How metabolism and development are intertwined in space and time.

Developmental transitions, occurring throughout the life cycle of plants, require precise regulation of metabolic processes to generate the energy and resources necessary for the committed growth processes. In parallel, the establishment of new cells, tissues, and even organs, alongside their differentiation provoke profound changes in metabolism. It is increasingly being recognized that there is a certain degree of feedback regulation between the components and products of metabolic pathways and developmental regulators. The generation of large-scale metabolomics datasets during developmental transitions, in combination with molecular genetic approaches has helped to further our knowledge on the functional importance of metabolic regulation of development. In this perspective article, we provide insights into studies that elucidate interactions between metabolism and development at the temporal and spatial scales. We additionally discuss how this influences cell growth-related processes. We also highlight how metabolic intermediates function as signaling molecules to direct plant development in response to changing internal and external conditions.

Regulatory mechanisms behind the phenotypic plasticity associated with Setaria italica water deficit tolerance.

Drought is one of the main environmental stresses that negatively impacts vegetative and reproductive yield. Water deficit responses are determined by the duration and intensity of the stress, which, together with plant genotype, will define the chances of plant survival. The metabolic adjustments in response to water deficit are complex and involve gene expression modulation regulated by DNA-binding proteins and epigenetic modifications. This last mechanism may also regulate the activity of transposable elements, which in turn impact the expression of nearby loci. Setaria italica plants submitted to five water deficit regimes were analyzed through a phenotypical approach, including growth, physiological, RNA-seq and sRNA-seq analyses. The results showed a progressive reduction in yield as a function of water deficit intensity associated with signaling pathway modulation and metabolic adjustments. We identified a group of loci that were consistently associated with drought responses, some of which were related to water deficit perception, signaling and regulation. Finally, an analysis of the transcriptome and sRNAome allowed us to identify genes putatively regulated by TE- and sRNA-related mechanisms and an intriguing positive correlation between transcript levels and sRNA accumulation in gene body regions. These findings shed light on the processes that allow S. italica to overcome drought and survive under water restrictive conditions.

Assessment and characterization of tomato lipophilic electrophiles and their potential contribution to nutraceutical properties via SKN-1/Nrf2 signaling activation.

Phytochemical electrophiles are drawing significant attention due to their properties to modulate signaling pathways related to cellular homeostasis. The aim of this study was to develop new tools to examine the electrophilic activity in food and predict their beneficial effects on health. We developed a spectrophotometric assay based on the nitrobenzenethiol (NBT) reactivity, as a thiol-reactive nucleophile, to screen electrophiles in tomato fruits. The method is robust, simple, inexpensive, and could be applied to other types of food. We quantified the electrophile activity in a tomato collection and associated this activity with the pigment composition. Thus, we identified lycopene, ß- and γ-carotenes, 16 by-products of carotenoid oxidation and 18 unknown compounds as NBT-reactive by HPLC-MS/MS. The potential benefits of NBT-reactive compounds on health were evaluated in the in vivo model of C. elegans where they activated the SKN-1/Nrf2 pathway, evidencing the ability of electrophilic compounds to induce a biological response.

Multiomics analyses reveal the roles of the ASR1 transcription factor in tomato fruits.

The transcription factor ASR1 (ABA, STRESS, RIPENING 1) plays multiple roles in plant responses to abiotic stresses as well as being involved in the regulation of central metabolism in several plant species. However, despite the high expression of ASR1 in tomato fruits, large scale analyses to uncover its function in fruits are still lacking. In order to study its function in the context of fruit ripening, we performed a multiomics analysis of ASR1-antisense transgenic tomato fruits at the transcriptome and metabolome levels. Our results indicate that ASR1 is involved in several pathways implicated in the fruit ripening process, including cell wall, amino acid, and carotenoid metabolism, as well as abiotic stress pathways. Moreover, we found that ASR1-antisense fruits are more susceptible to the infection by the necrotrophic fungus Botrytis cinerea. Given that ASR1 could be regulated by fruit ripening regulators such as FRUITFULL1/FRUITFULL2 (FUL1/FUL2), NON-RIPENING (NOR), and COLORLESS NON-RIPENING (CNR), we positioned it in the regulatory cascade of red ripe tomato fruits. These data extend the known range of functions of ASR1 as an important auxiliary regulator of tomato fruit ripening.

