A kiwellin disarms the metabolic activity of a secreted fungal virulence factor.

Fungi-induced plant diseases affect global food security and plant ecology. The biotrophic fungus Ustilago maydis causes smut disease in maize (Zea mays) plants by secreting numerous virulence effectors that reprogram plant metabolism and immune responses1,2. The secreted fungal chorismate mutase Cmu1 presumably affects biosynthesis of the plant immune signal salicylic acid by channelling chorismate into the phenylpropanoid pathway3. Here we show that one of the 20 maize-encoded kiwellins (ZmKWL1) specifically blocks the catalytic activity of Cmu1. ZmKWL1 hinders substrate access to the active site of Cmu1 through intimate interactions involving structural features that are specific to fungal Cmu1 orthologues. Phylogenetic analysis suggests that plant kiwellins have a versatile scaffold that can specifically counteract pathogen effectors such as Cmu1. We reveal the biological activity of a member of the kiwellin family, a widely conserved group of proteins that have previously been recognized only as important human allergens.

The shikimate pathway: gateway to metabolic diversity.

Covering: 1997 to 2023The shikimate pathway is the metabolic process responsible for the biosynthesis of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Seven metabolic steps convert phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P) into shikimate and ultimately chorismate, which serves as the branch point for dedicated aromatic amino acid biosynthesis. Bacteria, fungi, algae, and plants (yet not animals) biosynthesize chorismate and exploit its intermediates in their specialized metabolism. This review highlights the metabolic diversity derived from intermediates of the shikimate pathway along the seven steps from PEP and E4P to chorismate, as well as additional sections on compounds derived from prephenate, anthranilate and the synonymous aminoshikimate pathway. We discuss the genomic basis and biochemical support leading to shikimate-derived antibiotics, lipids, pigments, cofactors, and other metabolites across the tree of life.

The influence of model building schemes and molecular dynamics sampling on QM-cluster models: the chorismate mutase case study.

Most QM-cluster models of enzymes are constructed based on X-ray crystal structures, which limits comparison to in vivo structure and mechanism. The active site of chorismate mutase from Bacillus subtilis and the enzymatic transformation of chorismate to prephenate is used as a case study to guide construction of QM-cluster models built first from the X-ray crystal structure, then from molecular dynamics (MD) simulation snapshots. The Residue Interaction Network ResidUe Selector (RINRUS) software toolkit, developed by our group to simplify and automate the construction of QM-cluster models, is expanded to handle MD to QM-cluster model workflows. Several options, some employing novel topological clustering from residue interaction network (RIN) information, are evaluated for generating conformational clustering from MD simulation. RINRUS then generates a statistical thermodynamic framework for QM-cluster modeling of the chorismate mutase mechanism via refining 250 MD frames with density functional theory (DFT). The 250 QM-cluster models sampled provide a mean ΔG‡ of 10.3 ± 2.6 kcal mol-1 compared to the experimental value of 15.4 kcal mol-1 at 25 °C. While the difference between theory and experiment is consequential, the level of theory used is modest and therefore "chemical" accuracy is unexpected. More important are the comparisons made between QM-cluster models designed from the X-ray crystal structure versus those from MD frames. The large variations in kinetic and thermodynamic properties arise from geometric changes in the ensemble of QM-cluster models, rather from the composition of the QM-cluster models or from the active site-solvent interface. The findings open the way for further quantitative and reproducible calibration in the field of computational enzymology using the model construction framework afforded with the RINRUS software toolkit.

Biosensors for the detection of chorismate and cis,cis-muconic acid in Corynebacterium glutamicum.

