Comparative constraint-based modelling of fruit development across species highlights nitrogen metabolism in the growth-defence trade-off.

Although primary metabolism is well conserved across species, it is useful to explore the specificity of its network to assess the extent to which some pathways may contribute to particular outcomes. Constraint-based metabolic modelling is an established framework for predicting metabolic fluxes and phenotypes and helps to explore how the plant metabolic network delivers specific outcomes from temporal series. After describing the main physiological traits during fruit development, we confirmed the correlations between fruit relative growth rate (RGR), protein content and time to maturity. Then a constraint-based method is applied to a panel of eight fruit species with a knowledge-based metabolic model of heterotrophic cells describing a generic metabolic network of primary metabolism. The metabolic fluxes are estimated by constraining the model using a large set of metabolites and compounds quantified throughout fruit development. Multivariate analyses showed a clear common pattern of flux distribution during fruit development with differences between fast- and slow-growing fruits. Only the latter fruits mobilise the tricarboxylic acid cycle in addition to glycolysis, leading to a higher rate of respiration. More surprisingly, to balance nitrogen, the model suggests, on the one hand, nitrogen uptake by nitrate reductase to support a high RGR at early stages of cucumber and, on the other hand, the accumulation of alkaloids during ripening of pepper and eggplant. Finally, building virtual fruits by combining 12 biomass compounds shows that the growth-defence trade-off is supported mainly by cell wall synthesis for fast-growing fruits and by total polyphenols accumulation for slow-growing fruits.

The Tomato Guanylate-Binding Protein SlGBP1 Enables Fruit Tissue Differentiation by Maintaining Endopolyploid Cells in a Non-Proliferative State.

Cell fate maintenance is an integral part of plant cell differentiation and the production of functional cells, tissues, and organs. Fleshy fruit development is characterized by the accumulation of water and solutes in the enlarging cells of parenchymatous tissues. In tomato (Solanum lycopersicum), this process is associated with endoreduplication in mesocarp cells. The mechanisms that preserve this developmental program, once initiated, remain unknown. We show here that analysis of a previously identified tomato ethyl methanesulfonate-induced mutant that exhibits abnormal mesocarp cell differentiation could help elucidate determinants of fruit cell fate maintenance. We identified and validated the causal locus through mapping-by-sequencing and gene editing, respectively, and performed metabolic, cellular, and transcriptomic analyses of the mutant phenotype. The data indicate that disruption of the SlGBP1 gene, encoding GUANYLATE BINDING PROTEIN1, induces early termination of endoreduplication followed by late divisions of polyploid mesocarp cells, which consequently acquire the characteristics of young proliferative cells. This study reveals a crucial role of plant GBPs in the control of cell cycle genes, and thus, in cell fate maintenance. We propose that SlGBP1 acts as an inhibitor of cell division, a function conserved with the human hGBP-1 protein.

Respiration climacteric in tomato fruits elucidated by constraint-based modelling.

Tomato is a model organism to study the development of fleshy fruit including ripening initiation. Unfortunately, few studies deal with the brief phase of accelerated ripening associated with the respiration climacteric because of practical problems involved in measuring fruit respiration. Because constraint-based modelling allows predicting accurate metabolic fluxes, we investigated the respiration and energy dissipation of fruit pericarp at the breaker stage using a detailed stoichiometric model of the respiratory pathway, including alternative oxidase and uncoupling proteins. Assuming steady-state, a metabolic dataset was transformed into constraints to solve the model on a daily basis throughout tomato fruit development. We detected a peak of CO2 released and an excess of energy dissipated at 40 d post anthesis (DPA) just before the onset of ripening coinciding with the respiration climacteric. We demonstrated the unbalanced carbon allocation with the sharp slowdown of accumulation (for syntheses and storage) and the beginning of the degradation of starch and cell wall polysaccharides. Experiments with fruits harvested from plants cultivated under stress conditions confirmed the concept. We conclude that modelling with an accurate metabolic dataset is an efficient tool to bypass the difficulty of measuring fruit respiration and to elucidate the underlying mechanisms of ripening.

Hyperpolarized NMR of plant and cancer cell extracts at natural abundance.

