Plant Physiology and Biochemistry 84 (2014) 115e124

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Plant Physiology and Biochemistry

journal homepage: www.elsevier.com/locate/plaphy

Research article

Gibberellin secreting rhizobacterium, Pseudomonas putida H-2-3

modulates the hormonal and stress physiology of soybean to improve
the plant growth under saline and drought conditions
Sang-Mo Kang a, 1, Ramalingam Radhakrishnan a, 1, Abdul Latif Khan b, Min-Ji Kim a,
Jae-Man Park a, Bo-Ra Kim a, Dong-Hyun Shin a, In-Jung Lee a, *
a
School of Plant Biosciences, Kyungpook National University, Daegu 702-701, Republic of Korea
b
Department of Biological Sciences and Chemistry, University of Nizwa, Nizwa, Oman

a r t i c l e i n f o a b s t r a c t

Article history: The physiological changes in tolerant soybean plants under salt and drought stress conditions with
Received 16 April 2014 Pseudomonas putida H-2-3 were investigated. A bacterial isolate H-2-3 was isolated from soil and
Accepted 1 September 2014 identified as Pseudomonas putida H-2-3 by 16S rDNA sequences. The treatment of P. putida H-2-3
Available online 24 September 2014
significantly increased the length, fresh and dry weight of shoot and chlorophyll content in gibberellins
(GAs) deficient mutant Waito-c rice seedlings over the control, it might be the presence of GA1, GA4, GA9
Keywords:
and GA20. The soybean plant growth was retarded in salt (120 mM sodium chloride) and drought (15%
Antioxidants
polyethylene glycol) stress conditions at 10 days treatments, while P. putida H-2-3 effectively enhanced
Drought
Hormones
the shoot length and fresh weight of plants suffered at salt and drought stress. The chlorophyll content
Pseudomonas was lower in abiotic stress conditions and bacterial inoculant P. putida H-2-3 mitigated the stress ef-
Salt fects by an evidence of higher quantity of chlorophyll content in plants exposed to salt and drought.
Soybean The stress hormonal analysis revealed that individual treatment of P. putida H-2-3, salt and drought
significantly enhanced the abscisic acid and salicylic acid content than their control. P. putida H-2-3
applied to salt and drought stressed plants showed a lower level of abscisic acid and salicylic acid and a
higher level of jasmonic acid content. Under stress condition induced by salt and drought in plants
expressed higher level of total polyphenol, superoxide dismutase and radical scavenging activity and
no significant changes in flavonoids. The bio-inoculant, P. putida H-2-3 modulated those antioxidants
by declining superoxide dismutase, flavonoids and radical scavenging activity. P. putida H-2-3 induced
tolerance against abiotic stress was confirmed by a reduction of Na content in abiotic stressed plants.
The results suggest that P. putida H-2-3 application reprograms the chlorophyll, stress hormones and
antioxidants expression in abiotic stress affected soybean plant and improves their growth under stress
environment.
© 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction drought are considered as osmotic stress, it disturbs the ho-

Salinization of agricultural land is steadily increasing in many
parts of the world (Al-Karaki, 2006) because of low rainfall and
high potential evaporation in arid and semiarid regions, and it
generates a low water potential in the soil, which makes it
difficult to acquire water and nutrients for the plants (Porcel et al.,
2012). Therefore, salt stress results in a water-deficit or drought
stress condition (Mahajan and Tuteja, 2005). Both salinity and
* Corresponding author. Tel.: þ82 53 950 5708; fax: þ82 53 953 6972.
E-mail addresses: ijlee@knu.ac.kr, glyrrkri@gmail.com (I.-J. Lee).
1
These authors contributed equally to the work.
meostasis of ion distribution in the plant cell (Serrano et al., 1999;
Zhu, 2001). Osmotic stress affects the morphology, anatomy and
metabolism of plant (Prat and Fathi-Ettai, 1990; Silveira et al.,
2003) leads to decrease the plant growth and yield. Plants have
developed a range of physiological mechanisms to cope with
drought and salt stress including stomatal control, osmotic
adjustment and photoprotective effects (Yordanov et al., 2000;
Valladares and Pearcy, 2002; Martínez-Ferri et al., 2004), and
production of secondary metabolites and phytohormones
(Radhakrishnan and Lee, 2013).
Plants are colonized by microorganisms in their natural envi-
ronment. Plant growth-promoting bacteria can promote the plant

