Characterisation of recombinant epithiospecifier protein and its over-expression in Arabidopsis thaliana

Epithiospecifier protein (ESP) is a protein that catalyses formation of epithionitriles during glucosinolate hydrolysis. In vitro assays with a recombinant ESP showed that the formation of epithionitriles from alkenylglucosinolates is ESP and ferrous ion dependent. Nitrile formation in vitro however does not require ESP but only the presence of Fe(II) and myrosinase. Ectopic expression of ESP in Arabidopsis thaliana Col-5 under control of the strong viral CaMV 35S promoter altered the glucosinolate product profile from isothiocyanates towards the corresponding nitriles.     

Universally occurring phenylpropanoid and species-specific indolic metabolites in infected and uninfected Arabidopsis thaliana roots and leaves

A total of eleven alkali-released, aromatic compounds were identified by HPLC, MS and NMR analyses in cell wall extracts from Arabidopsis thaliana roots. Nine of them together constituted the three complete series of 4-hydroxy-, 4-hydroxy-3-methoxy, and 4-hydroxy-3,5-dimethoxy-substituted benzaldehydes, benzoic acids and cinnamic acids. The other two were indolic metabolites: indole-3-carboxylic acid and indole-3-carbaldehyde. Qualitatively similar, but quantitatively distinct profiles were obtained using cell-wall extracts from A. thaliana leaves. Several of these compounds, particularly indole-3-carboxylic acid, 4-hydroxybenzoic acid and all four aldehydes, increased considerably in concentration upon infection of roots with Pythium sylvaticum, as did at least some of them upon infection of leaves with Pseudomonas syringae pv tomato. Comparison of these results with analogous data on a variety of different plant species suggests a remarkable structural uniformity among the majority of constitutive as well as infection-induced, aromatic cell wall-bound compounds throughout the entire plant kingdom-in sharp contrast to the highly species-specific, chemically highly divers bouquets of soluble aromatic metabolites.     

Variation of glucosinolate accumulation among different organs and developmental stages of Arabidopsis thaliana

The glucosinolate content of various organs of the model plant Arabidopsis thaliana (L.) Heynh., Columbia (Col-0) ecotype, was analyzed at different stages during its life cycle. Significant differences were noted among organs in both glucosinolate concentration and composition. Dormant and germinating seeds had the highest concentration (2.5-3.3% by dry weight), followed by inflorescences, siliques (fruits), leaves and roots. While aliphatic glucosinolates predominated in most organs, indole glucosinolates made up nearly half of the total composition in roots and late-stage rosette leaves. Seeds had a very distinctive glucosinolate composition. They possessed much higher concentrations of several types of aliphatic glucosinolates than other organs, including methylthioalkyl and, hydroxyalkyl glucosinolates and compounds with benzoate esters than other organs. From a developmental perspective, older leaves had lower glucosinolate concentrations than younger leaves, but this was not due to decreasing concentrations in individual leaves with age (glucosinolate concentration was stable during leaf expansion). Rather, leaves initiated earlier in development simply had much lower rates of glucosinolate accumulation per dry weight gain throughout their lifetimes. During seed germination and leaf senescence, there were significant declines in glucosinolate concentration. The physiological and ecological significance of these findings is briefly discussed.     

New insight into the biosynthesis and regulation of indole compounds in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

In spite of their silent and sessile life, plants are dynamic organisms that have developed advanced defence strategies in their adaptation to the pressure of herbivores and pathogens. Natural plant products play an important role as chemical weapons in this warfare. Characteristic of cruciferous plants is the synthesis of nitrogen- and sulphur-rich compounds, such as glucosinolates (Mikkelsen et al. 2002) and indole alkaloids (Pedras et al. 2000). Glucosinolates are believed to be largely non-toxic, but upon tissue disruption, they are hydrolyzed by endogenous -thioglucosidases (myrosinases) (Rask et al. 2000) to primarily isothiocyanates and nitriles, which have many biological activities. These include not only important roles as repellents against herbivorous insects and microorganisms, but also as volatile attraction of specialized insects (Wittstock and Halkier 2002). For humans, these compounds serve as cancer-preventive agents, biopesticides, and flavor compounds (Talalay and Fahey 2001). Indole alkaloids are phytoalexins and production of specific alkaloids is usually limited to only a few species. Cruciferous plants include the model plant Arabidopsis, which produces the indole alkaloid camalexin. This review will focus on the central role of indole-3-acetaldoxime (IAOx) in the biosynthesis of indole glucosinolates, camalexin, and the phytohormone IAA.     

