Sphingolipids with 2-hydroxy fatty acids aid in plasma membrane nanodomain organization and oxidative burst

Plant sphingolipids mostly possess 2-hydroxy fatty acids (HFA), the synthesis of which is catalyzed by FA 2-hydroxylases (FAHs). In Arabidopsis (Arabidopsis thaliana), two FAHs (FAH1 and FAH2) have been identified. However, the functions of FAHs and sphingolipids with HFAs (2-hydroxy sphingolipids) are still unknown because of the lack of Arabidopsis lines with the complete deletion of FAH1. In this study, we generated a FAH1 mutant (fah1c) using CRISPR/Cas9-based genome editing. Sphingolipid analysis of fah1c, fah2, and fah1cfah2 mutants revealed that FAH1 hydroxylates very long-chain FAs (VLCFAs), whereas the substrates of FAH2 are VLCFAs and palmitic acid. However, 2-hydroxy sphingolipids are not completely lost in the fah1cfah2 double mutant, suggesting the existence of other enzymes catalyzing the hydroxylation of sphingolipid FAs. Plasma membrane (PM) analysis and molecular dynamics simulations revealed that hydroxyl groups of sphingolipid acyl chains play a crucial role in the organization of nanodomains, which are nanoscale liquid-ordered domains mainly formed by sphingolipids and sterols in the PM, through hydrogen bonds. In the PM of the fah1cfah2 mutant, the expression levels of 26.7% of the proteins, including defense-related proteins such as the pattern recognition receptors (PRRs) brassinosteroid insensitive 1-associated receptor kinase 1 and chitin elicitor receptor kinase 1, NADPH oxidase respiratory burst oxidase homolog D (RBOHD), and heterotrimeric G proteins, were lower than that in the wild-type. In addition, reactive oxygen species (ROS) burst was suppressed in the fah1cfah2 mutant after treatment with the pathogen-associated molecular patterns flg22 and chitin. These results indicated that 2-hydroxy sphingolipids are necessary for the organization of PM nanodomains and ROS burst through RBOHD and PRRs during pattern-triggered immunity.