Effect of methyl jasmonate and salicylic acid on sesquiterpene lactone accumulation in hairy roots of <Emphasis Type="Italic">Cichorium intybus</Emphasis>

The effects of methyl jasmonate (MJ) or salicylic acid (SA) on the sesquiterpene lactone content and biomass accumulation were investigated in a hairy root culture of Cichorium intybus. The guaianolides crepidiaside B (1), 8-deoxylactucin (2), and the germacranolide sonchuside A (3) were quantified by RP-HPLC. Neither MJ nor SA affected the growth of examined hairy root culture. Jasmonate up-regulated biosynthesis of the analysed sesqiterpene lactones in the culture (maximum after 72 h). SA caused a transient increase in sonchuside A accumulation in the roots (up to twofold increase compared with the control) and decrease of guaianolide content.     

Overexpression of a soybean salicylic acid methyltransferase gene confers resistance to soybean cyst nematode

Salicylic acid plays a critical role in activating plant defence responses after pathogen attack. Salicylic acid methyltransferase (SAMT) modulates the level of salicylic acid by converting salicylic acid to methyl salicylate. Here, we report that a SAMT gene from soybean (GmSAMT1) plays a role in soybean defence against soybean cyst nematode (Heterodera glycines Ichinohe, SCN). GmSAMT1 was identified as a candidate SCN defence‐related gene in our previous analysis of soybean defence against SCN using GeneChip microarray experiments. The current study started with the isolation of the full‐length cDNAs of GmSAMT1 from a SCN‐resistant soybean line and from a SCN‐susceptible soybean line. The two cDNAs encode proteins of identical sequences. The GmSAMT1 cDNA was expressed in Escherichia coli. Using in vitro enzyme assays, E. coli‐expressed GmSAMT1 was confirmed to function as salicylic acid methyltransferase. The apparent Km value of GmSAMT1 for salicylic acid was approximately 46 μm . To determine the role of GmSAMT1 in soybean defence against SCN, transgenic hairy roots overexpressing GmSAMT1 were produced and tested for SCN resistance. Overexpression of GmSAMT1 in SCN‐susceptible backgrounds significantly reduced the development of SCN, indicating that overexpression of GmSAMT1 in the transgenic hairy root system could confer resistance to SCN. Overexpression of GmSAMT1 in transgenic hairy roots was also found to affect the expression of selected genes involved in salicylic acid biosynthesis and salicylic acid signal transduction.     

Positive selection for single amino acid change promotes substrate discrimination of a plant volatile-producing enzyme

We used a combined evolutionary and experimental approach tobetter understand enzyme functional divergence within the SABATHgene family of methyltransferases (MTs). These enzymes catalyzethe formation of a variety of secondary metabolites in plants,many of which are volatiles that contribute to floral scentand plant defense such as methyl salicylate and methyl jasmonate.A phylogenetic analysis of functionally characterized membersof this family showed that salicylic acid methyltransferase(SAMT) forms a monophyletic lineage of sequences found in severalflowering plants. Most members of this lineage preferentiallymethylate salicylic acid (SA) as compared with the structurallysimilar substrate benzoic acid (BA). To investigate if positiveselection promoted functional divergence of this lineage ofenzymes, we performed a branch-sites test. This test showedstatistically significant support (P < 0.05) for positiveselection in this lineage of MTs (d_N/d_S = 10.8). A high posteriorprobability (pp = 0.99) identified an active site methionineas the only site under positive selection in this lineage. Toinvestigate the potential catalytic effect of this positivelyselected codon, site-directed mutagenesis was used to replaceMet with the alternative amino acid (His) in a Datura wrightiifloral–expressed SAMT sequence. Heterologous expressionof wild-type and mutant D. wrightii SAMT in Escherichia colishowed that both enzymes could convert SA to methyl salicylateand BA to methyl benzoate. However, competitive feeding withequimolar amounts of SA and BA showed that the presence of Metin the active site of wild-type SAMT resulted in a >10-foldhigher amount of methyl salicylate produced relative to methylbenzoate. The Met156His-mutant exhibited little differentialpreference for the 2 substrates because nearly equal amountsof methyl salicylate and methyl benzoate were produced. Evolutionof the ability to discriminate between the 2 substrates by SAMTmay be advantageous for efficient production of methyl salicylate,which is important for pollinator attraction as well as pathogenand herbivore defense. Because BA is a likely precursor forthe biosynthesis of SA, SAMT might increase methyl salicylatelevels directly by preferential methylation and indirectly byleaving more BA to be converted into SA.     

