SUPPORTING INFORMATION
Drought enhances folivory by shifting foliar metabolomes in Quercus ilex trees.
RIVAS-UBACH et al., New Phytologist
Methods S1.
Chemical analyses
C and N analyses: For each sample, 1.4 mg of powder were analyzed. C and N concentrations
were determined by elemental analysis using combustion coupled to gas chromatography with
a CHNS-O Elemental Analyser (EuroVector, Milan, Italy).
P and K analyses: The P and K analyses were performed using acid digestion in a microwave
reaction system under high pressure and temperature. Briefly, 250 mg of leaf powder were
transferred to a Teflon tube, and 5 mL of nitric acid and 2 mL of H2O2 were added (7). A
MARSXpress microwave reaction system (CEM, Mattheus, USA) was used to perform the acid
digestions. The temperature was increased to 130 ºC with a 10-min ramp, samples were
maintained at this temperature for 5 min, then the temperature was increased to 200 ºC with
a 10 min ramp where the samples were maintained for 20 min. To end the digestions, the
temperature was increased to 220 ºC with a 5 min ramp and maintained for a further 20 min.
All digested contents were transferred to 50-mL flasks and resuspended in Milli-Q water
to a final volume of 50 mL. After digestion, the P and K concentrations were determined by
ICP-OES (Optic Emission Spectrometry with Inductively Coupled Plasma) in an Optima 2300RL
spectrometer (The Perkin-Elmer Corporation, Norwalk, USA).
Methods S2.
NMR Elucidation.
The polar and nonpolar metabolic profiles of Quercus ilex leaf are shown in Figs. S2a and S2b
respectively and in Tables S1 and S2.
The detailed 1H NMR metabolic profile of the polar extract is shown in Figs. S3. The signals in
the region between 3.2 and 5.9 ppm correspond mainly to sugars. Among them, α-glucose and
β-glucose, with anomeric proton resonances at 5.29 ppm (d, J = 3.8 Hz) and at 4.70 ppm (d, J =
8.2 Hz) respectively, were identified. The disaccharide sucrose was also observed, with its
characteristic anomeric proton doublet of the glucose unit at 5.45 ppm (d, J = 3.8 Hz). The
proton singlet at 3.19 ppm is correlated via HSQC to the carbon signal at 53.8 ppm,
corresponding to choline which is an important osmolyte. In the aliphatic region between 2.2
and 1.7 ppm, the secondary metabolites quinic acid and quercitol were identified, showing
very intense signals. Among amino acids, valine, alanine, isoleucine, threonine and lysine were
identified. Some organic acids such as formic acid (sharp singlet at 8.50 ppm), citric acid, acetic
acid and lactic acid were identified as well. Finally, signals at the 6.5 - 7.4 ppm region, area
mainly of aromatic compounds, were also observed. All the identifications were based on the
1
H and 13C NMR complete or partial assignment of the molecules based on 1D and 2D NMR
experiments and on the comparison with reported data. For the complete description see
Table S1.
Fig. S2b shows the 1H NMR metabolic profile of the nonpolar extract samples. Saturated and
unsaturated fatty acid chains were identified, which come from free fatty acids, fatty alcohols,
diacylglycerols and triacylglycerols. Also, p-coumaric acid derivatives were observed in the
aromatic region. The complete description of nonpolar metabolites is shown in Table S2.
Table S1. NMR assignments of the metabolites identified in polar samples. Samples dissolved
in D2O (pH 6.0) and 1H and 13C NMR chemical shifts referenced to TSP.
Polar Metabolites
Bibliographical
Metabolite
Assignment
NMR chemical shift ()
References
Atom
1H
id.
13C
a, b, c
Id.
Name
molec
(ppm)
m,d J (Hz)e
(ppm)
1
5.29
d; 3,8
92.6
2
-
-
-
3
3.68
m*
73.0
4
3.45
m*
70.5
5
3.88
m*
72.8
6
3.95
m*
61.2
1
4.70
d; 8,2
96.4
2
3.29
dd; 9,3; 8,3
73.9
3
3.59
m*
77.0
.
