Sterol and sesquiterpenoid biosynthesis during a growth cycle of tobacco cell suspension cultures

The accumulation and biosynthesis of sterols and fungal elicitor-inducible sesquiterpenoids by tobacco (Nicotiana tabacum) cell suspension cultures were examined as a function of a 10 day culture cycle. Sterols accumulated concomitantly with fresh weight gain. The rate of sterol biosynthesis, measured as the incorporation rate of [¹⁴C]acetate and [³H]mevalonate, was maximal when the cultures entered into their rapid phase of growth. Changes in squalene synthetase enzyme activity correlated more closely with thein vivo synthesis rate and accumulation of sterols than 3-hydroxy-3-methylglutaryl CoA reductase (HMGR) enzyme activity. Cell cultures entering into the rapid phase of growth also responded maximally to fungal elicitor as measured by the production of capsidiol, an extracellular sesquiterpenoid. However, the rate of sesquiterpenoid biosynthesis, measured as the incorporation rate of [¹⁴C]acetate and [³H]mevalonate, could not be correlated with elicitor-inducible HMGR or sesquiterpene cyclase enzyme activities, nor elicitor-suppressible squalene synthetase enzyme activity.Abbreviations FPP farnesyl diphosphate - HMGR 3-hydroxy-3-methylglutaryl coenzyme A reductase     

Involvement of de Novo Protein Synthesis,Protein Kinase,Extracellular Ca2+, and Lipoxygenase in Arachidonic Acid Induction of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Genes and Isoprenoid Accumulation in Potato (Solanum tuberosum L.)

A series of inhibitors were tested to determine the participation of de novo protein synthesis, protein kinase activity, extracellular Ca2+, and lipoxygenase activity in arachidonic acid elicitation of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) gene expression and sesquiterpene phytoalexin biosynthesis in potato (Solanum tuberosum L. cv Kennebec). Gene-specific probes were used to discriminate effects on the expression of two HMGR genes (hmg1 and hmg2) that respond differentially in tuber tissue following wounding or elicitor treatment. Inhibition of protein synthesis with cycloheximide completely blocked arachidonate-induced hypersensitive necrosis and browning, including HMGR gene induction and phytoalexin accumulation. This suggests that proteins necessary for coupling arachidonic acid reception to HMGR mRNA accumulation are either rapidly turned over or not present constitutively and are induced following elicitor treatment. Staurosporin, a potent inhibitor of protein kinases, and ethyleneglycol-bis([beta]-aminoethyl ether)-N,N[prime]-tetraacetic acid, a Ca2+ chelator, inhibited arachidonate-induction of hmg2 gene expression and phytoalexin accumulation but did not inhibit the wound-induced expression of hmg1. However, staurosporin inhibited arachidonate's suppression of hmg1 gene expression. Eicosatetraynoic acid, a lipoxygenase inhibitor that suppresses elicitor-induced phytoalexin accumulation, also inhibited arachidonate's suppression of hmg1 and induction of hmg2. The results indicate that arachidonate's suppression of hmg1 and activation of hmg2 depend on a common intermediate or set of intermediates whose generation is sensitive to the inhibitors tested.     

Inhibition of elicitor-induced phytoalexin formation in cotton and soybean cells by citrate

