In vitro cultures of grape tissues: new possibilities to study grape berry physiology

Grape berries suffer important morphological, biochemical and physiological changes during its development and maturation. It is known that photoassimilates translocated from leaves serve as the major source of carbon and energy to support fruit needs, but recent findings revealed that, at least in the green phase, grape berries show high photosynthetic activity especially in the exocarp. The contribution of fruit photosynthesis for fruit growth and production of organic compounds is far from being understood. In this study photomixotrophic cell suspensions were established as an in vitro model to complement the study grape berry photosynthesis. Calli of CSB (Cabernet Sauvignon Berry) cells derived from the inner tissues of the grape berry were sub-cultured in liquid modified MS medium supplemented with 2% sucrose and different hormonal combinations, one auxin (NAA) and three cytokinins (BAP, ZEA and KIN) at two different final concentrations (0.5 and 1 g mL-1). Two different growth light intensities (45- 60 and 80-105 mol m-1 s-1) were also tested. Chlorophyll fluorescence PAM fluorometry was used to evaluate the photochemical efficiency (Fv/Fm) of all suspensions and chlorophyll content was also determined. Results showed that the cytokinin type was crucial to induce the photosynthetic phenotype, but Fv/Fm was low when compared to the value exhibited by grape berry skin. To further study the photoautrophy of the grape berry tissues new callus cultures were established from the exocarp tissues of the fruit, harvested at the green stage (Alvarinho cv). Portions of detached exocarp were cultured on B5 solid medium supplemented with different hormonal combinations (NAA or 2,4-D with BAP or ZEA) at different concentrations (0.1 or 0.2 g mL-1 for auxins and 0.2 or 0.4 g mL-1 for cytokinins). Only two combinations were responsive but both induced pale green calli with higher growth rates. To our knowledge, this is a pioneer study on calli production from the exocarp of the grape berry from the Portuguese variety Alvarinho. This approach opens good perspectives to study in more detail the physiology of these cells, namely the role of photosynthesis on cell growth and metabolite production. Moreover, it will allow to investigate the impact of several environmental factors on fruit photosynthesis, such as temperature, light and water potential.Fundação para a Ciência e a Tecnologia (research project no. PTDC/AGR-ALI/100636/2008

A 2d in vivo approach to study photosynthesis in grape berry

Is argued that fruit photosynthesis serves mainly as a respiratory CO2 refixation mechanism [1] but its contribution to growth and metabolism, localization and dynamics during fruit development are poorly known. Unlike the leaves, fruit volume imposes a constraint to photosynthesis by limiting light penetration. However, the patterns of chlorophyll distribution are apparently independent of a light intensity gradient. Microscopic observations of transversal slices of green stage grape berries (6-8 weeks after fruit set) of Alvarinho cultivar, revealed that exocarp cells, mesocarp cells next to vascular bundles, and seed coat cells present higher chlorophyll contents than inner mesocarp cells. The photosynthetic activity was determined on this material by Imaging-PAM fluorometry, a powerful tool for 2D mapping of in vivo photosynthesis. In 2 mm-thick grape berry discs, chlorophyll fluorescence parameters were estimated (Fv/Fm and II), and rapid light curves (RLC) were performed. Exocarp and seed coats of green berries showed the highest Fv/Fm values (ca. 0.6-0.7), and mesocarp cells around 85% of that value. Exocarp from mature grapes maintained Fv/Fm values during maturation, but in mesocarp and seed coats this value strongly decreased. ETRr were very sensitive to increasing light intensities and decreased with grape berry maturation. Our future prospects include the implication of photosynthesis on grape berry solute contents (sugars, acids), fruit and seed development.Fundação para a Ciência e a Tecnologia (research project no. PTDC/AGR-ALI/100636/2008

