Responses to larval herbivory in the phenylpropanoid pathway of Ulmus minor are boosted by prior insect egg deposition

Main conclusion Elms, which have received insect eggs as a ‘warning’ of larval herbivory, enhance their anti-herbivore defences by accumulating salicylic acid and amplifying phenylpropanoid-related transcriptional and metabolic responses to hatching larvae. Abstract Plant responses to insect eggs can result in intensified defences against hatching larvae. In annual plants, this egg-mediated effect is known to be associated with changes in leaf phenylpropanoid levels. However, little is known about how trees—long-living, perennial plants—improve their egg-mediated, anti-herbivore defences. The role of phytohormones and the phenylpropanoid pathway in egg-primed anti-herbivore defences of a tree species has until now been left unexplored. Using targeted and untargeted metabolome analyses we studied how the phenylpropanoid pathway of Ulmus minor responds to egg-laying by the elm leaf beetle and subsequent larval feeding. We found that when compared to untreated leaves, kaempferol and quercetin concentrations increased in feeding-damaged leaves with prior egg deposition, but not in feeding-damaged leaves without eggs. PCR analyses revealed that prior insect egg deposition intensified feeding-induced expression of phenylalanine ammonia lyase (PAL), encoding the gateway enzyme of the phenylpropanoid pathway. Salicylic acid (SA) concentrations were higher in egg-treated, feeding-damaged leaves than in egg-free, feeding-damaged leaves, but SA levels did not increase in response to egg deposition alone—in contrast to observations made of Arabidopsis thaliana. Our results indicate that prior egg deposition induces a SA-mediated response in elms to feeding damage. Furthermore, egg deposition boosts phenylpropanoid biosynthesis in subsequently feeding-damaged leaves by enhanced PAL expression, which results in the accumulation of phenylpropanoid derivatives. As such, the elm tree shows similar, yet distinct, responses to insect eggs and larval feeding as the annual model plant A. thaliana. Supplementary Information The online version contains supplementary material available at 10.1007/s00425-021-03803-0.


Supplementary Information: Overview
The following supplementary data are available for this article in this document, except for Table S3 (see separate Excel file):  Table S1 Primers for putative Ulmus minor genes involved in the biosynthesis of phenylpropanoids • Table S2 Reference genes and their primer sequences • Table S3 MS_MS analytical data (not included in this document; see separate supplemental Excel file) • Table S4 Phytohormone levels in locally treated Ulmus minor leaves after 24 h of larval feeding and at an equivalent time point for treatments without larval feeding • Table S5 Phytohormone levels in Ulmus minor leaves adjacent to treated leaves ('systemic' leaves) after 24 h of larval feeding and at an equivalent time point for treatments without larval feeding •

Supplementary Information: Protocols / UHPLC/ESI-QTOFMS settings for untargeted analyses and quality control
Our analyses were performed on an Infinity 1290 series UHPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) consisting of a binary pump (G4220A), an autosampler (G4226A, 20 µL loop), an autosampler thermostat (G1330B) and a thermostatted column compartment (G1316C), which was interfaced to an iFunnel Q-TOF mass spectrometer (G6550A, Agilent Technologies) via a dual Agilent jet stream electrospray ion source. Extracts (1 µL injection volume) were separated on a Zorbax RRHD Eclipse Plus C18 column (100 × 2.1 mm, 1.8 µm particle size, Agilent Technologies) using 0.1 % (v/v) formic acid in water and 0.1 % (v/v) formic acid in acetonitrile as eluent A and B, respectively. The following binary gradient programme at a constant flow rate of 400 µL min -1 was applied: 0-12 min, linear from 5 % to 20 % B; 12-20 min, linear from 20 % to 50 % B; 20-23 min, isocratic, 95 % B; 23-25 min, isocratic, 5 % B. The column temperature was maintained at 40 °C and the autosampler temperature at 6 °C. The mass spectrometer was operated in low mass range and extended dynamic range (2 GHz) mode. Centroid mass spectra were acquired in negative ion mode from m/z 70-1200 using an acquisition rate of three spectra per second. The following instrument settings were applied: of purine (20 µM) and hexakis-(2,2,3,3-tetrafluoropropoxy)phosphazine (20 µM) in 95 % aqueous acetonitrile was continuously introduced through the second sprayer of the dual ion source at a flow rate of 20 µL min -1 using an external HPLC pump equipped with a 1:100 splitting device.
To monitor analytical performance, a pooled quality control (QC) sample was prepared by mixing 20 µL aliquots of each leaf extract. In addition, two blank extracts were prepared. The leaf and blank extracts were repeatedly analysed in random order. To check the quality of the obtained raw data, retention times and abundances of spiked internal standards were evaluated using MassHunter Quantitative Analysis software (Agilent Technologies).
Afterwards feature intensities were normalised by sample fresh weight and log2 transformed.

