LeFRK4, a novel tomato (Lycopersicon esculentum Mill.) fructokinase specifically expressed in stamens

A full-length cDNA clone encoding a novel fructokinase, LeFRK4, was isolated from a tomato (Lycopersicon esculentum Mill.) flower cDNA library. The putative protein shares 62.6 and 55.5% amino acid identity with the two known tomato fructokinases, LeFRK1 and LeFRK2, respectively, and possesses three signature patterns of the pfkB family of carbohydrate kinases, two substrate recognition sites and an ATP-binding domain. The identification of LeFRK4 as a fructokinase was confirmed by complementation of mutant yeast cells unable to phosphorylate or grow on either glucose or fructose. LeFRK4 complemented growth on fructose but not on glucose. Non-soluble crude protein extracts prepared from the transformed yeast cells exhibited fructose but not glucose phosphorylation activity. Expression analysis demonstrated that LeFRK4 is specifically expressed in stamens. Using quantitative RT-PCR, we examined the relative expression of the three known FRK genes in different tomato organs. LeFRK2, encoding a substrate-inhibited fructokinase, was by far the predominantly expressed FRK gene in all organs, except for flowers in which it shared the same expression level with LeFRK4. The exclusive expression of LeFRK4 in stamens may point to a specific role of LeFRK4 in pollen development, anthesis, and perhaps pollination.