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

[EN] 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.This work was supported by the Fundacao para a Ciencia e Tecnologia (FCT), under the strategic programmes UID/AGR/04033/2020 and UID/BIA/04050/2020. This work was also supported by FCT and European Funds (FEDER/POCI/COMPETE2020) through the research project "MitiVineDrought-Combining `omics' with molecular, biochemical, and physiological analyses as an integrated effort to validate novel and easy-to-implement drought mitigation strategies in grapevine while reducing water use" with ref. PTDC/BIA-FBT/30341/2017 and ref. POCI-01-0145-FEDER-030341, respectively; through the research project "BerryPlastid-Biosynthesis of secondary compounds in the grape berry: unlocking the role of the plastid" with ref. POCI-010145-FEDER-028165 and ref. PTDC/BIA-FBT/28165/2017, respectively; and also through the FCT-funded research project "GrapeInfectomics" (PTDC/ASPHOR/28485/2017). A.C. was supported with a post-doctoral researcher contract/position within the project "MitiVineDrought" (PTDC/BIA-FBT/30341/2017 and POCI-01-0145-FEDER-030341). R.B. was supported by a PhD student grant (PD/BD/113616/2015) under the Doctoral Programme "Agricultural Production Chains-from fork to farm" (PD/00122/2012) funded by FCT. H.B. was supported by a PhD fellowship funded by FCT (SFRH/BD/144638/2019). This work also benefited from the networking activities within the European Unionfunded COST Action CA17111 "INTEGRAPE-Data Integration to maximize the power of omics for grapevine improvement".Breia, R.; Conde, A.; Badim, H.; Fortes, AM.; Geros, H.; Granell Richart, A. (2021). Plant SWEETs: from sugar transport to plant-pathogen interaction and more unexpected physiological roles. Plant Physiology. 186(2):836-852. https://doi.org/10.1093/plphys/kiab127S836852186

VvSWEET7 Is a Mono- and Disaccharide Transporter Up-Regulated in Response to Botrytis cinerea Infection in Grape Berries

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 Botrytis-susceptible variety "Trincadeira," we thoroughly analyzed modifications in the gene expression profile of key SWEET genes in Botrytis cinerea-infected grape berries. VvSWEET7 and VvSWEET15 are likely to play an important role during fruit development and Botrytis infection as they are strongly expressed at the green and mature stage, respectively, and were clearly up-regulated in response to infection. Also, B. cinerea infection down-regulated VvSWEET17a expression at the green stage, VvSWEET10 and VvSWEET17d expression at the veraison stage, and VvSWEET11 expression at the mature stage. VvSWEET7 was functionally characterized by heterologous expression in Saccharomyces cerevisiae as a low-affinity, high-capacity glucose and sucrose transporter with a K (m) of 15.42 mM for glucose and a K (m) of 40.08 mM for sucrose. VvSWEET7-GFP and VvSWEET15-GFP fusion proteins were transiently expressed in Nicotiana benthamiana 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 Botrytis infection.The work was supported by National Funds by FCT-Portuguese Foundation for Science and Technology, under the strategic programmes UID/AGR/04033/2019 and UID/BIA/04050/2019. The work was also supported by FCT and European Funds (FEDER/POCI/COMPETE2020) through the research project "MitiVineDrought-Combining "omics" with molecular, biochemical, and physiological analyses as an integrated effort to validate novel and easy-to-implement droughtmitigation strategies in grapevine while reducing water use" with ref. PTDC/BIA-FBT/30341/2017 and ref. POCI-01-0145-FEDER-030341, respectively; through the research project "BerryPlastid-Biosynthesis of secondary compounds in the grape berry: unlocking the role of the plastid" with ref. POCI-01-0145-FEDER-028165 and ref. PTDC/BIA-FBT/28165/2017, respectively; and also through the FCT-funded research project "GrapeInfectomics" (PTDC/ASP-HOR/28485/2017). This work was also supported by the project "INTERACT - VitalityWine -ref. NORTE-01-0145-FEDER-000017 - (through FEDER/COMPETE and NORTE2020/CCDR-N). AC was supported with a post-doctoral fellowof the mentioned INTERACT/VitalityWine project with ref. BPD/UTAD/INTERACT/VW/218/2016 and by a post-doctoral researcher contract/position within the project "MitiVineDrought" (PTDC/BIA-FBT/30341/2017 and POCI-01-0145-FEDER-030341). RB was supported with a PhD student grant (PD/BD/113616/2015) under theDoctoral Programme 'Agricultural Production Chains-from fork to farm' (PD/00122/2012) funded by FCT. This work also benefited from the networking activities within the European Union-funded COST Action CA17111 -"INTEGRAPE-Data Integration to maximize the power of omics 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.This work was supported by the “Contrato-Programa” UIDB/04050/2020 and UIDB/04033/2020 funded by national funds through the FCT I.P. The work was also supported by FCT and European Funds (FEDER/POCI/COMPETE2020) through the research projects MitiVineDrought (PTDC/BIA-FBT/30341/2017 and POCI-01-0145-FEDER-030341), BerryPlastid (PTDC/BIA-FBT/28165/2017 and POCI-01-0145-FEDER-028165) and CherryCrackLess (PTDC/AGR-PRO/7028/2014). R.B. was supported with a PhD student grant (PD/BD/113616/2015) under the Agrichains Doctoral Program (PD/00122/2012) funded by FCT. H.N. was supported by an FCT postdoctoral grant (SFRH/BPD/115518/2016) and A.C. was supported by a contract in the MitiVineDrought project.info:eu-repo/semantics/publishedVersio

