Proteomic analysis of the interaction between the plant growth promoting fhizobacterium paenibacillus polymyxa e681 and arabidopsis thaliana

122 Proteomics 2016, 16, 122–135DOI 10.1002/pmic.201500196
RESEARCH ARTICLE
Proteomic analyses of the interaction between the
plant-growth promoting rhizobacterium Paenibacillus
polymyxa E681 and Arabidopsis thaliana
Young Sang Kwon1,5∗∗
, Dong Yeol Lee1∗∗
, Randeep Rakwal2
, Seong-Bum Baek3
, Jeom Ho Lee3
,
Youn-Sig Kwak4
, Jong-Su Seo5
, Woo Sik Chung1
, Dong-Won Bae6∗
and Sang Gon Kim3
1
Division of Applied Life Science (BK21 Plus Program), Plant Molecular Biology and Biotechnology Research
Center, Gyeongsang National University, Jinju, Republic of Korea
2
Faculty of Health and Sport Sciences and Tsukuba International Academy for Sport Studies (TIAS), University of
Tsukuba, Tsukuba, Ibaraki, Japan
3
Department of Central Area Crop Science, National Institute of Crop Science, Rural Development Administration,
Suwon, Republic of Korea
4
Institute of Agriculture and Life Science, Gyeongsang National University, Jinju, Republic of Korea
5
Environmental Biology and Chemistry Center, Korea Institute of Toxicology, Jinju, Republic of Korea
6
Center for Research Facilities, Gyeongsang National University, Jinju, Republic of Korea
Received: May 27, 2015
Revised: September 6, 2015
Accepted: October 6, 2015
Plant growth-promoting rhizobacteria (PGPR) facilitate the plant growth and enhance their
induced systemic resistance (ISR) against a variety of environmental stresses. In this study,
we carried out integrative analyses on the proteome, transcriptome, and metabolome to in-
vestigate Arabidopsis root and shoot responses to the well-known PGPR strain Paenibacillus
polymyxa (P. polymyxa) E681. Shoot fresh and root dry weights were increased, whereas root
length was decreased by treatment with P. polymyxa E681. 2DE approach in conjunction
with MALDI-TOF/TOF analysis revealed a total of 41 (17 spots in root, 24 spots in shoot)
that were differentially expressed in response to P. polymyxa E681. Biological process- and
molecular function-based bioinformatics analysis resulted in their classiﬁcation into seven dif-
ferent protein groups. Of these, 36 proteins including amino acid metabolism, antioxidant,
defense and stress response, photosynthesis, and plant hormone-related proteins were up-
regulated, whereas ﬁve proteins including three carbohydrate metabolism- and one amino
acid metabolism-related, and one unknown protein were down-regulated, respectively. A good
correlation was observed between protein and transcript abundances for the 12 differentially
expressed proteins during interactions as determined by qPCR analysis. Metabolite analysis
using LC-MS/MS revealed highly increased levels of tryptophan, indole-3-acetonitrile (IAN),
indole-3-acetic acid (IAA), and camalexin in the treated plants. Arabidopsis plant inoculated
P. polymyxa E681 also showed resistance to Botrytis cinerea infection. Taken together these
results suggest that P. polymyxa E681 may promote plant growth by induced metabolism and
activation of defense-related proteins against fungal pathogen.
Keywords:
Arabidopsis / Metabolites / MALDI-TOF/TOF / Paenibacillus polymyxa E681 / Plant
proteomics
Additional supporting information may be found in the online version of this article at
the publisher’s web-site
Correspondence: Dr. Sang Gon Kim, Department of Central Area
Crop Science, National Institute of Crop Science, Rural Develop-
ment Administration, Suwon 441-707, Republic of Korea
E-mail: sen600@korea.kr
Fax: +82-31-695-4045
Abbreviations: IAN, indole-3-acetonitrile; ISR, induced systemic
resistance; PDA, potato dextrose agar; PGPR, plant growth-
promoting rhizobacteria; qPCR, quantitative real time polymerase
chain reaction; SDW, sterile distilled water
∗Additional corresponding author: Dr. Dong-Won Bae
E-mail: bdwon@gnu.ac.kr
∗∗These authors have contributed equally to this work
Colour Online: See the article online to view Figs. 3 and 5 in colour.
C 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

Proteomics 2016, 16, 122–135 123
Significance of the study
The present study aimed at investigating genes, proteins,
and metabolites modulated by P. polymyxa E681 during
Arabidopsis seedling growth. P. polymyxa E681 could im-
prove plant biomass and immunity by activating antioxidant,
defense-related proteins, hormone (auxin) and phytoalexin
(camalexin) in Arabidopsis. These results may lead to a
broader understanding of the molecular response to rhi-
zobacteia in plants.
1 Introduction
Rhizospheric bacteria playing an important role in plant
growth promotion are termed as plant growth-promoting
rhizobacteria (abbreviated hereafter, PGPR). PGPR have
been found to promote plant growth and help in sustain-
able agricultural development, protecting plants from phy-
topathogens. In other words, PGPR are beneficial bacteria
inhabiting the plant rhizosphere that are directly or indi-
rectly involved in promoting plant growth and biological con-
trol of plant diseases [1]. PGPR can improve plant health
and increase crop productivity by a variety of mechanisms
that involve solubilization of otherwise unavailable mineral
nutrients, stimulation of root growth, suppression of plant
diseases, and the synthesis of various hormones [2]. PGPR-
mediated plant growth enhancement has been investigated
by many researchers [3–5]. Among the PGPRs, the best doc-
umented bacterial genera are Pseudomonas spp. [6, 7], and
Bacillus spp. [8–10].
