Poplar protease inhibitor expression differs in an herbivore specific manner

Abstract

Background
Protease inhibitors are defense proteins widely distributed in the plant kingdom. By reducing the activity of digestive enzymes in insect guts, they reduce the availability of nutrients and thus impair the growth and development of the attacking herbivore. One well-characterized class of protease inhibitors are Kunitz-type trypsin inhibitors (KTIs), which have been described in various plant species, including Populus spp . Long-lived woody perennials like poplar trees encounter a huge diversity of herbivores, but the specificity of tree defenses towards different herbivore species is hardly studied. We therefore aimed to investigate the induction of KTIs in black poplar ( P. nigra ) leaves upon herbivory by three different chewing herbivores, Lymantria dispar and Amata mogadorensis caterpillars, and Phratora vulgatissima beetles.

Results
We identified and generated full-length cDNA sequences of 17 KTIs that are upregulated upon herbivory in black poplar leaves, and analyzed the expression patterns of the eight most up-regulated KTI s via qRT-PCR. We found that beetles elicited higher transcriptional induction of KTI s than caterpillars, and that both caterpillar species induced similar KTI expression levels. Furthermore, KTI expression strongly correlated with the trypsin-inhibiting activity in the herbivore-damaged leaves, but was not dependent on damage severity, i.e. leaf area loss, for most of the genes.

Conclusions
We conclude that the induction of KTIs in black poplar is controlled at the transcriptional level in a threshold-based manner and is strongly influenced by the species identity of the herbivore. However, the underlying molecular mechanisms and ecological consequences of these patterns remain to be investigated.

Full text

R E S E A R C H A R T I C L E Open Access
Poplar protease inhibitor expression differs
in an herbivore specific manner
Franziska Eberl
1*
, Thomas Fabisch
1
, Katrin Luck
1
, Tobias G. Köllner
1
, Heiko Vogel
2
, Jonathan Gershenzon
1
and
Sybille B. Unsicker
1
Abstract
Background: Protease inhibitors are defense proteins widely distributed in the plant kingdom. By reducing the
activity of digestive enzymes in insect guts, they reduce the availability of nutrients and thus impair the growth and
development of the attacking herbivore. One well-characterized class of protease inhibitors are Kunitz-type trypsin
inhibitors (KTIs), which have been described in various plant species, including Populus spp. Long-lived woody
perennials like poplar trees encounter a huge diversity of herbivores, but the specificity of tree defenses towards
different herbivore species is hardly studied. We therefore aimed to investigate the induction of KTIs in black poplar
(P. nigra) leaves upon herbivory by three different chewing herbivores, Lymantria dispar and Amata mogadorensis
caterpillars, and Phratora vulgatissima beetles.
Results: We identified and generated full-length cDNA sequences of 17 KTIs that are upregulated upon herbivory in
black poplar leaves, and analyzed the expression patterns of the eight most up-regulated KTIs via qRT-PCR. We
found that beetles elicited higher transcriptional induction of KTIs than caterpillars, and that both caterpillar species
induced similar KTI expression levels. Furthermore, KTI expression strongly correlated with the trypsin-inhibiting
activity in the herbivore-damaged leaves, but was not dependent on damage severity, i.e. leaf area loss, for most of
the genes.
Conclusions: We conclude that the induction of KTIs in black poplar is controlled at the transcriptional level in a
threshold-based manner and is strongly influenced by the species identity of the herbivore. However, the
underlying molecular mechanisms and ecological consequences of these patterns remain to be investigated.
Keywords: Kunitz-type trypsin inhibitors; herbivore specificity; woody plants; tree defenses, Lepidoptera, Coleoptera,
Salicaceae, Induced defenses, Proteinase inhibitors
Background
Over millions of years plants have developed numerous
strategies to defend themselves against plant-feeding ani-
mals. Apart from indirect defenses, which involve the re-
cruitment of an herbivore’s natural enemies, plants can
harm their attackers directly by producing mechanical
barriers, chemical toxins and deterrents, or by using bio-
chemical defenses that interfere with the herbivore’s
enzymatic machinery. Among chemical defenses, most
emphasis has been placed on low molecular weight me-
tabolites, but defensive proteins exist, such as protease
inhibitors (PIs) that reduce the digestibility of plant tis-
sue for the feeding herbivore. By inhibiting proteolytic
enzymes in the midgut of the herbivore, PIs diminish
protein digestion and hence lower the availability of free
amino acids required for herbivore growth and develop-
ment [15]. The PIs found in plants are numerous and di-
verse, with 99 different inhibitor families currently
described [32]. Those PI families, as well as distinct
members within a family, vary in their activity towards
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* Correspondence: feberl@ice.mpg.de
1
Department of Biochemistry, Max Planck Institute for Chemical Ecology
(MPI-CE), Hans-Knöll-Str. 8, 07745 Jena, Germany
Full list of author information is available at the end of the article
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https://doi.org/10.1186/s12870-021-02936-4
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the four types of proteases found in herbivore guts,
namely serine -, cysteine -, aspartic acid -, and metallo-
proteases. In herbivorous insects, the most abundant
protein-degrading enzymes are the serine proteases [15].