-Glucose
2
(Fan & Lane,
2008, Fan,
1996, Walker
et al., 1982)
a [COSY, TOCSY,
HSQC, HMBC], b,
-Glucose
c
3
(Fan & Lane,
2008, Fan,
1996, Walker
et al., 1982)
a [COSY, TOCSY,
HSQC, HMBC], b,
Sucrose
4
*
*
*
5
3.53
m*
76.2
6
3.81
m*
61.5
1
3.73
s
61.6
2
*
*
*
c
1
a [COSY, TOCSY,
HSQC, HMBC], b,
3
4.26
d; 8,8
76.8
4
4.08
t; 8,6
74.4
c
(Breitmaier et
al., 1979, Fan
& Lane, 2008,
Fan, 1996)
Alanine
5
3.94
m
81.8
6
3.87
d, 3,4
62.9
1'
5.45
d; 3,8
92.7
2'
3.61
m
71.5
3'
3.82
t; 9,5
72.8
4'
3.51
t; 9,4
69.7
5'
3.88
m
72.8
6'
3.86
d; 3,4
60.5
1
-
-
178.0
2
3.84
*
52.5
5
(Breitmaier et
al., 1979, Fan
3
1.53
d; 7,3
a [COSY, HSQC,
& Lane, 2008,
HMBC], b, c
Fan, 1996, Iles
et al., 1985,
16.5
London, 1988)
Isoleucine
1
-
-
*/**
2
*
d*/**
*/**
3
1.99
m*/**
*/**
6
(Breitmaier et
al., 1979, Fan
Threonine
4a
1.25
m
16.9
4b
1.48
m
16.9
5
0.93
t; 7,3
26.5
6
1.02
d;8,1
23.2
1
-
-
2
3.58
*
& Lane, 2008,
Fan, 1996, Iles
et al., 1985)
a [COSY, HSQC],
b, c
(Bolinger et
al., 1984,
*/**
Breitmaier et
a [COSY, HSQC],
3
4.25
m
66.3
al., 1979, Fan
b, c
& Lane, 2008,
Citrate
4
1.32
d; 6,40
20.6
1
-
*
*
2a
2.58
d*
45.6
Fan, 1996)
a [COSY, HSQC],
b, c
4
(Fan & Lane,
2008, Fan,
1996, Iles et
Dimethylamine
2b
2.55
3
-
1
2.74
d*
45.6
al., 1985)
*
s
36.0
(Fan & Lane,
2008, Fan,
1996)
a [HSQC], b,c
Quercitol
2
2.74
1
3.77
2
3.57
3
3.74
s
36.0
68.8
8
(McCasland et
al., 1968)
t;9,5
74.3
70.8
a [COSY, TOCSY,
4
3.96
t*
72.2
5
4.04
q*
68.3
6a
2.00
dt;13,9;3,4
33.2
6b
1.82
td;13,9;3,4
33.2
1
3.99
m
69.6
2
3.52
m
58.0
HSQC, HMBC], b,
c
Choline
Lactate
Valine
3
3.19
s
53.8
1
-
*
*
2
4.20
q*
70.3
3
1.37
d*
17.0
1
-
-
*
2
3.61
d*
*
3
2.28
m
33.4
4
1.01
d*
17.6
5
1.05
d*
*
10
(Fan, 1996,
a [COSY, HSQC,
Govindaraju
HMBC], b, c
et al., 2000)
11
(Fan & Lane,
a [COSY,HSQC],
2008, Fan,
b, c
1996, Iles et
al., 1985)
7
(Fan & Lane,
2008, Fan,
H
4
H3C
1
3
5
H3C
NH2
1996)
OH
2
H
O
a [COSY], b, c
Quinic acid
1
-
-
*
2
-
-
77.5
3a
2.00
dt; **
37.3
3b
2.07
dd; **
37.3
9
(Corse &
Lundin, 1970,
Duarte et al.,
2006)
a [COSY, TOCSY,
4
4.18
dt; **
70.4
5
3.58
dd; **
63.9
6
4.04
m
63.1
7a
1.90
dd; **
41.0
7b
2.10
ddd; ***
41.0
HSQC, HMBC], b,
c
Formate
10
(Brown et al.,
1989, Fan &
1
8.50
s
*
Lane, 2008,
a,b,c
Fan, 1996,
Nicholson et
al., 1985)
Lysine
1
-
-
*
2
*
*
*
3a
1.87
m
32.15
11
(Castro &
Manetti,
2007, Castro
et al., 2008,
Fan & Lane,
3b
*
*
32.15
a [COSY, TOCSY,
4a
*
*
*
HSQC, HMBC], b,
c
4b
*
*
*
2008, Fan,
1996, Manetti
et al., 2006,
Walker et al.,
1982)
5a
1.75
m
29.11
5b
*
*
29.11
6
3.04
t;7,5
41.78
* Overlapped signal.
** Not enough signal.
a From 1D 1H NMR chemical shift and 2D 1H-1H and 1H-13C NMR couplings.
b Comparison with 1H and 13C NMR data from references indicated.
c Comparison with 1H and 13C NMR data from the Biological Magnetic Resonance Data Bank.
(http://www.bmrb.wisc.edu/).17
d Multiplicity: singlet (s), doublet (d), t (triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets), ddd
(doublet of doublet of doublets), m (multiplet).
e 1H-1H coupling constant.
Table S2. NMR assignments of the main peaks of nonpolar samples. Samples dissolved in CDCl3
and 1H and 13C NMR chemical shifts referenced to the residual solvent peak.
NMR Peak/Spectral Region
 1H
m, J
 13C
id.
(ppm)
(Hz)
(ppm)
spe
Assignment
Bibliographical References
metabolite (atom id.)/group
(Gunstone, 1995, Lie Ken Jie &
Lam, 1995, Llusia et al., 2008,
Nemethy & Calvin, 1982, Sacchi
ct.