Corynebacterium glutamicum ATCC 13032 is a promising microbial chassis for industrial production of valuable compounds, including aromatic amino acids derived from the shikimate pathway. In this work, we developed two whole-cell, transcription factor based fluorescent biosensors to track cis,cis-muconic acid (ccMA) and chorismate in C. glutamicum. Chorismate is a key intermediate in the shikimate pathway from which value-added chemicals can be produced, and a shunt from the shikimate pathway can divert carbon to ccMA, a high value chemical. We transferred a ccMA-inducible transcription factor, CatM, from Acinetobacter baylyi ADP1 into C. glutamicum and screened a promoter library to isolate variants with high sensitivity and dynamic range to ccMA by providing benzoate, which is converted to ccMA intracellularly. The biosensor also detected exogenously supplied ccMA, suggesting the presence of a putative ccMA transporter in C. glutamicum, though the external ccMA concentration threshold to elicit a response was 100-fold higher than the concentration of benzoate required to do so through intracellular ccMA production. We then developed a chorismate biosensor, in which a chorismate inducible promoter regulated by natively expressed QsuR was optimized to exhibit a dose-dependent response to exogenously supplemented quinate (a chorismate precursor). A chorismate-pyruvate lyase encoding gene, ubiC, was introduced into C. glutamicum to lower the intracellular chorismate pool, which resulted in loss of dose dependence to quinate. Further, a knockout strain that blocked the conversion of quinate to chorismate also resulted in absence of dose dependence to quinate, validating that the chorismate biosensor is specific to intracellular chorismate pool. The ccMA and chorismate biosensors were dually inserted into C. glutamicum to simultaneously detect intracellularly produced chorismate and ccMA. Biosensors, such as those developed in this study, can be applied in C. glutamicum for multiplex sensing to expedite pathway design and optimization through metabolic engineering in this promising chassis organism. ONE-SENTENCE SUMMARY: High-throughput screening of promoter libraries in Corynebacterium glutamicum to establish transcription factor based biosensors for key metabolic intermediates in shikimate and ß-ketoadipate pathways.

PBS3: a versatile player in and beyond salicylic acid biosynthesis in Arabidopsis.

AVRPPHB SUSCEPTIBLE 3 (PBS3) belongs to the GH3 family of acyl acid amido synthetases, which conjugates amino acids to diverse acyl acid substrates. Recent studies demonstrate that PBS3 in Arabidopsis plays a key role in the biosynthesis of plant defense hormone salicylic acid (SA) by catalyzing the conjugation of glutamate to isochorismate to form isochorismate-9-glutamate, which is then used to produce SA through spontaneous decay or ENHANCED PSEUDOMONAS SUSCEPTIBILITY (EPS1) catalysis. Consistent with its function as an essential enzyme for SA biosynthesis, PBS3 is well known to be a positive regulator of plant immunity in Arabidopsis. Additionally, PBS3 is also involved in the trade-off between abiotic and biotic stress responses in Arabidopsis by suppressing the inhibitory effect of abscisic acid on SA-mediated plant immunity. Besides stress responses, PBS3 also plays a role in plant development. Under long-day conditions, PBS3 influences Arabidopsis flowering time by regulating the expression of flowering regulators FLOWERING LOCUS C and FLOWERING LOCUS T. Taken together, PBS3 functions in the signaling network of plant development and responses to biotic and/or abiotic stresses, but the molecular mechanisms underlying its diverse roles remain obscure.

Metabolic flux analysis of coenzyme Q10 synthesized by Rhodobacter sphaeroides under the influence of different pH regulators.