Indole glucosinolate and auxin biosynthesis in Arabidopsis thaliana (L.) Heynh. glucosinolate mutants and the development of clubroot disease

Mutants and wild type plants of Arabidopsis thaliana were analysed for differences in glucosinolate accumulation patterns, indole-3-acetic acid (IAA) biosynthesis and phenotype. A previously identified series of mutants, termed TU, with altered glucosinolate patterns was used in this study. Only the line TU8 was affected in shoot phenotype (shorter stems, altered branching pattern). Synthesis of IAA and metabolism were not much affected in the TU8 mutant during seedling development, although the content of free IAA peaked earlier in TU8 during plant development than in the wild type. Indole glucosinolates and IAA may, however, be involved in the development of clubroot disease caused by the obligate biotrophic fungus Plasmodiophora brassicae since the TU3 line had a lower infection rate than the wild type, and lines TU3 and TU8 showed decreased symptom development. The decline in clubroot formation was accompanied by a reduced number of fungal structures within the root cortex and slower development of the fungus. Indole glucosinolates were lower in infected roots of TU3 and TU8 than in control roots of these lines, whereas in wild-type plants the differences were not as prominent. Free IAA and indole-3-acetonitrile (IAN) were increased in infected roots of the wild type and mutants with normal clubroot symptoms, whereas they were reduced in infected roots of mutants TU3 and TU8. These results indicate a role for indole glucosinolates and IAN/IAA in relation to symptom development in clubroot disease. Received: 23 July 1998 / Accepted: 12 January 1999     

Tryptophan-dependent indole-3-acetic acid biosynthesis by ‘IAA-synthase’ proceeds via indole-3-acetamide

Plants are suggested to produce their major growth promoting phytohormone, indole-3-acetic acid (IAA), via multiple redundantly operating pathways. Although great effort has been made and plenty of possible routes have been proposed based on experimental evidence, a complete pathway for IAA production has yet to be demonstrated. In this study, an in-vitro approach was taken to examine the conversion of l-tryptophan (l-trp) to IAA by gas chromatography-mass spectrometry (GC-MS). Especially the influence of putative reaction intermediates on the enzymatic conversion of l-trp to IAA was analyzed. Among the substances tested only indole-3-acetamide (IAM) showed a pronounced effect on the l-trp conversion. We additionally report that IAM is synthesized from l-trp and that it is further converted to IAA by the utilized cell free Arabidopsis extract. Together, our results underscore the functionality of an IAM-dependent auxin biosynthesis pathway in Arabidopsis thaliana.     

Molecular cloning and characterization of an amidase from Arabidopsis thaliana capable of converting indole-3-acetamide into the plant growth hormone,indole-3-acetic acid

Acylamidohydrolases from higher plants have not been characterized or cloned so far. AtAMI1 is the first member of this enzyme family from a higher plant and was identified in the genome of Arabidopsis thaliana based on sequence homology with the catalytic-domain sequence of bacterial acylamidohydrolases, particularly those that exhibit indole-3-acetamide amidohydrolase activity. AtAMI1 polypeptide and mRNA are present in leaf tissues, as shown by immunoblotting and RT-PCR, respectively. AtAMI1 was expressed from its cDNA in enzymatically active form and exhibits substrate specificity for indole-3-acetamide, but also some activity against L-asparagine. The recombinant enzyme was characterized further. The results show that higher plants have acylamidohydrolases with properties similar to the enzymes of certain plant-associated bacteria such as Agrobacterium-, Pseudomonas- and Rhodococcus-species, in which these enzymes serve to synthesize the plant growth hormone, indole-3-acetic acid, utilized by the bacteria to colonize their host plants. As indole-3-acetamide is a native metabolite in Arabidopsis thaliana, it can no longer be ruled out that one pathway for the biosynthesis of indole-3-acetic acid involves indole-3-acetamide-hydrolysis by AtAMI1.     

CYP79B1 from Sinapis alba converts tryptophan to indole-3-acetaldoxime

The cytochrome P450 CYP79B1 from Sinapis alba has been heterologously expressed in Escherichia coli and shown to catalyze the conversion of tryptophan to indole-3-acetaldoxime. Three expression constructs were made, one expressing the native protein and two expressing proteins with different N-terminal modifications. The native construct gave the highest yield as estimated by enzymatic activity per liter of culture. Spheroplasts of E. coli expressing CYP79B1 were reconstituted with the Arabidopsis thaliana NADPH:cytochrome P450 reductase ATR1 heterologously expressed in E. coli to obtain enzymatic activity. This indicates that the E. coli electron-donating system, flavodoxin/flavodoxin reductase, does not support CYP79B1 activity. Recombinant CYP79B1 has a K(m) for tryptophan of 29+/-2 microM and a V(max) of 36.5+/-0.7nmolh(-1)(mlculture)(-1). The identity at the amino acid level of CYP79B1 is, respectively, 93 and 84% to CYP79B2 and CYP79B3 from A. thaliana, and 96% to CYP79B5 (Accession No. AF453287) from Brassica napus. The CYP79B subfamily of cytochromes P450 is likely to constitute a group of orthologous genes in the biosynthesis of indole glucosinolates.     

