The mechanism behind tenuazonic acid-mediated inhibition of plant plasma membrane H+-ATPase and plant growth

Abstract

The increasing prevalence of herbicide-resistant weeds has led to a search for new herbicides that target plant growth processes differing from those targeted by current herbicides. In recent years, some studies have explored the use of natural compounds from microorganisms as potential new herbicides. We previously demonstrated that tenuazonic acid (TeA) from the phytopathogenic fungus Stemphylium loti inhibits the plant plasma membrane (PM) H⁺-ATPase, representing a new target for herbicides. In this study, we further investigated the mechanism by which TeA inhibits PM H⁺-ATPase and the effect of the toxin on plant growth using Arabidopsis thaliana. We also studied the biochemical effects of TeA on the PM H⁺-ATPases from spinach (Spinacia oleracea) and A. thaliana (AHA2) by examining PM H⁺-ATPase activity under different conditions and in different mutants. Treatment with 200 μM TeA-induced cell necrosis in larger plants and treatment with 10 μM TeA almost completely inhibited cell elongation and root growth in seedlings. We show that the isoleucine backbone of TeA is essential for inhibiting the ATPase activity of the PM H⁺-ATPase. Additionally, this inhibition depends on the C-terminal domain of AHA2, and TeA binding to PM H⁺-ATPase requires the Regulatory Region I of the C-terminal domain in AHA2. TeA likely has a higher binding affinity toward PM H⁺-ATPase than the phytotoxin fusicoccin. Finally, our findings show that TeA retains the H⁺-ATPase in an inhibited state, suggesting that it could act as a lead compound for creating new herbicides targeting the PM H⁺-ATPase.

Keywords: AHA2; Arabidopsis thaliana; fusicoccin; natural compounds; phytotoxin; plasma membrane H(+)-ATPase; tenuazonic acid.

Figure 1

Testing the potential use of tenuazonic acid (TeA) as an herbicide.A, A. thaliana plants were grown for 31 days and treated with 200 μM TeA + 0.1% Adigor, 0.1% Adigor, or water on days 0, 2, and 4. Adigor was used as an adjuvant to penetrate the leaf cuticle. Plants (n = 8) were treated on days 0, 2, and 4 and photographed right before treatment and on day 6. B, The number of necrotic spots on plants treated with 200 μM TeA + 0.1% Adigor or 0.1% Adigor on days 2, 4, and 6 post treatments. Data are presented as the mean number of necrotic spots per plant ±SEM. C, The amount of leaf death per plant treated with 200 μM TeA + 0.1% Adigor on days 2, 4, and 6 post treatments. Data are presented as the mean number of dead leaves per plant. Statistical data analysis was performed using two-way ANOVA with Bonferroni multiple comparison test in GraphPad Prism 9, p < 0.0332 (∗), p < 0.0021 (∗∗), p < 0.0002 (∗∗∗), p < 0.0001 (∗∗∗∗).

Figure 2

Testing the potency of a tenuazonic acid (TeA) analog on inhibitions of plasma membrane (PM) H⁺-ATPase activity.A, chemical structure of TeA and L-TeA. B, S. oleracea PM vesicles were treated with 25 μM TeA or TeA analog to investigate their effects on ATPase activity at pH 6.5. Assays were performed in six technical replicates from three biological replicates (n = 18), and activity is presented as the mean percentage of inhibition ±SEM. Statistical data analysis was performed using one-way ANOVA with Bonferroni multiple comparison test in GraphPad Prism 9, p < 0.0332 (∗), p < 0.0021 (∗∗), p < 0.0002 (∗∗∗), p < 0.0001 (∗∗∗∗).

Figure 3

Tenuazonic acid (TeA) prevents fusicoccin (FC)-induced activation of plasma membrane (PM) H⁺-ATPase.A, A. thaliana seedlings were grown for 6 days on ½× MS agar then transferred to fresh ½× MS agar containing 10 μM TeA, 10 μM FC, 10 μM TeA/FC, or control treatment, and incubated for 6 days. The root length of seedlings was measured every other day using Fiji ImageJ. Growth assays were performed in technical replicates of 20 from two biological replicates (n = 40), and root length is given in cm presented as mean ±SEM. B, A. thaliana seedlings on day 6 of treatment with 10 μM TeA, 10 μM FC, 10 μM TeA/FC, or control. Scale bar = 0.5 cm. C, root elongation zones of A. thaliana seedlings on day 6 of treatment with 20 μM TeA or control. Scale bar = 100 μm. D, length of cells in the elongation zone of seedlings treated with 20 μM TeA, 10 μM TeA, 10 μM FC, 10 μM TeA/FC, or control for 6 days. Cell length was measured using Fiji ImageJ, and growth assays were performed in replicates of 29 to 30 from two biological replicates (n = 59–60); root length is given in μm presented mean ±SEM. E, A. thaliana seedlings were grown for 6 days on ½× MS agar and transferred to fresh ½× MS plates with 20 μM TeA or control, incubated for 3 days, then transferred to Bromocresol Purple agar, and grown overnight before assessing acidification. The experiment was repeated three independent times with similar results. Scale bar = 1 cm. F, TeA/FC competition assay measuring the ATPase activity of S. oleracea PM H⁺-ATPase. Purified PM vesicles were pre-incubated with 15 μM TeA, FC, or TeA/FC at pH 6.5 for 10 min prior to assay start and assays were performed using 10 μM of phytotoxins. Assays were performed in technical replicates of three from two biological replicates (n = 6), and activity is presented as the mean percentage of non-treated samples ±SEM. Statistical data analysis was performed using one-way or two-way ANOVA with Bonferroni multiple comparison test in GraphPad Prism 9, p < 0.0332 (∗), p < 0.0021 (∗∗), p < 0.0002 (∗∗∗), p < 0.0001 (∗∗∗∗).

