Functional characterization of VvCAX3: a grapevine cation/H + exchanger that transports Ca2+ and other cations

Grapevines are economically one of the most important fruit species worldwide. Thus, it is essential for winegrowers to guarantee fruit quality upon adverse climate conditions, including heavy rains before harvest that cause severe skin cracking and fruit spoilage. A close relationship has been demonstrated between increased tomato fruit integrity, increased calcium levels and increased activity of CAX-type cation/H+ exchangers. Therefore, the identification and characterization of grapevine CAX transporters is a landmark towards understanding calcium dynamics in the grape berry. In this study we have identified and functionally characterized the Vitis vinifera calcium-H+ antiporter VvCAX3, by heterologous expression of a yeast strain deficient in Ca2+ detoxification. Results showed that the presence of VvCAX3 restored the ability of the strain to grow in high levels of Ca2+ (100-200 mM), validating the role of the protein in Ca2+ transport. In addition, evidence for its role as exchanger of other cations, namely Na+, Li+, Mn2+ and Cu2+ was obtained. The data was complemented by gene expression studies in grape cell suspensions (Cabernet Sauvignon Berry) that showed a regulation of transcript abundance by cation levels in the culture medium. Studies in planta demonstrated that the transporter is highly expressed in green berries (pea size), decreasing gradually as development progresses, in accordance to the pattern of calcium accumulation in the fruit. VvCAX3 transcripts were also detected in grapevine roots, stems and leaves. Altogether the present study provided powerful insights on the role of VvCAX3 in grapevine calcium homeostasis.his work is supported by European investment funds by FEDER/COMPETE/POCI-Operacional Competitiveness and Internationalization Programme, under the Projects INTERACT-NORTE-01-0145-FEDER-000017-Linha VitalityWine-ON 0013 and POCI-01-0145-FEDER-006958, and by National Funds by FCT–Portuguese Foundation for Science and Technology, under the project UID/AGR/ 04033/2013, and CherryCrackLess (PTDC/AGR-PRO/7028/2014). VM was supported by a FCT postdoctoral Grant (SFRH/BPD/107905/ 2015).info:eu-repo/semantics/publishedVersio

Copper impacts on grape berry cells: uptake and detoxification

Copper has been extensively used as the active principle of fungicides, since the late 1800s when the “Bordeaux mixture” was developed and its spectacular efficiency proved against fungal pathogens such as downy mildew, which is a large threat to winegrowers. Although initially it seemed to improve plant growth in unproductive lands, repeated use of copper-based fungicides has led to the accumulation of large concentrations of this metal ion in vineyard soils and raised concerns regarding phytotoxicity. As major targets for heavy metal stress, plants have developed a number of mechanisms to withstand the elevated metal levels. Such responses include exclusion, chelation and compartmentation of metal ions. Both the mitochondria and plastids are copper sinks, and the vacuole is believed to constitute a copper delivery pathway within the cell, and not just a sequestration compartment, due to the proximity of the tonoplast to the other organelles of the plant cell. In the present study, grape berry cells (cv. Cabernet Sauvignon) were used as a model system to study the effect of copper on cell growth and viability. In the concentration range of 0 (+ the copper chelator BCS) to 100 μM CuSO4 growth was virtually unaffected. However, concentrations from 100 to 500 μM caused a sharp decrease in cell growth. The viability of grown cells decreased with the increase in copper concentration in a dose-dependent manner. Studies with the copper-sensitive fluorescent probe PhenGreen™ SK allowed for the identification of copper sinks in grape berry cells. Furthermore, transport studies were performed in isolated intact protoplasts loaded with this probe. The initial velocities of fluorescence quenching upon addition of copper followed a Michaelis-Menten kinetics, suggesting the involvement of mediated transport with a Km= 0.7 mM. Isolated vacuoles labeled with the pH-dependent fluorescent dye ACMA were used to study copper compartmentation as a mean of metal tolerance. Results showed that CuCl2 dissipates a pre-established pH gradient across the tonoplast suggesting the involvement of a Cu2+/H+ antiport system. Eight putative VvCTr (Vitis vinifera Copper Transporter) genes were identified, among which VvCTr1 was isolated and cloned and its expression is currently being studied.VM is supported by a PhD grant (SFRH/BD/64587/2009). This work was supported by Fundação para a Ciência e a Tecnologia (research project no. PTDC/AGRALI/100636/2008)