Coenzyme Q10 (CoQ10) is crucial for human beings, especially in the fields of biology and medicine. The aim of this experiment was to investigate the conditions for increasing CoQ10 production. At present, microbial fermentation is the main production method of CoQ10, and the production process of microbial CoQ10 metabolism control fermentation is very critical. Metabolic flux is one of the most important determinants of cell physiology in metabolic engineering. Metabolic flux analysis (MFA) is used to estimate the intracellular flux in metabolic networks. In this experiment, Rhodobacter sphaeroides was used as the research object to analyze the effects of aqueous ammonia (NH3·H2O) and calcium carbonate (CaCO3) on the metabolic flux of CoQ10. When CaCO3 was used to adjust the pH, the yield of CoQ10 was 274.43 mg·L-1 (8.71 mg·g-1 DCW), which was higher than that of NH3·H2O adjustment. The results indicated that when CaCO3 was used to adjust pH, more glucose-6-phosphate (G6P) entered the pentose phosphate (HMP) pathway and produced more NADPH, which enhanced the synthesis of CoQ10. At the chorismic acid node, more metabolic fluxes were involved in the synthesis of p-hydroxybenzoic acid (pHBA; the synthetic precursor of CoQ10), enhancing the anabolic flow of CoQ10. In addition, Ca2+ produced by the reaction of CaCO3 with organic acids promotes the synthesis of CoQ10. In summary, the use of CaCO3 adjustment is more favorable for the synthesis of CoQ10 by R. sphaeroides than NH3·H2O adjustment. The migration of metabolic flux caused by the perturbation of culture conditions was analyzed to compare the changes in the distribution of intracellular metabolic fluxes for the synthesis of CoQ10. Thus, the main nodes of the metabolic network were identified as G6P and chorismic acid. This provides a theoretical basis for the modification of genes related to the CoQ10 synthesis pathway.

Validated MAGIC and GWAS population mapping reveals the link between vitamin E content and natural variation in chorismate metabolism in tomato.

Tocochromanols constitute the different forms of vitamin E (VTE), essential components of the human diet, and display a high membrane protectant activity. By combining interval mapping and genome-wide association studies (GWAS), we unveiled the genetic determinants of tocochromanol accumulation in tomato (Solanum lycopersicum) fruits. To enhance the nutritional value of this highly consumed vegetable, we dissected the natural intraspecific variability of tocochromanols in tomato fruits and genetically engineered their biosynthetic pathway. These analyses allowed the identification of a total of 25 quantitative trait loci interspersed across the genome pinpointing the chorismate-tyrosine pathway as a regulatory hub controlling the supply of the aromatic head group for tocochromanol biosynthesis. To validate the link between the chorismate-tyrosine pathway and VTE, we engineered tomato plants to bypass the pathway at the arogenate branch point. Transgenic tomatoes showed moderate increments in tocopherols (up to approximately 20%) and a massive accumulation of tocotrienols (up to approximately 3400%). Gene expression analyses of these plants reveal a trade-off between VTE and natural variation in chorismate metabolism explained by transcriptional reprogramming of specific structural genes of the pathway. By restoring the accumulation of alpha-tocotrienols (α-t3) in fruits, the plants produced here are of high pharmacological and nutritional interest.

Can the local electric field be a descriptor of catalytic activity? A case study on chorismate mutase.

The current theoretical perception of enzymatic activity is highly reliant on the determination of the activation energy of the reactions, which is often calculated using computationally demanding quantum mechanical calculations. With the ever-increasing use of bioengineering techniques that produce too many variants of the same enzyme, a fast and accurate way to study the relative efficiency of enzymes is currently in high demand. Here, we propose the local electric field (LEF) of the enzyme along the reaction axis as a descriptor for the enzymatic activity using the example of chorismate mutase in its native form and several variants (R90A, R90G, and R90K/C88S). The study shows a direct correlation between the calculated enzymatic EF and the enzymatic activity for all the complexes. MD simulations of the Michaelis complex and the transition state analog (TSA) show a stabilizing force on the TSA due to the enzymatic EF. QM/MM and QM-only DFT calculations in the presence of an external electric field (EEF) oriented along the reaction axis show that the electric field can interact with the dipole moment of the TS, thereby stabilizing it and thus lowering the activation energy.

Dual-Localized WHIRLY1 Affects Salicylic Acid Biosynthesis via Coordination of ISOCHORISMATE SYNTHASE1, PHENYLALANINE AMMONIA LYASE1, and S-ADENOSYL-L-METHIONINE-DEPENDENT METHYLTRANSFERASE1.

