Metabolic crosstalk between hydroxylated monoterpenes and salicylic acid in tomato defense response against bacteria

Abstract

Hydroxylated monoterpenes (HMTPs) are differentially emitted by tomato (Solanum lycopersicum) plants resisting bacterial infection. We have studied the defensive role of these volatiles in the tomato response to bacteria, whose main entrance is through stomatal apertures. Treatments with some HMTPs resulted in stomatal closure and pathogenesis-related protein 1 (PR1) induction. Particularly, α-terpineol induced stomatal closure in a salicylic acid (SA) and abscisic acid-independent manner and conferred resistance to bacteria. Interestingly, transgenic tomato plants overexpressing or silencing the monoterpene synthase MTS1, which displayed alterations in the emission of HMTPs, exhibited changes in the stomatal aperture but not in plant resistance. Measures of both 2-C-methyl-D-erythritol-2,4-cyclopyrophosphate (MEcPP) and SA levels revealed competition for MEcPP by the methylerythritol phosphate (MEP) pathway and SA biosynthesis activation, thus explaining the absence of resistance in transgenic plants. These results were confirmed by chemical inhibition of the MEP pathway, which alters MEcPP levels. Treatments with benzothiadiazole (BTH), a SA functional analog, conferred enhanced resistance to transgenic tomato plants overexpressing MTS1. Additionally, these MTS1 overexpressors induced PR1 gene expression and stomatal closure in neighboring plants. Our results confirm the role of HMTPs in both intra- and interplant immune signaling and reveal a metabolic crosstalk between the MEP and SA pathways in tomato plants.

Figure 1.

Effect of monoterpenoid treatments on the defensive response of MM tomato plants. A) Stomatal aperture ratio of nontreated (NT) tomato plants or treated with α-terpineol, limonene, linalool, and 4-terpineol. Violin plots represent the stomatal aperture ratio for each treatment of a total of 40 stomata from 3 biological replicates. Different letters indicate statistically significant differences for each treatment (ANOVA, P < 0.05). B) RT-qPCR analysis of the tomato PR1 gene expression in NT tomato plants or treated with α-terpineol, limonene, linalool, and 4-terpineol. The y axis represents the value of the Ct increment (ΔΔCt). Values were normalized to Actin gene. Expression levels are represented as mean ± Sd of 3 biological replicates of 1 representative experiment. Letters represent statistically significant differences (ANOVA, P < 0.05) between treatments. Bacterial content 24 h after infection in tomato plants pretreated with 5 µM α-terpineol or NT and then inoculated by C) immersion or D) injection 1 d later. Data are presented as mean (log cfu/cm²) ± Sd of a representative experiment (n = 4; n = 5). Statistically significant differences (t test, P < 0.001) between treated and NT plants are represented by triple asterisks in C), and no statistically significant differences between treated and NT plants were observed in D).

Figure 2.

HMTP mode of action. A) Weight loss of nontreated (NT) or α-terpineol-treated (α-terpineol) tomato seedlings at different time points during 3 h. The experiment was repeated 3 times obtaining similar values, and 25 seedlings were used as described in Materials and methods (t test, P < 0.0001). B) Dose–response analysis of α-terpineol (blue), ABA (red), and mock (water, black) in stomatal aperture. Data represent the mean ± Sd of a representative experiment (n = 40). Letters indicate statistically significant differences for each treatment at each time point (ANOVA, P < 0.05). Stomatal aperture ratio mean values ± Sd of a total of 40 stomata from 3 biological replicates of NT and α-terpineol-treated tomato. Stomatal aperture after α-terpineol treatment (blue points) and NT (gray points) ratio in C) ABA-deficient tomato mutants (flacca) and the corresponding parental (Lukullus) D)NahG transgenic tomato plants impaired in SA accumulation and the corresponding parental MM. Data represent the mean ± Sd of a representative experiment (n = 50). Different letters indicate statistically significant differences for each treatment (ANOVA, P < 0.05). E) Bacterial content 24 h after infection in NahG tomato plants pretreated with 5 µM α-terpineol or NT and then inoculated by immersion 1 d later. Data are presented as mean (log cfu/cm²) ± Sd of a representative experiment (n = 4). No statistically significant differences between treated and NT plants were observed after t test.

