Graduate School of Science and Engineering, Saitama University, Sakuraku, Saitama 338-8570, Japan.
College of Life Sciences, Ritsumeikan University, Kusatsu, Shiga 525-8577, Japan.
Plant Physiol. 2022 Jun 1;189(2):839-857. doi: 10.1093/plphys/kiac134.
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.
植物中的神经酰胺大多含有 2-羟基脂肪酸 (HFA)，其合成由 FA 2-羟化酶 (FAHs) 催化。在拟南芥 (Arabidopsis thaliana) 中，已经鉴定出两种 FAHs（FAH1 和 FAH2）。然而，由于缺乏完全缺失 FAH1 的拟南芥系，FAHs 和含 HFA 的神经酰胺（2-羟基神经酰胺）的功能仍然未知。在这项研究中，我们使用基于 CRISPR/Cas9 的基因组编辑生成了 FAH1 突变体 (fah1c)。fah1c、fah2 和 fah1cfah2 突变体的神经酰胺分析表明，FAH1 羟基化非常长链 FAs（VLCFAs），而 FAH2 的底物是 VLCFAs 和棕榈酸。然而，在 fah1cfah2 双突变体中，2-羟基神经酰胺并没有完全缺失，这表明存在其他酶催化神经酰胺 FAs 的羟基化。质膜（PM）分析和分子动力学模拟表明，神经酰胺酰基链上的羟基在纳米域的组织中起着关键作用，纳米域是主要由 PM 中的神经酰胺和固醇形成的纳米级有序液体域，通过氢键形成。在 fah1cfah2 突变体的 PM 中，包括防御相关蛋白如模式识别受体（PRRs）油菜素内酯不敏感 1 相关受体激酶 1 和几丁质受体激酶 1、NADPH 氧化酶呼吸爆发氧化酶同源物 D (RBOHD) 和异三聚体 G 蛋白在内的 26.7%的蛋白质的表达水平低于野生型。此外，在 flg22 和几丁质处理后，fah1cfah2 突变体中的活性氧 (ROS) 爆发受到抑制。这些结果表明，在模式触发免疫过程中，2-羟基神经酰胺通过 RBOHD 和 PRRs 对于 PM 纳米域的组织和 ROS 爆发是必要的。
