agronomy Article Differences in Metabolic and Physiological Responses between Local and Widespread Grapevine Cultivars under Water Deficit Stress Igor Florez-Sarasa 1, *,† , María José Clemente-Moreno 2, *,† , Josep Cifre 2 , Miquel Capó 2 , Miquel Llompart 2 , Alisdair R. Fernie 3 and Josefina Bota 2 1 Centre for Research in Agricultural Genomics (CRAG) CSIC-IRTA-UAB-UB, Campus UAB Bellaterra, 08193 Barcelona, Spain 2 Research Group on Plant Biology, Balearic Islands University, Ctra Valldemossa km 7.5, 07122 Palma de Mallorca, Spain; pep.cifre@uib.es (J.C.); miquel.capo@uib.es (M.C.); miqllc@gmail.com (M.L.); j.bota@uib.es (J.B.) 3 Max-Planck-Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany; Fernie@mpimp-golm.mpg.de * Correspondence: igor.florez@cragenomica.es (I.F.-S.); esojariam2@gmail.com (M.J.C.-M.); Tel.: +34-935-636-600 (I.F.-S.); +34-971-17-34-48 (M.J.C.-M.) † These authors contributed equally to the manuscript.  Received: 10 July 2020; Accepted: 17 July 2020; Published: 21 July 2020  Abstract: Climate change forecasts suggest temperature increases and lower rainfall rates, both of which challenge viticulture, particularly in semi-arid areas where water availability is critical. In this scenario, the use of the genetic variability in grapevine varieties reported around the world represents an important strategy for the selection of climate-resilient cultivars. In this work, physiological and metabolomics analyses were conducted to compare the water deficit stress (WDS) responses of red and white, local and widespread grapevines cultivars. Leaf gas exchange, water use efficiency (WUE) and water relation parameters were determined in plants under well-watered and WDS conditions alongside assessment of the levels of foliar primary metabolites using gas-chromatography coupled to mass-spectrometry. Results denote that red and white local cultivars displayed more adapted physiological performance under WDS as compared to the widely-distributed ones. Multivariate analyses and specific changes in leaf primary metabolites indicate genotype-specific responses of local cultivars as compared to widespread ones. Differences in ascorbate-related and shikimate/phenylpropanoid metabolism could explain the better physiological performance under WDS in red local as compared to widespread cultivars. On the other hand, coordinated changes in respiratory- and stress-related sugars and amino acids could underlie the better WUE under WDS in the white local cultivar. All these results suggest several metabolic targets that could be useful as metabolic markers or for metabolic engineering in grapevine breeding programs to improve drought tolerance. Keywords: water deficit stress; Vitis vinifera; genetic variability; local cultivars; metabolomics; water use efficiency 1. Introduction Water availability is one of the main limiting factors affecting crop production in arid and semi-arid regions. This situation has further worsened in recent years due to climate change, which represents a major threat to the future of our primary agricultural productivity [1]. In this context, a broad effort is needed to reduce the impacts of global change and optimize the use of irrigation, which is increasingly Agronomy 2020, 10, 1052; doi:10.3390/agronomy10071052 www.mdpi.com/journal/agronomy Agronomy 2020, 10, 1052 2 of 21 necessary in Mediterranean viticulture. Over decades, the responses of grapevine to environmental changes have been under continuous review, with special attention to those expected under water deficit [2–5]. The selection of more water-use efficient or more drought-tolerant genotypes has been proposed as an important strategy to overcome global change impacts [6–9]. Generally, grapevine is considered as a drought-tolerant species, however, physiological responses to environmental constraints including stomatal regulation, photosynthetic capacity and water use efficiency (WUE) vary among genotypes [10]. While there is a wide knowledge concerning the intra-specific variability in the physiological response to water deficit stress (WDS) and WUE [10–21], the underlying mechanisms associated with this variability are not fully understood. The binary classification isohydric/anisohydric was widely used to identify genotype behaviors based on differences in stomatal conductance patterns in response