Figure 3.

Characterization of transgenic plants with altered levels of monoterpenoids. A) DNA construction for the generation of transgenic plants RNAi_MTS1. B) Analysis of MTS1 expression by RT-qPCR of the different RNAi_MTS1 transgenic tomato lines (2.1 and 5.1) and its parental (RG) infected with bacterial (Pst) or noninoculated (Mock). RG and transgenic plants were subjected to infection with Pst by immersion. Samples were taken 24 h after the bacterial infection. The RT-qPCR values were normalized with the level of expression of Actin gene. The y axis represents the value of the Ct increment (ΔΔCt). The expression levels correspond to the mean ± Sd of a representative experiment (n = 3). Statistically significant differences (ANOVA, P < 0.05) between genotypes and Pst-infected or Mock plants are represented by different letters. Relative HMTP levels (arbitrary units [A.U.]) analyzed by GC–MS in tomato 35S:MTS1 leaves and their control transgenic plants with empty vector (MM; C) and lines of RNAi_MTS1 2.1 and 5.1 and their parental (RG; D) upon mock inoculation and Pst infection. Data are presented as means ± Sd of a representative experiment (n = 5). Statistically significant differences are represented with asterisk (*), double asterisk (**), triple asterisk (***) and quadruple asterisk (****) and indicate significant differences by t test with respect to genetic background (MM or RG) with P < 0.05, P < 0.01, P < 0.001 and P < 0.0001, respectively.

Figure 4.

Activation of the defensive response in tomato plants with altered levels of monoterpenoids. Stomatal aperture, bacterial infectivity, and PR1 gene expression were studied for transgenic tomato lines overexpressing A to C) or silencing MTS1 gene D to F). Stomatal aperture ratio mean values ± Sd of a total of 40 stomata from 3 biological replicates are shown in A), for 35S:MTS1 leaves and their control transgenic plants with empty vector (MM), and in D), for lines of RNAi_MTS1 2.1 and 5.1 and their parental (RG). Asterisks (****) indicate statistically significant differences between genotypes (t test, P < 0.0001). Growth of Pst are shown in leaves of B)35S:MTS1 plants and their parental (MM) and E) both silencing lines of RNAi_MTS1 and their parental RG. Tomato plants were inoculated with bacterial Pst by immersion, and leaf samples were taken 24 h after bacterial infection. Data are presented as means (log cfu/cm²) ± Sd of a representative experiment (n = 5 and n = 4, respectively). RT-qPCR expression analysis of the tomato PR1 gene are shown in C)35S:MTS1 plants and their parental (MM) and F) both silencing lines of RNAi_MTS1 and their parental RG. Mock represents the noninoculated plants. The y axis represents the value of the Ct increment (ΔΔCt). Values were normalized to Actin gene. Expression levels are represented as mean ± Sd of 3 biological replicates of 1 representative experiment. Statistically significant differences (ANOVA, P < 0.05) between genotypes and infected (Pst) or mock-treated plants are represented by different letters.

Figure 5.

Reduction of MEcPP levels and ICS expression in 35S:MTS1 tomato transgenic plants. A) MEcPP content was measured (nmol/g FW, fresh weight) in overexpressing 35S:MTS1 plants and their control MM transgenic plants (n = 4) carrying an empty vector (MM) upon infection with Pst. Mock represents the noninoculated plants. Statistically significant differences (ANOVA, P < 0.05) between genotypes and infected (Pst) or mock-treated plants are represented by different letters. Levels are represented as mean ± Sd of 4 biological replicates. B)ICS expression levels in infected 35S:MTS1 plants and their corresponding parentals with empty vector (MM; n = 3). The y axis represents the value of the Ct increment (ΔΔCt). Values were normalized to Actin gene. Expression levels are represented as mean ± Sd of 3 biological replicates of 1 representative experiment. Statistically significant difference (t test, P < 0.05) between treated and nontreated is represented by an asterisk (*).

