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Research

Drought enhances folivory by shifting foliar metabolomes in


Quercus ilex trees
Albert Rivas-Ubach1,2, Albert Gargallo-Garriga1,2,3, Jordi Sardans1,2, Michal Oravec4, Laia Mateu-Castell1,2,
Mıriam Perez-Trujillo3, Teodor Parella3, Roma Ogaya1,2, Otmar Urban4 and Josep Pe~ nuelas1,2
1
CSIC, Global Ecology Unit CREAF-CEAB-CSIC-UAB, Cerdanyola del Valles, 08913 Catalonia, Spain; 2CREAF, Cerdanyola del Valles, 08913 Catalonia, Spain; 3Servei de Ressonància
Magnètica Nuclear, Faculty of Sciences and Biosciences, Universitat Autonoma de Barcelona, Bellaterra, 08193 Barcelona, Catalonia, Spain; 4Global Change Research Centre, Academy of
Sciences of the Czech Republic, Bĕlidla 4a, CZ-603 00 Brno, Czech Republic

Summary
Author for correspondence:  At the molecular level, folivory activity on plants has mainly been related to the foliar con-
Albert Rivas-Ubach centrations of nitrogen (N) and/or particular metabolites.
Tel: +34 935 813 355  We studied the responses of different nutrients and the whole metabolome of Quercus
Email: a.rivas@creaf.uab.cat
ilex to seasonal changes and to moderate field experimental conditions of drought, and how
Received: 8 October 2013 this drought may affect folivory activity, using stoichiometric and metabolomic techniques.
Accepted: 17 December 2013
 Foliar potassium (K) concentrations increased in summer and consequently led to higher
foliar K : phosphorus (P) and lower carbon (C) : K and N : K ratios. Foliar N : P ratios were not
New Phytologist (2014) 202: 874–885 lowest in spring as expected by the growth rate hypothesis. Trees exposed to moderate
doi: 10.1111/nph.12687 drought presented higher concentrations of total sugars and phenolics and these trees also
experienced more severe folivory attack.
 The foliar increases in K, sugars and antioxidant concentrations in summer, the driest Medi-
Key words: drought, ecology,
ecometabolomics, folivory, metabolomics, terranean season, indicated enhanced osmoprotection under natural drought conditions.
stoichiometry. Trees under moderate drought also presented higher concentrations of sugars and phenolics;
a plant response to avoid water loss. These shifts in metabolism produced an indirect relation-
ship between increased drought and folivory activity.

more frequently in contact with the insects (Raffa et al., 2013).


Introduction
Several other studies have also demonstrated that plant primary
Climate change is expected to lead to longer dry spells, higher metabolism is shifted in response to tissue predation or infection
evaporative demand and more intense droughts in the coming (Ehness et al., 1997; Widarto et al., 2006; Sardans et al., 2013a).
decades in several regions of the world, including the Mediterra- Drought is also a potential driver of changes in the elemental
nean basin (IPCC, 2007). Increased drought has been demon- carbon (C) : N : phosphorus (P) : potassium (K) stoichiometries
strated to enhance tree mortality in forests (Martinez-Vilalta & of different plant organs and ecosystems (Rivas-Ubach et al.,
Pinol, 2002; Allen et al., 2010; Galiano et al., 2012) and may 2012; Sardans et al., 2012a,b) with consequent effects on ecologi-
have multiple effects on ecosystem biodiversity and ecosystem cal processes and ultimately the structure and function of ecosys-
structure and function (Anderegg et al., 2013; Pe~ nuelas et al., tems (Elser et al., 1996; Sterner & Elser, 2002; Sardans et al.,
2013a). Furthermore, both xeric and mesic forests may be 2011a, 2012b; Pe~ nuelas et al., 2013b). K has been demonstrated
hydraulically vulnerable to drought (Choat et al., 2012). to play an important role under low water availability conditions
Several studies showed that herbivores commonly increase to avoid water losses, especially in summer drought (Ingram &
their activity in response to increased concentrations of soluble Bartels, 1996; Sardans et al., 2012a; Wang et al., 2013), through
nitrogen (N) in foliage (Larsson, 1989; Larsson & Bjorkman, increases in its foliar concentrations (Sardans et al., 2013b). The
1993; Rouault et al., 2006). Plant water status also plays an study of foliar K concentrations under natural or experimental
important role in the resistance of trees against herbivorous attack drought conditions thus requires more attention in the context of
(Rouault et al., 2006). Drought may affect foliar nutritional qual- ecological stoichiometry (Sardans et al., 2012a). The large varia-
ity and indirectly stimulate insect folivory (White, 1984; Rouault tion in C : N : P : K biomass stoichiometries in plants, both in
et al., 2006). Also, plants are able to respond to folivory attack by time and in space (latitude and altitude), may be a significant fac-
producing chemical defenses such as alkaloids, terpenes and tor, among others, in the selection of foliage with high nutritional
phenolics (Bennett & Wallsgrove, 1994; Kessler & Baldwin, content by herbivores (Gusewell & Koerselman, 2002; Lindroth
2001; Ali & Agrawal, 2012), and these chemical defenses seem to et al., 2002; Oleksyn et al., 2002; Raubenheimer & Simpson,
be more concentrated in those populations of plants historically 2003; Sardans et al., 2012b). Additionally, the rates of folivory

