See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/305427054

Metabolomics and its application to phytopathology

Chapter · January 2011

CITATIONS READS

0 40

2 authors:

Lucio Rodríguez Sifuentes Nicolas Soto-Cruz

Autonomous University of Coahuila Instituto Tecnológico de Durango (ITD)
3 PUBLICATIONS   11 CITATIONS    40 PUBLICATIONS   197 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Cocomposting of hydrocarbon contaminated soil View project

All content following this page was uploaded by Nicolas Soto-Cruz on 19 July 2016.

The user has requested enhancement of the downloaded file.

 Research Signpost
37/661 (2), Fort P.O.
Trivandrum-695 023
Kerala, India

Phytopathology in the Omics Era, 2011: 175-190 ISBN: 978-81-308-0438-5

Editors: Raúl Rodríguez Herrera, Cristobal N. Aguilar, June Kilpatrick Simpson-Williamson
and Gerardo Gutierrez Sanchez

11. Metabolomics and its application to

phytopathology

Lucio Rodríguez-Sifuentes and Nicolás Óscar Soto Cruz

Department of Chemistry and Biochemistry Engineering, Technological Institute of Durango
Durango, 34080, Durango, Mexico

Abstract. Metabolite determination helps to understand a gene’s

function, and many analytical techniques and statistical software
programs have been developed for this purpose. When a
microorganism attacks a plant, a variety of metabolites are produced
by both the plant and the pathogen. Metabolomics helps identify
pathogen genes that play an important role in the infection event, as
well as the plant genes that play important roles in a plant’s defense
against pathogens. The present chapter presents an overview of
metabolomics technology, tools used in the study of phytopathology,
as well as examples of advances in the field of metabolomics.

Introduction
In a nutritive medium, cells, tissues, or organisms incorporate assimilable
nutrients which are processed by enzymes into different products. The set of

Correspondence/Reprint request: Dr. Nicolás Óscar Soto Cruz, Department of Chemistry and Biochemistry
Engineering, Technological Institute of Durango, Durango, 34080, Durango, Mexico
E-mail: soto@itdposgrado-bioquimica.com.mx
176 Lucio Rodríguez-Sifuentes & Nicolás Óscar Soto Cruz

reactions involved in the processing of each type of nutrient constitutes

different metabolic pathways. Each pathway has a defined sequence of
detectable biochemical reaction steps that connect a specified set of input and
output metabolites (Stephanopoulos et al., 1998). In a catabolic pathway,
substrates are degraded to obtain energy, which requires an initial input of
energy and reduces the amount of metabolites available. In contrast anabolic
pathways use metabolites previously formed in catabolic pathways to
synthesize building blocks (aminoacids, nucleotides, fatty acids). Therefore,
all metabolic pathways in a biological system can be divided into catabolic
pathways, involving degradation reactions, and anabolic pathways, involving
biosynthesis reactions.
The generation of metabolites represents the response of a system’s
genome to its environment. These metabolites are not solely the end product
of gene expression patterns, but rather can also serve as regulatory molecules
(Rochfort, 2005). The set of metabolites synthesized by a biological system
constitute its “metabolome” (Fiehn, 2002). Weckwerth and Morgenthal
(2005) defined a metabolome as the dynamic set of all small molecules
present in an organism or biological sample, while Oliver et al. (1998) and
Goodacre et al. (2004) had previously defined a metabolome as the total
quantitative collection of small molecular weight compounds (metabolites)
present in a cell or organism which participate in metabolic reactions required
for growth, maintenance, and normal function. In both definitions, a key
factor of the metabolome is its dynamic nature that depends on external
conditions or genetic modifications to define its state in a biological system.
The goal of metabolomics is to identify and quantitate the physiological
state of a biological system under certain conditions. Thus, metabolomics has
emerged as one of the new “omics” sciences, preceded by the fields of
genomics, transcriptomics, and proteomics that have been employed to
understand global systems biology. In this chapter, basic concepts of
metabolomics are reviewed and the use of metabolomics as a tool for
elucidating changes in metabolism as a result of plant/pathogen interactions
are discussed.

