In-depth analysis of isochorismate synthase-derived metabolism in plant immunity: Identification of meta-substituted benzoates and salicyloyl-malate.

Isochorismate-derived metabolism enables biosynthesis of the plant defense hormone salicylic acid (SA) and its derivatives. In Arabidopsis thaliana, the stress-induced accumulation of SA depends on ISOCHORISMATE SYNTHASE1 (ICS1) and also requires the presumed isochorismate transporter ENHANCED DISEASE SUSCEPTIBILITY5 (EDS5) and the GH3 enzyme avrPphB SUSCEPTIBLE3 (PBS3). By comparative metabolite and structural analyses, we identified several hitherto unreported ICS1- and EDS5-dependent, biotic stress-inducible Arabidopsis metabolites. These involve meta-substituted SA derivatives (5-formyl-SA, 5-carboxy-SA, 5-carboxymethyl-SA), their benzoic acid (BA) analogs (3-formyl-BA, 3-carboxy-BA, 3-carboxymethyl-BA), and besides the previously detected salicyloyl-aspartate (SA-Asp), the ester conjugate salicyloyl-malate (SA-Mal). SA functions as a biosynthetic precursor for SA-Mal and SA-Asp, but not for the meta-substituted SA- and BA-derivatives, which accumulate to moderate levels at later stages of bacterial infection. Interestingly, Arabidopsis leaves possess oxidizing activity to effectively convert meta-formyl- into meta-carboxy-SA/BAs. In contrast to SA, exogenously applied meta-substituted SA/BA-derivatives and SA-Mal exert a moderate impact on plant immunity and defence-related gene expression. While the isochorismate-derived metabolites are negatively regulated by the SA receptor NON-EXPRESSOR OF PR GENES1, SA conjugates (SA-Mal, SA-Asp, SA-glucose conjugates) and meta-substituted SA/BA-derivatives are oppositely affected by PBS3. Notably, our data indicate a PBS3-independent path to isochorismate-derived SA at later stages of bacterial infection, which does not considerably impact immune-related characteristics. Moreover, our results argue against a previously proposed role of EDS5 in the biosynthesis of the immune signal N-hydroxypipecolic acid and associated transport processes. We propose a significantly extended biochemical scheme of plant isochorismate metabolism that involves an alternative generation mode for benzoate- and salicylate-derivatives.

The OsICS1 is directly regulated by OsWRKY6 and increases resistance against Xanthomonas oryzae pv. oryzae.

KEY MESSAGE: OsICS1 but not OsICS1-L mediates the rice response to Xoo inoculation, with its overexpression increasing resistance against this pathogen. OsICS1 but not OsICS-L is directly upregulated by OsWRKY6. Rice (Oryza sativa) is a staple crop for about half of the global population and is particularly important in the diets of people living in Asia, Latin America, and Africa. This crop is continually threatened by bacterial leaf blight disease caused by Xanthomonas oryzae pv. oryzae (Xoo), which drastically reduces yields; therefore, it is needed to elucidate the plant's resistance mechanisms against Xoo. Isochorismate synthase (ICS1) generates salicylic acid (SA) and increases resistance against bacterial disease. The OsICS1 is differently annotated in rice genome databases and has not yet been functionally characterized in the context of Xoo infection. Here, we report that the expression of the OsICS1 is directly regulated by OsWRKY6 and increases plant resistance against Xoo. Inoculation with Xoo increased the expression of OsICS1 but not that of the long variant of OsICS1 (OsICS1-L). OsWRKY6 directly activated the OsICS1 promoter but not the OsICS1-L promoter. OsICS1 overexpression in rice increased resistance against Xoo through the induction of SA-dependent bacterial defense genes. These data show that OsICS1 promotes resistance against Xoo infection.

Enzymatic reconstitution of salicylate formation in promysalin biosynthesis.

Promysalin is an amphipathic antibiotic isolated from Pseudomonas promysalinigenes (previously Pseudomonas putida RW10S1) which shows potent antibacterial activities against Gram-negative pathogens by inactivating succinate dehydrogenase. Based on the in-vivo studies, promysalin is hypothesized to be assembled from three building blocks: salicylic acid, proline, and myristic acid via a proposed but uncharacterized hybrid NRPS-PKS biosynthetic pathway. So far, no in-vitro biosynthetic studies have been reported for this promising antibiotic. Here, we report the first in-vitro reconstitution and biochemical characterization of two early enzymes on the pathway: PpgH, an isochorismate synthase (IS), and PpgG, an isochorismate pyruvate lyase (IPL) which are involved in the biosynthesis of salicylic acid, the polar fragment of promysalin. We also report a secondary chorismate mutase (CM) activity for PpgG. Based on our biochemical experiments, preliminary mechanistic proposals have been postulated for PpgH and PpgG. We believe this study will lay a strong foundation for elucidating the functions and mechanisms of other intriguing enzymes of the promysalin biosynthesis pathway, which may potentially unravel interesting enzyme chemistries and promote pathway engineering in the future.

