Monoterpenes Support Systemic Acquired Resistance within and between Plants

Abstract

This study investigates the role of volatile organic compounds in systemic acquired resistance (SAR), a salicylic acid (SA)-associated, broad-spectrum immune response in systemic, healthy tissues of locally infected plants. Gas chromatography coupled to mass spectrometry analyses of SAR-related emissions of wild-type and non-SAR-signal-producing mutant plants associated SAR with monoterpene emissions. Headspace exposure of Arabidopsis thaliana to a mixture of the bicyclic monoterpenes α-pinene and β-pinene induced defense, accumulation of reactive oxygen species, and expression of SA- and SAR-related genes, including the SAR regulatory AZELAIC ACID INDUCED1 (AZI1) gene and three of its paralogs. Pinene-induced resistance was dependent on SA biosynthesis and signaling and on AZI1 Arabidopsis geranylgeranyl reductase1 mutants with reduced monoterpene biosynthesis were SAR-defective but mounted normal local resistance and methyl salicylate-induced defense responses, suggesting that monoterpenes act in parallel with SA The volatile emissions from SAR signal-emitting plants induced defense in neighboring plants, and this was associated with the presence of α-pinene, β-pinene, and camphene in the emissions of the "sender" plants. Our data suggest that monoterpenes, particularly pinenes, promote SAR, acting through ROS and AZI1, and likely function as infochemicals in plant-to-plant signaling, thus allowing defense signal propagation between neighboring plants.

Figure 1.

Correlation of VOCs to SAR. The volatile emissions from DEX-treated Col-0 DEX:AvrRpm1-HA and eds1-2 DEX:AvrRpm1-HA plants were measured by GC-MS and analyzed by orthogonal partial least square regression (OPLS). The left panels ([A], [C], and [E]) represent sampling period 1 (SP1; VOCs collected 1–4 h after DEX treatment) and the right panels ([B], [D], and [F]) represent sampling point 2 (SP2; VOCs collected 4–7 h after DEX treatment). (A) and (B) OPLS score plots (green circles, Col-0; red triangles, eds1-2). The ellipses indicate the model tolerance based on Hotelling’s t² with a significance level of a = 0.05. Each circle represents an individual measurement of leaf volatiles, given as VOC emission rate (pmol m⁻² s⁻¹). (C) and (D) OPLS loading plots shown with the correlation scaled. The outer and inner ellipses indicate 100% and 75% of explained variance, respectively. Circles represent the X-loadings (VOCs) and squares depict the Y-loadings (plant genotype). (E) and (F) Correlation coefficient plots of the VOC emission rates correlating VOC emissions with eds1-2. The correlation coefficients are scaled and centered, and the error bars are derived from the jackknife method. Bars represent the average ± se of 18 to 21 replicates. (A) to (F) OPLS model fitness for both the sampling periods (SP1/SP2): r² (x) = 61/54%, q² (cum) = 56/34% using 1 predictive component. RMSEE (root mean square error of estimation) = 0.35/0.31; RMSEcv (root mean square error of cross-validation) = 0.43/0.47; P = 0.012/<0.05 CV-ANOVA. Significant differences in VOC emissions between Col-0 and eds1-2 in (C) to (F) are highlighted in red (Student’s t test, P < 0.05). PC, principal component; α-Pin, α-pinene; β-Pin, β-pinene; Cam, camphene; Iso, isopropyl palmitate; Sab, sabinene. Numbers in (B) to (F) refer to tentatively identified VOCs (Supplemental Table 1).

Figure 2.

Structures and Emission Rates of VOCs Potentially Related to SAR. (A) Chemical structures of the VOCs used in this study. Structures were taken or adapted from Chemspider. (B) to (F) Emission rates of α-pinene (B), β-pinene (C), camphene (D), sabinene (E), and isopropyl palmitate (F) from DEX-treated Col-0 DEX:AvrRpm1-HA (Col-0) and eds1-2 DEX:AvrRpm1-HA (eds1-2) plants during sampling period 1 (SP1; 1–4 h after the DEX treatment) and SP2 (4–7 h after the DEX treatment). Emission rates are plotted relative to the projected rosette areas of the emitting plants. Bars represent the average of 18 to 21 biologically independent replicates as defined in the Methods ±se. Asterisks indicate statistically significant differences from the Col-0 control (Student’s t test, P < 0.05).

Figure 3.