Metabolism regulation
Production of intracellular metabolites is a controlled process in
biological systems. Even a few changes in a metabolite profile can represent
a new physiological state. Transcriptomes, proteomes, and metabolomes
represent different snapshots of the physiological state of a biological system.
By measuring changes in the concentrations of intracellular metabolites
Metabolomics and its application in phytopathology 177

present, insights into the regulation of transcription, translation, and enzyme

activity can be identified (Vemuri and Aristidou, 2005).
Proteins play a very important role in establishing the metabolism of a
biological system, particularly in regard to the transport of substrates and
enzymatic activity. In response to changes in external conditions (i.e. changes
in substrate and product concentrations, pH, temperature, oxygen
concentration, etc.), signaling pathways in the cell determine which proteins
and how much of these proteins need to be synthesized. These processes
require both constitutive proteins, whose functions are constantly employed
by the cell, and therefore these proteins are constantly being produced, and
inducible proteins, which are produced only when their function is required.
This combination of protein expression enables an organism to prevent the
wasteful production of unnecessary proteins, yet remain dynamic in its
reponse to changes in its environment (Stephanopoulos et al., 1998).
Protein production can be regulated during DNA transcription and/or
RNA translation, and regulatory proteins that detect external signals can
facilitate or prevent gene transcription. Control of metabolism is hierarchical
and originates at the level of transcription. Opportunities for metabolic
control include the induction or repression of gene expression, degradation of
mRNA, protein activation, proteolysis of proteins, and/or enzyme activity
(allostery). Usually a combination of these regulatory mechanisms are
employed by a cell (Vemuri and Aristidou, 2005).
Enzymes are also dynamically regulated by the feedback of products,
metabolic intermediates, or other compounds which can activate or inhibit
enzyme activity. Feedback inhibition occurs when the product of a
biochemical reaction suppresses the activity of the enzyme involved in its
synthesis. With allosteric control, the activity of the enzyme depends on
molecules other than the substances upon which the enzyme directly acts.
Therefore, changes in an organism’s metabolism represents changes in an
organism’s metabolome (Stephanopoulos et al, 1998).

Metabolomics
Functional genomics deciphers the function of unknown genes, while
systems biology attempts to characterize all processes in a biological system.
The combination of data from these two approaches can identify metabolic
networks. Metabolomics is the study of global metabolite profiles in a
biological system (i.e. cell, tissue, or organism) under a given set of
conditions (Goodacre et al., 2004). Dunn et al. (2005) expanded the definition
of metabolomics to include the analysis of intra- and extra-cellular
metabolites in simple biological systems (including microbial, plant, and
178 Lucio Rodríguez-Sifuentes & Nicolás Óscar Soto Cruz

mammalian systems). For these studies, different terminologies have been

used in metabolomic research depending on the metabolic approaches applied
(Dunn et al., 2005):

• Metabolomics: Non-biased identification and quantification of all

metabolites in a biological system. The analytical technique(s) must be
highly selective and sensitive. No individual analytical technique, or
combination of techniques, can currently determine all metabolites
involved in the metabolome of microbes, plants, or mammalian species.
• Metabonomics: The quantitative measurement of the dynamic,
multiparametric metabolic response of living systems to
pathophysiological stimuli or genetic modification.
• Metabolite target analysis: Quantitative determination of one or more
metabolites related to a specific pathway following extensive sample
preparation and sensitive detection of chromatographic separation.
• Metabolite/metabolic profiling: Analysis to identify and quantify
metabolites related through similar chemistries or metabolic pathways.
Chromatographic separation is normally employed prior to detection with
minimal metabolite isolation after sampling.
• Metabolic fingerprinting: Global, rapid, and high-throughput analysis
of crude samples or sample extracts for sample classification or screening
of samples. Identification and quantification is not performed. Minimal
sample preparation is involved.
• Metabolic footprinting: Global measurement of metabolites secreted from
the intracellular volume into the growth medium. This high-throughput
method does not require the rapid quenching and time consuming extraction
of intracellular metabolites involved in microbial metabolomics.

Metabolomics integrates information from genomics (DNA sequencing),

transcriptomics (gene expression analysis), and proteomics (protein analysis)
to elucidate metabolic networks of a biological system. Regulation of these
metabolic networks is then investigated by analyzing changes in genomic,
transcriptomic, and proteomic profiles in response to genetic modifications
and environmental perturbations. It is particularly informative when the
transcriptome and proteome do not undergo changes, yet the metabolome
does (Fiehn, 2002).
Mebatolomics is a field that has grown exponentially since 2000 and has
been applied to many areas of biology (Sumner et al., 2003). Sumner et al.
(2003) provide a brief history of the field. Many reviews have highlighted the
different applications of metabolomics which have included areas of drug
discovery, disease diagnostics and treatments, modification of biochemical
Metabolomics and its application in phytopathology 179

pathways using metabolic engineering, assessments of genetically modified

organisms, predictions of novel metabolic pathways, and the principal aim of
this chapter, plant-pathogen interactions. For more detail regarding these
applications of metabolomics, readers should consult references 3 and 4.
Linking the “omics” sciences has been difficult since not all expressed
genes are converted to mRNAs, and not all expressed mRNAs are converted
to proteins. There are also isoenzymes which can produce different
metabolites (Rochfort, 2005). Nevertheless, many studies have been able to
integrate data from the “omics” sciences (Kant et al., 2004) as a result of new
techniques that have become available (Weckwert et al., 2004).

