https://orcid.org/0000-0002-4212-3266
Abstract
Plants can improve their resistance to feeding damage by insects if they have perceived insect egg deposition prior to larval feeding. Molecular analyses of these egg-mediated defence mechanisms have until now focused on angiosperm species. It is unknown how the transcriptome of a gymnosperm species responds to insect eggs and subsequent larval feeding. Scots pine (Pinus sylvestris L.) is known to improve its defences against larvae of the herbivorous sawfly Diprion pini L. if it has previously received sawfly eggs. Here, we analysed the transcriptomic and phytohormonal responses of Scots pine needles to D. pini eggs (E-pine), larval feeding (F-pine) and to both eggs and larval feeding (EF-pine). Pine showed strong transcriptomic responses to sawfly eggs and—as expected—to larval feeding. Many egg-responsive genes were also differentially expressed in response to feeding damage, and these genes play an important role in biological processes related to cell wall modification, cell death and jasmonic acid signalling. EF-pine showed fewer transcriptomic changes than F-pine, whereas EF-treated angiosperm species studied so far showed more transcriptional changes to the initial phase of larval feeding than only feeding-damaged F-angiosperms. However, as with responses of EF-angiosperms, EF-pine showed higher salicylic acid concentrations than F-pine. Based on the considerable overlap of the transcriptomes of E- and F-pine, we suggest that the weaker transcriptomic response of EF-pine than F-pine to larval feeding damage is compensated by the strong, egg-induced response, which might result in maintained pine defences against larval feeding.
Introduction
Forests are often challenged by mass outbreaks of herbivorous insects. In addition to constitutively available resistance traits, trees have evolved multiple inducible defences to insects (Haukioja 2006, Büchel et al. 2016, Celedon and Bohlmann 2019, Whitehill et al. 2023). For example, feeding-damaged trees can enhance their levels of secondary plant compounds and the activities of enzymes that are harmful to attackers (Lämke and Unsicker 2018, Whitehill and Bohlmann 2019). Furthermore, trees are known to release damage-induced volatiles that repel herbivores or attract antagonists of feeding larvae (e.g., Mumm and Hilker 2006, Holopainen 2011, Suckling et al. 2012, Fabisch et al. 2019).
Trees do not need to ‘wait’ until they are exposed to larval feeding damage; they can defend themselves beforehand against the initial egg deposition on their leaves (Hilker and Fatouros 2015, Reymond 2022). These egg-induced tree defences act, for instance, by releasing leaf volatiles that attract egg parasitoids or by changes of leaf chemistry that are harmful to the eggs (Meiners and Hilker 2000, Hilker et al. 2005, Bittner et al. 2017). Thus, tree responses to insect eggs can reduce the number of surviving eggs.
In addition, there is increasing evidence that plant responses to insect eggs significantly improve plant defences against the impending feeding damage by hatching larvae. Larvae developing on previously egg-laden plants have been shown to gain less weight and suffer higher mortality (Hilker and Fatouros 2016, Lortzing et al. 2020). This egg-mediated, improved defence against herbivory may benefit the plant, as has been shown for Arabidopsis thaliana L. Heynh. Egg-laden and subsequently feeding-damaged A. thaliana plants produce a significantly higher seed weight when they regrow and flower after herbivory than egg-free, feeding-damaged A. thaliana (Valsamakis et al. 2022).
The transcriptomic and phytohormonal plant responses to insect egg deposition, and their effects on responses to subsequent insect larval feeding, have been well studied in angiosperm species, especially in herbaceous plants (Brassicaceae and Solanaceae), but also in a tree species, Ulmus minor L. (overview: Lortzing et al. 2020). These angiosperm species show some conserved, common transcriptomic and phytohormonal core responses to insect eggs and larval feeding (Lortzing et al. 2019, 2020, Valsamakis et al. 2020). According to De La Torre et al. (2020), gymnosperms show a 58–61% sequence similarity of expressed genes with those of angiosperms. The Coniferales, a well-studied major group of the Gymnospermae, show strong constitutive and also damage-inducible defences (Schmidt et al. 2005, Krokene 2015, Celedon and Bohlmann 2019, Whitehill and Bohlmann 2019, López-Goldar et al. 2020, Vázquez-González et al. 2020).
In the gymnosperm Pinus sylvestris L., several previous studies addressed the tree’s responses to egg deposition and larval feeding damage by the common pine sawfly Diprion pini L. (Hilker et al. 2002, Beyaert et al. 2012, Bittner et al. 2017, Blomqvist et al. 2022). The tree mounts its defences against infestation by this sawfly already after egg deposition on the needles. The egg phase takes about 2 weeks until the larvae hatch. Egg deposition by this sawfly is linked with pine needle damage. During oviposition, the female saws a longitudinal slit into the needle with its chitinous ovipositor valves and releases the eggs in a row into the slit. The mechanical slitting by the sawfly’s ovipositor alone does not induce the release of needle volatiles that attract egg parasitoids. However, the sawfly’s subsequent insertion of the eggs, which are covered with an egg secretion, induces the emission of terpenoids, which then attract egg parasitoids that kill the sawfly eggs (Hilker et al. 2002). A recent study showed that the elicitor of this indirect pine defence is an annexin-like protein, which is associated with the egg secretion that the sawfly female releases with her eggs into the needle pouch (Hundacker et al. 2022). In addition to this indirect defence, egg-laden Scots pine needles accumulate greater quantities of hydrogen peroxide, which might either directly harm the sawfly eggs or induce further pine reactions (such as lignification of needle tissue), which ultimately hinder egg survival (Bittner et al. 2017, 2019).
In addition to these pine defences targeting sawfly eggs, pine responses to D. pini eggs have also been shown to significantly impair the performance of sawfly larvae. When D. pini larvae feed upon pine with prior sawfly egg deposition, they suffer higher mortality and gain less weight than larvae feeding upon egg-free pine (Beyaert et al. 2012). These findings suggest that pine takes the egg deposition by D. pini as a ‘warning’ of impending larval herbivory and subsequently improves its anti-herbivore defences against the larvae.
However, the molecular mechanisms resulting in this ecological effect, especially the transcriptomic and phytohormonal responses of pine as a gymnosperm species are currently unknown. Here, we asked whether and how these responses differ from those of angiosperm species to insect eggs and subsequent larval feeding. Therefore, we studied the transcriptomic and phytohormonal changes of P. sylvestris exposed to D. pini eggs only, to larvae only, or to both eggs and subsequent larval feeding. With respect to the phytohormone analyses, we focused on salicylic acid (SA), jasmonic acid (JA), JA-isoleucine (JA-Ile) and abscisic acid (ABA). Quantitative analyses of the transcriptomes, especially Gene Ontology (GO) term analyses, provided insights into possible biological processes that might be involved in pine responses to eggs and larvae. Analyses of samples exposed to the same treatment and harvested after different lengths of time helped us to elucidate the dynamics of pine responses. Analyses of samples exposed to different treatments allowed us to detect similarities and differences between pine responses to sawfly eggs and larvae, as well as to uncover the effects that pine responses to eggs had on subsequent responses to feeding damage.
Materials and methods
Plants and insects
For the transcriptomic analysis, 3-year-old P. sylvestris trees (not taller than 50 cm) were acquired from a tree nursery (Schlegel & Co., Riedlingen, Germany). For the phytohormone and qPCR analysis, 3-year-old P. sylvestris trees were obtained from a forest northeast of Berlin, Germany (53°08′36.0″N 13°33'56.2″E). Trees of this age are known to show defensive responses to D. pini eggs (Bittner et al. 2019). In European forests, young trees as well as older ones up to 140 years were found to be infested by D. pini (Brauns 1991). Needles from both the nursery trees and the forest trees were of the Δ-3-carene chemotype (Thoss et al. 2007), as tested by gas chromatography–mass spectrometry analyses of the needles (data not shown).