To date, DNA microarray technology and proteomic anal-
ysis have been applied to improve our understanding of
plant-bacterial interactions. A study in Arabidopsis showed
that some putative auxin-regulated genes were up-regulated
and some ethylene-responsive genes were down-regulated,
following exposure to Pseudomonas fluorescens FPT9601-T5
[11]. In another study, rice proteins involved in plant growth
and defense were induced after exposure to Bacillus cereus
NMSL88 [12]. Proteins reported to be directly or indirectly in-
volved in growth promotion were differentially expressed in
rice following inoculation with P. fluorescens KH-1 [13]. Rel-
atively fewer studies have focused on the integrative omics
analyses during plant and bacterial interactions. Paenibacillus
polymyxa (P. polymyxa) is a widely distributed endospore-
forming and non-pathogenic bacterium in rhizosphere [14].
The main roles of P. polymyxa are to stimulate plant growth
through the production of various plant hormones [15] and
to promote immunity of the rhizosphere [16]. In addition,
P. polymyxa strains are also known to produce several antibi-
otics and hydrolytic enzymes, including polymyxins, fusa-
ricidins, colistin, proteases, β-1,3-glucanases, cellulases, xy-
lanase, chitinases, and so on, which play important roles in
the biocontrol of plant pathogens [17–21]. P. polymyxa E681
was isolated from the rhizosphere of winter barley grown
in South Korea [22]. The full genome of P. polymyxa E681
was sequenced by the Genome Research Center at the Korea
Research Institute of Bioscience and Biotechnology [23]. Pre-
vious reports have shown that E681 could promote growth
of cucumber and sesame, and increase biological control ca-
pacity [22,24]. Seul et al. (2007) reported the protein changes
in P. polymyxa E681 grown in the presence or absence of
barley for the application of microbial physiology [25]. Al-
though P. polymyxa E681 has these capabilities, the molecular
mechanisms underlying the host-PGPR interaction are little
understood.
Herein, this study was conducted to investigate genes, pro-
teins, and metabolites modulated by P. polymyxa E681 during
Arabidopsis seedling growth. A total 41 proteins that were up-
or down-regulated in response to P. polymyxa E681 inocula-
tion were identified and classified into multiple biological
functions. Among them, 12 transcripts and four metabo-
lites levels corresponding proteins showed good correlation
with the proteome data. This information provided further
insight into the molecular mechanism of plant growth pro-
motion and defense response activation by the plant-microbe
interaction.
2 Materials and methods
2.1 Growth and inoculation conditions of plant and
bacteria
P. polymyxa E681 was streaked onto tryptic soy agar (TSA,
Difco Laboratories, Detroit, MI, USA) plates and incubated
for 24 h in darkness at 28ЊC. For long-term storage, bacte-
rial cultures were maintained at -80ЊC in tryptic soy broth
(TSB, Difco Laboratories, Detroit, MI, USA) that contained
20% glycerol. For experimental use, the fully grown bacterial
colonies were scraped off the plates and resuspended in ster-
ilized distilled water (SDW). The bacterial suspensions were
adjusted to 108
cfu/ml (OD600 = 1) based on optical density.
Arabidopsis thaliana Columbia (Col-0) ecoptype seeds were
sterilized in 20% household bleach for 20 min and rinsed
three times with sterile distilled water. A. thaliana plants
were grown on Murashige and Skoog medium containing
0.8% phytagel (pH 5.8) and 1% sucrose, and vernalized for
three days at 4ЊC in the absence of light. Seedlings were trans-
ferred to a growth chamber set to a 16-h light/8-h dark cycle
at 21ЊC. Seven-day-old vertically-grown Arabidopsis seedlings
were treated with P. polymyxa E681. For each treatment, 20 ␮L
cell suspensions (108
cfu/mL) were inoculated on root tips of
the seedlings. Control treatments consisted of an equivalent

124 Y. S. Kwon et al. Proteomics 2016, 16, 122–135
volume of SDW followed by cultivation in a growth chamber
for seven days at 21ЊC before collection of plant samples for
proteome analysis.
2.2 Protein extraction, 2DE, and image analysis
Total proteins from Arabidopsis thaliana were extracted fol-
lowing a previously published protocol [26]. Briefly, each two
grams of root and shoot tissues (three bio-replicates) were
finely powdered in liquid nitrogen. The root and shoot pro-
teins were isolated from powdered tissues first using cold
TCA/acetone buffer (10% TCA, and 0.07% 2-ME), followed
by adding SDS extraction buffer [30% sucrose, 2% SDS, and
0.1 M Tris–HCl (pH 8.8)] and saturated phenol, and finally
precipitated by the addition of 0.1 M ammonium acetate in
methanol. The obtained protein pellet was dissolved in ly-
sis buffer [9 M urea, 4% CHAPS, 1 mM PMSF, 50 mM DTT,
and 0.5% IPG buffer (Amersham Biosciences, San Francisco,
CA, USA)] and used for determining the protein concentra-
tion, prior to 2DE analysis as in [27]. Proteins (150 ␮g) were
loaded onto the 17 cm IPG strip (pH 5–8) and in-gel rehy-
drated for 12 h. The isoelectric focusing (IEF) was carried
out using following steps: 250 V for 15 min, 10 000 V for 3 h,
and 80 000 V for 8 h. The strips were reduced in an equili-
brating solution [30% glycerol, 50 mM Tris–HCl (pH 8.8),
6 M urea, 2% SDS] containing 1% DTT and then alkylated
by 2.5% iodoacetamide. The 2DE analysis was carried out
on 13% SDS-polyacrylamide gels, after which the proteins
in gel were stained with silver stain [27]. Three biological
replicates were performed. Images of the silver stained 2DE
gels were obtained using a GS-800 Imaging Densitometer
(Bio-Rad, Hercules, CA, USA) and analyzed with PDQuest
version 7.2.0 software (Bio-Rad, Hercules, CA, USA). Each
spot volume was normalized as the average volume of spots
on the gels, and average spot values from triplicate data were
compared. Quantitative analysis sets for each control and
P. polymyxa E681-treated root and shoot sample were gener-
ated. The proteins with statistically significant difference in
level (p < 0.05) by Student’s t-test were opted for identifica-
tion. A 1.5-fold change in expression was used as cut-off for
differentially modulated proteins.
