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Environmental and Experimental Botany 133 (2017) 5869
Contents lists available at ScienceDirect
Environmental and Experimental Botany

journal homepage: www.elsevier.com/locate/envexpbot
Plant growth-promoting endophyte Sphingomonas sp. LK11 alleviates

salinity stress in Solanum pimpinellifolium
Abdul Latif Khan, Dr.a,* , Muhammad Waqasb,c , Sajjad Asafb , Muhammad Kamrand,
Raheem Shahzadb , Saqib Bilalb , Muhammad Aaqil Khanb , Sang-Mo Kangb ,
Yoon-Ha Kimb , Byung-Wook Yunb , Ahmed Al-Rawahia , Ahmed Al-Harrasi, Prof.a,* ,
In-Jung Lee, Prof.c,*
a
UoN Chair of Omans Medicinal Plants and Marine Natural Products, University of Nizwa, Nizwa, Oman
School of Applied Biosciences, Kyungpook National University, Daegu, Republic of Korea
c
Department of Agriculture, Abdul Wali Khan University, Mardan, Pakistan
d
Plant Research Centre, School of Agriculture, Food and Wine, University of Adelaide, 5064, Australia
b
A R T I C L E I N F O
Article history:
Received 13 May 2016
Received in revised form 30 September 2016
Accepted 30 September 2016
Available online 1 October 2016
Keywords:
Salinity stress
Plant-microbe interaction
Hormonal cross-talk
Wild-type and mutant Solanum
pimpinellifolium tomato
Exogenous jasmonic acid
A B S T R A C T
Salinity stress can be detrimental to the growth and yield of plants. The adverse effects of salinity can be
ameliorated with the application of plant growth-promoting endophytic bacteria (PGPEB). The
concomitant benecial effects of exogenous jasmonic acid (JA) in synergism with PGPEB have been
well-established; however, its physiological responses under salinity stress have least been understood.
In initial screening, PGPEB (Sphingomonas sp. LK11) was grown with or without JA and sodium chloride.
LK11 did not signicantly compromise its growth and physiological processes such as biosynthesis of cell
wall-related amino acids (aspartate, glutamate, and alanine) during JA treatment under salinity stress.
This improved growth performance during salinity stress was mainly due to the expression of
glutathione-related genes in LK11 genome. PGPEB counteracting salinity stress can be vital trait to
improve crops growth and tolerance. In plant-microbe interaction, the interplay of JA and LK11 and their
synergistic effects on the growth of wild-type Solanum pimpinellifolium and non-isogenic mutant (Got-3)
plants under salinity stress were assessed. Combined LK11-JA applications signicantly improved the
shoot/root growth in both wild-type and Got-3 tomato plants with or without salinity stress. Combined
treatment of LK11-JA responded well to salinity stress by signicantly regulating glutathione contents in
wild-type and Got-3 plants. Endogenous stress-responsive abscisic acid contents were signicantly low
in sole and combined LK11-JA applications during salinity as compared to sole saline conditions in both
types of plants. Relatively high levels of salicylic acid (SA) and low levels of JA showed the impaired stress
tolerance and growth with/without salinity stress in Got-3 plants. In contrast, wild-type plants showed
relatively higher JA and lower SA levels in response to combined LK11-JA treatments under salinity stress.
These ndings indicate that combination of PGPEB and JA is not essentially harmful to plants; however,
both can reprogramme crop plant responses to overcome the adverse effects of salinity stress.
2016 Elsevier B.V. All rights reserved.
1. Introduction
Salinity is a well-known limiting factor that affects plant
growth and development. Increasing soil salinization is an
impediment to achieving the goal of sustainable agricultural
production. Salt stress increases ionic inuxes, oxidant imbalances,
* Corresponding authors.
E-mail addresses: Latifepm78@yahoo.co.uk (A.L. Khan),
aharrasi@unizwa.edu.om (A. Al-Harrasi), ijlee@knu.ac.kr (I.-J. Lee).
http://dx.doi.org/10.1016/j.envexpbot.2016.09.009
0098-8472/ 2016 Elsevier B.V. All rights reserved.
cell-division impairment, and membrane degeneration (Munns

and Tester, 2008; Gill and Tuteja, 2010; Shabala and Munns, 2012).
Plants respond to salt stress conditions by activating different
signalling pathways, which assist plants in adjusting their
metabolism to maintain a defensive regime (Khan et al., 2013a,
2015). This strategy involves variances in the signalling of stomatal
closure, osmotic/ionic carriers, and secondary metabolites. Phytohormones, such as salicylic acid (SA), jasmonic acids (JA), and
abscisic acid (ABA), also play vital roles in plant response to salt
stress (Kazan, 2015). These phytohormones and the cross-talk
between their regulatory pathways have often been analysed
A.L. Khan et al. / Environmental and Experimental Botany 133 (2017) 5869
under various environmental stress conditions, including salinity