Salicylic acid (SA) influences developmental senescence and is spatiotemporally controlled by various mechanisms, including biosynthesis, transport, and conjugate formation. Altered localization of Arabidopsis WHIRLY1 (WHY1), a repressor of leaf natural senescence, in the nucleus or chloroplast causes a perturbation in SA homeostasis, resulting in adverse plant senescence phenotypes. WHY1 loss-of-function mutation resulted in SA peaking 5 d earlier compared to wild-type plants, which accumulated SA at 42 d after germination. SA accumulation coincided with an early leaf-senescence phenotype, which could be prevented by ectopic expression of the nuclear WHY1 isoform (nWHY1). However, expressing the plastid WHY1 isoform (pWHY1) greatly enhanced cellular SA levels. Transcriptome analysis in the WHY1 loss-of-function mutant background following expression of either pWHY1 or nWHY1 indicated that hormone metabolism-related genes were most significantly altered. The pWHY1 isoform predominantly affected stress-related gene expression, whereas nWHY1 primarily controlled developmental gene expression. Chromatin immunoprecipitation-quantitative PCR assays indicated that nWHY1 directly binds to the promoter region of isochorismate synthase1 (ICS1), thus activating its expression at later developmental stages, but that it indirectly activates S-adenosyl- l -Met-dependent methyltransferase1 (BSMT1) expression via ethylene response factor 109 (ERF109). Moreover, nWHY1 repressed expression of Phe ammonia lyase-encoding gene (PAL1) via R2R3-MYB member 15 (MYB15) during the early stages of development. Interestingly, rising SA levels exerted a feedback effect by inducing nWHY1 modification and pWHY1 accumulation. Thus, the alteration of WHY1 organelle isoforms and the feedback of SA are involved in a circularly integrated regulatory network during developmental or stress-induced senescence in Arabidopsis.

Characterization of two 3-deoxy-d-Arabino-Heptulosonate 7-phosphate synthases from Bacillusmethanolicus.

3-Deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase catalyzes the condensation of phosphoenolpyruvate (PEP) with d-erythrose 4-phosphate (E4P) and plays an important role in regulating carbon flux toward aromatic amino acid biosynthesis in bacteria and plants. Sequence analysis of the DAHP synthases AroG1 and AroG2 from Bacillus methanolicus MGA3 suggested this thermophilic, methylotrophic bacterium possesses two type Iß DAHP synthases. This study describes production of AroG1 and AroG2 in Escherichia coli as hexa-histidine fused proteins, which were purified by affinity chromatography. Treatment with TEV protease afforded native proteins for characterization and kinetic analysis. AroG1 and AroG2 are, respectively, 30.1 kDa and 40.0 kDa proteins. Both enzymes have maximal activity over a pH range of 6.3-7.2. The apparent kinetic parameters at 50 °C and pH 7.2 for AroG1 are KmPEP 1100 ± 100 µM, KmE4P 530 ± 100 µM, and kcat 10.3 ± 1.2 s-1. The kinetic parameters for AroG2 are KmPEP 90 ± 20 µM, KmE4P 130 ± 40 µM, and kcat 2.0 ± 0.2 s-1. At 50 °C AroG2 retains 50% of its activity after 96 min whereas AroG1 retains less than 5% of its activity after 10 min. AroG2, which contains an N-terminal regulatory domain, is inhibited by chorismate and prephenate but not l-phenylalanine, l-tyrosine, or l-tryptophan. AroG1 is not inhibited by any of the molecules examined. Understanding DAHP synthase regulation in B. methanolicus is a first step toward generating biocatalysts that exploit the target-rich aromatic amino acid biosynthetic pathway for synthesis of chemicals from methanol.

In vitro characterization of 3-chloro-4-hydroxybenzoic acid building block formation in ambigol biosynthesis.