Natural abundance (13)C NMR spectra of biological extracts are recorded in a single scan provided that the samples are hyperpolarized by dissolution dynamic nuclear polarization combined with cross polarization. Heteronuclear 2D correlation spectra of hyperpolarized breast cancer cell extracts can also be obtained in a single scan. Hyperpolarized NMR of extracts opens many perspectives for metabolomics.

Deciphering genetic diversity and inheritance of tomato fruit weight and composition through a systems biology approach.

Integrative systems biology proposes new approaches to decipher the variation of phenotypic traits. In an effort to link the genetic variation and the physiological and molecular bases of fruit composition, the proteome (424 protein spots), metabolome (26 compounds), enzymatic profile (26 enzymes), and phenotypes of eight tomato accessions, covering the genetic diversity of the species, and four of their F1 hybrids, were characterized at two fruit developmental stages (cell expansion and orange-red). The contents of metabolites varied among the genetic backgrounds, while enzyme profiles were less variable, particularly at the cell expansion stage. Frequent genotype by stage interactions suggested that the trends observed for one accession at a physiological level may change in another accession. In agreement with this, the inheritance modes varied between crosses and stages. Although additivity was predominant, 40% of the traits were non-additively inherited. Relationships among traits revealed associations between different levels of expression and provided information on several key proteins. Notably, the role of frucktokinase, invertase, and cysteine synthase in the variation of metabolites was highlighted. Several stress-related proteins also appeared related to fruit weight differences. These key proteins might be targets for improving metabolite contents of the fruit. This systems biology approach provides better understanding of networks controlling the genetic variation of tomato fruit composition. In addition, the wide data sets generated provide an ideal framework to develop innovative integrated hypothesis and will be highly valuable for the research community.

(Homo)glutathione deficiency impairs root-knot nematode development in Medicago truncatula.

Root-knot nematodes (RKN) are obligatory plant parasitic worms that establish and maintain an intimate relationship with their host plants. During a compatible interaction, RKN induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells essential for nematode growth and reproduction. These metabolically active feeding cells constitute the exclusive source of nutrients for the nematode. Detailed analysis of glutathione (GSH) and homoglutathione (hGSH) metabolism demonstrated the importance of these compounds for the success of nematode infection in Medicago truncatula. We reported quantification of GSH and hGSH and gene expression analysis showing that (h)GSH metabolism in neoformed gall organs differs from that in uninfected roots. Depletion of (h)GSH content impaired nematode egg mass formation and modified the sex ratio. In addition, gene expression and metabolomic analyses showed a substantial modification of starch and γ-aminobutyrate metabolism and of malate and glucose content in (h)GSH-depleted galls. Interestingly, these modifications did not occur in (h)GSH-depleted roots. These various results suggest that (h)GSH have a key role in the regulation of giant cell metabolism. The discovery of these specific plant regulatory elements could lead to the development of new pest management strategies against nematodes.

Effects of long-term cadmium exposure on growth and metabolomic profile of tomato plants.

The response of tomato plants to long-term cadmium exposure was evaluated after a 90-days long culture in hydroponic conditions (0, 20, and 100 µM CdCl(2)). Cadmium preferentially accumulated in roots, and to a lower extent in upper parts of plants. Absolute quantification of 28 metabolites was obtained through (1)H NMR, HPLC-PDA, and colorimetric methods. The principal component analysis showed a clear separation between control and Cd treated samples. Proline and total ascorbate amounts were reduced in Cd-treated leaves, whereas α-tocopherol, asparagine, and tyrosine accumulation increased, principally in 100 µM Cd treated leaves. Carotenoid and chlorophyll contents decreased only in 100 µM Cd-mature-leaves, which correlate with a reduced expression of genes essential for isoprenoid and carotenoid accumulations. Our results show that tomato plants acclimatize during long-term exposure to 20 µM Cd. On the contrary, 100µM Cd treatment results in drastic physiological and metabolic perturbations leading to plant growth limitation and fruit set abortion.

Metabolic acclimation to hypoxia revealed by metabolite gradients in melon fruit.

