Wounding Triggers Wax Biosynthesis in Arabidopsis Leaves in an Abscisic Acid and Jasmonoyl-Isoleucine Dependent Manner.

Wounding caused by insects or abiotic factors such as wind and hail can cause severe stress for plants. Intrigued by the observation that wounding induces expression of genes involved in surface wax synthesis in a jasmonoyl-isoleucine (JA-Ile)-independent manner, the role of wax biosynthesis and respective genes upon wounding was investigated. Wax, a lipid-based barrier, protects plants both from environmental threats as well as from an uncontrolled loss of water. Its biosynthesis is described to be regulated by abscisic acid (ABA), whereas the main wound-signal is the hormone JA-Ile. We show in this study, that genes coding for enzymes of surface wax synthesis are induced upon wounding in Arabidopsis thaliana leaves in a JA-Ile-independent but ABA-dependent manner. Furthermore, the ABA-dependent transcription factor MYB96 is a key regulator of wax biosynthesis upon wounding. On the metabolite level, wound-induced wax accumulation is strongly reduced in JA-Ile-deficient plants, but this induction is only slightly decreased in ABA-reduced plants. To further analyze the ABA-dependent wound response, we conducted wounding experiments in high humidity. They show that high humidity prevents the wound-induced wax accumulation in A. thaliana leaves. Together the data presented in this study show that wound-induced wax accumulation is JA-Ile-dependent on the metabolite level, but the expression of genes coding for enzymes of wax synthesis is regulated by ABA.

Ex vivo metabolomics-A hypothesis-free approach to identify native substrate(s) and product(s) of orphan enzymes.

Over the past decade, the number of fully sequenced genomes has increased at an awe-inspiring pace. Similarly, the quality and scope of tools for the prediction of both protein structure and function has seen vast improvements. However, to pinpoint the exact function of a protein, for instance the exact reaction catalyzed by an enzyme, experimental evidence is crucial. At the same time, this step is the main bottleneck when generating a conclusive model for the function of an enzyme and to interpret its function in a physiological context. Hence, a comprehensive experimental strategy for functional annotation of enzymes that is as efficient as possible is required. Ex vivo metabolomics is a powerful non-targeted approach that overcomes several of the challenges inherent to in vitro characterization of enzymes with unknown functions. By incubating the recombinant enzyme of interest in a quasi-native metabolite extract from its tissue of origin under specific environmental and developmental conditions, the complete native substrate range can be tested in a single assay. This unlocks compounds that are commercially unavailable or otherwise difficult to procure. Coupled with non-targeted metabolomics analysis, ex vivo has the capability to test for and identify even unexpected substrates and assign the respective products of the enzymatic reaction.

Metabolite fingerprinting: A powerful metabolomics approach for marker identification and functional gene annotation.

Non-targeted metabolome approaches aim to detect metabolite markers related to stress, disease, developmental or genetic perturbation. In the later context, it is also a powerful means for functional gene annotation. A prerequisite for non-targeted metabolome analyses are methods for comprehensive metabolite extraction. We present three extraction protocols for a highly efficient extraction of metabolites from plant material with a very broad metabolite coverage. The presented metabolite fingerprinting workflow is based on liquid chromatography high resolution accurate mass spectrometry (LC-HRAM-MS), which provides suitable separation of the complex sample matrix for the analysis of compounds of different polarity by positive and negative electrospray ionization and mass spectrometry. The resulting data sets are then analyzed with the software suite MarVis and the web-based interface MetaboAnalyst. MarVis offers a straightforward workflow for statistical analysis, data merging as well as visualization of multivariate data, while MetaboAnalyst is used in our hands as complementary software for statistics, correlation networks and figure generation. Finally, MarVis provides access to species-specific metabolite and pathway data bases like KEGG and BioCyc and to custom data bases tailored by the user to connect the identified markers or features with metabolites. In addition, identified marker candidates can be interactively visualized and inspected in metabolic pathway maps by KEGG pathways for a more detailed functional annotation and confirmed by mass spectrometry fragmentation experiments or coelution with authentic standards. Together this workflow is a valuable toolbox to identify novel metabolites, metabolic steps or regulatory principles and pathways.

