtNAC72.A/B 5' untranslated region (5'-UTR). Finally, the overexpression of Ptlinc-NAC72 produces a hypersensitive phenotype under salt stress. Altogether, our results shed light on the cis- and trans-regulation of gene expression by lncRNAs in Populus and provides an example of long-term salt-induced Ptlinc-NAC72 that could be used to mitigate growth costs by conferring plant resilience to salt stress.

Drought-Responsive NAC Transcription Factor RcNAC72 Is Recognized by RcABF4, Interacts with RcDREB2A to Enhance Drought Tolerance in Arabidopsis.

RcNAC72, a key transcription factor that may respond to drought stress in Rosa chinensis 'Old Blush', was selected in our previous study. In the present study, we found that RcNAC72 is localized in the nucleus and is a transcriptional activator. RcNAC72 expression could be significantly induced by drought, low temperature, salt as well as abscisic acid (ABA) treatment. Analysis of the promoter revealed that multiple abiotic stress and hormone response elements were located in the promoter region. The promoter could respond to drought, low temperature, salt and ABA treatments to activate GUS gene expression. Overexpressing RcNAC72 in Arabidopsis thaliana enhanced sensitivity to ABA and tolerance to drought stress. Silencing of RcNAC72 by virus-induced gene silencing (VIGS) in rose leaves significantly reduced leaf water loss tolerance and leaf extension capacity. Physical interaction of RcNAC72 with RcDREB2A was shown by means of the yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. RcABF4 was demonstrated to be able to bind to the promoter of RcNAC72 by means of the yeast one-hybrid (Y1H) assay. These results provide new insights into the regulatory network of RcNAC72 response to drought stress in roses.