The phosphorylated pathway of serine biosynthesis links plant growth with nitrogen metabolism.

Because it is the precursor for various essential cellular components, the amino acid serine is indispensable for every living organism. In plants, serine is synthesized by two major pathways: photorespiration and the phosphorylated pathway of serine biosynthesis (PPSB). However, the importance of these pathways in providing serine for plant development is not fully understood. In this study, we examine the relative contributions of photorespiration and PPSB to providing serine for growth and metabolism in the C3 model plant Arabidopsis thaliana. Our analyses of cell proliferation and elongation reveal that PPSB-derived serine is indispensable for plant growth and its loss cannot be compensated by photorespiratory serine biosynthesis. Using isotope labeling, we show that PPSB-deficiency impairs the synthesis of proteins and purine nucleotides in plants. Furthermore, deficiency in PPSB-mediated serine biosynthesis leads to a strong accumulation of metabolites related to nitrogen metabolism. This result corroborates 15N-isotope labeling in which we observed an increased enrichment in labeled amino acids in PPSB-deficient plants. Expression studies indicate that elevated ammonium uptake and higher glutamine synthetase/glutamine oxoglutarate aminotransferase (GS/GOGAT) activity causes this phenotype. Metabolic analyses further show that elevated nitrogen assimilation and reduced amino acid turnover into proteins and nucleotides are the most likely driving forces for changes in respiratory metabolism and amino acid catabolism in PPSB-deficient plants. Accordingly, we conclude that even though photorespiration generates high amounts of serine in plants, PPSB-derived serine is more important for plant growth and its deficiency triggers the induction of nitrogen assimilation, most likely as an amino acid starvation response.

The Lotus japonicus acyl-acyl carrier protein thioesterase FatM is required for mycorrhiza formation and lipid accumulation of Rhizophagus irregularis.

Arbuscular mycorrhiza (AM) fungi establish symbiotic interactions with plants, providing the host plant with minerals, i.e. phosphate, in exchange for organic carbon. Arbuscular mycorrhiza fungi of the order Glomerales produce vesicles which store lipids as an energy and carbon source. Acyl-acyl carrier protein (ACP) thioesterases (Fat) are essential components of the plant plastid-localized fatty acid synthase and determine the chain length of de novo synthesized fatty acids. In addition to the ubiquitous FatA and FatB thioesterases, AM-competent plants contain an additional, AM-specific, FatM gene. Here, we characterize FatM from Lotus japonicus by phenotypically analyzing fatm mutant lines and by studying the biochemical function of the recombinant FatM protein. Reduced shoot phosphate content in fatm indicates compromised symbiotic phosphate uptake due to reduced arbuscule branching, and the fungus shows reduced lipid accumulation accompanied by the occurrence of smaller and less frequent vesicles. Lipid profiling reveals a decrease in mycorrhiza-specific phospholipid forms, AM fungal signature fatty acids (e.g. 16:1ω5, 18:1ω7 and 20:3) and storage lipids. Recombinant FatM shows preference for palmitoyl (16:0)-ACP, indicating that large amounts of 16:0 fatty acid are exported from the plastids of arbuscule-containing cells. Stable isotope labeling with [13 C2 ]acetate showed reduced incorporation into mycorrhiza-specific fatty acids in the fatm mutant. Therefore, colonized cells reprogram plastidial de novo fatty acid synthesis towards the production of extra amounts of 16:0, which is in agreement with previous results that fatty acid-containing lipids are transported from the plant to the fungus.

Specific membrane lipid composition is important for plasmodesmata function in Arabidopsis.

Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of "native" PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the ß-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes.

Lipids in plant-microbe interactions.