Calcium- and hormone-driven regulation of secondary metabolism and cell wall enzymes in grape berry cells

Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.jplph.2018.08.011.The efficacy of calcium sprays for improving fleshy fruit resistance to abiotic/biotic stress and enhancement of fruit shelf life has increasingly been explored. However, because calcium is a powerful secondary messenger in many signaling pathways, including those driven by abscisic acid (ABA) and jasmonates, it may interfere with the biosynthesis of specialized metabolites highly important for fruit and wine quality, such as phenolic compounds. In this study, a combination of biochemical and molecular biology approaches were applied to grape cell cultures and detached grape berries, in order to investigate the effect of calcium in the modulation of enzymes involved in the biosynthesis of phenolic compounds and in cell wall organization. Concentrations up to 10mM CaCl2 did not affect cell growth, size or viability, but triggered modifications in total phenolics content, particularly in anthocyanin levels in grape cell suspensions. The effects of calcium applied alone or in combination with ABA or methyl jasmonate (MeJA) were visible in several branches of specialized metabolic pathways, confirming that the calcium-hormone interplay regulates the expression of phenylalanine ammonia lyase (PAL), stilbene synthase (STS), dihydroflavonol reductase (DFR) and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT). The activity of PAL and UFGT enzymes was also specifically modulated by calcium, ABA and MeJA. These results closely correlated to the modifications observed in the expression of VvAM1 and VvABCC1 encoding vacuolar anthocyanin transporters. Modulation of the expression and activity of pectin methyl esterases (PME) and polygalacturonases (PG) by calcium was also evident, confirming an important role of calcium in cell wall organization via the regulation of enzyme activity, besides its well-known role in the formation of cross links between pectin molecules. Overall, this study uncovers important biochemical mechanisms induced by calcium and stress hormones on grape berries, and highlights the need to consider the consequences of calcium treatments and stress for fruit quality.This work was supported by European investment funds by FEDER/COMPETE/POCI-Operacional Competitiveness and Internationalization Programme, under the Projects INTERACT-NORTE-01-0145-FEDER-000017-Linha VitalityWine-ON 0013 and POCI-01-0145-FEDER-006958, and by National Funds by FCT-Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2013, and CherryCrackLess (PTDC/AGR-PRO/7028/2014). VM was supported by a FCT postdoctoral Grant (SFRH/BPD/107905/2015).info:eu-repo/semantics/publishedVersio

Calcium- and hormone-driven regulation of secondary metabolism and cell wall enzymes in grape berry cells

Supplementary material related to this article can be found, in the online version, at doi: https://doi.org/10.1016/j.jplph.2018.08.011.The efficacy of calcium sprays for improving fleshy fruit resistance to abiotic/biotic stress and enhancement of fruit shelf life has increasingly been explored. However, because calcium is a powerful secondary messenger in many signaling pathways, including those driven by abscisic acid (ABA) and jasmonates, it may interfere with the biosynthesis of specialized metabolites highly important for fruit and wine quality, such as phenolic compounds. In this study, a combination of biochemical and molecular biology approaches were applied to grape cell cultures and detached grape berries, in order to investigate the effect of calcium in the modulation of enzymes involved in the biosynthesis of phenolic compounds and in cell wall organization. Concentrations up to 10mM CaCl2 did not affect cell growth, size or viability, but triggered modifications in total phenolics content, particularly in anthocyanin levels in grape cell suspensions. The effects of calcium applied alone or in combination with ABA or methyl jasmonate (MeJA) were visible in several branches of specialized metabolic pathways, confirming that the calcium-hormone interplay regulates the expression of phenylalanine ammonia lyase (PAL), stilbene synthase (STS), dihydroflavonol reductase (DFR) and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT). The activity of PAL and UFGT enzymes was also specifically modulated by calcium, ABA and MeJA. These results closely correlated to the modifications observed in the expression of VvAM1 and VvABCC1 encoding vacuolar anthocyanin transporters. Modulation of the expression and activity of pectin methyl esterases (PME) and polygalacturonases (PG) by calcium was also evident, confirming an important role of calcium in cell wall organization via the regulation of enzyme activity, besides its well-known role in the formation of cross links between pectin molecules. Overall, this study uncovers important biochemical mechanisms induced by calcium and stress hormones on grape berries, and highlights the need to consider the consequences of calcium treatments and stress for fruit quality.This work was supported by European investment funds by FEDER/COMPETE/POCI-Operacional Competitiveness and Internationalization Programme, under the Projects INTERACT-NORTE-01-0145-FEDER-000017-Linha VitalityWine-ON 0013 and POCI-01-0145-FEDER-006958, and by National Funds by FCT-Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2013, and CherryCrackLess (PTDC/AGR-PRO/7028/2014). VM was supported by a FCT postdoctoral Grant (SFRH/BPD/107905/2015).info:eu-repo/semantics/publishedVersio

