Biology subjects

Herrfurth, C.

Publications and source records attributed to Herrfurth, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Glycosylceramide assembly and function in a model bryophyte

Research ConductedTo elucidate the functions of glycosylceramides, we generated and characterized mutants deficient in multiple steps contributing to their assembly in the model moss Physcomitrium patens. We mutagenized SPHINGOLIPID {Delta}8-DESATURASE, whose products are preferentially incorporated into glycosylceramides, and a suite of higher-order mutants combining sphingolipid {Delta}4-desaturase and glycosyl ceramide synthase. MethodsWe used targeted lipidomics to describe the chemotypes of all mutants. We used quantitative phenotype analysis, transcriptomics, and phytohormone profiling to understand the effects of these chemotypes on development and physiology. Key ResultsThese mutants present a range of phenotypes that collectively indicate that in P. patens (1) glycosylceramide deficiency impairs development, largely due to imbalance in free ceramide homeostasis (2) the synthesis of glycosylceramides is dependent upon the presence of a specific free ceramide profile (3) the {Delta}4-, but not the {Delta}8-desaturation, is strictly required for glycosylceramide synthesis, (4) cell division and differentiation, but not cell expansion, are affected by sphingolipid imbalance, and (5) sphingolipid imbalance results in oxylipin accumulation. ConclusionCollectively, our results elucidate the assembly and functions of glycosylceramides in a model bryophyte, and highlight conserved and specialized aspects of sphingolipid metabolism among plants.

plant biology↗

bioRxiv2025

Setaphyte VERY-LONG-CHAIN FATTY ACYL DESATURASES are desaturases that impact glycerolipid and sphingolipid metabolism

Desaturases in plants are diverse. They vary in localization, source of reducing power, and substrate preference, accepting glycerolipids, long-chain bases, acyl-CoAs, and acyl-ACPs, in varying states of (un)saturation and chain length. Their products are incorporated into membrane glycerolipids, sphingolipids, or storage lipids. We previously characterized a desaturase from Physcomitrium patens that predominantly affects the monounsaturation of very-long-chain fatty acyl (VLCFA) moieties of sphingolipids, naming this desaturase SPHINGOLIPID FATTY ACYL DESATURASE (SFD). Among embryophytes, candidate SFDs were only identified in setaphytes, including one paralog in P. patens and an ortholog in Marchantia polymorpha. Here, we characterize the P. patens paralog, and clarify via mutant analysis that SFDs affect not only sphingolipid metabolism, but also glycerolipid metabolism. We express both paralogs, as well as the candidate gene from M. polymorpha, in Saccharomyces cerevisiae, and show they desaturate VLCFAs incorporated into sphingolipids, triacylglycerols, and acyl-CoAs. The simplest explanation is that "SFDs" likely accept an acyl-CoA, rather than a sphingolipid substrate, as initially proposed. We suggest renaming these desaturases VERY-LONG-CHAIN FATTY ACYL DESATURASES (VFADs). The physiological functions of VFADs and analogous enzymes from other plant systems are discussed, as are the challenges with classifying desaturase activities. HighlightVFADs from P. patens and M. polymorpha produce monounsaturated very-long-chain fatty acids that are incorporated into sphingolipids and glycerolipids, likely via desaturation of an acyl-CoA substrate

plant biology↗

bioRxiv2025

Identification of INOSITOL PHOSPHORYLCERAMIDE SYNTHASE 2 (IPCS2) as a new rate-limiting component in Arabidopsis pathogen entry control

