Critical Reviews in Biotechnology, 2011; 31(2): 153–169

© 2011 Informa Healthcare USA, Inc.

ISSN 0738-8551 print/ISSN 1549-7801 online
DOI: 10.3109/07388551.2010.505908

REVIEW ARTICLE

Metabolomics: Creating new potentials for unraveling the

mechanisms in response to salt and drought stress and for the
biotechnological improvement of xero-halophytes
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Cheng-Jiang Ruan1, and Jaime A. Teixeira da Silva2

Key Laboratory of Biotechnology & Bio-Resources Utilization, Dalian Nationalities University, Dalian City, Liaoning,
1

China, and 2Faculty of Agriculture and Graduate School of Agriculture, Kagawa University, Miki-cho, Kagawa, Japan

Abstract
Breeders have long been interested in understanding the biological function and mechanism of xero-halophytes
and their ability for growth in drought-stricken and salinized environments. However, the mechanisms in response
to stress have been difficult to unravel because their defenses require regulatory changes to the activation of
multiple genes and pathways. Metabolomics is becoming a key tool in comprehensively understanding the cellular
response to abiotic stress and represents an important addition to the tools currently employed in genomics-assisted
selection for plant improvement. In this review, we highlight the applications of plant metabolomics in characterizing
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metabolic responses to salt and drought stress, and identifying metabolic quantitative trait loci (QTLs). We also
discuss the potential of metabolomics as a tool to unravel stress response mechanisms, and as a viable option for
the biotechnological improvement of xero-halophytes when no other genetic information such as linkage maps and
QTLs are available, by combining with germplasm-regression-combined marker-trait association identification.
Keywords:  Metabolomics, stress, mechanism, mQTL, GRC, marker-trait association, assisted selection,
xero-halophytes

Introduction of all arable lands by 2050 (Ashraf, 1994). In the face of a

The evolution of mankind has reached the point where
varieties from new resources need to be captured in or-
der to fill our basic needs for food, feed, and freshwater.
However, agriculturally utilizable areas are being lost as
a consequence of desertification and salinization, to the
extent that 11% of the earth’s fertile soils were degraded
in the last decades and 3% of the soil will not be able to
fulfil any biological functions in the future (Lieth, 1999;
Rozema and Flowers, 2008). The United Nations Food and
Agriculture Organization estimates that there are currently
4 million square kilometers of salinized land and a similar
area that is affected by sodicity, a condition in which Na+
ions represent more than 15% of the exchangeable cat-
ions (www.fao.org/agl/agl1/terrastat). Soil degradation
is already widespread in many regions (Figure 1), and is
expected to cause serious salinization of more than 50%
global scarcity of water resources, the increased saliniza-
tion of soil and water and the loss of arable lands, abiotic
stress is already a major limiting factor in plant growth
and will soon become even more severe as desertifica-
tion and salinization covers more and more of the world’s
­terrestrial area (Rozema and Flowers, 2008).
Scientists have long been developing sustainable bi-
ological production systems which can tolerate higher
water salinity, develop water desalinisation systems to
supply animals and humans with drinking water, and
provide technology for the water supply using renew-
able energy resources. The use of drought- and salt
resistant plant species may be extremely interesting in
such a context because they may contribute to the use
of desertic or marginal lands where no other plants are
able to grow and to save good quality water for human

Address for Correspondence:  Cheng-Jiang Ruan, 1Key Laboratory of Biotechnology & Bio-Resources Utilization, Dalian Nationalities
University, Dalian City, Liaoning, China. Tel.: +86 411 87656015; Fax: +86 411 87618179, E-mail: ruancj@yahoo.com.cn
(Received 13 March 2010; revised 21 May 2010; accepted 25 May 2010)

153
 154  Cheng-Jiang Ruan, and Jaime A. Teixeira da Silva
Abbreviations
ABA : abscisic acid;
AFLP: amplified fragment length polymorphism;
CE: capillary electrophoresis;
Da: dalton;
SPME : solid-phase microextraction;
eQTL: expression QTL;
FAIMS: field asymmetric waveform ion mobility spectrometry;
FT-ICR: Fourier transform-ion cyclotron resonance;
FT-IR: Fourier transform infrared;
GC: gas chromatography;
GLM : general linear model;
GRC: germplasm-regression-combined;
HPLC: high-performance liquid chromatography;
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IL: introgression line;
ISSR: inter-simple sequence repeats;
LC: liquid chromatography;
MAS: marker-assisted selection;
consumption, provide human nutrition, and green
­biofuel, to reduce CO2 emission, assimilate, and se-
quester carbon into biomass. For example, deserts are
currently underexploited for biomass production but
could act as a biospheric sink if adapted plant material
is selected: more than 22.5% of the world’s arid region
was estimated to be usable for planting halophytes
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(Glenn et  al., 1992). Salt-tolerant plants may be used
in land that has not been forested or farmed and can
afford beneficial side effects since they can aid in reveg-
etating arid regions, restore ecological biodiversity, and
improve mesoscale climatic parameters.
Plant breeders have long been interested in improv-
ing plants adapted to water and salt stress (Shao et al.,
2009), and several tolerant plants with potential inter-
est for agriculture and environmental management
have already been identified, such as Atriplex halimus
(Lutts et  al., 2004), Kosteletzkya virginica (Ruan et  al.,
2008a), and Salicornia bigelovii (Zerai et  al., 2010).
However, their rational use is greatly hampered by
insufficient knowledge about the mechanism of physi-
ological and biochemical mechanisms for plant toler-
ance to abiotic stress. In addition, since tolerant (e.g.
xero-halophyte) species were not previously domesti-
cated (until recently, for only some), they still exhibit
a high level of heterogeneity. Further selection steps
are therefore required before introducing them in ag-
ricultural schemes. Additional information concerning
their genotypes as well as a thorough estimation of the
discrimination of quality traits and their ability to adapt
to abiotic stress is a prerequisite for any improvement
strategy. Stress in plants can be defined as any change
in growth condition(s) that affects metabolic homeosta-
sis and requires an adjustment of metabolic pathways
in a process that is usually referred to as acclimation
(Mittler, 2006; Suzuki and Mittler, 2006). Metabolomics
could contribute significantly to the study of stress biol-
ogy in plants by identifying different compounds, such
 Critical Reviews in Biotechnology
miRNAs: micro RNA;
MLM: mixed linear model;
mQTL: metabolomic QTL;
MRA: multiple regression analysis;
MS: mass spectrometry;
NMR: nuclear magnetic resonance;
QTL: quantitative trait locus;
RAPD: random amplified polymorphic DNA;
RFLP: restriction fragment length polymorphism;
RIL: recombinant inbred line;
SAMPL: selective amplification of microsatellite polymorphic
loci;
siRNAs: small interfering RNA;
SLA: single linear regression analysis;
SNP: single nucleotide polymorphism;
SSR: simple sequence repeat;
TOF: time-of-flight;
UV-ESI: ultraviolet-electrospray ionization.
as the by-products of stress metabolism, stress signal
transduction molecules or molecules that are part of the
acclimation response of plants (Shulaev et al., 2008).
Plant metabolomics (Table 1) is a fast-growing technol-
ogy (Table 2) through qualitative and quantitative analysis
of metabolites to define the profile of cells or organs, then
to comprehensively understand the cellular response to
biotic and abiotic conditions (Schauer and Fernie, 2006).
As a branch of science concerned with the quantitative
understandings of the metabolite complement of inte-
grated living systems and its dynamic responses to the
changes of both endogenous factors (e.g. physiology and
development) and exogenous factors (e.g. environmental
factors and xenobiotics) (Tang and Wang, 2006), metabo-
lomics is bursting with vitality, with about 3045 (web of
science) and 1274 (PubMed) scientific papers related to
metabolomics (Figure 2) having been published during
2000–2009. Since the first example of the use of metabo-
lite profiling as a diagnostic tool was to determine the
mode of action of various herbicides (Sauter et al., 1988),
metabolomics has been used (i) to ascertain the differ-
ences between genetically modified and conventional
crops (Catchpole et  al., 2005) and the classification of
genotypes (Tikunov et  al., 2005; Laurentin et  al., 2008),
(ii) to test pathogenic infection (Allwood et al., 2006) and
characterize the metabolic response to biotic (Desbrosses
et al., 2005) or abiotic (Bohnert et al., 2006) stress, (iii) to
identify the genetic determinants of biochemical com-
position (Hall et  al., 2008), (iv) to discover novel genes
(Hagel and Facchini, 2008) and annotate gene function,
(v) to investigate the importance of environmental con-
ditions on quality (Carrari et al., 2006), (vi) to design tools
and markers for monitoring quality (Schauer and Fernie,
2006) and (vii) to identify metabolic quantitative trait loci
(mQTLs) (Fernie and Schauer, 2009).
The rapid development of plant metabolomics has
been covered by several reviews and books, which have
documented (i) the fundamental principles and state of
 Metabolomics: Creating new potentials for unraveling the mechanisms in response to salt  155
DNA