Stresse biótico em videira – elucidação do papel dos transportadores SWEET na interação planta-patógeno

Tese de Doutoramento em Ciências (Especialidade em Biologia)Os açúcares desempenham funções vitais nos seres vivos, principalmente como fontes de carbono e de energia, mas também como reguladores osmóticos e como moléculas sinalizadoras. Em particular, na videira (Vitis vinífera L.), a qualidade do vinho depende dos níveis de açúcar nos bagos de uva porque determinam a concentração em etanol e influenciam a síntese de compostos secundários (incluindo pigmentos). Diferentes famílias de transportadores membranares presentes no genoma das plantas desempenham um papel essencial na translocação de açúcares entre os tecidos fotossintéticos e os tecidos de armazenamento. Entre eles, os transportadores denominados SWEET (Sugars Will Eventually be Exported Transporter), recentemente identificados, têm revelado diferentes papéis em mecanismos fisiológicos onde o efluxo de açúcar é fundamental, como nos nectários. Na videira, a família SWEET compreende 17 membros. No presente estudo pretendeu-se elucidar o papel dos VvSWEETs na resposta da videira à infeção por fungos (Botrytis cinerea e Erysiphe necator) e ao stresse abiótico, incluindo a secura. Além disso, para estudar os papéis fisiológicos do VvSWEET7 e VvSWEET15, foram aplicadas diferentes técnicas de engenharia genética de plantas, tal como CRISPR-Cas9. Em particular, na variedade Trincadeira, susceptível ao fungo B. cinerea, e na variedade Carignan, susceptível a E. necator, foram analisadas em detalhe as modificações no perfil de expressão dos SWEETs em bagos de uva infetados. Os resultados mostraram que a infeção por E. necator causa modificações mais pronunciadas na expressão dos VvSWEETs do que a infecção por Botrytis. Por outro lado, a maioria dos SWEETs da videira foram regulados negativamente em bagos de uva em resposta à secura, no entanto, o VvSWEET10 e VvSWEET11 foram regulados positivamente. Foi também observado que a expressão do VvSWEET1, VvSWEET4 e VvSWEET11 é regulada positivamente em folhas tratadas com caulino (filme inerte usado para proteger as videiras em situações de deficit hídrico, de radiação solar extrema e de ondas de calor), sugerindo que este mineral estimula a capacidade de transporte de sacarose entre os tecidos fotossintéticos e os tecidos de armazenamento. Estudos subsequentes mostraram que os genes VvSWEET11 e VvSWEET15 são positivamente regulados em bagos de uva submetidos a temperaturas de 50ºC durante 7 dias, tratamento normalmente usado para a produção de uvas passas. Uma vez que os níveis de transcritos dos genes VvSWEET7 e VvSWEET15 foram elevados nos bagos de uva e aumentaram em resposta à infeção por Botrytis, as proteínas VvSWEET7 e VvSWEET15 foram alvo de estudos adicionais para se avaliar a sua localização sub-celular e função. As proteínas de fusão VvSWEET7-GFP e VvSWEET15- GFP foram transitoriamente expressas em células da epiderme de Nicotiana benthamiana e os resultados de microscopia confocal mostraram que ambas as proteínas se localizam claramente na membrana plasmática. Após expressão heteróloga numa estirpe mutante de Saccharomyces cerevisiae (hxt-null), a proteína VvSWEET7 foi caracterizada funcionalmente como um transportador de glucose e de sacarose (Km =15,4 mM glucose e Km = 40,1 mM sacarose). Ensaios de inibição competitiva mostraram que o manitol e o sorbitol inibem o transporte de D-[14C(U)]- glucose, sugerindo que, além de mono- e de dissacarídeos, o VvSWEET7 medeia o transporte de polióis. No presente trabalho foram ainda identificados no genoma da videira 18 membros da família de transportadores de açúcares denominada ERD6like e a proteína VvERD6l13 foi alvo de um estudo mais aprofundado. A proteína de fusão VvERD6l13-GFP foi transitoriamente expressa em folhas de N. benthamiana após transformação mediada por Agrobacterium e os resultados de microscopia de fluorescência mostraram que se localiza na membrana plasmática. Estudos de transporte de açúcares marcados radioativamente, após expressão heteróloga em leveduras mutantes (hxt-null), mostraram que a proteína VvERD6l13 é um transportador de sacarose com protões (Km = 33 mM). O gene VvERD6l13 é fortemente regulado em bagos de uva infetados com Botrytis ou E. necator, sugerindo que a proteína VvERD6l13 tem um papel importante durante a interação planta-patógeno. Além disso, o VvERD6l13 é expresso em diferentes tecidos da videira, em particular na raiz. Genericamente, os resultados mostraram que os transportadores VvSWEET e o VvERD6l desempenham um papel importante na mobilização de açúcares durante o desenvolvimento dos bagos de uva e que a sua expressão é regulada ao nível da transcrição em resposta ao stresse biótico e abiótico. No seu conjunto, estes resultados ajudam a compor o puzzle complexo dos mecanismos de resposta da videira aos stresses biótico e abiótico, abrindo ainda caminhos novos e desafiadores no tópico do transporte transmembranar em plantas.Sugars perform vital functions in the living world, primarily as sources of carbon and energy, but also as osmotic regulators and signaling molecules, among others. This is particularly relevant in the grapevine (Vitis vinifera L.) as the quality of the wine depends on the sugar concentration in the grape berry as it determines the final concentration in ethanol, but is also tightly