0.86-0.89
t
14.1
A
et al., 1997, The AOCS Lipid
-CH3 ; FAa chains
Library, Ulrich & Zhulin, 2007,
Vlahov, 1999)
0.93-0.96
t
14.4
B
-CH3 ; linoleyl FA chains
1.24-1.27
br
29.1-29.9
C
-CH2 ; FA chains
1.37
t; 7,2
13.8
CH3-CH2-OR; Ethoxy group
1.57
m
33.5
HO-CH2-CH2- ; FAlb
1.58-1.64
m
25.0
D
-CO-CH2-CH2- ; FA chains
2.02-2.10
m
27.0
E
=CH-CH2- ; UFAc chains
2.24-2.37
t
34.3
F
-CO-CH2- ; FA chains
2.33-2.36
t
33.9
G
-CO-CH2- ; FFAd
2.76
*
25.6
=CH-CH2-CH=; linoleyl and/or
linolenyl chain
H
=CH-CH2-CH= ; PUFAe chains
2.78-2.83
m
25.7
3.63
t
63.1
HO-CH2-; FAl
4.37
q; 7,2
61.7
CH3-CH2-OR; Ethoxy group
3.75
dd
68.4
I
-CH2OH; 1,2-DAGf
3.89
dd
68.4
I
-CH2OH; 1,2-DAG
4.15
dd
62.1
I
-CH2O-; TAGg
4.21
dd
62.7
I
-CH2O-; 1,2-DAG
4.29
dd
62.1
I
-CH2O-; TAG
4.40
dd
62.7
I
-CH2O-; 1,2-DAG
5.26
*
68.9
J
-CHO-; TAG
5.29-5.42
m
127.7-132.0
K
-CH= ; UFA chains
5.31
*
70.2
L
-CHO-; 1,2-DAG
6.29
d; 16,0
116.3
M
PCADh(2)
6.83
d; 8,5
115.8
M
PCAD(6/8)
7.43
d; 8,5
129.9
M
PCAD(5/9)
7.60
d; 16,0
143.6
M
PCAD(3)
-
166.9
PCAD(1)
-
127.1
PCAD(4)
-
157.4
PCAD(7)
a. FA: fatty acid; b. FAl: fatty alcohol; c. UFA: unsaturated fatty acid; d. FFA: free fatty acid; e. PUFA:polyunsaturated
fatty acid; f. 1,2-DAG: 1,2-diacylglycerol; g. TAG: triacylglycerol; h. PCAD: p-coumaric acid derivative
* Overlapped signal.
Table S3. LC-MS chromatogram processing. Chromatograms obtained by LC-MS and processed
by MzMine 2.0 (Pluskal et al., 2010). The following table summarizes the different processes
and parameters applied to foliar Q. ilex chromatograms.
(+H) Chromatograms
1
2
3
4
5
6
7
8
Baseline correction
Chromatogram type
MS level
Smoothing
Asymmetry
Mass detection (Exact Mass )
Noise level
Chromatogram builder
Min time span
Min height
m/z tolerance
Smoothing
Filter width
Chromatogram deconvolution
(Local minimum search)
Chromatographic threshold
Search minimum in RT range
(min)
Minimum relative height
Minimum absolute height
Min ratio of peak top/edge
Peak duration range
Chromatogram alignment (join
alignment)
m/z tolerance
weight for m/z
RT tolerance
Weight for RT
Gap filling (Peak Finder)
Intensity tolerance
m/z tolerance
Retention time tolerance
RT correction
Filtering
Minimum peaks in a row
Ions excluded from database
(-H) Chromatograms
TIC
1
10E7
0.001
TIC
1
10E7
0.001
5 × 105
4 × 105
0.05
25000
0.002
0.05
25000
0.002
5
5
65%
0.1
65%
0.1
5.0%
30000
2
0.0-2.0
5.0%
30000
2
0.0-2.0
0.001
80
0.15
20
0.001
80
0.2
20
20%
0.001
0.1
marked
20%
0.001
0.1
marked
8
8
<75
83.05
102.05
114.09
227.17
607.29
Between 0.0 and 1 min
Between 28.5 and 30 min
<85
119.035
223.082
391.196
159.25
186.186
Between 0.0 and 1,1 min
Between 27.0 and 30 min
Table S4. Metabolite assignment by LC-MS. The assignment of the metabolites was based on
the standards. The following table summarizes the retention time (RT) and mass (m/z) of the
assigned metabolites in both positive and negative ionization modes.