2.3 Trypsin digestion and MALDI-TOF/TOF-MS
Silver-stained protein spots were de-stained, and in-gel
trypsin digestion was carried out according to a previous
method [28,29]. Briefly, the de-stained [with15 mM Fe(CN)6
and 50 mM Na2S2O3 for a few minutes] gel pieces were
washed five times with 500 ␮L of MWA solution (50%
methanol : 40% water : 10% acetic acid) for 30 min. The
gels were mixed with 500 ␮L of 50 mM NH4HCO3 and
500 ␮L of ACN (5 min), and vacuum dried. Reduction
of the de-stained gel pieces was done with a solution of
10 mM DTT/0.1 M NH4HCO3 for 45 min at 56ЊC, alkylated in
55 mM C2H4INO/0.1 M NH4HCO3 for 30 min under dark-
ness at room temperature, and completely vacuum dried.
Next the dried gel pieces were re-hydrated in 3 ␮L of a di-
gestion buffer (25 mM NH4HCO3, 0.1% n-octyl glucoside)
containing 50 ng/mL trypsin. Post-rehydration, 5–15 ␮L di-
gestion buffer (minus the trypsin) was added to the gel
pieces. Peptides extraction was done twice with one volume
of ACN/H2O/CF3COOH (66:33:0.1, v/v/v) solution, follow-
ing which the sample was sonicated, centrifuged, and speed-
vacuum dried. A 50% ACN and 0.1% TFA solution was added
to dried protein sample, dissolved, and stored at −20ЊC until
further use.
2.4 Protein identification
The digested peptide solution (above) was carefully spotted
onto the MALDI-TOF/TOF target plate using a micro-pipette.
Analysis was carried out on an ABI 4800 Plus TOF-TOF
Mass Spectrometer (Applied Biosystems, Framingham, MA,
USA). Running conditions: 200 Hz ND: 355 nm YAG laser
operations; signal/noise ratios >25; 10 higher intense ions
were used for MS/MS analysis in 1 kV mode, 1000–1250
consecutive laser exposure. Spectral data (MS and MS/MS)
were unpacked using UniProt database (version 20131104;
30 938 908 sequences) and Protein Pilot V.3.0 database at
100 ;ppm of mass tolerance. MS/MS spectra search crite-
ria in the databases were – single missing pick, oxidation of
methionines, and carbamidomethylation of cysteines. A sta-
tistically significant threshold value of p = 0.05 was used for
searching individual peptide ions scores.
2.5 Protein functional classification
Gene ontology (GO) analysis of identified proteins was per-
formed using the following databases: TAIR (http://www.
arabidopsis.org/); KEGG (http://www.genome.jp/kegg/);
PIR (http://pir.georgetown.edu/); UniProtKB (http://www.
uniprot.org/). Based on the GO categories, proteins were
classified according to biological processes, molecular func-
tions, and cellular components.
2.6 Quantitative real time reverse transcription PCR
(qPCR)
Total RNA was extracted from Arabidopsis shoot and root
treated with P. polymyxa E681 for seven days using the LiCl
method [30]. The qPCR was performed using a Mx3000P
QPCR System (Agilent, Santa Clara, CA, USA) with SYBR
Green QPCR Master Mix (LPS Solution, Daejeon, Korea)
as described by Bae et al. (2008) [31]. Gene specific primers
for the 12 genes were designed using primer 3.0 soft-
ware (Supporting Information Table S1). The expression
level of the target transcript was normalized to that of the

Proteomics 2016, 16, 122–135 125
internal control, the Gapdh gene, and the relative expres-
sion ratio. Differences between means were tested using Stu-
dent’s t-test, and were considered statistically significant at
p < 0.05.
2.7 Metabolites analysis using LC-MS/MS
Leaf material (100 mg) was harvested seven days after
P. polymixa E681 inoculation and frozen in liquid nitrogen.
Extraction of the metabolites was performed essentially as
previously described [32]. Briefly, 500 ␮L of 70% methanol
supplemented with biochanin A at 1 mg/L (internal stan-
dard, IS) was added to 0.1 g of frozen leaf powder. After
10 min of sonication, samples were incubated for 15 min at
80ЊC in a water bath to stop myrosinase activity. Extracts were
allowed to cool down at room temperature and centrifuged
at 10 000 g for 10 min at 4ЊC. The supernatants were fil-
tered through a 0.22 ␮m filter (Merck Millipore, Darmstadt,
Germany) and LC-MS was performed and modified as done
by Zandalinas et al. (2012) [33]. A 1260 series LC system was
used for the HPLC analysis with the following specifications
and instruments: Zorbax Eclipse XDR-C18 column (4.6 ×
150 mm, 5 ␮m, Agilent Technologies, MD, USA), G1322A
degasser, G1330B autosampler, G1312B pump, and G1316A
oven (Agilent Technologies, CA, USA). The binary solvent
system was prepared as follows: 0.1% HCOOH in water (A)
and 0.1% HCOOH in methanol (B) with gradient of the mo-
bile phase from 5 to 95% B over 1 min, isocratic elution for
7 min, decreasing to 5% B over 0.1 min, and then isocratic
elution for 7 min. For each experiment, an injection volume
10 ␮L was used with a flow rate of 0.5 mL/min, and a col-
umn temperature of 40ЊC. MS/MS experiments were carried
out on the API 4000 LC-MS/MS system (Applied Biosystems,
Forster, CA, USA) which included a Turbo VTM
source and
a Turbo ion spray probe, and operating in the positive mode
with selected ion monitoring (SIM). For instrumental con-
trol and data acquisition, the BioAnalystTM
, version 1.4.2 and
the analyst software, version 1.4.2 were used. Nitrogen, as
nebulizing and drying gas was used at a pressure of 75 psi.