SIGNIFICANCE STATEMENTPolarized transport of executive defense gene products to sites of attempted microbial invasion is important for plant pathogen entry control and disease resistance. Here, we provide evidence that INOSITOL PHOSPHORYLCERAMIDE SYNTHASE 2 (IPCS2)-dependent sphingolipid production contributes to the role of the trans-Golgi network as a multi-domain sorting compartment, and mediates proper delivery of an ATP-binding cassette transporter to polarized plasma membrane domains at plant-microbe interaction sites. INOSITOL PHOSPHORYLCERAMIDE SYNTHASE 2 (IPCS2) is involved in the biosynthesis of complex sphingolipids at the trans-Golgi network (TGN). Here, we demonstrate a role of IPCS2 in penetration resistance against non-adapted powdery mildew fungi. A novel ipcs2W205* mutant was recovered from a forward genetic screen for Arabidopsis plants with enhanced epidermal cell entry success of the non-adapted barley fungus Blumeria graminis f. sp. hordei (Bgh). A yeast complementation assay and a sphingolipidomic approach revealed that the ipcs2W205* mutant represents a knock-out and lacks IPCS2-specific enzymatic activity. Further mutant analyses suggested that IPCS2-derived glycosyl inositol phosphorylceramides (GIPCs) are required for cell entry control of non-adapted fungal intruders. Confocal laser scanning microscopy (CLSM) studies indicated that upon pathogen attack, IPCS2 remains at the TGN to produce GIPCs, while focal accumulation of the defense cargo PENETRATION 3 (PEN3) at Bgh penetration sites was reduced in the ipcs2W205* mutant background. Thus, we propose a model in which sorting events at the TGN are facilitated by complex sphingolipids, regulating polar secretion of PEN3 to host-pathogen contact sites to terminate fungal ingress.

plant biology↗

bioRxiv2024

Complex sphingolipids are essential for cell division and plasmodesmal development in the moss Physcomitrium patens

Developmental patterning and organ structure are elegantly simple in the moss Physcomitrium patens. In molecular genetic studies, this facilitates both the cultivation of severe mutant alleles and their phenotypic characterization. Essential membrane lipids, such as complex phosphosphingolipids (in plants, glycosyl inositol phosphorylceramides, GIPCs), have been difficult to functionally characterize due to non-viable and pleiotropic phenotypes of mutants affected in their synthesis in Arabidopsis thaliana. Following the isolation and biochemical characterization of mutants affected in GIPC synthesis in P. patens, including sphinganine-C4-hydroxylase (s4h/sbh) and inositol phosphorylceramide synthase (ipcs), we now report some of their morphological, histological, and cytological phenotypes. We observed alteration in cell division, expansion, and differentiation. Specifically, the s4h knock-out mutant had abnormal cell division planes, as well as irregular depositions attached to cell walls. Severe ipcs mutant alleles showed frequent incomplete cell divisions, causing compromised cell autonomy as demonstrated by intercellular motility assays. These phenotypes suggest that sphingolipids impact both the orientation and proper formation of the cell plate during cytokinesis. Transmission electron microscopy revealed dramatic plasmodesmal structural defects in all three mutants, however, qualitative aspects of plasmodesmal transport do not seem to be severely impacted. Our methods can be used as a toolkit for quantifying growth, and specifically cell division and plasmodesmal phenotypes in mosses; our present results elucidate the specific contributions of GIPCs to fundamental cell functions. Finally, the severity of the observed defects in cell functions and ultrastructure highlight the resilience and utility of P. patens for studying basic cellular functions and severe mutant phenotypes.

plant biology↗

bioRxiv2024

Wax ester synthase overexpression affects stomatal development, water consumption and growth of poplars

Poplars are important fast-growing biomass crops. Their water-spending lifestyle renders them susceptible to drought and threatens plantations under global climate change with extended periods of water deprivation. The cuticle and stomatal regulation are major traits to protect plants from uncontrolled water loss. Here, we targeted the wax biosynthesis pathway of Populus x canescens by overexpressing jojoba (Simmondsia chinensis) wax ester synthase (ScWS) to improve cuticular properties. ScWS expression caused accumulation of lipid droplets inside the cells, decreased transcript levels of endogenous wax biosynthetic genes, and moderate shifts in surface wax composition but did not affect non-stomatal water loss. During short- and long-term drought scenarios under greenhouse and outdoor conditions, ScWS lines showed decreased stomatal conductance and increased water-use-efficiencies leading to a water-saving phenotype and delayed leaf shedding. This phenotype was caused by a high fraction (80%) of wax-occluded or semi-occluded stomata, and was accompanied by suppression of OCCLUDED STOMATAL PORE1 (OSP1), known to cause abberant wax accumulation at the stomatal ledges as found here. Occluded stomata limited poplar photosynthesis under high but not under low light intensities. Leaf damage and insect scores did not reveal differences compared with wild-type plants. Biomass production of ScWS lines was unaffected in short-term experiments but dropped below that of wild-type poplars at the end of two field seasons, indicating a growth trade-off. In conclusion, our study pinpoints a tight connection between wax biosynthesis and stomatal features and opens a new avenue to improve poplar water consumption by optimizing stomatal ledges with refined biotechnological approaches.

