Mycotoxin Ochratoxin A-induced cell death and changes in oxidative metabolism of Arabidopsis thaliana

We evaluated the phytotoxicity of mycotoxin ochratoxin A (OTA) from Aspergillus and Penicillium strains on Arabidopsis thaliana. The results demonstrate that the growth of Arabidopsis thaliana on media containing OTA was inhibited significantly. Moreover, OTA induced necrotic lesions in detached leaves, which are reminiscent of hypersensitive response lesions that are activated during plant–pathogen interactions and other abiotic stress factors. From our study, we can see that OTA exposure stimulated a biphasic oxidative burst in the leaves, resulting in the generation of hydrogen peroxide (H₂O₂) and superoxide anion radicals (O₂^·−) and in the concomitant down-regulation of antioxidant enzyme defense responses and up-regulation of lipid peroxidation. These results suggested that OTA damage might result from reactive oxygen species pathways. Our experiments provide a useful model plant system for research on OTA-induced plant cell death.     

Myzus persicae (green peach aphid) salivary components induce defence responses in Arabidopsis thaliana

Myzus persicae (green peach aphid) feeding on Arabidopsis thaliana induces a defence response, quantified as reduced aphid progeny production, in infested leaves but not in other parts of the plant. Similarly, infiltration of aphid saliva into Arabidopsis leaves causes only a local increase in aphid resistance. Further characterization of the defence-eliciting salivary components indicates that Arabidopsis recognizes a proteinaceous elicitor with a size between 3 and 10 kD. Genetic analysis using well-characterized Arabidopsis mutants shows that saliva-induced resistance against M. persicae is independent of the known defence signalling pathways involving salicylic acid, jasmonate and ethylene. Among 78 Arabidopsis genes that were induced by aphid saliva infiltration, 52 had been identified previously as aphid-induced, but few are responsive to the well-known plant defence signalling molecules salicylic acid and jasmonate. Quantitative PCR analyses confirm expression of saliva-induced genes. In particular, expression of a set of O -methyltransferases, which may be involved in the synthesis of aphid-repellent glucosinolates, was significantly up-regulated by both M. persicae feeding and treatment with aphid saliva. However, this did not correlate with increased production of 4-methoxyindol-3-ylmethylglucosinolate, suggesting that aphid salivary components trigger an Arabidopsis defence response that is independent of this aphid-deterrent glucosinolate.     

Jasmonic acid does not mediate root growth responses to wounding in Arabidopsis thaliana

Jasmonic acid (JA) is a crucial plant defence signalling substance that has recently been shown to mediate herbivory-induced root growth reduction in the ecological model species Nicotiana attenuata . To clarify whether JA-induced reduction of root growth might be a general response increasing plant fitness under biotic stress, a suite of experiments was performed with the model plant Arabidopsis thaliana . JA bursts were elicited in leaves of A. thaliana in different ways. Root growth reduction was neither induced by foliar application of herbivore oral secretions nor by direct application of methyl jasmonate to leaves. Root growth reduction was observed when leaves were infected with the pathogen Pseudomonas syringae pv. tomato, which persistently induces the JA signalling pathway. Yet, high resolution growth analyses of this effect in wild type and JA biosynthesis knock-out mutants showed that it was elicited by the bacterial toxin coronatine that suggests ethylene- but not JA-induced root growth reduction in A. thaliana . Overall, the results demonstrate that the reaction of root growth to herbivore-induced JA signalling differs among species, which is discussed in the context of different ecological defence strategies among species.     

A novel methyltransferase from the intracellular pathogen Plasmodiophora brassicae methylates salicylic acid

The obligate biotrophic pathogen Plasmodiophora brassicae causes clubroot disease in Arabidopsis thaliana, which is characterized by large root galls. Salicylic acid (SA) production is a defence response in plants, and its methyl ester is involved in systemic signalling. Plasmodiophora brassicae seems to suppress plant defence reactions, but information on how this is achieved is scarce. Here, we profile the changes in SA metabolism during Arabidopsis clubroot disease. The accumulation of SA and the emission of methylated SA (methyl salicylate, MeSA) were observed in P. brassicae‐infected Arabidopsis 28 days after inoculation. There is evidence that MeSA is transported from infected roots to the upper plant. Analysis of the mutant Atbsmt1, deficient in the methylation of SA, indicated that the Arabidopsis SA methyltransferase was not responsible for alterations in clubroot symptoms. We found that P. brassicae possesses a methyltransferase (PbBSMT) with homology to plant methyltransferases. The PbBSMT gene is maximally transcribed when SA production is highest. By heterologous expression and enzymatic analyses, we showed that PbBSMT can methylate SA, benzoic and anthranilic acids.     

