Professional Documents
Culture Documents
REVIEW ARTICLE
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
For personal use only.
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
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
consumption, provide human nutrition, and green as the by-products of stress metabolism, stress signal
biofuel, to reduce CO2 emission, assimilate, and se- transduction molecules or molecules that are part of the
quester carbon into biomass. For example, deserts are acclimation response of plants (Shulaev et al., 2008).
currently underexploited for biomass production but Plant metabolomics (Table 1) is a fast-growing technol-
could act as a biospheric sink if adapted plant material ogy (Table 2) through qualitative and quantitative analysis
is selected: more than 22.5% of the world’s arid region of metabolites to define the profile of cells or organs, then
was estimated to be usable for planting halophytes to comprehensively understand the cellular response to
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(Glenn et al., 1992). Salt-tolerant plants may be used biotic and abiotic conditions (Schauer and Fernie, 2006).
in land that has not been forested or farmed and can As a branch of science concerned with the quantitative
afford beneficial side effects since they can aid in reveg- understandings of the metabolite complement of inte-
etating arid regions, restore ecological biodiversity, and grated living systems and its dynamic responses to the
improve mesoscale climatic parameters. changes of both endogenous factors (e.g. physiology and
Plant breeders have long been interested in improv- development) and exogenous factors (e.g. environmental
ing plants adapted to water and salt stress (Shao et al., factors and xenobiotics) (Tang and Wang, 2006), metabo-
2009), and several tolerant plants with potential inter- lomics is bursting with vitality, with about 3045 (web of
est for agriculture and environmental management science) and 1274 (PubMed) scientific papers related to
have already been identified, such as Atriplex halimus metabolomics (Figure 2) having been published during
(Lutts et al., 2004), Kosteletzkya virginica (Ruan et al., 2000–2009. Since the first example of the use of metabo-
2008a), and Salicornia bigelovii (Zerai et al., 2010). lite profiling as a diagnostic tool was to determine the
However, their rational use is greatly hampered by mode of action of various herbicides (Sauter et al., 1988),
insufficient knowledge about the mechanism of physi- metabolomics has been used (i) to ascertain the differ-
ological and biochemical mechanisms for plant toler- ences between genetically modified and conventional
ance to abiotic stress. In addition, since tolerant (e.g. crops (Catchpole et al., 2005) and the classification of
xero-halophyte) species were not previously domesti- genotypes (Tikunov et al., 2005; Laurentin et al., 2008),
cated (until recently, for only some), they still exhibit (ii) to test pathogenic infection (Allwood et al., 2006) and
a high level of heterogeneity. Further selection steps characterize the metabolic response to biotic (Desbrosses
are therefore required before introducing them in ag- et al., 2005) or abiotic (Bohnert et al., 2006) stress, (iii) to
ricultural schemes. Additional information concerning identify the genetic determinants of biochemical com-
their genotypes as well as a thorough estimation of the position (Hall et al., 2008), (iv) to discover novel genes
discrimination of quality traits and their ability to adapt (Hagel and Facchini, 2008) and annotate gene function,
to abiotic stress is a prerequisite for any improvement (v) to investigate the importance of environmental con-
strategy. Stress in plants can be defined as any change ditions on quality (Carrari et al., 2006), (vi) to design tools
in growth condition(s) that affects metabolic homeosta- and markers for monitoring quality (Schauer and Fernie,
sis and requires an adjustment of metabolic pathways 2006) and (vii) to identify metabolic quantitative trait loci
in a process that is usually referred to as acclimation (mQTLs) (Fernie and Schauer, 2009).