Genome Genomics

Microarrays

mRNA

Transcriptome Transcriptomics

Protein

2D Gels+MS
Proteome Proteommics
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LC-MS/MSn

Metabolite

Metabolome Metabolomics NMR, LC-MS

Cell
Environmental factors
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Tissue

Organ
Genotype × Environment

Individual Serious soil degradation Soil degradation

Soil stability Lack vegetation

Phenotype

Mechanisms in response to abiotic stress

Improvement of plant tolerance, quality and yield

Figure 1.  Globe system biology and potential of metabolomics to understand response to water and salt stress and for tolerant plants
improvement.

the art technical aspects of metabolomics (Sumner et al.,
2003; Fernie et  al., 2004; Saito et  al., 2006; Ward et  al.,
2007; Allwood et  al., 2008), (ii) the standards for envi-
ronmental context biological samples (Morrison et  al.,
2007) and for plant biology context information in me-
tabolomic studies (Fiehn et al., 2007), (iii) sample prepa-
ration and data acquisition and interpretation (Fiehn,
2001), (iv) metabolic diversity (Harrigan et  al., 2007a)
and its fundamental insights into important ­biological
­ rocesses (Steinfath et  al., 2008), (v) the dynamics of
p
metabolite changes during stress (Zuther et  al., 2007;
Shulaev et al., 2008) (e.g. temperature, Guy et al., 2008a;
salinity, Sanchez et al., 2008a), (vi) the characterization
of plant metabolites of nutritional importance and sig-
nificance in human health (Hall et al., 2008), (vii) func-
tional genomics through the combination of proteomic

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 156  Cheng-Jiang Ruan, and Jaime A. Teixeira da Silva

Table 1.  Glossary in plant metabolomics, cited from Fiehn (2001), Harrigan and Goodacre (2003), Goodacre et al. (2004), Dunn et al.
(2005), Hall (2006), Last et al. (2007), and Fernie and Schauer (2009).
Term Definition
Metabolome The total quantitative collection of small molecular weight compounds present in a cell, tissue or
organism.
Metabolomics The measurement of the small molecular metabolite complement of the cell. The non-targeted
identification and quantification of all metabolites within an organism or system, under a given set
of conditions. It is a term usually used in non-plant systems referring to the quantitative detection of
metabolites that are dynamically altered by a living system in response to pathophysiological stimuli or
genetic modification. Such metabolites are commonly monitored in biofluids.
Metabolic profiling The identification and quantification of metabolites related through their metabolic pathway(s) or
similarities in their chemistry. Crude sample extracts are generally separated by chromatography prior to
their detection by MS.
Metabolite fingerprinting Rapid and high-throughput methods where global metabolite profiles are obtained from crude samples
or simple cellular extracts. In general, metabolites are neither quantified nor identified.
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Metabolite footprinting The measurement of metabolites secreted from the intracellular complement of an organism into its
extracellular growth medium. Sampling is rapid because metabolite quenching and extraction are not
required, and this approach has commonly been employed in microbial metabolomics.
Targeted metabolite analysis Quantitative analysis of a pre-selected list of metabolites. Either based on existing knowledge or following
broad-scope metabolomic analysis, in-depth biochemical profiling may be based on pre-defined groups
of metabolites. Such an approach relies on optimised metabolite, extraction, separation and detection.
GC Method for the separation of volatile compounds in a gas phase through differential binding to a column
at elevated temperatures.
HPLC Method for separating compounds in solution through their differential interactions with a column.
HPLC with electrochemical or Method for the separation of metabolites by HPLC followed by the detection of the metabolites based on
photodiode-array detection their redox behaviour or spectral absorption properties.
FT-IR Spectroscopy that enables the identification of classes of compound by analysing their interactions with
infrared light. Mass resolution is the ability to distinguish ions of different masses, usually expressed as a
ratio of the ion mass to the difference in mass that the mass spectrometer can reliably distinguish.
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FT-ICR mass spectrometry A method for obtaining accurate measurements of the mass-to-charge ratio of ions in a complex mixture,
allowing the identification and measurement of the molecules.

