Ismej 2013196 A
Ismej 2013196 A
Ismej 2013196 A
& 2014 International Society for Microbial Ecology All rights reserved 1751-7362/14
www.nature.com/ismej
ORIGINAL ARTICLE
Rhizosphere microbiome assemblage is affected by
plant development
Jacqueline M Chaparro, Dayakar V Badri and Jorge M Vivanco
Center for Rhizosphere Biology, Department of Horticulture and Landscape Architecture, Colorado State
University, Fort Collins, CO, USA
There is a concerted understanding of the ability of root exudates to influence the structure of
rhizosphere microbial communities. However, our knowledge of the connection between plant
development, root exudation and microbiome assemblage is limited. Here, we analyzed the structure
of the rhizospheric bacterial community associated with Arabidopsis at four time points
corresponding to distinct stages of plant development: seedling, vegetative, bolting and flowering.
Overall, there were no significant differences in bacterial community structure, but we observed that
the microbial community at the seedling stage was distinct from the other developmental time
points. At a closer level, phylum such as Acidobacteria, Actinobacteria, Bacteroidetes, Cyanobac-
teria and specific genera within those phyla followed distinct patterns associated with plant
development and root exudation. These results suggested that the plant can select a subset of
microbes at different stages of development, presumably for specific functions. Accordingly,
metatranscriptomics analysis of the rhizosphere microbiome revealed that 81 unique transcripts
were significantly (Po0.05) expressed at different stages of plant development. For instance, genes
involved in streptomycin synthesis were significantly induced at bolting and flowering stages,
presumably for disease suppression. We surmise that plants secrete blends of compounds and
specific phytochemicals in the root exudates that are differentially produced at distinct stages of
development to help orchestrate rhizosphere microbiome assemblage.
The ISME Journal (2014) 8, 790–803; doi:10.1038/ismej.2013.196; published online 7 November 2013
Subject Category: Microbial population and community ecology
Keywords: Arabidopsis thaliana; development; microbiome; rhizosphere; root exudation
Root exudation
Results
Root exudate data was obtained from Chaparro et al. Plant development influences the soil microbial
(2013) from in vitro grown Arabidopsis plants that community
were grown to the designated plant developmental We analyzed the influence of plant development
time points (seedling, vegetative, bolting and on the rhizosphere microbial community by 454
flowering). Briefly, 7-day old seedlings (see above pyrosequencing and obtained 55 921 high-quality
for seedling growth) were transferred to Magenta 16S rRNA sequence reads. After equalizing sampling
boxes containing 10 ml of MS media supplemented effort, 33 228 reads were retained for analysis. These
with 1% sucrose. Plants were grown until they were 7, reads clustered into 7452 OTUs at 3% distance
14, 21 or 28 days and were subsequently transferred sequence dissimilarity (Supplementary Table S1).
to new Magenta boxes containing 10 ml of sterilized We visualized the Bray–Curtis distances between
Millipore water. Root exudation was collected after samples using PCoA to determine how dissimilar
3 days of constant secretion (10, 17, 24 or 31 days) the soil microbial communities were at each plant
where solutions were filtered using nylon filters developmental time point. The rhizospheric micro-
(0.45 mm; Millipore, MA, USA). Root exudates were bial community of seedling was statistically
freeze-dried, dissolved in 5 ml of 80% methanol, (Po0.05) distinct from that of all other time points
dried under N gas and sent to the Genome Center (vegetative, bolting and flowering; Figure 1a). On the
Core Services at the University of California, Davis other hand, the rhizosphere microbial community
for GC–MS analysis on an Agilent 6890 gas established at vegetative, bolting and flowering were
chromatograph (see Chaparro et al., 2013). similar to each other (Figure 1a). We used PCA to
identify the factors that influence the soil microbial
community at each time point (Figure 1b). The first
Statistical analyses two principle components explain 90.6% of the
All statistical analyses were done using SAS (ver. variability in the data. Principle component 1
9.3; SAS Institute). The PROC MIXED function was explains 86.4% of the data while principle compo-
used to implement a two-way ANOVA analysis with nent 2 explains 4.2% of the data. These data clearly
a Tukey post-hoc adjustment to determine pairwise show that the soil microbial community found at
differences between the microbial communities at seedling was distinct from bolting and flowering.
