Comprehensive analysis of AP2/ERFs reveals their functional divergence in safflower flavonoid biosynthesis and stress...

Safflower (Carthamus tinctorius L.) is a multi-purpose cash crop known for its abundant flavonoids and fatty acids, widely used in agriculture and industry. However, as a transcription factor superfamily, AP2/ERF is widely involved in plant stress response and regulation of secondary metabolism. However, the systematic characterization and functional analysis of safflower AP2/ERF in flavonoid biosynthesis and stress stimulation is unknown, which limited the AP2/ERF's application in genetic improvement of safflower varieties. Here, a total of 103 CtAP2/ERF genes were identified in the safflower genome. Gene duplication analysis indicated that WGD or segmental duplication events and dispersed duplication events collectively contributed to the expansion of this gene family. Phylogenetic analysis revealed they were widely distributed in green plants. Gene structure analysis revealed distinct exon-intron organizations and conserved domains among different subfamilies. Compared with the other subfamilies, CtDREBs and CtERFs exhibited a high percentage of stress-response cis-acting elements, including abscisic acid, drought, hypoxia, and low temperature. Then, the fluorescence microscopy of N. benthamiana leaves transiently expressing six fusion proteins revealed that the CtERFs mainly localized to the nucleus, while the CtDREBs localized to both the cytoplasm and the nucleus, suggesting they were genuine transcription factors. Finally, the functional investigation of three proteins demonstrated that overexpression of CtERF_6, CtERF_32, and CtDREB_15 significantly enhanced flavonoid accumulation. However, compared to the two CtERFs (CtERF_6, CtERF_32), CtDREB_15 significantly inhibited seed germination and root elongation. These results indicated functional differentiation of safflower ERFs and DREBs subfamilies in flavonoid synthesis and stress resistance. Our study provides a new theoretical understanding of the diverged function of safflower CtAP2/ERFs, highlighting their potential applications in the crop improvement.