Cloning, expression and characterization of LeFRK3, the fourth tomato (Lycopersicon esculentum Mill.) gene encoding fructokinase

A full-length cDNA encoding a novel fourth fructokinase, LeFRK3, was cloned from green tomato (Lycopersicon esculentum Mill.) fruits. The putative protein shares 70, 65.5 and 69% amino acid homology with the three previously identified tomato fructokinases encoded by LeFRK1, LeFRK2 and LeFRK4, respectively. This fourth fructokinase has signature patterns of the pfkB family of carbohydrate kinases as well as substrate recognition sites and an ATP-binding domain. Confirmation for its fructokinase activity was obtained by complementation of triple mutant yeast cells that are unable to phosphorylate or grow on either glucose or fructose as LeFRK3 complemented growth on fructose but not on glucose. Moreover, soluble crude protein extracts prepared from the transformed yeast cells revealed fructose but not glucose phosphorylation activity. In contrast to the LeFRK1 gene product which is inhibited neither by fructose nor by Mg, and to LeFRK2 gene product which is inhibited by both fructose and Mg, the LeFRK3 product is inhibited by fructose but not by Mg. Separation by HPLC-ion exchange chromatography pointed to the gene product of LeFRK3 as the protein responsible for the third peak of fructokinase activity (FKIII), sharing the same pattern of fructose inhibition previously identified with FKIII in tomato fruits. Mapping of tomato fructokinases indicated that LeFRK3 is located on chromosome 2, unlike LeFRK1 (chromosome 3), LeFRK2 (chromosome 6), and LeFRK4 (chromosome 10). The relative expression levels of the four known FRK genes in different tomato organs were analyzed by quantitative RT-PCR. LeFRK2 and LeFRK3 are the predominant genes expressed in all organs with LeFRK3 having the highest level of expression in leaves and apices. LeFRK4 is expressed only in stamens. This differential expression patterns combined with the different biochemical characteristics of the four FRK isozymes suggest that each plays a different role in plant development.