(Weckwerth, 2008) or transcriptomic (Nikiforova, 2008)
and ­metabolomic data sets, and (viii) the trends and
coming of age in plant metabolomics (Last et  al., 2007;
Guy et  al., 2008b). In contrast, in this review we first
survey briefly plant metabolomic technologies. Then,
we focus on the metabolomics used to (1) analyze the
characterization of the metabolic response to salt and
drought stress; and (2) identify mQTLs. Finally, we dis-
cuss the prospect of the potential role for metabolomics
as a tool to unravel the mechanisms of biochemistry and
molecular biology in response to salt and drought stress
of plants and as a viable option for the improvement of
plant tolerance, yield, and quality in the future, especially
for orphan xero-halophyte plants, most of which have rel-
atively no genetic information compared to conventional
crops with known genetic information (e.g. sequences,
linkage maps, QTLs, etc.).
Plant metabolomics technologies
Metabolomics became a standard laboratory technique in
the past decade (Fernie et al., 2004), although it has been
carried out since the mid 1970s (Jellum, 1977). Currently,
a number of metabolomics techniques are used in plant
studies and these are shown in Table 2 and Figure 3.
Among these, both mass spectrometry (MS) and nuclear
magnetic resonance (NMR) dominate the metabolite
strategies. Gas chromatography (GC)-MS has long been
used for plant metabolomics, such as in the analysis of
terpenoids (Aharoni et  al., 2003; Chen et  al., 2003) and
volatiles (Tikunov et  al., 2005), mutant classification
(Messerli et  al., 2007), functional genomics (Schauer
and Fernie, 2006), and the integration of metabolic and
transcript datasets (Carrari et al., 2006; Baxter et al., 2007;
Kaplan et al., 2007). Liquid chromatography (LC)-MS is
better suited to the targeted profiling of specific types of
compounds that display similar ionizing behavior (e.g.
alkaloids) and is generally not used for broad-scope me-
tabolomic studies (Halket et al., 2005). The combination
of LC-MS and GC-MS data certainly provides a more
complete perspective of a plant metabolome, resulting
in the establishment of a tomato-specific LC-MS data-
base (Catchpole et  al., 2005; Moco et  al., 2006). These
techniques facilitate analyte identification by providing
information on both the chemical nature and mass of
individual molecules. In contrast, direct-infusion mass
spectrometry (DIMS) or fourier ­transform-ion cyclotron
resonance (FT-ICR)-MS yield less chemical information.
FT-ICR-MS, which has more recently been applied to me-
tabolomics, is the most effective in terms of mass resolu-
tion (i.e. the ability to distinguish two ions with similar
masses) and mass accuracy (i.e. how close a measured
mass is to the true mass) (Aharoni et  al., 2002; Tohge
et al., 2005; Zulak et al., 2008), and has been used to iden-
tify adulterants in vegetable oils (Wu et al., 2004), investi-
gate nitrogen metabolism in transgenic tobacco (Munger
et al., 2005), study the effects of herbicide and light/dark
treatments in Arabidopsis (Oikawa et al., 2006; Nakamura

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Table 2.  Plant metabolomics technologies and optimal materials selection, cited from Yin et al. (2005), Hagel and Facchini (2008) and Fernie and Schauer (2009).
Technology Properties Application Relative advantages and disadvantages References
GC-MS Accuracy: <50 ppm Analyses of relative low molecular weight, hydrophobic There are stable protocols for machine set-up, maintenance and Fiehn et al., 2000;
Mass range: <350 Da compounds (e.g. sugars, organic acids, tocopherols and usage, because it has been described as the ‘gold standard’ of Roessner et al.,
vitamins). metabolomics. However, the chromatographic procedures are in 2001; Harrigan
general slow and time-consuming, and it require derivatization and Goodacre,
prior to analysis, which waste extra time and even lead to changes 2003

© 2011 Informa Healthcare USA, Inc.