Enhanced production of valerenic acid in hairy root culture of Valeriana officinalis by elicitation

Valerenic acid (VA) is a pharmacologically-active sesquiterpene found in valerian (Valeriana officinalis L., Valerianaceae) roots and rhizomes. The plant produces only small amounts of this metabolite naturally. So, induction of hairy roots as well as elicitation can be useful to increase its commercial production. In this study, Wild-type strain ‘A13’ of Agrobacterium rhizogenes was used to induce hairy roots in valerian. The influence of three different elicitors including Fusarium graminearum extract (FE), methyl jasmonate (MJ) and salicylic acid (SA) on VA production in the selected hairy root line ‘LeVa-C4’ was also investigated. The 23-day-old cultures were treated with different concentrations of the elicitors at exposure time of 3 and 7 days. FE (1%) and MJ (100 µM L^?1) highly promoted VA production at 7 days after elicitation, to a level of 12.31- and 6-fold higher than that of non-elicited controls, respectively, and FE did not exert any negative effects on biomass yield of hairy root. SA did not significantly increase the production of VA. This is the first time study to assess the elicitation of hairy root cultures to promote VA biosynthesis in valerian and the resulting experiments demonstrated that F. graminearum extract and MJ were indeed a potent inducer of VA biosynthesis.     

Biochemical and structural characterization of benzenoid carboxyl methyltransferases involved in floral scent production in Stephanotis floribunda and Nicotiana suaveolens

Flower-specific benzenoid carboxyl methyltransferases from Stephanotis floribunda and Nicotiana suaveolens were biochemically and structurally characterized. The floral scents of both these species contain higher levels of methyl benzoate and lower levels of methyl salicylate. The S. floribunda enzyme has a 12-fold lower K(m) value for salicylic acid (SA) than for benzoic acid (BA), and results of in silico modeling of the active site of the S. floribunda enzyme, based on the crystal structure of Clarkia breweri salicylic acid methyltransferase (SAMT), are consistent with this functional observation. The enzyme was therefore designated SAMT. The internal concentration of BA in S. floribunda flowers is three orders of magnitude higher than the SA concentration, providing a rationale for the observation that these flowers synthesize and emit more methyl benzoate than methyl salicylate. The N. suaveolens enzyme has similar K(m) values for BA and SA, and the in silico modeling results are again consistent with this in vitro observation. This enzyme was therefore designated BSMT. However, the internal concentration of BA in N. suaveolens petals was also three orders of magnitude higher than the concentration of SA. Both S. floribunda SAMT and N. suaveolens BSMT are able to methylate a range of other benzenoid-related compounds and, in the case of S. floribunda SAMT, also several cinnamic acid derivatives, an observation that is consistent with the larger active site cavity of each of these two enzymes compared to the SAMT from C. breweri, as shown by the models. Broad substrate specificity may indicate recent evolution or an adaptation to changing substrate availability.     

Novel S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase,an enzyme responsible for biosynthesis of methyl salicylate and methyl benzoate,is not involved in floral scent production in snapdragon flowers

Using a functional genomic approach we have isolated and characterized a cDNA that encodes a salicylic acid carboxyl methyltransferase (SAMT) from Antirrhinum majus. The sequence of the protein encoded by SAMT has higher amino acid identity to Clarkia breweri SAMT than to snapdragon benzoic acid carboxyl methyltransferase (BAMT) (55 and 40% amino acid identity, respectively). Escherichia coli-expressed SAMT protein catalyzes the formation of the volatile ester methyl salicylate from salicylic acid with a K(m) value of 83 microM. It can also methylate benzoic acid to form methyl benzoate, but its K(m) value for benzoic acid is 1.72 mM. Snapdragon flowers do not emit methyl salicylate. The potential involvement of SAMT in production and emission of methyl benzoate in snapdragon flowers was analyzed by RNA gel blot analysis. SAMT mRNA was not detected in floral tissues by RNA blot hybridization, but low levels of SAMT gene expression were detected after real-time RT-PCR in the presence of SAMT-specific primers, indicating that this gene does not contribute significantly, if at all, in methyl benzoate production and emission in snapdragon flowers. Expression of SAMT in petal tissue was found to be induced by salicylic and jasmonic acid treatments.     

