Biology subjects

Feussner, I.

Publications and source records attributed to Feussner, I..

At least 37 records · Page 2Linked to original sources

bioRxiv2023

Viable mutants of essential genes in Physcomitrium patens as tools for studying primary metabolic processes

Sphingolipids are essential components of plant cells, which have been notoriously difficult to study in part due to pleiotropic or lethal knock-out mutant phenotypes. By relying on alternative end-joining of double stranded breaks, we successfully used CRISPR/Cas9 mutagenesis to generate a population of diverse, viable mutant alleles of genes required for sphingolipid assembly in totipotent protoplasts of the moss Physcomitrium patens. We targeted the INOSITOL PHOSPHORYLCERAMIDE SYNTHASE (IPCS) gene family, which catalyzes the committed step in the synthesis of glycosyl inositol phosphorylceramides (GIPCs), the most abundant class of sphingolipids found in plants. We isolated knock-out single mutants and knock-down higher-order mutants showing a spectrum of deficiencies in GIPC content. Remarkably, we also identified two mutant alleles accumulating inositol phosphorylceramides, the direct products of IPCS activity, and provide our best explanation for this unexpected phenotype. Our approach is broadly applicable for studying essential genes and gene families, and for obtaining unusual lesions within a gene of interest.

plant biology↗

bioRxiv2023

Caleosin 1 contributes to seed lipid droplet degradation by interaction with autophagy-related protein ATG8

Triacylglycerols (TAGs) accumulate in lipid droplets (LDs) of seed tissues to provide energy and carbon for seedling establishment. In the major route of LD degradation (lipolysis), TAGs are mobilized by lipases. However, LDs may be also degraded via lipophagy, a type of selective autophagy, which mediates LDs delivery to vacuoles or lysosomes. The exact mechanism of this process in plants still remains unresolved. Here, we provide evidence that during Arabidopsis thaliana seed germination, LDs are degraded by microlipophagy and that this process requires caleosin 1 (CLO1), a LD surface protein. We show co-localization of autophagy-related protein 8b (ATG8b) and LDs during seed germination and localization of lipidated ATG8 (ATG8-PE) to the LD fraction. We further demonstrate that CLO1, CLO2 and CLO3 interact with ATG8 proteins via their ATG8-interacting motifs (AIMs). Deletion of AIM localized directly before the proline knot disrupts CLO1 interaction with ATG8b, suggesting the essential role of this region in the interaction between the two proteins. Collectively, we provide new insights into the molecular mechanisms governing the interaction of LDs with the autophagy machinery in plant cells, contributing to understanding of the role of structural LD proteins in lipid mobilization.

plant biology↗

bioRxiv2022

Contrasting and conserved roles of NPR proteins in diverged land plant lineages

The NPR proteins function as salicylic acid (SA) receptors in Arabidopsis thaliana. AtNPR1 plays a central role in SA-induced transcriptional reprogramming whereby positively regulates SA-mediated defense. NPRs are found in the genomes of nearly all land plants. However, we know little about the molecular functions and physiological roles of NPRs in most plant species. Our phylogenetic and alignment analyses show that Brassicaceae NPR1-like proteins have characteristically gained or lost functional residues or motifs identified in AtNPRs, pointing to the possibility of a unique evolutionary trajectory for the Brassicaceae NPR1-like proteins that has resulted in peculiar functions. In line with this observation, we find that the only NPR in Marchantia polymorpha, MpNPR, is not the master regulator of SA-induced transcriptional reprogramming and negatively regulates bacterial resistance in this species. Interspecies complementation analysis indicated that the molecular properties of AtNPR1 and MpNPR are partially conserved, implying the diversification of NPR-associated pathways contributed to distinct roles of NPR in different species. The Mpnpr transcriptome suggested potential roles of MpNPR in heat and far-red light responses. We identify both Mpnpr and Atnpr1-1 display enhanced thermomorphogenesis. NPRs and NPR-associated pathways clearly have evolved distinctively in diverged land plant lineages to cope with different terrestrial environments.

plant biology↗

bioRxiv2022

Defining the lipidome of Arabidopsis leaf mitochondria: Specific lipid complement and lipid biosynthesis capacity

