microM Methyl Jasmonate Research Articles

Role of hydrogen sulfide in the methyl jasmonate response to cadmium stress in foxtail millet.

Methyl jasmonate (MeJA), a vital cellular regulator, mediates diverse developmental processes and defense responses against environmental stresse. Recently, a novel gasotransmitter, hydrogen sulfide (H2S), was found to have similar functions, but the interactions between H2S and MeJA in the acquisition of cadmium (Cd) tolerance have not been reported. Treating foxtail millet with 1 microM MeJA not only enhanced Cd tolerance and alleviated growth inhibitions but also decreased the contents of hydrogen peroxide, malondialdehyde and Cd in seedlings under 200 microM of Cd stress. Exogenous application of MeJA inhibited the transcript levels of the Natural Resistance-Associated Macrophage Protein (NRAMP1 and NRAMP6) and intensified Cd-induced expression of the homeostasis-related genes (MTP1, MTP12, CAX2 and ZIP4, besides HMA3). In addition, treatment with MeJA induced the production of endogenous H2S. Fumigation with sodium hydrosulfide (H2S donor) significantly enhanced MeJA-induced Cd tolerance, but this ability was weakened when H2S biosynthesis was inhibited with hydroxylamine. These results suggest that pretreatment with MeJA alleviated Cd stress and that this improvement was mediated by H2S in foxtail millet.

Cloning and Characterization of Phenylalanine Ammonia-Lyase and Cinnamate 4-Hydroxylase and Pyranocoumarin Biosynthesis in Angelica gigas

Phenylalanine ammonia-lyase (PAL) and cinnamate 4-hydroxylase (C4H) are important enzymes in the phenylpropanoid pathway and also in the accumulation of decursin (1) and decursinol angelate (2), which are major secondary metabolites in Angelica gigas. Using PCR with degenerate primers targeted to conserved regions of available orthologous PAL and C4H sequences, cDNAs encoding PAL and C4H from A. gigas were isolated. Both genes were used to show the comparative developmental and inducible accumulation of mRNAs in different organs and in suspension cells of A. gigas. PAL and C4H were induced most strongly in response to 300 microM methyl jasmonate treatment at 6 and 12 h, respectively, and were highly expressed in the fine roots of A. gigas. Similarly, the production of 1 and 2 was most prolific in the fine roots of the plant.

Adventitious rooting is enhanced by methyl jasmonate in tobacco thin cell layers

Adventitious roots (ARs) are induced by auxins. Jasmonic acid (JA) and methyl jasmonate (MeJA) are also plant growth regulators with many effects on development, but their role on ARs needs investigation. To this aim, we analyzed AR formation in tobacco thin cell layers (TCLs) cultured with 0.01-10 microM MeJA, either under root-inductive conditions, i.e., on medium containing 10 microM indole-3-butyric acid (IBA) and 0.1 microM kinetin, or without hormones. The explants were excised from the cultivars Samsun, Xanthii and Petite Havana, and from genotypes with altered AR-forming ability in response to auxin, namely the non-rooting rac mutant and the over-rooting Agrobacterium rhizogenes rolB transgenic line. Results show that NtRNR1 (G1/S) and Ntcyc29 (G2/M) gene activity, cell proliferation and meristemoid formation were stimulated in hormone-cultured TCLs by submicromolar MeJA concentrations. The meristemoids developed either into ARs and xylogenic nodules, or into xylogenic nodules only (rac TCLs). MeJA-induced meristemoid over-production characterized rolB TCLs. No rooting or xylogenesis occurred under hormone-free conditions, independently of MeJA and genotype. Endogenous JA progressively (days 1-4) increased in hormone-cultured TCLs in the absence of MeJA. JA levels were enhanced by 0.1 microM MeJA, on both days 1 and 4. Endogenous IBA was the only auxin detected, both in the free form and as IBA-glucose. Free IBA increased up to day 2, remaining constant thereafter (day 4). Its level was enhanced by 0.1 microM MeJA only on day 1, while IBA conjugation was not affected by MeJA. Taken together, these results show that an interplay between jasmonates and auxins regulates AR formation and xylogenesis in tobacco TCLs.

