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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.

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.

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.

Not only leaves but also other plant organs and structures typically considered as carbon sinks, including stems, roots, flowers, fruits and seeds, may exhibit photosynthetic activity. There is still a lack of a coherent and systematized body of knowledge and consensus on the role(s) of photosynthesis in these “sink” organs. With regard to fruits, their actual photosynthetic activity is influenced by a range of properties, including fruit anatomy, histology, physiology, development and the surrounding microclimate. At early stages of development fruits generally contain high levels of chlorophylls, a high density of functional stomata and thin cuticles. While some plant species retain functional chloroplasts in their fruits upon subsequent development or ripening, most species undergo a disintegration of the fruit chloroplast grana and reduction in stomata functionality, thus limiting gas exchange. In addition, the increase in fruit volume hinders light penetration and access to CO2, also reducing photosynthetic activity. This review aimed to compile information on aspects related to fruit photosynthesis, from fruit characteristics to ecological drivers, and to address the following challenging biological questions: why does a fruit show photosynthetic activity and what could be its functions? Overall, there is a body of evidence to support the hypothesis that photosynthesis in fruits is key to locally providing: ATP and NADPH, which are both fundamental for several demanding biosynthetic pathways (e.g., synthesis of fatty acids); O2, to prevent hypoxia in its inner tissues including seeds; and carbon skeletons, which can fuel the biosynthesis of primary and secondary metabolites important for the growth of fruits and for spreading, survival and germination of their seed (e.g., sugars, flavonoids, tannins, lipids). At the same time, both primary and secondary metabolites present in fruits and seeds are key to human life, for instance as sources for nutrition, bioactives, oils and other economically important compounds or components. Understanding the functions of photosynthesis in fruits is pivotal to crop management, providing a rationale for manipulating microenvironmental conditions and the expression of key photosynthetic genes, which may help growers or breeders to optimize development, composition, yield or other economically important fruit quality aspects.

The newly-identified SWEETs are high-capacity, low-affinity sugar transporters with important roles in numerous physiological mechanisms where sugar efflux is critical. SWEETs are desirable targets for manipulation by pathogens and their expression may be transcriptionally reprogrammed during infection. So far, few plant SWEET transporters have been functionally characterized, especially in grapevine. In this study, in the -susceptible variety \"Trincadeira,\" we thoroughly analyzed modifications in the gene expression profile of key genes in -infected grape berries. VvSWEET7 and VvSWEET15 are likely to play an important role during fruit development and infection as they are strongly expressed at the green and mature stage, respectively, and were clearly up-regulated in response to infection. Also, infection down-regulated expression at the green stage, and expression at the veraison stage, and expression at the mature stage. VvSWEET7 was functionally characterized by heterologous expression in as a low-affinity, high-capacity glucose and sucrose transporter with a of 15.42 mM for glucose and a of 40.08 mM for sucrose. VvSWEET7-GFP and VvSWEET15-GFP fusion proteins were transiently expressed in epidermal cells and confocal microscopy allowed to observe that both proteins clearly localize to the plasma membrane. In sum, VvSWEETs transporters are important players in sugar mobilization during grape berry development and their expression is transcriptionally reprogrammed in response to infection.

Lipids and oils determine the quality and industrial value of grape seeds. Studies with legume seeds demonstrated the influence of light on lipid metabolism and its association with seed photosynthesis. Grape berry seeds are photosynthetically active till mature stage, but mostly during the green stage and veraison. The objective of this work was to compare the lipid profiles of seeds from white grape berries (cv. Alvarinho) growing at two contrasting light microclimates in the canopy (low and high light, LL and HL respectively), previously reported to have distinct photosynthetic competences. Berries were collected at three developmental stages (green, veraison and mature) and from both microclimates, and the seeds were analyzed for their lipid profiles in an untargeted manner using liquid chromatography coupled to high resolution mass spectrometry (LCMS). The seed lipid profiles differed greatly among berry developmental stages, and to a lesser extend between microclimates. The LL microclimate coincided with a higher relative levels of fatty acids specifically at mature stage, while the HL microclimate led to an up-regulation of ceramides at green stage and of triacylglycerols and glycerophospholipids at mature stage. The seed transcript levels of four key genes (VvACCase1, VvΔ9FAD, VvFAD6 and VvLOXO) involved in fatty acid metabolism were analyzed using real-time qPCR. The lipoxygenase gene (VvLOXO) was down- and up-regulated by HL, as compared to LL, in seeds at green and veraison stages, respectively. These results suggest that seed photosynthesis may play distinct roles during seed growth and development, possibly by fueling different lipid pathways: at green stage mainly towards the accumulation of membrane-bound lipid species that are essential for cell growth and maintenance of the photosynthetic machinery itself; and at veraison and mature stages mainly towards storage lipids that contribute to the final quality of the grape seeds.

