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The importance of the alternative donation of electrons to the ubiquinol pool via the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) complex has been demonstrated. However, the functional significance of this pathway during seed development and germination remains to be elucidated. To assess the function of this pathway, we performed a detailed metabolic and transcriptomic analysis of Arabidopsis mutants to test the molecular consequences of a dysfunctional ETF/ETFQO pathway. We demonstrate that the disruption of this pathway compromises seed germination in the absence of an external carbon source and also impacts seed size and yield. Total protein and storage protein content is reduced in dry seeds, whilst sucrose levels remain invariant. Seeds of ETFQO and related mutants were also characterized by an altered fatty acid composition. During seed development, lower levels of fatty acids and proteins accumulated in the etfqo-1 mutant as well as in mutants in the alternative electron donors isovaleryl-CoA dehydrogenase (ivdh-1) and d-2-hydroxyglutarate dehydrogenase (d2hgdh1-2). Furthermore, the content of several amino acids was increased in etfqo-1 mutants during seed development, indicating that these mutants are not using such amino acids as alternative energy source for respiration. Transcriptome analysis revealed alterations in the expression levels of several genes involved in energy and hormonal metabolism. Our findings demonstrated that the alternative pathway of respiration mediated by the ETF/ETFQO complex affects seed germination and development by directly adjusting carbon storage during seed filling. These results indicate a role for the pathway in the normal plant life cycle to complement its previously defined roles in the response to abiotic stress.

This work aims to determine the effect of genotype x environment (GxE) interaction that influence blackcurrant (Ribes nigrum) fruit quality. We applied metabolomics-driven analysis on fruits from four cultivars grown in contrasting European-locations over two seasons. By integrating metabolomics and sensory analysis, we also defined specific metabolic signatures associated with consumer acceptance. Our results showed that rainfall is a crucial factor associated with accumulation of delphinidin- and cyanidin-3-O-glucoside, the two mayor blackcurrant pigments meanwhile temperature affects the main organic acid levels which can be decisive for fruit taste. Sensorial analysis showed that increases in terpenoid and acetate ester volatiles were strongly associated with higher appreciation score, while proacacipetalin, a cyanogenic-glycoside, was positively associated to bitter taste. Our results pave the way for the selection of high-quality cultivars and suitable production sites for blackcurrant cultivation.

To identify potential strategies for increasing the efficiency of tomato leaf metabolism, with a focus on the links between nitrogen/carbon metabolism, we explored a diel Flux Balance Analysis (FBA) model of a source leaf in which the metabolic output was varied up to the theoretically-achievable maximum. We noticed a potentially interesting switch in the use of glutamine synthetase (GS) isoforms –from the chloroplast isoform to the mitochondrial one- for nitrogen assimilation. To further explore this prediction, we characterized transgenic tomato plants over-expressing two tomato GS genes, GS1 and GS2, targeted to mitochondria. Both sets of transgenic plants were characterized as displaying faster growth rate, early flowering and increased fruit yield. In leaves, metabolomic profiling and enzyme activity analysis pointed that GS activity in mitochondrial plays a role in increasing the intracellular synthesis and subsequent export of sugar. Consistent with these changes, higher sucrose concentration in leaf exudates and reduced activities of enzymes involved in leaf starch synthesis were observed. Moreover, mitochondrial GS activity affected chloroplast redox status in a manner that modulated photorespiration and nitrogen metabolism. The combined data reveal the influence of mitochondrial GS activity on both foliar carbon/nitrogen balance and regulation of source-sink metabolism in tomato plants.

Plant quality trait improvement has become a global necessity due to the world overpopulation. In particular, producing crop species with enhanced nutrients and health-promoting compounds is one of the main aims of current breeding programs. However, breeders traditionally focused on characteristics such as yield or pest resistance, while breeding for crop quality, which largely depends on the presence and accumulation of highly valuable metabolites in the plant edible parts, was left out due to the complexity of plant metabolome and the impossibility to properly phenotype it. Recent technical advances in high throughput metabolomic, transcriptomic and genomic platforms have provided efficient approaches to identify new genes and pathways responsible for the extremely diverse plant metabolome. In addition, they allow to establish correlation between genotype and metabolite composition, and to clarify the genetic architecture of complex biochemical pathways, such as the accumulation of secondary metabolites in plants, many of them being highly valuable for the human diet. In this review, we focus on how the combination of metabolomic, transcriptomic and genomic approaches is a useful tool for the selection of crop varieties with improved nutritional value and quality traits.

Gibberellins (GAs) play a crucial role in modulating developmental processes throughout the plant life cycle. Among the processes in which GAs are involved, they are of significance during the transition and maintenance of the reproductive meristem, as well as in allowing the floral organs development. GAs are also able to regulate, alongside with auxin and cytokinin, the initial processes of fruit development, most likely because they are responsible for both division and cell expansion. It is unknown whether fluctuations in the endogenous content of GAs impact fruit development and metabolism during ripening. To investigate these questions, tomato mutant plants deficient in GAs biosynthesis (gib3, moderately deficient; gib2, intermediate deficiency and gib1, extremely deficient in GA) were used. Notably, gib2 and gib1 mutants were characterized by a complete interruption of their reproductive development at the floral bud level. Although gib3 plants displayed a slightly delay in fruit development, at the end of fruit ripening both WT and gib3 fruits were highly similar. Little differences were found between WT and gib3 mutant plants during floral development and total fruit yield. We demonstrated that reduced GAs in gib3 mutant did not promote morphological modifications in fruits and relatively few metabolic changes were observed between genotypes during fruit ripening. Overall, typical metabolic changes, including increments in amino acids and soluble sugars coupled with reductions in starch, were observed during ripening. Collectively, we demonstrate that GAs plays a significant role on change vegetative to reproductive stage, as well as on the binging of set fruit.