Figure 6.

Reduction of MEcPP levels and ICS expression in MM tomato plants with alterations in the MEP pathway upon bacterial infection. A) MEcPP content after FSM treatment and their control nontreated (NT) plants (n = 4). The media levels of nmol MEcPP/g FW (fresh weight) ± Sd are represented, and statistically significant difference is represented by an asterisk (*; t test, P < 0.05). B)ICS expression levels after FSM (n = 3) treatment and in NT plants. The y axis represents the value of the Ct increment (ΔΔCt). Values were normalized to Actin gene. Expression levels are represented as mean ± Sd of 3 biological replicates of 1 representative experiment. Statistically significant difference (t test, P < 0.05) between treated and NT is represented by an asterisk (*).

Figure 7.

Metabolic crosstalk between monoterpenoids and SA biosynthesis. Levels of free and glycosylated salicylic (SA; left panels) and GA (right panels) were analyzed by fluorescence–HPLC in transgenic tomato plants with alterations in MTS1 expression, 24 h after bacterial (Pst) infection. A) and B) show the phenolic content in overexpressing 35S:MTS1 tomato plants and their control plants with the empty vector (MM) and C) and D) in silencing lines of RNAi_MTS1 tomato plants and their parental (RG). Mock represents the noninoculated plants. Bars represent the mean (nmol SA/g FW, fresh weight) ± Sd of total levels of a representative experiment (n = 4). Significant differences between genotypes and infected or mock-inoculated plants are represented by different letters (ANOVA, P < 0.05).

Figure 8.

Pharmacological validation of the crosstalk between monoterpenoids and SA biosynthesis. Free and glycosylated A) SA (left panels) and B) GA (right panels) levels in FSM pretreated and nontreated (NT) infected MM tomato plants. The extracts were analyzed by fluorescence–HPLC. In both figures, bars represent the mean (nmol/g FW, fresh weight) ± Sd of total levels of a representative experiment (n = 4). Statistically significant differences (t test, P < 0.05) between treated and NT are represented by asterisks (*) of total levels of a representative experiment.

Figure 9.

Role of the MEP pathway on the tomato resistance to bacteria. Tomato pretreated plants were inoculated with Pst by immersion, and leaf samples were taken 24 h after bacterial infection. Data are presented as means ± Sd of a representative experiment. Bacterial content in infected A) MM tomato plants nontreated (NT, n = 4) and pretreated with FSM (n = 4). Statistically significant differences (t test, P < 0.01) between treated and NT are represented by asterisks (**). B)35S:MTS1 transgenic tomato plants and their corresponding MM background with the empty vector (MM) NT (n = 5) and pretreated with BTH (n = 4). Data are presented as means (log cfu/cm²) ± Sd of a representative experiment. Statistically significant differences (ANOVA, P < 0.05) between genotypes and BTH-treated or NT plants are represented by different letters.

Figure 10.

Interplant communication using 35S:MTS1 plants as emitters. Tomato MM plants (“Receivers”) were placed in closed chambers in the presence of MTS1 overexpressing plants (“Emitters”) or their corresponding control plants with the empty vector MM as control Emitters, and stomatal aperture ratio of 3 biological replicates was measured in receivers of tomato plants after cohabitation for 24 h. Two emitters (2x) vs. 2 receivers were used in A), and 4 emitters (4x) vs. 2 receivers were used in B). The relative expression of tomato PR1 gene C) was analyzed by RT-qPCR in MM receiver plants after cohabitation either with 35S:MTS1 or MM emitters. Two emitters vs. 2 receivers were used. The y axis represents the value of the Ct increment (ΔΔCt). Values were normalized to Actin gene. Expression levels are represented as mean ± Sd of 3 biological replicates of 1 representative experiment. Statistically significant differences (t test, P < 0.05 or P < 0.01) between treated and nontreated plants are represented by asterisks (*) or double asterisks (**), respectively.