Addition of an elicitor preparation from Verticillium dahliae to soybean or cotton cell suspension cultures induces the formation of the phytoalexins, glycelollin or sesquiterpene aldehydes, respectively. Recent work (PS Low, PF Heinstein 1986 Arch Biochem Biophys 249: 472-479) has shown that the induction of phytoalexin biosynthesis in these cells is preceded by rapid changes in the plant cell membrane which can be conveniently monitored by membrane associated fluorescent probes. Using this elicitation assay, we have found that citrate, a common metabolite of higher plants, acts as a potent inhibitor of elicitation when added prior to treatment with elicitor. The citrate concentration required to obtain a 50% inhibition of the elicitor-induced fluorescence transition in cultured cotton cells was found to be about 2 millimolar, while the concentration of citrate observed to inhibit elicitor-induced sesquiterpene aldehyde formation in the same cell suspensions was also 2 millimolar. Curiously, in the presence of elicitor, citrate at less than ID₅₀ concentrations increased cell mass accumulation significantly above control incubations without elicitor. A similar inhibition of glyceollin formation with an increase in cell mass accumulation was also observed upon addition of 1 to 5 millimolar citrate to soybean cell suspension cultures. The physiological significance of the inhibition by citrate of phytoalexin formation in plant cell suspensions was supported by the observation that a similar inhibition of sesquiterpene aldehyde formation occurs in cotton plantlets elicited by cold shock or V. dahliae stress. The specificity of citrate as an inhibitor of phytoalexin formation was demonstrated by data showing that other di- and tricarboxylic-hydroxy acids did not inhibit, with the exception of malate which inhibited phytoalexin formation in soybean cells with roughly half the potency of citrate. These experiments not only demonstrate that citrate can act as a specific inhibitor of elicitation, but they further confirm the validity of monitoring elicitation and its modulation with fluorescent probes.     

Induction of sesquiterpenoid biosynthesis in tobacco cell suspension cultures by fungal elicitor

Large amounts of the sesquiterpenoid capsidiol accumulated in the media of tobacco (Nicotiana tabacum L. cv KY14) cell suspension cultures upon addition of fungal elicitor. Capsidiol accumulation was proportional to the amount of elicitor added. The accumulation of capsidiol was preceded by a transient increase in the capsidiol de novo synthesis rate as measured by the incorporation of exogenous [¹⁴C]acetate. Changes in 3-hydroxy-3-methylglutaryl-CoA reductase activity (HMGR; EC 1.1.1.34), an enzyme of general isoprenoid metabolism, paralleled the changes in [¹⁴C]acetate incorporation into capsidiol. Incubation of the cell cultures with mevinolin, a potent in vitro inhibitor of the tobacco HMGR enzyme activity, inhibited the elicitor-induced capsidiol accumulation in a concentration dependent manner. [¹⁴C]Acetate incorporation into capsidiol was likewise inhibited by mevinolin treatment. Unexpectedly, [³H] mevalonate incorporation into capsidiol was also partially inhibited by mevinolin, suggesting that mevinolin may effect secondary sites of sesquiterpenoid biosynthesis in vivo beyond HMGR. The data indicated the importance of the induced HMGR activity for capsidiol production in elicitor-treated tobacco cell suspension cultures.     

Rapid induction by fungal elicitor of the synthesis of cinnamyl-alcohol dehydrogenase, a specific enzyme of lignin synthesis

A fivefold increase in the extractable activity of cinnamyl-alcohol dehydrogenase, an enzyme of phenylpropanoid metabolism specific for lignin synthesis, was observed within 10 h of treatment of cell-suspension cultures of bean (Phaseolus vulgaris L.) with a high-molecular-mass elicitor preparation heat-released from mycelial cell walls of the bean pathogen Colletotrichum lindemuthianum. Elicitor caused a rapid, marked but transient increase in the synthesis of cinnamyl-alcohol dehydrogenase with maximum rates 2-3 h after elicitation, concomitant with the phase of rapid increase in enzyme activity. There is a close correspondence between increased polysomal mRNA activity encoding cinnamyl-alcohol dehydrogenase, as measured by incorporation of [35S]methionine into immunoprecipitable enzyme subunits in vitro, and the stimulation of enzyme synthesis in vivo in response to elicitor. This marked increase in polysomal mRNA activity represents an increase as a proportion of total cellular mRNA activity, indicating that elicitor does not stimulate synthesis of this enzyme by selective recruitment from the total pool of cellular mRNA. Elicitor stimulation of cinnamyl-alcohol dehydrogenase activity and enzyme synthesis is more rapid than previously observed for other proteins involved inducible defense mechanisms, such as enzymes of phytoalexin biosynthesis or the apoproteins of cell-wall hydroxyproline-rich glycoproteins.     

