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Engineering the plant rhizosphere
Yanxia Zhang1, Carolien Ruyter-Spira1,2 and
Harro J Bouwmeester1
Plant natural products are low molecular weight compounds
playing important roles in plant survival under biotic and abiotic
stresses. In the rhizosphere, several groups of plant natural
products function as semiochemicals that mediate the
interactions of plants with other plants, animals and
microorganisms. The knowledge on the biosynthesis and
transport of these signaling molecules is increasing fast. This
enables us to consider to optimize plant performance by
changing the production of these signaling molecules or their
exudation into the rhizosphere. Here we discuss recent
advances in the understanding and metabolic engineering of
these rhizosphere semiochemicals.
Addresses
1
Laboratory of Plant Physiology, Wageningen University, the
Netherlands
2
Plant Research International, Droevendaalsesteeg 1, 6708 PB
Wageningen, the Netherlands
Corresponding author: Bouwmeester, Harro J
(harro.bouwmeester@wur.nl)
Current Opinion in Biotechnology 2015, 32:136–142
This review comes from a themed issue on Plant biotechnology
Edited by Inge Broer and George N Skaracis
http://dx.doi.org/10.1016/j.copbio.2014.12.006
0958-1669/# 2014 Published by Elsevier Ltd.
Introduction
The rhizosphere is a complex ecosystem consisting of a
narrow zone around the plant root. Many studies have
shown that this narrow soil zone encompasses a wide
range of organisms including fungi, bacteria, oomycetes
and nematodes [1], as well as root parasitic plants [2]. All
these rhizosphere organisms may have an, positive or
negative, effect on plant fitness. Plants, in turn, produce
and secrete a variety of metabolites into the rhizosphere
to affect the rhizosphere-ecosystem [3]. This includes so-
called primary metabolites such as organic acids, carbo-
hydrates and amino acids but also a large array of second-
ary metabolites, nowadays preferably called plant natural
products.
Plant natural products are low molecular weight com-
pounds such as alkaloids, terpenoids and phenolics that
Current Opinion in Biotechnology 2015, 32:136–142
often have beneficial effects for humans, such as phar-
maceutical activity [4]. Their production in plants is
sometimes constitutive, sometimes elicited upon expo-
sure to stress conditions [5]. Some of these plant natural
products have a strong impact in the rhizosphere as they
mediate the interaction of plants with other plants, ani-
mals and micro-organisms [6].
In this review, we will discuss the use of some of the
‘omics’ technologies to improve our understanding of
what processes shape the rhizosphere as well as the use
of plant metabolic engineering to affect the rhizosphere.
The use of ‘omics’ to analyze the rhizosphere
A challenging aspect of the study of the rhizosphere is its
analysis. Recent advances in the analytical chemistry,
particularly gas chromatography-mass spectrometry
(GC–MS), liquid chromatography–mass spectrometry
(LC–MS) and capillary electrophoresis–mass spectrome-
try (CE–MS) now allow for untargeted approaches (called
metabolomics) with greatly enhanced quantitative and
qualitative analysis of the chemical composition of any
plant part including the rhizosphere [7]. Although still
with less sensitivity, nuclear magnetic resonance (NMR)
based metabolomics also is valuable as it allows not only
quantification but also structural elucidation of com-
pounds [7]. However, the rhizosphere is by definition
not sterile and that implies that the metabolites we
measure may be produced or modified by micro-organ-
isms [8]. Root exudate collection under sterile conditions
should therefore be considered as an option to study the
true plant ‘exudome’. The large scale analysis of metab-
olites in roots or the root exudate can also be combined with
transcriptomics to elucidate the genes involved in the
production of certain rhizosphere signaling molecules.
To integrate the data generated from these different
‘omics’ profiling technologies, tools such as Plant MetGen-
MAP, omeSOM and PRIme Update have been developed
to find the genes that encode metabolic pathways [9–11].
For the analysis of the rhizosphere itself, the emergence of
metagenomics — in which total DNA extracts of samples
such as the rhizosphere are used to quantitatively and
qualitatively analyze the composition of certain (sub)
groups of organisms such as fungi or bacteria — provides
a powerful tool to elucidate the community composition of
the rhizosphere [12]. Moreover, RNA-based metatran-
scriptomics as well as metaproteomics could provide
insights into the expression and translation of genes in-
duced by the interactions of plants with other organisms
[13,14]. To capture the full complexity and dynamics of
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 Engineering the plant rhizosphere Zhang, Ruyter-Spira and Bouwmeester 137
the rhizosphere, systems biology approaches will be re-
quired to establish dynamic network models [15].
Metabolic engineering of the rhizosphere
Using the above-mentioned approaches more and more
insight has been provided in the role of plant natural
products in rhizosphere signaling. In parallel there has
also been large progress in the elucidation and metabolic
engineering of the biosynthesis of plant natural products
[16]. To illustrate the exciting new possibilities of using
this knowledge to change the rhizosphere interactions