of sampling.
GC×GC-MS Accuracy: <50 ppm Similar to GC-MS With better separation of co-eluting compounds and increased Ma et al., 2007
Mass range: <350 Da sensitivity owing to GC× GC.
GC-TOF-MS Analysis of high molecular weight molecules and Fast scan times, which give rise to either improved peak Fraser et al., 2007
compounds recalcitrant to other approaches (e.g. deconvolution (the ability to resolve partially co-eluting peaks) or
carotenoids) higher sample throughput.
SPME GC-MS Accuracy: <50 ppm Analyses of volatile compounds (e.g. aroma components SPME is an innovative, solvent-free extraction procedure, involving Kusch and Knupp,
Mass range: <350 Da and repellents). immersing a phase-coated fused silica fiber into the liquid sample 2004
or the headspace above the liquid or solid sample, to adsorb/absorb
the analytes of interest
CE-MS Accuracy: <50 ppm Analyses of polar compounds (e.g. amino acids, CoA- A highly sensitive methodology that can detect low abundance Oikawa et al., 2008
Mass range: <1000 Da Derivates, sugars, organic acids, tocopherols and metabolites and that provides good analyte separation. In addition,
vitamins). it is about only 10 min to detect one sample.
LC-MS Accuracy: 50–100 ppm Analyses of mainly secondary metabolites with low to Measure a far broader range of metabolites encompassing both Tohge et al., 2005;
Mass range: <1500 Da moderate molecular weight (e.g. carotenoids, flavonoids, primary and secondary metabolites. But, it needs longer analysis Huhman et al.,
glucosinolates and vitamins). time. 2005
FT-ICR-MS Accuracy: <1 ppm High-resolution MS in combination with LC is highly Relies solely on very high resolution mass analysis, which Oikawa et al., 2008
Mass range: <1500 Da powerful. Enables the identification of unknown potentially enables the measurement of the empirical formula for
metabolites by mass to charge ratio. thousands of metabolites, however, it is somewhat limited by the
lack of chromatographic separation.
Direct- Accuracy: 50–100 ppm Non separative technique giving a fingerprint of the It is possible to determine naphthalene and total Pavón et al., 2007
injection-MS Mass range: <1500 Da metabolic content in a biological sample methylnaphthalenes without the need to perform chromatographic
separation, such that the time of analysis per sample is reduced
from 13 to only 2.5 min.
FAIMS-MS Accuracy: 50–100 ppm Next generation hyphenation technology to MS. Enables FAIMS is an ideal ion processing tool for mass spectrometry. Guevremont, 2004
Mass range: <1500 Da selection of specific ions, reducing ion suppression and Since ion separation is based on properties of the ions that are not
matrix effects. FAIMS enables the separation of isobaric related to mass-to-charge-ratio, the combined FAIMS-MS system
compounds in combination with selective MS. effectively separates in two dimensions. The separation of many
types of isobaric ions, including diastereoisomers and positional
isomers, has been demonstrated.
NMR Mass range: <∼50 kDa Non destructive analyses of abundant metabolites in a NMR is a key method for high-throughput comparative analysis of Fernie et al., 2004;
sample. plant extractscan, providing subcellular information and it is easier Ward et al., 2007
to derive atomic information for flux modelling from NMR than
from MS-based approaches.
Metabolomics: Creating new potentials for unraveling the mechanisms in response to salt  157
 158  Cheng-Jiang Ruan, and Jaime A. Teixeira da Silva
et  al., 2007), and to examine the metabolic ­response of
opium poppy cell cultures to elicitor treatment (Zulak
et  al., 2008). However, the high instrumentation cost of
FT-ICR-MS has hindered its more widespread use (Hagel
and Facchini, 2008).
Compared to the ‘hyphenated’ analytical platforms
based on MS with a major drawback of the time re-
quired for sample separation (Hagel and Facchini,
2008), NMR spectroscopy offers several advantages over
­high-throughput metabolite analyses, such as relatively
simple sample preparation and the ­non-destructive
analysis of samples. A list of over 300 common plant
metabolites and their characteristic 1H NMR shifts have
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been published, although the values are restricted to
aqueous samples (Verpoorte et  al., 2007). 2H, 13C or
15
N can be used to label samples prior to NMR analy-
sis, which is particularly suited to the elucidation of
biosynthetic pathways and metabolic flux analyses
(Kruger et  al., 2003; Ratcliffe and Shachar-Hill, 2005;
Ward and Beale, 2006). For example, transcript profiles
of Arabidopsis albino mutants were compared with
metabolite profiles obtained by NMR analysis of 13C6-
glucose-fed plants revealing distinct labeling patterns
in the albino lines compared with controls and an al-
teration in carbon/nitrogen homeostasis caused by a
loss of chloroplast function (Tian et al., 2007).
Separation-based methods coupled with MS are
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relatively sensitive (e.g. 106–109-fold more sensitive than
NMR; Sumner et al., 2003), but their sensitivities are not
uniform for all metabolites and can be pretty low for
certain analytes under the given analytical conditions.
For metabolomics purposes, both chromatography and
MS methods suffer from some common disadvantages
in terms of metabolite identification and quantification.
Both techniques require fairly extensive sample prepara-
tions and are invasive and destructive (to samples), hence
are not suitable for in vivo or in situ studies. Both of these
techniques require prior knowledge about the samples
and have high recurrent expenditure though chromato-
graphic methods are reasonably inexpensive. In contrast,
the NMR-based methods are user independent, require
little or no sample preparation, and have excellent reso-
lution and reproducibility (Ward et al., 2007; Hagel and
Facchini, 2008; Zulak et al., 2008). In addition, NMR of-
fers rich molecular information including metabolite
structure, concentration, molecular dynamics, interac-
tions, pH, and compartmentation when diffusion-editing
techniques are employed (Tang and Wang, 2006; Allwood
et al., 2008; Hagel and Facchini, 2008). Furthermore NMR
is non-invasive and non-destructive to samples, thus
making it possible to facilitate in vivo and in situ studies.
Hence, it is clear that the best choice at present may lie
in the combination of a number of existing techniques.
The hybrid method, chromatography, MS and NMR has
already played an important role in metabolite identi-
fication, such as LC-SPE-NMR (Christophoridou et  al.,
2005), LC-NMR-MS (Ward and Beale, 2006), FT-ICR-MS
and NMR (Hagel and Facchini, 2008). It is particularly
 Critical Reviews in Biotechnology
exciting to note that a statistical heterospectroscopy
method has been reported (Crockfor et al., 2006) which
takes advantages of both NMR and UPLC-MS. It is pre-
dictable that such correlation techniques will become
increasingly important in the future.
Application of metabolomics in plant
tolerance to salt and drought stress
Homeostasis, a set-value for metabolism under optimal
conditions, is rarely achieved by plants because of the
cost exerted by external stress factors: climatic, biotic,
and nutrient imbalances (Bohnert et  al., 2006). Among
these, stresses caused by abiotic conditions, such as tem-
perature extremes (freezing, cold, and heat), water avail-
ability (drought and ion excess), and ion toxicity (salinity
and heavy metals), have been difficult to dissect because
the defense responses to abiotic factors require regulatory
changes to the activation of multiple genes and pathways.
In the past several years, there has been relative success
of the over- or under-expression of single or several genes
by targeting genes, proteins or enzymatic reactions that
had been indicated as important components of stress
tolerance or sensitivity (Sung et al., 2003; Baniwal et al.,
2004; Mittler et al., 2004; Bohnert et al., 2006); in contrast,
for effectively improving plant tolerance to abiotic stress,
a shift should be emphasized and practiced in the future:
away from focusing on the end-points of response chains
to engineering genes governing upstream reactions that
affect entire pathways and groups of genes (Vinocur
and Altman, 2005). However, our limited knowledge of
stress-associated metabolism remains a major gap in
our understanding of the mechanisms of plant toler-
ance to abiotic stress; therefore, comprehensive profiling
of stress-associated metabolites is most relevant to the
successful biotechnological breeding of stress-tolerant
plants.
In the past decade, the application of omics-type
technologies (e.g. genome, transcriptome, proteome
and metabolome) has enhanced our understanding
of plant responses to abiotic stresses that disturb the
homeostatic equilibrium. Among these techniques,
attention to metabolite analysis is obviously growing
in the development of metabolite sensors and data-
bases (Cook et  al., 2004; Hirai et  al., 2004; 2005) and
has been used to study temperature (Cook et al., 2004;
Kaplan et  al., 2004; Kaplan et  al., 2007), water and sa-
linity (Johnson et  al., 2003; Brosche et  al., 2005; Gong
et  al., 2005; Cramer et  al., 2007; Kim et  al., 2007), bo-
ron (Roessner et  al., 2006), sulfur (Nikiforova et  al.,
2004; Nikiforova et  al., 2005a; Nikiforova et  al., 2005b;
Nikiforova 2008), phosphorus (Hernandez et al., 2007),
oxidative (Baxter et al., 2007), and heavy metal (Le Lay
et al., 2006) stress as well as a combination of multiple
stresses (Rizhsky et al., 2004) in plants (Figure 4). Here,
we summarize the application of plant metabolomics
in analyzing the mechanisms in response to salt and
drought stress in different plants, such as Arabidopsis,
 Metabolomics: Creating new potentials for unraveling the mechanisms in response to salt  159
A 900

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500

Papers
400

300

200

100

0
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
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Year

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200
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0
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Year

Figure 2.  Scientific publications on metabolomics searched in the Web of science (A) using topic search (Nov. 22, 2009) and in PubMed (B)
using a search of Title/Abstract (Nov. 27, 2009).

Whole organism, Organs, Tissues, Whole cells tomato, rice, grape, pea, Lotus japonicus, Lolium perenn,
legume, Populus euphratica, Thellungiella halophila,
in vitro and Limonium l­ atifolium (Table 3).
in vivo, ex vivo Based on a measure of the soluble amino acids ex-
Extracts tracted from the leaves of three grass species using
high-performance liquid chromatography of derivatives
formed with o-phthaldialdehyde and β-mercaptoethanol,
1
H NMR and GC–MS, 2-amino-5-hydroxypentanoic acid
(5-hydroxynorvaline, 5-HNV) increased in amount with
drought stress had a retention time not corresponding to
FT-ICR any common amino acid, the amount of 5-HNV in the
Sensitivity -MS Throughput leaves of the more drought tolerant C4 grasses, Cynodon
dactylon and Zoysia japonica, increased with increasing
water deficit (Carmo-Silva et  al., 2009). Fumagalli et  al.
?
(2009) investigated the metabolic profile in the shoots
GC/LC-
MS/(MS) NMR and roots of two rice cultivars (Arborio and Nipponbare)
through H-1-high-resolution magic angle spinning and
liquid-state NMR experiments. Drought and salt stress
Metabolomic data
experiments on shoot and root growth showed Arborio
Identification seedlings to be more sensitive than those of Nipponbare
to these abiotic stresses. Principal component analysis
highlighted a significant accumulation of amino acids
and sugars in the shoots and roots under stress condi-
tions and the existence of clear differences between the
Figure 3.  Platforms of technologies used in metabolomics studies
two analysed rice cultivars. In particular, Arborio seed-
(adapted from Dunn et al., 2005; Tang and Wang, 2006; Hagel and
Facchini, 2008). lings accumulated a higher concentration of amino acids

© 2011 Informa Healthcare USA, Inc.