It is therefore not surprising that serine PIs are widely
distributed in the plant kingdom [20,21]. One of the
best characterized classes of serine PIs are the Kunitz-
type trypsin inhibitors (KTIs; also Kunitz-type protease
inhibitors, KPI), of which some are also able to inhibit
cysteine proteases [2,6]. KTIs are relatively small pro-
teins with a mass of 20 to 25 kDa [39], with a β-trefoil
structure, consisting of a β-barrel and several loops, of
which one is binding to the active site of the target pro-
tease [42]. The biological activity of KTIs has been dem-
onstrated by using gut extracts in in vitro assays [16,29],
as well as monitoring the fitness of herbivores feeding
on KTI-enriched diets [2,6,22,25,26,30]. Since the
first description of a KTI in soybean [19,24], most sub-
sequent studies have also focused on KTIs from legume
species [16,17,22,30,36,43]. However, KTIs in trees
have gained more attention in past years. In species of
the genus Populus, several KTIs have been identified and
characterized [7,27,29,37,39], and some shown to be
inducible by mechanical wounding or insect herbivory
[27–29,39]. For example, feeding by the forest tent cat-
erpillar, a generalist herbivore, increased KTI transcript
abundance locally and systemically in hybrid poplar
leaves [28]. In fact, genes encoding for KTIs belong to
the most up-regulated ones in systemic poplar leaves
upon mechanical wounding [9]. In a study by Philippe
et al. [39] it was shown that the transcriptional induction
triggered by wounding varies among the KTIs and in a
time-dependent manner. So far, most studies used
Malacosoma disstria, a generalist lepidopteran species,
to investigate herbivore-triggered KTI responses in pop-
lar [27,28,39]. To our knowledge, the specificity of pop-
lar KTI induction towards other herbivore species has
not yet been investigated.
Specificity of response to different herbivores may be
especially important for large, long-lived woody peren-
nials like trees, which encounter a vast diversity of herbi-
vores in their lifetimes. For example, it is well known
that plants react differently to leaf-chewing herbivores
than herbivores feeding on phloem-sap [13,23]. Specifi-
city of anti-herbivore defenses can also be observed
within the same feeding guild, and even within the same
species depending on the insect’s developmental stage.
For example, early instar generalist caterpillars induced a
stronger defense reaction in black poplar leaves than late
instar caterpillars of the same species [31]. The under-
lying mechanism might be explained by HAMPs or
DAMPS (herbivore- or damage-associated molecular
patterns, respectively) that plants perceive when being
attacked [13]. These are influenced by the physical
attributes of herbivory, such as leaf area removal or the
timing of tissue damage, but also by chemical cues such
as salivary compounds of the herbivores [33]. All of
these traits can be herbivore species-specific and may
allow plants to distinguish among attackers and mount
adequate and effective defenses against specific herbi-
vores. In black poplar trees, such herbivore-specific reac-
tions could be shown for signaling molecules [14], as
well as chemical defense traits such as volatile emission
[14,31,47]. In a recent study by Fabisch et al. [14], total
PI activity against trypsin was more strongly induced by
beetle feeding than by caterpillar feeding on black poplar
leaves. However, to date, we do not know which specific
genes are responsible for the observed differences in PI
activity and whether or not transcription of PI-encoding
genes differs between beetle- and caterpillar-fed leaves.
In this study, we therefore tested the hypothesis that
different herbivore species induce KTI genes in a
species-specific manner. We identified 17 KTI genes
from a transcriptome of black poplar and generated full-
length cDNA sequences of the most up-regulated ones.
Gene expression patterns of these KTI genes as deter-
mined by qRT-PCR upon herbivory by three different
insect species (Fig. 1) show striking differences among
the species.