Filter.
To narrow down features of interest, we applied the following four filters. (I) We were mainly interested in compounds deriving from the phenylpropanoid pathway, which mainly consists of carbon-based, oxidized molecules. Due to the specific molecular weights of carbon, oxygen and hydrogen, precise molecular weights in the range of interest show a positive molecular mass shift.
Therefore, we omitted features with mass shifts towards a negative weight, expressed via the first decimal place between 0.5 and 0.99. Crude plant extracts consist of many plant compounds, among them many salty or protein compounds. Usually, these compounds are either very polar or non-polar and reach the mass-spectrometer very early or towards the end of the chromatographic separation.
Since these substances cause many misleading signals, we omitted features acquired during (II) the first 70 sec and (III) after 1000 sec from the results. (IV) Mean areas of features per treatment below an area of 13 (log2 transformed) were often masked by matrix effects due to their low intensity.
Consequently, we omitted features, which were below a log2 transformed area of 13 in all four treatments. Applying all four filters, 1395 features were considered for further statistical analysis.  (Guijas et al. 2018) and MassBank (Horai et al. 2010). In case of no match, CID mass spectra were manually interpreted in order to refine or confirm initial hits from compound database or literature search. By this, 20 compounds could be identified to an annotation level of 1-4 according to Sumner et al. (2007). By comparison of chromatographic and mass spectral data, putative annotations of two metabolites (esculin, compound #22, Fig. 4, main text) and suberic acid (#31) were verified using commercially available reference compounds.  Table S3). Identity of aglycones was confirmed by analysis of pseudo-MS3 spectra obtained from protonated aglycone ions, whose formation was induced by in-source fragmentation (funnel exit DC 140 V). The obtained spectra were referenced against CID mass spectra obtained from [M+H] + ions of authentic kaempferol (K), quercetin (Q) and isorhamnetin (I). Among the compounds shown in Fig. 3, main text, identities of K-3-Rut (compound #7), K-3-Glc (#8), Q-3-Rut (#14), Q-3-GlcA (#15), Q-3-Glc (#16) and I-3-Rut (#18) were verified by comparison of chromatographic and mass spectral data obtained from commercially available reference compounds. Flavan-3-ols and derived dimeric and trimeric proanthocyanidins were annotated based on accurate tandem mass spectral data. Identity of catechin and epicatechin were verified by authentic reference compounds.  Table S3. Right side: abbreviations of detected compounds, (E)C = (Epi)catechin, C = Catechin, EC = Epicatechin, DeoxyHex=Deoxyhexose; Bottom of heatmap: C = untreated control leaves, E = locally egg-treated leaves, F = locally feeding-damaged leaves, 24 h feeding period, EF = locally egg-treated, feeding-damaged leaves, 24 h feeding period. Log2 fold change relative to control was calculated by log2 of the ratio of mean peak area per metabolite in a treated leaf relative to the mean of the respective metabolite peak area in the control. Statistics: ANOVA did not show significant differences at P<0.05; n=9-10  Altmann et al. (2018) derived from a mapping of the elm transcriptome against the Arabidopsis transcriptome (TAIR10 annotation), and from Perdiguero et al. (2015), based on blast queries (BlastN, threshold E < 10−6). b Contigs from TSA (Transcriptome shotgun assembly) BioProjectID 312302 (Altmann et al. 2018)