Analytical techniques in mebatolomics

The detection, identification, and quantification of metabolites can be very
complicated. For example, metabolites include a variety of chemical
compounds with different complexities. Dunn and co-workers (2005)
demonstrated that metabolite concentrations can vary over nine orders of
magnitude (pM-mM), which represents a significant challenge for the
analytical technologies used to detect these changes over such a wide scale. In
addition, the number of metabolites produced in a biological system is
daunting. Oksman-Caldentey and Inzé (2004) have estimated the number of
metabolites in the plant kingdom to range from 100 000 - 200 000. As a result,
special attention must be given to sample preparation, analytical methods, and
data processing involved with metabolomics. As a whole, these considerations
present significant challenges in the field of plant metabolomics.
In the study of plant/pathogen interactions, sample preparation is a key step
and can be very complicated. For example, extraction of metabolites from
plants or pathogens requires processing of different plant tissues (i.e. flowers,
leaves, roots, petioles, etc.) and the use of different solvents. While methanol
would be used for the extraction of highly soluble metabolites (i.e.
carbohydrates), chloroform is used to extract metabolites with low solubility
(i.e. membrane lipids, fatty acids) (Allwood et al., 2008). Once samples have
been prepared, the metabolites present need to be detected, quantified, and
ideally, identified. No single analytical technique can fulfill all three
requirements simultaneously, therefore, a combination of different analytical
techniques is needed. As a result, many new analytical techniques have been
developed, and the most widely used technologies are briefly described below.
Spectroscopic techniques are based on the absorbance of electromagnetic
radiation (EM) to generate radiation or induce the transition atoms to a higher
energy state (Allwood et al., 2008). Table 1 presents principal spectroscopic
techniques, including Fourier transform spectroscopy (FT-IR) and nuclear
 180

Table 1. Spectroscopy techniques used in the study of the metabolomics of plants and plant-host interactions.
Lucio Rodríguez-Sifuentes & Nicolás Óscar Soto Cruz
Metabolomics and its application in phytopathology 181

magnetic resonance (NMR), that are commonly used in characterizing the

metabolomics of plants and plant-pathogen interactions. The techniques are
summarized according to Allwood et al. (2008).
Mass spectrometry (MS) is commonly used in metabolomics to analyze
plants and plant-host interactions. During MS, ions are separated based on
their mass-to-charge (m/z) ratio and detected. M/z ratios are recorded along
with their relative abundance and presented as a mass spectrum (Allwood et al.,
2008). Mass analyzers frequently used for MS include (Weckwerth and
Morgenthal, 2005):
• Single quadrupole: A mass-selective ion filter that consists of four
electronically operated metal rods.
• Triple quadrupole: Three quadrupole devices coupled in a linear array.
In the single reaction monitoring mode (SMR), selected “parent ions”
pass through the first quadrupole and enter a second quadrupole device
that is used as a collision cell to generate product ions. The third
quadrupole serves as a selective mass filter for the determination of the
resulting “daughter ions”.
• Ion trap: Collects and stores ions by forcing them into stable orbits and
subsequently releases them based on mass. Collected and stored “parent
ions” can also be fragmented and thus produce “daughter ions”.
• Time-of-flight (TOF): Ions are simultaneously accelerated to generate
the same kinetic energy for any given ion. Using a long and evacuated
flight tube, fixed distance ions are separated by their m/z ratio and
velocity.
The resolution of MS analysis, i.e. the sensitivity and selectivity, is
improved by fractionating samples prior to analysis using chromatographic
techniques. Allwood et al. (2008) have described the use of gas
chromatography (GC) and liquid chromatography (LC) in combination with
MS analysis for metabolomic studies of plants and plant-pathogen
interactions. A brief summary is presented here:

