Methyl Jasmonate

We obtained callus, cell suspension and hairy root cultures of Tropaeolum majus and we demonstrated their ability to produce glucotropaeolin and myrosinase. In hairy roots glucotropaeolin content and myrosinase activity were higher in comparison with callus, cell suspension and leaves of intact plants. In hairy root cultures the highest glucotropaeolin contents were detected on the 9th day of culture. In

In this work, a cDNA encoding a defensin from Picea glauca, named PgD1, was isolated. The corresponding PgD1 cDNA encodes a putative protein of 83 amino acids with a calculated molecular mass of 8.9 kDa. The mature protein of 50 amino acids is characterized by the presence of specific highly conserved residues common to all plant defensins. PgD1 is developmentally expressed during seed germination and is also up-regulated by wounding and by jasmonic acid treatment, suggesting a role in both the constitutive and induced defense mechanisms. The recombinant protein was found to cause extensive growth inhibition of three fungal pathogens (Cylindrocladium floridanum, Fusarium oxysporum and Nectria galligena) at 2.5 mM. Crown

Pathogenesis-related (PR) proteins are plant proteins that are induced in response to pathogen attack. PR proteins are grouped into independent families based on their sequences and properties. The PR-4 family comprises class I and class II chitinases. We have isolated a full-length cDNA encoding a chitinase from maize which shares a high degree of nucleotide and amino acid sequence homology with the class II chitinases of the PR-4 family of PR proteins. Our results indicate that fungal infection, and treatment either with fungal elicitors or with moniliformin, a mycotoxin produced by the fungus Fusarium moniliforme, increase the level of ZmPR4 mRNA. In situ mRNA hybridization analysis in sections obtained from fungus-infected germinating embryos revealed that ZmPR4 mRNA accumulation occurs in those cell types that first establish contact with the pathogen. ZmPR4 mRNA accumulation is also stimulated by treatment with silver nitrate whereas the application of the hormones gibberellic acid or acetylsalicylic acid has no effect. Wounding, or treatment with abscisic acid or methyl jasmonate, results in accumulation of ZmPR4 mRNA in maize leaves. Furthermore, the ZmPR4 protein was expressed in Escherichia coli, purified and used to obtain polyclonal antibodies that specifically recognized ZmPR4 in protein extracts from fungus-infected embryos. Accumulation of ZmPR4 mRNA in fungus-infected maize tissues was accompanied by a significant accumulation of the corresponding protein. The possible implications of these findings as part of the general defence response of maize plants against pathogens are discussed.

The effects of methyl jasmonate elicitation were investigated in embryogenic suspension cultures of Eleutherococcus sessiliflorus in bioreactors for the production of eleutherosides and chlorogenic acid. Eleutherosides and chlorogenic acid increased with methyl jasmonate elicitation (in the range 50-400 M); however, fresh weight, dry weight and growth ratio of the cells were inhibited by increasing methyl jasmonate concentration. There were 3.5-, 2.6-, 2.5-and 3.2-fold increments in eleutherosides B, E, E1 and chlorogenic acids were evident, respectively, with 200 M methyl jasmonate treatment. These results suggest that methyl jasmonate elicitation is beneficial for eleutheroside accumulation in the embryogenic suspension cultures.

Methyl jasmonate (MJ) is a hormone released by plants in response to external stress, injury or pathogenic invasions. This present investigation evaluated the antidepressant effect of intraperitoneal doses of MJ in mice. Mice were given MJ in the doses of 10, 25 and 50 mg/kg daily for 7 days and then subjected to forced swim test (FST), tail suspension test (TST) and yohimbine lethality test (YLT). The results showed that MJ produced a significant decrease in the period of immobility in the FST and TST, indicating antidepressant activity. MJ potentiated the toxic effect of yohimbine in the YLT, which further suggests antidepressant property and also indicates facilitatory effect on both serotonergic and noradrenergic systems respectively. However, MJ did not significantly alter the spontaneous motor activity of the animals, which indicates a lack of central nervous system stimulant effect. Taken together, these findings suggest that MJ has antidepressant activity and the mechanisms underlying this effect may involve serotonergic and noradrenergic systems.

A mini-hydroponic growing system was employed for seedlings of kudzu vine (Pueraria montana) and contents of isoflavones (daidzein, genistein, daidzin, genistin, and puerarin) from shoot and root parts of seedlings were analyzed quantitatively. In addition, exogenous cork pieces, polymeric adsorbent, XAD-4, and universal elicitor, methyl jasmonate (MeJA), were used to regulate the production of these isoflavones. It was shown that cork pieces up-regulate the production of daidzein and genistein up to seven- and eight-fold greater than the levels obtained for control roots. In contrast, levels of glucosyl conjugates, daidzin and genistin, decrease up to five- and eight-fold, respectively. Cork treatment also induces the excretion of the root isoflavone constituents into the growth medium. Minimal levels of isoflavones are absorbed by the cork pieces. XAD-4 stimulates the production of glucosyl conjugates, daidzin and genistin, in root parts about 1.5-fold greater than that obtained in control roots. These are the highest amounts of daidzin and genistin that are observed (5.101 and 6.759 mg g−1 dry weight, respectively). In contrast to these two adsorbents, MeJA increases the accumulation of isoflavones in shoot rather than in root parts of seedlings, about three- to four-fold over control levels, with the exception of genistein. These studies reveal new observations on the regulation of isoflavone production in hydroponically grown Pueraria montana plants by two adsorbents (cork pieces and XAD-4) and MeJA elicitor.

