Abstract
L-Galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) catalyzes the last step in the main pathway of vitamin C (L-ascorbic acid) biosynthesis in higher plants. In this study, we first characterized the spatial and temporal expression of SlGalLDH in several organs of tomato (Solanum lycopersicum) plants in parallel with the ascorbate content. P(35S):Slgalldh(RNAi) silenced transgenic tomato lines were then generated using an RNAi strategy to evaluate the effect of any resulting modification of the ascorbate pool on plant and fruit development. In all P(35S):Slgalldh(RNAi) plants with reduced SlGalLDH transcript and activity, plant growth rate was decreased. Plants displaying the most severe effects (dwarf plants with no fruit) were excluded from further analysis. The most affected lines studied exhibited up to an 80% reduction in SlGalLDH activity and showed a strong reduction in leaf and fruit size, mainly as a consequence of reduced cell expansion. This was accompanied by significant changes in mitochondrial function and altered ascorbate redox state despite the fact that the total ascorbate content remained unchanged. By using a combination of transcriptomic and metabolomic approaches, we further demonstrated that several primary, like the tricarboxylic acid cycle, as well as secondary metabolic pathways related to stress response were modified in leaves and fruit of P(35S):Slgalldh(RNAi) plants. When taken together, this work confirms the complexity of ascorbate regulation and its link with plant metabolism. Moreover, it strongly suggests that, in addition to ascorbate synthesis, GalLDH could play an important role in the regulation of cell growth-related processes in plants.
Highlights
L-Galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) catalyzes the last step in the main pathway of vitamin C (L-ascorbic acid) biosynthesis in higher plants
By using a combination of transcriptomic and metabolomic approaches, we further demonstrated that several primary, like the tricarboxylic acid cycle, as well as secondary metabolic pathways related to stress response were modified in leaves and fruit of P35S:SlgalldhRNAi plants
Since L-GalLDH catalyzes the terminal step of the major pathway of ascorbate biosynthesis in plants (Wheeler et al, 1998), this enzyme is a good candidate for controlling the variations in ascorbate content in plants (Tamaoki et al, 2003; Bartoli et al, 2005; Tokunaga et al, 2005)
Summary
L-Galactono-1,4-lactone dehydrogenase (EC 1.3.2.3) catalyzes the last step in the main pathway of vitamin C (L-ascorbic acid) biosynthesis in higher plants. The most affected lines studied exhibited up to an 80% reduction in SlGalLDH activity and showed a strong reduction in leaf and fruit size, mainly as a consequence of reduced cell expansion This was accompanied by significant changes in mitochondrial function and altered ascorbate redox state despite the fact that the total ascorbate content remained unchanged. Agius et al (2003) showed that during the ripening phase of strawberry (Fragaria spp.) fruit, ascorbate is produced via the reduction of D-GalUA to L-galactonic acid, which in turn is spontaneously converted to L-galactono-lactone, the immediate precursor of ascorbate This pathway shares the requirement of L-GalLDH activity as the terminal step in ascorbate synthesis. Transcriptome and metabolome analyses of P35S:SlgalldhRNAi transgenic lines further suggest that phenotypic changes can be attributed to the resulting modifications in secondary and primary metabolism
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