Molecular dissection of two homoeologous wheat genes encoding RING H2-type E3 ligases: TaSIRFP-3A and TaSIRFP-3B

Two homoeologous wheat genes, TaSIRFP-3A and TaSIRFP-3B, encode the RING-HC-type E3 ligases that play an inhibitory role in sucrose metabolism in response to cold stress. In higher plants, the attachment of ubiquitin (Ub) and the subsequent recognition and degradation by the 26S proteasome affects a variety of cellular functions that are essential for survival. Here, we characterized the two homoeologous wheat genes encoding the really interesting new gene (RING) HC-type E3 ligases: TaSIRFP-3A and TaSIRFP-3B (Triticum aestivum SINA domain including RING finger protein 1 and 2), which regulate target proteins via the Ub/26S proteasome system. The TaSIRFP-3A gene was highly expressed under cold stress. In contrast, its homoeologous gene, TaSIRFP-3B, showed only a slight increase in expression levels in shoots. Despite these differences, both the proteins exhibited E3 ligase activity with the cytosol- and nucleus-targeted localization, demonstrating their conserved molecular function. Heterogeneous overexpression of TaSIRFP-3A or TaSIRFP-3B in Arabidopsis showed delayed plant growth causing a reduction in sucrose synthase enzymatic activity and photosynthetic sucrose synthesis, by regulating sucrose synthase proteins. TaSIRFP-3A- or TaSIRFP-3B-overexpressing plants showed higher hypersensitivity under cold stress than WT plants with an accumulation of reactive oxygen species (ROS). These results suggest that the negative regulation of TaSIRFP-3A and TaSIRFP-3B in response to cold stress is involved in sucrose metabolism.