through alteration of plant metabolic fluxes, we will
discuss three representatives of such compounds, the
strigolactones, flavonoids and the terpenoid, b-caryophyl-
lene [17–19].
Strigolactones
Strigolactones (SLs) are plant hormones as well as rhizo-
sphere signaling compounds [20,21]. In the rhizosphere,
SLs play a positive as well as a negative role. On the one
hand, they are the hyphal branching factors of the arbus-
cular mycorrhizal (AM) fungi [22]. AM fungi are symbi-
otic organisms, that improve the water and nutrient
uptake of plants. On the other hand, they are germination
stimulants of root parasitic witchweeds and broomrapes
— Striga, Phelipanche and Orobanche spp. — that are
causing severe yield losses in many crops [23]. With
the exception of the latter role, the SLs generally act
as positive mediators that improve stress tolerance of
plants under unfavorable soil conditions. SLs also play
a role in harnessing plants to drought and salt stress
[24,25]. Under phosphorous (P) and nitrogen (N) limit-
ing soil conditions, the biosynthesis and exudation of SLs
is up-regulated [26,27], which is beneficial for the inter-
action of the plant with AM fungi, but also for the
symbiosis with Rhizobium spp., which induce root nodu-
lation of legumes and are responsible for atmospheric
nitrogen fixation [22,28]. Adequate P and N fertiliza-
tion, on the other hand, will limit the secretion of SLs and
hence reduces the germination rates of the root parasitic
plants [29].
An important feature of the SLs is their structural diver-
sity and the existence of different stereoisomers in nature
[30,31]. The over 20 different reported natural SLs differ
in their capacity to stimulate the germination of parasitic
plant seeds, induce hyphal branching in AM fungi and
control endogenous processes in plants such as branching
[32,33,34,35]. For instance, the SLs 5-deoxystrigol, 4-
deoxyorobanchol (also known as ent-20 -epi-5-deoxystrigol
[36]), strigol and sorgomol have a stronger (60% higher)
effect on stimulation of Striga hermonthica seed germina-
tion, while orobanchol is the most potent SL in promoting
hyphal branching in AM fungi [33]. The multitude of
functions of different SLs in different biological processes
makes the SLs an attractive target for rhizosphere engi-
neering. This is supported by the fact that conventional
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breeding in sorghum has resulted in the selection of
varieties that have strongly reduced induction of germi-
nation of the parasitic weed Striga hermonthica without any
other obvious changes in stress tolerance or plant archi-
tecture [37].
For genetic modification of the SL rhizosphere profile
knowledge of the SL biosynthetic pathway in plants is
required. In the past six years a large part of the SL
pathway was elucidated. The biosynthetic genes DWARF
27 (D27), CAROTENOID CLEAVAGE DIOXYGENASES
7 and 8 (CCD7 and CCD8) and the cytochrome P450 gene
MORE AXILLARY GROWTH 1 (MAX1) were shown to be
required to produce SLs [38,39] (Figure 1). In rice, the
enzymatic activity of MAX1 is key for the formation and
structural diversification of SLs, with two MAX1 homologs
(carlactone oxidase and 4-deoxyorobanchol-4-hydroxy-
lase) converting carlactone into 4-deoxyorobanchol and
then further into orobanchol [40] (Figure 1). In several
studies, using SL biosynthetic mutants or transgenic
knock-down lines, it was demonstrated that reduction of
the SL levels results in decreased germination of parasitic
plant seeds [20,21,41]. As a side effect of reduced SL
levels, AM fungal symbiosis was also reduced in transgenic
tomato, but the decrease in parasitic plant seed germina-
tion was larger than the decrease in AM fungal symbiosis,
offering potential for the optimization of SL levels in the
rhizosphere [41]. Natural variation in SL levels or compo-
sition between various cultivars could also be exploited in
breeding programs, and be used in association or mapping
studies to identify new genes involved in SL biosynthesis,
its regulation or SL diversification. In the model crop rice, a
successful genetic mapping approach was used to identify a
major quantitative trait locus (QTL) controlling several SL
related traits (tillering, Striga seed germination, SL pro-
duction) and led to the discovery of the two above-men-
tioned MAX1 homologs that are involved in SL production
[42]. Further biochemical characterization of these genes
was achieved by using heterologous transient expression in
Nicotinana benthamiana, a powerful tool to study plant
metabolism [43]. This showed that the genes mapped in
the QTL encode carlactone oxidase and 4-deoxyoroban-
chol-4-hydroxylase, as described above [40]. This com-
bined approach of gene discovery and subsequent
biochemical characterization provides a powerful tool to
study how various SLs are produced in plants, and will help
to identify more genes involved in SL diversification and to
design rhizosphere metabolic engineering strategies.
Finally, the secretion of SLs into the rhizosphere is
actively regulated. A petunia ABC transporter, PDR1,
was identified to be a SL cellular transporter, exporting
orobanchol from petunia roots into the rhizosphere [44].
Heterologous expression of PDR1 in Arabidopsis resulted
in improved export of exogenously supplied synthetic
strigolactone, GR24 [44]. Considering the different
structures of the SLs, it will be important to study the
Current Opinion in Biotechnology 2015, 32:136–142
138 Plant biotechnology