Bacteria and fungi can undergo symbiotic or pathogenic interactions with plants. Membrane lipids and lipid-derived molecules from the plant or the microbial organism play important roles during the infection process. For example, lipids (phospholipids, glycolipids, sphingolipids, sterol lipids) are involved in establishing the membrane interface between the two organisms. Furthermore, lipid-derived molecules are crucial for intracellular signaling in the plant cell, and lipids serve as signals during plant-microbial communication. These signal lipids include phosphatidic acid, diacylglycerol, lysophospholipids, and free fatty acids derived from phospholipase activity, apocarotenoids, and sphingolipid breakdown products such as ceramide, ceramide-phosphate, long chain base, and long chain base-phosphate. Fatty acids are the precursors for oxylipins, including jasmonic acid, and for azelaic acid, which together with glycerol-3-phosphate are crucial for the regulation of systemic acquired resistance. This article is part of a Special Issue titled "Plant Lipid Biology," guest editors Kent Chapman and Ivo Feussner.

Arbuscular mycorrhiza-specific enzymes FatM and RAM2 fine-tune lipid biosynthesis to promote development of arbuscular mycorrhiza.

During arbuscular mycorrhizal symbiosis (AMS), considerable amounts of lipids are generated, modified and moved within the cell to accommodate the fungus in the root, and it has also been suggested that lipids are delivered to the fungus. To determine the mechanisms by which root cells redirect lipid biosynthesis during AMS we analyzed the roles of two lipid biosynthetic enzymes (FatM and RAM2) and an ABC transporter (STR) that are required for symbiosis and conserved uniquely in plants that engage in AMS. Complementation analyses indicated that the biochemical function of FatM overlaps with that of other Fat thioesterases, in particular FatB. The essential role of FatM in AMS was a consequence of timing and magnitude of its expression. Lipid profiles of fatm and ram2 suggested that FatM increases the outflow of 16:0 fatty acids from the plastid, for subsequent use by RAM2 to produce 16:0 ß-monoacylglycerol. Thus, during AMS, high-level, specific expression of key lipid biosynthetic enzymes located in the plastid and the endoplasmic reticulum enables the root cell to fine-tune lipid biosynthesis to increase the production of ß-monoacylglycerols. We propose a model in which ß-monoacylglycerols, or a derivative thereof, are exported out of the root cell across the periarbuscular membrane for ultimate use by the fungus.

GLUCAN SYNTHASE-LIKE8 and STEROL METHYLTRANSFERASE2 are required for ploidy consistency of the sexual reproduction system in Arabidopsis.

In sexually reproducing plants, the meiocyte-producing archesporal cell lineage is maintained at the diploid state to consolidate the formation of haploid gametes. In search of molecular factors that regulate this ploidy consistency, we isolated an Arabidopsis thaliana mutant, called enlarged tetrad2 (et2), which produces tetraploid meiocytes through the stochastic occurrence of premeiotic endomitosis. Endomitotic polyploidization events were induced by alterations in cell wall formation, and similar cytokinetic defects were sporadically observed in other tissues, including cotyledons and leaves. ET2 encodes GLUCAN SYNTHASE-LIKE8 (GSL8), a callose synthase that mediates the deposition of callose at developing cell plates, root hairs, and plasmodesmata. Unlike other gsl8 mutants, in which defects in cell plate formation are seedling lethal, cytokinetic defects in et2 predominantly occur in flowers and have little effect on vegetative growth and development. Similarly, mutations in STEROL METHYLTRANSFERASE2 (SMT2), a major sterol biosynthesis enzyme, also lead to weak cytokinetic defects, primarily in the flowers. In addition, SMT2 allelic mutants also generate tetraploid meiocytes through the ectopic induction of premeiotic endomitosis. These observations demonstrate that appropriate callose and sterol biosynthesis are required for maintaining the ploidy level of the premeiotic germ lineage and that subtle defects in cytokinesis may lead to diploid gametes and polyploid offspring.

Fatty acid synthesis and lipid metabolism in the obligate biotrophic fungus Rhizophagus irregularis during mycorrhization of Lotus japonicus.