Nuclear genes that promote splicing of group I introns in the chloroplast 23S rRNA and psbA genes in Chlamydomonas reinhardtii

Defence against pathogens in Arabidopsis is orchestrated by at least three signalling molecules: salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). The hrl1 (hypersensitive response-like lesions 1) mutant of Arabidopsis is characterized by spontaneous necrotic lesions, accumulation of reactive oxygen species, constitutive expression of SA- and ET/JA-responsive defence genes, and enhanced resistance to virulent bacterial and oomycete pathogens. Epistasis analyses of hrl1 with npr1, etr1, coi1 and SA-depleted nahG plants revealed novel interactions between SA and ET/JA signalling pathways in regulating defence gene expression and cell death. RNA gel-blot analysis of RNA isolated separately from the lesion+ and the lesion- leaves of double mutants of hrl1 revealed different signalling requirements for the expression of defence genes in these tissues. Expression of the ET/JA-responsive PDF1.2 gene was markedly reduced in hrl1 npr1 and in SA-depleted hrl1 nahG plants. In hrl1 nahG plants, expression of PDF1.2 was regulated by benzathiadiazole in a concentration-dependent manner: induced at low concentration and suppressed at high concentration. The hrl1 etr1 plants lacked systemic PR-1 expression, and exhibited compromised resistance to virulent Pseudomonas syringae and Peronospora parasitica. Inhibiting JA responses in hrl1 coi1 plants lead to exaggerated cell death and severe stunting of plants. Finally, the hrl1 mutation lead to elevated expression of AtrbohD, which encodes a major subunit of the NADPH oxidase complex. Our results indicate that defence gene expression and resistance against pathogens in hrl1 is regulated synergistically by SA and ET/JA defence pathways.     

Assessment on pollution of Ochratoxin A in grain in China and its apoptosis effect on vitro-cultured human tubular kidney cells

Ochratoxin A (OTA) is nephrotoxic, immunosuppressive, and teratogenic in many species and is a possible human carcinogen. In this study, we investigated the OTA pollution situations of grains in northern China and the signaling pathway that mediated OTA-induced apoptosis in human tubular kidney cells (HKCs). Samples of grains collected from three representative areas were determined by using high-performance liquid chromatography fluorescence method. The effects of OTA on cell apoptosis, caspase-3, Bax, and Bcl-2 expression, and phosphorylation of c-Jun NH(2) terminal kinase (JNK) were detected in cultured HKCs via flow cytometry (FCM), Hoechst 33258 staining, and Western blot. It showed that OTA pollution of edible grains was very common in north China. OTA could affect caspase-3, Bax, and Bcl-2 expression and increased cell apoptosis in cultured HKCs. The JNK signalling pathway might play an important role during these cellular events.     

The cellular redox state as a modulator in cadmium and copper responses in Arabidopsis thaliana seedlings

The cellular redox state is an important determinant of metal phytotoxicity. In this study we investigated the influence of cadmium (Cd) and copper (Cu) stress on the cellular redox balance in relation to oxidative signalling and damage in Arabidopsis thaliana. Both metals were easily taken up by the roots, but the translocation to the aboveground parts was restricted to Cd stress. In the roots, Cu directly induced an oxidative burst, whereas enzymatic ROS (reactive oxygen species) production via NADPH oxidases seems important in oxidative stress caused by Cd. Furthermore, in the roots, the glutathione metabolism plays a crucial role in controlling the gene regulation of the antioxidative defence mechanism under Cd stress. Metal-specific alterations were also noticed with regard to the microRNA regulation of CuZnSOD gene expression in both roots and leaves. The appearance of lipid peroxidation is dual: it can be an indication of oxidative damage as well as an indication of oxidative signalling as lipoxygenases are induced after metal exposure and are initial enzymes in oxylipin biosynthesis.In conclusion, the metal-induced cellular redox imbalance is strongly dependent on the chemical properties of the metal and the plant organ considered. The stress intensity determines its involvement in downstream responses in relation to oxidative damage or signalling.     