Cell expansion is a significant contributor to organ growth and is driven by the accumulation of osmolytes to increase cell turgor pressure. Metabolic modelling has the potential to provide insights into the processes that underpin osmolyte synthesis and transport, but the main computational approach for predicting metabolic network fluxes, flux balance analysis (FBA), often uses biomass composition as the main output constraint and ignores potential changes in cell volume. Here we present GrOE-FBA (Growth by Osmotic Expansion - Flux Balance Analysis), a framework that accounts for both the metabolic and ionic contributions to the osmotica that drive cell expansion, as well as the synthesis of protein, cell wall and cell membrane components required for cell enlargement. Using GrOE-FBA, the metabolic fluxes in dividing and expanding cell were analyzed, and the energetic costs for metabolite biosynthesis and accumulation in the two scenarios were found to be surprisingly similar. The expansion phase of tomato fruit growth was also modelled using a multi-phase single optimization GrOE-FBA model and this approach gave accurate predictions of the major metabolite levels throughout fruit development as well as revealing a role for transitory starch accumulation in ensuring optimal fruit development.

Although autophagy is a conserved mechanism operating across eukaryotes, its effects on crops and especially their metabolism has received relatively little attention. Indeed, whilst a few recent studies have used systems biology tools to look at the consequences of lack of autophagy in maize these focused on leaf tissues rather than the kernels. Here we utilized RNA interference (RNAi) to generate tomato plants that were deficient in the autophagy-regulating protease ATG4. Plants displayed an early senescence phenotype yet relatively mild changes in the foliar metabolome and were characterized by a reduced fruit yield phenotype. Metabolite profiling indicated that metabolites of ATG4-RNAi tomato leaves just exhibited minor alterations while that of fruit displayed bigger difference compared to the WT. In detail, many primary metabolites exhibited decreases in the ATG4-RNAi lines, such as proline, tryptophan and phenylalanine, while the representative secondary metabolites (quinic acid and 3-trans-caffeoylquinic acid) were present at substantially higher levels in ATG4-RNAi green fruits than in WT. Moreover, transcriptome analysis indicated that the most prominent differences were in the significant upregulation of organelle degradation genes involved in the proteasome or chloroplast vesiculation pathways, which was further confirmed by the reduced levels of chloroplastic proteins in the proteomics data. Furthermore, integration analysis of the metabolome, transcriptome and proteome data indicated that ATG4 significantly affected the lipid metabolism, chlorophyll binding proteins and chloroplast biosynthesis. These data collectively lead us to propose a more sophisticated model to explain the cellular co-ordination of the process of autophagy.

Fruit composition determines the fruit quality and, consequently, consumer acceptance. As fruit quality can be modified by environmental conditions, it will be impacted by future alterations produced by global warming. Therefore, agricultural activities will be influenced by the changes in climatological conditions in cultivable areas, which could have a high socioeconomic impact if fruit production and quality decline. Currently, different stresses are being applied to several cultivated species to evaluate their impact on fruit metabolism and plant performance. With the use of metabolomic tools, these changes can be precisely measured, allowing us to determine changes in the patterns of individual compounds. As these changes depend on both the stress severity and the specific species involved and even on the specific cultivar, individual analysis must be conducted. To date, the most-studied crops have mainly been crops that are widely cultivated and have a high socioeconomic impact. In the near future, with the development of these metabolomic strategies, their implementation will be extended to other species, which will allow the adaptation of cultivation conditions and the development of varieties with high adaptability to climatological changes.

The ability to adapt to the environment is crucial for plant survival and thus a refined communication system capable of integrating endogenous and exogenous signals and further relaying this information to different parts of the plant is a key component of such adaptability. Given that they grow in highly distinct environments it is arguably unsurprising that roots and shoots display different responses to a given environmental condition. Accordingly, a higher sensitivity of roots to gibberellins (GAs) allows rapid adjustments in growth and development possibly triggering (a) stress tolerance mechanism(s). Here we investigated the differential metabolic responses between root and shoot following reductions of the endogenous GA content using tomato (Solanum lycopersicum L.) plants deficient in GA biosynthesis (gib3, moderately deficient, gib2, intermediate deficiency and gib1, extremely deficient in GAs). GA depletion impedes shoot growth to a greater extent than root growth in all mutants. Moreover, the greater the reduction in GA content the greater the extent of a disturbance at the metabolic level. Low leaf carbohydrate contents were observed in plants displaying higher root growth, suggesting an enhanced flow of photoassimilate to support root growth. Large increases in amino acids contents of either roots or shoot were observed. The increased amino acid content was coupled to reduced levels of TCA cycle intermediates suggesting that these changes are directly linked to early reactions of nitrogen assimilation. The combined data are discussed in terms of our current understanding of the interaction between GA and primary metabolism and their crosstalk in environmental responses.