Arbuscular mycorrhiza formation with fungi of the Glomeromycota represents a widespread symbiotic interaction of vascular plants. Different signaling events and metabolic adaptations are required for the close interaction between the two partners. Membrane lipid synthesis is a prerequisite for symbiosis, and membrane properties depend on lipid composition. Lipid profiling was performed by liquid chromatography mass spectrometry to study the role of triacylglycerol, diacylglycerol, phospholipids, galactolipids, sterols and sphingolipids during the colonization of Lotus japonicus roots with Rhizophagus irregularis (syn. Glomus intraradices). Mycorrhization leads to an increased phosphate supply and suppresses the increase in galactolipids commonly observed in phosphate-deprived plants. In addition to free sterols and sterol esters, R. irregularis contains sterol glucosides and acylated sterol glucosides. Glycosylated sphingolipids (glucosylceramide, dihexosylceramide) and inositolphosphorylceramide were detected in the fungus. Lyso-phosphatidylcholine, a lipid previously implicated in mycorrhiza signaling, is present in low amounts in mock-infected and mycorrhized roots. The composition of fungal phospholipids changes after mycorrhization because molecular species with palmitvaccenic (di-16:1) or tetracosenoic (24:1) acyl groups decrease in intraradical mycelium. This adaptation of lipid metabolism during intraradical growth is likely a prerequisite for symbiosis, achieving functional compatibility between the fungal and the periarbuscular membrane. Data mining in genomic and transcript databases revealed the presence of genes encoding enzymes of lipid biosynthesis in R. irregularis. However, no gene encoding multidomain fatty acid de novo synthase was detected in the genome sequence of this obligate biotrophic fungus.

Fatty acid phytyl ester synthesis in chloroplasts of Arabidopsis.

During stress or senescence, thylakoid membranes in chloroplasts are disintegrated, and chlorophyll and galactolipid are broken down, resulting in the accumulation of toxic intermediates, i.e., tetrapyrroles, free phytol, and free fatty acids. Chlorophyll degradation has been studied in detail, but the catabolic pathways for phytol and fatty acids remain unclear. A large proportion of phytol and fatty acids is converted into fatty acid phytyl esters and triacylglycerol during stress or senescence in chloroplasts. We isolated two genes (PHYTYL ESTER SYNTHASE1 [PES1] and PES2) of the esterase/lipase/thioesterase family of acyltransferases from Arabidopsis thaliana that are involved in fatty acid phytyl ester synthesis in chloroplasts. The two proteins are highly expressed during senescence and nitrogen deprivation. Heterologous expression in yeast revealed that PES1 and PES2 have phytyl ester synthesis and diacylglycerol acyltransferase activities. The enzymes show broad substrate specificities and can employ acyl-CoAs, acyl carrier proteins, and galactolipids as acyl donors. Double mutant plants (pes1 pes2) grow normally but show reduced phytyl ester and triacylglycerol accumulation. These results demonstrate that PES1 and PES2 are involved in the deposition of free phytol and free fatty acids in the form of phytyl esters in chloroplasts, a process involved in maintaining the integrity of the photosynthetic membrane during abiotic stress and senescence.

Impaired sterol ester synthesis alters the response of Arabidopsis thaliana to Phytophthora infestans.

Non-host resistance of Arabidopsis thaliana against Phytophthora infestans, the causal agent of late blight disease of potato, depends on efficient extracellular pre- and post-invasive resistance responses. Pre-invasive resistance against P. infestans requires the myrosinase PEN2. To identify additional genes involved in non-host resistance to P. infestans, a genetic screen was performed by re-mutagenesis of pen2 plants. Fourteen independent mutants were isolated that displayed an enhanced response to Phytophthora (erp) phenotype. Upon inoculation with P. infestans, two mutants, pen2-1 erp1-3 and pen2-1 erp1-4, showed an enhanced rate of mesophyll cell death and produced excessive callose deposits in the mesophyll cell layer. ERP1 encodes a phospholipid:sterol acyltransferase (PSAT1) that catalyzes the formation of sterol esters. Consistent with this, the tested T-DNA insertion lines of PSAT1 are phenocopies of erp1 plants. Sterol ester levels are highly reduced in all erp1/psat1 mutants, whereas sterol glycoside levels are increased twofold. Excessive callose deposition occurred independently of PMR4/GSL5 activity, a known pathogen-inducible callose synthase. A similar formation of aberrant callose deposits was triggered by the inoculation of erp1 psat1 plants with powdery mildew. These results suggest a role for sterol conjugates in cell non-autonomous defense responses against invasive filamentous pathogens.

