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.
Highlights
Grapevines are well adapted to semi-arid climate, the increasingly more frequent combined effect of drought, high air temperature and high evaporative demand has a negative impact in grapevine yield (Chaves et al, 2010) and, if severe, in berry quality (Teixeira et al, 2013)
We identified miR164c as a putative negative regulator of transcription factor VvMYBPA1, involved in regulation of the PA pathway and proceeded to screen it in miRbase for their stem-loop sequence or pri-miRNA sequence, the non-mature sequence of the regulatory miRNA possibly harboring small open reading frames (ORF) corresponding to regulatory miRNA-encoded peptides (miPEPs)
An in silico analysis for micropeptide screening led to the selection of miPEP164c, a candidate miPEP with putative regulatory function in grape berry flavonoid biosynthesis metabolic pathway, in the branch of PA synthesis. miR164c was predicted in silico to post-transcriptionally inhibit grapevine transcription factor VvMYBPA1, involved in the activation of flavonoid synthesis, of PA synthesis
Summary
Grapevines are well adapted to semi-arid climate, the increasingly more frequent combined effect of drought, high air temperature and high evaporative demand has a negative impact in grapevine yield (Chaves et al, 2010) and, if severe, in berry quality (Teixeira et al, 2013). Berry and wine quality depend strongly on the grapevine adaptability to drought, heat and light/UV intensity. This abiotic stressors impact highly regulated molecular mechanisms underlying the synthesis of several quality-related compounds, such as anthocyanins, proanthocyanidins (PAs), flavanols, and flavonols (Downey et al, 2006; Teixeira et al, 2013). Catechin synthesis is catalyzed by leucoanthocyanidin reductase (LAR) that uses leucoanthocyanidins as substrate. Leucoanthocyanidins can be catalyzed by leucoanthocyanidin oxygenase (LDOX), continuing the flavonoid pathway and resulting in the formation of anthocyanidins, a substrate of both UDP-glucose flavonoid 3-O-glucosyltransferase (UFGT), in the synthesis of anthocyanins, and anthocyanidin reductase (ANR), in the synthesis of epicatechin, another building block of PAs (Gagné et al, 2009)
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