The table grape exporting industry faces two major problems associated with long distance refrigerated transportation: damage due to mold growth and unexpected temperature rise, that accelerate mold proliferation and causes water loss in the fruit. To overcome mold growth, a usual practice points toward the use of gas generators.Thermal damage due to refrigeration brake down could be predicted with the

The important anticancer pharmaceuticals, vinblastine and vincristine, are produced by Catharanthus roseus. Given their cytotoxicity, these valuable alkaloids are produced in very small quantities within the aerial parts of the plant. The high cost of isolating the drugs has led to research efforts to increase the alkaloid content of C. roseus cell cultures, tissue cultures, and seedlings. The metabolic engineering

To date enormous attempts have been devoted to improve Taxol production exploiting various methodologies from bioprocess engineering to biotechnological and synthetic approaches. We have developed a 2-stage suspension cell culture of Taxus baccata L. using modified B5 medium in order to improve cell growth as well as productivity. After callus induction and cell line selection, B5 medium was supplemented with vanadyl sulfate (0.1 mg/l), silver nitrate (0.3 mg/l) and cobalt chloride (0.25 mg/l) at the first day of stage I culture to maximize cell growth. This medium was further supplemented with sucrose (1%) and ammonium citrate (50 mg/l) on day 10 and sucrose (1%) and phenylalanine (0.1 mM) on day 20 (i.e., biomass growth medium). At stage II (day 25), two different concentrations of several elicitors such as methyl jasmonate (10 or 20 mg/l), salicylic acid (50 or 100 mg/l) and fungal elicitor (25 or 50 mg/l) were added to the biomass growth medium with the aim of improving cellular productivity.

In plant, basic leucine zipper (bZIP) transcription factors play various roles in response to biotic and abiotic stimuli, as well as in developmental processes. In the present study, a novel bZIP gene, designated as TabZIP1, was isolated from wheat leaves infected by Puccinia ...

Jasmonate and related signalling compounds have a crucial role in both host immunity and development in plants, but the molecular details of the signalling mechanism are poorly understood. Here we identify members of the jasmonate ZIM-domain (JAZ) protein family as key regulators of jasmonate signalling. JAZ1 protein acts to repress transcription of jasmonate-responsive genes. Jasmonate treatment causes JAZ1 degradation and this degradation is dependent on activities of the SCF COI1 ubiquitin ligase and the 26S proteasome. Furthermore, the jasmonoyl-isoleucine (JA-Ile) conjugate, but not other jasmonate-derivatives such as jasmonate, 12-oxo-phytodienoic acid, or methyl-jasmonate, promotes physical interaction between COI1 and JAZ1 proteins in the absence of other plant proteins. Our results suggest a model in which jasmonate ligands promote the binding of the SCF COI1 ubiquitin ligase to and subsequent degradation of the JAZ1 repressor protein, and implicate the SCF COI1 -JAZ1 protein complex as a site of perception of the plant hormone JA-Ile.

Triterpenoid saponins are a class of plant secondary metabolites with structure derived from the precursor oxidosqualene in which one or more sugar residues are added. They have a wide range of pharmacological applications, such as antiplatelet, hypocholesterolemic, antitumoral, anti-HIV, immunoadjuvant, anti-inflammatory, antibacterial, insecticide, fungicide and anti-leishmanial agents. Their accumulation in plant cells is stimulated in response to changes mediated by biotic and abiotic elicitors. The enhancement of saponin yields by methyl jasmonate in plants and cell cultures in several species indicates the involvement of these metabolites in plant defence mechanisms. The elucidation of their biosynthesis at the molecular level has advanced recently. Most studies to date have focused on the participation of early enzymes in the pathway, including oxidosqualene cyclase, squalene synthase and dammarenediol synthase, as well as in isolating and characterizing genes that encode b-amyrin synthase. Yields of bioactive saponins in various plant species and experimental systems have been successfully increased by treating cells and tissues with jasmonate or by exposing these to oxidative stress. These elicitation and molecular studies are consolidating a robust knowledge platform from which to launch the development of improved sources for commercial supply of bioactive saponins.

The results presented in this paper describe for the first time the presence of an inducible enzyme in the biosynthetic pathway leading to the anticancer drug paclitaxel. Methyl jasmonate, when administered to Taxus baccata cell suspension cultures, was found to rapidly induce geranylgeranyl diphosphate (GGPP) synthase activity. Experiments showed that after elicitation at day 7 of the subculturing period, the enzyme activity is rapidly induced during the first 6 h and reaches its maximum of 60.7 μkat/kg of protein after 5 days. The enzyme’s induction was followed by a sharp increase in taxane accumulation in the cells, which started 24 h after elicitation and reached its maximum after 5 days. In addition taxine-B was found in the medium of unelicited cultures but not in the medium of elicited cells.

The potential of three externally applied chemical plant activators, Bion, BABA and methyl jasmonate, known to act only through the plant defence system and not on the pathogen directly, to induce resistance against wild-type Pectobacterium carotovorum was examined in white-flowered calla lily ( Zantedeschia aethiopica ). Following a 24-h induction period, plants were challenge-inoculated with P. carotovorum , originally isolated from calla lily or potato plants, previously transformed using a gfp broad-host-range promoter-probe vector. After another 24 h, Bion treatment (10 µ g mL -1 , as a drench) reduced disease symptoms more than sixfold and bacterial proliferation by four orders of magnitude. BABA treatment (5-10 µ g mL -1 , also as a drench) reduced the rate of infection by 75-85%. However, the protection afforded by both inducers did not persist. Also, at higher concentrations both displayed a phytotoxic effect. By contrast, methyl jasmonate (10 m m , applied as a leaf spray) completely inhibited P. carotovorum development in calla lily leaves and afforded a long-lasting effect. It is suggested that the defence response of calla lily against P. carotovorum involves the SA-signalling pathway in the short term, but the jasmonate/ethylene-signalling pathway is required for durable protection.

