Functional characterization of wheat GSK kinase TaSK41 and screening for interacting proteins

SK41, a plant glycogen synthase kinase 3 (GSK3) /SHAGGY-like kinases gene, plays an important role in grain development and grain weight formation. To further elucidate the biological function of <italic>TaSK41</italic> in regulating wheat grain development and its underlying molecular mechanisms, this study examined the expression patterns of <italic>TaSK41</italic> across different tissues, its subcellular localization, the grain phenotypes of transgenic rice overexpressing <italic>TaSK41</italic>, and its interacting proteins. qRT-PCR analysis revealed that <italic>TaSK41</italic> was ubiquitously expressed, with particularly high expression in spikes, early developing grains, ovaries, and seed coats. Subcellular localization assays showed that the TaSK41-GFP fusion protein was predominantly localized in both the cytoplasm and the nucleus. Overexpression of <italic>TaSK41</italic> in transgenic rice led to a reduction in thousand-grain weight, accompanied by significant decreases in both grain length and width. Through yeast two-hybrid screening, 17 candidate proteins were identified as potential interactors with TaSK41. Among these, full-length interaction validation was conducted for <italic>TaARF4</italic>and <italic>TaBSK3</italic>, both of which are associated with grain development. Point-to-point assays confirmed that TaSK41 interacts with the full-length TaARF4 protein. This interaction was further validated <italic>in vivo</italic> using a luciferase complementation assay, confirming that TaSK41 physically interacts with TaARF4 in plant cells. These findings provide a theoretical basis for future investigations into the molecular mechanisms by which TaSK41 regulates grain weight formation in wheat.