874 New Phytologist (2014) 202: 874–885 Ó 2014 The Authors


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Phytologist Research 875

have been observed to be directly related to the relative was to understand: (1) the stoichiometric responses of Q. ilex, the
concentrations of foliar structural compounds such as lignin most dominant tree in the forests of the Mediterranean basin, to
rather than to foliar N concentrations, with high relative concen- seasonal changes and to experimental conditions of moderate
trations of these structural compounds usually being avoided drought; (2) the foliar metabolomic responses of Q. ilex to differ-
(Choong et al., 1992; Williams et al., 1998). Thus, C : N ratios ent seasons and moderate drought; (3) how the foliar stoichiome-
have been shown to play a key role in folivory (Ji et al., 2011) tric and metabolic shifts of Q. ilex may affect the selection of food
through the effects of structural compounds or N concentrations. by herbivores, and (4) how Q. ilex is able to shift its metabolome
Furthermore, changes in plant stoichiometry may influence the to respond to herbivore attack. For this purpose, the Q. ilex forest
coevolution of insects by shifting the composition and was used to test our hypothesis that drought, simulated in experi-
concentration of defensive chemical compounds of plants mental plots, could alter folivorous activity by altering the foliar
(Raubenheimer & Simpson, 2003), as most elements, such as C, metabolome. Our results could thus help to predict the impact of
N and P, generally do not act in isolation but as components of climate change on trophic webs (Pe~ nuelas & Sardans, 2009a).
molecular compounds (Pe~ nuelas & Sardans, 2009a) such as lig-
nin and cellulose in lignified structures or various compounds
Materials and Methods
that defend against herbivorous attack (Bennett & Wallsgrove,
1994; Kessler & Baldwin, 2001).
Study site
Most studies examining the relationships between plant metab-
olites and herbivory predation at the molecular level have gener- This study was performed in a natural Quercus ilex L. forest in
ally focused on the identification of single compounds or families the Prades Mountains in southern Catalonia (see Ogaya &
of metabolites (Sardans et al., 2013a). The application of the new Pe~nuelas, 2007 for details; 41°21′N, 1°2′E). All sampled plots
metabolomic techniques in ecology and plant physiology (eco- faced south-southeast on a 25% slope at 930 m above sea level.
metabolomics) allows the study of the complete metabolome of The climate of the region is mesic-Mediterranean with a marked
an organism, the total set of metabolites in an organism at a spe- summer drought for 3 months (Supporting Information Fig.
cific moment (Fiehn, 2002), and its response to abiotic and biotic S1a). The vegetation consists of a forest dominated by Q. ilex,
environmental shifts (Pe~ nuelas & Sardans, 2009b; Sardans et al., followed by Phillyrea latyfolia and Arbutus unedo, among others.
2011b; Sardans & Pe~ nuelas, 2012; Pe~ nuelas et al., 2013a;
Rivas-Ubach et al., 2013). Recent ecometabolomic studies have
Experimental design
reported metabolomic changes in plants exposed to a biotic stress
such as herbivorous predation (Jansen et al., 2009; Leiss et al., Four plots (15 9 10 m) of mature Q. ilex forest were established
2009) or an abiotic stress such as drought (Charlton et al., 2008; 15 m apart in March 1999 (Ogaya et al., 2003). Two randomly
Lugan et al., 2009; Sardans et al., 2011b; Rivas-Ubach et al., 2012). assigned plots received a drought treatment, and the other two
The link between shifts in foliar stoichiometries and shifts in foliar served as control plots. The drought treatment consisted of cover-
metabolomes throughout the seasons and under experimental ing c. 30% of the soil surface with 1 9 14 m PVC strips oriented
drought in the field has been recently demonstrated (Rivas-Ubach down the slope and 0.5–0.8 m above the soil surface to prevent
et al., 2012), so the nutritional quality of food for herbivores may irrigation by rainwater. PVC strips do not produce an increase in
thus also change under stress conditions (Rouault et al., 2006). soil temperature (Fig. S1d). A ditch 0.8–1 m in depth was dug
The relationship between plant stoichiometry and metabolism along the entire top edge of the treatment plots to intercept ups-
and herbivorous attack under drought conditions, however, is lope runoff. All water intercepted by the strips was channeled to
still unclear and warrants further study (Raubenheimer & Simp- the bottom edge of the drought plots. Soil moisture, air humid-
son, 2003). The rates of folivory can thus be influenced directly ity, precipitation and air and soil temperatures were monitored
by food quality and indirectly by plant water status in what could every 30 min. The drought treatment resulted in an average
be a cascade effect of drought. As a broader and better under- annual reduction of 18% in relative soil moisture (Fig. S1b,c; see
standing of the metabolomic shifts in plants grown under single Barbeta et al., 2013 for details). This reduction in soil moisture
and interacting stress factors such as drought and herbivory is has increased tree mortality and reduced growth during the last
greatly needed, we performed this study where we hypothesized 10 yr (Fig. S2).
that shifts in both foliar elemental stoichiometry and metabolo-
mics produced by drought would influence folivory activity. By
Sampling of leaves
affecting the quality of the food available to herbivores, drought-
induced shifts in foliar chemical composition may lead to Five adult Q. ilex individuals 25–50 cm in diameter were ran-
long-term cascade effects that could alter trophic webs. domly selected from each plot as study cases (total n = 20). Leaves
Once per season, we sampled leaves of Quercus ilex trees from a were sampled once each season: in February (winter), May
mature forest in Catalonia (northeastern Iberian Peninsula) (spring), August (summer) and November (autumn). A small
exposed to conditions of moderate experimental drought similar branch exposed to the sun was removed from each tree with a
to those projected for the coming decades. The metabolomes and pole, and a sample of the youngest well-developed leaves was fro-
elemental compositions of all samples were analyzed. The rates of zen in liquid nitrogen for the stoichiometric and metabolomic
folivory were also determined for each sampled tree. Our goal analyses. The remaining leaves were stored in bags at 6–8°C for