We are grateful to Prof. Dr. Lothar Willmitzer for his support and advice. We thank Prof. Dr. Ralph Bock (Max Planck Institute of Molecular Plant physiology, Golm, Germany) and Dr. Caterina D’Ambrosio (Centro Ricerche Metapontum Agrobios, ALSIA, Italy) for kindly providing the transplastomic pNLyc#2 and LCe seeds and the homozygous nuclear High Caro (H.C.) lines, respectively. We thank Dr. Camila Caldana and Anne Michaelis for providing the GC facility and running the GC samples, respectively, and Maria Rosa Rodriguez-Goberna for technical support related with pigment analysis (supported by grant BIO2017-84041-P from the Spanish AEI). In addition, we thank Hana Martinkova and Petra Amakorova for their help with phytohormone analyses. The hormonomics work was funded by the Ministry of Education, Youth and Sports of the Czech Republic (European Regional Development Fund-Project “Plants as a tool for sustainable global development” No. CZ.02.1.01/0.0/0.0/16_019/0000827), and the Internal Grant Agency of Palacký University (IGA_PrF_2021_011)

Plant respiration provides metabolic flexibility under changing environmental conditions by modulating the activity of the nonphosphorylating alternative pathways from the mitochondrial electron transport chain, which bypass the main energy-producing components of the cytochrome oxidase pathway (COP). While adjustments in leaf primary metabolism induced by changes in day length are well studied, possible differences in the in vivo contribution of the COP and the alternative oxidase pathway (AOP) between different photoperiods remain unknown. In our study, in vivo electron partitioning between AOP and COP and expression analysis of respiratory components, photosynthesis, and the levels of primary metabolites were studied in leaves of wild-type (WT) plants and cytochrome c (CYTc) mutants, with reduced levels of COP components, under short- and long-day photoperiods. Our results clearly show that differences in AOP and COP in vivo activities between WT and cytc mutants depend on the photoperiod likely due to energy and stress signaling constraints. Parallel responses observed between in vivo respiratory activities, TCA cycle intermediates, amino acids, and stress signaling metabolites indicate the coordination of different pathways of primary metabolism to support growth adaptation under different photoperiods.

This article belongs to the Special Issue Antioxidant Metabolic Pathways in Plants 2.0, Salinity has a negative impact on plant growth, with photosynthesis being downregulated partially due to osmotic effect and enhanced cellular oxidation. Redox signaling contributes to the plant response playing thioredoxins (TRXs) a central role. In this work we explore the potential contribution of Arabidopsis TRXo1 to the photosynthetic response under salinity analyzing Arabidopsis wild-type (WT) and two Attrxo1 mutant lines in their growth under short photoperiod and higher light intensity than previous reported works. Stomatal development and apertures and the antioxidant, hormonal and metabolic acclimation are also analyzed. In control conditions mutant plants displayed less and larger developed stomata and higher pore size which could underlie their higher stomatal conductance, without being affected in other photosynthetic parameters. Under salinity, all genotypes displayed a general decrease in photosynthesis and the oxidative status in the Attrxo1 mutant lines was altered, with higher levels of H2O2 and NO but also higher ascorbate/glutathione (ASC/GSH) redox states than WT plants. Finally, sugar changes and increases in abscisic acid (ABA) and NO may be involved in the observed higher stomatal response of the TRXo1-altered plants. Therefore, the lack of AtTRXo1 affected stomata development and opening and the mutants modulate their antioxidant, metabolic and hormonal responses to optimize their adaptation to salinity., This research was funded by Spanish grants MINECO/FEDER (BFU2017-86585-P and PGC2018-093824-B-C41), MINECO/ERDF (CTM2014-53902-C2-1-P), and Seneca Excellence Project (19876/GERM/15). ASG and SDV were supported by FPI grants from MINECO. MN was supported by a predoctoral fellowship BES-2015- 072578 from MINECO, Spain co-financed by the European Social Fund (ESF). IFS received funding from the European Union’s Horizon 2020 Research and Innovation Program under the Marie Skłodowska-Curie grant agreement Nº 753301.

This article belongs to the Section Plant Physiology and Metabolism., Plant respiration provides metabolic flexibility under changing environmental conditions by modulating the activity of the nonphosphorylating alternative pathways from the mitochondrial electron transport chain, which bypass the main energy-producing components of the cytochrome oxidase pathway (COP). While adjustments in leaf primary metabolism induced by changes in day length are well studied, possible differences in the in vivo contribution of the COP and the alternative oxidase pathway (AOP) between different photoperiods remain unknown. In our study, in vivo electron partitioning between AOP and COP and expression analysis of respiratory components, photosynthesis, and the levels of primary metabolites were studied in leaves of wild-type (WT) plants and cytochrome c (CYTc) mutants, with reduced levels of COP components, under short- and long-day photoperiods. Our results clearly show that differences in AOP and COP in vivo activities between WT and cytc mutants depend on the photoperiod likely due to energy and stress signaling constraints. Parallel responses observed between in vivo respiratory activities, TCA cycle intermediates, amino acids, and stress signaling metabolites indicate the coordination of different pathways of primary metabolism to support growth adaptation under different photoperiods., This research was funded by FONDECYT No. 1191118 from the National Agency for Research and Development (ANID) and by the Agencia Nacional de Promoción Científica y Tecnológica, grant PICT-2018-01439. In addition, I.F.-S. has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement no. 753301, the ‘Ramon y Cajal’ contract RYC2019-027244-I/AEI/10.13039/501100011033 and the European Social Fund.

Fruit quality, that remains crucial to human nutrition and health, is closely related to biochemical composition, mostly, although not only, due to low molecular-weight metabolites. Final fruit quality results from coordinated physiological processes during development from anthesis to growth and ripe stages. Fruit development and quality may be modulated in response to (a)biotic stress, as well as through compositional evolution postharvest. Biotic stress effects are not addressed here. Comprehensive biochemical analyses of the metabolome have been used to describe and generate knowledge concerning fruit biochemical composition and metabolism, and their developmental and spatial changes before and after harvest. They have involved NMR- or MS-based fingerprinting, profiling or imaging strategies, possibly combined with targeted analyses (e.g., antioxidant metabolites). In this chapter, we first stress analytical aspects crucial for fruit metabolomics. Then, recent examples concerning metabolomics of temperate and tropical fleshy fruits (fresh, not processed), whether they are models or species of agronomical interest, are discussed in the context of possible impacts on fruit agricultural practices or breeding. These cases concern metabolism reprogramming during fruit set, development and postharvest, environmental effects, gene functional analysis, genetics and systems biology. Major current challenges of specific interest for fruit metabolomics are also discussed as examples of the problems and possibilities for this exciting research field.

