Priming of Plant Resistance to Heat Stress and Tomato Yellow Leaf Curl Thailand Virus With Plant-Derived Materials

Abstract

Plants are often simultaneously exposed to diverse environmental stresses, and can tune suitable responses to them through hormones. Salicylic acid (SA) and jasmonic acid (JA) signaling pathways are known to enhance resistance against heat stress and tomato yellow leaf curl Thailand virus (TYLCTHV) infection. However, there is limited information regarding alternative natural priming agents against heat stress and viruses. In this study, two plant-derived priming agents, eugenol and anise oil, were tested for their roles in conferring thermotolerance and virus resistance in tomato plants. Under heat stress, the survival rates and average fresh weight were higher in plants treated with eugenol or anise oil than in control plants. These two priming agents were further tested for antiviral activities. After TYLCTHV infection, the disease incidence and relative abundance of TYLCTHV were lower in anise oil- and eugenol-treated plants than in control plants. Further analyses revealed that a few SA, JA, and RNA silencing genes were enhanced in the former. Moreover, SA, JA, and H₂O₂ contents increased considerably after eugenol and anise oil treatments. Our findings imply that anise oil and eugenol initiated SA- and JA-mediated defenses to promote thermotolerance and antiviral responses of tomato plants.

Keywords: anise oil; eugenol; jasmonic acid; salicylic acid; thermotolerance; tomato (Solanum lycopersicum); tomato yellow leaf curl Thailand virus (TYLCTHV).

FIGURE 1

Effects of the pre-application of eugenol and anise oil on the thermotolerance of tomato plants. (A) Schedule for the application of plant-derived materials, heat treatment, and harvest. Phenotypes were analyzed at the indicated time points. The concentrations of the plant-derived materials, eugenol and anise oil, were 200 μg mL^–1. After an exposure to heat stress and a 5-day recovery period, tomato plants with different treatments were photographed (B). The survival rates (C) and fresh weights (D) of these plants were also recorded based on 18 technical replicates in three independent experiments. Additionally, the relative leakage of leaf electrolytes (E) was measured after a 3-day recovery period. Bars represent the mean of three experiments ( ± standard error of the mean). Significant differences between control and anise oil- or eugenol-treated plants were assessed with Student’s t-test (^*P < 0.05; ^∗∗P < 0.01).

FIGURE 2

Effects of anise oil and eugenol on the expression of HSFs and HSPs in tomato plants under heat stress. Fourteen-day-old tomato seedlings treated with a control solution (CK), anise oil (AO), or eugenol (EG) for 24 h were subjected to heat stress at 45°C for 12 h. After heat treatment (0 hpt) and 8-h recovery (8 hpt), total RNA was extracted from these plants (A). The SlHSFA2 (B), SlHSFB1 (C), SlHSP101 (D), SlHSP90 (E), and SlHSP17.6 (F) expression levels were analyzed by RT-qPCR. The β-actin expression level was used as the internal control. Relative gene expression levels were normalized against the expression level in untreated tomato plants. Data were analyzed with the 2^{–Δ⁢Δ⁢Ct} method. Bars represent the mean ( ± standard error of the mean) of three independent biological replicates each with three technical replicates. Significant differences between control and anise oil- or eugenol-treated plants were assessed with Student’s t-test (^*P < 0.05; ^∗∗P < 0.01).

FIGURE 3

Antiviral effects of anise oil and eugenol on TYLCTHV. Tomato plants were treated with foliar applications of eugenol (100, 200, or 400 μg mL^–1) or anise oil (100, 200, or 400 μg mL^–1) 24 h before TYLCTHV inoculations. The plants were photographed at 15 days post-inoculation (dpi) (A). The disease incidence was evaluated by detecting TYLCTHV-positive plants at 15 dpi. Fisher’s least significant difference test (LSD) was employed examining significant differences (P < 0.05) among different treatments. (B). Additionally, the relative abundances of TYLCTHV DNA-A (C) and DNA-B (D) in anise oil- and eugenol-treated plants and control were estimated by a quantitative real-time PCR assay. Samples were collected every 5 days after the onset of the viral infection (0, 5, 10, and 15 dpi). Five individual samples for each treatment were analyzed. Relative TYLCTHV DNA amounts in tomato plants were normalized against the DNA content at 0 dpi. In addition, relative expression of viral RNA, AC1 (E) and BC1 (F), at 5 dpi was also analyzed by RT-qPCR. The β-actin expression level was used as the internal control. Relative gene expression levels were normalized against the expression level in control. Data were analyzed with the 2^{–Δ⁢Δ⁢Ct} method. Bars represent the mean ( ± standard error of the mean) of at least three independent biological replicates each with three technical replicates. Significant differences between control and anise oil- or eugenol-treated plants were assessed with Student’s t-test (^*P < 0.05; ^∗∗P < 0.01).