Ó 2014 The Authors New Phytologist (2014) 202: 874–885


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876 Research Phytologist

determination of water content and for photographic analysis of 50-ml centrifuge tubes were labeled: set A for the extraction of
the extent of consumption by herbivores. metabolites and set B for lyophilization. A set of crystal jars for
the organic fractions were also labeled. The tubes of set A
received 200 mg of powdered leaf material of each sample. Six
Calculation of the proportion of leaves consumed by
milliliters of water/methanol (1 : 1) and 6 ml of chloroform were
herbivores
added to each tube. The samples were vortexed for 15 s and then
Fifteen randomly selected leaves stored at 6–8°C from each tree sonicated for 2 min at room temperature. All tubes were centri-
were placed on a flat white surface and photographed with a fuged at 1100 g for 15 min. Four milliliters of each fraction
Nikon D80 camera with a Nikkor AF-S 18-135/3.5-5.6 G DX (aqueous and organic) was collected independently; aqueous frac-
lens to calculate the percentage of folivory. The area of the leaves tions were transferred to the centrifuge tubes of set B, and organic
consumed was calculated using Adobe Photoshop CS2 (Adobe fractions were transferred to the crystal jars. This procedure was
Systems Incorporated, San Jose, CA, USA) by following the repeated twice for two extractions of the same sample. The aque-
method of Pe~ nuelas et al. (2013c). The assigned value of con- ous samples, previously resuspended in water to reduce the pro-
sumed area for each tree was the mean of the 15 leaves analyzed portion of methanol (< 15% methanol), were lyophilized, and
(see Fig. S3 for details). The values of folivory for each tree were then 4 ml of water was added to each tube, which was vortexed
then standardized by the total foliar biomass of Q. ilex in its plot. and centrifuged at 23 000 g for 3 min. The samples were frozen
All values were thereafter transformed for normality (arcsin at 80°C and lyophilized again. The organic fractions were
(square root(percentage))). Additionally, the fresh leaves were transferred to round-bottomed evaporation flasks and dried in a
also used for calculation of fresh weight, dry weight, foliar water rotary vacuum evaporator. Finally, 1 ml of KH2PO4-NaOD-
content, foliar size and leaf mass per unit area (LMA). buffered D2O + 0.01% trimethylsilyl propionic acid sodium salt
(TSP; pH 6.0) was added to the dried aqueous fractions,
and 1 ml of chloroform-D + 0.01% tetramethylsilyl (TMS) was
Foliar processing for elemental and metabolomic analyses
added to the dried organic fractions. TSP and TMS were used as
The processing of the leaves is described in detail in Rivas-Ubach internal standards. All solutions were resuspended with a micro-
et al. (2013). Briefly, leaves frozen in liquid nitrogen were lyophi- pipette, transferred to 2-ml centrifuge tubes and centrifuged at
lized and stored in plastic cans at 20°C. Samples were ground 23 000 g for 3 min. The supernatants were transferred to NMR
with a ball mill at 1600 rpm for 6 min (Mikrodismembrator-U; sample tubes.
B. Braun Biotech International, Melsungen, Germany), produc-
ing a fine powder that was then stored at 80°C until the extrac-
Extraction of metabolites for liquid chromatography–mass
tion of the metabolites.
spectrometry (LC-MS) analysis
The extraction of metabolites followed the protocol of t’Kindt
Elemental analysis
et al. (2008) with minor modifications. Two sets of Eppendorf
For the C and N analyses, 1.4 mg of the powder from each sample tubes were labeled: set A for extractions and set B for the extracts
was analyzed. The C and N concentrations were determined by ele- from set A.
mental analysis using combustion coupled to gas chromatography The tubes of set A received 100 mg of sample powder from
with a CHNS-O Elemental Analyser (EuroVector, Milan, Italy). each sample, and then 1 ml of MeOH/H2O (80 : 20) was added
Macroelements (P and K) were extracted by acid digestion in a to each tube. The tubes were vortexed for 15 min, sonicated for
microwave reaction system under high pressure and temperature 5 min at room temperature and then centrifuged at 23 000 g for
(Sardans et al., 2010). Briefly, 250 mg of leaf powder was placed 5 min. After centrifugation, 0.6 ml of the supernatant from each
in a Teflon tube with 5 ml of nitric acid and 2 ml of H2O2. tube was transferred to the corresponding Eppendorf tubes of
A MARSXpress microwave reaction system (CEM, Mattheus, set B. This procedure was repeated for two extractions of the
NC, USA) was used for the acid digestions (see the ‘Chemical same sample. The tubes of set B were centrifuged at 23 000 g
analyses’ section of Methods S1 for details). The digested mate- for 5 min. The supernatants were collected using crystal syrin-
rial was transferred to 50-ml flasks and resuspended in Milli-Q ges, filtered through 0.22-lm pore microfilters and transferred
water (EMD Millipore, Darmstadt, Germany) to a final volume to a labeled set of high-performance liquid chromatography
of 50 ml. After digestion, the P and K concentrations were deter- (HPLC) vials. The vials were stored at 80°C until the LC-MS
mined by optic emission spectrometry with inductively coupled analysis.
plasma (ICP-OES) (Optima 4300; Perkin-Elmer Corporation,
Norwalk, CT, USA; see Methods S1 for details).
LC-MS analysis
LC-MS chromatograms were obtained using a Dionex Ultimate
Extraction of metabolites for analysis by nuclear magnetic
3000 HPLC system (Thermo Fisher Scientific/Dionex RSLC,
resonance (NMR)
Waltham, MA, USA) coupled to an LTQ Orbitrap XL high-
The extraction of foliar metabolites for NMR analysis is resolution mass spectrometer (Thermo Fisher Scientific)
described in detail in Rivas-Ubach et al. (2013). First, two sets of equipped with a heated electrospray ionization (HESI II) source.

