Carotenoid is a tetraterpene pigment beneficial for human health. Although the carotenoid biosynthesis pathway has been extensively studied in plants, relatively little is known about their regulation in sweet potato. Previously, we conducted the transcriptome database of differentially expressed genes between the sweet potato (Ipomoea batatas) cultivar ‘Weiduoli’ and its high-carotenoid mutant ‘HVB-3’. In this study, we selected one of these candidate genes, IbNAC29, for subsequent analyses. IbNAC29 belongs to the plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factor family. Relative IbNAC29 mRNA level in the HVB-3 storage roots was ~1.71-fold higher than Weiduoli. Additional experiments showed that the contents of α-carotene, lutein, β-carotene, zeaxanthin, and capsanthin are obviously increased in the storage roots of transgenic sweet potato plants overexpressing IbNAC29. Moreover, the levels of carotenoid biosynthesis genes in transgenic plants were also up-regulated. Nevertheless, yeast one-hybrid assays indicated that IbNAC29 could not directly bind to the promoters of these carotenoid biosynthesis genes. Furthermore, the level of IbSGR1 was down-regulated, whose homologous genes in tomato can negatively regulate carotene accumulation. Yeast three-hybrid analysis revealed that the IbNAC29-IbMYB1R1-IbAITR5 could form a regulatory module. Yeast one-hybrid, electrophoretic mobility shift assay, quantitative PCR analysis of chromatin immunoprecipitation and dual-luciferase reporter assay showed that IbAITR5 directly binds to and inhibits the promoter activity of IbSGR1, up-regulating carotenoid biosynthesis gene IbPSY. Taken together, IbNAC29 is a potential candidate gene for the genetic improvement of nutritive value in sweet potato.

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