cellulase and capsidiol

S-Carvone has been described as a negative regulator of mevalonic acid (MVA) production by interfering with 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) activity, a key player in isoprenoid biosynthesis. The impact of this monoterpene on the production of capsidiol in Nicotiana tabacum, an assumed MVA-derived sesquiterpenoid phytoalexin produced in response to elicitation by cellulase, was investigated. As expected, capsidiol production, as well as early stages of elicitation such as hydrogen peroxide production or stimulation of 5-epi-aristolochene synthase activity, were repressed. Despite the lack of capsidiol synthesis, apparent HMGR activity was boosted. Feeding experiments using (1-13C)Glc followed by analysis of labeling patterns by 13C-NMR, confirmed an MVA-dependent biosynthesis; however, treatments with fosmidomycin, an inhibitor of the MVA-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) isoprenoid pathway, unexpectedly down-regulated the biosynthesis of this sesquiterpene as well. We postulated that S-carvone does not directly inhibit the production of MVA by inactivating HMGR, but possibly targets an MEP-derived isoprenoid involved in the early steps of the elicitation process. A new model is proposed in which the monoterpene blocks an MEP pathway-dependent protein geranylgeranylation necessary for the signaling cascade. The production of capsidiol was inhibited when plants were treated with some inhibitors of protein prenylation or by further monoterpenes. Moreover, S-carvone hindered isoprenylation of a prenylable GFP indicator protein expressed in N. tabacum cell lines, which can be chemically complemented with geranylgeraniol. The model was further validated using N. tabacum cell extracts or recombinant N. tabacum protein prenyltransferases expressed in Escherichia coli. Our study endorsed a reevaluation of the effect of S-carvone on plant isoprenoid metabolism.

Topics: Alcohol Oxidoreductases; Biomass; Cell Death; Cellulase; Cyclohexane Monoterpenes; Dimethylallyltranstransferase; Down-Regulation; Erythritol; Fosfomycin; Mevalonic Acid; Models, Biological; Monoterpenes; Nicotiana; Plant Leaves; Protein Prenylation; Secondary Metabolism; Sesquiterpenes; Signal Transduction; Stress, Physiological; Sugar Phosphates

In the Solanaceae, biotic and abiotic elicitors induce de novo synthesis of sesquiterpenoid stress metabolites known as phytoalexins. Because plant hormones play critical roles in the induction of defense-responsive genes, we have explored the effect of abscisic acid (ABA) on the synthesis of capsidiol, the major wild tobacco (Nicotiana plumbaginifolia) sesquiterpenoid phytoalexin, using wild-type plants versus nonallelic mutants Npaba2 and Npaba1 that are deficient in ABA synthesis. Npaba2 and Npaba1 mutants exhibited a 2-fold higher synthesis of capsidiol than wild-type plants when elicited with either cellulase or arachidonic acid or when infected by Botrytis cinerea. The same trend was observed for the expression of the capsidiol biosynthetic genes 5-epi-aristolochene synthase and 5-epi-aristolochene hydroxylase. Treatment of wild-type plants with fluridone, an inhibitor of the upstream ABA pathway, recapitulated the behavior of Npaba2 and Npaba1 mutants, while the application of exogenous ABA reversed the enhanced synthesis of capsidiol in Npaba2 and Npaba1 mutants. Concomitant with the production of capsidiol, we observed the induction of ABA 8'-hydroxylase in elicited plants. In wild-type plants, the induction of ABA 8'-hydroxylase coincided with a decrease in ABA content and with the accumulation of ABA catabolic products such as phaseic acid and dihydrophaseic acid, suggesting a negative regulation exerted by ABA on capsidiol synthesis. Collectively, our data indicate that ABA is not required per se for the induction of capsidiol synthesis but is essentially implicated in a stress-response checkpoint to fine-tune the amplification of capsidiol synthesis in challenged plants.

Topics: Abscisic Acid; Arachidonic Acid; Botrytis; Cellulase; Molecular Sequence Data; Mutation; Nicotiana; Plant Proteins; Pyridones; Sesquiterpenes; Signal Transduction

When growth-phase cell suspension cultures of Capsicum annuum were treated with cellulase-elicitor preparation at 3 microg/ml, the level of capsidiol was transiently increased in the culture media rather than in the cells reaching its maximum approx 24 h after treatment. With methyl jasmonate it took 18 h. Elicitor treatment doubled phospholiphase A(2) (PLA(2)) activity but simultaneous treatment with aristolochic acid, a PLA(2) inhibitor, inhibited sesquiterpenoid accumulation as well as PLA(2) activity. Mastoparan, a G protein activator, treatment also increased PLA(2) activity and capsidiol production. Taken together, the present study shows that induction of capsidiol production in the C. annuum is mediated by PLA(2) activation.

Topics: Acetates; Aristolochic Acids; Capsicum; Cells, Cultured; Cellulase; Chromatography, Gas; Cyclopentanes; Intercellular Signaling Peptides and Proteins; Oxylipins; Peptides; Phospholipase A2 Inhibitors; Phospholipases A2; Sesquiterpenes; Time Factors; Wasp Venoms

Jasmonates are well documented for their ability to modulate the expression of plant genes and to influence specific aspects of disease/pest resistance traits. We and others have been studying the synthesis of sesquiterpene phytoalexins in elicitor/pathogen-challenged plants and have sought to determine if methyl jasmonate (MeJA) could substitute for fungal elicitors in the induction of capsidiol accumulation by tobacco cell cultures. The current results demonstrate that MeJA does in fact induce phytoalexin accumulation, but with a much more delayed induction time course than elicitor. While elicitor treatment induced strong but transient changes in key enzymes of sesquiterpene biosynthesis, sesquiterpene cyclase, and aristolochene/deoxy-capsidiol hydroxylase, MeJA did not. Instead, MeJA caused a protracted induction of cyclase activity and only a low level of hydroxylase activity. MeJA induced the expression of at least two sesquiterpene cyclase genes, including one that had not been observed previously in elicitor-induced mRNA populations. Only a small portion of the total sesquiterpene cyclase mRNA induced by MeJA was associated with polysomal RNA, suggesting that the MeJA treatment imposed both transcriptional and posttranscriptional regulation in tobacco cells. These results are not consistent with MeJA playing a role in orchestrating defense responses in elicitor-treated tobacco cells, but do provide evidence that MeJA induces a subset of genes coding for the biosynthesis of sesquiterpene phytoalexins.

Topics: Acetates; Amino Acid Sequence; Base Sequence; Carbon-Carbon Lyases; Cells, Cultured; Cellulase; Cyclopentanes; DNA Primers; DNA, Plant; Fungal Proteins; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Genes, Plant; Molecular Sequence Data; Nicotiana; Oxylipins; Plant Growth Regulators; Plants, Toxic; RNA, Messenger; RNA, Plant; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Sesquiterpenes