 160  Cheng-Jiang Ruan, and Jaime A. Teixeira da Silva

Abiotic stresses

Osmotic stress Other stresses

Disiccation (water deficit) Ion toxicity (metals, Na/Cl)

Water and soil salinity Heat, anaerobiosis (anoxia)
Cold (cell freezing) Oxidation, UV, wounding
Macro- and micro-nutrients
N, P, S, O, C

Tolerance (cellular and SOS, protein kinases, Membrane

molecular mechanisms)
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of South Carolina on 10/05/11

phospholipids, LEA-type genes

Avoidance (morphological, Growth regulation, ABA

anatomical and Metabolic reaponse to
physiological changes) abiotic stress based on
metabolomics techniques Detoxification (i.e.,
(GC/LC-MS, FT-ICR-MS damage control and repair)
Discovering and and NMR)
annotating new genes Unraveling of plant
sensing and signaling
systems
Identification of mQTL

Discriminant adaptation Mechanism of plant

(the ability of a genotype to responses and
survive and produce adaptation for
For personal use only.

biomass relative to other abiotic stress

species)

Improvement of plant tolerance

Figure 4.  Utilization of plant metabolomics in unraveling mechanisms in response to abiotic stress (abiotic stress of dashed box adapted
from Zhu, 2002; Vinocur and Altman, 2005). C: carbon; N: nitrate; O: oxygen; P, phosphate; S, sulfur; SOS, salt overly sensitive.

and sugars than Nipponbare seedlings (Fumagalli et al. Identification of mQTLs

2009).
Studies on the metabolic mechanisms of plants in
salt and drought stress indicated that metabolomics
provides a wealth of data and is already providing a
better understanding of the changes in cellular me-
tabolism that are induced by salt and drought stresses
(Table 3, Figure 4). Based on a multivariate correla-
tion study on the metabolome, ionome and biomass
changes of Lotus japonicus challenged by salt stress,
Sanchez et al. (2008a) showed that it may be possible in
the future to use metabolic fingerprinting as a breeding
tool to select the individual plants that best cope with
salt stress. Despite being a relatively new approach
in plant biology, metabolomics is becoming a viable
tool in studying plant stress responses, and provides
insight into the features of stress-induced changes in
higher plants, revealing not only conserved but also
­species-specific responses during salt acclimation. In
the future, detailed analyses of the effects of salt ac-
climation and related stress factors in time and dose
may help to direct xero-halophyte breeding programs
toward increased salt tolerance (Sanchez et al., 2008a).
Although humans have cultivated crops for over 5000
years, we have only recently developed the capacity to
select for more than a handful of traits, and cultivated
varieties of plants contain only a small fraction of the
variation present in the gene pool (Fernie and Schauer,
2009). In the natural habitats of cultivated orphan plants,
the wild ancestors or genotypes can still be found and
germplasm centres have been set up worldwide to con-
serve these valuable resources in the form of seed banks
(Tanksley et  al., 1997), providing a source of genetic
variation for crop improvement.
The wide-spread development of molecular-marker
techniques in the early 1980s revolutionized plant
breeding, with enormous potential to improve the effi-
ciency and precision of conventional plant breeding via
­marker-assisted selection (MAS) (Collard and Mackill,
2008). The large number of QTLs mapping studies for
diverse crops species have provided an abundance of
DNA marker-trait associations, and have been success-
fully used in the MAS for the improvement of quantita-
tive traits in cereal crops (Hansen et al., 2008; Hospital,

 Critical Reviews in Biotechnology

 Metabolomics: Creating new potentials for unraveling the mechanisms in response to salt  161

Table 3.  Applied examples of metabolomics used to investigate salt and drought stress.
Metabolomic
Species technique Results References
Arabidopsis GC-MS and LC-MS Bioinformatics analysis of the data using PCA and batchlearning self- Kim et al.,
organizing mapping analysis revealed that short-term responses to salt stress 2007
included the induction of the methylation cycle for the supply of methyl
groups, the phenylpropanoid pathway for lignin production and glycine
betaine production. The longterm effects were the coinduction of glycolysis
and sucrose metabolism and coreduction of the methylation cycle.
GC-TOF-MS and Metabolite profiling revealed that accumulation of amino acids depended Urano
CE-MS on ABA production, but the level of the oligosaccharide raffinose was et al., 2009
regulated by ABA independently under dehydration stress. Metabolic network
analysis showed that global metabolite-metabolite correlations occurred in
dehydration-increased amino acids in wild-type, and strong correlations with
raffinose were reconstructed in nc3-2. This metabolomics analysis revealed
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of South Carolina on 10/05/11

new molecular mechanisms of dynamic metabolic networks in response to

dehydration stress.
Thellungiella halophila GC-MS The authors found drastic differences in metabolic profiles of the two species. Gong et al.,
and Arabidopsis Generally, Thellungiella had a higher metabolite levels both without and with 2005
salt stress when compared with Arabidopsis. In Arabidopsis, 150 mM salt stress
caused increase in sucrose, proline and an unknown metabolite (putative
complex sugar). In Thellungiella, the response was more complex. In addition
to having higher levels of many metabolites before stress, changes in other
sugars, sugar alcohols, organic acids and phosphate were detected.
Arabidopsis thaliana, GC-TOF-MS The authors present evidence for conserved and divergent metabolic Sanchez
Lotus japonicus and responses among these three species and conclude that a change in the et al.,
Oryza sativa balance between amino acids and organic acids may be a conserved 2008a
metabolic response of plants to salt stress
Rice GC-MS A detailed analysis of the root response to salt acclimation showed a strong Zuther
depletion of tri-carboxylic acids (TCA) cycle intermediates, pyruvic, citric, et al., 2007
For personal use only.

