Chloroplast ATP-dependent metalloprotease FtsH5/VAR1 confers cold-stress tolerance through singlet oxygen and salicylic acid signaling

Cold stress substantially affects plant growth and productivity. Chloroplasts are primary sites for the production of reactive oxygen species (ROS) and biosynthesis of the defense hormone salicylic acid (SA) under environmental stress conditions. However, the mechanisms by which plants integrate ROS and SA signaling to adapt to stress remain elusive. Here, we report that Arabidopsis FILAMENTOUS TEMPERATURE-SENSITIVE H5/YELLOW VARIEGATED1 (FtsH5/VAR1), a thylakoid-localized ATP-dependent zinc metalloprotease, is essential for plant cold-stress tolerance. The var1-1 mutant exhibits pronounced chlorosis and variegation, as well as retarded growth under cold stress conditions. We observed a strong correlation between elevated SA biosynthesis/signaling and the cold-sensitive phenotype of var1. Reducing SA accumulation, either by overexpressing the salicylate hydroxylase gene (NahG) or knocking out SA biosynthesis-related genes (ICS1, EDS1, or PAD4), partially suppressed the chlorosis phenotype of var1. Furthermore, we demonstrated that EXECUTOR1 (EX1)-mediated singlet oxygen (¹O₂) signaling acts upstream of EDS1 to regulate the expression of SA-responsive genes (SARGs) in var1 under cold stress. Notably, we identified a critical role for EX2, in which the mutation in EX2 significantly suppressed the cold-sensitive phenotype of var1, in activating the expression of SARGs while repressing photosynthesis-associated nuclear genes. Collectively, our results suggest a vital role for VAR1 in plant cold tolerance and highlight the tight connection between ¹O₂ and SA signaling, elucidating a previously unheeded function of EX2, which likely operates independently of EX1-mediated ¹O₂ signaling.