(Mittler, 2006; Suzuki and Mittler, 2006). Metabolomics The rapid development of plant metabolomics has
could contribute significantly to the study of stress biol- been covered by several reviews and books, which have
ogy in plants by identifying different compounds, such documented (i) the fundamental principles and state of
Genome Genomics
Microarrays
mRNA
Transcriptome Transcriptomics
Protein
2D Gels+MS
Proteome Proteommics
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LC-MS/MSn
Metabolite
Cell
Environmental factors
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Tissue
Organ
Genotype × Environment
Phenotype
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., and its fundamental insights into important biological
2003; Fernie et al., 2004; Saito et al., 2006; Ward et al., rocesses (Steinfath et al., 2008), (v) the dynamics of
p
2007; Allwood et al., 2008), (ii) the standards for envi- metabolite changes during stress (Zuther et al., 2007;
ronmental context biological samples (Morrison et al., Shulaev et al., 2008) (e.g. temperature, Guy et al., 2008a;
2007) and for plant biology context information in me- salinity, Sanchez et al., 2008a), (vi) the characterization
tabolomic studies (Fiehn et al., 2007), (iii) sample prepa- of plant metabolites of nutritional importance and sig-
ration and data acquisition and interpretation (Fiehn, nificance in human health (Hall et al., 2008), (vii) func-
2001), (iv) metabolic diversity (Harrigan et al., 2007a) tional genomics through the combination of proteomic
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) terpenoids (Aharoni et al., 2003; Chen et al., 2003) and
and metabolomic data sets, and (viii) the trends and volatiles (Tikunov et al., 2005), mutant classification
coming of age in plant metabolomics (Last et al., 2007; (Messerli et al., 2007), functional genomics (Schauer
Guy et al., 2008b). In contrast, in this review we first and Fernie, 2006), and the integration of metabolic and
survey briefly plant metabolomic technologies. Then, transcript datasets (Carrari et al., 2006; Baxter et al., 2007;
we focus on the metabolomics used to (1) analyze the Kaplan et al., 2007). Liquid chromatography (LC)-MS is
characterization of the metabolic response to salt and better suited to the targeted profiling of specific types of
drought stress; and (2) identify mQTLs. Finally, we dis- compounds that display similar ionizing behavior (e.g.
cuss the prospect of the potential role for metabolomics alkaloids) and is generally not used for broad-scope me-
as a tool to unravel the mechanisms of biochemistry and tabolomic studies (Halket et al., 2005). The combination
molecular biology in response to salt and drought stress of LC-MS and GC-MS data certainly provides a more
of plants and as a viable option for the improvement of complete perspective of a plant metabolome, resulting
plant tolerance, yield, and quality in the future, especially in the establishment of a tomato-specific LC-MS data-
for orphan xero-halophyte plants, most of which have rel- base (Catchpole et al., 2005; Moco et al., 2006). These
atively no genetic information compared to conventional techniques facilitate analyte identification by providing
crops with known genetic information (e.g. sequences, information on both the chemical nature and mass of
linkage maps, QTLs, etc.). individual molecules. In contrast, direct-infusion mass
spectrometry (DIMS) or fourier transform-ion cyclotron
resonance (FT-ICR)-MS yield less chemical information.
Plant metabolomics technologies FT-ICR-MS, which has more recently been applied to me-
Metabolomics became a standard laboratory technique in tabolomics, is the most effective in terms of mass resolu-
the past decade (Fernie et al., 2004), although it has been tion (i.e. the ability to distinguish two ions with similar
carried out since the mid 1970s (Jellum, 1977). Currently, masses) and mass accuracy (i.e. how close a measured
a number of metabolomics techniques are used in plant mass is to the true mass) (Aharoni et al., 2002; Tohge
studies and these are shown in Table 2 and Figure 3. et al., 2005; Zulak et al., 2008), and has been used to iden-
Among these, both mass spectrometry (MS) and nuclear tify adulterants in vegetable oils (Wu et al., 2004), investi-
magnetic resonance (NMR) dominate the metabolite gate nitrogen metabolism in transgenic tobacco (Munger
strategies. Gas chromatography (GC)-MS has long been et al., 2005), study the effects of herbicide and light/dark
used for plant metabolomics, such as in the analysis of treatments in Arabidopsis (Oikawa et al., 2006; Nakamura
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
been published, although the values are restricted to these, stresses caused by abiotic conditions, such as tem-
aqueous samples (Verpoorte et al., 2007). 2H, 13C or perature extremes (freezing, cold, and heat), water avail-
15
N can be used to label samples prior to NMR analy- ability (drought and ion excess), and ion toxicity (salinity
sis, which is particularly suited to the elucidation of and heavy metals), have been difficult to dissect because
biosynthetic pathways and metabolic flux analyses the defense responses to abiotic factors require regulatory
(Kruger et al., 2003; Ratcliffe and Shachar-Hill, 2005; changes to the activation of multiple genes and pathways.