Elicitors are compounds, which activate chemical defences in plants. Various biosynthetic pathways are activated in treated plants depending on the compound used. The most intensively studied elicitor for manipulating defence pathways in plants is methyl jasmonate, which modifies e.g. the production of terpenoids, the main constituents of conifer oleoresin. Other commonly tested chemical elicitors are salicylic acid, methyl salicylate and benzothiadiazole, which affect production of phenolic compounds in plants. Both jasmonate-based and salicylate-based elicitors have been shown to have suppressive effects on fungal diseases and insect pests of plants. So far, knowledge regarding the efficiency of elicitor treatments for enhancing pest and fungal disease resistance of conifer seedlings is very limited. We review current knowledge of the effect of these elicitor compounds on pest and disease resistance in plants, and we analyze the potential pros and cons of using elicitors for future pest management strategies in forest nurseries.

Plant defence can be induced by exposing plants to the plant hormone jasmonic acid (JA) or its volatile ester, methyl jasmonate (MeJA). Carrageenans (Carr) -sulphated d-galactans extracted from red algae -can also induce plant defences. In this study, the effects of exogenous MeJA and Carr application (concentration 300 and 12.7 lmol, respectively) on volatile emissions from two widespread evergreen woody species, Pinus sylvestris (nine Turkish and one Finnish provenance) and Quercus ilex (Italian provenance) were investigated. We collected headspace samples from seedlings and analysed the quality and quantity of volatile compounds emitted by treated and control plants. In total, 19 monoterpenes, 10 sesquiterpenes, 10 green leaf volatiles (GLVs) and two aromatic compounds were emitted by P. sylvestris from all the provenances studied. Foliar MeJA application clearly affected the volatile profiles of trees from all the provenances. Effects of Carr were genotype specific. In Q. ilex, emissions of sesquiterpenes, GLVs and the homoterpene (E)-DMNT were all induced by MeJA application. However, emissions of most constitutively emitted monoterpenes were significantly reduced. Carr application also led to a significant reduction in monoterpene emissions, but without corresponding increases in other emissions. Our results indicate that exogenously applied MeJA and Carr can both significantly modify the volatile profiles of P. sylvestris and Q. ilex, but also that there are important provenance-and species-specific differences in the overall degree of elicitation and compositions of elicited compounds.

The plant defensin PDF1.2 has previously been shown to accumulate systemically via a salicylic acid-independent pathway in leaves of Arabidopsis upon challenge by fungal pathogens. To further investigate the signalling and transcriptional processes underlying plant defensin induction, a DNA fragment containing 1184 bp and 1232 bp upstream of the transcriptional and translational start sites, respectively, was cloned by inverse PCR. To test for promoter activity this DNA fragment was linked to the β-glucuronidase (GUS)-encoding region of the UidA gene as a translational fusion and introduced into Arabidopsis ecotype C-24. Challenge of the transgenic plants with the fungal pathogens Alternaria brassicicola and Botrytis cinerea resulted in both local and systemic induction of the reporter gene. Wounding of the transgenic plants had no effect on GUS activity. Treatment of the transgenic plants with either jasmonates or the active oxygen generating compound paraquat strongly induced the reporter gene. In contrast, neither salicylate nor its functional analogues 2,6-dichloroisonicotinic acid and 1,2,3-benzothiodiazole-7-carbothioic acid S-methyl ester resulted in reporter gene induction. These results are consistent with the existence of a salicylic acid-independent signalling pathway, possibly involving jasmonates as regulators, that is triggered by pathogen challenge but not by wounding. The transgenic plants containing the PDF1.2-based promoter-reporter construct will provide useful tools for future genetic dissection of this novel systemic signalling pathway.

ost of elicitors are cheap, available, easy to use and environmentally safe, beside their ability to induce resistance in many plants against pathogens attack. In this connection, exogenously foliar application of chitosan (CHI), salicylic acid (SA) and methyl jasmonate (MeJA) proved to be effective in reducing downy mildew (caused by Pseudoperonospora cubensis) occurrence (DO) and severity (DS) in cucumber plants under field conditions. Most of concentrations used of the tested elicitors significantly increased cucumber growth parameters (plant height, no. of branches, shoot dry weight and leaf area) and physiological aspects (nitrogen, potassium, phosphorus, calcium, total phenols and photosynthetic pigments) in shoot as well as yield and its quality. CHI at 0.05% and SA at 100mg/l were the most effective in this concern. Application of elicitors increased leaf anatomical characters, i.e. thickness of leaf blade, thickness of palisade and spongy parenchyma as well as thickness of midrib region and dimension of vascular bundles. Results indicate that CHI and SA can be used in the field as a means of protection against cucumber downy mildew as well as improving its growth and yield in addition to decrease of the fungicides used.

Hydroponically cultivated Nigella sativa L. plants treated with methyl jasmonate (MeJA) showed a twelvefold increase in levels of the monodesmosidic triterpene saponins a-hederin and kalopanaxsaponin I (KsI) in the leaves. We will demonstrate that these two saponins accounted for approximately 10% of the dry plant matter, of which 93% was KsI and 7% a-hederin. To address the molecular basis of saponin induction by MeJA, we cloned and characterized the b-amyrin synthase gene (NsbAS1) encoding one of the key enzymes in triterpene saponin biosynthesis. As expected, NsbAS1 transcription was induced by MeJA and led to the production of b-amyrin when over-expressed in yeast.

