Effects of Ca2+ signaling inhibition on cold acclimation in winter rapeseed

• Inhibition of Ca²⁺ influx reduces proBrAFP1 activity in stems and leaves. • Ca²⁺ suppression altered hormone levels, with elevated ABA and SA contents, while IAA, GA3, and ZT were reduced. • Ca²⁺ chelation also compromised membrane integrity. Winter rapeseed bolting is a highly nutritious and health-beneficial vegetable. It is also one of the earliest harvested leafy greens in northern China, providing an important source of fresh produce in the early spring. However, freezing during overwintering the most severe challenge to its production. In this study, we focused on the effects of Ca²⁺ influx inhibitors (EGTA, La³⁺, Gd³⁺, and RuR) on the cold adaptation of winter rapeseed. Our results demonstrate that cold treatment at 4 °C and -4 °C significantly enhanced proBrAFP1 activity and upregulated the expression of cold-responsive genes, including CNGC, CDPK, OST1, ICE1 , and CBFs , in both winter rapeseed and transgenic Arabidopsis . However, Inhibition of Ca²⁺ influx reduces proBrAFP1 activity in stems and leaves of transgenic Arabidopsis lines. Additionally, Ca²⁺ suppression altered hormone levels, with elevated ABA and SA contents, while IAA, GA3, and ZT were reduced. Furthermore, the study revealed that Ca²⁺ inhibition exacerbated oxidative stress. Ca²⁺ chelation also compromised membrane integrity, with increased electrolyte leakage and MDA content. Notably, some inhibitors, such as Gd³⁺ and RuR, mitigated ROS accumulation and membrane injury to a certain extent. Overall, These findings provide valuable insights for developing strategies to enhance cold tolerance in rapeseed bolting production.