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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.

Pedranzani, H.; Sierra-de-Grado, R.; Vigliocco, A.; Miersch, O.; Abdala, G.; Cold and water stresses produce changes in endogenous jasmonates in two populations of Pinus pinaster Ait Plant Growth Regul. 52, 111-116, (2007) DOI: 10.1007/s10725-007-9166-2

There is considerable evidence suggesting that jasmonates (JAs) play a role in plant resistance against abiotic stress. It is well known that in Angiosperms JAs are involved in the defense response, however there is little information about their role in Gymnosperms. Our proposal was to study the involvement of JAs in Pinus pinaster Ait. reaction to cold and water stress, and to compare the response of two populations of different provenances (Gredos and Bajo Tiétar) to these stresses. We detected 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and the hydroxylates 11-hydroxyjasmonate and 12-hydroxyjasmonate in foliage and shoots of P. pinaster plants. The response of the Gredos population to cold stress differed from that of Bajo Tiétar. Gredos plants showed a lower JA-basal level than Bajo Tiétar; under cold stress JA increased twofold at 72 h, while it decreased in Bajo Tiétar plants. The hydroxylates slightly increased in both populations due to cold stress treatment. Under water stress, plants from Gredos showed a remarkable JA-increase; thus the JA-response was much more prominent under water stress than under cold stress. In contrast, no change was found in JA-level in Bajo Tiétar plants under water stress. The level of JA-precursor, OPDA, was very low in control plants from Gredos and Bajo Tiétar. Under water stress OPDA increased only in plants from Bajo Tiétar. Therefore, we inform here of a different JAs-accumulation pattern after the stress treatment in P. pinaster from two provenances, and suggest a possible correlation with adaptations to diverse ecological conditions.

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.

Abdala, G.; Miersch, O.; Kramell, R.; Vigliocco, A.; Agostini, E.; Forchetti, G.; Alemano, S.; Jasmonate and octadecanoid occurrence in tomato hairy roots. Endogenous level changes in response to NaCl Plant Growth Regul. 40, 21-27, (2003) DOI: 10.1023/A:1023016412454

Jasmonic acid biosynthesis occurs in leaves and there is also evidence of a similar pathway in roots. The expression of lipoxygenase, allene oxide cyclase and low amounts of transcripts of allene oxide synthase in tomato roots indicates that some steps of the jasmonate synthesis may occur in these organs. Thus, the aim of the present work was to study the jasmonate and octadecanoid occurrence in tomato roots using isolated cultures of hairy roots. These were obtained by the transformation of cv. Pera roots with Agrobacterium rhyzogenes. Also we investigated the effect of NaCl stress on the endogenous levels of these compounds. Jasmonic acid, 12-oxophytodienoic acid and their methylated derivatives, as well as a jasmonate-isoleucine conjugate, were present in control hairy roots of 30 d of culture. The 12-oxophytodienoic acid and its methylated derivative showed higher levels than jasmonic acid and its methylated form, although the content of the conjugate was the same as that of jasmonic acid. After salinization of hairy roots for 14, 20 and 30 d, free jasmonates and octadecanoids were measured. Fourteen days after salt treatment, increased levels of these compounds were found, jasmonic acid and 12-oxophytodienoic acid showed the most remarkable rise. 11-OH-jasmonic acid was found at 14 d of culture in control and salt-treated hairy roots; whereas the 12-OH- form of jasmonic acid was only detected in the salt-treated hairy roots. Agrobacterium rhizogenes cultures did not produce jasmonates and/or octadecanoids.

Abdala, G.; Castro, G.; Miersch, O.; Pearce, D.; Changes in jasmonate and gibberellin levels during development of potato plants (Solanum tuberosum) Plant Growth Regul. 36, 121-126, (2002) DOI: 10.1023/A:1015065011536

Among the multiple environmental signals and hormonal factors regulatingpotato plant morphogenesis and controlling tuber induction, jasmonates (JAs)andgibberellins (GAs) are important components of the signalling pathways in theseprocesses. In the present study, with Solanum tuberosum L.cv. Spunta, we followed the endogenous changes of JAs and GAs during thedevelopmental stages of soil-grown potato plants. Foliage at initial growthshowed the highest jasmonic acid (JA) concentration, while in roots the highestcontent was observed in the stage of tuber set. In stolons at the developmentalstage of tuber set an important increase of JA was found; however, in tubersthere was no change in this compound during tuber set and subsequent growth.Methyl jasmonate (Me-JA) in foliage did not show the same pattern as JA; Me-JAdecreased during the developmental stages in which it was monitored, meanwhileJA increased during those stages. The highest total amount of JAs expressed asJA + Me-JA was found at tuber set. A very important peak ofJA in roots was coincident with that observed in stolons at tuber set. Also, aprogressive increase of this compound in roots was shown during the transitionof stolons to tubers. Of the two GAs monitored, gibberellic acid(GA3) was the most abundant in all the organs. While GA1and GA3 were also found in stolons at the time of tuber set, noothermeasurements of GAs were obtained for stolons at previous stages of plantdevelopment. Our results indicate that high levels of JA and GAs are found indifferent tissues, especially during stolon growth and tuber set.

Ortel, B.; Atzorn, R.; Hause, B.; Feussner, I.; Miersch, O.; Wasternack, C.; Jasmonate-induced gene expression of barley (Hordeum vulgare) leaves - the link between jasmonate and abscisic acid Plant Growth Regul. 29, 113-122, (1999) DOI: 10.1023/A:1006212017458

In barley leaves a group of genes is expressed in response to treatment with jasmonates and abscisic acid (ABA) [21]. One of these genes coding for a jasmonate-induced protein of 23 kDa (JIP-23) was analyzed to find out the link between ABA and jasmonates by recording its expression upon modulating independently, the endogenous level of both of them. By use of inhibitors of JA synthesis and ABA degradation, and the ABA-deficient mutant Az34, as well as of cultivar-specific differences, it was shown that endogenous jasmonate increases are necessary and sufficient for expression of this gene. The endogenous rise of ABA did not induce synthesis of JIP-23, whereas exogenous ABA did not act via jasmonates. Different signalling pathways are suggested and discussed.

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.

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.

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.

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.
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