methyl-jasmonate and Wounds-and-Injuries

Natural rubber production in Hevea brasiliensis is determined by both tapping and ethephon frequencies. It is affected by a complex physiological disorder called tapping panel dryness. This syndrome is likely to be induced by environmental and latex harvesting stresses. Defence responses, including rubber biosynthesis, are dramatically mediated by wounding, jasmonate and ethylene (ET), among other factors. Using real-time RT-PCR, the effects of wounding, methyl jasmonate (MeJA) and ET on the relative transcript abundance of a set of 25 genes involved in their signalling and metabolic pathways were studied in the bark of 3-month-old epicormic shoots. Temporal regulation was found for 9 out of 25 genes. Wounding treatment regulated the transcript abundance of 10 genes. Wounding-specific regulation was noted for the HbMAPK, HbBTF3b, HbCAS1, HbLTPP and HbPLD genes. MeJA treatment regulated the transcript abundance of nine genes. Of these, the HbMYB, HbCAS2, HbCIPK and HbChi genes were shown to be specifically MeJA inducible. ET response was accompanied by regulation of the transcript abundance of eight genes, and six genes, HbETR2, HbEIN2, HbEIN3, HbCaM, HbPIP1 and HbQM, were specifically regulated by ET treatment. Additionally, the transcript level of the HbGP and HbACR genes was enhanced by all three treatments simultaneously. Overall, a large number of genes were found to be regulated 4 h after the treatments were applied. This study nevertheless revealed some jasmonic acid-independent wound signalling pathways in H. brasiliensis, provided a general characterization of signalling pathways and will serve as a new base from which to launch advanced studies of the network of pathways operating in H. brasiliensis.

Topics: Acetates; Cyclopentanes; DNA Primers; Ethylenes; Gene Expression Profiling; Hevea; Oxylipins; Plant Bark; Plant Diseases; Plant Proteins; Plant Shoots; Reverse Transcriptase Polymerase Chain Reaction; Transcription, Genetic; Wounds and Injuries

Wounding caused local and systemic induction of lipoxygenase (LOX) activity in passion fruit (Passiflora edulis f. flavicarpa) leaves, while exposing intact plants to methyl jasmonate (MJ) vapor provoked a much stronger response. Western blot analysis of these leaf protein extracts using polyclonal antibodies against cucumber LOX, revealed an accumulation of a 90 kDa protein, consistent with LOX enzymatic assays. The inducible LOX was purified to apparent homogeneity, and in vitro analysis of LOXactivity using linoleic acid as substrate showed that it possesses C-13 specificity. Immunocytochemical localization studies using leaf tissue from MJ-treated plants demonstrated that the inducible LOX was compartmented in large quantities in the chloroplasts of mesophyll cells, associated with the stroma. The results suggest that the wound response in passion fruit plants may be mediated by a chloroplast 13-LOX, a key enzyme of the octadecanoid defense-signaling pathway.

Topics: Acetates; Antigens; Chloroplasts; Cucumis sativus; Cyclopentanes; Enzyme Induction; Lipoxygenase; Oxylipins; Passiflora; Plant Growth Regulators; Plant Leaves; Wounds and Injuries

The tobacco retrotransposon Tto1, one of a few active retrotransposons of plants, has been shown to be activated by tissue culture. Its transposition is regulated mainly at the transcriptional level. It is shown here that expression of Tto1 can be induced in leaves of tobacco by wounding stress. Exogenous supply of methyl jasmonate, which is known to be a potent inducer of certain wound-responsive genes in plants, also induces Tto1 RNA expression. Tto1 RNA was detected within 2 to 4 h after wounding/cutting treatment, and increased levels of Tto1 RNA were observed during subsequent incubation periods for 48 h. Expression of Tto1 RNA after cutting treatment was induced more significantly in young expanding leaves rather than in older mature leaves, suggesting that developmental or physiological factors may be required for the strong response to Tto1 transcription to wounding stimuli. Experiments with transgenic tobacco plants carrying the Tto1-LTR: beta-glucuronidase fusion gene (LTR:GUS) revealed that Tto1 actually contains cis-regulatory regions in response to wounding and methyl jasmonate. These findings are discussed in relation to the mechanism of transcriptional activation and the evolutionary role played by retrotransposons.