Multiple signalling pathways mediate fungal elicitor-induced beta-thujaplicin biosynthesis in Cupressus lusitanica cell cultures

The biosynthesis of a phytoalexin, beta-thujaplicin, in Cupressus lusitanica cell cultures can be stimulated by a yeast elicitor, H(2)O(2), or methyl jasmonate. Lipoxygenase activity was also stimulated by these treatments, suggesting that the oxidative burst and jasmonate pathway may mediate the elicitor-induced accumulation of beta-thujaplicin. The elicitor signalling pathway involved in beta-thujaplicin induction was further investigated using pharmacological and biochemical approaches. Treatment of the cells with calcium ionophore A23187 alone stimulated the production of beta-thujaplicin. A23187 also enhanced the elicitor-induced production of beta-thujaplicin. EGTA, LaCl(3), and verapamil pretreatments partially blocked A23187- or yeast elicitor-induced accumulation of beta-thujaplicin. These results suggest that Ca(2+) influx is required for elicitor-induced production of beta-thujaplicin. Treatment of cell cultures with mastoparan, melittin or cholera toxin alone or in combination with the elicitor stimulated the production of beta-thujaplicin or enhanced the elicitor-induced production of beta-thujaplicin. The G-protein inhibitor suramin inhibited the elicitor-induced production of beta-thujaplicin, suggesting that receptor-coupled G-proteins are likely to be involved in the elicitor-induced biosynthesis of beta-thujaplicin. Indeed, both GTP-binding activity and GTPase activity of the plasma membrane were stimulated by elicitor, and suramin and cholera toxin affected G-protein activities. In addition, all inhibitors of G-proteins and Ca(2+) flux suppressed elicitor-induced increases in lipoxygenase activity whereas activators of G-proteins and the Ca(2+) signalling pathway increased lipoxygenase activity. These observations suggest that Ca(2+) and G-proteins may mediate elicitor signals to the jasmonate pathway, and the jasmonate signalling pathway may then lead to the production of beta-thujaplicin.     

Induction of sesquiterpene cyclase and suppression of squalene synthetase activities in plant cell cultures treated with fungal elicitor

Addition of elicitor, cell wall fragments of the fungus Phytophthora parasitica, to tobacco cell suspension cultures (Nicotiana tabacum) resulted in the rapid synthesis and secretion of large amounts of antibiotic sesquiterpenoids. Pulse-labeling experiments with [¹⁴C]acetate and [³H] mevalonate demonstrated that the induction of sesquiterpenoid biosynthesis, maximal by 6 to 9 hours after elicitor addition to the cell cultures, was paralleled by a rapid and large decline in the incorporation rate of radioactivity into sterols. Consequently, sterol accumulation was also inhibited upon addition of elicitor to the cell cultures. Sesquiterpene cyclase activity was absent from control cell cultures but induced to a maximum within 10 hours of elicitor addition to the cell cultures. The cyclase activity remained elevated for an additional 30 hours before declining. In contrast, squalene synthetase activity was suppressed to less than 15% of that found in control cells within 7 hours of elicitor addition. Our results suggest that the channeling of isoprenoid intermediates, and especially farnesyl diphosphate, into sesquiterpenoids occurred by a coordinated increase in the sesquiterpene cyclase and a decrease in the squalene synthetase enzyme activities. A reexamination of the data pertaining to the transient induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity (EC 1.1.1.34) in elicitor-treated cells suggested that, while the reductase activity was necessary for sesquiterpenoid biosynthesis, it functioned more to maintain a sufficient level of intermediates between mevalonate and farnesyl diphosphate rather than as a rate limiting step controlling the synthesis rate of any one class of isoprenoids.     

Biosynthesis of the sesquiterpenic phytoalexin capsidiol in elicited root cultures of chili pepper (Capsicum annuum)

The biosynthesis of the sesquiterpenic phytoalexin capsidiol was investigated using in vitro root cultures of chili pepper (Capsicum annuum) elicited with cellulase. Optimal concentrations of cellulase and sucrose for capsidiol production were established. A simple spectrophotometric procedure to quantify capsidiol was improved. Monoclonal antibodies against a tobacco sesquiterpene cyclase were used to detect a similar protein in pepper root extracts. We found that capsidiol was secreted to the medium and the maximal production was achieved at 24 h after elicitation. In contrast, the maximal amount of the elicitor inducible sesquiterpene cyclase was found between 6 and 8 h. Addition of small amounts of polyvinylpyrrolidone was necessary for sesquiterpene cyclase enzyme activity assays.Abbreviations AP alkaline phosphatase - BCIP 5-bromo-4-chloro-3-indolylphosphate - DMF dimethyl-formamide - FPP farnesyl pyrophosphate - MAb monoclonal antibodies - NBT nitro blue tetrazolium - PVP polyvinylpyrrolidone - SC sesquiterpene cyclase     