Figure 1

MEP pathway MVA pathway Phenylpropanoid pathway

IPP DMAPP
4-coumaroyl CoA + malony CoA
IPP DMAPP

GGPPS CHS
CHI,IFS
FPPS
Chalcones Isoflavonoids
GGPP phytoene lycopene β-carotene
CHI
D27 FPP

CCD7 Naringenin Flavones

TPS (caryophyllene synthase)
CCD8 F3H

carlactone Dihydroflavonols
sesquiterpenes
MAX1 : carlactone oxidase (caryophyllene) ANS FLS

4-deoxyorobanchol Anthocyanins Fiavonols Condensed tannins

(strigolactone)

MAX1:4-deoxyorobanchol-4-hydroxylase

orobanchol
(orobanchol-typestrigolactone)

Current Opinion in Biotechnology

The biosynthetic routes to strigolactones (SLs), flavonoids and b-caryophyllene. SLs are synthesized from carotenoids which are derived from the
plastidial methylerythritol 4-phosphate (MEP) pathway. The biosynthetic genes D27, CCD7 and CCD8 encode the production of the SL precursor
carlactone. The rice homolog of the Arabidopsis cytochrome P450 gene MAX1, carlactone oxidase, is involved in the BC-ring closure to form the
SL 4-deoxyorobanchol. Another MAX1 homolog, 4-deoxyorobanchol-4-hydroxylase catalyzes the hydroxylation of 4-deoxyorobanchol to
orobanchol. The sesquiterpene b-caryophyllene is a product of the cytosolic mevalonic acid (MVA) pathway, and its formation is catalyzed by the
terpene synthase (TPS), b-caryophyllene synthase from farnesyl diphosphate (FPP). The flavonoids are derived from the central phenylpropanoid
pathway. Abbreviations: IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; GGPP, geranylgeranyl diphosphate; GGPPS, GGPP
synthase; FPPS, FPP synthase. Solid arrows represent single enzymatic steps. Dashed arrows represent multiple enzymatic steps.