New Phytologist (2014) 202: 874–885 Ó 2014 The Authors


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Chromatography was performed on a reversed-phase C18 Hyper- function equivalent to 0.2-Hz line broadening before the Fourier
sil gold column (150 9 2.1 mm, 3 lm particle size; Thermo transform. The experimental time was c. 8 min per sample.
Fisher Scientific) at 30°C. The mobile phases consisted of aceto-
nitrile (A) and water (0.1% acetic acid) (B). Both mobile phases NMR metabolite identification Standard 2D NMR experi-
were filtered and degassed for 10 min in an ultrasonic bath before ments (1H–1H correlated spectroscopy (COSY), 1H–1H total cor-
use. At a flow rate of 0.3 ml min 1, the elution gradient began at relation spectroscopy (TOCSY), 1H–13C heteronuclear single
10% A (90% B) and was maintained for 5 min, and then was quantum correlation (HSQC) and 1H–13C heteronuclear multiple
changed to 10% B (90% A) for the next 20 min. The initial pro- bond correlation (HMBC)), and 1D selective 1H TOCSY
portions (10% A; 90% B) were gradually recovered over the next experiments were performed on representative polar and
5 min, and the column was then washed and stabilized for 5 min nonpolar samples for the identification of metabolites. For the
before injecting the next sample. The injection volume of the water–methanol extract samples, 2D experiments were carried
samples was 5 ll. All samples were injected twice, once with the out with standard presaturation of the residual water peak during
HESI operating in negative ionization mode ( H) and once in the relaxation delay. Experiments were acquired using standard
positive ionization mode (+H). The Orbitrap mass spectrometer Bruker pulse sequences and routine conditions (see Methods S2
was operated in Fourier transform mass spectrometry for details). The procedure followed was that described by
(FTMS) full-scan mode with a mass range of 50–1000 m/z and Rivas-Ubach et al. (2013). All elucidated metabolites were further
high-resolution (60 000). The resolution and sensitivity of the confirmed using data reported in the literature (see Tables S1 and
spectrometer were monitored by injecting a standard of caffeine S2 for references).
after every 10 samples, and the resolution was further monitored
with lock masses (phthalates). Blank samples were also analyzed
Processing of LC-MS chromatograms
during the sequence.
The raw data files from the spectrometer were processed using
MZMINE 2.10 (Pluskal et al., 2010). Chromatograms were base-
NMR analysis
line-corrected, deconvoluted, aligned and filtered before the
NMR experiments were performed on a Bruker AVANCE 600 numerical database was exported in CSV format (Table S3 for
spectrometer working at a magnetic field of 14.1 T (1H and 13C details). Metabolites were assigned by exact mass and retention
NMR frequencies of 600.13 and 150.13 MHz, respectively) and time from the measurements of the standards in the MS and
equipped with an automatic sample changer, a multinuclear tri- MSn modes of the spectrometer (Table S4 for details). Different
ple resonance TBI probe and a temperature control unit (Bruker assigned variables corresponding to the same molecular com-
Biospin, Rheinstetten, Germany). The temperature into the pro- pounds were summed. The numerical values of the variables
behead was previously calibrated and maintained constant for all extracted from the LC-MC chromatograms correspond to the
the experiments at 298.0 K; for this purpose a temperature absolute peak areas of the chromatograms detected by the spec-
equilibration delay of 2 min is left for each sample before the trometer. The area is directly proportional to the concentration
shimming process. All NMR sample handling, automation, of the variable, so we use the term ‘concentration’ in this article
acquisition and processing was controlled using TOPSPIN 3.1 soft- when referring to changes in the amount of metabolites between
ware (Bruker Biospin). Spectra were referenced to TSP (1H and the studied factors (control vs drought or between seasons).
13
C at d 0.00 ppm) in the case of polar samples and to the signal
of the residual solvent, CHCl3 (1H at d 7.26 ppm and 13C at d
NMR bucketing
77.16 ppm), in the case of nonpolar samples.
The processing of 1H NMR spectra is detailed in Rivas-Ubach
1 et al. (2013). Briefly, before the exportation of the 1H NMR
H NMR fingerprinting
numerical databases, all spectra were phased, baseline-corrected
All extract samples were analyzed by high-resolution one- and referenced to the resonance of the internal standard (TSP for
dimensional (1D) 1H NMR spectroscopy following the condi- polar and TMS for nonpolar samples) at d 0.00 ppm with TOP-
1
tions described in Rivas-Ubach et al. (2013) and using standard SPIN 3.1. A variable-size bucketing was thus applied to all H
pulse-acquisition 1D 1H-NMR experiments. In the case of the NMR spectra using AMIX software (Bruker Biospin), scaling the
water/methanol extract samples, proton spectra were acquired buckets relative to the internal standard (TMS or TSP). The
with suppression of the residual water resonance. The water reso- output was a data set containing the integral values for each
nance signal was presaturated at a power level of 55 dB, corre- assigned 1H NMR spectral peak in the described pattern. The
sponding to an effective field of 30 Hz during a relaxation delay buckets corresponding to the same molecular compound were
of 2 s. Each experiment was acquired as a set of 32 000 data summed.
points, over a spectral width of 16 ppm, as the sum of 128 tran-
sients and with an acquisition time of 1.7 s. The resulting free
Statistical analyses
induction decays (FIDs) were Fourier-transformed and the spec-
tra obtained were phased and baseline-corrected. The FIDs of To test for differences between seasons and drought treatments in
polar samples were multiplied by an exponential apodization foliar elemental stoichiometry and metabolome, the LC-MS and

