Salicylic acid biosynthesis is not from phenylalanine in Arabidopsis

The phytohormone salicylic acid(SA) regulates biotic and abiotic stress responses in plants. Two distinct biosynthetic pathways for SA have been well documented in plants: the isochorismate(IC)pathway in the chloroplast and the phenylalanine ammonia-lyase(PAL) pathway in the cytosol.However, there has been no solid evidence that the PAL pathway contributes to SA biosynthesis. Here,we report that feeding Arabidopsis thaliana with Ring-¹³C-labeled phenylalanine(¹³C₆     

Accumulation of Isochorismate-derived 2,3-Dihydroxybenzoic 3-O-β-d-Xyloside in Arabidopsis Resistance to Pathogens and Ageing of Leaves

An intricate network of hormone signals regulates plant development and responses to biotic and abiotic stress. Salicylic acid (SA), derived from the shikimate/isochorismate pathway, is a key hormone in resistance to biotrophic pathogens. Several SA derivatives and associated modifying enzymes have been identified and implicated in the storage and channeling of benzoic acid intermediates or as bioactive molecules. However, the range and modes of action of SA-related metabolites remain elusive. In Arabidopsis, Enhanced Disease Susceptibility 1 (EDS1) promotes SA-dependent and SA-independent responses in resistance against pathogens. Here, we used metabolite profiling of Arabidopsis wild type and eds1 mutant leaf extracts to identify molecules, other than SA, whose accumulation requires EDS1 signaling. Nuclear magnetic resonance and mass spectrometry of isolated and purified compounds revealed 2,3-dihydroxybenzoic acid (2,3-DHBA) as an isochorismate-derived secondary metabolite whose accumulation depends on EDS1 in resistance responses and during ageing of plants. 2,3-DHBA exists predominantly as a xylose-conjugated form (2-hydroxy-3-β-O-d-xylopyranosyloxy benzoic acid) that is structurally distinct from known SA-glucose conjugates. Analysis of DHBA accumulation profiles in various Arabidopsis mutants suggests an enzymatic route to 2,3-DHBA synthesis that is under the control of EDS1. We propose that components of the EDS1 pathway direct the generation or stabilization of 2,3-DHBA, which as a potentially bioactive molecule is sequestered as a xylose conjugate.     

An unconventionally secreted effector from the root knot nematode Meloidogyne incognita,Mi-ISC-1, promotes parasitism by disrupting salicylic acid biosynthesis in host plants

Plant-parasitic nematodes need to deliver effectors that suppress host immunity for successful parasitism. We have characterized a novel isochorismatase effector from the root-knot nematode Meloidogyne incognita, named Mi-ISC-1. The Mi-isc-1 gene is expressed in the subventral oesophageal glands and is up-regulated in parasitic-stage juveniles. Tobacco rattle virus-induced gene silencing targeting Mi-isc-1 attenuated M. incognita parasitism. Enzyme activity assays confirmed that Mi-ISC-1 can catalyse hydrolysis of isochorismate into 2,3-dihydro-2,3-dihydroxybenzoate in vitro. Although Mi-ISC-1 lacks a classical signal peptide for secretion at its N-terminus, a yeast invertase secretion assay showed that this protein can be secreted from eukaryotic cells. However, the subcellular localization and plasmolysis assay revealed that the unconventional secretory signal present on the Mi-ISC-1 is not recognized by the plant secretory pathway and that the effector was localized within the cytoplasm of plant cells, but not apoplast, when transiently expressed in Nicotiana benthamiana leaves by agroinfiltration. Ectopic expression of Mi-ISC-1 in N. benthamiana reduced expression of the PR1 gene and levels of salicylic acid (SA), and promoted infection by Phytophthora capsici. The cytoplasmic localization of Mi-ISC-1 is required for its function. Moreover, Mi-ISC-1 suppresses the production of SA following the reconstitution of the de novo SA biosynthesis via the isochorismate pathway in the cytoplasm of N. benthamiana leaves. These results demonstrate that M. incognita deploys a functional isochorismatase that suppresses SA-mediated plant defences by disrupting the isochorismate synthase pathway for SA biosynthesis to promote parasitism.     

