Acylated monogalactosyl diacylglycerol: prevalence in the plant kingdom and identification of an enzyme catalyzing galactolipid head group acylation in Arabidopsis thaliana.

The lipid phase of the thylakoid membrane is mainly composed of the galactolipids mono- and digalactosyl diacylglycerol (MGDG and DGDG, respectively). It has been known since the late 1960s that MGDG can be acylated with a third fatty acid to the galactose head group (acyl-MGDG) in plant leaf homogenates. In certain brassicaceous plants like Arabidopsis thaliana, the acyl-MGDG frequently incorporates oxidized fatty acids in the form of the jasmonic acid precursor 12-oxo-phytodienoic acid (OPDA). In the present study we further investigated the distribution of acylated and OPDA-containing galactolipids in the plant kingdom. While acyl-MGDG was found to be ubiquitous in green tissue of plants ranging from non-vascular plants to angiosperms, OPDA-containing galactolipids were only present in plants from a few genera. A candidate protein responsible for the acyl transfer was identified in Avena sativa (oat) leaf tissue using biochemical fractionation and proteomics. Knockout of the orthologous gene in A. thaliana resulted in an almost total elimination of the ability to form both non-oxidized and OPDA-containing acyl-MGDG. In addition, heterologous expression of the A. thaliana gene in E. coli demonstrated that the protein catalyzed acylation of MGDG. We thus demonstrate that a phylogenetically conserved enzyme is responsible for the accumulation of acyl-MGDG in A. thaliana. The activity of this enzyme in vivo is strongly enhanced by freezing damage and the hypersensitive response.

The activity of HYDROPEROXIDE LYASE 1 regulates accumulation of galactolipids containing 12-oxo-phytodienoic acid in Arabidopsis.

Arabidopsis produces galactolipids containing esters of 12-oxo-phytodienoic acid (OPDA) and dinor-12-oxo-phytodienoic acid (dnOPDA). These lipids are referred to as arabidopsides and accumulate in response to abiotic and biotic stress. We explored the natural genetic variation found in 14 different Arabidopsis accessions to identify genes involved in the formation of arabidopsides. The accession C24 was identified as a poor accumulator of arabidopsides whereas the commonly used accession Col-0 was found to accumulate comparably large amounts of arabidopsides in response to tissue damage. A quantitative trait loci analysis of an F2 population created from a cross between C24 and Col-0 located a region on chromosome four strongly linked to the capacity to form arabidopsides. Expression analysis of HYDROPEROXIDE LYASE 1 (HPL1) showed large differences in transcript abundance between accessions. Transformation of Col-0 plants with the C24 HPL1 allele under transcriptional regulation of the 35S promoter revealed a strong negative correlation between HPL1 expression and arabidopside accumulation after tissue damage, thereby strengthening the view that HPL1 competes with ALLENE OXIDE SYNTHASE (AOS) for lipid-bound hydroperoxide fatty acids. We further show that the last step in the synthesis of galactolipid-bound OPDA and dnOPDA from unstable allene oxides is exclusively enzyme-catalyzed and not the result of spontaneous cyclization. Thus, the results presented here together with previous studies suggest that all steps in arabidopside biosynthesis are enzyme-dependent and apparently all reactions can take place with substrates being esterified to galactolipids.

Role of the penetration-resistance genes PEN1, PEN2 and PEN3 in the hypersensitive response and race-specific resistance in Arabidopsis thaliana.

Plants are highly capable of recognizing and defending themselves against invading microbes. Adapted plant pathogens secrete effector molecules to suppress the host's immune system. These molecules may be recognized by host-encoded resistance proteins, which then trigger defense in the form of the hypersensitive response (HR) leading to programmed cell death of the host tissue at the infection site. The three proteins PEN1, PEN2 and PEN3 have been found to act as central components in cell wall-based defense against the non-adapted powdery mildew Blumeria graminis fsp. hordei (Bgh). We found that loss of function mutations in any of the three PEN genes cause decreased hypersensitive cell death triggered by recognition of effectors from oomycete and bacterial pathogens in Arabidopsis. There were considerable additive effects of the mutations. The HR induced by recognition of AvrRpm1 was almost completely abolished in the pen2 pen3 and pen1 pen3 double mutants and the loss of cell death could be linked to indole glucosinolate breakdown products. However, the loss of the HR in pen double mutants did not affect the plants' ability to restrict bacterial growth, whereas resistance to avirulent isolates of the oomycete Hyaloperonospora arabidopsidis was strongly compromised. In contrast, the double and triple mutants demonstrated varying degrees of run-away cell death in response to Bgh. Taken together, our results indicate that the three genes PEN1, PEN2 and PEN3 extend in functionality beyond their previously recognized functions in cell wall-based defense against non-host pathogens.

Arabidopsis phospholipase dδ is involved in basal defense and nonhost resistance to powdery mildew fungi.

Plants have evolved a complex array of defensive responses against pathogenic microorganisms. Recognition of microbes initiates signaling cascades that activate plant defenses. The membrane lipid phosphatidic acid, produced by phospholipase D (PLD), has been shown to take part in both abiotic and biotic stress signaling. In this study, the involvement of PLD in the interaction between Arabidopsis (Arabidopsis thaliana) and the barley powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) was investigated. This nonadapted pathogen is normally resisted by a cell wall-based defense, which stops the fungal hyphae from penetrating the epidermal cell wall. Chemical inhibition of phosphatidic acid production by PLD increased the penetration rate of Bgh spores on wild-type leaves. The analysis of transfer DNA knockout lines for all Arabidopsis PLD genes revealed that PLDδ is involved in penetration resistance against Bgh, and chemical inhibition of PLDs in plants mutated in PLDδ indicated that this isoform alone is involved in Bgh resistance. In addition, we confirmed the involvement of PLDδ in penetration resistance against another nonadapted pea powdery mildew fungus, Erysiphe pisi. A green fluorescent protein fusion of PLDδ localized to the plasma membrane at the Bgh attack site, where it surrounded the cell wall reinforcement. Furthermore, in the pldδ mutant, transcriptional up-regulation of early microbe-associated molecular pattern response genes was delayed after chitin stimulation. In conclusion, we propose that PLD is involved in defense signaling in nonhost resistance against powdery mildew fungi and put PLDδ forward as the main isoform participating in this process.