Molecular cloning and characterization of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (CaHDR) from Camptotheca acuminata and its functional identification in Escherichia coli

Camptothecin is an anti-cancer monoterpene indole alkaloid. The gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (designated as CaHDR), the last catalytic enzyme of the MEP pathway for terpenoid biosynthesis, was isolated from camptothecin-producing Camptotheca acuminata. The full-length cDNA of CaHDR was 1686 bp encoding 459 amino acids. Comparison of the cDNA and genomic DNA of CaHDR revealed that there was no intron in genomic CaHDR. Southern blot analysis indicated that CaHDR belonged to a low-copy gene family. RT-PCR analysis revealed that CaHDR expressed constitutively in all tested plant organs with the highest expression level in flowers, and the expression of CaHDR could be induced by 100 microM methyl-jasmonate (MeJA), but not by 100 mg/L salicylic acid (SA) in the callus of C. acuminata. The complementation of CaHDR in Escherichia coli ispH mutant MG1655 demonstrated its function.

Combined effects of phytohormone, indole-3-butyric acid, and methyl jasmonate on root growth and ginsenoside production in adventitious root cultures of Panax ginseng C.A. Meyer

Indole-3-butyric acid at 25 microM with methyl jasmonate (MJ) at 100 microM in Panax ginseng synergistically stimulated both root growth and ginsenoside accumulation compared with 100 microM MJ alone. Productivity of ginsenoside was 10 mg l(-1) d(-1) compared to 7.3 mg l(-1) d(-1) with MJ elicitation alone.

Methyl jasmonate and salicylic acid induced oxidative stress and accumulation of phenolics in Panax ginseng bioreactor root suspension cultures.

To investigate the enzyme variations responsible for the synthesis of phenolics, 40 day-old adventitious roots of Panax ginseng were treated with 200 microM methyl jasmonate (MJ) or salicylic acid (SA) in a 5 L bioreactor suspension culture (working volume 4 L). Both treatments caused an increase in the carbonyl and hydrogen peroxide (H2O2) contents, although the levels were lower in SA treated roots. Total phenolic, flavonoid, ascorbic acid, non-protein thiol (NPSH) and cysteine contents and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical reducing activity were increased by MJ and SA. Fresh weight (FW) and dry weight (DW) decreased significantly after 9 days of exposure to SA and MJ. The highest total phenolics (62%), DPPH activity (40%), flavonoids (88%), ascorbic acid (55%), NPSH (33%), and cysteine (62%) contents compared to control were obtained after 9 days in SA treated roots. The activities of glucose 6-phosphate dehydrogenase, phenylalanine ammonia lyase, substrate specific peroxidases (caffeic acid peroxidase, quercetin peroxidase and ferulic acid peroxidase) were higher in MJ treated roots than the SA treated ones. Increased shikimate dehydrogenase, chlorogenic acid peroxidase and beta-glucosidase activities and proline content were observed in SA treated roots than in MJ ones. Cinnamyl alcohol dehydrogenase activity remained unaffected by both MJ and SA. These results strongly indicate that MJ and SA induce the accumulation of phenolic compounds in ginseng root by altering the phenolic synthesis enzymes.

Microarray-based screening of jasmonate-responsive genes in Arabidopsis thaliana

Jasmonates comprise a family of plant hormones that regulate gene expression to modulate diverse developmental and defensive processes. To screen a set of jasmonate-responsive Arabidopsis genes, we performed a microarray analysis using an Affymetrix GeneChip containing about 8,300 gene probes synthesized in situ. External treatment with 100 microM methyl jasmonate resulted in significant changes (more than twofold increases or decreases) in the expression levels of 137 genes in the rosette leaves of 5-week-old Arabidopsis plants. Of these, 74 genes were up-regulated, including those involved in jasmonate biosynthesis, defense responses, oxidative stress responses, senescence, and cell wall modification. In contrast, the expression of genes involved in chlorophyll constitution and photosynthesis was down-regulated. Most importantly, the jasmonate treatment significantly reduced transcripts of abscisic acid-responsive cold/drought-stress genes, which suggests that an antagonistic interaction occurs between the jasmonate and abscisic acid signaling pathways in abiotic stress responses. Northern blot analysis of some selected genes revealed that the jasmonate-responsive genes exhibited unique time-course expression patterns after the external jasmonate treatment. Based on the basic clustering of the genes, we established a likely regulation scenario: the genes induced early after treatment are involved in signaling mechanisms that activate or repress other genes, whereas intermediate- and late-accumulating genes are activated by the signaling mechanisms and are subsequently involved in the ultimate jasmonate-modulated cellular responses.