Biosynthesis of salicylic acid in plants

Salicylic acid (SA) is an important signal molecule in plants. Two pathways of SA biosynthesis have been proposed in plants. Biochemical studies using isotope feeding have suggested that plants synthesize SA from cinnamate produced by the activity of phenylalanine ammonia lyase (PAL). Silencing of PAL genes in tobacco or chemical inhibition of PAL activity in Arabidopsis, cucumber and potato reduces pathogen-induced SA accumulation. Genetic studies, on the other hand, indicate that the bulk of SA is produced from isochorismate. In bacteria, SA is synthesized from chorismate through two reactions catalyzed by isochorismate synthase (ICS) and isochorismate pyruvate lyase (IPL). Arabidopsis contains two ICS genes but has no gene encoding proteins similar to the bacterial IPL. Thus, how SA is synthesized in plants is not fully elucidated. Two recently identified Arabidopsis genes, PBS3 and EPS1, are important for pathogen-induced SA accumulation. PBS3 encodes a member of the acyl-adenylate/thioester-forming enzyme family and EPS1 encodes a member of the BAHD acyltransferase superfamily. PBS3 and EPS1 may be directly involved in the synthesis of an important precursor or regulatory molecule for SA biosynthesis. The pathways and regulation of SA biosynthesis in plants may be more complicated than previously thought.Key words: salicylic acid biosynthesis, isochorismate synthase, phenylalanine ammonia lyase     

Establishing a novel biosynthetic pathway for the production of 3,4-dihydroxybutyric acid from xylose in Escherichia coli

3-Hydroxy-γ-butyrolactone (3HBL) is an attractive building block owing to its broad applications in pharmaceutical industry. Currently, 3HBL is commercially produced by chemical routes using petro-derived carbohydrates, which involves hazardous materials and harsh processing conditions. Only one biosynthetic pathway has been reported for synthesis of 3HBL and its hydrolyzed form 3,4-dihydroxybutyric acid (3,4-DHBA) using glucose and glycolic acid as the substrates and coenzyme A as the activator, which involves multiple steps (>10 steps) and suffers from low productivity and yield. Here we established a novel five-step biosynthetic pathway for 3,4-DHBA generation from D-xylose based on the non-phosphorylative D-xylose metabolism, which led to efficient production of 3,4-DHBA in Escherichia coli. Pathway optimization by incorporation of efficient enzymes for each step and host strain engineering by knocking out competing pathways enabled 1.27 g/L 3,4-DHBA produced in shake flasks, which is the highest titer reported so far. The novel pathway established in engineered E. coli strain demonstrates a new route for 3,4-DHBA biosynthesis from xylose, and this engineered pathway has great potential for industrial biomanufacturing of 3,4-DHBA and 3HBL.     

Labeling Patterns of Chloroplastidic Isoprenoids in Cultured Cells of Liverwort Ptychanthus striatus

Incorporation studies administering ²H- and ¹³C- labeled mevalonate (MVA) and ¹³C-labeled glucose to suspension cultured cells of the liverwort, Ptychanthus striatus, were carried out in order to examine the biosynthesis of the phytyl side-chain of chlorophyll a. Administration of ¹³C- and ²H-labeled MVA provided evidence for the involvement of the MVA pathway in the phytyl side-chain biosynthesis and preferential labeling of the farnesyl diphosphate (FPP)-derived portion. An alternate labeling pattern in the phytyl side-chain was observed which was slightly different to the nonequivalent labeling in other liverworts, such as Heteroscyphus planus and Lophocolea heterophylla and in the hornwort, Anthoceros punctatus. The labeling pattern observed after the administration of ¹³C-labeled glucose revealed the simultaneous involvement of the non-MVA pathway in the phytol biosynthesis of P. striatus cells.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.     

A study on biosynthesis of “citral” in lemongrass (<Emphasis Type="Italic">C. flexuosus</Emphasis>) cv. Suvarna

