Dierk Scheel

Mitogen-activated protein kinase cascades in plants: a new nomenclature

Mitogen-activated protein kinase (MAPK) cascades are universal signal transduction modules in eukaryotes, including yeasts, animals and plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. In plants, MAPK cascades are involved in responses to various biotic and abiotic stresses, hormones, cell division and developmental processes. Completion of the Arabidopsis genome-sequencing project has revealed the existence of 20 MAPKs, 10 MAPK kinases and 60 MAPK kinase kinases. Here, we propose a simplified nomenclature for Arabidopsis MAPKs and MAPK kinases that might also serve as a basis for standard annotation of these gene families in all plants.

Pre- and postinvasion defenses both contribute to nonhost resistance in Arabidopsis

Nonhost resistance describes the immunity of an entire plant species against nonadapted pathogen species. We report that Arabidopsis PEN2 restricts pathogen entry of two ascomycete powdery mildew fungi that in nature colonize grass and pea species. The PEN2 glycosyl hydrolase localizes to peroxisomes and acts as a component of an inducible preinvasion resistance mechanism. Postinvasion fungal growth is blocked by a separate resistance layer requiring the EDS1-PAD4-SAG101 signaling complex, which is known to function in basal and resistance (R) gene–triggered immunity. Concurrent impairment of pre- and postinvasion resistance renders Arabidopsis a host for both nonadapted fungi.

Receptor-Mediated Increase in Cytoplasmic Free Calcium Required for Activation of Pathogen Defense in Parsley

Transient influx of Ca2+ constitutes an early element of signaling cascades triggering pathogen defense responses in plant cells. Treatment with the Phytophthora sojae–derived oligopeptide elicitor, Pep-13, of parsley cells stably expressing apoaequorin revealed a rapid increase in cytoplasmic free calcium ([Ca2+]cyt), which peaked at ∼1 μM and subsequently declined to sustained values of 300 nM. Activation of this biphasic [Ca2+]cyt signature was achieved by elicitor concentrations sufficient to stimulate Ca2+ influx across the plasma membrane, oxidative burst, and phytoalexin production. Sustained concentrations of [Ca2+]cyt but not the rapidly induced [Ca2+]cyt transient peak are required for activation of defense-associated responses. Modulation by pharmacological effectors of Ca2+ influx across the plasma membrane or of Ca2+ release from internal stores suggests that the elicitor-induced sustained increase of [Ca2+]cyt predominantly results from the influx of extracellular Ca2+. Identical structural features of Pep-13 were found to be essential for receptor binding, increases in [Ca2+]cyt, and activation of defense-associated responses. Thus, a receptor-mediated increase in [Ca2+]cyt is causally involved in signaling the activation of pathogen defense in parsley.

Signal transmission in the plant immune response

Genetic and biochemical dissection of signaling pathways regulating plant pathogen defense has revealed remarkable similarities with the innate immune system of mammals and Drosophila. Numerous plant proteins resembling eukaryotic receptors have been implicated in the perception of pathogen-derived signal molecules. Receptor-mediated changes in levels of free calcium in the cytoplasm and production of reactive oxygen species and nitric oxide constitute early events generally observed in plant-pathogen interactions. Positive and negative regulation of plant pathogen defense responses has been attributed to mitogen-activated protein kinase cascades. In addition, salicylic acid, jasmonic acid and ethylene are components of signaling networks that provide the molecular basis for specificity of plant defense responses. This article reviews recent advances in our understanding of early signaling events involved in the establishment of plant disease resistance.

Pep-13, a plant defense-inducing pathogen-associated pattern from Phytophthora transglutaminases.

