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.

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.

The grapevine CAX-interacting protein VvCXIP4 is exported from the nucleus to activate the tonoplast Ca2+/H+ exchanger VvCAX3.

MAIN CONCLUSION: The nuclear-localized CAX-interacting protein VvCXIP4 is exported to the cytosol after a Ca2+ pulse, to activate the tonoplast-localized Ca2+/H+ exchanger VvCAX3. Vacuolar cation/H+ exchangers (CAXs) have long been recognized as 'housekeeping' components in cellular Ca2+ and trace metal homeostasis, being involved in a range of key cellular and physiological processes. However, the mechanisms that drive functional activation of the transporters are largely unknown. In the present study, we investigated the function of a putative grapevine CAX-interacting protein, VvCXIP4, by testing its ability to activate VvCAX3, previously characterized as a tonoplast-localized Ca2+/H+ exchanger. VvCAX3 contains an autoinhibitory domain that drives inactivation of the transporter and thus, is incapable of suppressing the Ca2+-hypersensitive phenotype of the S. cerevisiae mutant K667. In this study, the co-expression of VvCXIP4 and VvCAX3 in this strain efficiently rescued its growth defect at high Ca2+ levels. Flow cytometry experiments showed that yeast harboring both proteins effectively accumulated higher Ca2+ levels than cells expressing each of the proteins separately. Bimolecular fluorescence complementation (BiFC) assays allowed visualization of the direct interaction between the proteins in tobacco plants and in yeast, and also showed the self-interaction of VvCAX3 but not of VvCXIP4. Subcellular localization studies showed that, despite being primarily localized to the nucleus, VvCXIP4 is able to move to other cell compartments upon a Ca2+ stimulus, becoming prone to interaction with the tonoplast-localized VvCAX3. qPCR analysis showed that both genes are more expressed in grapevine stems and leaves, followed by the roots, and that the steady-state transcript levels were higher in the pulp than in the skin of grape berries. Also, both VvCXIP4 and VvCAX3 were upregulated by Ca2+ and Na+, indicating they share common regulatory mechanisms. However, VvCXIP4 was also upregulated by Li+, Cu2+ and Mn2+, and its expression increased steadily throughout grape berry development, contrary to VvCAX3, suggesting additional physiological roles for VvCXIP4, including the regulation of VvCAXs not yet functionally characterized. The main novelty of the present study was the demonstration of physical interaction between CXIP and CAX proteins from a woody plant model by BiFC assays, demonstrating the intracellular mobilization of CXIPs in response to Ca2+.

The grapevine NIP2;1 aquaporin is a silicon channel.

Silicon (Si) supplementation has been shown to improve plant tolerance to different stresses, and its accumulation in the aerial organs is mediated by NIP2;1 aquaporins (Lsi channels) and Lsi2-type exporters in roots. In the present study, we tested the hypothesis that grapevine expresses a functional NIP2;1 that accounts for root Si uptake and, eventually, Si accumulation in leaves. Own-rooted grapevine cuttings of the cultivar Vinhão accumulated >0.2% Si (DW) in leaves when irrigated with 1.5 mM Si for 1 month, while Si was undetected in control leaves. Real-time PCR showed that VvNIP2;1 was highly expressed in roots and in green berries. The transient transformation of tobacco leaf epidermal cells mediated by Agrobacterium tumefaciens confirmed VvNIP2;1 localization at the plasma membrane. Transport experiments in oocytes showed that VvNIP2;1 mediates Si and arsenite uptake, whereas permeability studies revealed that VvNIP2;1 expressed in yeast is unable to transport water and glycerol. Si supplementation to pigmented grape cultured cells (cv. Gamay Freáux) had no impact on the total phenolic and anthocyanin content, or on the growth rate and VvNIP2;1 expression. Long-term experiments should help determine the extent of Si uptake over time and whether grapevine can benefit from Si fertilization.