A metabolomics approach using (1)H NMR and GC-MS profiling of primary metabolites and quantification of adenine nucleotides with luciferin bioluminescence was employed to investigate the spatial changes of metabolism in melon fruit. Direct (1)H NMR profiling of juice collected from different locations in the fruit flesh revealed several gradients of metabolites, e.g. sucrose, alanine, valine, GABA or ethanol, with increase in concentrations from the periphery to the center of the fruit. GC-MS profiling of ground samples revealed gradients for metabolites not detected using (1)H NMR, including pyruvic and fumaric acids. The quantification of adenine nucleotides highlighted a strong decrease in both ATP and ADP ratios and the adenylate energy charge from the periphery to the center of the fruit. These concentration patterns are consistent with an increase in ethanol fermentation due to oxygen limitation and were confirmed by observed changes in alanine and GABA concentrations, as well as other markers of hypoxia in plants. Ethanol content in melon fruit can affect organoleptic properties and consumer acceptance. Understanding how and when fermentation occurred can help to manage the culture and limit ethanol production.

Transcriptional and metabolic adjustments in ADP-glucose pyrophosphorylase-deficient bt2 maize kernels.

During the cloning of monogenic recessive mutations responsible for a defective kernel phenotype in a Mutator-induced Zea mays mutant collection, we isolated a new mutant allele in Brittle2 (Bt2), which codes for the small subunit of ADP-glucose pyrophosphorylase (AGPase), a key enzyme in starch synthesis. Reverse transcription-polymerase chain reaction experiments with gene-specific primers confirmed a predominant expression of Bt2 in endosperm, of Agpsemzm in embryo, and of Agpslzm in leaf, but also revealed considerable additional expression in various tissues for all three genes. Bt2a, the classical transcript coding for a cytoplasmic isoform, was almost exclusively expressed in the developing endosperm, whereas Bt2b, an alternative transcript coding for a plastidial isoform, was expressed in almost all tissues tested with a pattern very similar to that of Agpslzm. The phenotypic analysis showed that, at 30 d after pollination (DAP), mutant kernels were plumper than wild-type kernels, that the onset of kernel collapse took place between 31 and 35 DAP, and that the number of starch grains was greatly reduced in the mutant endosperm but not the mutant embryo. A comparative transcriptome analysis of wild-type and bt2-H2328 kernels at middevelopment (35 DAP) with the 18K GeneChip Maize Genome Array led to the conclusion that the lack of Bt2-encoded AGPase triggers large-scale changes on the transcriptional level that concern mainly genes involved in carbohydrate or amino acid metabolic pathways. Principal component analysis of (1)H nuclear magnetic resonance metabolic profiles confirmed the impact of the bt2-H2328 mutation on these pathways and revealed that the bt2-H2328 mutation did not only affect the endosperm, but also the embryo at the metabolic level. These data suggest that, in the bt2-H2328 endosperms, regulatory networks are activated that redirect excess carbon into alternative biosynthetic pathways (amino acid synthesis) or into other tissues (embryo).

Sugar import and phytopathogenicity of Spiroplasma citri: glucose and fructose play distinct roles.

We have shown previously that the glucose PTS (phosphotransferase system) permease enzyme II of Spiroplasma citri is split into two distinct polypeptides, which are encoded by two separate genes, crr and ptsG. A S. citri mutant was obtained by disruption of ptsG through homologous recombination and was proved unable to import glucose. The ptsG mutant (GII3-glc1) was transmitted to periwinkle (Catharanthus roseus) plants through injection to the leaf-hopper vector. In contrast to the previously characterized fructose operon mutant GMT 553, which was found virtually nonpathogenic, the ptsG mutant GII3-glc1 induced severe symptoms similar to those induced by the wild-type strain GII-3. These results, indicating that fructose and glucose utilization were not equally involved in pathogenicity, were consistent with biochemical data showing that, in the presence of both sugars, S. citri used fructose preferentially. Proton nuclear magnetic resonance analyses of carbohydrates in plant extracts revealed the accumulation of soluble sugars, particularly glucose, in plants infected by S. citri GII-3 or GII3-glc1 but not in those infected by GMT 553. From these data, a hypothetical model was proposed to establish the relationship between fructose utilization by the spiroplasmas present in the phloem sieve tubes and glucose accumulation in the leaves of S. citri infected plants.