Summary: 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.

NIN-like proteins (NLPs) are conserved, plant-specific transcription factors that play crucial roles in the nitrate signaling response, plant growth and development, and abiotic stress responses. However, their functions have not been explored in sweet potato. In this study, we identified 7 NLPs in cultivated hexaploid sweet potato (Ipomoea batatas, 2n = 6x = 90), 9 NLPs in the diploid relative Ipomoea trifida (2n = 2x = 30), and 12 NLPs in Ipomoea triloba (2n = 2x = 30) via genome structure analysis and phylogenetic characterization, respectively. The protein physiological properties, chromosome localization, phylogenetic relationships, syntenic analysis maps, gene structure, promoter cis-acting regulatory elements, and protein interaction networks were systematically investigated to explore the possible roles of homologous NLPs in the nitrate signaling response, growth and development, and abiotic stress responses in sweet potato. The expression profiles of the identified NLPs in different tissues and treatments revealed tissue specificity and various expression patterns in sweet potato and its two diploid relatives, supporting differences in the evolutionary trajectories of the hexaploid sweet potato. These results are a critical first step in understanding the functions of sweet potato NLPs and offer more candidate genes for improving nitrogen use efficiency and increasing yield in cultivated sweet potato.

The sweet potato albino mutant “Snowwhite” serves as an auxiliary platform for studying gene function and editing efficiency.