Table S4
Phytohormone levels in locally treated Ulmus minor leaves after 24 h of larval feeding and at an equivalent time point for treatments without larval feeding. C = untreated control leaves, E = eggtreated leaves, F = feeding-damaged leaves, EF = egg-treated, feeding-damaged leaves. Concentration in ng/g FW as mean ± SE, n=9-10. ANOVA + Tukey of log-transformed data, P<0.05. Different letters below means ± SE indicate significant differences between treatments

Table S5
Phytohormone levels in Ulmus minor leaves adjacent to treated leaves ('systemic' leaves) after 24 h of larval feeding and at an equivalent time point for treatments without larval feeding. C = untreated control leaves, E = egg-treated leaves, F = feeding-damaged leaves, EF = egg-treated, feeding-damaged leaves. Concentration in ng/g FW as mean ± SE, n=9-10. ANOVA + Tukey of logtransformed data, P<0.05. Different letters below means ± SE indicate significant differences between treatments  Gene expression was measured in treated leaves after 6 h or 24 h of larval feeding and at an equivalent time point for treatments without larval feeding. C = untreated control leaves, E = egg-treated leaves, F = feedingdamaged leaves, EF = egg-treated, feeding-damaged leaves.  Table S7 Expression of homologues of phenylpropanoid synthesis-related genes in systemic Ulmus minor leaves. Gene expression was measured in leaves adjacent to treated leaves after 24 h of larval feeding and at an equivalent time point for treatments without larval feeding. C = untreated control leaves, E = egg-treated leaves, F = feeding-damaged leaves, EF = egg-treated, feeding-damaged leaves. Medians (25 %, 75 % quartiles) of relative expression (fold change, normalised to C) are shown. Different letters next to medians indicate significant differences between treatments. Kruskal-Wallis test + Wilcoxon rank-sum test, P<0.05; n=7-10  Table S8 Concentrations of kaempferol and quercetin aglycones derived from acidified (hydrolysed) Ulmus minor extracts. HPLC-DAD analysis of acid-hydrolysed methanolic leaf extracts. C = untreated control leaves, E = egg-treated leaves, F = locally feeding-damaged leaves, 24 h feeding period, EF = locally egg-treated, feedingdamaged leaves, 24 h feeding period. Concentration in µg/g FW as mean ± SE, n=9-10. Different letters below means ± SE indicate significant differences between treatments. ANOVA + Tukey, P<0.05  Table S9 Relative quantification of flavonol glycosides, flavan-3-ols and derived proanthocyanidins. Metabolites detected in methanolic leaf extracts by UHPLC/-ESI-QTOFMS analysis. C = untreated control leaves, E = egg-treated leaves, F = locally feeding-damaged leaves, 24 h feeding period, EF = locally egg-treated, feeding-damaged leaves, 24 h feeding period. Mean peak area ± SD, n=9-10, different letters below means ± SD indicate significant differences between treatments at P<0.05 (Tukey post-hoc test); ANOVA P-values: see this table. For compounds #20-30 and #31, see Table S10 and  Table S10 Relative quantification of phenylpropanoid metabolites as determined by non-targeted UHPLC/ESI-QTOFMS analyses of methanolic Ulmus minor extracts. C = untreated control leaves, E = egg-treated leaves, F = locally feeding-damaged leaves, 24 h feeding period, EF = locally egg-treated, feeding-damaged leaves, 24 h feeding period. Mean peak area ± SD, n=9-10, different letters below means ± SD indicate significant differences between treatments at P<0.05 (Tukey post-hoc test)  Table S3 .