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.

Figure 1

C4H and branching in the upper phenylpropanoid pathway. PAL, Phe ammonia-lyase; 4CL, 4-hydroxycinnamate CoA ligase; AOPP, amino-β-phenyl-propionic acid is an inhibitor of PAL (Amrhein et al., 1983); MDCA, methylene dioxocinnamic acid is an inhibitor of 4CL (Funk and Brodelius, 1990).

Figure 2

Molecules tested as inhibitors of CYP73A1.

Figure 3

Time- and concentration-dependent inactivation of CYP73A1 by 4PB, 3PB, and 4PO. Recombinant CYP73A1 in W(R) yeast microsomes was preincubated in the presence of NADPH and different concentrations of the propynyl derivatives before assaying residual C4H activity. A, Time course of the C4H inactivation measured upon incubation with 4PB. Similar pseudo-first-order kinetics of inactivation were obtained with 3PB and 4PO. B, Plot of the half-inactivation times, estimated from linear regression analysis of curves shown in A, versus reciprocal of inhibitor concentration. C and D, Half-inactivation time plots, as in B, for 3PB and 4PO.

Figure 4

Inhibition of C4H activity by 3PA. Lineweaver-Burk representation of the inhibition of C4H activity of recombinant CYP73A1 (3 nm in the assay) by 3PA (0, 1, 3, or 10 μm). Data are means of duplicate experiments.

Figure 5

Time course of the conversion of CA into p-coumarate in a tobacco BY cell suspension culture. One-milliliter aliquots of 5-d-old cells were incubated with 20 μm [¹⁴C]CA in Eppendorf tubes at 30°C. CA to p-coumarate conversion was evaluated after TLC analysis of extracted free phenolics, and identity of radiolabeled spots was confirmed by HPLC-diode array analysis. Data are expressed as a proportion of initial CA recovered as p-coumarate at the end of the incubation.

Figure 6

Inhibition of C4H activity in tobacco BY cells. Inhibitors in DMSO were added to a 5-d-old suspension culture. DMSO was added to the control. Final concentrations in the culture were 0.06% (v/v) DMSO, 40 μm 2HN, 40 μm 3PA, 100 μm 4PB, and 10 μm PIP. Aliquots collected after 0.5, 4, 22, and 46 h of incubation were assayed for conversion of [¹⁴C]CA (20 μm). Data are means ± sd of four measurements.

Figure 7

Inhibition of PAL activity. Whole extracts of β-megaspermin pretreated BY cells were diluted 5-fold in 100 mm borate buffer, pH 8.8, and incubated with 85 μm [¹⁴C]Phe and various concentrations of inhibitors for 1 h at 37°C. Radiolabeled cinnamic acid was extracted and counted by liquid scintillation. Data are means ± sd of two experiments. 100% activity = 19 pmol h⁻¹μL⁻¹.

Figure 8

Accumulation of total SA in inhibitor-treated cells. A 5-d-old tobacco BY cell culture was treated with 10 μm PIP, 10 μm 4PB, or DMSO. Identical treatment was performed 30 min after elicitation with β-megaspermin (β-meg). Free and conjugated SA were extracted from aliquots and hydrolyzed, and total SA was purified and quantified by fluorometry. The experiment was performed on two subcultures maintained independently to evaluate variations of cell response.

Figure 9

General synthetic route for the synthesis of propynyl inhibitors of CYP73A1.