Non-Mature miRNA-Encoded Micropeptide miPEP166c Stimulates Anthocyanin and Proanthocyanidin Synthesis in Grape Berry Cells.

The phenylpropanoid and flavonoid pathways exhibit intricate regulation, not only influenced by environmental factors and a complex network of transcription factors but also by post-transcriptional regulation, such as silencing by microRNAs and miRNA-encoded micropeptides (miPEPs). VviMYBC2-L1 serves as a transcriptional repressor for flavonoids, playing a crucial role in coordinating the synthesis of anthocyanin and proanthocyanidin. It works in tandem with their respective transcriptional activators, VviMYBA1/2 and VviMYBPA1, to maintain an equilibrium of flavonoids. We have discovered a miPEP encoded by miR166c that appears to target VviMYBC2-L1. We conducted experiments to test the hypothesis that silencing this transcriptional repressor through miPEP166c would stimulate the synthesis of anthocyanins and proanthocyanidins. Our transcriptional analyses by qPCR revealed that the application of exogenous miPEP166c to Gamay Fréaux grape berry cells resulted in a significant upregulation in flavonoid transcriptional activators (VviMYBA1/2 and VviMYBPA1) and structural flavonoid genes (VviLDOX and VviDFR), as well as genes involved in the synthesis of proanthocyanidins (VviLAR1 and VviANR) and anthocyanins (VviUFGT1). These findings were supported by the increased enzyme activities of the key enzymes UFGT, LAR, and ANR, which were 2-fold, 14-fold, and 3-fold higher, respectively, in the miPEP166c-treated cells. Ultimately, these changes led to an elevated total content of anthocyanins and proanthocyanidins.

An overview on miRNA-encoded peptides in plant biology research.

MicroRNAs (miRNAs) are short (21-23 nt) regulatory RNA molecules present in plants and animals which are known for regulating the mRNA target gene expression either by cleavage or translational repression. With the advancements in miRNAs research in plants towards their biogenesis and applications has directed the recent discovery of pri-miRNAs encoding functional peptides or microRNA peptides (miPEPs). These miPEPs are encoded by 5' of pri-miRs containing short ORFs (miORFs). miPEPs are known to enhance the activity of their associated miRNAs by increasing their accumulation and hence downregulating the target genes. Since miPEPs are very specific for each miRNA, they are considered as novel and effective tools for improving traits of interest for plant growth promotion and plant-microbe interaction. Entire peptidome research is the need of the hour. This review thus summarizes recent advancements in miPEP research and its applications as a technology with important agronomical implications with miRNAs augmentation.

Internalization of miPEP165a into Arabidopsis Roots Depends on Both Passive Diffusion and Endocytosis-Associated Processes.

MiPEPs are short natural peptides encoded by microRNAs in plants. Exogenous application of miPEPs increases the expression of their corresponding miRNA and, consequently, induces consistent phenotypical changes. Therefore, miPEPs carry huge potential in agronomy as gene regulators that do not require genome manipulation. However, to this end, it is necessary to know their mode of action, including where they act and how they enter the plants. Here, after analyzing the effect of Arabidopsis thaliana miPEP165a on root and aerial part development, we followed the internalization of fluorescent-labelled miPEP165a into roots and compared its uptake into endocytosis-altered mutants to that observed in wild-type plants treated or not with endocytosis inhibitors. The results show that entry of miPEP165a involves both a passive diffusion at the root apex and endocytosis-associated internalization in the differentiation and mature zones. Moreover, miPEP165a is unable to enter the central cylinder and does not migrate from the roots to the aerial part of the plant, suggesting that miPEPs have no systemic effect.

miRNA-encoded peptides (miPEPs): A new tool to analyze the roles of miRNAs in plant biology.

MicroRNAs (miRNAs) are short RNA molecules negatively regulating the expression of many important genes in plants and animals. We have recently shown that plant primary transcripts of miRNAs encode peptides (miPEPs) able to increase specifically the transcription of their associated miRNA.(1) We discuss here the possibility of using miPEPs as a new tool for functional analysis of single members of miRNA families in plants, including in non-model plants, that could avoid transgenic transformation and minimize artifactual interpretation. We also raise several fundamental and crucial questions that need to be address for a deeper understanding of the cellular and molecular mechanisms underlining the regulatory activity of miPEPs.

Biological Activity of Artificial Plant Peptides Corresponding to the Translational Products of Small ORFs in Primary miRNAs and Other Long "Non-Coding" RNAs.

Generally, lncPEPs (peptides encoded by long non-coding RNAs) have been identified in many plant species of several families and in some animal species. Importantly, molecular mechanisms of the miPEPs (peptides encoded by primary microRNAs, pri-miRNAs) are often poorly understood in different flowering plants. Requirement for the additional studies in these directions is highlighted by alternative findings concerning positive regulation of pri-miRNA/miRNA expression by synthetic miPEPs in plants. Further extensive studies are also needed to understand the full set of their roles in eukaryotic organisms. This review mainly aims to consider the available data on the regulatory functions of the synthetic miPEPs. Studies of chemically synthesized miPEPs and analyzing the fine molecular mechanisms of their functional activities are reviewed. Brief description of the studies to identify lncORFs (open reading frames of long non-coding RNAs) and the encoded protein products is also provided.