We extend our analysis of the transcriptional reorganization that occurs when the native tobacco, Nicotiana attenuata, is attacked by Manduca sexta larvae by cloning 115 transcripts by mRNA differential display reverse transcription-polymerase chain reaction and subtractive hybridization using magnetic beads (SHMB) from the M. sexta-responsive transcriptome. These transcripts were spotted as cDNA with eight others, previously confirmed to be differentially regulated by northern analysis on glass slide microarrays, and hybridized with Cy3-and Cy5-labeled probes derived from plants after 2, 6, 12, and 24 h of continuous attack. Microarray analysis proved to be a powerful means of verifying differential expression; 73 of the cloned genes (63%) were differentially regulated (in equal proportions from differential display reverse transcriptionpolymerase chain reaction and SHMB procedures), and of these, 24 (32%) had similarity to known genes or putative proteins (more from SHMB). The analysis provided insights into the signaling and transcriptional basis of direct and indirect defenses used against herbivores, suggesting simultaneous activation of salicylic acid-, ethylene-, cytokinin-, WRKY-, MYB-, and oxylipin-signaling pathways and implicating terpenoid-, pathogen-, and cell wall-related transcripts in defense responses. These defense responses require resources that could be made available by decreases in four photosynthetic-related transcripts, increases in transcripts associated with protein and nucleotide turnover, and increases in transcripts associated with carbohydrate metabolism. This putative up-regulation of defense-associated and down-regulation of growth-associated transcripts occur against a backdrop of altered transcripts for RNA-binding proteins, putative ATP/ADP translocators, chaperonins, histones, and water channel proteins, responses consistent with a major metabolic reconfiguration that underscores the complexity of response to herbivore attack. ; fax 49 -0 -3641-571102.

It has been suggested that the rol genes of Agrobacterium rhizogenes could play an essential role in the activation of secondary metabolite production in plant transformed cultures. This study investigated whether the content of anthraquinone phytoalexins was changed in callus cultures of Rubia cordifolia transgenic for the 35S-rolB and 35S-rolC genes in comparison with a non-transformed callus culture. The anthraquinone content was shown to be significantly increased in transgenic cultures, thus providing further evidence that the rol-gene transformation can be used for the activation of secondary metabolism in plant cells. Methyl jasmonate and salicylic acid strongly increased anthraquinone accumulation in both transgenic and non-transgenic R. cordifolia calluses, whereas ethephon did not. A treatment of the cultures by cantharidin, the protein phosphatase 2A inhibitor, resulted in massive induction of anthraquinone accumulation in the transgenic cultures only. We suggest the involvement of a cantharidin-sensitive protein phosphorylation mechanism in anthraquinone biosynthesis in transgenic cultures.

The effect of atmospheric methyl jasmonate on the oxylipin pathway was investigated in leaves of tobacco (Nicotiana tabacum L.), cucumber (Cucumis sativa L.), and Arabidopsis thaliana (L.). Differential sensitivities of test plants to methyl jasmonate were observed. Thus, different concentrations of methyl jasmonate were required for induction of changes in the oxylipin pathway. Arabidopsis was the least and cucumber the most sensitive to methyl jasmonate. Methyl jasmonate induced the accumulation of lipoxygenase protein and a corresponding increase in extractable lipoxygenase activity. Atmospheric methyl jasmonate additionally induced hydroperoxide lyase activity and the enhanced production of severa1 volatile six-carbon products. It is interesting that lipid hydroperoxidase activity, which is a measure of hydroperoxide lyase plus allene oxide synthase plus possibly other lipid hydroperoxidemetabolizing activities, was not changed by methyl jasmonate treatment. Methyl jasmonate selectively altered the activity of certain enzymes of the oxylipin pathway (lipoxygenase and hydroperoxide lyase) and increased the potential of leaves for greatly enhanced six-carbon-volatile production.

To date enormous attempts have been devoted to improve Taxol production exploiting various methodologies from bioprocess engineering to biotechnological and synthetic approaches. We have developed a 2-stage suspension cell culture of Taxus baccata L. using modified B5 medium in order to improve cell growth as well as productivity. After callus induction and cell line selection, B5 medium was supplemented with vanadyl sulfate (0.1 mg/l), silver nitrate (0.3 mg/l) and cobalt chloride (0.25 mg/l) at the first day of stage I culture to maximize cell growth. This medium was further supplemented with sucrose (1%) and ammonium citrate (50 mg/l) on day 10 and sucrose (1%) and phenylalanine (0.1 mM) on day 20 (i.e., biomass growth medium). At stage II (day 25), two different concentrations of several elicitors such as methyl jasmonate (10 or 20 mg/l), salicylic acid (50 or 100 mg/l) and fungal elicitor (25 or 50 mg/l) were added to the biomass growth medium with the aim of improving cellular productivity. For morphological analysis, microscopic inspection was carried out during cultivation. Cell-associated and extracellular amount of Taxol were detected and measured using HPLC methodology. At stage I, overall Taxol amount of biomass growth medium was 13.75 mg/l (i.e., 5.6-fold higher than that of untreated B5 control). At stage II, treated cells with methyl jasmonate (10 mg/l), salicylic acid (100 mg/l) and fungal elicitor (25 mg/l) produced the highest amount of Taxol (39.5 mg/l), which is 16-fold higher than that of untreated B5 control (2.45 mg/l). Microscopic analyses of Taxus cells in suspension cultures showed various positional auto-fluorescence showing direct correlation with Taxol production. Our studies revealed that intervallic supplementation of B5 medium with combination of biomass growth factors at stage I and mixture of elicitors at stage II could significantly increase Taxol production. Thus, we suggest that the exploitation of this methodology may improve the production of Taxol since demands for Taxol pharmaceuticals are increasingly growing and resource paucities have limited its direct harvesting from Taxus trees.

