The combined effects of Golden2-like 2 and Pseudo-Response Regulator 2 control the fruit pigmentation in pepper

Fruit pigmentation mainly controlled by chlorophyll, carotenoids, and flavonoids is an important phenomenon for fortifying the nutritional and esthetic value of humans. Our previous study identified Pseudo-Response Regulator 2 (PRR2) as a regulator of fruit pigmentation through its association with the c1 locus. A single variation of glk2 or prr2 did not affect the pigmentation of immature fruit in Capsicum accessions, while accessions harboring both glk2/prr2 showed pale-green immature fruits. The immature fruit color in the F₂ population derived from a cross between "Milyang" (GLK2/PRR2, green fruit) and "Goshiki" (glk2/prr2, pale-green fruit) was highly associated with GLK2 and PRR2. Dual variation in GLK2 and PRR2 caused by natural variation and gene silencing resulted in altered pigmentation in both immature and ripe fruits of "Numex halloween" and "Habanero orange." Furthermore, co-silencing of GLK2 and PRR2 in "Royal black" (GLK2/PRR2) resulted in pale-green immature fruits. Reduced pigmentation was associated with a decrease in plastid size and thylakoid number, and the expression of chlorophyll and carotenoid biosynthesis genes was downregulated in the dual-variated fruits. Quadruple variation by co-silencing of GLK2 and CCS in "Numex halloween" (prr2/psy) resulted in white ripe fruits. Additionally, GLK2 was shown to physically interact with PRR2 in yeast two-hybrid and bimolecular fluorescence complementation assays. Taken together, the combined effects of GLK2 and PRR2 determine c1 and fruit pigmentation, and our findings provide a genetic basis for improving fruit quality traits.