each plant developmental time point. To ensure that However, we observed that the soil microbial
the data followed the assumptions of normality, community corresponding to the vegetative stage
sequences were log2 transformed. To identify if overlapped with the microbial communities estab-
developmentally dependent root exudation influ- lished at seedling and with those at bolting and
enced the soil microbial community, Pearson corre- flowering. This suggests that there is a transition
lation analysis was performed with the compounds state of the microbial community between seedling
released as root exudates (identified in Chaparro and bolting/flowering (Figure 1b).
et al. (2013)) and the corresponding phylogenetic We also determined the total OTU richness,
data. To ensure that the data abided by the evenness and diversity of the sequencing data for
assumptions of normality, the sequencing data was each time point (seedling, vegetative, bolting and
log2 transformed and the metabolomics data was log flowering; Table 1). Although there were no statis-
transformed. To determine how the functional tically significant differences between the time
microbiome changes with plant growth pairwise points with respect to overall community character-
comparisons between log2 transformed and istics, we observed that vegetative had the largest
Figure 1 Multivariate analyses of the rhizosphere microbial community through plant development analyzed by 454 pyrosequencing.
(a) Principal Coordinate Analysis (PCoA) for the visualization of pairwise community dissimilarity (Bray–Curtis index) of the
rhizosphere microbial community at each plant developmental stage (seedling, vegetative, bolting and flowering). 95% confidence
ellipses are shown around each developmental stage. (b) Principal Component Analysis (PCA) of the rhizosphere microbial community
at each plant developmental stage. 95% confidence ellipses are shown around each developmental stage.
Evenness Diversity
Figure 3 Bacterial phyla that significantly (Po0.05) change with plant development. (a) Acidobacteria, (b) Actinobacteria,
(c) Bacteroidetes and (d) Cyanobacteria. The bars with different letters are significantly different (ANOVA Tukey post-hoc Po0.05)
from one another. Each point represents one repetition and graphs show mean±SE.
development (Supplementary Figure S4A-H). For Table 2 Pearson correlation analysis of the OTUs classified as
example, Streptomyces increased in abundance from Acidobacteria, Actinobacteria, Bacteroidetes or Cyanobacteria
seedling to vegetative when the highest abundance with the compounds released as root exudates
was reached and then significantly decreased at
Phylum Number of Number of Total number
bolting stage to levels below those observed at positive negative of correlations
seedling and remaining at these levels throughout correlations correlations
flowering (Supplementary Figure S4E). On the other
hand, six genera belonging to the Bacteroidetes Acidobacteria 156 75 231
phylum significantly increased with plant aging Actinobacteria 103 46 149
(Supplementary Figures S5A-F). In general, bacteria Bacteroidetes 8 16 24
Cyanobacteria 130 243 373
classified as Cyanobacteria were significantly more
abundant at bolting than at seedling and vegetative The values indicate the number of significant (Po0.05) Pearson
and then decreased in abundance at flowering correlations for each phyla.
(Supplementary Figures S6A-C).
categorized as amino acids, phenolics, sugars or
Plant root exudation correlates with rhizosphere sugar alcohols. Phenolic compounds significantly
microbes through plant development correlated with the soil microbial community of
To determine how developmentally dependent root Acidobacteria, Actinobacteria, Bacteroidetes and
exudate changes may influence the soil microbiome, Cyanobacteria (412 significant correlations; Table 3),
Pearson correlation analysis was performed between followed by amino acids (151), sugars (137) and sugar
the compounds released as root exudates (data from alcohols (77).
Chaparro et al. (2013)) and the rhizosphere bacteria
that significantly changed through the life span of
the plant (Supplementary Table S2). We observed Plant development influences the functional
that Cyanobacteria significantly (Po0.05) correlated microbiome
with the most root exudate compounds (373 correla- To determine the functional genes that were differ-
tions; Table 2) while Bacteroidetes correlated with entially expressed at early (seedling and vegetative)
the least (24 correlations). To determine whether a compared to late plant development (bolting and
specific class of root exudate compound was flowering) two-sample t-tests on log2 transformed
involved in soil microbial community, dynamics and standardized data were performed on the
root exudates from Chaparro et al. (2013) were metatranscriptomics data from Chaparro et al.