Salinity has a negative impact on plant growth, with photosynthesis being downregulated partially due to osmotic effect and enhanced cellular oxidation. Redox signaling contributes to the plant response playing thioredoxins (TRXs) a central role. In this work we explore the potential contribution of Arabidopsis TRXo1 to the photosynthetic response under salinity analyzing Arabidopsis wild-type (WT) and two Attrxo1 mutant lines in their growth under short photoperiod and higher light intensity than previous reported works. Stomatal development and apertures and the antioxidant, hormonal and metabolic acclimation are also analyzed. In control conditions mutant plants displayed less and larger developed stomata and higher pore size which could underlie their higher stomatal conductance, without being affected in other photosynthetic parameters. Under salinity, all genotypes displayed a general decrease in photosynthesis and the oxidative status in the Attrxo1 mutant lines was altered, with higher levels of H2O2 and NO but also higher ascorbate/glutathione (ASC/GSH) redox states than WT plants. Finally, sugar changes and increases in abscisic acid (ABA) and NO may be involved in the observed higher stomatal response of the TRXo1-altered plants. Therefore, the lack of AtTRXo1 affected stomata development and opening and the mutants modulate their antioxidant, metabolic and hormonal responses to optimize their adaptation to salinity.

Phenylpropanoids are a large class of plant secondary metabolites, which play essential roles in human health mainly associated with their antioxidant activity. Strawberry (Fragaria × ananassa) is a rich source of phytonutrients, including phenylpropanoids, which have been shown to have beneficial effects on human health. In this study, using the F. × ananassa ‘232’ × ‘1392’ F1 segregating population, we analyzed the genetic control of individual phenylpropanoid metabolites, total polyphenol content (TPC) and antioxidant capacity (TEAC) in strawberry fruit over two seasons. We have identified a total of 7, 9, and 309 quantitative trait loci (QTL) for TPC, TEAC and for 77 polar secondary metabolites, respectively. Hotspots of stable QTL for health-related antioxidant compounds were detected on linkage groups LG IV-3, LG V-2 and V-4, and LG VI-1 and VI-2, where associated markers represent useful targets for marker-assisted selection of new varieties with increased levels of antioxidant secondary compounds. Moreover, differential expression of candidate genes for major and stable mQTLs was studied in fruits of contrasting lines in important flavonoids. Our results indicate that higher expression of FaF3′H, which encodes the flavonoid 3′-hydroxylase, is associated with increased content of these important flavonoids.

23 pags., 6 figs., Endoplasmic reticulum–plasma membrane contact sites (ER–PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER–PM protein tether synaptotagmin1 (SYT1) exhibit decreased PM integrity under multiple abiotic stresses, such as freezing, high salt, osmotic stress, and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER–PM tether that also functions in maintaining PM integrity. The ER–PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild-type while the levels of most glycerolipid species remain unchanged. In addition, the SYT1-green fluorescent protein fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work, This work was supported by the Ministerio de Economıa y Competitividad, co-financed by the European Regional Development Fund (grant no. BIO2017-82609-R to M.A.B.), the Ministerio de Ciencia, Innovacion y Universidades (grant no. PGC2018-098789-B-I00 to N.R.-L.) UMA-FEDER (grant UMA18-FEDERJA-154 to N.R.-L.), and the Marie SkłodowskaCurie actions (grant no. H2020-655366-IIF- PLICO to M.A.B. and N.R.-L.). N.R.L. was supported by the Ramon y Cajal program RYC-2013-12699 (MINECO, Spain). J.P.-S. and S.G.-H. were funded by the Ministerio de Economıa y Competitividad in Formacion del Personal Investigador Fellowship (grant no. BES-2012-052324) and (PRE2018- 085284), respectively. R.P.H. and J.A.N. received support from the Biotechnology and Biological Sciences Research Council (BBSRC, UK) in the form of an Institute Strategic Programme Grant (grant no. BBS/E/C/000I0420). J.L. is supported by the Program of Introducing Talents of Discipline to Universities (111 Project, grant no. B13007). A.P.M. and J.P.-S. were supported by the Shanghai Center for Plant Stress Biology (Chinese Academy of Sciences), Chinese 1000 Talents Program. A.R. was supported by the Natural Sciences and Engineering Research Council of Canada (NSERCDiscovery Grant no. RGPIN-2019-05568). Support was also provided by AEI/FEDER, UE (grant nos. BIO2016-79187-R and PID2019-106987RB-I00 to J.P.-S.) and by the European Research Council under the European Union’s Seventh Framework Programme (grant no. FP7/2007-2013)/ERC grant agreement no. 742985 to J.F. and T-Rex (project number 682436 to D.V.D.).

Sugars play a key role in fruit quality, as they directly influence taste, and thus consumer acceptance. Carbohydrates are the main resources needed by the plant for carbon and energy supply and have been suggested to be involved in all the important developmental processes, including embryogenesis, seed germination, stress responses, and vegetative and reproductive growth. Recently, considerable progresses have been made in understanding regulation of fruit ripening mechanisms, based on the role of ethylene, auxins, abscisic acid, gibberellins, or jasmonic acid, in both climacteric and non-climacteric fruits. However, the role of sugar and its associated molecular network with hormones in the control of fruit development and ripening is still poorly understood. In this review, we focus on sugar signaling mechanisms described up to date in fruits, describing their involvement in ripening-associated processes, such as pigments accumulation, and their association with hormone transduction pathways, as well as their role in stress-related responses.