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NMR metabolomic fingerprints of the Q. ilex leaves were sub- (a)


jected to PERMANOVA analysis (Anderson et al., 2008) using
the Euclidean distance, with season (spring, summer, autumn
and winter) and treatment (control and drought) as fixed factors
and folivory as a covariable. The number of permutations was
set at 999. The PERMANOVA analysis was conducted with
PERMANOVA+ for the PRIMER v.6 software (Anderson et al.,
2008). One-way ANOVAs were performed for each individual
stoichiometric or metabolite variable (Tables S5, S6). Statistically
significant results of those ANOVAs are indicated by asterisks in
Figs 1 and 2.
Additionally, to understand how the foliar stoichiometry and
metabolome of Q. ilex shifted with the factors studied (season
and climatic treatment), the foliar stoichiometric and metabolo-
mic fingerprints were also subjected to principal component
analysis (PCA) and partial least squares discriminant analysis
(PLS-DA). The seasonal PCA and PLS-DA included the finger-
prints of all seasons combined (Figs 1, S4). Trees in summer
season present the accumulated foliar signals of folivory in the
Mediterranean basin; for this reason, in order to study the effects
of drought on folivory, the fingerprints from summer leaves were
additionally submitted to separate PCAs and PLS-DA (Figs 2, (b)
S5). The PCAs and PLS-DA were performed using the pca and
pls.da functions, respectively, of the mixOmics package of R (R
Development Core Team, 2011). The score coordinates of the
variables of the PCAs were subjected to one-way ANOVAs

Fig. 1 Component 1 vs component 2 of the partial least squares


discriminant analysis (PLS-DA) of the elemental, stoichiometric and
metabolomic variables in Quercus ilex leaves for data of all seasons. (a)
Component 1 and (b) component 2 of the stoichiometric and metabolomic
variables. Carbon (C) : nitrogen (N) : phosphorus (P) : potassium (K) ratios
are shown in red. Different metabolomic families are indicated by different
colors: blue, sugars; green, amino acids; cyan, nucleotides; violet, (c)
phenolics; yellow, related compounds of amino acid and sugar metabolism
(RCAAS); brown, terpenes; dark blue, overlapped nuclear magnetic
resonance (NMR) signals, and light orange, nonpolar metabolites.
Acronyms and abbreviations are used for some of the variables and a
number has been assigned to each metabolite forming part of the
overlapped NMR signals: sucrose (Suc; 1), a-glucose (aG; 2), b-glucose
(bG; 3), hexose (Hex), pentose (Pent), disaccharide (Disacch), lactic acid
(11), citric acid (4), alanine (Ala; 5), isoleucine (Ile; 6), threonine (Thr),
valine (Val; 7), leucine (Leu), phenylalanine (Phen), proline (Pro), arginine
(Arg), tryptophan (Trp), tyrosine (Tyr), quercitol (8), quinic acid (9),
choline (10), N-acetyl group (12), polyphenol (13), fatty acids (FA),
unsaturated fatty acids (UFA), polyunsaturated fatty acids (PUFA),
diacylglycerides (DGA), triacylglyceride 1 (TGA1), triacylglyceride 2
(TGA2), aldehyde group (Ald), acetyl group (Acetyl), linoleyl fatty acid
(Linoleyl FA), overlapped NMR signals (O1–O10): O1, 5 + 10 + 2 + 1; O2,
10 + 2 + 1; O3, 10 + 3 + 1; O4, 4 + 13; O5, 6 + 7; O6, 11 + unknown, O7,
11 + 12, O8, 8 + 9; O9, 8 + 9 + 2 + 3 + 1 and O10, 8 + 1. Unassigned
metabolites are not represented in the variable plot. (c) Samples
categorized by season and drought treatment. Seasons are indicated by
different colors (green, spring; red, summer; yellow, autumn; blue,
winter). Control and drought trees are indicated by asterisks and triangles to detect statistical differences among groups (see supporting
respectively. Capital letters indicate the mean component 1 vs component
information of Rivas-Ubach et al., 2013). A Kolmogorov–
2 scores for the treatments in each season (C, control trees; D, droughted
trees). Variables marked with asterisks showed statistical significance Smirnov (KS) test was performed on each variable to test for
(P < 0.05) or marginal statistical significance (P < 0.1) in one-way ANOVAs normality. All assigned and identified metabolites were normally
(Supporting Information Table S5). distributed, and any unidentified metabolomic variable that was

New Phytologist (2014) 202: 874–885 Ó 2014 The Authors


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Phytologist Research 879

not normally distributed was removed from the data set. The KS scores of this PCA, including the entire elemental, stoichiometric
tests were performed with the ks.test function of the tuncogof and metabolomic variation of the case of trees in summer, were
package of R (R Development Core Team, 2011). plotted against the degree of folivory (Fig. 3). Folivory was
Folivory begins in spring and early summer, and trees do not analyzed with a general linear model (GLM) as a function of the
produce new leaves in summer, so the sampled leaves had accu- climatic treatment and the metabolomic variation (PC1 scores).
mulated signs of folivory by the middle of the summer. An addi- Another GLM analyzed the total foliar sugar and phenolic con-
tional PCA was thus conducted with summer data excluding the centrations as functions of climatic treatment and season. STATIS-
proportions of herbivorous consumption (Fig. S6). The PC1 TICA v8.0 (StatSoft, Tulsa, OK, USA) was used for the one-way
ANOVAs and the post hoc tests of the score coordinates of the
PCAs. GLM analyses were performed with the lme function of
the nlme package of R (R Development Core Team, 2011). All
(a)
statistical analyses were performed at the individual level.

Results
The PERMANOVA analysis performed with all elemental, stoi-
chiometric and metabolomic data (assigned and unassigned
metabolites) indicated that leaves in different seasons, under
drought treatment and with different degrees of folivory had dif-
ferent foliar chemistries and metabolisms (folivory: pseudo-
F = 2.4832; P < 0.001; season: pseudo-F = 2.4749; P < 0.001;
and treatment: pseudo-F = 3.1031; P < 0.001).

