Glutathione-indole-3-acetonitrile is required for camalexin biosynthesis in Arabidopsis thaliana

Camalexin, a major phytoalexin in Arabidopsis thaliana, consists of an indole ring and a thiazole ring. The indole ring is produced from Trp, which is converted to indole-3-acetonitrile (IAN) by CYP79B2/CYP79B3 and CYP71A13. Conversion of Cys(IAN) to dihydrocamalexic acid and subsequently to camalexin is catalyzed by CYP71B15. Recent studies proposed that Cys derivative, not Cys itself, is the precursor of the thiazole ring that conjugates with IAN. The nature of the Cys derivative and how it conjugates to IAN and subsequently forms Cys(IAN) remain obscure. We found that protein accumulation of multiple glutathione S-transferases (GSTs), elevation of GST activity, and consumption of glutathione (GSH) coincided with camalexin production. GSTF6 overexpression increased and GSTF6-knockout reduced camalexin production. Arabidopsis GSTF6 expressed in yeast cells catalyzed GSH(IAN) formation. GSH(IAN), (IAN)CysGly, and γGluCys(IAN) were determined to be intermediates within the camalexin biosynthetic pathway. Inhibitor treatments and mutant analyses revealed the involvement of γ-glutamyl transpeptidases (GGTs) and phytochelatin synthase (PCS) in the catabolism of GSH(IAN). The expression of GSTF6, GGT1, GGT2, and PCS1 was coordinately upregulated during camalexin biosynthesis. These results suggest that GSH is the Cys derivative used during camalexin biosynthesis, that the conjugation of GSH with IAN is catalyzed by GSTF6, and that GGTs and PCS are involved in camalexin biosynthesis.     

ROS perception in Arabidopsis thaliana: the ozone-induced calcium response

Ozone is responsible for more crop losses than any other air pollutant. The changes in gene expression, which occur in plants in response to ozone, have been well characterized, yet little is known about how ozone is perceived or the signal transduction steps that follow. The earliest characterized response to ozone is an elevation in cytosolic-free calcium, which takes place within seconds of exposure. In this study, the calcium response to ozone was investigated in Arabidopsis thaliana seedlings using a variety of fumigation protocols. Ozone elicited distinct calcium responses in the aerial tissue and roots of seedlings. The calcium response in the cotyledons and leaves was biphasic and sensitive to the rate at which the ozone concentration increased. The response in the root was monophasic and insensitive to the rate of increase in ozone concentration. Experiments utilizing inhibitors of antioxidant metabolism demonstrated that the magnitude of the first peak in calcium in the aerial tissues was dependent upon the redox status of the plant. Seedlings were shown to be able to distinguish between ozone and hydrogen peroxide, producing a calcium signal in response to one of these reactive oxygen species (ROS) when they had become refractory to the other. Pre-treatment with ozone altered the calcium response to hydrogen peroxide and vice versa, indicating that the calcium response to a given ROS may reflect the stress history of the plant. These data suggest ROS signalling is more sophisticated than previously realized and raise questions over current models of ozone perception.     

Quantification of camalexin, a phytoalexin from Arabidopsis thaliana: a comparison of five analytical methods

Camalexin is a phytoalexin of Arabidopsis thaliana and an important component of inducible defenses. Accurate quantification of low concentrations suffers from interference by structurally related metabolites. A. thaliana plants were induced with silver nitrate and camalexin was extracted using methanol and identified and quantified by (i) TLC as a blue fluorescent band, (ii) microtiter plate-based fluorescence spectroscopy, (iii) GC on a midpolar column coupled to flame ionization detection, (iv) C₁₈ HPLC coupled to a photodiode detector, and (v) UPLC coupled to a mass spectrometer detector. Standard curves over the range of 0.1–15 μg ml⁻¹ gave R² values from 0.996 to 0.999. The different methods were compared and evaluated for their ability to detect and quantify increasing concentrations (<0.4–8 μg g⁻¹ FW) of camalexin. Each of the techniques presented advantages and disadvantages with regard to accuracy, precision, interference, analytical sensitivity, and limits of detection. TLC is a good qualitative technique for the identification of camalexin and fluorescence spectroscopy is subject to quenching when performed on crude extracts. Comparable results were obtained with GC–FID, HPLC–PDA, and UPLC–MS, with UPLC–MS having the added advantage of short analysis times and detection based on accurate mass.     

Origin of the thiazole ring of camalexin, a phytoalexin from Arabidopsis thaliana.

