Studying plant–microbe interactions using stable
isotope technologies
Jim I Prosser, J Ignacio Rangel-Castro and Ken Killham
Interactions between plants and microorganisms in the
rhizosphere are complex and varied. They include the general
transfer of nutrients and specific interactions mediated by the
release of signalling molecules from plant roots. Until recently,
understanding the nature of these interactions was limited by a
reliance on traditional, cultivation-based techniques. Stable
isotope probing provides the potential for cultivationindependent characterisation of organisms actively
assimilating carbon derived from plant root exudate or added
to the soil. Current applications have focused on interactions
with relatively low-level specificity, but there is significant
potential for mechanistic studies of more specific interactions,
particularly if the sensitivity of the technique can be increased.
growth on root exudates are supplemented by more
specific interactions. Examples of specific interactions
include specific strains of nitrogen-fixing bacteria (rhizobia) that colonise particular groups of plants [5], mycorrhizal associations (between fungi and plant roots) that
improve uptake of phosphorus and trace elements [6],
and the production of antibiotics by specific rhizosphere
bacteria that inhibit growth of plant pathogens [7]. These
interactions are of critical economic importance and
are central to nutrient and pest-control management
strategies in agriculture and forestry; they are also essential for the function and maintenance of many natural
ecosystems [8].
Addresses
School of Biological Sciences, University of Aberdeen, St Machar Drive,
Aberdeen AB24 3UU, United Kingdom
Plant–microbe interactions are potentially controlled
through bulk rhizosphere carbon flow and through more
subtle, but potentially powerful, modifications generated
by signalling molecules and blockers of signals. The main
constituents of rhizosphere carbon flow, in terms of their
potential selective role, are low molecular weight carbon
compounds (dominating young rhizospheres), polymeric
and structural compounds (dominating mature rhizospheres) and signalling and signal-blocking compounds
(see [9]), such as furanones that mimic bacterial N-acyl
homoserine lactone signals and affect population-densitydependent behaviour in rhizobacteria [10]. Understanding how carbon flow constituents activate key functional
genes for interactions between plants and bacterial/fungal
plant pathogens, and their antagonists, and between
rhizobial/mycorrhizal microbial partners is critical for
sustainable management of crops. This knowledge may
ultimately provide the information that will enable plant
genetic engineering for manipulation of rhizosphere carbon flow to optimise plant and crop growth.
Corresponding author: Prosser, Jim I ([email protected])
Current Opinion in Biotechnology 2006, 17:98–102
This review comes from a themed issue on
Analytical biotechnology
Edited by Jan Roelof van der Meer and J Colin Murrell
Available online 18th January 2006
0958-1669/$ – see front matter
# 2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.copbio.2006.01.001
Introduction
The rhizosphere, the region of soil surrounding a plant
root, is the site of highest microbial biomass and activity
and the area of greatest complexity within the soil environment. It is here that interactions between plants and
microorganisms are most intense and most varied. The
plant exerts a major influence on microbial communities
through the release of a range of organic compounds, as
root exudates, and eventually through nutrients released
when the roots die and are degraded. Plants benefit from
the microbial turnover of root exudates and other soil
organic and inorganic matter, which releases nutrients
and enhances the soil structure. In some cases, correlations have been reported between particular plants, or
plant communities, and the species composition of microbial communities colonising the rhizosphere (e.g. [1,2]),
but these links are less clear in complex natural ecosystems [3,4]. Non-specific effects resulting from microbial
Current Opinion in Biotechnology 2006, 17:98–102
Plant–microorganism interactions have been studied
extensively, but major questions remain unanswered
because of a reliance on cultivation-based techniques
and an inability to determine the origin and fate of
mediating organic compounds. Stable isotope probing
(SIP) provides this facility. SIP involves the addition of
a compound (typically a carbon substrate) labelled with a
stable isotope. Cell components of organisms that assimilate this substrate will be ‘heavier’, owing to the incorporation of the stable isotope. ‘Light’ and ‘heavy’ cell
components can then be analysed separately to distinguish ‘total’ and active members of the community. The
compounds can be added directly, as 13C-labelled compounds, or assimilation of root exudates can be studied by
growing plants in the presence of 13CO2. Assimilation can
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Studying plant–microbe interactions Prosser, Rangel-Castro and Killham 99
be measured by extraction and analysis of either phospholipid fatty acids (PLFAs), DNA or RNA. Below we
describe how SIP has been used to study the influence of
plant root exudates on rhizosphere communities in grassland and rice wetlands and discuss its future potential.