Our experimental trees grew in pots filled with potting soil Classic T (Einheitserde, Uetersen, Germany). When potting the trees, we very gently placed the roots of the young trees into the pots, thus paying attention to avoid damage of the roots. Prior to the experiments, all trees were first kept in a greenhouse under long-day conditions (18 h:6 h light:dark, average temperature 20 °C) for at least 2 months. At least 3 days prior to treatments with eggs and/or larvae, the potted trees were transferred to a climate chamber for acclimation to the experimental abiotic conditions (20 °C, 18 h:6 h light:dark, 70% relative humidity, 100-μmol photons m−2 s−1).
Diprion pini was reared according to established protocols of Bombosch and Ramakers (1976) and Eichhorn (1976) with minor changes. Branches from P. sylvestris trees (at least 10 years old) were cut in forests in the surroundings of Berlin. Prior to offering them to D. pini, they were kept in water and stored in a cool climate chamber (10 °C, 18 h:6 h light:dark, 70% relative humidity, 100-μmol photons m−2 s−1). For D. pini rearing, the branches were transferred into a warm climate chamber (20 °C, 18 h:6 h light:dark, 70% relative humidity, 100-μmol photons m−2 s−1). Here, the branches were offered to D. pini adults for mating and egg deposition. The egg incubation time until hatching of larvae takes 10–14 days under the abiotic conditions used here. Diprion pini larvae fed upon the needles of these pine branches. They progress through five (male) to six (female) larval stages until pupation. Each pupa was placed individually in a small glass vial (5 ml) that was closed with a perforated lid. The pupae were kept in darkness at 7 °C until needed for further rearing or for the experiments.
To obtain adults for further rearing, the pupae were transferred to a warm climate chamber (20 °C, 18 h:6 h light:dark, 70% relative humidity, 100-μmol photons m−2 s−1). Adults emerging from the pupae were exposed to pine branches again for further rearing.
To obtain age-synchronized adults for starting the treatment of experimental trees, we also transferred a set of the individually kept pupae from the cool climate chamber to the warm chamber. Since the adults emerged in the small vials, males and females could not mate prior to their exposure to experimental trees. We only used adults that were not older than 5 days for the experiments.
Plant treatments
All plant treatments were conducted in a climate chamber at 20 °C, 18 h:6 h light:dark, 70% relative humidity, 100-μmol photons m−2 s−1. For the treatment, an acclimatized, potted tree was placed in a PLEXIGLAS cylinder (60-cm height, 9.5 L). The cylinder was closed at the bottom and the top with a PLEXIGLAS lid. The lids had small openings for insertion of a tube through which charcoal-filtered air was introduced into the cylinder from the bottom and allowed to leave the cylinder from the top (airflow about 200 mL × min−1).
Each tree was exposed to D. pini egg deposition (E), to D. pini larval feeding (F) or to both egg deposition and subsequent larval feeding (EF). We also kept trees untreated for control (C) in PLEXIGLAS cylinders. We simultaneously placed E-, F-, EF- and C-trees (n = 5 of each type) in the climate chamber and collected their needles after a certain treatment period (Figure 1). For each treatment period, a new set of trees was treated, and new control trees were included. Two experiments were conducted, one for harvesting needles for the RNA sequencing analysis and another one for the qPCR and phytohormone analysis. The schedule for needle harvesting after different treatment periods is outlined below (Figure 1, section ‘sampling of needle material’).
Scheme of P. sylvestris treatments and sampling time points. Needles of 3-year-old P. sylvestris trees were treated with natural egg deposition by D. pini (E-pine), larval feeding (F-pine) or natural egg deposition with subsequent feeding (EF-pine). Untreated control (C-pine) trees (grey arrow, no treatment) were included into the experiments. Needles were harvested from E-, F-, EF- and C-pine at different time points after treatments. A new set of trees was used for each sampling time point, thereby avoiding the possibility that sampling at an early time point affects the tree’s response at a later time point. Needles were harvested at 1 h, 24 h and 10 days after egg deposition (yellow arrow). Eleven days after egg deposition, which is an early possible hatching time point after development of D. pini eggs under the abiotic conditions used, 10 D. pini larvae were placed each on egg-free and previously egg-laden pine trees (green arrow and brown arrow, respectively). Needles were harvested after a 1- and 24-h larval feeding period. At equivalent time points, we also harvested needles from egg-laden E-pine trees that had not received any larvae. Needles from control pine trees were harvested at all above-mentioned sampling time points. For the RNA sequencing and phytohormone analysis, n = 4–5 trees were used for each treatment and time point. For the qPCR, we used n = 3–5 trees per treatment and time point.
To obtain egg-treated (E) pine, two virgin male and two virgin female adults were placed on a tree and left there for 24 h to allow mating and egg deposition on the pine needles. Thereafter, the adults were removed, and the egg-laden pine was left in the cylinder for the treatment periods outlined in Figure 1. The natural egg incubation time of D. pini takes about 11–14 days under the abiotic condition used here.
To obtain pine exposed to larval feeding damage (F-treatment), 10 young larvae (L2 to L3) were taken from ‘provider’ trees and placed on the needles of egg-free pine. No first instar larvae (L1) were transferred to the experimental trees; these larvae are too vulnerable and mortality was always high after transfer. Pine needles with larvae were in a position equivalent to those where females had deposited their eggs on trees in the E-treatment setup.
To obtain pine exposed to eggs and standardized larval feeding (EF-treatment), trees were first exactly treated as E-pine. On Day 11 after experimental start, we placed 10 young larvae (L2 to L3) on the trees. Thus, the larvae were placed here briefly before the egg incubation time ended and before larvae would hatch naturally (Figure 1). If larvae had already hatched naturally from the eggs laid on a tree, this tree was excluded from the experiment.
This experimental procedure allowed us to standardize the onset of larval feeding as well as the number of feeding larvae in the F- and EF-treatment.
Sampling of needle material
We harvested locally treated needles from E-, F- and EF-pine trees and from the respective control C-pine trees after different treatment periods (Figure 1). The entire treatment period lasted 12 days. Needles were always harvested during daytime (9:00–12:00 h).
Egg-laden needles were harvested 1 and 24 h after egg deposition to analyse early responses to eggs. Furthermore, egg-laden needles were harvested toward the end of the egg phase, i.e., 10 days after egg deposition, to determine transcriptomic pine responses just prior to larval hatching.
On Day 11, larvae were transferred to the plants and could feed there for either 1 or 24 h. Feeding-damaged needles were harvested 1 and 24 h after the onset of feeding damage from F-trees and EF-trees. Additionally, we sampled needles from E-trees at time points equivalent to those at which needles were sampled from F- and EF-trees; we collected needles only from those E-trees from which no larvae had hatched yet.
Needles from the untreated control (C) trees were harvested at the same time points and from equivalent positions as needles that were taken from E-, F- and EF-trees.
The harvested needles were frozen in liquid nitrogen and stored at −80 °C. Frozen needles were ground to a fine powder under liquid nitrogen with heat-sterilized mortars. We ground the entire intact needles from C-trees and the entire locally treated needles from F- and EF-trees. The egg-laden needles from E-trees were processed by cutting out the egg row and grinding only needle parts with a length of 2 cm maximum directly next to both sides of the egg rows. Needles were kept frozen during this process to exclude responses to the mechanical removal of the egg rows from the needles.
