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

Journal of Experimental Botany, Vol. 66, No. 11 pp. 3391–3404, 2015 doi:10.1093/jxb/erv151 Advance Access publication 11 April 2015 This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details) RESEARCH PAPER Metabolomic profiling in tomato reveals diel compositional changes in fruit affected by source–sink relationships Camille Bénard1,2, Stéphane Bernillon2,3, Benoît Biais2, Sonia Osorio4,5, Mickaël Maucourt3,6, Patricia Ballias2,3, Catherine Deborde2,3, Sophie Colombié2, Cécile Cabasson3,6, Daniel Jacob2,3, Gilles Vercambre1, Hélène Gautier1, Dominique Rolin3,6, Michel Génard1, Alisdair R. Fernie4, Yves Gibon2,3 and Annick Moing2,3,* INRA, UR1115 Plantes et Systèmes de culture Horticoles, Domaine St Paul, Site Agroparc, 84914 Avignon, France INRA, UMR1332, Biologie du Fruit et Pathologie, 71 av Edouard Bourlaux, 33140 Villenave d’Ornon, France 3 Plateforme Métabolome du Centre de Génomique Fonctionnelle Bordeaux, MetaboHUB, IBVM, Centre INRA Bordeaux, 71 av Edouard Bourlaux, 33140 Villenave d’Ornon, France 4 Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany 5 Instituto de Hortofruticultura Subtropical y Mediterranea (IHSM), Universidad de Málaga-Consejo Superior de Investigaciones Científicas, Departamento de Biología Molecular y Bioquímica, Málaga, Spain 6 Univ. Bordeaux, UMR1332, Biologie du Fruit et Pathologie, 71 av Edouard Bourlaux, 33140 Villenave d’Ornon, France 2 * To whom correspondence should be addressed. E-mail: Received 22 December 2014; Revised 20 February 2015; Accepted 9 March 2015 Abstract 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. Key words: Diurnal changes, fruit metabolism, 1H-NMR, MS, metabolomics, Solanum lycopersicum. Abbreviations: ANOVA, analysis of variance; CV, coefficient of variation; DPA, days post-anthesis; DW, dry weight; FW, fresh weight; GABA, γ-aminobutyric acid; GC-MS, gas chromatography coupled with mass spectrometry; GGM, Gaussian graphical model; 1H-NMR, proton NMR; LC-MS, liquid chromatography coupled with mass spectrometry; MS, mass spectrometry; PAR, photosynthetically active radiation; PCA, principal component analysis; SLA, specific leaf area; TOF, time of flight. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 1 3392 | Bénard et al. Introduction extracts give access to a range of primary metabolites. Liquid chromatography coupled with mass spectrometry (LC-MS) of semi-polar extracts provides relative quantification of secondary metabolites belonging to several families of compounds including flavonoids, hydroxycinnamates, and glycoalkaloids. Such analytical approaches have largely been used recently for crop species including tomato (de Vos et al., 2011). However, source–sink studies involving metabolomics remain rare for fruit crops. As most works about changes in metabolites in source–sink interactions have been derived from Arabidopsis, it is now meaningful to study crop species of economic importance such as tomato plants that have multiple fruits that serve as strong sinks. In the present work, diurnal compositional changes were measured in greenhouse-grown tomato expanding fruits and the closest mature leaves using a combination of metabolomics approaches based on NMR and MS, and on robotized microplate measurements of starch, proteins, and total free amino acids. Since shading has been shown to affect both yield and fruit quality in tomato (Gent, 2007), we also investigated the leaves and fruits of plants experiencing different light regimes (cloudy vs sunny day, and control vs shading condition). With contrasted carbon availability at the plant level, leaf and fruit diel variations were investigated to study the relationships between the composition of the mature leaves close to the harvested fruit truss and that of the fruit pericarp using metabolite networks. These approaches provided information about possible metabolic regulations in the context of the relationships between source leaf and sink fruit. Materials and methods Plant material and growth conditions Tomato (Solanum lycopersicum L. cv. Moneymaker) plants were grown in a greenhouse in south-west France from June to September according to commercial practices as detailed in Supplementary text at JXB online. Fruit load was set at six fruits per truss when needed. The entire fruit development from anthesis to the red-ripe stage lasted about 55 d. Two conditions were applied: ‘control’ (276 plants), and low-light conditions, referred to as ‘shaded’ [138 plants with a shadow net stopping 60% of incident light, with limited effects on temperature, installed in early July when fruits of truss 3 were at about 6 d post-anthesis (DPA)]. During the plant culture, we focused on two diel cycles hereafter referred to as ‘Experiments’. Experiment 1 (Exp. 1) was performed in late July on an overcast day, with expanding fruits located on truss 3 (23 ± 1 DPA), and Exp. 2 was performed in late A (...truncated)