Metabolomics in Plant-Microbe Interactions in The Root

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Metabolomics in plant-microbe
interactions in the roots
Li Chen, Melina Schwier, Jenna Krumbach, Stanislav Kopriva∗, and
Richard P. Jacoby
Institute for Plant Sciences, Cluster of Excellence on Plant Sciences (CEPLAS), University of Cologne,
Cologne, Germany
∗
Corresponding author: e-mail address: skopriva@uni-koeln.de
Contents
1. Introduction 2
2. Metabolites as signals in plant symbiosis 4
3. Metabolites involved in plant pathogen interaction 6
4. Metabolomics for identification of plant metabolites shaping root microbiome 9
5. Exometabolomics 13
6. Challenges of metabolomics of plant-microbe systems 18
6.1 Collection of root exudates 18
6.2 Determining the origin of metabolites in plant-microbe interactions 19
7. Concluding remarks 21
References 22
Abstract
In the last decade the importance of microbiota for plant performance has been over-
whelmingly demonstrated. Plant microbiomes, both in roots and shoots, fulfill a pleth-
ora of functions that can strongly influence various plant traits. Metabolites are the main
tools that plants use to actively shape their microbiome. Mechanistically, plants exude a
complex mix of primary and secondary metabolites from roots, which serve as growth
substrates for certain microbial strains, exert toxic effects on others, or act as signals in
mediating the plant microbe interactions. Flavonoids and strigolactones have long
been known to play important roles in enabling microbial symbiosis, even though their
full complexity may not yet be fully elucidated. Recently, several other plant metabolites
have also been implicated as important players mediating the interaction with the root
microbiome, including molecules such as coumarins, camalexin and triterpenes.
Because plants use such a wide range of chemical classes to shape their microbiome,
mechanistic studies of plant-microbe interactions are increasingly applying high-
throughput metabolomics techniques. In this review, we describe how metabolomics
approaches have profiled the chemical complexity of plant rhizodeposits, and discuss
how metabolomics can be utilized to functionally characterize specific metabolites that
influence plant-microbe interactions. We will also discuss the advanced metabolite
Advances in Botanical Research # 2020 Elsevier Ltd 1

ISSN 0065-2296 All rights reserved.
https://doi.org/10.1016/bs.abr.2020.09.018
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2 Li Chen et al.
analyses that are needed to disentangle the complex metabolic interactions between
plant hosts and their associated microbial communities.
1. Introduction
Plants in their natural environments live surrounded by a vast number
and variety of other organisms. Some of them use the plants as their source of
food, some compete with plants for resources, and some help plants to
acquire nutrients or to overcome the competition. Microorganisms, i.e.,
bacteria, fungi, and protists form the largest and most diverse group of plant
neighbors. Often these microorganisms and their roles in the plant ecosys-
tems are characterized as “the good, the bad, and the ugly” to represent ben-
eficial microbiota, plant pathogens, and the soil-borne human pathogens
(Mendes, Garbeva, & Raaijmakers, 2013). Indeed, some microbial species
in rhizosphere soil are beneficial to plants, functioning as plant growth pro-
moting rhizobacteria (PGPR) or symbionts to improve plant health and
nutrition. Other microbes display either commensalism, by maintaining a
neutral relationship with host plants or pathogenicity, resulting in infection
symptoms, such as plant tissue degradation, rusts, or powdery mildews
(Bulgarelli, Schlaeppi, Spaepen, Ver Loren van Themaat, & Schulze-
Lefert, 2013; van der Heijden, de Bruin, Luckerhoff, van Logtestijn, &
Schlaeppi, 2016). It is thus of great importance to uncover mechanisms
behind these complex interactions and as they might underpin agricultural
approaches to improving food production and sustainability.
As plants are sessile, they have developed a number of mechanisms to
interact with surrounding microorganisms. These include defense mecha-
nisms against pathogens and synthesis of attractants for symbiotic organisms.
Although genetically encoded, the plant-microbe communications rely on
metabolites: their synthesis, transport, and metabolism. For successful inter-
action with soil microorganisms, plants excrete metabolites into the soil in
the form of root exudates (Sasse, Martinoia, & Northen, 2018). Metabolite
analysis in the multi-organism systems, however, is far from trivial and there-
fore the knowledge on metabolic interactions lags behind the understanding
of the genetics of the interactions, particularly with respect to plant immu-
nity. Nevertheless, the last decade brought a significant progress in awareness
of the role metabolites play in plant microbe interactions and in adapting
metabolomics approaches to further dissect these interactions (Fig. 1).
Here we describe some of the challenges of studying metabolic interactions
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Metabolomics in plant-microbe interactions in the roots 3
Fig. 1 Metabolic dialogues between plants and microbes. Plant-microbe interactions

range from pathogenic to mutualistic, but a common theme is the important role
played by metabolites. This figure highlights some of the key metabolic exchanges
known to underpin plant-microbe interactions in the roots. In symbiotic interactions,
plants exude a set of signaling molecules that recruit AMF and BNF symbionts. These
microbes boost plant nutrition by mobilizing N and P. Pathogenic interactions between
plants and microbes are characterized by a chemical warfare, whereby both parties
either attack one another, manipulate or defend by releasing a set of bioactive second-
ary metabolites. In the commensal interactions, root exudation provides carbon sub-
strates to fuel the proliferation of a diverse microbial community. Metabolomics
provides a high-throughput platform to characterize these metabolic exchanges in
greater depth. The figure was created with BioRender.com.
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4 Li Chen et al.
between plants and microbes and provide examples of successful identifica-

tion of the role of specific metabolites in these interactions. We will also dis-
cuss the ways metabolomics approaches may help to disentangle the
complex metabolic interactions between plant hosts and their associated
microbial communities. We will focus this discussion to the interactions
of the roots, as the area of the most significant increase in knowledge.
2. Metabolites as signals in plant symbiosis