FIGURE 4

Effects of anise oil and eugenol on the expression of SA- and JA-related genes in tomato plants. Fourteen-day-old tomato seedlings were treated with a control solution (CK), anise oil (AO), or eugenol (EG). Total RNA was extracted from these plants at 8 h post-treatment (hpt) and 24 hpt (A). The expression levels of SA-related defense genes, SlPR1 (B), SlPR1b (C), SlNPR1 (D), SlAOX1a (E), and SlAOX1c (F), and JA-related defense genes, SlPI-II (G), SlPPO (H), SlMAPK3 (I), SlLoxD (J), and SlCOI1 (K), were analyzed by qRT-PCR. The β-actin expression level was used as the internal control. Relative gene expression levels were normalized against the expression level in untreated tomato plants. Data were analyzed with the 2^{–Δ⁢Δ⁢Ct} method. Bars represent the mean ( ± standard error of the mean) of three independent biological replicates each with three technical replicates. Significant differences between control and anise oil- or eugenol-treated plants were assessed with Student’s t-test (^*P < 0.05; ^∗∗P < 0.01).

FIGURE 5

Effects of anise oil and eugenol on the expression of RNA silencing genes in tomato plants. Fourteen-day-old tomato seedlings were treated with a control solution (CK), anise oil (AO), or eugenol (EG). Total RNA was extracted from these plants at 8 h post-treatment (hpt) and 24 hpt. The expression levels of RNA silencing genes, SlDCL2 (A), SlDCL4 (B), Ty-1 (C), SlRDR1 (D), SlAGO1A (E), SlAGO2A (F), SlAGO1B (G), and SlAGO2B (H) were analyzed by RT-qPCR. The β-actin expression level was used as the internal control. Relative gene expression levels were normalized against the expression level in untreated tomato plants. Data were analyzed with the 2^{–Δ⁢Δ⁢Ct} method. Bars represent the mean ( ± standard error of the mean) of three independent biological replicates each with three technical replicates. Significant differences between control and anise oil- or eugenol-treated plants were assessed with Student’s t-test (^*P < 0.05; ^∗∗P < 0.01).

FIGURE 6

Effects of anise oil and eugenol on the expression of defense-related genes in tomato plants under TYLCV infection with/without heat stress. Fourteen-day-old tomato seedlings treated with a control solution (CK), eugenol (EG), or anise oil (AO) for 24 h were inoculated TYLCTHV. Then, some plants were incubated at 45°C for 12 h. Total RNA was extracted from these plants. The expression levels of SA-related defense genes, SlPR1 (A), SlPR1b (B), SlNPR1 (C), and SlAOX1a (D), RNA silencing genes, SlDCL2 (E), SlDCL4 (F), SlAGO2A (G), and Ty-1 (H), and JA-related defense genes, SlPI-II (I) and SlMAPK3 (J), were analyzed by RT-qPCR. The β-actin expression level was used as the internal control. Relative gene expression levels were normalized against the expression level in control. Data were analyzed with the 2^{–Δ⁢Δ⁢Ct} method. Bars represent the mean ( ± standard error of the mean) of three independent biological replicates each with three technical replicates. Significant differences between control and anise oil- or eugenol-treated plants were assessed with Student’s t-test (^*P < 0.05; ^∗∗P < 0.01).

FIGURE 7

Effects of anise oil and eugenol on the endogenous SA and JA contents. Fourteen-day-old tomato seedlings were treated with a control solution (CK), eugenol (EG), or anise oil (AO). The treated leaves were harvested just before the treatments (0 h) and at 8, 24, and 48 h after treatments. The endogenous SA (A) and JA (B) contents were detected. The SA and JA levels of the untreated tomato plants was treated as the normalized reference, with a value of one. Bars represent the mean ( ± standard deviation) from two measurements (each with triplicate samples). Significant differences between control and anise oil- or eugenol-treated plants were assessed with Student’s t-test (^*P < 0.05; ^∗∗P < 0.01).

FIGURE 8

Effects of anise oil and eugenol on the accumulation of H₂O₂ and the expression of antioxidant genes in tomato plants. 3′-Diaminobenzidine (DAB) staining was used to detect H₂O₂ in control, anise oil-, and eugenol-treated tomato plants at 8 hpt and 24 hpt (A). Furthermore, H₂O₂ content was quantified by titanium chloride method (B). In addition, expression of antioxidant genes, SlAPX2 (C) and SlCAT2 (D), was also analyzed by RT-qPCR. The β-actin expression level was used as the internal control. Relative gene expression levels were normalized against the expression level in untreated tomato plants. Data were analyzed with the 2^{–Δ⁢Δ⁢Ct} method. Bars represent the mean (± standard error of the mean) of three independent biological replicates each with three technical replicates. Significant differences between control and anise oil- or eugenol-treated plants were assessed with Student’s t-test (^*P < 0.05; ^∗∗P < 0.01).