Ward and Beale, 2006). For example, transcript profiles In the past several years, there has been relative success
of Arabidopsis albino mutants were compared with of the over- or under-expression of single or several genes
metabolite profiles obtained by NMR analysis of 13C6- by targeting genes, proteins or enzymatic reactions that
glucose-fed plants revealing distinct labeling patterns had been indicated as important components of stress
in the albino lines compared with controls and an al- tolerance or sensitivity (Sung et al., 2003; Baniwal et al.,
teration in carbon/nitrogen homeostasis caused by a 2004; Mittler et al., 2004; Bohnert et al., 2006); in contrast,
loss of chloroplast function (Tian et al., 2007). for effectively improving plant tolerance to abiotic stress,
Separation-based methods coupled with MS are a shift should be emphasized and practiced in the future:
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relatively sensitive (e.g. 106–109-fold more sensitive than away from focusing on the end-points of response chains
NMR; Sumner et al., 2003), but their sensitivities are not to engineering genes governing upstream reactions that
uniform for all metabolites and can be pretty low for affect entire pathways and groups of genes (Vinocur
certain analytes under the given analytical conditions. and Altman, 2005). However, our limited knowledge of
For metabolomics purposes, both chromatography and stress-associated metabolism remains a major gap in
MS methods suffer from some common disadvantages our understanding of the mechanisms of plant toler-
in terms of metabolite identification and quantification. ance to abiotic stress; therefore, comprehensive profiling
Both techniques require fairly extensive sample prepara- of stress-associated metabolites is most relevant to the
tions and are invasive and destructive (to samples), hence successful biotechnological breeding of stress-tolerant
are not suitable for in vivo or in situ studies. Both of these plants.
techniques require prior knowledge about the samples In the past decade, the application of omics-type
and have high recurrent expenditure though chromato- technologies (e.g. genome, transcriptome, proteome
graphic methods are reasonably inexpensive. In contrast, and metabolome) has enhanced our understanding
the NMR-based methods are user independent, require of plant responses to abiotic stresses that disturb the
little or no sample preparation, and have excellent reso- homeostatic equilibrium. Among these techniques,
lution and reproducibility (Ward et al., 2007; Hagel and attention to metabolite analysis is obviously growing
Facchini, 2008; Zulak et al., 2008). In addition, NMR of- in the development of metabolite sensors and data-
fers rich molecular information including metabolite bases (Cook et al., 2004; Hirai et al., 2004; 2005) and
structure, concentration, molecular dynamics, interac- has been used to study temperature (Cook et al., 2004;
tions, pH, and compartmentation when diffusion-editing Kaplan et al., 2004; Kaplan et al., 2007), water and sa-
techniques are employed (Tang and Wang, 2006; Allwood linity (Johnson et al., 2003; Brosche et al., 2005; Gong
et al., 2008; Hagel and Facchini, 2008). Furthermore NMR et al., 2005; Cramer et al., 2007; Kim et al., 2007), bo-
is non-invasive and non-destructive to samples, thus ron (Roessner et al., 2006), sulfur (Nikiforova et al.,
making it possible to facilitate in vivo and in situ studies. 2004; Nikiforova et al., 2005a; Nikiforova et al., 2005b;
Hence, it is clear that the best choice at present may lie Nikiforova 2008), phosphorus (Hernandez et al., 2007),
in the combination of a number of existing techniques. oxidative (Baxter et al., 2007), and heavy metal (Le Lay
The hybrid method, chromatography, MS and NMR has et al., 2006) stress as well as a combination of multiple
already played an important role in metabolite identi- stresses (Rizhsky et al., 2004) in plants (Figure 4). Here,
fication, such as LC-SPE-NMR (Christophoridou et al., we summarize the application of plant metabolomics
2005), LC-NMR-MS (Ward and Beale, 2006), FT-ICR-MS in analyzing the mechanisms in response to salt and
and NMR (Hagel and Facchini, 2008). It is particularly drought stress in different plants, such as Arabidopsis,
800
700
600
500
Papers
400
300
200
100
0
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
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B 350
300
250
200
Papers
150
100
50
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0
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009
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
Abiotic stresses
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.