The cytosolic invertase NI6 affects vegetative growth, flowering, fruit set, and yield in tomato.

Sucrose metabolism is important for most plants, both as the main source of carbon and via signaling mechanisms that have been proposed for this molecule. A cleaving enzyme, invertase (INV) channels sucrose into sink metabolism. Although acid soluble and insoluble invertases have been largely investigated, studies on the role of neutral invertases (A/N-INV) have lagged behind. Here, we identified a tomato A/N-INV encoding gene (NI6) co-localizing with a previously reported quantitative trait locus (QTL) largely affecting primary carbon metabolism in tomato. Of the eight A/N-INV genes identified in the tomato genome, NI6 mRNA is present in all organs, but its expression was higher in sink tissues (mainly roots and fruits). A NI6-GFP fusion protein localized to the cytosol of mesophyll cells. Tomato NI6-silenced plants showed impaired growth phenotype, delayed flowering and a dramatic reduction in fruit set. Global gene expression and metabolite profile analyses of these plants revealed that NI6 is not only essential for sugar metabolism, but also plays a signaling role in stress adaptation. We also identified major hubs, whose expression patterns were greatly affected by NI6 silencing; these hubs were within the signaling cascade that coordinates carbohydrate metabolism with growth and development in tomato.

Validated MAGIC and GWAS population mapping reveals the link between vitamin E content and natural variation in chorismate metabolism in tomato.

Tocochromanols constitute the different forms of vitamin E (VTE), essential components of the human diet, and display a high membrane protectant activity. By combining interval mapping and genome-wide association studies (GWAS), we unveiled the genetic determinants of tocochromanol accumulation in tomato (Solanum lycopersicum) fruits. To enhance the nutritional value of this highly consumed vegetable, we dissected the natural intraspecific variability of tocochromanols in tomato fruits and genetically engineered their biosynthetic pathway. These analyses allowed the identification of a total of 25 quantitative trait loci interspersed across the genome pinpointing the chorismate-tyrosine pathway as a regulatory hub controlling the supply of the aromatic head group for tocochromanol biosynthesis. To validate the link between the chorismate-tyrosine pathway and VTE, we engineered tomato plants to bypass the pathway at the arogenate branch point. Transgenic tomatoes showed moderate increments in tocopherols (up to approximately 20%) and a massive accumulation of tocotrienols (up to approximately 3400%). Gene expression analyses of these plants reveal a trade-off between VTE and natural variation in chorismate metabolism explained by transcriptional reprogramming of specific structural genes of the pathway. By restoring the accumulation of alpha-tocotrienols (α-t3) in fruits, the plants produced here are of high pharmacological and nutritional interest.

The conserved brassinosteroid-related transcription factor BIM1a negatively regulates fruit growth in tomato.

Brassinosteroids (BRs) are steroid hormones that play key roles in plant development and defense. Our goal is to harness the extensive knowledge of the Arabidopsis BR signaling network to improve productivity in crop species. This first requires identifying components of the conserved network and their function in the target species. Here, we investigated the function of SlBIM1a, the closest tomato homolog of AtBIM1, which is highly expressed in fruit. SlBIM1a-overexpressing lines displayed severe plant and fruit dwarfism, and histological characterization of different transgenic lines revealed that SlBIM1a expression negatively correlated with fruit pericarp cell size, resulting in fruit size modifications. These growth phenotypes were in contrast to those found in Arabidopsis, and this was confirmed by the reciprocal ectopic expression of SlBIM1a/b in Arabidopsis and of AtBIM1 in tomato. These results determined that BIM1 function depends more on the recipient species than on its primary sequence. Yeast two-hybrid interaction studies and transcriptomic analyses of SlBIM1a-overexpressing fruit further suggested that SlBIM1a acts through its interaction with SlBZH1 to govern the transcriptional regulation of growth-related BR target genes. Together, these results suggest that SlBIM1a is a negative regulator of pericarp cell expansion, possibly at the crossroads with auxin and light signaling.

