Plants have evolved complex signaling networks to regulate their growth and development. Some of these signaling components also play a crucial role in secondary metabolite biosynthesis. Among the signaling components identified to date, ELONGATED HYPOCOTYL 5 (HY5), a bZIP family transcription factor is the most investigated and known as the center of transcriptional network hub. However, HY5 has not been characterized from plants known to synthesize important secondary metabolites. In this study, based on homology search and phylogenetic analysis, HY5 has been identified from Nicotiana tobaccum, and characterized for its role in secondary plant product biosynthesis and stress response through developing overexpressing lines and CRISPR/Cas9-based knockout mutant plants. NtHY5 was able to complement the Arabidopsis thaliana hy5 mutant at molecular, morphological and biochemical levels. Overexpression of NtHY5 in tobacco led to the up-regulation of the phenylpropanoid pathway genes and enhanced the flavonoid content, whereas mutant plants had the opposite effect. Electrophoretic Mobility Shift Assay (EMSA) suggested that NtHY5 interacts with the promoter of NtMYB12, a transcription factor known to regulate flavonoid biosynthesis. In addition, NtHY5 enhanced the abiotic stress tolerance as evident by the salt tolerance ability of HY5 overexpressing lines by diminishing the ROS accumulation after salt treatment. These data provide credible evidence about the potential role of NtHY5 in light-mediated flavonoid biosynthesis, plant growth and abiotic stress tolerance in tobacco. The photomorphogenic mutant, Nthy5, developed in this study, will help in elucidating the role of the HY5 in different biological processes in tobacco.
MicroRNAs (miRNAs) are small non-coding RNAs that play a central role in regulating various developmental and biological processes. The expression of miRNAs is differentially modulated in response to various stresses. Based on the recent findings, it has been shown that some of the pri-miRNAs encode small regulatory peptides, microRNA-encoded peptides (miPEP). miPEPs are reported to regulate the growth and development of plants by modulating corresponding miRNA expression; however, the role of these peptides in different stresses has not been explored yet. Here, we reported that pri-miR408 encodes a small peptide, miPEP408, that regulates the expression of miR408, its targets, and associated phenotype in Arabidopsis. Plants overexpressing miR408 showed severe sensitivity under low sulphur (LS), Arsenite As(III) and LS+As(III) stress, while miR408 mutant developed through the CRISPR/Cas9 approach showed tolerance. Transgenic lines showed phenotypic alteration and modulation in the expression of genes involved in the sulphur reduction pathway and affect sulphate and glutathione accumulation. Similar to miR408 overexpressing lines, the exogenous application of synthetic miPEP408 or miPEP408 overexpression led to sensitivity in plants under LS, As(III) and combined LS+As(III) stress compared to control. This study suggests the involvement of miR408 and miPEP408 in heavy metal and nutrient deficiency responses.
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