Efficient seed detachment from glumes is crucial for cereal threshing. Naked grains, lacking substantial glume coverage, significantly improve threshing efficiency and seed quality. Sorghum, a major crop domesticated early in human history, exhibits considerable variation in glume coverage, with naked grains being prevalent among modern cultivars. Hulled sorghum presents significant challenges for mechanized planting and threshing, leading to substantial production losses. Conversely, naked sorghum varieties are favored due to higher efficiency in mechanized processes. Although glume coverage has been a key trait in sorghum classification, its underlying genetic basis remained unknown. G protein complexes, composed of α, β, and γ subunits, play a critical role in signal transduction in plants. Phospholipases are key components of the G-protein cycle, involved in plant defense and cell growth. This research aimed to identify the genetic basis of glume coverage in sorghum, focusing on the potential role of G proteins and phospholipases in glume development. The study hypothesized that a gene encoding a G protein γ subunit regulates glume coverage, with variations in this gene leading to the naked grain phenotype.

Previous research highlights the importance of efficient threshing in cereal production. The transition from hulled to naked grains has been a significant milestone in the domestication of various cereals, including maize, where a mutation in *tga1* was identified as a causal factor. In wheat, the *Tg* and *Sog* loci affect glume toughness and threshing efficiency. Glume coverage has been used to classify sorghum subspecies, but the underlying genetic mechanisms remained poorly understood. Studies on G protein signaling in plants demonstrate their diverse roles in various physiological processes, including cell growth and development. Phospholipases are also implicated in G protein-mediated signaling pathways. However, the specific connection between G protein subunits, phospholipases, and glume development in sorghum remained elusive prior to this research.

The study employed a multi-pronged approach. First, a diverse sorghum panel (915 accessions) was used to analyze glume coverage morphology and its correlation with threshing efficiency. A genome-wide association study (GWAS) was conducted using a sorghum association panel (SAP; 352 inbred lines) to identify genomic loci associated with glume coverage. Map-based cloning was employed in an F2 population derived from a cross between hulled and naked sorghum lines to fine-map a major locus linked to this trait. To refine the locus, marker-assisted selection (MAS) was used to screen subsequent generations of offspring plants. Genetic variations within the candidate gene region were analyzed in a set of sorghum inbred lines, associating these variations with glume coverage. Near-isogenic lines (NILs) were developed to confirm the role of the candidate gene (*GC1*). Transgenic plants overexpressing *GC1* and *gc1* (truncated versions) were generated using the ubiquitin promoter in a sorghum recipient line. Additionally, a *GC1* knockout mutant was created via genome editing. To investigate the interaction between *GC1* and potential downstream regulatory components, the study utilized immunoprecipitation coupled with mass spectrometry (IP-MS). Luciferase complementation imaging (LCI) and co-immunoprecipitation (Co-IP) assays were used to verify protein interactions. Histological analysis (longitudinal and transverse paraffin sections) and RNA-seq were employed to investigate the cellular and molecular mechanisms underlying glume development. Finally, geographic distribution analysis and selection signature analysis were performed to examine the evolutionary trajectory of *GC1* alleles.

GWAS and map-based cloning identified *GC1*, encoding an atypical G protein γ subunit, as a major gene controlling glume coverage. Natural variations in the *GC1* C-terminus resulted in truncated alleles. The truncated *gc1* alleles accumulate at significantly higher protein levels than the full-length *GC1*. *GC1* negatively regulates glume coverage, while *gc1* alleles show a reduction in glume length and coverage, leading to increased threshing efficiency. Overexpression of *GC1* led to reduced glume coverage, whereas *GC1* knockout resulted in increased glume coverage, confirming its negative regulatory role. The truncated *gc1* alleles promote the degradation of the patatin-related phospholipase SbpPLAII-1, affecting glume cell proliferation. Histological analysis revealed a reduction in glume cell number in *gc1* lines. Transcriptome analysis showed that Cyclin-CDK genes involved in cell proliferation are downregulated in *gc1* lines. Geographic distribution analysis revealed that the truncated *gc1* alleles are prevalent in regions known for sorghum domestication, suggesting positive selection for naked grains. The selection analysis revealed a strong positive selection signal for truncated *GC1* alleles in naked sorghum varieties, indicating human selection targeted these alleles during domestication.

This study definitively identifies *GC1* as a key regulator of glume coverage in sorghum. The findings highlight the importance of subtle variations in the C-terminus of the atypical G protein γ subunit in determining this critical agricultural trait. The increased stability of truncated *gc1* proteins and their effect on SbpPLAII-1 degradation provides a mechanistic explanation for the reduced glume size in naked sorghum varieties. The geographic distribution and selection signature analyses support the hypothesis that these variations were positively selected during sorghum domestication. The results of this study can inform future breeding programs aimed at developing improved sorghum varieties with increased threshing efficiency. The interaction between *GC1* and SbpPLAII-1 suggests a novel regulatory module involving G-protein signaling and phospholipase activity in controlling cell proliferation during glume development. Future studies could focus on further elucidating this pathway and identifying additional interacting components.

This research uncovers the genetic basis of glume coverage variation in sorghum, identifying *GC1* as a major gene controlling this important agricultural trait. The study demonstrates that naturally occurring variations in the *GC1* C-terminus lead to increased protein stability, reduced glume size, and enhanced threshing efficiency. The findings offer valuable insights into the domestication process of sorghum and provide a target for genetic improvement strategies. Future research could focus on dissecting the downstream components of the *GC1*-SbpPLAII-1 pathway and exploring the potential for utilizing *GC1* alleles to improve other aspects of sorghum production.

While the study comprehensively investigated *GC1*'s role, it lacks double knockout mutants for *GC1* and *SbpPLAII-1*, which would strengthen the genetic interaction claim. The study focuses on a specific set of *GC1* alleles; additional variation and wider genomic context could be explored to refine the understanding of glume development regulation. The geographic distribution analysis relies on existing germplasm collections, which may not fully represent the historical diversity of sorghum. Finally, the study predominantly focuses on the effects on glume coverage; the impact on other aspects of grain development and overall yield needs further investigation.