Plant roots do not exist alone underground; their rhizosphere is occu-
pied by a vast array of microorganisms. The rhizosphere is thus a hot spot of
plant-microbe interactions as root exudates are the main source of carbon to
fuel microorganisms’ growth and to shape microbial community ( Jacoby,
Peukert, Succurro, Koprivova, & Kopriva, 2017; Mendes et al., 2013;
Sasse et al., 2018). Two well characterized groups of symbiont can be found
among the rhizosphere microbes, Rhizobia, that form nitrogen-fixing
symbiosis in legumes, and mycorrhizal fungi, which enter a symbiotic asso-
ciation with plant roots (Smith & Smith, 2012; Udvardi & Poole, 2013).
Biological nitrogen fixation is an important source of nitrogen to plants with
great impact on agricultural practice, since this process potentially provides
nitrogen fertilizer without depleting the finite fossil fuel resources nor
producing greenhouse gases. This functional symbiosis is mediated by flavo-
noids exuded from plant roots and nod factors released by the Rhizobia.
Flavonoids, secondary metabolites released by plant roots and seed
coats, act as primary signals to rhizobia (Bladergroen & Spaink, 1998).
They are inducers of the rhizobacterial nod genes through binding to mem-
brane associated nodD protein, which forms complex with the flavonoids
and act as a transcriptional regulator of other nodulation genes, which are
responsible for rhizobia synthesis of nod factors (Schlaman, Spaink,
Okker, & Lugtenberg, 1989). Nod factors are lipochitooligosaccharides,
often sulfated, which act as signals that can be detected by plant roots
(Long, 1996). They induce a signaling cascade that triggers changes in root
hair structure and cell wall division. This aids to trap rhizobia and guide them
inside the roots, resulting in formation of nodules, in which symbiosis and
nitrogen fixation take place (Cullimore, Ranjeva, & Bono, 2001; Long,
1996; Oldroyd, Murray, Poole, & Downie, 2011). Nod factors are highly
specific, ensuring a strict specificity in nodulation between a particular plant
species and rhizobial strain (Oldroyd et al., 2011). However, flavonoids play
a broader role in plant symbiosis with Rhizobia. They are essential for
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nodule initiation due to regulation of auxin transport (Wasson, Pellerone, &

Mathesius, 2006). In addition, flavonoids and isoflavonoids are also impor-
tant for the other type of nodulation, in actinorhizal symbiosis with Frankia
(Abdel-Lateif et al., 2013; Auguy et al., 2011).
The main type of mycorrhiza of herbaceous plants is formed by
arbuscular mycorrhizal fungi (AMF) of the phylum Glomeromycota, which
colonize many economically important crops, including cereals, vegetables,
and horticultural plants (Smith & Smith, 2012). Mycorrhiza improve crops
yield, supplying nutrition and alleviating abiotic stress. Mycelium of AMF
scavenges effectively for phosphorus (P), which increases P uptake of plants
and then leads to positive host plant growth. AMF could even improve plant
growth under P deficiency (Harrison, Dewbre, & Liu, 2002; Smith,
Smith, & Jakobsen, 2003). AMF can also significantly improve host plant
resistance to several abiotic stresses like salinity, drought, cold/heat, nutrient
deficiency and metal toxic stress ( Jung, Martinez-Medina, Lopez-Raez, &
Pozo, 2012). The signaling leading to AMF symbiosis is similar to signaling
in nodulation, a plant signal triggers fungal hyphae differentiation and syn-
thesis of a “myc” factor that together with other signals initiates a symbiotic
signaling cascade, that has many common elements with the signaling in
nodulation (Schmitz & Harrison, 2014). The primary plant signals are the
sesquiterpenes named strigolactones, phytohormones with function in
control of shoot and root branching, inducers of germination of parasitic
plants, such as Striga (Akiyama, Matsuzaki, & Hayashi, 2005; Waters,
Gutjahr, Bennett, & Nelson, 2017). Strigolactones are exuded from plant
roots particularly during phosphate limitation and are triggering extensive
branching in the AMF (Schmitz & Harrison, 2014). Strigolactones are, how-
ever, not the only plant metabolic signals to enable the symbiosis, addition-
ally, C16 aliphatic fatty acids associated with cutin are necessary for forming
appressoria and penetration of plant cells (Wang et al., 2012) and flavonoids
also promote spore germination (Tsai & Phillips, 1991). Also for the long
elusive myc signal, two groups of metabolites have been found that exert
the same effect on plant roots as mycorrhiza fungi, lipochitooligosaccharides
and chitin oligomers (Genre et al., 2013; Maillet et al., 2011). While the
myc-lipochitooligosaccharides have a similar structure as nod factors and
are also often sulfated, the chitin oligomers are common with various fungal
pathogens (Schmitz & Harrison, 2014). How these signals are distinguished
is still an open question, as is the number and specificity of the various mol-
ecules in fungal exudates, due to a very low abundance. An intriguing recent
development is the identification of hydroxy- and carboxyblumenol
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6 Li Chen et al.
glucosides in leaves of mycorrhized plants, accumulation of which responds

quantitatively to the level of colonization (Wang et al., 2018). The blumenol
derivatives have been detected in number of plants species and were induced
by diverse mycorrhiza fungi. They might prove to be a valuable tool to esti-
mate rapidly and in high throughput the mycorrhiza colonization levels, e.g.,
in genetic screens (Wang et al., 2018).
3. Metabolites involved in plant pathogen interaction