Multi-gene metabolic engineering of tomato plants results in increased fruit yield up to 23%.

The capacity to assimilate carbon and nitrogen, to transport the resultant sugars and amino acids to sink tissues, and to convert the incoming sugars and amino acids into storage compounds in the sink tissues, are key determinants of crop yield. Given that all of these processes have the potential to co-limit growth, multiple genetic interventions in source and sink tissues, plus transport processes may be necessary to reach the full yield potential of a crop. We used biolistic combinatorial co-transformation (up to 20 transgenes) for increasing C and N flows with the purpose of increasing tomato fruit yield. We observed an increased fruit yield of up to 23%. To better explore the reconfiguration of metabolic networks in these transformants, we generated a dataset encompassing physiological parameters, gene expression and metabolite profiling on plants grown under glasshouse or polytunnel conditions. A Sparse Partial Least Squares regression model was able to explain the combination of genes that contributed to increased fruit yield. This combinatorial study of multiple transgenes targeting primary metabolism thus offers opportunities to probe the genetic basis of metabolic and phenotypic variation, providing insight into the difficulties in choosing the correct combination of targets for engineering increased fruit yield.

Phytochrome-Dependent Temperature Perception Modulates Isoprenoid Metabolism.

Changes in environmental temperature influence many aspects of plant metabolism; however, the underlying regulatory mechanisms remain poorly understood. In addition to their role in light perception, phytochromes (PHYs) have been recently recognized as temperature sensors affecting plant growth. In particular, in Arabidopsis (Arabidopsis thaliana), high temperature reversibly inactivates PHYB, reducing photomorphogenesis-dependent responses. Here, we show the role of phytochrome-dependent temperature perception in modulating the accumulation of isoprenoid-derived compounds in tomato (Solanum lycopersicum) leaves and fruits. The growth of tomato plants under contrasting temperature regimes revealed that high temperatures resulted in coordinated up-regulation of chlorophyll catabolic genes, impairment of chloroplast biogenesis, and reduction of carotenoid synthesis in leaves in a PHYB1B2-dependent manner. Furthermore, by assessing a triple phyAB1B2 mutant and fruit-specific PHYA- or PHYB2-silenced plants, we demonstrated that biosynthesis of the major tomato fruit carotenoid, lycopene, is sensitive to fruit-localized PHY-dependent temperature perception. The collected data provide compelling evidence concerning the impact of PHY-mediated temperature perception on plastid metabolism in both leaves and fruit, specifically on the accumulation of isoprenoid-derived compounds.

Effects of chlorogenic acid on thermal stress tolerance in C. elegans via HIF-1, HSF-1 and autophagy.

BACKGROUND: Chlorogenic acid (CGA) is a polyphenol widely distributed in plants and plant-derived food with antioxidant and protective activities against cell stress. Caenorhabditis elegans is a model organism particularly useful for understanding the molecular and biochemical mechanisms associated with aging and stress in mammals. In C. elegans, CGA was shown to improve resistance to thermal, while the underlying mechanisms that lead to this effect require further understanding. PURPOSE: The present study was conducted to investigate the underlying molecular mechanisms behind CGA response conferring thermotolerance to C. elegans. METHODS AND RESULTS: Signaling pathways that could be involved in the CGA-induced thermotolerance were evaluated in C. elegans strains with loss-of-function mutation. CGA-induced thermotolerance required hypoxia-inducible factor HIF-1 but no insulin pathway. CGA exposition (1.4 µM CGA for 18 h) before thermal stress treatment increased HIF-1 levels and activity. HIF-1 activation could be partly attributed to an increase in radical oxygen species and a decrease in superoxide dismutase activity. In addition, CGA exposition before thermal stress also increased autophagy just as hormetic heat condition (HHC), worms incubated at 36 °C for 1 h. RNAi experiments evidenced that autophagy was increased by CGA via HIF-1, heat-shock transcription factor HSF-1 and heat-shock protein HSP-16 and HSP-70. In contrast, autophagy induced by HHC only required HSF-1 and HSP-70. Moreover, suppression of autophagy induction showed the significance of this process for adapting C. elegans to cope with thermal stress. CONCLUSION: This study demonstrates that CGA-induced thermotolerance in C. elegans is mediated by HIF-1 and downstream, by HSF-1, HSPs and autophagy resembling HHC.