Solute transport across plant cell membranes

CanaBQ is a Journal of the Portuguese Biochemical Society (http://canalbq.spb.pt/index.html)The transport of solutes across cell membranes, including organic nutrients, such as sugar, osmolytes, ions or metabolic waste products, is of extreme importance in all living systems. Up to 14% of the genome of all organisms represents information for transport proteins, which reflects the importance of such process. Transporters are also involved in the transduction of environmental and endogenous signals. Several transport systems have been identified and fully characterised at both molecular and biophysical levels in a wide variety of living organisms, from bacteria to humans, with the bacterial lactose permease (LacY) being a good example of such successful studies. The majority of transporter proteins are very well conserved throughout living systems, and some of them, such as sugar transporters, belong to a large family (SP, Sugar Porter). In higher plants, the photoassimilated carbon is transported from mature leaves throughout the phloem, mainly in the form of sucrose, as in the grapevine, or mannitol, as in the olive tree, to heterotrophic organs such as developing leaves, flowers, fruits and roots, which rely on its supply for their growth and development. Thus, the unlocking of the mechanisms of photoassimilate transport into plant sink tissues, as well as their regulation, has an important basic and applied relevance. Moreover, as most living organisms, plants also face a continuous battle against adverse environmental factors like increasing soil salinity, heat and drought. In this context, solute transport also has a relevant role in plant defence. For instance, the efficient exclusion of Na+ excess from the cytoplasm and vacuolar Na+ accumulation are the most important steps towards the maintenance of ion homeostasis under salt stress. The production, transport and accumulation of compatible solutes like mannitol are also important plant responses to salinity and drought. Like animals, where important diseases such as depression and hypertension are commonly treated with drugs targeted to specific transporters, plants have also benefited from the extensive and ongoing study of membrane transport. The present review provides an overview on the investigation that has been conducted in our laboratory under the scope of this fascinating topic.This work was supported by the Portuguese Foundation for Science and Technology (FCT) (research project ref. PTDC/AGR-ALI/100636/2008; to A. Conde, grant ref. SFRH/BD/47699/2008; to V. Martins, grant ref. SFRH/BD/64587/2009; to H. Noronha, grant ref. SFRH/BD/75257/2010, to N. Fontes, grant ref. SFRH/ BD/23169/2005, and to C. Conde, grant ref. SFRH/ BPD/34998/2007). We are also grateful to BabeliUM, the Language Centre of the University of Minho, namely Ana Teresa Correia, for revising the English version of the manuscript

Impacto do cobre na videira (Vitis vinifera L.): abordagens moleculares, bioquímicas e biotecnológicas