Identification of salicylic acid-independent responses in an Arabidopsis phosphatidylinositol 4-kinase beta double mutant.

BACKGROUND AND AIMS: We have recently shown that an Arabidopsis thaliana double mutant of type III phosphatidylinositol-4-kinases (PI4Ks), pi4kß1ß2, constitutively accumulated a high level of salicylic acid (SA). By crossing this pi4kß1ß2 double mutant with mutants impaired in SA synthesis (such as sid2 impaired in isochorismate synthase) or transduction, we demonstrated that the high SA level was responsible for the dwarfism phenotype of the double mutant. Here we aimed to distinguish between the SA-dependent and SA-independent effects triggered by the deficiency in PI4Kß1 and PI4Kß2. METHODS: To achieve this we used the sid2pi4kß1ß2 triple mutant. High-throughput analyses of phytohormones were performed on this mutant together with pi4kß1ß2 and sid2 mutants and wild-type plants. Responses to pathogens, namely Hyaloperonospora arabidopsidis, Pseudomonas syringae and Botrytis cinerea, and also to the non-host fungus Blumeria graminis, were also determined. Callose accumulation was monitored in response to flagellin. KEY RESULTS: We show here the prominent role of high SA levels in influencing the concentration of many other tested phytohormones, including abscisic acid and its derivatives, the aspartate-conjugated form of indole-3-acetic acid and some cytokinins such as cis-zeatin. We show that the increased resistance of pi4kß1ß2 plants to the host pathogens H. arabidopsidis, P. syringae pv. tomato DC3000 and Bothrytis cinerea is dependent on accumulation of high SA levels. In contrast, accumulation of callose in pi4kß1ß2 after flagellin treatment was independent of SA. Concerning the response to Blumeria graminis, both callose accumulation and fungal penetration were enhanced in the pi4kß1ß2 double mutant compared to wild-type plants. Both of these processes occurred in an SA-independent manner. CONCLUSIONS: Our data extensively illustrate the influence of SA on other phytohormone levels. The sid2pi4kß1ß2 triple mutant revealed the role of PI4Kß1/ß2 per se, thus showing the importance of these enzymes in plant defence responses.

Identification of the heme acquisition system in Vibrio vulnificus M2799.

Vibrio vulnificus, the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V. vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V. vulnificus. Further investigation showed that hupA, hvtA, and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA, but not hvtA. In addition, ptrB was co-transcribed with hvtA, and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C.

Cooperative functioning between phenylalanine ammonia lyase and isochorismate synthase activities contributes to salicylic acid biosynthesis in soybean.

Salicylic acid (SA), an essential regulator of plant defense, is derived from chorismate via either the phenylalanine ammonia lyase (PAL) or the isochorismate synthase (ICS) catalyzed steps. The ICS pathway is thought to be the primary contributor of defense-related SA, at least in Arabidopsis. We investigated the relative contributions of PAL and ICS to defense-related SA accumulation in soybean (Glycine max). Soybean plants silenced for five PAL isoforms or two ICS isoforms were analyzed for SA concentrations and SA-derived defense responses to the hemibiotrophic pathogens Pseudomonas syringae and Phytophthora sojae. We show that, unlike in Arabidopsis, PAL and ICS pathways are equally important for pathogen-induced SA biosynthesis in soybean. Knock-down of either pathway shuts down SA biosynthesis and abrogates pathogen resistance. Moreover, unlike in Arabidopsis, pathogen infection is associated with the suppression of ICS gene expression. Pathogen-induced biosynthesis of SA via the PAL pathway correlates inversely with phenylalanine concentrations. Although infections with either virulent or avirulent strains of the pathogens increase SA concentrations, resistance protein-mediated response to avirulent P. sojae strains may function in an SA-independent manner. These results show that PAL- and ICS-catalyzed reactions function cooperatively in soybean defense and highlight the importance of PAL in pathogen-induced SA biosynthesis.

