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.

Seed germination and seedling development assisted by ultrasound: gaps and future research directions.

Since the early 1930s, when the first corn hybrids were grown commercially, innovations in the agriculture industry have had an unprecedent impact worldwide, helping to meet the demands for food of an exponentially growing population. In particular, seed technology research has contributed substantially to the improvement of crop performance over the years. Ultrasonic treatment of seeds is a green technology that promises to have an impact on the food industry, enhancing germination and seedling development in different species through the stimulation of water and oxygen uptake and seed metabolism. The increase in starch degradation has been associated with the stimulation of the α-amylases of the endosperm, but relatively few reports focus on how ultrasound affects seed germination at the biochemical and molecular levels. For instance, the picture is still unclear regarding the impact of ultrasound on transcriptional reprogramming in seeds. The purpose of this review is to assess the literature on ultrasound seed treatment accurately and critically, ultimately aiming to encourage new scientific and technological breakthroughs with a real impact on worldwide agricultural production while promoting sustainable practices on biological systems. © 2023 Society of Chemical Industry.

Grapevine woody tissues accumulate stilbenoids following bud burst.

MAIN CONCLUSION: After bud burst, a transcriptional reprogramming of the shikimate and phenylpropanoid pathways occurs in grapevine canes resulting in the accumulation of stilbenoids like resveratrol and viniferin. Stilbenoids are phenylpropanoid compounds with important biological properties and biotechnological applications that are synthesized in grapevine in response to different stresses. Although they are found in woody tissues, such as canes and buds, their biosynthesis and accumulation have been essentially described in berries. We have previously shown that transcripts encoding secondary metabolism enzymes accumulate in grapevine canes following the transition from dormancy (E-L 1) to bud burst (E-L 4) suggesting that secondary metabolites may accumulate in grapevine canes during this transition. In the present study, using UPLC-MS we demonstrate the accumulation of important metabolites such as ferulic acid and the stilbenoids E-resveratrol, E-piceatannol and E-ε-viniferin. Stilbenoids accumulation correlated with the increased expression of several stilbene synthase genes and of VviMYB14, encoding a transcription factor that regulates stilbene biosynthesis. In addition, a general stimulation of the plastidial shikimate pathway was observed. Taken together, results show that important secondary metabolites accumulate in the woody canes during bud burst. These findings may aid biotechnological approaches aimed at extracting biologically active phenolic compounds, including stilbenoids, from grapevine woody tissues.

Combined omics approaches expose metabolite-microbiota correlations in grape berries of three cultivars of Douro wine region.

This study hypothesized the existence of cultivar-associated correlations between grape berry metabolites and its microbial residents, in Douro wine region. Integrated metabolomics with metabarcoding showed that the microbial biodiversity is not associated to berry sugar concentration, but closely connected to the profile of amino acids, flavonoids and wax compounds, which drove cultivar differentiation together with the prevalence of pathogenic fungi, yeasts and bacteria, mainly Dothideomycetes and Gammaproteobacteria. Over 7000 metabolite-microbiota correlations with ρ >|0.99| exposed a core of 15 metabolites linked to 11 microbial taxa. Serine, oxalate, cyanidin-3-O-glucoside, petunidin-3-O-glucoside, gallic acid, germanicol, sitosterol and erythrodiol correlated negatively to the abundance of most taxa, including Alternaria, Aureobasidium, Pseudopithomyces, Pseudomonas and Sphingomonas. In contrast, phenylalanine, asparagine, alanine, (epi)gallocatechin and procyanidin gallate mediated positive metabolite-OTU correlations. E. necator and A. carbonarius correlated negatively with stigmasterol and amyrin. Complex fungi-bacteria relationships ruled by Dothideomycetes and Alphaproteobacteria further suggest tight host-microbe interactions at the carposphere.

Biosynthesis of Chlorophyll and Other Isoprenoids in the Plastid of Red Grape Berry Skins.