Cell-autonomous defense, re-organization and trafficking of membranes in plant-microbe interactions.

Plant cells dynamically change their architecture and molecular composition following encounters with beneficial or parasitic microbes, a process referred to as host cell reprogramming. Cell-autonomous defense reactions are typically polarized to the plant cell periphery underneath microbial contact sites, including de novo cell wall biosynthesis. Alternatively, host cell reprogramming converges in the biogenesis of membrane-enveloped compartments for accommodation of beneficial bacteria or invasive infection structures of filamentous microbes. Recent advances have revealed that, in response to microbial encounters, plasma membrane symmetry is broken, membrane tethering and SNARE complexes are recruited, lipid composition changes and plasma membrane-to-cytoskeleton signaling is activated, either for pre-invasive defense or for microbial entry. We provide a critical appraisal on recent studies with a focus on how plant cells re-structure membranes and the associated cytoskeleton in interactions with microbial pathogens, nitrogen-fixing rhizobia and mycorrhiza fungi.

Heterologous Production of Plant Terpenes in the Photosynthetic Bacterium Rhodobacter capsulatus.

Terpenes are one of the largest classes of secondary metabolites that occur in all kingdoms of life and offer diverse valuable properties for food and pharma industry including pleasant odor or taste as well as antimicrobial or anticancer activities. A multitude of terpene biosynthesis pathways are known, but their efficient biotechnological exploitation requires an adequate microorganism as host which can ideally provide an optimal supply with biosynthetic isoprene precursors. Rhodobacter capsulatus, a Gram-negative, facultative anaerobic, photosynthetic non-sulfur purple bacterium belonging to the α-proteobacteria represents such a host particularly suitable for terpene production. Here, we describe methods for the expression of terpene biosynthetic enzymes in R. capsulatus and the extraction of products for analysis. At the same time, we summarize the current strategies to adjust the biosynthetic precursor supply via isoprenoid biosynthetic pathways.

A processive glycosyltransferase involved in glycolipid synthesis during phosphate deprivation in Mesorhizobium loti.

Natural habitats are often characterized by a low availability of phosphate. In plants and many bacteria, phosphate deficiency causes different physiological responses, including the replacement of phosphoglycerolipids in the membranes with nonphosphorous lipids. We describe here a processive glycosyltransferase (Pgt) in Mesorhizobium loti (Rhizobiales) involved in the synthesis of di- and triglycosyldiacylglycerols (DGlycD and TGlycD) during phosphate deprivation. Cells of the corresponding Δpgt deletion mutant are deficient in DGlycD and TGlycD. Additional Pgt-independent lipids accumulate in Mesorhizobium after phosphate starvation, including diacylglyceryl trimethylhomoserine (DGTS) and ornithine lipid (OL). The accumulation of the nonphosphorous lipids during phosphate deprivation leads to the reduction of phosphoglycerolipids from 90 to 50%. Nodulation experiments of Mesorhizobium wild type and the Δpgt mutant with its host plant, Lotus japonicus, revealed that DGlycD and TGlycD are not essential for nodulation under phosphate-replete or -deficient conditions. Lipid measurements showed that the Pgt-independent lipids including OL and DGTS accumulate to higher proportions in the Δpgt mutant and therefore might functionally replace DGlycD and TGlycD during phosphate deprivation.

Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry.