Topics: Acetates; Caulimovirus; Cells, Cultured; Cyclopentanes; Genes, Reporter; Glucuronidase; Nicotiana; Oxylipins; Plant Diseases; Plant Growth Regulators; Plant Leaves; Plants, Toxic; Recombinant Fusion Proteins; Regulatory Sequences, Nucleic Acid; Repetitive Sequences, Nucleic Acid; Retroelements; RNA, Plant; Transcriptional Activation; Transfection; Wounds and Injuries

A cDNA clone CaH2B, which is highly expressed in floral buds, was isolated from hot pepper plants (Capsicum annuum) by the mRNA differential display method. Sequence analysis of CaH2B revealed that the clone contains an open reading frame of 145 amino acid residues, which are 77% identical to a maize H2B histone. The CaH2B mRNA was barely detectable in roots, was more abundant in anthers than in seedlings, and was expressed highest in floral buds and fruits. An in situ analysis of CaH2B in floral buds indicated that the transcript is highly present in the pollen and petals. Northern analysis of CaH4, a pepper H4 histone cDNA, which was obtained during the expressed sequence tag (EST) analysis of anther tissues, showed that the expression pattern was very similar to that of CaH2B, although the expression level was slightly different. Both histone genes were examined for inducibility by wounding, methyl jasmonate (MJ), or phytohormones. CaH2B and CaH4 were induced by wounding, and maximally induced ca. 3 h after wound treatment both in vitro and in planta. Airborne MJ greatly induced the expression of the genes as well. The inducing effect by wounding was suppressed by MJ, suggesting that wounding and MJ might have different roles in signal transduction for the histone gene induction. Southern blot hybridization showed that both H2B and H4 genes are comprised of multigene families in the hot pepper.

Topics: Acetates; Amino Acid Sequence; Base Sequence; Capsicum; Cyclopentanes; DNA, Complementary; DNA, Plant; Gene Expression Regulation, Plant; Gene Library; Genes, Plant; Histones; Molecular Sequence Data; Oxylipins; Plant Growth Regulators; Plants, Medicinal; Pollen; RNA, Messenger; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Tissue Distribution; Transcriptional Activation; Wounds and Injuries

We investigated the relationship between the expression of lipoxygenase (LOX) genes and the systemin-dependent wound response in tomato (Lycopersicon esculentum) leaves. A polymerase chain reaction-based approach was used to isolate two tomato Lox cDNAs, called TomLoxC and TomLoxD. Both TomLOXC and TomLOXD amino acid sequences possess an N-terminal extension of about 60 residues that were shown by in vitro uptake to function as transit peptides, targeting these proteins into the chloroplast. Within 30 to 50 min following wounding or systemin or methyl jasmonate treatments, the TomLoxD mRNA level increased and reached a maximum between 1 and 2 h. TomLoxC mRNA was not detectable in leaves and was not found following wounding, but it was found in ripening fruits, indicating that the two tomato Lox genes are regulated in different tissues by different processes. The results suggest that the TomLoxD gene is up-regulated in leaves in response to wounding and encodes a chloroplast LOX that may play a role as a component of the octadecanoid defense-signaling pathway.

