Overexpression of a low-affinity fructokinase MdFRK1 leads to enhanced accumulation of fructose in apple calli.

Fructokinase (FRK) (EC 2.7.1.4) activates fructose through phosphorylation, which guides the activated fructose into primary metabolism and regulates the abilities of fructose to serve as a signal in plants. It was previously shown that the apple (Malus domestica) gene MdFRK1 is highly expressed in mature fruit and significantly correlates with the accumulation of fructose. In this study, the coding sequences of MdFRK1 were isolated from 'Royal Gala' apples. The amino acid sequence of MdFRK1 (MD00G1004100) significantly resembles that of AtFRK1 (AT5G51830) in Arabidopsis thaliana, which contains the di-Gly (GG) and GAGD motifs characteristic of the pfkB family of carbohydrate kinases. Treatment with exogenous sugar showed that the application of 3% fructose significantly enhanced the expression of MdFRK1. This suggests that MdFRK1 is also induced by high levels of sugar. The ORF of MdFRK1 contained 1074 bp, and the protein is primarily localized in the cytoplasm. MdFRK1 had a particularly low affinity to fructose (Km = 0.54 mM), and its overexpression in transgenic calli resulted in significantly higher concentrations of fructose. These results highlight the pivotal role of the highly specific fructokinase MdFRK1 in the accumulation of fructose that is associated with the ripening of apples. This study furnishes valuable insights to enhance the flavor of apples in the future and enriches comprehension of the molecular mechanisms that govern the levels of fructose in plants and modulate signaling.