Methods
Plants and insects
Populus nigra L. (Salicaceae) trees were grown from cut-
tings obtained from trees in a common garden near Jena,
Germany. These trees were originally derived from a sin-
gle female genotype from a P. nigra population (species
identified by Sybille Unsicker based on morphological
features) located in Küstrin-Kietz, Germany (52°34′1“N,
14°38’3”E). Since cuttings for this study were taken from
trees in a common garden, no permission was necessary
for collecting plant material; a voucher specimen will be
deposited in spring 2021 in the Herbarium Haussknecht
(JE) in Jena, Germany. The cuttings were potted in 2 L
pots, grown in the greenhouse (18/20 °C, night/day, rela-
tive humidity 60%, natural light with 9–14 h photo-
period, supplemented light for 12 h) and transferred to a
climate chamber (18/20 °C, night/day; relative humidity
60%; photoperiod 16 h) 2 days before the onset of the ex-
periment. Trees were either grown for 4 months to ap-
proximately 0.5 m (Transcriptome samples) or grown to
a height of 1.6 m (approximately 6 months) and pruned
back to 0.8 m 4 weeks before treatment (Gene expression
samples).
Lymantria dispar L. (Erebidae, Lepidoptera) caterpil-
lars are generalist feeders with a broad host range, pref-
erably deciduous trees. L. dispar caterpillars were
hatched from eggs kindly provided by the US Depart-
ment of agriculture (USDA, Buzzards Bay, MA, USA)
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and reared on artificial diet (MP Biomedicals LLC, Ill-
kirch, France) in a climate chamber (14/10 h, light/dark,
20–23 °C, relative humidity 60%) until they reached the
third instar, the stage used for the experiments. This
species is reared continuously at the MPI-CE.
Amata mogadorensis Blachier (Erebidae, Lepidoptera)
caterpillars are also generalists with a preference for
woody plants and shrubs. A. mogadorensis caterpillars
were hatched from eggs provided by a private breeder
(www.entomologenportal.de) and reared on black poplar
foliage until they reached the third instar, the stage used
for the experiment. Individuals were reared until adult
stage to confirm the species identity.
Phratora vulgatissima L. (Chrysomelidae, Coleoptera)
beetles are specialists, feeding on a narrow range of
hosts within the Salicaceae. Beetles (taxonomically deter-
mined by Lars Möckel; individuals in alcohol available at
the MPI-CE) were reared in the laboratory on black
poplar trees.
Experimental designs and sampling
Plant material from two different experiments was used
to analyze the transcriptome (see Transcriptome sam-
ples) or the gene expression of Kunitz-type trypsin in-
hibitors (KTIs; see Gene expression samples).
Transcriptome samples
A leaf pool (8 leaves from the stem of a young black
poplar tree (n= 4) was wrapped with gauze and then
infested with L. dispar caterpillars (4 individuals per
tree), adult P. vulgatissima beetles (6 individuals per
tree), or left untreated (control). Due to time differences
in the availability of the experimental insects, the beetle
treatment was conducted two weeks earlier than the cat-
erpillar treatment; both treatments had their own re-
spective control group (n= 4), which was treated and
sampled at the same time as the herbivore-treated
plants, but was not exposed to herbivores. After 2 d, the
treated leaves were flash-frozen in liquid nitrogen and
stored at −80 °C.
Gene expression samples
For gene expression analysis by qRT-PCR, leaf material
from an experiment described in Fabisch et al. [14] was
used, where further details on the methods are de-
scribed. In short, a leaf pool (5 leaves) of black poplar
trees (n= 10, but a random selection of 6 was used for
gene expression analysis) was wrapped with PET bags
(Bratschlauch, Toppits, Minden, Germany) and then
infested with L. dispar caterpillars (10 per tree), A.
mogadorensis (10 per tree), P. vulgatissima beetles (50
per tree), or left untreated (control). After 1 d, the num-
ber of caterpillars was reduced to prevent excessive leaf
loss. After a total feeding period of 2 d, the leaves were
photographed to assess the damage and subsequently
flash-frozen in liquid nitrogen and stored at −80 °C. The
damage was quantified as leaf area loss from the photo-
graphs by reconstructing the original leaf area in the pic-
ture and counting the number of pixels representing the
total and the removed leaf areas (Photoshop, Version
15.0.0, Adobe Systems Incorporated, San Francisco,
USA). Pixels were converted to area (cm
2
) using a refer-
ence field in the photograph.
RNA isolation and cDNA synthesis
Frozen leaves were ground in liquid nitrogen and RNA
was isolated using the InviTrap Spin Plant Mini Kit
(Stratec Biomedical AG, Birkenfeld, Germany), including
DNase digestion. RNA concentration was measured with
a NanoDrop 2000c spectrophotometer (Peqlab Biotech-
nologie GmbH, Erlangen, Germany). For transcriptome
samples, an additional quality check was conducted with
the RNA 6000 Nano Kit on a Bioanalyzer (Agilent, Santa
Clara, CA, USA). cDNA was synthesized from RNA
Fig. 1 Insects used in this study and their damage pattern after 2 d feeding on black poplar leaves. Amata mogadorensis and Lymantria dispar
(gypsy moth) remove large areas from the leaves, whereas Phratora vulgatissima (blue willow beetle) causes small, but numerous lesions
Eberl et al. BMC Plant Biology (2021) 21:170 Page 3 of 11
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using SuperScript-III reverse transcriptase and oligo-dT
primers (Thermo Fisher Scientific, Waltham, MA, USA).
