Upon jasmonate treatment barley leaf segments express a putative ribosome-inactivating protein (JIP60). The influence of this protein on translation in planta has been analysed by using barley plants and tobacco plants transformed with a barley cDNA encoding JIP60. In both plant systems JIP60 exhibited N-glycosidase activity in vivo. The depurination of the 25S rRNA of tobacco and barley ribosomes led to accumulation of translationally inactive polysomes.

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.

A new jasmonate, N-[(—)-jasmonoyl]-tyramine, was identified from petunia pollen in which (—)-jasmonic acid was detected and quantified.

In this paper we report the in-planta activity of the ribosome-inactivating protein JIP60, a 60-kDa jasmonate-induced protein from barley (Hordeum vulgare L.), in transgenic tobacco (Nicotiana tabacum L.) plants. All plants expressing the complete JIP60 cDNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter exhibited conspicuous and similar phenotypic alterations, such as slower growth, shorter internodes, lanceolate leaves, reduced root development, and premature senescence of leaves. Microscopic inspection of developing leaves showed a loss of residual meristems and higher degree of vacuolation of mesophyll cells as compared to the wild type. When probed with an antiserum which was immunoreactive against both the N- and the C-terminal half of JIP60, a polypeptide with a molecular mass of about 30 kDa, most probably a processed JIP60 product, could be detected. Phenotypic alterations could be correlated with the differences in the detectable amount of the JIP60 mRNA and processed JIP60 protein. The protein biosynthesis of the transformants was characterized by an increased polysome/monosome ratio but a decreased in-vivo translation activity. These findings suggest that JIP60 perturbs the translation machinery in planta. An immunohistological analysis using the JIP60 antiserum indicated that the immunoreactive polypeptide(s) are located mainly in the nucleus of transgenic tobacco leaf cells and to a minor extent in the cytoplasm.

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. (C) 1997 Federation of European Biochemical Societies.

Developmental expression of a 23 kDa jasmonate-induced protein (JIP-23) of barley leaves (Hordeum vulgare cv. Salome) was studied by measuring the time-dependent accumulation of transcript and protein during germination. Tissue-specific expression of JIP-23 was analyzed immunocytochemically and by in situ hybridizations, respectively. During seed germination JIP-23 mRNA was found to accumulate transiently with a maximum at 32 h, whereas the protein was steadily detectable after the onset of expression. The occurrence of new isoforms of JIP-23 during germination in comparison to jasmonate-treated leaves suggests, that the JIP-23 gene family of barley is able to express different subsets of isoforms dependent on the developmental stage. JIP-23 and its transcript were found mainly in the scutellum, the scutellar nodule and in lower parts of the primary leaf of 6 days old seedings. All these tissues exhibited high levels of endogenous jasmonates. In situ hybridization revealed specific accumulation of JIP-23 mRNA in companion cells of the phloem in the nodule plate of the scutellum. In accordance with that, JIP-23 was detected immunocytochemically in phloem cells of the root as well as of the scutellar nodule and in parenchymatic cells of the scutellum. The cell type-specific occurrence of JIP-23 was restricted to cells, which are known to be highly stressed osmotically by active solute transport. This observation suggests, that the expression of this protein might be a response to osmotic stress during development.

ABSTRACTIn barley leaves, there is a dramatic alteration of gene expression upon treatment with jasmonates leading to the accumulation of newly formed proteins, designated as jasmonate‐inducible proteins (JIPs). In the present study, a new jasmonate‐inducible cDNA, designated pHvJS37, has been isolated by differential screening of a γgt10 cDNA library constructed from mRNA of jasmonate‐treated barley leaf segments. The open reading frame (ORF) encodes a 39‐9 kDa polypeptide which cross‐reacts with antibodies raised against the in vivo JIP‐37. The hydropathic plot suggests that the protein is mainly hydrophilic, containing two hydrophilic domains near the C‐terminus. Database searches did not show any sequence homology of pHv.JS37 to known sequences. Southern analysis revealed at least two genes coding for JIP‐37 which map to the distal portion of the long arm of chromosome 3 and are closely related to genes coding for JIP‐23. The expression pattern of the JIP‐37 genes over time shows differential responses to jasmonate, abscisic acid (ABA), osmotic stress (such as sorbitol treatment) and desiccation stress. No expression was found under salt stress. From experiments using an inhibitor and intermediates of jasmonate synthesis such as α‐linolenic acid and 12‐oxophytodienoic acid, we hypothesize that there is a stress‐induced lipid‐based signalling pathway in which an endogenous rise of jasmonate switches on JIP‐37 gene expression. Using immunocytochemical techniques, JIP‐37 was found to be simultaneously located in the nucleus, the cytoplasm and the vacuoles.