Fruit metabolic and transcriptional programs differentiate among Andean tomato (Solanum lycopersicum L.) accessions.

MAIN CONCLUSION: Andean tomatoes differed from the wild ancestor in the metabolic composition and the expression of genes related with mitochondrial functions, and environmental stresses, making them potentially suitable for breeding programmes. Traditional landraces or "criollo" tomatoes (Solanum lycopersicum L.) from Andean areas of Argentina, selected for their fruit quality, were analysed in this study. We explored the metabolome and transcriptome of the ripe fruit in nine landrace accessions representing the seven genetic groups and compared them to the mature fruit of the wild progenitor Solanum pimpinellifolium. The content of branched- (isoleucine and valine) and aromatic (phenylalanine and tryptophan) amino acids, citrate and sugars were significantly different in the fruit of several "criollo" tomatoes compared to S. pimpinellifolium. The transcriptomic profile of the ripe fruit showed several genes significantly and highly regulated in all varieties compared to S. pimpinellifolium, like genes encoding histones and mitochondrial proteins. Additionally, network analysis including transcripts and metabolites identified major hubs with the largest number of connections such as constitutive photomorphogenic protein 1 (a RING finger-type ubiquitin E3 ligase), five Zn finger transcription factors, ascorbate peroxidase, acetolactate synthase, and sucrose non-fermenting 1 kinase. Co-expression analysis of these genes revealed a potential function in acquiring tomato fruit quality during domestication.

Metabolic responses to arbuscular mycorrhizal fungi are shifted in roots of contrasting soybean genotypes.

Modern breeding programs have reduced genetic variability and might have caused a reduction in plant colonization by arbuscular mycorrhizal fungi (AM). In our previous studies, mycorrhizal colonization was affected in improved soybean genotypes, mainly arbuscule formation. Despite substantial knowledge of the symbiosis-related changes of the transcriptome and proteome, only sparse clues regarding metabolite alterations are available. Here, we evaluated metabolite changes between improved (I-1) and unimproved (UI-4) soybean genotypes and also compare their metabolic responses after AM root colonization. Soybean genotypes inoculated or not with AM were grown in a chamber under controlled light and temperature conditions. At 20 days after inoculation, we evaluated soluble metabolites of each genotype and treatment measured by GC-MS. In this analysis, when comparing non-AM roots between genotypes, I-1 had a lower amount of 31 and higher amount of only 4 metabolites than the UI-4 genotype. When comparing AM roots, I-1 had a lower amount of 36 and higher amount of 4 metabolites than UI-4 (different to those found altered in non-AM treated plants). Lastly, comparing the AM vs non-AM treatments, I-1 had increased levels of three and reduced levels of 24 metabolites, while UI-4 only had levels of 12 metabolites reduced by the effect of mycorrhizas. We found the major changes in sugars, polyols, amino acids, and carboxylic acids. In a targeted analysis, we found lower levels of isoflavonoids and alpha-tocopherol and higher levels of malondialdehyde in the I-1 genotype that can affect soybean-AM symbiosis. Our studies have the potential to support improving soybean with a greater capacity to be colonized and responsive to AM interaction.

Identification and characterization of metabolite quantitative trait loci in tomato leaves and comparison with those reported for fruits and seeds.

