Effect of yeast elicitor and salicylic acid on the fluctuation of phytohormone contents in Ti-transformed Salvia miltiorrhiza cell cultures

When Ti transformed Salvia miltiorrhiza cells werecultured in a MS-NH₄ medium (MS without ammonium nitrate, containing30 g/L sucrose) at 25 °C in darkness for 18d, the total tanshinone (cryptotanshinone and tashinone IIA)contents in cultures were 12.23 mg/L and 15.07 mg/Lfor yeast elicitor (4 g/L), and yeast elicitor plus 200mol/L salicylic acid (SA) treated cultures, respectively,whereas only trace amounts of tanshinone were detected in the control or SAtreated cells. To explore the hormonal background concerning these phenomena,endogenous phytohormones were determined using ELISA kits. We found that ABA andiPAs contents in yeast elicitor plus SA treated cell cultures were increased 2.8to 9.8-fold and 3.6 to 5.8-fold respectively, while contents of GA₁and IAA were decreased by 13.2%–56.9% and 34.8%–74.6% respectively.This suggests that higher levels of ABA and iPAs combined with lower levels ofGA₁ and IAA inhibit the growth of cells, then probably stimulate thetanshinone production.     

Characterization and expression profiling of 4-hydroxyphenylpyruvate dioxygenase gene (<Emphasis Type="Italic">Smhppd</Emphasis>) from <Emphasis Type="Italic">Salvia</Emphasis> <Emphasis Type="Italic">miltiorrhiza</Emphasis> hairy root cultures

A novel 4-hydroxyphenylpyruvate dioxygenase gene (designated as Smhppd) was cloned from hairy roots of Salvia miltiorrhiza Bung. The full-length cDNA of Smhppd was 1,736 bp long with an ORF (open reading frame) that putatively encoded a polypeptide of 481 amino acids, with a predicted molecular mass of 52.54 kDa. The deduced amino acid sequence of the Smhppd gene shared high homology with other known HPPDs. Analysis of Smhppd genomic DNA revealed that it contained two exons and one intron. The analysis of Smhppd promoter region was also presented. Southern-blot analysis revealed that the Smhppd was a low-copy gene in S. miltiorrhiza. Real-time quantitative PCR analysis indicated that Smhppd was constitutively expressed in roots, stems and leaves of S. miltiorrhiza, with the high expression in roots. In addition, Smhppd expreession level under different stress condition was also analyzed during the hairy root culture period, including signaling components for plant defence responses, such as methyl jasmonate and salicylic acid, as well as an abiotic elicitor, Ag⁺ and a biotic elicitor, yeast extract. This study will enable us to further understand the role Smhppd plays in the synthesis of active pharmaceutical compounds in S. miltiorrhiza at molecular level.     

RNAi-mediated suppression of the phenylalanine ammonia-lyase gene in Salvia miltiorrhiza causes abnormal phenotypes and a reduction in rosmarinic acid biosynthesis

Medicinal Salvia miltiorrhiza contains two main groups of active pharmaceutical ingredients: lipid-soluble tanshinones and water-soluble phenolic acids, including rosmarinic acid and salvianolic acid B. Phenylalanine ammonia-lyase (PAL) catalyzes the first step in the phenylpropanoid pathway and is assumed to be closely related to the accumulation of rosmarinic acid and its derivatives. We selected a 217-bp fragment, located at the 3′ end of the coding region of PAL1, to establish an RNA interference construct that was introduced into S. miltiorrhiza via Agrobacterium tumefaciens-mediated transformation. PAL-suppressed plants exhibited several unusual phenotypes such as stunted growth, delayed root formation, altered leaves, and reduced lignin deposition. The total phenolic content was decreased by 20–70% in PAL-suppressed lines, and was accompanied by lower PAL activity. Down-regulation of PAL also affected the expression of C4H, 4CL2, and TAT, which are related genes in the rosmarinic acid pathway. Moreover, rosmarinic acid and salvianolic acid B were markedly reduced in PAL-suppressed lines, as detected by HPLC analysis. Our results indicate that PAL is very important for the synthesis of major water-soluble pharmaceutical ingredients within S. miltiorrhiza.     

