Engineering seed-specific gossypol-free cotton for human-safe consumption by harnessing the dominant-negative effect...

The global yield of cottonseed could meet the annual protein requirements of approximately half a billion people if gossypol were absent from the seeds. Here, we characterize the molecular mechanism by which the Gl₂^e mutation exerts a dominant-negative effect on gland development, providing a mechanistic basis for engineering seed-specific gossypol-free (SSGF) cotton. We show that Gl₂/Gl₃ form multimers-likely tetramers-that function as master regulators within the transcriptional network controlling gossypol gland development. Further analyses demonstrate that Gl₂^e, a dominant mutant allele of Gl₂, induces a glandless phenotype through its dominant-negative effect. In addition, multimers composed of Gl₂^e and Gl₂/Gl₃ retain E-box binding activity but lack transcriptional activation capacity, thereby inhibiting gland organogenesis. Guided by these insights, we engineered SSGF cotton by driving Gl₂^e expression specifically during seed development, effectively suppressing gossypol gland formation in seeds. Multi-year, multi-location field trials of the SSGF cotton confirmed the stable production of gossypol-free seeds without compromising fiber yield or other key agronomic traits. Notably, completely gossypol-free oil and flour can be produced directly from SSGF seeds without the need for degossypolization. This work establishes a mechanistic foundation for understanding gland development and offers a sustainable path toward enhancing global plant-derived protein and oil resources.