Exogenous Application of Non-mature miRNA-Encoded miPEP164c Inhibits Proanthocyanidin Synthesis and Stimulates Anthocyanin Accumulation in Grape Berry Cells

Abstract

Secondary metabolic pathways in grape berries are tightly regulated by an array of molecular mechanisms, including microRNA-mediated post-transcriptional regulation. As recently discovered, before being processed into mature microRNAs (miRNAs), the primary transcripts of miRNAs (pri-miRNAs) can encode for small miRNA-encoded peptides (micropeptides – miPEPs) that ultimately lead to an accentuated downregulation of the respective miRNA-targeted genes. Although few studies about miPEPs are available, the discovery of miPEPs reveals a new layer of gene regulation at the post-transcriptional level that opens the possibility to regulate plant metabolism without resorting to gene manipulation. Here, we identified a miPEP encoded in non-mature miR164c putatively targeting grapevine transcription factor VvMYBPA1 (miPEP164c/miPEP-MYBPA1), a positive regulator of key genes in the proanthocyanidin (PA)-biosynthetic pathway, a pathway that competes directly for substrate with the anthocyanin-biosynthetic pathway. Thus, the objective of this work was to test the hypothesis that the exogenous application of miPEP164c (miPEP-MYBPA1) can modulate the secondary metabolism of grape berry cells by inhibiting PA biosynthetic pathway while simultaneously stimulating anthocyanin synthesis. The exogenous application of miPEP164c to suspension-cultured cells from grape berry (cv. Gamay) enhanced the transcription of its corresponding non-mature miR164c, with a maximum effect at 1 μM and after a period of 10 days, thus leading to a more pronounced post-transcriptional silencing of its target VvMYBPA1. This led to a significant inhibition of the PA pathway, mostly via inhibition of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) enzymatic activities and VvLAR1 downregulation. In parallel, the anthocyanin-biosynthetic route was stimulated. Anthocyanin content was 31% higher in miPEP164c-treated cells, in agreement with the observed upregulation of VvUFGT1 transcripts and UFGT enzyme activity levels.

Interplay between R2R3 MYB‐type activators and repressors regulates proanthocyanidin biosynthesis in banana (Musa acuminata)

“…As the EAR motif by forming complex with chromatin‐altering proteins regulates the expression of different genes at epigenetic level (Kagale & Rozwadowski, 2011), and in our analysis, the C2 motif, a type of EAR motif is present in all identified MaMYBPR proteins, these chromatin‐altering proteins might form a complex with these repressors to modulate their repressor activity in banana. The grapevine gene VvMYBPA1 transcripts are targeted by microRNA164c (miR164c) for mRNA cleavage, resulting in lower expression of LAR and ANR , and therefore lower proanthocyanidin accumulation in grape berries (Vale et al ., 2021). The varying accumulation of the transcripts of regulatory R2R3‐MYB genes in banana tissues may be attributed to these regulators acting at different levels, which are yet to be identified.…”

Constitutive expression of VviNAC17 transcription factor significantly induces the synthesis of flavonoids and other phenolics in transgenic grape berry cells

“…The extraction of anthocyanins was performed using 100 mg of grape berry cells from each experimental condition as described in Vale et al (2021). The extraction process was performed using 90% (v/v) methanol and 10% (v/v) deionized water.…”

Integrative Analysis of miRNAs and Their Targets Involved in Ray Floret Growth in Gerbera hybrida

“…In this study, ghy-miR156 accumulated to high levels during S6, which implies an association with anthocyanin biosynthesis. In addition, miR164-encoded peptides (miPEP164), which are encoded by primary transcripts of miR164, positively regulate the proanthocyanidin biosynthetic pathway [97]. In this study, levels of the mature sequence of ghy-miR164 were shown to increase with ray petal growth, and thus it will be interesting to investigate the role of miPEP164 in the anthocyanin biosynthesis pathway in gerbera ray petals.…”