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Publikationen - Molekulare Signalverarbeitung

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Publikation

Gross, H.B.; Dalebout, T.; Grubb, C.D.; Abel, S. Functional detection of chemopreventive glucosinolates in <em>Arabidopsis thaliana</em> Plant Science 159, 265 - 272, (2000)

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Publikation

Miersch, O.; Wasternack, C. Octadecanoid and jasmonate signaling in tomato leaves (<EM>Lycopersicon esculentum</EM> Mill.): Endogenous jasmonates do not induce jasmonate biosynthesis Biol. Chem. 381, 715-722, (2000)

Jasmonates and their precursors, the octadecanoids, are signals in stress-induced alteration of gene expression. Several mRNAs coding for enzymes of jasmonic acid (JA) biosynthesis are up-regulated upon JA treatment or endogenous rise of JA level. Here we inspected the positive feed back of endogenous JA on JA formation as well as its beta-oxidation steps. JA responsive gene expression was recorded in terms of proteinase inhibitor2 (pin2) mRNA accumulation. JA formed upon treatment of tomato (Lycopersicon esculentum cv. Moneymaker) leaves with JA derivatives carrying different length of the carboxylic acid side chain was quantified by gas chromatography-mass spectrometry (GC-MS). The data reveal that beta-oxidation of the side chain occurs up to a butyric acid moiety. The amount of JA formed from side-chain modified JA derivatives, correlated with pin2-mRNA accumulation. JA derivatives with a carboxylic side chain of 3, 5 or 7 carbon atoms were unable to form JA and to express on pin2, whereas even numbered derivatives were active. After treatment of tomato leaves with (10-2H)-(-)-12-oxophytoenoic acid, (4-2H)-(-)-JA and its methyl ester were formed and could be quantified separately from the endogenously unlabeled JA pool by GC-MS analysis via isotopic discrimination. The level of 8 nmol per g f.w. JA and its methyl ester originated exclusively from labeled 12-oxophytoenic acid. This and further data indicate that endogenous synthesis of the JA precursor 12-oxophytodienoic acid as well as of JA and its methyl ester are not induced in tomato leaves, suggesting that positive feedback in JA biosynthesis does not function in vivo.
Publikation

Kramell, R.; Miersch, O.; Atzorn, R.; Parthier, B.; Wasternack, C. Octadecanoid-derived alteration of gene expression and the 'oxylipin signature' in stressed barley leaves - implications for different signalling pathways Plant Physiol. 123, 177-186, (2000)

Stress-induced gene expression in barley (Hordeum vulgare cv. Salome) leaves has been correlated with temporally changing levels of octadecanoids and jasmonates, quantified by means of gas chromatography/mass spectrometry-single ion monitoring. Application of sorbitol-induced stress led to a low and transient rise of jasmonic acid (JA), its precursor 12-oxophytodienoic acid (OPDA) and the methyl esters JAME and OPDAME, respectively, followed by a large increase in their levels. JA and JAME peaked between 12 and 16 h, about 4 hours before OPDA and OPDAME. However, OPDA accumulated up to a 2.5-fold higher level than the other compounds. Dihomo-jasmonic acid and 9,13-didehydro-12- oxophytoenoic acid were identified as minor components. Kinetic analyses revealed that a transient threshold of jasmonates or octadecanoids is necessary and sufficient to initiate JA responsive gene expression. Although OPDA and OPDAME applied exogenously were metabolized to JA in considerable amounts, both of them can induce gene expression per se as evidenced by those genes which do not respond to endogenously formed JA. Also, coronatine induces JA-responsive genes independently from endogenous JA. As evidenced by application of deuterated JA, endogenous synthesis of JA is not induced by JA treatment. The data are discussed in terms of distinct signalling pathways.
Publikation

Abel, S.; Nürnberger, T.; Ahnert, V.; Krauss, G.J.; Glund, K. Induction of an extracellular cyclic nucleotide phosphodiesterase as an accessory ribonucleolytic activity during phosphate starvation of cultured tomato cells Plant Physiology 122, 543 - 532, (2000)

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Publikation

Wasternack, C.; Hause, B. Jasmonate - Signale zur Stressabwehr und Entwicklung in Pflanzen Biologie in unserer Zeit 30, 312-319, (2000)

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Publikation

Colon-Carmona, A.; Chen, D.L.; Yeh, K.C.; Abel, S. Aux/IAA proteins are phosphorylated by phytochrome in vitro Plant Physiology 124, 1728-1738, (2000)