Innate immunity, an ancient form of defense against microbial infection, is well described for animals and is also suggested to be important for plants. Discrimination from self is achieved through receptors that recognize pathogen‐associated molecular patterns (PAMPs) not found in the host. PAMPs are evolutionarily conserved structures which are functionally important and, thus, not subject to frequent mutation. Here we report that the previously described peptide elicitor of defense responses in parsley, Pep‐13, constitutes a surface‐exposed fragment within a novel calcium‐dependent cell wall transglutaminase (TGase) from Phytophthora sojae. TGase transcripts and TGase activity are detectable in all Phytophthora species analyzed, among which are some of the most destructive plant pathogens. Mutational analysis within Pep‐13 identified the same amino acids indispensable for both TGase and defense‐eliciting activity. Pep‐13, conserved among Phytophthora TGases, activates defense in parsley and potato, suggesting its function as a genus‐specific recognition determinant for the activation of plant defense in host and non‐host plants. In summary, plants may recognize PAMPs with characteristics resembling those known to trigger innate immune responses in animals.

High Throughput Identification of Potential Arabidopsis Mitogen-activated Protein Kinases Substrates

Mitogen-activated protein kinase (MAPK) cascades are universal and highly conserved signal transduction modules in eucaryotes, including plants. These protein phosphorylation cascades link extracellular stimuli to a wide range of cellular responses. However, the underlying mechanisms are so far unknown as information about phosphorylation substrates of plant MAPKs is lacking. In this study we addressed the challenging task of identifying potential substrates for Arabidopsis thaliana mitogen-activated protein kinases MPK3 and MPK6, which are activated by many environmental stress factors. For this purpose, we developed a novel protein microarray-based proteomic method allowing high throughput study of protein phosphorylation. We generated protein microarrays including 1,690 Arabidopsis proteins, which were obtained from the expression of an almost nonredundant uniclone set derived from an inflorescence meristem cDNA expression library. Microarrays were incubated with MAPKs in the presence of radioactive ATP. Using a threshold-based quantification method to evaluate the microarray results, we were able to identify 48 potential substrates of MPK3 and 39 of MPK6. 26 of them are common for both kinases. One of the identified MPK6 substrates, 1-aminocyclopropane-1-carboxylic acid synthase-6, was just recently shown as the first plant MAPK substrate in vivo, demonstrating the potential of our method to identify substrates with physiological relevance. Furthermore we revealed transcription factors, transcription regulators, splicing factors, receptors, histones, and others as candidate substrates indicating that regulation in response to MAPK signaling is very complex and not restricted to the transcriptional level. Nearly all of the 48 potential MPK3 substrates were confirmed by other in vitro methods. As a whole, our approach makes it possible to shortlist candidate substrates of mitogen-activated protein kinases as well as those of other protein kinases for further analysis. Follow-up in vivo experiments are essential to evaluate their physiological relevance.

Ozone-Induced Programmed Cell Death in the Arabidopsis radical-induced cell death1 Mutant

Short, high-concentration peaks of the atmospheric pollutant ozone (O3) cause the formation of cell death lesions on the leaves of sensitive plants. Numerous similarities between the plant responses to O3 and pathogens suggest that O3 triggers hypersensitive response-like programmed cell death (PCD). We examined O3 and superoxide-induced cell death in the O3-sensitive radical-induced cell death1 (rcd1) mutant. Dying cells in O3-exposed rcd1 exhibited several of the typical morphological characteristics of the hypersensitive response and PCD. Double-mutant analyses indicated a requirement for salicylic acid and the function of the cyclic nucleotide-gated ion channel AtCNGC2 in cell death. Furthermore, a requirement for ATPases, kinases, transcription, Ca2+ flux, caspase-like proteolytic activity, and also one or more phenylmethylsulfonyl fluoride-sensitive protease activities was shown for the development of cell death lesions in rcd1. Furthermore, mitogen-activated protein kinases showed differential activation patterns in rcd1 and Columbia. Taken together, these results directly demonstrate the induction of PCD by O3.

Parsley protoplasts retain differential responsiveness to u. v. light and fungal elicitor.