The influence of plant root exudates on active
microbial community structure
Grassland soil rhizosphere communities
Pulse-labelling of photosynthesising plants with 13CO2 is
the most direct approach to determine which organisms
utilize plant root exudates [11,12]. Experimental systems employing this approach for SIP range from microcosm/pot systems [13], through monoliths [14], to field
studies [12] in which 13CO2 is supplied to closed-top
chambers [15]. Transfer of organic carbon assimilated
following CO2 fixation during photosynthesis (photoassimilate) to root exudates and microbial biomass occurs
rapidly. Ostle et al. [14] found 13C incorporation into DNA
and RNA within several hours and estimated a residence
time of 15–20 days for 13C within RNA. This demonstrates the requirement for rapid sampling after labelling,
before significant turnover of label and secondary utilization of the 13C can occur.
A major challenge in applying SIP to rhizosphere studies
is achieving sufficient sensitivity to detect very low levels
of activity, and initial studies employed the highly sensitive PLFA-SIP approach. PLFA-SIP targets cell membrane components, which constitute a relatively high
proportion of the cell biomass. Butler et al. [13] pulselabelled annual ryegrass (Lolium multiflorum) with 13CO2
in a greenhouse experiment at two stages of plant growth:
during the transition between active root growth and
rapid shoot growth, and during rapid root growth. Incorporation of labelled photoassimilate into microbial biomass was rapid and was greatest in fungal PLFAs,
whereas Gram-positive and Gram-negative bacteria
showed greater relative activity during the first and second growth stages, respectively. Treonis et al. [16] also
used PLFA-SIP to determine how active grassland rhizosphere communities were influenced by liming; lime is
added to the soil to raise pH and increase nutrient
availability. PLFAs were analysed 4 and 8 days after field
application of 13CO2 for 5 h, at ambient concentration, to
closed-top chambers placed over limed or unlimed plots.
Markers for fungi and Gram-negative bacteria showed
greatest enrichment and highest turnover. However, liming did not affect calculated turnover rates and did not
affect which organisms used recent photoassimilate.
Variation in gene sequences between different microorganisms is much greater than that between different
PLFAs, and DNA- and RNA-SIP therefore provide much
greater taxonomic resolution. RNA-SIP also provides
greater sensitivity, owing to higher target copy number.
Griffiths et al. [17] used RNA-SIP to study the influence
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of drying and wetting on active rhizosphere communities
in intact turfs, in pots, following a 4 h pulse of 13CO2.
Unexpectedly, RNA yields showed no consistent pattern
with time or wetting regime; 13C enrichment was low, and
insignificant in rewetted and dried soils. In addition, no
clear band was obtained in 13C-labelled heavy RNA
fractions, suggesting that enrichment was insufficient,
that significant turnover occurred before the first (1
day) measurement or that correlation between cellular
RNA and activity was insufficient.
RNA-SIP has been used successfully, however, to distinguish active microbial communities in limed and unlimed
soils. Rangel-Castro et al. [12] repeated the field experiment of Treonis et al. [16], pulse-labelling limed and
unlimed grassland soils with 13CO2 for 6 h. RNA-SIP of
samples taken 3 h after labelling was followed by denaturing gradient gel electrophoresis (DGGE) analysis of RT–
PCR (reverse transcriptase polymerase chain reaction)amplified bacterial, archaeal and fungal small subunit
ribosomal RNA genes. DGGE profiles from fractions along
the 12C–13C-RNA gradient from limed soils were similar,
but significant differences were observed in unlimed soils
for bacteria (Figure 1) and fungi. This indicates that most
active organisms in limed soils are active through the
utilization of root exudates, whereas in unlimed soils
(where root exudation was lower) activity results from
utilization of both root exudates and other components
of soil organic matter. Archaea were not detected in
Figure 1
Distinguishing active and inactive microbial communities in limed and
unlimed soils. Limed and unlimed grassland soil plots were supplied with
13
C-CO2 for 6 h. 13C-labelled photoassimilate was released as root
exudates and incorporated by microorganisms able to use these
compounds. Microbial communities were then characterised by
denaturing gradient gel electrophoresis (DGGE) profiles of bacterial
small subunit rRNA partial sequences of heavy (13C) fractions (4–6) and
light (12C) fractions (8–10) obtained from RNA extracted from (a) limed
and (b) unlimed grassland soils labelled in the field with 13CO2. M
represents markers. Differences in 12C and 13C profiles were evident in
unlimed samples, but not in samples from limed soil. (Figure reproduced
from [12] with permission).