INTRODUCTION: To date, most studies of natural variation and metabolite quantitative trait loci (mQTL) in tomato have focused on fruit metabolism, leaving aside the identification of genomic regions involved in the regulation of leaf metabolism. OBJECTIVE: This study was conducted to identify leaf mQTL in tomato and to assess the association of leaf metabolites and physiological traits with the metabolite levels from other tissues. METHODS: The analysis of components of leaf metabolism was performed by phenotypying 76 tomato ILs with chromosome segments of the wild species Solanum pennellii in the genetic background of a cultivated tomato (S. lycopersicum) variety M82. The plants were cultivated in two different environments in independent years and samples were harvested from mature leaves of non-flowering plants at the middle of the light period. The non-targeted metabolite profiling was obtained by gas chromatography time-of-flight mass spectrometry (GC-TOF-MS). With the data set obtained in this study and already published metabolomics data from seed and fruit, we performed QTL mapping, heritability and correlation analyses. RESULTS: Changes in metabolite contents were evident in the ILs that are potentially important with respect to stress responses and plant physiology. By analyzing the obtained data, we identified 42 positive and 76 negative mQTL involved in carbon and nitrogen metabolism. CONCLUSIONS: Overall, these findings allowed the identification of S. lycopersicum genome regions involved in the regulation of leaf primary carbon and nitrogen metabolism, as well as the association of leaf metabolites with metabolites from seeds and fruits.

Clustermatch: discovering hidden relations in highly diverse kinds of qualitative and quantitative data without standardization.

MOTIVATION: Heterogeneous and voluminous data sources are common in modern datasets, particularly in systems biology studies. For instance, in multi-holistic approaches in the fruit biology field, data sources can include a mix of measurements such as morpho-agronomic traits, different kinds of molecules (nucleic acids and metabolites) and consumer preferences. These sources not only have different types of data (quantitative and qualitative), but also large amounts of variables with possibly non-linear relationships among them. An integrative analysis is usually hard to conduct, since it requires several manual standardization steps, with a direct and critical impact on the results obtained. These are important issues in clustering applications, which highlight the need of new methods for uncovering complex relationships in such diverse repositories. RESULTS: We designed a new method named Clustermatch to easily and efficiently perform data-mining tasks on large and highly heterogeneous datasets. Our approach can derive a similarity measure between any quantitative or qualitative variables by looking on how they influence on the clustering of the biological materials under study. Comparisons with other methods in both simulated and real datasets show that Clustermatch is better suited for finding meaningful relationships in complex datasets. AVAILABILITY AND IMPLEMENTATION: Files can be downloaded from https://sourceforge.net/projects/sourcesinc/files/clustermatch/ and https://bitbucket.org/sinc-lab/clustermatch/. In addition, a web-demo is available at http://sinc.unl.edu.ar/web-demo/clustermatch/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

A Tomato Tocopherol-Binding Protein Sheds Light on Intracellular α-Tocopherol Metabolism in Plants.

Tocopherols are non-polar compounds synthesized in the plastids, which function as major antioxidants of the plant cells and are essential in the human diet. Both the intermediates and final products of the tocopherol biosynthetic pathway must cross plastid membranes to reach their sites of action. So far, no protein with tocopherol binding activity has been reported in plants. Here, we demonstrated that the tomato SlTBP protein is targeted to chloroplasts and able to bind α-tocopherol. SlTBP-knockdown tomato plants exhibited reduced levels of tocopherol in both leaves and fruits. Several tocopherol deficiency phenotypes were apparent in the transgenic lines, such as alterations in photosynthetic parameters, dramatic distortion of thylakoid membranes and significant variations in the lipid profile. These results, along with the altered expression of genes related to photosynthesis, and tetrapyrrole, lipid, isoprenoid, inositol/phosphoinositide and redox metabolism, suggest that SlTBP may act in conducting tocopherol (or its biosynthetic intermediates) between the plastid compartments and/or at the interface between chloroplast and endoplasmic reticulum membranes, affecting interorganellar lipid metabolism.

Contrasting metabolic profiles of tasty Andean varieties of tomato fruit in comparison with commercial ones.