Transcriptome analysis
Sequencing was done at the Max Planck-Genome-
Center (Köln, Germany) on a HiSeq 2500 (Illumina, San
Diego, CA, USA) with 9 Mio reads per sample. Detailed
information on quality control measures, the assembly
of the de novo transcriptome and the annotation can be
found in Eberl et al. [12], but the most relevant informa-
tion will be summarized here. The annotation was done
using, among others, BLAST, Gene Ontology (GO) and
InterPro terms (InterProScan, EBI). Contigs encoding
for potential KTI proteins were identified based on a
positive BLAST hit against a known KTI in the NCBI nr
database, GO terms associated with serine proteinase in-
hibitors and/or a hit against the Pfam domain PF00197
(Kunitz STI protease inhibitor), or InterPro domains
IPR011065 (Kunitz inhibitor STI-like superfamily) and
IPR002160 (Proteinase inhibitor I3, Kunitz legume). In
order to identify further KTI candidates, the P. nigra
transcriptome was uploaded in an internal database and
used for BLAST analysis of poplar KTI sequences from
NCBI (www.ncbi.nlm.nih.gov/) and Phytozome (https://
phytozome.jgi.doe.gov/pz/portal.html). Digital gene ex-
pression analysis was carried out using CLC Genomics
Workbench v9.1 to generate BAM (mapping) files, and
expression levels were then estimated using QSeq Soft-
ware (DNAStar Inc., Madison, WI, United States). The
log2 (RPKM) values (normalized mapped read values;
geometric means of the biological replicate samples)
were used to calculate fold-change values. Differentially
expressed genes were identified using the Student’st-
test (as implemented in Qseq) corrected for multiple
testing using the Benjamini–Hochberg procedure to
check the false discovery rate (FDR). With an FDR-
corrected p-value less than 0.05 a gene was considered
significantly differentially expressed.
In addition to the KTI gene sequences in the transcrip-
tome of the herein described experiment, another KTI
gene (PnKTI B1) was identified from an additional leaf
transcriptome from the same P. nigra genotype and
comparable L. dispar herbivory treatment (unpublished).
Furthermore, another sequence encoding a KTI (PnKTI
A4, or SQ33325–2), which was not present in the tran-
scriptome, was identified during amplification from
cDNA (see below) with primers originally designed for
PnKTI A13 (SQ33325).
Cloning and sequencing of PI genes
Full-length open reading frames (ORF) were amplified
from a mix of cDNA originating from herbivore-induced
samples in a PCR using Phusion High Fidelity polymer-
ase in HF-buffer according to the manufacturer’s manual
(New England Biolabs GmbH, Frankfurt/Main,
Germany). Primers were designed based on the putative
ORF from the transcriptome whenever available, or with
the ORF of the homologous genes retrieved from the
NCBI data base (https://www.ncbi.nlm.nih.gov/). PCR
products were cloned into a PCR4-blunt TOPO vector
(Thermo Fisher) and fully sequenced using the Sanger
protocol and capillary sequencing with an ABI Prism-
Gene- Analyser 3130xl (Applied Biosystems).
Sequence alignments and phylogenetic analysis
Homologs of P. nigra KTI sequences were identified
using the BLAST-search of the NCBI data base (https://
blast.ncbi.nlm.nih.gov/Blast.cgi) and the P. trichocarpa
genome v3.0 (https://phytozome.jgi.doe.gov/). Align-
ments and similarity calculations were done with Gen-
eious software (Biomatters, Auckland, New Zealand).
An amino acid alignment of poplar KTI proteins was
constructed using the MUSCLE algorithm implemented
in MEGA6 [46]. Tree reconstruction was done with
MEGA6 using the Neighbor-Joining method and the
JTT matrix-based method. All positions with less than
80% site coverage were eliminated.
Gene expression analysis by qRT-PCR
cDNA (diluted 1:3 with water) from the Gene expression
samples was used for quantitative real-time PCR (qRT-
PCR), which was performed in a Brilliant III Ultra-Fast
SYBR reaction mixture (Agilent) on a CFX Connect
Real-Time PCR Detection System (Bio-Rad Laboratories,
Hercules, CA, USA) with 40 2-step cycles (95 °C, 30s +
60 °C, 30s) and a melting curve from 53 to 95 °C. Primer
sequences can be found in Table S2. The PCR products
were verified by cloning and sequencing as described
above. Gene expression was calculated using CFX Man-
ager 3.1 (Bio-Rad) using the ΔΔc
q
method and taking
primer efficiencies into account. Values were normalized
to Actin as a reference gene [41] and expressed relative
to a control sample.
