Epistasis of two classical color genes, B and L-2, synergistically controls carotenoid accumulation in squash

Carotenoid accumulation underlies fruit color and nutritional quality in squash (Cucurbita pepo). One pair of dominant genes, B and L-2, have been long known to interact epistatically, substantially boosting carotenoid accumulation and producing intensely orange-fleshed fruit. However, their molecular identities and regulatory mechanism are unknown. Here, we show that B encodes a truncated H subunit of magnesium chelatase (CpCHLHB) and L-2 encodes a homolog of Arabidopsis Pseudo-Response Regulator 2 (CpAPRR2-A). Significantly, expression of phytoene synthase (CpPSY-A), which encodes the major rate-limiting enzyme in carotenoid biosynthesis, was dramatically upregulated in fruit of B/B L-2/L-2 plants compared with b/b L-2/L-2 or B/B l-2/l-2, showing that the B and L-2 interaction affects CpPSY-A transcription. A similar upregulation was also observed in Arabidopsis gun5 L-2 transgenic plants, where gun5 is a genetic mimic of the C. pepo B gene. The wild-type CpCHLHb physically interacted with CpAPRR2-A, attenuating the CpAPRR2-A-mediated activation of CpPSY-A. In contrast, the truncated CpCHLHB lost its ability to interact with CpAPRR2-A, enabling CpAPRR2-A to activate CpPSY-A and produce intensely orange fruit. These findings uncover the mechanism underlying the epistatic interaction through which B and L-2 act synergistically to boost carotenoid production, offering novel mechanistic insights and key targets for improving crop quality. One-sentence summarySynergistic epistasis between B and L-2 arises from loss of interaction between their encoded proteins, resulting in dramatically upregulating the key rate-limiting enzyme in carotenoid biosynthesis pathway to produce intensely orange-fleshed fruit in squash.