to water potentials [12,20]. However, recent studies suggest that these two strategies can occur within the same variety depending on the experimental conditions (e.g., climatic conditions, growing season, water treatment, field or controlled conditions) so, even if a continuum between isohydric to anisohydric behavior exists, different strategies in response to drought have been identified and its implications in WUE deserve to be further studied [10,22–25]. In this sense, local grapevine varieties from the Balearic Islands have been described as physiologically better adapted to deficient irrigation, particularly with regard to WUE [10,13]. These local cultivars are documented as ancient pre-phylloxera vines cultivated in the Balearic Islands [26] and have been widely and thoroughly studied at the level of genetic characterization, population structure and pedigree [27,28]. However, a better understanding of the genotype-specific molecular responses to WDS and their link to physiological responses is still necessary in order to achieve a more efficient viticulture in the future. Better performance under drought and higher temperatures involves specific morpho-anatomical, physiological and metabolic adaptations [20,29]. Metabolite profiling approaches have been successfully employed to study environmental responses in wild, model and crop plants (reviewed in Obata and Fernie, Peters et al. and Alseekh et al. [30–32]). More recently, the combination of ecophysiological and metabolomics approaches has improved our current understanding of plant responses to environmental clues [33–35]. In the particular case of grapevine and other Mediterranean woody crops, the use of metabolomics together with transcriptomics and proteomics approaches has been recently proposed as key for advancing in the characterization of plant genotypes exhibiting physiological and/or genetic traits conferring abiotic stress tolerance [36]. While different studies have investigated the genotypic variability of grapevine metabolic responses to WDS, most of them have focused on berry development and quality composition [37–41]. However, the regulation of grapevine leaf metabolism under WDS is key for understanding berry development, since the berry is a sink tissue tightly depending on leaf metabolism [42]. This issue can be relevant for breeding programs, especially when considering the different physiological behavior of varieties from arid and semi-arid areas that could diminish the effect of water deficit and support productivity [4,42]. That said, while some studies have compared grapevine genotypic differences on leaf metabolic responses to drought [42–44], the number of genotypes used has been limited (i.e., no more than two) and, to our knowledge, none of them included red and white cultivars locally adapted to the Mediterranean climate. In this work, we have studied grapevine WDS responses in local and widely-distributed cultivar (four red and two white) at the leaf level by measuring several ecophysiological parameters in combination with metabolite profiling analysis. Given evidence of a higher WUE in local cultivars from the Balearic Islands, as introduced above, we hypothesize that local cultivars should display metabolic adaptations related to their better responses to WDS. Common physiological and metabolic responses to WDS were observed in all genotypes. Nevertheless, multivariate analyses of metabolite data separated locally from widely-distributed cultivars and some metabolites displayed specific patterns in these cultivars. The associations between specific metabolic features of local cultivars and their physiological differences to widespread cultivars are discussed. Agronomy 2020, 10, 1052 3 of 21 2. Materials and Methods 2.1. Plant Material and Treatments Six grapevine cultivars were chosen to perform the present study; two red and one white local cultivar(s): Escursac, Callet and Giró Ros; and two red and one white widespread ones: Merlot, Syrah and Moscatel. The experiment was performed in summer 2016 (from 4th July to 22th July) at the experimental field site of Universitat de les Illes Balears (39◦ 350 N, 2◦ 390 E) (Balearic Islands, Spain). Eight plants per cultivar were maintained without fruits (manually cut) since grape development and ripening induces large changes in the metabolic activity