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Publikationen - Molekulare Signalverarbeitung

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Publikation

Moss, B. L.; Mao, H.; Guseman, J. M.; Hinds, T. R.; Hellmuth, A.; Kovenock, M.; Noorassa, A.; Lanctot, A.; Calderón Villalobos, L. I. A.; Zheng, N.; Nemhauser, J. L.; Rate Motifs Tune Auxin/Indole-3-Acetic Acid Degradation Dynamics Plant Physiol. 169, 803-813, (2015) DOI: 10.1104/pp.15.00587

Ubiquitin-mediated protein degradation is a common feature in diverse plant cell signaling pathways; however, the factors that control the dynamics of regulated protein turnover are largely unknown. One of the best-characterized families of E3 ubiquitin ligases facilitates ubiquitination of auxin (aux)/indole-3-acetic acid (IAA) repressor proteins in the presence of auxin. Rates of auxin-induced degradation vary widely within the Aux/IAA family, and sequences outside of the characterized degron (the minimum region required for auxin-induced degradation) can accelerate or decelerate degradation. We have used synthetic auxin degradation assays in yeast (Saccharomyces cerevisiae) and in plants to characterize motifs flanking the degron that contribute to tuning the dynamics of Aux/IAA degradation. The presence of these rate motifs is conserved in phylogenetically distant members of the Arabidopsis (Arabidopsis thaliana) Aux/IAA family, as well as in their putative Brassica rapa orthologs. We found that rate motifs can act by enhancing interaction between repressors and the E3, but that this is not the only mechanism of action. Phenotypes of transgenic plants expressing a deletion in a rate motif in IAA28 resembled plants expressing degron mutations, underscoring the functional relevance of Aux/IAA degradation dynamics in regulating auxin responses.
Publikation

Gasperini, D.; Chauvin, A.; Acosta, I. F.; Kurenda, A.; Stolz, S.; Chételat, A.; Wolfender, J.-L.; Farmer, E. E.; Axial and Radial Oxylipin Transport Plant Physiol. 169, 2244-2254, (2015) DOI: 10.1104/pp.15.01104

Jasmonates are oxygenated lipids (oxylipins) that control defense gene expression in response to cell damage in plants. How mobile are these potent mediators within tissues? Exploiting a series of 13-lipoxygenase (13-lox) mutants in Arabidopsis (Arabidopsis thaliana) that displays impaired jasmonic acid (JA) synthesis in specific cell types and using JA-inducible reporters, we mapped the extent of the transport of endogenous jasmonates across the plant vegetative growth phase. In seedlings, we found that jasmonate (or JA precursors) could translocate axially from wounded shoots to unwounded roots in a LOX2-dependent manner. Grafting experiments with the wild type and JA-deficient mutants confirmed shoot-to-root oxylipin transport. Next, we used rosettes to investigate radial cell-to-cell transport of jasmonates. After finding that the LOX6 protein localized to xylem contact cells was not wound inducible, we used the lox234 triple mutant to genetically isolate LOX6 as the only JA precursor-producing LOX in the plant. When a leaf of this mutant was wounded, the JA reporter gene was expressed in distal leaves. Leaf sectioning showed that JA reporter expression extended from contact cells throughout the vascular bundle and into extravascular cells, revealing a radial movement of jasmonates. Our results add a crucial element to a growing picture of how the distal wound response is regulated in rosettes, showing that both axial (shoot-to-root) and radial (cell-to-cell) transport of oxylipins plays a major role in the wound response. The strategies developed herein provide unique tools with which to identify intercellular jasmonate transport routes.
Publikation

Goetz, S.; Hellwege, A.; Stenzel, I.; Kutter, C.; Hauptmann, V.; Forner, S.; McCaig, B.; Hause, G.; Miersch, O.; Wasternack, C.; Hause, B.; Role of cis-12-Oxo-Phytodienoic Acid in Tomato Embryo Development Plant Physiol. 158, 1715-1727, (2012) DOI: 10.1104/pp.111.192658