Tese de doutoramento em Programa Doutoral em Biologia de PlantasSince the development of Bordeaux mixture in the late 1800’s, copper-based fungicides have been widely used against grapevine (Vitis vinifera L.) diseases, mainly in organic but also in conventional viticulture. Although they initially seemed to improve plant growth in unproductive lands, their intensive use has raised concerns regarding toxicity to plants and soil contamination, and research has emerged to characterize copper effects on grapevine physiology. However, at grape berry level the effects of plant exposure to copper remain largely unexplored. In the present study molecular, biochemical and biotechnological approaches were combined to investigate the mechanisms mediating copper transport and compartmentation in grape cells and the consequences of altered copper status in grape berry composition and wine quality. Toxicological assessments in grape cell suspensions (CSB, Cabernet Sauvignon Berry) showed that copper reduced the growth and cell viability in a dose-dependent manner above 100 μM and was accumulated in specific metal ion sinks. Copper transport across the plasma membrane of grape cells was characterized with the copper-sensitive probe Phen Green SK, and results showed that copper uptake was mediated by a high capacity saturable transport system (Km = 583 μM; Vmax = 177 x10-6 %ΔF min-1 protoplast-1), that was regulated by copper availability in the culture medium. The compartmentation of copper in the vacuole of grape cells was studied by fluorescence microscopy and spectrofluorimetry. The pH-sensitive fluorescent probe ACMA showed the involvement of H+-dependent copper transport across the tonoplast, which was dependent on the transmembrane pH gradient generated by both V-H+-ATPase and V-H+-PPase, but apparently not regulated by copper availability in the culture medium. Eight putative COPT/Ctr-type Vitis vinifera copper transporters were identified in the grapevine genome, and expression studies in CSB suspensions showed that VvCTr1 and VvCTr8 were distinctly affected by CuSO4 availability. The in silico analysis of all VvCTr members showed a close phylogenetic relationship to COPT proteins from Arabidopsis and rice, and typical features of Ctr transporters, including the presence of three transmembrane domains. In a field trial, the expression of each VvCTr was studied by qPCR in grape berries and leaves cv. “Vinhão” throughout the fructification season. In plants treated with a triazol-based fungicide, transcripts of VvCTr1 and VvCTr8 were the most abundant in leaves and berries, respectively, while VvCTr4, VvCTr5 and VvCTr7 were the least expressed. The application of Bordeaux mixture induced a transcriptional reprogramming of VvCTr gene expression depending on the developmental stage, which was associated to increased copper levels in grape berries (33.3 μg g-1 DW in the mature phase – washed berries). Further characterization of Ctr-mediated transport was achieved with the functional characterization of VvCTr1. Fusions with fluorescent proteins demonstrated that VvCTr1 monomers are self-interacting and the protein is located to the vacuolar membrane. The expression of VvCTr1 in yeast ctrΔ strains and Arabidopsis copt5 seedlings allowed the validation of its involvement in intracellular copper transport. The presence of VvCTr1 transcripts in grapevine roots, stem and leaves further supported its essential role in copper homeostasis. The effect of Bordeaux mixture on the metabolomics profile of grape berries and leaves cv. “Vinhão” was studied throughout fruit development by GC-TOF-MS, UV-visible spectrophotometry. While copper-treated berries accumulated 7 to 14-fold more copper than control fruits, the levels of sugars, essentially fructose, organic acids and flavan-3-ols were modified at specific developmental stages. A 40% decrease of free natural amino acids including arginine and proline was observed in mature fruits, together with a decrease in mineral nitrogen and protein content. This shift in grape berry composition is likely to have implications in fruit and wine quality. The effect of Bordeaux mixture on the volatile composition of wines cv. “Vinhão” was evaluated by Liquid-Liquid Microextraction GC-FID and SPE GC-Ion Trap-MS analyses. Laboratory-scale vinifications were performed, and a significant delay in the degradation of reducing sugars was observed in musts from copper-treated grapes, which contained 12.6 mg L-1 copper at the end of fermentation. Increased copper levels promoted a decrease in the levels of higher alcohols, including isoamyl alcohol, aldehydes, including acetaldehyde, esters of organic acids, lactones, volatile phenols and ketones, while increasing the levels of acetates, volatile fatty acids, fatty acid ethyl esters and terpenes, namely linalool. Besides its importance at a scientific standpoint, the present study contributed to elucidate the effect of copper-based fungicides in grape berry composition and wine quality.Desde o desenvolvimento da calda bordalesa no final do século XIX, os fungicidas à base de cobre têm sido amplamente usados para combater doenças da videira (Vitis vinifera L.), especialmente