Genome Wide Identification, Molecular Characterization, and Gene Expression Analyses of Grapevine NHX Antiporters Suggest Their Involvement in Growth, Ripening, Seed Dormancy, and Stress Response.

Plant NHX antiporters are critical for cellular pH, Na+, and K+ homeostasis and salt tolerance. Even though their genomic and functional studies have been conducted in many species, the grapevine NHX family has not been described yet. Our work highlights the presence of six VvNHX genes whose phylogenetic analysis revealed their classification in two distinct groups: group I vacuolar (VvNHX1-5) and group II endosomal (VvNHX6). Several cis-acting regulatory elements related to tissue-specific expression, transcription factor binding, abiotic/biotic stresses response, and light regulation elements were identified in their promoter. Expression profile analyses of VvNHX genes showed variable transcription within organs and tissues with diverse patterns according to biochemical, environmental, and biotic treatments. All VvNHXs are involved in berry growth, except VvNHX5 that seems to be rather implicated in seed maturation. VvNHX4 would be more involved in floral development, while VvNHX2 and 3 display redundant roles. QPCR expression analyses of VvNHX1 showed its induction by NaCl and KNO3 treatments, whereas VvNHX6 was induced by ABA application and strongly repressed by PEG treatment. VvNHX1 plays a crucial role in a bunch of grape developmental steps and adaptation responses through mechanisms of phyto-hormonal signaling. Overall, VvNHX family members could be valuable candidate genes for grapevine improvement.

Sweet Cherry (Prunus avium L.) PaPIP1;4 Is a Functional Aquaporin Upregulated by Pre-Harvest Calcium Treatments that Prevent Cracking.

The involvement of aquaporins in rain-induced sweet cherry (Prunus avium L.) fruit cracking is an important research topic with potential agricultural applications. In the present study, we performed the functional characterization of PaPIP1;4, the most expressed aquaporin in sweet cherry fruit. Field experiments focused on the pre-harvest exogenous application to sweet cherry trees, cultivar Skeena, with a solution of 0.5% CaCl2, which is the most common treatment to prevent cracking. Results show that PaPIP1;4 was mostly expressed in the fruit peduncle, but its steady-state transcript levels were higher in fruits from CaCl2-treated plants than in controls. The transient expression of PaPIP1;4-GFP in tobacco epidermal cells and the overexpression of PaPIP1;4 in YSH1172 yeast mutation showed that PaPIP1;4 is a plasma membrane protein able to transport water and hydrogen peroxide. In this study, we characterized for the first time a plasma membrane sweet cherry aquaporin able to transport water and H2O2 that is upregulated by the pre-harvest exogenous application of CaCl2 supplements.

Resveratrol-Loaded Lipid Nanocarriers Are Internalized By Endocytosis in Yeast.

Different positive pharmacological effects have been attributed to the natural product resveratrol (RSV), including antioxidant, antiaging, and cancer chemopreventive properties. However, its low bioavailability and rapid metabolic degradation has led to the suspicion that many of the biological activities of this compound observed in vitro may not be attainable in humans. To improve its bioavailability and pharmacokinetic profile, attempts have been made to encapsulate RSV into lipid-based nanocarrier systems. Here, the dioctadecyldimethylammonium bromide (DODAB):monoolein (MO) liposomal system (1:2) loaded with RSV revealed appropriate characteristics for drug release purposes: reduced size for cellular uptake (157 ± 23 nm), stability up to 80 days, positive surface charge (ζ ≈ +40 mV), and a controlled biphasic release of RSV from the lipid nanocarriers over a period of almost 50 h at pH 5.0 and 7.4. Moreover, the encapsulation efficiency of the nanocarrier ranged from 70% to 92% and its RSV loading capacity from 9% to 14%, when [RSV] was between 100 and 200 µM. The partition coefficient ( Kp) of RSV between lipid and aqueous phase was log Kp = 3.37 ± 0.10, suggesting moderate to high lipophilicity of this natural compound and reinforcing the lipid nanocarriers' suitability for RSV incorporation. The thermodynamic parameters of RSV partitioning in the lipid nanocarriers at 37 °C (Δ H = 43.76 ± 5.68 kJ mol-1; Δ S = 0.20 ± 0.005 kJ mol-1; and Δ G = -18.46 ± 3.48 kJ mol-1) reflected the spontaneity of the process and the establishment of hydrophobic interactions. The cellular uptake mechanism of the RSV-loaded nanocarriers labeled with the lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH) was studied in the eukaryotic model system Saccharomyces cerevisiae. Thirty minutes after incubation, yeast cells readily internalized nanocarriers and the spots of blue fluorescence of DPH clustered around the central vacuole in lipid droplets colocalized with the green fluorescence of the lipophilic endocytosis probe FM1-43. Subsequent studies with the endocytosis defective yeast deletion mutant ( end3Δ) and with the endocytosis inhibitor methyl-ß-cyclodextrin supported the involvement of an endocytic pathway. This novel nanotechnology approach opens good perspectives for medical applications.

