Untargeted Metabolomics Profiling of Arabidopsis WT, lbr-2-2 and bak1-4 Mutants Following Treatment with Two LPS Chemotypes

Abstract

Plants perceive pathogenic threats from the environment that have evaded preformed barriers through pattern recognition receptors (PRRs) that recognise microbe-associated molecular patterns (MAMPs). The perception of and triggered defence to lipopolysaccharides (LPSs) as a MAMP is well-studied in mammals, but little is known in plants, including the PRR(s). Understanding LPS-induced secondary metabolites and perturbed metabolic pathways in Arabidopsis will be key to generating disease-resistant plants and improving global plant crop yield. Recently, Arabidopsis LPS-binding protein (LBP) and bactericidal/permeability-increasing protein (BPI)-related proteins (LBP/BPI related-1) and (LBP/BPI related-2) were shown to perceive LPS from Pseudomonas aeruginosa and trigger defence responses. In turn, brassinosteroid insensitive 1 (BRI1)-associated receptor kinase 1 (BAK1) is a well-established co-receptor for several defence-related PRRs in plants. Due to the lack of knowledge pertaining to LPS perception in plants and given the involvement of the afore-mentioned proteins in MAMPs recognition, in this study, Arabidopsis wild type (WT) and mutant (lbr2-2 and bak1-4) plants were pressure-infiltrated with LPSs purified from Pseudomonas syringae pv. tomato DC3000 (Pst) and Xanthomonas campestris pv. campestris 8004 (Xcc). Metabolites were extracted from the leaves at four time points over a 24 h period and analysed by UHPLC-MS, generating distinct metabolite profiles. Data analysed using unsupervised and supervised multivariate data analysis (MVDA) tools generated results that reflected time- and treatment-related variations after both LPS chemotypes treatments. Forty-five significant metabolites were putatively annotated and belong to the following groups: glucosinolates, hydroxycinnamic acid derivatives, flavonoids, lignans, lipids, oxylipins, arabidopsides and phytohormones, while metabolic pathway analysis (MetPA) showed enrichment of flavone and flavanol biosynthesis, phenylpropanoid biosynthesis, alpha-linolenic acid metabolism and glucosinolate biosynthesis. Distinct metabolite accumulations depended on the LPS chemotype and the genetic background of the lbr2-2 and bak1-4 mutants. This study highlights the role of LPSs in the reprogramming Arabidopsis metabolism into a defensive state, and the possible role of LBR and BAK1 proteins in LPSs perception and thus plant defence against pathogenic bacteria.

An efficient protocol for extracting thylakoid membranes and total leaf proteins from Posidonia oceanica and other polyphenol-rich plants

“…B Phenolic compounds profiling. Heatmap of the major phenolic compounds (identified with available standards (a) or annotated from literature (b: [ 45 ], c: [ 47 ], d: [ 48 ]) in the five species ( Q. pubescens , O. basilicum , P. oceanica , A. thaliana , V. vinifera ). Common logarithm of the peak area means (area/mL) for each condition (five technical replicates) and each metabolite is expressed per one microliter of the extract solution injected (one mg of the starting plant dry material per mL).…”

“…Extraction and UHPLC-ESI-HRMS profiling analysis was carried out according to [ 46 ]. The major phenolic compounds in each species were identified with available standards or annotated with bibliographic data [ 45 – 48 ]. Common logarithm of the peak area means (> 19,000 area/mL) for each condition (five technical replicates) and for each metabolite expressed per one microliter injected of the extract solution (one mg of the starting plant dry material per mL).…”

The oomycete MAMP, arachidonic acid, and anAscophyllum nodosum-derived plant biostimulant induce defense metabolome remodeling in tomato

“…In the same study, SA and JA were also positively correlated with LPS treatment, as we also observed in tomato following treatment with AA (Finnegan et al 2016; Lewis et al 2022). Recent work in A. thaliana wild-type and receptor mutants treated with two chemotypes of LPS showed increases in hydroxycinnamic acid and derivatives and enrichment of the associated phenylpropanoid pathway (Offor et al 2022). Work in tobacco similarly found treatment with LPS, chitosan, and flg22 all induced accumulation of hydroxycinnamic acid and derivatives, and that defense responses elicited by these MAMPs were modulated by both SA and JA (Mhlongo et al 2016).…”

Plasma Membrane-Associated Proteins Identified in Arabidopsis Wild Type, lbr2-2 and bak1-4 Mutants Treated with LPSs from Pseudomonas syringae and Xanthomonas campestris

“…Flavone 3 -O-methyltransferase 1 identified at 12 h in LPS Pst -treated lbr2-2 is involved in flavonol and lignin biosynthesis process needed for ultraviolet (UV) radiation protection, herbivore feeding deterrent and defence against pathogens [171]. These results suggest that LPS Pst and LPS Xcc triggered the synthesis of enzymes that catalyse the accumulation of defence-related proteins and secondary metabolites [172], in response to bacterial pathogen attacks.…”