A New Role For Green Leaf Volatile Esters in Tomato Stomatal Defense Against Pseudomonas syringe pv. tomato

Abstract

The volatile esters of (Z)-3-hexenol with acetic, propionic, isobutyric, or butyric acids are synthesized by alcohol acyltransferases (AAT) in plants. These compounds are differentially emitted when tomato plants are efficiently resisting an infection with Pseudomonas syringae pv. tomato. We have studied the defensive role of these green leaf volatile (GLV) esters in the tomato response to bacterial infection, by analyzing the induction of resistance mediated by these GLVs and the phenotype upon bacterial infection of tomato plants impaired in their biosynthesis. We observed that treatments of plants with (Z)-3-hexenyl propionate (HP) and, to a greater extent with (Z)-3-hexenyl butyrate (HB), resulted in stomatal closure, PR gene induction and enhanced resistance to the bacteria. HB-mediated stomatal closure was also effective in several plant species belonging to Nicotiana, Arabidopsis, Medicago, Zea and Citrus genus, and both stomatal closure and resistance were induced in HB-treated NahG tomato plants, which are deficient in salicylic acid (SA) accumulation. Transgenic antisense AAT1 tomato plants, which displayed a reduction of ester emissions upon bacterial infection in leaves, exhibited a lower ratio of stomatal closure and were hyper-susceptible to bacterial infection. Our results confirm the role of GLV esters in plant immunity, uncovering a SA-independent effect of HB in stomatal defense. Moreover, we identified HB as a natural stomatal closure compound with potential agricultural applications.

Keywords: AAT1; GLV esters; bacteria; defense; stomata; tomato.

Figure 1

Effects of (Z)-3-hexenol acetic (HA), propionic (HP), isobutyric (HiB) or butyric (HB) esters on stomatal aperture and PR gene expression of Flora-Dade tomato leaves. Samples were collected 24 h after treatments. (A) Stomatal aperture ratio mean values ± SD of three biological replicates from a representative experiment. Asterisks (^***) indicate statistically significance differences from control (p < 0.0010) and treated or P. syringae pv. tomato DC3000 infected (Inf) plants. (B) Representative images of stomata of control and HB-treated plants. (C) and (D) Expression of the tomato PR1 and P23 genes in plants after treatments with the different esters. Values were normalized to Actin. Expression levels are represented as mean ± SD of three biological replicates of one representative experiment. Double asterisk (^**) and triple asterisk (^***) indicate statistically significance differences between control and treated plants with p < 0.01 and p < 0.001, respectively.

Figure 2

Effect of GLV ester treatments on resistance to Pst and involvement of SA on stomatal closure defense. (A) Growth of Pst on leaves of control and treated-plants with (Z)-3-hexenyl acetate (HA), (Z)-3-hexenyl propionate (HP), (Z)-3-hexenyl butyrate (HiB) and (Z)-3-hexenyl-butyrate (HB). Plants were treated or not (Control) for 24 h with HA, HP, HiB or HB, and then infected with Pst. Samples were collected at 24 h post-infection and CFU were measured. Each bar represents the mean ± SD of three biological replicates of one representative experiment. (B) Representative symptomatology of control and HB-treated tomato plants at 3 days post inoculation with Pst. (C) and (D) Effects of treatments with (Z)-3-hexenyl-butyrate (HB) on the stomatal aperture (C) and on the resistance against Pst (D) of Moneymaker and NahG leaves. Plants were treated (HB) or not (CONTROL) for 24 h with HB, and then infected with Pst. Samples were collected 24 h after treatments for the stomatal aperture analysis. The growth of Pst was evaluated 1 day upon infection, by measuring the colony forming units (CFU). Asterisk (^*) and triple asterisk (^***) indicate significance differences with respect to control plants with p < 0.05 and p < 0.001, respectively.

Figure 3

Susceptibility of antisense as-AAT1 plants to Pst. (A) Growth of Pst on leaves of Flora-Dade (FD) and antisense AAT transgenic plants (AAT). Leaf colony-forming units (CFU) of bacteria were measured in leaves from wild-type (FD) and transgenic lines AAT 3677 and AAT 3936. Bacterial growth was measured 24 h after inoculation. Results correspond to means ± SE of five independent plants from a representative experiment. Data are derived from three independent experiments. (B) Cell death of FD and antisense AAT transgenic tomato plants (AAT) infected with Pst. Cell death of tomato 24 h after challenge with virulent Pst in wild-type (FD) plants and transgenic lines AAT 3677 and AAT 3936. Cell death was measured in the form of percent ion leakage in plants treated with a mock challenge or challenged with virulent bacteria. Data are derived from three independent experiments. Asterisks (^*) and (^***) indicate significance with a p < 0.05 and p < 0.001 with respect to WT plants, respectively.

Figure 4

Stomatal opening in Flora-Dade (FD) and AAT antisense transgenic plants infected with Pst. (A) Ratio of stomatal opening (r/R) in leaves of AAT1 lines (AAT3677 and AAT3936) and FD leaves 24 h after inoculation with Pst. An ANOVA test was performed and different letters indicate the statistical significances with a p-value < 0.05 (B) Representative images of stomata of FD and AAT infected plants.

Figure 5

Growth of Pst on leaves of non-treated and HB-treated Flora Dade plants, and antisense AAT transgenic tomato plants after infiltration. Leaf colony-forming units (CFU) of bacteria were measured at 24 h after inoculation with a syringe in Flora Dade non-treated (FD) or HB-treated (FD-HB) plants and antinsense transgenic lines AAT 3677 and AAT 3936. Results correspond to means ± SE of six independent plants from a representative experiment. A t-test analysis was performed with the data coming from three independent experiments. Asterisks (^*) and (^***) indicate significance with a p-value < 0.05 and p-value < 0.001, respectively, with respect to control plants.

Figure 6

Stomatal opening in plants treated with (Z)-3-hexenyl-butyrate (HB). (A) Time course analysis of the (HB) effect on tomato stomata closure. Stomata ratios were analyzed in non-treated (control) and HB-treated (HB) tomato leaves at 0 (T0), 6, 10, 24, 48, 72 h post-treatment (h), and at 7 and 10 days post-treatment (d). (B) Effectivity of HB treatments in different species. Samples were collected 24 or 48 h after treatments. Asterisk (^*), double asterisks (^**) and triple asterisks (^***) indicate significant differences between control and HB-treated plants with p < 0.05, p < 0.01 and p < 0.001, respectively.

Figure 7

Zig-zag model for stomatal defense. Analogously to the previously proposed zig-zag model for plant immunity (Jones and Dangl, 2006), three different phases are proposed for the stomatal defense. In phase 1, upon perception of the pathogen-associated molecular patterns (PAMPs), plants close stomata in an ABA-dependent manner, contributing to the PAMP-triggered immunity (PTI). In phase 2, bacterial coronatine provokes a JA-dependent reopening of the stomata, interfering with PTI. In phase 3, the bacterial effectors are recognized by NB-LRR proteins activating effector-triggered immunity (ETI), and then the plant recloses the stomata in a GLV-dependent and SA-independent process.