A molecular perspective on starch metabolism in woody tissues.

MAIN CONCLUSION: The elucidation of the molecular mechanisms of starch synthesis and mobilization in perennial woody tissues is of the utmost scientific and agricultural importance. Starch is the main carbohydrate reserve in plants and is fundamental in human nutrition and several industrial processes. In leaves, starch accumulated during the day is degraded throughout the night and the resulting sugars, glucose and maltose, are exported to the cytosol by the specialized transmembrane translocators pGT and MEX, respectively. Nevertheless, the degradation of the starch granule is a complex process not completely elucidated. While the mechanisms of starch mobilization during germination in the dead endosperm of cereal seeds are well described, the molecular and biochemical mechanisms involved in starch storage in the heterotrophic tissues of woody plants and its utilization in spring and winter are still puzzling. It is known that some biochemical steps of starch synthesis are conserved in heterotrophic tissues and in the leaves, but some aspects are particular to sink organs. From an agronomic standpoint, the knowledge on starch storage and mobilization in woody tissues is pivotal to understand (and to optimize) some common practices in the field that modify source-sink relationships, such as pruning and defoliation. Soluble sugars resulting from starch are also pivotal to cold adaptation, and in several fruits, such as banana and kiwifruit, starch may provide soluble sugars during ripening. In this review, we explore the recent advances on the molecular mechanisms and regulations involved in starch synthesis and mobilization, with a focus on perennial woody tissues.

Correction to: A molecular perspective on starch metabolism in woody tissues.

The original article was corrected.

Low source-sink ratio reduces reserve starch in grapevine woody canes and modulates sugar transport and metabolism at transcriptional and enzyme activity levels.

MAIN CONCLUSION: Severe leaf removal decreases storage starch and sucrose in grapevine cv. Cabernet Sauvignon fruiting cuttings and modulates the activity of key enzymes and the expression of sugar transporter genes. Leaf removal is an agricultural practice that has been shown to modify vineyard efficiency and grape and wine composition. In this study, we took advantage of the ability to precisely control the number of leaves to fruits in Cabernet Sauvignon fruiting cuttings to study the effect of source-sink ratios (2 (2L), 6 (6L) and 12 (12) leaves per cluster) on starch metabolism and accumulation. Starch concentration was significantly higher in canes from 6L (42.13 ± 1.44 mg g DW-1) and 12L (43.50 ± 2.85 mg g DW-1) than in 2L (22.72 ± 3.10 mg g DW-1) plants. Moreover, carbon limitation promoted a transcriptional adjustment of genes involved in starch metabolism in grapevine woody tissues, including a decrease in the expression of the plastidic glucose-6-phosphate translocator, VvGPT1. Contrarily, the transcript levels of the gene coding the catalytic subunit VvAGPB1 of the VvAGPase complex were higher in canes from 2L plants than in 6L and 12L, which positively correlated with the biochemical activity of this enzyme. Sucrose concentration increased in canes from 2L to 6L and 12L plants, and the amount of total phenolics followed the same trend. Expression studies showed that VvSusy transcripts decreased in canes from 2L to 6L and 12L plants, which correlated with the biochemical activity of insoluble invertase, while the expression of the sugar transporters VvSUC11 and VvSUC12, together with VvSPS1, which codes an enzyme involved in sucrose synthesis, increased. Thus, sucrose seems to control starch accumulation through the adjustment of the cane sink strength.