Electron spray voltage was 5.5 kV with source temperature
of 600ЊC. Quadrupole and ion trap resolutions were between
0.6 and 0.8 (unit resolution).
2.8 Fungal growth and plant inoculation
Botrytis cinerea was grown on agar PDA medium for 14 days at
27ЊC in darkness. Spores were collected with distilled water.
The Columbia (Col-0) ecoptype of A. thaliana superficially
sterilized seeds were germinated and grown in Murashige
and Skoog agar medium. At four weeks after germination,
rosette leaves were inoculated with a 5 ␮L drop of a suspen-
sion of 5×105
cfu/mL on the surface of leaves. The disease
symptoms on inoculated leaves and the extension of necrotic
lesion diameter measured at seven days after inoculation.
Figure 1. Effect of P. E681 inoculation on the growth of Arabdiop-
sis thaliana ecotype Col-0. Representative photograph of rosettes
(A), shoot fresh weight (B), root length (C) and root dry weight (D)
of three-weeks-old Arabdiopsis after 7 days of E681 inoculation.
Data represent the means ± SEM of three replicates. Asterisks in-
dicate significant difference (P < 0.05) between E681 and control
treatments.
3 Results and discussion
3.1 Effect of P. polymixa E681 inoculation on
arabidopsis growth
The effect of P. polymixa E681 inoculation on plant growth
was investigated in Arabidopsis seedlings (Fig. 1). Arabidopsis
plants were grown both in vitro and in sterilized soil. The inoc-
ulation with PGPR strain P. polymixa E681 enhanced seedling
growth (Fig. 1A). This result was in accordance with previ-
ous reports showing that P. polymixa E681 promoted seedling
growth of Arabidopsis [34, 35]. P. polymixa E681 inoculation
dramatically increased about 52% shoot fresh weight and 24%
root dry weight (Fig. 1B and D), respectively, whereas primary
root length was 29% repressed (Fig. 1C). Many previous re-
searches reported that low concentrations of indole-3-acetic
acid (IAA), which is the best-characterized auxin produced
by many plant-associated bacteria [36], can stimulate primary
root elongation, whereas high IAA levels stimulate the for-
mation of lateral roots, thereby decreasing the primary root
length [37–39]. These results suggest that P. polymixa E681
may produce phytohormones and secondary metabolites to
boost Arabidopsis growth [40].
3.2 Differential proteomic analysis of Arabidopsis
shoot and root tissues after inoculation with
P. polymyxa E681
To better understand the direct or indirect interactions be-
tween Arabidopsis root and P. polymyxa E681, root proteome
profiles were analyzed at seven days after inoculating the Ara-
bidopsis root with E681 strain (108
cfu/mL). From the 2DE

Figure 2. Heat map of differentilally expressed proteins in
A. thaliana root (A) and shoot (B) by P. polymixa E681. Clustering
of dataset was performed using MeV4.9 software. All quantitative
information is transmitted using a color scale in which the color
ranges from green for the highest down-regulation (–5) to red for
the highest up-regulation (+5).
analysis, about 800 reproducible protein spots were detected
and representative images were obtained from control and
P. polymixa E681-inoculated root and shoot (Fig. 2, Support-
ing Information Fig. 1). Both Arabidopsis root (Fig. 2A, Sup-
porting Information Fig. 1A and B) and shoot (Fig. 2B, Sup-
porting Information Fig. 1C and D) revealed 41 differentially
expressed protein spots, post-inoculation. Among these pro-
tein spots, differentially expressed proteins were presented by
means of a heat map. Sixteen protein spots were up-regulated
and one protein spot were down-regulated in roots (Fig. 2A),
19 protein spots were up-regulated and five protein spots were
down-regulated in shoot (Fig. 2B). All protein spots with a dif-
ferential intensity and showing statistically significant differ-
ences between control and P. polymixa E681-inoculated root
and shoot were selected for further identification.
3.3 Identification and classification of the
P. polymyxa E681-responsive proteins in
Arabidopsis
Differentially expressed protein spots were excised from
silver-stained gels, in-gel digested by trypsin and identified
by MALDI-TOF/TOF MS. The lists of differentially ex-
pressed proteins in root and shoot were summarized in
Tables 1 and 2, respectively. In root, proteins modulated by
P. polymyxa E681 were categorized according to biological
process into classes corresponding to antioxidant (23%),
defense and stress (23%), amino acid metabolism (18%),
carbohydrate metabolism (12%), photosynthesis (12%), and
plant hormone (6%) (Fig. 3A). In shoot, which was not in
direct contact with strain E681, major identified proteins
were involved in antioxidant (26%), amino acid metabolism
(21%), carbohydrate metabolism (17%), lipid metabolism
(8%), defense and stress (8%), photosynthesis (8%), and
plant hormone (8%) related functions (Fig. 3B). These results
imply that P. polymyxa E681 strain might promote resistance
to various environments stressors by causing over expression
of antioxidant- and stress-related proteins in root, and
stimulate shoot growth and disease resistance by indirectly
inducing antioxidant- and metabolism-related proteins.
3.4 Biological mechanisms of interaction between
Arabidopsis and P. polymyxa E681
In Arabidopsis root and shoot, the most proteins with signifi-
cant changes in quantities were those involved in amino acid
metabolism (20%), including carbohydrate (15%) and lipid
metabolism (5%) (Fig. 3C). Ten proteins were up-regulated
(spots S4, S12, S13, S14, S18, S23, R12, R13, R15, and R17),
while six proteins were down-regulated (spots S11, S16, S19,
S17, S22, and R6) (Tables 1 and 2). This demonstrated that
metabolism is modulated systemically due to by P. polymyxa
E681 effect on Arabidopsis root and shoot.