Auxin/indole-3-acetic acid (Aux/IAA) genes encode short-lived transcription factors that are induced as a primary response to the plant growth hormone IAA or auxin. Gain-of-function mutations in Arabidopsis genes,SHY2/IAA3, AXR3/IAA17, andAXR2/IAA7 cause pleiotropic phenotypes consistent with enhanced auxin responses, possibly by increasing Aux/IAA protein stability. Semidominant mutations shy2-1D,shy2-2, axr3-1, and axr2-1induce ectopic light responses in dark-grown seedlings. Because genetic studies suggest that the shy2-1D andshy2-2 mutations bypass phytochrome requirement for certain aspects of photomorphogenesis, we tested whether SHY2/IAA3 and related Aux/IAA proteins interact directly with phytochrome and whether they are substrates for its protein kinase activity. Here we show that recombinant Aux/IAA proteins from Arabidopsis and pea (Pisum sativum) interact in vitro with recombinant phytochrome A from oat (Avena sativa). We further show that recombinant SHY2/IAA3, AXR3/IAA17, IAA1, IAA9, and Ps-IAA4 are phosphorylated by recombinant oat phytochrome A in vitro. Deletion analysis of Ps-IAA4 indicates that phytochrome A phosphorylation occurs on the N-terminal half of the protein. Metabolic labeling and immunoprecipitation studies with affinity-purified antibodies to IAA3 demonstrate increased in vivo steady-state levels of mutant IAA3 in shy2-2 plants and phosphorylation of the SHY2-2 protein in vivo. Phytochrome-dependent phosphorylation of Aux/IAA proteins is proposed to provide one molecular mechanism for integrating auxin and light signaling in plant development.
Publikation

Quint, M.; Melchinger, A.E.; Dussle, C.M.; Lübberstedt, T. Breeding for virus resistance in maize Genetika 32, 283-291, (2000)

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Publikation

Chen, D.L.; Delatorre,.C.A.; Bakker, A.; Abel, S. Conditional identification of phosphate starvation-response mutants in <span style="font-style: italic;">Arabidopsis thaliana</span> Planta 211, 13 - 22, (2000)

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Publikation

Ziegler, J.; Stenzel, I.; Hause, B.; Maucher, H.; Miersch, O.; Hamberg, M.; Grimm, M.; Ganal, M.; Wasternack, C. Molecular cloning of allene oxide cyclase: The enzyme establishing the stereochemistry of octadecanoids and jasmonates J. Biol. Chem. 275, 19132-19138, (2000) DOI: 10.1074/jbc.M002133200

Allene oxide cyclase (AOC) catalyses the stereospecific cyclisation of an unstable allene oxide to 9(S),13(S)-12-oxo-10,15(Z)-phytodienoic acid, the ultimate precursor of jasmonic acid. This enzyme has previously been purified, and two identical N-terminal peptides were found suggesting AOC to be a homodimeric protein. Furthermore, the native protein was N-terminal processed. Using degenerate primers, a PCR fragment could be generated from tomato, which was further used to isolate a full length cDNA clone of 1kb coding for a protein with 245 amino acids with a molecular mass of 26 kDa. Whereas expression of the whole coding region failed to detect AOC activity, a 5-'truncated protein showed high activity, suggesting that additional amino acids impair the enzymatic function. Steric analysis of the 12-oxo-phytodienoic acid formed by the recombinant AOC revealed exclusive (>99%) formation of the 9(S),13(S) enantiomer. Exclusive formation of this enantiomer was also found in wounded tomato leaves. Southern analysis and genetic mapping revealed the existence of a single gene for AOC located on chromosome 2 of tomato. Inspection of the N-terminus revealed the presence of a chloroplastic transit peptide, and the location of AOC protein in that compartment could be shown by immunohistochemical methods. Concomitant with the jasmonate levels, the accumulation of AOC mRNA was transiently induced after wounding of tomato leaves.
Publikation

Abel, S.; Theologis, A. Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression Plant Journal 5, 421-427, (1994)

An improved protocol is reported to isolate and transiently transform mesophyll protoplasts of Arabidopsis thaliana. Transfected leaf protoplasts support high levels of expression of the bacterial reporter gene coding for β-glucuronidase (GUS), under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transient expression of GUS activity was monitored spectrophotometrically and reached a maximum between 18 and 48 h after polyethylene glycol (PEG)-mediated DNA uptake. Histochemical staining for GUS activity revealed reproducible transformation frequencies between 40 and 60%, based on the number of protoplasts survived. To demonstrate the applicability of the transient expression system, the subcellular localization of GUS proteins tagged with different nuclear polypeptides was studied in transfected mesophyll protoplasts, revealing nuclear compartmentalization of the chimeric GUS enzymes. Furthermore, Arabidopsis mesophyll protoplasts support auxin-mediated induction of chloramphenicol acetyl-transferase (CAT) activity when transfected with a transcriptional fusion between the CAT reporter gene and the early auxin-inducible PS-IAA4/5 promoter. Hence, the method allows in vivo analysis of promoter activity and subcellular localization of fusion proteins in a homologous transformation system.
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