The cyanobacterium Fischerella ambigua is a natural producer of polychlorinated aromatic compounds, the ambigols A-E. The biosynthetic gene cluster (BGC) of these highly halogenated triphenyls has been recently identified by heterologous expression. It consists of 10 genes named ab1-10. Two of the encoded enzymes, i.e. Ab2 and Ab3, were identified by in vitro and in vivo assays as cytochrome P450 enzymes responsible for biaryl and biaryl ether formation. The key substrate for these P450 enzymes is 2,4-dichlorophenol, which in turn is derived from the precursor 3-chloro-4-hydroxybenzoic acid. Here, the biosynthetic steps leading towards 3-chloro-4-hydroxybenzoic acid were investigated by in vitro assays. Ab7, an isoenzyme of a 3-deoxy-7-phosphoheptulonate (DAHP) synthase, is involved in chorismate biosynthesis by the shikimate pathway. Chorismate in turn is further converted by a dedicated chorismate lyase (Ab5) yielding 4-hydroxybenzoic acid (4-HBA). The stand alone adenylation domain Ab6 is necessary to activate 4-HBA, which is subsequently tethered to the acyl carrier protein (ACP) Ab8. The Ab8 bound substrate is chlorinated by Ab10 in meta position yielding 3-Cl-4-HBA, which is then transfered by the condensation (C) domain to the peptidyl carrier protein and released by the thioesterase (TE) domain of Ab9. The released product is then expected to be the dedicated substrate of the halogenase Ab1 producing the monomeric ambigol building block 2,4-dichlorophenol.

Induction of pre-chorismate, jasmonate and salicylate pathways by Burkholderia sp. RR18 in peanut seedlings.

AIMS: To characterize the mechanisms by which bacteria in the peanut rhizosphere promote plant growth and suppress Aspergillus niger, the fungus that causes collar rot of peanut. METHODS AND RESULTS: In all, 131 isolates cultured from the peanut rhizosphere were assayed for growth promotion in a seedling germination assay. The most effective isolate, RR18, was identified as Burkholderia sp. by 16S sequencing analysis. RR18 reduced collar rot disease incidence and increased the germination rate and biomass of peanut seeds, and had broad-spectrum antifungal activity. Quantitative analyses showed that RR18 induced long-lasting accumulation of jasmonic acid, salicylic acid and phenols, and triggered the activity of six defence enzymes related to these changes. Comparative proteomic analysis of treated and untreated seedlings revealed a clear induction of four abundant proteins, including a member of the pre-chorismate pathway, a regulator of clathrin-coated vesicles, a transcription factor and a hypothetical protein. CONCLUSION: Burkholderia sp. RR18 promotes peanut growth and disease resistance, and stably induces two distinct defence pathways associated with systemic resistance. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates that a strain of the Burkholderia cepacia complex can elicit both salicylic- and jasmonic-acid-mediated defences, in addition to having numerous other beneficial properties.

Brassicaceae-specific Gretchen Hagen 3 acyl acid amido synthetases conjugate amino acids to chorismate, a precursor of aromatic amino acids and salicylic acid.

To modulate responses to developmental or environmental cues, plants use Gretchen Hagen 3 (GH3) acyl acid amido synthetases to conjugate an amino acid to a plant hormone, a reaction that regulates free hormone concentration and downstream responses. The model plant Arabidopsis thaliana has 19 GH3 proteins, of which 8 have confirmed biochemical functions. One Brassicaceae-specific clade of GH3 proteins was predicted to use benzoate as a substrate and includes AtGH3.7 and AtGH3.12/PBS3. Previously identified as a 4-hydroxybenzoic acid-glutamate synthetase, AtGH3.12/PBS3 influences pathogen defense responses through salicylic acid. Recent work has shown that AtGH3.12/PBS3 uses isochorismate as a substrate, forming an isochorismate-glutamate conjugate that converts into salicylic acid. Here, we show that AtGH3.7 and AtGH3.12/PBS3 can also conjugate chorismate to cysteine and glutamate, which act as precursors to aromatic amino acids and salicylic acid, respectively. The X-ray crystal structure of AtGH3.12/PBS3 in complex with AMP and chorismate at 1.94 Å resolution, along with site-directed mutagenesis, revealed how the active site potentially accommodates this substrate. Examination of Arabidopsis knockout lines indicated that the gh3.7 mutants do not alter growth and showed no increased susceptibility to the pathogen Pseudomonas syringae, unlike gh3.12 mutants, which were more susceptible than WT plants, as was the gh3.7/gh3.12 double mutant. The findings of our study suggest that GH3 proteins can use metabolic precursors of aromatic amino acids as substrates.