Mitochondria are often considered the power stations of the cell, playing critical roles in various biological processes such as cellular respiration, photosynthesis, stress responses and programmed cell death. To maintain the structural and functional integrities of mitochondria, it is crucial to achieve a defined membrane lipid composition between different lipid classes wherein specific proportions of individual lipid species are present. Although mitochondria are capable of self-synthesizing a few lipid classes, many phospholipids are synthesized in the endoplasmic reticulum and transferred to mitochondria via membrane contact sites, as mitochondria are excluded from the vesicular transportation pathway. However, knowledge on the capability of lipid biosynthesis in mitochondria and the precise mechanism of maintaining the homeostasis of mitochondrial lipids is still scarce. Here we describe the lipidome of mitochondria isolated from Arabidopsis leaves, including the molecular species of glycerolipids, sphingolipids and sterols to depict the lipid landscape of mitochondrial membranes. In addition, we define proteins involved in lipid metabolism by proteomic analysis and compare our data with mitochondria from cell cultures since they still serve as model system. Proteins putatively localized to the membrane contact sites are proposed based on the proteomic results and online databases. Collectively, our results suggest that leaf mitochondria are capable - with the assistance of membrane contact site-localised proteins - of generating several lipid classes including phosphatidylethanolamines, cardiolipins, diacylgalactosylglycerols and free sterols. We anticipate our work to be a foundation to further investigate the functional roles of lipids and their involvement in biochemical reactions in plant mitochondria. One sentence summaryThe lipid landscape of plant mitochondria suggests that they are capable in generating several phospholipid classes with the assistant of membrane contact site-localized proteins.

plant biology↗

bioRxiv2022

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. Exact mass information and fragmentation by mass spectrometry (MSMS) suggest a methylated derivative of NHP (MeNHP), a NHP-OGlc-hexosyl conjugate (NHP-OGlc-Hex) and an additional NHP-OGlc-derivative. All three compounds were formed in wildtype leaves but 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 an UV-C treatment. Interestingly, the signal intensity of MeNHP and other NHP-derived metabolites increased in ugt76b1-1 mutant plants. This suggests a detour, for the inability to synthesize NHP-O-glucoside. For MeNHP, we unequivocally determined the site of methylation at the carboxylic acid function. 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 additional storage or transport form of NHP. HighlightIn this work, we identify N-hydroxy pipecolic acid (NHP) metabolites including methyl ester and complex glycosides. The application of methyl ester is able to rescue the disease phenotype of the biosynthesis deficient mutant of NHP.

plant biology↗

bioRxiv2021

Heat stress leads to rapid lipid remodelling and transcriptional adaptations in Nicotiana tabacum pollen tubes

After reaching the stigma, pollen grains germinate and form a pollen tube that transports the sperm cells to the ovule. Due to selection pressure between pollen tubes, they likely evolved mechanisms to quickly adapt to temperature changes to sustain an elongation at the highest possible rate. We investigated these adaptions in Nicotiana tabacum pollen tubes grown in vitro under 22 {degrees}C and 37 {degrees}C by a multi-omic approach including lipidomic, metabolomic and transcriptomic analysis. Both glycerophospholipids and galactoglycerolipids increased in saturated acyl chains under heat stress while triacylglycerols changed less in respect to desaturation but showed higher levels. Free sterol composition was altered, and sterol ester levels decreased. The levels of sterylglycosides and several sphingolipid classes and species were augmented. Most amino acids increased during heat stress, including the non-codogenic amino acids {gamma}-amino butyrate and pipecolate. Furthermore, the sugars sedoheptulose and sucrose showed higher levels. Also the transcriptome underwent pronounced changes with 1,570 of 24,013 genes being differentially up- and 813 being downregulated. Transcripts coding for heat shock proteins and many transcriptional regulators were most strongly upregulated, but also transcripts that have so far not been linked to heat stress. Transcripts involved in triacylglycerol synthesis were increased, while the modulation of acyl chain desaturation seemed not to be transcriptionally controlled indicating other means of regulation. One sentence summaryNicotiana tabacum pollen tubes react to heat stress with metabolomic adaptations, transcriptional adjustments, and a rapid and reversible lipid remodelling.