na agricultura biológica. Diversos estudos têm sido direcionados para avaliar o impacto da aplicação de cobre na fisiologia da videira, embora permaneça pouco estudado o seu efeito ao nível do fruto. No presente trabalho foram combinadas abordagens moleculares, bioquímicas e de carácter biotecnológico para investigar os mecanismos envolvidos no transporte e compartimentação do cobre nas células do bago de uva, bem como os efeitos da alteração dos níveis de cobre na composição do bago e na qualidade do vinho. A sonda fluorescente Phen Green SK foi utilizada para estudar o transporte de cobre através da membrana plasmática de células CSB (Cabernet Sauvignon Berry). Os resultados mostraram que a entrada de cobre é mediada por um sistema de transporte saturável de elevada capacidade, regulado pela disponibilidade de cobre no meio de cultura. Os resultados obtidos com a sonda fluorescente ACMA, sensível ao pH, mostraram que o movimento do cobre através da membrana vacuolar é mediado por um sistema de antiporte com H+, dependente do gradiente de H+ gerado pelas bombas V-H+-ATPase e V-H+-PPase. A análise do genoma da videira permitiu identificar oito genes codificantes de transportadores de cobre do tipo COPT/Ctr (VvCTr1-VvCTr8). Estudos in silico mostraram um elevado grau de semelhança entre as sequências aminoacídicas dos VvCTr e dos COPT de Arabidopsis e de Oryza sativa, bem como a presença de motivos típicos dos Ctrs. O trabalho subsequente incluiu ensaios de campo que possibilitaram o estudo do efeito da aplicação da calda bordalesa na expressão dos VvCTrs em bagos e em folhas de videira da variedade Vinhão. Os resultados de qPCR mostraram que em plantas tratadas unicamente com fungicidas à base de triazol, os genes VvCTr1 e VvCTr8 foram os mais expressos em folhas e bagos, respectivamente, enquanto os genes VvCTr4, VvCTr5 e VvCTr7 foram os menos expressos. Em plantas tratadas com calda bordalesa ocorreu uma reprogramação da expressão dos VvCTrs, cujo padrão foi dependente do estado de desenvolvimento do fruto. O tratamento com a calda bordalesa provocou aumentos de 7 a 14 vezes dos níveis de cobre em bagos lavados. Os estudos prosseguiram com o intuito de se caracterizar funcionalmente os transportadores de cobre da videira. Neste âmbito, o gene VvCTr1 foi clonado a partir de cDNA de bago e a sua função como transportador intracelular de cobre foi demonstrada por complementação de mutantes de levedura (ctr1Δctr3Δctr2Δ e ctr1Δctr3Δ) e de Arabidopsis (copt5). Adicionalmente, estudos de localização subcelular com proteínas de fusão fluorescentes mostraram que os monómeros de VvCTr1 são capazes de interagir entre si e que o transportador se localiza na membrana vacuolar. O efeito da calda bordalesa no metaboloma do bago e de folhas de videira da variedade Vinhão sujeitas ao protoloco de tratamento descrito anteriormente foi também alvo de estudo. A análise dos extratos por “GC-TOF-MS” mostrou que os bagos de plantas tratadas com cobre sofreram alterações metabólicas, traduzidas por níveis diferentes de açúcares, de aminoácidos, de ácidos orgânicos e de alguns metabolitos secundários em fases específicas do desenvolvimento. Em particular, em bagos maduros de plantas sujeitas ao tratamento com cobre o conteúdo em aminoácidos livres sofreu uma redução de 40%, acompanhada por uma redução menos significativa dos níveis de proteína total e de azoto mineral. No presente trabalho foi ainda estudado o efeito dos níveis de cobre no mosto - resultantes da aplicação de calda bordalesa -, na constituição volátil do vinho. Para o efeito, foram efectuadas vinificações à escala laboratorial, tendo-se observado um atraso significativo na conversão de açúcares redutores em mostos de bagos tratados com cobre. Os níveis elevados de cobre (12.6 mg L-1) promoveram uma diminuição do conteúdo em álcoois, aldeídos, estéres de ácidos orgânicos, lactonas, fenóis voláteis e cetonas. Em paralelo, foi observado um aumento dos níveis de acetatos, ácidos gordos voláteis, ésteres etílicos de ácidos gordos e terpenos. O presente estudo revestiu-se de uma dimensão fundamental e aplicada e contribuiu para se compreenderem melhor os efeitos dos fungicidas à base de cobre na constituição química do bago de uva e no perfil aromático do vinho.Fundação para a Ciência e a Tecnologia - Bolsa de Doutoramento SFRH/BD/64587/2009; Fundação para a Ciência e a Tecnologia – projeto “GrapeBerryFactory: Sugars, acids, phenolics and water on grape development and ripening” (FCOMP-01-0124-FEDER-008760; PTDC/AGR-ALI/100636/2008); Fundação para a Ciência e a Tecnologia - projeto de cooperação científica Portugal-Tunísia (FCT/5955/27/5/2013/S); FEDER/COMPETE - Operational Competitiveness Programme - projeto FCOMP-01-0124-FEDER-022692; Projeto Europeu INNOVINE – “Combining innovation in vineyard management and genetic diversity for a sustainable European viticulture” (FP7-KBBE-2012-6-311775); COST Action FA1106 – QualityFruit, “An integrated systems approach to determine the developmental mechanisms controlling fleshy fruit quality in tomato and grapevine”; COST Action FA1003 - "East-west collaboration for grapevine diversity exploration and mobilization of adaptive traits for breeding”