Purification and characterization of an inducible sesquiterpene cyclase from elicitor-treated tobacco cell suspension cultures

An elicitor-inducible sesquiterpene cyclase, which catalyzes the conversion of farnesyl diphosphate to 5-epi-aristolochene (IM Whitehead, DR Threlfall, DF Ewing [1989] Phytochemistry 28:775-779) and representing a committed step in the phytoalexin biosynthetic pathway in tobacco, was purified by a combination of hydrophobic interaction, anion exchange, hydroxylapatite, and chromatofocusing chromatography. From 2 kilograms of elicited tobacco (Nicotiana tabacum) cells, approximately 500 micrograms of cyclase protein was purified, representing greater than a 130-fold increase in the specific activity of the enzyme and a 4% recovery of the starting activity. The purified enzyme resolved as two major polypeptides of 60 and 62 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Biochemical characterization of the enzyme activity included an absolute requirement for magnesium, an isoelectric point of 4.5 to 4.9, and a K_m for farnesyl diphosphate of 2 to 5 micromolar. The purified cyclase protein was used to generate mouse polyclonal antibodies which efficiently inhibited cyclase activity in an in vitro assay. Electrophoresis of extracts from elicitor-treated cells or purified cyclase enzyme on native polyacrylamide gels separated the cyclase enzyme into four polypeptides as shown by immunoblot analysis using poly- and monoclonal antibodies. Proportionate cyclase enzyme activity comigrated with those polypeptides. No cyclase polypeptides were detectable in extracts of control cells by immunoblot analysis. However, immunoblot analysis of proteins from elicitor-treated cells using four independent monoclonal antibody lines and the polyclonal antibodies detected the same polypeptides, regardless of whether the proteins were separated by native or SDS-PAGE. The results suggest an induction of multiple cyclase polypeptides in elicitor-treated cells resulting from either the expression of multiple genes or multiple post-translational processing events.     

Methylation reactions and the phytoalexin response in alfalfa suspension cultures

 In order to determine why the activated methyl cycle is up-regulated in plants undergoing defence responses to fungal pathogens we have monitored the utilisation of methyl groups derived from methionine in cell-suspension cultures of alfalfa (Medicago sativa L.) treated for various times with fungal elicitor, by carrying out a parallel labelling study with [³⁵S]methionine and [methyl-³H]methionine. The distribution of the two radiolabels among the medium, soluble cellular components and cell wall was then determined. In the absence of elicitor the utilisation of the two radiolabels was similar. However, in the presence of the elicitor the total incorporation of radioactivity from [methyl-³H]methionine into metabolites was far greater than from [³⁵S]methionine, indicating that the methyl label had been utilised in methylation reactions. Elicitor treatment resulted in up to a sixfold increase in the use of ³H-methyl groups in the methylation of hydrophobic metabolites. In the period 0–24 h after elicitor treatment, increased methylation was directed largely into the synthesis of the isoflavonoid phytoalexin medicarpin and related metabolites. Newly synthesized phytoalexins were exported into the medium, while a significant proportion of the medicarpin accumulating in the cell in the early stages of elicitation was derived from the hydrolysis of its respective conjugate. Elicitor treatment also modified the incorporation of ³H-methyl groups into the cell wall. Between 0 and 24 h after elicitor treatment the methylation of pectin in the cell wall declined. After 24 h, pectin methylation recovered and was associated with an increase in the methylation of other wall-bound polysaccharide components. Since no other major metabolic sink for the increased methylation was determined we conclude that the increased activity of the activated methyl cycle during defence interactions in alfalfa is required to support phytoalexin synthesis and cell wall modifications. Received: 1 August 1996 / Accepted: 24 October 1996     

