Perfil metabólico primario de hojas de encina en respuesta al estrés hídrico por sequía

Antonio, Carla

Gil Sanchez, Luis Alfonso

Rodrigues, Ana M.

Rodríguez Calcerrada, Jesús

Servicio de Coordinación de Bibliotecas de la Universidad Politécnica de Madrid

GC-TOF-MS Metabolite Profiling of Drought Tolerant Quercus ilex
Ana M. Rodrigues, Jesús Rodríguez-Calcerrada, Luis Gil, Carla António
 Leaf sampling from 2 year-old Q. ilex seedlings was carried out at mild (LS), moderate (MS), severe (SS) and very
severe (VS) drought stress conditions. Stress conditions were established as function of predawn (Ψpd) and
midday (Ψmd) leaf water potential as LS: -0.5 < Ψpd < -1 MPa and -2 < Ψmd < -2.5 MPa; MS: -1 < Ψpd < -4 MPa and -
2.5 < Ψmd < -5 MPa; SS: -4 < Ψpd < -7 MPa and -5 < Ψmd < -7.5 MPa; VS: Ψpd < -7 MPa and Ψmd < -7.5 MPa.
 Primary metabolites were extracted and analysed using a well-established protocol for metabolite profiling as in
[2]. Statistical analyses were performed using R software [3-5].
The authors are thankful to Guillermo G. Gordaliza and
Meng Li. A.M. Rodrigues acknowledges FCT for the PhD
fellowship (PD/BD/114417/2016) and the ITQB NOVA
International PhD Programme “Plants for Life”
(PD/00035/2013). J. Rodríguez-Calcerrada acknowledges
funding by the project OLMOS (AGL2012-35580). C.
António acknowledges FCT Investigator Programme
(IF/00376/2012/CP0165/CT0003), ITQB NOVA R&D GreenIT
‘Bioresources for sustainability’ (UID/Multi/04551/2013),
and LabMet metabolomics facility at CTBE (Campinas,
Brazil) for GC-TOF-MS metabolite profiling services.
III International Symposium on Profiling—ISPROF 2017. 4-7 September 2017, Caparica, Portugal
Holm oak (Quercus ilex L.) is one of the dominant tree species in natural forest ecosystems of the Western Mediterranean Basin. This species is well
adapted to summer droughts but might not be able to cope with future increases in drought severity, duration, and/or frequency as the climate becomes
warmer and the water availability decreases [1]. To better understand the mechanisms underlying drought tolerance in Q. ilex, a metabolite profiling
analysis was performed with gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) using leaves of 2-year-old Q. ilex seedlings subjected to
increasing drought severity.
INTRODUCTION
RESULTS & DISCUSSION
METHODS REFERENCES
 A set of 31 primary metabolites was detected in Q. ilex leaves.
 Amino acids and derivatives were the most abundant metabolites
with a total of 15 chemical species, followed by sugars and sugar-
alcohols (seven and three chemical species, respectively) and six
organic acids.
 Mild water stress caused most sugars and sugar alcohols to increase
through moderate to very severe water stress conditions, suggesting
a role of these metabolites in stress signaling and osmoregulation.
 At very severe water stress conditions most amino acids
dramatically increased, especially γ-aminobutyric acid (GABA) and
proline, which have a role in protection against oxidative damage.
[1] A Barbeta, J Peñuelas, Plant Ecol Divers (2016) 
DOI:10.1080/17550874.2016.1212288
[2] J Lisec, et al., Nat Protoc. 1 (2006) 387-396
[3] R Core Team (2016) https://www.R-project.org
[4] GR Warnes, et al. (2016) https://CRAN.R-
project.org/package=gplots
[5] KA Le Cao, et al. (2016) https://CRAN.R-
project.org/package=mixOmics
LS M
S
S
S
V
S
Tyrosine 
Glycine
Phenylalanine 
Isoleucine
Valine
Glutamate
Raffinose
Ornithine
Malate
Trehalose
Succinate
Sucrose
Cystine
Urea
Serine
Threonine
myo-Inositol
Fumarate
Xylose
Threonate
Glycerol
Glycolate
Asparagine
beta-Alanine
Glycerate
Erythritol
Proline
GABA
Fructose
Glucose 
Fucose
-1 0 1
S
AA
OA
GC-TOF-MS
The high drought tolerance of Q. ilex might rely on early water stress signaling and osmoregulation by hexoses and polyols, and enhanced protection
against oxidative damage by amino acids at severe water stress. Q. ilex has shown mechanisms of acclimation to drought, which can be useful for its
persistence under a future drier climate.
Fig. 1. Heat map of fold changes in leaf primary metabolite relative values in Quercus ilex
2 year-old seedlings subjected to increasing levels of drought stress (mild, LS; moderate, MS;
severe, SS; and very severe, VS) in relation to their well-watered controls. Relative values (as
means ± SE of four independent measurements) were normalized to the internal standard (ribitol)
and fresh weight of the samples; false color imaging was performed on
log10-transformed GC-TOF-MS metabolite data. Values corresponded to means ± SE of four
independent measurements. Significant changes using Student’s t test are indicated as
○ p < 0.05; ◊ p < 0.01; △ p < 0.001, with respect to controls. Metabolites grouped in sugars & sugar
alcohols (S), amino acids & derivatives (AA) and organic acids (OA). Heat maps and clustering were
performed in R software [3] using the “heatmap.2” function from “gplots” package [4]
Fig. 2 Supervised PLS-DA score plot (a) and corresponding loadings scatter plot (b)
derived from the normalized primary metabolite relative values (log10 transformed) in
Quercus ilex 2 year-old seedlings subjected to increasing levels of drought stress (mild,
LS; moderate, MS; severe, SS; and very severe, VS) in relation to their well-watered
controls. Plots performed in R software [3] using the “mixOmics” package [5]
 Very severe stress group was clearly separated from the others.
 The characteristics of PLS-DA score plot and respective loadings scatter
plot were consistent with the result of cluster analysis in the heat map
(Fig. 1).


GC-TOF-MS metabolite profiling of drought tolerant Quercus ilex

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GC-TOF-MS metabolite profiling of drought tolerant Quercus ilex

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