Figure 4

Tenuazonic acid (TeA)-induced inhibition is dependent on the C-terminal domain of the plasma membrane (PM) H⁺-ATPase.A, amino acid sequence of the AHA2 C-terminal domain and truncated mutants aha2Δ77, aha2Δ66, and aha2Δ61. Amino acid residue R880 is marked with an asterisk (∗). Regulatory Region I (R-I) and II (R-II) are marked in bold. B, TeA-induced inhibition of the ATPase activity of AHA2, aha2Δ61, aha2Δ66, and aha2Δ77 purified from the PM fraction from S. cerevisiae. Membrane fractions were treated with 50 μM TeA or 50 μM vanadate (P-type ATPase inhibitor) at pH 6.5 for AHA2 and pH 7 for aha2Δ77, aha2Δ66, and aha2Δ61. The assays were performed in technical replicates of three from two biological replicates (n = 6), and activity is presented as the mean percentage compared to non-treated samples ±SEM. C, TeA-induced inhibition of the ATPase activity of AHA2 and aha2R880A purified from the PM fraction from S. cerevisiae. PM fractions were treated with 50 μM TeA in pH 6.5 for AHA2 and pH 7 for aha2R880A. Assays were performed in technical replicates of three from two biological replicates (n = 6) and activity is presented as mean percentage compared to non-treated samples ±SEM. Statistical data analysis was performed using Two-way ANOVA with Bonferroni multiple comparison test in GraphPad Prism 9, p < 0.0332 (∗), p < 0.0021 (∗∗), p < 0.0002 (∗∗∗), p < 0.0001 (∗∗∗∗).

Figure 5

Tenuazonic acid (TeA)-induced inhibition depends on the activity state of the plasma membrane (PM) H⁺-ATPase.A, TeA-induced inhibition of the ATPase activity of AHA2 and aha2R913A purified from the PM and internal membrane (IM) fractions from S. cerevisiae. Membrane fractions were treated with 50 μM TeA or 50 μM vanadate (P-type ATPase inhibitor) at pH 6.5 for AHA2 and pH 7 for aha2R913A. Assays were performed in technical replicates of three from two biological replicates (n = 6), and activity is presented as the mean percentage compared to non-treated samples ±SEM. B, immunoblot and 14-3-3 overlay blot of the AHA2 and aha2R913A PM and IM fractions to detect the binding ability of 14-3-3 proteins to these proteins. C, relative binding of 14-3-3 protein towards AHA2 and aha2R913A purified from the PM fraction. Overlay assays were performed in technical replicates of three (n = 3), and values are presented as relative ratios of the lane intensity from overlay blots and multiplied by protein levels from the immunoblot ±SEM. D, detection of PM H⁺-ATPase and pThr residues in the PM H⁺-ATPase by immunoblotting using PM vesicles from S. oleracea treated with and without λ-phosphatase (λpp). E, TeA inhibition assay of S. oleracea PM H⁺-ATPase treated with and without λ-phosphatase (λpp). PM vesicles were treated with 25 μM TeA at pH 6.5, and all assays were performed with technical replicates of six (n = 6). Non-detectable (n.d.) activity is indicated. Statistical data analysis was performed using two-way ANOVA with Bonferroni multiple comparison test in GraphPad Prism 9, p < 0.0332 (∗), p < 0.0021 (∗∗), p < 0.0002 (∗∗∗), p < 0.0001 (∗∗∗∗).

Figure 6

Model of the role of tenuazonic acid (TeA) in inhibiting plant plasma membrane (PM) H⁺-ATPase activity.A, the binding affinity of TeA to the PM H⁺-ATPase is higher than the binding affinity of fusicoccin (FC). TeA maintains the enzyme in a tight conformation, preventing it from binding to FC to activate the pump. B, TeA inhibits the activity of full-length PM H⁺-ATPase by locking the C-terminal regulatory domain, keeping the pump in an autoinhibited state. C, truncation of the C-terminal regulatory domain of PM H⁺-ATPase removes its TeA binding site, preventing the pump from being inhibited by TeA. D, phosphorylation of the penultimate threonine residue and binding of 14-3-3 protein to the C-terminal domain of PM H⁺-ATPase keeps the enzyme in a conformation where the C-terminal domain is released and flexible. This prevents TeA from binding to PM H⁺-ATPase, resulting in no inhibition.

Supporting Figures 1

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