Enhanced Ginsenoside Productivity by Combination of Ethephon and Methyl Jasmoante in Ginseng (Panax ginseng C.A. Meyer) Adventitious Root Cultures

Ethephon at 50 microM enhanced both root growth and ginsenoside accumulation in ginseng (Panax ginseng C.A. Meyer) adventitious root cultures, but at 100 microM it inhibited only ginsenoside accumulation. Ginsenoside productivity with 50 microM ethephon was the highest at 1.7 mg l(-1) d(-1) after 8 days of elicitation. However, elicitation with 50 microM ethephon and 100 microM methyl jasmonate (MJ) improved productivity (6.3 mg l(-1) d(-1)) whereas elicitation with 100 microM MJ alone gave only 2.9 mg l(-1) d(-1).

Methyl jasmonate and salicylic acid elicitation induces ginsenosides accumulation, enzymatic and non-enzymatic antioxidant in suspension culture Panax ginseng roots in bioreactors

The effects of methyl jasmonate (MJ) and salicylic acid (SA) on changes of the activities of major antioxidant enzymes, superoxide anion accumulation (O2-), ascorbate, total glutathione (TG), malondialdehyde (MDA) content and ginsenoside accumulation were investigated in ginseng roots (Panax ginseng L.) in 4 l (working volume) air lift bioreactors. Single treatment of 200 microM MJ and SA to P. ginseng roots enhanced ginsenoside accumulation compared to the control and harvested 3, 5, 7 and 9 days after treatment. MJ and SA treatment induced an oxidative stress in P. ginseng roots, as shown by an increase in lipid peroxidation due to rise in O2- accumulation. Activity of superoxide dismutase (SOD) was inhibited in MJ-treated roots, while the activities of monodehydroascorbate reductase (MDHAR), dehydroascorbate reductase (DHAR), SOD, guaiacol peroxidase (G-POD), glutathione peroxidase (GPx) and glutathione reductase (GR) were induced in SA-treated roots. A strong decrease in the activity of catalase (CAT) was obtained in both MJ- and SA-treated roots. Activities of ascorbate peroxidase (APX) and glutathione S transferase (GST) were higher in MJ than SA while the contents of reduced ascorbate (ASC), redox state (ASC/(ASC+DHA)) and TG were higher in SA- than MJ-treated roots while oxidized ascorbate (DHA) decreased in both cases. The result of these analyses suggests that roots are better protected against the O2- stress, thus mitigating MJ and SA stress. The information obtained in this work is useful for efficient large-scale production of ginsenoside by plant-root cultures.

Methyl Jasmonate Stimulates Jaceosidin and Hispidulin Production in Cell Cultures of Saussurea medusa

Cell cultures of Saussurea medusa produce valuable secondary metabolites, and jaceosidin and hispidulin are the major bioactive compounds. In the present study, the cultures were challenged by methyl jasmonate (MJ). The highest jaceosidin and hispidulin concentrations (65.2 +/- 3.67 mg/L and 12.3 +/- 0.47 mg/L) were achieved with 5 microM MJ added to 9-d-old subcultures, being 2.2-fold and 4.2-fold, respectively, higher than those from controls. The elicitor had little influence on cell growth, indicating that the changed biological processes did not include alterations in cell division. Furthermore, we observed that the activities of phenylalanine ammonia lyase were transiently increased after treatment with MJ, which suggests that this elicitor modifies jaceosidin and hispidulin production by regulating the phenylpropanoid pathway.

Enhancement of ginsenoside biosynthesis in high-density cultivation of Panax notoginseng cells by various strategies of methyl jasmonate elicitation