Mode
Compound
RT (min)
m/z
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Negative
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Positive
Catechin
Chlorogenic acid
Citric acid
Deoxy-hexose
Disaccharide
Epicatechin
Epigallocatechin
Gallic acid
Hexose
Homoorientin
Kampferol
Lactic acid
Malic acid
Pentose
Pyruvate
Quercetin
Quinic acid
Rhamnetin
Sodium salicylate
Succinic acid
Adenine
Adenosine
α-humulene
Alanine
Arginine
Aspartic acid
Caryophyllene
Catechin
Chlorogenic acid
Epigallocatechin
Glutamic acid
Glutamine
Kampferol
Leucine
Luteolin
Phenylalanine
Proline
Pyridoxine
Quercetin
Rhamnetin
Tryptophan
Tyrosine
Valine
3.44
3.11
1.77
1.42
1.43
4.93 - 5.2
1.54 - 2.64
1.55 - 1.83
1.44
9.45
14.82
1.52; 1.75
1.51; 1.78
1.43
1.65
13.72
1.47
15.98
10.51
1.74; 1.78
1.42; 1.77
1.49; 1.75
20.27
1.43
1.34
1.5
21.46
3.44
3.11
1.54 - 2.64
1.41
1.46
14.82
1.76
13.68
1.91
1.49
1.38
13.72
15.95
2.49
1.54 - 1.77
1.53
289.0718
353.0873
191.0196
163.0618
341.108
289.0713
305.0667
169.0147
179.056
447.0923
285.0404
89.0245
133.0143
149.0456
87.0089
301.0355
191.056
315.0509
137.0245
117.0194
136.0614
268.1038
205.1949
90.054
175.119
134.044
221.1899
291.0863
355.084
307.0812
148.0604
147.076
287.0552
132.101
287.0551
166.086
116.07
170.0812
303.0498
317.0653
205.097
182.081
118.086
Table S5. Data for one-way ANOVAs of all stoichiometry and assigned metabolites extracted
from Q. ilex leaves for seasons (summer, autumn, winter, spring). Integral mean values (mM
relative to initial TSP or TMS concentrations (0.01%)) are given for the RMN variables, marked
by asterisks (*). Integral mean values of deconvoluted total intensities are given for the LC-MS
variables, marked by crosses (†). Different letters beside the values indicate significant
seasonal differences detected by LSD post-hoc tests (p < 0.05).
Variable
C/N
N/P
C/P
C/K
K/P
N/K
N (mg/g dry weight)
P (mg/g dry weight)
K (mg/g dry weight)
C (mg/g dry weight)
aG *
BG *
Suc *
Deoxy-Hex †
Hex †
Pent †
Disacch †
Ile *
Thr *
Ala †
Val †
Leu †
Phen †
Pro †
Arg †
Trp †
Tyr †
Adenine †
Adenosine †
Choline †
Quercitol *
Polyphenol *
Phenolic group *
Quinic acid †
Catechin †
Chlorogenic acid †
Epicatechin †
Epigallocatechin †
Gallic acid †
Homoorientin †
Kampferol †
Luteolin †
Pyridoxine †
Quercetin †
Rhamnetin †
Asp.ac †
Lac.ac †
SEASON
AUTUMN
WINTER
SUMMER
42.29829
13.62614
571.9214
77.1155
7.869561
1.856670
11.88250
0.885445
6.862471
496.7660
6.017010
4.150334
8.975502
11312267
83095558
6887232
14132326
0.800511
0.437575
3567865
7.486852E+13
10421698
9931861
3865724
1761996
7733855
2071404
5498695
18420795
1.540490
27.25285
15.39138
21.28824
1.531992E+09
1.953379E+09
86723.69
3887947
42592593
3790056
63611951
25974094
140290.3
697662.3
8599422
5939953
5210008
6369585
a
a
b
a
d
b
b
a
b
b
a
a
b
b
c
ab
ab
a
b
b
b
c
b
a
b
a
a
a
a
a
a
a
a
c
39.16987
12.90552
502.6113
84.7965
6.490965
2.202410
12.97053
1.033817
6.437835
501.5189
5.627649
4.488954
5.342097
10177827
67722637
7031891
22054521
0.606389
0.325627
2011520
7.411317E+13
8477753
16308864
5529905
2530282
1136389
1457088
4228099
10891533
1.043819
24.22621
11.97778
16.30085
1.290526E+09
1.541045E+09
82417.51
2982179
47130408
3212265
55919822
22547824
47045.9
172048.3
8508000
4893556
7241072
6621801
b
b
b
b
cd
a
a
a
a
ab
b
ab
b
b
b
b
b
d
b
b
b
c
b
b
b
b
b
b
b
b
b
b
b
b
42.18622
13.58659
570.9746
116.2141
5.374841
2.785448
11.96500
0.892095
4.656478
501.2745
5.823781
4.179343
7.530463
9503434
122136094
8693196
26564959
0.931038
0.441336
2927839
1.522798E+14
23590504
30909308
10163191
3002304
94926108
2042432
9963002
10847325
1.295577
24.74710
15.74066
22.17597
1.113824E+09
1.696778E+09
93842.30
2703103
36217531
3511846
55675633
22040560
73569.5
343072.7
9478483
5395996
7503356
6110383
SPRING
a
a
a
b
ab
b
b
b
a
b
a
bc
a
a
a