For the RNA sequencing and phytohormone analyses, we obtained n = 4–5 samples, and for the qPCR analyses, we had n = 3–5 samples of each treatment (E, F, EF and C) and each sampling time point. For the vast majority of treatments and time points, we obtained n = 5 samples as expected from the number of trees used. The irregular number of replicates is due to the rare exclusion of trees from sampling because (i) larvae hatched earlier than 10 days after egg deposition, (ii) larvae escaped from treated needles or died for unknown reasons, (iii) the number of available sawfly females was limited or (iv) the extraction of RNA or phytohormones was unsuccessful.
RNA extraction
RNA was extracted from ground frozen pine needles with the InviTrap Spin Plant RNA Mini Kit (Stratec, Berlin, Germany) according to the manual. Further details about extraction, purification and quality control are provided in Method S1 available as Supplementary data at Tree Physiology Online.
RNA sequencing
A volume of 25-μl RNA (dissolved in nuclease-free H2O) of each sample was sent on dry ice for sequencing (Novogene Co., Ltd, Beijing, China). From this volume, 1-μg RNA per sample was used. The company conducted the following steps for sequencing. In short, first the RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). Thereafter, RNA integrity and quantitation were checked using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Finally, sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturer’s recommendations, and index adapters were added to attribute sequences to each sample. The library preparations were sequenced on an Illumina platform with a 150-bp paired end sequencing protocol. Further details of the company’s purification, sequencing and library preparation are provided in Method S2 available as Supplementary data at Tree Physiology Online.
Transcriptome de novo assembly and annotation
Quality control of RNA sequencing raw reads and transcriptome de novo assembly from RNA sequencing was performed at Novogene.
For QC, reads containing adapter sequences, reads with more than 10% of uncertain nucleotides (labelled ‘N’ from the Illumina sequencing machine) and reads with more than 50% low-quality bases (≤5) were removed. In total, 18.5 × 106 to 27.3 × 106 clean reads for each sample were obtained after QC and used for analysis.
For transcriptome de novo assembly, Trinity version 2.6.6 (Grabherr et al. 2011) was used, followed by hierarchical contig clustering with Corset version 4.6 (Davidson and Oshlack 2014) to remove redundant contigs. Reads from all samples were used to generate the assembly. The longest transcript of each cluster was then assigned as a unigene.
For annotation of the resulting unigene transcripts, we performed blast analysis on the Galaxy Europe platform (The Galaxy Community 2022) with its built-in tools. We constructed a blast database with the makeblastdb tool using release 55 of the TAIR10 Arabidopsis peptide annotation file from ENSEMBL plants (Yates et al. 2022). Unigene transcripts were annotated with blast against this database with a threshold of 10−5. The highest-ranked hit was used for further analysis. In total, 60,295 (35.5%) of the pine unigene transcripts could be annotated to A. thaliana transcripts.
We used the built-in analysis tools of the BLAST2GO version 6.0.3 suite (Conesa and Götz 2008) to retrieve functional GO terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways from the annotated transcripts.
Differential gene expression analysis
For read counting, we used kallisto version 0.46.0 (Bray et al. 2016) with 100 bootstraps. The index file was created with the unigene transcript file from the de novo assembly pipeline. For the (putative) transcripts detected here in treated pine needles, we refer to differentially expressed genes (DEGs) as standard terminology while keeping in mind that the number of genes does not necessarily match the number of transcripts (Niu et al. 2022). The DEG analysis was conducted in R (version 3.6.1) (R Development Core Team 2015) with the DESeq2 package (Bioconductor version 3.9) (Love et al. 2014). R basic syntax was extended with the tidyverse package (version 1.3.0) (Wickham et al. 2019). Prior to importing the kallisto count files to DESeq2, tximport (Bioconductor version 3.9) (Soneson et al. 2015) was used to convert count files to the DESeq data format.
All genes with a read count sum greater than five in each sample were considered valid for further DEG analyses. In addition, we excluded all transcripts from statistical analysis that were not considered to be related to plant species. To identify the taxonomy of the unigene transcripts, we performed a Diamond Blast analysis with a rigid threshold of 10−9 against the complete ncbi_nr_2021_01 database included in the Galaxy server. To identify the taxonomic relationship between the blast results identified, we used the R package taxonomizr version 0.9.2 (https://cran.r-project.org/web/packages/taxonomizr/index.html).
cDNA synthesis and qPCR
To validate the RNA sequencing data, we conducted qPCR expression analyses of selected genes in needles from untreated trees and trees exposed to egg deposition, to larval feeding or to both egg deposition and larval feeding. In total, gene expression levels in control trees were compared with those in the seven following sample types: egg-treated needles 1 h, 24 h and 10 days after egg deposition; feeding-treated needles 1 and 24 h after the onset of feeding and egg-treated plus subsequently feeding-damaged needles 1 and 24 h after the onset of feeding (Figure 1). Samples for the qPCR analyses were collected from trees (n = 3–5) treated in an experimental setup independent of the setup used for RNA sequencing. For each sampling time point, new trees were used, thus avoiding the possibility that sampling at an early time point affected the tree’s responses at a later time point.
We normalized the C(t) values of E-, F- and EF-samples to those of untreated C-samples and to the three housekeeping genes ubiquitin (PsUBI), cytochrome subunit 6 (PsPetB) and chloroplast ATPase beta subunit (PsC-ATP) according to Pfaffl (2001) and Vandesompele et al. (2002). Further details about the methods of the qPCR analyses are provided in Method S3 available as Supplementary data at Tree Physiology Online.
To validate the RNA sequencing data, we focused (i) on genes that might be involved in defence against insects, i.e., genes involved in cell wall modification, in phenylpropanoid and terpenoid biosynthesis, chitinase activity, Ca2+ signalling and phytohormone biosynthesis/signalling, and (ii) on genes that, according to the results of the RNA sequencing analysis, were significantly differentially expressed due to the treatment in at least three of the seven aforementioned sample types (Table S1 available as Supplementary data at Tree Physiology Online). The primer sequences of these genes and of the three selected housekeeping genes are presented in Table S2 available as Supplementary data at Tree Physiology Online.
Phytohormone analyses
In order to elucidate the phytohormonal responses of pine to D. pini eggs and larvae, we analysed concentrations of salicylic acid (SA), jasmonic acid (JA), jasmonic acid isoleucine (JA-Ile) and abscisic acid (ABA) in needles from untreated control trees and trees exposed to the E-, F- and EF-treatments (Figure 1). Samples for the phytohormone analyses were collected from trees that were also used for the qPCR analysis, i.e., from an experiment independent of that used for the RNA sequencing analysis. Phytohormone extraction and analyses were conducted following the methods described by Bandoly et al. (2016) and Drok et al. (2018). In short, ethyl acetate (spiked with deuterated phytohormones as internal standards) was used as extraction buffer. Extracted phytohormones were dried and resolved in 70% methanol. Phytohormones were analysed by UPLC-MS/MS (Q-ToF-ESI) and normalized to the respective internal standards and the weight of the extracted plant material. Further details are provided in Method S4 available as Supplementary data at Tree Physiology Online.
Data visualization and statistical analysis
Statistical analyses of RNA sequencing data were performed with DESeq2 (Wald test) for comparison of gene expression in control needles to those subjected to different treatments. Genes were considered to be DEGs at a significance level of P ≤ 0.05 after Benjamini–Hochberg correction for multiple testing. The total number of DEGs per treatment and time point is given in Table S3 available as Supplementary data at Tree Physiology Online.
