Abstract
Organic acid content is regarded as one of the most important quality traits of fresh tomato (Solanum lycopersicum). However, the complexity of carboxylic acid metabolism and storage means that it is difficult to predict the best way to engineer altered carboxylic acid levels. Here, we used a biochemical analysis of a tomato introgression line with increased levels of fruit citrate and malate at breaker stage to identify a metabolic engineering target that was subsequently tested in transgenic plants. Increased carboxylic acid levels in introgression line 2-5 were not accompanied by changes in the pattern of carbohydrate oxidation by pericarp discs or the catalytic capacity of tricarboxylic acid cycle enzymes measured in isolated mitochondria. However, there was a significant decrease in the maximum catalytic activity of aconitase in total tissue extracts, suggesting that a cytosolic isoform of aconitase was affected. To test the role of cytosolic aconitase in controlling fruit citrate levels, we analyzed fruit of transgenic lines expressing an antisense construct against SlAco3b, one of the two tomato genes encoding aconitase. A green fluorescent protein fusion of SlAco3b was dual targeted to cytosol and mitochondria, while the other aconitase, SlAco3a, was exclusively mitochondrial when transiently expressed in tobacco (Nicotiana tabacum) leaves. Both aconitase transcripts were decreased in fruit from transgenic lines, and aconitase activity was reduced by about 30% in the transgenic lines. Other measured enzymes of carboxylic acid metabolism were not significantly altered. Both citrate and malate levels were increased in ripe fruit of the transgenic plants, and as a consequence, total carboxylic acid content was increased by 50% at maturity.
Highlights
Organic acid content is regarded as one of the most important quality traits of fresh tomato (Solanum lycopersicum)
We focused on IL2-5, in which fruit citrate and malate contents were increased (Fig. 1)
In comparison with the tomato ‘M82’ parent line, citrate was significantly increased at each of the developmental ages, with the difference between IL2-5 and cv M82 increasing during development such that ripe fruit of IL2-5 contained 60% more citrate than those of cv M82
Summary
Organic acid content is regarded as one of the most important quality traits of fresh tomato (Solanum lycopersicum). Accumulation in the vacuole is a function of both the influx and efflux of citrate from the vacuole (Shimada et al, 2006) and subsequent metabolism by cytosolic isoforms of aconitase and isocitrate dehydrogenase Given this complexity and the variety of flux modes within carboxylic acid metabolism (Sweetlove et al, 2010), it is not obvious what the best strategy for engineering increased accumulation of carboxylic acids in fleshy fruits would be. This is reflected in the range of enzymes that have been proposed to control fruit citrate accumulation, including phosphoenolpyruvate carboxylase (Guillet et al, 2002), phosphoenolpyruvate carboxykinase (Famiani et al, 2005), citrate synthase (Sadka et al, 2000a), and aconitase (Sadka et al, 2000b; Degu et al, 2011)
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