of plants acting as the most active sink for photosynthates [42,45]. Therefore, plants were grown without fruits avoiding the effect of other major factors, such as differences among cultivars in sink source ratios. Also, non-grafted plants were used in order to avoid confounding effects of rootstock scion interactions. All plants were obtained by direct rooting from 0.3 m dormant cuttings with at least 3 leaf nodes. Cuttings were soaked in 0.3% Captan solution for 3 h and hydrated for 24 h. After that, rooting was induced by indole-3-butyric acid (IBA) 2g L−1 , and plants were maintained in a greenhouse under controlled conditions. Soil temperature at 2 cm deep was 26–28 ◦ C. Air temperature was 23 ◦ C and air humidity about 80%. When cuttings presented 4–5 expanded leaves, they were transplanted into pots and grown outdoors in 15 L pots filled with organic substrate and perlite mixture (3:1) and placed in a completely randomized design. They were irrigated daily from May until the start of the experiment and supplemented every 3–5 days with organic-mineral fertilizer NPK containing (%): N, 5; P2 O5 , 8; K2 O 15; MgO, 2; organic C, 17.4, humic acid, 5; SO3 , 15; Fe, 1; Zn 2 × 10−3 ; Mn 1 × 10−2 . To avoid soil evaporation during the experiment a blanket of acrylic fibers of 7 cm thickness was placed in each pot and was only removed during water supply. Two irrigation regimes were established on the basis of stomatal conductance (gs ) measurements: field capacity or well-watered (gs > 200 mmol H2 O m−2 s−1 ) and moderate water stress (50 0.7 associated to the principal component 1 (orange). Data used was taken 2 increases in all cultivars from Figure(red)1 under WDS, and Table S4.and Suc,with a r > Glc, 2 sucrose; 0.7 associated to the glucose; Fru, principal fructose; component G6P, 1 (orange). Data glucose-6-phosphate; F6P, used was taken from Figure 1 and Table S4. Suc, sucrose; Glc, glucose; Fru, fructose; fructose-6-phospate; E4P, erythrose-4-phospate; 3PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate; G6P, glucose-6- phosphate; Pyr, pyruvate; F6P,Cit, fructose-6-phospate; E4P, 2-OG, citrate; Isocit, isocitrate; erythrose-4-phospate; 2-oxo-glutarate; 3PG, Succ, 3-phosphoglycerate; succinate; Fum, fumarate;PEP, phosphoenolpyruvate; Pyr, pyruvate; Cit, citrate; Isocit, isocitrate; 2-OG, 2-oxo-glutarate; Mal, malate; OOA, oxaloacetate; Asp, aspartate; Lys, lysine; Thr, threonine; b-Ala, beta-alanine; Val, Succ, succinate; valine; Ile,Fum, fumarate; isoleucine; Ala,Mal, malate; alanine; OOA, Ser, oxaloacetate; serine; Asp, Gly, glycine; aspartate; Lys, Glycerol-3P, lysine; Thr, threonine; glycerol-3-phosphate; Glu, b-Ala, beta-alanine; glutamate; Val, valine; Ile, isoleucine; GABA, gamma-aminobutyrate; Pro, Ala, alanine; proline; Ser, 4-hydroxy-proline; OH-Pro, serine; Gly, glycine; Glycerol-3P, Orn, ornithine; glycerol-3-phosphate; Glu, glutamate; Gln, glutamine; Arg, arginine; GABA, Trp, Put, putrescine; gamma-aminobutyrate; Pro, proline; tryptophan; Tyr, tyrosine; OH-Pro, 4- Phe, phenylalanine; hydroxy-proline; Orn, ornithine; Gln, glutamine; Arg, arginine; Put, putrescine; Trp, OH-Cinamate; 4-hydroxy-cinnamate; L-Gal-1,4-lactone, L-galactone-1,4-lactone; Asc, ascorbate; DhAsc,tryptophan; Tyr, tyrosine; Phe, phenylalanine; dehydroascorbate. *MetabolitesOH-Cinamate; significantly (p <4-hydroxy-cinnamate; 0.05) increasing under WDSL-Gal-1,4-lactone, L-galactone- with the exception of Callet. 1,4-lactone; Asc, ascorbate; DhAsc, dehydroascorbate. *Metabolites significantly (p < 0.05) increasing 3.3. Metabolite under WDSProfiling with theand Responses exception under Well-Watered and Water Deficit Stress Treatments of Callet. As for the physiological data, we calculated the metabolite fold-change responses to WDS in all Next, we performed cultivars (Figure a multivariate 1) in order to evaluate cluster analysis in order the responsiveness among to identify which the different genotypes cultivars. display In general, similar metabolic several amino profiles acids, as welland whether as sugars andthis sugarseparation alcohols, is in line were with theincreased significantly observedinphysiological all genotypes differences while organic acids were less responsive. The following common and significant(Callet, (Figure 2B). In this sense, dendrogram separated the local cultivars