A number of nematodes, fungi, oomycetes and bacteria are notorious
soil-borne plant pathogens (Back, Haydock, & Jenkinson, 2002; Kamoun
et al., 2015; Mansfield et al., 2012). Plants infected by soil-borne pathogens
suffer from root rot, root blackening, wilt, stunting or seedling damping-off.
For example, Phellinus weirii, a widespread indigenous fungal pathogen in the
coniferous forests of western North America, can cause laminated root rot in
several conifer species and even kill some vigorous conifer trees (Hansen &
Goheen, 2000). P. weirii penetrates roots and utilizes nutrients in the phloem
and cambium. So even though the canopy of the tree looks green, the roots
are irreparably damaged. This fungus is considered as the most economically
damaging pathogen in the conifer forest in the west of the Cascade
Mountains (Hansen & Goheen, 2000). Another typical pathogen is the fun-
gal pathogen of soybeans, cereals, tomatoes, and other crops, Fusarium spp.,
which colonizes roots and causes root rot symptoms and wilting of the plants
(Arias, Leandro, & Munkvold, 2013; Gordon, 2017).
Chemical cues in the rhizosphere are important also for root pathogens
to recognize and infect root tissues. For example, zoospores and hyphae of
Phytophthora sojae, an oomycete pathogen infecting soybean root, recognize
trace amount of isoflavones daidzein and genistein, chemicals secreted by
soybean, and use them as chemotrophic signals to direct their growth
(Morris, Bone, & Tyler, 1998). Similarly, Aphanomyces cochlioides causing
root rot disease in spinach is attracted by several flavonoids from spinach root
exudates, such as Cochliophilin A (Tahara, Ohkawa, Takayama, & Ogawa,
2001). Root exudates generally play an important role in attracting patho-
gens to the roots as well as in their deterring or in triggering microbiome
functions that help plants to defend against the pathogens (Haney,
Samuel, Bush, & Ausubel, 2015; Jacoby, Chen, Schwier, Koprivova, &
Kopriva, 2020; Sasse et al., 2018) and see below).
Multiple metabolites are part of the plant immune response to protect
plants against a variety of pests. Most of our knowledge on these metabolites
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comes from experiments with leaf pathogens. One of the best examples are
the tryptophan derived defense compounds in Arabidopsis, indolic
glucosinolates and camalexin. Glucosinolates are sulfur rich secondary com-
pounds of the Caparales that form the basis of the “mustard oil bomb”
(Bones & Rossiter, 1996). Upon tissue damage, glucosinolates stored in
the vacuoles come into contact with the enzyme myrosinase which cleaves
a glucose residue and the remaining instable intermediate rearranges into
organic isothiocyanates, nitriles, or thiocyanates (Halkier & Gershenzon,
2006). These pungent volatiles are a deterrent to herbivores but act also
in immunity against fungal and bacterial pathogens (Bednarek et al.,
2009; Clay, Adio, Denoux, Jander, & Ausubel, 2009). Glucosinolates are
synthesized from amino acids, the main contributors being methionine
for aliphatic glucosinolates, phenylalanine for aromatic, and tryptophan
for indolic ones (Halkier & Gershenzon, 2006). Tryptophan is a precursor
for another defense compounds of the Brassicaceae, the phytoalexin
camalexin. Camalexin was first isolated from a plant of the Brassicaceae fam-
ily, camelina (Camelina sativa), but detected and studied primarily in
Arabidopsis, where it is formed upon infection with fungal pathogens, some
bacterial strains, and also abiotic factors (Glawischnig, 2007). Variation in
induction of camalexin synthesis has been shown to largely control the resis-
tance with necrotrophic pathogen Botrytis cinerea in leaves of Arabidopsis
accessions (Rowe & Kliebenstein, 2008). Both compounds have only
recently been found in infected roots and in root exudates with important
roles in interaction with plant growth promoting microorganisms (Iven
et al., 2012; Koprivova et al., 2019; Lahrmann et al., 2015). However, apart
from a clear inhibition of the growth of root fungal pathogen Verticillium lon-
gisporum by camalexin, little is known about the extent of the importance of
indolic phytoalexins in root pathogen defense. One challenge in under-
standing of the mechanisms of action of plant derived indolic compounds
is the variety of secondary modifications these compounds undergo.
A number of hydroxylation, methylation, and glycosylation reactions con-
tribute to the large structural diversity of indolic glucosinolates and other
indole compounds in plant roots (Fahey, Zalcmann, & Talalay, 2001;
Stahl et al., 2016). Thus, one opportunity for high-throughput plant met-
abolomics involves the development of new analysis pipelines to quantify
and exactly determine the structures of such compounds. Phytoalexins,
however, are not limited to glucosinolates and indoles. For example, flavo-
noids were shown to play an important role in protection of number of plant
species against Fusarium and other fungal pathogens (reviewed in Mierziak,
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8 Li Chen et al.
Kostyn, & Kulma, 2014). Accumulation of phenolic compounds was the

major difference between hops cultivars resistant or susceptible to soilborne
pathogen Verticillium nonalfalfae (Kunej, Mikulic-Petkovsek, Radisek, &
Stajner, 2020). For more details on how metabolomics helps to dissect
the metabolic networks in plant defense see Chapter “Untangling plant
immune responses through metabolomics” by Petriacq.
An interesting aspect of plant immunity is priming, i.e., intensifying
immune response by previous triggers. For example, β-aminobutyric acid
(BABA), a priming agent in Arabidopsis, increases plants resistance to nec-
rotrophic fungus Plectosphaerella cucumerina by stimulating callose-rich cell
wall depositions (Ton & Mauch-Mani, 2004). BABA-induced resistance
in Arabidopsis might be based on formation of stable hormone conjugates,
which can be rapidly hydrolyzed into active signals to synthesize defensive
compounds upon attack (Dietz, Sauter, Wichert, Messdaghi, & Hartung,
2000). Therefore, it is essential to identify such hormone conjugates, which
function in plant immunity system. Salicylic acid, jasmonic acid, abscisic acid
(ABA), and indole derivatives are good candidates for studying plant immu-
nity and a targeted metabolomics workflow can help to identify which are
responsible for mediating the priming response. Revealing accumulation of
indole-3-carboxylic acid in BABA-primed Arabidopsis plants and its func-
tion in defense against P. cucumerina is a good example for further studies
(Gamir, Pastor, Cerezo, & Flors, 2012).
An additional important task for metabolomic analysis in plant microbe
interactions involves characterizing the full repertoire of bioactive com-
pounds released by plant pathogens. These molecules can be non-protein
effectors used to attack the plant (Pusztahelyi, Holb, & Pocsi, 2015), and fur-
thermore some of these metabolites also exert toxicity upon humans
(Adeniji, Babalola, & Loots, 2020). A chemically diverse variety of toxins
are produced by pathogens, including aflatoxin, alternariol, toxoflavin,
fumonisin, or coronatine, synthesized and released by pathogenic species
of Aspergillus, Alternaria, Burkholderia, Fusarium, and Pseudomonas, respec-
tively (Adeniji et al., 2020; Lee & Ryu, 2017). It is estimated that globally
25% of crops are contaminated with mycotoxins, with negative conse-
quences for human health. Metabolomics, particularly LC-MS technologies
are available to detect mycotoxins in plant material and food with a sensi-
tivity of 0.03 μg kg 1 (Eshelli, Qader, Jambi, Hursthouse, & Rateb,
2018). There is a clear opportunity for metabolomics to provide new
insights into pathogen biology, but further development of the methodol-
ogy is necessary in order to minimize matrix effects and reduce interference
of other plant metabolites (Eshelli et al., 2018).
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4. Metabolomics for identification of plant metabolites

