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.

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

VviNAC17 is a grapevine transcription factor activated by ABA. Because ABA has been proposed as the main signal modulating the secondary metabolism in grape berry skins, here we postulated VviNAC17 as a positive regulator of secondary metabolism in grape cells. To validate the hypothesis, VviNAC17 was constitutively and stably overexpressed in grape berry suspension-cultured cells of Gamay Fréaux cv. by Agrobacterium-mediated transformation. Targeted transcriptional analyses by qPCR showed that several genes involved the phenylpropanoid (VviPAL1), stilbenoid (VviSTS1) and flavonoid pathways (VviDFR, VviLAR1, VviANR, VviLDOX, and VviUFGT1), as well as anthocyanin vacuolar transport and accumulation (VviGST4 and VvMATE1) were significantly upregulated in VviNAC17-overexpressing transgenic cells, which translated in the stimulation of a number of enzymatic activities in those pathways. This was the case of phenylalanine ammonia lyase (PAL) and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) that were about 2-fold and 3.5-fold higher in VviNAC17-overexpressing cells than in control cells. VviNAC17-overexpressing cells accumulated significantly higher amounts of anthocyanins, proanthocyanidins, total flavonoids and total phenolics. These findings confirmed that VviNAC17 is an important positive regulator of secondary metabolism in grapevine contributing to the accumulation of important berry quality-related secondary metabolites.

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

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.

Chemical Characterization of Sambucus nigra L. Flowers Aqueous Extract and Its Biological Implications.

The main goal of this study was to chemically characterize an aqueous S. nigra flower extract and validate it as a bioactive agent. The elderflower aqueous extraction was performed at different temperatures (50, 70 and 90 °C). The extract obtained at 90 °C exhibited the highest phenolic content and antiradical activity. Therefore, this extract was analyzed by GC-MS and HPLC-MS, which allowed the identification of 46 compounds, being quercetin and chlorogenic acid derivatives representative of 86% of the total of phenolic compounds identified in hydrophilic fraction of the aqueous extract. Naringenin (27.2%) was the major compound present in the lipophilic fraction. The antiproliferative effects of the S. nigra extract were evaluated using the colon cancer cell lines RKO, HCT-116, Caco-2 and the extract's antigenotoxic potential was evaluated by the Comet assay in RKO cells. The RKO cells were the most susceptible to S. nigra flower extract (IC50 = 1250 µg mL-1). Moreover, the extract showed antimicrobial activity against Gram-positive bacteria, particularly Staphylococcus aureus and S. epidermidis. These results show that S. nigra-based extracts can be an important dietary source of bioactive phenolic compounds that contribute to health-span improving life quality, demonstrating their potential as nutraceutical, functional foods and/or cosmetic components for therapeutic purposes.

Plant SWEETs: from sugar transport to plant-pathogen interaction and more unexpected physiological roles.

Sugars Will Eventually be Exported Transporters (SWEETs) have important roles in numerous physiological mechanisms where sugar efflux is critical, including phloem loading, nectar secretion, seed nutrient filling, among other less expected functions. They mediate low affinity and high capacity transport, and in angiosperms this family is composed by 20 paralogs on average. As SWEETs facilitate the efflux of sugars, they are highly susceptible to hijacking by pathogens, making them central players in plant-pathogen interaction. For instance, several species from the Xanthomonas genus are able to upregulate the transcription of SWEET transporters in rice (Oryza sativa), upon the secretion of transcription-activator-like effectors. Other pathogens, such as Botrytis cinerea or Erysiphe necator, are also capable of increasing SWEET expression. However, the opposite behavior has been observed in some cases, as overexpression of the tonoplast AtSWEET2 during Pythium irregulare infection restricted sugar availability to the pathogen, rendering plants more resistant. Therefore, a clear-cut role for SWEET transporters during plant-pathogen interactions has so far been difficult to define, as the metabolic signatures and their regulatory nodes, which decide the susceptibility or resistance responses, remain poorly understood. This fuels the still ongoing scientific question: what roles can SWEETs play during plant-pathogen interaction? Likewise, the roles of SWEET transporters in response to abiotic stresses are little understood. Here, in addition to their relevance in biotic stress, we also provide a small glimpse of SWEETs importance during plant abiotic stress, and briefly debate their importance in the particular case of grapevine (Vitis vinifera) due to its socioeconomic impact.