Glycerolipids, sphingolipids, and sterol lipids constitute the major lipid classes in plants. Sterol lipids are composed of free and conjugated sterols, i.e., sterol esters, sterol glycosides, and acylated sterol glycosides. Sterol lipids play crucial roles during adaption to abiotic stresses and plant-pathogen interactions. Presently, no comprehensive method for sterol lipid quantification in plants is available. We used nanospray ionization quadrupole-time-of-flight mass spectrometry (Q-TOF MS) to resolve and identify the molecular species of all four sterol lipid classes from Arabidopsis thaliana. Free sterols were derivatized with chlorobetainyl chloride. Sterol esters, sterol glycosides, and acylated sterol glycosides were ionized as ammonium adducts. Quantification of molecular species was achieved in the positive mode after fragmentation in the presence of internal standards. The amounts of sterol lipids quantified by Q-TOF MS/MS were validated by comparison with results obtained with TLC/GC. Quantification of sterol lipids from leaves and roots of phosphate-deprived A. thaliana plants revealed changes in the amounts and molecular species composition. The Q-TOF method is far more sensitive than GC or HPLC. Therefore, Q-TOF MS/MS provides a comprehensive strategy for sterol lipid quantification that can be adapted to other tandem mass spectrometers.

Urine metabolites for the identification of Onchocerca volvulus infections in patients from Cameroon.

BACKGROUND: The tropical disease onchocerciasis (river blindness), caused by Onchocerca volvulus filarial nematodes, is targeted for elimination by mass treatment with nematocidal and antimicrobial drugs. Diagnosis of O. volvulus infections is based on counts of skin-borne microfilariae, but additional diagnostic tools, e.g. worm- or host-derived small RNAs, proteins or metabolites, are required for high-throughput screening. N-acetyltyramine-O,ß-glucuronide (NATOG) was suggested as a biomarker for onchocerciasis but its viability as diagnostic tool has been challenged. METHODS: We performed a screening program of urine samples from individuals from Cameroon infected with O. volvulus, Loa loa, Mansonella perstans or a combination thereof. Urine metabolites were measured by liquid chromatography-mass spectrometry (LC-MS). Principle component analysis (PCA) revealed that onchocerciasis causes complex changes of the urine metabolome. RESULTS: The mean NATOG content was elevated in urine of O. volvulus-infected compared with non-infected individuals, but NATOG levels showed considerable variation. However, 13.8% of all O. volvulus-infected individuals had high NATOG levels never reached by individuals without filarial infections or only infected with L. loa or M. perstans. Therefore, the identification of individuals with high NATOG levels might be used to screen for the elimination of onchocerciasis after mass drug application. Additional metabolites, including a compound identified as cinnamoylglycine, had high PC1/PC2 loadings in the data set. Mean levels of cinnamoylglycine were increased in O. volvulus-infected individuals, and 17.2% of all O. volvulus individuals had elevated cinnamoylglycine levels not reached by the controls. CONCLUSIONS: On an individual level, NATOG alone had poor discriminative power distinguishing infected from non-infected individuals. However, 13.8% of all O. volvulus-infected individuals had NATOG levels never reached by individuals without filarial infections or infected with only L. loa or M. perstans. Discrimination of O. volvulus infections from controls or individuals suffering from multiple infections was improved by the measurement of additional metabolites, e.g. cinnamoylglycine. Thus, measuring a combination of urine metabolites may provide a way to assess onchocerciasis on the population level. This provides the possibility to design a strategy for large-scale onchocerciasis epidemiological screening programs based on urine rather than invasive techniques.

Biosynthesis of cycloartenol by expression of plant and bacterial oxidosqualene cyclases in engineered Rhodobacter capsulatus.

Cyclic triterpenes are a large group of secondary metabolites produced by plants, fungi and bacteria. They have diverse biological functions, and offer potential health benefits for humans. Although various terpenes from the mono-, sesqui- and diterpene classes are easy to produce in engineered bacteria, heterologous synthesis of cyclic triterpenes is more challenging. We have recently shown that the triterpene cycloartenol can be produced in Rhodobacter capsulatus SB1003 but initial titers were low with 0.34mgL-1. To assess, if this phototrophic α-proteobacterium can be engineered for enhanced triterpene production, we followed two alternative strategies by comparing the performance of the R. capsulatus SB1003 wildtype strain with two recombinant strains carrying either a mevalonate pathway implemented from Paracoccus zeaxanthinifaciens or a deletion in the intrinsic carotenoid biosynthesis gene crtE. These strains are thus engineered for an enhanced isoprenoid biosynthesis or a suppressed precursor conversion by the competing carotenoid pathway. Moreover, three different cycloartenol synthase (CAS) genes from Arabidopsis thaliana or the myxobacterial strains Stigmatella aurantiaca Sga15 and DW4/3-1 were tested for heterologous cycloartenol synthesis. We found that the heterologous expression of mevalonate pathway enzymes had little impact on cycloartenol levels irrespective of the chosen CAS. In contrast, the use of the newly constructed carotenoid-deficient crtE deletion strain showed threefold increased cycloartenol product titers. We conclude that R. capsulatus is a promising alternative host for the functional expression of triterpene biosynthetic enzymes from plants and microbes. Apparently, product titers can also be improved by suppression of competing precursor consumption.