shaping root microbiome
As sessile organisms, plants have adapted to make use of their micro-
biome to support their development in terms of health and growth. The
impact of the microbial assemblages ranges from detrimental effects of soil
borne pathogens to mutualistic or beneficial interactions that ameliorate abi-
otic stress tolerance, host nutrient acquisition and resistance against pests,
pathogens and diseases (Bulgarelli et al., 2013). A shift in balance of the
microbiota can thus substantially affect these essential roles, having impact
on crop production in agricultural ecosystems. This has been demonstrated
in a recent study whereby both plant development and nitrogen fertilization
fueled changes in the structure of root-associated microbiomes of wheat
(Chen et al., 2019). Manipulating root microbiome has the potential to
address the challenges of increased demand for food due to expanding
human population, with the underlining issues of climate change, reduced
agricultural productivity and cultivable land, and assist to optimize crop
yield, agricultural sustainability and reduce losses by biotic and abiotic
stresses (Hartman et al., 2018). Therefore, the unprecedented attention to
understand the microbiome composition and dynamics to encourage
plant-beneficial relationships has become increasingly relevant and
attractive.
The literature on understanding the microbiome assembly has long been
dominated by geneticists. The emphasis has been on describing the taxo-
nomic composition of the microbiota and on the changes in this composi-
tion through different factors (Bai et al., 2015; Bergelson, Mittelstrass, &
Horton, 2019; Bulgarelli et al., 2012; Bulgarelli et al., 2013; Lundberg
et al., 2012; Muller, Vogel, Bai, & Vorholt, 2016). These studies demon-
strated clearly that while soil is the major determinant of microbiome com-
position, plants significantly shape their microbiome. For example,
Lundberg et al. (2012) showed that different Arabidopsis accessions have dis-
tinct microbiomes and that its composition also depends on the develop-
mental stage; Bai et al. (2015) found similarities between leaf and root
microbiomes; and Bergelson et al. (2019), found evidence that there is a
stronger influence of host effects on plant-fungal communities than bacterial
taxa. These findings made apparent the important roles of plant genotypes in
determining the composition and function of the microbiome ( Jacoby et al.,
2020). The primary mechanism by which plants may affect the microbiota is
through root exudation. Indeed, root exudates play an important role in
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10 Li Chen et al.
mediating plant interactions with rhizosphere symbionts and pathogens

modulating their contribution to plant performance, nutrient uptake,
defense, and response to abiotic stress. In order to take advantages of these
interactions in the field of agriculture and ecology and reduce fertilizer
inputs, it is essential to elucidate qualitative and quantitative composition
of root exudates and microorganisms’ metabolome and functions of their
components. Root exudates include a diversity of chemical compounds
which can be divided into primary and secondary metabolites, such as sugars,
amino acids, organic acids, phenolics, and phytohormones (Monchgesang
et al., 2016; Sasse et al., 2018; Zhalnina et al., 2018). To better understand
the rhizosphere chemical environment and plant microbe interactions,
underground metabolites should be able to be traced and analyzed. Due
to the huge diversity of root exudates, metabolomics must be applied to
identify and annotate the rhizosphere metabolome. Correspondingly, and
given the importance of metabolites in the communication between plants
and microbes, the role of metabolomics in their dissection is getting increas-
ing attention.
Metabolomics provides an unbiased assessment of a cell’s physiology and
metabolite distribution in response to its environment. Metabolomics
approaches have been efficacious in elucidating defense mechanisms such
as induced resistance (Schwachtje, Fischer, Erban, & Kopka, 2018), allelop-
athy (Kong et al., 2018), or nutrient deficiency (Ziegler et al., 2016).
Metabolomics studies of the rhizosphere can be challenging due to the
complexity and dynamic interactive microenvironment. The composition
of this underground ecosystem is rich in primary and secondary metabolites
as forms of chemical communication to surrounding soil organisms. The
highly diverse species- and genotype-specific metabolites, particularly sec-
ondary ones, enable the respective organism to adapt to demanding condi-
tions, by recruiting beneficial and growth promoting microorganisms or
activating defense mechanisms against pathogens ( Jacoby et al., 2020).
Thus, there is a constant shift of microbial assemblages, manipulated by root
exudates that respond to the biotic and abiotic challenges (Kong et al., 2018;
Monchgesang et al., 2016; Sasse et al., 2018). The root exudates play a cen-
tral role as a source of metabolites for complex signaling systems, meaning
that characterizing the composition and dynamics of root exudates would
enhance our understanding of the plant-microbe network that is established
for the overall fitness of the plant. Unbiased analytical metabolomics tools
such as HPLC, LC-MS and NMR spectroscopy allows researchers to iden-
tify and quantify the metabolites of root exudates in response to various
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treatments to further understand complex mechanisms of the plant and their

network with microorganisms (Monchgesang et al., 2016; Yuan et al., 2018;
Zhalnina et al., 2018; Ziegler et al., 2016).
LC-MS based metabolome approaches has been widely used for detec-
tion and characterization of biologically active chemical compounds.
Feussner and Feussner (2019) originally developed a methodology for the
rapid and sensitive quantification of the well-known plant hormone
jasmonate and its biosynthetic precursors from minimum plant material.
This method of metabolite fingerprinting however, also allowed applica-
tions on metabolites in a great variety of plant species and respective plant
organs as well as mycelia of various fungi and fluids derived from both plant
and fungi (Feussner & Feussner, 2019). The workflow allows a broad metab-
olite coverage via two-phase extraction, separate analysis of polar and
non-polar compounds by Ultra-performance liquid chromatography
(UPLC), which is then subjected to both positive and negative electrospray
ionization (ESI) time-of-flight (TOF)-MS analysis. For data analysis and
visualizations, the researchers recommend MarVis-toolbox, which also
allows for implementing custom databases. This flexible methodology grants
the ability to apply a broader database for comparison to uncover metabolites
responsible for triggering crucial signaling mechanisms. This metabolic fin-
gerprinting approach was successful, for example, in identifying the antimi-
crobial coumarin scopoletin to be dominantly produced and secreted from
Arabidopsis roots (Stringlis et al., 2018). Scopoletin is produced under iron
deficiency as its potent chelator to improve uptake in plants. This study iden-
tified a transcription factor MYB72 and a β-glucosidase BGLU42 as essential
for scopoletin exudation. In addition, the report found that scopoletin affects
the association of Arabidopsis with rhizosphere bacterium Pseudomonas
simiae WCS417, and is thus involved in shaping of plant microbiome
(Stringlis et al., 2018; Voges, Bai, Schulze-Lefert, & Sattely, 2019).
Along with coumarins, triterpenes have been recently found to be
involved in shaping of the microbiome. Triterpenes are natural plant prod-
ucts involved in antimicrobial activity and defense against various pathogens
and pests (Papadopoulou, Melton, Leggett, Daniels, & Osbourn, 1999;
Thimmappa, Geisler, Louveau, O’Maille, & Osbourn, 2014). They are syn-
thesized via the mevalonate pathway and can accumulate inside the plant
tissue as triterpene glycosides. Very recently, Huang et al. (2019), discovered
that the disruption of synthesis of three Arabidopsis triterpenes, derived from
thalianol and arabidiol gene clusters and accumulating in roots, resulted in
altered root microbiota. The results of metabolite analyses and microbiome
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12 Li Chen et al.
profiling suggest that triterpenes contribute significantly to selective modu-