The NtAMI1 gene functions in cell division of tobacco BY-2 cells in the presence of indole-3-acetamide

Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cells can be grown in medium containing indole-3-acetamide (IAM). Based on this finding, the NtAMI1 gene, whose product is functionally equivalent to the AtAMI1 gene of Arabidopsis thaliana and the aux2 gene of Agrobacterium rhizogenes, was isolated from BY-2 cells. Overexpression of the NtAMI1 gene allowed BY-2 cells to proliferate at lower concentrations of IAM, whereas suppression of the NtAMI1 gene by RNA interference (RNAi) caused severe growth inhibition in the medium containing IAM. These results suggest that IAM is incorporated into plant cells and converted to the auxin, indole-3-acetic acid, by NtAMI1.     

A chromatin-dependent mechanism regulates gene expression at the core of the Arabidopsis circadian clock

The mechanisms of circadian clock function in Arabidopsis rely on the complex relationships among core clock components. The current model of the Arabidopsis oscillator comprises a myriad of repressors but the mechanisms responsible for activation remain largely unknown. In our recent studies, we have demonstrated that the rhythms in H3 acetylation (H3ac) and H3K4 trimethylation (H3K4me3) are a key mechanism at the positive arm of the oscillator. H3K4me3 rhythmic accumulation is delayed compared to that of H3ac, which opens the possibility for separate roles for each mark. Indeed, the use of inhibitors that block H3K4me3 accumulation was concomitant with increased clock repressor binding, suggesting that H3K4me3 might control the timing from activation to repression. Plants mis-expressing the histone methyltransferase SET DOMAIN GROUP 2 (SDG2/ATXR3) displayed altered H3K4me3 accumulation, oscillator gene expression and clock repressor binding, suggesting that SDG2/ATXR3 is a key component contributing to proper circadian expression.     

Knock-out of Arabidopsis AtNHX4 gene enhances tolerance to salt stress

AtNHX4 belongs to the monovalent cation:proton antiporter-1 (CPA1) family in Arabidopsis. Several members of this family have been shown to be critical for plant responses to abiotic stress, but little is known on the biological functions of AtNHX4. Here, we provide the evidence that AtNHX4 plays important roles in Arabidopsis responses to salt stress. Expression of AtNHX4 was responsive to salt stress and abscisic acid. Experiments with CFP-AtNHX4 fusion protein indicated that AtNHX4 is vacuolar localized. The nhx4 mutant showed enhanced tolerance to salt stress, and lower Na⁺ content under high NaCl stress compared with wild-type plants. Furthermore, heterologous expression of AtNHX4 in Escherichia coli BL21 rendered the transformants hypersensitive to NaCl. Deletion of the hydrophilic C-terminus of AtNHX4 dramatically increased the hypersensitivity of transformants, indicating that AtNHX4 may function in Na⁺ homeostasis in plant cell, and its C-terminus plays a role in regulating the AtNHX4 activity.     

Benzoic acid glucosinolate esters and other glucosinolates from Arabidopsis thaliana

The spectacular recent progress in Arabidopsis thaliana molecular genetics furnishes outstanding tools for studying the formation and function of all metabolites in this cruciferous species. One of the major groups of secondary metabolites in A. thaliana is the glucosinolates. These hydrophilic, sulfur-rich glycosides appear to serve as defenses against some generalist herbivores and pathogens, and as feeding and oviposition stimulants to specialist herbivores. To help study their biosynthesis and role in plant-insect interactions, we wanted to determine the complete glucosinolate content of A. thaliana. In previous studies, 24 glucosinolates had been identified from ecotype Columbia. We reinvestigated Columbia as well as additional ecotypes and mutant lines, and identified 12 further glucosinolates, including five novel compounds. Structures were elucidated by MS and NMR spectroscopy of their desulfated derivatives, and by enzymatic cleavage of the attached ester moieties. Four of the novel glucosinolates are benzoate esters isolated from the seeds. In all but one of these compounds, esterification is on the glucose moiety rather than the side chain, a very unusual feature for glucosinolates. Among additional glucosinolates identified were the first non-chain elongated, methionine-derived glucosinolate from A. thaliana and the first compounds that appear to be derived from leucine.     

