The CtMYB63 -CtU-box1-CtUCH1 module regulates cold tolerance and Hydroxysafflor yellow A accumulation in Carthamus tinctorius

Abstract

Low-temperature chilling injury poses a formidable threat to plant growth, development, and crop productivity. MYB transcription factors have emerged as key players, orchestrating a symphony of positive regulation in the realms of cold tolerance and flavonoid accumulation. However, the intricate regulatory mechanisms underlying safflower's response to such stressors still remain unclear. In the present study, a putative CtMYB63 was discovered to positively regulate low-temperature tolerance and hydroxysafflor yellow A accumulation in safflower. CtMYB63 exhibited up-regulation in the face of low-temperature stress. Similarly, the silencing of CtMYB63 via RNAi showed down-regulation of key genes involved in cold stress pathway emphasized their susceptibility to low temperature stress unlike overexpressed and wild-type plants. In addition, the interaction between CtMYB63 and a deubiquitin enzyme (CtUCH1) was also confirmed through yeast two-hybrid analysis. Remarkably, the overexpression of CtUCH1 in safflower rebounded the positive effects on low-temperature tolerance, further corroborating its regulatory prowess. Interestingly, while the accumulation of Hydroxysafflor yellow A remained unaffected in CtUCH1 overexpressed plants, it showed positive impact on cold tolerance. Furthermore, the overexpression of CtUCH1 in MYB63 mutant Arabidopsis leads to the restoration of MYB63 function and enhance its ability to regulate downstream target genes. This restoration of MYB function highlighted the intricate interplay between CtUCH1 and CtMYB63 in shaping safflower's transcriptional landscape. The detrimental effects on cold tolerance and Hydroxysafflor yellow A accumulation became evident as the U-box family of E3 ubiquitin ligases, particularly CtU-box1, targeted CtMYB63 for ubiquitination. In this complex interplay, the deubiquitinating prowess of CtUCH1 emerged as a vital modulator, preserving the stability of CtMYB63 by counteracting the adverse effects of CtU-box1. By elucidating this novel regulatory module, consisting of CtMYB63-CtU-box1-CtUCH1, our study pioneers an innovative pathway for enhancing cold stress resilience and optimizing Hydroxysafflor yellow A accumulation in safflower. These findings not only deepen our understanding of safflower's intricate adaptive mechanisms in response to cold stress but also pave the way for the development of novel strategies in crop improvement and pharmaceutical applications.