In response to insect feeding, tomato plants (Lycopersicon esculentum) release elevated levels of volatile organic compounds; that is, monoterpenes and sesquiterpenes are released both locally and systemically with wounding while C 6 green leaf volatiles are released only from damaged leaves. With the exogenous application (100 nmol) of the C 6 -tomato-volatile (E)-2-hexenal, an increase in the release of local and systemic terpenes was observed, while an equimolar amount of methyl jasmonate triggered only local emissions of terpenes. Labeling studies with 13 CO 2 showed that de novo synthesis was not required for monoterpene or sesquiterpene release immediately following chemical treatment or insect feeding. Comparative measurements were made between aldehyde doses applied to the plant and levels naturally released from leaves with insect damage. #

The effects of two elicitors: jasmonic acid and methyl jasmonate on cell growth as well as on rosmarinic acid accumulation in cell suspension cultures of Mentha 9 piperita were investigated. The highest rosmarinic acid accumulation 117.95 mg g -1 DW (12% DW) was measured 24 h after addition of 100 lM methyl jasmonate. A similar concentration 110.12 mg g -1 DW was detected 48 h after application of 200 lM jasmonic acid. Those values were nearly 1.5 times higher compared to the control sample, without elicitation. There was no substantial influence of elicitors on rosmarinic acid secretion into the culture media. Extracellular concentrations of rosmarinic acid were similar to the values from the control variants. It was documented that suspension cultures of M. piperita treated with elicitors showed a decrease in biomass accumulation when compared to the control.

In this research, effects of aminoethoxyvinylglycine (AVG) and methyl jasmonate (MeJA) applied 2 weeks ahead of estimated harvest date on fruit colour, firmness and individual phenolic compounds of three plum varieties were investigated during the cold storage. Fruits of ‘Black Amber’, ‘Black Beauty’ and ‘Fortune’ were kept at 0±0.5 °C temperature and 90±5% relative humidity for 4 weeks. During the storage, decreases in L* (lightness), chroma and hue angle values were observed. AVG treatments differed significantly (P<0.05) by control and MeJA treatment. In all plum varieties, AVG treatment resulted in significant increases in flesh firmness at the end of storage. MeJA increased significantly the firmness of ‘Black Beauty’ and ‘Fortune’ fruit at the end of storage. Chlorogenic acid was detected as major phenolic compound in plum varieties. AVG treatment to ‘Black Amber’ fruit increased the contents of chlorogenic and p-coumaric acid during storage, whereas it reduced chlorogenic and p-coumaric acid content in ‘Fortune’ fruit. MeJA treatment led to not only increases of rutin, ferulic acid and naringenin contents in the fruits of ‘Black Beauty’ and ‘Fortune’ varieties during the storage, but also significant increases of kaempferol content in ‘Black Amber’ and ‘Black Beauty’ fruits. While AVG treatment compared to control significantly decreased chlorogenic acid, quercetin and naringenin contents of ‘Black Amber’ fruits at the end of cold storage, it increased content of p-coumaric acid. At the end of storage, AVG and MeJA treatments reduced significantly the content of ferulic acid in ‘Black Beauty’ fruits. In addition, MeJA treatment significantly increased chlorogenic acid, ferulic acid and kaempferol contents in ‘Fortune’ fruits.

1988) The NH,terminal 21 amino acid residues are not essential for the papaininhibitory activity of oryzacystatin, a member of the cystatin superfamily. J Biol Chem 263: 7655-7659 Abe K, Emori Y, Kondo H, Suzuki K, Arai S (1987a) Molecular cloning of a cysteine proteinase inhibitor of rice (oryzacystatin). Homology with animal cystatins and transient expression in the ripening process of rice seeds. J Biol Chem 262: 16793-16797 Abe K, Kondo H, Arai S (198%) Purification and characterization of a rice cysteine proteinase inhibitor. Agric Biol Chem 51: Abe M, Abe K, Kuroda M, Arai S (1992) Corn kernel cysteine proteinase inhibitor as a nove1 cystatin superfamily member of plant origin. Eur J Biochem 209: 933-937 Baumgartner 8, Chrispeels MJ (1976) Partia1 characterization of a protease inhibitor which inhibits the major endopeptidase present in the cotyledons of mung beans. Plant Physiol 58: 1-6 Baumgartner, Chrispeels MJ (1977) Purification and characterization of vicilin peptidohydrolase, the major endopeptidase in the cotyledons of mung-bean seedlings. Eur J Biochem 77: 223-233 Bertl PJ, Storer AC (1995) Alignment/phylogeny of the papain superfamily of cysteine proteases. J Mo1 Biol 246: 273-283 Bode W, Engh R, Musil D, Thiele U, Huber R, Karshikov A, Brzin J, Kos J, Turk V (1988) The 2.0 A X-ray crystal structure of chicken egg white cystatin and its possible mode of interaction with cysteine proteinases. EMBO J 7: 2593-2599 Bolter CJ (1993) Methyl jasmonate induces papain inhibitor(s) in tomato leaves. Plant Physiol 103: 1347-1353 Botella MA, Quesada MA, Kononowicz AK, Bressan RA, Pliego F, Hasegawa PM, Valpuesta V (1994a) Characterization and in situ localization of a salt-induced tomato peroxidase mRNA. Plant Mo1 Biol 25: 105-114 Botella MA, Quesada MA, Medina MI, Pliego F, Valpuesta V (1994b) Induction of a tomato peroxidase gene in vascular tissue. FEBS Lett 347: 195-198 Brattsten LB (1991) Bioengineering of crop plants and resistant biotype evolution in insects: counteracting coevolution. Arch Insect Biochem Physiol 17: 253-267 Brown WE, Ryan CA (1984) Isolation and characterization of a wound-inducible trypsin inhibitor from alfalfa leaves. Biochem Brzin J, Ritonja A, Popovic T, Turk V (1990) Low molecular mass protein inhibitor of cysteine proteinases from soybean. Biol Chem Hoppe Seyler Suppl 371: 167-170 Ceros M, Carbonell J (1993) Purification and characterization of a thiol-protease induced during senescence of unpollinated ovaries of Pisum sativum. Physiol Plant 88: 267-274 Chen M-S, Johnson B, Wen L, Muthukrishnan S , Kramer KJ, Morgan TD, Reeck GR (1992) Rice cystatin: bacterial expression, purification, cysteine proteinase inhibitory activity, and insect growth suppressing activity of a truncated form of the protein.