BACKGROUND: The fruits of most commercial tomato cultivars (Solanum lycopersicum L.) are deficient in flavour. In contrast, traditional 'criollo' tomato varieties are appreciated for fruit of excellent organoleptic quality. Small farmers from the Andean valleys in Argentina have maintained their own tomato varieties, which were selected mainly for flavour. This work aims to correlate the chemical composition of the fruit with the sensory attributes of eight heirloom tomato varieties. The long-term goal is to identify potential candidate genes capable of altering the chemicals involved in flavour. RESULTS: A sensory analysis was conducted and the metabolomics of fruit were determined. The data revealed that defined tomato aroma and sourness correlated with citrate and several volatile organic compounds (VOC), such as α-terpineol, p-menth-1-en-9-al, linalool and 3,6-dimethyl-2,3,3a,4,5,7a-hexahydrobenzofuran (DMHEX), a novel volatile recently identified in tomato. Two sensory attributes - sweetness and a not-acidic taste - correlated with the characteristic tomato taste, and also with fructose, glucose, and two VOCs, benzaldehyde, and 2-methyl-2-octen-4-one. CONCLUSIONS: These data provide new evidence of the complex chemical combination that induced the flavour and aroma of the good-tasting 'criollo' tomato fruit. That is, the compounds that correlated with defined tomato aroma and acidic taste did not correlate with sweetness, or with characteristic tomato taste. © 2018 Society of Chemical Industry.

The genetic architecture of photosynthesis and plant growth-related traits in tomato.

To identify genomic regions involved in the regulation of fundamental physiological processes such as photosynthesis and respiration, a population of Solanum pennellii introgression lines was analyzed. We determined phenotypes for physiological, metabolic, and growth related traits, including gas exchange and chlorophyll fluorescence parameters. Data analysis allowed the identification of 208 physiological and metabolic quantitative trait loci with 33 of these being associated to smaller intervals of the genomic regions, termed BINs. Eight BINs were identified that were associated with higher assimilation rates than the recurrent parent M82. Two and 10 genomic regions were related to shoot and root dry matter accumulation, respectively. Nine genomic regions were associated with starch levels, whereas 12 BINs were associated with the levels of other metabolites. Additionally, a comprehensive and detailed annotation of the genomic regions spanning these quantitative trait loci allowed us to identify 87 candidate genes that putatively control the investigated traits. We confirmed 8 of these at the level of variance in gene expression. Taken together, our results allowed the identification of candidate genes that most likely regulate photosynthesis, primary metabolism, and plant growth and as such provide new avenues for crop improvement.

Neuronal network analyses reveal novel associations between volatile organic compounds and sensory properties of tomato fruits.

INTRODUCTION: The process of tomato (Solanum lycopersicum) breeding has affected negatively the fruit organoleptic properties and this is evident when comparing modern cultivars with heirloom varieties. Flavor of tomato fruit is determined by a complex combination of volatile and nonvolatile metabolites that is not yet understood. OBJECTIVES: The aim of this work was to provide an alternative approach to exploring the relationship between tomato odour/taste and volatile organic compounds (VOCs). METHODS: VOC composition and organoleptic properties of seven Andean tomato landraces along with an edible wild species (Solanum pimpinellifolium) and four commercial varieties were characterized. Six hedonic traits were analyzed by a semitrained sensory panel to describe the organoleptic properties. Ninety-four VOCs were analyzed by headspace solid phase microextraction/gas chromatography-mass spectrometry (HS/SPME/GC-MS). The relationship between sensory data and VOCs was explored using an Artificial Neural Networks model (Kohonen Self Organizing Maps, omeSOM). RESULTS AND CONCLUSION: The results showed a strong preference by panelists for tomatoes of landraces than for commercial varieties and wild species. The predictive analysis by omeSOM showed 15 VOCs significantly associated to the typical and atypical tomato odour and taste. Moreover, omeSOM was used to predict the relationship of VOC ratios with sensory data. A total of 108 VOC ratios out of 8837 VOC ratios were predicted to be contributing to the typical and atypical tomato odour and taste. The metabolic origin of these flavor-associated VOCs and the metabolic point or target for breeding strategies were discussed.

Crop metabolomics: from diagnostics to assisted breeding.

BACKGROUND: Until recently, plant metabolomics have provided a deep understanding on the metabolic regulation in individual plants as experimental units. The application of these techniques to agricultural systems subjected to more complex interactions is a step towards the implementation of translational metabolomics in crop breeding. AIM OF REVIEW: We present here a review paper discussing advances in the knowledge reached in the last years derived from the application of metabolomic techniques that evolved from biomarker discovery to improve crop yield and quality. KEY SCIENTIFIC CONCEPTS OF REVIEW: Translational metabolomics applied to crop breeding programs.