Indole-3-acetic acid is synthesized from L-tryptophan in roots of Arabidopsis thaliana

The promoter of the nit1 gene, encoding the predominantly expressed isoform of the Arabidopsis thaliana (L.) Heynh. nitrilase isoenzyme family, fused to the β-glucuronidase gene (uidA) drives β-glucuronidase expression in the root system of transgenic A. thaliana and tobacco plants. This expression pattern was shown to be controlled developmentally, suggesting that the early differentiation zone of root tips and the tissue surrounding the zone of lateral root primordia formation may constitute sites of auxin biosynthesis in plants. The root system of A. thaliana was shown to express functional nitrilase enzyme. When sterile roots were fed [²H]₅-L-tryptophan, they converted this precusor to [²H]₅-indole-3-acetonitrile and [²H]₅-indole-3-acetic acid. This latter metabolite was further metabolized into base-labile conjugates which were the predominant form of [²H]₅-indole-3-acetic acid extracted from roots. When [1-¹³C]-indole-3-acetonitrile was fed to sterile roots, it was converted to [1-¹³C]-indole-3-acetic acid which was further converted to conjugates. The results prove that the A. thaliana root system is an autonomous site of indole-3-acetic acid biosynthesis from L-tryptophan. Received: 3 February 1998 / Accepted: 17 April 1998     

Arabidopsis AtIscA-I is affected by deficiency of Fe-S cluster biosynthetic scaffold AtCnfU-V

IscA has been proposed to be a scaffold protein of the iron-sulfur cluster biosynthetic machinery. We have identified the IscA homolog to be localized to plastids, termed AtIscA-I, in Arabidopsis thaliana. The AtIscA-I protein was apparently constitutively expressed in all tissues analyzed in Arabidopsis. The AtIscA-I protein exists in the stroma as a soluble protein which tends to form a homo-dimer and can host a [2Fe-2S]-like cluster. Complete loss of the protein from plastids did not cause any significant defect either in normal plant growth or in biogenesis of major iron-sulfur proteins, indicating this protein is not essential or redundant for these functions. In contrast, loss of one of the three plastid-localized CnfU scaffold proteins, AtCnfU-V, caused significant reduction in the level of AtIscA-I. These data suggest that efficient biogenesis of AtIscA-I scaffold requires function of another essential scaffold protein CnfU.     

Xylogalacturonan exists in cell walls from various tissues of Arabidopsis thaliana

Evidence is presented for the presence of xylogalacturonan (XGA) in Arabidopsis thaliana. This evidence was obtained by extraction of pectin from the seeds, root, stem, young leaves and mature leaves of A. thaliana, followed by treatment of these pectin extracts with xylogalacturonan hydrolase (XGH). Upon enzymatic treatment, XGA oligosaccharides were primarily produced from pectin extracts obtained from the young and mature leaves and to a lesser extent from those originating from the stem of A. thaliana. The oligosaccharide GalA(3)Xyl was predominantly formed from these pectin extracts. No XGA oligosaccharides were detected in digests of pectin extracts from the seeds and roots. A low number of XGA oligosaccharides was obtained from pectins of A. thaliana. This indicates a uniform distribution of xylose in XGA from A. thaliana. The predominant production of GalA(3)Xyl, as well as the release of linear GalA oligosaccharides pointed to a lower degree of xylose substitution in XGA from A. thaliana than in XGA from apple and potato. The estimated amount of XGA accounted for approximately 2.5%, 7% and 6% (w/w) of the total carbohydrate in the pectin fraction of the stem, young leaves and mature leaves, respectively.     

ESP and ESM1 mediate indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate in Arabidopsis

Glucosinolates are plant secondary metabolites that act as direct defenses against insect herbivores and various pathogens. Recent analysis has shown that methionine-derived glucosinolates are hydrolyzed/activated into either nitriles or isothiocyanates depending upon the plants genotype at multiple loci. While it has been hypothesized that tryptophan-derived glucosinolates can be a source of indole-acetonitriles, it has not been explicitly shown if the same proteins control nitrile production from tryptophan-derived glucosinolates as from methionine-derived glucosinolates. In this report, we formally test if the proteins involved in controlling aliphatic glucosinolate hydrolysis during tissue disruption can control production of nitriles during indolic glucosinolate hydrolysis. We show that myrosinase is not sufficient for indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate and requires the presence of functional epithospecifier protein in planta and in vitro to produce significant levels of indol-3-acetonitrile. This reaction is also controlled by the Epithiospecifier modifier 1 gene. Thus, like formation of nitriles from aliphatic glucosinolates, indol-3-acetonitrile production following tissue disruption is controlled by multiple loci raising the potential for complex regulation and fine tuning of indol-3-acetonitrile production from indol-3-ylmethyl glucosinolate.