related to the amount of secondary compounds (including pigments) synthesized during ripening. Different sugar transporter families are present in the genome of plants to fulfill the task of transmembrane sugar transport, which is pivotal for long distance transport between sources and sinks. Among these, the newly identified SWEETs transporters (from Sugars Will Eventually be Exported Transporter) have important roles in numerous physiological mechanisms where sugar efflux is critical. In grapevine, the SWEET family comprises 17 members. In this study, the main objective was to elucidate the role of VvSWEETs in grapevine response to fungal attack (Botrytis cinerea or Erysiphe necator infection) and abiotic stress, including drought. Also, to further study the physiological roles of VvSWEET7 and VvSWEET15, different plant genetic engineering techniques, such as CRISPRCas9, were used. In the B. cinerea-susceptible cv. Trincadeira and in the E. necator-susceptible cv. Carignan, modifications in the gene expression profile of SWEETs in infected grape berries were thoroughly analyzed. Overall, results showed that E. necator infection caused more pronounced modifications in VvSWEET gene expression than Botrytis infection. Moreover, the majority of grapevine SWEET genes were down-regulated in berries from droughtstressed vines of cv. Tempranillo, while VvSWEET10 and VvSWEET11 were up-regulated. In kaolin-treated leaves the expression of VvSWEET1, VvSWEET4 and VvSWEET11 was up-regulated, suggesting that this chemically inert mineral used to protect vines from radiation, drought and heat stimulates sucrose transport capacity improving source-to-sink transport of sucrose. Results also showed that VvSWEET11 and VvSWEET15 were strongly up-regulated in berries subjected to 50ºC during 7 days, a protocol normally used to produce raisins. Following the observation that VvSWEET7 and VvSWEET15 were strongly expressed in berries and clearly up-regulated in response to Botrytis infection in cv. Trincadeira, they were subjected to additional studies to evaluate the subcellular localization and function of the encoded proteins. VvSWEET7-GFP and VvSWEET15-GFP fusion proteins were transiently expressed in Nicotiana benthamiana epidermal cells after Agrobacterium-mediated transformation and both proteins clearly localized to the plasma membrane, as assessed by confocal microscopy. VvSWEET7 was functionally characterized after overexpression in an hxt-null Saccharomyces cerevisiae strain as a low-affinity, high-capacity glucose and sucrose transporter, with a Km of 15.4 mM for glucose and 40.1 mM for sucrose. Competitive inhibition experiments showed that mannitol and sorbitol also inhibited D-[14C(U)]-glucose transport, suggesting that, besides mono- and disaccharides, VvSWEET7 mediates the transport of polyols. In the grapevine genome 18 members of the sugar transporter family ERD6l were identified and VvERD6l13 was selected for further characterization. The fusion protein VvERD6l13-GFP was transiently expressed in N. benthamiana leaves after Agrobacterium-mediated transformation. VvERD6l13 is localized in the plasma membrane. When VvERD6l13 was heterologously expressed in an hxt-null S. cerevisiae strain, it was observed that the protein mediates H+-dependent sucrose transport with a Km = 33 mM. VvERD6l13 is strongly up-regulated in infected grape berries with Botrytis or E. necator, suggesting that it plays an important role during pathogen-host plant interaction. Moreover, VvERD6l13 is expressed in different grapevine tissues, but its steady-state transcript levels were particularly high in roots. In sum, VvSWEET and VvERD6l transporters are important players in sugar mobilization during grape berry development and their expression is transcriptionally reprogrammed in response to biotic and abiotic stress. Together, these results constitute a new piece of the complex puzzle that is grapevine interaction with its surrounding environment and existing biological threats, while also opening new and exciting pathways in the plant sugar transporter research topic.Fundação para a Ciência e Tecnologia (FCT) - Bolsa de Doutoramento (PD/BD/113616/2015), integrada no Programa Doutoral “Agricultural Production Chains-From Fork to Farm (AgriChains) (PD/00122/2012). Fundação para a Ciência e Tecnologia (FCT) e Fundos Europeus (FEDER/POCI /COMPETE2020) – Projecto “MitiVineDrought - Combining "omics" with molecular, biochemical and physiological analyses as an integrated effort to validate novel and easy-to-implement drought mitigation strategies in grapevine while reducing water use” (PTDC/BIA-FBT/30341/2017 and POCI-01-0145-FEDER-030341). Fundação para a Ciência e Tecnologia (FCT) e Fundos Europeus (FEDER/POCI /COMPETE2020) – Projecto “BerryPlastid - Biosynthesis of secondary compounds in the grape berry: unlocking the role of the plastid” (POCI-01-0145-FEDER-028165 and PTDC/BIA-FBT/28165/2017). Fundação para a Ciência e Tecnologia (FCT) – Projecto “GrapInfectomics - Transcriptome and metabolome reprogramming in Vitis vinifera cv. Aragonês and Vitis rupestris berries upon infection with Erysiphe necator” (PTDC/ASP-HOR/28485/2017). Fundação para a Ciência e Tecnologia (FCT) – Projecto “CherryCrackLess” - Cherry cracking & mitigation strategies: towards their understanding using a functional metabolomic approach” (PTDC/AGRPRO/ 7028/2014)