Lipid profiling of the filarial nematodes Onchocerca volvulus, Onchocerca ochengi and Litomosoides sigmodontis reveals the accumulation of nematode-specific ether phospholipids in the host.

Onchocerciasis, a neglected tropical disease prevalent in western and central Africa, is a major health problem and has been targeted for elimination. The causative agent for this disease is the human parasite Onchocerca volvulus. Onchocerca ochengi and Litomosoides sigmodontis, infectious agents of cattle and rodents, respectively, serve as model organisms to study filarial nematode infections. Biomarkers to determine infection without the use of painful skin biopsies and microscopic identification of larval worms are needed and their discovery is facilitated by an improved knowledge of parasite-specific metabolites. In addition to proteins and nucleic acids, lipids may be suitable candidates for filarial biomarkers that are currently underexplored. To fill this gap, we present the phospholipid profile of the filarial nematodes O. ochengi, O. volvulus and L. sigmodontis. Direct infusion quadrupole time-of-flight (Q-TOF) mass spectrometry was employed to analyze the composition of phospholipids and their molecular species in the three nematode species. Analysis of the phospholipid profiles of plasma or serum of uninfected and infected hosts showed that nematode-specific phospholipids were below detection limits. However, several phospholipids, in particular ether lipids of phosphatidylethanolamine (PE), were abundant in O. ochengi worms and in bovine nodule fluid, suggesting that these phospholipids might be released from O. ochengi into the host, and could serve as potential biomarkers.

Ergothioneine stands out from hercynine in the reaction with singlet oxygen: Resistance to glutathione and TRIS in the generation of specific products indicates high reactivity.

The candidate vitamin ergothioneine (ET), an imidazole-2-thione derivative of histidine betaine, is generally considered an antioxidant. However, the precise physiological role of ET is still unresolved. Here, we investigated in vitro the hypothesis that ET serves specifically to eradicate noxious singlet oxygen (1O2). Pure 1O2 was generated by thermolysis at 37°C of N,N'-di(2,3-dihydroxypropyl)-1,4-naphthalenedipropanamide 1,4-endoperoxide (DHPNO2). Assays of DHPNO2 with ET or hercynine (= ET minus sulfur) at pH 7.4 were analyzed by LC-MS in full scan mode to detect products. Based on accurate mass and product ion scan data, several products were identified and then quantitated as a function of time by selected reaction monitoring. All products of hercynine contained, after a [4+2] cycloaddition of 1O2, a carbonyl at position 2 of the imidazole ring. By contrast, because of the doubly bonded sulfur, we infer from the products of ET as the initial intermediates a 4,5-dioxetane (after [2+2] cycloaddition) and hydroperoxides at position 4 and 5 (after Schenck ene reactions). The generation of single products from ET, but not from hercynine, was fully resistant to a large excess of tris(hydroxymethyl)aminomethane (TRIS) or glutathione (GSH). This suggests that 1O2 markedly favors ET over GSH (at least 50-fold) and TRIS (at least 250-fold) for the initial reaction. Loss of ET was almost abolished in 5mM GSH, but not in 25mM TRIS. Regeneration of ET seems feasible, since some ET products - by contrast to hercynine products - decomposed easily in the MS collision cell to become aromatic again.

The photosynthetic bacteria Rhodobacter capsulatus and Synechocystis sp. PCC 6803 as new hosts for cyclic plant triterpene biosynthesis.