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
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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
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
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2009; Jannink et al., 2009). However, the results for MAS/ 181 metabolites to elucidate the biological phenomenon
map-based QTLs have been modest. This has been due of heterosis at the metabolic level. They determined the
to several factors including the absence of tight linkage position and effect of 147 QTLs for metabolite absolute
QTLs, non-availability of mapping populations and the mid-parent heterosis (aMPH), and identified 153 and
substantial time needed to develop such populations. 83 QTLs for augmented additive (Z(1)) and dominance
In addition, mapping populations often display limited effects (Z(2)), respectively.
genetic variability, which presents a significant barrier in Schauer et al. (2008) showed that the mean hereditabil-
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crop improvement. ity of the mQTLs was in a range that would be regarded as
A metabolomic approach might be useful in the intermediate (i.e. between 0.20 and 0.35 – this also found
breeding of crops, since valuable plant traits such as taste in Arabidopsis; Rowe et al., 2008). However, a handful of
and yield are closely related to metabolic conditions the traits were nevertheless highly correlated and dis-
(Oikawa et al., 2008). Recent advances in MS-based me- played a reasonable heritability (a mean r of between 0.3
tabolomics and data processing techniques should now and 0.69). A similar finding was observed in a maize study,
allow large-scale QTL analyses of untargeted metabolic which revealed a great influence of the environment on
profiles, which may uncover previously unknown regu- the metabolite profiles of the three genotypes studied
latory functions of loci in metabolic pathways (Tikunov (Harrigan et al. 2007b). A comparative study of tomato in-
et al., 2005; Vorst et al., 2005; Keurentjes et al., 2006). trogression lines (ILs) and heterozygous ILs revealed that
Importantly, and in contrast to other complex traits, such most of the metabolic QTLs were dominantly inherited
as morphological or physiological traits, the molecular with a considerable number displaying an additive or re-
structure of the analyzed metabolites and the knowl- cessive mode of action and only a negligible amount dis-
edge of the pathway architecture may already suggest playing the characteristics of overdominant inheritance
the function of the gene underlying the mQTL (Morreel (Fernie and Schauer, 2009). Interestingly, the mode of
et al., 2006). In addition, broad-spectrum metabolite inheritance was quantitatively different between diverse
analyses now also allow QTL mapping of an expanded classes of compounds with, for example, sugars and acids
portion of the plant metabolome (Keurentjes et al., 2006; displaying significantly different patterns of inheritance.
Schauer et al., 2006; Meyer et al., 2007). Moreover, several metabolite pairs belonging to the same
Recently, metabolomics has come to represent an pathway displayed a similar mode of inheritance at the
important addition to the tools currently employed same chromosomal loci (Schauer et al., 2008), indicating
in genomics-assisted selection for crop improvement that the variation in both metabolites is probably medi-
(Fernie and Schauer, 2009; Kliebenstein, 2009), some ated by enzymes involved in their interconversion.
