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

Abstract

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.

Molecular evidence for new foodways in the early colonial Caribbean: organic residue analysis at Isla de Mona, Puerto Rico

“…These three classes of compounds are diagnostic for plant-derived foodstuffs. Plant sphingolipids, for example, are mostly composed of 2-hydroxy fatty acids (Ukawa et al 2022 ), and these compounds are widely present in the samples in Lipid Profile 3. Plant waxes such as Candelilla wax are produced by leaves of various Euphorbia species which are native to the Americas and are present on Isla de Mona today and contain a mixture of odd-numbered n- alkanes with 29–31 carbon atoms (Colombini and Modugno 2009 , p. 99).…”

“…Some plants from the Brassica family are native to Isla de Mona, including Cakile lanceolata and Lepidium virginicum (Britton 1915 ), so the identification of erucic acid may indicate the cooking and consumption of these native plants. In addition, the 2-hydroxy fatty acids and long-chain dicarboxylic acids found in Lipid Profile 2 are further evidence for plant products, as 2-hydroxy fatty acids are widely present in plant sphingolipids (Ukawa et al 2022 ), while mid- to long-chain dicarboxylic acids are common oxidations product of erucic and oleic acids, both of which are found in high abundance in plants (Colombini and Modugno 2009 , p. 8). This strengthens the interpretation of oleic acid and erucic acid as being derived from the past vessel contents rather than contamination.…”

Chloroplastic Sec14-like proteins modulate growth and phosphate deficiency responses in Arabidopsis and rice

“…Then, DNA fragments obtained by the digestion of resultant plasmids with Asc I and Pac I were cloned into the CRISPR/Cas9 vector, pZH_OsU6gRNA_PubiMMCas9 ( Mikami et al 2015 ), to produce 2 single-guide RNAs (sgRNAs). Transformation of the rice cultivar Nipponbare with Agrobacterium tumefaciens (strain EHA105) harboring the constructed CRISPR/Cas9 vector, followed by the selection of transformed calli with antibiotics, was carried out as described in Toki et al (2006) . DNA sequencing of the target sites and the 3 possible off-target sites predicted by CRISPR-P v2.0 ( http://crispr.hzau.edu.cn/CRISPR2/ ) was performed to confirm that the T0-generation plants of 3 independent lines carried biallelic mutations at the target locus but no undesired editing.…”

New Paradigms in Brassinosteroids, Strigolactones, Sphingolipids, and Nitric Oxide Interaction in the Control of Lateral and Adventitious Root Formation

“…In accordance, hydroxyl groups of SPL chains affect the abundance of the transmembrane co-receptor BRASSINOSTEROID INSENSITIVE 1 -ASSOCIATED RECEPTOR KINASE 1 (BAK1), involved in controlling the early events of the BR-signaling pathway [ 152 ]. In Arabidopsis, where 2-hydroxy SPLs are necessary for the organization of plasma membrane nanodomains, these compounds affect the abundance of BAK1, demonstrating the existence of an interaction between SPL and BR signaling in plant immunity [ 153 ]. It has been very recently suggested that BRs affect the specific orientation of unsaturated/saturated fatty acids in the membranes.…”