Monoterpene-Induced Resistance in Arabidopsis against Pst. (A) and (C) Plants were exposed as described in the Methods to hexane (negative control; Mock), 1.6 µmole of MeSA (positive control), or different concentrations of a mixture (±)α-pinene and (−)β-pinene (1:1 v:v) (Pin; [A]) or camphene (Cam; [C]) as indicated below the panels. (B) Plants were exposed to hexane (Mock) for 3 d or to 0.6 µmole of Pin for 1, 2, or 3 d followed by 2, 1, or 0 d in the growth chamber (Air) as indicated below the panel. (D) Plants were exposed to hexane or to 0.6 µmole of Pin, 0.1 µmole of Cam, or 0.6 µmole of Pin + 0.1 µmole of Cam (Pin + Cam). (E) and (F) Plants were exposed to hexane or to 0.6 µmole of the following pinenes (Pin): (±)α-pinene and (−)β-pinene (1:1 v/v) [(±)α/(−)β], (±)α-pinene, and (+)β-pinene (1:1 v/v) [(±)α/(+)β], (±)α-pinene [(±)α], or (−)β-pinene [(−)β] as indicated below the panels. (A) to (F) After 3 d of treatment, the plants were inoculated with Pst, and the resulting in planta Pst titers were determined at 4 dpi. Bars represent the average of three replicates ± sd, and asterisks ([A] to [C] and [E]) indicate significant differences from the mock controls (Student’s t test, P < 0.05). Different letters above the bars in (D) indicate statistically significant differences (Student’s t test, P < 0.05). These experiment were repeated two ([B] and [D]) to at least three times ([A], [C], [E], and [F]) with comparable results.

Figure 4.

Monoterpene-Induced Resistance Related to SA Signaling. Plants were exposed to hexane (negative control; Mock), 1.6 µmole of MeSA (positive control), or 0.6 µmole of (±)α-pinene:(−)β-pinene (1:1 v/v) (Pin) as described in Methods. (A) and (B) After 3 d, the treated Col-0, eds1-2, npr1-1 (A), and sid2-1 (B) plants were inoculated with Pst, and the resulting in planta Pst titers were determined at 4 dpi. Bars represent the average of three replicates ± sd, and asterisks indicate significant differences from the mock controls (Student’s t test, P < 0.05). (C) After 3 d of treatment, PR1 transcript accumulation was determined relative to that of UBIQUITIN in Col-0 plants by RT-qPCR. Bars represent the average of three replicates ± sd. These experiments were repeated at least three times with comparable results.

Figure 5.

Pinene-Triggered Changes in the Arabidopsis Gene Expression Profile. (A) Significantly enriched GO terms (TAIR) among the upregulated genes obtained from microarray data (P < 0.05 and log₂ fold change >1). The circle size indicates the number of genes annotated with the respective term. Identical color indicates related GO terms originating from a specific ancestral node. (B) Heat map of all regulated genes (P < 0.05 and absolute log₂ fold change > 1) with consistent direction of change across the four biological replicates. Asterisks indicate genes, whose transcriptional regulation by the pinene mixture was confirmed by RT-qPCR (Supplemental Figure 5).

Figure 6.

Monoterpene-Induced Resistance in Col-0 Wild-Type, azi1-2, and gly1-3 Mutant Plants. The plants were exposed to hexane (negative control/mock) or to 0.6 µmole of (±)α-pinene:(−)β-pinene (1:1 v/v) (Pin) as described in Methods. After 3 d, the plants were inoculated with Pst, and the resulting in planta Pst titers were determined at 4 dpi. Bars represent the average of three replicates ± sd, and asterisks indicate significant differences from the mock controls (Student’s t test, P < 0.05). This experiment was repeated three times with comparable results.

Figure 7.

Superoxide Anion Radical Accumulation in Response to Pinene Treatment. Col-0 wild-type plants were exposed to hexane (negative control; Mock) or to 0.6 µmole of (±)α-pinene:(−)β-pinene (1:1 v/v) (Pin) as described in Methods. After 3 d, the accumulation of superoxide anion radicals (O_2·−) was visualized with nitroblue tetrazolium (A). The pixel intensity of the pinene-treated leaves was quantified relative to that of the mock-treated leaves, which was set at 100% (B). Data in (A) and (B) stem from two out of four biologically independent replicate experiments with comparable results. Black bars in (A) indicate 1 cm. Bars in (B) represent the average of 10 replicates ± se, and asterisks indicate a statistically significant difference from the Mock control (Student’s t test, ***P < 0.0001).

Figure 8.