Despite current knowledge showing that fruits like tomato and grape berries accumulate different components of the light reactions and Calvin cycle, the role of green tissues in fruits is not yet fully understood. In mature tomato fruits, chlorophylls are degraded and replaced by carotenoids through the conversion of chloroplasts in chromoplasts, while in red grape berries, chloroplasts persist at maturity and chlorophylls are masked by anthocyanins. To study isoprenoid and lipid metabolism in grape skin chloroplasts, metabolites of enriched organelle fractions were analyzed by high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) and the expression of key genes was evaluated by real-time polymerase chain reaction (PCR) in berry skins and leaves. Overall, the results indicated that chloroplasts of the grape berry skins, as with leaf chloroplasts, share conserved mechanisms of synthesis (and degradation) of important components of the photosynthetic machinery. Some of these components, such as chlorophylls and their precursors, and catabolites, carotenoids, quinones, and lipids have important roles in grape and wine sensory characteristics.

A proteomic analysis shows the stimulation of light reactions and inhibition of the Calvin cycle in the skin chloroplasts of ripe red grape berries.

The role of photosynthesis in fruits still challenges scientists. This is especially true in the case of mature grape berries of red varieties lined by an anthocyanin-enriched exocarp (skin) almost impermeable to gases. Although chlorophylls are degraded and replaced by carotenoids in several fruits, available evidence suggests that they may persist in red grapes at maturity. In the present study, chloroplasts were isolated from the skin of red grape berries (cv. Vinhão) to measure chlorophyll levels and the organelle proteome. The results showed that chloroplasts (and chlorophylls) are maintained in ripe berries masked by anthocyanin accumulation and that the proteome of chloroplasts from green and mature berries is distinct. Several proteins of the light reactions significantly accumulated in chloroplasts at the mature stage including those of light-harvesting complexes of photosystems I (PSI) and II (PSII), redox chain, and ATP synthase, while chloroplasts at the green stage accumulated more proteins involved in the Calvin cycle and the biosynthesis of amino acids, including precursors of secondary metabolism. Taken together, results suggest that although chloroplasts are more involved in biosynthetic reactions in green berries, at the mature stage, they may provide ATP for cell maintenance and metabolism or even O2 to feed the respiratory demand of inner tissues.

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.

Impact of Deficit Irrigation on Grapevine cv. 'Touriga Nacional' during Three Seasons in Douro Region: An Agronomical and Metabolomics Approach.

The introduction of irrigation in vineyards of the Mediterranean basin is a matter of debate, in particular in those of the Douro Demarcated Region (DDR), due to the limited number of available studies. Here, we aimed to perform a robust analysis in three consecutive vintages (2018, 2019, and 2020) on the impact of deficit irrigation on the yield, berry quality traits, and metabolome of cv. 'Touriga Nacional'. Results showed that in the peaks of extreme drought, irrigation at 30% crop evapotranspiration (ETc) (R30) was able to prevent a decay of up to 0.4 MPa of leaf predawn water potential (ΨPd), but irrigation at 70% ETc (R70) did not translate into additional protection against drought stress. Following three seasons of irrigation, the yield was significantly improved in vines irrigated at R30, whereas irrigation at R70 positively affected the yield only in the 2020 season. Berry quality traits at harvest were not significantly changed by irrigation, except for Total Soluble Solids (TSS) in 2018. A UPLC-MS-based targeted metabolomic analysis identified eight classes of compounds, amino acids, phenolic acids, stilbenoid DP1, stilbenoid DP2, flavonols, flavan-3-ols, di-OH- and tri-OH anthocyanins, and showed that anthocyanins and phenolic acids did not change significantly with irrigation. The present study showed that deficit irrigation partially mitigated the severe summer water deficit conditions in the DDR but did not significantly change key metabolites.

Saccharomyces cerevisiae Cells Lacking the Zinc Vacuolar Transporter Zrt3 Display Improved Ethanol Productivity in Lignocellulosic Hydrolysates.

Yeast-based bioethanol production from lignocellulosic hydrolysates (LH) is an attractive and sustainable alternative for biofuel production. However, the presence of acetic acid (AA) in LH is still a major problem. Indeed, above certain concentrations, AA inhibits yeast fermentation and triggers a regulated cell death (RCD) process mediated by the mitochondria and vacuole. Understanding the mechanisms involved in AA-induced RCD (AA-RCD) may thus help select robust fermentative yeast strains, providing novel insights to improve lignocellulosic ethanol (LE) production. Herein, we hypothesized that zinc vacuolar transporters are involved in vacuole-mediated AA-RCD, since zinc enhances ethanol production and zinc-dependent catalase and superoxide dismutase protect from AA-RCD. In this work, zinc limitation sensitized wild-type cells to AA-RCD, while zinc supplementation resulted in a small protective effect. Cells lacking the vacuolar zinc transporter Zrt3 were highly resistant to AA-RCD, exhibiting reduced vacuolar dysfunction. Moreover, zrt3Δ cells displayed higher ethanol productivity than their wild-type counterparts, both when cultivated in rich medium with AA (0.29 g L-1 h-1 versus 0.11 g L-1 h-1) and in an LH (0.73 g L-1 h-1 versus 0.55 g L-1 h-1). Overall, the deletion of ZRT3 emerges as a promising strategy to increase strain robustness in LE industrial production.