a
a
ab
a
a
a
b
a
a
a
b
ab
a
a
c
b
b
b
b
43.55859
12.65678
550.5556
109.6029
5.393000
2.533329
11.59750
0.930850
4.922463
501.2185
6.496155
5.279338
4.695855
8283399
79844162
6937933
30164246
0.727391
0.376471
2775083
1.600834E+14
11044350
26103989
14756314
2594525
11048345
628666
4429600
11418633
1.471539
25.69583
14.14173
19.24353
1.116493E+09
1.957675E+09
88329.01
2438935
35222684
3789569
57063388
26668875
112639.2
206236.2
8926141
4495051
9116571
6424977
a
ab
a
b
bc
b
b
b
a
a
b
c
b
b
a
b
ab
b
a
b
a
a
b
c
b
b
ab
ab
ab
c
a
a
b
a
F
p
3.58477
1.11242
2.84708
9.68552
6.07256
6.69686
4.61285
4.13922
8.64768
2.54490
1.04830
3.53452
10.77758
5.24351
8.52596
3.27374
26.29805
3.71506
4.00130
6.76460
23.08100
36.63706
16.71035
13.30101
1.27475
63.73468
12.04537
3.56145
2.98152
1.64612
0.78888
2.95328
3.08414
10.83631
5.56307
0.05330
1.03349
1.53078
0.81244
0.82253
1.05755
4.36493
5.93158
0.30910
0.56062
12.35343
0.45129
0.017607
0.349497
0.043165
0.000018
0.000932
0.000458
0.005130
0.009028
0.000053
0.062412
0.376300
0.018712
0.000006
0.002438
0.000061
0.025675
0.000000
0.015041
0.010654
0.000424
0.000000
0.000000
0.000000
0.000000
0.289214
0.000000
0.000002
0.018111
0.036640
0.185925
0.503864
0.037923
0.032335
0.000005
0.001679
0.983657
0.382744
0.213484
0.490959
0.485512
0.372323
0.006892
0.001096
0.818735
0.642668
0.000001
0.717134
Succ.ac †
Cit.ac †
Glut.ac †
Pir †
Mal.ac †
α-humulene
Caryophyllene
Overlapping1 *
Overlapping2 *
Overlapping3 *
Overlapping4 *
Overlapping5 *
Overlapping6 *
Overlapping7 *
Overlapping8 *
Overlapping9 *
Overlapping10 *
UFA *
TAG+DAG *
PUFA *
P*
Linoleyl FA *
FA *
DAG *
Ald *
Acetyl *
16570697
158063505
30798422
10597297
288704758
1506341
31458.1
43.43188
11.12391
12.12945
3.051565
0.432328
2.263595
0.406019
15.11812
19.20380
12.41667
22.74433
2.546622
8.631549
3.755380
7.683955
116.2851
13.18280
0.432162
6.316552
a
b
b
b
ab
b
b
a
a
a
ab
ab
b
a
b
a
a
a
a
ab
a
a
a
12678641
223779176
41275713
14067267
266570535
2605868
262900.0
31.36382
9.68886
9.24635
2.270426
0.314493
1.949481
0.400394
13.89496
16.80289
8.81738
27.29784
1.237474
9.566383
2.835052
7.735614
129.7729
14.18169
0.399634
4.948125
b
a
ab
a
b
b
ab
b
d
c
b
b
b
bc
a
b
a
b
a
a
a
a
b
15682285
135408263
33262197
11211737
228916618
9315889
385043.0
38.04842
10.23706
11.22037
3.490667
0.485281
2.497122
0.555244
13.14657
17.58093
11.00373
17.95084
1.187863
5.288433
1.839125
5.941504
106.1379
8.40722
0.268933
4.383811
a
b
b
b
c
a
a
a
abc
b
a
a
a
abc
c
b
b
c
b
b
b
b
b
17165800
147593431
50578093
9607654
301694902
2894857
304277.0
30.10467
9.25318
10.07431
2.933038
0.424704
2.061469
0.502439
15.23863
18.05957
8.87950
23.60898
1.532120
9.476447
2.868086
7.695507
107.6017
14.29089
0.318003
5.384305
a
b
a
b
a
b
a
b
cd
a
ab
ab
ab
bc
b
b
a
b
a
b
a
b
ab
3.85842
14.86171
5.15844
6.99700
7.49909
5.20789
2.96437
5.55420
1.47215
3.73908
7.78369
2.35644
2.08579
3.21761
1.00354
0.95513
5.00243
6.57121
13.31292
18.50884
8.76008
4.10045
3.21462
15.86459
6.75223
4.51887
0.012653
0.000000
0.002694
0.000326
0.000186
0.002542
0.037414
0.001683
0.228837
0.014556
0.000134
0.078432
0.109099
0.027410
0.395996
0.418413
0.003215
0.000522
0.000000
0.000000
0.000046
0.009415
0.027510
0.000000
0.000425
0.005705
Table S6. Data for one-way ANOVAs of all stoichiometry and assigned metabolites extracted
from Q. ilex leaves for treatments (control, drought) with data from all seasons. Integral mean
values (mM relative to initial TSP or TMS concentrations (0.01%)) are given for the NMR
variables, marked by asterisks (*). Integral mean values of deconvoluted total intensities are
given for the LC-MS variables, marked by crosses (†).Different letters beside the values indicate
significant seasonal differences detected by LSD post-hoc tests (p < 0.05) between treatments.