• Gas chromatography: Prior to ionization, separation is achieved using

gas as the mobile phase. GC-MS ion formation is achieved by electron
impact (EI) and involves the independent elimination of the sample’s
solvent prior to the vaporized sample being swept into the ionization
source where it is impacted by a steady flow of electrons with sufficient
energy to ionize the vaporized molecule. Mass detection is achieved
using a single quadrupole, TOF, or ion trap mass analysers. Metabolites
are identified by comparing and matching the retention times and/or
retention index of each sample with chromatograms of chemical
182 Lucio Rodríguez-Sifuentes & Nicolás Óscar Soto Cruz

standards. This technique is relatively inexpensive, can resolve complex

mixtures, and is used for volatile metabolites (up to 250°C). Two
important disadvantages of the technique are that metabolites which are
heat-labile are not able to be detected, and this technique can not be
applied to non-volatile high molecular weight metabolites.
• Liquid chromatography: Prior to ionization, separation is achieved
using liquid as the mobile phase. High-performance liquid
chromatography (HPLC) refers to the use of small packing particles and
a relatively high pressure, while ion formation in LC-MS is achieved by
electrospray ionization (ESI) or atmospheric pressure chemical ionization
(APCI). During ESI, a sample in a suitable solvent at atmospheric
pressure is sprayed out of a small needle to which a high charge has been
applied. Small charged droplets are therefore produced and following
rapid solvent evaporation, the charged ions are swept into the MS where
a TOF analyzer detects the mass of the ions and their intensity.
Metabolite quantification using LC-MS may be achieved by using
chemical standards to determine peak area ratios or provide calibration.
While this technique has good reproducibility, complex mixtures are not
well separated due to the generally poor resolving power of LC columns.
Another alternative technology is the coupling of capillary
electrophoresis to MS (CE-MS). For this approach, electrically charged
analytes are separated by their mobility in a capillary filled with an
electrolyte under the influence of an electric field (Weckwerth and
Morgenthal, 2005).
Chromatographic techniques coupled with spectroscopic or spectrometric
detection can enhance the resolution, sensitivity, and selectivity of metabolite
detection. In each case, the combination of analytical techniques employed
depends on the type of sample to be characterized.

Data analysis
Once metabolites have been separated and characterized, data processing
and statistical analysis are performed to correlate changes in metabolites to
genetic changes or changes in external stimuli. Various software tools are
available that can take the raw data collected from different analytical
techniques and deconvolute overlapping chromatogram peaks to align
different chromatograms for the comparison of different data sets (Duran et al.,
2003; Vorst et al., 2005).
Metabolite profiles can contain hundreds or thousands of data points, and
therefore, represent a large quantity of information that needs to be organized
Metabolomics and its application in phytopathology 183

and interpreted in order to understand the changes in metabolism that have

occurred. Computer-based statistical methods have been developed that
reduce the number of data and allow the data to be simplified according to
specific variables.
Principal component analysis (PCA) is a method in which the variance in
a set of multivariate data is described in order to generate a new set of linear
combinations of the original variables, metabolites, or spectral data, called
principal components (PCs). PCs are orthogonal to each other and form a
new coordinate system for the separation of samples. Each PC accounts for a
portion of the total variance of the data set. Often, a small set of PCs (2 or 3)
account for over 90% of the total variance of a data set, and in such
circumstances, one can resynthesize the data from those few PCs and reduce
the dimensions of the original data set. Independent component analysis
(ICA) is a method comparable to PCA, except that the PCs do not have to be
orthogonal to each other, and based on their independence, allows better
optimization (Sumner et al., 2003; Weckwerth and Morgenthal, 2005).
In Hierarchical cluster analysis (HCA), multivariate data are grouped by
pairs in a data set according to their similarity. Distance measures of the data
can include Euclidean distance, Manhattan distance, or correlation. HCA then
presents a dendogram, or tree, to visualize the distribution of the data, and
branch lengths can be made proportional to the distances between groups. As
a result, data can be represented visually to quickly convey the similarities
between samples of various data sets (Sumner et al., 2003).
Another approach is that referred to as previous methods, which are
unsupervised because they do not require previous information. In contrast,
supervised methods require creation calibration to be performed. For
example, in a protein concentration assay, standards are used to establish
protein concentration curves from which protein concentrations of unknown
samples can be calculated. The functional analyses by co-response in yeast
(FANCY) approach is another supervised method based on co-response
analysis which measures how two metabolite concentrations (or fluxes)
respond to the same perturbation. This measurement is estimated from the
ratio of change in one metabolite concentration with respect to changes in a
second metabolite concentration (Sumner et al, 2003).