aconitic, malic and 2-oxoglutaric acids, and of shikimic and quinic acids. This
depletion was accompanied by increases in amino acids.
Tomato FT-IR spectroscopy Metabolic fingerprints were analyzed using unsupervised (PCA) and Johnson
supervised (DFA) algorithms. PCA was not able to discriminate between et al., 2003
control and salt-treated groups in any variety, while DFA was able to classify
control and salt-treated groups in both varieties.
Grape GC-MS Concentration of glucose, malate and proline is higher in water-deficit-treated Cramer
plants, than in salt-stressed plants. These differences in metabolite levels et al. 2007
were correlated with differences in transcript levels of many genes involved in
energy metabolism and nitrogen assimilation, suggesting a higher demand in
water-deficit-treated plants to adjust osmotically, detoxify ROS and cope with
photoinhibition than in salt-stressed plants.
GC-MS, HPLC The metabolic responses of grapes to water deficit varied with the cultivar Deluc
and fruit pigmentation. Chardonnay berries, which lack any significant et al., 2009
anthocyanin content, exhibited increased photoprotection mechanisms
under water deficit conditions. Water deficit increased ABA, proline, sugar
and anthocyanin concentrations in Cabernet Sauvignon, but not Chardonnay
berries.
Limonium latifolium Sugars, inositols and proline behaved as osmobalancers, while organic Gagneul
acids decreased upon salt stress. In addition, metabolic responses during et al., 2007
acclimation to salinity suggested that many changes in organic solute
composition are controlled by constitutive developmental programs,
further supporting the hypothesis on a constitutive, adaptive mechanism of
halophytes, which may be interpreted as a metabolic anticipation of stress.
Pea (Pisum sativum L.) 1D and 2D NMR The metabolites that were present at significantly higher concentrations in Charlton
spectroscopy drought-stressed plants under all growth conditions included proline, valine, et al., 2008
threonine, homoserine, myoinositol, gamma-aminobutyrate (GABA) and
trigonelline (nicotinic acid betaine). Metabolites that were altered in relative
amounts in different experiments, but not specifically associated with drought-
stress, were also identified. These included glutamate, asparagine and malate,
with the last being present at up to 5-fold higher concentrations in plants
grown in field experiments.
Legume GC-MS Metabolic changes in legume were characterized by a general increase in the Sanchez
steady-state levels of many amino acids, sugars and polyols, with a concurrent et al.,
decrease in concentration of most organic acids. 2008b
Table 3. continued on next page

© 2011 Informa Healthcare USA, Inc.

 162  Cheng-Jiang Ruan, and Jaime A. Teixeira da Silva
Table 3. Continued.

Metabolomic
Species technique Results References
Soybean HPLC-UV-ESI-MS Comparative targeted metabolic profiling revealed marked differences in Wu et al.,
and HPLC-ESI-MSn the metabolite composition between salt-sensitive and salt-tolerant soybean 2008
varieties. PCA clearly demonstrated that it is possible to use secondary
metabolites, for example, isoflavones and saponins, to discriminate between
closely related soybean genotypes. Genistin and group B saponins were
identified as the key secondary metabolites correlated with salt tolerance.
Lolium perenne L. GC-MS Difference in the metabolic profiles of the leaf tissue under different water Foito et al.,
stress were principally due to a reduction in fatty acid levels in the more 2009
susceptible Cashel genotype and an increase in sugars and compatible solutes
in the more tolerant PI462336 genotype. Sugars with a significant increase
included: raffinose, trehalose, glucose, fructose and maltose. Increasing the
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of South Carolina on 10/05/11

ability of perennial ryegrass to accumulate these sugars in response to a water

deficit may lead to more tolerant varieties.
Populus euphratica GC-MS Increased amino acid levels, specifically proline, valine and b-alanine, and Brosche
changes in sugar and polyol metabolism, which appeared to be related to high et al., 2005
sodium conditions in the field. The levels of glycerol, glyceric acid and myo-
inositol increased while those of fructose and mannitol decreased slightly.

2009; Jannink et al., 2009). However, the results for MAS/
map-based QTLs have been modest. This has been due
to several factors including the absence of tight linkage
QTLs, non-availability of mapping populations and the
substantial time needed to develop such populations.
In addition, mapping populations often display limited
genetic variability, which presents a significant barrier in
For personal use only.
181 metabolites to elucidate the biological phenomenon
of heterosis at the metabolic level. They determined the
position and effect of 147 QTLs for metabolite absolute
mid-parent heterosis (aMPH), and identified 153 and
83 QTLs for augmented additive (Z(1)) and dominance
­effects (Z(2)), respectively.
Schauer et al. (2008) showed that the mean hereditabil-

crop improvement.
A metabolomic approach might be useful in the
breeding of crops, since valuable plant traits such as taste
and yield are closely related to metabolic conditions
(Oikawa et al., 2008). Recent advances in MS-based me-
tabolomics and data processing techniques should now
allow large-scale QTL analyses of untargeted metabolic
profiles, which may uncover previously unknown regu-
latory functions of loci in metabolic pathways (Tikunov
et  al., 2005; Vorst et  al., 2005; Keurentjes et  al., 2006).
Importantly, and in contrast to other complex traits, such
as morphological or physiological traits, the molecular
structure of the analyzed metabolites and the knowl-
edge of the pathway architecture may already suggest
the function of the gene underlying the mQTL (Morreel
et  al., 2006). In addition, broad-spectrum metabolite
analyses now also allow QTL mapping of an expanded
portion of the plant metabolome (Keurentjes et al., 2006;
Schauer et al., 2006; Meyer et al., 2007).
Recently, metabolomics has come to represent an
important addition to the tools currently employed
in genomics-assisted selection for crop improvement
(Fernie and Schauer, 2009; Kliebenstein, 2009), some
mQTLs have been identified in Arabidopsis, tomato and
Populus for 11 studies on metabolomics (Table 4). Rowe
et  al. (2008) used statistical analysis to identify a large
number of mQTLs with moderate phenotypic effects and
found frequent epistatic interactions controlling a major-
ity of the variation. Based on two mapping populations
of a cross between the Arabidopsis thaliana accessions
Col-0 and C24, Lisec et al. (2009) analyzed the levels of
ity of the mQTLs was in a range that would be regarded as
intermediate (i.e. between 0.20 and 0.35 – this also found
in Arabidopsis; Rowe et al., 2008). However, a handful of
the traits were nevertheless highly correlated and dis-
played a reasonable heritability (a mean r of between 0.3
and 0.69). A similar finding was observed in a maize study,
which revealed a great influence of the environment on
the metabolite profiles of the three genotypes studied
(Harrigan et al. 2007b). A comparative study of tomato in-
trogression lines (ILs) and heterozygous ILs revealed that
most of the metabolic QTLs were dominantly inherited
with a considerable number displaying an additive or re-
cessive mode of action and only a negligible amount dis-
playing the characteristics of overdominant inheritance
(Fernie and Schauer, 2009). Interestingly, the mode of
inheritance was quantitatively different between diverse
classes of compounds with, for example, sugars and acids
displaying significantly different patterns of inheritance.
Moreover, several metabolite pairs belonging to the same
pathway displayed a similar mode of inheritance at the
same chromosomal loci (Schauer et al., 2008), indicating
that the variation in both metabolites is probably medi-
ated by enzymes involved in their interconversion.
Comparative analysis of metabolite and developmen-
tal variation suggests an integral link between the plant
central metabolism and development/physiology, but
QTLs for metabolite and developmental traits were not
co-localized more than expected by chance (Keurentjes
et al., 2006; Meyer et al., 2007). This lack of overlap be-
tween the known development and mQTLs may indi-
cate that genetic regulation of plant metabolism is more

