Transcription factors CpSPL5 and CpSPL8 negatively regulate salt tolerance in Codonopsis pilosula by inhibiting SOS pathway

Environmental stresses such as salt and drought severely affect plant growth and development. SQUAMOSA-promoter binding protein-like (SPL) transcription factors (TFs) play critical roles in the regulation of diverse processes; however, reports describing the SPL regulation of plant responses to abiotic stress are relatively few. In this study, two stress-responsive TFs from Codonopsis pilosula (CpSPL5 and CpSPL8) are reported, which confer salt stress sensitivity. CpSPL5 and CpSPL8 are expressed in almost all tissues and localized in the nucleus, where the CpSPL5 transcript level is relatively higher than that of CpSPL8. Their expression levels are significantly suppressed in hairy roots treated with ABA, NaCl, PEG-6000, and under high temperature stress. Compared with the control, CpSPL5, or CpSPL8-overexpressed hairy roots increased salt stress sensitivity, and exhibited higher levels of O²⁻ and MDA, as well as lower superoxide dismutase and peroxidase activities. Further, the CpSPL5 or CpSPL8 interference transgenic hairy roots enhanced salt tolerance and exhibited contrasting phenotype and antioxidant indices. Although all genotypes revealed significantly increased Na⁺ and decreased K⁺ contents under salt stress, the physiological indicators of CpSPL5 or CpSPL8-interference transgenic hairy roots could be partially restored, where CpSPL5 was more sensitive to salt stress than CpSPL8. A yeast one-hybrid and dual-luciferase assay revealed that CpSPL5 and CpSPL8 directly targeted and inhibited the expression of CpSOS2 in the salt overly sensitive (SOS) pathway, which promoted salt stress sensitivity. Our findings suggest that CpSPL5 and CpSPL8 served as negative regulators of salt tolerance, which indicate that members of the SPL family participate in the plant SOS pathway.