The Evolution of Leaf Function during Development Is Reflected in Profound Changes in the Metabolic Composition of the Vacuole.

During its development, the leaf undergoes profound metabolic changes to ensure, among other things, its growth. The subcellular metabolome of tomato leaves was studied at four stages of leaf development, with a particular emphasis on the composition of the vacuole, a major actor of cell growth. For this, leaves were collected at different positions of the plant, corresponding to different developmental stages. Coupling cytology approaches to non-aqueous cell fractionation allowed to estimate the subcellular concentrations of major compounds in the leaves. The results showed major changes in the composition of the vacuole across leaf development. Thus, sucrose underwent a strong allocation, being mostly located in the vacuole at the beginning of development and in the cytosol at maturity. Furthermore, these analyses revealed that the vacuole, rather rich in secondary metabolites and sugars in the growth phases, accumulated organic acids thereafter. This result suggests that the maintenance of the osmolarity of the vacuole of mature leaves would largely involve inorganic molecules.

Fruit setting rewires central metabolism via gibberellin cascades.

Fruit set is the process whereby ovaries develop into fruits after pollination and fertilization. The process is induced by the phytohormone gibberellin (GA) in tomatoes, as determined by the constitutive GA response mutant procera However, the role of GA on the metabolic behavior in fruit-setting ovaries remains largely unknown. This study explored the biochemical mechanisms of fruit set using a network analysis of integrated transcriptome, proteome, metabolome, and enzyme activity data. Our results revealed that fruit set involves the activation of central carbon metabolism, with increased hexoses, hexose phosphates, and downstream metabolites, including intermediates and derivatives of glycolysis, the tricarboxylic acid cycle, and associated organic and amino acids. The network analysis also identified the transcriptional hub gene SlHB15A, that coordinated metabolic activation. Furthermore, a kinetic model of sucrose metabolism predicted that the sucrose cycle had high activity levels in unpollinated ovaries, whereas it was shut down when sugars rapidly accumulated in vacuoles in fruit-setting ovaries, in a time-dependent manner via tonoplastic sugar carriers. Moreover, fruit set at least partly required the activity of fructokinase, which may pull fructose out of the vacuole, and this could feed the downstream pathways. Collectively, our results indicate that GA cascades enhance sink capacities, by up-regulating central metabolic enzyme capacities at both transcriptional and posttranscriptional levels. This leads to increased sucrose uptake and carbon fluxes for the production of the constituents of biomass and energy that are essential for rapid ovary growth during the initiation of fruit set.

Model-assisted analysis of sugar metabolism throughout tomato fruit development reveals enzyme and carrier properties in relation to vacuole expansion.

A kinetic model combining enzyme activity measurements and subcellular compartmentation was parameterized to fit the sucrose, hexose, and glucose-6-P contents of pericarp throughout tomato (Solanum lycopersicum) fruit development. The model was further validated using independent data obtained from domesticated and wild tomato species and on transgenic lines. A hierarchical clustering analysis of the calculated fluxes and enzyme capacities together revealed stage-dependent features. Cell division was characterized by a high sucrolytic activity of the vacuole, whereas sucrose cleavage during expansion was sustained by both sucrose synthase and neutral invertase, associated with minimal futile cycling. Most importantly, a tight correlation between flux rate and enzyme capacity was found for fructokinase and PPi-dependent phosphofructokinase during cell division and for sucrose synthase, UDP-glucopyrophosphorylase, and phosphoglucomutase during expansion, thus suggesting an adaptation of enzyme abundance to metabolic needs. In contrast, for most enzymes, flux rates varied irrespectively of enzyme capacities, and most enzymes functioned at <5% of their maximal catalytic capacity. One of the major findings with the model was the high accumulation of soluble sugars within the vacuole together with organic acids, thus enabling the osmotic-driven vacuole expansion that was found during cell division.

¹4C pulse labeling to estimate external fluxes and turnovers in primary metabolism.

Steady state (13)C-MFA is classically used to measure fluxes in complex metabolic networks. However, the modeling of steady state labeling allows the quantification of internal fluxes only and requires the estimation, by other methods, of the external fluxes, corresponding to substrate uptake (carbon input into the network) and to the production rate of compounds that accumulate within plant cells (network output). Additionally, it is not always possible to discriminate between different pathways that lead to the same label distribution. Methods to measure fluxes, based on direct measurements of pool size and on (14)C short-time labeling experiments, are described in this chapter. To illustrate this approach, we focus on the quantification of sucrose and starch turnovers.

Effect of hexokinase activity on tomato root metabolism during prolonged hypoxia.

Hypoxically induced tolerance to anoxia in roots of tomato (Solanum lycopersicum) was previously shown to depend on sucrose and the induction of sucrose synthase. In contrast to maize, root hexokinase (HXK) activities did not increase during hypoxia and glucose was unable to sustain glycolytic flux under anoxia. In this paper, we asked whether hypoxic metabolism in roots would be altered in transgenic tomato plants overexpressing either a plant (Arabidopsis) or a yeast (Saccharomyces cerevisiae) HXK and whether such modifications could be related to improved energy metabolism and consequently root tolerance under anoxia. Tomato plants grown hydroponically with shoots always maintained in air were submitted to a 7 d hypoxic treatment applied by stopping air bubbling. A combination of techniques including (1)H-nuclear magnetic resonance spectroscopy, RT-PCR and enzyme analyses was used to obtain a broad picture of hypoxic root metabolism. In normoxic conditions, HXK overexpression resulted in higher ADP and AMP levels only in roots of AtHXK1 transgenic plants. During hypoxic treatment, oxygen levels in the hydroponic tank decreased rapidly to 5 kPa within the first 2 d and then remained at 5 kPa throughout the 7 d experiment. Oxygen levels were similar at 5 and 20 cm below the water surface. A decline of the adenylate energy status was observed after 2 d of hypoxic treatment, with a further decrease by 7 d in roots of non-transgenic (WT) and ScHXK2, but not in AtHXK1 transgenic plants. Sucrose synthase activity increased to comparably higher levels at 7 d of hypoxic treatment in WT and ScHXK2 compared with AtHXK1 roots. Differences between WT and the transgenic plants are discussed with respect to the metabolic response to low (hypoxia) but not zero (anoxia) oxygen.