Current Opinion in Biotechnology 2006, 17:98–102
100 Analytical biotechnology
unlimed soils and either did not utilize root exudates or
utilization was below the detection limit. Seeding experiments indicated that at least 106, fully labelled cells per
gram of soil are required in this system for detection by
RNA-SIP [12].
Figure 2
Rice wetland rhizosphere communities
Lu et al. [18] used PLFA-SIP to investigate seasonal
patterns in the microbial utilization of photoassimilate
following 13CO2 pulse-labelling of rice plants. The relative abundance of different PLFAs varied between, but
not within, early and late growth stages — with a switch
during greatest plant growth activity. The results indicate
that different members of the root-exudate-utilizing community were active during different stages of growth, but
analysis of PLFA restricted the identification of active
organisms.
RNA-SIP has also been used to study active methanogens
in the rice rhizosphere, where exudates can provide
carbon for methanogenesis. Lu and Conrad [19] supplied
rice plants with 49 short pulses of 13CO2 over a period of 7
days. Labelled methane was detected rapidly, demonstrating close linkage to photosynthesis. RNA-SIP was
carried out and amplified archaeal 16S rRNA genes were
analysed by terminal restriction fragment length polymorphism (T-RFLP). Profiles from control (unlabelled)
rhizospheres and from light DNA fractions from labelled
rhizospheres were similar, indicating that labelling did
not introduce bias. However, one fragment from
labelled plant microcosms increased in frequency in
high buoyant density fractions (Figure 2). Analysis of
clone libraries from light and heavy bands indicated
that this fragment was derived from the Rice Cluster
I (RC-I), which has no cultivated representative, and
that these organisms were responsible for methanogenic
activity in these soils.
Using RNA-SIP to study active methanogens in the rhizosphere. Rice
plants were grown with 13CO2 for 7 days. RNA was then extracted from
the soil and ‘heavy’ and ‘light’ RNA were fractionated by
ultracentrifugation. Microbial communities utilising labelled root
exudates were characterised by T-RFLP analysis of 16S rRNA genes
amplified from different fractions of the RNA buoyant density gradient.
Changes in the relative abundance of different terminal restriction
fragments (T-RFs) provide evidence of relative activities of different
methanogens (listed below) on root exudates. (Figure reproduced from
[18] with permission).
vation (i.e. in situ physiology) and to characterise the
complex interactions between environmental factors and
physiological diversity.
Future potential
Non-specific interactions
Lu et al. [20] extended this work to distinguish organisms producing methane through hydrogenotrophic
methanogenesis, linked to reduction of CO2, and through
acetate cleavage. Excised rice roots were incubated
under an atmosphere of 13CO2 with either H2 or N2,
and either phosphate or carbonate buffer. DNA-SIP
revealed changes in the relative abundance of the three
dominant members of the community during incubation
for 16 days and revealed differences in community
dynamics (Figure 3). Analysis of clone libraries identified
terminal restriction fragments of 91 base pairs (bp),
185 bp and 392 bp, originating from Methanobacteriaceae,
Methanosarcinaceae and RC-I, respectively. The results
indicate that RC-I is responsible for production of
methane from H2/CO2 and that Methanosarcinaceae might
have contributed to both hydrogen- and acetate-dependent methanogenesis. The study demonstrates the ability of SIP to determine the in situ function of different
physiological groups without the requirement for cultiCurrent Opinion in Biotechnology 2006, 17:98–102
SIP has been used to investigate the influence of root
exudates on microbial communities through 13CO2 pulselabelling of growing plants. An alternative approach,
which may provide greater sensitivity and control, is
the amendment of soil with 13C-labelled organic carbon
compounds typical of plant root exudates. The potential
for this approach has been demonstrated by several SIP
studies to determine the utilization of a range of organic
compounds [21,22] by soil communities. This approach
could also be used to assess functional diversity and
redundancy within soil microbial communities, with
respect to organic carbon, and utilization of decaying
organic matter.