Cyclic triterpenes constitute one of the most diverse groups of plant natural products. Besides the intriguing biochemistry of their biosynthetic pathways, plant triterpenes exhibit versatile bioactivities, including antimicrobial effects against plant and human pathogens. While prokaryotes have been extensively used for the heterologous production of other classes of terpenes, the synthesis of cyclic triterpenes, which inherently includes the two-step catalytic formation of the universal linear precursor 2,3-oxidosqualene, is still a major challenge. We thus explored the suitability of the metabolically versatile photosynthetic α-proteobacterium Rhodobacter capsulatus SB1003 and cyanobacterium Synechocystis sp. PCC 6803 as alternative hosts for biosynthesis of cyclic plant triterpenes. Therefore, 2,3-oxidosqualene production was implemented and subsequently combined with different cyclization reactions catalyzed by the representative oxidosqualene cyclases CAS1 (cycloartenol synthase), LUP1 (lupeol synthase), THAS1 (thalianol synthase) and MRN1 (marneral synthase) derived from model plant Arabidopsis thaliana. While successful accumulation of 2,3-oxidosqualene could be detected by LC-MS analysis in both hosts, cyclase expression resulted in differential production profiles. CAS1 catalyzed conversion to only cycloartenol, but expression of LUP1 yielded lupeol and a triterpenoid matching an oxidation product of lupeol, in both hosts. In contrast, THAS1 expression did not lead to cyclic product formation in either host, whereas MRN1-dependent production of marnerol and hydroxymarnerol was observed in Synechocystis but not in R. capsulatus. Our findings thus indicate that 2,3-oxidosqualene cyclization in heterologous phototrophic bacteria is basically feasible but efficient conversion depends on both the respective cyclase enzyme and individual host properties. Therefore, photosynthetic α-proteo- and cyanobacteria are promising alternative candidates for providing new bacterial access to the broad class of triterpenes for biotechnological applications.

Lipid transfer from plants to arbuscular mycorrhiza fungi.

Arbuscular mycorrhiza (AM) symbioses contribute to global carbon cycles as plant hosts divert up to 20% of photosynthate to the obligate biotrophic fungi. Previous studies suggested carbohydrates as the only form of carbon transferred to the fungi. However, de novo fatty acid (FA) synthesis has not been observed in AM fungi in absence of the plant. In a forward genetic approach, we identified two Lotus japonicus mutants defective in AM-specific paralogs of lipid biosynthesis genes (KASI and GPAT6). These mutants perturb fungal development and accumulation of emblematic fungal 16:1ω5 FAs. Using isotopolog profiling we demonstrate that 13C patterns of fungal FAs recapitulate those of wild-type hosts, indicating cross-kingdom lipid transfer from plants to fungi. This transfer of labelled FAs was not observed for the AM-specific lipid biosynthesis mutants. Thus, growth and development of beneficial AM fungi is not only fueled by sugars but depends on lipid transfer from plant hosts.

Quadrupole time-of-flight mass spectrometry analysis of glycerophospholipid molecular species in the two halophyte seed oils: Eryngium maritimum and Cakile maritima.

Future applications of lipids in clinical cohort studies demand detailed glycerophospholipid molecule information and the application of high-throughput lipidomics platforms. In the present work, a novel sensitive technique with high mass resolution and accuracy was applied to accomplish phospholipid analysis. Nanospray ionization quadrupole time-of-flight mass spectrometry was used to separate and quantify the glycerophospholipid classes as well as molecular species in two halophyte seed oils from Cakile maritima and Eryngium maritimum. Precursor or neutral loss scans of their polar head groups allowed the detection of molecular species within particular glycerophospholipid classes. Phosphatidylcholine was found to be the most abundant glycerophospholipid in both seed oils whereas phosphatidylethanolamine and phosphatidic acid were less abundant. Phosphatidylinositol, phosphatidylserine and phosphatidylglycerol were minor glycerophospholipids. Several molecular species within each class were detected and the main molecular species (C36:4, C36:3, C36:2, 34:2 and C34:1) were quantitatively different between the two halophytes and the different glycerophospholipids.