Analytical and Fluorimetric Methods for the Characterization of the Transmembrane Transport of Specialized Metabolites in Plants

The characterization of membrane transport of specialized metabolites is essential to understand their metabolic fluxes and to implement metabolic engineering strategies towards the production of increased levels of these valuable metabolites. Here, we describe a set of procedures to isolate tonoplast membranes, to check their purity and functionality, and to characterize their transport properties. Transport is assayed directly by HPLC analysis and quantification of the metabolites actively accumulated in the vesicles, and indirectly using the pH sensitive fluorescent probe ACMA (9-amino-6- chloro-2-methoxyacridine), when a proton antiport is involved.1) Fundo Europeu de Desenvolvimento Regional funds through the Operational Competitiveness Programme COMPETE and by National Funds through Fundação para a Ciência e a Tecnologia (FCT) under the projects FCOMP-01-0124-FEDER-037277 (PEst-C/SAU/LA0002/2013) and FCOMP-01-0124-FEDER-019664 (PEst-C/SAU/LA0002/2010); (2) by the FCT scholarships co-supported by FCT and POPH-QREN (European Social Fund), SFRH/BD/41907/2007 (IC) and SFRH/BD/74257/2010 (HN); (3) by a Postdoctoral fellowship financed by national funds through FCT under the project Incentivo/SAU/LA0002/2014 (VM); (4) by a Scientific Mecenate Grant from Grupo Jerónimo Martins.info:eu-repo/semantics/publishedVersio

Heterologous expression of the human Copper Transporters hCTR1 and hCTR2 in yeast for endocytic trafficking analysis

SPB2016info:eu-repo/semantics/draf

Study of the human copper transporters hCtr1 and hCtr2 using a yeast host

info:eu-repo/semantics/draf

Functional characterization of the Human Copper Transporters hCTR1 and hCTR2 in the yeast Saccharomyces cerevisiae

Copper (Cu) is an essential trace element for both eukaryotic and prokaryotic organisms. It plays a crucial role as co-factor of metalloenzymes that participate in important cellular processes, such as growth, development and physiology. Although its importance in maintaining cell health, high level of this ion is extremely toxic (Wang et al., 2011). Therefore, cells possess tight regulated systems to conserve copper homeostasis. One of these mechanisms includes the endocytosis of the Copper Transporter 1 (Ctr1) at high Cu levels, a process already verified in yeast and human cells (Liu et al., 2007; Maryon et al., 2013). Besides these new advances, the molecular mechanisms that are behind the intracellular trafficking of the hCtr1 protein are still poor understood. So, to get new insights into this mechanism, an heterologous expression system was created using the yeast Saccharomyces cerevisiae as host (Pereira et al., 2016)). Human CTR1 and CTR2 genes tagged with GFP were cloned into pYPKpw plasmid and transformed into a S. cerevisiae strain disrupted for copper transporters. Importantly, phenotypic assays demonstrated that human Ctr1 complemented the yeast ctr-mutant strain for the ability to grow in a medium containing nonfermentable carbon sources. Moreover, hCtr1 and hCtr2 were localized at the plasma membrane and intracellularly. Data will be presented regarding the expression of hCTRs in different conditionsFCT (PD/BD/135208/2017); Centro de Biologia Molecular e Ambiental (CBMA