mQTLs have been identified in Arabidopsis, tomato and Comparative analysis of metabolite and developmen-
Populus for 11 studies on metabolomics (Table 4). Rowe tal variation suggests an integral link between the plant
et al. (2008) used statistical analysis to identify a large central metabolism and development/physiology, but
number of mQTLs with moderate phenotypic effects and QTLs for metabolite and developmental traits were not
found frequent epistatic interactions controlling a major- co-localized more than expected by chance (Keurentjes
ity of the variation. Based on two mapping populations et al., 2006; Meyer et al., 2007). This lack of overlap be-
of a cross between the Arabidopsis thaliana accessions tween the known development and mQTLs may indi-
Col-0 and C24, Lisec et al. (2009) analyzed the levels of cate that genetic regulation of plant metabolism is more
of metabolite profiling and MAS could prove highly in- as linkage maps and QTLs are available.
formative in understanding and influencing the chemi-
cal composition of crop species (Schauer et al., 2006;
Potential of metabolomics in
Hall, 2006; Fernie et al., 2006). However, identification of
biotechnological improvement of
map-based QTLs is not feasible for orphan, long-juvenile
xero-halophytes
woody, and xero-halophyte plants when no other ge-
netic information such as linkage maps and QTLs are In the face of a drought and salinized world (Munns,
available. For example, sea buckthorn (Hippophae L.), 2005), cultivation of salt-tolerant crops can help address
regarding drought tolerance is highly heterozygous and the threat of irreversible global salinization of fresh wa-
has a juvenile period from 3∼5 years. Dried-shrink dis- ter and soils (Rozema and Flowers, 2008), and initiatives
ease (DSD) is a dangerous pathogen that destroys this are being undertaken on a world-wide basis to develop
species and halts commercial production, but it is always saline vegetable crops for fuel, food, and fiber (Ahmad
a difficult process for sea buckthorn to select materials and Malik, 2002). Today, only about 1% of species of land
with DSD-resistance in conventional breeding programs. plants can grow and reproduce in coastal or inland sa-
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This is because DSD infects plants that are a minimum line sites (Flowers and Colmer, 2008), only some of them
of three years old, whereas all plants infected by DSD are are tolerant to seawater salinity; more halophytes resist
more than 3 years old and in general at least 3∼5 years lower salinity concentrations. Potentially, many of these
are required for the sea buckthorn plant to express DSD salt-adapted plants could become salt-tolerant crops
symptoms to its full potential (Ruan et al., 2008b). A QTL in a saline agriculture in which soil salinity is less than
linkage map is unknown for sea buckthorn, thus the con- (perhaps half ) that of seawater (Yeo, 1998; Flowers, 2004;
struction of mapping populations (even F1 crosses) are Colmer et al., 2005; Flowers and Flowers, 2005; Rozema
time-consuming and labor-intensive. and Flowers, 2008), by biotechnological improvements
To overcome the limitations of map-based QTL, and before introducing them in agricultural schemes.
as an alternative to planned populations, molecular Modern biotechnology might speed up the process
marker-trait associations have been identified by the of achieving conventional crops that are resistant to
combination between germplasm and the regression high concentrations of Na+ and Cl− in saline agriculture.
technique. For example, biotechnology has generated traditional
Germplasm-regression-combined (GRC) marker-trait crops that are resistant to plant pests and diseases, such
association identification has been successful in different as genetically modified corn and cotton. However, over
quantitative traits of many plants (Mishra and Sen-Mandi, the past 15 years, bioengineering has not delivered
2004), including Asia rice (Oryza sativa) (Virk et al., 1996), salt-tolerant cultivars of conventional crops such as wheat
alfalfa (Medicago sativa L.) (Obert et al., 2000; Maureira- or rice for release to farmers (Glenn et al., 1999; Rozema
Bulter et al., 2007), tea (Camellia sinensis) (Mishra and and Flowers, 2008). So, although between 1996 and 2006
Sen-Mandi, 2004), bread wheat (Roy et al., 2006), Mulberry there were more than 30 reports of the transformation of
(Morus indica L.) (Vijayan et al., 2006), coconut (Cocos nu- rice with different genes aimed at increasing salt toler-
cifera L.) (Shalini et al., 2007), birch (Betula platyphylla) ance, transgenic salt tolerant rice is not close to release.