SUMMARYEndoplasmic Reticulum-Plasma Membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to WT while the levels of most glycerolipid species remain unchanged. Additionally, SYT1-GFP preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a crucial SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

Anthocyanins are the principal color-producing compounds synthesized in developing fruits of strawberry (Fragariaspp.). Substantial natural variation in color have been observed in fruits of diploid and octoploid accessions, resulting from distinct accumulation and distribution of anthocyanins in fruits. Anthocyanin biosynthesis is controlled by a clade of R2R3 MYB transcription factors, among whichMYB10has been shown as the main activator in strawberry fruit. Here, we show thatMYB10mutations cause most of the anthocyanin variation observed in diploid woodland strawberry(F. vesca)and octoploid cultivated strawberry (F. ×ananassa). Using a mapping-by-sequencing approach, we identified agypsy-transposon insertion inMYB10that truncates the protein and knocks out anthocyanin biosynthesis in a white-fruitedF. vescaecotype. Two additional loss-of-functionMYB10mutations were identified among geographically diverse white-fruitedF. vescaecotypes. Genetic and transcriptomic analyses in octoploidFragaria spp.revealed thatFaMYB10-2,one of threeMYB10homoeologs identified, residing in theF. iinumae-derivedsubgenome, regulates the biosynthesis of anthocyanins in developing fruit. Furthermore, independent mutations inMYB10-2are the underlying cause of natural variation in fruit skin and flesh color in octoploid strawberry. We identified a CACTA-like transposon(FaEnSpm-2)insertion in theMYB10-2promoter of red-fleshed accessions that was associated with enhanced expression and anthocyanin accumulation. Our findings suggest that putative cis regulatory elements provided byFaEnSpm-2are required for high and ectopicMYB10-2expression and induction of anthocyanin biosynthesis in fruit flesh. We developedMYB10-2(sub-genome) specific DNA markers for marker-assisted selection that accurately predicted anthocyanin phenotypes in octoploid segregating populations.

Grapes, one of the oldest agricultural crops, are cultivated to produce table fruits, dried fruits, juice, and wine. Grapevine variety is composed of clones that share common morphological traits. However, they can differ in minor genetic mutations which often result in not only notorious morphological changes but also in other non-visible sensorial distinctive attributes. In the present work, we identified three Vitis vinifera cv. Pinot noir clones grown under identical field conditions that showed different grape cluster types. Here, sensorial analysis together with non-targeted metabolite profiles by Ultra High performance Liquid Chromatography (UPLC) couples to Ultra High Resolution Mass Spectrometry (FT-ICR-MS) of wines elaborated from the three different grape cluster types was studied with the aim of (i) finding sensorial differences among these three types of wines, and, if there were, (ii) determining the molecular features (metabolites) associated with these sensorial attributes by a multivariate statistical approach.

© 2019 The Authors., FaMADS9 is the strawberry (Fragaria x ananassa) gene that exhibits the highest homology to the tomato (Solanum lycopersicum) RIN gene. Transgenic lines were obtained in which FaMADS9 was silenced. The fruits of these lines did not show differences in basic parameters, such as fruit firmness or colour, but exhibited lower Brix values in three of the four independent lines. The gene ontology MapMan category that was most enriched among the differentially expressed genes in the receptacles at the white stage corresponded to the regulation of transcription, including a high percentage of transcription factors and regulatory proteins associated with auxin action. In contrast, the most enriched categories at the red stage were transport, lipid metabolism and cell wall. Metabolomic analysis of the receptacles of the transformed fruits identified significant changes in the content of maltose, galactonic acid‐1,4‐lactone, proanthocyanidins and flavonols at the green/white stage, while isomaltose, anthocyanins and cuticular wax metabolism were the most affected at the red stage. Among the regulatory genes that were differentially expressed in the transgenic receptacles were several genes previously linked to flavonoid metabolism, such as MYB10, DIV, ZFN1, ZFN2, GT2, and GT5, or associated with the action of hormones, such as abscisic acid, SHP, ASR, GTE7 and SnRK2.7. The inference of a gene regulatory network, based on a dynamic Bayesian approach, among the genes differentially expressed in the transgenic receptacles at the white and red stages, identified the genes KAN1, DIV, ZFN2 and GTE7 as putative targets of FaMADS9. A MADS9‐specific CArG box was identified in the promoters of these genes., This work was supported by grants BIO2013-44199-R, AGR12- 40066-CO2-02, and RTI2018-099797-B-100 (MINECO, Spain), and the European Union’s H2020 Programme (GoodBerry; grant number 679303) and ERC-2014-StG638134. DP and SO acknowledge the support by Spanish Ministry of Science and Innovation (Ramon and Cajal contracts, RYC2011-09170 and RYC2013-12699).