lation of the microbial assemblage of Arabidopsis roots (Huang et al., 2019).
The complex pattern of triterpene in root exudates makes it difficult to iden-
tify single active metabolite, requiring combination of several methods,
including GC-MS, lC-MS, and NMR. Accordingly, quantitative
H NMR has demonstrated its value as a powerful method for analyzing
triterpene content in crude plant extracts of roots and rhizomes as well as
aerial sections of Actaea racemosa, A. podocarpa, and A. cordifolia (Imai,
Lankin, Godecke, Chen, & Pauli, 2020). This methodology does not
require reference compounds for all targets for quantitative analysis, but
the hydrogen signals allow the quantification of less common derivates of
cycloartane directly in the extracts.
Benzoxazinoids are a class of bioactive indole metabolites produced by
grasses, which have long been implicated as important for influencing plant-
microbe interactions. Recent work by Cotton et al. (2019) has applied high-
throughput metabolomics to investigate maize mutants impaired in
benzoxazinoid synthesis. Data analysis revealed that mutant plants exhibit
widespread alterations to root metabolite profiles, which could mechanisti-
cally explain why these mutants recruit an altered microbial community.
Methodologically, this study reports a promising approach to link metabolite
abundance with microbiome composition, by searching for correlative links
between metabolite peaks acquired via LC-MS versus OTUs acquired via
amplicon sequencing. This integrative approach has strong potential to
bridge the gap between biochemical analyses of plant metabolite composi-
tion and taxonomic analyses of microbial community composition.
Interestingly, metabolites have been shown to mediate also epigenetic
regulation of plant-microbe interactions (Vilchez et al., 2020). The obser-
vation that a hypermetylation Arabidopsis mutant rdd, deficient in the
DNA demethylases REPRESSOR OF SILENCING 1 (ROS1),
DEMETER-LIKE 2 (DML2), and DML3, loses the plant growth promo-
tion effect of Bacillus megaterium YC4 led to identification of myo-inositol as
the modulator of this interaction. Myo-inositol was highly depleted in the
exudates of rdd mutant compared to Col-0 and also other mutants with
lower accumulation of this metabolite showed altered interaction with
B. megaterium YC4 (Vilchez et al., 2020). Thus, metabolomics analyses
might be necessary also for dissection of genetic and epi-genetic mechanisms
controlling plant microbe interactions.
It is a high priority to identify the biochemical changes induced in non-
exposed tissues following microbial inoculation at a distal position on the
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plant. These molecules could mediate signaling processes, either to commu-

nicate immune responses within the plant, or alternatively to modulate the
surrounding microbial community by exuding substrates that nourish com-
mensals or toxins that kill pathogens. Untargeted metabolomics offers a
high-throughput platform to deepen our knowledge of microbe-induced
plant signaling processes, either by identifying novel chemical agents that
transduce these signaling events, or by characterizing the metabolic alter-
ations occurring in distal tissues. Recently, Korenblum et al. (2020) mea-
sured the exudate profiles of tomato roots that were distally positioned
from the microbial inoculation, by using a split-root growth system that
physically separated the two root sections. A major conclusion was that ace-
tylsugars and various glycosides are strongly responsive to long-distance sig-
nals imparted by microbial inoculation. These plant metabolic responses are
not uniform, because the distal patterns of root exudation observed in the
sterile compartment were significantly altered by the microbial treatment
applied to local roots. This work shows that the combination of split-root
techniques with untargeted metabolomics provides an excellent platform
to identify new aspects of plant systemic signaling.
5. Exometabolomics
A promising new application of metabolomics in the field of plant-
microbe interactions is exometabolomics, which involves measuring the
metabolic footprint that microbes imprint upon their chemical environ-
ment. This approach has strong potential to deliver new mechanistic insights
into plant microbiome research, because microbial substrate uptake is a cen-
tral mechanism shaping the assembly of the plant-associated microbial com-
munity (Sasse et al., 2018). However, there is an incomplete knowledge of
which plant metabolites are consumed by which microbial strains.
Exometabolomics has the potential to fill this knowledge gap, by characterizing
the nutritional niches occupied by individual microbes. This information about
microbial substrate preferences can then be used to mechanistically explain
microbial community assembly, mediated via processes such as niche differen-
tiation and competitive exclusion ( Jacoby & Kopriva, 2019).
Generally, exometabolomic workflows deploy high-throughput tech-
niques to measure the specific plant metabolites that are utilized by microbial
strains as growth substrates (Fig. 2). The derived datasets can deliver new
insights into the nutritional interdependence between plants and their asso-
ciated microbiome, by defining the specific plant metabolites used as a
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14 Li Chen et al.
Fig. 2 Exometabolomics applied to study plant-microbe interactions in the roots.

Exometabolomics is a methodological approach that uses high-throughput met-
abolomics techniques to measure microbial substrate preferences. In the field of
plant-microbe interactions, exometabolomics has strong potential to deliver new infor-
mation about the metabolic niches occupied by root-associated microbes. Here we pre-
sent a schematic illustration showing how exometabolomics workflows can be applied
to study plant-microbe interactions in the roots. First, root metabolites are provided as
the sole carbon source in the growth medium, using either root extracts or root exu-
dates. Next, microbes are inoculated into this medium, using either an individual strain
or a microbial community. Following microbial proliferation, mass spectrometry is used
to profile the metabolite composition of the culture supernatant. Data analysis involves
identifying metabolite peaks with differential abundance between the inoculated
medium versus the sterile medium. Here, the two blue peaks represent metabolites that
were consumed as microbial growth substrates, which give information about the met-
abolic niche that this microbe occupies in the rhizosphere. Additionally, the pink peak
represents a metabolite that was released by the microbe, which could play a role in
cross-feeding, antibiosis or signaling.
carbon source for microbial proliferation. It has long recognized that the
plant rhizosphere provides a rich habitat for microbial growth, because com-
pared to the surrounding soil, the root zone is highly enriched with carbon
substrates arising from root exudation and sloughed-off root cells ( Jones,
Nguyen, & Finlay, 2009). Biochemically, this substrate mixture is highly
complex, containing both primary and secondary plant metabolites
(Dennis, Miller, & Hirsch, 2010). Although it is widely accepted that the
microbial consumption of these root metabolites plays a significant role in
shaping the composition of the root microbiome, there is a relatively poor
knowledge about the metabolic interdependencies that have coevolved
between plants and microbes. For instance, it has been suggested that micro-
bial specialization onto distinct plant hosts is mediated by specialized cata-
bolic pathways to utilize distinct secondary metabolites only produced by
that species (Chagas, Pessotti, Caraballo-Rodriguez, & Pupo, 2018).
However, there have been relatively few studies that have characterized
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these nutrient exchanges in terms of individual metabolites. Meanwhile, it is