Metabolomics in phytopathology
Metabolic changes can occur in genetically modified plants, during
plant/pathogen interactions, as well as in response to a plant’s environment.
Plant metabolomics not only provides information about changes in plant
184 Lucio Rodríguez-Sifuentes & Nicolás Óscar Soto Cruz

metabolites in response to environmental conditions or genetic modifications,

but also provides phenotypic characteristics of the plant.
Plant pathogens have been classified as biotrophics, necrotrophics, or
hemibiotrophs. Biotrophic pathogens need living plant tissue in order to grow
and reproduce, however, biotrophic pathogens will kill the plant tissue in the
later stages of infection. In contrast, necrotrophic pathogens kill their host
plant tissue in the early stages of infection and feed on the dead tissue.
Hemibiotrophs pathogens, on the other hand, will initiate their infection cycle
similar to biotrophic pathogens and then will convert to being necrotrophic
pathogens when the vegetal tissues decay (Berger et al., 2007; Keon et al.,
2007).
When a plant pathogen, including fungi, bacteria, or virus, invades a
plant, it feeds on the tissues of the plant. As a result, a plant’s defense
responses are activated and the metabolism of the plant changes. Mechanisms
for these processes have been described and provide valuable insight into the
ability of plants to survive in their environment, as well as their ability to
regulate their metabolism and physiology. Similarly, pathogens also undergo
changes in metabolism during the infection of plants. Therefore,
plant/pathogen interactions can trigger changes in the metabolism of both
plant and pathogen. In work by Berger et al. (2007), a model was established
to investigate the process by which a biotrophic pathogen can attack a plant.
In this model, the presence of the pathogen resulted in a decrease in
photosynthesis due to an accumulation of oxylipin 12-oxo-phytodienoic acid,
and an increase in respiration and invertase enzyme activity in the plant.
Hexose is the product released by the invertase enzyme, and in plants hexose
is both a nutrient and a signal for defense activation. However, hexose is also
a nutrient and signaling protein for pathogens. Some pathogens actually
produce invertases and other extracellular sucrolytic enzymes on their own as
well. While this model does not allow the study of all plant/biotrophic
pathogen interactions possible, it is well–suited for these studies.
Keon et al. (2007) described the combination of two “omics”
technologies, metabolomics and transcriptomics, to describe the infection of
wheat leaves by the fungal pathogen, Mycosphaerella graminicola
(M. graminicola). As a hemibiotrophic pathogen, M. graminicola causes
septoria leaf blotch disease in many grain cultures. Infection of wheat leaves
initially involves a latent period, which can extend up to several weeks,
where the pathogen obtains nutrients from the living tissues (biotrophic
mechanism) of the plant before symptoms of the disease are detected. When
chlorotic and necrotic lesions appear, M. graminicola has become a
necrotrophic pathogen and its growth rate increases. In this study, when
wheat leaves were inoculated with M. graminicola, the first visible symptoms
Metabolomics and its application in phytopathology 185

appeared on day 9, and leaf necrosis appeared on days 14 and 15. The fungus
remained apoplastic throughout the infection. Analysis of the apoplastic
fluids by 1H-NMR was performed at different disease stages in order to
determine the nutrients present in the apoplaste, particularly sugars and
amino acids. On day 6 in the absence of symptoms, no differences in sugar
and amino acid content of the wheat leaves were detected. On day 9 when
symptoms first appeared, a slight increase in the sugar and amino acid
content was detected. By day 13, a large increase in the sugar and amino acid
content was detected. PCA of the full spectra was performed and PC1 was
found to account for 99.3% of the variance in the data observed. This result
indicated that samples from days 9 and 13 were the most distinct, leading the
authors to hypothesize that the overall increase in apoplastic nutrients
detected was due to their release from wheat leaf cells. Gene microarrays
were used to correlate differences in gene expression in M. graminicola as a
consequence of changes in nutrient levels in the environment. The results
indicated that the genes that were normally repressed in high nutrient
environments were highly expressed during days 9 and 10 when the
availability of nutrients had decreased. Accordingly, genes involved in
energy production were highly expressed by day 14. These results were
consistent with previous studies that had observed obvious differences in host
physiology during the infection of M. graminicola.
In studies by Vikram et al. (2004), GC/MS was used to detect the
presence of volatile metabolites in samples of headspace gas collected from
apples (Malus domestica Borkh) incubated with four different fungi post-
harvest pathogens. These pathogens included: Botrytis cinerea pers
responsible for gray mold rot, Penicillium expansum link responsible for blue
mold rot, Mucor piriformis fischer responsible for mucor rot, and Monilinia
sp. Apples inoculated with the different pathogens presented visible
symptoms of disease and produced volatile compounds. The abundance and
frequency of occurrence of these metabolites differed depending on the
pathogens present. For example, Mucor produced 4-methyl-1-hexene,
2-methyltetrazole and butanoic acid butyl ester; Penicillium produced
fluoroethene and 3,4-dimethyl-1-hexene; Botrytis produced acetic acid
methyl ester; and Monilinia produced fluoroethane. None of these volatile
compounds were identified in the absence of pathogens (controls). Based on
these results, the authors propose that GC/MC could be used to identify
pathogens present in storage rooms and possibly even detect apple diseases in
their early stages.
When Bednarek and co-workers (2005) studied changes in the levels of
indolic and phenylpropanoid secondary metabolites in Arabidopsis
(Arabidopsis thaliana) root in response to infection by the root-pathogenic
186 Lucio Rodríguez-Sifuentes & Nicolás Óscar Soto Cruz