The grapevine VvCAX3 is a cation/H+ exchanger involved in vacuolar Ca2+ homeostasis.

MAIN CONCLUSION: The grapevine VvCAX3 mediates calcium transport in the vacuole and is mostly expressed in green grape berries and upregulated by Ca 2+ , Na + and methyl jasmonate. Calcium is an essential plant nutrient with important regulatory and structural roles in the berries of grapevine (Vitis vinifera L.). On the other hand, the proton-cation exchanger CAX proteins have been shown to impact Ca2+ homeostasis with important consequences for fruit integrity and resistance to biotic/abiotic stress. Here, the CAX gene found in transcriptomic databases as having one of the highest expressions in grapevine tissues, VvCAX3, was cloned and functionally characterized. Heterologous expression in yeast showed that a truncated version of VvCAX3 lacking its NNR autoinhibitory domain (sCAX3) restored the ability of the yeast strain to grow in 100-200 mM Ca2+, demonstrating a role in Ca2+ transport. The truncated VvCAX3 was further shown to be involved in the transport of Na+, Li+, Mn2+ and Cu2+ in yeast cells. Subcellular localization studies using fluorescently tagged proteins confirmed VvCAX3 as a tonoplast transporter. VvCAX3 is expressed in grapevine stems, leaves, roots, and berries, especially at pea size, decreasing gradually throughout development, in parallel with the pattern of calcium accumulation in the fruit. The transcript abundance of VvCAX3 was shown to be regulated by methyl jasmonate (MeJA), Ca2+, and Na+ in grape cell suspensions, and the VvCAX3 promotor contains several predicted cis-acting elements related to developmental and stress response processes. As a whole, the results obtained add new insights on the mechanisms involved in calcium homeostasis and intracellular compartmentation in grapevine, and indicate that VvCAX3 may be an interesting target towards the development of strategies for enhancement of grape berry properties.

Identification and functional characterization of grapevine transporters that mediate glucose-6-phosphate uptake into plastids.

MAIN CONCLUSION: Two grapevine glucose-6-Pi plastidial transporters differently expressed in plant organs and in response to environmental and hormonal signals are characterized. They are involved in starch accumulation in berries and canes. In grapevine, starch accumulation in the trunk is important for winter storage of carbon and in the flower for reproductive development. Berries also accumulate starch in their plastids, which are also involved in the synthesis of aroma compounds important for fruit quality. The present work characterizes two glucose-phosphate translocators (VvGPT1, VvGPT2) that control the accumulation of starch in grape amyloplasts. Three different splicing variants identified for VvGPT2 (VvGPT2α, VvGPT2ß and VvGPT2Ω) were more expressed in the leaves than in other organs. In contrast, VvGPT1 transcripts were more abundant in mature berries, canes and flowers than in the leaves. Expression of 35S-VvGPT1-GFP and 35S-VvGPT2Ω-GFP in tobacco leaf epidermal cells showed that the fusion proteins localized at the plastidial envelope. Complementation of the Arabidopsis pgi1-1 mutant impaired in leaf starch synthesis restored its ability to synthesize starch, demonstrating that VvGPT1 and VvGPT2Ω mediate the transport of glucose-6-Pi across the plastidial envelope. In grape cell suspensions, ABA, light and galactinol, together with sucrose and fructose, significantly increased the transcript abundance of VvGPT1, whereas VvGPT2Ω expression was affected only by sucrose. In addition, elicitation with methyl jasmonate strongly upregulated VvGPT1, VvGPT2Ω and VvPAL1, suggesting a role for GPTs in the production of secondary compounds in grapevine. Moreover, in grapevines cultivated in field conditions, VvGPT1 expression was higher in berries more exposed to the sun and subjected to higher temperatures. Although both VvGPT1 and VvGPT2 mediate the same function at the molecular level, they exhibit different expression levels and regulation in plant organs and in response to environmental and hormonal signals.