often asserted that plants might modulate their exudate chemistries in order to
recruit cooperative strains into their rhizosphere (Lambers, Mougel, Jaillard, &
Hinsinger, 2009), but again there is relatively little information about the spe-
cific molecules involved. Here, we argue that exometabolomics is well suited
to provide new information that will help to answer these outstanding
questions.
The datasets derived from exometabolomics workflows provide much
more detailed information about microbial substrate uptake compared to
alternative techniques, such as minimal medium growth assays or phenotype
microarrays. In the recent literature, multiple studies have deployed
exometabolomics to study plant-associated microbes. Two recent studies have
investigated the substrate uptake preferences of nitrogen-fixing symbionts
cultivated on laboratory growth media, using LC-MS (Fei, diCenzo,
Bowdish, McCarry, & Finan, 2016) and NMR (Montes-Grajales, Esturau-
Escofet, Esquivel, & Martinez-Romero, 2019). One disadvantage of using
laboratory growth media for exometabolomics studies is, however, that these
media do not fully represent the biochemical environment of the plant host,
particularly because they lack secondary metabolites specific to the target spe-
cies. Therefore, we will focus on two recent studies that have applied
exometabolomics to study the metabolic footprints of plant-associated
microbes cultivated either on medium containing root exudates (Zhalnina
et al., 2018) or root extracts ( Jacoby, Martyn, & Kopriva, 2018). Here we will
review these studies in detail, focusing on the methodological approaches and
key biological insights.
Zhalnina et al. (2018) utilized untargeted metabolomics workflow to
assess changes of root exudates during growth stages of an annual grass
(Avena barbata), coupled with exometabolomics to reveal the relationship
between root exudates and rhizosphere microbial community. This data
was generated via HILIC-LC-MS/MS, by detecting metabolites present
in root exudates at four different plant developmental stages, as well as mea-
suring whether these metabolites were utilized as growth substrates by a
panel of 16 bacterial strains when cultivated using root exudates as the sole
carbon source. This set of bacterial isolates was chosen to encompass taxa
that were either enriched or depleted in response to plant growth. Data anal-
ysis showed that bacterial strains enriched in the rhizosphere were signifi-
cantly more likely to uptake aromatic organic acids and amino acids
compared to strains that were depleted by plant growth. A broader analysis
of root exudate consumption by isolated strains showed that the uptake of
ARTICLE IN PRESS
16 Li Chen et al.
most root exudate components was generally quite similar among diverse
bacterial isolates. However, a small set of metabolites including salicylic acid
were more likely to be utilized via strains that successfully colonize the rhi-
zosphere, which positions these metabolites as key mediators of microbial
community assembly. Assessing the study, a key strength is that it combines
the temporal analysis of plant root exudation traits with exometabolomic
measurement of bacterial substrate uptake preferences. Integrating these
two datasets, it can be confidently concluded that rhizosphere microbial
assembly is shaped by root exudates, whereby younger plants recruit their
microbiome by exuding the preferred metabolic substrates of strains that
successfully colonize the rhizosphere. This work showcases the advantages
of applying exometabolomics to study microbial community assembly,
because the derived data pinpoints the adaptive metabolite exchanges that
have coevolved between plants and microbes to confer a fitness benefit
for both parties.
Jacoby et al. (2018) utilized untargeted metabolomics to investigate the
substrate preferences of bacterial strains cultivated on Arabidopsis root
extract, in order to compare the metabolic niches occupied by two bacterial
strains isolated from field-grown Arabidopsis plants versus E. coli isolated
from the human gut. Exometabolomic profiles were acquired by analyzing
culture filtrates using C18-LC-MS, following cultivation on growth
medium where Arabidopsis root extracts were provided as the sole carbon
source. Data analysis showed that all three strains took up a common set of
62 MS-features, with m/z values mainly corresponding to primary metab-
olites such as amino acids. However, the two bacterial strains isolated from
field-grown Arabidopsis roots were both capable of consuming a wide vari-
ety of MS-features matching to plant secondary metabolites such as
scopoletin and xylobiose, none of which were catabolized by E. coli. This
suggests that E. coli is poorly adapted to colonize the Arabidopsis rhizo-
sphere, because it cannot establish a differential metabolic niche.
Furthermore, there was relatively little overlap in the substrate preferences
of the two plant-associated strains, implying that these two strains avoid
direct competition by occupying complementary metabolic niches.
Additionally, the study reports that all three strains release a significant num-
ber of novel MS-features into their growth media, which could potentially
be important in rhizosphere ecology for microbe-microbe interactions such
as cross-feeding. For the wider research community, the major takeaway
from this work is the development of a relatively straightforward method-
ological approach for conducting rhizosphere exometabolomics in the
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model plant Arabidopsis. This workflow can be easily adapted to profile var-
ious Arabidopsis mutants or diverse microbial strains, with the major advan-
tage that it is relatively straightforward to generate root extracts compared to
root exudates. Potentially, this methodology will be a useful tool for
pinpointing the molecular mechanisms that underpin root colonization
and niche differentiation in the Arabidopsis microbiome.
Mass spectrometry imaging is a complementary technique to
exometabolomics, because it provides information about the spatial distribu-
tion of metabolic substrates in the environment (Silva & Northen, 2015).
Multiple recent studies have deployed either MALDI-imaging or
LAESI-MS to investigate the in situ metabolomic changes that nitrogen-
fixing bacteria induce upon legume root nodules (Gemperline,
Jayaraman, Maeda, Ane, & Li, 2015; Stopka et al., 2017; Velickovic
et al., 2018). Another study has recently applied LAESI-MS to analyze
the in situ metabolic changes in Setaria viridis roots inoculated with
growth-promoting bacteria (Agtuca et al., 2020). Compared to standard
metabolomics approaches that involve sample homogenization, a key
advantage of these spatially-resolved workflows is the ability to dissect
how metabolite composition varies according to the plant’s anatomy and
histology. For research into plant-microbe interactions, spatially-resolved
metabolomics approaches can give new insights into the metabolic alter-
ations occurring at the distinct site of microbial colonization. There is an
opportunity for future studies to couple mass spectrometry imaging with
exometabolomics, because the imaging datasets provide spatial information
about metabolite availability on the plant root, whereas the exometabolomic
information defines the substrate preferences of microbial colonists. These
two approaches can be integrated together, to give new information about
the site-specific metabolic exchanges occurring between plants and
microbes located at different anatomical regions along the root.
Assessing the literature applying exometabolomics to study plant-
microbe interactions, it is clear that the field is still in its early stages of devel-
opment, with publications only starting to emerge from 2016 onwards.
However, we anticipate that the field will grow quickly over the coming
years, because this methodological approach is the most appropriate tool
for analyzing the microbial consumption of specific root metabolites. The
derived data provide complementary information to genetic and taxonomic
studies that dominate the literature, because these techniques are poorly
suited to identifying the underlying mechanisms that shape microbial
community assembly. Methodologically, exometabolomics is incredibly
ARTICLE IN PRESS
18 Li Chen et al.
versatile, with various mass spectrometry platforms as well as NMR being

