Natural allelic variation of basic helix–loop–helix transcription factor 25 regulates carotenoid biosynthesis in sweet potato

Carotenoid-rich orange-fleshed sweet potato (OFSP) is an important staple diet and source of nutrition in developing countries, including Africa and Asia. However, the regulation of carotenoid biosynthesis remains to be better understood. A natural allelic variation closely linked to carotenoid biosynthesis was identified in the promoter region of the IbbHLH25 gene that encodes a basic helix-loop-helix (bHLH) transcription factor, by transcriptome and haplotype analyses of different flesh colour sweet potato accessions. An 86-bp deletion reduced the transcription of the IbbHLH25 promoter in white- and yellow-fleshed sweet potatoes; however, the deletion was absent in OFSP. IbbHLH25 was highly expressed in the storage roots of carotenoid-rich sweet potato. The overexpression of IbbHLH25 significantly increased the carotenoid contents (by 2.5-fold-6.0-fold) and proportions, especially β-carotene and β-cryptoxanthin; their contents increased by 21.2-fold-55.7-fold and 4.6-fold-9.5-fold, respectively, and their proportions increased by 48.5% and 13.0%, respectively, and the silencing of IbbHLH25 had opposite effects. IbbHLH25 formed heterodimers with IbbHLH66 to directly and synergistically activate the transcription of carotenoid biosynthesis key genes IbGGPPS, IbLCYB and IbBCH. The overexpression of IbbHLH66 significantly increased the carotenoid contents (by 2.3-fold-3.8-fold) and proportions, especially β-carotene and β-cryptoxanthin; their contents increased by 15.2-fold-25.6-fold and 3.1-fold-5.1-fold, respectively, and their proportions increased by 31.1% and 9.6%, respectively. These findings expand our understanding of bHLHs in regulating carotenoid biosynthesis and suggest additional roles in affecting carotenoid component proportions, providing candidate genes for nutritional biofortification of agricultural products.