The principal phytoalexin that accumulates in Arabidopsis thaliana after infection by fungi or bacteria is 3-thiazol-2'-yl-indole (camalexin). Detached noninoculated leaves of Arabidopsis and leaves inoculated with the fungus Cochliobolus carbonum were fed [35S]cysteine (Cys) and [35S]methionine. Inoculated leaves incorporated more than a 200-fold greater amount of radioactivity from [35S]Cys into camalexin, as compared with noninoculated leaves. The amount of radioactivity from [35S]Cys that was incorporated into camalexin from inoculated Arabidopsis leaves was 10-fold greater than the amount of radioactivity that was incorporated into camalexin from [35S]methionine. Additional labeling experiments were performed to determine whether other atoms of Cys are incorporated into camalexin. [14C]Cys and [35S]Cys were incorporated into camalexin with approximately the same efficiency. Cys labeled either with deuterium (D3-Cys[2,3,3]) or 13C and 15N ([U-13C,15N]Cys) was also fed to inoculated leaves of Arabidopsis; camalexin was analyzed by mass spectroscopic analysis. The average ratio of molecular ion intensities of 203/200 for [U-13C,15N]Cys-labeled camalexin was 4.22, as compared with 0.607 for the average 203/200 ratio for unlabeled camalexin. The mass fragment-ion intensity ratios of 60/58 (thiazole ring ion fragment) and 143/142 were also higher for [U-13C,15N]Cys-labeled camalexin, as compared with unlabeled camalexin. The 59/58 and 201/200 ratios were higher for D3-Cys-labeled camalexin as compared with unlabeled camalexin. These data are consistent with the predicted formation of the thiazole ring of camalexin from Cys.     

Regulation of the host response to bacterial lipopolysaccharides

Bacterial endotoxins or lipopolysaccharides (LPS) are unique glycolipids present in the outer cell membrane of all gram-negative bacteria. It is now generally recognized that LPS is of primary importance in initiating the pathophysiological changes that often accompany gram-negative bacillary infections in humans including hypotensive shock, disseminated intravascular coagulation, and metabolic abnormalities. Although the biochemical mechanisms of these changes are not well understood, increasing emphasis has been placed on defining the biochemical response of the macrophage (M phi) to LPS. In this paper we describe two M phi-derived factors induced by LPS that may be important in the expression of endotoxic activity in the host. These are a procoagulant activity, which is present on the cell membrane of LPS-treated rabbit liver M phi and acts by directly activating coagulation factor X, and a factor released into the supernatant by LPS-treated peritoneal exudate M phi, which suppresses steroidogenesis in explanted adrenocortical cells. The potential role of the M phi in regulating the binding of LPS to high-density lipoproteins through the induction of acute phase proteins is also considered.     

Characterisation of the first enzymes committed to lysine biosynthesis in Arabidopsis thaliana

In plants, the lysine biosynthetic pathway is an attractive target for both the development of herbicides and increasing the nutritional value of crops given that lysine is a limiting amino acid in cereals. Dihydrodipicolinate synthase (DHDPS) and dihydrodipicolinate reductase (DHDPR) catalyse the first two committed steps of lysine biosynthesis. Here, we carry out for the first time a comprehensive characterisation of the structure and activity of both DHDPS and DHDPR from Arabidopsis thaliana. The A. thaliana DHDPS enzyme (At-DHDPS2) has similar activity to the bacterial form of the enzyme, but is more strongly allosterically inhibited by (S)-lysine. Structural studies of At-DHDPS2 show (S)-lysine bound at a cleft between two monomers, highlighting the allosteric site; however, unlike previous studies, binding is not accompanied by conformational changes, suggesting that binding may cause changes in protein dynamics rather than large conformation changes. DHDPR from A. thaliana (At-DHDPR2) has similar specificity for both NADH and NADPH during catalysis, and has tighter binding of substrate than has previously been reported. While all known bacterial DHDPR enzymes have a tetrameric structure, analytical ultracentrifugation, and scattering data unequivocally show that At-DHDPR2 exists as a dimer in solution. The exact arrangement of the dimeric protein is as yet unknown, but ab initio modelling of x-ray scattering data is consistent with an elongated structure in solution, which does not correspond to any of the possible dimeric pairings observed in the X-ray crystal structure of DHDPR from other organisms. This increased knowledge of the structure and function of plant lysine biosynthetic enzymes will aid future work aimed at improving primary production.     

A single locus determines sensitivity to bacterial flagellin in Arabidopsis thaliana

Peptides corresponding to the most conserved domain of eubacterial flagellin act as potent elicitors in cells of different plant species. In intact Arabidposis thaliana seedlings these peptides (flg22 and flg15) caused callose deposition, induction of genes coding for pathogenesis-related proteins and a strong inhibition of growth. Half-maximal growth inhibition occurred at peptide concentrations of approximately 100 nM. In contrast, peptides representing the corresponding flagellin domains of the plant-associated bacteria A. tumefaciens and R. meliloti were inactive even at concentrations of 10 microM. With the exception of Ws-0, all ecotypes of A. thaliana tested were sensitive to flg22. Crosses of Ws-0 with the sensitive ecotypes Col-0 and La-er, respectively, resulted in sensitive F1 seedlings. In the F2 generation of both crosses, sensitivity segregated as a single trait with markers of chromosome 5 and a ratio of 3:1. Dominance of the locus sensing flagellin, termed FLS-1, suggests that it encodes an element which is important for the perception of the flagellin signal.     