 Critical Reviews in Biotechnology

 Metabolomics: Creating new potentials for unraveling the mechanisms in response to salt  163
complex than presumed, such that current studies lack
sufficient power to detect the majority of mQTLs pres-
ent in a population. The level of genetic complexity
regulating plant metabolism will determine the size of
the structured mapping populations necessary for ef-
fective mQTL analyses and could potentially affect the
methodology and interpretation of association mapping
studies.
Combination of metabolomics- and
marker-assisted selection
Some reports have also suggested that the combination
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of South Carolina on 10/05/11
of metabolite profiling and MAS could prove highly in-
formative in understanding and influencing the chemi-
cal composition of crop species (Schauer et  al., 2006;
Hall, 2006; Fernie et al., 2006). However, identification of
map-based QTLs is not feasible for orphan, long-juvenile
woody, and xero-halophyte plants when no other ge-
netic information such as linkage maps and QTLs are
available. For example, sea buckthorn (Hippophae L.),
regarding drought tolerance is highly heterozygous and
has a juvenile period from 3∼5 years. Dried-shrink dis-
ease (DSD) is a dangerous pathogen that destroys this
species and halts commercial production, but it is always
a difficult process for sea buckthorn to select materials
with DSD-resistance in conventional breeding programs.
For personal use only.
This is because DSD infects plants that are a minimum
of three years old, whereas all plants infected by DSD are
more than 3 years old and in general at least 3∼5 years
are required for the sea buckthorn plant to express DSD
symptoms to its full potential (Ruan et al., 2008b). A QTL
linkage map is unknown for sea buckthorn, thus the con-
struction of mapping populations (even F1 crosses) are
time-consuming and labor-intensive.
To overcome the limitations of map-based QTL, and
as an alternative to planned populations, molecular
marker-trait associations have been identified by the
combination between germplasm and the regression
technique.
Germplasm-regression-combined (GRC) marker-trait
association identification has been successful in different
quantitative traits of many plants (Mishra and Sen-Mandi,
2004), including Asia rice (Oryza sativa) (Virk et al., 1996),
alfalfa (Medicago sativa L.) (Obert et al., 2000; Maureira-
Bulter et  al., 2007), tea (Camellia sinensis) (Mishra and
Sen-Mandi, 2004), bread wheat (Roy et al., 2006), Mulberry
(Morus indica L.) (Vijayan et al., 2006), coconut (Cocos nu-
cifera L.) (Shalini et al., 2007), birch (Betula platyphylla)
(Wang, 2007; Wang et al., 2008; Xia et al., 2008), oat (Avena
sativa L.) (Achleitner et al., 2008), mulberry (Morus ssp.)
(Kar et  al., 2008), sea buckthorn (Hippophae L.) (Ruan
et al., 2009; Li et al., 2010). Ruan et al. (2010) have enlisted
the utilization of this strategy in identifying the markers
associated with desirable traits, such as RAPD markers as-
sociated with various quantitative traits in Asia rice (Virk
et al., 1996) and protein content in wheat (Dholakia et al.,
2001), SCAR markers associated with birch fiber length
© 2011 Informa Healthcare USA, Inc.
(Wang et  al., 2008), ISSR markers associated with yield
traits (Vijayan et al., 2006) and ­protein content (Kar et al.,
2008) in mulberry and SSR markers associated with seed
size in soybean (Hoeck et al., 2003) and mite resistance in
coconut (Shalini et al., 2007). In resistance, based on 52
accessions of 16 sea buckthorn varieties and lines, Ruan
et  al. (2009) identified four ISSR markers that were sig-
nificantly correlated with resistance to DSD, using GRC-
based mark-trait association identification. Hence, mark-
ers associated with interesting traits, which are identified
by the GRC-based method, will play an important role in
plant MAS/QTL breeding programs, especially for orphan
xero-halophytes when no other genetic information such
as ­linkage maps and QTLs are available.
Potential of metabolomics in
biotechnological improvement of
xero-halophytes
In the face of a drought and salinized world (Munns,
2005), cultivation of salt-tolerant crops can help address
the threat of irreversible global salinization of fresh wa-
ter and soils (Rozema and Flowers, 2008), and initiatives
are being undertaken on a world-wide basis to develop
saline vegetable crops for fuel, food, and fiber (Ahmad
and Malik, 2002). Today, only about 1% of species of land
plants can grow and reproduce in coastal or inland sa-
line sites (Flowers and Colmer, 2008), only some of them
are tolerant to seawater salinity; more halophytes resist
lower salinity concentrations. Potentially, many of these
salt-adapted plants could become salt-tolerant crops
in a saline agriculture in which soil salinity is less than
(perhaps half ) that of seawater (Yeo, 1998; Flowers, 2004;
Colmer et al., 2005; Flowers and Flowers, 2005; Rozema
and Flowers, 2008), by biotechnological improvements
before introducing them in agricultural schemes.
Modern biotechnology might speed up the process
of achieving conventional crops that are resistant to
high concentrations of Na+ and Cl− in saline agriculture.
For example, biotechnology has generated traditional
crops that are resistant to plant pests and diseases, such
as genetically modified corn and cotton. However, over
the past 15 years, bioengineering has not delivered
­salt-tolerant cultivars of conventional crops such as wheat
or rice for release to farmers (Glenn et al., 1999; Rozema
and Flowers, 2008). So, although between 1996 and 2006
there were more than 30 reports of the transformation of
rice with different genes aimed at increasing salt toler-
ance, transgenic salt tolerant rice is not close to release.
The likely explanation is that salt tolerance is a complex
trait determined by many different genes, so that transfor-
mation of multiple genes into a plant is required (Stanton
et  al., 1992; Flowers, 2004). Because salt resistance has
already evolved in halophytes, domestication of these
plants is an approach that should be considered (Stanton
et al., 1992; Flowers, 2004; Colmer et al., 2005; Flowers and
Flowers, 2005; Rozema and Flowers, 2008). However, as
occurred with traditional crops such as rice, wheat, corn,
 Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of South Carolina on 10/05/11
For personal use only.