Induction and potentiation of diterpenoid tanshinone accumulation in Salvia miltiorrhiza hairy roots by β-aminobutyric acid

The non-protein amino acid -aminobutyric acid (BABA) is a proven inducer of plant defense against pathogens. This work examines its effect on the production of diterpenoid tanshinones in Salvia miltiorrhiza hairy root cultures, both separately and in combination with a yeast elicitor (YE, the carbohydrate fraction of yeast extract). In the absence of YE, BABA at 0.1, 1 and 2 mM caused a dose-dependent enhancement of tanshinone accumulation, with up to a 4.5-fold increase (from 0.24 to 1.09 mg/g DW) in total content of three major tanshinones (cryptotanshinone, tanshinone I and tanshinone IIA) in the hairy roots. The combination of BABA with YE treatment further enhanced tanshinone production, but only when the BABA treatment was applied to the culture a few days before the YE treatment. Compared with methyl jasmonate, BABA was more effective in enhancing tanshinone production. A 3-day pretreatment with 1 mM BABA followed by YE-treatment, increased the total tanshinone content of roots by 9.4 times to 2.26 mg/g cells, and the volumetric tanshinone yield of culture by 6.3 times (from 3.2 to 20.1 mg/l). The results suggest that BABA can strongly potentiate elicitor-induced secondary metabolism in plant tissue cultures.     

Effects of methyl jasmonate and salicylic acid on cell growth and cryptotanshinone formation in Ti transformed Salvia miltiorrhiza cell suspension cultures

Exogenous methyl jasmonate (MJ) or salicylic acid (SA) when applied alone failed to induce cryptotanshinone (a phytoalexin) formation in Ti-transformed Salvia miltiorrhiza cell suspension cultures. However, when applied in combination with yeast elicitor, SA at 50–500 M enhanced the yeast elicitor-induced cryptotanshinone formation while MJ reduced the yeast elicitor-induced cryptotanshinone formation. Ibuprofen at 100 M did not inhibit the yeast elicitor-induced cryptotanshinone formation. DMSO was superior to ethanol as a solvent for introducing MJ to the cell cultures.     

Induction of phytoalexin formation in crown gall and hairy root culture of Salvia miltiorrhiza by methyl viologen

Methyl viologen (MV) (20–150 M), a generator of superoxide anion (O₂ ^–), but not hydrogen peroxide (H₂O₂) (10 M–2 mM) triggered the formation of cryptotanshinone (a phytoalexin) in cultures of both crown galls and hairy roots of Salvia miltiorrhiza. MV also inhibited the biomass formation and decreased the contents of phenolic acids in both cultures whereas H₂O₂ did not. In addition, MV and yeast elicitor induced cryptotanshinone formation synergistically only in crown gall cultures. Treatment of the cultures with 3.3 M diphenylene iodonium, an inhibitor of NAD(P)H oxidase, did not exhibit any detrimental effect on the yeast elicitor-induced cryptotanshinone formation in hairy root cultures whereas 1 M diphenylene iodonium was inhibitory on yeast elicitor-induced cryptotanshinone formation in crown gall cultures.     

Elicitors and priming agents initiate plant defense responses

Biotic elicitors produced by plant pathogens or herbivore pests rapidly activate a range of plant chemical defenses when translocated to plant tissue. The fatty acid conjugate volicitin has proven to be a robust elicitor model for studying herbivore-induced plant defense responses. Here we review the role of insect-derived volicitin (N-[17-hydroxylinolenoyl]-L-glutamine) as an authentic elicitor of defense responses, specifically as an activator of signal volatiles that attract natural enemies of herbivore pests. Comparisons are drawn between volicitin as an elicitor of plant defenses and two other classes of signaling molecules, C₆ green-leaf volatiles and C₄ bacterial volatiles that appear to prime plant defenses thereby enhancing the capacity to mobilize cellular defense responses when a plant is faced with herbivore or pathogen attack.     

Tanshinones extend chronological lifespan in budding yeast Saccharomyces cerevisiae

Natural products with anti-aging property have drawn great attention recently but examples of such compounds are exceedingly scarce. By applying a high-throughput assay based on yeast chronological lifespan measurement, we screened the anti-aging activity of 144 botanical materials and found that dried roots of Salvia miltiorrhiza Bunge have significant anti-aging activity. Tanshinones isolated from the plant including cryptotanshione, tanshinone I, and tanshinone IIa, are the active components. Among them, cryptotanshinone can greatly extend the budding yeast Saccharomyces cerevisiae chronological lifespan (up to 2.5 times) in a dose- and the-time-of-addition-dependent manner at nanomolar concentrations without disruption of cell growth. We demonstrate that cryptotanshinone prolong chronological lifespan via a nutrient-dependent regime, especially essential amino acid sensing, and three conserved protein kinases Tor1, Sch9, and Gcn2 are required for cryptotanshinone-induced lifespan extension. In addition, cryptotanshinone significantly increases the lifespan of SOD2-deleted mutants. Altogether, those data suggest that cryptotanshinone might be involved in the regulation of, Tor1, Sch9, Gcn2, and Sod2, these highly conserved longevity proteins modulated by nutrients from yeast to humans.     