Random insertion transposon mutagenesis of Mycobacterium fortuitum identified mutant defective in biofilm formation.

Mycobacterium fortuitum has emerged as a nosocomial infectious agent and biofilm formation attributed for the presence of this bacterium in hospital environment. Transposon random mutagenesis was used to identify membrane-proteins for biofilm formation in M. fortuitum. Ten mutants were shortlisted from a library of 450 mutants for examine their biofilm forming ability. Comparative biofilm ability with respect to wild type M. fortuitum ATCC 6841 showed an altered and delayed biofilm formation in one mutant namely, MT721. Sequence analysis revealed mutation in anthranilate phosphoribosyl transferase (MftrpD), which is associated with tryptophan operon. Functional interaction study of TrpD protein through STRING showed its interaction with chorismate utilizing proteins, majorly involved in synthesis of aromatic amino acid and folic acid, suggesting that biofilm establishment and maintenance requires components of central metabolism. Our study indicates important role of MftrpD in establishment and maintenance of biofilm by M. fortuitum, which may further be explored for drug discovery studies against mycobacterial infections.

Metabolic engineering of Escherichia coli for production of chemicals derived from the shikimate pathway.

The shikimate pathway is indispensable for the biosynthesis of natural products with aromatic moieties. These products have wide current and potential applications in food, cosmetics and medicine, and consequently have great commercial value. However, compounds extracted from various plants or synthesized from petrochemicals no longer satisfy the requirements of contemporary industries. As a result, an increasing number of studies has focused on this pathway to enable the biotechnological manufacture of natural products, especially in E. coli. Furthermore, the development of synthetic biology, systems metabolic engineering and high flux screening techniques has also contributed to improving the biosynthesis of high-value compounds based on the shikimate pathway. Here, we review approaches based on a combination of traditional and new metabolic engineering strategies to increase the metabolic flux of the shikimate pathway. In addition, applications of this optimized pathway to produce aromatic amino acids and a range of natural products is also elaborated. Finally, this review sums up the opportunities and challenges facing this field.

Alteration of the Route to Menaquinone towards Isochorismate-Derived Metabolites.

Chorismate and isochorismate constitute branch-point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli. We propose a model for the binding of isochorismate to the active site of MenD ((1R,2S, 5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α-ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double-variant Asn117Arg-Leu478Thr preferentially converts (5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHD), the hydrolysis product of isochorismate, with a >70-fold higher ratio than that for the wild type. The single-variant Arg107Ile uses (5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHA) as substrate with >6-fold conversion compared to wild-type MenD. The novel compounds have been made accessible in vivo (up to 5.3 g L-1 ). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild-type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.

Chorismatases - the family is growing.

Chorismatases catalyse the cleavage of chorismate, yielding (dihydroxy-)benzoate derivatives, which often constitute starter units for pharmaceutically relevant secondary metabolites. Depending on their products, chorismatases have been classified into three different subfamilies. These can be assigned using a set of amino acid residues in the active site. Here, we describe five new chorismatases, two of them members of a new subfamily, which has been discovered through correlation analysis of homologous protein sequences. The enzymes from the new subfamily produce exclusively 4-hydroxybenzoate, the same compound as produced by the structurally unrelated chorismate lyases. This showcase of convergent evolution is an example of the existence of more than one pathway to central building blocks. In contrast to chorismate lyases, however, chorismatases do not suffer from product inhibition (up to 2 mM 4-HBA), while the remaining kinetic parameters are in the same range; this makes them an interesting alternative for biocatalytic applications.

Isolation and characterization of mutants corresponding to the MENA, MENB, MENC and MENE enzymatic steps of 5'-monohydroxyphylloquinone biosynthesis in Chlamydomonas reinhardtii.