N-Hydroxy pipecolic acid methyl ester is involved in Arabidopsis immunity.

The biosynthesis of N-hydroxy pipecolic acid (NHP) has been intensively studied, though knowledge on its metabolic turnover is still scarce. To close this gap, we discovered three novel metabolites via metabolite fingerprinting in Arabidopsis thaliana leaves after Pseudomonas infection and UV-C treatment. Exact mass information and fragmentation by tandem mass spectrometry (MS/MS) suggest a methylated derivative of NHP (MeNHP), an NHP-OGlc-hexosyl conjugate (NHP-OGlc-Hex), and an additional NHP-OGlc-derivative. All three compounds were formed in wild-type leaves but were not present in the NHP-deficient mutant fmo1-1. The identification of these novel NHP-based molecules was possible by a dual-infiltration experiment using a mixture of authentic NHP and D9-NHP standards for leaf infiltration followed by UV-C treatment. Interestingly, the signal intensity of MeNHP and other NHP-derived metabolites increased in ugt76b1-1 mutant plants. For MeNHP, we unequivocally determined the site of methylation at the carboxylic acid moiety. MeNHP application by leaf infiltration leads to the detection of a MeNHP-OGlc as well as NHP, suggesting MeNHP hydrolysis to NHP. This is in line with the observation that MeNHP infiltration is able to rescue the fmo1-1 susceptible phenotype against Hyaloperonospora arabidopsidis Noco 2. Together, these data suggest MeNHP as an additional storage or transport form of NHP.

Multi-omics analysis of xylem sap uncovers dynamic modulation of poplar defenses by ammonium and nitrate.

Xylem sap is the major transport route for nutrients from roots to shoots. In the present study, we investigated how variations in nitrogen (N) nutrition affected the metabolome and proteome of xylem sap and the growth of the xylem endophyte Brennaria salicis, and we also report transcriptional re-wiring of leaf defenses in poplar (Populus × canescens). We supplied poplars with high, intermediate or low concentrations of ammonium or nitrate. We identified 288 unique proteins in xylem sap. Approximately 85% of the xylem sap proteins were shared among ammonium- and nitrate-supplied plants. The number of proteins increased with increasing N supply but the major functional categories (catabolic processes, cell wall-related enzymes, defense) were unaffected. Ammonium nutrition caused higher abundances of amino acids and carbohydrates, whereas nitrate caused higher malate levels in xylem sap. Pipecolic acid and N-hydroxy-pipecolic acid increased, whereas salicylic acid and jasmonoyl-isoleucine decreased, with increasing N nutrition. Untargeted metabolome analyses revealed 2179 features in xylem sap, of which 863 were differentially affected by N treatments. We identified 124 metabolites, mainly from specialized metabolism of the groups of salicinoids, phenylpropanoids, phenolics, flavonoids, and benzoates. Their abundances increased with decreasing N, except coumarins. Brennaria salicis growth was reduced in nutrient-supplemented xylem sap of low- and high- NO3- -fed plants compared to that of NH4+ -fed plants. The drastic changes in xylem sap composition caused massive changes in the transcriptional landscape of leaves and recruited defenses related to systemic acquired and induced systemic resistance. Our study uncovers unexpected complexity and variability of xylem composition with consequences for plant defenses.

Plastidic membrane lipids are oxidized by a lipoxygenase in Lobosphaera incisa.

Green microalgae can accumulate neutral lipids, as part of a general lipid remodeling mechanism under stress such as nitrogen starvation. Lobosphaera incisa is of special interest because of its unique TAG acyl chain composition, especially 20:4 (n-6) can reach up to 21% of dry weight after nitrogen starvation. In order to identify factors that may influence the accumulation of polyunsaturated fatty acids (PUFAs), we identified recently a linoleate 13-lipoxygenase (LiLOX). It shares highest identity with plastidic enzymes from vascular plants and is induced upon nitrogen starvation. Here, we confirmed the localization of LiLOX in the stroma of plastids via transient expression in epithelial onion cells. In order to further characterize this enzyme, we focused on the identification of the endogenous substrate of LiLOX. In this regard, an ex vivo enzymatic assay, coupled with non-targeted analysis via mass spectrometry allowed the identification of MGDG, DGDG and PC as three substrate candidates, later confirmed via in vitro assays. Further investigation revealed that LiLOX has preferences towards the lipid class MGDG, which seems in agreement with its localization in the galactolipid rich plastid. Altogether, this study shows the first characterization of plastidic LOX from green algae, showing preference for MGDGs. However, lipidomics analysis did neither reveal an endogenous LiLOX product nor the final end product of MGDG oxidation. Nevertheless, the latter is a key to understanding the role of this enzyme and since its expression is highest during the degradation of the plastidic membrane, it is tempting to assume its involvement in this process.