plant biology↗

bioRxiv2024

The GPAT4/6/8 clade functions in Arabidopsis root suberization non-redundantly with the GPAT5/7 clade required for suberin lamellae

SummaryLipid polymers such as cutin and suberin strengthen the diffusion barrier properties of the cell wall in specific cell types and are essential for water relations, mineral nutrition, and stress protection in plants. Land plant-specific glycerol-3-phosphate acyltransferases (GPATs) of different clades are central players in cutin and suberin monomer biosynthesis. Here, we show that the GPAT4/6/8 clade in Arabidopsis thaliana, which is known to mediate cutin formation, is also required for developmentally regulated root suberization, in addition to the established roles of GPAT5/7 in suberization. The GPAT5/7 clade is mainly required for abscisic acid-regulated suberization. In addition, the GPAT5/7 clade is crucial for the formation of the typical lamellated suberin ultrastructure observed by transmission electron microscopy, as distinct amorphous globular polyester structures were deposited in the apoplast of the gpat5 gpat7 double mutant, in contrast to the thinner but still lamellated suberin deposition in the gpat4 gpat6 gpat8 triple mutant. The intrinsic phosphatase activity of GPAT4, GPAT6, and GPAT8, which leads to monoacylglycerol biosynthesis, may be important for suberin biosynthesis. GPAT5/7 lack an active phosphatase domain. Notably, gpat5 gpat7 phenotypes were partially reverted by treatment with a phosphatase inhibitor or the expression of phosphatase-dead variants of GPAT4/6/8. Thus, GPATs that lack an active phosphatase domain, which are predicted to synthetize lysophosphatidic acids, might be crucial for the formation of the lamellated structure of suberin. GPATs with active and non-active phosphatase domains appear to have non-redundant functions and must cooperate to achieve the efficient biosynthesis of correctly structured suberin. Significance statementThe establishment of proper lamellated suberin in roots plays essential roles in regulating mineral nutrition and water relations in plants. The basis for the macromolecular arrangement determining the ultrastructure and properties of suberin lamellae is unknown. Here, we report that both the GPAT4/6/8 and GPAT5/7 clades of glycerol-3-phosphate acyltransferases (GPATs) contribute to suberin formation in Arabidopsis roots. In addition, we reveal that the GPAT5/7 clade is required for the formation of the lamellated suberin ultrastructure. Several lines of evidence suggest that the loss of phosphatase activity in GPATs might have played a crucial role in the formation of suberin lamellae during evolution.

plant biology↗

bioRxiv2024

Time-resolved oxidative signal convergence across the algae-embryophyte divide

The earliest land plants faced a significant challenge in adapting to environmental stressors. Stress on land is unique in its dynamics, entailing swift and drastic changes in light and temperature. While we know that land plants share with their closest streptophyte algal relatives key components of the genetic makeup for dynamic stress responses, their concerted action is little understood. Here, we combined time-course stress profiling using photophysiology, transcriptomics on 2.7 Tbp of data, and metabolite profiling analyses on more than 270 distinct samples, to study stress kinetics across three 600-million-year-divergent streptophytes. Through co-expression analysis and Granger causal inference we predict a gene regulatory network that retraces a web of ancient signal convergence at ethylene signaling components, osmosensors, and chains of major kinases. These kinase hubs already integrated diverse environmental inputs since before the dawn of plants on land.

evolutionary biology↗

bioRxiv2024

RfaA (YqhY), a novel adaptor protein, controls metabolite-sensitive protein degradation in Bacillus subtilis