Polyols are important metabolites that often function as carbon and energy sources and/or osmoprotective solutes in some plants. In grapevine, and in the grape berry in particular, the molecular aspects of polyol transport and metabolism and their physiological relevance are virtually unknown to date. Here, the biochemical function of a grapevine fruit mesocarp polyol transporter (VvPLT1) was characterized after its heterologous expression in yeast. This H⁺-dependent plasma membrane carrier transports mannitol (K m=5.4 mM) and sorbitol (K m=9.5 mM) over a broad range of polyols and monosaccharides. Water-deficit stress triggered an increase in the expression of VvPLT1 at the fully mature stage, allowing increased polyol uptake into pulp cells. Plant polyol dehydrogenases are oxireductases that reversibly oxidize polyols into monosaccharides. Mannitol catabolism in grape cells (K m=30.1 mM mannitol) and mature berry mesocarps (K m=79 mM) was, like sorbitol dehydrogenase activity, strongly inhibited (50–75%) by water-deficit stress. Simultaneously, fructose reduction into polyols via mannitol and sorbitol dehydrogenases was stimulated, contributing to their higher intracellular concentrations in water-deficit stress. Accordingly, the concentrations of mannitol, sorbitol, galactinol, myo-inositol, and dulcitol were significantly higher in berry mesocarps from water-deficit-stressed Tempranillo grapevines. Metabolomic profiling of the berry pulp by GC-TOF-MS also revealed many other changes in its composition induced by water deficit. The impact of polyols on grape berry composition and plant response to water deficit stress, via modifications in polyol transport and metabolism, was analysed by integrating metabolomics with transcriptional analysis and biochemical approaches.

Climate change increasingly challenges viticulture, demanding innovative and sustainable strategies to preserve grapevine productivity and grape quality. MicroRNA-encoded peptides (miPEPs) have emerged as natural regulators of gene expression, providing a novel mechanism for fine-tuning plant metabolism. Here, we evaluated whether exogenous application of miPEP164c, previously shown to repress VviMYBPA1 in vitro, can modulate flavonoid pathways in field-grown grapevines (Vitis vinifera L. cv. Vinhão). Grape clusters were sprayed with 1 µM miPEP164c before and during véraison, and molecular, biochemical, and metabolomic analyses were performed at harvest. miPEP164c treatment significantly upregulated pre-miR164c transcripts, leading to post-transcriptional silencing of VviMYBPA1 and strong downregulation of the proanthocyanidin-related genes VviLAR1, VviLAR2, and VviANR. Correspondingly, LAR and ANR activities were reduced by up to 75%, and total proanthocyanidin content decreased by nearly 30%. Metabolomic profiling showed reduced flavan-3-ols and moderate shifts in phenolic acids and stilbenoids, while anthocyanins increased slightly. Overall, miPEP164c reprogrammed flavonoid metabolism under vineyard conditions, selectively lowering tannin biosynthesis without affecting other key phenolics. These findings establish miPEPs as promising biostimulants for precise modulation of grape berry composition, offering new tools for urgently needed sustainable and precision viticulture and improved wine quality under climate change and the increasing environmental challenges it poses.

Drought, elevated air temperature, and high evaporative demand are increasingly frequent during summer in grape growing areas like the Mediterranean basin, limiting grapevine productivity and berry quality. The foliar exogenous application of kaolin, a radiation-reflecting inert mineral, has proven effective in mitigating the negative impacts of these abiotic stresses in grapevine and other fruit crops, however, little is known about its influence on the composition of the grape berry and on key molecular mechanisms and metabolic pathways notably important for grape berry quality parameters. Here, we performed a thorough molecular and biochemical analysis to assess how foliar application of kaolin influences major secondary metabolism pathways associated with berry quality-traits, leading to biosynthesis of phenolics and anthocyanins, with a focus on the phenylpropanoid, flavonoid (both flavonol- and anthocyanin-biosynthetic) and stilbenoid pathways. In grape berries from different ripening stages, targeted transcriptional analysis by qPCR revealed that several genes involved in these pathways-VvPAL1, VvC4H1, VvSTSs, VvCHS1, VvFLS1, VvDFR, and VvUFGT-were more expressed in response to the foliar kaolin treatment, particularly in the latter maturation phases. In agreement, enzymatic activities of phenylalanine ammonia lyase (PAL), flavonol synthase (FLS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) were about two-fold higher in mature or fully mature berries from kaolin-treated plants, suggesting regulation also at a transcriptional level. The expression of the glutathione S-transferase VvGST4, and of the tonoplast anthocyanin transporters VvMATE1 and VvABCC1 were also all significantly increased at véraison and in mature berries, thus, when anthocyanins start to accumulate in the vacuole, in agreement with previously observed higher total concentrations of phenolics and anthocyanins in berries from kaolin-treated plants, especially at full maturity stage. Metabolomic analysis by reverse phase LC-QTOF-MS confirmed several kaolin-induced modifications including a significant increase in the quantities of several secondary metabolites including flavonoids and anthocyanins in the latter ripening stages, probably resulting from the general stimulation of the phenylpropanoid and flavonoid pathways.

Correspondence to Dr Rita Gama; ritarocha@campus.ul.pt Description A 72-year-old man presented to the emergency department with a 3-week history of fatigue, weight loss, night sweats, fever and severe nasal obstruction with nasal discharge. CT scan images revealed a neoformative lesion occupying the anterior portion of the left nasal cavity with 1×1.4 cm, showing heterogeneous contrast uptake, and conditioning right septum deviation and total obliteration of the anterior left nasal cavity (figure 2). ENNKTL, nasal type, also known as lethal midline granuloma, is an Epstein-Barr virus-associated lymphoma, and represents about 0.44% of the extranodal sinonasal lymphomas.1 2 The nose is the most common initial involved site, and can manifest as an intranasal lesion conditioning obstructive symptoms, with bleeding and purulent nasal discharge.3 Commonly it manifests with unspecific symptoms of rhinitis or sinusitis, making a malignant diagnosis difficult to suspect.