Abstract:

Fructokinase (FRK) serves as a pivotal enzyme in sugar conversion through fructose phosphorylation in cereal grains. However, its role in modulating wheat grain size and starch accumulation remains unclear. This study characterized the wheat TaFRK2-7B1 gene, elucidating its critical role in regulating grain development. qRT-PCR revealed high expression levels of TaFRK2-7B1 in young spikes, particularly in the endosperm. The protein was localized in both nucleus and cytoplasm. In vitro assays with purified TaFRK2-7B1-GST proteins confirmed its fructokinase activity. Transgenic rice overexpressing TaFRK2-7B1 had higher thousand-grain weight (TGW), larger grains, increased fructokinase activity, lower fructose levels, and more starch content than wild types. A kompetitive allele-specific PCR (KASP) marker distinguished two haplotypes of the TaFRK2-7B1 gene in 247 wheat accessions. The TaFRK2-7B1b haplotype exhibited higher fructokinase activity in vitro and in vivo than that of the TaFRK2-7B1a haplotype, and was associated with higher TGW and starch content. The TaFRK2-7B1b haplotype is more prevalent in China's major wheat-producing regions, with its frequency increasing over breeding generations, suggesting it has been preferentially selected in Chinese wheat breeding programs. The findings will provide valuable insights into how wheat fructokinase TaFRK2-7B1 regulates grain weight and highlight its potential application for wheat yield improvement.