Csa-miR160 targets transcriptional repressor CsARF18 and modulates auxin-mediated crocin biosynthesis in Crocus...

Auxin plays a central regulatory role in plant development and secondary metabolism. Crocins are key compounds responsible for the pharmacological effects of saffron, and their content is reduced by exogenous IAA. However, the mechanism underlying this regulation remains unclear. In this study, we identified an auxin response factor, ARF18, whose gene expression is induced by IAA and negatively correlates with crocin biosynthesis. Silencing of CsARF18 resulted in increased crocin content, whereas overexpression of CsARF18 led to a decrease in crocin content. CsARF18 was shown to bind to AuxREs within the promoters of CsPSY1 and CsALDH5, consequently inhibiting their transcription. Furthermore, the microRNA Csa-miR160 was identified as a regulator of CsARF18, promoting its degradation. Co-overexpression of Csa-miR160 and CsARF18 in Crocus sativus L. alleviated the inhibitory effect of CsARF18 on crocin biosynthesis, whereas silencing Csa-miR160 failed to alleviate this inhibition. Our results demonstrate that Csa-miR160 targets and degrades CsARF18, thereby weakening its inhibitory regulation on CsPSY1 and CsALDH5. Based on these findings, we propose the "CsARF18-CsPSY/CsALDH-crocin" regulatory model, in which Csa-miR160 modulates auxin signaling during crocin biosynthesis in C. sativus. This study lays the foundation for further research on the secondary metabolites of saffron.