The biosynthesis of the flavonolignan silymarin, a constitutive compound of the fruits of Silybum marianum with strong antihepatotoxic and hepatoprotective activities, is severely reduced in cell cultures of this species. It is well known that elicitation is one of the strategies employed to increase accumulation of secondary metabolites. Our study here reports on the effect of several compounds on the production of silymarin in S. marianum cultures. Yeast extract (YE), chitin and chitosan were compared with respect to their effects on silymarin accumulation in S. marianum suspensions and only yeast extract stimulated production. Jasmonic acid (JA) potentiated the yeast extract effect. One of the jasmonic acid derivatives, methyl jasmonate (MeJA), strongly promoted the accumulation of silymarin. Methyl jasmonate acted in a number of steps of the metabolic pathway of flavonolignans and its stimulating effect was totally dependent of "de novo" protein synthesis. Chalcone synthase (CHS) activity was enhanced by methyl jasmonate; however there did not appear to be a temporal relationship between silymarin accumulation and increase in enzyme activity. Also, this increase was not blocked by the protein synthesis inhibitor cycloheximide (CH). This study indicates that elicitor treatment promotes secondary metabolite production in S. marianum cultures and that jasmonic acid and its functional analogue plays a critical role in elicitation.

Treatment of Silybum marianum cell cultures with methyl jasmonate elicits the production of the antihepatotoxic drug silymarin and its release into the culture medium. In this work, we investigated the involvement of peroxidases (EC 1.11.1.7; donor hydrogen peroxidase oxido-reductase) in silymarin turnover in cell cultures as well as the influence of elicitation on the activity towards several substrates. Peroxidases from cell extracts and, to a higher degree from the spent medium, used the silymarin precursors taxifolin and coniferyl alcohol as substrates. Silymarin compounds were also degraded by suspension culture peroxidases; however, the oxidation efficiency was not modified by elicitation. S. marianum peroxidases were able to catalyse the oxidative coupling of taxifolin and coniferyl alcohol to silybinins. The synthetic activity was mainly associated with the extracellular compartment and as before, methyl jasmonate did not modify oxidative coupling activity. Changes in the isoenzyme profiles were not observed in elicited cultures.

Dioscorea fordii Prain et Burk., a member of the family Dioscoreaceae, is an important tuberous food in China owing to its edible and medicinal functions. However, the lack of healthy planting material has restricted its production. The present study was an attempt to develop a protocol for in vitro propagation of D. fordii. In an initial two by two (22) factorial experiment, the effects of culture system and activated charcoal have been observed for axillary shoot proliferation and tuberization of in vitro plantlets. Shoot length, frequency of proliferation, fresh weight and dry weight of shoots, frequency of tuberization and mean number of tubers per plantlet (NTPs) were significantly (P ≤ 0.05) greater in liquid medium compared to semi-solid media. Secondly, an orthogonal experimental design [L9 (34)] was used to investigate the effects of the ratio of naphthalene acetic acid (NAA) to 6-benzyladenine (BA) (NAA/BA), paclobutrazol, methyl jasmonate and sucrose concentration on tuberization. Sucrose concentration had the record effect on frequency of tuberization, NTPs and frequency of proliferation. The preferred medium for axillary shoot proliferation and tuberization of D. fordii found to be MS basal medium (Murashige and Skoog in Physiol Plant 15:473–479, 1962) supplemented with 1.0 mg l−1 BA, 0.1 mg l−1 NAA, 30 g l−1 sucrose and 1.5 g l−1 AC in liquid culture.

Citrus plants are currently facing biotic and abiotic stresses. Therefore, the characterization of molecular traits involved in the response mechanisms to stress could facilitate selection of resistant varieties. Although large cDNA microarray profiling has been generated in citrus tissues, the available protein expression data are scarce. In this study, to identify differentially expressed proteins in Citrus clementina leaves after infestation by the two-spotted spider mite Tetranychus urticae, a proteome comparison was undertaken using two-dimensional gel electrophoresis. The citrus leaf proteome profile was also compared with that of leaves treated over 0-72 h with methyl jasmonate, a compound playing a key role in the defense mechanisms of plants to insect/arthropod attack. Significant variations were observed for 110 protein spots after spider mite infestation and 67 protein spots after MeJA treatments. Of these, 50 proteins were successfully identified by liquid chromatography-mass spectrometry-tandem mass spectrometry. The majority constituted photosynthesis-and metabolism-related proteins. Five were oxidative stress associated enzymes, including phospholipid glutathione peroxidase, a salt stressed associated protein, ascorbate peroxidase and Mn-superoxide dismutase. Seven were defense-related proteins, such as the pathogenesis-related acidic chitinase, the protease inhibitor miraculin-like protein, and a lectin-like protein. This is the first report of differentially regulated proteins after T. urticae attack and exogenous MeJA application in citrus leaves.