http://dx.doi.org/10.1016/j.plaphy.2014.09.001
0981-9428/© 2014 Elsevier Masson SAS. All rights reserved.
116 S.-M. Kang et al. / Plant Physiology and Biochemistry 84 (2014) 115e124
growth by several mechanisms i.e., production of plant hormones,
asymbiotic N2 fixation, ammonia production, solubilization of
mineral phosphate and other essential nutrients, and control of
phytopathogenic microorganisms (Tank and Saraf, 2003). Non-
pathogenic bacteria associate with plant roots and can increase
plant tolerance to abiotic stresses (Dimkpa et al., 2009). Root
colonization by rhizobacteria are expressing an ACC (1-
aminocyclopropane-1-carboxylate) -deaminase, which lowers
ethylene level under unfavorable conditions and confers resistance
to stress, by catalyzing the conversion of ACC into ammonia and a-
ketobutyrate (Glick et al., 1998). Additionally, bacteria induce
resistance through defense system where plant cells produce
different antioxidants such as superoxide dismutase, total poly-
phenol, flavones, DPPH antioxidant activities and plant hormones
(Karthikeyan et al., 2007; Cho et al., 2008).
The growth of soybean plants has been affected by soil salinity
and drought stress (Radhakrishnan et al., 2013; Radhakrishnan and
Lee, 2013). Soybean (Glycine max L.) seeds are rich in protein, oil,
carbohydrates, minerals and other phytochemicals. It is not only
important for its nutritional content, but improves the soil fertility
by the ability of nitrogen fixation. The rhizobacteria are playing a
vital role in nitrogen fixation. The symbiotic relationship of rhizo-
bacteria with soybean roots is well studied for nodulation, but the
mechanism of plantebacterial interaction study is still under
steady progress. Very few studies have been performed on secre-
tion of plant growth regulators from bacterial culture and their
interaction with crop plants (Kang et al., 2014a). Some of the bac-
teria such as Acetobacter diazotrophicus, Acinetobacter calcoaceticus,
Azospirillum brasilense, Herbaspirillum seropedicae, Bacillus lichen-
iformis, Bacillus macrolides, Bacillus pumilus, Burkholderia sp. KCTC
11096BP, Leifsonia soli SE134 and Rhizobium phaseoli have identified
as GAs producing bacteria (Janzen et al., 1992; Atzorn et al., 1988;
Bastian et al., 1998; Gutierrez-Manero et al., 2001; Joo et al.,
2005, 2009; Kang et al., 2009; Kang et al., 2014a).
The number of bacteria belongs to Acetobacter, Azospirillum,
Azotobacter, Bacillus, Burkholderia, Klebsiella, Pseudomonas, and
Serratia have been reported as plant growth promoting bacteria
(Glick, 1995; Jones et al., 2007). Specifically, the soil-borne Pseu-
domonas has received particular attention for plant growth pro-
motion because of their excellent root-colonizing attribute and
produces a wide range of enzymes and metabolites that help the
plant withstand at environmental stress conditions (Vessey, 2003).
The plant hormone IAA (Indole-3-acetic acid) was recorded in
bacterial isolates Pseudomonas aureantiaca TSAU22, Pseudomonas
extremorientalis TSAU6 and Pseudomonas extremorientalis TSAU20
(Egamberdieva, 2009), and there are several reports on the pro-
duction of GA by Pseudomonas species (Afzal et al., 2010; Karakoc
and Aksoz, 2004). In this study was aimed to elucidate the GA
producing Pseudomonas putida H-2-3 induced physiological
changes on endogenous hormones, antioxidants and ionic balance
in abiotic stress affected soybean plants improve the stress
tolerance.
2. Materials and methods
2.1. Isolation, identification and phylogenetic analysis of bacterial
isolate H-2-3
Soil samples were collected from various agricultural fields
(brown to dark brown loamy; moderate, fine and medium granular
structure) of chungcheongbuk-do, Danyang, Republic of Korea. One
gram of soil sample was added to 50 ml of sterile saline solution,
and resulting suspensions were serially diluted (104) and 0.1 ml
aliquots were inoculated on plates containing tryptic soy/agar (TSA;
Merck Co., Germany) medium. The plates were incubated for
48 h at 30  C and bacterial colonies were differentiated by their
morphology, pigmentation and growth rate.
The isolated numbers of bacterial colonies were separately
cultured and tested their plant growth promoting effect on soy-
bean. An efficient plant growth promoting bacterial strain (H-2-3)
was identified on the basis of partial 16S ribosomal DNA (rDNA)
sequence. The chromosomal DNA was isolated through standard
procedures (Sambrook and Russel, 2001). The 27F primer (50 -
AGAGTTTGATC(AC)TGGCTCAG-30 ) and 1492R primer (50 -CGG (CT)
TACCTTGTTACGACTT-30 ) were used to PCR amplification of 16S
rDNAs. The amplification reaction was performed as previously
described (Adachi et al., 1996). The BLAST search program (http://
www.ncbi.nlm.nih.gov/BLAST/) was helped to find the nucleotide
sequence homology of this bacterial isolate. The closely related
nucleotide sequences were aligned by ClustalW and MEGA (version
5.0) software, and the neighbor-joining tree was generated using
same software. Bootstrap replication (1000 replications) was used
for a statistical support for the nodes in the phylogenetic tree.
2.2. Bioassay on gibberellins biosynthesis mutant Waito-C rice
The plant growth promoting metabolites production from
P. putida H-2-3 was analyzed by performing screening bioassay on
gibberellins biosynthesis deficient mutant Waito-C rice. Its seeds
were surface sterilized with 2.5% sodium hypochlorite for 30 min,
rinsed with autoclaved distilled water, and incubated for 24 h with
20-ppm uniconazol (Ikeda et al., 2001). The germinated seeds were
grown on agar medium (0.8%) in a growth chamber (day/night
cycle: 14 h; 28  C/10 h; 18  C; relative humidity 60e70%; light in-
tensity 1000 mmm2 s1 using natrium lamps) for 10 days. After
attaining two leaves stage, 10 ml culture filtrate of bacterial isolate
P. putida H-2-3 was applied at the apex of Waito-C rice seedlings.
The length, fresh and dry weight of shoot and chlorophyll content
were recorded in rice plants after a week.
2.3. Extraction and quantification of gibberellins in culture
The diterpene plant hormones, gibberellins (GAs) were extrac-
ted from P. putida H-2-3 culture filtrates after 3 days of incubation,
according to an established protocol (Lee et al., 1998). The extracted
GAs were subjected to reverse-phase C18-HPLC and were chro-
matographed on a 3.9  300 m Bondapak, C18 column (Waters
Corp., USA) and eluted at 1.5 ml/min with the following gradient:
0e5 min, isocratic 28% methanol (MeOH) in 1% aqueous acetic acid;
5e35 min, linear gradient from 28 to 86% MeOH; 35 to 36 min, 86
to 100% MeOH; 36 to 40 min, isocratic 100% MeOH. The forty eight
fractions (each fraction contains 1.5 ml) were collected and pre-
pared for injection to gas chromatography/mass spectrometer (GC/
MS) with selected ion monitoring mode (SIM) (6890N network GC
system, and5973 network mass selective detector; Agilent Tech-
nologies, USA). For each GA type, 1 ml of sample was injected to a
30 m  0.25 mm (i.d.), 0.25 mm film thickness DB-1 capillary col-
umn (J & W Scientific Co., USA). The GC oven temperature was
programmed for a 1 min hold at 60  C, then to rise at 15  C/min to
200  C followed by 5  C/min to 285  C. Helium carrier gas was
maintained at a head pressure of 30 kPa. The GC was directly
interfaced to a Mass Selective Detector with an interface and source
temperature of 280  C, an ionizing voltage of 70 eV and a dwell time
of 100 ms. Full scan mode (the first trial) and three major ions of the
supplemented [2H2] GAs internal standards (obtained from Prof.
Lewis N. Mander, Australian National University, Canberra,
Australia) and the bacterial gibberellins were monitored simulta-
neously. The retention time was determined using hydrocarbon
standards to calculate the KRI (Kovats Retention Index) value, while
 S.-M. Kang et al. / Plant Physiology and Biochemistry 84 (2014) 115e124
the GAs quantification was based on peak area ratios of non-
deuterated (extracted) GAs to deuterated GAs.
2.4. Bacterial inoculation and plant growth under abiotic stress
condition
Soybean (Glycine max. L. cv. Taekwang) seeds were donated by
Prof. Dong-Jang Lee, School of applied biosciences, Kyungpook
national university, Republic of Korea and surface sterilized with 1%
Tween 80 solution and 2% perchloric acid and washed thoroughly
with sterile distilled water for 5 min in shaking incubator
(120 rpm). Seeds were sown in autoclaved pots containing peat
moss (13e18%), perlite (11%), coco-peat (63e68%) and zeolite
(6e8%) with macro-nutrients present as: NHþ 1
4 ~0.09 mg ; NO3

