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Publikationen - Molekulare Signalverarbeitung

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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

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

Kramell, R.; Miersch, O.; Schneider, G.; Wasternack, C.; Liquid chromatography of jasmonic acid amine conjugates Chromatographia 49, 42-46, (1999) DOI: 10.1007/BF02467185

Racemic jasmonic acid (3R,7R/3S,7S)-(±)-JA) was chemically conjugated with different biogenic amines originating from aliphatic and aromatic α-amino acids by decarboxylation. The resulting isomeric compounds were subjected to reversed-phase high-performance liquid chromatography (HPLC) and to HPLC on the chiral stationary phases Chiralpak AS and Nucleodex β-PM. Under reversed-phase conditions, all the homologous amine derivatives tested could be separated from each other except the JA-conjugates containing 2-phenyl-ethylamine and 3-methylbutylamine. On both chiral supports the (3R,7R)-(−)-JA conjugates eluted earlier than those of the enantiomeric counterpart (3S,7S)-(+)-JA. On Chiralpak AS all the isomers studied could be separated to baseline with a mobile phase containingn-hexane and 2-propanol. The calculated resolution factors were between 1.80 and 4.17. The pairs of isomers were also chromatographed on the cyclodextrin stationary phase Nucleodex β-PM with methanol-triethylammonium acetate buffer as mobile phase. Under these conditions resolution factors were between 0.74 and 1.29. The individual isomers were chiroptically characterized by measurement of their circular dichroism.
Publikation

Bohlmann, H.; Vignutelli, A.; Hilpert, B.; Miersch, O.; Wasternack, C.; Apel, K.; Wounding and chemicals induce expression of the Arabidopsis thaliana gene Thi2.1, encoding a fungal defense thionin, via the octadecanoid pathway FEBS Lett. 437, 281-286, (1998) DOI: 10.1016/S0014-5793(98)01251-4

In seedlings of Arabidopsis thaliana the thionin gene Thi2.1 is inducible by methyl jasmonate, wounding, silver nitrate, coronatine, and sorbitol. We have used a biochemical and genetic approach to test the signal transduction of these different inducers. Both exogenously applied jasmonates and jasmonates produced endogenously upon stress induction, lead to GUS expression in a Thi2.1 promoter-uidA transgenic line. No GUS expression was observed in a coi1 mutant background which lacks jasmonate perception whereas methyl jasmonate and coronatine but not the other inducers were able to overcome the block in jasmonic acid production in a fad3-2 fad7-2 fad8 mutant background. Our results show conclusively that all these inducers regulate Thi2-1 gene expression via the octadecanoid pathway.
Publikation

Vörös, K.; Feussner, I.; Kühn, H.; Lee, J.; Graner, A.; Löbler, M.; Parthier, B.; Wasternack, C.; Characterization of a methyljasmonate-inducible lipoxygenase from barley (Hordeum vulgare cv. Salome) leaves Eur. J. Biochem. 251, 36-44, (1998) DOI: 10.1046/j.1432-1327.1998.2510036.x

We found three methyl jasmonate−induced lipoxygenases with molecular masses of 92 kDa, 98 kDa, and 100 kDa (LOX‐92, ‐98 and ‐100) [Feussner, I., Hause, B., Vörös, K., Parthier, B. & Wasternack, C. (1995) Plant J. 7 , 949−957]. At least two of them (LOX‐92 and LOX‐100), were shown to be localized within chloroplasts of barley leaves. Here, we describe the isolation of a cDNA (3073 bp) coding for LOX‐100, a protein of 936 amino acid residues and a molecular mass of 106 kDa. By sequence comparison this lipoxygenase could be identified as LOX2‐type lipoxygenase and was therefore designated LOX2 : Hv : 1 . The recombinant lipoxygenase was expressed in Escherichia coli and characterized as linoleate 13‐LOX and arachidonate 15‐LOX, respectively. The enzyme exhibited a pH optimum around pH 7.0 and a moderate substrate preference for linoleic acid. The gene was transiently expressed after exogenous application of jasmonic acid methyl ester with a maximum between 12 h and 18 h. Its expression was not affected by exogenous application of abscisic acid. Also a rise of endogenous jasmonic acid resulting from sorbitol stress did not induce LOX2 : Hv : 1 , suggesting a separate signalling pathway compared with other jasmonate‐induced proteins of barley. The properties of LOX2 : Hv : 1 are discussed in relation to its possible involvement in jasmonic acid biosynthesis and other LOX forms of barley identified so far.
Publikation

Kramell, R.; Schneider, G.; Miersch, O.; Chiral separation of amide conjugates of jasmonic acid by liquid chromatography Chromatographia 45, 104-108, (1997) DOI: 10.1007/BF02505545

Synthetic amide conjugates of (−)-jasmonic acid and its (+)-enantiomer were resolved by means of chiral liquid chromatography. The diastereomeric pairs prepared by chemical reaction of (±)-jasmonic acid with a series of (S)- or (R)-amino acids and with some (S)-amino acid alcohols were completely separated on Chiralpak AS using a mixture of n-hexane/2-propanal as mobile phase. The retention data indicate that the (−)-jasmonic acid conjugates eluted faster than those of the (+)-enantiomer, independent on the configuration of the bound amino acid. Likewise, enantiomeric derivatives of (±)-jasmonic acid and non-chiral amino acids were completely separated on the chiral stationary phase and showed the same elution sequence. The resolution factors,Rs, were found to range between 1.13 and 6.64. The separated compounds were chiropatically analyzed by measurement of the circular dichroism.
Publikation

Kramell, R.; Miersch, O.; Hause, B.; Ortel, B.; Parthier, B.; Wasternack, C.; Amino acid conjugates of jasmonic acid induce jasmonate-responsive gene expression in barley (Hordeum vulgare L.) leaves FEBS Lett. 414, 197-202, (1997) DOI: 10.1016/S0014-5793(97)01005-3

Leaves of barley (Hordeum vulgare L. cv. Salome ) treated with jasmonic acid (JA), its methyl ester (JM), or its amino acid conjugates exhibit up‐regulation of specific genes and down‐regulation of house‐keeping genes. This transcriptional regulation exhibits several specificities. (i) The (−)‐enantiomers are more active, and conjugates are mainly active if they carry an l ‐amino acid moiety. (ii) The various JA‐responsive genes respond differentially to enantiomeric and chiralic forms. (iii) Both JA and its amino acid conjugates exhibiting no or negligible interconversion induce/repress genes.
Publikation

Hertel, S. C.; Knöfel, H.-D.; Kramell, R.; Miersch, O.; Partial purification and characterization of a jasmonic acid conjugate cleaving amidohydrolase from the fungus Botryodiplodia theobromae FEBS Lett. 407, 105-110, (1997) DOI: 10.1016/S0014-5793(97)00307-4

A protein preparation from the mycelium of the tropical pathogenic fungus Botryodiplodia theobromae revealed a novel peptidase activity. This enzyme was capable of cleaving conjugates of jasmonic acid with α-amino acids. The protein was enriched 108-fold by gel filtration, ion exchange and hydrophobic interaction chromatography. The enzyme was found to be a glycoprotein with a molecular mass of about 107 kDa. The amidohydrolase seems to be very specific with regard to (−)-jasmonic acid and α-amino acids with (S)-configuration.
  • Die Leibniz-Gemeinschaft
  • Digitalisierung in der Landwirtschaft
  • Martin-Luther Universität Halle
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