plant biology↗

bioRxiv2021

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. Here, we investigated how variations in nitrogen (N) nutrition affected the metabolome and proteome of xylem sap, growth of the xylem endophyte Brennaria salicis and report transcriptional re-wiring of leaf defenses in poplar (Populus x canescens). We supplied poplars with high, intermediate or low concentrations of ammonium or nitrate. We identified 288 unique proteins in xylem sap. About 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, while 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 122 metabolites, mainly from specialized metabolism of the groups of salicinoids, phenylpropanoids, phenolics, flavonoids, and benzoates. Their abundances increased with decreasing N. Endophyte growth was stimulated in xylem sap of high N- and suppressed in that of low N-fed plants. The drastic changes in xylem sap composition caused massive changes in the transcriptional landscape of leaves and recruited defense pathways against leaf feeding insects and biotrophic fungi, mainly under low nitrate. Our study uncovers unexpected complexity and variability of xylem composition with consequences for plant defenses. Significance statementThis study reports the largest, currently available plant xylem sap proteome and metabolome databases and highlights novel discoveries of specialized metabolites and phytohormones in the xylem sap. This is the first multi-omics study linking differences in nitrogen supply with changes xylem sap composition, endophyte growth and transcriptional defenses in leaves.

plant biology↗

bioRxiv2021

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.

plant biology↗

bioRxiv2021

Sphingolipid-Induced Programmed Cell Death Is a Salicylic Acid and EDS1-Dependent Phenotype in Arabidopsis

Ceramides and long chain bases (LCBs) are plant sphingolipids involved in the induction of plant programmed cell death (PCD). The fatty acid hydroxylase mutant fah1 fah2 exhibits high ceramide levels and moderately elevated LCB levels. Salicylic acid (SA) is strongly induced in these mutants, but no cell death is visible. To determine the effect of ceramides with different chain lengths, fah1 fah2 was crossed with ceramide synthase mutants longevity assurance gene one homologue1-3 (loh1, loh2 and loh3). Surprisingly, only triple mutants with loh2 show a cell death phenotype under the selected conditions. Sphingolipid profiling revealed that the greatest differences between the triple mutant plants are in the LCB and LCB-phosphate (LCB-P) fraction. fah1 fah2 loh2 plants accumulate LCB d18:0 and LCB-P d18:0. Crossing fah1 fah2 loh2 with the SA synthesis mutant sid2-2, and with the SA signaling mutants enhanced disease susceptibility 1-2 (eds1-2) and phytoalexin deficient 4-1 (pad4-1), revealed that lesions are SA- and EDS1-dependent. These quadruple mutants also suggest that there may be a feedback loop between SA and sphingolipid metabolism as they accumulated less ceramides and LCBs. In conclusion, PCD in fah1 fah2 loh2 is a SA and EDS1-dependent phenotype, which is likely due to accumulation of LCB d18:0.

biochemistry↗

bioRxiv2021

An ancient route towards salicylic acid and its implications for the perpetual Trichormus-Azolla symbiosis

Despite its small size, the water fern Azolla is a giant among plant symbioses. Within each of its leaflets, a specialized leaf cavity is home to a population of nitrogen-fixing cyanobacteria (cyanobionts). While examples of nitrogen fixing cyanobionts are found across the land plant tree of life, Azolla is unique in that its symbiosis is perpetual: the cyanobionts are inherited during sexual and vegetative propagation of the fern. What underpins the communication between the two partners? In angiosperms, the phytohormone salicylic acid (SA) is a well-known regulator of plant-microbe interactions. Using HPLC-MS/MS, we pinpoint the presence of SA in the fern; using comparative genomics and phylogenetics, we mined homologs of SA biosynthesis genes across Chloroplastida (Viridiplantae). While canonical isochorismate synthase (ICS) sequences are largely limited to angiosperms, homologs for the entire Phenylalanine ammonia-lyase (PAL)-dependent pathway likely existed in the last common ancestor of land plants. Indeed, A. filiculoides secondarily lost its ICS, but has the genetic competence to derive SA from benzoic acid. Global gene expression data from cyanobiont-containing and -free A. filiculoides unveil a putative feedback loop: SA appears to induce cyanobacterial proliferation, which in turn down-regulates genes in SA biosynthesis and its responses.

plant biology↗

bioRxiv2020

An integrative approach points to membrane composition as a key factor in E. coli persistence

Many diverse bacteria can enter non- or slow-growing states where they are transiently tolerant to antibiotics. Despite its medical importance, the genetic mechanisms underlying this persistence remain largely unknown, especially for spontaneous (type II) persistence that arise during exponential growth in rich medium. To address this challenge, here we combine genomic, transcriptomic and lipidomic analysis to identify the persistence mechanisms. We first analyzed the genome of the high-persistence mutant Escherichia coli DS1 (hipQ) to identify candidate genes for the high persistence phenotype. We then compared the gene expression profile of spontaneous persisters to normally growing cells with RNAseq and find that the activation of stress response mechanisms is likely not very important in the entrance into hipQ-driven spontaneous persistence. Transcriptomic results also suggest that modifications in the cell membrane play an important role, as further corroborated by lipidomic profiles showing a higher level of unsaturated fatty acids in spontaneous persisters compared to induced persisters or normally growing cells. Taken together, our results indicate that changing membrane composition is a key process in persistence, and further our understanding of spontaneous persister cells from the DS1 (hipQ) context.