Coculture of genetically transformed roots and shoots for synthesis, translocation, and biotransformation of secondary metabolites

Genetically transformed shooty teratomas of Atropa belladonna and a Duboisia leichhardtii x D. myoporoides hybrid were studied for biotransformation of tropane alkaloids in shake flasks and bioreactors. Although de novo synthesis of hyoscyamine and scopolamine was limited, shoots of both species were able to translocate and accumulate significant quantities of exogenous alkaloid. The maximum yield of scopolamine from hyoscyamine fed to the Duboisia hybrid shoots was 35% w/w; the yield of the scopolamine precursor, 6beta-hydroxyhyoscyamine, was 37% w/w. Biotransformation activity was poor in A. belladonna shooty teratomas provided with exogenous hyoscyamine; however, scopolamine levels comparable with those in leaves of the whole plant accumulated in shoots fed with hairy root extract. Coculture of A. belladonna shooty teratomas and hairy roots in the same hormone-free medium was investigated as a means of providing a continuous source of hyoscyamine for conversion to scopolamine. Of the biotransformation systems tested with A. belladonna, coculture produced the highest levels of scopolamine and the highest scopolamine: hyoscyamine ratios. Cocultured shoots accumulated up to 0.84 mg g(-1) dry weight scopolamine, or 3-11 times the average concentrations found in leaves of the whole plant. The scopolamine: hyoscyamine ratio in coculture ranged from 0.07 to 1.9, a significant improvement over levels of 0-0.03 normally found in A. belladonna hairy roots. Addition of Pluronic F-68 or copper sulfate to the medium and variation in initial medium pH did not improve hyoscyamine release from hairy roots. Scopolamine levels were increased using 1 muM copper sulfate or initial medium pH between 6.0 and 8.0; however, results from elicitation of hairy roots could not match the beneficial effect on scopolamine synthesis of root-shoot coculture. Addition of 0.001-1.0% (w/v) Pluronic F-68 to the roots reduced hyoscyamine release but postponed necrosis in the root tissue for up to 60 d. (c) 1996 John Wiley & Sons, Inc.     

Production of some benzylisoquinoline alkaloids in Papaver armeniacum L. hairy root cultures elicited with salicylic acid and methyl jasmonate

Papaver armeniacum hairy roots were induced by four Rhizobium rhizogenes strains on three explants (shoot, root, and hypocotyl). Also, the effects of two concentrations (100 and 200 μM) of methyl jasmonate (MJ) and salicylic acid (SA) were assessed on productions of papaverine, noscapine, thebaine, morphine, and codeine and expression of some related genes (TYDC, DBOX, BBE, SalAT, T6ODM, and COR) in P. armeniacum L. hairy root culture at 24 and 48 h after elicitation. R. rhizogenes strain C58C1 induced the highest hairy root rate on hypocotyl explant. Application of 100 μM MJ resulted in the highest contents of thebaine, codeine, and morphine by enhancing the expression of SalAT, COR, and T6ODM genes, respectively, while application of 100 μM SA resulted in the highest contents of papaverine and noscapine by upregulating DBOX and BBE genes, respectively. 100 μM MJ can be used as an effective elicitor in P. armeniacum hairy root culture to increase studied morphinan alkaloids. Also, SA can be suggested for enhancing papaverine and noscapine contents in P. armeniacum hairy root culture. It may be due to that there is a SA- and MJ-signaling crosstalk, which results in reciprocal antagonism between SA and MJ signaling pathways. The effects of MJ and SA elicitors on benzylisoquinoline alkaloids (BIAs) production were level-dependent.

     

Rosmarinic acid production by <Emphasis Type="Italic">Coleus forskohlii</Emphasis>hairy root cultures

Coleus forskohlii hairy root cultures were found to produce forskolin and rosmarinic acid (RA) as the main metabolites. The growth and RA production by C. forskohlii hairy root cultures in various liquid media were examined. The hairy root cultures showed good growth in hormone-free Murashige and Skoog medium containing 3% (w/v) sucrose (MS medium), and Gamborg B5 medium containing 2% (w/v) sucrose (B5 medium). RA yield reached 4.0 mg (MS medium) and 4.4 mg (B5 medium) after 5 weeks of culture in a 100 ml flask containing 20 ml of each medium. Hairy root growth and RA were also investigated after treatment with various concentrations of yeast extract (YE), salicylic acid (SA) and methyl jasmonic acid (MJA). RA production in a 100 ml flask containing 20 ml B5 medium reached 5.4 mg (1.9 times more than control) with treatment of 0.01 or 1% (w/v) YE, 5.5 mg (2.0 times more than control) with treatment of 0.1 mM SA, and the maximum RA content with 9.5 mg per flask (3.4 times more than control) was obtained in the hairy roots treated with 0.1 mM MJA. These results suggest that MJA is an effective elicitor for production of RA in C. forskohlii hairy root cultures.     