In plants, the amino acids serve as precursors for a variety
of plant hormones [41, 42]. Auxins of plant hormones have
a crucial role in plant growth and lateral root development,
and have also been involved in plant defense [43]. Recently,
it was reported that the P. polymyxa E681 could promote
plant growth by producing indole-3-acetic acid (IAA) depen-
dent on exogenous tryptophan involved in auxin biosynthesis
[44]. From our proteomic results, nitrilase 1 (NIT1, spot S4),
anthranilate synthase beta subunit 1 (ASB1, spot S12), indole-
3-glycerol phosphate synthase (IGPS, spot S13), and auxin-
responsive protein (spot R9) enzymes could be involved in the
tryptophan-dependent pathway and 5-enolpyruvylshikimate-
3-phospate synthase (EPSPS, spots S23 and R15) could par-
ticipate in the shikimate pathway during process of auxin
biosynthesis or auxin signaling pathway. NIT activities may
have a significant impact on the outcome of plant–microbe
interactions [45]. ASB1 and IGPS have an important role
in plant defense responses [46, 47]. Previous DNA microar-
ray analysis also showed that NIT1, ASB1, and IGPS in the
tryptophan-dependent IAA biosynthesis pathway were up-
regulated in Arabidopsis by PGPR strain GB03 [48]. The EP-
SPS is the target of the glyphosate herbicide, and non-plant
EPSPS provides the herbicide-resistance trait in a number of
commercial transgenic crops [49]. Additionally, up-regulated
actin 7 (ACT7, spot S21) was the only Arabidopsis actin gene
to respond strongly to auxin, other hormone treatments,
light regime, and wounding, and may be the primary actin
gene responding to external stimuli [50]. Other proteins in-
volved in amino acids biosynthesis, include two cysteine syn-
thase (CYSD1, spots S14 and R12) members, and those are

Proteomics 2016, 16, 122–135 127
Table 1. Identification of the differentially expressed proteins in root inoculated with P. polymyxa E681
Noa) Description Gene ID Mr/pI b) MPc) SCd) Folde) Biological function
(UniProt) (Theoretical) (%) change
R1 Defensin-like protein 16 PDF1.2 Q9FI23 8968/8.14 4 39 +7.13 Plant defense
R2 2-Cys peroxiredoxin BAS1 BAS1 Q96291 29118/6.92 4 19 +2.58 Plant defense
R3 Glutathione peroxidase 6 GPX6 O48646 25739/9.38 5 18 +4.83 Antioxidant
R4 Photosystem I reaction center
subunit II-2
PSAD2 Q9SA56 22350/9.78 4 18 +2.00 Photosynthesis
R5 Peroxiredoxin-2B PRXIIB Q9XEX2 17531/5.17 3 20 +6.71 Antioxidant
R6 3-isopropylmalate dehydratase
small subunit 3
IPMI Q9ZW85 27059/6.33 5 28 –2.22 Amino acid metabolism
R7 ATP synthase subunit O,
mitochondrial
At5g13450 Q96251 26362/9.25 8 30 +7.07 Photosynthesis
R8 Glutathione S-transferase
DHAR1
GST Q9FWR4 23554/5.56 6 44 +1.88 Antioxidant
R9 Auxin-responsive protein IAA9 E1A7B5 32755/5.17 6 28 +14.13 Plant hormone signal
R10 Putative uncharacterized
protein
At1g18060 Q9LM40 25353/9.68 7 36 +1.65 Unknown
R11 Annexin D6 ANN6 Q9LX08 36661/7.72 6 20 +2.08 Stress response
R12 Cysteine synthase D1 CYSD1 Q9S6Z7 34501/5.23 4 15 +4.65 Amino acid metabolism
R13 Fructose-bisphosphate aldolase FBA8 Q9LF98 38858/6.05 11 33 +1.80 Carbohydrate metabolism
R14 Peroxidase 43 PER43 Q9SZH2 35798/5.68 6 17 +5.24 Antioxidant
R15 3-phosphoshikimate
1-carboxyvinyltransferase
(5-enolpyruvylshikimate-3-
phosphate
synthase)
EPSPS P05466 56269/6.30 11 19 +2.59 Amino acid metabolism
R16 Cytochrome P450 CYP71B15 Q9LW27 56482/8.45 9 29 +2.34 Plant defense
R17 Pyruvate dehydrogenase
complex component E2
PDCE2 Q5M729 58887/7.95 9 17 +1.78 Carbohydrate metabolism
a) Numbers correspond to the spot numbers marked on the 2D gels shown in Supporting Information Fig. 1.
b) Theoretical MW (kDa) and pI values.
c) The number of matched peptides (MP).
d) The percentage of sequence coverage (SC).
e) ‘+’ up-regulation; ‘–‘down-regulation.
responsible for the final step in biosynthesis of cysteine. This
enzyme was highly tolerant to toxic sulfur dioxide and sul-
fite as well as the herbicide paraquat in tobacco [51]. The
3-isopropylmalate dehydratase (IPMI, spot R6) is involved in
the biosynthesis of leucine. The current evidence based on
our proteome data suggests that the enhancement of auxin
biosynthesis and auxin signaling is essential for the promo-
tion of Arabidopsis plant growth in response to P. polymyxa
E681.
Carbohydrate metabolism utilizes the captured photosyn-
thetic energy providing the plant with required carbon, which
is critical for the production of new tissues, and also can have
profound effects on plant growth through modulation of cell
division and expansion [52]. In this study, two proteins sig-
nificantly up-regulated by P. polymyxa E681 are related to
the glycolysis pathway, including fructose-bisphosphate al-
dolase (FBA8, spot R13) and pyruvate dehydrogenase com-
plex component E2 (PDCE2, spot R17) in root. However,
glyoxylate/succinic semialdehyde reductase 1 (GLYR1, spot
S11), L-galactose dehydrogenase (LGALDH, spot S16), alco-
hol dehydrogenase (ADH1, spot S19), which are involved
in redox homeostasis or oxidative stress tolerance [53–55],
were down-regulated by P. polymyxa E681. Recently, Carval-
hais et al. (2013) reported that soil microbial communities
suppressed the expression of marker genes involved in ox-
idative stress/redox signaling while up-regulating photosyn-
thesis [56]. B. phytofirmans strain PsJN inoculation stimulates
plant growth, photosynthesis, and carbohydrate metabolism
in grapevine [57]. These enzymes may be differentially mod-
ulated for supplying enough energy and stress/redox home-
ostasis during interaction with the rhizobateria.