Sugar and hormone connections

Sugars modulate many vital processes that are also controlled by hormones during plant growth and development. Characterization of sugar-signalling mutants in Arabidopsis has unravelled a complex signalling network that links sugar responses to two plant stress hormones--abscisic acid and ethylene--in opposite ways. Recent molecular analyses have revealed direct, extensive glucose control of abscisic acid biosynthesis and signalling genes that partially antagonizes ethylene signalling during seedling development under light. Glucose and abscisic acid promote growth at low concentrations but act synergistically to inhibit growth at high concentrations. The effects of sugar and osmotic stress on morphogenesis and gene expression are distinct. The plasticity of plant growth and development are exemplified by the complex interplay of sugar and hormone signalling.     

Toxicokinetics and toxicodynamics of ochratoxin A, an update

Ochratoxin A (OTA) is a mycotoxin produced by fungi of two genera: Penicillium and Aspergillus. OTA has been shown to be nephrotoxic, hepatotoxic, teratogenic and immunotoxic to several species of animals and to cause kidney and liver tumours in mice and rats. Because of differences in the physiology of animal species, wide variations are seen in the toxicokinetic patterns of absorption, distribution and elimination of the toxin. Biotransformation of OTA has not been entirely elucidated. At present, data regarding OTA metabolism are controversial. Several metabolites have been characterized in vitro and/or in vivo, whereas other metabolites remain to be characterized. Several major mechanisms have been shown as involved in the toxicity of OTA: inhibition of protein synthesis, promotion of membrane peroxidation, disruption of calcium homeostasis, inhibition of mitochondrial respiration and DNA damage. The contribution of metabolites in OTA genotoxicity and carcinogenicity is still unclear. The genotoxic status of OTA is still controversial because contradictory results were obtained in various microbial and mammalian tests, notably regarding the formation of DNA adducts. More recent studies are focused on the OTA ability to disturb cellular signalling and regulation, to modulate physiological signals and thereby to influence cells viability and proliferation. The present paper offers an update on these different issues. In addition since humans and animals are likely to be simultaneously exposed to several mycotoxins, especially through their diet, the little information available on the combined effects of OTA and other mycotoxins has also been reviewed.     

Priming for enhanced defence responses by specific inhibition of the Arabidopsis response to coronatine

The priming agent β-aminobutyric acid (BABA) is known to enhance Arabidopsis resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 by potentiating salicylic acid (SA) defence signalling, notably PR1 expression. The molecular mechanisms underlying this phenomenon remain unknown. A genome-wide microarray analysis of BABA priming during Pst DC3000 infection revealed direct and primed up-regulation of genes that are responsive to SA, the SA analogue benzothiadiazole and pathogens. In addition, BABA was found to inhibit the Arabidopsis response to the bacterial effector coronatine (COR). COR is known to promote bacterial virulence by inducing the jasmonic acid (JA) response to antagonize SA signalling activation. BABA specifically repressed the JA response induced by COR without affecting other plant JA responses. This repression was largely SA-independent, suggesting that it is not caused by negative cross-talk between SA and JA signalling cascades. Treatment with relatively high concentrations of purified COR counteracted BABA inhibition. Under these conditions, BABA failed to protect Arabidopsis against Pst DC3000. BABA did not induce priming and resistance in plants inoculated with a COR-deficient strain of Pst DC3000 or in the COR-insensitive mutant coi1-16. In addition, BABA blocked the COR-dependent re-opening of stomata during Pst DC3000 infection. Our data suggest that BABA primes for enhanced resistance to Pst DC3000 by interfering with the bacterial suppression of Arabidopsis SA-dependent defences. This study also suggests the existence of a signalling node that distinguishes COR from other JA responses.     

NPR1 and EDS11 contribute to host resistance against Fusarium culmorum in Arabidopsis buds and flowers

The cereal ear blight fungal pathogen Fusarium culmorum can infect Arabidopsis floral tissue, causing disease symptoms and mycotoxin production. Here we assessed the effect of seven mutants and one transgenic overexpression line, residing in either the salicylic acid (SA), jasmonic acid (JA) or ethylene (ET) defence signalling pathways, on the outcome of the Fusarium –Arabidopsis floral interaction. The bacterial susceptiblity mutant eds11 was also assessed. Flowering plants were spray inoculated with F. culmorum conidia to determine the host responses to initial infection and subsequent colonization. Enhanced susceptibility and higher concentrations of deoxynivalenol mycotoxin were observed in buds and flowers of the npr1 and eds11 mutants than in the wild-type Col-0 plants. An effect of the other two defence signalling pathways on disease was either absent (ET/JA combined), absent/minimal (ET) or inconclusive (JA). Overall, this study highlights a role for NPR1 and EDS11 in basal defence against F. culmorum in some floral organs. This is the first time that any of these well-characterized defence signalling mutations have been evaluated for a role in floral defence in any plant species.     