The restructuring of grape berry waxes by calcium changes the surface microbiota.

The observation that exogenous Ca shifted the polyphenolic profile of grape berries and conferred a glossy appearance to mature fruits led us to hypothesize that the composition of grape berry waxes and thus surface microbiota are modified. In two cultivars sharing the same microclimate, the triterpenoid and steroid profile of berry cuticle was characterized by a targeted metabolomic approach, and surface microbial communities were surveyed by ITS and 16S metabarcoding. Results showed that Ca strongly decreased the levels of oleanolic acid, while steroids and neutral triterpenoids were affected in a cultivar-dependent manner. A total of 174 fungi and 192 bacteria OTUs were identified, with Dothideomycetes, Leotiomycetes, Alphaproteobacteria and Gammaproteobacteria comprising the most abundant classes. Ca decreased fungi biodiversity, favoring the growth of Basidiomycetes, and shifting fungi-bacteria relationships. Metabolite-microbiota networks revealed a tight relationship between microbial communities and triterpenoid components of fruit waxes, mainly stigmasterol, tremulone and oleanolic acid.

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.

Molecular reprogramming in grapevine woody tissues at bud burst.

Perennial woody plants undergo a period of dormancy from the beginning of autumn until the end of spring. Whereas the molecular and physiological events that characterize dormancy release of buds have been described in detail, those occurring in woody tissues underneath the buds are mostly unknown. To bridge this gap, the mRNA populations of cane segments located underneath the bud were analyzed at bud dormancy (E-L 1) and at bud burst (E-L 4). They revealed an important reprogramming of gene expression suggesting that cell division, cell wall metabolism and the mobilization of sugars are the main metabolic and cellular events occurring in cane woody tissues at bud burst. Also, the upregulation of several genes of sugar metabolism, encoding starch- and sucrose-degrading enzymes and sugar transporters, correlates with the decrease in starch and soluble sugars in woody tissues concomitant with increased sucrose synthase and α-amylolytic biochemical activities. The latter is likely due to the VviAMY2 gene that encodes a functional α-amylase as observed after its heterologous expression in yeast. Taken together, these results are consistent with starch and sugar mobilization in canes being primarily involved in grapevine secondary growth initiation and supporting the growth of the emerging bud.

Exogenous Calcium Delays Grape Berry Maturation in the White cv. Loureiro While Increasing Fruit Firmness and Flavonol Content.

Vineyard calcium (Ca) sprays have been increasingly used by grape growers to improve fruit firmness and thus maintain quality, particularly in periods of heavy rains and hail. The observation that Ca visibly modified berry size, texture, and color in the most prominent white cultivar of the DOC region 'Vinhos Verdes', cultivar (cv.) Loureiro, led us to hypothesize that Ca induced metabolic rearrangements that resulted in a substantial delay in fruit maturation. Targeted metabolomics by ultra-performance liquid chromatography coupled to mass spectrometry and directed transcriptomics were thus combined to characterize the metabolic and transcriptional profiles of cv. Loureiro berries that, together with firmness, °Brix, and fruit weight measurements, allowed to obtain an integrated picture of the biochemical and structural effects of Ca in this cultivar. Results showed that exogenous Ca decreased amino acid levels in ripe berries while upregulating PAL1 expression, and stimulated the accumulation of caftaric, coutaric, and fertaric acids. An increase in the levels of specific stilbenoids, namely E-piceid and E-ω-viniferin, was observed, which correlated with the upregulation of STS expression. Trace amounts of anthocyanins were detected in berries of this white cultivar, but Ca treatment further inhibited their accumulation. The increased berry flavonol content upon Ca treatment confirmed that Ca delays the maturation process, which was further supported by an increase in fruit firmness and decrease in weight and °Brix at harvest. This newly reported effect may be specific to white cultivars, a topic that deserves further investigation.

