Light-dependent expression and accumulation of miR408-encoded peptide, miPEP408, is regulated by HY5 in Arabidopsis.

Recent studies propose that primary transcripts of miRNAs (pri-miRNAs) contain small Open Reading Frames (ORFs) capable of encoding miRNA-encoded peptides (miPEPs). These miPEPs can function as transcriptional regulators for their corresponding pri-miRNAs, ultimately enhancing mature miRNA accumulation. Notably, pri-miR408 encodes the functional peptide miPEP408, regulating expression of miR408 and its target genes, providing plant tolerance to stresses. While miPEPs are crucial regulators, the factors governing them are have not been studied in detail. Here, we explored the light-dependent regulation of miPEP408 in Arabidopsis. Expression analysis during dark-light transitions revealed light-induced transcription and accumulation of the miPEP408. As the promoter of miR408 contains cis-acting elements responsible for binding to the bZIP-type transcription factor ELONGATED HYPOCOTYL5 (HY5), known for light-mediated regulation in plants, we studied its involvement in the regulation of miR408. Analysis of HY5 mutant (hy5-215), complemented line (HY5OX/hy5), and CONSTITUTIVE PHOTOMORPHOGENIC 1 mutant (cop1-4) plants supported HY5's positive regulation of miPEP408. Grafting and GUS assays further suggested the role of HY5 as a shoot-root mobile signal inducing light-dependent miPEP408 expression. This study underscores the regulatory impact of light on small peptides, exemplified by miPEP408, mediated by the key transcription factor HY5.

Small but mighty: Peptides regulating abiotic stress responses in plants.

Throughout evolution, plants have developed strategies to confront and alleviate the detrimental impacts of abiotic stresses on their growth and development. The combat strategies involve intricate molecular networks and a spectrum of early and late stress-responsive pathways. Plant peptides, consisting of fewer than 100 amino acid residues, are at the forefront of these responses, serving as pivotal signalling molecules. These peptides, with roles similar to phytohormones, intricately regulate plant growth, development and facilitate essential cell-to-cell communications. Numerous studies underscore the significant role of these small peptides in coordinating diverse signalling events triggered by environmental challenges. Originating from the proteolytic processing of larger protein precursors or directly translated from small open reading frames, including microRNA (miRNA) encoded peptides from primary miRNA, these peptides exert their biological functions through binding with membrane-embedded receptor-like kinases. This interaction initiates downstream cellular signalling cascades, often involving major phytohormones or reactive oxygen species-mediated mechanisms. Despite these advances, the precise modes of action for numerous other small peptides remain to be fully elucidated. In this review, we delve into the dynamics of stress physiology, mainly focusing on the roles of major small signalling peptides, shedding light on their significance in the face of changing environmental conditions.

Mutation in shoot-to-root mobile transcription factor, ELONGATED HYPOCOTYL 5, leads to low nicotine levels in tobacco.

Tobacco remains one of the most commercially important crops due to the parasympathomimetic alkaloid nicotine used in cigarettes. Most genes involved in nicotine biosynthesis are expressed in root tissues; however, their light-dependent regulation has not been studied. Here, we identified the ELONGATED HYPOCOTYL 5 homolog, NtHY5, from Nicotiana tabacum and demonstrated that NtHY5 could complement the Arabidopsis thaliana hy5 mutant at molecular, morphological and biochemical levels. We report the development of CRISPR/Cas9-based knockout mutant plants of tobacco, NtHY5CR, and show down-regulation of the nicotine and phenylpropanoid pathway genes leading to a significant reduction in nicotine and flavonol content, whereas NtHY5 overexpression (NtHY5OX) plants show the opposite effect. Grafting experiments using wild-type, NtHY5CR, and NtHY5OX indicated that NtHY5 moves from shoot-to-root to regulate nicotine biosynthesis in the root tissue. Shoot HY5, directly or through enhancing expression of the root HY5, promotes nicotine biosynthesis by binding to light-responsive G-boxes present in the NtPMT, NtQPT and NtODC promoters. We conclude that the mobility of HY5 from shoot-to-root regulates light-dependent nicotine biosynthesis. The CRISPR/Cas9-based mutants developed, in this study; with low nicotine accumulation in leaves could help people to overcome their nicotine addiction and the risk of death.

microRNA408 and its encoded peptide regulate sulfur assimilation and arsenic stress response in Arabidopsis.

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 biotic and abiotic stresses. Recent findings have shown that some pri-miRNAs encode small regulatory peptides known as microRNA-encoded peptides (miPEPs). miPEPs regulate the growth and development of plants by modulating corresponding miRNA expression; however, the role of these peptides under different stress conditions remains unexplored. Here, we report that pri-miR408 encodes a small peptide, miPEP408, that regulates the expression of miR408, its targets, and associated phenotype in Arabidopsis. We also report that miR408, apart from Plantacyanin (ARPN) and Laccase3 (LAC3), targets a glutathione S-transferase (GSTU25) that plays a role in sulfur assimilation and exhibits a range of detoxification activities with the environmental pollutant. Plants overexpressing miR408 showed severe sensitivity under low sulfur (LS), arsenite As(III), and LS + As(III) stress, while miR408 mutants developed using the CRISPR/Cas9 approach showed tolerance. Transgenic lines showed phenotypic alteration and modulation in the expression of genes involved in the sulfur reduction pathway and affect sulfate and glutathione accumulation. Similar to miR408 overexpressing lines, the exogenous application of synthetic miPEP408 and miPEP408OX lines led to sensitivity in plants under LS, As(III), and combined LS + As(III) stress compared to the control. This study suggests the involvement of miR408 and miPEP408 in heavy metal and nutrient deficiency responses through modulation of the sulfur assimilation pathway.