Maize metabolome and proteome responses to controlled cold stress partly mimic early-sowing effects in the field and differ from those of Arabidopsis.

In Northern Europe, sowing maize one-month earlier than current agricultural practices may lead to moderate chilling damage. However, studies of the metabolic responses to low, non-freezing, temperatures remain scarce. Here, genetically-diverse maize hybrids (Zea mays, dent inbred lines crossed with a flint inbred line) were cultivated in a growth chamber at optimal temperature and then three decreasing temperatures for 2 days each, as well as in the field. Leaf metabolomic and proteomic profiles were determined. In the growth chamber, 50% of metabolites and 18% of proteins changed between 20 and 16°C. These maize responses, partly differing from those of Arabidopsis to short-term chilling, were mapped on genome-wide metabolic maps. Several metabolites and proteins showed similar variation for all temperature decreases: seven MS-based metabolite signatures and two proteins involved in photosynthesis decreased continuously. Several increasing metabolites or proteins in the growth-chamber chilling conditions showed similar trends in the early-sowing field experiment, including trans-aconitate, three hydroxycinnamate derivatives, a benzoxazinoid, a sucrose synthase, lethal leaf-spot 1 protein, an allene oxide synthase, several glutathione transferases and peroxidases. Hybrid groups based on field biomass were used to search for the metabolite or protein responses differentiating them in growth-chamber conditions, which could be of interest for breeding.

Impacts of Paraburkholderia phytofirmans Strain PsJN on Tomato (Lycopersicon esculentum L.) Under High Temperature.

Abnormal temperatures induce physiological and biochemical changes resulting in the loss of yield. The present study investigates the impact of the PsJN strain of Paraburkholderia phytofirmans on tomato (Lycopersicon esculentum Mill.) in response to heat stress (32°C). The results of this work showed that bacterial inoculation with P. phytofirmans strain PsJN increased tomato growth parameters such as chlorophyll content and gas exchange at both normal and high temperatures (25 and 32°C). At normal temperature (25°C), the rate of photosynthesis and the photosystem II activity increased with significant accumulations of sugars, total amino acids, proline, and malate in the bacterized tomato plants, demonstrating that the PsJN strain had a positive effect on plant growth. However, the amount of sucrose, total amino acids, proline, and malate were significantly affected in tomato leaves at 32°C compared to that at 25°C. Changes in photosynthesis and chlorophyll fluorescence showed that the bacterized tomato plants were well acclimated at 32°C. These results reinforce the current knowledge about the PsJN strain of P. phytofirmans and highlight in particular its ability to alleviate the harmful effects of high temperatures by stimulating the growth and tolerance of tomato plants.

Metabotyping of 30 maize hybrids under early-sowing conditions reveals potential marker-metabolites for breeding.

INTRODUCTION: In Northern Europe, maize early-sowing used to maximize yield may lead to moderate damages of seedlings due to chilling without visual phenotypes. Genetic studies and breeding for chilling tolerance remain necessary, and metabolic markers would be particularly useful in this context. OBJECTIVES: Using an untargeted metabolomic approach on a collection of maize hybrids, our aim was to identify metabolite signatures and/or metabolites associated with chilling responses at the vegetative stage, to search for metabolites differentiating groups of hybrids based on silage-earliness, and to search for marker-metabolites correlated with aerial biomass. METHODS: Thirty genetically-diverse maize dent inbred-lines (Zea mays) crossed to a flint inbred-line were sown in a field to assess metabolite profiles upon cold treatment induced by a modification of sowing date, and characterized with climatic measurements and phenotyping. RESULTS: NMR- and LC-MS-based metabolomic profiling revealed the biological variation of primary and specialized metabolites in young leaves of plants before flowering-stage. The effect of early-sowing on leaf composition was larger than that of genotype, and several metabolites were associated to sowing response. The metabolic distances between genotypes based on leaf compositional data were not related to the genotype admixture groups, and their variability was lower under early-sowing than normal-sowing. Several metabolites or metabolite-features were related to silage-earliness groups in the normal-sowing condition, some of which were confirmed the following year. Correlation networks involving metabolites and aerial biomass suggested marker-metabolites for breeding for chilling tolerance. CONCLUSION: After validation in other experiments and larger genotype panels, these marker-metabolites can contribute to breeding.