A non-targeted metabolomics analysis identifies wound-induced oxylipins in Physcomitrium patens.

Plant oxylipins are a class of lipid-derived signaling molecules being involved in the regulation of various biotic and abiotic stress responses. A major class of oxylipins are the circular derivatives to which 12-oxo-phytodienoic acid (OPDA) and its metabolite jasmonic acid (JA) belong. While OPDA and its shorter chain homologue dinor-OPDA (dnOPDA) seem to be ubiquitously found in land plants ranging from bryophytes to angiosperms, the occurrence of JA and its derivatives is still under discussion. The bryophyte Physcomitrium patens has received increased scientific interest as a non-vascular plant model organism over the last decade. Therefore, we followed the metabolism upon wounding by metabolite fingerprinting with the aim to identify jasmonates as well as novel oxylipins in P. patens. A non-targeted metabolomics approach was used to reconstruct the metabolic pathways for the synthesis of oxylipins, derived from roughanic, linoleic, α-linolenic, and arachidonic acid in wild type, the oxylipin-deficient mutants of Ppaos1 and Ppaos2, the mutants of Ppdes being deficient in all fatty acids harboring a Δ6-double bond and the C20-fatty acid-deficient mutants of Ppelo. Beside of OPDA, iso-OPDA, dnOPDA, and iso-dnOPDA, three additional C18-compounds and a metabolite being isobaric to JA were identified to accumulate after wounding. These findings can now serve as foundation for future research in determining, which compound(s) will serve as native ligand(s) for the oxylipin-receptor COI1 in P. patens.

The evolution of the phenylpropanoid pathway entailed pronounced radiations and divergences of enzyme families.

Land plants constantly respond to fluctuations in their environment. Part of their response is the production of a diverse repertoire of specialized metabolites. One of the foremost sources for metabolites relevant to environmental responses is the phenylpropanoid pathway, which was long thought to be a land-plant-specific adaptation shaped by selective forces in the terrestrial habitat. Recent data have, however, revealed that streptophyte algae, the algal relatives of land plants, have candidates for the genetic toolkit for phenylpropanoid biosynthesis and produce phenylpropanoid-derived metabolites. Using phylogenetic and sequence analyses, we here show that the enzyme families that orchestrate pivotal steps in phenylpropanoid biosynthesis have independently undergone pronounced radiations and divergence in multiple lineages of major groups of land plants; sister to many of these radiated gene families are streptophyte algal candidates for these enzymes. These radiations suggest a high evolutionary versatility in the enzyme families involved in the phenylpropanoid-derived metabolism across embryophytes. We suggest that this versatility likely translates into functional divergence, and may explain the key to one of the defining traits of embryophytes: a rich specialized metabolism.

The glycosyltransferase UGT76B1 modulates N-hydroxy-pipecolic acid homeostasis and plant immunity.