Fungal elicitor-induced retardation and its restoration of root growth in tobacco seedlings

In this study, we investigated responses of growing and intact tobacco (N. tabacum cv Xanthi) seedlings to a fungal elicitor, a xylanase from Trichoderma viride (TvX). In addition to the induction of defense gene expression, TvX treatment caused the retardation of growth of seedlings. In the TvX-treated seedlings, growth of primary roots was markedly reduced through repression of cell division and longitudinal cell elongation in a meristematic zone and an elongation zone, respectively. However, cell differentiation to form vascular bundles and root hairs continued. In the TvX-treated root cap, disappearance of starch granules in columella cells and aggregation of border cells were observed. Furthermore, the TvX-induced growth retardation was restored after removal of the elicitor, resulting in a plastic alteration of root architecture. Therefore, the fungal elicitor might act as an environmental cue that regulates root growth and development as well as the ordinary defense responses in plant seedlings. These findings suggest a novel aspect of plant growth regulation via a plant–microbe interaction in the rhizosphere.     

MAP kinase cascades in elicitor signal transduction

 Protein kinases play important roles in elicitor signal transduction. In this article, I describe the current view of the role of mitogen-activated protein kinase (MAPK) cascades in elicitor signal transduction of plant cells based on our own research and recent developments in this field. In the past several years, it has become apparent that MAPK cascades play important roles in elicitor signal transduction in plants. Our early studies demonstrated the identification of p47 MAPK in tobacco as an elicitor-responsive protein kinase and possible involvement of p47 MAPK in elicitor signal transduction to induce defense responses, including defense gene expression and hypersensitive cell death. However, the molecular identity of p47 MAPK is still unclear. Recent important studies suggest that tobacco MAPK cascades that include SIPK, and/or WIPK, and NtMEK2, an upstream kinase for both SIPK and WIPK, have a crucial function in induction of defense responses and hypersensitive cell death. The orthologs of these protein kinases in Arabidopsis and alfalfa are also suggested to have similar functions. Furthermore, the identification of loss-of-function mutation in Arabidopsis reveals a negative regulatory role for putative MAPK cascades in plant defense mechanisms. Received: February 7, 2002 / Accepted: February 25, 2002     

BAK1 is required for the attenuation of ethylene‐inducing xylanase (Eix)‐induced defense responses by the decoy receptor LeEix1

Elicitor recognition plays a key role in the reaction of plants to pathogens and the induction of plant defense responses. Furthermore, plant–microbe interactions involve numerous regulatory systems essential for plant defense against pathogens. Ethylene‐inducing xylanase (Eix) is a potent elicitor of plant defense responses in specific cultivars of tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum). The Eix receptors (LeEix1 and LeEix2) belong to a superclade of leucine‐rich repeat receptor‐like proteins (RLP) with a signal for receptor‐mediated endocytosis, which was shown to be essential for proper induction of defense responses. Both receptors are able to bind Eix, while only LeEix2 mediates defense responses. Here we demonstrate that LeEix1 heterodimerizes with LeEix2 upon application of the Eix elicitor. We show that LeEix1 attenuates Eix‐induced internalization and signaling of the LeEix2 receptor. Furthermore, we demonstrate, using yeast two‐hybrid and in planta bimolecular fluorescence complementation assays, that the brassinosteroid co‐receptor, BAK1, binds LeEix1 but not LeEix2. In BAK1‐silenced plants, LeEix1 was no longer able to attenuate plant responses to Eix, indicating that BAK1 is required for this attenuation. We suggest that LeEix1 functions as a decoy receptor for LeEix2, a function which requires BAK1.     