Polyols in grape berry: transport and metabolic adjustments as a physiological strategy for water-deficit stress tolerance in grapevine.

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=5.4mM) and sorbitol (K m=9.5mM) 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=30.1mM mannitol) and mature berry mesocarps (K m=79mM) 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.

Copper homeostasis in grapevine: functional characterization of the Vitis vinifera copper transporter 1.

MAIN CONCLUSION: The Vitis vinifera copper transporter 1 is capable of self-interaction and mediates intracellular copper transport. An understanding of copper homeostasis in grapevine (Vitis vinifera L.) is particularly relevant to viticulture in which copper-based fungicides are intensively used. In the present study, the Vitis vinifera copper transporter 1 (VvCTr1), belonging to the Ctr family of copper transporters, was cloned and functionally characterized. Amino acid sequence analysis showed that VvCTr1 monomers are small peptides composed of 148 amino acids with 3 transmembrane domains and several amino acid residues typical of Ctr transporters. Bimolecular fluorescence complementation (BiFC) demonstrated that Ctr monomers are self-interacting and subcellular localization studies revealed that VvCTr1 is mobilized via the trans-Golgi network, through the pre-vacuolar compartment and located to the vacuolar membrane. The heterologous expression of VvCTr1 in a yeast strain lacking all Ctr transporters fully rescued the phenotype, while a deficient complementation was observed in a strain lacking only plasma membrane-bound Ctrs. Given the common subcellular localization of VvCTr1 and AtCOPT5 and the highest amino acid sequence similarity in comparison to the remaining AtCOPT proteins, Arabidopsis copt5 plants were stably transformed with VvCTr1. The impairment in root growth observed in copt5 seedlings in copper-deficient conditions was fully rescued by VvCTr1, further supporting its involvement in intracellular copper transport. Expression studies in V. vinifera showed that VvCTr1 is mostly expressed in the root system, but transcripts were also present in leaves and stems. The functional characterization of VvCTr-mediated copper transport provides the first step towards understanding the physiological and molecular responses of grapevines to copper-based fungicides.

Vacuolar transport of the medicinal alkaloids from Catharanthus roseus is mediated by a proton-driven antiport.

Catharanthus roseus is one of the most studied medicinal plants due to the interest in their dimeric terpenoid indole alkaloids (TIAs) vinblastine and vincristine, which are used in cancer chemotherapy. These TIAs are produced in very low levels in the leaves of the plant from the monomeric precursors vindoline and catharanthine and, although TIA biosynthesis is reasonably well understood, much less is known about TIA membrane transport mechanisms. However, such knowledge is extremely important to understand TIA metabolic fluxes and to develop strategies aimed at increasing TIA production. In this study, the vacuolar transport mechanism of the main TIAs accumulated in C. roseus leaves, vindoline, catharanthine, and α-3',4'-anhydrovinblastine, was characterized using a tonoplast vesicle system. Vindoline uptake was ATP dependent, and this transport activity was strongly inhibited by NH4(+) and carbonyl cyanide m-chlorophenyl hydrazine and was insensitive to the ATP-binding cassette (ABC) transporter inhibitor vanadate. Spectrofluorimetry assays with a pH-sensitive fluorescent probe showed that vindoline and other TIAs indeed were able to dissipate an H(+) gradient preestablished across the tonoplast by either vacuolar H(+)-ATPase or vacuolar H(+)-pyrophosphatase. The initial rates of H(+) gradient dissipation followed Michaelis-Menten kinetics, suggesting the involvement of mediated transport, and this activity was species and alkaloid specific. Altogether, our results strongly support that TIAs are actively taken up by C. roseus mesophyll vacuoles through a specific H(+) antiport system and not by an ion-trap mechanism or ABC transporters.