~0.205 mg g1; P2O5 ~0.35 mg g1and K2O ~0.1 mg g1. The isolated
bacterial strain (H-2-3) was inoculated on tryptic soy broth for
bacterial culture and incubated for 48 h at 30  C. Three-week-old
soybean seedlings were treated with 5 ml of bacterial culture
(108 cfu/ml) suspension. Plants were grown in a greenhouse
(30 ± 2  C) (50 plants per treatment) and irrigated with sterile
distilled water. At seven days later, 120 mM sodium chloride and
15% polyethylene glycol were applied separately to plants to induce
salt and drought stress respectively. The shoot length, fresh weight
and chlorophyll content were measured at 38-days-old plants and
the collected plant samples were used for hormones, antioxidants
and nutrient analysis.
2.5. Quantification of abscisic acid (ABA)
P. putida H-2-3 induced changed on endogenous ABA content in
soybean plants under abiotic stress conditions was quantified from
the lyophilized leaf samples by following the protocols of Qi et al.
(1998) and Kamboj et al. (1999). Leaf samples were ground with
30 ml of extraction solution containing 95% isopropanol, 5% glacial
acetic acid, and 20 ng of ABA. The filtrate was concentrated and
residue was dissolved in 4 ml of 1 N sodium hydroxide solution, and
then washed three times with 3 ml of methylene chloride. The
aqueous phase was adjusted to a pH of 3.5 and was partitioned
three times into ethyl acetate (EtOAc). EtOAc extracts were then
combined and evaporated. The dried residue was dissolved in
phosphate buffer (pH 8.0) and then run through a poly-
vinylpolypyrrolidone (PVPP) column. The phosphate buffer (pH 3.5)
was added and partitioned three times into EtOAc. EtOAc extracts
were combined again and evaporated. The residue was dissolved in
dichloromethane (CH2Cl2), and passed through a silica cartridge
(Sep-Pak; Water Associates, Milford, Massachusetts, USA) pre-
washed with 10 ml of diethyl ether: methanol (3:2, v/v) and
10 ml of dichloromethane. ABA was recovered from the cartridge by
elution with 10 ml of diethyl ether (CH3eCH2)2O: methanol
(MeOH) (3:2, v/v). The extracts were dried and methylated by
adding diazomethane for GC/MS-SIM (6890 N network GC system,
and the 5973 network mass-selective detector; Agilent Technolo-
gies, Palo Alto, CA, USA) analysis. For quantification, the Lab-Base
(ThermoQuset, Manchester, UK) data system software was used
to monitor responses to ions with a m/e of 162 and 190 for Me-ABA
and 166 and 194 for Me-[2H6]-ABA.
2.6. Quantification of jasmonic acid (JA)
The endogenous JA level in soybean plants was quantified ac-
cording to the protocol of McCloud and Baldwin (1997). The hun-
dred milligrams of powdered leaf samples were suspended in a
solution of acetone and 50 mM citric acid (70:30, v/v), and [9,10-
2H2]-9,10-dihydro-JA (20 ng) was added. The extracts were evap-
orated resulting aqueous solutions were filtered and extracted
117
three times with 10 ml diethyl ether. The pooled extracts were then
loaded on a solid phase extraction cartridge (500 mg of sorbent,
aminopropyl) and the cartridges were washed with 7.0 ml of tri-
chloromethane and 2-propanol (2:1, v/v). The bound JA and the
pertinent standard were eluted with 10 ml of diethyl ether and
acetic acid (98:2, v/v). After evaporation of the solvents and ester-
ification of the residue with excess diazomethane, the sample was
adjusted to 50 mL with dichloromethane. The extracts were then
analyzed by GCeMS (6890 N network GC system and the 5973
network mass selective detector; Agilent Technologies, Palo Alto,
CA, USA). To enhance the sensitivity of the method, spectra were
recorded in the selected-ion mode. For JA determination, the
fragment ion was monitored at m/z ¼ 83 amu corresponding to the
base peaks of JA and [9, 10-2H2]-9, 10-dihydro-JA. The amount of
endogenous JA was calculated from the peak areas of JA compared
to the corresponding standards.
2.7. Quantification of salicylic acid (SA)
SA was extracted and quantified as described by method of
Enyedi et al. (1992) and Seskar et al. (1998). The hundred milli-
grams of leaf samples were extracted with 90% and 100% methanol
by centrifuging at 10,000  g. The combined methanol extracts
were vacuum-dried and resuspended in 2.5 ml of 5% trichloroacetic
acid, and the supernatant was partitioned with ethyl acetate/
cyclopentane/isopropanol (49.5:49.5:1, v/v). The top organic layer
was transferred to a 4 ml vial for drying with nitrogen gas and again
suspended in 1 ml of 70% methanol. HPLC analyses were carried out
on a Shimadzu fluorescence detector (Shimadzu RF-10AXL, exci-
tation and emission detected at 305 and 365 nm, respectively)
fitted with a C18 reverse-phase HPLC column (HP hypersil ODS).
The flow rate was 1.0 ml/min. The SA content in samples was
calculated by authentic standard peak values.
2.8. DPPH radical scavenging activity
The free radical scavenging activity of leaf samples was deter-
mined using DPPH assay followed by an established method (Blois,
1958). Leaf samples were extracted with MeOH and reaction
mixture contained 5 mg diphenyl-1-picrylhydrazyl (DPPH) dis-
solved in 50 ml MeOH. The reaction mixture and MeOH extract
(1:1) were allowed to incubate at room temperature in the dark
condition for 30 min. The absorbance of samples was detected in
517 nm. The radical scavenging activity was calculated and
expressed as percentage.
2.9. SOD assay
SOD activity in P. putida H-2-3 treated and untreated soybean
plants were determined by the method of Marklund and Marklund
(1974). The leaf tissues were ground with 1.3 ml of buffer solution
contains 50 mM TriseHCl and 10 mM EDTA, pH 8.5. The extracted
sample (100 ㎕) and 100 ㎕ pyrogallol (7.2 mM) were mixed and
allowed at 25  C for 10 min. The reaction was stopped by adding 50
㎕of 1 N HCl and then it was measured at 420 nm. The percentage of
SOD activity was calculated from the given formula:
SOD activityð%Þ ¼ ½1  ðA  BÞ=CÞ  100
The extracted sample added with (A) or without pyrogallol (B),
and control (C) was represented buffer solution added with
pyrogallol.
118 S.-M. Kang et al. / Plant Physiology and Biochemistry 84 (2014) 115e124

