Physalis pubescens and Physalis alkekengi, members of the Physalis genus, are valued for their delicious and medicinal fruits as well as their different ripened fruit colors-golden for P. pubescens and scarlet for P. alkekengi. This study aimed to elucidate the pigment composition and genetic mechanisms during fruit maturation in these species. Fruit samples were collected at four development stages, analyzed using spectrophotometry and high-performance liquid chromatography (HPLC), and complemented with transcriptome sequencing to assess gene expression related to pigment biosynthesis. β-carotene was identified as the dominant pigment in P. pubescens, contrasting with P. alkekengi, which contained both lycopene and β-carotene. The carotenoid biosynthesis pathway was central to fruit pigmentation in both species. Key genes pf02G043370 and pf06G178980 in P. pubescens, and TRINITY_DN20150_c1_g3, TRINITY_DN10183_c0_g1, and TRINITY_DN23805_c0_g3 in P. alkekengi were associated with carotenoid production. Notably, the MYB-related and bHLH transcription factors (TFs) regulated zeta-carotene isomerase and β-hydroxylase activities in P. pubescens with the MYB-related TF showing dual regulatory roles. In P. alkekengi, six TF families-bHLH, HSF, WRKY, M-type MADS, AP2, and NAC-were implicated in controlling carotenoid synthesis enzymes. Our findings highlight the intricate regulatory network governing pigmentation and provide insights into Physalis germplasm's genetic improvement and conservation.
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