The second major group of differentially regulated proteins
in response to P. polymyxa E681 inoculation is the antioxidant-
related proteins associated with antioxidant properties and
oxidative burst pathway (Fig. 3C). A total of 10 proteins (spots
S2, S7, S8, S9, S10, S15, R3, R5, R8 and R14) were identified
as antioxidant proteins and showed an increase in their abun-
dance in P. polymyxa E681-treated Arabidopsis root and shoot.
In normal physiological conditions all of these antioxidant en-
zymes act as scavenging enzymes that remove ROS, thus pro-
tecting cells from oxidative damage. ROS also influence the
gene or protein expression involved in growth, cell cycle, pro-
grammed cell death (PCD), abiotic stress responses, pathogen
defense, systemic signaling, and development [58]. In this

Table 2. Identification of the differentially expressed proteins in shoot following P. polymyxa E681 inoculation in root
Noa) Description Gene ID Mr/pI b) MPc) SCd) Folde) Biological function
(UniProt) (Theoretical) (%) change
S1 Calcium-binding protein CML42 CML42 Q9SVG9 21191/4.59 7 25 +2.15 Plant defense
S2 Glutathione S-transferase U21 GSTU21 F4IA73 25857/5.42 5 22 +3.18 Antioxidant
S3 Phytochromobilin:ferredoxin
oxidoreductase
HY2 F4IZU7 29208/5.74 6 20 +5.78 Photosynthesis
S4 Nitrilase 1 NIT1 Q8LFU8 25328/5.44 3 21 +5.64 Amino acid metabolism
S5 Carbonic anhydrase CA1 Q56×90 28509/5.29 7 35 +1.77 Photosynthesis
S6 Putative uncharacterized protein At3g42760 Q9M191 26945/5.50 4 16 –2.17 Unknown
S7 Glutathione S-transferase
DHAR2
GST Q9FRL8 23506/5.79 7 37 +1.92 Antioxidant
S8 L-ascorbate peroxidase 1 APX1 F4HU93 27788/5.85 6 29 +1.56 Antioxidant
S9 Glutathione S-transferase F6 GSTF6 P42760 23471/5.80 6 30 +1.71 Antioxidant
S10 NADPH-dependent thioredoxin
reductase C
NTR3 Q8W4M1 26216/5.26 8 19 +1.55 Antioxidant
S11 Glyoxylate/succinic
semialdehyde reductase 1
GLYR1 Q9LSV0 30957/5.88 7 32 –1.73 Carbohydrate metabolism
S12 Anthranilate synthase beta
subunit 1
ASB1 Q42565 30762/7.67 3 14 +5.05 Amino acid metabolism
S13 Indole-3-glycerol phosphate
synthase
IGPS P49572 41032/5.92 6 20 +3.31 Amino acid metabolism
S14 Cysteine synthase D1 CYSD1 Q9S6Z7 34501/5.23 4 12 +2.24 Amino acid metabolism
S15 Thioredoxin reductase 2 NTR2 Q39242 40895/6.26 6 24 +1.70 Antioxidant
S16 L-galactose dehydrogenase LGALDH O81884 34738/5.49 2 12 –1.86 Carbohydrate metabolism
S17 Dihydroxyacetone kinase,
putative
DAK Q8L7L9 34635/5.65 3 12 +4.37 Lipid metabolism
S18 Probable
6-phosphogluconolactonase 5
PGL Q84WW2 35912/7.66 4 23 +5.70 Carbohydrate metabolism
S19 Alcohol dehydrogenase class-P ADH1 P06525 41836/5.83 6 21 –1.76 Carbohydrate metabolism
S20 N-acetyltransferase HLS1 HLS1 Q42381 45532/8.85 8 21 +2.52 Plant hormone signal
S21 Actin-7 ACT7 P53492 41937/5.31 19 59 +5.42 Response to auxin
S22 3-ketoacyl-CoA thiolase 1 KAT1 Q8LF48 47152/8.59 7 25 +2.33 Lipid metabolism
S23 5-enolpyruvylshikimate-3-
phosphate
synthase
EPSPS P05466 56269/6.30 10 18 +2.87 Amino acid metabolism
S24 TPR repeat-containing
thioredoxin TDX
TDX Q8VWG7 42055/5.67 6 24 +3.16 Stress response
a) Numbers correspond to the spot numbers marked on the 2D gels shown in Supporting Information Fig. 1.
b) Theoretical MW (kDa) and pI values.
c) The number of matched peptides (MP).
d) The percentage of sequence coverage (SC).
e) ‘+’ up-regulation; ‘–’ down-regulation.
study, four glutathione S-transferases (GSTs, spots S2, S7, S9
and R8) were significantly up-regulated in P. polymyxa E681-
treated Arabidopsis. GSTs have been reported to enhance re-
sistance to a virulent bacterial pathogen Pseudomonas syringae
pv. tomato DC3000 in Arabidopsis [59] and were increased in
response to various hormones such as salicylic acid, ethylene,
methyl jasmonate, auxin as well as biotic and abiotic stress
[60, 61]. In addition, five antioxidant enzymes including L-
ascorbate peroxidase 1 (APX1, spot S8), NADPH-dependent
thioredoxin reductase C (NTR3, spot S10), thioredoxin reduc-
tase 2 (NTR2, spot S15), peroxiredoxin-2B (PRXIIB, spot R5),
and peroxidase 43 (PER43, spot R14) were also up-regulated
by P. polymyxa E681. A previous study reported that PGPR en-
hanced abiotic stress tolerance in Solanum tuberosum through
changes in the expression of ROS-scavenging enzymes [62].