2.10. Total polyphenol content Table 1

Effect of P. putida H-2-3 on growth of GA-deficient mutant rice Waito-C. Control
refers to distilled water; Medium refers to LB broth; H-2-3 refers to P. putida H-2-3
Total polyphenol content was determined by Folin-Ciocalteu bacterial culture. Data represent the mean ± stand error of three replicates. The
colorimetric method (Amerine and Ough, 1980). Leaf tissues were means denoted by the same letter in each column are not significantly different
extracted with 80% MeOH and 50 ㎕ of extract was mixed with 1 ml (p < 0.05).
of 2% Na2CO3 and 50 ㎕ of 1 N Folin-Ciocalteu reagent at room Treatment Shoot length Shoot fresh Shoot dry Chlorophyll content
temperature for 30 min. The absorbance of total polyphenol was (cm) weight (g) weight (g) (SPAD)
measured at 750 nm. Gallic acid was used as standard to quantify
Control 7.03 ± 0.24b 0.72 ± 0.02b 0.11 ± 0.005b 28.28 ± 0.29ab
the total polyphenol. Medium 6.88 ± 0.14b 0.70 ± 0.01b 0.11 ± 0.006b 27.95 ± 0.39b
P. putida 7.71 ± 0.17a 0.79 ± 0.02a 0.13 ± 0.004a 29.74 ± 0.35a
H-2-3
2.11. Total flavonoid content

The leaf samples were extracted with 80% MeOH for total fla-
was injected to ICP-MC to quantify the contents of sodium (Na) and
vonoids quantification as per the method followed by Zhishen et al.
phosphate (P).
(1999). The crude extract (100 ㎕) was added with reaction mix-
tures, 500 ㎕ MeOH, 50 ㎕ 10% AlCl₃, 50 ㎕ 1 M NaOH and 300 ㎕
dH2O at room temperature for 30 min. The total flavonoids were
measured at 510 nm and quantified by quercetin standard.
2.13. Statistical analysis

2.12. Elemental analysis
The plant samples were immediately shifted into liquid nitrogen
and freeze dried (55  C; Virtis Freeze Dryer, Gardiner, USA). The
dried samples (0.2 g) were digested with HNO3 and H2O2 in a mi-
crowave and 3% HNO3 was added to digested samples, and then it
All of the biochemical data are mean values of a representative
experiment (n ¼ 3) and shown as the mean ± SE. The data were
analyzed using Sigma Plot Software (10.0) and analysis of variance
(ANOVA) was used to compare the statistical difference based on
Duncan's multiple range test (DMRT), at significance level of
p  0.05.