The tradeoff between growth and defense is a critical aspect of plant immunity. Therefore, the plant immune response needs to be tightly regulated. Salicylic acid (SA) is an important plant hormone regulating defense against biotrophic pathogens. Recently, N-hydroxy-pipecolic acid (NHP) was identified as another regulator for plant innate immunity and systemic acquired resistance (SAR). Although the biosynthetic pathway leading to NHP formation is already been identified, how NHP is further metabolized is unclear. Here, we present UGT76B1 as a uridine diphosphate-dependent glycosyltransferase (UGT) that modifies NHP by catalyzing the formation of 1-O-glucosyl-pipecolic acid in Arabidopsis thaliana. Analysis of T-DNA and clustered regularly interspaced short palindromic repeats (CRISPR) knock-out mutant lines of UGT76B1 by targeted and nontargeted ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) underlined NHP and SA as endogenous substrates of this enzyme in response to Pseudomonas infection and UV treatment. ugt76b1 mutant plants have a dwarf phenotype and constitutive defense response which can be suppressed by loss of function of the NHP biosynthetic enzyme FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1). This suggests that elevated accumulation of NHP contributes to the enhanced disease resistance in ugt76b1. Externally applied NHP can move to distal tissue in ugt76b1 mutant plants. Although glycosylation is not required for the long-distance movement of NHP during SAR, it is crucial to balance growth and defense.

Convergence of sphingolipid desaturation across over 500 million years of plant evolution.

For plants, acclimation to low temperatures is fundamental to survival. This process involves the modification of lipids to maintain membrane fluidity. We previously identified a new cold-induced putative desaturase in Physcomitrium (Physcomitrella) patens. Lipid profiles of null mutants of this gene lack sphingolipids containing monounsaturated C24 fatty acids, classifying the new protein as sphingolipid fatty acid denaturase (PpSFD). PpSFD mutants showed a cold-sensitive phenotype as well as higher susceptibility to the oomycete Pythium, assigning functions in stress tolerance for PpSFD. Ectopic expression of PpSFD in the Atads2.1 (acyl coenzyme A desaturase-like 2) Arabidopsis thaliana mutant functionally complemented its cold-sensitive phenotype. While these two enzymes catalyse a similar reaction, their evolutionary origin is clearly different since AtADS2 is a methyl-end desaturase whereas PpSFD is a cytochrome b5 fusion desaturase. Altogether, we suggest that adjustment of membrane fluidity evolved independently in mosses and seed plants, which diverged more than 500 million years ago.

Verticillium longisporum Elicits Media-Dependent Secretome Responses With Capacity to Distinguish Between Plant-Related Environments.

Verticillia cause a vascular wilt disease affecting a broad range of economically valuable crops. The fungus enters its host plants through the roots and colonizes the vascular system. It requires extracellular proteins for a successful plant colonization. The exoproteomes of the allodiploid Verticillium longisporum upon cultivation in different media or xylem sap extracted from its host plant Brassica napus were compared. Secreted fungal proteins were identified by label free liquid chromatography-tandem mass spectrometry screening. V. longisporum induced two main secretion patterns. One response pattern was elicited in various non-plant related environments. The second pattern includes the exoprotein responses to the plant-related media, pectin-rich simulated xylem medium and pure xylem sap, which exhibited similar but additional distinct features. These exoproteomes include a shared core set of 221 secreted and similarly enriched fungal proteins. The pectin-rich medium significantly induced the secretion of 143 proteins including a number of pectin degrading enzymes, whereas xylem sap triggered a smaller but unique fungal exoproteome pattern with 32 enriched proteins. The latter pattern included proteins with domains of known pathogenicity factors, metallopeptidases and carbohydrate-active enzymes. The most abundant proteins of these different groups are the necrosis and ethylene inducing-like proteins Nlp2 and Nlp3, the cerato-platanin proteins Cp1 and Cp2, the metallopeptidases Mep1 and Mep2 and the carbohydrate-active enzymes Gla1, Amy1 and Cbd1. Their pathogenicity contribution was analyzed in the haploid parental strain V. dahliae. Deletion of the majority of the corresponding genes caused no phenotypic changes during ex planta growth or invasion and colonization of tomato plants. However, we discovered that the MEP1, NLP2, and NLP3 deletion strains were compromised in plant infections. Overall, our exoproteome approach revealed that the fungus induces specific secretion responses in different environments. The fungus has a general response to non-plant related media whereas it is able to fine-tune its exoproteome in the presence of plant material. Importantly, the xylem sap-specific exoproteome pinpointed Nlp2 and Nlp3 as single effectors required for successful V. dahliae colonization.

Heat stress response in the closest algal relatives of land plants reveals conserved stress signaling circuits.