Metabolic correlation networks have been used in several instances to obtain a deeper insight into the complexity of plant metabolism as a whole. In tomato (Solanum lycopersicum), metabolites have a major influence on taste and overall fruit quality traits. Previously a broad spectrum of metabolic and phenotypic traits has been described using a Solanum pennellii introgression-lines (ILs) population. To obtain insights into tomato fruit metabolism, we performed metabolic network analysis from existing data, covering a wide range of metabolic traits, including lipophilic and volatile compounds, for the first time. We provide a comprehensive fruit correlation network and show how primary, secondary, lipophilic, and volatile compounds connect to each other and how the individual metabolic classes are linked to yield-related phenotypic traits. Results revealed a high connectivity within and between different classes of lipophilic compounds, as well as between lipophilic and secondary metabolites. We focused on lipid metabolism and generated a gene-expression network with lipophilic metabolites to identify new putative lipid-related genes. Metabolite–transcript correlation analysis revealed key putative genes involved in lipid biosynthesis pathways. The overall results will help to deepen our understanding of tomato metabolism and provide candidate genes for transgenic approaches toward improving nutritional qualities in tomato.

The strawberry fruit is perishable due to its high water content and soft texture, yet exhibits pleasant organoleptic and nutritional profile. Here we conducted a metabolomics-driven analysis followed by linear modelling to dissect the molecular processes in strawberry postharvest. Fruits from five cultivars were harvested and refrigerated during a ten-day period under three different atmospheres: ambient, CO2-enriched and O3-enriched. These analyses revealed that metabolites involved in, (i) organoleptic and nutritional properties; (ii) stress tolerance displayed duration and postharvest treatment-dependent levels. Ozone-enriched atmosphere appears to counteract postharvest negative effects, with fruits exhibiting lower levels of fermentative metabolites when compared to fruits kept in an ambient atmosphere. Furthermore, metabolic reconfiguration towards the synthesis of protective metabolites of those fruits can possibly confer enhanced tolerance to postharvest abiotic stresses. Finally, results from the linear modelling identified metabolites which could be used as biomarkers to assess strawberry quality during its postharvest shelf life.

It has been hypothesized that plants can get beneficial trade-offs from viral infections when grown under drought conditions. However, experimental support for a positive correlation between virus-induced drought tolerance and increased host fitness is scarce. We investigated whether increased virulence exhibited by the synergistic interaction involving Potato virus X (PVX) and Plum pox virus (PPV) improves tolerance to drought and host fitness in Nicotiana benthamiana and Arabidopsis thaliana. Infection by the pair PPV/PVX and by PPV expressing the virulence protein P25 of PVX conferred an enhanced drought-tolerant phenotype compared with single infections with either PPV or PVX. Decreased transpiration rates in virus-infected plants were correlated with drought tolerance in N. benthamiana but not in Arabidopsis. Metabolite and hormonal profiles of Arabidopsis plants infected with the different viruses showed a range of changes that positively correlated with a greater impact on drought tolerance. Virus infection enhanced drought tolerance in both species by increasing salicylic acid accumulation in an abscisic acid-independent manner. Viable offspring derived from Arabidopsis plants infected with PPV increased relative to non-infected plants, when exposed to drought. By contrast, the detrimental effect caused by the more virulent viruses overcame potential benefits associated with increased drought tolerance on host fitness.

Advances in analytical chemistry, computation and biotechnology have led to the recent development of methodologies for broad metabolite profiling. Metabolomics now plays a significant role in fundamental plant biology and applied biotechnology. The study of plant metabolism has been one of the first beneficiaries of these technologies, and applications have focused on plant metabolite engineering, functional genomics and physiology. Metabolomics has gained importance in biotechnology applications, as exemplified by quantitative loci analysis. Integration of different broad-range metabolite profiling into genomics approaches (system biology) driven by metabolome data will bring a change in our understanding of the regulation of metabolic networks and, therefore, facilitate improvements in our understanding of plant breeding. Key Concepts Metabolomics is defined as the quantitative complement of low-molecular-weight metabolites present in a cell under a given set of physiological conditions. Systems biology is the combination of molecular and system-level studies applying a synergistic approach involving modelling, theory and experiment. Metabolite marker is a metabolite that is objectively measured and evaluated as an indicator of normal biological processes or specific trait. Metabolic phenotype is the products of interactions among a variety of factors such as environment or genetic perturbation. Functional genomics is the field of molecular biology that uses many possible approaches (i.e. genomics, transcriptomics, proteomics and metabolomics) to understand the properties and function of the entirety of an organism's genes and gene products. Keywords: metabolite profiling; metabolomics; metabolism; functional genomics; mass spectrometry

Salinity stress limits plant growth and has a major impact on agricultural productivity. Here, we identify NAC transcription factor SlTAF1 as a regulator of salt tolerance in cultivated tomato (Solanum lycopersicum). While overexpression of SlTAF1 improves salinity tolerance compared with wild-type, lowering SlTAF1 expression causes stronger salinity-induced damage. Under salt stress, shoots of SlTAF1 knockdown plants accumulate more toxic Na+ ions, while SlTAF1 overexpressors accumulate less ions, in accordance with an altered expression of the Na+ transporter genes SlHKT1;1 and SlHKT1;2. Furthermore, stomatal conductance and pore area are increased in SlTAF1 knockdown plants during salinity stress, but decreased in SlTAF1 overexpressors. We identified stress-related transcription factor, abscisic acid metabolism and defence-related genes as potential direct targets of SlTAF1, correlating it with reactive oxygen species scavenging capacity and changes in hormonal response. Salinity-induced changes in tricarboxylic acid cycle intermediates and amino acids are more pronounced in SlTAF1 knockdown than wild-type plants, but less so in SlTAF1 overexpressors. The osmoprotectant proline accumulates more in SlTAF1 overexpressors than knockdown plants. In summary, SlTAF1 controls the tomato’s response to salinity stress by combating both osmotic stress and ion toxicity, highlighting this gene as a promising candidate for the future breeding of stress-tolerant crops.

