During maize (Zea mays L.) domestication, seed protein content sharply declined1,2. In plants, glutamine and asparagine levels are closely correlated with protein content3,4. Asparagine is synthesized from glutamine, a process catalysed by asparagine synthase5. Teosinte harbours a superior haplotype of asparagine synthase 4 (ASN4)2. Here, we report that teosinte also possesses a superior haplotype gene promoting glutamine synthesis. We identify and clone teosinte high protein 3 (THP3), which encodes glutamate-oxaloacetate transaminase 1 (GOT1), a key enzyme involved in nitrogen assimilation and carbon–nitrogen balance. The superior THP3-T allele, subjected to negative selection during domestication, has natural variations that boost both its expression and enzymatic activity. Overexpressing THP3-T, but not the modern THP3-B allele, significantly increases seed protein, representing altered carbon–nitrogen composition. Pyramiding THP3-T with THP9-T (the latter encoding asparagine synthase 4 (ASN4)) synergistically elevates both seed and whole-plant protein in elite hybrids while maintaining yield. Our findings demonstrate a powerful strategy for crop improvement by reintroducing beneficial rare alleles disfavoured during domestication from wild relatives.