From our previous research, the expression of antioxidants
and oxidative stress-related proteins including thioredoxin-
dependent peroxidase 1 (TPX1), dehydroascorbate reduc-
tase (DHAR), Fe-superoxide dismutase (SOD), and GSTs in
Arabidopsis were induced by Bacillus subtilis GB03 volatile
emissions [27]. These suggest that antioxidant enzymes in-
duced by effect of P. polymyxa E681 could play a role in in-
creasing tolerance to various environmental stresses in the
plant.
Many differentially expressed proteins induced by
P. polymyxa E681 inoculation were identified to be de-
fense and stress-related proteins in shoot and root. Calcium-
binding protein CML42 (spot S1), TPR repeat-containing
thioredoxin TDX (spot S24), putative defensin-like protein
258 (spot R1), 2-cys peroxiredoxin BAS1 (spot R2), annexin D6

Proteomics 2016, 16, 122–135 129
Figure 3. Functional classification of P. polymixa E681-responsive proteins in (A) root, (B) shoot and (C) total A. thaliana seedlings. Identified
annotated proteins are included and presented according to biological process. The percentage distributions of the Gene ontology (GO)
terms were conducted using the iProClass GO tool in the Protein Information Resource (PIR) database.
(spot R11), cytochrome P450 (spot R16) were all significantly
up-regulated by P. polymyxa E681. Among them, the CML42
is an important CaM-related protein that participates in cal-
cium cell signaling during the plant immune response to
bacterial pathogens [63]. Cytochrome P450 (spot R16), a mul-
tifunctional defense enzyme, was involved the biosynthesis
of the indole-derived phytoalexin camalexin [64], resistance to
fungal pathogen [65], and activated by the transcription fac-
tor WRKY33 upon infection with P. syringae [66]. These two
enzymes are common defense-related proteins that are usu-
ally involved in different plant–pathogen interactions. The
increased expression of these two proteins indicates that the
protection of plants from pathogens through an ISR response
is initiated upon the inoculation of rhizobacteria.
In addition, the up-regulated proteins by P. polymyxa E681
include photosynthesis proteins such as phytochromobilin
(spot S3), photosystem I reaction center subunit II-2 (spot
R4) and ATP synthase subunit O (spot R7), suggesting the
enhancement of photosynthesis. It has been previously
shown that PGPR treatment was associated with an increase
in photosynthetic capacity [13,67]. These proteins (spot S3 and
R7) were annotated in various biological functions, such as
chlorophyll, photosystem I (PSI) reaction center, and energy
metabolism. These results imply that overexpression of pho-
tosynthesis enzyme may lead to an increase in the photosyn-
thetic activity of P. polymyxa E681-treated plant. Alternatively,
P. polymyxa E681 possibly stimulates energy production and
protein biosynthesis required for the increase in plant growth,
which might be linked with plant defense response.
Other proteins such as carbonic anhydrase (spot S5),
putative dihydroxyacetone kinase (spot S17), probable 6-
phosphogluconolactonase 5 (spot S18), and 3-ketoacyl-CoA
thiolase 1 (spot S22) exhibited an increased expression in
response to inoculation with P. polymixa E681. Among the

Figure 4. Effect of P. polymixa E681 on proteome and transcript levels of A. thaliana shoot and root. Comparative views of 12 spots
representing shoot and root show changes in abundance after P. polymixa E681-inoculation (A). Total RNA was extracted from shoot and
root and subjected to qPCR (B). Gene expression data were normalized against expression of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). Values were analyzed by Student’s t-test. Asterisk indicates statistically significant difference (p < 0.05).
identified proteins, two differentially expressed proteins
(spots S6 and R10) were identified as unknown functional
proteins, and will require further study in the future.
3.5 qPCR analysis
To further confirm the relationship of the protein and tran-
script level, the transcript levels corresponding proteins such
as PDF1.2 (spot R1), GPX6 (spot R3), IAA9 (spot R9), FBA8
(spot R13), EPSPS (spot R15), CYP71B15 (spot R16), CML42
(spot S1), HY2 (spot S3), NIT1 (spot S4), GSTF6 (spot S9),
ASB1 (spot S12), and ADH1 (spot S19) (Fig. 4A) were eval-
uated by qPCR analysis as shown in Fig. 4B. The expression
of genes corresponding to the identified proteins increased
significantly after P. polymyxa E681 inoculation, consistent
with the patterns of transcript accumulation and proteome
levels to be very similar. In contrast, the genes encoding
HY2 (spot S3) and GSTF6 (spot S9) showed increased and
decreased expression, respectively, after P. polymyxa E681
inoculation, for its corresponding protein expression. These
results suggest that some of the identified proteins are reg-
ulated at the transcriptional level, while others are post-
transcriptionally regulated.
3.6 Protein and metabolite levels in Arabidopsis
plant response to P. polymyxa E681
Characteristic for plants is their production of phytoalex-
ins, with growth promotion and disease resistance, previ-
ously proposed to be synthesized via the shikimate pathway.