Oxylipins including jasmonates are signaling compounds in plant growth, development, and responses to biotic and abiotic stresses. In Arabidopsis (Arabidopsis thaliana) most mutants affected in jasmonic acid (JA) biosynthesis and signaling are male sterile, whereas the JA-insensitive tomato (Solanum lycopersicum) mutant jai1 is female sterile. The diminished seed formation in jai1 together with the ovule-specific accumulation of the JA biosynthesis enzyme allene oxide cyclase (AOC), which correlates with elevated levels of JAs, suggest a role of oxylipins in tomato flower/seed development. Here, we show that 35S::SlAOC-RNAi lines with strongly reduced AOC in ovules exhibited reduced seed set similarly to the jai1 plants. Investigation of embryo development of wild-type tomato plants showed preferential occurrence of AOC promoter activity and AOC protein accumulation in the developing seed coat and the embryo, whereas 12-oxo-phytodienoic acid (OPDA) was the dominant oxylipin occurring nearly exclusively in the seed coat tissues. The OPDA- and JA-deficient mutant spr2 was delayed in embryo development and showed an increased programmed cell death in the developing seed coat and endosperm. In contrast, the mutant acx1a, which accumulates preferentially OPDA and residual amount of JA, developed embryos similar to the wild type, suggesting a role of OPDA in embryo development. Activity of the residual amount of JA in the acx1a mutant is highly improbable since the known reproductive phenotype of the JA-insensitive mutant jai1 could be rescued by wound-induced formation of OPDA. These data suggest a role of OPDA or an OPDA-related compound for proper embryo development possibly by regulating carbohydrate supply and detoxification.
Publikation

Weigelt, K.; Küster, H.; Rutten, T.; Fait, A.; Fernie, A. R.; Miersch, O.; Wasternack, C.; Emery, R. J. N.; Desel, C.; Hosein, F.; Müller, M.; Saalbach, I.; Weber, H.; ADP-Glucose Pyrophosphorylase-Deficient Pea Embryos Reveal Specific Transcriptional and Metabolic Changes of Carbon-Nitrogen Metabolism and Stress Responses Plant Physiol. 149, 395-411, (2009) DOI: 10.1104/pp.108.129940

We present a comprehensive analysis of ADP-glucose pyrophosphorylase (AGP)-repressed pea (Pisum sativum) seeds using transcript and metabolite profiling to monitor the effects that reduced carbon flow into starch has on carbon-nitrogen metabolism and related pathways. Changed patterns of transcripts and metabolites suggest that AGP repression causes sugar accumulation and stimulates carbohydrate oxidation via glycolysis, tricarboxylic acid cycle, and mitochondrial respiration. Enhanced provision of precursors such as acetyl-coenzyme A and organic acids apparently support other pathways and activate amino acid and storage protein biosynthesis as well as pathways fed by cytosolic acetyl-coenzyme A, such as cysteine biosynthesis and fatty acid elongation/metabolism. As a consequence, the resulting higher nitrogen (N) demand depletes transient N storage pools, specifically asparagine and arginine, and leads to N limitation. Moreover, increased sugar accumulation appears to stimulate cytokinin-mediated cell proliferation pathways. In addition, the deregulation of starch biosynthesis resulted in indirect changes, such as increased mitochondrial metabolism and osmotic stress. The combined effect of these changes is an enhanced generation of reactive oxygen species coupled with an up-regulation of energy-dissipating, reactive oxygen species protection, and defense genes. Transcriptional activation of mitogen-activated protein kinase pathways and oxylipin synthesis indicates an additional activation of stress signaling pathways. AGP-repressed embryos contain higher levels of jasmonate derivatives; however, this increase is preferentially in nonactive forms. The results suggest that, although metabolic/osmotic alterations in iAGP pea seeds result in multiple stress responses, pea seeds have effective mechanisms to circumvent stress signaling under conditions in which excessive stress responses and/or cellular damage could prematurely initiate senescence or apoptosis.
Publikation

Guranowski, A.; Miersch, O.; Staswick, P. E.; Suza, W.; Wasternack, C.; Substrate specificity and products of side-reactions catalyzed by jasmonate:amino acid synthetase (JAR1) FEBS Lett. 581, 815-820, (2007) DOI: 10.1016/j.febslet.2007.01.049