Trypsin-inhibiting activity assay
In order to correlate gene expression with protease in-
hibitor activity, the trypsin-inhibiting activity assay was
performed as described in Fabisch et al. [14]. In short,
10 mg freeze-dried leaf material was extracted with
400 μL buffer (25 mM Hepes-KOH, pH 7.2, 3% PVPP,
2% PVP, 1 mM EDTA) and the extract tested for
trypsin-inhibiting activity in a colorimetric (cleavage of
N-acetyl-DL-phenylalanine beta-naphthyl ester) in-gel
diffusion assay.
Statistical analysis
All data were checked for statistical assumptions, i.e.
homogeneity of variances and normal distribution. Gene
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expression data for all KTI genes had to be log
10
-trans-
formed to meet the statistical assumptions for paramet-
ric testing. For gene expression data, a one-way
MANOVA (multivariate analysis of variance) coupled to
a Tukey’s post-hoc test was applied. All statistical ana-
lyzes were conducted using SPSS 17.0 (SPSS, Chicago,
IL, USA).
Results
Identification of herbivore-induced Kunitz-type trypsin
inhibitors
The transcriptome of black poplar leaves with and with-
out herbivory by two different insect species, Lymantria
dispar (Lepidoptera) and Phratora vulgatissima (Coleop-
tera), was used to identify genes encoding herbivore-
induced Kunitz-type trypsin inhibitors (KTIs). Among
all sequences in the transcriptome, 45 were identified as
protease inhibitor genes (PIs), of which 30 were up-
regulated upon both caterpillar and beetle herbivory,
seven showed different regulation patterns depending on
herbivore identity, and eight were down-regulated upon
herbivory by either of the herbivores (Table S3). Among
the 45 PI genes, 15 belong to the KTIs, and were all up-
regulated upon herbivory (Fig. 2). These 15 KTI se-
quences, plus two additionally identified KTI genes, were
compared to previously described poplar KTIs (Table
S4) and named according to the nomenclature of Ma
et al. [27].
A phylogenetic analysis based on the amino acid align-
ment revealed that the KTIs cluster into 4 subfamilies
(Fig. 3). Most of the 17 KTIs belong to the subfamilies A
and C, whereas only one protein belongs to subfamily D.
Interestingly, all members of the C-subfamily showed a
low expression and were only marginally up-regulated
upon herbivory in comparison to members of the other
three subfamilies (Fig. 2). Therefore, KTIs from the sub-
family C were not considered in further analysis. Out of
the remaining KTI genes, those with the highest expres-
sion levels in herbivore-induced samples were chosen
for cDNA sequencing, yielding the full-length open read-
ing frames of ten PnKTI genes (Fig. 3).
Herbivore-specific induction of KTI gene expression
To study the specificity of KTI gene expression, we used
three different herbivore species that exhibit either simi-
lar (L. dispar, Amata mogadorensis) or different (P. vul-
gatissima) damage patterns on black poplar leaves (Fig.
1), but all cause similar leaf area loss (Table S1). In a
previous study, we showed that total trypsin inhibitor ac-
tivity in black poplar leaves is induced upon herbivory
by three different herbivores, especially by P. vulgatis-
sima [14]. To study this phenomenon at the transcrip-
tional level, the relative gene expression of nine
candidate PnKTIs was analyzed by qRT-PCR, using ran-
domly selected samples from this previous study.
While PnKTI A2 could not be amplified in the qPCR
reaction and was therefore excluded from further ana-
lysis, all of the remaining eight PnKTI genes showed sig-
nificant up-regulation upon herbivory by all of the tested
insects (Fig. 4). A multivariate analysis including damage
severity as covariate revealed that the herbivory treat-
ment had the strongest effect on PnKTI gene expression
(F
(24)
= 7.230; P< 0.001).