The Prenyltransferase GGR Is Essential for SAR. (A) GGR transcript accumulation in wild-type, ggr1-1, and ggr1-2 plants was determined relative to that of UBIQUITIN by RT-qPCR. Bars represent the average of three replicates ± sd. (B) SAR in wild-type, ggr1-1, and ggr1-2 plants. Plants were treated in two lower leaves with 10 mM MgCl₂ (Mock; M) or with Pst AvrRpm1 (SAR; S). Three days later, the systemic (2nd) leaves were inoculated with Pst, and the resulting in planta Pst titers were determined at 4 dpi. (C) Monoterpene-induced resistance in wild-type, ggr1-1, and ggr1-2 plants. Plants were exposed to hexane (negative control; Mock), 1.6 µmole of MeSA (positive control), or 0.6 µmole of (±)α-pinene:(−)β-pinene (1:1 v/v) (Pin) as described in Methods. After 3 d, the plants were inoculated with Pst, and the resulting in planta Pst titers were determined at 4 dpi. (D) Chemical complementation of the SAR-deficient phenotype of ggr1-1 plants with pinene. As a primary treatment (1°), Col-0 wild-type and ggr1-1 plants were either treated as in (B) in the lanes marked with M and S or left untreated (−). Simultaneously, plants were exposed to hexane (Mock; M) or Pin for 3 d (3d) as in (C) or to Pin for 1 d either on the first (T1), second (T2), or third (T3) day of the normal treatment. Subsequently, all plants were inoculated with Pst, and the resulting in planta Pst titers were determined at 4 dpi. Bars in (B) to (D) represent the average of three replicates ± sd, and asterisks indicate significant differences from the mock controls (Student’s t test, P < 0.05). These experiments were repeated two ([A] and [D]) to at least three times ([B] and [C]) with comparable results.

Figure 9.

Monoterpenes Are Important for Plant-to-Plant SAR Signaling. Eight wild-type receiver plants were incubated in gas-tight desiccators together with 12 mock-treated (M) or Pst AvrRpm1-infected (S) Col-0 wild-type, eds1-2, or ggr1-1 sender plants as indicated below the panel. After 3 d, the receiver plants (recipients) were inoculated with Pst, and the resulting in planta Pst titers were determined at 4 dpi. Bars represent the average of four replicates ± sd, and the asterisk indicates a significant difference from the mock control (Student’s t test, P < 0.005). This experiment was repeated three times with comparable results.

Figure 10.

Arabidopsis Monoterpene Emissions Are Induced by Pst AvrRpm1 and Dependent on GGR. (A) to (C) Emission rates of α-pinene (A), β-pinene (B), and camphene (C) from Col-0 wild-type and ggr1-1 mutant plants 1 d before (T0) and during the first (T1), second (T2), and third (T3) day after spray inoculation of the plants with Pst AvrRpm1 or a corresponding mock treatment. VOCs were collected during the day for 8 h per sampling period. Emission rates are plotted relative to the projected rosette areas of the emitting plants. Bars represent the average of 6 to 7 (Col-0 Mock) to 9 (Col-0 Pst AvrRpm1) or 10 (ggr1-1 Pst AvrRpm1) biologically independent replicates as defined in the Methods ±se. Asterisks indicate statistically significant differences to the corresponding mock controls (Student’s t test, P < 0.05). ND, not detectable. (D) PCA biplot of α-pinene, β-pinene, and camphene emission rates (pmol m⁻² s⁻¹) from Pst AvrRpm1-infected Col-0 (green) and ggr1-1 (red) plants and from mock-treated Col-0 plants (gray) at T0 (circles), T1 (diamonds), T2 (squares), and T3 (triangles). The ellipses denote 100, 75, and 50% (outer to inner, respectively) explained variance. The arrows were added to indicate the directions of the VOC variables (in black squares) projected into the 2-d plane of the biplot. The variances explained by principal components (PC) 1 and 2 are given in parentheses.

Figure 11.

Working Model of the Role of Pinenes in Plant Immunity. Pinenes accumulate downstream of EDS1 and trigger immunity via EDS1, SA biosynthesis (possibly via CBP60g), and NPR1-mediated SA signaling. Also, pinenes trigger the accumulation of superoxide anion radicals (O_2·−) that might themselves induce immunity or induce the accumulation of AzA promoting SAR together with AZI1 and EARLI1. Finally, pinenes induce the expression of AZI1 and its paralogs EARLI1, AZI3, and AZI4 and act through AZI1 to enhance immunity. Established interactions are depicted in black, and hypothetical interactions are depicted in gray. Solid arrows indicate induction or activation, broken arrows indicate signaling, and the rounded arrows indicate the EDS1-SA positive feedback loop. Proteins are circled. Genes are in italics, and compounds are in plain lettering.