The GO term enrichment analysis and KEGG pathway enrichment analysis were performed on biological processes with DAVID version 2021 (https://david.ncifcrf.gov) (Da Huang et al. 2009). All GO terms and KEGG pathways containing at least three genes were considered enriched at P-value < 0.05 after using Fisher’s exact test. The GO terms used in the enrichment analysis are given in Table S4 available as Supplementary data at Tree Physiology Online.
Calculation, visualization and statistical analyses of the qPCR and phytohormone data were performed using the software R version 3.6.1 (R Development Core Team 2015), SigmaPlot version 11.0 (Systat Software GmbH 2008) and Excel version 16.0 (Microsoft Corporation 2019). Data were tested for normal distribution with the Shapiro–Wilk test and for homogeneity of variances with Levene’s test. Since a new set of trees was used for each sampling time point, samples taken at different time points were independent from each other. Pairwise comparisons of phytohormone and qPCR data obtained from treated needles with those of their respective controls were analysed using the Mann–Whitney U test. Multiple comparisons of phytohormone data obtained from feeding-damaged F- and EF-trees, as well as from E-pine trees and controls at equivalent times points, were analysed using the Kruskal–Wallis test followed by a Tukey post hoc test.
Results
RNA sequencing: transcript abundance, validation and overview of DEG analyses
The de novo assembly of the transcriptomes of untreated P. sylvestris needles (C), egg-treated needles (E), feeding-damaged needles (F) and those exposed to both eggs and subsequent larval feeding (EF) resulted in 169,750 putative transcripts (here referred to as DEGs) with a mean length of 1036 bp and an N50 length of 1511 bp (Table S5 and Item SI1 available as Supplementary data at Tree Physiology Online). Completeness of the transcriptome was assessed with BUSCO version 3.0.2 (Simão et al. 2015) and resulted in 69.9% complete matches, 7.4% duplicate matches, 6.0% fragmented matches and 16.7% missing matches with the pine unigene transcripts.
Overall, 13,344 genes were differentially expressed in treated trees when compared with control plants. Of these, 7510 were upregulated and 5834 downregulated (Figure 2, Table S3 available as Supplementary data at Tree Physiology Online).
Number of DEGs. Needles of P. sylvestris were treated with D. pini egg deposition (E; yellow bars), larval feeding on previously egg-free pine (F; green bars) or natural egg deposition with subsequent feeding (EF; brown bars). Needles were sampled 1 h, 24 h and 10 days after egg deposition, as well as 1 and 24 h after the onset of larval feeding. The DEGs were differentially expressed to a significant degree when compared with untreated controls (C; Wald test; corrected P-value ≤ 0.05). Bars above (below) the zero x-axis show the number of upregulated (downregulated) DEGs. Number of replicates: N = 4–5 for each treatment and time point.
The differential expression detected by the RNA sequencing analysis was validated by performing a qPCR analysis of 13 DEGs detected in differently treated samples harvested at different time points after treatment (seven sample types in total, see ‘Materials and methods’, section ‘cDNA synthesis and qPCR’). These 91 comparisons of qPCR and RNA sequencing data resulted in about 87% of DEGs being regulated in the same direction, and about 69% that did not differ by more than 50% in their expression levels, while still being regulated in the same direction (Table S1 available as Supplementary data at Tree Physiology Online).
In the following sections, the transcriptomic responses of Scots pine are considered separately according to the different treatments applied and as compared with the untreated control. In addition, we subjected all genes that were differentially expressed in treated trees as compared with untreated control trees to a GO term analysis, as well as to a KEGG pathway analysis. We further analysed how the transcriptomes of the differently treated trees overlap.
Scots pine responds to sawfly egg deposition with strong transcriptomic changes and higher JA concentrations
To determine how the transcriptome and concentrations of phytohormones of a gymnosperm species change in response to sawfly egg deposition, we analysed the transcriptome and phytohormone levels of P. sylvestris at early and late time points after egg deposition. When analysing how many of the DEGs detected in all of the treatments were already regulated during the egg treatment of pine needles, we found that about 66% of all upregulated and about 69% of all downregulated DEGs were egg-responsive (Table S3 available as Supplementary data at Tree Physiology Online).
More than 3200 genes were significantly differentially expressed 1 h after egg deposition (Figure 2). This number more than doubled (to more than 6600 genes) 24 h after egg deposition. Following this strong, rapid transcriptomic response, the number of DEGs decreased to almost the control level during the egg incubation phase.
Overall, the pine trees showed a strong transcriptomic response especially in the 24 h following egg deposition. Thereafter, gene expression levels returned to almost the control level at the end of the egg phase.
A qualitative analysis of the egg-responsive genes (E vs C) by GO term enrichment analysis (Figure 3, Tables S4 and S6 available as Supplementary data at Tree Physiology Online) revealed that photosynthesis-related GO terms were enriched with downregulated genes 24 h after egg deposition. The GO terms involved in cell wall modification, lignin biosynthesis and cell death—including hypersensitive response (HR)—were mostly enriched with upregulated genes. Many GO terms related to secondary metabolites such as terpenes, flavonoids and other phenylpropanoids were enriched with upregulated genes at the first three time points during egg treatment. The GO terms involved in responses to chitin were also enriched in egg-treated pine. Among the phytohormone-related GO terms, those that were auxin-related were mostly enriched with upregulated genes, but only 1 h and 10 days after egg deposition. Ethylene-related terms were enriched with upregulated genes at all three time points during egg treatment. Among the ABA-related GO terms, some were enriched with upregulated DEGs (see Figure 3, top, ABA slot), but several were also enriched with downregulated DEGs 1 and 24 h after egg deposition (see Figure 3, bottom, ABA slot). Jasmonic acid-related terms were only enriched with upregulated genes; the number of enriched JA-related terms decreased during the egg phase. Salicylic acid-related GO terms were enriched with both up- and downregulated genes mostly 1 h after egg deposition.
Gene Ontology term enrichment. Shown are significantly DEGs in needles of P. sylvestris 1 h, 24 h and 10 days after D. pini egg deposition, and 1 and 24 h after the onset of larval feeding. Top figure: enrichment with upregulated genes; bottom figure: enrichment with downregulated genes. Differently coloured horizontal bars below the figure show groups of GO terms related to similar biological processes, i.e., GO terms related to ‘photosynthesis’, ‘cell wall modification’ (cell wall mod.) (including lignin), ‘cell death’ (CD) (including ‘hypersensitive response’ (HR)), ‘secondary metabolites’ (sec. Metabolites) (including ‘phenylpropanoids’ (PP), ‘flavonoids’ (FL), ‘terpenes’ (TP) and ‘others’ (OT)), ‘response to chitin’ (RC) and those related to ‘phytohormones’ (including ‘jasmonic acid’ (JA), ‘salicylic acid’ (SA), ‘abscisic acid’ (ABA), ‘auxin’ (AUX), ‘ethylene’ (ET) and ‘others’ (OT)) are grouped here. The GO term identities included in these groups are listed in Table S4 available as Supplementary data at Tree Physiology Online (compare GO term ID numbers given above the figure with numbers in Table S4 available as available as Supplementary data at Tree Physiology Online). The enrichment of each GO term is shown by different circles for each treatment and sampling time point. The fold enrichment is illustrated by the size of each circle (highest enrichment = 25 in top figure; highest enrichment = 10 in bottom figure). The P-value (modified Fisher’s exact test; P < 0.05) is visualized by the colour of each circle. Numbers in the yellow (egg deposition)/green/brown (feeding) arrows on the left side of the figure indicate the different sampling time points. The enrichments of GO terms for the treatments of egg deposition (E), larval feeding (F) and natural egg deposition with subsequent feeding (EF) were all compared to the respective, untreated control (C). Additionally, EF was compared with F. Horizontal, dashed lines separate data from E samples from those of EF and F samples, and data from EF and F samples at the 1 and 24 h sampling time points. Vertical, dashed lines separate the different GO term groups. A list of all significantly enriched GO terms is provided in Table S6 available as Supplementary data at Tree Physiology Online.