Elevated salicylic acid levels conferred by increased expression of ISOCHORISMATE SYNTHASE 1 contribute to hyperaccumulation of SUMO1 conjugates in the Arabidopsis mutant early in short days 4.

Post-translational modification of proteins by attachment of small ubiquitin-like modifier (SUMO) is essential for plant growth and development. Mutations in the SUMO protease early in short days 4 (ESD4) cause hyperaccumulation of conjugates formed between SUMO and its substrates, and phenotypically are associated with extreme early flowering and impaired growth. We performed a suppressor mutagenesis screen of esd4 and identified a series of mutants called suppressor of esd4 (sed), which delay flowering, enhance growth and reduce hyperaccumulation of SUMO conjugates. Genetic mapping and genome sequencing indicated that one of these mutations (sed111) is in the gene salicylic acid induction-deficient 2 (SID2), which encodes ISOCHORISMATE SYNTHASE I, an enzyme required for biosynthesis of salicylic acid (SA). Analyses showed that compared with wild-type plants, esd4 contains higher levels of SID2 mRNA and about threefold more SA, whereas sed111 contains lower SA levels. Other sed mutants also contain lower SA levels but are not mutant for SID2, although most reduce SID2 mRNA levels. Therefore, higher SA levels contribute to the small size, early flowering and elevated SUMO conjugate levels of esd4. Our results support previous data indicating that SUMO homeostasis influences SA biosynthesis in wild-type plants, and also demonstrate that elevated levels of SA strongly increase the abundance of SUMO conjugates.

A critical role of a plant-specific TFIIB-related protein, BRP1, in salicylic acid-mediated immune response.

An integral part of plant immunity is transcription reprogramming by concerted action of specific transcription factors that activate or repress genes through recruitment or release of RNA polymerase II (Pol II). Pol II is assembled into Pol II holoenzyme at the promoters through association with a group of general transcription factors including transcription factor IIB (TFIIB) to activate transcription. Unlike other eukaryotic organisms, plants have a large family of TFIIB-related proteins with 15 members in Arabidopsis including several plant-specific TFIIB-related proteins (BRPs). Molecular genetic analysis has revealed important roles of some BRPs in plant reproductive processes. In this study, we report that Arabidopsis knockout mutants for BRP1, the founding member of the BRP protein family, were normal in growth and development, but were hypersusceptible to the bacterial pathogen Psuedomonas syringae. The enhanced susceptibility of the brp1 mutants was associated with reduced expression of salicylic acid (SA) biosynthetic gene ISOCHORISMATE SYNTHASE 1 (ICS1) and SA-responsive PATHOGENESIS-RELATED (PR) genes. Pathogen-induced SA accumulation was reduced in the brp1 mutants and exogenous SA rescued the brp1 mutants for resistance to the bacterial pathogen. In uninfected plants, BRP1 was primarily associated with the plastids but pathogen infection induced its accumulation in the nucleus. BRP1 acted as a transcription activator in plant cells and binded to the promoter of ICS1. These results collectively indicate that BRP1 is a functionally specialized transcription factor that increasingly accumulates in the nucleus in response to pathogen infection to promote defense gene expression.

Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities.

The isochorismate and salicylate synthases are members of the MST family of enzymes. The isochorismate synthases establish an equilibrium for the conversion chorismate to isochorismate and the reverse reaction. The salicylate synthases convert chorismate to salicylate with an isochorismate intermediate; therefore, the salicylate synthases perform isochorismate synthase and isochorismate-pyruvate lyase activities sequentially. While the active site residues are highly conserved, there are two sites that show trends for lyase-activity and lyase-deficiency. Using steady state kinetics and HPLC progress curves, we tested the "interchange" hypothesis that interconversion of the amino acids at these sites would promote lyase activity in the isochorismate synthases and remove lyase activity from the salicylate synthases. An alternative, "permute" hypothesis, that chorismate-utilizing enzymes are designed to permute the substrate into a variety of products and tampering with the active site may lead to identification of adventitious activities, is tested by more sensitive NMR time course experiments. The latter hypothesis held true. The variant enzymes predominantly catalyzed chorismate mutase-prephenate dehydratase activities, sequentially generating prephenate and phenylpyruvate, augmenting previously debated (mutase) or undocumented (dehydratase) adventitious activities.

Lysine221 is the general base residue of the isochorismate synthase from Pseudomonas aeruginosa (PchA) in a reaction that is diffusion limited.