A single addition of 200 microM methyl jasmonate (MJA) to high-density cell cultures of Panax notoginseng enhanced ginsenoside production in both shake-flask (250 ml) and airlift bioreactor (ALR; 1 l working volume). Repeated elicitation with two additions of 200 microM MJA during cultivation further induced the ginsenoside biosynthesis in both cultivation vessels. The content of ginsenosides Rg1, Re, Rb1 and Rd in the ALR was increased from, respectively, 0.18+/-0.01, 0.21+/-0.01, 0.21+/-0.02 and 0 mg per100 mg dry cell weight (DW) in untreated cell cultures (control) to 0.32+/-0.02, 0.36+/-0.02, 0.72+/-0.06 and 0.08+/-0.01 mg per100 mg DW with a single addition of MJA and further increased to 0.43+/-0.02, 0.46+/-0.03, 1.09+/-0.07 and 0.14+/-0.02 mg per100 mg DW with two additions of MJA. Interestingly, the activity of the Rb1 biosynthetic enzyme (UDPG-ginsenoside Rd glucosyltransferase), was also increased with a single elicitation by MJA and increased again by a repeated elicitation, which coincided well with the trend in the increase in Rb(1) content. In order to further improve the cell density and ginsenoside production, a strategy of MJA repeated elicitation combined with sucrose feeding was adopted. The final cell density and total ginsenoside content in the ALR reached 27.3+/-1.5 g/l and 2.02+/-0.06 mg per100 mg DW; and the maximum production of ginsenoside Rg1, Re, Rb1 and Rd was 111.8+/-4.7, 117.2+/-4.6, 290.2+/-5.1 and 32.7+/-8.1 mg/l, respectively. The strategies demonstrated and the information obtained in this work are useful for the efficient large-scale production of bioactive ginsenosides by plant cell cultures.

Methyl jasmonate elicitation enhanced synthesis of ginsenoside by cell suspension cultures of Panax ginseng in 5-l balloon type bubble bioreactors

The effects of methyl jasmonate (MJ) elicitation on the cell growth and accumulation of ginsenoside in 5-l bioreactor suspension cultures of Panax ginseng were investigated. Ginsenoside accumulation was enhanced by elicitation by MJ (in the range 50-400 microM); however, fresh weight, dry weight and growth ratio of the cells was strongly inhibited by increasing MJ concentration. The highest ginsenoside yield was obtained at 200 microM MJ. In the second experiment, 200 microM MJ was added on day 15 during the cultivation. The ginsenoside, Rb group, and Rg group ginsenoside content increased 2.9, 3.7, and 1.6 times, respectively, after 8 days of MJ treatment. Rb group gisnsenosides accumulated more than Rg group ginsenosides. Among Rb group ginsenosides, Rb1 content increased significantly by four times but the contents of Rb2, Rc and Rd increased only slightly. Among Rg group ginsenosides, Rg1 and Re showed 2.3-fold and 3.0-fold increments, respectively, whereas there was only a slight increment in Rf group ginsenosides. These results suggest that MJ elicitation is beneficial for ginsenoside production using 5-l bioreactor cell suspension cultures.

Adventitious root growth and ginsenoside accumulation in Panax ginseng cultures as affected by methyl jasmonate

Adventitious roots of ginseng were treated with methyl jasmonate (MJ) up to 150 microM and cultured for 40 days. Up to 100 microM MJ inhibited the root growth but increase ginsenoside accumulation. In a two-stage bioreactor culture, total ginsenosides, after elicitation with 100 microM MJ peaked after 10 days at 48 mg g(-1) dry wt and then dropped sharply. Of the two groups of ginsenosides (Rb and Rg), higher amounts of Rb accumulated in the adventitious roots.

Methyl jasmonate disrupts shoot formation in tobacco thin cell layers by over-inducing mitotic activity and cell expansion.

The aim of the present study was to determine early cyto-histological events associated with the reduced number of shoots formed at the end of culture in tobacco (Nicotiana tabacum L.) thin cell layers treated with methyl jasmonate (MJ) [S. Biondi et al. (2001) J Exp Bot 52:1-12]. The results show that 0.1-10 microM MJ strongly stimulated mitotic activity early in culture relative to untreated controls. Treatment with MJ also induced anomalous mitoses. Enhanced proliferative growth was confirmed by northern analysis and in situ hybridisation using cDNA probes of the G1/S phase-specific genes ubiquitin carboxyl-extension protein (ubi-CEP), topoisomerase 1 (top1) and ribonucleotide reductase (RNR). The formation of meristematic cell clusters on day 5 was also enhanced by 1 muM MJ, but subsequent development of these cell clusters into meristemoids and shoot primordia was reduced by all MJ concentrations in a dose-dependent manner. Cell expansion was stimulated by MJ concentrations ranging from 0.001 to 10 microM; expanded cells frequently occurred around and within meristemoids and shoot primordia, and displayed thickened and suberised cell walls; cell wall thickness increased with increasing MJ concentration. These cytological events caused alterations in the tunica and stem differentiation of the shoot dome. The apparently paradoxical role of MJ, which deregulates shoot formation through a stimulation of growth events, i.e., mitotic activity and cell expansion, is discussed.

Production of biologically active taxoids by a callus culture of Taxus cuspidata.

