AbstractUV-B regulation of anthocyanin biosynthesis in vegetative and grapevine berry tissues has been extensively described. However, its relation with UV-B-regulated microRNAs (miRNAs) has not been addressed before in this species. We explored by deep sequencing of small RNA libraries the developmental dynamics and UV-B effects on miRNAs and associated phased small interfering RNA (phasi-RNAs)-producing loci abundances inin vitro-grown plantlets, in field-grown berry skins of cv. Cabernet Sauvignon, and low- and high UV-B fluence treatments of greenhouse-grown berries at several time points around veraison. We observed by RNA blotting a differential effect of low-versus high-fluence UV-B on miR828 abundances (an effector of anthocyanins and UV-absorbing polyphenolics) across berry development, and identified other miRNAs that correlated with miR828 dynamics. The functional significance of the observed UV-coordinated miRNA responses to UV was supported by degradome evidences of AGO-programmed slicing of mRNAs. Inverse co-expression of the up-regulated miRNAs miR156, miR482, miR530, and miR828 with cognate target gene expressions in response to high fluence UV-B measured by quantitative real-time PCR. These UV-response relationships were also corroborated by analyzing three published transcriptome datasets (berries subjected to UV-C for 1 hr [at pre-veraison], UV-B for five weeks post-veraison, and five red-skinned varieties across four berry development time points). Based on observed significant changes by UV-B on miRNA and derivative phasi-RNA abundances, we propose a regulatory network model of UV responses impacting anti-oxidant and stress-associated polyphenolic compound biosynthesis. In this model high-fluence UV-B increases miR168 (validated in a UV-B small RNA-derived degradome library to targetARGONAUTE1, which spawns phasi-RNAs) and miR530 (targets a novel Plus-3 domain mRNA), while decreasing miR403 abundances (validated to targetARGONAUTE2), thereby coordinating post-transcriptional gene silencing activities by different AGOs. Up-regulation of miR3627/4376 (validated to target Ca2+-transporting ATPase10 that spawns phasi-RNAs) could facilitate anthocyanin accumulation. miR395 and miR399, induced by sulfur and phosphorus starvation in other species (conditions known to trigger anthocyanin accumulation) respond positively to UV-B radiation and are shown to slice cognate targets in grapevine. miR156/miR535 is shown to targetSQUAMOSA PROMOTER-BINDINGtranscription factor genes that potentially regulate the activities of MYB-bHLH-WD40 complexes and thereby anthocyanin biosynthesis. Increases in MYB-bHLH-WD40 TFs could also contribute to the observed up-regulation of miR828 via the conserved and degradome-validated auto-regulatory loop involving miR828/TAS4abcto regulateMYBA6/A7/A5-MYB113-likelevels and thereby anthocyanin levels. These results and meta-analysis provide a basis for systems approaches to better understand non-coding RNA functions in response to UV.
Omics Approaches for Understanding Grapevine Berry Development: Regulatory Networks Associated with Endogenous Processes and Environmental Responses