Topics: Acetates; Amino Acid Sequence; Chloroplasts; Cyclopentanes; Enzyme Induction; Gene Expression Regulation, Plant; Genes, Plant; Kinetics; Lipoxygenase; Molecular Sequence Data; Oxylipins; Peptides; Plant Growth Regulators; Plant Proteins; RNA, Messenger; Sequence Alignment; Sequence Homology, Amino Acid; Solanum lycopersicum; Time Factors; Transcription, Genetic; Wounds and Injuries

Myrosinase is regarded as a defense-related enzyme in the Brassicaceae and is capable of hydrolyzing glucosinolates into various compounds, some of which are toxic. Several myrosinase isoenzymes exist, and some of them have been found in association with nonmyrosinase proteins. One of these associated proteins, myrosinase-associated protein (MyAP), was purified from seeds of Brassica napus both in complexes with myrosinase and in a free form. MyAP is a glycosylated, 40-kD protein with at least one intramolecular disufide bridge. A monoclonal anti-MyAP antibody precipitated myrosinase activity from B. napus seed extracts and in these complexes both a 65- and a 70-kD myrosinase were present. The subsequent cloning and analysis revealed the existence of a gene family encoding MyAP or MyAP-related protein and that transcripts corresponding to MyAP in nonwounded plants are found predominantly in seeds. At least some members of the gene family exhibited responsiveness toward wounding and methyl jasmonate vapor. MyAP displayed considerable similarity to an early nodulin (ENOD8) from Medicago sativa and to a proline-rich protein (APG), described as another specific, from Arabidopsis thaliana and B. napus. Similarity to expressed sequence tags from both A. thaliana and Oryza sativa has also been found.

Topics: Acetates; Amino Acid Sequence; Base Sequence; Brassica; Chromatography, Affinity; Chromatography, Ion Exchange; Cloning, Molecular; Cyclopentanes; Electrophoresis, Polyacrylamide Gel; Enzyme Induction; Genes, Plant; Glycoproteins; Glycoside Hydrolases; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Molecular Sequence Data; Molecular Weight; Oligodeoxyribonucleotides; Oxylipins; Plant Growth Regulators; Plant Proteins; Recombinant Proteins; RNA, Messenger; Sequence Homology, Amino Acid; Transcription, Genetic; Wounds and Injuries

Three cysteine proteinase inhibitor cDNA clones (pL1, pR1, and pN2) have been isolated from a soybean (Glycine max L. Merr.) embryo library. The proteins encoded by the clones are between 60 and 70% identical and contain the consensus QxVxG motif and W residue in the appropriate spatial context for interaction with the cysteine proteinase papain. L1, R1, and N2 mRNAs were differentially expressed in different organs of plants (juvenile and mature) and seedlings, although N2 mRNA was constitutive only in flowers. R1 and N2 transcripts were induced by wounding or methyl jasmonate (M-JA) treatment in local and systemic leaves coincident with increased papain inhibitory activity, indicating a role for R1 and N2 in plant defense. The L1 transcript was constitutively expressed in leaves and was induced slightly by M-JA treatment in roots. Unlike the chymotrypsin/trypsin proteinase inhibitor II gene (H. Peña-Cortés, J. Fisahn, L. Willmitzer [1995] Proc Natl Acad Sci USA 92: 4106-4113), expression of the soybean genes was only marginally induced by abscisic acid and only in certain tissues. Norbornadiene, a competitive inhibitor of ethylene binding, abolished the wounding or M-JA induction of R1 and N2 mRNAs but not the accumulation of the wound-inducible vspA transcript. Presumably, ethylene binding to its receptor is involved in the wound inducibility of R1 and N2 but not vspA mRNAs. Bacterial recombinant L1 and R1 proteins, expressed as glutathione S-transferase fusion proteins, exhibited substantial inhibitory activities against vicilin peptidohydrolase, the major thiol endopeptidase in mung bean seedlings. Recombinant R1 protein had much greater cysteine proteinase inhibitor activity than recombinant L1 protein, consistent with the wound inducibility of the R1 gene and its presumed role in plant defense.