Ten known taxoids, paclitaxel, 7-epi-taxol, taxol C, baccatin VI, taxayuntin C, taxuyunnanine C and its analogues (2-5), and yunnanxane (6), and an abietane, taxamairin A, were produced in the callus culture of Taxus cuspidata cultivated on a modified Gamborg's B5 medium in the presence of 0.5 mg/L NAA. After stimulation with 100 microM methyl jasmonate, five more taxoids, cephalomannine, 1beta-dehydroxybaccatin VI, taxinine NN-11 (1), baccatin I, and 2alpha-acetoxytaxusin, and one more abietane, taxamairin C, were found in addition to the above-mentioned compounds. It was also observed that the content of the products increased over three times. Taxinine NN-11 (1) is a new taxane whose structure was assigned as 5alpha,13alpha-diacetoxy-9alpha-cinnamoyloxy-4(20),11-taxadien-10beta-ol by analysis of its spectral data. Taxinine NN-11 (1) exhibited significant MDR reversal activity toward 2780 AD tumor cells. The results of primary screening based on 39 human cancer cell lines suggest that 1 also belongs to a new mechanistic class. Efficient production of 1 was investigated using the callus culture of T. cuspidata.

Redox-related peroxidative responses evoked by methyl-jasmonate in axenically cultured aeroponic sunflower (Helianthus annuus L.) seedling roots.

Methyl-jasmonate (MeJA) has been proposed to be involved in the evocation of defense reactions, as the oxidative burst in plants, substituting the elicitors or enhancing their effect. 48 h dark- and sterilely cultured (axenic) aeroponic sunflower seedling roots excised and treated with different concentrations of MeJA showed a strong and quick depression of the H(+) efflux rate, 1.80 microM MeJA totally stopping it for approximately 90 min and then reinitiating it again at a lower rate than controls. These results were wholly similar to those obtained with nonsterilely cultured roots and have been interpreted as mainly based on H(+) consumption for O(2)(*-) dismutation to H(2)O(2). Also K(+) influx was strongly depressed by MeJA, even transitorily reverting to K(+) efflux. These results were consistent with those associated to the oxidative burst in plants. MeJA induced massive H(2)O(2) accumulation in the middle lamella and intercellular spaces of both the root cap cells and the inside tissues of the roots. The native acidic extracellular peroxidase activity of the intact (nonexcised) seedling roots showed a sudden enhancement (by about 52%) after 5 min of MeJA addition, maintained for approximately 15 min and then decaying again to control rates. O(2) uptake by roots gave similar results. These and other results for additions of H(2)O(2) or horseradish peroxidase, diphenylene iodonium, and sodium diethyldithiocarbamate trihydrate to the reaction mixture with roots were all consistent with the hypothesis that MeJA induced an oxidative burst, with the generation of H(2)O(2) being necessary for peroxidase activity. Results with peroxidase activity of the apoplastic fluid were in accordance with those of the whole root. Finally, MeJA enhanced NADH oxidation and inhibited hexacyanoferrate(III) reduction by axenic roots, and diphenylene iodonium cancelled out these effects. Redox activities by CN(-)- preincubated roots were also studied. All these results are consistent with the hypothesis that MeJA enhanced the NAD(P)H oxidase of a redox chain linked to the oxidative burst, so enhancing the generation of O(2)(*-) and H(2)O(2), O(2) uptake, and peroxidase activity by roots.

Enhancement of taxol production and release in Taxus chinensis cell cultures by ultrasound, methyl jasmonate and in situ solvent extraction.

This study evaluates the use of a novel mechanical stimulus, ultrasound (US), and a putative chemical elicitor, methyl jasmonate (MJ), combined with in situ solvent extraction (two-phase culture), to enhance taxol production by Taxus chinensis cells in suspension culture. The volumetric taxol yield was increased 1.5- to 1.8-fold with 2 min US treatment once or twice during a 4-week culture period, about 5-fold with 60-120 microM MJ, and 7- to 9-fold by in situ solvent extraction of taxol with dibutyl phthalate (DBP) (11% v/v). The percent of extracellular taxol or taxol release was also significantly increased. The combined use of US (day 5 or 9) and MJ treatment (day 7) resulted in taxol yields 20-50% higher than each of the treatments used alone. The most favorable strategy for taxol production was the application of US or MJ treatment, followed by in situ solvent extraction, giving rise to a taxol yield of 33-35 mg/l, about 17-fold higher than the control, at 1.9 mg/l. It was found that the organic solvent DBP, as well as US and MJ, stimulated the enzyme activity of secondary metabolic pathways, which was partially responsible for the enhanced taxol production.

