Priming of Arabidopsis resistance to herbivory by insect egg deposition depends on the plant's developmental stage

Abstract

While traits of plant resistance to herbivory often change during ontogeny, it is unknown whether the primability of this resistance depends on the plant's developmental stage. Resistance in non-flowering Arabidopsis thaliana against Pieris brassicae larvae is known to be primable by prior egg deposition on leaves. We investigated whether this priming effect is maintained in plants at the flowering stage. Larval performance assays revealed that flowering plants' resistance to herbivory was not primable by egg deposition. Accordingly, transcriptomes of flowering plants showed almost no response to eggs. In contrast, egg deposition on non-flowering plants enhanced the expression of genes induced by subsequent larval feeding. Strikingly, flowering plants showed constitutively high expression levels of these genes. Larvae performed generally worse on flowering than on non-flowering plants, indicating that flowering plants constitutively resist herbivory. Furthermore, we determined the seed weight in regrown plants that had been exposed to eggs and larvae during the non-flowering or flowering stage. Non-flowering plants benefitted from egg priming with a smaller loss in seed yield. The seed yield of flowering plants was unaffected by the treatments, indicating tolerance towards the larvae. Our results show that the primability of anti-herbivore defences in Arabidopsis depends on the plant's developmental stage.

Keywords: herbivory; insect eggs; larval feeding; plant ontogeny; plant resistance; plant tolerance; priming; salicylic acid; seed yield; transcriptome.

Fig. 1.

Impact of Pieris brassicae eggs on non-flowering and flowering Arabidopsis on P. brassicae larval weight. Larval weight in mg (means ±SE) of larvae after 2 d feeding on previously egg-laden (EF, yellow) or egg-free (F, green) plants in the vegetative (left side) or reproductive stage (right side). Chronological age of plants when treated with eggs: (A) 5 weeks old; (B) 7 weeks old; (C) 9 weeks old, (D, E): 12 weeks old (performance on leaves and flowers); (F) 10 weeks old. (A-E) Plant growth and treatment under short day conditions (8 h/16 h light/dark cycle). (F) Plants grew for 7 weeks under short day conditions and were treated under long day conditions (16 h/8 h light/dark cycle; see Table 1, Exp. 1-6). Statistically significant differences between treatments (***P<0.001) and non-significant differences (‘ns’, P>0.05) are shown, as assessed with Student’s t-test; n=7–10 (plants) per treatment. Statistical details and data for larval weight measurements at different time points are listed in Supplementary Table S3.

Fig. 2.

Impact of Pieris brassicae eggs on the transcriptomes of feeding-damaged or non-feeding-damaged leaves or flowers of Arabidopsis plants in the vegetative or reproductive stage. (A) Scatter plots of PCA showing group patterns of samples from each treatment according to the first two principal components. Ellipses indicate 95% confidence intervals. (B) Number of differentially expressed genes (red: UP: up-regulated, blue: DOWN: down-regulated) for the following treatment comparisons: E versus C (orange), F versus C (green), EF versus C (yellow) and EF versus F (dark red). The length of the lines depicts the differences in the number of differentially expressed genes; n=3–5 samples per treatment. Plants in the vegetative stage were 7 weeks old; those in the reproductive stage were 12 weeks old (see Table 1, Exp. 7). The plants were exposed to P. brassicae eggs (E, orange), feeding for 1 d by P. brassicae larvae (F, green), egg deposition followed by feeding for 1 d (EF, yellow), or were left as untreated controls (C, grey).

Fig. 3

Transcriptional response of non-flowering Arabidopsis plants (vegetative stage) to Pieris brassicae eggs and larval feeding. Plants in the vegetative stage were 7 weeks old; those in the reproductive stage were 12 weeks old (see Table 1, Exp. 7). The comparisons include up-regulated (red) and down-regulated genes (blue) of the transcriptional response to the eggs (E versus C, orange), the response to 1 d of larval feeding (F versus C, green) and the response to larval feeding on leaves with and without prior egg deposition (EF versus F, dark red).

Fig. 4.

Characterization of the transcriptional priming response of Arabidopsis plants in the vegetative and reproductive stages. (A) Heatmaps of the transcriptional priming response (EF versus F) of non-flowering plants (vegetative stage) compared with (i) the transcriptional changes of the same genes in similarly treated flowering plants (EF versus F reproductive stage) and (ii) the transcriptional changes of the same genes when comparing untreated control leaves from flowering plants to untreated control leaves from non-flowering plants (Crep versus Cveg). (B) Venn diagrams showing the number of commonly up-regulated and down-regulated genes in the differentially expressed transcriptome of egg-laden, feeding-induced, non-flowering plants in the vegetative stage (EF versus F vegetative stage) and the differentially expressed transcriptome of untreated plants in the reproductive stage (Crep versus Cveg). Plants in the vegetative stage were 7 weeks old; those in the reproductive stage were 12 weeks old (see Table 1, Exp. 7). Plants were treated with Pieris brassicae larval feeding (F), eggs and subsequent larval feeding (EF), or were left as untreated controls (C). Log₂FC in expression is shown in red = up-regulation, blue = down-regulation.