Fig. 1. Phylogenetic tree based on the sequence obtained from 27F and 1492R primers of 16S rDNA of H-2-3 (KF731678) and related fungi. Percentage of confidence levels generated
from 1000 bootstrap trees is indicated in each node.
 S.-M. Kang et al. / Plant Physiology and Biochemistry 84 (2014) 115e124
3. Results
3.1. Plant growth promoting ability of P. putida H-2-3
P. putida H-2-3 was isolated from soil and identified by phylo-
genetic analysis (Fig. 1). Result of BLAST revealed that bacterial
strain H-2-3 had 100% sequence homology with P. putida H-2-3.
The sequence was submitted to the NCBI GenBank and was given
accession no. KF731678. The bacterial isolate, P. putida H-2-3 was
tested on GA deficient mutant Waito-C rice to detect their plant
growth promoting ability by the secretion of bioactive metabolites
including GA. The results of this study were given in Table 1. The
culture filtrates of P. putida H-2-3 treatment showed a significant
increase in length of shoot (9.6%), fresh (9.7%) and dry weight of
shoot (18.2%) and chlorophyll content (5.3%) over the control. The
medium composition of bacterial culture did not influence the
growth of rice plants as conformed via the separate treatment of LB
medium on rice plants.
The presence of gibberellins in culture filtrates of P.putida H-2-3
was analyzed using GC/MS selected ion monitor (SIM) and HPLC.
The obtained results showed that bioactive GAs i.e. GA1 and GA4
and inactive GAs i.e. GA9 and GA20 were occurred in culture filtrates
(Fig. 2). Among these four GAs, the secretion of GA4 was higher than
other GAs detected in P. putida H-2-3 culture filtrates.
3.2. Ameliorative effect of P. putida H-2-3 against abiotic stress
condition
P. putida H-2-3 was tested on soybean plants grown under 15%
PEG induced drought and 120 mM NaCl induced salt stress (Fig. 3;
Table 2) to know their ameliorative effect against abiotic stresses.
The shoot length (17.3%), plant fresh weight (8.6%) and chlorophyll
content (11.5%) had higher in plants treated with P. putida H-2-3
than bacteria untreated controls. The drought stress significantly
reduced the shoot length, plant fresh weight and chlorophyll con-
tent, while P. putida H-2-3 recovered the damaging effect of
drought stress by increasing shoot length, plant fresh weight and
chlorophyll content. Moreover, a significant reduction of shoot
length (18.0%), plant fresh weight (11.7%) and chlorophyll content
(10.8%) was recorded in salt stress affected plants when compared
to their control, and P. putida H-2-3 enhanced the shoot length,
plant fresh weight and chlorophyll content.
20
16
GAs content (ng 100ml )
-1
12
8
4
0
GA4 GA9 GA20
GA1
Fig. 2. GAs in P. putida H-2-3 culture medium. Bars represent the mean þ stand error
of three replicates.
119
3.3. Hormonal changes
The environmental factors such as drought and saline soil
whereas bacterial interaction influenced the endogenous hor-
mones of soybean plants (Fig. 4). P. putida H-2-3 treated plants had
higher concentration of ABA than bacteria non-treated plants. The
drought and salt stresses caused a significantly higher level of ABA
content in plants over the control, while the additional treatment of
P. putida H-2-3 to drought and salt affected plants ameliorated the
stress effects by reducing the ABA content. Another stress hormone,
JA was lower in P. putida H-2-3 inoculated plants when compared to
their control. The similar quantity of JA was recorded in salt
stressed plants, but it was reduced in plants at drought stress.
P. putida H-2-3 interaction significantly stimulated the greater level
of JA content in plants subjected to drought and salt stresses.
Moreover, SA concentration was also altered by abiotic stresses and
bacterial interaction. The significantly higher concentration of SA
was noticed in plants affected by individual treatment of salt stress
followed by drought stress than other treatments and control.
Application of P. putida H-2-3 to soybean plants showed higher
level of SA content even under drought stress, while that content
was lower in salt stressed plants co-inoculated with P. putida H-2-3.
3.4. Antioxidants changes
To avoid the abiotic stress induced damage, the plants regulate
their antioxidants such as total polyphenol, flavonoids, SOD activity
and DPPH scavenging activity. P. putida H-2-3 interaction mitigated
the drought and salt stresses damage on soybean plants by
changing stress induced antioxidants profile (Fig. 5). Total poly-
phenol content was significantly higher in drought and salt
stressed plants than bacteria non-treated plants. When compared
to abiotic stress affected plants, the co-inoculation of P. putida H-2-
3 to drought and salt stressed plants showed higher and lower
concentration of total polyphenol content, respectively. Flavonoids
were higher in plants by P. putida H-2-3 interaction at non-stress
and abiotic stresses, and flavonoids accumulation was almost
similar in control, drought and salt stressed plants. Moreover, the
plants grown under drought and salt stress condition showed sig-
nificant higher activity of SOD than their control and this stress
induced an increase of SOD activity was declined by application of
P. putida H-2-3. Similarly, drought and salt stress caused a higher
DPPH scavenging activity in soybean plants and P. putida H-2-3
involved to lower the DPPH scavenging activity in non-stressed and
abiotic stressed plants.
3.5. Elemental changes
The detection of sodium content in plants denotes the level of
toxicity of abiotic stress. The elements such as sodium (Na) and
phosphorus (P) were quantified in soybean plants treated with
P. putida H-2-3, PEG and NaCl, and results were given in Fig. 6. The
drought and salt stress affected plants had higher Na accumulation,
i.e. 18.7% and 89.4%, respectively over the control plants. The bac-
teria P. putida H-2-3 associated with soybean plants and showed a
reduction of Na content in abiotic stressed and non-stressed plants.
The P. putida H-2-3 inoculation significantly reduced Na content in
drought and salt stressed plants and mitigates the stress effects. In
addition, P. putida H-2-3 interaction helped to increase the P con-
tent in soybean plants over the control. Drought stress also slightly
enhanced the P content and the significantly higher concentration
of P was found in salt stressed plants to the comparison with other
treatments. The co-inoculation of P. putida H-2-3 with abiotic
stressed plants showed a higher level of P content in drought stress
and a lower level of P content in salt stress conditions.
120 S.-M. Kang et al. / Plant Physiology and Biochemistry 84 (2014) 115e124

Fig. 3. P. putida H-2-3 induced changes on the growth of soybean plants under abiotic stresses. The growth variation of soybean plants interaction without (Control) or with
bacterial isolate P. putida H-2-3 (H-2-3) at salt (NaCl) or drought (PEG) stresses.

