Um tributo ao manitol em Olea europaea

A elevada salinidade e a secura são factores ambientais que afectam negativamente o crescimento das plantas e, consequentemente, a sua produtividade. O excesso de sal e o deficit hídrico podem causar citotoxicidade iónica, stresse hiperosmótico e oxidativo e desequilíbrios nutricionais. Contudo, as plantas de habitats salinos ou secos, como a oliveira (Olea europaea), exibem diversas adaptações morfológicas, fisiológicas e moleculares, de onde se destacam a capacidade de acumular no espaço intracelular solutos osmoprotectores, como os polióis. Com efeito, o manitol é um dos principais produtos da fotossíntese em O. europaea e na polpa da azeitona o seu conteúdo aumenta durante o amadurecimento até 8 mg g-1 peso seco no fruto maduro. Será apresentado e discutido o trabalho desenvolvido no seio do grupo de investigação do Departamento de Biologia da Universidade do Minho que visa a elucidação do papel do manitol como fonte de carbono e de energia e como soluto protetor contra os stresses biótico e abiótico na oliveira.Fundação para a Ciência e a Tecnologia (FCT) [POCI/AGR/56378/2004; PTDC/AGR-ALI/100636/2008; bolsa de doutoramento SFRH/BD/47699/2008 atribuída a A.C.

Exogenous application of non-mature miRNA-encoded miPEP164c inhibits proanthocyanidin synthesis and stimulates anthocyanin accumulation in grape berry cells

Secondary metabolic pathways in grape berries are tightly regulated by an array of molecular mechanisms, including microRNA-mediated post-transcriptional regulation. As recently discovered, before being processed into mature microRNAs (miRNAs), the primary transcripts of miRNAs (pri-miRNAs) can encode for small miRNA-encoded peptides (micropeptides miPEPs) that ultimately lead to an accentuated downregulation of the respective miRNA-targeted genes. Although few studies about miPEPs are available, the discovery of miPEPs reveals a new layer of gene regulation at the post-transcriptional level that opens the possibility to regulate plant metabolism without resorting to gene manipulation. Here, we identified a miPEP encoded in non-mature miR164c putatively targeting grapevine transcription factor VvMYBPA1 (miPEP164c/miPEP-MYBPA1), a positive regulator of key genes in the proanthocyanidin (PA)-biosynthetic pathway, a pathway that competes directly for substrate with the anthocyanin-biosynthetic pathway. Thus, the objective of this work was to test the hypothesis that the exogenous application of miPEP164c (miPEP-MYBPA1) can modulate the secondary metabolism of grape berry cells by inhibiting PA biosynthetic pathway while simultaneously stimulating anthocyanin synthesis. The exogenous application of miPEP164c to suspension-cultured cells from grape berry (cv. Gamay) enhanced the transcription of its corresponding non-mature miR164c, with a maximum effect at 1 M and after a period of 10 days, thus leading to a more pronounced post-transcriptional silencing of its target VvMYBPA1. This led to a significant inhibition of the PA pathway, mostly via inhibition of leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) enzymatic activities and VvLAR1 downregulation. In parallel, the anthocyanin-biosynthetic route was stimulated. Anthocyanin content was 31% higher in miPEP164c-treated cells, in agreement with the observed upregulation of VvUFGT1 transcripts and UFGT enzyme activity levels.This work was supported by Fundação para a Ciência e Tecnologia (FCT), under the strategic programs 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 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). AC was supported with a post-doctoral researcher contract/position within the project “MitiVineDrought” (PTDC/BIA-FBT/30341/2017 and POCI-01-0145-FEDER 030341). FEDER/COMPETE also supported this work through the project “AgriFoodXXI” (ref. NORTE-01-0145-FEDER 000041). MV was supported by a Ph.D. fellowship funded by FCT (SFRH/BD/144637/2019). HB was supported by a Ph.D. fellowship funded by FCT (SFRH/BD/144638/2019). This work also benefited from the networking activities within the European COST Action CA17111 “INTEGRAPE—Data Integration to maximize the power of omics for grapevine improvement,” the CoLAB VINES & WINES, and the CoLAB 4FOOD – Collaborative Laboratory for Innovation in the Food Industry.info:eu-repo/semantics/publishedVersio