(ALL YEAR) TREATMENT
C/N
N/P
C/P
C/K
K/P
N/K
N (mg/g dry weight)
P (mg/g dry weight)
K (mg/g dry weight)
C (mg/g dry weight)
aG *
BG *
Suc *
Deoxy-Hex †
Hex †
Pent †
Disacch †
Ile *
Thr *
Ala †
Val †
Leu †
Phen †
Pro †
Arg †
Trp †
Tyr †
Adenine †
Adenosine †
Choline †
Quercitol *
Polyphenol *
Phenolic group *
Quinic acid †
Catechin †
Chlorogenic acid †
Epicatechin †
Epigallocatechin †
Gallic acid †
Homoorientin †
Kampferol †
Luteolin †
CONTROL
40.84257
13.48905
548.2077
98.65126
6.124151
2.429144
12.32400
0.927450
5.549705
498.1220
5.437045
4.295323
4.840646
9279617
77861033
7345000
23872520
0.699775
0.408277
3017774
1.229186E+14
12616352
18206920
8482520
3120388
23320247
1329974
3237191
13303293
1.209234
22.91172
13.49634
18.03568
1.237052E+09
1.640474E+09
88017.60
2765944
43916566
3728851
53809793
23043107
90516.89
b
b
b
b
b
b
b
b
a
b
b
b
b
b
b
a
b
DROUGHT
42.85607
12.89829
551.0343
95.48032
6.438726
2.261255
11.85590
0.941341
5.875869
502.2862
6.568781
4.760449
8.510523
10363487
99328335
7440337
22599121
0.838697
0.383679
2639069
1.086165E+14
14296263
23602431
8755692
1806060
34947376
1777840
8940312
12526731
1.477316
28.14730
15.21025
21.60112
1.289336E+09
1.944040E+09
87772.54
3246753
36396699
3428421
62489855
25650127
97517.36
a
a
a
a
a
a
a
a
b
a
a
a
a
a
a
b
a
F
4.04553
1.62914
0.02034
0.20098
0.35378
0.94057
2.58445
0.15366
0.58808
8.66700
10.93840
2.53508
40.47144
3.35992
5.82383
0.03515
0.44361
3.67636
0.72229
1.99605
1.14476
0.91341
3.46258
0.02748
8.83947
1.28109
3.79370
17.73076
0.12016
2.43858
14.51730
2.91242
5.76095
0.54184
12.05120
0.00015
0.58999
2.79446
0.98913
4.62151
1.29770
0.11088
p
0.047788
0.205659
0.886955
0.655192
0.553726
0.335168
0.112011
0.696148
0.445507
0.004282
0.001433
0.115439
0.000000
0.070667
0.018188
0.851776
0.507375
0.058898
0.398030
0.161739
0.287990
0.342202
0.066590
0.868762
0.003934
0.261209
0.055091
0.000068
0.729804
0.122484
0.000278
0.091930
0.018802
0.463907
0.000852
0.990310
0.444773
0.098650
0.323070
0.034713
0.258166
0.740054
Pyridoxine †
Quercetin †
Rhamnetin †
Asp.ac †
Lac.ac †
Succ.ac †
Cit.ac †
Glut.ac †
Pir †
Mal.ac †
α-humulene
Caryophyllene
Overlapping1 *
Overlapping2 *
Overlapping3 *
Overlapping4 *
Overlapping5 *
Overlapping6 *
Overlapping7 *
Overlapping8 *
Overlapping9 *
Overlapping10 *
UFA *
TAG+DAG *
PUFA *
P*
Linoleyl FA *
FA *
DAG *
Ald *
Acetyl *
Herbivory
447097.6
7963006
4830741
7682559
6184678
14838437
175769858
41941804
11980111
267766088
3037257
321757.2
27.50030
8.06413
8.94536
2.811879
0.374319
2.071991
0.460949
15.03701
16.00664
7.74314
23.52921
1.601675
8.245113
2.757615
7.024638
115.5635
12.46900
0.336018
5.241531
0.239194
a
b
a
a
b
b
b
b
b
b
b
264729.2
9825966
5547896
6842992
6577590
16300829
154931129
35880529
10677113
275398005
5188794
167701.9
43.97410
12.08737
12.38988
3.060969
0.454084
2.313843
0.471099
13.66212
19.81695
12.81551
22.27179
1.650365
8.236293
2.891207
7.503652
114.3353
12.56230
0.373348
5.274866
0.384876
b
a
b
b
a
a
a
a
a
a
a
2.93551
6.09596
0.73926
2.36981
1.63022
1.93747
2.83074
2.05088
2.74878
0.33713
1.65242
2.87427
57.15315
65.29019
36.19355
1.51780
2.83613
2.04606
0.05271
1.89619
16.43988
66.89548
0.57681
0.05315
0.00021
0.19493
1.08310
0.03754
0.01125
1.39237
0.00657
23.07889
0.090675
0.015765
0.392566
0.127801
0.205510
0.167951
0.096527
0.156165
0.101398
0.563188
0.202483
0.094046
0.000000
0.000000
0.000000
0.221654
0.096164
0.156595
0.819017
0.172445
0.000118
0.000000
0.449854
0.818272
0.988454
0.660067
0.301221
0.846876
0.915819
0.241591
0.935597
0.000007
Table S7. Data for one-way ANOVAs of all stoichiometry and assigned metabolites extracted
from Q. ilex leaves for treatments (control, drought) in summer. Integral mean values (mM
relative to initial TSP or TMS concentrations (0.01%)) are given for the NMR variables, marked
by asterisks (*). Integral mean values of deconvoluted total intensities are given for the LC-MS
variables, marked by crosses (†). Different letters beside the values indicate significant
differences detected by LSD post hoc tests (p < 0.05) between treatments.