Involvement of lipid transfer proteins in resistance against a non-host powdery mildew in Arabidopsis thaliana.

Non-specific lipid transfer proteins (LTPs) are involved in the transport of lipophilic compounds to the cuticular surface in epidermal cells and in the defence against pathogens. The role of glycophosphatidylinositol (GPI)-anchored LTPs (LTPGs) in resistance against non-host mildews in Arabidopsis thaliana was investigated using reverse genetics. Loss of either LTPG1, LTPG2, LTPG5 or LTPG6 increased the susceptibility to penetration of the epidermal cell wall by Blumeria graminis f. sp. hordei (Bgh). However, no impact on pre-penetration defence against another non-host mildew, Erysiphe pisi (Ep), was observed. LTPG1 was localized to papillae at the sites of Bgh penetration. This study shows that, in addition to the previously known functions, LTPGs contribute to pre-invasive defence against certain non-host powdery mildew pathogens.

Redundancy among phospholipase D isoforms in resistance triggered by recognition of the Pseudomonas syringae effector AvrRpm1 in Arabidopsis thaliana.

Plants possess a highly sophisticated system for defense against microorganisms. So called MAMP (microbe-associated molecular patterns) triggered immunity (MTI) prevents the majority of non-adapted pathogens from causing disease. Adapted plant pathogens use secreted effector proteins to interfere with such signaling. Recognition of microbial effectors or their activity by plant resistance (R)-proteins triggers a second line of defense resulting in effector triggered immunity (ETI). The latter usually comprises the hypersensitive response (HR) which includes programmed cell death at the site of infection. Phospholipase D (PLD) mediated production of phosphatidic acid (PA) has been linked to both MTI and ETI in plants. Inhibition of PLD activity has been shown to attenuate MTI as well as ETI. In this study, we systematically tested single and double knockouts in all 12 genes encoding PLDs in Arabidopsis thaliana for effects on ETI and MTI. No single PLD could be linked to ETI triggered by recognition of effectors secreted by the bacterium Pseudomonas syringae. However, repression of PLD dependent PA production by n-butanol strongly inhibited the HR following Pseudomonas syringae effector recognition. In addition some pld mutants were more sensitive to n-butanol than wild type. Thus, the effect of mutations of PLDs could become detectable, and the corresponding genes can be proposed to be involved in the HR. Only knockout of PLDδ caused a loss of MTI-induced cell wall based defense against the non-host powdery mildew Erysiphe pisi. This is thus in stark contrast to the involvement of a multitude of PLD isoforms in the HR triggered by AvrRpm1 recognition.

Formation of oxidized phosphatidylinositol and 12-oxo-phytodienoic acid containing acylated phosphatidylglycerol during the hypersensitive response in Arabidopsis.

Plant membranes are composed of a wide array of polar lipids. The functionality of these extends far beyond a pure structural role. Membrane lipids function as enzyme co-factors, establish organelle identity and as substrates for enzymes such as lipases and lipoxygenases. Enzymatic degradation or oxidation (enzymatic or non-enzymatic) of membrane lipids leads to the formation of a diverse group of bioactive compounds. Plant defense reactions provoked by pathogenic microorganisms are often associated with substantial modifications of the lipidome. In this study, we profiled changes in phospholipids during the hypersensitive response triggered by recognition of the bacterial effector protein AvrRpm1 in Arabidopsis thaliana. A simple and robust LC-MS based method for profiling plant lipids was designed to separate all the major species of glycerolipids extracted from Arabidopsis leaf tissue. The method efficiently separated several isobaric and near isobaric lipid species, which otherwise are difficult to quantify in direct infusion based profiling. In addition to the previously reported OPDA-containing galactolipids found to be induced during hypersensitive response in Arabidopsis, three OPDA-containing sulfoquinovosyl diacylglycerol species, one phosphatidylinositol species as well as two acylated OPDA-containing phosphatidylglycerol species were found to accumulate during the hypersensitive response in Arabidopsis. Our study confirms and extends on the notion that the hypersensitive response in Arabidopsis triggers a unique profile of Allene Oxide Synthase dependent oxidation of membrane lipids. Primary targets of this oxidation seem to be uncharged and anionic lipid species.

Oxo-phytodienoic acid (OPDA) is formed on fatty acids esterified to galactolipids after tissue disruption in Arabidopsis thaliana.

Biotic and abiotic stress induces the formation of galactolipids esterified with the phytohormones 12-oxo-phytodienoic acid (OPDA) and dinor-oxo-phytodienoic acid (dnOPDA) in Arabidopsis thaliana. The biosynthetic pathways of free (dn)OPDA is well described, but it is unclear how they are incorporated into galactolipids. We herein show that (dn)OPDA containing lipids are formed rapidly after disruption of cellular integrity in leaf tissue. Five minutes after freeze-thawing, 60-70% of the trienoic acids esterified to chloroplast galactolipids are converted to (dn)OPDA. Stable isotope labeling with (18)O-water provides strong evidence for that the fatty acids remain attached to galactolipids during the enzymatic conversion to (dn)OPDA.