Constitutive expression levels in undamaged leaves dif-
fered among the PnKTI genes, with members of the A
subfamily generally displaying higher expression levels
than those of the B and D subfamilies (Table S5). Upon
herbivory, however, the genes showed even more appar-
ent differences in their inducibility (Fig. 4). Caterpillar
herbivory by L. dispar and A. mogadorensis resulted in
an up-regulation of all KTI genes by approximately 10
(PnKTI A6) to 2000-fold (PnKTI D2) in comparison to
the constitutive levels. All KTI genes were induced to
Fig. 2 Expression of Kunitz-type trypsin inhibitor (KTI) genes in black
poplar leaves after feeding by gypsy moth caterpillars (L. dispar)or
blue willow beetles (P. vulgatissima) compared to their respective
controls (Control 1 and 2), and compared to actin (ACT) and
elongation factor 1-α(EF1-α) as constitutively expressed ‘house-
keeping genes’. Shown are the mean RPKM (reads per kilobase of
transcript per million mapped reads; n= 4) as result of the
transcriptome analysis
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similar levels by these two lepidopteran herbivores. Bee-
tle herbivory by P. vulgatissima, however, caused a much
stronger induction of KTI gene expression than caterpil-
lar herbivory. Expression levels in beetle-damaged leaves
increased up to 40,000-fold (PnKTI D2) compared to
undamaged controls. Nevertheless, the induction levels
differed substantially among the individual genes, ran-
ging from approximately 40 (PnKTI A6) and several
hundred (PnKTI A15,B1,B5) up to several thousand-
fold (PnKTI A13,A14,D2). Interestingly, PnKTI D2,a
gene with one of the lowest constitutive expression
levels, showed by far the strongest relative induction
upon both caterpillar and beetle herbivory (Fig. 4h).
When considering herbivore treatments only (exclud-
ing the undamaged control group), we found that the
damage severity (% leaf area loss) did not have a sub-
stantial effect on expression levels of most of the
PnKTIs. Only two genes, PnKTI A7 and PnKTI B1, were
significantly influenced by this factor in their expression
(ANCOVA; PnKTI A7:F
(1)
= 9.348, P= 0.009; PnKTI B1:
F
(1)
= 5.012; P= 0.042). Accordingly, the expression of
the PnKTIs also did not correlate with the damage sever-
ity, except for PnKTI A7, which showed a positive rela-
tionship with leaf area loss (Spearman’s rank correlation:
ρ= 0.556, P= 0.017). The total trypsin-inhibiting activity
(Table S1[14];), on the other hand, strongly correlated
with the expression of all PnKTIs with a positive rela-
tionship (Table 1).
Discussion
Here we describe sequence analyses and expression pat-
terns of Kunitz-type trypsin inhibitors (KTIs) in black
poplar (Populus nigra), including ten full-length cDNA
sequences, of which six had not been described before in
Fig. 3 Phylogenetic tree of poplar KTI proteins. PnKTIs identified in this study are shown in blue and asterisks mark full-length cDNA sequences.
The tree was inferred using the Neighbor-Joining method and the JTT matrix-based method. Bootstrap values (n= 500) are shown next to each
node. The tree is drawn to scale, with branch lengths in the units of the number of amino acid substitutions per site
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P. nigra. Eight of these PnKTIs were studied in the con-
text of herbivore species-specific induction patterns in
leaves and we could show that beetle herbivory elicits a
much stronger transcriptional response than caterpillar
herbivory of the same magnitude.
Expression levels and inducibility of individual black
poplar KTI genes
The up-regulation of protease inhibitor (PI) transcrip-
tion and activity has been described previously, also in
black poplar [27,38]. However, both constitutive
expression levels and amplitude of induction vary be-
tween studies. In our study, members of the C-subfamily
generally showed low expression levels and little or no
up-regulation upon herbivory (Fig. 2). In contrast, Ma
et al. [27] observed stronger herbivore induction for
most of the genes in this subfamily. This suggests that
the regulation of KTI transcription depends on more
factors than herbivore feeding or wounding alone. Cer-
tain traits of the plants, such as age, genotype [44]or
previously experienced damage may play a role, but also
the experimental conditions such as abiotic conditions,
Fig. 4 Transcript accumulation of Kunitz-type trypsin inhibitor genes (KTIs) of the a,b, and dsubfamily in black poplar leaves after herbivory by
two caterpillar species (L. dispar, A. mogadorensis) and one beetle species (P. vulgatissima). Shown are the gene expression normalized to Actin
and relative to a control sample as boxplots (median with upper and lower quartile as bars; n= 6); results of the ANOVA are given in each graph.
Different letters indicate significant differences among groups (P< 0.05; Tukey’s post-hoc test)
Eberl et al. BMC Plant Biology (2021) 21:170 Page 7 of 11
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

timing [27,39] or damage severity could potentially in-
fluence expression levels. However, there are also con-
sistent patterns among the different studies. In our
study, PnKTI D2, the only member of the D subfamily,
showed the highest inducibility, i.e. relative change upon
herbivory (Fig. 4). The same gene was amongst the most
up-regulated KTIs upon herbivory and mechanical
wounding in another black poplar study [27]. Similarly,
the high herbivore-induced expression levels of PnKTI
A14 in our experiments (Fig. 2; Table S5) match well
with the results obtained for the corresponding ortholog
in a hybrid poplar species (P. trichocarpa xdeltoides)
after herbivory and mechanical wounding [39]. However,
this gene also showed relatively high transcript abun-
dance in undamaged controls, assuming also a role in
constitutive defense or primary metabolism. On the con-
trary, PnKTI D2, which displays minimal expression
levels in undamaged tissue in our and a similar study
[27], seems to act exclusively in induced anti-herbivore
defense.