Camalexin and IAA are synthesized from tryptophan via
indole-3-acetaldoxime (IAOx) in a reaction that was most
probably catalyzed by ASB1 (spot S12), GSTF6 (spot S9),
CYP71B15 (spot R16), and NIT1 (spot S4), which were highly
increased at both the gene and protein levels by P. polymyxa
E681 (Fig. 5A). To find some correlation with metabolites
involved in the camalexin and IAA biosynthesis pathway,
we further performed LC-MS/MS analysis using Arabidop-
sis response to P. polymyxa E681. The contents of trypto-
phan (Fig. 5B), IAN (Fig. 5C), IAA (Fig. 5D), and camalexin

Proteomics 2016, 16, 122–135 131
Figure 5. Activation of tryptophan-
derived glucosinolates and camalexin
biosynthesis pathway related proteins
or metabolites in A. thaliana response
to P. polymixa E681. The putative path-
way leads to the phytoalexin camalexin
and IAA (A). Proteins in blue were
identified by MALDI-TOF/TOF and a
minimum twofold increase in expres-
sion by P. polymixa E681. Tryptophan,
IAN, IAA, and camalexin were ana-
lyzed by LC-MS/MS (B). Analysis of LC-
MS/MS was carried out using seven-
day-old Arabidopsis seedlings leaves
(100 mg) after P. polymixa E681 inoc-
ulation. IAOx = indole-3-acetaldoxime,
IAN = indole-3-acetonitrile, IAA = in-
dole acetic acid, GSH = Glutathione.
(Fig. 5E) in Arabidopsis leaves at seven days after treatment
with P. polymyxa E681 were more clearly increased compared
to control plants. Thus, these results suggest that the assumed
correlation among integrative omics data can be effectively
considered as a general rule, and were consistent with previ-
ous researches. Lebuhn et al. (1997) reported that P. polymyxa
E681 was involved in the production of plant hormones and
secondary metabolites to enhance plant growth and inhibit
plant diseases [40].
3.7 Effect of P. polymyxa E681 inoculation on
Arabidopsis resistance to Botrytis cinerea
To determine whether P. polymyxa E681 could effectively
activate defense mechanisms that lead to pathogen resis-
tance, we tested the responses of leaves from four-week-old
Arabidopsis seedlings to the necrotrophic pathogen Botrytis
cinerea. As shown in Fig. 6, Arabidopsis plants treated with
P. polymyxa E681 showed significant improvement in resis-
tance to B. cinerea over untreated plants (Fig. 6A and B). The
lengths of lesions were significantly reduced in treated plants
compared with water control (Fig. 6C). Recently, Contreras-
Cornejo et al. (2011) reported that immunity induced by Tri-
choderma involves both hormone and camalexin dependent
mechanism against Botrytis cinerea [68]. These data demon-
strate that P. polymyxa E681 treatment also renders enhanced
resistance to B. cinerea in Arabidopsis.
3.8 Emerging view of Arabidopsis protein responses
with P. polymyxa E681 interaction through an
integrative omics analyses approach
In this study, we identified 41 proteins from Arabidopsis root
and shoots, which were differentially expressed during the
A. thaliana-P. polymyxa E681 interaction. P. polymyxa E681 is
capable of promoting plant growth and improved resistance to
the phytopathogen. An overview of proposed pathways from
Arabidopsis response to P. polymyxa E681 was generated to
highlight the major proteins (Fig. 7). One pathway might be
involved in auxin signaling (IAA9, HLS1, and ACT7) based
on accumulation of energy (FBA8, PGL, CA1, and PSAD2),
amino acid (CYSD1, IGPS), and tryptophan metabolism
(NIT1, EPSPS) to plant growth promotion (Fig. 7). An-
other pathway might be associated with improving resis-
tance to the phytopathogen. Antioxidant/stress (GST, APX1,
Figure 6. P. polymixa E681 confers resistance to Botrytis cinerea
attack in A. Thaliana. Visual comparison of disease symptoms on
Arabidopsis leaves 72 h after inoculation with B. cinerea (A and
B). Size of lesions formed in Arabidopsis leaves seven days after
inoculation with B. cinerea (C). Data points represent average
lesion size ± SE from 60 independent leaves, asterisks denote a
significant difference from control leaves (p < 0.05) as determined
by t-test.

Figure 7. Proposed model of the growth
promotion and defense response in
A. thaliana caused by P. polymixa E681
inoculation. Most of proteins identified
in this proteomic analysis were highly
increased. P. polymixa E681 was shown
to be capable of providing activated
growth promotion and improved toler-
ance to biotic stress.
GPX6, PER43, CML42, and BAS1) and tryptophan/camalexin
(ASB1, GSTF6, and CYP71B15) biosynthesis-related proteins
were activated by P. polymyxa E681. That in turn might induce
the JA signaling-related protein, PDF1.2 and finally enhance
ISR against fungal pathogen.
4 Concluding remarks
To our knowledge, this is the first proteomic-based research
focused on the interaction between Arabidopsis and PGPR
strain P. polymyxa E681. A total 41 proteins were identified to
be differentially expressed in root and shoot by P. polymyxa
E681. The majority of these proteins were related to de-
fense/stress, antioxidant, photosynthesis and plant hormone-
related functions. Our results showed that the improved plant
growth and defense by P. polymyxa E681 could be associated
with the following steps: (i) activation of antioxidant, defense-
related proteins, and phytoalexin such as camalexin to im-
prove plant immunity; (ii) regulation of the plant metabolism
and hormone such as auxin to increase biomass of the plant.
This research also provides a comprehensive overview of the
applications of proteomics in plant-microbe interactions that
may lead to a broader understanding of the molecular re-
sponse to rhizobacteia in plants.
This work was carried out with the support of ‘‘Cooperative
Research Program for Agriculture Science & Technology Develop-
ment (Project title: Maintenance and characteristics evaluation
of corn genetic resources, Project No. PJ00874701)’’ and a grant
from the Next-Generation BioGreen 21 Program (#PJ01109101)
funded of Rural Development Administration, Republic of Korea.
SGK was supported by 2016 Postdoctoral Fellowship of National
Institute of Crop Science, Rural Development Administration
(RDA), Republic of Korea. YSK and DYL were supported by
a scholarship from the BK21 plus program funded by MOEST
in Korea.
The authors have declared no conflict of interest.
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