oomycete, Pythium sylvaticum, MS/NMR analysis identified 16 indolic, one

heterocyclic, and three phenylpropanoid compounds in both infected and
uninfected roots. An analysis of root extracts by HPLC showed that a
decrease in the three phenylpropanoids and an increase in most of the indolic
compounds was the result of infection by P. sylvaticum. When secondary
metabolites secreted by infected and uninfected roots were also analyzed,
similar metabolite profiles were found. Consistent with these data,
transcriptomic analysis showed that most of the mRNAs induced encoded
enzymes for indolic and phenylpropanoid biosynthetic pathway components.
Therefore, plant and pathogen metabolites, target or whole metabolites, must
be differentiated in order to identify the pathogen response of the plant versus
the infection mechanisms of the pathogen. In this study, production of indolic
and phenylpropanoids compounds by P. sylvaticum was rejected, and the
biological function of these compounds as secondary metabolites remains
unclear. A decrease in phenylpropanoid levels during infection would be
predicted to be associated with the rapid mobilization of precursors needed
for the synthesis of defense-related compounds, while an increase in indolic
metabolites during infection would indicate a role as signaling molecules or
antimicrobial properties. When metabolomics was applied to the Arabidopsis
mutants, an alternative pathway for the use of the indolic compound,
camalexin, was identified, which differed from the pathway reported by other
authors.
Stagonospora nodorum (S. nodorum) is a necrotrophic fungal pathogen
that causes blotches on wheat and requires asexual sporulation to maintain its
infection cycle. Lowe et al. (2009) performed metabolomic studies to
determine the role of trehalose during sporulation in S. nodorum. Polar
metabolites extracted in vitro and sporulation assays performed in planta
were analyzed by GC-MS. In both samples, the amount of trehalose increased
with the progression of sporulation. Trehalose 6-phosphate dephosphate
synthase (TPS) is an enzyme involved in the first commited step of trehalose
biosynthesis. Deletion of this gene produced a S. nodorum strain that was
unable to synthesize trehalose, which severely affected spore formation.
Correspondingly, the authors suggested that trehalose is a sporulation-
associated metabolite, and biocontrol agents that degrade trehalose could be a
strategy for the reduction of S. nodorum infection.
Metabolomic analysis was used by Jobic et al. (2007) to study
interactions between the necrotrophic fungus, Scleorotinia sclerotiorum
(S. sclerotiorum), and the sunflower, Helianthus annuus L. Perchloric acid
extracts from infected plants were analyzed using 13C- and 31P-NMR
spectroscopy and a decrease in fructose and sucrose content during infection
was detected, indicating that plant sugars were used by S. sclerotiorum.
Metabolomics and its application in phytopathology 187

Using proteomic and transcriptomic analyses, expression of invertase and

hexose tranporters by S. sclerotiorum was also confirmed. Mannitol, a
pathogen-protective polyol, was also found to increase during infection.
Given that the mannitol cycle could use the degradation of sucrose by the
plant as a substrate for the production of fructose, the increase in
glycerilphosphoryl-choline (GPC) and glycerilphosphoryl-ethanolamine
(GPE) detected in the infected plant samples could be the result of an attack
of the host membrane polar lipids by S. sclerotiorum to induce cell death. In
contrast, trehalose, a sugar found to accumulate in S. sclerotiorum,
consistently remained at a low concentration during infection. Based on these
data, the authors suggested that an increased production of protective polyols,
such as glycerol, could prevent infection by S. sclerotiorum in sunflowers.
Overall, these studies demonstrate the importance of identifying and
quantifying metabolites produced during plant/pathogen interactions,
information which can be used to establish new control strategies for
different plant pathogens.