Fig. 5.

Impact of Piers brassicae eggs on 11-week-old flowering and non-flowering Arabidopsis plants (see Table 1, Exp. 8). Larval weight in mg (means ±SE) of P. brassicae larvae after 2 d feeding on previously egg-laden (EF, yellow) or egg-free (F, green), non-flowering (left side) or flowering (right side), wild-type (WT) plants. Significant differences between treatments (***P<0.001) and non-significant differences (‘ns’, P>0.05, Student’s t-test) are shown. Different lowercase letters indicate significant differences between larval weights on egg-free flowering and non-flowering WT (P<0.001, Student’s t-test); n=8 plants per treatment. Statistical details are provided in Supplementary Table S3.

Fig. 6.

Transcriptional response of flowering and non-flowering Arabidopsis plants of identical chronological age, i.e. 11 weeks old (see Table 1, Exp. 8). Expression level (Log₂FC, means ±SE) of priming marker genes (PR5, PR2, PR1, CAX3, PDF1.4) and jasmonic acid- or ethylene-responsive genes (MYC2, VSP1, and PR4) in untreated, non-flowering and flowering wild-type plants [Cveg and Crep, respectively] are shown. Significant differences are indicated between treatments (**P<0.01, ***P<0.001), differences by trend (+, P<0.1) and non-significant differences (‘ns’, P>0.1), as assessed with Student’s t-test; n=8–10 (plants) per treatment. Statistical details are provided in Supplementary Table S2. C: untreated control; veg: plants in the vegetative stage; rep: plants in the reproductive stage.

Fig. 7.

Impact of Pieris brassicae eggs on 7-week-old flowering and non-flowering Arabidopsis plants (see Table 1, Exp. 9). (A, B) Larval weight in mg (means ±SE) of P. brassicae larvae after (A) 2 d, and (B) 6 d of feeding on previously egg-laden (EF, yellow) or egg-free (F, green), non-flowering (left side) or flowering (right side), plants. Significant differences between treatments (***P<0.001) and non-significant differences (‘ns’, P>0.05, Student’s t-test) are shown. Different lowercase letters indicate significant differences between larval weights on untreated controls of non-flowering, flowering WT, and svp-32, plants (P<0.05, Student’s and Welch t-test). P values were FDR corrected; n=8 (plants) per treatment. Statistical details are provided in Supplementary Table S3.

Fig. 8.

Transcriptional response of flowering and non-flowering Arabidopsis plants of identical chronological age, i.e. 7 weeks old (see Table 1, Exp. 9). Expression level (Log₂FC, means ±SE) of priming marker genes (PR5, PR2, PR1, CAX3, PDF1.4) and jasmonic acid- or ethylene-responsive genes (MYC2, VSP1 and PR4) in flowering and non-flowering wild-type (WT) and flowering svp-32 mutant plants after treatment with Pieris brassicae larval feeding with (EF) or without (F) prior P. brassicae egg deposition, or plants were left untreated (C). Different letters above the bars indicate significant differences between the treatments for each WT stage and svp-32 (P<0.05; ANOVA with Tukey’s test post-hoc); Significant differences are shown between untreated controls of non-flowering WT compared to flowering WT and svp-32 (**P<0.01, ***P<0.001) and non-significant differences (‘ns’, P>0.05), as assessed with Student’s t-test and FDR P value adjustment; n=7–8 (WT plants) and 5–8 (svp-32) plants per treatment. Statistical details are provided in Supplementary Table S2.

Fig. 9.

Impact of Pieris brassicae egg deposition and larval feeding damage on total seed weight of subsequently regrown Arabidopsis plants. (A) Treatments of leaves with eggs and feeding in the vegetative, non-flowering plant stage (left, 7-week-old plants) or in the reproductive, flowering plant stage (right, 12-week-old plants). Plants produced seeds in climate chambers under long day conditions (see Table 1, Exp. 10). The treatments effects were statistically evaluated with a linear mixed model with eggs, feeding and their interaction as fixed factors and experimental block as random factor. The asterisks represent significant effects of the factors (***P<0.001, *0.01<P<0.05), non-significant differences (ns, P>0.05); n=6–10 (plants) per treatment. (B) Plants were treated in the vegetative stage and were 7 weeks old. In contrast to the experiment shown in Fig. 9A, the leaf tissue that had been left after larval feeding was cut away. After recovery, plants produced seeds in the greenhouse under long day conditions (see Table 1, Exp. 12). Total seed weight per plant in mg (means ±SE); plants were treated with P. brassicae eggs (E, orange bars), P. brassicae larval feeding (F, green bars), with eggs followed by feeding (EF, yellow bars), or were left untreated as controls (C, grey bars). Non-significant differences (‘ns’ P>0.05) are shown, as assessed with Student’s t-test; n=9–10 (plants) per treatment. Statistical details are listed in Supplementary Table S2.