All land plants (embryophytes) share a common ancestor that likely evolved from a filamentous freshwater alga. Elucidating the transition from algae to embryophytes - and the eventual conquering of Earth's surface - is one of the most fundamental questions in plant evolutionary biology. Here, we investigated one of the organismal properties that might have enabled this transition: resistance to drastic temperature shifts. We explored the effect of heat stress in Mougeotia and Spirogyra, two representatives of Zygnematophyceae - the closest known algal sister lineage to land plants. Heat stress induced pronounced phenotypic alterations in their plastids, and high-performance liquid chromatography-tandem mass spectroscopy-based profiling of 565 transitions for the analysis of main central metabolites revealed significant shifts in 43 compounds. We also analyzed the global differential gene expression responses triggered by heat, generating 92.8 Gbp of sequence data and assembling a combined set of 8905 well-expressed genes. Each organism had its own distinct gene expression profile; less than one-half of their shared genes showed concordant gene expression trends. We nevertheless detected common signature responses to heat such as elevated transcript levels for molecular chaperones, thylakoid components, and - corroborating our metabolomic data - amino acid metabolism. We also uncovered the heat-stress responsiveness of genes for phosphorelay-based signal transduction that links environmental cues, calcium signatures and plastid biology. Our data allow us to infer the molecular heat stress response that the earliest land plants might have used when facing the rapidly shifting temperature conditions of the terrestrial habitat.

Lolium perenne apoplast metabolomics for identification of novel metabolites produced by the symbiotic fungus Epichloë festucae.

Epichloë festucae is an endophytic fungus that forms a symbiotic association with Lolium perenne. Here we analysed how the metabolome of the ryegrass apoplast changed upon infection of this host with sexual and asexual isolates of E. festucae. A metabolite fingerprinting approach was used to analyse the metabolite composition of apoplastic wash fluid from uninfected and infected L. perenne. Metabolites enriched or depleted in one or both of these treatments were identified using a set of interactive tools. A genetic approach in combination with tandem MS was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using MarVis in combination with the BioCyc and KEGG databases, and an in-house Epichloë metabolite database. We were able to identify the known endophyte-specific metabolites, peramine and epichloëcyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novel Epichloë-derived metabolites, we deleted a gene encoding a NRPS (lgsA) that is highly expressed in planta. Comparative MS analysis of apoplastic wash fluid from wild-type- vs mutant-infected plants identified a novel Leu/Ile glycoside metabolite present in the former.

Mapping Cellular Microenvironments: Proximity Labeling and Complexome Profiling (Seventh Symposium of the Göttingen Proteomics Forum).

Mass spectrometry-based proteomics methods are finding increasing use in structural biology research. Beyond simple interaction networks, information about stable protein-protein complexes or spatially proximal proteins helps to elucidate the biological functions of proteins in a wider cellular context. To shed light on new developments in this field, the Göttingen Proteomics Forum organized a one-day symposium focused on complexome profiling and proximity labeling, two emerging technologies that are gaining significant attention in biomolecular research. The symposium was held in Göttingen, Germany on 23 May, 2019, as part of a series of regular symposia organized by the Göttingen Proteomics Forum.

Isochorismate-derived biosynthesis of the plant stress hormone salicylic acid.

The phytohormone salicylic acid (SA) controls biotic and abiotic plant stress responses. Plastid-produced chorismate is a branch-point metabolite for SA biosynthesis. Most pathogen-induced SA derives from isochorismate, which is generated from chorismate by the catalytic activity of ISOCHORISMATE SYNTHASE1. Here, we ask how and in which cellular compartment isochorismate is converted to SA. We show that in Arabidopsis, the pathway downstream of isochorismate requires only two additional proteins: ENHANCED DISEASE SUSCEPTIBILITY5, which exports isochorismate from the plastid to the cytosol, and the cytosolic amidotransferase avrPphB SUSCEPTIBLE3 (PBS3). PBS3 catalyzes the conjugation of glutamate to isochorismate to produce isochorismate-9-glutamate, which spontaneously decomposes into SA and 2-hydroxy-acryloyl-N-glutamate. The minimal requirement of three compartmentalized proteins controlling unidirectional forward flux may protect the pathway against evolutionary forces and pathogen perturbations.