microbiology↗

bioRxiv2020

The glycosyltransferase UGT76B1 is critical for plant immunity as it governs the homeostasis of N-hydroxy-pipecolic acid

The trade-off between growth and defense is a critical aspect of plant immunity. Therefore, plant immune response needs to be tightly regulated. The hormone regulating plant defense against biotrophic pathogens is salicylic acid (SA). Recently, N-hydroxy-pipecolic acid (NHP) was identified as second regulator for plant innate immunity and systemic acquired resistance. Although the biosynthetic pathway leading to NHP formation has already been identified, the route how NHP is further metabolized was unclear. Here, we present UGT76B1 as a UDP-dependent glycosyltransferase that modifies NHP by catalyzing the formation of 1-O-glucosyl-pipecolic acid (NHP-OGlc). Analysis of T-DNA and CRISPR knock-out mutant lines of UGT76B1 by targeted and non-targeted UHPLC-HRMS underlined NHP and SA as endogenous substrates of this enzyme in response to Pseudomonas infection and UV treatment. UGT76B1 shows similar KM for NHP and SA. ugt76b1 mutant plants have a dwarf phenotype and a constitutive defense response which can be suppressed by loss of function of the NHP biosynthetic enzyme 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 systemic acquired resistance, it is crucial to balance growth and defense.Competing Interest StatementThe authors have declared no competing interest.View Full Text

plant biology↗

bioRxiv2019

Reprogramming of the apoplast metabolome of Lolium perenne upon infection with the mutualistic symbiont Epichloë festucae

Epichloe festucae is an endophytic fungus that forms a mutualistic 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 non-infected 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 mass spectrometry was used to identify a novel product of a secondary metabolite gene cluster. Metabolites likely to be present in the apoplast were identified using the MarVis Pathway in combination with the BioCyc and KEGG databases, and an in-house Epichloe metabolite database. We were able to identify the known endophyte-specific metabolites, peramine and epichloecyclins, as well as a large number of unknown markers. To determine whether these methods can be applied to the identification of novel Epichloe-derived metabolites, we deleted a gene encoding a NRPS (lgsA) that is highly expressed in planta. Comparative mass spectrometric analysis of apoplastic wash fluid from wild-type- versus mutant- infected plants identified a novel Leu/Ile glycoside metabolite present in the former.

microbiology↗

bioRxiv2019

Ectomycorrhizal fungi induce systemic resistance against insects on a non-mycorrhizal plant in a CERK1-dependent manner

Below-ground microbes can induce systemic resistance (ISR) against foliar pests and pathogens on diverse plant hosts. The prevalence of ISR among plant-microbe-pest systems raises the question of host specificity in microbial induction of ISR. To test whether ISR is limited by plant host range, we tested the ISR-inducing ectomycorrhizal (ECM) fungus Laccaria bicolor on the non-mycorrhizal plant Arabidopsis. We found that root inoculation with L. bicolor triggered ISR against the insect herbivore Trichoplusia ni and induced systemic susceptibility (ISS) against the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (Pto). We found that L. bicolor-triggered ISR against T. ni was dependent on jasmonic acid (JA) signaling and salicylic acid (SA) biosynthesis and signaling. We found that heat killed L. bicolor and chitin are sufficient to trigger ISR against T. ni and ISS against Pto and that the chitin receptor CERK1 is necessary for L. bicolor-mediated effects on systemic immunity. Collectively our findings suggest that some ISR responses might not require intimate co-evolution of host and microbe, but rather might be the result of root perception of conserved microbial signals.

plant biology↗

bioRxiv2019

The cell-wall-localised BETA-XYLOSIDASE 4 contributes to immunity of Arabidopsis against Botrytis cinerea