Flavonoid diversity and biosynthesis in seed of Arabidopsis thaliana

Functional characterization of genes involved in the flavonoid metabolism and its regulation requires in-depth analysis of flavonoid structure and composition of seed from the model plant Arabidopsis thaliana. Here, we report an analysis of the diverse and specific flavonoids that accumulate during seed development and maturation in wild types and mutants. Wild type seed contained more than 26 different flavonoids belonging to flavonols (mono and diglycosylated quercetin, kaempferol and isorhamnetin derivatives) and flavan-3-ols (epicatechin monomers and soluble procyanidin polymers with degrees of polymerization up to 9). Most of them are described for the first time in Arabidopsis. Interestingly, a novel group of four biflavonols that are dimers of quercetin-rhamnoside was also detected. Quercetin-3-O-rhamnoside (the major flavonoid), biflavonols, epicatechin and procyanidins accumulated in the seed coat in contrast to diglycosylated flavonols that were essentially observed in the embryo. Epicatechin, procyanidins and an additional quercetin-rhamnoside-hexoside derivative were synthesized in large quantities during seed development, whereas quercetin-3-O-rhamnoside displayed two peaks of accumulation. Finally, 11 mutants affected in known structural or regulatory functions of the pathway and their three corresponding wild types were also studied. Flavonoid profiles of the mutants were consistent with previous predictions based on genetic and molecular data. In addition, they also revealed the presence of new products in seed and underlined the plasticity of this metabolic pathway in the mutants.     

Arabidopsis PAD3, a gene required for camalexin biosynthesis, encodes a putative cytochrome P450 monooxygenase

Phytoalexins are low molecular weight antimicrobial compounds that are synthesized in response to pathogen attack. The phytoalexin camalexin, an indole derivative, is produced by Arabidopsis in response to infection with the bacterial pathogen Pseudomonas syringae. The phytoalexin deficient 3 (pad3) mutation, which causes a defect in camalexin production, has no effect on resistance to P. syringae but compromises resistance to the fungal pathogen Alternaria brassicicola. We have now isolated PAD3 by map-based cloning. The predicted PAD3 protein appears to be a cytochrome P450 monooxygenase, similar to those from maize that catalyze synthesis of the indole-derived secondary metabolite 2,4-dihydroxy-1, 4-benzoxazin-3-one. The expression of PAD3 is tightly correlated with camalexin synthesis and is regulated by PAD4 and PAD1. On the basis of these findings, we conclude that PAD3 almost certainly encodes an enzyme required for camalexin biosynthesis. Moreover, these results strongly support the idea that camalexin does not play a major role in plant resistance to P. syringae infection, although it is involved in resistance to a fungal pathogen.     

Phospholipase D is a negative regulator of proline biosynthesis in Arabidopsis thaliana

Accumulation of proline has been observed in a large number of plant species in response to drought and salt stresses, suggesting a key role of this amino acid in plant stress adaptation. Upstream components of the proline biosynthesis signal transduction pathways are still poorly defined. We provide experimental evidence that phospholipase D (PLD) is involved in the regulation of proline metabolism in Arabidopsis thaliana. The application of primary butyl alcohols, which divert part of PLD-derived phosphatidic acid by transphosphatidylation, stimulated proline biosynthesis even without hyperosmotic constraints. Moreover, application of primary butyl alcohols enhanced the proline responsiveness of seedlings to mild hyperosmotic stress. These data indicate that some PLDs are negative regulators of proline biosynthesis and that plants present a higher proline responsiveness to hyperosmotic stress when this regulator is abolished. We clearly demonstrate that PLD signaling for proline biosynthesis is similar to RD29A gene expression and different from the abscisic acid-dependent RAB18 gene expression. Our data reveal that PLDs play positive and negative roles in hyperosmotic stress signal transduction in plants, contributing to a precise regulation of ion homeostasis and plant salt tolerance.     