The KEGG pathway analysis supported the results obtained by the GO term analysis and revealed highly significant enrichment of downregulated genes involved in ‘carbon fixation in photosynthetic organisms’ and highly significant enrichment of upregulated genes involved in ‘linolenic acid metabolism’ and ‘phenylpropanoid biosynthesis’. ‘Biosynthesis of secondary metabolites’ was strongly enriched with upregulated genes 1 h after egg deposition; however, 24 h after egg deposition, this category was strongly enriched with downregulated genes (Table S7 available as Supplementary data at Tree Physiology Online).
The phytohormone measurements (Figure 4) revealed a clear trend for an enhanced SA concentration 10 days after egg deposition. The JA concentration increased significantly 1 h after egg deposition; at later time points, JA levels no longer significantly differed between egg-laden and egg-free control needles. Concentrations of JA-Ile increased significantly 1 and 24 h after egg deposition. The ABA concentration was significantly higher 10 days after egg deposition. In contrast, just 1 h after egg deposition ABA levels were significantly lower than in the control needles.
Phytohormone concentrations in needles of P. sylvestris. Shown are the results 1 h, 24 h and 10 days after D. pini egg deposition, as well as 1 and 24 h after the onset of larval feeding. The non-normally distributed data are visualized as boxplots with the median as centre and all data points as dots. (a) Salicylic acid (SA), (b) jasmonic acid (JA), (c) jasmonic acid-isoleucine (JA-Ile) and (d) abscisic acid (ABA). Treatments were: natural egg deposition (E; yellow), larval feeding (F; green), egg deposition with subsequent feeding (EF; brown) and an untreated control (C; grey). Significant differences between concentrations in C-pine and E-pine 1 h, 24 h and 10 days after egg deposition are indicated by asterisks (Mann–Whitney U test; *P < 0.05, ***P < 0.001). Significant differences between control, E-, F- and EF-pine at the 1 and 24 h time points of larval feeding (and equivalent time points in C- and E-pine) are indicated by different letters (P < 0.05; Kruskal–Wallis test with Tukey post hoc test). For each treatment and time point: N = 4–5 replicates. In some cases, fewer dots than four are visible per treatment; these dots (data) are overlapping.
Taken together, pine showed strong transcriptomic changes in response to sawfly egg deposition. Gene Ontology terms related to photosynthesis were enriched with downregulated genes, while GO terms related to cell wall modification, phenylpropanoids, terpenes and JA signalling were especially enriched with upregulated genes. The changes in phytohormone concentrations in response to the egg treatment were moderate, but significant, for JA, JA-Ile and ABA.
Pine transcriptomic responses to sawfly larval feeding largely overlap with responses to sawfly egg deposition
To address the question of how insect egg deposition on a gymnosperm species affects the plant’s responses to subsequent larval feeding, we first analysed the transcriptomic and phytohormonal responses of pine to larval feeding on egg-free pine and compared them with the responses to egg deposition.
Feeding by sawfly larvae on egg-free pine needles caused the differential expression of 71% of all upregulated, and 55% of all downregulated, DEGs (Table S3 available as Supplementary data at Tree Physiology Online). Almost 4000 genes were differentially expressed 1 h after feeding upon egg-free needles, i.e., just a few more than the number of DEGs briefly after egg deposition (Figure 2). More than 6700 genes were differentially expressed 24 h after larval feeding. This was about the same number as was detected in response to a 24-h egg phase.
The GO term analysis of feeding-responsive genes in F-pine (F vs C) revealed enrichment at both sampling time points after the onset of larval feeding; these GO terms are related to photosynthesis, lignin, cell wall modification, HR, cell death, several classes of secondary metabolites, response to chitin and to phytohormones, especially JA (Figure 3, Tables S4 and S6 available as Supplementary data at Tree Physiology Online). In contrast, few GO terms (photosynthesis, cell wall modification, phenylpropanoids, SA, ABA and auxin) were enriched with downregulated genes in F-pine (Figure 3, Tables S4 and S6 available as Supplementary data at Tree Physiology Online).
According to the KEGG pathway analysis, the categories most significantly enriched with upregulated genes in response to feeding damage were ‘phenylpropanoid biosynthesis’, ‘biosynthesis of secondary metabolites’ and ‘plant–pathogen interaction’. ‘Zeatin biosynthesis’ was strongly enriched with downregulated genes after a 1-h feeding period, but after a 24-h feeding period, this pathway was significantly enriched with upregulated genes (Table S7 available as Supplementary data at Tree Physiology Online).
The phytohormone analysis revealed that SA levels did not significantly change in response to larval feeding. Jasmonic acid and JA-Ile levels slightly increased already after a 1-h feeding period and strongly increased after a 24-h feeding period. ABA levels were significantly enhanced in needles of F-trees 24 h after the onset of larval feeding (Figure 4).
When comparing the pine responses to larval feeding (F vs C) with those to egg deposition (E vs C), our transcriptomic data revealed that many of the egg-responsive genes were also differentially expressed in response to feeding damage (Figure 5a). The upregulated DEGs in E- and F-pine overlap by 40.2% of the total number of upregulated DEGs, while the downregulated DEGs overlap by 28.1% of the total downregulated DEG number (Table S3 available as Supplementary data at Tree Physiology Online). Both E-pine and F-pine showed especially strong transcriptomic responses to genes involved in cell wall modification and JA signalling. Accordingly, both E- and F-pine showed increases in JA and JA-Ile concentrations (Figure 4).
Overlapping DEGs in differently treated P. sylvestris trees. Venn diagrams are showing the number of pine genes uniquely and commonly (overlapping) differentially expressed in trees that were treated with natural egg deposition (E; yellow), larval feeding (F; green) or natural egg deposition with subsequent feeding (EF; brown). (a) Differentially expressed genes of E-trees and F-trees at all sampling time points; (b) DEGs in F- and EF-pine 1 and 24 h after the onset of larval feeding and in E-trees at equivalent time points; (c) DEGs in F- and EF-pine 1 h and 24 h after the onset of larval feeding and in E-trees 1 h, 24 h and 10 days after egg deposition. Black numbers show upregulated, and blue numbers downregulated, genes, all normalized to untreated controls (C).
Egg-laden, feeding-damaged pine shows weaker transcriptomic responses, but higher SA levels, than egg-free, feeding-damaged pine
To elucidate the impact of insect egg deposition on the transcriptomic and phytohormonal responses of a gymnosperm species to larval feeding damage, we analysed the transcriptome and phytohormone concentrations of previously egg-laden pine after a 1- and 24-h larval feeding period. In a further step, we compared the responses of these EF-pine trees to those of egg-free, feeding-damaged F-pines.
