Genetic and Biochemical Characterization of Fructose Accumulation: A Strategy to Improve Fruit Quality

The goal of the research project was to evaluate the potential to genetically modify or engineer carbohydrate metabolism in tomato fruit to enhance levels of fructose, a sugar with nearly twice the sweetness value of other sugars. The specific research objectives to achieve that goal were to: 1. Establish the inheritance of a fructose-accumulating trait identified in F1 hybrids of an inferspecific cross between L. hirsutum XL. esculentum and identify linked molecular markers to facilitate its introgression into tomato cultivars. This objective was completed with the genetic data indicating a single major gene, termed Fgr (Fructose glucose ratio), that controlled the partitioning of hexose in the mature fruit. Molecular markers for the gene, were developed to aid introgression of this gene into cultivated tomato. In addition, a second major gene encoding fructokinase 2 (FK2) was found to be a determinant of the fructose to glucose ratio in fruit. The relationship between FK2 and Fgr is epistatic with a combined synergistic effect of the two hirsutum-derived genes on fructose/glucose ratios. 2. Characterize the metabolic and transport properties responsible for high fructose/glucose ratios in fructose-accumulating genotypes. The effect of both the Fgr and FK2 genes on the developmental accumulation of hexoses was studied in a wide range of genetic backgrounds. In all backgrounds the trait is a developmental one and that the increase in fructose to glucose ratio occurs at the breaker stage of fruit development. The following enzymes were assayed, none of which showed differences between genotypes, at either the breaker or ripe stage: invertase, sucrose synthase, FK1, FK2, hexokinase, PGI and PGM. The lack of effect of the FK2 gene on fructokinase activity is surprising and at present we have no explanation for the phenomenon. However, the hirsutum derived Fgr allele was associated with significantly lower levels of phosphorylated glucose, G1c-1-P and G1c-6-P and concomitantly higher levels of the phosphorylated fructose, Fru-6-P, in both the breaker and ripe stage. This suggests a significant role for the isomerase reaction. 3. Develop and implement molecular genetic strategies for the production of transgenic plants with altered levels of enzymes that potentially control fructose/glucose ratios in fruit. This objective focused on manipulating hexokinase and fructokinase expression in transgenic plants. Two highly divergent cDNA clones (Frk1 and Frk2), encoding fructokinase (EC 2.7.1.4), were isolated from tomato (Lycopersicon esculentum) and a potato fructokinase cDNA clone was obtained from Dr. Howard Davies. Following expression in yeast, each fructokinase was identified to code for one of the tomato or potato fructokinase isoforms Transgenic tomato plants were generated with the fructokinase cDNA clone in both sense and antisense orientations and the effect of the gene on tomato plants is currently being studied.