Light microclimate-driven changes at transcriptional level in photosynthetic grape berry tissues

Viticulture practices that change the light distribution in the grapevine canopy can interfere with several physiological mechanisms, such as grape berry photosynthesis and other metabolic pathways, and consequently impact the berry biochemical composition, which is key to the final wine quality. We previously showed that the photosynthetic activity of exocarp and seed tissues from a white cultivar (Alvarinho) was in fact responsive to the light microclimate in the canopy (low and high light, LL and HL, respectively), and that these different light microclimates also led to distinct metabolite profiles, suggesting a berry tissue-specific interlink between photosynthesis and metabolism. In the present work, we analyzed the transcript levels of key genes in exocarps and seed integuments of berries from the same cultivar collected from HL and LL microclimates at three developmental stages, using real-time qPCR. In exocarp, the expression levels of genes involved in carbohydrate metabolism (VvSuSy1), phenylpropanoid (VvPAL1), stilbenoid (VvSTS1), and flavan-3-ol synthesis (VvDFR, VvLAR2, and VvANR) were highest at the green stage. In seeds, the expression of several genes associated with both phenylpropanoid (VvCHS1 and VvCHS3) and flavan-3-ol synthesis (VvDFR and VvLAR2) showed a peak at the véraison stage, whereas that of RuBisCO was maintained up to the mature stage. Overall, the HL microclimate, compared to that of LL, resulted in a higher expression of genes encoding elements associated with both photosynthesis (VvChlSyn and VvRuBisCO), carbohydrate metabolism (VvSPS1), and photoprotection (carotenoid pathways genes) in both tissues. HL also induced the expression of the VvFLS1 gene, which was translated into a higher activity of the FLS enzyme producing flavonol-type flavonoids, whereas the expression of several other flavonoid pathway genes (e.g., VvCHS3, VvSTS1, VvDFR, and VvLDOX) was reduced, suggesting a specific role of flavonols in photoprotection of berries growing in the HL microclimate. This work suggests a possible link at the transcriptional level between berry photosynthesis and pathways of primary and secondary metabolism, and provides relevant information for improving the management of the light microenvironment at canopy level of the grapes.This research and the APC was funded by FCT—Portuguese Foundation for Science and Technology, grant provided to Andreia Garrido (PD/BD/128275/2017), under the Doctoral Programme “Agricultural Production Chains—from fork to farm” (PD/00122/2012), and by the European Social Funds and the Regional Operational Programme Norte 2020.info:eu-repo/semantics/publishedVersio

Kaolin foliar application has a stimulatory effect on phenylpropanoid and flavonoid pathways in grape berries