Impact of the light microclimate on photosynthetic activity of grape berry (Vitis vinifera): insights for radiation absorption mitigations' measures

IPCC's predicted rise in mean temperatures, increase in the frequency of summer heat waves and decrease in soil water availability for the Mediterranean regions will have an impact on foliar and fruit photosynthesis. But mitigation measures aiming reducing radiation absorption by the vine canopy may pose light limitations to grape berry photosynthesis. This work focused on the influence of the light level of the canopy microenvironment where clusters develop on the photosynthetic competence of grape berry tissues (exocarp and seed integument) throughout fruit growing season by imaging PAM fluorometry. Clusters from low (LL), medium (ML) and high light (HL) microclimates were sampled from green to mature stages. Both tissues showed high maximum quantum efficiency (Fv/Fm) and photosynthetic capacity (ETRm) at the green stage, exocarp extending to mature stages while seed photosynthetic activity was more restricted to green stage. The light microclimate had a significant effect on the photosynthesis of both tissues but also in their photosynthetic phenotypes along the season. In LL, both tissues showed lower activity in all stages, higher susceptibility to photoinhibition and lack of response to short-term light acclimation; ML and HL grapes adjust their activity peaking at different light intensities, were more responsive to light changing conditions, recover better from high light. Overall, our results suggest that not only light/temperature stress conditions imposed by climate changes but also viticulture practices causing changes in canopy light microclimates may have significant impacts on grape berry photosynthesis and hence in fruit development and quality.This work is supported by: European Investment Funds by FEDER/ COMPETE/POCI– Operacional Competitiveness and Internacionalization Programme, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2013.info:eu-repo/semantics/publishedVersio