The effect of yeast elicitor on the growth and secondary metabolism of hairy root cultures of Salvia miltiorrhiza

Hairy root cultures of Salvia miltiorrhiza transformed with Agrobacterium rhizogenes ATCC 15834 produced a tiny amount of tanshinones and a constituent level of phenolic acids under normal growth conditions. Upon elicitation with yeast elicitor, the production of both phenolic acids and tanshinones was enhanced. For example, the contents of two phenolic acids, rosmarinic acid and lithospermic acid B were elevated from 1.24% and 2.59% to 2.89% and 2.98% of dry wt, respectively while the intracellular content of cryptotanshinone increased from 0.001% to as much as 0.096% of dry wt. Yeast elicitor also improved the growth of hairy roots (from 3.9 g/l to 7.3 g/l on a dry wt basis). Liquid chromatography-mass spectrometry (LC-MS) was developed for simultaneous detection and identification of phenolic acids and tanshinones in the extracts of S. miltiorrhiza. Rosmarinic acid, lithospermic acid B, cryptotanshinone, tanshinone I, tanshinone IIA and tanshinone IIB were identified by comparison with standards available. Dihydrotanshinone I and methylenetanshiquinone were tentatively identified by the molecular weights and the elution comparable with the literature. An unknown compound with a molecular weight of 280 was found in yeast-elicitor treated hairy root cultures, which was one of the major tanshinones induced.     

The phenylalanine ammonia-lyase gene family in Salvia miltiorrhiza: genome-wide characterization, molecular cloning and expression analysis

Salvia miltiorrhiza Bunge is a well-known material of traditional Chinese medicine. Hydrophilic phenolic acids, such as rosmarinic acid and salvianolic acid B, are a group of pharmaceutically important compounds in S. miltiorrhiza. The biosynthesis of rosmarinic acid requires the coordination of the phenylpropanoid pathway and the tyrosine-derived pathway. Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenylpropanoid pathway. Systematic analysis of the SmPAL gene family has not been carried out. We report here the identification of three SmPALs through searching the recently obtained working draft of the S. miltiorrhiza genome and full-length cDNA cloning. Bioinformatic and phylogenetic analyses showed that SmPAL1 and SmPAL3 clustered in a sub-clade of dicot PALs, whereas SmPAL2 fell into the other one. Some important cis-elements were conserved in three SmPAL promoters, whereas the others were not. SmPAL1 and SmPAL3 were highly expressed in roots and leaves of S. miltiorrhiza, but SmPAL2 were predominately expressed in stems and flowers. It indicates that SmPAL1 and SmPAL3 function redundantly in rosmarinic acid biosynthesis. All SmPALs were induced in roots treated with PEG and MeJA, but the time and degree of responses were different, suggesting the complexity of SmPAL-associated metabolic network in S. miltiorrhiza. This is the first comprehensive study dedicated to SmPAL gene family characterization. The results provide a basis for elucidating the role of SmPAL genes in the biosynthesis of bioactive compounds.     

Development of a Colorimetric Assay for Determining the Amount of H2O2 Generated in Tobacco Cells in Response to Elicitors and Its Application to Study of the Structure-Activity Relationship of Flagellin-Derived Peptides

Plants exhibit various defense responses after recognizing elicitor molecules derived from pathogenic microorganisms and host plants. In this study, we developed an improved colorimetric assay for quantifying the generation of H₂O₂ in plant cells, one of the defense responses, to evaluate elicitor activity quantitatively. H₂O₂ is detected using a dye, N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)-diphenylamine sodium salt (DA-64), which can be measured by conventional spectrometers in a highly sensitive and quantitative manner. Using this method, we successfully measured the elicitor activity of flagellin-derived peptides in cultured tobacco cells, and identified several structural features of the peptides important for the elicitor activity. The results suggest that the structural factors required for expression of the elicitor activity differ slightly among plant species. The efficient and sensitive assay developed in this study should be useful not only for studying structure-activity relationships, but also for the screening of novel compounds that can induce defense responses.     

Roles of reactive oxygen species in methyl jasmonate and nitric oxide-induced tanshinone production in Salvia miltiorrhiza hairy roots

Salvia miltiorrhiza is one of the most popular traditional Chinese medicinal plants for treatment of coronary heart disease. Tanshinones are the main biological active compounds in S. miltiorrhiza. In this study, effects of exogenous methyl jasmonate (MJ) and nitric oxide (NO) on tanshinone production in S. miltiorrhiza hairy roots were investigated and the roles of reactive oxygen species (ROS) in MJ and NO-induced tanshinone production were elucidated further. The results showed that contents of four tanshinone compounds were significantly increased by 100 μM MJ when compared to the control. Application of 100 μM sodium nitroprusside (SNP), a donor of NO, also resulted in a significant increase of tanshinone production. Expression of two key genes encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) was up-regulated by MJ and SNP. Generations of O₂ ⁻ and H₂O₂ were triggered by MJ, but not by SNP. The increase of tanshinone production and up-regulation of HMGR and DXR expression induced by MJ were significantly inhibited by ROS scavengers, superoxide dismutase (SOD) and catalase (CAT). However, neither SOD nor CAT was able to suppress the SNP-induced increase of tanshinone production and expression of HMGR and DXR gene. In conclusion, tanshinone production was significantly stimulated by MJ and SNP. Of four tanshinone compounds, cryptotanshinone accumulation was most affected by MJ elicitation, while cryptotanshinone and tanshinone IIA accumulation was more affected by SNP elicitation. ROS mediated MJ-induced tanshinone production, but SNP-induced tanshinone production was ROS independent.     

