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

Journal of Experimental Botany Metabolomic profiling in tomato reveals diel compositional changes in fruit affected by source-sink relationships Camille Bénard 1 2 Stéphane Bernillon 0 1 Benoît Biais 1 Sonia Osorio 4 5 Mickaël Maucourt 0 3 Patricia Ballias 0 1 Catherine Deborde 0 1 Sophie Colombié 1 Cécile Cabasson 0 3 Daniel Jacob 0 1 Gilles Vercambre 2 Hélène Gautier 2 Dominique Rolin 0 3 Michel Génard 2 Alisdair R. Fernie 5 Yves Gibon 0 1 Annick Moing 0 1 0 Plateforme Métabolome du Centre de Génomique Fonctionnelle Bordeaux , MetaboHUB, IBVM , Centre INRA Bordeaux , 71 av Edouard Bourlaux, 33140 Villenave d'Ornon , France 1 INRA, UMR1332, Biologie du Fruit et Pathologie , 71 av Edouard Bourlaux, 33140 Villenave d'Ornon , France 2 INRA, UR1115 Plantes et Systèmes de culture Horticoles , Domaine St Paul, Site Agroparc, 84914 Avignon , France 3 Univ. Bordeaux, UMR1332, Biologie du Fruit et Pathologie , 71 av Edouard Bourlaux, 33140 Villenave d'Ornon , France 4 Instituto de Hortofruticultura Subtropical y Mediterranea (IHSM), Universidad de Málaga-Consejo Superior de Investigaciones Científicas, Departamento de Biología Molecular y Bioquímica , Málaga , Spain 5 Max-Planck-Institut für Molekulare Pflanzenphysiologie , Am Mühlenberg 1, 14476 Potsdam-Golm , Germany 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. Diurnal changes; fruit metabolism; 1H-NMR; MS; metabolomics; Solanum lycopersicum - Introduction Source-to-sink relationships are central for growth and performance in plants (Lemoine et al., 2013; Ruan et al., 2013), especially fruit crops. After fruit set, fruit growth and development depend largely on the import of metabolites, mineral elements, and water from other organs. Most metabolites are imported from photosynthetic leaves, but fruit photosynthesis also contributes to carbon nutrition, especially in young fruits (Lytovchenko et  al., 2011). The study of source–sink relationships, and especially assimilate transport and partitioning into competing organs, is therefore of special interest in relation to the improvement of fruit yield and quality (Ho, 1996). For fleshy fruits, source–sink relationships have been studied using a range of approaches, such as wholeplant physiology including measurements of biomass allocation and modelling (Heuvelink, 1995), labelling experiments (Minchin et al., 1997), photosynthetic rates and carbohydrate levels (Blanke, 2009), measurements of enzyme activities in fruit (Wang et  al., 1993), genetics (Yelle et  al., 1991), ecophysiological modelling (Liu et al., 2007), and, more recently, transcriptomics (Pastore et  al., 2011). Moreover, systems biology is an emerging approach for source and sink studies. In source–sink studies, the source and sink balance is often modified by changing alternatively the source or the sink. The photosynthesizing source providing carbon can be modified using for instance changes in light intensity or duration (Vasseur et  al., 2011) or leaf thinning (Arnold et  al., 2004), and/or the fruit sink utilizing carbon can be modified through fruit thinning (Do et al., 2010). Irrespective of the experimental design and approach chosen for their study, source–sink relationships depend on the diurnal behaviour of source leaves, i.e. exporting leaves. Diurnal changes in the biochemical composition of mature leaves have been investigated intensively, for example in Arabidopsis (Gibon et  al., 2006) and potato (UrbanczykWochniak et al., 2005). Synchronization of leaf metabolism with diel environmental changes contributes to the regulation of plant growth and increases in plant fitness (Harmer, 2009). During the day, photosynthesis in mature leaves fuels carbohydrate synthesis and sucrose export to the growing vegetative or reproductive sink organs. At night, remobilization of starch stored during the day contributes to maintain sucrose export. Diel changes also occur for nitrogen metabolism including nitrate assimilation in leaves (Scheible et  al., 2000). While the growth rate of fleshy fruit has been shown to vary diurnally in tomato (Guichard et al., 2005), in relation to water potential variations, very few studies describe diel compositional changes in fruits. A work on apple fruit during the growing phase revealed no changes in sugar content (Klages et al., 2001). An earlier work on tomato fruit during the expansion phase showed no significant changes in hexose and malate content (Pearce et al., 1992). Nowadays, leaf and fruit compositional changes can be described in detail using metabolomics combining several analytical strategies (Hall, 2011). Gas chromatography coupled with mass spectrometry (GC-MS) and proton nuclear magnetic resonance spectroscopy (1H-NMR) of polar 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 (...truncated)