substrate specificity of the SL cellular transporter(s) in
different plant species to establish whether they can help
with or allow for modification of the SL exudation pro-
files.
Flavonoids
Flavonoids are the major class of phenylpropanoid path-
way compounds in plant root exudates. This group of
compounds commonly consists of a C6-C3-C6 (C15)
carbon skeleton (Figure 2). They function as chemical
messengers to mediate the interaction between plants
and other organisms, for example Rhizobia. Host plant
secreted flavonoids are elicitors for the expression of
nodulation (nod) genes in the rhizobial symbiont, leading
to the induction of nodulation factors (Nod factors)
[45,46], which are signaling compounds that are recog-
nized by the plant and activate the symbiosis signaling
pathway [46]. Some root exudate flavonoids have been
shown to stimulate spore germination of arbuscular my-
corrhizal fungi and to be involved in defense against root
pathogens and cyst nematodes [47]. In addition, flavo-
noids are allelochemicals in the interaction of parasitic
weeds and their hosts. The host plant Desmodium unci-
natum secretes isoflavones such as uncinanone B that
stimulates Striga germination, while uncinanone C inhi-
bits the post-germination attachment of Striga to the host
[48]. Therefore Desmodium uncinatum is utilized as inter-
crop to control Striga infection in the field [49].
The biosynthetic pathway of flavonoids has been largely
unraveled [50]. Chalcone synthase is regulating the first
committed step in the biosynthesis of flavonoids, catalyz-
ing the formation of chalcone from 4-coumaroyl-CoA and
malonyl-CoA [17]. From chalcone, six other major classes
of flavonoids are derived: flavones, flavonols, flavandiols,
anthocyanins, condensed tannins and isoflavonoids [51].
The main enzymes involved in the biosynthesis of these
flavonoids have been identified, such as chalcone isom-
erase (CHI), flavanone-3-hydroxylase (F3H), flavonol
synthase (FLS) and the anthocyanidin synthase (ANS)
[50] (Figure 1). These genes form the key for manipulat-
ing flavonoid biosynthesis which could have great impact
in the rhizosphere and hence in agriculture. For example,
in the legumes Medicago truncatula and soybean, silencing
of chalcone synthase (CHS) and isoflavone synthase (IFS)
resulted in flavonoid deficiency, which prevented the
formation of root nodules [52,53]. Similar as for SLs,
the exudation of flavonoids has also been shown to be
mediated by ABC transporters. In M. truncatula, silencing
of the ABCG transporter MtABCG10 in hairy roots de-
creased the level of several isoflavonoids in the root
exudate, which resulted in faster infection by the root
pathogen Fusarium oxysporum [54].

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 Engineering the plant rhizosphere Zhang, Ruyter-Spira and Bouwmeester 139

Figure 2

negative regulation

positive regulation

increasing

no
du
lat
ion

OH
HO O

caryophyllene
OH
strigolactones
OH O

flavonoids
)
igh

L↑
oh

i, S
nt t

–N
cie

,S
–P
uffi

L↑
i (s
+P

root insects

rhizobia bacteria
nematode
seeds of root parasitic weeds

AM Fungi
Tolerance to abiotic stress (drought, salt, etc.)
Current Opinion in Biotechnology

Examples of interactions in the rhizosphere between plants and other organisms. Plant roots release chemical signals into the rhizosphere, such
as SLs, flavonoids and b-caryophyllene to mediate the interactions with other organisms as indicated. These interactions help plants to deal with
suboptimal conditions, such as limitations in nutrients (P and N), drought and salt stress and biotic stresses such as root herbivorous insects.