The isochorismate synthase from Pseudomonas aeruginosa (PchA) catalyzes the conversion of chorismate to isochorismate, which is subsequently converted by a second enzyme (PchB) to salicylate for incorporation into the salicylate-capped siderophore pyochelin. PchA is a member of the MST family of enzymes, which includes the structurally homologous isochorismate synthases from Escherichia coli (EntC and MenF) and salicylate synthases from Yersinia enterocolitica (Irp9) and Mycobacterium tuberculosis (MbtI). The latter enzymes generate isochorismate as an intermediate before generating salicylate and pyruvate. General acid-general base catalysis has been proposed for isochorismate synthesis in all five enzymes, but the residues required for the isomerization are a matter of debate, with both lysine221 and glutamate313 proposed as the general base (PchA numbering). This work includes a classical characterization of PchA with steady state kinetic analysis, solvent kinetic isotope effect analysis and by measuring the effect of viscosogens on catalysis. The results suggest that isochorismate production from chorismate by the MST enzymes is the result of general acid-general base catalysis with a lysine as the base and a glutamic acid as the acid, in reverse protonation states. Chemistry is determined to not be rate limiting, favoring the hypothesis of a conformational or binding step as the slow step.

Structure of isochorismate synthase DhbC from Bacillus anthracis.

The isochorismate synthase DhbC from Bacillus anthracis is essential for the biosynthesis of the siderophore bacillibactin by this pathogenic bacterium. The structure of the selenomethionine-substituted protein was determined to 2.4 Šresolution using single-wavelength anomalous diffraction. B. anthracis DhbC bears the strongest resemblance to the Escherichia coli isochorismate synthase EntC, which is involved in the biosynthesis of another siderophore, namely enterobactin. Both proteins adopt the characteristic fold of other chorismate-utilizing enzymes, which are involved in the biosynthesis of various products, including siderophores, menaquinone and tryptophan. The conservation of the active-site residues, as well as their spatial arrangement, suggests that these enzymes share a common Mg(2+)-dependent catalytic mechanism.

Salicylic Acid Biosynthesis and Metabolism: A Divergent Pathway for Plants and Bacteria.

Salicylic acid (SA) is an active secondary metabolite that occurs in bacteria, fungi, and plants. SA and its derivatives (collectively called salicylates) are synthesized from chorismate (derived from shikimate pathway). SA is considered an important phytohormone that regulates various aspects of plant growth, environmental stress, and defense responses against pathogens. Besides plants, a large number of bacterial species, such as Pseudomonas, Bacillus, Azospirillum, Salmonella, Achromobacter, Vibrio, Yersinia, and Mycobacteria, have been reported to synthesize salicylates through the NRPS/PKS biosynthetic gene clusters. This bacterial salicylate production is often linked to the biosynthesis of small ferric-ion-chelating molecules, salicyl-derived siderophores (known as catecholate) under iron-limited conditions. Although bacteria possess entirely different biosynthetic pathways from plants, they share one common biosynthetic enzyme, isochorismate synthase, which converts chorismate to isochorismate, a common precursor for synthesizing SA. Additionally, SA in plants and bacteria can undergo several modifications to carry out their specific functions. In this review, we will systematically focus on the plant and bacterial salicylate biosynthesis and its metabolism.

Overexpression of isochorismate synthase enhances salt tolerance in barley.

Isochorismate synthase (ICS) is a key enzyme for the synthesis of salicylic acid (SA) in plants. SA plays an important role in the response of plants to abiotic stress. In this study, transgenic barley was constructed to evaluate the function of ICS under salt stress. ICSOE lines showed obvious salt stress tolerance, this results from the increased outward Na+ flux and inward K+ flux in roots, thereby maintaining a lower cytosolic Na+/K+ ratio under salt stress. Overexprssion of ICS also improved Na+ sequestration in shoots under salt stress. In addition, ICSOE lines displayed less accumulation of reactive oxygen species and oxidative damage, accompanied by higher activity of antioxidant enzymes. The improved Na+/K+ ratio, Na+ sequestration, and antioxidative competence play an important role in the enhanced salt tolerance of ICSOE lines. These findings help to elucidate the abiotic stress resistance of the ICS pathway in barley.

Overexpression of isochorismate synthase enhances drought tolerance in barley.