Elicitor-induced activation of phospholipases plays an important role for the induction of defense responses in suspension-cultured rice cells

Biphasic generation of reactive oxygen species (ROS) induced by N-acetylchitooligosaccharide elicitor in rice cells was associated with the activation of phopholipase C (PLC) and phospholipase D (PLD). The activation of both enzymes was observed for the first phase of ROS generation, but only the activation of PLD was evident for the second response. Activation of PLD was associated with its recruitment to the membrane. Enzymatic products of these phospholipases, diacylglycerol (DG) and phosphatidic acid (PA), could induce ROS generation by themselves. Moreover, the addition of these lipids compensated the inhibition of the second phase of ROS generation by cycloheximide, indicating the involvement of the synthesis of PLD or related proteins in the second phase of ROS generation. DG and PA also induced the expression of elicitor-responsive genes in the absence of the elicitor. They could not induce phytoalexin biosynthesis by themselves but greatly enhanced the elicitor-induced phytoalexin accumulation. Further, the inhibition of PLD by 1-butanol inhibited the elicitor-induced phytoalexin accumulation, indicating the involvement of PLD and its reaction product, PA, in the induction of phytoalexin biosynthesis. These results indicated the importance of phospholipid signaling, especially by PLD and its product PA, in plant defense responses.     

The Lipoxygenase Pathway is Involved in Elicitor-induced Phytoalexin Accumulation in Plane Tree (Platanus acerifolia) Cell-suspension Cultures

The Ceratocystis fimbriata f.sp. platani 66 kDa glycoprotein elicitor-induced secretion of soluble coumarins by plane tree (Platanus acerifolia (Aiton) Wild) cell-suspension cultures was investigated by studying the possible involvement of the octadecanoid pathway in the cell response. When cell-suspension cultures were treated with the glycoprotein elicitor, the cells exhibited a rapid and transient increase in lipoxygenase activity, in synthesis of endogenous jasmonic acid prior to the accumulation of coumarin phytoalexins. The treatment of cells with an inhibitor of lipoxygenase (ETYA) before elicitor addition, drastically reduced the lipoxygenase activity, the production of endogenous jasmonic acid and phytoalexin accumulation. The results demonstrate the role of the jasmonate pathway in the intracellular signal cascade.     

L-Phenylalanine ammonia-lyase fromPhaseolus vulgaris: Modulation of the levels of active enzyme bytrans-cinnamic acid

The extractable activity ofl-phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) in cell suspension cultures of bean (Phaseolus vulgaris) is greatly induced following exposure to an elicitor preparation from the cell walls of the phytopathogenic fungusColletotrichum lindemuthianum. Following exogenous application oftrans-cinnamic acid (the product of the PAL reaction) to elicitor-induced cells, the activity of the enzyme rapidly declines. Loss of enzyme activity is accompanied by inhibition of the rate of synthesis of PAL subunits, as determined by [³⁵S]methionine pulse-labelling followed by specific immunoprecipitation; this is insufficient to account for the rapid loss of PAL enzyme activity. Pulse-chase and immune blotting experiments indicate that cinnamic acid does not affect the rate of degradation of enzyme subunits, but rather mediates inactivation of the enzyme. A non-dialysable factor from cinnamicacid-treated bean cells stimulates removal of PAL activity from enzyme extracts in vitro; this effect is dependent on the presence of cinnamic acid. Such loss of enzyme activity in vitro is accompanied by an apparent loss or reduction of the dehydroalanine residue of the enzyme's active site, as detected by active-site-specific tritiation, although levels of immunoprecipitable enzyme subunits do not decrease. Furthermore, cinnamic-acid-mediated loss of enzyme activity in vivo is accompanied, in pulse-chase experiments, by a greater relative loss of³⁵S-labelled enzyme subunits precipitated by an immobilised active-site affinity ligand than of subunits precipitated with anti-immunoglobulin G. It is therefore suggested that a possible mechanism for cinnamic-acid-mediated removal of PAL activity may involve modification of the dehydroalanine residue of the enzyme's active site.Abbreviations AOPP l--aminoxy--phenylpropionic acid - CA trans-cinnamic acid - PAGE polyacrylamide gel electrophoresis - PAL l-phenylalanine ammonia-lyase - SDS sodium dodecyl sulphate     