Grapevine aquaporins: Diversity, cellular functions, and ecophysiological perspectives.

High-scored premium wines are typically produced under moderate drought stress, suggesting that the water status of grapevine is crucial for wine quality. Aquaporins greatly influence the plant water status by facilitating water diffusion across the plasma membrane in a tightly regulated manner. They adjust the hydraulic conductance of the plasma membrane rapidly and reversibly, which is essential in specific physiological events, including adaptation to soil water scarcity. The comprehension of the sophisticated plant-water relations at the molecular level are thus important to optimize agricultural practices or to assist plant breeding programs. This review explores the recent progresses in understanding the water transport in grapevine at the cellular level through aquaporins and its regulation. Important aspects, including aquaporin structure, diversity, cellular localization, transport properties, and regulation at the cellular and whole plant level are addressed. An ecophysiological perspective about the roles of grapevine aquaporins in plant response to drought stress is also provided.

Calcium and methyl jasmonate cross-talk in the secondary metabolism of grape cells.

In grape cell cultures cv. Gamay Fréaux var. Teinturier, Ca was shown to decrease cell pigmentation through the inhibition of anthocyanin biosynthesis, while stimulating stilbenoids accumulation. Because methyl jasmonate (MeJA) is a well-known inducer of secondary metabolism in grape cells, and Ca antagonizes its stimulatory effect over several enzymes of core metabolic branches, in the present study we hypothesized that Ca and MeJA signaling pathways interact to regulate specific secondary metabolism routes. Grape cultured cells were elicited with MeJA or with MeJA + Ca and an UPLC-MS-based targeted metabolomic method was implemented to characterize their polyphenolic profiles. Results were compared with the profile of cells elicited with Ca only, previously reported. Data was complemented with gene expression analysis, allowing the assembly of a metabolic map that unraveled routes specifically regulated by both elicitors. MeJA + Ca specifically boosted E-resveratrol and E-ε-viniferin levels by 180% and 140%, respectively, in comparison to cells treated with MeJA only, while the stimulatory effect of MeJA over flavonoid synthesis was inhibited by Ca. In parallel, Ca downregulated most flavonoid pathway genes, including LAR1, ANS, BAN and ANR. Ca was able to mimic or potentiate the effect of MeJA on the expression of JA signaling genes, including JAR1, PIN and PR10. Transcript/metabolite correlation networks exposed the central influence of FLS1,STS,CDPK17 and COI1 in polyphenolic biosynthetic routes. This study highlights the potential of the MeJA-Ca combination for diverting polyphenolic metabolism towards the production of specific metabolites of interest, highly relevant in a biotechnological perspective.

Endocytosis of nutrient transporters in fungi: The ART of connecting signaling and trafficking.

Plasma membrane transporters play pivotal roles in the import of nutrients, including sugars, amino acids, nucleobases, carboxylic acids, and metal ions, that surround fungal cells. The selective removal of these transporters by endocytosis is one of the most important regulatory mechanisms that ensures a rapid adaptation of cells to the changing environment (e.g., nutrient fluctuations or different stresses). At the heart of this mechanism lies a network of proteins that includes the arrestin-related trafficking adaptors (ARTs) which link the ubiquitin ligase Rsp5 to nutrient transporters and endocytic factors. Transporter conformational changes, as well as dynamic interactions between its cytosolic termini/loops and with lipids of the plasma membrane, are also critical during the endocytic process. Here, we review the current knowledge and recent findings on the molecular mechanisms involved in nutrient transporter endocytosis, both in the budding yeast Saccharomyces cerevisiae and in some species of the filamentous fungus Aspergillus. We elaborate on the physiological importance of tightly regulated endocytosis for cellular fitness under dynamic conditions found in nature and highlight how further understanding and engineering of this process is essential to maximize titer, rate and yield (TRY)-values of engineered cell factories in industrial biotechnological processes.

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.

Vineyard calcium sprays reduce the damage of postharvest grape berries by stimulating enzymatic antioxidant activity and pathogen defense genes, despite inhibiting phenolic synthesis.