In total, 43% of all detected DEGs were upregulated, and 20% downregulated, in previously egg-laden and subsequently feeding-damaged (EF) trees (Table S3 available as Supplementary data at Tree Physiology Online). When combining the number of DEGs in EF- and F-trees over the two sampling time points during feeding damage, they made up 75% of the upregulated DEGs and 63% of the downregulated DEGs detected overall.
The number of DEGs in EF-pine after 1 h of feeding damage was about 2000, which was almost half the number of DEGs in egg-free, feeding-damaged F-pine (Figure 2). In summary, the number of DEGs was surprisingly much higher in feeding-damaged F-pine without prior egg deposition than in feeding-damaged EF-pine with prior egg deposition.
To address the question of how many of the DEGs in EF-pine are uniquely expressed in these trees and how many are also differentially expressed in F-pine, we conducted two overlap analyses.
First, we conducted an overlap analysis that included the DEGs in E-pine sampled at equivalent time points as in F- and EF-pine (compare Figures 1 and 5b). This comparison allowed us to detect how many of the genes that were still regulated by the egg treatment at these time points overlap with those in EF- and F-pine. One hour after the onset of larval feeding, there were fewer than 400 genes uniquely expressed in EF-treated plants, while almost 1600 genes were expressed in both F- and EF-pine. About 2400 genes were additionally uniquely expressed in F-pine. This pattern was even clearer after 24 h, with about 300 genes uniquely expressed in EF-pine, but around 2600 expressed in both treatments and about 4000 uniquely expressed genes in F-treated pine. Therefore, while F- and EF-pine had many DEGs in common, F-pine had more uniquely expressed DEGs. There was minimal overlap of DEGs in F- and EF-pines with those in E-pines because of the low number of DEGs that were detected at these time points (see Figure 2, sampling time points for E-pine equivalent to 1 and 24 h after the onset of feeding upon F- and EF-pine).
In a second overlap analysis, we compared the DEGs in F- and EF-pine and additionally included the DEGs in E-pine detected 1 h, 24 h and 10 days after egg deposition. This comparison allowed us to determine the number of DEGs that were uniquely expressed only during the feeding phase, but not during the egg phase (Figure 5c). When comparing the overlap of these DEGs just between E-pine and F-pine after a 1-h feeding period (~1200) with the overlap of DEGs just between E- and EF-pine after a 1-h feeding period (~160), the number of overlapping DEGs in E- and F-pine was almost 10-fold higher. When doing the same analysis after 24 h of feeding, the difference was even stronger, with more than a 20-fold higher number of DEGs in the E- and F-pine overlap (~2100 vs ~ 90). In spite of this huge overlap of DEGs in E- and F-pine, there were still many genes uniquely expressed during the egg phase when comparing them to the feeding-damaged plants at both time points. This analysis again revealed a substantial overlap of DEGs in F- and EF-pine. It further showed that the number of common DEGs in E- and EF-pine was much smaller than in E- and F-pine.
A qualitative comparison of the DEGs in EF-pine and controls (EF vs C) revealed many GO terms enriched with upregulated genes at both sampling time points (Figure 3, Tables S4 and S6 available as Supplementary data at Tree Physiology Online). These GO terms include all those mentioned in Figure 3. For GO terms enriched with downregulated genes, we found a conspicuous enrichment of photosynthesis-related GO terms early (1 h) after the onset of feeding. This finding is supported by the KEGG analysis, which also showed significant enrichment with downregulated genes, such as in the category ‘carbon fixation of photosynthetic organisms’ at this time point (Table S7 available as Supplementary data at Tree Physiology Online).
When directly comparing the GO term enrichment in EF- and F-pine (EF vs F, Figure 3), the most prominent differences were detected for GO terms related to photosynthesis, lignin, HR and cell death, secondary metabolites, responses to chitin and to JA. These GO terms were significantly more enriched with downregulated genes in EF- than F-pine after 1 h of feeding. These differences vanished after 24 h of feeding. At this time point, three GO terms related to cell wall modification were significantly more enriched with upregulated genes in EF-pine than F-pine (GO terms ‘xyloglucan metabolic process’, ‘plant epidermis development’ and ‘cell wall organization’; Figure 3, Table S4 available as Supplementary data at Tree Physiology Online).
The KEGG pathway enrichment analysis also showed highly significant enrichment with upregulated genes involved in ‘biosynthesis of secondary metabolites’ as well as in ‘phenylpropanoid biosynthesis’ for EF-pine, which were also enriched in F-pine (Table S7 available as Supplementary data at Tree Physiology Online). A further pathway highly enriched with upregulated genes in EF-pine was ‘alpha-linolenic acid metabolism’ in EF-pine after a 24-h feeding period.
With respect to phytohormone concentrations, JA concentrations were significantly higher 1 h after larval feeding in EF-pine needles than in C-pine needles, whereas the concentrations in F-pine were only tentatively higher. At the same time point, JA-Ile concentrations were higher in both EF-pine and F-pine compared with C-pine, but EF-pine and F-pine did not differ from each other. After 24 h of larval feeding, all phytohormone concentrations were significantly higher in EF-pine and F-pine compared with C-pine, except for SA which was only significantly higher in EF-pine. However, none of the phytohormone concentrations differed between EF-pine and F-pine needles.
Overall, sawfly egg deposition changed the transcriptomic responses to feeding damage by attenuating the feeding-induced transcriptomic response and by enriching especially GO terms related to cell wall modification with upregulated genes. With respect to phytohormonal changes in response to feeding damage, egg deposition affected only the SA concentrations in feeding-damaged pine, but none of the other analysed phytohormones.
Discussion
Our study demonstrated that P. sylvestris showed strong and rapid transcriptomic responses to egg deposition of the sawfly D. pini. The differential expression of genes in response to egg deposition almost reverted to control levels toward the end of the egg phase. Feeding by young larvae upon egg-free pine needles induced a strong transcriptomic response that largely overlapped with the response to egg deposition. The transcriptomic response to larval feeding was much weaker when needles had been previously exposed to egg deposition. While both EF-pine and F-pine showed significantly enhanced levels of JA, JA-Ile and ABA, only EF-pine had significantly enhanced SA levels when compared with untreated control pine. We found the enrichment of phenylpropanoid-related GO terms and of the KEGG pathway ‘phenylpropanoid biosynthesis’ with upregulated genes after egg deposition in E-pine, but also in feeding-damaged F- and EF-pine.
To highlight the responses of a gymnosperm species to insect egg deposition and feeding compared with the known responses of angiosperm species, we will first contrast the transcriptomic and phytohormonal responses of P. sylvestris to sawfly egg deposition with the known responses of angiosperms to egg deposition. Then, we will compare the effects of insect egg deposition on pine responses to larval feeding damage with the impact of insect eggs on responses of angiosperm plants to larval feeding.
Pine responses to insect egg deposition: a comparison with angiosperm plant responses
When considering the dynamics of pine transcriptomic responses to D. pini egg deposition, the intense gene expression observed 24 h after egg deposition then declined until it had almost vanished by the end of the egg phase (Figure 2). Similarly, in elm (U. minor) leaves, the highest number of egg-responsive genes has been detected 1 h after elm leaf beetle egg deposition (Altmann et al. 2018). Other angiosperm plants such as A. thaliana (Little et al. 2007, Valsamakis et al. 2022), bittersweet nightshade Solanum dulcamara L. (Geuss et al. 2017) and tobacco plants (Nicotiana attenuata Torr. ex S. Watson) (Drok et al. 2018) have shown a considerable number of DEGs 1–3 days after egg deposition. Similar to the response of pine to sawfly eggs, egg-induced differential expression of genes in elm had almost reverted to the control level by the end of the egg phase (Altmann et al. 2018). Thus, these perennial wooden plant species of pine and elm show similar dynamics of transcriptomic responses to insect egg deposition.
