supervision of: Dr. Artur A.Schaffer, Prof. Joseph Riov

Starch accumulates transiently in the young tomato fruits and can contribute approximately 20% to the dry weight of the fruit tissue. The starch degradation in the developing tomato fruit leads to the accumulation of soluble sugars in the ripening fruit, which serve as the main determinant of fruit quality. The goal of the present work was the analysis of the sucrose-to-starch metabolic pathways and the molecular control of starch synthesis in developing tomato fruit, in order to shed light on strategies of increasing fruit starch content. In a preliminary study comparing the activities of eleven enzymes of the sucrose to starch metabolic pathway, four enzymes showed a coordinated pattern of developmental loss of activity correlated with the cessation of starch synthesis. Two of these enzymes, fructokinase (FK) and ADP-Glc pyrophosphorylase (AGPase) were suggested to be potentially limiting the flux of starch synthesis in tomato fruit starch accumulation. Accordingly, the focus of the research was directed towards these two enzymatic reactions. Three fructokinase isozymes (FKI, FKII, FKIII), including a novel FKIII, and two hexokinase isozymes (HK1, HK2) from developing tomato fruit were chromatographically separated, partially purified and kinetically characterized. The three FK isozymes could be distinguished from one another with respect to affinity to fructose, Mg+2 and nucleosides. However, results from genetically transformed tomato plants with modified FK activities indicated that this enzymatic reaction is not directly limiting starch synthesis in tomato fruit. AGPase catalyzes the synthesis of ADP-Glc and functions as a limiting enzyme in starch synthesis. The enzyme functions as a heterotetramer consisting of two large subunits (encoded by three AgpL genes, AgpL1, AgpL2 and AgpL3) and two small subunits (encoded by a single AgpS1 gene). A set of near isogenic tomato plants was developed differing in the origin of the AgpL1 allele, derived from an interspecific cross between the cultivated S. lycopersicum L. (formerly L. esculentum Mill., source of the AgpL1E allele) and wild species tomato S. habrochaites S. Knapp and D.M. Spooner (formerly L. hirsutum Humb. and Bonpl., source of the AgpL1H allele). The fruit carrying the AgpL1H allele is characterized by increased AGPase activity and increased immature fruit starch content, as well as higher total soluble solids (TSS) in the mature fruit, following by the breakdown of the transient starch. The introgression harboring the AgpL1 locus mapped to the distal portion of chromosome 1, was delimited to ca. 1 cM and does not include other previously reported QTLs for TSS. In order to determine the cause of the increased enzyme activity due to the AgpL1H allele, we compared the expression of all subunits (L1, L2, L3 and S1) in developing tomato fruit in the two near isogenic lines. The AgpL1H allele in the high starch line was expressed for a prolonged period of fruit development, compared to the AgpL1E allele in the normal starch line. This correlates with increases in AGPase activity, AGPase subunit protein level and starch accumulation patterns during fruit development of the high starch line. The results suggest that the increased activity of AGPase in the AgpL1H tomatoes is related to the increased expression of the regulatory large subunit and subsequent maintenance of the active heterotetramer. Comparative analysis of partially purified enzymes from both genotypes did not show differences in biochemical characteristics. Both enzymes are thermoactivated at 56OC with activity increasing up to 90%, whereas the AGPase-L1H protein appears to be slightly more thermostable. Post-translational redox activation of AGPase is similar in the high and standard starch lines. No significant differences were observed in the kinetic properties of the partially purified AGPase enzymes from the two genotypes, i.e., substrate affinity or sensitivity to 3- PGA/PPi regulation. Furthermore, no significant differences between the metabolite concentrations in the green fruit of the two genotypes were observed. Analysis of full length sequences of the two alleles of AgpL1 gene did not indicate significant differences: the two alleles, AgpL1E and AgpL1H are 4500bp and 4497bp long, respectively and both consist of 15 exons with 97% homology between the coding regions, and 14 introns. A number of small differences (SNPs and indels) were found between the two sequences that were specific for AgpL1H. Sequencing of 2000pb long putative promoter region showed 86% homology between the two genotypes. The results of this research indicate that the increased activity of AGPase in tomato fruits harboring the wild tomato L. hirsutum AgpL1 introgression is due to the increased expression and protein levels of the regulatory large subunit, which subsequently leads to an extended stability and activity of the functional holoenzyme. It serves as an example of intra-molecular heterosis in which the alleles for the individual subunits of the enzyme tetramer complement each other in a heterotic manner.