Fruit flavor and nutritional characteristics are key quality traits and ones of the main factors influencing consumer preference. Central carbon metabolism, also known as primary metabolism, contributes to the synthesis of intermediate compounds that act as precursors for plant secondary metabolism. Specific and specialized metabolic pathways that evolved from primary metabolism play a key role in the plant's interaction with its environment. In particular, secondary metabolites present in the fruit serve to increase its attractiveness to seed dispersers and to protect it against biotic and abiotic stresses. As a consequence, several important organoleptic characteristics, such as aroma, color, and fruit nutritional value, rely upon secondary metabolite content. Phenolic and terpenoid compounds are large and diverse classes of secondary metabolites that contribute to fruit quality and have their origin in primary metabolic pathways, while the delicate aroma of ripe fruits is formed by a unique combination of hundreds of volatiles that are derived from primary metabolites. In this review, we show that the manipulation of primary metabolism is a powerful tool to engineer quality traits in fruits, such as the phenolic, terpenoid, and volatile content. The enzymatic reactions responsible for the accumulation of primary precursors are bottlenecks in the transfer of metabolic flux from central to specialized metabolism and should be taken into account to increase the yield of the final products of the biosynthetic pathways. In addition, understanding the connection and regulation of the carbon flow between primary and secondary metabolism is a key factor for the development of fruit cultivars with enhanced organoleptic and nutritional traits.

Gibberellins (GAs) have been shown to be involved in the tolerance of plants to a range of environmental stresses. However, the physiological and metabolic implications of altered levels of GAs in plants under drought stress remain largely unknown. To understand the contribution of GAs for the responses to water deficit conditions, physiological and metabolic parameters were evaluated in tomato deficient mutants in GAs biosynthesis, gib1, gib2, and gib3. Interestingly, the mutants maintained leaf water content over a longer time period and recovered photosynthesis faster than wild-type (WT) plants. Furthermore, gib1 and gib2 plants showed no apparent wilt even after reaching low leaf water potential (-1.3 MPa). Additionally, mutants plants partitioned more biomass to the roots than shoots compared to WT. The maintenance of leaf water content and consequent increased water use efficiency observed in the mutants can be explained by the osmotic adjustment of leaf and root cells, mainly due to amino acid accumulation. Collectively, our results suggest that plants with reduced GAs content are able to cope with water deprivation by increasing proline and other amino acid levels. This response, alongside partitioning of carbon to roots allows GAs-deficient plants to maintain the leaf turgor for a longer time and thereby promotes water deficit tolerance.

68 p.-7 fig.-10 fig. supl.-1 tab. supl., BIR1 is a receptor‐like kinase that functions as a negative regulator of basal immunity and cell death in Arabidopsis.Using Arabidopsis thaliana and Tobacco rattle virus (TRV), we investigate the antiviral role of BIR1, the molecular mechanisms of BIR1 gene expression regulation during viral infections, and the effects of BIR1 overexpression on plant immunity and development.We found that SA acts as a signal molecule for BIR1 activation during infection. Inactivating mutations of BIR1 cause strong antiviral resistance that is not due to constitutive cell death or SA defense priming in the bir1‐1 mutant. RNA‐directed DNA methylation (RdDM) and post‐transcriptional silencing are both required to negatively regulate BIR1 expression upon viral induction. BIR1 overexpression causes severe developmental defects, cell death and premature death that correlate with the constitutive activation of plant immune responses.Our findings suggest that BIR1 acts as a negative regulator of antiviral defense in plants, and indicate that RNA silencing contributes, alone or in conjunction with other regulatory mechanisms, to define a threshold expression for proper BIR1 function beyond which an autoimmune response may occur. This work provides novel mechanistic insights into the regulation of BIR1 homeostasis that may be common for other plant immune components., This work has been supported by FPI fellowships (BES-2013-063138 and EEBB-I-16-10815) to IG-B, by a Ramon y Cajal grant (RyC-2011-07006) to VR-F and by National Research grants (BIO2012-39973 and BIO2015-70752-R) to CL from Ministerio de Economía y Competitividad (MINECO/FEDER), Spain, and by a National Institutes of Health (USA) grant R01GM108722 to ATW.

Summary Tomato (Solanum lycopersicum) fruit ripening is regulated co‐operatively by the action of ethylene and a hierarchy of transcription factors, including RIPENING INHIBITOR (RIN) and NON‐RIPENING (NOR). Mutations in these two genes have been adopted commercially to delay ripening, and accompanying textural deterioration, as a means to prolong shelf life. However, these mutations also affect desirable traits associated with colour and nutritional value, although the extent of this trade‐off has not been assessed in detail. Here, we evaluated changes in tomato fruit pericarp primary metabolite and carotenoid pigment profiles, as well as the dynamics of specific associated transcripts, in the rin and nor mutants during late development and postharvest storage, as well of those of the partially ripening delayed fruit ripening (dfd) tomato genotype. These profiles were compared with those of the wild‐type tomato cultivars Ailsa Craig (AC) and M82. We also evaluated the metabolic composition of M82 fruit ripened on or off the vine over a similar period. In general, the dfd mutation resulted in prolonged firmness and maintenance of quality traits without compromising key metabolites (sucrose, glucose/fructose and glucose) and sectors of intermediary metabolism, including tricarboxylic acid cycle intermediates. Our analysis also provided insights into the regulation of carotenoid formation and highlighted the importance of the polyamine, putrescine, in extending fruit shelf life. Finally, the metabolic composition analysis of M82 fruit ripened on or off the vine provided insights into the import into fruit of compounds, such as sucrose, during ripening.