Incorporation of [1-¹³C]-glucose and fosmidomycin was achieved in young and rapidly expanding (aged 15 days) leaves of lemongrass (C. flexuosus) cv. suvarna to elucidate biosynthetic origin of citral (3,7-dimethyl-2,6-octadienal). Analyses of the resultant ¹³C-labeling patterns of citral by quantitative ¹³C-NMR spectroscopy revealed significant %¹³C enrichment at carbons C-3, C-5, C-7 and C-9 in citral. This labeling pattern of the citral is in accordance with their biosynthesis via 2C-methyl-d-erythritol-4-phosphate (MEP) pathway. However, incorporation of [1-¹³C]-glucose achieved in the presence of fosmidomycin resulted in a ¹³C-labeling pattern of citral which did not match with labeling pattern characteristic of the MEP pathway. In addition, we studied the activity pattern of the DXR enzyme following fosmidomycin (25, 50, 75 and 100 μM concentrations) treatment of the young (aged 15 days) leaves for 48 h. The results revealed that fosmidomycin (100 μM) caused drastic inhibition (>50 %) of the DXR enzyme activity. The levels of the citral measured in the fosmidomycin treated leaves were also found to be reduced with decrease the activity of DXR enzyme. In conclusion, the results of the present work revealed the presence of the MEP pathway and its role in the biosynthesis of citral in lemongrass. In addition, the critical role of the DXR enzyme in the citral biosynthesis is highlighted. This is the first report on elucidation of the MEP pathway in lemongrass and may help in deeper understanding of the monoterpene biosynthesis and regulation in the genus Cymbopogon of high industrial significance.     

Salicylic acid production in response to biotic and abiotic stress depends on isochorismate in Nicotiana benthamiana

Salicylic acid (SA) is an important signal involved in the activation of defence responses against abiotic and biotic stress. In tobacco, benzoic acid or glucosyl benzoate were proposed to be precursors of SA. This is in sharp contrast with studies in Arabidopsis thaliana, where SA derives from isochorismate. We have determined the importance of isochorismate for SA biosynthesis in Nicotiana benthamiana using virus-induced gene silencing of the isochorismate synthase (ICS) gene. Plants with silenced ICS expression do not accumulate SA after exposure to UV or to pathogen stress. Plants with silenced ICS expression also exhibit strongly decreased levels of phylloquinone, a product of isochorismate. Our data provide evidence for an isochorismate-derived synthesis of SA in N. benthamiana.     

3-Hydroxy-3-phenylpropanoic acid is an intermediate in the biosynthesis of benzoic acid and salicylic acid but benzaldehyde is not

Stable-isotope-labelled (²H₆,¹⁸O) 3-hydroxy-3-phenylpropanoic acid, a putative intermediate in the biosynthesis of benzoic acid (BA) and salicylic acid (SA) from cinnamic acid, has been synthesized and administered to cucumber (Cucumis sativus L.) and Nicotiana attenuata (Torrey). Analysis of the products by gas chromatography-mass spectrometry revealed incorporation of labelling into BA and SA, but not into benzaldehyde. In a separate experiment, 3-hydroxy- 3-phenylpropanoic acid was found to be a metabolite of phenylalanine, itself the primary metabolic precursor of BA and SA. These data suggest that cinnamic acid chain shortening is probably achieved by β-oxidation, and that the proposed “non-oxidative” pathway of side-chain degradation does not function in the biosynthesis of BA and SA, in cucumber and N. attenuata. Received: 10 February 2000 / Accepted: 18 April 2000     

Spermidine: an indirect precursor of the pyrrolidine rings of nicotine and nornicotine in Nicotiana glutinosa

Spermidine, which was labeled asymmetrically in its four-carbon moiety ([6-¹⁴C]-1,5,10-triazadecane), was administered to Nicotiana glutinosa plants. After 7 days the plants were harvested, yielding radioactive nicotine (0.43 % incorporation) and nornicotine (0.07 % inc.). A systematic degradation of the alkaloids indicated that they were labelled equally at C-2′ and C-5′ of their pyrrolidine rings. These results are consistent with the hypothesis that spermidine is degraded to putrescine prior to its incorporation into the pyrrolidine rings of nicotine and nornicotine.     

The incorporation of [5,6-13C2]Nicotinic acid into the tobacco alkaloids examined by the use of 13C nuclear magnetic resonance

[5,6-¹⁴C,¹³C₂]Nicotinic acid was prepared from [¹⁴C,¹³C]methyl iodide via nitromethane, 2-nitroacetaldehyde oxime, 3-nitroquinoline, 3-aminoquinoline, and quinoline in 20% overall yield. Administration of this material to Nicotiana tabacum and N. glauca afforded labeled anabasine, anatabine, nicotine, and nornicotine. Qualitative and quantitative incorporation (0.07–4.5% specific incorporation) was determined by radioactive assay and by examination of the ¹³C NMR spectra of these alkaloids. Satellites due to spin-spin coupling of the incorporated contiguous ¹³C atoms were observed at the resonances due to C-5 and C-6 in anabasine, nicotine, and nornicotine. In anatabine, satellites were found at C-5, C-6, C-5′, and C-6′.     