Elemental, stoichiometric and metabolomic shifts across


seasons and experimental drought treatments
The PCA conducted with all seasons, including all the elemental,
stoichiometric and metabolomic data, indicated that more than
50% of the total variance was already gathered by the first four
PCs (PC1 = 15.2%, PC2 = 14.7%, PC3 = 14.1% and
PC4 = 12.7%; Fig. S4). Differences between seasons were
explained by PC1 (P < 0.001), PC3 (P < 0.05) and PC4

(b)

Fig. 2 Component 1 of the partial least squares discriminant analysis


(PLS-DA) of the metabolomic and stoichiometric variables for the summer
data. (a) Variable plot and (b) case plot. Carbon (C) : nitrogen
(N) : phosphorus (P) : potassium (K) ratios are shown in red and herbivory
is shown in dark red. Different metabolomic families are indicated by
different colors: blue, sugars; green, amino acids; cyan, nucleotides; violet,
phenolics; yellow, related compounds of amino acid and sugar metabolism
(RCAAS); brown, terpenes; dark blue, overlapped nuclear magnetic
resonance (NMR) signals; light orange, nonpolar metabolites. Unassigned Fig. 3 Principal component 1 (PC1) scores of a principal component
metabolites are not represented in the graph. Details of the variables are analysis (PCA), excluding the degree of folivory, for summer metabolomic
given in Fig. 1. Control trees, asterisks; droughted trees, triangles. and stoichiometric data (Supporting Information Fig. S1) vs the proportion
Variables marked with asterisks showed statistical significance (P < 0.05) or of foliar consumption (arcsin(square root(percentage of consumption))).
marginal statistical significance (P < 0.1) in one-way ANOVAs (Supporting Gray circles, control trees; gray crosses, droughted trees. Black circle, mean
Information Table S7). of control trees  SE; black cross, mean of droughted trees  SE.

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(P < 0.001). Post hoc analysis of the score coordinates of the dif- (a)
ferent case trees showed that the stoichiometries and metabolo-
mes of summer leaves differed significantly from those of leaves
in other seasons on the PC1 axis. Droughted trees differed from
control trees in PC1 (P < 0.001), PC2 (P < 0.05) and PC4
(P < 0.001). PLS-DA with all seasons including all the elemental,
stoichiometric data showed clear separation between seasons
(Fig. 1).
The C, N, P and K foliar concentrations and the ratios C : N,
C : P, C : K, N : K and K : P, but not N : P, changed with the sea-
sons (Fig. 1, Table S5). The highest K : P and K : N concentra-
(b)
tion ratios occurred in summer, coinciding with the highest foliar
K concentrations. Foliar N and P concentrations were highest in
autumn, thus resulting in the lowest C : N and C : P ratios (see
Table S5 and Sardans et al., 2013b for more details).
Most assigned and identified metabolites in the leaves of
Q. ilex shifted across the seasons (Fig. 1, Table S5). The main
changes were between spring and summer, two of the most criti-
cal seasons in the Mediterranean basin, because spring is the
period of growth and summer is the driest season. Summer leaves
generally had higher concentrations of polyphenolics and sucrose,
whereas spring leaves had higher concentrations of amino acids, (c)
some related compounds of amino acid and sugar metabolism
(RCAAS) and some sugars such as pentoses and disaccharides,
products directly related to growth. The effects of experimental
drought were also detected on the seasonal PLS-DA and PCA
plots (Figs 1, S4, Tables S6, S7). In the case plot for both analy-
ses, PLS-DA and PCA, droughted trees tended to be distributed
in the same direction as the summer trees; summer is the driest
season in Mediterranean climates (Fig. S1a).

Total concentrations of amino acids, sugars and Fig. 4 Bar graphs of the total foliar concentrations of amino acids (a),
polyphenolics sugars (b) and phenolics (c)  SE. Open bars, control plots; closed bars,
droughted plots. The statistically significant differences between seasons
The concentrations of the assigned and identified variables of the and treatments were detected by Bonferroni post hoc tests (P < 0.05).
different metabolite families (amino acids, sugars and phenolics) Seasonal statistical differences are indicated by different letters, and
climatic statistical differences are indicated by asterisks. The concentration
were summed (Fig. 4). The integral values of 1H NMR spectra
of metabolites on the y-axis is explained in detail in the Materials and
relative to the internal standard were standardized to sum with Methods section.
the peak area of the LC-MS chromatograms. Factorial ANOVAs
indicated that trees presented higher concentrations of foliar
Folivory and drought
amino acids in spring and winter (Fig. 4a). The concentrations of
sugars were higher in winter than in the other seasons (Fig. 4b). We did not detect significant differences between leaves of con-
Trees had higher concentrations of total phenolics in summer, trol trees and droughted trees in foliar fresh weight (F = 0.74;
and the drought treatments increased the phenolic concentrations P = 0.398), foliar dry weight (F = 0.64; P = 0.43), foliar water
significantly in summer and winter (Fig. 4c). The drought treat- content (F = 1.45; P = 0.25) and foliar size (F = 0.33; P = 0.57).
ment affected the trees in all seasons, increasing the concentra- Mean values of LMA was 13.6% lower in droughted plants
tions of total sugars, but the concentrations in summer were not (19 mg DW cm 2) than in control plants (22 mg DW cm 2;
significantly different from those of the control trees (Fig. 4b). P < 0.05) in summer, the analyzed season for folivory. The PLS-
The GLM analysis with total sugars as a dependent variable, DA and PCA conducted to investigate the relationships of foli-
season and climatic treatment as fixed independent variables and vory with drought summer also indicated a difference between
individuals as a random variable showed statistical significance control and droughted trees in the multidimensional space
for both climatic treatment (P < 0.001) and season (P < 0.05). (Figs 2, S5). PC1 of the PCA of summer explained 18.7% of the
The GLM analysis with the same independent variables but with total variance and separated significantly control from droughted
total phenolics as a dependent variable also showed statistical sig- trees (P < 0.001; Fig. S5).
nificance for both climatic treatment (P < 0.05) and season The GLM conducted with the summer data, including the
(P < 0.05). accumulated proportions of folivory and with folivory as a