Table 2
Effect of P. putida H-2-3 on PEG and NaCl stressed soybean plants. Control refers to
phytohormones, and phosphate solubilization and nitrogen fixa-
distilled water; PEG refers to 15% polyethylene glycol; NaCl refers to 120 mM sodium tion (Naz and Bano, 2010; Sgroy et al., 2009; Stearns et al., 2012;
chloride. Data represent the mean ± stand error of three replicates. DMRT analysis Viruel et al., 2011). Several Pseudomonas strains survive under
was carried out between bacterial treated and untreated plants at each stress con- environmental stress conditions by their production of exopoly-
ditions. The means denoted by the same letter in each stress or no stress conditions
saccharide, which protects the bacteria from water stress and in-
are not significantly different (p < 0.05).
creases water retention and regulates the diffusion of organic
Treatment Shoot length Plant fresh weight Chlorophyll carbon (Hepper, 1975; Wilkinson, 1958; Roberson and Firestone,
(cm) (g) (SPAD)
1992; Chenu, 1993; Chenu and Roberson, 1996). Particularly, salt
Control No bacteria 30.75 ± 0.44b 16.28 ± 0.34b 33.10 ± 0.77b and drought stresses adversely affect soybean plants and retard
P. putida H-2-3 36.00 ± 0.56a 17.64 ± 0.39a 36.90 ± 0.36a their yield. The inoculation of beneficial Pseudomonas sp would be
PEG No bacteria 26.60 ± 0.54b 14.36 ± 0.45b 31.96 ± 0.42a
P. putida H-2-3 30.22 ± 0.90a 16.20 ± 0.43a 32.58 ± 0.74a
improved the plant growth under stress condition. It was observed
NaCl No bacteria 25.17 ± 1.00b 14.34 ± 0.32b 29.55 ± 0.83a that P. putida H-2-3 associated plants had higher shoot length, plant
P. putida H-2-3 27.98 ± 0.86a 16.32 ± 0.34a 30.82 ± 0.63a fresh weight and chlorophyll content as compared to non-
inoculated control. The mitigating effect of P. putida against salt
stress in canola seedlings and an increase of chlorophyll content
were determined by Glick et al. (1997), Hamdia and El-komy
4. Discussion (1998), and Jalili et al. (2009). An elevation of chlorophyll content
in plants leads to higher photosynthetic rate and starch production,
The soil living plant growth promoting rhizobacteria have been which might be supported the plant growth improvement under
reported to promote plant growth by nitrogen fixation, phosphate unfavorable salinity and drought environment.
solubilization, plant growth promoting metabolites production Abiotic stresses stimulate the ABA accumulation in plants
and disease suppression (Kang et al., 2009; Richardson et al., (Zhang et al., 2006), we also found a similar mechanism in our
2009). There are some studies have done on identification of findings. Plants grown under drought and salt stresses, the
gibberelline (GA) producing rhizobacterium, Pseudomonas species endogenous ABA content induces stomatal closure to minimize
(Afzal et al., 2010; Karakoc and Aksoz, 2004; Kang et al., 2014a). In water loss through transpiration (Leung and Giraudat, 1998) and
current study, we isolated and identified GA producing P. putida H- alleviates stress effects and increase the plant stress tolerance
2-3 from the agricultural field soil of chungcheongbuk-do, through activation of many stress-responsive genes (Herrera-
Danyang, Republic of Korea. The release of GAs from microbes to Medina et al., 2007). Previously, several studies have demon-
rhizosphere or plant root is a source of GA for plant growth strated an increase in ABA content in soybean plants under osmotic
(Radhakrishnan et al., 2013). The exogenous application of GAs can stress conditions (Khan et al., 2011; Radhakrishnan and Lee, 2013)
enhances the plant growth even under salt stress condition and, microbial interaction mitigates the stress effects in plants by
(Hamayun et al., 2010). The growth promoting ability of P. putida reducing the ABA content (Radahakrishnan et al., 2013). Although,
H-2-3 was determined by significantly increasing the growth of the role of ABA in plants have been well characterized under stress
Waito-C rice, it is a known dwarf and GA-deficient rice cultivar conditions, there are few studies concerning the role of endogenous
with blocked C13-hydroxylation pathway for GA biosynthesis plant ABA at plant growth promoting bacterial interaction (Kang
(Ikeda et al., 2001). The endogenous GAs involve in seed germi- et al., 2012). In current study, an association of P. putida H-2-3 to
nation, dormancy, stem elongation, flowering, sex expression and salt and drought stressed soybean plants helped to inhibit the
senescence (Daviere and Achard, 2013). Although, several studies stress induced ABA accumulation and increased the plant growth.
have been done to understand the role and biosynthetic pathway Previously, we observed the Burkholdera cepacia SE4, Prom-
of GAs in plants, the similar report on bacteria are scanty. The icromonospora sp. SE188 and A. calcoaceticus SE370 bacterial
culture filtrates of P. putida H-2-3 having bioactive GAs (GA1 and treatments promoted the plant growth and reduced the ABA con-
GA4) and physiologically inactive GAs (GA9 and GA20) suggest an tent in salt and drought stress affected cucumber plants (Kang et al.,
active GAs biosynthesis pathway of bacteria indirectly stimulated 2014b).
the plant growth. In addition to ABA, the plant hormone jasmonic acid (JA) has
P. putida strains have been reported as plant growth promoting been shown to prevent the oxidative stress damage and protects
bacteria by producing ACC deaminase, siderophores, the plants (Pedranzani et al., 2003). The JA is involved in a complex
 S.-M. Kang et al. / Plant Physiology and Biochemistry 84 (2014) 115e124 121

