Phosphorylation fine‐tunes ceramide synthase activity and stability to modulate sphingolipid biosynthesis and immune responses

Ceramide synthases (CerSs) are central to sphingolipid biosynthesis and influence plant development and defense responses. However, how CerS activity is regulated in plants remains unclear. Here, we discovered that LAG ONE HOMOLOG 2 (LOH2), the sole long-chain CerS in Arabidopsis (Arabidopsis thaliana), is post-translationally regulated by the ubiquitous kinase casein kinase 2 (CK2). CK2 interacts with LOH2 and phosphorylates serine residues S289 and S291 within its C-terminal region. We mutated these two serines to alanines and expressed the resulting non-phosphorylatable LOH2 variant in transgenic plants and protoplasts. We found that phosphorylation enhances LOH2 enzymatic activity, partially by increasing its substrate-binding affinity, but concurrently promotes LOH2 polyubiquitination and degradation via the 26S proteasome without affecting its subcellular localization. Plants expressing a non-phosphorylatable LOH2 variant showed diminished cell death, reduced C16 ceramide biosynthesis and salicylic acid (SA) accumulation, and compromised resistance to the fungal toxin Fumonisin B1 and the bacterial pathogen Pseudomonas syringae. Pathogen infection induces LOH2 phosphorylation, promoting C16 ceramide accumulation, SA production, and resistance gene expression. Collectively, our findings demonstrate that CK2 fine-tunes LOH2 enzymatic activity and stability, and thus the production of long-chain ceramides through phosphorylation, thereby regulating plant development and defense responses.