Biochemical effects of miconazole on fungi. II. Inhibition of ergosterol biosynthesis in Candida albicans.

The effects of the antifungal agent miconazole nitrate on the ergosterol biosynthesis in Candida albicans were investigated after in vitro contact with the drug for 1, 4, 16 and 24 h. A time- and dose-(2.10^?10–10^?4 M) dependent inhibition of [¹⁴C]acetate incorporation into ergosterol was observed. Fifty percent inhibition of the acetate incorporation into ergosterol was found after 1 h incubation in the presence of 10^?9 M miconazole. Simultaneously 24-methylenedihydrolanosterol, lanosterol, obtusifoliol, 4,14-dimethylzymosterol and 14-methylfecosterol accumulated.The accumulation of 14 α-methyl sterols suggests that this antifungal agent is a potent inhibitor of one of the metabolic steps involved in the demethylation at C-14. The absence of 24-methyl sterols and of sterols with a C-22 [23] double bond in miconazole treated C. albicans indicates that miconazole also inteferes with the reduction of the 24(28)-double bond and with the introduction of the 22(23)-double bond.Miconazole also intervenes to a small extent in triglyceride synthesis. However, in all circumstances studied, ergosterol biosynthesis was affected at lower doses than those interfering with the acetate incorporation into triglycerides. 16 and 24 h of incubation in the presence of miconazole (≥ 10^?6 M) also resulted in an increased fatty acid synthesis.It is suggested that the miconazole-induced inhibition of the C-14 demethylation may be at the origin of the previously observed permeability changes in miconazole treated C. albicans.     

Evidence for a universal pathway of abscisic Acid biosynthesis in higher plants from o incorporation patterns

Previous labeling studies of abscisic acid (ABA) with ¹⁸O₂ have been mainly conducted with water-stressed leaves. In this study, ¹⁸O incorporation into ABA of stressed leaves of various species was compared with ¹⁸O labeling of ABA of turgid leaves and of fruit tissue in different stages of ripening. In stressed leaves of all six species investigated, avocado (Persea americana), barley (Hordeum vulgare), bean (Phaseolus vulgaris), cocklebur (Xanthium strumarium), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum), ¹⁸O was most abundant in the carboxyl group, whereas incorporation of a second and third ¹⁸O in the oxygen atoms on the ring of ABA was much less prominent after 24 h in ¹⁸O₂. ABA from turgid bean leaves showed significant ¹⁸O incorporation, again with highest ¹⁸O enrichment in the carboxyl group. The ¹⁸O-labeling pattern of ABA from unripe avocado mesocarp was similar to that of stressed leaves, but in ripe fruits there was, besides high ¹⁸O enrichment in the carboxyl group, also much additional ¹⁸O incorporation in the ring. In ripening apple fruit tissue (Malus domestica), singly labeled ABA was most abundant with more ¹⁸O incorporated in the tertiary hydroxyl group than in the carboxyl group of ABA. Smaller quantities of this monolabeled product (C-1′-¹⁸OH) were also detected in the stressed leaves of barley, bean, and tobacco, and in avocado fruits. It is postulated that a large precursor molecule yields an aldehyde cleavage product that is, in some tissues, rapidly converted to ABA with retention of ¹⁸O in the carboxyl group, whereas in ripening fruits and in the stressed leaves of some species the biosynthesis of ABA occurs at a slower rate, allowing this intermediate to exchange ¹⁸O with water. On the basis of ¹⁸O-labeling patterns observed in ABA from different tissues it is concluded that, despite variations in precursor pool sizes and intermediate turnover rates, there is a universal pathway of ABA biosynthesis in higher plants which involves cleavage of a larger precursor molecule, presumably an oxygenated carotenoid.     

Biosynthesis of alkane-2, 3-diols: chemical synthesis of 3-hydroxy-[3-14C]octadecane-2-one and its reduction to [3-14C]octadecane-2, 3-diol in the uropygial glands of ring-necked pheasants (Phasianus colchicus).