Plant cell walls constitute physical barriers that restrict access of microbial pathogens to the contents of plant cells. The primary cell wall of multicellular plants predominantly consists of cellulose, hemicellulose and pectin. In Arabidopsis, a cell wall-localised protein, BETA-XYLOSIDASE 4 (BXL4) that belongs to a seven-member BETA-XYLOSIDASE (BXL) gene family was induced upon infection with the necrotrophic fungal pathogen Botrytis cinerea and mechanical wounding in a jasmonoyl isoleucine (JA-Ile) dependent manner. Ectopic expression of the BXL4 gene in Arabidopsis seed coat epidermal cells was able to rescue a bxl1 mutant phenotype suggesting that like BXL1, BXL4, had both xylosidase and arabinosidase activity and acts in mura on cell wall polysaccharides. bxl4 mutants show a compromised resistance to B. cinerea. Upon infection, bxl4 mutants accumulated reduced levels of JA-Ile and camalexin. Conditional overexpression of BXL4 resulted in enhanced expression of PDF1.2 and PAD3 transcripts both before and after B. cinerea infection. This was associated with reduced susceptibility of the transgenic lines to B. cinerea. These data suggest that remodelling or degradation of one or more cell wall polysaccharides is important for plant immunity against B. cinerea and plays a role in pathogen-induced JA-Ile and camalexin accumulation. One-sentence summaryBXL4 is a putative bifunctional xylosidase/arabinofuranisodase localising to the apoplast, important for immunity against the necrotrphic pathogen B. cinerea.

plant biology↗

bioRxiv2019

Fungal effector Jsi1 hijacks plant JA/ET signaling through Topless

Ustilago maydis (U. maydis) is the causal agent of maize smut disease. During the colonization process, the fungus secretes effector proteins which suppress immune responses and redirect the host-metabolism in favor of the pathogen. Here we describe a novel strategy by which U. maydis induces plant jasmonate/ethylene (JA/ET) hormone signaling and thereby biotrophic susceptibility. The U. maydis effector Jasmonate/Ethylene signaling inducer 1 (Jsi1) possesses an ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif, DLNxxP, which interacts with the second WD40 domain of the conserved plant co-repressor family Topless/Topless related (TPL/TPR). Jsi1 interaction with TPL/TPRs leads to derepression of the ethylene response factor (ERF) branch of the JA/ET signaling pathway, supporting biotrophic susceptibility. Jsi1 likely activates the ERF branch by interfering with the binding of endogenous DLNxxP motif-containing ERF transcription factors to TPL/TPR proteins. The identification of effector proteins possessing a DLNxxP motif in different fungal species with biotrophic and hemibiotrophic lifestyles together with the validation of the interaction between such effectors from Magnaporthe oryzae (M. oryzae), Sporisorium scitamineum (S. scitamineum), and S. reilianum with TPL proteins indicates the convergent evolution of effectors for modulating the TPL/TPR co-repressor hub.

plant biology↗

bioRxiv2019

Enhanced Disease Susceptibility 5 (EDS5) is required for N-hydroxy pipecolic acid formation

Pipecolic acid is essential for the establishment of systemic acquired resistance in plants. It is synthesized in the plastid and further processed in the cytosol to its active form N-hydroxy pipecolic acid. Here we provide strong evidence that the exporter Enhanced Disease Susceptibility 5 is required for the biosynthesis of not only salicylic acid, but also N-hydroxy pipecolic acid, suggesting that it represents a convergent point of plant immunity.

plant biology↗

bioRxiv2019

From isochorismate to salicylate: a new reaction mechanism for salicylic acid biosynthesis

The phytohormone salicylic acid (SA) is a central regulator of plant immunity. Despite such functional importance, our knowledge of its biosynthesis is incomplete. Previous work showed that SA is synthesized from chorismic acid in plastids. The bulk of pathogen-induced SA derives from isochorismate generated by the catalytic activity of ISOCHORISMATE SYNTHASE1 (ICS1). How and in which cellular compartment isochorismate is converted to SA is unknown. Here we show that the pathway downstream of isochorismate requires only two additional proteins: the plastidial isochorismate exporter ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5) and the cytosolic amido-transferase AvrPphB SUSCEPTIBLE3 (PBS3). PBS3 catalyzes the conjugation of glutamate to isochorismate. The reaction product isochorismate-9-glutamate spontaneously decomposes into enolpyruvyl-N-glutamate and SA. This previously unknown reaction mechanism appears to be conserved throughout the plant kingdom.\n\nOne Sentence SummarySalicylic acid is synthesized via isochorismate-9-glutamate by PBS3.

plant biology↗