There was no correlation between gene expression
for most of the PnKTI genes and damage severity,
which suggests a threshold-based activation of PnKTI
transcription rather than continuous control, in which
more damage would lead to higher KTI transcript
levels. Furthermore, we found a strong positive rela-
tionship between the trypsin-inhibiting activity in
poplar leaves and the transcription levels for all
PnKTI genes. This indicates that P. nigra KTI activity
is predominantly controlled at the transcriptional level
and hence by de novo biosynthesis. The importance
of de novo biosynthesis of stress-induced PIs has
already been demonstrated in rice [40].
Herbivore specificity in PnKTI induction
When we analyzed the transcription of KTIs in leaves
damaged by different insect herbivores, it became evi-
dent that beetles elicited a much stronger induction of
all tested KTIs than caterpillars (Fig. 4). Similar observa-
tions come from pine trees [35] and milkweed [1,48],
where beetle herbivory induced stronger defense re-
sponses (resins and terpenes, or latex, respectively) com-
pared to caterpillar herbivory. Species-specificity has
been reported for the induction of PIs in other systems,
though not in poplar trees. In soybean, damage by fall
armyworm caterpillars increased the activity of PIs,
whereas thrips damage did not [43]. De Oliveira et al.
[11] even observed varying response of tomato PIs to
damage by herbivores of the same genus. They showed
that PI activity was induced by the spider mite Tetrany-
chus urticae, but was suppressed by T. evansi [11]. Inter-
estingly, feeding damage by lepidopteran and
coleopteran herbivores in tomato yielded opposite re-
sults to our study in black poplar. Here, gene expression
and trypsin inhibiting activity was more strongly induced
by the tobacco hornworm than by the Colorado potato
beetle [10].
The difference in PnKTI expression between beetle
(Phratora vulgatissima) and caterpillar (Lymantria dis-
par and Amata mogadorensis) herbivory might be based
on the different damage pattern these insects cause, even
though all three of them are leaf chewers and removed
the same total leaf area. While caterpillars removed large
chunks of the leaves, the beetles caused small but nu-
merous lesions in the leaves (Fig. 1). The number of le-
sions was found to be a key factor determining the
emission of volatiles, another important anti-herbivore
defense trait in black poplar [31]. Other factors, such as
the duration of damage or the chemical compounds de-
posited on the plant may also be important. When artifi-
cial damage was administered to lima bean with a
mechanical caterpillar, changes in the amount of time
that damage lasted as well as the area damaged affected
the emission of volatiles [34]. Furthermore, species-
specific compounds in the saliva could trigger distinct
defense responses or the magnitude of response as re-
ported here. The importance of insect-derived elicitors
for PI induction has been demonstrated in another pop-
lar species, where mechanical wounding and simultan-
eous application of oral secretions from forest tent
caterpillars suppressed the induction of PIs [39]. It is
likely that oral secretions of the insects used in this
study also exhibit a suppressive effect, maybe with vary-
ing efficacy on PI induction. Whether herbivore host
range, comparing generalists such as L. dispar and A.
mogadorensis versus specialists such as P. vulgatissima,
plays a role in the induction of PIs, is not clear. Special-
ists usually possess a higher tolerance towards specific
chemical defenses of their hosts, such as salicinoids in
black poplar trees [3]. An increased induction of a
defense, such as the PIs, to specialist herbivores could
therefore be a more effective way to defend against these
Table 1 Correlations of individual PnKTI gene expression versus
total foliar trypsin-inhibiting activity (μgg
−1
DW; data from [14])
in all herbivore-treated (L. dispar, A. mogadorensis, and P.
vulgatissima feeding) samples of black poplar leaves. Spearman
rank-correlation, significant values are highlighted in bold font
PnKTI Spearman’sρP
PnKTIA6 0.707 0.001
PnKTI A7 0.648 0.004
PnKTI A13 0.646 0.004
PnKTI A14 0.730 0.001
PnKTI A15 0.700 0.001
PnKTI B1 0.710 0.001
PnKTI B5 0.597 0.009
PnKTI D2 0.582 0.011
Eberl et al. BMC Plant Biology (2021) 21:170 Page 8 of 11
Content courtesy of Springer Nature, terms of use apply. Rights reserved.

insects. Future studies using more herbivore species, or
generalists and specialists that are more closely related
to each other and cause similar feeding patterns, are ne-
cessary to determine if herbivore host range influences
PI induction.