The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fpls.2016. 01150Drought, elevated air temperature, and high evaporative demand are increasingly frequent during summer in grape growing areas like the Mediterranean basin, limiting grapevine productivity and berry quality. The foliar exogenous application of kaolin, a radiation-reflecting inert mineral, has proven effective in mitigating the negative impacts of these abiotic stresses in grapevine and other fruit crops, however, little is known about its influence on the composition of the grape berry and on key molecular mechanisms and metabolic pathways notably important for grape berry quality parameters. Here, we performed a thorough molecular and biochemical analysis to assess how foliar application of kaolin influences major secondary metabolism pathways associated with berry quality-traits, leading to biosynthesis of phenolics and anthocyanins, with a focus on the phenylpropanoid, flavonoid (both flavonol- and anthocyanin-biosynthetic) and stilbenoid pathways. In grape berries from different ripening stages, targeted transcriptional analysis by qPCR revealed that several genes involved in these pathways—VvPAL1, VvC4H1, VvSTSs, VvCHS1, VvFLS1, VvDFR, and VvUFGT—were more expressed in response to the foliar kaolin treatment, particularly in the latter maturation phases. In agreement, enzymatic activities of phenylalanine ammonia lyase (PAL), flavonol synthase (FLS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) were about two-fold higher in mature or fully mature berries from kaolin-treated plants, suggesting regulation also at a transcriptional level. The expression of the glutathione S-transferase VvGST4, and of the tonoplast anthocyanin transporters VvMATE1 and VvABCC1 were also all significantly increased at véraison and in mature berries, thus, when anthocyanins start to accumulate in the vacuole, in agreement with previously observed higher total concentrations of phenolics and anthocyanins in berries from kaolin-treated plaThe work was supported by European Union Funds (FEDER/COMPETE-Operational Competitiveness Programme —INNOVINE—ref. 311775, Enoexcel—Norte—07-0124FEDER-000032 and INTERACT - NORTE-01-0145-FEDER000017 - Linha VitalityWine - ON 0013), and by Portuguese national funds (FCT-Portuguese Foundation for Science and Technology) under the project FCOMP-01-0124FEDER-022692. AC was supported by Enoexcel—Norte— 07-0124-FEDER-000032 and INTERACT - NORTE-010145-FEDER-000017.This work also benefited from the networking activities within the European funded COST ACTION FA1106 “QualityFruit.”info:eu-repo/semantics/publishedVersio

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.

Mannitol transport and oxidation are synchronized in Olea europaea under salt and drought stresses

The intracellular accumulation of organic compatible solutes acting as osmoprotectants, such as polyols, is an essential response mechanism of several plants to drought and salinity. In Olea europaea a mannitol transport system (OeMaT1) has been previously characterised as a key player in plant response to salinity, and here, heterotrophic sink models, such as olive suspension-cultured cells and fruit tissues, and source leaves were used for analytical, biochemical and molecular studies focused on mannitol metabolism to better understand its involvement in drought and salt stress tolerance. The kinetic parameters of mannitol dehydrogenase (MTD) determined in mannitol-growing cells, at 25 ºC and pH 9.0, were as follows: Km, 54.5 mM mannitol and Vmax, 0.47 µmol h-1 mg-1 protein. The corresponding cDNA was cloned and subsequently named OeMTD1. OeMTD1 expression was correlated with MTD activity, OeMaT1 expression and carrier-mediated mannitol transport, in both mannitol- and sucrose-growing cells. Moreover, sucrose-growing cells showed only residual OeMTD activity, even though high levels of OeMTD1 transcription were observed. The results support that OeMTD is regulated at both transcriptional and post-transcriptional levels due to substrate influence. Remarkably, MTD activity and OeMTD1 expression were dramatically repressed after Na+, K+ and PEG treatments, both in mannitol- and sucrose-growing cells. In contrast, salt and drought significantly increased mannitol transport activity and OeMaT1 expression, thus allowing for the intracellular accumulation of this polyol. Altogether, the results strongly suggest that olive tree copes with drought and salinity by tightly coordinating mannitol transport with intracellular metabolism. Taking into account the protective role of mannitol in olive tree, it would be extremely interesting to investigate if a criterious exogenous addition of this polyol during usual agricultural practices could be beneficial for olive tree development and productivity, and consequently for the whole olive-based industry.This work was supported by the Fundação para a Ciência e Tecnologia (FCT) (research project ref. PTDC/AGR-ALI/100636/2008 and A. Conde grant ref. SFRH/BD/47699/2008

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

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

Constitutive expression of VviNAC17 transcription factor significantly induces the synthesis of flavonoids and other phenolics in transgenic grape berry cells