(E)-b-Caryophyllene compound attracts the natural enemy of D. virgifera, an

(E)-b-Caryophyllene is a sesquiterpene, present in many
floral volatile blends and produced as defense signal
against pathogens [55]. In addition, (E)-b-caryophyllene
is exuded by maize roots upon herbivory by the larvae of
Diabrotica virgifera. Interestingly, in the rhizosphere, this
entomopathogenic nematode [56,57]. (E)-b-Caryophyl-
lene is a terpenoid and is hence produced from isopen-
tenyl diphosphate (IPP). All terpenoids, including
monoterpenes (C10), sesquiterpenes (C15) and diterpenes
(C20), are derived from either the cytosolic mevalonate

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140 Plant biotechnology
(MVA) pathway or the plastidial 2-C-methyl-D-erythritol-
4-phosphate (MEP) pathway [58] (Figure 1). Terpene
synthases are the key enzymes in terpene biosynthesis, as
they account for the formation of the terpenoid skeleton
from the isoprenoid precursors geranyl diphosphate (C10),
farnesyl diphosphate (C15) and geranylgeranyl diphos-
phate (C20) [58]. Several terpene synthases were identi-
fied as (E)-b-caryophyllene synthases, for example from
Arabidopsis, cucumber, rice and maize, which all catalyze
the formation of caryophyllene from farnesyl diphosphate
[55,57,59,60]. In American maize lines — that do not
produce (E)-b-caryophyllene and are therefore highly
sensitive to D. virgifera — overexpression of a (E)-b-
caryophyllene synthase resulted in constitutive emission
of (E)-b-caryophyllene into the rhizosphere, which en-
hanced the attracting of the entomopathogenic nematode
and decreased damage by the root herbivore [61,62].
Moreover, the negative effect of D. virgifera on maize
yield was reduced in the engineered b-caryophyllene
synthase overexpressing lines [61].
Perspectives
The important recent advances in the elucidation of plant
natural product pathways has resulted in the discovery of
many of the enzymes and corresponding genes required
for the biosynthesis and transport of a variety of rhizo-
sphere signaling molecules, enabling us to start trying to
use metabolic engineering to affect the rhizosphere. The
rapid developments in multi-gene introduction into
plants hold promise for the possibility to introduce even
multi-step pathways [63]. The concomitant advent of -
omics technologies such as metabolomics and metage-
nomics also allows us to study the consequences of these
engineering approaches in much greater detail than pos-
sible so far. Even though rhizosphere metabolic engineer-
ing is still in its infancy we assume this technology is going
to have a great impact on agriculture as we should in the
future be able to selectively stimulate or inhibit the
development of certain rhizosphere organisms and thence
the performance of our crops. Undoubtedly there are
many challenges to overcome. These include the discov-
ery of novel rhizosphere signaling compounds, which is
not trivial, as they may be present in extremely low
concentrations as was demonstrated for the strigolactones.
Another challenge is the identification of the genes in-
volved in the production of these novel signaling mole-
cules and the mechanisms involved in the understanding
(perception) of the molecular dialog between plants and
other organisms. To solve this, on the one hand, the
continuously improving sensitivity of metabolomics will
provide more information about the structures of the
rhizosphere compounds and their intermediates, while
genetic mapping and transcriptomics analysis through
RNA sequencing will offer the opportunity to discover
the corresponding genes that can then be used for rhizo-
sphere engineering. On the other hand, the recent devel-
opments in next generation sequencing technologies will
Current Opinion in Biotechnology 2015, 32:136–142
also provide us with more information about the organ-
isms that are affected by the plant exudate, and will help
us to unravel the perception and interaction mechanisms
involved [64,65]. A final challenge is that alteration of
these plant metabolic pathways through genetic modifi-
cation, may also affect other plant processes. For example,
both SLs and flavonoids also affect transport of the master
plant hormone auxin [66,67], and SL itself is a plant
hormone regulating plant architecture [23]. Changes in
the metabolism of these signaling molecules therefore
need to be carefully tuned. Nevertheless, metabolic
engineering of the rhizosphere is likely going to be an
important tool to optimize crop plant performance by
tuning the rhizosphere community composition.
Acknowledgements
We acknowledge funding by the Netherlands Organization for Scientific
Research (NWO); (VICI grant, 865.06.002 and Equipment grant,
834.08.001).
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