Whether the herbivore specific induction patterns of
PnKTIs have ecological relevance is another open ques-
tion. One factor that plays an important role in this con-
text is ‘effect specificity’[20]. PIs possess varying
effectiveness in defense against different herbivores, as
could be observed in the performance of five different
herbivores that had been reared on PI-supplemented di-
ets [8]. Similarly, the cotton bollworm exhibited distinct
preference and performance towards different classes of
protease inhibitors [25]. This can be explained by the
fact that PIs, on the one hand, vary in their ability to in-
hibit different proteases, i.e. trypsin, chymotrypsin and
elastase [29], and that insects, on the other hand, vary in
their gut protease activities [8,20]. Additionally, the gut
pH, which differs substantially between Lepidoptera and
Coleoptera [20], also influences the inhibitory activity of
PIs [49]. It would therefore be interesting to dissect the
role of individual KTIs in black poplar towards different
insect herbivores, for example by using transgenic trees
or diet supplementation of recombinant KTIs. ‘Response
specificity’towards herbivore species is believed to be
more cost-effective for a plant than a similar response
to all herbivores [20]. Keeping in mind the fitness
costs that are linked to the biosynthesis of PIs [18], a
plant might aim to induce a subset of PIs to which a
herbivore is most sensitive. In this context, PI activity
should not be evaluated independently of other plant
defense compounds. In tobacco, PIs function synergis-
tically with the chemical defense compound nicotine,
which becomes more toxic when herbivores have to
compensate for nutritional deficits by increased feed-
ing activity [45]. Black poplar contains toxic defense
compounds called salicinoids, which have been shown
to negatively influence herbivore performance and
survival [4,5]. Therefore, possible synergistic effects
between salicinoids and PIs, the two main compo-
nents of direct defense in this tree should be investi-
gated in future studies.
Conclusion
Our major conclusion is that PI induction in black pop-
lar leaves depends on the identity of the feeding herbi-
vore, with beetles inducing a stronger response than
caterpillars. Furthermore, PI activity is regulated at the
level of transcription and most likely in a threshold-
based fashion. However, most of the molecular mecha-
nisms underlying the patterns observed and their eco-
logical consequences remain to be elucidated.
Abbreviations
KTI: Kunitz-type trypsin inhibitors; qRT-PCR: Quantitative real-time PCR;
cDNA: Complementary DNA; PI: Protease inhibitor; HAMPs: Herbivore-
associated molecular patterns; DAMPs: Damage-associated molecular
patterns; ORF: Open reading frame; PCR: Polymerase chain reaction;
MANOVA: Multivariate analysis of variance
Supplementary Information
The online version contains supplementary material available at https://doi.
org/10.1186/s12870-021-02936-4.
Additional file 1: Table S1. Feeding damage by the three herbivores
and trypsin-inhibiting activity in poplar. Table S2. Primer sequences used
for cloning and qRT-PCR. Table S3. Differential expression of contigs an-
notated as protease inhibitors in the transcriptome of black poplar leaves.
Table S4. Nomenclature of KTI homologs in this and other studies.
Table S5. Quantification cycles of the qRT-PCR analysis for individual KTI
genes.
Acknowledgements
We thank Almuth Hammerbacher and Chhana Ullah for assistance during
the transcriptome experiment, Bettina Raguschke for the plasmid
sequencing, and Beate Rothe for her help in sample analysis.
Authors’contributions
FE, TF, JG, TGK, SBU conceived the project; TF conducted the biological
experiments and leaf sample collection; FE analyzed the gene expression; KL
and FE cloned and sequenced all candidate genes; TGK created the
phylogenetic tree; HV assembled and analyzed the transcriptome; FE wrote
the manuscript; all authors reviewed and commented on the manuscript.
The authors read and approved the final manuscript.
Funding
This research was funded by the Max Planck Society. Thomas Fabisch was
funded by the Gerhard and Ellen Zeidler Stiftung. The funding bodies
provided financial support to the research project, but were not involved in
the design of the study and collection, analysis, and interpretation of data
and in writing the manuscript. Open Access funding enabled and organized
by Projekt DEAL.
Availability of data and materials
The short-read data have been deposited in the EBI short read archive (SRA)
with the following sample accession numbers: ERS5844847- ERS5844862. The
complete study can also be accessed directly using the following URL:
http://www.ebi.ac.uk/ena/data/view/PRJEB43369.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Department of Biochemistry, Max Planck Institute for Chemical Ecology
(MPI-CE), Hans-Knöll-Str. 8, 07745 Jena, Germany.
2
Department of
Entomology, MPI-CE, Hans-Knöll-Str. 8, 07745 Jena, Germany.
Received: 30 October 2020 Accepted: 25 March 2021
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