Spearmint targets microtubules by (-)-carvone

Abstract

Allelopathy can provide sustainable alternatives to herbicides because it is based on specific signals rather than generic toxicity. We show that the allelopathic activity of Spearmint and Watermint is linked with their main compounds, (-)-carvone and (+)-menthofuran, both deriving from (-)-limonene. Germination of Poppy and Cress, and root growth of Arabidopsis thaliana are inhibited by very low concentrations of (-)-carvone, acting even through the gas phase. (+)-Menthofuran is active as well, but at lower efficacy. Using fluorescently tagged marker lines in tobacco BY-2 cells and Arabidopsis roots, we demonstrate a rapid degradation of microtubules and a remodeling of actin filaments in response to (-)-carvone and, to a milder extent, to (+)-menthofuran. This cytoskeletal response is followed by cell death. By means of a Root Chip system, we can follow the tissue dependent response of the cytoskeleton and show a cell-type dependent gradient of sensitivity between meristem and distal elongation zone, accompanied by programmed cell death.

Figure 1

Differential composition of essential oil in three closely related Mentha species via GC–MS. Blue circles represent the parental species M. aquatica, yellow circles the parental species M. spicata var. crispa, green circles their natural hybrid, M. x piperita. (A) Relative contents are given as % v/v of the extracted oil. The compounds detected via GC–MS are ordered according to their retention time from bottom to top. Six biological replicates for each species were analyzed. Arrows indicate carvone, menthofuran, and limonene. (B) Pathway of the main components. Relative abundance is shown by the different size of the circles representing the different species.

Figure 2

(−)-Carvone inhibits germination of Cress. (A) Representative image of the phenomenon. The upper image shows the outcome after applying the solvent control n-hexane, the lower image after application of 100 ppm (−)-carvone, both administered through the gas phase. (B) Dose–response of germination inhibition over the concentration of (−)-carvone as compared to the structurally related monoterpenes (+)-menthofuran and (−)-limonene, as well as compared to the solvent control after incubation in darkness at 25°C for either 3 or for 5 days. A linear trend line is indicated. Data represent means and SD from biological triplicates.

Figure 3

(−)-Carvone inhibits germination of Poppy. (A) Representative image of the phenomenon. The upper image shows the outcome after applying the solvent control n-hexane, the lower image after application of 100 ppm (−)-carvone, both administered through the gas phase. (B) Dose–response of germination inhibition over the concentration of (−)-carvone as compared to the structurally related monoterpene (+)-menthofuran, as well as compared to the solvent control after incubation in a 12 h light/12 h dark cycle at 20°C for 2 or 6 days. A saturation curve is indicated. Data represent means and SD from biological triplicates.

Figure 4

(−)-Carvone and (+)-menthofuran inhibit root growth in Arabidopsis thaliana. Relative growth rates (DL/L in %^.d⁻¹) during the first 2 days (0–2 d, white bars) and the subsequent 2 days (2–4 d, grey bars) of exposure to the respective compound mixed into the agar. Time zero is defined as 2 days of stratification in the dark and in the cold followed by 5 days of germination. Data represent means and standard errors from two independent series of experiments (separate Petri dishes) with 15–22 individual roots per Petri dish. Differences from the water control were tested by the non-parametric Wilcox test. Significant differences are represented by different lettering, whereby ab (AB) meaning P < 0.05, and b (B) meaning P < 0.01 compared to the water control (a or A, respectively).

Figure 5

Cytotoxicity of (−)-carvone and (+)-menthofuran. Cell mortality in response to (−)-carvone and (+)-menthofuran was measured in non-transformed BY-2 cells (WT) with and without 10 μM Taxol, a specific inhibitor of microtubule disassembly, as well as in BY-2 cells expressing the actin-binding domain 2 of fimbrin fused to GFP (GF11) and cells expressing tubulin α3 from tobacco in fusion with GFP (TuA3). The values for the solvent control, n-hexane, are added and marked by -, while + means the respective compound (using a concentration of 0.5% (v/v)). In this experiment, the compounds were mixed with the cell suspension. Mortality was scored 30 min after addition of the compound. Data represent means and standard errors from three independent biological replications with a population of 2500 individual cells per replication. The significance was tested by a one-tailed paired t-test with ^*  P < 0.05, ^**  P < 0.01, and ^***  P < 0.001 and indicated by asterisks.

Figure 6

Response of microtubules to (−)-carvone and (+)-menthofuran. Representative BY-2 cells expressing tubulin α3 in fusion with GFP followed by spinning disc confocal microscopy after treatment with either 1 μL of n-hexane as solvent control, (−)-carvone or (+)-menthofuran at proliferation phase, day 3 after subcultivation. Maximal intensity projections of confocal z-stacks are shown. Compounds were administered through the gas phase. The volume of the solvent control, n-hexane, was identical (1 μL). The volume of the cell suspension was 20 μL in all experiments.

Figure 7

Response of actin filaments to (−)-carvone and (+)-menthofuran. Representative BY-2 cells expressing the actin-binding domain 2 of fimbrin in fusion with GFP followed by spinning disc confocal microscopy after treatment with either 1 μL of n-hexane as solvent control, (−)-carvone or (+)-menthofuran at proliferation phase, day 3 after subcultivation. Maximal intensity projections of confocal z-stacks are shown. Compounds were administered through the gas phase. The volume of the solvent control, n-hexane, was identical (1 μL). The volume of the cell suspension was 20 μL in all experiments.

Figure 8

Response of microtubules to the essential oil of M. x piperita in Arabidopsis. Representative Arabidopsis roots expressing TuB6-GFP followed by laser scanning confocal microscopy after treatment with 4 μL^.mL⁻¹ of essential oil of M. x piperita at day 7 postgermination. Compounds were administered through ½ MS medium containing 0.5% sucrose. Propidium iodide was added to 2 μg^.mL⁻¹ to label the cell walls and follow cell death. The images are individual confocal sections. Observations were conducted in the multi-tracking mode using 488 and 555 nm laser excitations.

Figure 9

Response of microtubules to (−)-carvone in Arabidopsis. Representative Arabidopsis roots expressing TuB6-GFP followed by laser scanning confocal microscopy after treatment with 1 μL^.mL⁻¹ of (−)-carvone at day 7 (root tip) or day 10 (elongation zone) postgermination. Compounds were administered through ½ MS medium containing 0.5% sucrose. Propidium iodide was added to 2 μg^.mL⁻¹ to label the cell walls and follow cell death. The images are individual confocal sections. Observations were conducted in the multi-tracking mode using 488 and 555 nm laser excitations.

Figure 10

Response of microtubules to (+)-menthofuran in Arabidopsis. Representative Arabidopsis roots expressing TuB6-GFP followed by laser scanning confocal microscopy after treatment with 2 μL^.mL⁻¹ of (+)-menthofuran at day 7 postgermination. Compounds were administered through ½ MS medium containing 0.5% sucrose. Propidium iodide was added to 2 μg^.mL⁻¹ to label the cell walls and follow cell death. The images are individual confocal sections. Observations were conducted in the multi-tracking mode using 488 and 555 nm laser excitations.

Figure 11

Design and fabrication of the Root Chip. (A) Design of the chip with three parallel channels equipped with inlets and outlets for supply flow and entry points for the roots. The channels are covered by a glass slide. (B) A specimen of the Root Chip ready for use. (C) Design of the mould for casting. (D) A PDMS cast generated by the mould in (C) as intermediate step of fabrication.