(SUMMER) TREATMENT
C/N
N/P
C/P
C/K
K/P
N/K
N (mg/g dry weight)
P (mg/g dry weight)
K (mg/g dry weight)
C (mg/g dry weight)
aG *
BG *
Suc *
Deoxy-Hex †
Hex †
Pent †
Disacch †
Ile *
Thr *
Ala †
Val †
Leu †
Phen †
Pro †
Arg †
Trp †
Tyr †
Adenine †
Adenosine †
Choline †
Quercitol *
Polyphenol *
Phenolic group *
Quinic acid †
Catechin †
Chlorogenic acid †
Epicatechin †
Epigallocatechin †
Gallic acid †
Homoorientin †
Kampferol †
Luteolin †
CONTROL
41.66038
13.77995
569.3676
81.59398
7.235808
1.969276
11.97600
0.880722
6.317420
494.0180
5.233182
4.007798
6.52583
11665376
84067267
7127894
15342556
0.744576
0.500795
4158994
7.523658E+13
8918857
8742734
3580016
2477675
5981268
2093373
2826604
21049369
1.764591
23.21481
12.98213
17.21579
1.484741E+09
1.734808E+09
43031.3
2170140
47459630
3909556
61644091
25383765
136963.8
b
b
b
a
a
a
b
b
b
b
b
b
DROUGHT
42.93619
13.47232
574.4752
72.63693
8.503313
1.744063
11.78900
0.890169
7.407521
499.5140
6.800838
4.292870
11.42518
10959158
82123850
6646569
12922096
0.856447
0.374355
2976737
7.450046E+13
11924538
11120989
4151432
1046317
9486441
2049436
8170786
15792222
1.316388
31.29090
17.80063
25.36068
1.579244E+09
2.171950E+09
130416.1
5605754
37725555
3670557
65579812
26564422
143616.8
a
a
a
b
b
b
a
a
a
a
a
a
F
0.34966
0.09652
0.01958
1.07595
1.55022
0.86426
0.09529
0.02510
1.84736
3.65572
4.70655
0.33796
21.92706
0.42219
0.01415
0.32928
2.77051
0.37981
2.99581
5.07479
0.00411
1.70889
0.71659
0.53599
7.68947
1.55074
0.00983
4.40172
1.09683
1.54677
6.48586
6.58725
10.23488
0.52287
7.92429
3.01184
3.28101
1.47933
0.11656
0.28566
0.06162
0.01792
p
0.561660
0.759615
0.890258
0.313341
0.229069
0.364851
0.761102
0.875882
0.190877
0.071929
0.043682
0.568219
0.000185
0.524053
0.906632
0.573187
0.113326
0.545423
0.100586
0.036992
0.949575
0.207583
0.408379
0.473527
0.012541
0.228994
0.922129
0.050284
0.308822
0.229567
0.020237
0.019415
0.004971
0.478912
0.011462
0.099746
0.086807
0.239589
0.736744
0.599558
0.806765
0.894986
Pyridoxine †
Quercetin †
Rhamnetin †
Asp.ac †
Lac.ac †
Succ.ac †
Cit.ac †
Glut.ac †
Pir †
Mal.ac †
α-humulene
Caryophyllene
Overlapping1 *
Overlapping2 *
Overlapping3 *
Overlapping4 *
Overlapping5 *
Overlapping6 *
Overlapping7 *
Overlapping8 *
Overlapping9 *
Overlapping10 *
UFA *
TAG+DAG *
PUFA *
P*
Linoleyl FA *
FA *
DAG *
Ald *
Acetyl *
Herbivory
1005105
7823142
5209374
5597393
6191870
17165656
157678450
37458234
10913495
277676155
1184114
48176.16
33.00544
8.73067
10.08155
2.816203
0.430013
1.862727
0.438479
14.62969
16.48671
9.64605
23.61925
2.507704
8.687640
3.484175
7.363597
114.9628
12.99469
0.371887
6.428194
0.206635
a
b
b
b
b
b
b
b
b
390220
9375702
6670531
4822622
6547300
15975739
158448559
24138609
10281099
299733361
1828568
14740.12
53.85831
13.51716
14.17735
3.286928
0.434643
2.664463
0.373560
15.60654
21.92088
15.18729
21.86941
2.585541
8.575459
4.026586
8.004314
117.6074
13.37092
0.492436
6.204910
0.452817
b
a
a
a
a
a
a
a
a
3.44019
0.77178
0.47712
1.13350
0.27299
0.42050
0.00167
2.05407
0.16723
1.19513
0.53673
1.92285
17.70423
13.77425
9.24243
1.31184
0.00171
5.94843
0.59953
0.22186
6.17741
15.26398
0.45029
0.01841
0.01487
0.55731
0.68387
0.03839
0.08809
3.42695
0.04476
26.35006
0.080084
0.391244
0.498542
0.301104
0.607707
0.524873
0.967844
0.168942
0.687411
0.288713
0.473225
0.182478
0.000529
0.001598
0.007042
0.267062
0.967429
0.025315
0.448808
0.643287
0.022993
0.001033
0.510719
0.893590
0.904284
0.464985
0.419085
0.846865
0.770019
0.080620
0.834815
0.000070
Fig. S1 a) Ten-year average (1999-2009) of soil-moisture (v.v. ±SE) of control plots for each
year season. b) Ten-year average (1999-2009) reduction of soil moisture of droughted plots
relative to control plots (% ±SE). c) Reduction of soil moisture of droughted plots relative to
control plots in 2009 (%±SE). d) Four-year average (2007-2010) soil temperature of control
and droughted plots (ºC ±SE)
Fig. S2 a) Ten-year average (1999-2009) of stem mortality (%). b) Ten-year average (1999-2009)
of annual aboveground biomass increment (Mg/ha).