Effects of biotic and abiotic elicitors on cell growth and tanshinone accumulation in Salvia miltiorrhiza cell cultures

This study examined the effects of biotic and abiotic elicitors on the production of diterpenoid tanshinones in Salvia miltiorrhiza cell culture. Four classes of elicitors were tested, heavy metal ions (Co²⁺, Ag⁺, Cd²⁺), polysaccharides (yeast extract and chitosan), plant response-signaling compounds (salicylic acid and methyl jasmonate), and hyperosmotic stress (with sorbitol). Of these, Ag (silver nitrate), Cd (cadmium chloride), and polysaccharide from yeast extract (YE) were most effective to stimulate the tanshinone production, increasing the total tanshinone content of cell by more than ten-fold (2.3 mg g^-1 versus 0.2 mg g^-1 in control). The stimulating effect was concentration-dependent, most significant at 25 μM of Ag and Cd and 100 mg l^-1 (carbohydrate content) of YE. Of the three tanshinones detected, cryptotanshinone was stimulated most dramatically by about 30-fold and tanshinones I and IIA by no more than 5-fold. Meanwhile, most of the elicitors suppressed cell growth, decreasing the biomass yield by about 50% (5.1–5.5 g l^-1 versus 8.9 g l^-1 in control). The elicitors also stimulated the phenylalanine ammonia lyase activity of cells and transient increases in the medium pH and conductivity. The results suggest that the elicitor-stimulated tanshinone accumulation was a stress response of the cells.     

Elicitation of 2,3-Dihydroxybenzoic Acid and Ajmalicine in a Catharanthus roseus Suspension Culture*

Addition of an elicitor preparation from cell walls of Phytophthora megasperma f. sp. glycinea (Pmg elicitor) to a newly established cell suspension culture of Catharanthus roseus induced extracellular free 2,3-dihydroxybenzoic acid, suggesting its role in pathogen defense. The same substance also accumulated intracellularly in a bound form. Treatment of the crude Pmg elicitor preparation with trypsin abolished elicitor activity, suggesting that the active fraction is proteinaceous. The cells became more sensitive to low but not to elevated elicitor concentrations when they were pretreated with 2,6-dichloroisonicotinic (DCIA) or 5-chlorosalicylic (5CSA) acid for about 1 day before addition of the elicitor. This indicates that the elicitor reception/transduction system becomes improved by these compounds known to be related to systemic acquired resistance against plant pathogens. The newly established cell line initially accumulated also the indole alkaloid ajmalicine, a process enhanced by Pmg elicitor. This potency was lost during subculturing for about 1 year and was also not restored by preincubation with DCIA or 5CSA. In contrast, elicitation of 2,3-dihydroxybenzoic acid synthesis was undiminished, suggesting that the Pmg elicitor perception system was still functioning and not the cause for the decline in elicited indole alkaloid production.     

Fungal elicitor preparations and methyl jasmonate enhance rosmarinic acid accumulation in suspension cultures of Coleus blumei

bstract Suspension cultures of Coleus blumei (Lamiaceae) treated with either an elicitor preparation from the culture medium of the phytopathogenic oomycete Pythium aphanidermatum or with methyl jasmonate enhanced accumulation of rosmarinic acid approximately threefold. The specific activities of phenylalanine ammonia lyase and rosmarinic acid synthase were also enhanced after addition of the fungal elicitor. The addition of methyl jasmonate transiently increased activities of phenylalanine ammonia lyase and hydroxyphenylpyruvate reductase, whereas the activity of rosmarinic acid synthase was not stimulated and the activity of tyrosine aminotransferase was slightly and constantly enhanced. Methyl jasmonate stimulated rosmarinic acid accumulation not only when added directly to the culture medium, but also when it could reach the cells only via the gas phase. Received: 2 April 1997 / Revision received:16 June 1997 / Accepted: 15 September 1997