Acyloin has been proposed to be an intermediate in the biosynthesis of long chain alkane-2,3-diols. In order to test this possibility, specifically labeled 3-hydroxyoctadecane-2-one (acyloin) was synthesized by coupling 2-methyl-1,3-dithiane with [1-¹⁴C]hexadecanal followed by cleaving of the thioketal. Injection of the synthetic 3-hydroxy [3-¹⁴C]octadecane-2-one into the uropygial gland of the ring-necked pheasant resulted in the formation of labeled octadecane-2,3-diol. Chemical degradation of this diol showed that all of the ¹⁴C was contained in C-3 of the diol showing direct conversion of acyloin to the diol. These observations support the hypothesis that alkane-2,3-diols might be biosynthesized by reduction of the acyloin derived from a condensation between hydroxyethyl thiamine pyrophosphate and fatty aldehyde. Gas-liquid chromatographic analysis of the alkane-2,3-diols, as their isopropylidene derivatives, of the pheasant strongly suggests that they are of the erythro-configuration; however, alkane-2,3-diol enzymatically formed from the racemic acyloin injected into the gland contained 59.5% erythro- and 40.5% threo-diastereoisomers. This distribution was identical to that produced by chemical reduction of the synthetic racemic acyloin. These results clearly show that the reduction step does not show a preference for either of the enantiomers of the acyloin and that the stereospecificity in diol biosynthesis probably resides in the condensation step.     

Biosynthesis of (−)-ent-kaurenoic acid in Smallanthus sonchifolius and its effect against microbial biofilms

The biosynthetic pathway of (–)-ent-kaurenoic acid (1) was investigated by incorporation of 1-d-¹³C-glucose in Smallanthus sonchifolius (Asteraceae) plantlets. The ¹³C-enrichment pattern indicated that methylerythritol-4-phosphate (MEP) pathway is the biosynthetic pathway involved in diterpenoid biosynthesis. Our studies in S. sonchifolius reinforce that the biosynthesis of different classes of terpenes should not be compartmentalized into cytosol and plastids. Additionally, (–)-ent-kaurenoic acid showed antimicrobial activity against Staphylococcus aureus biofilm.     

Biosynthesis of gallic and ellagic acids with14C-labeled compounds inAcer andRhus leaves

The biosynthetic pathway for gallic and ellagic acids in young, mature and autumn leaves ofAcer buergerianum andRhus succedanea was examined by tracer experiments, and also by isotope competition, withd-shikimic acid-¹⁴C,l-phenylalanine-U-¹⁴C,l-phenyllactic acid-U-¹⁴C, gallic acid-G-¹⁴C and their unlabeled compounds. In young leaves of both plants, the incorporation rate of labeled shikimic acid into gallic acid was significantly higher than that of labeled phenylalanine, whereas in the mature and autumn leaves the latter was a good precursor rather than the former for the gallic acid biosynthesis. Therefore, two pathways for gallic acid formation, through β-oxidation of phenylpropanoid and through dehydrogenation of shikimic acid, could be operating inAcer andRhus leaves, and the preferential pathway is altered by leaf age. In both plants, the incorporation rate of labeled phenyllactic acid during a 24 hr metabolic period was almost the same as that of labeled phenylalanine. The incorporation ofd-skikimic acid-G-¹⁴C,l-phenylalanine-U-¹⁴C andl-phenyllactic acid-U-¹⁴C into ellagic acid was very similar to the case of the radioactive gallic acid formation. Furthermore, regardless of the presence of unlabeled shikimic acid and/or phenylalanine, incorporation of the radioactivity of labeled gallic acid into ellagic acid occurred at a very high rate, suggesting the reciprocal radical reaction of gallic acid for the ellagic acid formation. The incorporation of labeled compounds into ellagitanins was also examined and their biosynthesis discussed further.     

Inter-organ defense networking: Leaf whitefly sucking elicits plant immunity to crown gall disease caused by Agrobacterium tumefaciens

Plants have elaborate defensive machinery to protect against numerous pathogens and insects. Plant hormones function as modulators of defensive mechanisms to maintain plant resistance to natural enemies. Our recent study suggests that salicylic acid (SA) is the primary phytohormone regulating plant responses to Agrobacterium tumefaciens infection. Tobacco (Nicotiana benthamiana Domin.) immune responses against Agrobacterium-mediated crown gall disease were activated by exposure to the sucking insect whitefly, which stimulated SA biosynthesis in aerial tissues; in turn, SA synthesized in aboveground tissues systemically modulated SA secretion in root tissues. Further investigation revealed that endogenous SA biosynthesis negatively modulated Agrobacterium-mediated plant genetic transformation. Our study provides novel evidence that activation of the SA-signaling pathway mediated by a sucking insect infestation has a pivotal role in subsequently attenuating Agrobacterium infection. These results demonstrate new insights into interspecies cross-talking among insects, plants, and soil bacteria.