(E)-Nerolidol is a volatile signal that induces defenses against insects and pathogens in tea plants

Abstract

Plants release large amounts of volatile organic compounds (VOCs) in response to attackers. Several VOCs can serve as volatile signals to elicit defense responses in undamaged tissues and neighboring plants, but many questions about the ecological functions of VOCs remain unanswered. Tea plants are impacted by two harmful invaders, the piercing herbivore Empoasca (Matsumurasca) onukii Matsuda and the pathogen Colletotrichum fructicola. To determine the VOC signals in tea, we confirmed CsOPR3 as a marker gene and set up a rapid screening method based on a 1.51 kb CsOPR3 promoter fused with a β-glucuronidase (GUS) reporter construct (OPR3p::GUS) in Arabidopsis. Using this screening system, a terpenoid volatile (E)-nerolidol was identified as a potent signal that elicits plant defenses. The early responses triggered by (E)-nerolidol included the activation of a mitogen-activated protein kinase and WRKY, an H₂O₂ burst, and the induction of jasmonic acid and abscisic acid signaling. The induced plants accumulated high levels of defense-related chemicals, which possessed broad-spectrum anti-herbivore or anti-pathogen properties, and ultimately triggered resistance against Empoasca onukii and Colletotrichum fructicola in tea. We propose that these findings can supply an environmentally friendly management strategy for controlling an insect pest and a disease of tea plants.

Keywords: Plant biotechnology; Plant immunity.

Fig. 1. Protein and transcript levels of CsOPR3 in tea leaves under different treatments.

a Western blot analysis of CsOPR3 accumulation in tea leaves subjected to the following treatments: mechanical wounding (W), jasmonic acid (JA, 150 μg ml⁻¹), and E. onukii (TLH) infestation. b Relative expression level of CsOPR3 in TLH-infested tea leaves and controls (Con). Values are means + SEs for five independent biological replicates. Asterisks indicate significant differences between TLH-treated and control plants (t test, *P < 0.05; **P < 0.01)

Fig. 2. Analysis of the products of reactions catalyzed by the CsOPR3-His protein.

a SDS-PAGE analysis of recombinant CsOPR3. The CsOPR3-His protein was expressed in E. coli and purified by ion-exchange chromatography. 1 molecular marker, 2 crude protein not induced by IPTG, 3 crude protein induced by IPTG, and 4 purified enzyme. b Total ion chromatograms of the products of the reactions of enantiomeric cis-OPDA with the CsOPR3-His protein. c Total ion chromatograms of products of the reaction of enantiomeric cis-OPDA with the control protein. peak 1: (+)-cis-OPC-8:0; peak 2: (−)-cis-OPC-8:0; peak 3: (+)-cis-OPDA; peak 4: (−)-cis-OPDA. d Relative OPDA consumption by the soluble protein fraction with or without the recombinant CsOPR3 protein. The reactions were carried out in 0.5 ml assay solution at 25 °C for 0.5 h with (+)-cis-OPDA or (−)-cis-OPDA as a substrate

Fig. 3. Complementary experimental analysis of wound-induced JA levels in the Arabidopsis opr3 mutant following the overexpression of CsOPR3.

a Growth phenotypes of WT, opr3 and opr3-overexpressing CsOPR3 (oeL1 and oeL2) lines in 20-day-old plants. b Identification of transgene copies in the two independent transgenic lines (oeL1 and oeL2). Total genomic DNA was extracted from transgenic Arabidopsis leaves and digested using EcoR I (E) or Xba I (X). (c) Confirmation of CsOPR3 expression in Arabidopsis WT, opr3, oeL1, and oeL2 lines using RT-PCR. d Endogenous JA contents in Arabidopsis WT, opr3, oeL1, and oeL2 leaves (20 days old) with or without wounding treatment. The values are the means + SEs for five independent biological replicates. Letters indicate significant differences among JA levels in different Arabidopsis lines (ANOVA, P < 0.05).

Fig. 4. CsOPR3 and jasmonic acid (JA) are involved in the defense of tea plants against C. fructicola.

a, b Transcript level and protein levels of CsOPR3 in tea leaves after C. fructicola treatment (Cfr). c, d Contents of JA (c) and JA-Ile (d) in tea leaves after C. fructicola treatment (Cfr) or the controls (Con). Values are presented as the means + SEs for five biological replicates. e Trypan blue staining for cell death in tea leaves at 6 days post inoculation. ddH₂O, tea plants treated with ddH₂O as the control for Cfr treatment; Buf, tea plants treated with 50 mM sodium phosphate buffer of pH 8 as the control for JA treatment; Cfr, tea plants infected with C. fructicola suspension at a concentration of 2 × 10⁵ spores per ml; JA + Cfr, tea plants treated with both JA and C. fructicola. Asterisks indicate significant differences between Cfr-treated and control plants (t test, *P < 0.05; **P < 0.01).