Phylloquinone (PhQ), or vitamin K1 , is an essential electron carrier (A1 ) in photosystem I (PSI). In the green alga Chlamydomonas reinhardtii, which is a model organism for the study of photosynthesis, a detailed characterization of the pathway is missing with only one mutant deficient for MEND having been analyzed. We took advantage of the fact that a double reduction of plastoquinone occurs in anoxia in the A1 site in the mend mutant, interrupting photosynthetic electron transfer, to isolate four new phylloquinone-deficient mutants impaired in MENA, MENB, MENC (PHYLLO) and MENE. Compared with the wild type and complemented strains for MENB and MENE, the four men mutants grow slowly in low light and are sensitive to high light. When grown in low light they show a reduced photosynthetic electron transfer due to a specific decrease of PSI. Upon exposure to high light for a few hours, PSI becomes almost completely inactive, which leads in turn to lack of phototrophic growth. Loss of PhQ also fully prevents reactivation of photosynthesis after dark anoxia acclimation. In silico analyses allowed us to propose a PhQ biosynthesis pathway in Chlamydomonas that involves 11 enzymatic steps from chorismate located in the chloroplast and in the peroxisome.

An E. coli biosensor for screening of cDNA libraries for isochorismate pyruvate lyase-encoding cDNAs.

Salicylic acid (SA) is an essential hormone for development and induced defense against biotrophic pathogens in plants. The formation of SA mainly derives from chorismate via demonstrated isochorismate synthase (ICS) and presumed isochorismate pyruvate lyase (IPL)-mediated steps in Arabidopsis thaliana, but so far no plant enzyme displaying IPL activity has been identified. Here, we developed an E. coli SA biosensor to screen for IPL activity based on the SalR regulator/salA promoter combination from Acinetobacter sp ADP1, to control the expression of the reporter luxCDABE. The biosensor was responsive to micromolar concentrations of exogenous SA, and to endogenous SA produced after transformation with a plasmid permitting IPTG-inducible expression of bacterial IPL in this biosensor strain. After screening a cDNA library constructed from turnip crinkle virus (TCV)-infected Arabidopsis ecotype Di-17, we identified an enzyme, PRXR1, as a putative IPL that converts isochorismate into SA. Our results provide a new experimental approach to identify IPL and new insights into the SA biosynthesis pathway in Arabidopsis.

ENO2 knock-out mutants in Arabidopsis modify the regulation of the gene expression response to NaCl stress.

There is a growing awareness that some dual-function enzymes may provide a directly evidence that metabolism could feed into the regulation of gene expression via metabolic enzymes. However, the mechanism by which metabolic enzymes control gene expression to optimize plant stress responses remains largely unknown in Arabidopsis thaliana. LOS2/ENO2 is a bifunctional gene transcribed a functional RNA that translates a full-length version of the ENO2 protein and a truncated version of the MBP-1 protein. Here, we report that eno2 negatively regulates plant tolerance to salinity stress. NaCl treatment caused the death of the mutant eno2/eno2 homozygote earlier than the wild type (WT) Arabidopsis. To understand the mechanism by which the mutant eno2 had a lower NaCl tolerance, an analysis of the expressed sequence tag (EST) dataset from the WT and mutant eno2 Arabidopsis was conducted. Firstly, the most identified up- and down-regulated genes are senescence-associated gene 12 (SAG12) and isochorismate mutase-related gene, which are associated with salicylic acid (SA) inducible plant senescence and endogenous SA synthesis, respectively. Secondly, the differentially regulated by salt stress genes in mutant eno2 are largely enriched Gene Ontology(GO) terms associated with various kinds of response to stimulations. Thirdly, in the Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping, we find that knocking out ENO2-influenced genes were most enriched into metabolite synthesis with extra plant-pathogen interaction pathway and plant hormone signal transduction pathway. Briefly, with the translation shifting function, LOS2/ENO2 not only influenced the genes involved in SA synthesis and transduction, but also influenced genes that participate in metabolite synthesis in cytoplasm and gene expression variation in nuclear under salt stress.