Isochorismate synthase (ICS) is a key enzyme for the synthesis of salicylic acid (SA) in plants. SA mediates plant responses to both biotic and abiotic stresses. In previous studies, we found that overexpression of ICS (ICSOE) or suppression of ICS (ICSRNAi) affected the host response to Fusarium graminearum in barley. However, whether the barley ICS gene plays a role in adapting to abiotic stresses remains to be determined. In the present study, expression of the ICS gene was upregulated when treated with 20 % PEG6000, and ICSOE lines were more drought tolerant than wild type (WT) and ICSRNAi. In addition, the abscisic acid (ABA) levels in the ICSOE lines were higher than those in the WT and ICSRNAi lines under drought stress. High ABA levels significantly reduced Gs and E, which may impact water retention under drought stress. Under drought conditions, the activity of antioxidant enzymes was significantly higher in the ICSOE lines, correlating with a lower levels of reactive oxygen species (ROS) and malondialdehyde (MDA). Enhanced antioxidant competence also contributed to drought tolerance in ICSOE lines. These findings help elucidate the abiotic stress resistance of the ICS pathway in barley.

Regulation of Postharvest Tomato Fruit Ripening by Endogenous Salicylic Acid.

Exogenous application of salicylic acid (SA) has been known for delaying ripening in many fruit and vegetables. But the function of endogenous SA in relation to postharvest fruit performance is still unexplored. To understand the role of endogenous SA in postharvest fruit ripening of tomato, 33 tomato lines were examined for their endogenous SA content, membrane stability index (MSI), and shelf life (SL) at turning and red stages of tomato fruit ripening. Six tomato lines having contrasting shelf lives from these categories were subjected further for ethylene (ET) evolution, 1-aminocyclopropane-1-carboxylic acid synthase (ACS), 1-aminocyclopropane-1-carboxylic acid oxidase (ACO), polygalacturonase (PG), pectin methyl esterase (PME), antioxidant assays and lipid peroxidation. It was found that high endogenous SA has a direct association with low ET evolution, which leads to the high SL of fruit. High lycopene content was also found to be correlated with high SA. Total antioxidants, PG, and PME decreased and lipid peroxidation increased from turning to red stage of tomato fruit development. Furthermore, these lines were subjected to expression analysis for SA biosynthesis enzymes viz. Solanum lycopersicum Isochorismate Synthase (SlICS) and SlPAL. Real-time PCR data revealed that high SL lines have high SlPAL4 expression and low SL lines have high SlPAL6 expression. Based on the results obtained in this study, it was concluded that endogenous SA regulates ET evolution and SL with the aid of the antioxidative defense system, and SlPAL4 and SlPAL6 genes play significant but opposite roles during fruit ripening.

Salicylic Acid Biosynthesis in Plants.

Salicylic acid (SA) is an important plant hormone that is best known for mediating host responses upon pathogen infection. Its role in plant defense activation is well established, but its biosynthesis in plants is not fully understood. SA is considered to be derived from two possible pathways; the ICS and PAL pathway, both starting from chorismate. The importance of both pathways for biosynthesis differs between plant species, rendering it hard to make generalizations about SA production that cover the entire plant kingdom. Yet, understanding SA biosynthesis is important to gain insight into how plant pathogen responses function and how pathogens can interfere with them. In this review, we have taken a closer look at how SA is synthesized and the importance of both biosynthesis pathways in different plant species.

Barley isochorismate synthase mutant is phylloquinone-deficient, but has normal basal salicylic acid level.

Salicylic acid (SA) is an important signaling hormone in plant immunity. It can be synthesized by either the phenylpropanoid pathway or the isochorismate pathway, but mutant studies of this have been scarce in other species than Arabidopsis. Here we identified a mutation that introduced a stop-codon early in the barley gene for isochorismate synthase (ICS). We found that homozygous ics plants wilted if not sprayed with 1,4-dihydroxy-2-naphthoic acid, a precursor of phylloquinone, also synthesized via the isochorismate pathway. Interestingly, ics had unchanged SA, suggesting that the basal level of SA is synthesized via the phenylpropanoid pathway. Previous studies have failed seeing increased SA levels in barley after attack by the powdery mildew fungus, Blumeria graminis f.sp. hordei (Bgh), and indeed, we saw no changes in the interaction of ics with this fungus. Overall, we hope this mutant will be useful for other studies of SA in barley.

Comparative analysis of plant isochorismate synthases reveals structural mechanisms underlying their distinct biochemical properties.