The effect of osmotically active substances on the alteration of endogenous jasmonates was studied in barley (Hordeum vulgare L. cv. Salome) leaf tissue. Leaf segments were subjected to solutions of d-sorbitol, d-mannitol, polyethylene glycol 6000, sodium chloride, or water as a control. Alterations of endogenous jasmonates were monitored qualitatively and quantitatively using immunoassays. The structures of jasmonates isolated were determined on the basis of authentic substances by capillary gas chromatography-mass spectrometry. The stereochemistry of the conjugates was confirmed by high performance liquid chromatography with diastereoisomeric references. In barley leaves, jasmonic acid and its amino acid conjugates, for example, with valine, leucine, and isoleucine, are naturally occurring jasmonates. In untreated leaf segments, only low levels of these native jasmonates were found. After treatment of the leaf tissues with sorbitol, mannitol, as well as with polyethylene glycol, an increase of both jasmonic acid and its conjugates could be observed, depending on the stress conditions used. In contrast, salt stress was without any stimulating effect on the levels of endogenous jasmonates. From barley leaf segments exposed to sorbitol (1m) for 24 h, jasmonic acid was identified as the major accumulating compound. Jasmonic acid-amino acid conjugates increased likewise upon stress treatment.

Biologia plantarum, an international journal for experimental botany founded in 1959 by Professor Bohumil Němec. Covers all branches of experimental botany ranging from molecular biology and biotechnology to whole-plant and stand functioning.

Abscisic acid (ABA) and jasmonic acid (JA) induce upon exogenous addition to barley leaf segments synthesis of a similar set of proteins, so-called jasmonate-induced proteins (JIPs). The relationship between both compounds in inducing JIPs was studied with inhibitors and mutants of their metabolism. Taking advantage of the sorbitol-induced increase of endogenous content of jasmonates and ABA, ABA and jasmonate biosynthesis were inhibited by fluridone and anti-inflammatory drugs such as aspirin, propyl gallate, or ibuprofene, respectively. Therefore, large differences in the endogenous content of JA and ABA were achieved. As measured by the appearance of JIPs or their mRNAs, JIP expression occurred in the ABA-deficient mutant Az34 and the carotenoid-deficient mutant albozonata under sorbitol stress, which leads to high level of jasmonates, but a low level of ABA as compared to the wild type. In all cases of experimentally changed content of ABA and jasmonates, JIP gene expression took place only if jasmonates were present at high levels. Possible signalling pathways in JIP gene expression are discussed.

A. J. Boyd, S. M. Wahrlich, P. R. Poole, P. Brabcová, V. Čapková, J. LuŠtinec, A. Březinová, V. Motyka, C. Auer, J. Holík, M. Kamínek, Z. Burdach, W. Karcz, N. Chauvaux, L. Poduje, R. Child, H. Van Onckelen, G. N. Cherepneva, J. A. Dieleman, D. Kuiper, S. D. Dimova-Terziivanova, T. D. Tsonev, D. N. Terziivanov, F. A. Gudumak, A. I. Derendovskaya, R. D. Hammerton, B. Nicander, E. Tillberg, J. Holub, G. Ievinsh, O. Kreicbergs, T. Iturriagatoitia-Bueno, P. John, M. Kovač, M. Ravnikar, R. Kurtyka, J. Stolarek, V. V. Kuznetsov, R. Oelmuller, R. G. Herrmann, O. N. Kulaeva, J. Lehmann, R. Atzorn, J. Leopold, C. Wasternack, B. Parthier, L. A. Lutova, I. S. Buzovkina, S. O. Shishkova, O. N. Tikhodeev, A. Yu. Makoveychuk, E. Malkowski, R. Martinez, M. Parra, D. Valero, A. I. M. Gomes, M. A. Moya-Leon, L. I. Musatenko, A. A. Padun, A. R. Mustafina, G. R. Kudoyarova, L. V. Nazarenko, V. E. Semenenko, V. Ördög, Z. Molnár, A. A. Padun, A. Pastor, L. Alegre, V. -P. Pelkonen, A. Kauppi, O. Junttila, R. Reski, S. Kruse, B. Kasten, K. Reutter, M. Wehe, M. Faust, G. Gorr, W. O. Abel, I. Rukasz, T. Schmülling, M. Faiss, M. Strnad, C. Gatz, Gernot Schneider, O. P. Serdyuk, L. D. Smolygna, T. V. Laurinavichene, A. A. Tsyganskov, F. M. Shakirova, M. V. Bezrukova, L. D. Smolygina, O. P. Serdyuk, T. V. Laurinavichene, A. A. Tsyganskov, R. Stikic, S. Pekic, W. J. Davies, S. A. Quarrie, L. Tomljanovic, Z. Jovanovic, M. Strnad, P. Redig, J. Hanus, V. Van Dongen, H. Van Onckelen, M. Szabó, S. E. Köves, I. Stefanov, J. Molnár, M. D. Titika, V. A. Vasjuk, T. V. Andrianova, C. Vuylsteker, B. Palms, O. Leleu, S. Rambour, Y. Z. Wang, M. D. Cramer, A. Schierholt, S. H. Lips, E. Zažímalová, W. Zhong, W. Hartung, C. Schobert, E. Komor