Concluding remarks
Metabolomics has become a robust science, and therefore, many
recommendations for standardization of its associated techniques have been
published (Castle et al., 2006; Fiehn et al., 2007). Furthermore, Lindon et al.
(2005) have recommended standards for conducting and reporting
metabolomics studies, as well as proposed minimum requirements for the
recording of results, analytical techniques, and statistical methods.
In the study of plant/pathogen interactions, sample heterogeneity is a
significant problem. Since different tissues of a plant can all respond to a
pathogen stimulus, sample preparations must take into consideration the goal of
optimizing metabolite information specific to compartmentalization. In
addition, different extraction methods may be necessary for different tissues,
and sometimes more than one extraction method is necessary (Gullberg, 2004).
Analytical techniques for the determination and quantification of
metabolites are critical to the field of metabolomics. Development of these
methods is fundamental to being able to identify and quantify large quantities
of metabolites, with the interpretation of the resulting data being dependent
on statistical tools that are currently available, as well as new tools that are
being developed. All of these factors contribute to the ability of scientists to
obtain accurate and functional metabolomics data.
“Omics” technologies have greatly impacted phytopathology, and in
particular, metabolomics has been an area of growth with the development of
188 Lucio Rodríguez-Sifuentes & Nicolás Óscar Soto Cruz

new analytical techniques and data analysis methodologies. With the

available data, metabolomics has enabled scientists to better understand the
mechanisms involved in plant/pathogen interactions. Metabolomics has been
especially useful in determining which metabolites are associated with the
pathogen versus metabolites contributed by defense mechanisms of the plant.
Metabolite profiles of a biological system are based on gene expression,
the activity and interactions of enzymes, and the regulation of these sets of
molecules. Regulatory control is a very complex process in biological systems
(in this case, plants and pathogens), and begins with gene transcription (the
transcriptome) that is based on the interactions of various proteins and
metabolites with DNA. When the resulting mRNAs are translated to proteins,
generating the proteome, these proteins carry out the processes necessary for
the production of metabolites (i.e. the metabolome) (Vemuri and Aristidou,
2005). It is important to keep in perspective the hierarchy of omics technologies
and their interactions in phytopathology, or any biological system, so that a
holistic view of plant and pathogen physiology can be used to identify relevant
pathways and their components in order to select specific targets to increase
plant defense mechanisms or prevent pathogen diseases. In the context of the
“omics” technologies, it could be said that metabolomics provides the last piece
of the puzzle in establishing the connections between the “omic” data sets to
provide a complete perspective for functional genomics.

References
1. Allwood, J.W., Ellis, D.I., and Goodacre, R. (2008) Metabolomic technologies
and their application to the study of plants and plant-host interactions Physiol
Plant 132,117-135
2. Bednarek, P., Schneider, B., Svatos, A., Oldman, N.J., and Hahlbrock Klaus.
(2005). Structural complexity, differential response to infection, and tissue
specificity of indolic and phenylpropanoid secondary metabolism in arabidopsis
roots Plant Physiol 138,1058-1070.
3. Berger, S., Sinha, A.K., and Roitsch, T. (2007) Plant physiology meets
phytopathology: plant primary metabolism and plant-pathogen interactions J Exp
Bot 58,4019-4026.
4. Castle, A.L., Fiehn, O., Kaddurah-Daouk, R., and Lindon, J.C. (2006)
Metabolomics standard workshop and the development of international standards
for reporting metabolomics experimental results Brief Bioinform 7,159-165.
5. Dunn, W.B., Bailey, N.J.C., and Johnson, H.E. (2005) Measuring the
metabolome: current analytical technologies Analyst 130,606-625.
6. Duran, A.L., Yang, J., Wang, L., and Sumner, L.W. (2003) Metabolomics
spectral formatting, alignment and conversion tools (MSFACTs) Bioinformatics
19,2283-2293.
Metabolomics and its application in phytopathology 189