3500 Control Another stress responsive plant hormone, salicylic acid (SA) was
a a
P.putida H-2-3 differentially expressed in plants at environmental stresses. The
3000
a defense role of SA is well known in plant responses to abiotic
stresses (Borsani et al., 2001; Iqbal and Ashraf, 2010). Yang et al.
b
ABA content (ng g-1 DW)

2500
b (2004) reported that endogenous SA plays an important anti-
b
oxidative role in plants against oxidative stress. During
2000
plantebacterial interaction showed various degrees of SA expres-
1500
sion in crop plants (Kang et al., 2014b). We found P. putida H-2-3
associated with soybean plants at drought or salt stress comforted
1000 to improve the plant growth by increasing or decreasing the
endogenous SA level, respectively. The rhizobacteria induced sys-
500 temic resistance including SA accumulation in plants under salt and
drought stress was elucidated in cucumber plants and suggested
0
NS DS SS that exogenous application of beneficial bacterial cultures are able
5 to counteract the inhibitory effects of different adverse environ-
a mental conditions in plant growth through modulation of SA
4
biosynthesis (Kang et al., 2014b).
Plants produce various secondary metabolites to prevent the
JA content (ng g-1 DW)

a oxidative stress induced by biotic and abiotic factors. Polyphenols

a
3 are originated from the phenyl propanoid pathway and flavo-
b b
noids are biosynthesized from phenyl alanine and malonyl-CoA,
b and which act as potential indicator for osmotic stress tolerance
2
(Cheruiyot et al., 2007). It protects the plants against ROS pro-
duction by different environmental stresses (Sreenivasulu et al.,
1
2000). In present study, total polyphenol content was higher
and flavonoids content was not changed in drought and salt
0 stress affected plants. P. putida H-2-3 association regulates the
NS DS SS soybean plant growth by re-programming the synthesis and
degradation of total polyphenol and flavonoids content at abiotic
100
stress condition. Rojas-Tapias et al. (2012) reported that the
polyphenols content was significantly higher in salt stress injured
80 a maize leaves and Azotobacter inoculation also increased the
amount of polyphenols in injured plants than their control.
SA content (ng g-1 DW)

Similarly, Nautiyal et al. (2008) found that Bacillus lentimorbus

60
a NRRL B-30488 inoculation mediated an elevation of total poly-
a
b phenol in vegetables. These studies are elucidated the plant
40 b growth promoting bacteria could be involved in endogenous
b regulation of antioxidants and enhance the plant growth under
20
unfavorable environment.
The stress related antioxidant enzyme, superoxide dismutase
(SOD) and DPPH scavenging activity were higher in soybean
0 plants infected by drought and salt stresses than their control.
NS DS SS
The over production of SOD at stress condition indicates that
Fig. 4. Effect of P. putida H-2-3 on ABA, JA and SA content in soybean plants affected by specific plant variety might be tolerance to drought and salt
drought and salt stresses. NS- no stress, DS- drought stress, SS- salt stress. Bars stresses. Acar et al. (2001) observed the SOD activity was
represent the mean þ stand error of three replicates. DMRT analysis was carried out
increased by the effect of drought treatments in the leaves of
between bacterial treated and untreated plants at each stress conditions. Means fol-
lowed by the same letter in each stress or no stress conditions are not significantly drought resistant barley varieties TOKAK-157/37 and 56000/
different (p < 0.05). MISC-233 as compared to sensitive variety ERGINEL-90. Antioxi-
dant activity depends on the hydrogen radical dissociation from
phenolic compounds to form a stable compound with DPPH
signal transduction pathway to regulate the plants growth when radical. The DPPH-radical scavenging activity and antioxidant
plants wounded by insects and microorganisms (Hause et al., enzymes were increased in rice seedlings due to chilling and heat
2007). JA is beneficial to bacteria and induces nod gene expres- shock condition (Kang and Saltveit, 2002). Moreover, P. putida H-
sion by flavonoid biosynthesis pathway in rhizobacteria (Zhang 2-3 mitigated the drought and salt stress in soybean by reducing
et al., 2007). Moreover, osmotic stress condition declined the JA SOD and DPPH-radical scavenging activity in stress infected
contents in soybean plants (Radhakrishnan and Lee, 2013). The plants. Similar results were described by Wu et al. (2014) and
various plant growth promoting substances and fungi were used to suggested that Klebsiella oxytoca Rs-5 interaction with cotton
attenuate the abiotic stress effect in plant by reprogramming the JA alleviated the stress effect against salt stress by decreasing SOD
synthesis (Hamayun et al., 2010; Khan et al., 2011; Radhakrishnan activity and improved the plant growth performance.
and Lee, 2013). Similarly in current study, low level of JA in Soil salinity affects many physiological activities related to
abiotic stressed soybean plants was elevated by P. putida H-2-3 plant nutrient uptake (Lee et al., 2008), especially it increases the
treatment to drought and salt affected plants and suggests that uptake of Naþ, which leads to nutritional imbalances (Neel et al.,
P. putida H-2-3 interaction has not only reprogrammed the soybean 2002). Higher concentration of Naþ in tissues is considered as
for higher growth while significantly mitigated the osmotic stress the most critical factor responsible for salt toxicity and soybean
induced by NaCl and PEG. plants had higher level of Naþ at salt and drought stresses and
122 S.-M. Kang et al. / Plant Physiology and Biochemistry 84 (2014) 115e124

Fig. 5. Influence of P. putida H-2-3 on total polyphenol, flavonoids content, SOD and DPPH scavenging activities in drought and salt stressed soybean plants. NS- no stress, DS-
drought stress, SS- salt stress. Bars represent the mean þ stand error of three replicates. DMRT analysis was carried out between bacterial treated and untreated plants at each stress
conditions. Means followed by the same letter in each stress or no stress conditions are not significantly different (p < 0.05).