Both enantiomers of the trans stereoisomer of magnolione 4 have been prepared by asymmetric Michael addition of ethyl acetoacetate to 2-pentylcyclopentenone, followed by hydrolysis/decarboxylation. The Michael reaction occurs under solid/liquid phase transfer catalysis in the presence of N-methylanthracenylquininium (or quinidinium) chloride. Enantiomeric excesses up to 76% are obtained. The trans structure of the compounds has been fully established by a careful NMR analysis, while the absolute configuration has been assigned as (2S,3S) for (þ)-4 by the analysis of the CD spectrum. q

Grapevine is subject to a number of diseases that affect yield and wine quality. To limit the excessive use of phytochemicals in the vineyard, alternative strategies have to be developed. Plant treatment with signaling molecules like elicitors stimulates their natural defense mechanisms. To improve grapevine tolerance against fungal pathogens, Vitis vinifera plants were treated with a natural exogenous elicitor, methyl jasmonate (MeJA). MeJA-treated leaves (Cabernet Sauvignon foliar cuttings) reacted by increasing transcript levels coding pathogenesis-related proteins (acidic class IV chitinase, serine protease inhibitor, polygalacturonase-inhibiting protein, and -1,3-glucanase) and coding enzymes involved in phytoalexin biosynthesis (one phenylalanine ammonia lyase and one stilbene synthase). This was correlated with the accumulation of stilbenes (antimicrobial compounds). The eliciting activity of MeJA was confirmed by enhanced tolerance of grapevine foliar cuttings and vineyard against powdery mildew (75% and 73%, respectively). On the basis of these original results, MeJA can therefore act as an efficient elicitor in an alternative strategy of grapevine protection.

Hot water dips (45 Á/60 8C for 2.5 Á/60 min) were evaluated as potential treatments to reduce sprout and root growth in peeled or unpeeled cloves in which sprouts had begun internal development but not yet emerged. Water dips at 5/50 8C did not reduce sprout and adventitious root growth in cloves stored at 10 8C and !/95% RH, while treatments at 55 8C for 10 min were effective. Several dips at 60 8C inhibited sprout and root growth, although only a 2.5 min treatment was both effective and non-injurious. Respiration rates of heat-treated garlic were higher than those of untreated cloves. L* color value (lightness) of the peeled cloves was sometimes decreased by heat treatment, but chroma and hue were not affected. The hot water dips had no effect on firmness or pungency (thiosulfinate concentrations). Methyl jasmonate dips at 10 (3 and 10 (4 M were ineffective for sprout control but did reduce root development. A dip at 60 8C 2.5 min was as effective as 1% O 2 '/10% CO 2 atmosphere to retard sprout and root growth during 6 months at 0 Á/1 8C.

The LTR retrotransposons are the most abundant mobile elements in the plant genome and seem to play an important role in genome reorganization induced by environmental challenges. Their success in this function depends on the ability of their promoters to respond to different signaling pathways that regulate plant adaptation to biotic and abiotic stresses. The promoter of the TLC1.1 retrotransposon from Solanum chilense contains two primary ethylene-responsive elements (PERE boxes) that are essential for its response to ethylene and for the stressinduced expression. Here, we describe that a 270 bp fragment (P270), derivative of this retroelement promoter, is also able to activate the transcription of the GUS reporter gene in transgenic plants in response to salicylic acid (SA), abscisic acid (ABA), methyl jasmonate (MeJA), hydrogen peroxide (H 2 O 2 ) and the synthetic auxin 2,4-D. PERE boxdependent and independent routes are involved in the response of P270 to these signal molecules. MeJA, H 2 O 2 and 2,4-D activate this promoter through cis-acting elements other than PERE boxes, whereas ABA and SA act via a PERE box-independent pathway but require this element for maximal activation. Three putative cis-acting elements MRE, GCN4 and GT1/TCA identified in the P270 promoter may be involved in the PERE box-independent activation pathway. These results suggest that the promoter of TLC1.1 may act as an integrator of different signal transduction pathways, allowing this member of the TLC1 retrotransposon family to be activated in response to multiples challenges.

Physical (geometric, volumetric and colour) and mechanical properties of apple (cv. Fuji) as affected by Methyl Jasmonate treatments were analyzed. Geometric mean diameter, fruit mass, sphericity and surface area ranged from 71.36 to 69.97 mm, 196.53 to 194.83 g, 1.126 to 1.129,
and 161.32 to 158.11 cm2 with MeJA doses, respectively. The bulk and fruit densities varied from 383.65 to 364.22 kg/m3 and from 967.96 to 954.63 kg/m3, with MeJA treatments. L*, a*, b* value of skin apple changed from 50.76 to 46.89, from 28.06 to 30.35, and from 24.58 to 22.43, respectively with MeJA doses. The skin and flesh firmnesses varied from 101.49 to 112.60 N and 74.18 to 72.71 N, respectively. The sphericity and projected area increased with MeJA doses used.
As the MeJA doses increased, the bulk and fruit densities decreased, whereas volume increased. L*, b* and hue angle characteristics of skin apple were found to decrease, whereas a* was found to increase with MeJA doses. The fruit-removal-force was decreased linearly with increasing MeJA doses. The rubber surface offered the maximum friction followed by plywood and galvanized metal for MeJA doses.