7. Fiehn, O. (2002) Metabolomics – the link between genotypes and phenotypes

Plant Mol Biol 48,155-171.
8. Fiehn, O., Robertson, D., Griffin, J., van der Werf, M. Nikolau, B.,
Morrison, N., Summer, L.W., Goodacre, R., Hardy, N.W., Taylor, C., Fostel,
J., Kristal, B., Kaddurah-Daouk, R., Mendes, P., van Ommen, B., Lindone,
J.C., and Sansone, S.A. (2007) The metabolomics standards initiative (MSI)
Metabolomics 3,175-178.
9. Goodacre, R., Vaidyanathan, S., Dunn, W.B., Harrigan, G.G., and Kell, D.B.
(2004) Metabolomics by numbers: acquiring and understanding global metabolite
data Trends Biotechnol 22,245-252.
10. Gullberg, J., Jonson, P., Nordström, A., Sjöström, M., and Moritz, T. (2004)
Design of experiments: an efficient strategy to identify factors influencing
extraction and derivatization of Arabidopsis thaliana samples in metabolomic
studies with gas chromatography/mass spectrometry Anal Biochem 331,283-295.
11. Jobic, C., Boisson, A.M., Gout, E., Rascle, C., Fèvre, M., Cotton, P., and
Bligny, R. (2007) Metabolic processes and carbon nutrient exchanges between
host and pathogen sustain the disease development during sunflower infection by
Sclerotinia sclerotiorum Planta 226,251-265.
12. Kant, M.R., Ament, K., Sabelis, M.W., Haring, M.A., and Schuurink, R.C.
(2004) Differential timing of spider mite-induced direct and indirect defenses in
tomato plants Plant Physiol 135,483-495.
13. Keon, J., Antoniw, J., Carzaniga, R., Deller, S., Ward, J.L., Baker, J.M.,
Beale, M.H., Hammond-Kosack, K., and Rudd, J.J. (2007) Transcriptional
adaptation of Mycosphaerella graminicola to programmed cell death (PCD) of its
susceptible wheat host MPMI 20,178-193.
14. Lindon, J.C., Nicholson, J.K., Holmes, E., Keun, H.C., Craig, A., Pearce,
J.T.M., Bruce, S.J., Hardy, N., Sansone, S., Antti, H., Jonsson, P., Daykin,
C., Navarange, M., Berger, R.D., Verheij, E.R., Amberg, A., Baunsgaard, D.,
Cantor, G.H., Lehman-McKeeman, L., Earll, M., Wold, S., Johansson, E.,
Haselden, J.N., Kramer, K., Thomas, C., Lindberg, J., Schuppe-Koistinen,
I., Wilson, I.D., Reily, M.D., Robertson, D.G., Senn, H., Krotzky, A., Kochar,
S., Powell, J., van der Ouderaa, F., Plumb, R., Schaefer, H., and Spraul, M.
(2005) Summary recommendations for standardization and reporting of
metabolic analyses Nat Biotechnol 23,833-838.
15. Lowe, R.G.T., Lord, M., Rybak, K., Trengove, R.D., Oliver, R.P., and
Solomon, P.S. (2009) Trehalose biosynthesis is involved in sporulation of
Stagonospora nodorum Fungal genetics and biology 46,381-389.
16. Oksman-Caldentey, K.M., and Inzé, D. (2004) Plant cell factories in the post-
genomics era: new ways to produce designer secondary metabolites Trends Plant
Sci 9,433-440.
17. Oliver, S.G., Winson, M.K., Kell, D.B., and Baganz, F. (1998) Systematic
functional analysis of the yeast genome Trends Biotechnol 16,373-378.
18. Rochfort, S. (2005) Metabolomics reviewed: A new “omics” platform
technology for systems biology and implications for natural products research J
Nat Prod 68,1813-1820.
 190 Lucio Rodríguez-Sifuentes & Nicolás Óscar Soto Cruz

19. Stephanopoulos, G.N., Aristidou, A.A., and Nielsen, J. (1998) Metabolic

engineering. Principles and methodologies Academic Press, San Diego.
20. Sumner, L.W., Mendes, P., and Dixon, R.A. (2003) Plant metabolomics: large-
scale photochemistry in the functional genomics era Phytochemistry 62,817-836.
21. Vemuri, G., and Aristidou, A. (2005) Metabolic engineering in the –omics era:
elucidating and modulating regulatory networks Microbiol Mol Biol Rev 69,
197-216.
22. Vikram, A., Prithiviraj, B., Hamzehzarghani, H., and Kushalappa A.C.
(2004) Volatile metabolite profiling to discriminate diseases of McIntosh apple
inoculated with fungal pathogens J Sci Food Agric 84,1333-1340.
23. Vorst, O.F., de Vos, C.H.R., Lommen, A., Staps, R.V., Visser, R.G.F., Bino,
R.J., and Hall, R.D. (2005) A non-directed approach to the differential analysis
of multiple LC/MS – derived metabolic profiles Metabolomics 1,169-180.
24. Weckwerth, W., and Morgenthal, K. (2005) Metabolomics: from pattern
recognition to biological interpretation DDT 10,1551-1558.
25. Weckwert, W., Wenzel, K., and Fiehn, O. (2004) Process for the integrated
extraction, identification and quantification of metabolites, proteins and RNA to
reveal their co-regulation in biochemical networks Proteomics 4,78-83.

View publication stats