Isochorismate synthase (ICS) converts chorismate into isochorismate, a precursor of primary and secondary metabolites including salicylic acid (SA). SA plays important roles in responses to stress conditions in plants. Many studies have suggested that the function of plant ICSs is regulated at the transcriptional level. In Arabidopsis thaliana, the expression of AtICS1 is induced by stress conditions in parallel with SA synthesis, and AtICS1 is required for SA synthesis. In contrast, the expression of NtICS is not induced when SA synthesis is activated in tobacco, and it is unlikely to be involved in SA synthesis. Studies on the biochemical properties of plant ICSs are limited, compared with those on transcriptional regulation. We analyzed the biochemical properties of four plant ICSs: AtICS1, NtICS, NbICS from Nicotiana benthamiana, and OsICS from rice. Multiple sequence alignment analysis revealed that their primary structures were well conserved, and predicted key residues for ICS activity were almost completely conserved. However, AtICS1 showed much higher activity than the other ICSs when expressed in Escherichia coli and N. benthamiana leaves. Moreover, the levels of AtICS1 protein expression in N. benthamiana leaves were higher than the other ICSs. Construction and analysis of chimeras between AtICS1 and OsICS revealed that the putative chloroplast transit peptides (TPs) significantly affected the levels of protein accumulation in N. benthamiana leaves. Chimeric and point-mutation analyses revealed that Thr531, Ser537, and Ile550 of AtICS1 are essential for its high activity. These distinct biochemical properties of plant ICSs may suggest different roles in their respective plant species.

Isochorismate-based salicylic acid biosynthesis confers basal resistance to Fusarium graminearum in barley.

Salicylic acid (SA) plays an important role in signal transduction and disease resistance. In Arabidopsis, SA can be made by either of two biosynthetic branches, one involving isochorismate synthase (ICS) and the other involving phenylalanine ammonia-lyase (PAL). However, the biosynthetic pathway and the importance of SA remain largely unknown in Triticeae. Here, we cloned one ICS and seven PAL genes from barley, and studied their functions by their overexpression and suppression in that plant. Suppression of the ICS gene significantly delayed plant growth, whereas PAL genes, both overexpressed and suppressed, had no significant effect on plant growth. Similarly, suppression of ICS compromised plant resistance to Fusarium graminearum, whereas similar suppression of PAL genes had no significant effect. We then focused on transgenic plants with ICS. In a leaf-based test with F. graminearum, transgenic plants with an up-regulated ICS were comparable with wild-type control plants. By contrast, transgenic plants with a suppressed ICS lost the ability to accumulate SA during pathogen infection and were also more susceptible to Fusarium than the wild-type controls. This suggests that ICS plays a unique role in SA biosynthesis in barley, which, in turn, confers a basal resistance to F. graminearum by modulating the accumulation of H2 O2 , O2- and reactive oxygen-associated enzymatic activities. Although SA mediates systemic acquired resistance (SAR) in dicots, there was no comparable SAR response to F. graminearum in barley. This study expands our knowledge about SA biosynthesis in barley and proves that SA confers basal resistance to fungal pathogens.

Molecular cloning, characterization, and promoter analysis of the isochorismate synthase (AaICS1) gene from Artemisia annua.

Isochorismate synthase (ICS) is a crucial enzyme in the salicylic acid (SA) synthesis pathway. The full-length complementary DNA (cDNA) sequence of the ICS gene was isolated from Artemisia annua L. The gene, named AaICS1, contained a 1710-bp open reading frame, which encoded a protein with 570 amino acids. Bioinformatics and comparative study revealed that the polypeptide protein of AaICS1 had high homology with ICSs from other plant species. Southern blot analysis suggested that AaICS1 might be a single-copy gene. Analysis of the 1470-bp promoter of AaICS1 identified distinct cis-acting regulatory elements, including TC-rich repeats, MYB binding site (MBS), and TCA-elements. An analysis of AaICS1 transcript levels in multifarious tissues of A. annua using quantitative real-time polymerase chain reaction (qRT-PCR) showed that old leaves had the highest transcription levels. AaICS1 was up-regulated under wounding, drought, salinity, and SA treatments. This was corroborated by the presence of the predicted cis-acting elements in the promoter region of AaICS1. Overexpressing transgenic plants and RNA interference transgenic lines of AaICS1 were generated and their expression was compared. High-performance liquid chromatography (HPLC) results from leaf tissue of transgenic A. annua showed an increase in artemisinin content in the overexpressing plants. These results confirm that AaICS1 is involved in the isochorismate pathway.