Metabolic Profiling of Transgenic Tomato Plants Overexpressing Hexokinase Reveals That the Influence of Hexose Phosphorylation Diminishes during Fruit Development

Abstract

We have conducted a comprehensive metabolic profiling on tomato (Lycopersicon esculentum) leaf and developing fruit tissue using a recently established gas chromatography-mass spectrometry profiling protocol alongside conventional spectrophotometric and liquid chromatographic methodologies. Applying a combination of these techniques, we were able to identify in excess of 70 small-M r metabolites and to catalogue the metabolite composition of developing tomato fruit. In addition to comparing differences in metabolite content between source and sink tissues of the tomato plant and after the change in metabolite pool sizes through fruit development, we have assessed the influence of hexose phosphorylation through fruit development by analyzing transgenic plants constitutively overexpressing Arabidopsis hexokinase AtHXK1. Analysis of the total hexokinase activity in developing fruits revealed that both wild-type and transgenic fruits exhibit decreasing hexokinase activity with development but that the relative activity of the transgenic lines with respect to wild type increases with development. Conversely, both point-by-point and principal component analyses suggest that the metabolic phenotype of these lines becomes less distinct from wild type during development. In summary, the data presented in this paper demonstrate that the influence of hexose phosphorylation diminishes during fruit development and highlights the importance of greater temporal resolution of metabolism.Hexokinase (E.C. 2.7.1.1) catalyzes the phosphorylation of hexoses to form hexose monophosphates. This reaction is especially important in plants because the use of free phosphates is particularly complex in higher plants ( Kruger, 1997). There have been many reports on the presence of glucokinase and hexokinase enzymes in a wide variety of plant species including tomato (Lycopersicon esculentum; Martinez-Barajaz and Randall, 1998), maize (Zea mays; Doehlert, 1989;Schnarrenberger, 1990; Galina et al., 1995), potato (Solanum tuberosum; Renz and Stitt, 1993;Veramendi et al., 1999), pea (Pisum sativum; Turner et al., 1977;Turner and Copeland, 1981) Recently, transgenic manipulations of the activity of hexokinase have been carried out in tomato, potato, and Arabidopsis (Jang et al., 1997; Dai et al., 1999;Veramendi et al., 1999Veramendi et al., , 2002. The results of these manipulations varied greatly between species. Transgenic Arabidopsis seeds that exhibited decreased or increased activities of hexokinase 1 displayed hyposensitive or hypersensitive responses to growth on high (6% [w/v]) Glc containing agar (Jang et al., 1997). The authors concluded that the hexokinase protein acts as a sensor for Glc in an analogous manner to those operating in yeast (Saccharomyces cerevisiae) and that the modulation in the abundance of this sensor led to changes in gene expression that were responsible for the phenotype observed. The overexpression of this Arabidopsis hexokinase isoform in tomato plants led to growth inhibition, reduced photosynthesis, and a...

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