Jasmonate:amino acid synthetase (JAR1) is involved in the function of jasmonic acid (JA) as a plant hormone. It catalyzes the synthesis of several JA‐amido conjugates, the most important of which appears to be JA‐Ile. Structurally, JAR1 is a member of the firefly luciferase superfamily that comprises enzymes that adenylate various organic acids. This study analyzed the substrate specificity of recombinant JAR1 and determined whether it catalyzes the synthesis of mono‐ and dinucleoside polyphosphates, which are side‐reaction products of many enzymes forming acyl ∼ adenylates. Among different oxylipins tested as mixed stereoisomers for substrate activity with JAR1, the highest rate of conversion to Ile‐conjugates was observed for (±)‐JA and 9,10‐dihydro‐JA, while the rate of conjugation with 12‐hydroxy‐JA and OPC‐4 (3‐oxo‐2‐(2Z ‐pentenyl)cyclopentane‐1‐butyric acid) was only about 1–2% that for (±)‐JA. Of the two stereoisomers of JA, (−)‐JA and (+)‐JA, rate of synthesis of the former was about 100‐fold faster than for (+)‐JA. Finally, we have demonstrated that (1) in the presence of ATP, Mg2+, (−)‐JA and tripolyphosphate the ligase produces adenosine 5′‐tetraphosphate (p4A); (2) addition of isoleucine to that mixture halts the p4A synthesis; (3) the enzyme produces neither diadenosine triphosphate (Ap3A) nor diadenosine tetraphosphate (Ap4A) and (4) Ap4A cannot substitute ATP as a source of adenylate in the complete reaction that yields JA‐Ile.
Publikation

Ederli, L.; Morettini, R.; Borgogni, A.; Wasternack, C.; Miersch, O.; Reale, L.; Ferranti, F.; Tosti, N.; Pasqualini, S.; Interaction between Nitric Oxide and Ethylene in the Induction of Alternative Oxidase in Ozone-Treated Tobacco Plants Plant Physiol. 142, 595-608, (2006) DOI: 10.1104/pp.106.085472

The higher plant mitochondrial electron transport chain contains, in addition to the cytochrome chain, an alternative pathway that terminates with a single homodimeric protein, the alternative oxidase (AOX). We recorded temporary inhibition of cytochrome capacity respiration and activation of AOX pathway capacity in tobacco plants (Nicotiana tabacum L. cv BelW3) fumigated with ozone (O3). The AOX1a gene was used as a molecular probe to investigate its regulation by signal molecules such as hydrogen peroxide, nitric oxide (NO), ethylene (ET), salicylic acid, and jasmonic acid (JA), all of them reported to be involved in the O3 response. Fumigation leads to accumulation of hydrogen peroxide in mitochondria and early accumulation of NO in leaf tissues. Although ET accumulation was high in leaf tissues 5 h after the start of O3 fumigation, it declined during the recovery period. There were no differences in the JA and 12-oxo-phytodienoic acid levels of treated and untreated plants. NO, JA, and ET induced AOX1a mRNA accumulation. Using pharmacological inhibition of ET and NO, we demonstrate that both NO- and ET-dependent pathways are required for O3-induced up-regulation of AOX1a. However, only NO is indispensable for the activation of AOX1a gene expression.
Publikation

Mur, L. A.; Kenton, P.; Atzorn, R.; Miersch, O.; Wasternack, C.; The Outcomes of Concentration-Specific Interactions between Salicylate and Jasmonate Signaling Include Synergy, Antagonism, and Oxidative Stress Leading to Cell Death Plant Physiol. 140, 249-262, (2006) DOI: 10.1104/pp.105.072348

Salicylic acid (SA) has been proposed to antagonize jasmonic acid (JA) biosynthesis and signaling. We report, however, that in salicylate hydroxylase-expressing tobacco (Nicotiana tabacum) plants, where SA levels were reduced, JA levels were not elevated during a hypersensitive response elicited by Pseudomonas syringae pv phaseolicola. The effects of cotreatment with various concentrations of SA and JA were assessed in tobacco and Arabidopsis (Arabidopsis thaliana). These suggested that there was a transient synergistic enhancement in the expression of genes associated with either JA (PDF1.2 [defensin] and Thi1.2 [thionin]) or SA (PR1 [PR1a-β-glucuronidase in tobacco]) signaling when both signals were applied at low (typically 10–100 μm) concentrations. Antagonism was observed at more prolonged treatment times or at higher concentrations. Similar results were also observed when adding the JA precursor, α-linolenic acid with SA. Synergic effects on gene expression and plant stress were NPR1- and COI1-dependent, SA- and JA-signaling components, respectively. Electrolyte leakage and Evans blue staining indicated that application of higher concentrations of SA + JA induced plant stress or death and elicited the generation of apoplastic reactive oxygen species. This was indicated by enhancement of hydrogen peroxide-responsive AoPR10-β-glucuronidase expression, suppression of plant stress/death using catalase, and direct hydrogen peroxide measurements. Our data suggests that the outcomes of JA-SA interactions could be tailored to pathogen/pest attack by the relative concentration of each hormone.
Publikation