The carboxylation of acetyl-CoA is the committed step of fatty acid synthesis catalyzed by the multisubunit acetyl-CoA carboxylase (ACCase). However, the mechanisms that control the activity of this enzyme are poorly understood. Here, we identify the so far unknown protein RfaA (YqhY) of the model bacterium Bacillus subtilis as a regulator of fatty acid acquisition that targets the AccC subunit of ACCase for degradation. RfaA interacts with both AccC and the ClpE unfoldase subunit of the ClpEP protease complex. While the former interaction is not sensitive to the physiological conditions, RfaA interacts with ClpE only in the absence of the global amino group donor glutamate. This results in the degradation of AccC in the absence of glutamate. The inactivation of the rfaA gene results in the accumulation of fatty acids in the cell and in the formation of lipid droplets which are toxic for the bacteria. The reduced fatty acid synthesis in the absence of glutamate as a result of RfaA-and ClpEP-dependent degradation of AccC thus prevents intoxication of the cells by fatty acids. Our findings suggest that the degradation of enzymes that catalyze the committed step of biosynthetic pathways might be an important mechanism to control cellular homeostasis. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/580884v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1f09555org.highwire.dtl.DTLVardef@1dc12e8org.highwire.dtl.DTLVardef@15d6961org.highwire.dtl.DTLVardef@1b1923e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Plasticity of the Arabidopsis leaf lipidome and proteome in response to pathogen infection and heat stress

Plants must cope with a variety of stressors during their life cycle, and the adaptive responses to these environmental cues involve all cellular organelles. Among them, comparatively little is known about the contribution of cytosolic lipid droplets (LDs) and their core set of neutral lipids and associated surface proteins to the rewiring of cellular processes in response to stress. Here, we analyzed the changes that occur in the lipidome and proteome of Arabidopsis leaves after pathogen infection with Botrytis cinerea or Pseudomonas syringae, or after heat stress. Analyses were carried out in wild-type plants and the oil-rich double mutant tgd1-1 sdp1-4 that allowed for an allied study of the LD proteome in stressed leaves. Using liquid chromatography-tandem mass spectrometry-based methods, we show that a hyperaccumulation of the primary LD core lipid triacylglycerol is a general response to stress and that acyl chain and sterol composition are remodeled during cellular adaptation. Likewise, comparative analysis of the LD protein composition in stress-treated leaves highlighted the plasticity of the LD proteome as part of the general stress response. We further identified at least two additional LD-associated proteins, whose localization to LDs in leaves was confirmed by confocal microscopy of fluorescent protein fusions. Taken together, these results highlight LDs as dynamic contributors to the cellular adaptation processes that underlie how plants respond to environmental stress. One sentence summaryBiotic and heat stress strongly alters the lipidome and proteome of Arabidopsis leaves including the proteome of lipid droplets.

plant biology↗

bioRxiv2024

Dual action of sphinganine in the plant disease resistance to bacteria.

Sphingolipids are ubiquitous, highly diverse molecules constituting at least 40% of plant plasma membranes. Initially known as modulators of membrane integrity, they now emerge as important players in plant responses to (a)biotic stresses. The interaction between Arabidopsis thaliana and the bacterium Pseudomonas syringae pv. tomato DC3000 AvrRpm1 (Pst AvrRpm1) culminates in the activation of a programmed cell death known as the hypersensitive response, which is part of the plant immune response. In this study, we showed that the co-infiltration of Pst AvrRpm1 and sphinganine (d18:0) in Arabidopsis leaves suppress the hypersensitive response. This suppression phenotype is also observed with bacteria carrying the effectors AvrB and AvrPphB but not with the ones carrying AvrRpt2 and AvrRps4. Sphingolipid-induced hypersensitive response suppression by Pst AvrRpm1 is correlated with the down-regulation of the gene AtNMT1 encoding a N-myristoyltransferase. d18:0 does not have a direct antibacterial effect and its co-infiltration in plants does not display typical signs of immune response such as activation of salicylic acid signaling pathway and extracellular reactive oxygen species production. Biophysical studies showed that d18:0 interacts with plant plasma membrane lipids. More specifically, d18:0 disturbs plant plasma membrane organization and mechanical properties. Our results demonstrate that sphingolipids play an important role in plant resistance, especially by interfering with the plasma membrane organization and effector localization and thus disturbing their function and subsequent immune responses.

plant biology↗

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↗

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↗

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

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↗