Table 4  Identification of metabolomic QTL.
Species (materials)
Arabidopsis thaliana [Landsberg erecta
(Ler) × Cape Verde Islands (Cvi ) RIL
population]
Arabidopsis [Columbia (Col) × C24 RIL
Metabolomic
technique Results References
HPLC QTL analysis identified six loci determining total aliphatic glucosinolate accumulation, six loci Kliebenstein et al., 2001
controlling total indolic glucosinolate concentration, and three loci regulating benzylic glucosinolate
levels. The results showed that two of the loci controlling total aliphatic glucosinolates map to
biosynthetic loci that interact epistatically to regulate aliphatic glucosinolate accumulation. In addition
to the six loci regulating total indolic glucosinolate concentration, mapping of QTL for the individual
indolic glucosinolates identified five additional loci that were specific to subsets of the indolic
glucosinolates.
LC-QTOF MS QTL analysis of more than 2000 mass peaks enabled the identification of QTLs for about 75% of the Keurentjes et al., 2006
mass signals. More than one-third of the signals were not detected in either parent, indicating the large
potential for modification of metabolic composition through classical breeding. For 1592 mass signals
(74.8%), the authors detected at least one significant QTL using a two-part parametric model. On
average, the authors found nearly 2.0 QTLs per analyzed mass, leading to a total of 4213 QTLs.
GC-MS Significant QTLs were detected for 9 out of 11 of the metabolite level traits. Keurentjes et al., 2008
GC-MS Six biomass QTLs and 157 mOTLs were identified. Two of the biomass QTL coincided with significantly Meyer et al., 2007
164  Cheng-Jiang Ruan, and Jaime A. Teixeira da Silva

population and an IL population]
Arabidopsis thaliana (IL population derived GC-MS
from accessions Col-0 and C24)
Tomato (a population of 74 Solanum
lycopersicum × S. pennellii ILs)
GC-MS
Tomato (ILs population)
Tomato [a series of crosses lines between a GC-MS
cherry tomato and three independent large
fruit cultivar (Levovil, VilB, and VilD)]
Tomato (A IL population derived from GC-MS
a cross between the cultivated tomato
Solanum lycopersicum and its wild relative, S.
habrochaites)
Populus (two full-sib poplar families, HPLC
Populus deltoides cv. S9-2 × P. nigra cv. Ghoy
and P. deltoides cv. S9-2 × P. trichocarpa cv.
V24)
more mQTL than statistically expected, supporting the notion that the metabolic profile and
biomass accumulation of a plant are linked. Furthermore, three of the six biomass QTLs could be
mathematically predicted based purely on their metabolite composition.
QTL based on analysis of the introgression lines showed that five of six biomass QTL and 55% of the Lisec et al., 2008
mQTL found by Meyer et al. (2007) in the RIL population were also found in the IL population
A broad profiling of tomato volatiles yielded 100 QTL that were conserved across harvests. 25 loci were Tieman et al., 2006
identified that are significantly altered in one or more of 23 different volatiles and four were altered in
citric acid content.
Identification of 889 QTL governing the accumulation of 74 metabolites, including important primary Fernie and Schauer, 2009
metabolites, such as sugars, organic acids, essential amino acids, intermediate metabolites and
vitamins.
The authors identified 889 quantitative fruit metabolic loci and 326 loci that modify yield-associated Schauer et al., 2006
traits. The mapping analysis indicates that at least 50% of the metabolic loci are associated with QTLs
that modify whole-plant yield-associated traits.
35 single-trait mQTLs were identified in the L background and 16 in the B background. Zanor et al., 2009
A total of 30 QTLs affecting the emission of one or more volatiles were mapped. A metabolite tree Mathieu et al., 2009
derived from these data provides new insights into the pathways for the synthesis of several of these
volatiles. One QTL is a novel locus affecting fruit carotenoid content on chromosome 2. Volatile
emissions from this and other lines indicate that the linear and cyclic apocarotenoid volatiles are
probably derived from separate carotenoid pools.
Four robust mQTLs, associated with rate-limiting steps in flavonoid biosynthesis, were mapped. Morreel et al., 2006
Each mQTL was involved in the flux control to one or two flavonoids. Based on the identities of the
affected metabolites and the flavonoid pathway structure, a tentative function was assigned to three of
these mQTL, and the corresponding candidate genes were mapped.

Critical Reviews in Biotechnology

 Metabolomics: Creating new potentials for unraveling the mechanisms in response to salt  165

Broad germplasm

Phenotype values

Phenotype-based Mapping GRC-based mark-trait

Metabolomics Transcriptomics Sequencing
selection population association identification

Phenotype- and Map-based Markers Gene ‘Next generation’

mQTL eQTL SNP
enzyme-based QTL associated with identification sequencing
selection interesting trait
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by University of South Carolina on 10/05/11

III III IV IV IV IV

I II Metabolomics- and marker- siRNAs and eQTL- and

V
assisted breeding miRNAs SNP-based MAS

Improvement for trait of interest in plant breeding programs

Figure 5.  Breeding technology from past to present to future (parts of adapted from Fernie and Schauer, 2009), especially for improvement
of xero-halophytes using the combination of metabolomics- and marker- assisted breeding (dash-dotted lines). Number: I indicates
breeding programs has been begun since 1980, II since 1990, III since 2000, IV indicates breeding programs will begin after 2010, and V after
2020. In the future, the market introduction of new varieties will be shortened within 4∼5 years by multiple advanced biotechnologies.
For personal use only.

and potatoes, domestication and improvement of wild
halophytic plant species is needed to convert them into
viable crops with high yields. Such a process can begin by
screening collections for the most productive genotypes
based on modern biotechnology (e.g. molecular markers
associated with interesting traits, metabolomic mecha-
nisms of tolerance, quality and yield in stress conditions,
and transgenic engineering). However, compared to con-
ventional crops, most xero-halophytic plants are orphan
because there is no genetic information for improve-
ment, and how to select and improve these plants is still a
­challenge for plant breeders in the future.
In the twenty first century, science has moved for-
ward from simple method development studies to mul-
tiple techniques to answer actual biological questions
(e.g. mechanisms in response to salt and drought stress)
and to improve the tolerance, quality, and yield of
plants that can grow under stress conditions (Figure 5).
The ongoing efforts to elucidate the metabolic response
to abiotic stresses indicate that metabolomics-assisted
breeding might be useful in the development of plants
that are more resistant to these stresses. The combined
use of metabolomics and GRC-based markers associ-
ated with interesting traits will probably considerably
shorten the time required for the production of elite
lines (Figure 5). This combination will provide us with a
holistic view of how plants respond to abiotic stress and
enable us to develop advanced strategies to enhance
the tolerance, quality, and yield of xero-halophytic
plants to abiotic stress conditions. Hence, modern
biotechnological methods based on a combination
of mQTL and GRC-based markers associated with
interesting traits will create a new and viable option for
MAS improvement of plant tolerance, yield and qual-
ity, especially for orphan xero-halophyte plants when
no other genetic information such as ­linkage maps and
QTLs are available.
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Baxter CJ, Redestig H, Schauer N, Repsilber D, Patil KR, Nielsen J,
Selbig J, Liu J, Fernie AR, Sweetlove LJ. (2007). The metabolic
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