Alternative Splicing of TaGS3 Differentially Regulates Grain Weight and Size in Bread Wheat

The heterotrimeric G-protein mediates growth and development by perceiving and transmitting signals in multiple organisms. Alternative splicing (AS), a vital process for regulating gene expression at the post-transcriptional level, plays a significant role in plant adaptation and evolution. Here, we identified five splicing variants of G_γ subunit gene <i>TaGS3</i> (<i>TaGS3.1</i> to <i>TaGS3.5</i>), which showed expression divergence during wheat polyploidization, and differential function in grain weight and size determination. <i>TaGS3.1</i> overexpression significantly reduced grain weight by 5.89% and grain length by 5.04%, while <i>TaGS3.2</i>-<i>3.4</i> overexpression did not significantly alter grain size compared to wild type. Overexpressing <i>TaGS3.5</i> significantly increased the grain weight by 5.70% and grain length by 4.30%. Biochemical assays revealed that <i>TaGS3</i> isoforms (TaGS3.1-3.4) with an intact OSR domain interact with WGB1 to form active G_βγ heterodimers that further interact with WGA1 to form inactive G_αβγ heterotrimers. Truncated isoforms <i>TaGS3.2-3.4</i> , which lack the C-terminal Cys-rich region but have enhanced binding affinity to WGB1, antagonistically compete with <i>TaGS3.1</i> to bind WGB1, while <i>TaGS3.5</i> with an incomplete OSR domain does not interact with WGB1. Taking these observations together, we proposed that <i>TaGS3</i> differentially regulates grain size via AS, providing a strategy by which the grain size is fine-tuned and regulated at the post-transcriptional level.