Effect of Mixing Time on Taxoid Production Using Suspension Cultures of Taxus chinensis in a Centrifugal Impeller Bioreactor

The effects of fluid mixing on the cell growth and secondary metabolite production of plant cells were investigated in a low-shear centrifugal impeller bioreactor (CIB) system. Suspension cultures of Taxus chinensis cells producing taxuyunnanine C (Tc), a physiologically active secondary metabolite, were used as a model system for this investigation. The mixing time (t(m)) and volumetric oxygen transfer coefficient (k(L)a) in the bioreactor were characterized at various cell densities and operating conditions. A constant t(m) of 5 s or 10 s was maintained during cultivation by adjusting the impeller agitation speed with no detrimental effect on the cultured cells. A higher cell density, Tc content and total Tc production were obtained under the shorter mixing time of 5 s. The favorable effect of more rapid mixing on Tc production was also confirmed when the Tc accumulation was significantly increased through culture elicitation using 100 microM methyl jasmonate (MJA). The lower Tc production at the longer t(m) of 10 s was mainly attributed to oxygen transfer limitation in the dead zones and larger cell aggregates resulting from poor mixing.

Efficient production of saikosaponins in Bupleurum falcatum root fragments combined with signal transducers.

An efficient system to produce saikosaponins (saikosaponin-a and -d) in Bupleurum falcatum adventitious root fragments combined with signal transducers was developed. The roots are heterogeneous in terms of size and shape and sometimes form aggregates during cultivation. When the roots were cut to lengths of about 5 mm using a scalpel and cultivated, the root fragments did not form the aggregates, and root growth and saikosaponin production were not inhibited. After screening various signal transducers, it was clear that methyl jasmonate (MeJA) markedly promoted saikosaponin production. By comparing the effect of MeJA and related substances on saikosaponin production, we conclude that both the pentenyl and carboxylmethyl group of MeJA play an important role in the promotion of saikosaponin production. Addition of both 100 microM MeJA and 20 mM CaCl2 to the medium stimulated the content of saikosaponin in the root, with levels reaching 31.7 mg/g-dry root for 15 days of cultivation. A large amount of root fragments were prepared using a blender and cultivated (23 g-dry root/l) with 400 microM MeJA and 20 mM CaCl2, resulting in a high concentration of saikosaponins (747.3 mg/l).

Methyl jasmonate upregulates biosynthetic gene expression, oxidation and conjugation of polyamines, and inhibits shoot formation in tobacco thin layers

The effect of methyl jasmonate (MJ) on de novo shoot formation and polyamine metabolism was investigated in thin layer explants of tobacco (Nicotiana tabacum L. cv. Samsun). A relatively low concentration of MJ (0.1 microM) enhanced explant fresh weight, but had no effect on the final number of shoots per explant while higher concentrations (1 and 10 microM) significantly inhibited organogenesis. The histological study revealed that, with increasing concentrations of MJ, the formation of meristemoids and shoot domes declined and the incidence of cell hypertrophy increased. In explants cultured with 0.1, 1 or 10 microM MJ, the endogenous levels of free putrescine, spermidine and spermine generally declined compared with controls, after 7 and 15 d. Perchloric acid-soluble conjugated polyamines accumulated dramatically during culture, but much more so in the presence of MJ than in controls. Acid-insoluble conjugated spermidine alone increased in response to the elicitor. Activities of the putrescine biosynthetic enzymes arginine decarboxylase (ADC, EC 4.1.1.19) and ornithine decarboxylase (ODC, EC 4.1.1.17) in the soluble fraction of MJ-treated explants displayed up to 3-fold increases relative to control explants. However, the most relevant increases in these enzyme activities occurred in the particulate fraction. The activity of S:-adenosylmethionine decarboxylase (SAMDC, EC 4.1.1.21), an enzyme involved in spermidine and spermine biosynthesis, was also stimulated by exposure to MJ. Northern analyses revealed MJ-induced, generally dose-dependent, increases in the mRNA levels of all three enzymes. Diamine oxidase (DAO, EC 1.4.3.6) activity was stimulated by MJ mainly in the cell wall fraction. The upregulation of polyamine metabolism is discussed in relation to the morphogenic behaviour of MJ-treated explants.