In pine, more than half of all DEGs in E-pine were also regulated in F-pine. A similar overlap was found in elm trees infested by elm leaf beetle eggs or elm leaf beetle larvae (Altmann et al. 2018). The overlaps may be due to the oviposition mode of these two herbivorous insect species. Diprion pini slits a needle longitudinally, cutting the parenchymatic tissue, and inserts its eggs in a row into the slit needle (Hilker et al. 2002). The egg deposition of the elm leaf beetle is also associated with leaf wounding; the beetle removes the leaf epidermis at the oviposition site and lays its eggs on parenchymatic tissue (Hilker and Meiners 2006). The leaf wounding associated with egg deposition by D. pini and the elm leaf beetle might explain (i) that the egg deposition process induces a similar set of genes in the host plants of these insect species as larval feeding does and (ii) that the dynamics of the transcriptomic responses to insect egg deposition are similar in pine and elm. However, an overlap of egg- and feeding-responsive genes was also found in A. thaliana (Valsamakis et al. 2022), in black mustard plants (Brassica nigra L. W. D. J. Koch) (Bonnet et al. 2017) and in tobacco plants (N. attenuata) (Drok et al. 2018) infested with eggs or larvae of lepidopteran species, which do not damage the leaf tissue during oviposition. Furthermore, a Generally Applicable Gene set Enrichment analysis of four angiosperm species treated with insect eggs and larval feeding also revealed a large overlap of insect egg- and feeding-induced responses (Lortzing et al. 2020). Thus, regardless of ovipositional wounding, plant transcriptomic responses to eggs and to larvae obviously share a common and conserved core response.
The type of GO terms and KEGG pathways enriched with DEGs in response to sawfly egg deposition on pine suggests that this gymnosperm species shares several similarities with angiosperm species in its response to insect eggs (Figure 3, Table S7 available as Supplementary data at Tree Physiology Online). In the following, we will focus on GO terms related to photosynthesis, hypersensitive responses (HR), response to chitin, phenylpropanoid biosynthesis and terpenoid biosynthesis.
The enrichment of photosynthesis-related GO terms and of the KEGG pathway ‘carbon fixation in photosynthetic organisms’ with downregulated pine genes supports previous studies that have shown reduced photosynthetic activity in egg-laden P. sylvestris (Schröder et al. 2005). When considering that pine increased its JA levels briefly after sawfly egg deposition, it is an interesting parallel that the downregulation of photosynthesis-related genes was also found in other conifers (e.g., Pinus albicaulis Engelm., Picea abies L. H. Karst.) treated with methyl jasmonate (Liu et al. 2017, Wilkinson et al. 2022). Downregulation of photosynthetic activity or related genes was found as well in angiosperm species responding to methyl jasmonate (Lee and Zwiazek 2019) or insect egg deposition (Little et al. 2007, Valsamakis et al. 2022). The downregulation of photosynthetic activity may be considered a trade-off of defence against the eggs (Schröder et al. 2005).
Enrichment of the GO term ‘plant type hypersensitive response’ with upregulated genes in egg-laden pine corroborates previous studies that have shown the accumulation of ROS and necrotic plant tissue in P. sylvestris laden with D. pini eggs (Bittner et al. 2017, 2019). Accumulation of ROS and the formation of HR-like symptoms have also been shown in A. thaliana (Little et al. 2007, Gouhier-Darimont et al. 2013), several brassicacean species other than A. thaliana (Bruessow and Reymond 2007, Griese et al. 2021, Caarls et al. 2023) and a solanaceous species (S. dulcamara) (Geuss et al. 2017). Responses such as these might result in desiccation of the eggs (Hilker and Fatouros 2015, Griese et al. 2021). Furthermore, the oviposition mode of the sawfly results in considerable disruption of cell wall integrity. Such a change in cell wall architecture induced by stress is well known to be linked with hydrogen peroxide accumulation—which might lead to HR and lignin deposition (Rui and Dinneny 2020, Baez et al. 2022). Enrichment of the GO term ‘chitin response’ in egg-laden pine may be caused by the chitinous ovipositor valves of the sawfly female, and possibly abrased, minute particles of the saw teeth. A previous study by Davis et al. (2002) revealed that chitinases are inducible by exogenous application of JA onto slash pine, a finding that is interesting in light of the induction of JA in P. sylvestris early after sawfly egg deposition.
One early response of pine to egg deposition showed a clear enrichment of phenylpropanoid-related GO terms and the KEGG pathway ‘phenylpropanoid biosynthesis’ with upregulated genes. Egg-induced concentrations of phenylpropanoids or egg-induced expression of genes involved in phenylpropanoid synthesis have been observed in angiosperm species, e.g., A. thaliana (Little et al. 2007, Lortzing et al. 2019) and bittersweet nightshade (Geuss et al. 2017). The defensive function of these increased concentrations of phenylpropanoids, such as flavonoids, against eggs remains unclear. However, if hatching larvae encounter enhanced the concentrations of phenylpropanoids produced during the egg phase, these compounds might harm those larvae. A defensive function of phenylpropanoids against the feeding stages of insects has been shown in numerous studies (War et al. 2018, Singh et al. 2021).
Our data show egg-induced expression of a sesquiterpene synthase (Table S1 available as Supplementary data at Tree Physiology Online) and enrichment of the GO term ‘diterpenoid biosynthetic process’ with upregulated genes (Figure 3, Table S4 available as Supplementary data at Tree Physiology Online). Gymnosperms are rich in terpenes, which serve as defensive compounds against many herbivorous insects (Mumm and Hilker 2006). So far, we do not know whether the enrichment of the GO term ‘diterpenoid biosynthetic process’ in the pine trees used here would result in an enhanced production of viscous, sticky diterpenes (Keeling and Bohlmann 2006) that might harm the gas exchange of developing sawfly eggs. It has been shown that egg-induced changes in the emission of mono- and sesquiterpenes in some angiosperm species, e.g., elm (Büchel et al. 2011) and black mustard (Fatouros et al. 2012), serve as an attraction of egg parasitoids to host eggs.
The pine phytohormonal responses to D. pini eggs show some parallels to the responses of A. thaliana to Pieris brassicae eggs. Egg-induced increase in JA and JA-Ile levels was found in both plant species (Valsamakis et al. 2020), although A. thaliana leaves are not wounded by P. brassicae egg deposition. Levels of SA were enhanced by trend in egg-laden pine, whereas A. thaliana and S. dulcamara laden with eggs showed significantly higher SA levels (Bruessow et al. 2010, Geuss et al. 2017). While the ABA concentrations in A. thaliana did not change in response to egg deposition, P. sylvestris did have increased ABA levels by the end of the egg incubation phase. Future studies need to quantify whether this increase in ABA concentrations is related to increased abscission of egg-laden needles. We observed very little abscission of egg-laden needles in our experiments and the abiotic laboratory conditions used here.
Impact of egg deposition on pine responses to feeding damage
When comparing the effect of egg deposition on the transcriptomic response of pine and angiosperms to the one of feeding damage, one difference is immediately apparent. While EF-pine showed strikingly less differential expression of genes than F-pine, previously egg-laden angiosperm plants have been found to respond to larval feeding damage with more transcriptomic activity than egg-free plants, at least when the feeding damage began (Bonnet et al. 2017, Altmann et al. 2018, Drok et al. 2018, Lortzing et al. 2019). The higher number of DEGs in EF- than F-angiosperm plants at the onset of larval feeding was found regardless of whether the oviposition was associated with leaf wounding or not. This suggests that the attenuated transcriptional response of EF-pine to larval feeding when compared with F-pine is not only due to the particular D. pini oviposition mode.
