Tomato Fructokinases Exhibit Differential Expression and Substrate Regulation

Two divergent genes encoding fructokinase, Frk1 and Frk2, have been previously shown to be expressed in tomato (Lycopersicon esculentum L.) and have now been further characterized with regard to their spatial expression and the enzymic properties of the encoded proteins. Frk1 and Frk2 mRNA levels were coordinately induced by exogenous sugar, indicating that both belong to the growing class of sugar-regulated genes. However, in situ hybridization indicated that Frk1 and Frk2 were expressed in a spatially distinct manner, with Frk2 mRNA primarily localized in cells of the fruit pericarp, which store starch, and Frk1 mRNA distributed ubiquitously in pericarp tissue. To evaluate the biochemical characteristics of the products of the Frk1 and Frk2 genes, each cDNA was expressed in a mutant yeast (Saccharomyces cerevisiae) line defective in hexose phosphorylation and unable to grow on glucose or fructose (Fru). Both Frk1 and Frk2 proteins expressed in yeast conferred the ability to grow on Fru and exhibited fructokinase activity in vitro. Although both Frk1 and Frk2 both utilized Fru as a substrate, only Frk2 activity was inhibited at high Fru concentrations. These results indicate that Frk2 can be distinguished from Frk1 by its sensitivity to substrate inhibition and by its temporal and spatial pattern of expression, which suggests that it plays a primary role in plant cells specialized for starch storage.

此前已有研究证实，编码果糖激酶（fructokinase）的两个基因Frk1与Frk2可在番茄（Lycopersicon esculentum L.）中表达，本研究进一步针对二者的空间表达模式及编码蛋白的酶学特性展开了系统表征。Frk1与Frk2的mRNA水平可被外源糖类协同诱导，表明二者均属于日益扩增的糖调控基因家族。然而，原位杂交结果显示，Frk1与Frk2的空间表达模式存在显著差异：Frk2的mRNA主要定位于储存淀粉的果实果皮细胞中，而Frk1的mRNA则在果皮组织中广泛分布。为解析Frk1与Frk2基因编码产物的生化特性，研究人员将两条互补脱氧核糖核酸（complementary DNA，cDNA）分别转入己糖磷酸化功能缺陷、无法在葡萄糖或果糖（Fru）培养基上生长的酿酒酵母（Saccharomyces cerevisiae）突变菌株中进行异源表达。经酵母异源表达的Frk1与Frk2蛋白均能使该突变菌株恢复在果糖培养基上的生长能力，且在体外实验中均表现出果糖激酶活性。尽管Frk1与Frk2均能以果糖作为底物，但仅Frk2的酶活会在高浓度果糖条件下受到抑制。上述结果表明，Frk2可通过其对底物抑制的敏感性以及时空表达模式与Frk1区分开来，这提示Frk2在特化用于淀粉储存的植物细胞中发挥主要功能。