successfully applied across different microbes (Pinu & Villas-Boas, 2017).
In the context of plant root metabolites, HILIC chromatography provides
better separation of the polar primary metabolites that presumably represent
the bulk of nutrient exchange between plants and microbes (Zhalnina et al.,
2018), whereas C18 chromatography offers sharp and reproducible elution
profiles for hydrophobic secondary metabolites that could play key roles in
microbial niche differentiation ( Jacoby et al., 2018). Computationally,
exometabolomics datasets can be analyzed using online repositories for
natural product mass spectra (Wang et al., 2016) as well as microbial
cross-feeding (Kosina et al., 2018), enabling researchers to integrate their
primary data with results of pre-existing studies. In the longer term, it is
tempting to speculate that exometabolomics could assist in strategies to
rationally design the plant microbiome, by matching the substrate uptake
profile of high-performing microbial inoculants versus the root exudation
profile of new crop varieties.
6. Challenges of metabolomics of plant-microbe

systems
6.1 Collection of root exudates
Studies of metabolite abundance and function in plant-microbe interactions
pose several challenges beyond the classical use of plant metabolomics. The
first problem is already the sampling of root exudates. Given the complexity
of root architecture and soil structure, recovering root exudates from soil-
grown plants unaltered by sorption and microbial activity is very difficult
(Oburger & Jones, 2018). In addition, the exudates have to avoid contam-
ination by metabolites released from soil organic matter and from dead root
cells. Therefore, most of our knowledge on root exudates is derived from a
hydroponics approach and collection of root exudation in nutrient solution
or water (Carvalhais et al., 2013; Strehmel, Bottcher, Schmidt, & Scheel,
2014; Zhalnina et al., 2018; Ziegler et al., 2016). As this is obviously a lim-
itation, Oburger and Jones (2018) questioned how ecologically relevant
exudation results obtained under hydroponic conditions are in comparison
to the natural conditions of soil. Unfortunately, so far this is a rhetorical
question since even the best systems for exudate collection from soil grown
plants are still only partly natural, e.g., through using a membrane or mesh
separating the roots from the soil (Oburger et al., 2014) or sampling tubes
absorbing specific compound classes (Weidenhamer, Mohney, Shihada, &
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Rupasinghe, 2014). A growth system designed by Petriacq et al. (2017), in

which plants grow in tubes filled with soil perforated at the bottom and exu-
dates are extracted by a rapid flush of the tube with different concentrations
of methanol, seems to offer a good solution to describe metabolic compo-
sition of the rhizosphere. However, to distinguish between plant exudates
and soil organic compounds metabolized by the microbiota is not trivial
and even isotopic labeling of plant metabolites might not solve this limitation
due to rapid metabolic processes in the rhizosphere. Thus, unfortunately, a
perfect universal extraction protocol does not yet exist and for most research
questions the hydroponics approach with all its disadvantages still remains
the method of choice.
Some questions on root exudation, particularly its dynamics, can be
addressed by labeling techniques. For example, diverse metabolic sensors
can be introduced into the rhizosphere to quantify the exuded metabolites.
In a pioneering study Pini et al. (2017) developed biosensors based on
Rhizobium leguminosarum bv viciae expressing luciferase under control of var-
ious chemically inducible promoters. These biosensors were able to detect
amino acids, organic acids, sugars, and flavonoids and monitor the quanti-
tative and spatial exudation pattern of these metabolites (Pini et al., 2017).
Alternatively, sensors can be based on enzymes immobilized on roots
producing colored or fluorescent products as successfully tested to image
glucose (Voothuluru, Braun, & Boyer, 2018). Another possibility is to
use isotope labeling, e.g., exposure of the leaves to 13CO2 or 14CO2, to
quantify root exudation or for 14C also image the spatial pattern of the exu-
dation (Oburger & Jones, 2018). While the labeling approaches can bring
some answers, they do not allow to assess the whole diversity of metabolites
in the exudates.
6.2 Determining the origin of metabolites in plant-microbe

interactions
When a metabolite is found in rhizosphere accumulating upon interaction of
plant with specific microbes, it is often impossible to distinguish whether it
was synthesized and exuded by the plant, the microbe, or both. This is par-
ticularly problematic during analyses of tissues with high proportion of
microbes, e.g. nodules or highly arbusculated roots, or in experiments in
which plant tissues are infiltrated with large titres of microbes (Allwood,
Heald, Lloyd, Goodacre, & Mur, 2012). If the microbes are less abundant,
it is possible to test for the presence and quantity of compounds not synthe-
sized by plants and estimate the microbial contribution to the total
ARTICLE IN PRESS
20 Li Chen et al.
metabolome. One example is ergosterol found almost exclusively in fungi

