Inhibition of plant asparagine synthetase by monoterpene cineoles

Abstract

Asparagine (Asn) synthetase (AS) is the key enzyme in Asn biosynthesis and plays an important role in nitrogen mobilization. Despite its important physiological function, little research has been done documenting inhibitors of plant AS. Plant growth inhibition caused by the natural monoterpene 1,4-cineole and its structurally related herbicide cinmethylin was reversed 65% and 55%, respectively, by providing 100 microM Asn exogenously. Reversion of the phytotoxic effect was dependent on the concentration of Asn. The presence of either 1,4-cineole or cinmethylin stimulated root uptake of [(14)C]Asn by lettuce (Lactuca sativa) seedlings. Although the physiological responses suggested that both compounds affected Asn biosynthesis, biochemical analysis of AS activity showed that the natural monoterpene was a potent inhibitor (I(50) = approximately 0. 5 microM) of the enzyme, whereas the commercial product was not inhibitory up to levels of 10 mM. Analysis of the putative metabolite, 2-hydroxy-1,4-cineole, showed that the cis-enantiomer was much more active than the trans-enantiomer, suggesting that the hydroxyl group was involved in the specific ligand/active site interaction. This is the first report that AS is a suitable herbicide target site, and that cinmethylin is apparently a proherbicide that requires metabolic bioactivation via cleavage of the benzyl-ether side chain.

Figure 1

Dose-response curves and structures for cinmethylin (A) and 1,4-cineole (B). Error bars represent ±1 sd.

Figure 2

Percent reversal of cinmethylin-induced root growth inhibition by exogenous supply of amino acids (see “Materials and Methods”). All 20 essential amino acids were tested individually at 100-μm final concentrations (except for Tyr, Trp, and Met at 50 μm). Asterisks denote significance (**, P ≤ 0.0001; *, P ≤ 0.01); error bars = 1 sd.

Figure 3

Dose-response curve illustrating the reversal effect of exogenous Asn on the phytotoxic effect of 1 μm cinmethylin and 1,4-cineole. Error bars = 1 sd.

Figure 4

Root uptake of [¹⁴C]Asn (DPM ×10,000) by untreated seedlings and 1 μm cinmethylin. Error bars = 1 sd.

Figure 5

Relative activity (percent of control) of AS at different concentrations of 1,4-cineole, 1,8-cineole, and cinmethylin. See “Materials and Methods” for assay conditions. AS activity varied between 1,400 and 1,600 nmol mg⁻¹ h⁻¹ in the control treatments.

Figure 6

Relative activity (percent of control) of AS at different concentrations of cis- and trans-2-hydroxy-1,4-cineole. See “Materials and Methods” for assay conditions. AS activity varied between 1,400 and 1,600 nmol mg⁻¹ h⁻¹ in the control treatments.

Figure 7

Model illustrating the proposed mechanism of action of cinmethylin and monoterpene cineoles.