The grape aquaporin VvSIP1 transports water across the ER membrane.

Water diffusion through biological membranes is facilitated by aquaporins, members of the widespread major intrinsic proteins (MIPs). In the present study, the localization, expression, and functional characterization of a small basic intrinsic protein (SIP) from the grapevine were assessed. VvSIP1 was expressed in leaves and berries from field-grown vines, and in leaves and stems from in vitro plantlets, but not in roots. When expressed in tobacco mesophyll cells and in Saccharomyces cerevisiae, fluorescent-tagged VvSIP1 was localized at the endoplasmic reticulum (ER). Stopped-flow spectroscopy showed that VvSIP1-enriched ER membrane vesicles from yeast exhibited higher water permeability and lower activation energy for water transport than control vesicles, indicating the involvement of protein-mediated water diffusion. This aquaporin was able to transport water but not glycerol, urea, sorbitol, glucose, or inositol. VvSIP1 expression in Xenopus oocytes failed to increase the water permeability of the plasma membrane. VvSIP1-His-tag was solubilized and purified to homogeneity from yeast ER membranes and the reconstitution of the purified protein in phosphatidylethanolamine liposomes confirmed its water channel activity. To provide further insights into gene function, the expression of VvSIP1 in mature grapes was studied when vines were cultivated in different field conditions, but its transcript levels did not increase significantly in water-stressed plants and western-exposed berries. However, the expression of the aquaporin genes VvSIP1, VvPIP2;2, and VvTIP1;1 was up-regulated by heat in cultured cells.

The first insight into the metabolite profiling of grapes from three Vitis vinifera L. cultivars of two controlled appellation (DOC) regions.

The characterization of the metabolites accumulated in the grapes of specific cultivars grown in different climates is of particular importance for viticulturists and enologists. In the present study, the metabolite profiling of grapes from the cultivars, Alvarinho, Arinto and Padeiro de Basto, of two Portuguese Controlled Denomination of Origin (DOC) regions (Vinho Verde and Lisboa) was investigated by gas chromatography-coupled time-of-flight mass spectrometry (GC-TOF-MS) and an amino acid analyzer. Primary metabolites, including sugars, organic acids and amino acids, and some secondary metabolites were identified. Tartaric and malic acids and free amino acids accumulated more in grapes from vines of the DOC region of Vinho Verde than DOC Lisboa, but a principal component analysis (PCA) plot showed that besides the DOC region, the grape cultivar also accounted for the variance in the relative abundance of metabolites. Grapes from the cultivar, Alvarinho, were particularly rich in malic acid and tartaric acids in both DOC regions, but sucrose accumulated more in the DOC region of Vinho Verde.

Flow cytometry as a novel tool for structural and functional characterization of isolated yeast vacuoles.

The yeast vacuole is functionally analogous to the mammalian lysosome. Both play important roles in fundamental cellular processes such as protein degradation, detoxification, osmoregulation, autophagy and apoptosis which, when deregulated in humans, can lead to several diseases. Some of these vacuolar roles are difficult to study in a cellular context, and therefore the use of a cell-free system is an important approach to gain further insight into the different molecular mechanisms required for vacuolar function. In the present study, the potentialities of flow cytometry for the structural and functional characterization of isolated yeast vacuoles were explored. The isolation protocol resulted in a yeast vacuolar fraction with a degree of purity suitable for cytometric analysis. Moreover, isolated vacuoles were structurally and functionally intact and able to generate and maintain electrochemical gradients of ions across the vacuolar membrane, as assessed by flow cytometry. Proton and calcium gradients were dissipated by NH4Cl and calcimycin, respectively. These results established flow cytometry as a powerful technique for the characterization of isolated vacuoles. The protocols developed in this study can also be used to enhance our understanding of several molecular mechanisms underlying the development of lysosome-related diseases, as well as provide tools to screen for new drugs that can modulate these processes, which have promising clinical relevance.