The considerable overlap of DEGs in E- and F-pine, in combination with the initially high but subsequently diminishing response to egg deposition, suggests that EF-pine can afford a less powerful transcriptional response to larval feeding damage because many genes have already been expressed during the egg phase and might need regulation only in F-pine, i.e., plants that have not experienced egg deposition prior to larval feeding. This implies that processes induced by the differential expression of genes in the egg phase remain active or can be quickly reactivated, in response to feeding damage. If indeed processes induced by gene expression early after egg deposition remained in a ‘stand-by’ mode until the end of the egg phase but were activated upon feeding more sensitively and efficiently in EF-pine than feeding-inducible processes in F-pine, this would fit into the concept of priming, which here would occur on the posttranscriptional level (Conrath et al. 2015, Hilker et al. 2016, Martinez-Medina et al. 2016, Wilkinson et al. 2019). At the beginning of larval feeding (within 1 h), fewer ‘cell wall modification’- and ‘cell death’-related GO terms enriched with upregulated genes were detected in EF-pine than in F-pine when compared with C-pine. These GO terms were also found to be enriched with upregulated genes in egg-laden pine. Changes triggered by the differential expression of these genes might still be effective against feeding larvae. However, when considering GO terms related to ‘secondary metabolites’, and especially to ‘phenylpropanoids’ and ‘terpenes’, after 24 h of feeding, more GO terms were more strongly enriched with upregulated genes in EF-pine than F-pine. If these transcriptomic responses of EF-pine trees result in enhanced concentrations of phenylpropanoids, it would parallel the metabolic responses of egg-laden angiosperms to larval feeding damage. Several angiosperm plants increase their concentrations of distinct phenylpropanoids when exposed to insect egg deposition prior to larval feeding damage, e.g., caffeoyl putrescine in tobacco plants (Bandoly et al. 2015, 2016) and quercetin and kaempferol derivatives in elm and A. thaliana (Altmann et al. 2018, Lortzing et al. 2019).
Sawfly egg deposition on pine significantly affected the SA concentration in EF-pine after 24 h of larval feeding, whereas no such effect was found in F-pine. None of the other phytohormonal responses to larval feeding in pine was affected by prior egg deposition. The high concentration of SA in EF-pine seems not to be based on a maintained (high) egg-induced SA level concentration lasting into the end of the egg phase. Several EF-treated angiosperm plants have also shown higher levels of SA than controls (Bonnet et al. 2017, Lortzing et al. 2019, Schott et al. 2022). No antagonistic ecological effects of (feeding-induced) high JA and SA levels were detected in these EF-plants, as might be expected based on other studies of the interaction between JA and SA (Erb et al. 2012, Pieterse et al. 2012, Thaler et al. 2012, Caarls et al. 2015). However, the dynamics of concentration changes and the ratio of SA and JA(-Ile) might play a role in determining the ecological effects of JA and SA interactions. Rather than leading to the antagonistic interactions often observed, the elevated levels of SA and JA in EF-plants might result instead in coordinated interactions, thus contributing to improved plant defences. Several other studies addressing the interactions of JA and SA have also found neutral or positive interactions between JA and SA, both in angiosperms (e.g., Schenk et al. 2000, Mur et al. 2006, Lortzing et al. 2019, Zhang et al. 2020, Aerts et al. 2021, Ullah et al. 2022) and gymnosperms (Arnerup et al. 2013).
Conclusions
Our study revealed that P. sylvestris responds to D. pini egg deposition by remarkable changes in the expression of numerous genes. These responses affected later transcriptional responses to larval feeding damage. Pine transcriptional responses to both the egg deposition and larval feeding damage showed considerable overlaps and occurred rapidly, indicating a fast and sensitive perception of infestation-associated molecular patterns, which might be important to limit the infestation already in its initial phase.
A comparison of pine responses with those of angiosperms to insect egg deposition and subsequent larval feeding highlights several common features, among them the downregulation of photosynthesis and changes in cell wall structure in E-plants as well as a stronger upregulation of phenylpropanoid biosynthesis and a stronger increase in SA levels in EF-plants compared with F-plants. A striking difference between the transcriptomic responses of EF-pine and EF-angiosperms is the clearly attenuated response of EF-pine to larval feeding, while EF-angiosperms studied until now have shown stronger transcriptomic responses to the onset of larval feeding. A strong transcriptomic response of EF-pine to feeding damage might be redundant. Processes rapidly triggered by expression of the numerous genes induced by the sawfly’s severe ovipositional wounding might still be active, or easily be reactivated, when larvae start feeding. Thus, a more ‘relaxed’ transcriptomic response of egg-laden pine to feeding damage might help to avoid ‘hyper-immunity’ and benefit the ‘maintenance of signal homeostasis’, as recently discussed by Pontiggia et al. (2020) with regards to plant responses to stress. Future studies of gymnosperms infested by other insect species, which do not inflict severe ovipositional wounding to the needles, need to clarify whether the attenuated transcriptomic responses of P. sylvestris laden with D. pini eggs to larval feeding damage are characteristic of gymnosperm species, or whether this is due in pine to the severe ovipositional damage inflicted by this sawfly species.
Furthermore, since D. pini shares a long evolutionary history with its host plant species like many other herbivorous insects (Kergoat et al. 2017), more research is needed to elucidate the counteradaptations of these insects to the plant’s egg-mediated defences. Studies of possible suppressive effects of insect egg deposition on plant defences against larvae (Bruessow et al. 2010) as well as of avoidance behaviour of the insects (e.g., egg deposition on the bark or larval movements to egg-free needles) will shed further light on how insects can counteract egg-mediated plant defences.
Acknowledgments
We thank the technicians Beate Eisermann and Laura Hagemann, Institute of Biology, Freie Universität Berlin, for rearing the sawflies and taking care of the pine trees. Many thanks are due to Axel Schmidt, Max Planck Institute for Chemical Ecology in Jena, Germany, for his advice in the experimental design, DEG analysis and qPCR validation, and to Andreas Springer, Mass Spectrometry Core Facility, Freie Universität Berlin, Germany, for supporting the UPLC-ESI-MS/MS analysis.
Conflict of interest
None declared.
Funding
Deutsche Forschungsgemeinschaft, German Research Foundation (project Hi 416/23-1).
Authors’ contributions
J.H. and M.H. designed the study and planned the experiments. J.H. conducted the experiments for RNA sequencing and prepared the RNA samples for this analysis. N.B. conducted the transcriptome assembly and annotation and prepared the DEG data set. T.L. prepared the samples for the qPCR experiments and phytohormone analysis. He conducted the qPCR analysis for validating the results obtained by the RNA sequencing analysis. J.H. analysed the DEG data set and conducted a GO term enrichment and KEGG pathway enrichment analysis. J.H. conducted the phytohormone analysis. V.L. provided advice for the GO term and KEGG analysis and prepared Figure 3. J.H. wrote a first draft of the manuscript. M.H. revised the manuscript. All authors contributed to the writing of the manuscript and approved the submitted version.
Data availability statement
The transcriptomic data set is available at the BioStudies database under the accession no. S-BSST1074. Further data are available within the manuscript and/or its supplementary materials.