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Phytologist Research 881

dependent variable and climatic treatment and PC1 scores of the stem (James, 1984) that are able to store essential nutrients and
additional PCA (without the folivory variable; Fig. S6) as inde- metabolites such as carbohydrates to ensure rapid growth after
pendent variables (P < 0.001; R2 = 0.60), indicated significant severe stress (Canadell & Zedler, 1995). Our stoichiometric and
effects of the drought treatment (P < 0.05) and no significant metabolomic data support the idea that trees, with large woody
effect of the PC1 scores (P = 0.53). These results imply that the structures such as trunks, lignotubers, and/or extensive root
existence of a significant relationship between folivory and networks, are able to buffer the metabolomic changes in foliar
the PC1 score coordinates of the PCA is mainly attributable to ontogeny by storing large amounts of nutrients, allowing the allo-
the fixed effect of drought (Fig. 3). cation of essential resources to photosynthetic organs and thus
leaf growth and resistance to severe environmental disturbances
(Canadell & Zedler, 1995; Landhausser & Lieffers, 2002;
Discussion
Anderegg & Callaway, 2012; Galiano et al., 2012).
The largest stoichiometric shift between seasons appeared to
Seasonality and drought
be related to the responses to drought conditions (Figs 1, S4,
The foliar stoichiometry and metabolome of Q. ilex varied with Table S5). Foliar concentrations of K were higher in trees in sum-
season (PERMANOVA P < 0.001; Figs 1, S4). Seasonal PLS-DA mer, the driest season. The resulting higher foliar K : P and lower
and PCA identified significant stoichiometric and metabolomic N : K and C : K concentration ratios (Sardans et al., 2013b) were
shifts between summer and the other seasons, although the differ- thus in agreement with the findings of other Mediterranean sea-
ences between seasons were not as marked as those of an earlier sonal studies (Sardans et al., 2012a, 2013b). K participates in the
study with the Mediterranean shrub Erica multiflora (Rivas- water economy of plants (Babita et al., 2010) through osmotic
Ubach et al., 2012). Trees in spring, the Mediterranean growing control (Babita et al., 2010; Laus et al., 2011) and improves the
season, had high concentrations of metabolites directly related to functioning of foliar stomata (Khosravifar et al., 2008). These
growth such as amino acids, some RCAAS and some sugars such results demonstrate the important role of K in the ecology of
as pentoses and disaccharides, in accordance with the earlier study terrestrial plants (Cakmak, 2005; Sardans et al., 2012a, 2013b).
(Rivas-Ubach et al., 2012; Fig. 1), even though the total concen- Moreover, these shifts in K concentrations in summer leaves
tration of amino acids did not differ from the levels in winter were accompanied by higher concentrations of sucrose, which
(Fig. 4a), the coldest season in the Mediterranean basin. In can also act as an osmolyte that, together with K, can help to pre-
Q. ilex, foliar N : P ratios did not present the lowest values in vent the loss of water through osmotic control (Ingram & Bartels,
spring as expected from the growth rate hypothesis (Elser et al., 1996).
1996; Rivas-Ubach et al., 2012; Figs 1, S4, Table S5), which pro- As seen in previous studies, a decrease in growth and an
poses that organisms with high rates of growth require high con- increase in tree mortality are among the main consequences of
centrations of P (low N : P ratios) to meet the demands of this prolonged moderate drought (Barbeta et al., 2013; Fig. S2).
protein synthesis (Elser et al., 2003). Our results are nevertheless Our seasonal PLS-DA and PCA with metabolomic and stoichi-
in accordance with those of other studies in plants. The high ometric data also showed interesting differences between the trees
foliar concentrations of amino acids in winter trees might be in the drought plots and those in the control plots (Figs 1, S4;
attributable to accumulation under cold conditions. It has been PERMANOVA P < 0.001). Droughted trees tended to have the
observed that some amino acids such as proline and glycine are same pattern as summer trees on the Component 2 of case plot
able to buffer the NADP+ : NADPH ratio in plants (Xu et al., of PLS-DA (Fig. 1) and PC1 and PC2 of PCA (Fig. S4), in
2013) as a consequence of low photosynthetic activity in winter accordance with a previous study (Rivas-Ubach et al., 2012), thus
to provide reducing agents for the generation of ATP in mito- indicating a foliar elemental-metabolomic response to drought
chondria (Hare & Cress, 1997; Xu et al., 2013). However, Mat- independent of plant ontogeny. Summer trees and droughted
zek & Vitousek (2009) found no significant relationships trees in all seasons tended to have higher foliar concentrations of
between the N : P ratio and increased growth in 14 species of flavonoids (Figs 1, 4c, S4), the largest group of naturally occur-
pines, concluding that terrestrial plants must invest in fundamen- ring polyphenols (Strack et al., 1994), but the flavonoid composi-
tal functions other than growth, such as storage and defense, that tion differed between summer and the drought treatment.
often require different investments in N and P (Herms & Matt- Summer trees had higher concentrations of quinic acid, catechin
son, 1992; Pe~ nuelas & Estiarte, 1998). and luteolin, among other polyphenolics, whereas droughted
As observed in the multivariate ordination analyses, the con- trees had higher concentrations of catechin, quercitol, homoori-
centrations of the various sugar compounds detected were distrib- entin and quercetin (Figs 1, S4, Table S6). These differences cor-
uted differentially among the seasons (Figs 1, S4), but droughted related with the differential effects of drought treatment in the
trees in winter had the highest concentrations of total sugars different seasons (Fig. 4c). Many biological functions have been
(Fig. 4b), which may also be related to their accumulation and ascribed to flavonoids, especially their role as antioxidants (Burda
allocation from other organs (e.g. wood and lignotubers) in & Oleszek, 2001; Lee et al., 2003). Their antioxidant activity is
response to a cold environment to leaves or further preparation mainly a result of their role as electron donors (RiceEvans et al.,
for the growth season (Grimaud et al., 2013; Xu et al., 2013). 1997) and their ability to alter the kinetics of peroxidation (Arora
Quercus ilex is an evergreen tree with slow growth; it presents et al., 2000). As expected in a water-limited Mediterranean
large lignotubers, swollen woody structures at the base of the ecosystem, oxidative stress in plants tends to increase under