Escursac increases and after WDS Giró were Ros) from in observed the allwidespread genotypes: ones under both myo-inositol, WW and maltose, WDSand raffinose conditions. proline asThis well suggests that local as glutamate and cultivars have genotype-specific metabolic differences threonine (with the exception of Callet; Figures 1 and 3). that could underlie the observed different physiological responses to WDS. With regard to the stress treatment, all cultivars under WW With regard to red cultivars, Syrah was displaying, by far, the highest number (thirty-three) of conditions clearly clustered away from those under WDS. significantly changed metabolites after WDS treatment (Figure 1). In Merlot, fourteen metabolites significantly increased after WDS, being all these metabolites shared with Syrah except Serine and Valine. 3.3. Metabolite Profiling and Responses under Well-Watered and Water Deficit Stress Treatments Notably, changes in amino acid profiles were very similar in Merlot and Syrah while much less organic acids,As for the sugars physiological and sugar alcohols data, wesignificantly were calculated the metabolite changed fold-change in Merlot responses as compared to WDS to Syrah. Thein all local cultivars (Figure red cultivars 1) in order displayed lowerto evaluate numbers of the responsiveness significantly changed among the different metabolites, twelve cultivars. and fourteen In general, in Callet several aminorespectively and Escursac, acids, as well(Figureas 1). sugars Whileand sugar several alcohols, of the were changing metabolites significantly increased in local cultivarsinwereall genotypes whilechanging similar to those organic acids in thewere less responsive. widespread ones, thereThe following were common and some exceptions. significant Aspartate, increases isocitrate and after WDS(trans-caffeate shikimate were observed onlyininall genotypes: Merlot althoughmyo-inositol, Syrah displayedmaltose, a very raffinose and proline similar trend) as well as were significantly glutamate and threonine increased only in widespread(withcultivars the exception after of WDSCallet; Figures (Figures 1 and 1 and 4).3). On the other hand, lysine and With regard isoleucine to red cultivars, only increased in Callet Syrah was displaying, (a similar by far, theand trend in Escursac), highest number putrescine (thirty-three) only significantlyof significantly changed(Figures increased in Escursac metabolites 1 andafter WDS treatment 4). Finally, (Figure decreased urea significantly 1). In Merlot, onlyfourteen in Escursac metabolites while the significantly opposite wasincreased observed after WDS, in Syrah being1). (Figure all these metabolites shared with Syrah except Serine and Valine. Notably, changes in amino acid profiles were very similar in Merlot and Syrah while much less organic acids, sugars and sugar alcohols were significantly changed in Merlot as compared to Syrah. The local red cultivars displayed lower numbers of significantly changed metabolites, twelve Aspartate, isocitrate and shikimate (trans-caffeate only in Merlot although Syrah displayed a very similar trend) were significantly increased only in widespread cultivars after WDS (Figures 1 and 4). On the other hand, lysine and isoleucine only increased in Callet (a similar trend in Escursac), and putrescine only significantly increased in Escursac (Figures 1 and 4). Finally, urea significantly decreased Agronomy only 2020, in Escursac while the opposite was observed in Syrah (Figure 1). 10, 1052 11 of 21 Figure 4. Metabolic model showing differences in the water deficit stress (WDS) response of leaf Figure 4.metabolism primary Metabolic model between showing differences widespread in the and local water deficit cultivars. stress (WDS) Metabolites response highlighted are of leaf those primary significant showing metabolism (p 0.05) (green) cultivars as compared only to their in local red corresponding (black) widespread and white (grey) ones. cultivars Metabolites (i.e., not significantly only significantly changing changing (p > 0.05) in widespread only in local red (black) and white (grey) cultivars (i.e., only significantly changing cultivars) are also highlighted. Data used was taken from Figure 1. For the metabolite abbreviations in widespread cultivars) see Figureare also highlighted. 3. *Only significantlyData (p