The differential response of cultured parsley cells to u.v. irradiation and elicitor treatment is a paradigm for analysis of specific plant defense responses. We demonstrate that freshly isolated parsley protoplasts, in the absence of detectable cell wall, maintain fully the ability to be activated by these important environmental factors. Stimulated protoplasts synthesize typical qualitative patterns and amounts of potentially protective flavonoid glycosides and coumarin phytoalexins following either u.v. irradiation or treatment with fungal elicitor, respectively. Induced accumulation of mRNAs and enzymes of the phenylpropanoid biosynthetic pathways is nearly identical in protoplasts and cells. Stimulation of protoplasts with elicitor requires only a short period of contact, which is not sufficient for cell wall regeneration. Importantly, there is no activation of these pathways during protoplast preparation. These results establish that parsley protoplasts respond appropriately to two physically distinct stimuli and might serve as an especially suitable system for the analysis of signal transduction and gene activation.

Changes in the accumulation of soluble and cell wall-bound phenolics in elicitor-treated cell suspension cultures and fungus-infected leaves of Solanum tuberosum

Cell suspension cultures of potato (Solanum tuberosum cv. Datura) treated with an elicitor preparation from Phytophthora infestans and potato leaves infected with the same fungus were used to study changes in the accumulation patterns of soluble and cell wall-bound phenolics. The compounds were identified by chromatographic comparison with authentic substances and by spectroscopic methods (FAB mass spectrometry, 1H and 13C NMR). The soluble phenolics were 4-O-β-glucopyranosylhydroquinone (arbutin), 4-O-β-glucopyranosylbenzoate, 3-methoxy-4-O-β-glucopyranosylbenzoate (vanillate glucoside), N-(E)-caffeoylputrescine, 2-O-β-glucopyranosylbenzoate (salicylate glucoside), N-(E)-feruloylputrescine, and N-(E)-feruloylaspartate. The cell wall-bound phenolics were 4-hydroxybenzoate, 4-hydroxybenzaldehyde, 3-methoxy-4-hydroxybenzaldehyde (vanillin), 4-(E)-coumarate, (E)-ferulate, N-4-(E)-coumaroyltyramine, and N-(E)-feruloyltyramine. The most prominent phenolics showing elicitor- or fungus-induced increases in accumulation rates were the soluble putrescine amides and cell wall-bound 4-hydroxybenzaldehyde and tyramine amides. In addition, there was a secretion of large amounts of coumaroyltyramine into the cell culture medium.

Metabolome Analysis of Biosynthetic Mutants Reveals a Diversity of Metabolic Changes and Allows Identification of a Large Number of New Compounds in Arabidopsis

Metabolomics is facing a major challenge: the lack of knowledge about metabolites present in a given biological system. Thus, large-scale discovery of metabolites is considered an essential step toward a better understanding of plant metabolism. We show here that the application of a metabolomics approach generating structural information for the analysis of Arabidopsis (Arabidopsis thaliana) mutants allows the efficient cataloging of metabolites. Fifty-six percent of the features that showed significant differences in abundance between seeds of wild-type, transparent testa4, and transparent testa5 plants could be annotated. Seventy-five compounds were structurally characterized, 21 of which could be identified. About 40 compounds had not been known from Arabidopsis before. Also, the high-resolution analysis revealed an unanticipated expansion of metabolic conversions upstream of biosynthetic blocks. Deficiency in chalcone synthase results in the increased seed-specific biosynthesis of a range of phenolic choline esters. Similarly, a lack of chalcone isomerase activity leads to the accumulation of various naringenin chalcone derivatives. Furthermore, our data provide insight into the connection between p-coumaroyl-coenzyme A-dependent pathways. Lack of flavonoid biosynthesis results in elevated synthesis not only of p-coumarate-derived choline esters but also of sinapate-derived metabolites. However, sinapoylcholine is not the only accumulating end product. Instead, we observed specific and sophisticated changes in the complex pattern of sinapate derivatives.