Structural basis for substrate recognition in the salicylic acid carboxyl methyltransferase family

Recently, a novel family of methyltransferases was identified in plants. Some members of this newly discovered and recently characterized methyltransferase family catalyze the formation of small-molecule methyl esters using S-adenosyl-L-Met (SAM) as a methyl donor and carboxylic acid-bearing substrates as methyl acceptors. These enzymes include SAMT (SAM:salicylic acid carboxyl methyltransferase), BAMT (SAM:benzoic acid carboxyl methyltransferase), and JMT (SAM:jasmonic acid carboxyl methyltransferase). Moreover, other members of this family of plant methyltransferases have been found to catalyze the N-methylation of caffeine precursors. The 3.0-A crystal structure of Clarkia breweri SAMT in complex with the substrate salicylic acid and the demethylated product S-adenosyl-L-homocysteine reveals a protein structure that possesses a helical active site capping domain and a unique dimerization interface. In addition, the chemical determinants responsible for the selection of salicylic acid demonstrate the structural basis for facile variations of substrate selectivity among functionally characterized plant carboxyl-directed and nitrogen-directed methyltransferases and a growing set of related proteins that have yet to be examined biochemically. Using the three-dimensional structure of SAMT as a guide, we examined the substrate specificity of SAMT by site-directed mutagenesis and activity assays against 12 carboxyl-containing small molecules. Moreover, the utility of structural information for the functional characterization of this large family of plant methyltransferases was demonstrated by the discovery of an Arabidopsis methyltransferase that is specific for the carboxyl-bearing phytohormone indole-3-acetic acid.     

The role of salicylic acid in response of two rice cultivars to chilling stress

Two rice (Oryza sativa L.) cultivars differing in chilling sensitivity, Changbaijiu (chilling-tolerant) and Zhongjian (chilling-sensitive) were pre-treated with 0.5, 1.0 and 2.0 mM salicylic acid (SA) for 24 h before chilling at 5°C for 1 d. Chilling induced SA accumulation, particularly conjugated SA in both leaves and roots of the two rice cultivars. After SA administration, SA accumulated in the roots of both cultivars at a concentration-dependent manner, whereas only a slight increase was observed in their leaves. Conjugated SA accounted for most of the increase. The beneficial effect of SA treatment on protecting rice seedlings from chilling injury was not observed at any concentration in either cultivar. Pre-treatment with SA even decreased their chilling tolerance confirmed by increased electrolyte leakage and lipid peroxidation. Further, most of the activities of antioxidant enzymes decreased or remained unchanged in leaves and roots of SA pre-treated seedlings after chilling. These results implied that down-regulation of antioxidant defence might be involved in the reduction of chilling tolerance in SA-pre-treated plants.     

Influence of exogenous salicylic acid on flavonolignans and lipoxygenase activity in the hairy root cultures of Silybum marianum

Silymarin is one of the most potent antioxidant so far developed from plant sources used as hepatoprotectants. Influence of different concentrations (0, 1, 2, 4, 6 and 8 mg/50 ml culture) and exposure time (24, 48, 72, 96 and 120 h) of salicylic acid on lipoxygenase activity, linoleic acid content, growth and production of silymarin in hairy root cultures of S. marianum were investigated. Detection and identification of flavonolignans was carried out by high performance liquid chromatograph method. Salicylic acid enhanced silymarin production (1.89 mg g⁻¹ DW). The optimal feeding condition was the addition of salicylic acid (6 mg/50 ml culture) after 24 h in which the silymarin content was 2.42 times higher than the control (0.78 mg g⁻¹ DW). The content of silybin, isosilybin, silychristin, silydianin and taxifolin were 0.703, 0.017, 0.289, 0.02 and 0.863 mg g⁻¹ DW respectively in these samples, while in non-treated hairy roots were 0.027, 0.046, 0.23, 0.022 and 0.453 respectively. Lipoxygenase activity also affected by elicitation. lipoxygenase activity increased 24 h after treatment by ∼1.57- fold (0.21 Δ OD₂₃₄/mg protein min⁻¹). Upon elicitation with salicylic acid, linoleic acid content of hairy roots (38.26 mg g⁻¹ DW) were also elevated after 24 h, in which the linoleic acid content was 2.37 times higher than the control (16.1 mg g⁻¹ DW). It is feasible that elicitation with salicylic acid regulates the jasmonate pathway, which in turn mediates the elicitor-induced accumulation of silymarin.     

Investigation of liquid-solid hydrodynamic boundary layers and oxygen requirements in hairy root cultures.

