title: Chemical inactivation of the cinnamate 4-hydroxylase allows for the accumulation of salicylic acid in elicited cells.
abstract: The cinnamate (CA) 4-hydroxylase (C4H) is a cytochrome P450 that catalyzes the second step of the main phenylpropanoid pathway, leading to the synthesis of lignin, pigments, and many defense molecules. Salicylic acid (SA) is an essential trigger of plant disease resistance. Some plant species can synthesize SA from CA by a mechanism not yet understood. A set of specific inhibitors of the C4H, including competitive, tight-binding, mechanism-based irreversible, and quasi-irreversible inhibitors have been developed with the main objective to redirect cinnamic acid to the synthesis of SA. Competitive inhibitors such as 2-hydroxy-1-naphthoic acid and the heme-coordinating compound 3-(4-pyridyl)-acrylic acid allowed strong inhibition of C4H activity in a tobacco (Nicotiana tabacum cv Bright Yellow [BY]) cell suspension culture. This inhibition was however rapidly relieved either because of substrate accumulation or because of inhibitor metabolism. Substrate analogs bearing a methylenedioxo function such as piperonylic acid (PIP) or a terminal acetylene such as 4-propynyloxybenzoic acid (4PB), 3-propynyloxybenzoic acid, and 4-propynyloxymethylbenzoic acid are potent mechanism-based inactivators of the C4H. PIP and 4PB, the best inactivators in vitro, were also efficient inhibitors of the enzyme in BY cells. Inhibition was not reversed 46 h after cell treatment. Cotreatment of BY cells with the fungal elicitor beta-megaspermin and PIP or 4PB led to a dramatic increase in SA accumulation. PIP and 4PB do not trigger SA accumulation in nonelicited cells in which the SA biosynthetic pathway is not activated. Mechanism-based C4H inactivators, thus, are promising tools for the elucidation of the CA-derived SA biosynthetic pathway and for the potentiation of plant defense.
authors.affiliation: Department of Plant Stress Response, Institute of Plant Molecular Biology, Centre National de la Recherche Scientifique-Unité Propre de Recherche 2357, Université Louis Pasteur, 28 Rue Goethe, F-67000 Strasbourg, France.
authors.forename: Guillaume A
authors.initials: GA
authors.lastname: Schoch
authors.forename: Georgi N
authors.initials: GN
authors.lastname: Nikov
authors.forename: William L
authors.initials: WL
authors.lastname: Alworth
authors.forename: Danièle
authors.initials: D
authors.lastname: Werck-Reichhart
mesh_terms: D001565:Benzoates / Q000737:chemistry / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D002460:Cell Line; D002934:Cinnamates / Q000378:metabolism; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D062385:Hydroxybenzoates / Q000737:chemistry / Q000494:pharmacology; D007113:Immunity, Innate / Q000187:drug effects; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015394:Molecular Structure; D010840:Phytosterols; D020156:Salicylic Acid / Q000378:metabolism*; D013379:Substrate Specificity; D013997:Time Factors; D014026:Nicotiana / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D050564:Trans-Cinnamate 4-Monooxygenase
chemical_list: D001565:Benzoates; D002934:Cinnamates; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D062385:Hydroxybenzoates; D010840:Phytosterols; C059564:propynyl p-methoxybenzoic acid; C029010:cinnamic acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D050564:Trans-Cinnamate 4-Monooxygenase; D020156:Salicylic Acid; C005455:piperonylic acid