Calcium supplements have been increasingly used for decay prevention, sanitation and nutritional enrichment of fruits, as more environmentally friendly alternatives to fungicides. However, little is known on the effects of these supplements on grape berry biochemical and molecular properties during storage. In this study, we addressed the hypothesis that the application of calcium chloride (CaCl2) in grapevines throughout the fruiting season reduces damage (and decay) of postharvest grape berries, through several biochemical and transcriptional modifications in sugar transport, secondary metabolism, antioxidant activity, cell wall organization and pathogen defense. Results showed that calcium (Ca) treatments in cv. "Vinhão" vines increased fruit Ca content and significantly decreased fruit damage by 60%, 10-d after storage at 4 °C. Grape berries from Ca-treated vines displayed lower levels of total phenolics and anthocyanins, compared to control fruits, corroborating the downregulation of PAL1 and STS which resulted in decreased non-enzymatic antioxidant capacity estimated by FRAP assay. In contrast, a strong upregulation of CAT1, ASPX1, ASPX3, GLPX1, CSD3 and CSD6 encoding antioxidant enzymes was observed. Accordingly, catalase enzyme activity was stimulated, significantly reducing hydrogen peroxide (H2O2) levels by 36%. The overexpression of the cell wall and pathogen defense genes PME, PGIP, PIN and PR1 likely contributed to the reduction in fruit rot. This work suggested that preharvest Ca treatment is an efficient agronomical strategy that prolongs the shelf life of grape berries through modifications at molecular and biochemical levels, bringing further insight on the benefits and drawbacks of preharvest Ca applications on postharvest fruit quality attributes.

Lactoferrin perturbs lipid rafts and requires integrity of Pma1p-lipid rafts association to exert its antifungal activity against Saccharomyces cerevisiae.

Lactoferrin (Lf) is a bioactive milk-derived protein with remarkable wide-spectrum antifungal activity. To deepen our understanding of the molecular mechanisms underlying Lf cytotoxicity, the role of plasma membrane ergosterol- and sphingolipid-rich lipid rafts and their association with the proton pump Pma1p was explored. Pma1p was previously identified as a Lf-binding protein. Results showed that bovine Lf (bLf) perturbs ergosterol-rich lipid rafts organization by inducing intracellular accumulation of ergosterol. Using yeast mutant strains lacking lipid rafts-associated proteins or enzymes involved in the synthesis of ergosterol and sphingolipids, we found that perturbations in the composition of these membrane domains increase resistance to bLf-induced yeast cell death. Also, when Pma1p-lipid rafts association is compromised in the Pma1-10 mutant and in the absence of the Pma1p-binding protein Ast1p, the bLf killing activity is impaired. Altogether, results showed that the perturbation of lipid rafts and the inhibition of both Pma1p and V-ATPase activities mediate the antifungal activity of bLf. Since it is suggested that the combination of conventional antifungals with lipid rafts-disrupting compounds is a powerful antifungal approach, our data will help to pave the way for the use of bLf alone or in combination for the treatment/eradication of clinically and agronomically relevant yeast pathogens/fungi.

VviRafS5 Is a Raffinose Synthase Involved in Cold Acclimation in Grapevine Woody Tissues.

The accumulation of raffinose family oligosaccharides (RFOs) is a hallmark of plant response to different abiotic stresses, including cold. The synthesis of galactinol, by galactinol synthases (GolS), and raffinose, by raffinose synthases (RafS), are fundamental for stress-induced accumulation of RFOs, but the role of these enzymes in the cold response of grapevine (Vitis vinifera L.) woody tissues is still unclear. To address this gap in the literature, 1-year-lignified grapevine canes were incubated at 4°C for 7 and 14 days and tissues were analyzed for sugar content and gene expression. Results showed that, in parallel to starch breakdown, there was an increase in soluble sugars, including sucrose, glucose, fructose, raffinose, and stachyose. Remarkably, abscisic acid (ABA) levels increased during cold acclimation, which correlated with the increased expression of the key ABA-synthesis genes VviNCED2 and VviNCED3. Expression analysis of the VviGolS and VviRafS family allowed the identification of VviRafS5 as a key player in grapevine cold response. The overexpression of VviRafS5 in Saccharomyces cerevisiae allowed the biochemical characterization of the encoded protein as a raffinose synthase with a size of ~87 kDa. In grapevine cultured cells, VviRafS5 was upregulated by cold and ABA but not by heat and salt stresses. Our results suggest that ABA accumulation in woody tissues during cold acclimation upregulates VivRafS5 leading to raffinose synthesis.