Plant tissues treated with the naturally occurring cyclopentanone compound methyl jasmonate or exposed to stress causing in planta jasmonate accumulation express distinctive proteins and, concomitantly, reduce the synthesis of most preexisting proteins. One of the recently identified jasmonate-induced proteins, designated JIP60, in barley is a ribosome-inactivating protein that cleaves polysomes of both animal and plant origin into their ribosomal subunits. By attacking foreign and self ribosomes, respectively, JIP60 appears to be both a defense protein and a potent regulator of protein synthesis in stressed plant tissues.

Upon treatment with jasmonic acid methyl ester (methyl jasmonate, JaMe) detached leaf segments of in vitro-regenerated plants of Nicotiana plumbaginifolia accumulate novel abundant proteins and mRNAs. Among them are late embryogenesis abundant (Lea) transcripts, which appear in a temporally defined manner during somatic embryogenesis and normally do not occur in mature plant organs such as leaves or roots. To pursue this observation, the induction by methyl jasmonate of embryogenesis-related proteins and mRNAs in leaf and root tissues was investigated by comparing the two-dimensional patterns of in vivo- and in vitro-translated polypeptides. We found a selective reinduction by methyl jasmonate of several embryo-specific proteins and mRNAs, whose expression is associated with the formation of early and mature globular stages. Some of the jasmonate-induced embryo-specific proteins were also expressed in leaves, but not in roots, in response to abscisic acid (ABA) or osmotic stress (sorbitol) treatment, suggesting their role in osmoprotection of leaf and embryo tissues as discussed for LEA proteins. Western blot analyses with polyclonal antibodies raised against jasmonate-induced proteins (JIPs) of M(r) 23 000 from barley highlighted the tissue-specific reappearance of a group of closely related, evolutionarily conserved embryo-specific proteins in N. plumbaginfolia leaves after JaMe, ABA and sorbitol treatment. Actin and tubulin, for which the transcript contents were estimated, increased in amount in the course of somatic embryogenesis, but did not change in abundance under the various conditions of treatment. Our results imply a specific role of JaMe in controlling gene expression during somatic embryogenesis in N. plumbaginifolia.

The naturally occurring plant growth regulator (-)-jasmonic acid methyl ester (JaMe) induces the formation of novel abundant proteins in excised barley leaf segments. Concomitantly, this substance depresses the translation of most preexisting ("control") leaf mRNAs, including those for nuclear-encoded chloroplast proteins such as the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (SSU, rbcS gene product) and several light harvesting chlorophyll protein complex apoproteins (LHCPs, cab gene products). The changes in protein synthesis observed for SSU and LHCPs did not correspond to equivalent alterations in the rbcS and cab transcript levels. Analysis of polysome-associated in vitro translatable and hybridizable mRNAs, however, demonstrated a restriction of rbcS and cab transcripts to smaller polysomes in JaMe-exposed leaf tissues, in comparison to water-treated tissues. Since treatment of JaMe-incubated leaf segments with cycloheximide prior to harvest led to a shift of both transcripts toward larger polysomes, a hormone-induced impairment of chain initiation is assumed to lower translation of SSU and LHCP in situ. In contrast, the mRNA for plastid leucyl-tRNA synthetase (LRS1, lrs1 gene product) neither changed its abundance nor its association with polysomes in JaMe-treated leaves and was translated into the corresponding polypeptide. Together, our results highlight a remarkable variability of nuclear gene expression in response to plant growth regulators of the methyl jasmonate type.