Comprehensive LC-MS-Based Metabolite Fingerprinting Approach for Plant and Fungal-Derived Samples.

Liquid chromatography-mass spectrometry (LC-MS)-based nontargeted metabolome approaches aim to detect chemotypes as markers for stress, disease, developmental, or genetic perturbation. Herein, we present a metabolite fingerprinting workflow, which is applicable for the analysis of tissues and fluids derived from plants and fungi. This is based on a broad metabolite coverage by a two-phase extraction and the separate analysis of polar, and nonpolar compounds by positive as well as negative electrospray ionization. For analysis of the resulting comprehensive data sets, the interactive and user-friendly data mining software MarVis-Suite is used. It supports statistical analysis, adduct correction, data merging, as well as visualization of multivariate data. Finally, MarVis shapes marker identification to the organism of interest. Therefore, it provides access to the species-specific databases KEGG and BioCyc and to custom databases tailored by the user.

The glycosyltransferase UGT76E1 significantly contributes to 12-O-glucopyranosyl-jasmonic acid formation in wounded Arabidopsis thaliana leaves.

Jasmonoyl-isoleucine (JA-Ile) is a phytohormone that orchestrates plant defenses in response to wounding, feeding insects, or necrotrophic pathogens. JA-Ile metabolism has been studied intensively, but its catabolism as a potentially important mechanism for the regulation of JA-Ile-mediated signaling is not well-understood. Especially the enzyme(s) responsible for specifically glycosylating 12-hydroxy-jasmonic acid (12-OH-JA) and thereby producing 12-O-glucopyranosyl-jasmonic acid (12-O-Glc-JA) is still elusive. Here, we used co-expression analyses of available Arabidopsis thaliana transcriptomic data, identifying four UDP-dependent glycosyltransferase (UGT) genes as wound-induced and 12-OH-JA-related, namely, UGT76E1, UGT76E2, UGT76E11, and UGT76E12 We heterologously expressed and purified the corresponding proteins to determine their individual substrate specificities and kinetic parameters. We then used an ex vivo metabolite-fingerprinting approach to investigate these proteins in conditions as close as possible to their natural environment, with an emphasis on greatly extending the range of potential substrates. As expected, we found that UGT76E1 and UGT76E2 are 12-OH-JA-UGTs, with UGT76E1 contributing a major in vivo UGT activity, as deduced from Arabidopsis mutants with abolished or increased UGT gene expression. In contrast, recombinant UGT76E11 acted on an unidentified compound and also glycosylated two other oxylipins, 11-hydroxy-7,9,13-hexadecatrienoic acid (11-HHT) and 13-hydroxy-9,11,15-octadecatrienoic acid (13-HOT), which were also accepted by recombinant UGT76E1, UGT76E2, and UGT76E12 enzymes. UGT76E12 glycosylated 12-OH-JA only to a low extent, but also accepted an artificial hydroxylated fatty acid and low amounts of kaempferol. In conclusion, our findings have elucidated the missing step in the wound-induced synthesis of 12-O-glucopyranosyl-jasmonic acid in A. thaliana.

Integrative omics - from data to biology.

INTRODUCTION: Multi-omic approaches are promising a broader view on cellular processes and a deeper understanding of biological systems. with strongly improved high-throughput methods the amounts of data generated have become huge, and their handling challenging. Area Covered: New bioinformatic tools and pipelines for the integration of data from different omics disciplines continue to emerge, and will support scientists to reliably interpret data in the context of biological processes. comprehensive data integration strategies will fundamentally improve systems biology and systems medicine. to present recent developments of integrative omics, the göttingen proteomics forum (gpf) organized its 6th symposium on the 23rd of november 2017, as part of a series of regular gpf symposia. more than 140 scientists attended the event that highlighted the challenges and opportunities but also the caveats of integrating data from different omics disciplines. Expert commentary: The continuous exponential growth in omics data require similar development in software solutions for handling this challenge. Integrative omics tools offer the chance to handle this challenge but profound investigations and coordinated efforts are required to boost this field.