Fig. S3 Methodology used for the calculation of foliar herbivore attack. Foliar damages such as
necrosis caused by other factors than herbivores were not taken into account in the
calculation of folivory and those leaves were considered as no predated (Folivory = 0%).
Fig. S4 PC1 versus PC2 of the PCA of the elemental, stoichiometric and metabolomic variables
in Q. ilex leaves. Panel a shows the stoichiometric and metabolomic variables. C/N/P/K ratios
and folivory are shown in red. Colors indicate different metabolomic families: blue, sugars;
green, amino acids; orange, RCAAS; cyan, nucleotides; violet, phenolics; light orange, nonpolar
metabolites; dark blue, overlapped NMR signals and brown, terpenes. A number has been
assigned to each metabolite forming part of the overlapped NMR signals: sucrose (Suc; 1), αglucose (αG; 2), β-glucose (βG; 3), deoxy-hexose (Deoxy-Hex), hexose (Hex), pentose (Pent),
disaccharide (Disacch), aspartic acid (Asp.ac), lactic acid (Lac.ac; 11), succinic acid (Succ.ac),
citric acid (Cit.ac; 4), pyruvate (Pir), malic acid (Mal.ac), gallic acid (Gall.ac), alanine (Ala; 5),
isoleucine (Ile; 6), threonine (Thr), valine (Val; 7), leucine (Leu), phenylalanine (Phen), proline
(Pro), arginine (Arg), tryptophan (Trp), tyrosine (Tyr), quercitol (Q.OH; 8), quinic acid (Q.ac;
9), choline (Chol; 10), catechin (Cate), epicatechin (Epica), epigallocatechin (Epiga),
homoorientin (Hom), quercetin (Quer), rhamnetin (Rham), kampferol (Kamp), luteolin (Lut),
chlorogenic acid (Chlo.ac), N-acetyl group (12), polyphenol (Poly; 13), phenolic group (Phe.gr),
pyridoxine (Pyri), caryophyllene (Caryo), α-humulene (αHum), 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), polyphenol derived 1 (P.1), 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 represented by
small grey dots. Panel b shows the samples categorized by season and drought treatment.
Seasons are indicated by different colors (green, spring; red, summer; yellow, autumn and blue,
winter). Climatic treatment is indicated by geometric figures: circles, controls; triangles,
drought. The black symbols indicate the mean PC1 vs. PC2 scores for the treatments (control
trees, circle and droughted trees, cross). Arrows outside the plot indicate the mean PC for each
season (colored arrows) or treatment (black arrows). The statistically significant differences
between seasons were detected by Bonferroni post-hoc tests and are indicated by letters
(p<0.05).
Fig. S5 Plots of the PCAs of the metabolomic and stoichiometric variables for spring and
summer. (a) variable plot for spring, (b) case plot for spring, (c) variable plot for summer and (d)
case plot for summer. C/N/P/K ratios and herbivory are shown in red. Colors indicate different
metabolomic families: blue, sugars; green, amino acids; orange, RCAAS; cyan, nucleotides;
violet, phenolics; light orange, nonpolar metabolites; dark blue, overlapped NMR signals and
brown, terpenes. Unassigned metabolites are represented by small grey dots. The variables are
described in Fig. 1. Control trees are indicated by circles and blue colors, and droughted trees
are indicated by crosses and orange colors. Arrows outside the plots indicate the mean PC for
each treatment. The statistically significant differences between seasons were detected by
Bonferroni post-hoc tests and are indicated by letters (p<0.05).
Fig. S6 PCA plots of the metabolomic and stoichiometric variables, excluding folivory as a
variable, of all seasons (a) and case trees (b). Colors indicate different metabolomic families:
blue, sugars; green, amino acids; orange, RCAAS; cyan, nucleotides; violet, phenolics; light
orange, nonpolar metabolites; dark blue, overlapped NMR signals and brown, terpenes.
Unassigned metabolites are represented by small grey dots. Variable labels are described in Fig.
1 of the main text. Control trees are indicated by asterisks and blue colors, and droughted
trees are indicated by triangles and orange colors.
Fig. S7 Total concentration of polyphenolics of summer cases (Fig. 4 of the main article) versus
the proportion of foliar consumption.
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