Specific interactions
SIP has not yet been used to study interactions mediated
by signalling, but has potential if the methodology can be
developed to target functional genes: for example, those
encoding antibiotic synthesis by antagonists of soil-borne
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Studying plant–microbe interactions Prosser, Rangel-Castro and Killham 101
Figure 3
Using DNA-SIP to distinguish active methane-producing microorganisms. Different groups of methanogens produce methane using different
metabolic pathways. To determine which organisms were active under conditions favouring these different pathways, excised roots were incubated
with 13CO2 and either (a) nitrogen or (b,c) hydrogen with either (a,b) phosphate or (c) carbonate. Active methanogen communities were characterised
by T-RFLP analysis of 16S rRNA genes amplified from labelled and unlabelled DNA extracted from roots. Three groups were detected, represented by
terminal restriction fragments (T-RFs) of 392 bp, 185 bp and 91 bp, corresponding to Rice Cluster-I, the Methanosarcinaceae and
Methanobacteriaceae, respectively. Differences in their relative abundance indicates which organisms are favoured by the different incubation
conditions. (Figure reproduced from [19] with permission).
pathogens or genes involved in nitrogen fixation and
nutrient capture/transfer in rhizobial and mycorrhizal
microbial symbionts, respectively. At one level this is
possible, through DNA-SIP targeting of functional genes,
but the more exciting and important goal is analysis of
13
C-labelled mRNA for such genes. This will determine
whether specific genes are expressed, highlight factors
influencing their expression and, if sequence information
is available, identify the organisms hosting expressed
genes. This will be of greatest value if SIP can be used
to determine which carbon compounds are responsible for
gene expression, where expression may result from a
range of compounds.
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Limitations and challenges
SIP has added a new dimension to our understanding of
which microorganisms do what in the environment, and
its exploitation is at an early stage. It is currently limited
by several factors. Nucleic-acid-based SIP operates close
to detection limits, particularly in field studies. Sensitivity
is greater for RNA-SIP than DNA-SIP and can be
increased by analysis of fractions with specific primers
or probes or through the determination of 13C content in
specific RNA species using 16S rRNA gene probes and
streptavidin-coated magnetic beads [17]. Additional
rounds of centrifugation will increase separation of
labelled and unlabelled material and can help increase
Current Opinion in Biotechnology 2006, 17:98–102
102 Analytical biotechnology
sensitivity. SIP is also limited to the use of carbon stable
isotopes [23] and is of greatest value when studying
assimilation of low molecular weight, readily utilizable
root exudates. Added substrates will be turned over,
complicating analysis of primary utilizers, but time-course
studies enable tracking of carbon through different organisms/communities as degradation proceeds. It is much
more difficult, in theory, to deal with complex organic
material. This will degrade slowly, biomass (nucleic acid)
yield will be lower, and secondary utilizers will potentially utilize breakdown products rapidly. The extent of
these limitations depends, however, on the questions
being asked and the particular systems being studied.
9.
Conclusions
13. Butler JL, Williams MA, Bottomley PJ, Myrold DD: Microbial
community dynamics associated with rhizosphere carbon
flow. Appl Environ Microbiol 2003, 69:6793-6800.
PLFA-SIP analysis of microbial assimilation of root exudates in a greenhouse experiment.
This article has highlighted the power of SIP in resolving
plant–microorganism interactions in the rhizosphere.
Published studies have involved the investigation of
relatively low-specificity interactions, but there is potential for analysis of more specific interactions to increase
our understanding of the role of root/rhizosphere carbon
flow. Such knowledge will increase our potential to
manipulate plants to orchestrate their interactions with
microorganisms and to exploit microbial processes, ranging from nutrient supply to the suppression of soil-borne
pathogens.
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