VviNAC17 is a grapevine transcription factor activated by ABA. Because ABA has been proposed as the main signal modulating the secondary metabolism in grape berry skins, here we postulated VviNAC17 as a positive regulator of secondary metabolism in grape cells. To validate the hypothesis, VviNAC17 was constitutively and stably overexpressed in grape berry suspension-cultured cells of Gamay Fréaux cv. by Agrobacterium-mediated transformation. Targeted transcriptional analyses by qPCR showed that several genes involved the phenylpropanoid (VviPAL1), stilbenoid (VviSTS1) and flavonoid pathways (VviDFR, VviLAR1, VviANR, VviLDOX, and VviUFGT1), as well as anthocyanin vacuolar transport and accumulation (VviGST4 and VvMATE1) were significantly upregulated in VviNAC17-overexpressing transgenic cells, which translated in the stimulation of a number of enzymatic activities in those pathways. This was the case of phenylalanine ammonia lyase (PAL) and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) that were about 2-fold and 3.5-fold higher in VviNAC17-overexpressing cells than in control cells. VviNAC17-overexpressing cells accumulated significantly higher amounts of anthocyanins, proanthocyanidins, total flavonoids and total phenolics. These findings confirmed that VviNAC17 is an important positive regulator of secondary metabolism in grapevine contributing to the accumulation of important berry quality-related secondary metabolites.This work was supported by Fundação para a Ciência e Tecnologia (FCT), under the strategic program UIDB/ 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-toimplement drought mitigation strategies in grapevine while reducing water use” with ref. PTDC/BIA-FBT/30341/2017 and ref. POCI-01-0145-FEDER-030341; AC was supported with a post-doctoral researcher contract/position within the project “Miti VineDrought” (ref. PTDC/BIA-FBT/30341/2017 and ref. POCI01-0145-FEDER-030341). HB was supported by a Ph.D. fellowship funded by FCT (SFRH/BD/144638/2019). MV was supported by a Ph.D. fellowship funded by FCT (SFRH/BD/144637/2019).info:eu-repo/semantics/publishedVersio

Metabolomics of photosynthetically active tissues in white grapes: effects of light microclimate and stress mitigation strategies

The effects of climate change are becoming a real concern for the viticulture sector, with impacts on both grapevine physiology and the quality of the fresh berries and wine. Short-term mitigation strategies, like foliar kaolin application and smart irrigation regimes, have been implemented to overcome these problems. We previously showed that these strategies also influence the photosynthetic activity of the berries themselves, specifically in the exocarp and seed. In the present work, we assessed the modulating effects of both canopy-light microclimate, kaolin and irrigation treatments on the metabolic profiles of the exocarp and seed, as well as the potential role of berry photosynthesis herein. Berries from the white variety Alvarinho were collected at two contrasting light microclimate positions within the vine canopy (HLhigh light and LLlow light) from both irrigated and kaolin-treated plants, and their respective controls, at three fruit developmental stages (green, véraison and mature). Untargeted liquid chromatography mass spectrometry (LCMS) profiling of semi-polar extracts followed by multivariate statistical analysis indicate that both the light microclimate and irrigation influenced the level of a series of phenolic compounds, depending on the ripening stage of the berries. Moreover, untargeted gas chromatography mass spectrometry (GCMS) profiling of polar extracts show that amino acid and sugar levels were influenced mainly by the interaction of irrigation and kaolin treatments. The results reveal that both photosynthetically active berry tissues had a distinct metabolic profile in response to the local light microclimate, which suggests a specific role of photosynthesis in these tissues. A higher light intensity within the canopy mainly increased the supply of carbon precursors to the phenylpropanoid/flavonoid pathway, resulting in increased levels of phenolic compounds in the exocarp, while in seeds, light mostly influenced compounds related to carbon storage and seed development. In addition, our work provides new insights into the influence of abiotic stress mitigation strategies on the composition of exocarps and seeds, which are both important tissues for the quality of grape-derived products.This research and APC were funded by the FCT-Portuguese Foundation for Science and Technology, grant provided to Andreia Garrido (PD/BD/128275/2017), under the Doctoral Program "Agricultural Production Chains-from fork to farm" (PD/00122/2012).info:eu-repo/semantics/publishedVersio