Plants synthesize and emit a large range of volatile organic compounds (VOCs) that play important roles in their interactions with the environment, from attracting pollinators and seed dispersers to protectants such as repellants and pathogen inhibitors. As such, the development of techniques for headspace collection of volatiles in combination with gas chromatography-mass spectrometry (GC-MS) has an important impact on our understanding of the biosynthesis of plant VOCs. Furthermore, knowledge of the plant VOCs can be valuable in relation to plant breeding for improving fruit flavor or enhancing resistance to insects or pathogens. This chapter describes a reliable method for extracting volatile compounds by headspace solid-phase microextraction (HS-SPME), and separate and detect them by GC-MS.

Fruit development and ripening is a unique developmental process to flowering plants that ensures the propagation of seeds and plant survival. In addition, fruits are an essential part of human diet. In particular, strawberry is a rich source of nutraceuticals such as vitamin C, folate and phenolic compounds. Strawberry production and breeding is becoming an extremely competing area of economic development worldwide. Cost of production in many countries is increasing due to a number of challenges such as rising labour costs, pest control or water availability. One way to increase competiveness is increasing fruit quality of new strawberry cultivars. Amazing advances have been made in our knowledge of the different metabolic pathways that take place in the final stages of fruit development and that lead to a flavourful and ripe strawberry fruit. Similarly, different genes involved in gene regulation during ripening have been discovered and characterized. In parallel, the discovery of loci responsible for natural variation among strawberry germplasm is producing a growing amount of DNA markers that after validation could be used in accelerating the selection of new cultivars with improved fruit quality. This chapter summarizes main advances in the study of fruit ripening in the octoploid strawberry and QTL controlling fruit quality traits.

Summary The receptacle of the strawberry (Fragaria × ananassa) fruit accounts for the main properties of the ripe fruit for human consumption. As it ripens, it undergoes changes similar to other fruits in sugar : acid ratio, volatile production and cell wall softening. However, the main regulators of this process have not yet been reported. The white stage marks the initiation of the ripening process, and we had previously reported a peak of expression for a FaGAMYB gene. Transient silencing of FaGAMYB using RNAi and further determination of changes in global gene expression by RNAseq, and composition of primary and secondary metabolites have been used to investigate the role played by this gene during the development of the receptacle. Down-regulation of FaGAMYB caused an arrest in the ripening of the receptacle and inhibited colour formation. Consistent with this, several transcription factors associated with the regulation of flavonoid biosynthetic pathway showed altered expression. FaGAMYB silencing also caused a reduction of ABA biosynthesis and sucrose content. Interestingly, exogenous ABA application to the RNAI-transformed receptacle reversed most defects caused by FaGAMYB down-regulation. The study assigns a key regulatory role to FaGAMYB in the initiation of strawberry receptacle ripening and acting upstream of the known regulator ABA.

It has been hypothesized that plants can get beneficial trade-offs from viral infections when grown under drought conditions. However, experimental support for a positive correlation between virus-induced drought tolerance and increased host fitness is scarce. We investigated whether increased virulence exhibited by the synergistic interaction involving Potato virus X (PVX) and Plum pox virus (PPV) improves tolerance to drought and host fitness in Nicotiana benthamiana and Arabidopsis thaliana. Infection by the pair PPV/PVX and by PPV expressing the virulence protein P25 of PVX conferred an enhanced drought-tolerant phenotype compared with single infections with either PPV or PVX. Decreased transpiration rates in virus-infected plants were correlated with drought tolerance in N. benthamiana but not in Arabidopsis. Metabolite and hormonal profiles of Arabidopsis plants infected with the different viruses showed a range of changes that positively correlated with a greater impact on drought tolerance. Virus infection enhanced drought tolerance in both species by increasing salicylic acid accumulation in an abscisic acid-independent manner. Viable offspring derived from Arabidopsis plants infected with PPV increased relative to non-infected plants, when exposed to drought. By contrast, the detrimental effect caused by the more virulent viruses overcame potential benefits associated with increased drought tolerance on host fitness.

The role of auxin in ripening strawberry (Fragaria ×ananassa) fruits has been restricted to the early stages of development where the growth of the receptacle is dependent on the delivery of auxin from the achenes. At later stages, during enlargement of the receptacle, other hormones have been demonstrated to participate to different degrees, from the general involvement of gibberellins and abscisic acid to the more specific of ethylene. Here we report the involvement of auxin at the late stages of receptacle ripening. The auxin content of the receptacle remains constant during ripening. Analysis of the transcriptome of ripening strawberry fruit revealed the changing expression pattern of the genes of auxin synthesis, perception, signaling and transport along with achene and receptacle development from the green to red stage. Specific members of the corresponding gene families show active transcription in the ripe receptacle. For the synthesis of auxin, two genes encoding tryptophan aminotransferases, FaTAA1 and FaTAR2, were expressed in the red receptacle, with FaTAR2 expression peaking at this stage. Transient silencing of this gene in ripening receptacle was accompanied by a diminished responsiveness to auxin. The auxin activity in the ripening receptacle is supported by the DR5-directed expression of a GUS reporter gene in the ripening receptacle of DR5-GUS transgenic strawberry plants. Clustering by co-expression of members of the FaAux/IAA and FaARF families identified five members whose transcriptional activity was increased with the onset of receptacle ripening. Among these, FaAux/IAA11 and FaARF6a appeared, by their expression level and fold-change, as the most likely candidates for their involvement in the auxin activity in the ripening receptacle. The association of the corresponding ARF6 gene in Arabidopsis to cell elongation constitutes a suggestive hypothesis for FaARF6a involvement in the same cellular process in the growing and ripening receptacle.