Signalling mechanisms and agricultural applications of (Z)-3-hexenyl butyrate-mediated stomatal closure

Abstract

Biotic and abiotic stresses can severely limit crop productivity. In response to drought, plants close stomata to prevent water loss. Furthermore, stomata are the main entry point for several pathogens. Therefore, the development of natural products to control stomata closure can be considered a sustainable strategy to cope with stresses in agriculture. Plants respond to different stresses by releasing volatile organic compounds. Green leaf volatiles, which are commonly produced across different plant species after tissue damage, comprise an important group within volatile organic compounds. Among them, (Z)-3-hexenyl butyrate (HB) was described as a natural inducer of stomatal closure, playing an important role in stomatal immunity, although its mechanism of action is still unknown. Through different genetic, pharmacological, and biochemical approaches, we here uncover that HB perception initiates various defence signalling events, such as activation of Ca²⁺ permeable channels, mitogen-activated protein kinases, and production of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species. Furthermore, HB-mediated stomata closure was found to be independent of abscisic acid biosynthesis and signalling. Additionally, exogenous treatments with HB alleviate water stress and improve fruit productivity in tomato plants. The efficacy of HB was also tested under open field conditions, leading to enhanced resistance against Phytophthora spp. and Pseudomonas syringae infection in potato and tomato plants, respectively. Taken together, our results provide insights into the HB signalling transduction pathway, confirming its role in stomatal closure and plant immune system activation, and propose HB as a new phytoprotectant for the sustainable control of biotic and abiotic stresses in agriculture.

Figure 1

Transcriptomic analysis suggests a role of HB in plant immunity. Functional profiling analysis of upregulated (A) and downregulated (B) DEGs between HB- and control-treated plants.

Figure 2

HB-mediated stomatal closure requires Ca2+ signalling and ROS production by NADPH oxidases, but not ABA biosynthesis. (A) Lukullus (WT) and flacca tomato leaf discs were floated on liquid MS for 3 hours under light. Then, 1 μM flg22, 10 μM ABA, or 50 μM HB were applied, and stomatal aperture ratio was determined 2 hours after treatments. In the case of chemical inhibitors experiments, 2 mM EGTA (B), 20 μM DPI (C), or 2 mM SHAM (D) were added in the liquid medium for MoneyMaker tomato leaf discs before elicitor treatments. Violin plots represent the stomatal aperture ratio of 50 stomata for each treatment. Different letters indicate statistically significant differences for each genotype and treatment (P < 0.05, two-way ANOVA with Tukey HSD test).

Figure 3

Activation of MPK3 and MPK6 is involved in HB-mediated stomatal immunity. MPK activation assay in tomato (A) and Arabidopsis thaliana (B) leaf discs 15, 30 and 60 minutes after treatments with 1 μM flg22, 10 μM ABA and 50 μM HB. MPK activation was detected by immunoblot analysis using the Phospho-p44/42 MAPK (Erk1/2; Thr-202/Tyr204) rabbit monoclonal antibody. Western blot experiments were performed three times and yielded similar results. (C) Stomatal aperture ratio was measured in tomato leaf discs floated on MS liquid for 3 hours under light, pre- treated with the MPKs inhibitor PD98059 20 μM and followed by treatments with 1 μM flg22, 10 μM ABA and 50 μM HB for 2 hours. (D) Growth of Pst on tomato leaves of control (-HB) and HB-treated (+HB) after treatments with the MPKs inhibitor PD98059. Plants were sprayed with PD98059 100 μM for 3 hours, subsequently treated with HB 5 μM or water for 24 h into methacrylate chambers, and then dip inoculated with Pst. Bacterial growth measurements were done 24 hours after inoculation. (E) Stomatal aperture ratio of Arabidopsis Col-0, mpk3, and mpk6 mutants leaf discs 2 hours after treatments with flg22, ABA, and HB. (F) Growth of Pst on A. thaliana leaves of control (−HB) and HB-treated (+HB) 3 days after inoculation. Plants were treated with HB 5 μM or water for 24 hours into methacrylate chambers, and then infected by spray with Pst. In stomatal aperture experiments, 50 stomata were measured per each treatment and condition (violin plots). Data correspond to at least four independent plants ± SEM of a representative experiment. Statistically significant differences are represented by different letters (p < 0.05, two-way ANOVA with Tukey HSD).

Figure 4

HB treatments induce tolerance to drought in tomato plants and reduce proline and ABA contents. Differences related to stomatal aperture ratio (A), weight (B), ion leakage (C), proline content (D) and ABA content (E) in tomato plants treated (+HB) or not (−HB) with HB, in normal (+H₂0) or water stressed conditions (−H₂0). Samples were taken 3 or 6 days after drought exposure (DAD). Data in (A) corresponds to 50 stomata per each treatment and condition (violin plots). Data in (B), (C) and (D) correspond to six independent plants ± SEM of a representative experiment. Data in (E) correspond to the averages of three independent plants ± SEM of a representative experiment. Different letters indicate statistically significant differences for each genotype and treatment (p < 0.05, two-way ANOVA with Tukey HSD).

Figure 5

Transcriptomic analysis of HB-treated tomato plants under drought stress. Functional profiling analysis of upregulated (A) and downregulated (B) DEGs between HB- and control-treated plants under water deprivation.

Figure 6

Agricultural applications of HB treatments under open field experiments. Number of flowers (A) and number of set fruits (B) of tomato plants under limited water availability conditions (50%) and treated (+HB) or not (−HB) with 5 mM HB. Time units are referred as follows: X DA Z, being X day (0–18); DA days after; Z day of treatment (A–H). Points represent mean values. Different letters indicate significant differences at each time point (P < 0.05, two-way ANOVA with Tukey HSD). Efficacy on severity (C) and efficacy on incidence (D) of tomato plants naturally infected by Pseudomonas syringae and either untreated, HB weekly treated at different concentrations (0.05, 5, 50, 500 mM) or treated with the positive control OXICOOP (0.5%). Time units are referred as follows: X DA Z, being X day (0–8); DA days after; Z day of treatment (A–C). An ANOVA test was performed, and different letters indicate statistical significances with a P-value <0.05. Efficacy on severity, (E) and efficacy on incidence (F) of potato plants naturally infected by Phytophthora infestans and either untreated, HB weekly treated at different concentrations (0.05, 5, 50, 500 mM) or treated with the positive control OROCOBRE (0.3%). Time units are referred as follows: X DA Z, being X day (0–27); DA Days After; Z day of treatment (A–D). An ANOVA test was performed, and different letters indicate statistical significances with a p < 0.05.