(Wang, 2007; Wang et al., 2008; Xia et al., 2008), oat (Avena The likely explanation is that salt tolerance is a complex
sativa L.) (Achleitner et al., 2008), mulberry (Morus ssp.) trait determined by many different genes, so that transfor-
(Kar et al., 2008), sea buckthorn (Hippophae L.) (Ruan mation of multiple genes into a plant is required (Stanton
et al., 2009; Li et al., 2010). Ruan et al. (2010) have enlisted et al., 1992; Flowers, 2004). Because salt resistance has
the utilization of this strategy in identifying the markers already evolved in halophytes, domestication of these
associated with desirable traits, such as RAPD markers as- plants is an approach that should be considered (Stanton
sociated with various quantitative traits in Asia rice (Virk et al., 1992; Flowers, 2004; Colmer et al., 2005; Flowers and
et al., 1996) and protein content in wheat (Dholakia et al., Flowers, 2005; Rozema and Flowers, 2008). However, as
2001), SCAR markers associated with birch fiber length occurred with traditional crops such as rice, wheat, corn,
Table 4 Identification of metabolomic QTL.
Metabolomic
Species (materials) technique Results References
Arabidopsis thaliana [Landsberg erecta HPLC QTL analysis identified six loci determining total aliphatic glucosinolate accumulation, six loci Kliebenstein et al., 2001
(Ler) × Cape Verde Islands (Cvi ) RIL controlling total indolic glucosinolate concentration, and three loci regulating benzylic glucosinolate
population] 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
Arabidopsis [Columbia (Col) × C24 RIL 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] 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.
Arabidopsis thaliana (IL population derived GC-MS QTL based on analysis of the introgression lines showed that five of six biomass QTL and 55% of the Lisec et al., 2008
from accessions Col-0 and C24) mQTL found by Meyer et al. (2007) in the RIL population were also found in the IL population
Tomato (a population of 74 Solanum A broad profiling of tomato volatiles yielded 100 QTL that were conserved across harvests. 25 loci were Tieman et al., 2006
lycopersicum × S. pennellii ILs) identified that are significantly altered in one or more of 23 different volatiles and four were altered in
citric acid content.
GC-MS 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.
Tomato (ILs population) 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.
Tomato [a series of crosses lines between a GC-MS 35 single-trait mQTLs were identified in the L background and 16 in the B background. Zanor et al., 2009
cherry tomato and three independent large
fruit cultivar (Levovil, VilB, and VilD)]
Tomato (A IL population derived from GC-MS A total of 30 QTLs affecting the emission of one or more volatiles were mapped. A metabolite tree Mathieu et al., 2009
a cross between the cultivated tomato derived from these data provides new insights into the pathways for the synthesis of several of these
Solanum lycopersicum and its wild relative, S. volatiles. One QTL is a novel locus affecting fruit carotenoid content on chromosome 2. Volatile
habrochaites) emissions from this and other lines indicate that the linear and cyclic apocarotenoid volatiles are
probably derived from separate carotenoid pools.
Populus (two full-sib poplar families, HPLC Four robust mQTLs, associated with rate-limiting steps in flavonoid biosynthesis, were mapped. Morreel et al., 2006
Populus deltoides cv. S9-2 × P. nigra cv. Ghoy Each mQTL was involved in the flux control to one or two flavonoids. Based on the identities of the
and P. deltoides cv. S9-2 × P. trichocarpa cv. affected metabolites and the flavonoid pathway structure, a tentative function was assigned to three of
V24) these mQTL, and the corresponding candidate genes were mapped.
Broad germplasm
Phenotype values
III III IV IV IV IV
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 interesting traits will create a new and viable option for
halophytic plant species is needed to convert them into MAS improvement of plant tolerance, yield and qual-
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screening collections for the most productive genotypes no other genetic information such as linkage maps and
based on modern biotechnology (e.g. molecular markers QTLs are available.
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