and used as a marker for fungal biomass (Larsson & Saraf, 1997).
Combined with physical separation of the microbes away from plant tissues,
this would help to identify metabolites specifically enriched in one of the
fractions, as such compounds would allow to estimate the purity of the frac-
tion. The physical separation is similar to the pipeline allowing determina-
tion of bacterial transcriptome changes during plant immune response
(Nobori et al., 2018). While possibly applicable for metabolomics studies
of infiltrated bacteria in plant leaves, this approach has not been tested for
the roots and is not suitable for metabolites that are secreted and do not accu-
mulate inside the cells. The separation can be simplified by using plant cell
cultures instead of whole tissues (Allwood et al., 2012), but with the same
limitations. Nevertheless, for determination of early metabolic changes in
plant cells elicited by microbes this approach can be useful.
A much more promising approach to distinguish origin of specific metab-
olites is isotope labeling. Stable isotope probing (SIP) approaches have been
used to determine microbial activity in soil or water by using labeled substrates
and measuring isotopic enrichment of microbial cells, products or biomarkers
(Pett-Ridge & Firestone, 2017). SIP has been used to identify microorganisms
in a complex community capable of metabolizing methane, after feeding
13
CH4, isolation of 13C-enriched RNA, and metatranscriptomics (Dumont,
Pommerenke, & Casper, 2013). Similarly, biotraps loaded with 13C-labeled
naphthalene or fluorene were used to collect bacterial biofilms in contaminated
groundwater wells and 13C-enriched proteins were identified by comparison
with similar biotraps loaded with non-labeled hydrocarbons (Herbst et al.,
2013). The analysis detected Burkholderiales, Actinomycetales, and
Rhizobiales as the most active microorganisms for degradation of polycyclic
aromatic hydrocarbon contaminants in the groundwater communities. In ani-
mal gut microbiome research SIP was used, e.g., to identify Ruminococcus bromii
as the primary starch degrading bacterial strain (Kovatcheva-Datchary et al.,
2009) or Allobaculum spp. as particularly active glucose utilizer (Herrmann
et al., 2017). Similar approach can be used to study plant microbe interactions,
where the source of labeled metabolites is the plant, through growing in 13CO2
enriched atmosphere (Pett-Ridge & Firestone, 2017). Using this approach,
several bacterial strains, mainly of the order Burkholderiales, have been found
through strong 13C labeling of their RNA, indicating rapid uptake of root exu-
dates (Vandenkoornhuyse et al., 2007). Conceptually, heavy isotope labeling of
one of the interacting partners should enable identification of origins of specific
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metabolites at the site of interaction, however, this has been only very rarely
used in practice. In a pioneering study, Pang et al. (2018) used 13C and 15N
labeled Pseudomonas cultures to study early metabolic events during plant
immune response. Due to the isotope labeling the bacterial metabolites show
a mass shift at the same retention times and can be distinguished from the plant
derived counterparts. As a proof of principle, the phytotoxin coronatine that is
exported from the bacteria, was measured and confirmed to be composed of
the heavy isotopes, i.e., of bacterial origin (Pang et al., 2018). The analysis
allowed to determine plant metabolites affected by interaction with the bacteria
and showed their particular effect on amino acid synthesis. The promising
technology thus awaits further testing, particularly in the rhizosphere
environment.
7. Concluding remarks
In the last decade, it has become increasingly recognized that root-
dwelling microorganisms play a major role in shaping plant performance.
Metabolites are the primary means of communication between plants and
their microbiota, but there is an incomplete mechanistic knowledge about
how root exudates shape the microbiome. In our opinion, the next step
change in understanding plant-microbe interactions will be dependent on
applying metabolomics techniques to identify which specific rhizosphere
metabolites are involved in key biological processes such as mutualist
recruitment, pathogen suppression, and immune signaling. Several such sig-
nals have already been identified using metabolomics techniques, and the
corresponding analytic pipelines are becoming more widespread and easy
to use. Although there are inherent technical challenges to studying
plant-microbe interactions using metabolomics, new approaches such as
exometabolomics and stable isotope probing seem well-suited to deliver
mechanistic insights. Assessing the current state of the field, there seems
to be an opportune alignment of research questions with methodological
tools, because many important processes in plant-microbe metabolic inter-
actions are underpinned by poorly characterized metabolic exchanges, and
newly emerging metabolomic approaches provide a high-throughput plat-
form to identify the relevant molecules. Therefore, we are confident that the
next decade will witness a dramatic surge of new discoveries found using
metabolomics in plant-microbe interactions.
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22 Li Chen et al.
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Advances in Botanical Research # 2020 Elsevier Ltd 1

Metabolomics in plant-microbe interactions in the roots 3

Fig. 1 Metabolic dialogues between plants and microbes. Plant-microbe interactions

between plants and microbes and provide examples of successful identifica-

2. Metabolites as signals in plant symbiosis

Metabolomics in plant-microbe interactions in the roots 5

nodule initiation due to regulation of auxin transport (Wasson, Pellerone, &

glucosides in leaves of mycorrhized plants, accumulation of which responds

3. Metabolites involved in plant pathogen interaction

Metabolomics in plant-microbe interactions in the roots 7

Kostyn, & Kulma, 2014). Accumulation of phenolic compounds was the

Metabolomics in plant-microbe interactions in the roots 9

4. Metabolomics for identification of plant metabolites

mediating plant interactions with rhizosphere symbionts and pathogens

Metabolomics in plant-microbe interactions in the roots 11

treatments to further understand complex mechanisms of the plant and their

profiling suggest that triterpenes contribute significantly to selective modu-

Metabolomics in plant-microbe interactions in the roots 13

plant. These molecules could mediate signaling processes, either to commu-

Fig. 2 Exometabolomics applied to study plant-microbe interactions in the roots.

Metabolomics in plant-microbe interactions in the roots 15

these nutrient exchanges in terms of individual metabolites. Meanwhile, it is

Metabolomics in plant-microbe interactions in the roots 17

versatile, with various mass spectrometry platforms as well as NMR being

6. Challenges of metabolomics of plant-microbe

Metabolomics in plant-microbe interactions in the roots 19

Rupasinghe, 2014). A growth system designed by Petriacq et al. (2017), in

6.2 Determining the origin of metabolites in plant-microbe

metabolome. One example is ergosterol found almost exclusively in fungi

Metabolomics in plant-microbe interactions in the roots 21

Metabolomics in plant-microbe interactions in the roots 23

Ca2+ spiking in Medicago truncatula roots and their production is enhanced by

Metabolomics in plant-microbe interactions in the roots 25

Metabolomics in plant-microbe interactions in the roots 27

Metabolomics in plant-microbe interactions in the roots 29

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