P. putida H-2-3 association avoided the over accumulation of Naþ
and maintained the ion homeostasis. The results are agreement
with the report of Vaishnav et al. (2013), who found that Pseu-
domonas sp. VS-1 maintained lower level of Naþ in 200 mM NaCl
affected soybean plants. In current study, we found the higher
level of P in drought and the lower level of P in salt stressed plants
treated with P. putida H-2-3 than their corresponding stress
treatments. Soil living phosphate-solubilizing bacteria can in-
crease P availability to plants by solubilizing insoluble phosphates,
which might be improved P nutrition and the availability of other
nutrients (Goldstein, 1986; Gyaneshwar et al., 2002). The higher
concentration of P in plants co-inoculated with P. putida H-2-3
bacteria might be involved in mitigating the drought stress by
overcoming the limited P availability in saline soils. Several reports
are suggested that application of Pseudomonas bacteria positively
influence on plant nutrition under salt stress conditions and it is
helpful to improve their plant growth and suppress the adverse
effect of salinity (Ozawa et al., 2007; Nadeem et al., 2009; Lingua
et al., 2013).
In conclusion, the association of P. putida H-2-3 with plants was
found to enhance the plant growth as well as tolerance to drought
and salt stresses through an array of mechanisms. It was confirmed
by regulating phytohormones, antioxidants and nutritional content
in soybean plants under drought and salt stresses environment. The
obtained results of this study suggest the use of P. putida H-2-3
strain can induce tolerance to abiotic stresses in plants and
contribute to sustainable agriculture.
Acknowledgment
Authors are thankful to National Research Foundation of Korea
(NRF) founded by the Ministry of Education, Science and Technol-
ogy for providing financial support through a Basic Science
Research Program (2011-0022027).
Contributions
SMK and RR designed and performed the experiments. ALK,
MJK, JMP performed the plant hormonal analysis, and BRK and DHS
performed the antioxidants analysis for the work. SMK, RR and IJL
wrote the manuscript. All authors read and approved the final
manuscript.
 S.-M. Kang et al. / Plant Physiology and Biochemistry 84 (2014) 115e124 123

3.0 tolerance to drought in Arabidopsis thaliana. Mol. Plant Microbe Interact. 21,
a 1067e1075.
Control
P. putida H-2-3 Daviere, J.M., Achard, P., 2013. Gibberellin signaling in plants. Development 140,
2.5
1147e1151.
b Dimkpa, C., Weinand, T., Asch, F., 2009. Planterhizobacteria interactions alleviate
2.0 abiotic stress conditions. Plant Cell Environ. 32, 1682e1694.
Na (x 10-3 mg kg-1)

a Egamberdieva, D., 2009. Alleviation of salt stress by plant growth regulators and
a IAA producing bacteria in wheat. Acta Physiol. Plant. 31, 861e864.
b
1.5 Enyedi, A.J., Yalpani, N., Silverman, P., Raskin, I., 1992. Localization, conjugation and
b function of salicylic acid in tobacco during the hypersensitive reaction to to-
bacco mosaic virus. Proc. Natl. Acad. Sci. U. S. A. 89, 2480e2484.
1.0
Glick, B.R., 1995. The enhancement of plant growth by freeliving bacteria. Can. J.
Microbiol. 41, 109e117.
0.5 Glick, B.R., Liu, C., Ghosh, S., Dumbrov, E.B., 1997. Early development of canola
seedlings in the presence of the plant growth promoting rhizobacterium
Pseudomonas putida GR 12-2. Soil Biol. Biochem. 29, 1233e1239.
0.0 Glick, B.R., Penrose, D.M., Li, J.P., 1998. A model for the lowering of plant ethylene
NS DS SS concentrations by plant growth-promoting bacteria. J. Theor. Biol. 190, 63e68.
10
a Goldstein, A.H., 1986. Bacterial phosphate solubilization: historical perspective and
a future prospects. Am. J. Altern. Agric. 1, 57e65.
Gutierrez-Manero, F.J., Ramos-Solano, B., Probanza, A., Mehouachi, J., Tadeo, F.R.,
8 a b Talon, M., 2001. The plant-growthpromoting rhizobacteria Bacillus pumilis and
b Bacillus licheniformis produce high amounts of physiologically active gibberel-
lins. Physiol. Plant. 111, 206e211.
P (x 10-3 mg kg-1)

b
6 Gyaneshwar, P., Kumar, N., Parekh, L.J., Poole, P.S., 2002. Role of soil microorganisms
4
2
0 Hepper, C.M., 1975. Extracellular polysaccharides of soil bacteria. In: Walker, N.
NS DS SS
Fig. 6. Sodium (Na) and phosphate (P) content in soybean plants treated with P. putida
H-2-3, drought and salt stresses. NS- no stress, DS- drought stress, SS- salt stress. Bars
represent the mean þ stand error of three replicates. DMRT analysis was carried out
between bacterial treated and untreated plants at each stress conditions. Means fol-
lowed by the same letter in each stress or no stress conditions are not significantly
different (p < 0.05).
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