Ó 2014 The Authors New Phytologist (2014) 202: 874–885


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New
882 Research Phytologist

conditions of drought (Price et al., 1989; Dat et al., 2000; N concentrations (Choong et al., 1992; Williams et al., 1998). As
Munne-Bosch & Pe~ nuelas, 2004; Pe~ nuelas et al., 2004). Querci- discussed, droughted trees tended to have higher concentrations
tol has been associated with the avoidance of osmotic stress by of sugars and polyphenols (flavonoids) than did the controls
reducing the osmotic potential during drought in Quercus species (Fig. 4). Flavonoids such as quercetin have been shown to act as
(Passarinho et al., 2006; Spiess et al., 2012). Our study found no phagostimulants in herbivorous insects (Diaz Napal et al., 2010;
significant differences (P = 0.12) between choline concentrations Kosonen et al., 2012). Despite the scarcity of supporting evi-
in droughted and control trees but did identify a tendency toward dence, our results also indicated a higher folivorous activity in
higher concentrations in droughted plants (Table S6). Choline is droughted trees, supporting the premise of the high palatability
also involved in osmotic protection (McNeil et al., 2001). of some flavonoids that are not directly related to defense but act
Drought will probably have a significant impact on the metabol- as antioxidants (Diaz Napal et al., 2010; Kosonen et al., 2012).
omes of Mediterranean plants, because dry periods will become Also, droughted leaves had higher concentrations of sugars
more frequent and intense in the coming decades, as predicted by (Fig. 4), especially sucrose and a-glucose (Fig. 2), which could
projections of climate change (IPCC, 2007). thus provide a source of rapid energy for herbivores. The higher
Droughted trees also presented higher concentrations of total concentrations of sugars and some flavonoids found in the foliage
soluble sugars in their leaves (Fig. 4c), supporting the premise of droughted Q. ilex trees appeared to be related to the increase in
that sugars act as osmolytes to prevent the loss of water by regulat- herbivorous activity (Figs 3, S7).
ing water potential and draw water into leaves during periods of Not all the assigned polyphenols, however, provide only anti-
drought (Ingram & Bartels, 1996). Control trees in summer did oxidant protection in plants. The role of polyphenolics as a
not have significantly lower foliar concentrations of sugars relative chemical defense against predation by herbivores has been widely
to droughted trees (Fig. 4b), even though droughted trees had a reported (Berg, 2003; Lokvam & Kursar, 2005; Treutter, 2006;
tendency toward higher concentrations, presenting significantly Kosonen et al., 2012; Rani & Pratyusha, 2013). Moreover, Koso-
higher concentrations of sucrose and a-glucose. The small differ- nen et al. (2012) reported that some polyphenolics could be toxic
ences in foliar sugar concentrations between control and drough- to specialist herbivores but could increase the palatability of
ted leaves may be mainly attributable to the naturally dry plants for generalist herbivores and suggested that climate change
summers of the Mediterranean basin that increased the foliar may reduce the damage caused by specialist herbivores and
sugar concentrations in both control and droughted trees (Fig. 4). increase that caused by generalists. Phenolic acids could thus
potentially be used for pest management (Rani & Pratyusha,
2013). It has been reported that even mammals can be affected
Folivory
by the polyphenols of plants. For example, Berg (2003) reported
The highest herbivorous activity by insects occurs mainly in that elevated concentrations of catechin negatively affected the
spring and early summer in the Mediterranean basin (Powell & consumption of plants by collared lemmings. In our study, nearly
Logan, 2005; Bonal et al., 2010), and usually the leaves of trees all the assigned polyphenols were associated with drought.
have accumulated most signs of folivory by the middle of
the summer. Interestingly, folivorous activity was higher in the
droughted trees in summer (Figs 2, 3). We did not detect any sig-
nificant difference between control trees and droughted trees in
foliar fresh weight, foliar dry weight, foliar water content or foliar
size that could explain the higher folivory activity in summer.
The small LMA differences between controls and droughted trees
were attributable to differences in thickness and not to the foliar
density as found in past studies (Ogaya & Pe~ nuelas, 2006), which
should not influence consumption through folivory.
GLM analyses for the summer season indicated that the rela-
tionship between folivory and PC1 score coordinates of the addi-
tional PCA for foliar stoichiometry and metabolomics (Fig. S6)
was mainly attributable to the fixed effect of drought, because
folivory was not correlated with PC1 within treatments (control,
drought; P > 0.05; Fig. 3). Our results thus suggest that the stoi-
chiometric and metabolomic shifts were mainly caused by the
experimental drought, which subsequently indirectly increased
the degree of folivory. Fig. 5 Summary of the relationship between foliar metabolic shifts
Our stoichiometric results identified no significant relation- produced by drought and folivory activity. As drought increases (a) there is
ships between foliar N concentrations and foliar C : N ratios and a shift in the foliar metabolome of Quercus ilex, mainly by increasing the
concentrations of antioxidants and sugars (b). This increase in antioxidants
the degree of folivory, as reported in other studies (Larsson, and sugars stimulates folivory activity on droughted trees relative to
1989; Larsson & Bjorkman, 1993; Rouault et al., 2006), indicat- control trees (c). The increase in folivory in Q. ilex leaves may later cause
ing that, in our study, folivory was not influenced only by foliar an increase in the synthesis of defense compounds (d).

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New
Phytologist Research 883

Most of the assigned flavonoids have antioxidant activity, but


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