Rates of oxygen uptake, growth and alkaloid production by hairy roots in submerged culture were investigated using a recirculation reactor allowing operation at high liquid velocities for removal of hydrodynamic boundary layers. Measurements were performed at dissolved oxygen tensions of 31-450% air saturation. Critical oxygen concentrations for Atropa belladonna hairy roots were above air saturation, viz. 100-125% air saturation for oxygen uptake and 150% air saturation for growth, demonstrating that these roots cultivated in reactors with air sparging are oxygen-limited. The critical oxygen tension for oxygen uptake by Solanum aviculare hairy roots was 75% air saturation. Both the specific oxygen uptake rate and specific growth rate of A. belladonna hairy roots were dependent on the mass (g dry weight) of roots present; even in the absence of boundary layers, growth did not remain exponential over the entire culture period. Cryo-scanning electron microscopy showed that hairy roots grown submerged in liquid medium were covered with thick layers of hydrated mucilage and root hairs, representing a significant additional barrier to oxygen transfer. Roots protruding out of the liquid medium showed no evidence of mucilage accumulation. The specific oxygen demand of A. belladonna root tips was 3.3-11.5 times higher than for the remainder of the roots, the ratio increasing as the dissolved oxygen tension was reduced. Specific growth rates, biomass yields from sugar, and atropine levels were maximum at around 150% air saturation, but decreased significantly with oxygen concentrations above ca. 200%.     

Species-dependent expression of the hyoscyamine 6 beta-hydroxylase gene in the pericycle.

The tropane alkaloid scopolamine is synthesized in the pericycle of branch roots in certain species of the Solanaceae. The enzyme responsible for the synthesis of scopolamine from hyoscyamine is hyoscyamine 6 beta-hydroxylase (H6H). The gene for H6H was isolated from Hyoscyamus niger. It has an exon/intron organization very similar to those for ethylene-forming enzymes, suggesting a common evolutionary origin. The 827-bp 5' flanking region of the H6H gene was fused to the beta-glucuronidase (GUS) reporter gene and transferred to three solanaceous species by Agrobacterium-mediated transformation systems: H. niger and belladonna (Atropa belladonna), which have high and low levels, respectively, of H6H mRNA in the root, and tobacco (Nicotiana tabacum), which has no endogenous H6H gene. Histochemical analysis showed that GUS expression occurred in the pericycle and at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of transgenic H. niger hairy roots, but only at the root meristem of hairy roots and plants of transgenic tobacco. In transgenic hairy roots and regenerated plants of belladonna, the root meristem was stained with GUS activity, except for a few transformants in which the vascular cylinder was also stained. These studies indicate that the cell-specific expression of the H6H gene is controlled by some genetic regulation specific to scopolamine-producing plants.     

Effect of the engineered indole pathway on accumulation of phenolic compounds in Catharanthus roseus hairy roots

Catharanthus roseus has been well-known to contain indole alkaloids effective for treatment of diverse cancers. We examined the intracellular accumulation profiles of phenolic compounds in response to ectopic overexpression of tryptophan feedback-resistant anthranilate synthase holoenzyme (ASalphabeta) in C. roseus hairy roots. Among 13 phenolic compounds measured, 6 phenolic compounds were detected in late exponential phase ASalphabeta hairy roots. Uninduced and induced ASalphabeta hairy roots accumulated up to 1.2 and 4.5 mg/g DW over a 72-h period, respectively. Upon induction, in parallel with a rapid increase in tryptophan in the first 48 h, accumulation of phenolic compounds tended to increase to a maximum level (4.5 mg/g DW) at 48 h, after which phenolic levels decreased back to the uninduced level by 72 h. Naringin was a predominant form that comprised about 72% and 36% of the total content of phenolic compounds in the uninduced and induced lines, respectively. Upon induction, accumulation of catechin drastically increased with the highest level (3.6 mg/g) occurring at 48 h, whereas that of all others except for salicylic acid showed no statistical difference. Catechin is a final product of the flavonoid pathway, and thus metabolic flux into this pathway is transiently increased by overexpression of AS. Like catechin, salicylic acid is very sensitive to induction as it began to increase to 5-fold within 4 h of induction, but unlike catechin, no significant accumulation of salicylic acid was noted after 4 h of induction. The results suggest differential regulation of this particular biosynthesis branch within the phenolic pathway.     

Salicylic acid inhibits the biosynthesis of ethylene in detached rice leaves

The effects of salicylic acid (SA) on ethylene biosynthesis in detached rice leaves were investigated. SA at pH 3.5 effectively inhibited ethylene production within 2 h of its application. It inhibited the conversion of ACC to ethylene, but did not affect the levels of ACC and conjugated ACC. Thus, the inhibitory effect of SA resulted from the inhibition of both synthesis of ACC and the conversion of ACC to ethylene.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene-forming enzyme - SA salicylic acid