The treatment of Arabidopsis thaliana with methyl jasmonate was used to investigate the reaction of 2467 selected genes of primary and secondary metabolism by macroarray hybridization. Hierarchical cluster analysis allowed distinctions to be made between diurnally and methyl jasmonate regulated genes in a time course from 30 min to 24 h. 97 and 64 genes were identified that were up-or down-regulated more than 2-fold by methyl jasmonate, respectively. These genes belong to 18 functional categories of which sulfurrelated genes were by far strongest affected. Gene expression and metabolite patterns of sulfur metabolism were analysed in detail, since numerous defense compounds contain oxidized or reduced sulfur. Genes encoding key reactions of sulfate reduction as well as of cysteine, methionine and glutathione synthesis were rapidly up-regulated, but none of the known sulfur-deficiency induced sulfate transporter genes. In addition, increased expression of genes of sulfur-rich defense proteins and of enzymes involved in glucosinolate metabolism was observed. In contrast, profiling of primary and secondary sulfur metabolites revealed only an increase in the indole glucosinolate glucobrassicin upon methyl jasmonate treatment. The observed rapid mRNA changes were thus regulated by a signal independent of the known sulfur deficiency response. These results document for the first time how comprehensively the regulation of sulfur-related genes and plant defense are connected. This interaction is discussed as a new approach to differentiate between supply-and demand-driven regulation of the sulfate assimilation pathway.

Jasmonates and salicylic acid are considered to be signal molecules that induce a variety of plant genes involved in wound or defence response, as well as affecting nos promoter activity. In this paper we examined whether these chemicals could also affect nod genes from isogenic rhizobia strains. Isogenic strains contain the Rhizobium leguminosarum nodA promoter fused to the lacZ gene of Escherichia coli and differ only in the source of the regulatory nodD gene. Naringenin, jasmonic acid and methyl jasmonate induced expression of nod genes in strain RBL1284 and salicylic acid showed no activity alone or when used in combination with other compounds; addition of naringenin + jasmonic acid produced a synergistic effect. Results obtained with strain RBL5284 were similar to those for RBL1284 albeit the combination of naringenin with the other compounds markedly inhibited nod gene expression. Whereas RBL5283 responded to naringenin with a strong induction, jasmonic acid, methyl jasmonate or salicylic acid showed no significant responses. The inhibitory effect of salicylic acid on nod gene expression indicates that the induction mechanism of jasmonic acid, methyl jasmonate, N-propyldihydrojasmonate and naringenin is probably different from that of salicylic acid.

The beneficial effect of methyl jasmonate (MeJA) on the antagonistic yeast Pichia membranefaciens for control of anthracnose rot caused by Colletotrichum acutatum in postharvest loquat fruit and the possible mechanisms were investigated. The results indicated that the biocontrol activity of P. membranefaciens at 1 Â 10 8 colony-forming units ml À1 on anthracnose rot was enhanced by 10 lmol MeJA treatment. The combined treatment of P. membranefaciens with MeJA resulted in a remarkably improved control of the disease in comparison with the treatment of P. membranefaciens or MeJA alone. P. membranefaciens in combination with MeJA induced higher activities of two defense-related enzymes chitinase and b-1,3-glucanase in loquat fruit than applying the yeast or MeJA alone. The in vitro experiment showed that the addition of 10 lmol l À1 MeJA to the suspensions of P. membranefaciens significantly inhibited spore germination and germ tube elongation of C. acutatum than the yeast or MeJA alone. In addition, MeJA enhanced the population of P. membranefaciens both in vitro and in the wounds of the loquat fruit. These results suggested that MeJA could improve the biocontrol activity of P. membranefaciens on anthracnose rot in loquat fruit and the improved control of the disease by MeJA is directly because of the higher inhibitory effect on pathogen growth and the increased population size of antagonist, and indirectly because of the enhanced disease resistance in loquat fruit by the combination treatment.

Silymarin (Sm) from the fruit of Silybum marianum is an isomeric mixture of pharmacologically active flavonolignans which are formed by oxidative coupling of taxifolin (Tx) and coniferyl alcohol (CA). Suspension cultures of this plant constitutively secrete small amounts of Sm into the extracellular medium. Production can be increased by inclusion of cyclodextrins (CDs) in cultures. Both hydroxylated (RHCD) and dimethylated (RMCD) CDs strongly induced prompt accumulation of CA in the medium followed by a late production of flavonolignans. Simultaneous addition of methyl jasmonate (MJ) and RMCD to cells did not significantly modify CA release or flavonolignan accumulation. Delayed addition of MJ to cultures subcultivated in medium containing RMCD markedly influenced Sm production by promoting conversion of the previously formed CA precursor.

The effects of jasmonic acid (JA) and methyl jasmonate (MeJA) on potato (Solanum tuberosum L.) tuberization were studied in relation to cytokinins using single-node cuttings (SNCs) in vitro. In three potato cultivars differing in maturity levels, JA or MeJA (0.0, 2.5, 5.0, 7.5 and 10.0 μM) action was examined with N6-benzyladenine (BA: 0.0, 22.0 and 44.0 μM) under optimum tuber-inducing treatment with 80 g L−1 sucrose. Although jasmonates had a stimulatory role in root growth from SNCs, BA inhibited the root growth and antagonized the jasmonates action on root growth promotion. There was a strong inhibitory effect of BA on stoloniferous shoot growth, and in combination with jasmonates it could exert a much pronounced inhibitory effect. Jasmonates did not have any role in tuber induction in terms of tuber number and source-sink coordination (harvest index), rather their effects were counteracted in the presence of 22.0 μM BA in early cultivar. However, they had a promoting effect on tuber growth after induction in early cultivar, possibly due to its lower levels of endogenous gibberellins. Cytokinins detrimentally affected the tuber growth, and antagonized the jasmonates action irrespective of the maturity levels of the cultivars. It could, however, increase the tuber dry matter concomitant with a higher accumulation of starch. The promoting effect of jasmonates on tuber dry matter and starch accumulation was apparent only in early cultivar. There were significant interactions between cytokinin and jasmonates for accumulation of reducing and total sugars in tubers. The results show that exogenous cytokinins antagonize the jasmonates action, and the correlated effects of these two growth hormones interact with the maturing time of the cultivar during potato tuber formation in vitro.