Chemical genetic identification of glutamine phosphoribosylpyrophosphate amidotransferase as the target for a novel bleaching herbicide in Arabidopsis

A novel phenyltriazole acetic acid compound (DAS734) produced bleaching of new growth on a variety of dicotyledonous weeds and was a potent inhibitor of Arabidopsis (Arabidopsis thaliana) seedling growth. The phytotoxic effects of DAS734 on Arabidopsis were completely alleviated by addition of adenine to the growth media. A screen of ethylmethanesulfonate-mutagenized Arabidopsis seedlings recovered seven lines with resistance levels to DAS734 ranging from 5- to 125-fold. Genetic tests determined that all the resistance mutations were dominant and allelic. One mutation was mapped to an interval on chromosome 4 containing At4g34740, which encodes an isoform of glutamine phosphoribosylamidotransferase (AtGPRAT2), the first enzyme of the purine biosynthetic pathway. Sequencing of At4g34740 from the resistant lines showed that all seven contained mutations producing changes in the encoded polypeptide sequence. Two lines with the highest level of resistance (125-fold) contained the mutation R264K. The wild-type and mutant AtGPRAT2 enzymes were cloned and functionally overexpressed in Escherichia coli. Assays of the recombinant enzyme showed that DAS734 was a potent, slow-binding inhibitor of the wild-type enzyme (I(50) approximately 0.2 microm), whereas the mutant enzyme R264K was not significantly inhibited by 200 microm DAS734. Another GPRAT isoform in Arabidopsis, AtGPRAT3, was also inhibited by DAS734. This combination of chemical, genetic, and biochemical evidence indicates that the phytotoxicity of DAS734 arises from direct inhibition of GPRAT and establishes its utility as a new and specific chemical genetic probe of plant purine biosynthesis. The effects of this novel GPRAT inhibitor are compared to the phenotypes of known AtGPRAT genetic mutants.     

Long-distance CO(2) signalling in plants.

Stomatal numbers are tightly controlled by environmental signals including light intensity and atmospheric CO(2) partial pressure. This requires control of epidermal cell development during the early phase of leaf growth and involves changes in both the density of cells on the leaf surface and the proportion of cells that adopt a stomatal fate. This paper reviews the current understanding of how stomata develop and describes recent advances that have given insights into the regulatory mechanisms involved using mutant Arabidopsis plants that implicates a role for long-chain fatty acids in cell-to-cell communication. Evidence is presented which indicates that long-distance signalling from mature to newly developing leaves forms part of the mechanism by which stomatal development responds to environmental cues. Analysis of mutant plants suggests that the plant hormones abscisic acid, ethylene and jasmonates are implicated in the long-distance signalling pathway and that the action may be mediated by reactive oxygen species.     

Signalling requirements for Erwinia amylovora‐induced disease resistance,callose deposition and cell growth in the non‐host Arabidopsis thaliana

Erwinia amylovora is the causal agent of the fire blight disease in some plants of the Rosaceae family. The non‐host plant Arabidopsis serves as a powerful system for the dissection of mechanisms of resistance to E. amylovora. Although not yet known to mount gene‐for‐gene resistance to E. amylovora, we found that Arabidopsis activated strong defence signalling mediated by salicylic acid (SA), with kinetics and amplitude similar to that induced by the recognition of the bacterial effector avrRpm1 by the resistance protein RPM1. Genetic analysis further revealed that SA signalling, but not signalling mediated by ethylene (ET) and jasmonic acid (JA), is required for E. amylovora resistance. Erwinia amylovora induces massive callose deposition on infected leaves, which is independent of SA, ET and JA signalling and is necessary for E. amylovora resistance in Arabidopsis. We also observed tumour‐like growths on E. amylovora‐infected Arabidopsis leaves, which contain enlarged mesophyll cells with increased DNA content and are probably a result of endoreplication. The formation of such growths is largely independent of SA signalling and some E. amylovora effectors. Together, our data reveal signalling requirements for E. amylovora‐induced disease resistance, callose deposition and cell fate change in the non‐host plant Arabidopsis. Knowledge from this study could facilitate a better understanding of the mechanisms of host defence against E. amylovora and eventually improve host resistance to the pathogen.