Elicitor-inducible 3-hydroxy-3-methylglutaryl coenzyme a reductase activity is required for sesquiterpene accumulation in tobacco cell suspension cultures

Addition of cell wall fragments from Phytophthora species or cellulase from Trichoderma viride, but not pectolyase from Aspergillus japonicus, to tobacco (Nicotiana tabacum) cell suspension cultures induced the accumulation of the extracellular sesquiterpenoid capsidiol. Pulse-labeling experiments with [¹⁴C]acetate and [³H]mevalonate suggested that enzymatic steps preceding mevalonate were limiting capsidiol biosynthesis in the pectolyase-treated cell cultures. Treatment of the cell cultures with either Phytophthora cell wall fragments or cellulase induced 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and sesquiterpene cyclase activities, enzymes of the sesquiterpene biosynthetic pathway, and phenylalanine ammonia lyase activity, an enzyme of the general phenylpropanoid pathway. Pectolyase treatment induced sesquiterpene cyclase and phenylalanine ammonia lyase activities, but not HMGR activity. These results corroborate the importance of inducible HMGR enzyme activity for sesquiterpene accumulation.     

Rapid accumulation and metabolism of polyphosphoinositol and its possible role in phytoalexin biosynthesis in yeast elicitor-treated<Emphasis Type="Italic"> Cupressus lusitanica</Emphasis> cell cultures

Inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] rapidly accumulates in elicited Cupressus lusitanica Mill. cultured cells by 4- to 5-fold over the control, and then it is metabolized. Correspondingly, phospholipase C (PLC) activity toward phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] is stimulated to high levels by the elicitor and then decreases whereas Ins(1,4,5)P₃ phosphatase activity declines at the beginning of elicitation and increases later. These observations indicate that elicitor-induced biosynthesis and dephosphorylation of Ins(1,4,5)P₃ occur simultaneously and that the Ins(1,4,5)P₃ level may be regulated by both PtdIns(4,5)P₂–PLC and Ins(1,4,5)P₃ phosphatases. Studies on the properties of PLC and Ins(1,4,5)P₃ phosphatases indicate that PLC activity toward PtdIns(4,5)P₂ was optimal at a lower Ca²⁺ concentration than activity toward phosphatidylinositol whereas Ins(1,4,5)P₃ phosphatase activity is inhibited by high Ca²⁺ concentration. This suggests that Ins(1,4,5)P₃ biosynthesis and degradation may be regulated by free cytosolic Ca²⁺. In addition, a relationship between Ins(1,4,5)P₃ signaling and accumulation of a phytoalexin (-thujaplicin) is suggested because inhibition or promotion of Ins(1,4,5)P₃ accumulation by neomycin or LiCl affects elicitor-induced production of -thujaplicin. Moreover, ruthenium red inhibits elicitor-induced accumulation of -thujaplicin while thapsigargin alone induces -thujaplicin accumulation. These results suggest that Ca²⁺ released from intracellular calcium stores may mediate elicitor-induced accumulation of -thujaplicin via an Ins(1,4,5)P₃ signaling pathway, since it is widely accepted that Ins(1,4,5)P₃ can mobilize Ca²⁺ from intracellular stores. This work demonstrates an elicitor-triggered Ins(1,4,5)P₃ turnover, defines its enzymatic basis and regulation, and suggests a role for Ins(1,4,5)P₃ in elicitor-induced phytoalexin accumulation via a Ca²⁺ signaling pathway.Abbreviations Ins(1,4,5)P₃ Inositol-1,4,5-trisphosphate - Ins(1,4)P₂ Inositol-1,4-bisphosphate - Ins(4,5)P₂ Inositol-4,5-bisphosphate - Ins(1)P Inositol 1-phosphate - Ins(4)P Inositol 4-phosphate - PLC Phospholipase C - PtdIns Phosphatidylinositol - PtdIns(4,5)P₂ Phosphatidylinositol 4,5-bisphosphate - YE Yeast elicitor