Successful expression of a novel bacterial gene for pinoresinol reductase and its effect on lignan biosynthesis in transgenic Arabidopsis thaliana

Pinoresinol reductase and pinoresinol/lariciresinol reductase play important roles in an early step of lignan biosynthesis in plants. The activities of both enzymes have also been detected in bacteria. In this study, pinZ, which was first isolated as a gene for bacterial pinoresinol reductase, was constitutively expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Higher reductive activity toward pinoresinol was detected in the resultant transgenic plants but not in wild-type plant. Principal component analysis of data from untargeted metabolome analyses of stem, root, and leaf extracts of the wild-type and two independent transgenic lines indicate that pinZ expression caused dynamic metabolic changes in stems, but not in roots and leaves. The metabolome data also suggest that expression of pinZ influenced the metabolisms of lignan and glucosinolates but not so much of neolignans such as guaiacylglycerol-8-O-4′-feruloyl ethers. In-depth quantitative analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) indicated that amounts of pinoresinol and its glucoside form were markedly reduced in the transgenic plant, whereas the amounts of glucoside form of secoisolariciresinol in transgenic roots, leaves, and stems increased. The detected levels of lariciresinol in the transgenic plant following β-glucosidase treatment also tended to be higher than those in the wild-type plant. Our findings indicate that overexpression of pinZ induces change in lignan compositions and has a major effect not only on lignan biosynthesis but also on biosynthesis of other primary and secondary metabolites.     

Adaptation and growth response of Arabidopsis thaliana to deuterium

Summary Arabidopsis seeds were sown aseptically on mineral media containing between 0 and 90% of heavy water (D₂O). Initially, a D₂O level of over 50% was lethal for the plants. However, after culture for six successive generations on 50% D₂O, plants were capable of growing marginally on media containing up to 70% D₂O, but not higher. With increasing concentration, deuterium progressively delays germination, slows growth, reduces survival, results in bleaching of the leaves and delays flowering. Pollen fertility is not affected measurably but seed set is reduced with increasing levels of deuteration so that at 70% D₂O few seeds were obtained. The viability of the seeds harvested from plants grown on deuterated media is low. No chlorophyll or morphological mutants were observed among a large number of plant progenies. Seeds from plants cultured on D₂O media for several generations grow normally on proteated media in the very first generation.     

The biosynthesis of benzoic acid glucosinolate esters in Arabidopsis thaliana

The siliques and seeds of Arabidopsis thaliana accumulate a series of glucosinolates containing an alkyl side chain of varying length with a terminal benzoate ester function. The biosynthesis of these unusual nitrogen- and sulfur-containing natural products was investigated by feeding isotopically-labeled precursors to detached flowering stems. Glucosinolates were extracted, purified and analyzed by tandem mass spectrometry. Phenylalanine and benzoic acid were incorporated into the benzoate ester function, and methionine and acetate were incorporated into the aliphatic portion of the side chain in a position-specific manner. The labeling patterns observed were consistent with the chain extension of methionine by a three-step elongation cycle which begins with the condensation of acetyl-CoA with a 2-oxo acid derived from methionine and ends with an oxidative decarboxylation forming a new 2-oxo acid with an additional methylene group. Incorporation of desulfo-4-methylthiobutyl glucosinolate into 4-benzoyloxybutyl olucosinolate suggested chain-extended methionine derivatives are first converted to their corresponding methylthioalkyl glucosinolates with further side chain modification occurring later. Transformation of the methylthiol function to a hydroxyl group is followed by esterification with benzoic acid. The siliques appear to possess the complete machinery for carrying out all of the reactions in the biosyntheis of these complex glucosinolates.     

Phytoalexins and phytoanticipins from the wild crucifers Thellungiella halophila and Arabidopsis thaliana: rapalexin A, wasalexins and camalexin

Investigation of phytoalexin production using abiotic elicitation showed that the phytoalexin rapalexin A was produced by both Thellungiella halophila and Arabidopsis thaliana, but while A. thaliana produced camalexin, T. halophila produced wasalexins A and B and methoxybrassenin B. Considering that the genome of T. halophila is being sequenced currently and that the wasalexin pathway present in T. halophila is expected to involve a number of genes also present in Brassica species, our discovery should facilitate the isolation of genes involved in biosynthetic pathways of phytoalexins of the most economically important crucifer species.     

Fight to the death: Arabidopsis thaliana defense response to fungal necrotrophic pathogens

Necrotrophic fungi, being the largest class of fungal plant pathogens, pose a serious economic problem to crop production. They are the cause of heavy losses in agriculture worldwide. Understanding the process of plant infection by necrotrophic fungi, including subtle interaction networks connecting such evolutionarily distinct organisms has recently been given high research priority. Such studies are now possible mainly because of the utility of the model plant Arabidopsis thaliana. A. thaliana has a sequenced genome and thousands of mutants available, allowing investigation of virtually all aspects of plant pathogenesis. This review focuses on morphological and molecular changes in A. thaliana, which occur during response to infection by necrotrophic fungi. These responses in relation to resistance and susceptibility of the plant will be discussed.