Topics: Acetates; Amino Acid Sequence; Cloning, Molecular; Cyclopentanes; Cysteine Proteinase Inhibitors; DNA, Complementary; Gene Expression Regulation, Developmental; Gene Expression Regulation, Plant; Glycine max; Molecular Sequence Data; Oxylipins; Papain; Plant Growth Regulators; RNA, Messenger; Sequence Homology, Amino Acid; Transcription, Genetic; Wounds and Injuries

The soybean vegetative storage proteins, VSP alpha and VSP beta, are acid phosphatases that accumulate to very high levels in hypocotyls, young leaves and flowers and pods. The genes encoding the soybean VSP are activated by jasmonate, wounding, sugars and light and down regulated by phosphate and auxin. In this study, expression of an Arabidopsis thaliana gene (Atvsp) encoding a protein homologous to soybean Vsp alpha and Vsp beta, was examined and compared to expression of the soybean Vsp genes. Atvsp mRNA was present at high levels in flowers and buds and at low levels in roots, stems, leaves and siliques. Expression of Atvsp in leaves could be induced by wounding or by treatment of illuminated plants with methyl jasmonate and sucrose. Roots of plants with wounded leaves also accumulated Atvsp mRNA indicating that this gene can be regulated by a transmissible wound signal. Phosphate partially inhibited expression of Atvsp. Arabidopsis proteins of 29 and 30 kDa crossreacted with antibodies against soybean VSP. These proteins were very abundant in flowers and the proteins accumulated in leaves and roots of plants treated with methyl jasmonate. The level of these proteins in flowers was similar to the levels of soybean VSP in young soybean leaves. Overall, these data indicate that Arabidopsis Atvsp and soybean VspA/B genes are regulated similarly and that in both plants, the gene products can accumulate to high levels. This suggests that genes homologous to VspA/B may be of greater general significance than previously recognized.

Topics: Acetates; Acid Phosphatase; Antibodies; Arabidopsis; Arabidopsis Proteins; Blotting, Southern; Blotting, Western; Cyclopentanes; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Glycine max; Light; Oxylipins; Phosphates; Plant Proteins; RNA, Messenger; Sucrose; Wounds and Injuries

We have isolated a gene, hmg1, for 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) from Camptotheca acuminata, a Chinese tree that produces the anti-cancer monoterpenoid indole alkaloid camptothecin (CPT). HMGR supplies mevalonate for the synthesis of the terpenoid component of CPT as well as for the formation of many other primary and secondary metabolites. In Camptotheca, hmg1 transcripts were detected only in young seedlings and not in vegetative organs of older plants. Regulation of the hmg1 promoter was studied in transgenic tobacco using three translational fusions (-1678, -1107, -165) with the beta-glucuronidase (GUS) reporter gene. Histochemical analysis of plants containing each of the three promoter fusions showed similar developmental and spatial expression patterns. In vegetative tissues, GUS staining was localized to the epidermis of young leaves and stems, particularly in glandular trichomes. Roots showed intense staining in the cortical tissues in the elongation zone and light staining in the cortex of mature roots. hmg1::GUS expression was also observed in sepals, petals, pistils, and stamens of developing flowers, with darkest staining in the ovary wall, ovules, stigmas, and pollen. Leaf discs from plants containing each of the translational fusions showed a 15- to 20-fold wound induction of hmg1::GUS expression over 72 h; however, this increase in GUS activity was completely suppressed by treatment with methyl jasmonate. Taken together, these data show that a 165-bp fragment of Camptotheca hmg1 promoter is sufficient to confer developmental regulation as well as wound induction and methyl jasmonate suppression of GUS expression in transgenic tobacco.

Topics: Acetates; Cyclopentanes; Gene Expression Regulation; Gene Library; Glucuronidase; Molecular Sequence Data; Nicotiana; Oxylipins; Plants, Genetically Modified; Plants, Medicinal; Plants, Toxic; Plasmids; Promoter Regions, Genetic; Recombinant Fusion Proteins; Wounds and Injuries