Culture of the Tomato Micro-Tom Cultivar in Greenhouse.

Micro-Tom tomato cultivar is particularly adapted to the development of genomic approaches in tomato. Here, we describe the culture of this plant in greenhouse, including climate regulation, seed sowing and watering, vegetative development, plant maintenance, including treatment of phytosanitary problems, and reproductive development.

Metabolomic profiling in tomato reveals diel compositional changes in fruit affected by source-sink relationships.

A detailed study of the diurnal compositional changes was performed in tomato (Solanum lycopersicum cv. Moneymaker) leaves and fruits. Plants were cultivated in a commercial greenhouse under two growth conditions: control and shaded. Expanding fruits and the closest mature leaves were harvested during two different day/night cycles (cloudy or sunny day). High-throughput robotized biochemical phenotyping of major compounds, as well as proton nuclear magnetic resonance and mass spectrometry metabolomic profiling, were used to measure the contents of about 70 metabolites in the leaves and 60 metabolites in the fruits, in parallel with ecophysiological measurements. Metabolite data were processed using multivariate, univariate, or clustering analyses and correlation networks. The shaded carbon-limited plants adjusted their leaf area, decreased their sink carbon demand and showed subtle compositional modifications. For source leaves, several metabolites varied along a diel cycle, including those directly linked to photosynthesis and photorespiration. These metabolites peaked at midday in both conditions and diel cycles as expected. However, transitory carbon storage was limited in tomato leaves. In fruits, fewer metabolites showed diel fluctuations, which were also of lower amplitude. Several organic acids were among the fluctuating metabolites. Diel patterns observed in leaves and especially in fruits differed between the cloudy and sunny days, and between the two conditions. Relationships between compositional changes in leaves and fruits are in agreement with the fact that several metabolic processes of the fruit appeared linked to its momentary supply of sucrose.

Remarkable reproducibility of enzyme activity profiles in tomato fruits grown under contrasting environments provides a roadmap for studies of fruit metabolism.

To assess the influence of the environment on fruit metabolism, tomato (Solanum lycopersicum 'Moneymaker') plants were grown under contrasting conditions (optimal for commercial, water limited, or shaded production) and locations. Samples were harvested at nine stages of development, and 36 enzyme activities of central metabolism were measured as well as protein, starch, and major metabolites, such as hexoses, sucrose, organic acids, and amino acids. The most remarkable result was the high reproducibility of enzyme activities throughout development, irrespective of conditions or location. Hierarchical clustering of enzyme activities also revealed tight relationships between metabolic pathways and phases of development. Thus, cell division was characterized by high activities of fructokinase, glucokinase, pyruvate kinase, and tricarboxylic acid cycle enzymes, indicating ATP production as a priority, whereas cell expansion was characterized by enzymes involved in the lower part of glycolysis, suggesting a metabolic reprogramming to anaplerosis. As expected, enzymes involved in the accumulation of sugars, citrate, and glutamate were strongly increased during ripening. However, a group of enzymes involved in ATP production, which is probably fueled by starch degradation, was also increased. Metabolites levels seemed more sensitive than enzymes to the environment, although such differences tended to decrease at ripening. The integration of enzyme and metabolite data obtained under contrasting growth conditions using principal component analysis suggests that, with the exceptions of alanine amino transferase and glutamate and malate dehydrogenase and malate, there are no links between single enzyme activities and metabolite time courses or levels.

Analysis of enzyme activities.

The evaluation of enzyme activities, especially their capacities, represents an important step towards the modelling of biochemical pathways in living organisms. The implementation of microplate technology enables the determination of up to >50 enzymes in relatively large numbers of samples and in various biological materials. Most of these enzymes are involved in central metabolism and several pathways are entirely covered. Direct or indirect assays can be used, as well as highly sensitive assays, depending on the abundance of the enzymes under study. To exemplify such methods, protocols for UDP-glucose pyrophosphorylase (E.C. 2.7.7.9) operating in real time and for pyrophosphate:fructose-6-phosphate 1-phosphotransferase (E.C. 2.7.1.90) are presented.

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.