Emphasizing the Role of Long Non-Coding RNAs (lncRNA), Circular RNA (circRNA), and Micropeptides (miPs) in Plant Biotic Stress Tolerance.

Biotic stress tolerance in plants is complex as it relies solely on specific innate immune responses from different plant species combating diverse pathogens. Each component of the plant immune system is crucial to comprehend the molecular basis underlying sustainable resistance response. Among many other regulatory components, long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) have recently emerged as novel regulatory control switches in plant development and stress biology. Besides, miPs, the small peptides (100-150 amino acids long) encoded by some of the non-coding portions of the genome also turned out to be paramount regulators of plant stress. Although some studies have been performed in deciphering the role of miPs in abiotic stress tolerance, their function in regulating biotic stress tolerance is still largely elusive. Hence, the present review focuses on the roles of long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) in combating biotic stress in plants. The probable role of miPs in plant-microbe interaction is also comprehensively highlighted. This review enhances our current understanding of plant lncRNAs, circRNAs, and miPs in biotic stress tolerance and raises intriguing questions worth following up.

Characterization of plant microRNA-encoded peptides (miPEPs) reveals molecular mechanisms from the translation to activity and specificity.

MicroRNAs (miRNAs) are transcribed as long primary transcripts (pri-miRNAs) by RNA polymerase II. Plant pri-miRNAs encode regulatory peptides called miPEPs, which specifically enhance the transcription of the pri-miRNA from which they originate. However, paradoxically, whereas miPEPs have been identified in different plant species, they are poorly conserved, raising the question of the mechanisms underlying their specificity. To address this point, we identify and re-annotate multiple Arabidopsis thaliana pri-miRNAs in order to identify ORF encoding miPEPs. The study of several identified miPEPs in different species show that non-conserved miPEPs are only active in their plant of origin, whereas conserved ones are active in different species. Finally, we find that miPEP activity relies on the presence of its own miORF, explaining both the lack of selection pressure on miPEP sequence and the ability for non-conserved peptides to play a similar role, i.e., to activate the expression of their corresponding miRNA.

In silico identification of candidate miRNA-encoded Peptides in four Fabaceae species.

MicroRNAs (miRNAs) are one of the main regulators of gene expression. Recent studies have demonstrated that primary transcripts of miRNAs (pri-miRNAs) encode regulatory peptides, called miRNA-encoded peptides (miPEPs), capable of enhancing the expression of their associated miRNAs in plants. In this work, we aimed to computationally identify miPEPs produced by small open reading frames (ORFs) in pri-miRNAs from four species of Fabaceae. Five families of miRNAs were investigated, based on their role in plant-microorganism interaction. We used the miR171 family as a training dataset centered on the information about mtr-miPEP171b and vvi-miPEP171d already described. From the sequences of the pri-miRNAs and the genomic regions where they were located, ORFs encoding putative miPEPs were predicted. The 5'-most ORFs encoding peptides on pri-miRNAs were aligned and the amino acids conservation was observed. In total, 81 sequences of potential miPEPs were identified. We found conserved miPEPs inside pri-miRNAs from soybean and between soybean, common bean, and cowpea. Besides, our results showed few conserved miPEPs among isoforms of the same miRNA and no conservation among different miRNA families, which indicate the possible specificity of miPEPs in relation to their corresponding miRNAs. Our findings contribute to the understanding of miPEPs features in plants and provide the basis for studies aiming the biotechnological use of miPEPs in leguminous species.

Pervasive translation of small open reading frames in plant long non-coding RNAs.

Long non-coding RNAs (lncRNAs) are primarily recognized as non-coding transcripts longer than 200 nucleotides with low coding potential and are present in both eukaryotes and prokaryotes. Recent findings reveal that lncRNAs can code for micropeptides in various species. Micropeptides are generated from small open reading frames (smORFs) and have been discovered frequently in short mRNAs and non-coding RNAs, such as lncRNAs, circular RNAs, and pri-miRNAs. The most accepted definition of a smORF is an ORF containing fewer than 100 codons, and ribosome profiling and mass spectrometry are the most prevalent experimental techniques used to identify them. Although the majority of micropeptides perform critical roles throughout plant developmental processes and stress conditions, only a handful of their functions have been verified to date. Even though more research is being directed toward identifying micropeptides, there is still a dearth of information regarding these peptides in plants. This review outlines the lncRNA-encoded peptides, the evolutionary roles of such peptides in plants, and the techniques used to identify them. It also describes the functions of the pri-miRNA and circRNA-encoded peptides that have been identified in plants.

Noncoding but Coding: Pri-miRNA into the Action.

Some pri-miRNAs can code for short peptides called micropeptides (miPEPs) and it has been suggested that these peptides positively regulate the accumulation of their associated miRNAs. Recent data further support this model and point towards the potential for miPEPs to be used in the agricultural sector to improve crop agronomic traits.