Jasmonic acid methyl ester (methyl jasmonate, JaMe) causes accumulation of novel abundant proteins in excised leaf segments of barley, and concomitantly represses synthesis of most pre-existing ('control') proteins. The changes in control protein synthesis do not correspond to equivalent alterations at the in vitro translatable mRNA level, suggesting a post-transcriptional mode of regulation. Methyl jasmonate did not interfere with the in vitro translation of either control or JaMe-induced mRNAs. Polysome runoff translation, in combination with two-dimensional separations of the products formed, however, revealed a reduced synthesis of control proteins in JaMe-exposed leaf tissues, in contrast to a continued translation of these polypeptides in water-treated leaf segments. In vitro translation of polysomal RNAs demonstrated the preferential association of control mRNAs with polysomes of water-treated leaf tissues but not with polysomes of JaMe-treated tissues. Polysomes isolated from the latter leaves contained primarily JaMe-induced mRNAs, as shown by in vitro translation and Northern hybridization with gene-specific probes. Treatment of JaMe-incubated leaf tissues,with cycloheximide prior to harvesting caused an increase of in vitro translatable control mRNAs recovered from polysomes, thus highlighting an impairment of control protein synthesis by JaMe at the level of translation initiation.

The plant growth substance (−)‐jasmonic acid methyl ester (methyl jasmonate, JaMe) affects plastid gene expression at the protein and mRNA levels when applied exogenously to detached leaf segments of Hordeum vulgare L. cv. Salome. Translation of the large subunits of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (LSU, rbcL gene products) and of the 65 and 68 kDa proteins of photosystem I (psaA and psaB gene products, respectively) ceased, whereas synthesis of the 32 kDa photosystem II protein (D1, psbA gene product) continued in JaMe‐treated leaf tissues. These changes were not caused by corresponding alterations in transcript abundances. The loss of LSU protein synthesis, occurring within 24 h of JaMe treatment, correlated with a decline in the in vitro‐translatable rbcL mRNA, but contrasted with an almost constant transcript level. The 5′ ends of the rbcL transcripts shifted from ‘‐59’ in freshly harvested or water‐treated leaves to ‘‐94’ in JaMe‐treated leaf tissues. Transcripts ending at these positions presumably arise from alternative processing of the primary transcript ending at position ‘‐316’. The ‘‐94’ transcript contains, within the 5′ untranslated region, a 35‐base motif with remarkable complementarity to the extreme 3′ terminal part of the 16S rRNA, involved in intramolecular base pairing within the ribosome and can associate with 30S but not 70S complexes in organello, suggesting that intermolecular base pairing impairs translation initiation, probably by competing for ribosome binding at the Shine‐Dalgarno sequence. In contrast, transcripts ending at ‘‐59’ lack the 5′ terminal ‘extra’ sequence and are active in terms of translation initiation.

Upon treatment with abscisic acid (ABA) or as a result of water stress (desiccation) detached leaf segments of barley ( Hordeum vulgare L. cv. Salome) synthesized proteins of M r 66000, 37000, 30000 and 23000 which had previously been characterized as abundant methyl jasmonate (JaMe)‐induced proteins. The time course of appearance of mRNAs encoding JaMe‐induced proteins was compared under the three different treatments, i.e. JaMe, ABA and desiccation. From the overall analysis by in vitro translation, a complex alteration in the leaf mRNA population became evident that depended on the treatment employed. mRNAs likewise induced by the various treatments could be discriminated from mRNAs appearing specifically after JaMe‐ or ABA‐treatment or in response to desiccation. For two mRNAs induced by JaMe, ABA and desiccation, sequence homology was suggested to transcripts encoding late embryogenesis abundant (LEA) proteins. The two transcript species of 2.17 kb and 1.28 kb detected with a synthetic Lea gene‐specific probe in northern blot hybridizations appeared with different time courses and accumulated to different extents under the various treatments, highlighting the obvious diversity of Lea gene expression in response to ABA, JaMe and desiccation. We suggest a role of jasmonates in mediating water stress reactions in vegetative tissues of barley.