Bücking, H.; Förster, H.; Stenzel, I.; Miersch, O.; Hause, B.; Applied jasmonates accumulate extracellularly in tomato, but intracellularly in barley FEBS Lett. 562, 45-50, (2004) DOI: 10.1016/S0014-5793(04)00178-4

Jasmonic acid (JA) and its derivatives are well‐characterized signaling molecules in plant defense and development, but the site of their localization within plant tissue is entirely unknown. To address the question whether applied JA accumulates extracellularly or intracellularly, leaves of tomato and barley were fed with 14C‐labeled JA and the label was localized in cryofixed and lyophilized leaf tissues by microautoradiography. In tomato the radioactivity was detectable within the apoplast, but no label was found within the mesophyll cells. By contrast, in barley leaf tissues, radioactivity was detected within the mesophyll cells suggesting a cellular uptake of exogenously applied JA. JA, applied to leaves of both plants as in the labeling experiments, led in all leaf cells to the expression of JA‐inducible genes indicating that the perception is completed by JA signal transduction.
Publikation

O'Donnell, P. J.; Schmelz, E.; Block, A.; Miersch, O.; Wasternack, C.; Jones, J. B.; Klee, H. J.; Multiple Hormones Act Sequentially to Mediate a Susceptible Tomato Pathogen Defense Response Plant Physiol. 133, 1181-1189, (2003) DOI: 10.1104/pp.103.030379

Phytohormones regulate plant responses to a wide range of biotic and abiotic stresses. How a limited number of hormones differentially mediate individual stress responses is not understood. We have used one such response, the compatible interaction of tomato (Lycopersicon esculentum) and Xanthomonas campestris pv vesicatoria (Xcv), to examine the interactions of jasmonic acid (JA), ethylene, and salicylic acid (SA). The role of JA was assessed using an antisense allene oxide cyclase transgenic line and the def1 mutant to suppress Xcv-induced biosynthesis of jasmonates. Xcv growth was limited in these lines as was subsequent disease symptom development. No increase in JA was detected before the onset of terminal necrosis. The lack of a detectable increase in JA may indicate that an oxylipin other than JA regulates basal resistance and symptom proliferation. Alternatively, there may be an increase in sensitivity to JA or related compounds following infection. Hormone measurements showed that the oxylipin signal must precede subsequent increases in ethylene and SA accumulation. Tomato thus actively regulates the Xcv-induced disease response via the sequential action of at least three hormones, promoting expansive cell death of its own tissue. This sequential action of jasmonate, ethylene, and SA in disease symptom development is different from the hormone interactions observed in many other plant-pathogen interactions.
Publikation

Hause, B.; Maier, W.; Miersch, O.; Kramell, R.; Strack, D.; Induction of Jasmonate Biosynthesis in Arbuscular Mycorrhizal Barley Roots Plant Physiol. 130, 1213-1220, (2002) DOI: 10.1104/pp.006007

Colonization of barley (Hordeum vulgare cv Salome) roots by an arbuscular mycorrhizal fungus, Glomus intraradices Schenck & Smith, leads to elevated levels of endogenous jasmonic acid (JA) and its amino acid conjugate JA-isoleucine, whereas the level of the JA precursor, oxophytodienoic acid, remains constant. The rise in jasmonates is accompanied by the expression of genes coding for an enzyme of JA biosynthesis (allene oxide synthase) and of a jasmonate-induced protein (JIP23). In situ hybridization and immunocytochemical analysis revealed that expression of these genes occurred cell specifically within arbuscule-containing root cortex cells. The concomitant gene expression indicates that jasmonates are generated and act within arbuscule-containing cells. By use of a near-synchronous mycorrhization, analysis of temporal expression patterns showed the occurrence of transcript accumulation 4 to 6 d after the appearance of the first arbuscules. This suggests that the endogenous rise in jasmonates might be related to the fully established symbiosis rather than to the recognition of interacting partners or to the onset of interaction. Because the plant supplies the fungus with carbohydrates, a model is proposed in which the induction of JA biosynthesis in colonized roots is linked to the stronger sink function of mycorrhizal roots compared with nonmycorrhizal roots.
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