Fig. 5. GUS activities in OPR3p::GUS transgenic Arabidopsis plants.

a Diagram of the OPR3p::GUS fusion vector. The number indicates the CsOPR3 5’ promoter end point relative to the transcriptional start site. b Growth phenotypes of WT plants and OPR3p::GUS lines (pL1-1 and pL2-5) at 20 days of age. c, d GUS staining (c) and GUS activity (d) in 15-day-old OPR3p::GUS Arabidopsis seedlings, 12 h after treatment with jasmonic acid (JA), buffer (Buf), or the controls (Con). Values are presented as the means + SEs for five biological replicates. e GUS staining of 15-day-old OPR3p::GUS Arabidopsis seedlings 2 h after exposure to (E)-nerolidol or the controls (Con). f Western blot analysis of the accumulation of CsOPR3 in tea leaves exposed to (E)-nerolidol or the controls (Con). Letters indicate significant differences among treatments (ANOVA, P < 0.05).

Fig. 6. (E)-Nerolidol regulates the mitogen-activated protein kinase (MAPK) and WRKY genes in tea plants.

a, b Transcript levels of CsMAPK (a) and CsWRKY3 (b) in tea leaves exposed to (E)-nerolidol and the controls (Con). Values are presented as the means + SEs for five biological replicates. c Western blot analysis of the protein accumulation of CsMAPK and CsWRKY3 in tea leaves exposed to (E)-nerolidol. Asterisks indicate significant differences between tea leaves treated with (E)-nerolidol and the controls (t test, *P < 0.05; **P < 0.01).

Fig. 7. Effect of (E)-nerolidol on the levels of signaling molecules related to defense in tea plants.

a H₂O₂ contents in control (Con) and (E)-nerolidol-treated plants. The inset shows the DAB staining of H₂O₂ levels in tea leaves exposed to (E)-nerolidol and the controls (Con). b–e Contents of JA (b), JA-Ile (c), ABA (d), and SA (e) in control (Con) and (E)-nerolidol-treated plants. Values are presented as the means + SEs for five biological replicates. Asterisks indicate the significant differences between (E)-nerolidol-treated plants and controls (t test, *P < 0.05; **P < 0.01).

Fig. 8. Effect of (E)-nerolidol on the defense of tea plants against E. onukii.

a–c PPO activities (a), chitinase activities (b), and callose contents (c) in tea plants under Con, Nerolidol, TLH, and Nerolidol + TLH treatments. For the Nerolidol treatment, tea plants were exposed to (E)-nerolidol for 0.5 h and ventilated for 0.5 h, and the second leaves were collected for measurement 12 h after treatment. For the TLH treatment, tea plants were infested with15 E. onukii for 12 h. For the Nerolidol + TLH treatment, tea plants were exposed to (E)-nerolidol for 0.5 h, ventilated for 0.5 h, and infested with E. onukii for 12 h. Values are presented as the means + SEs for five biological replicates. d Callose deposition in control leaves (Con) and (E)-nerolidol-treated leaves. Aniline blue was used to stain tea leaves to detect callose. Scale bars represent 50 μm. e The numbers of E. onukii female adults on (E)-nerolidol-treated plants and controls. The inset shows the percentage of TLH eggs on pairs of plants, 3 days after TLH was released. Values are presented as the means + SEs for six independent biological replicates. f The survival rates of TLH nymphs fed on (E)-nerolidol-treated plants and control plants 3 days after the nymphs were placed on the plants. Values are the means + SEs for six biological replicates. g The amount of honeydew per day per TLH female adult fed on (E)-nerolidol-treated plants or control plants. Values are the means + SEs for fifteen biological replicates. Letters represent significant differences among the four treatments (ANOVA, P < 0.05). Asterisks indicate the significant differences between the treatments and controls (t test, *P < 0.05; **P < 0.01).

Fig. 9. Effect of (E)-nerolidol on the susceptibility of tea plants to C. fructicola.

a, b PAL activity (a) and lignin content (b) in tea plants under different treatments. Con, control; Nerolidol, tea plants were exposed to (E)-nerolidol for 0.5 h, ventilated for 0.5 h, and collected for measurement 24 h after treatment; Cfr, tea plants were infected with C. fructicola for 24 h; Nerolidol + Cfr, tea plants were exposed to (E)-nerolidol for 0.5 h, ventilated for 0.5 h, and infected with C. fructicola for 24 h. c Hyphal growth of C. fructicola under (E)-nerolidol treatment compared with the controls (Con) at different time points. d The infected surface areas of leaves at 9 d after C. fructicola treatment (Cfr) or C. fructicola and (E)-nerolidol treatment (Nerolidol + Cfr). Values are the means + SEs for nineteen biological replicates. e Trypan blue staining for cell death on tea leaves at 6 d after different treatments. Con, tea plants were treated with ddH2O as controls; Cfr, tea plants were infected with a C. fructicola suspension with a concentration of 2 × 10⁵ spores per ml; Nerolidol + Cfr, tea plants were treated with (E)-nerolidol and C. fructicola. Letters indicate significant differences among treatments (ANOVA, P < 0.05). Asterisks indicate the significant differences between two different treatments (t test, *P < 0.05; **P < 0.01).