Acidobacteria 35 12 60 38 24 18 37 7
Actinobacteria 3 21 58 25 24 0 18 0
Bacteroidetes 2 0 4 10 1 5 1 1
Cyanobacteria 67 11 60 157 0 65 3 10
Total Correlations 151 412 137 77
(2013). A total of 81 unique transcripts out of 1240 Table 4 Taxonomic assignments of the differentially expressed
assigned to the KEGG database were significantly (81) transcripts were categorized based on their activity and
(Po0.05) differentially expressed with plant age whether the corresponding transcript were significantly (t-test
Po0.05) expressed early or late in plant development
(Supplementary Table S3). iPATH2 (Yamada et al.,
2011) was used to map at what time point (early or Early Late Total
late) these 81 functional transcripts were more
abundant (Supplementary Figure S2). Of those Symbiotic N fixers 5 9 14
transcripts, 32 were more abundant during seed- Free N fixers 6 11 17
ling and vegetative stages, while 49 were more Antagonistic 5 6 11
Plant growth promoting 0 3 3
numerous during plant bolting and flowering. The Pathogen 8 4 12
majority of the transcripts that were differently Xenobiotic/metal detoxification 4 5 9
expressed between early and late development are Unclassified function 4 11 15
involved in metabolism and genetic information
processing. Two transcripts are involved in
toluene degradation (succinate dehydrogenase
iron-sulfur protein) and the transcript involved Pseudomonas syringae. Accordingly, we categorized
in nitrotoluene degradation (hydrogenase large these microbes into seven groups depending on
subunit) were significantly (Po0.05) more abun- their potential activity towards the plant (i.e.,
dant early in plant development. On the other symbiotic N fixation, free N fixation, antagonists,
hand, a transcript involved in bacterial chemo- PGPRs, pathogens, xenobiotic/metal detoxifiers or
taxis (two-component system, chemotaxis family, unclassified; Table 4). We observed that many of
response regulator) and one involved in streptomy- these transcripts aligned to symbiotic N fixers, free
cin biosynthesis (dTDP-glucose 4,6-dehydratase) N fixers or pathogens (14, 17 or 12 transcripts
were significantly more abundant in late develop- respectively; Table 4), such as Nitrobacter,
mental stages. Among five transcripts involved in Rhodospirillum, Nitrosospira, Mesorhizobium,
nitrogen metabolism, three and two were signifi- Cyanobacteria, Azorhizobium or Dickeya dadantii.
cantly expressed at early or late plant developmen- Interestingly, most of the transcripts that aligned to
tal stages, respectively. For example, the transcript pathogens were significantly expressed early during
formamidase, carbonic anhydrase and nitric-oxide development (8 genes) while transcripts that aligned
reductase NorQ protein were highly expressed to microbes involved in plant growth promotion or
during plant seedling and vegetative stages. Con- N fixation were more abundant late (23 genes;
versely, the transcripts nitrite reductase (NAD(P)H) Table 4). For example, the transcripts involved
large subunit and periplasmic nitrate reductase in bacterial chemotaxis (two-component system,
NapA were more abundant during plant bolting chemotaxis family, response regulator) were signifi-
and flowering. cantly more abundant at late time points and aligned
to the nitrogen fixing PGPR Methylobacterium
extorquens (Ivanova et al., 2001; Sy et al., 2001;
Beneficial microbes are more active during late plant Lidstrom and Chistoserdova, 2002). On the other
development hand, the transcript involved in amoebiasis
The 81 transcripts that were significantly expressed (Ras-related protein Rab-7A) was significantly more
through development were aligned to the BLAT abundant at early time points, and it aligned with
database (Kent, 2002) within MG-RAST (Meyer et al., the pathogen Moniliophthora perniciosa. Addition-
2008) to determine which microbes were carrying ally, microbes that are antagonistic and produce
out the expressed functions (Supplementary Table S3). fungicides or bactericides such as Streptosporan-
These transcripts were attributed to bacteria gium, Streptomyces avermitilis and Sorangium
such as Bradyrhizobia, Streptomyces, Azoarcus or cellulosum were transcriptionally active late
Table 5 Pearson correlation analysis of the significantly expressed transcripts at early or late plant development correlated with the
group of compounds released as root exudates
Supplementary Information accompanies this paper on The ISME Journal website (http://www.nature.com/ismej)