VvERD6l13 is a grapevine sucrose transporter highly up-regulated in response to infection by Botrytis cinerea and Erysiphe necator

The Early-Response to Dehydration six-like (ERD6l) is one of the largest families of sugar transporters in plants, however, is also one of the less studied with very few members characterized. In this work, we identified 18 members of the grapevine ERD6l family, analyzed their promoters and putative topology and additionally functionally characterized the member VvERD6l13. VvERD6l13 was strongly up-regulated in grape berries infected with Botrytis cinerea and Erysiphe necator in cv. Trincadeira and Carignan, respectively, suggesting an important role in grape berry-pathogen interaction, as we had hypothesized. In Cabernet Sauvignon Berry suspension cultured cells, VvERD6l13 was also up-regulated, by 4-fold, 48 h after elicitation with mycelium extract of B. cinerea. Besides being expressed in grape berries from various developmental stages, VvERD6l13 is also expressed in leaves, canes, flowers and, noticeably, in roots. Using tobacco and an hut-null Saccharomyces cerevisiae strain as heterologous expression models, we showed that VvERD6l13 is localized at the plasma membrane and mediates the H -dependent transport of sucrose (K-m = 33 mM) thus confirming VvERD6l13 as a bona fide sugar transporter involved in sugar mobilization in grapevine and transcriptionally induced in response to biotic stress.The authors would like to thank Cecilia Rego and Pedro Reis for help with infection procedures and sampling of samples infected with Botrytis cinerea and Cecilia Rego and Diana Pimentel for help in sampling of berries naturally infected with Erysiphe necator. The work was supported by National Funds by FCT-Portuguese Foundation for Science and Technology, under the strategic programmes UID/AGR/04033/2019 and UID/BIA/04050/2019. The work was also supported by FCT and European Funds (FEDER/POCI/COMPETE2020) through the research project "MitiVineDrought-Combining "omics" with molecular, biochemical, and physiological analyses as an integrated effort to validate novel and easy-to-implement drought mitigation strategies in grapevine while reducing water use" with ref. PTDC/BIA-FBT/30341/2017 and ref. POCI-01-0145-FEDER-030341, respectively; through the research project "BerryPlastid-Biosynthesis of secondary compounds in the grape berry: unlocking the role of the plastid" with ref. POCI-01-0145-FEDER-028165 and ref. PTDC/BIA-FBT/28165/2017, respectively; and also through the FCT-funded research project "GrapeInfectomics" (PTDC/ASPHOR/28485/2017). This work was also supported by the project "INTERACT - VitalityWine -ref. NORTE-010145-FEDER-000017 -(through FEDER/COMPETE and NORTE2020/CCDRN). AC was supported with a post-doctoral fellow of the mentioned INTERACT/Vitality Wine project with ref. BPD/UTAD/INTERACT/VW/218/2016 and by a post-doctoral researcher contract/position within the project "MitiVineDrought" (PTDC/BIA-FBT/30341/2017 and POCI-01-0145-FEDER-030341). RB was supported with a PhD student grant (PD/BD/113616/2015) under the Doctoral Programme 'Agricultural Production Chains-from fork to farm' (PD/00122/2012) funded by FCT. This work also benefited from the networking activities within the European Union-funded COST Action CA17111 "INTEGRAPE-Data Integration to maximize the power of omics for grapevine improvement"

Kaolin particle film application stimulates photoassimilate synthesis and modifies the primary metabolome of grape leaves

Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.jplph.2018.02.004.Water scarcity is associated with extreme temperatures and high irradiance, and significantly and increasingly affects grapevine yield and quality. In this context, the foliar application of kaolin, a chemically inert mineral that greatly reflects ultraviolet and infrared radiations, as well as, in part, photosynthetically active radiation, has recently been shown to decrease photoinhibition in mature leaves. Here, the influence of this particle film on grapevine leaf metabolome and carbohydrate metabolism was evaluated. Molecular mechanisms underlying photoassimilate synthesis, metabolism and transport capacity were assessed by targeted transcriptional analyses and enzymatic activity assays. Kaolin application increased sucrose concentration in leaves and sucrose transport/phloem loading capacity, as suggested by the stimulation of the transcription of sucrose transporters VvSUC12 and VvSUC27 in these source organs. While the biosynthesis of sucrose increased, as evidenced by higher sucrose content and sucrose phosphate synthase (SPS) activity in leaves, the concentration of transitory starch before the dark period remained unaltered, despite a higher total amylolytic activity in the leaves of kaolin-treated plants. Metabolomic analysis by GC-TOF-MS showed that the application of kaolin enhanced the amounts of simple sugars, including fructose, maltose, xylulose, xylose, sophorose, ribose and erythrose; sugars-phosphate, like mannose-6-Pi, hexose-6-Pi, glucose-6-Pi, glucose-1-Pi, glycerol--Pi and fructose-6-Pi; polyols, like xylitol, maltitol, lactitol, glycerol, galactinol and erythritol; organic acids and amino acids.The work was supported by European Union Funds (FEDER/ COMPETE-Operational Competitiveness Programme – INNOVINE – ref. 311775, Enoexcel – Norte – 07-0124-FEDER-000032, and INTERACT – NORTE-01- 0145-FEDER-000017 – Linha VitalityWine – ON 0013), and by Portuguese national funds (FCT-Portuguese Foundation for Science and Technology) under the project UID/AGR/04033/2013. Artur Conde was supported by INTERACT – NORTE-01- 0145-FEDER-000017 – Linha VitalityWine – ON 0013 (BPD/UTAD/INTERACT/VW/218/ 2016). Richard Breia was supported by FCT under the fellowship PD/ BD/113616/2015.info:eu-repo/semantics/publishedVersio

Postharvest dehydration induces variable changes in the primary metabolism of grape berries

Postharvest dehydration causes changes in texture, color, taste and nutritional value of food due to the high temperatures and long drying times required. In grape berries, a gradual dehydration process is normally utilized for raisin production and for making special wines. Here we applied a raisin industry-mimicking dehydration process for eleven days at 50 °C to intact berry clusters from cv. Sémillon plants, and a set of molecular, cellular and biochemical analyses were performed to study the impact of postharvest dehydration in the primary metabolism. Transcriptional analyses by real time qPCR showed that several aquaporins (VvTIP1;2 and VvSIP1) and sugar transporters (VvHT1, VvSWEET11, VvSWEET15, VvTMT1, VvSUC12) genes were strongly upregulated. Moreover, the study of key enzymes of osmolytes metabolism, including mannitol dehydrogenase (VvMTD) and sorbitol dehydrogenase (VvSDH), at gene expression and protein activity level, together with the transcriptional analysis of the polyol transporter gene VvPLT1, showed an enhanced polyol biosynthesis capacity, which was supported by the detection of sorbitol in dehydrated grapes only. The metabolism of organic acids was also modulated, by the induction of transcriptional and biochemical activity modifications in malate dehydrogenases and malic enzymes that led to organic acid degradation, as demonstrated by HPLC analysis. Taken together, this study showed that primary metabolism of harvested berries was severely influenced in response to dehydration treatments towards lower organic acid and higher sorbitol concentrations, while sugar transporter and aquaporin genes were significantly upregulated.The work was supported by European Union Funds (FEDER/ COMPETE-Operational Competitiveness Programme - INNOVINE - ref. 311775, and INTERACT - NORTE-01-0145-FEDER-000017 - Linha VitalityWine - ON 0013). This work was also supported by: European Investment Funds by FEDER/COMPETE/POCI – Operacional Competitiveness and Internacionalization Programme, under Project POCI-01-0145-FEDER006958 andNationalFundsbyFCT-PortugueseFoundationforScience and Technology, under the project UID/AGR/04033/2013. Artur Conde was supported by INTERACT - NORTE-01- 0145FEDER-000017 - Linha VitalityWine - ON 0013 (BPD/UTAD/ INTERACT/VW/218/2016). Richard Breia was supported by FCT under the fellowship PD/BD/113616/2015.info:eu-repo/semantics/publishedVersio