(Caarls et al., 2015). During salinity, the ionic imbalance causes the
production of superoxide anion and hydroxyl radicals, among
others, which are regulated by antioxidants, including glutathione,
to counteract the effects of radicals on plant cells (Manai et al.,
2014; Cheng et al., 2015). Moreover, current literature suggests that
thiol-based antioxidant systems can improve the defence mechanisms as well as photosynthetic activities of plants during stress
conditions, especially induced by chemicals such as salt and
polyethylene glycol (Cheng et al., 2015; Nahar et al., 2015; Dietz,
2016). Lower activation of glutathione alike active metabolites/
enzymes can be detrimental to crop growth and development
during stress conditions.
Previous studies have reported the use of plant growthpromoting rhizobacteria (PGPR) for improving crop performance
under adverse saline conditions (Mayak et al., 2004; Bashan et al.,
2014; Egamberdieva and Lugtenberg, 2014; Kang et al., 2014).
However, a few plant growth-promoting bacteria are known to aid
in ameliorating the impacts of salt stress. Plant growth-promoting
endophytic bacteria (PGPEB) form symptomless colonies in
healthy host plant tissues. They are generally considered the most
effective class of microorganisms in assisting host plants to cope
with abiotic and biotic stresses (Khan et al., 2013a; Mandyam and
Jumpponen, 2014). Furthermore, endophytic symbiosis within the
roots of host plants helps in regulating the uptake of mineral
nutrients and exudation of defensive metabolites from the roots,
thereby maintaining phytohormone balance (Khan et al., 2013a;
Bashan et al., 2014). However, very few endophytic bacteria have
been reported to confer such positive effects to host plants.
The role of PGPEB during phytohormonal application to
commercially cultivated crop plants under salinity stress is largely
unknown. Among plant hormones, jasmonic acid (JA) has been
shown to trigger the activation of inducible immune responses,
which are activated in response to pathogenic microbes and
herbivorous insects, in plants (Carvalhais et al., 2013; Caarls et al.,
2015). PGPR are known to play a role in triggering the induced
systemic response (ISR) that prepares the host plant to respond to
pathogens (Bordiec et al., 2011). For example, Pseudomonas
uorescens colonizing the root zones of Arabidopsis thaliana
triggers ISR through a JA-dependent but salicylic acid (SA)independent mechanism (Iavicoli et al., 2003; Carvalhais et al.,
2013). However, some specic strains of PGPR are known to trigger
ISR via SA signalling (Bordiec et al., 2011). Although the systemic
induction of resistance by PGPR is well documented, insufcient
data are available regarding the effects of PGPEB and exogenous JA
on the regulation of endogenous phytohormones and antioxidants,
especially glutathiones.
The synergism between potential PGPR and exogenous
phytohormonal treatment should be explored further, as some
PGPR might have benecial effects when applied in combination
with exogenous phytohormones. To investigate this further, we
used the PGPEB, Sphingomonas sp. LK11, a bacterial strain recently
isolated and identied from a medicinal plant growing in Oman
(Khan et al., 2014). Sphingomonas are gram-negative bacteria that
occur in a diverse range of environments and form yellowpigmented colonies on solid growth media. They are metabolically
exible, and capable of consuming a variety of environmental
contaminants and naturally occurring compounds (Miyauchi et al.,
1998; Aylward et al., 2013; Pukrov et al., 2013). They have been
reported to assist in the degradation of metabolites in the
environment (Qu et al., 2013; Liu et al., 2014). Genetic analysis
of Sphingomonas sp. has revealed the presence of carbazole
degradation genes in its genome (Kilbane et al., 2002). Furthermore, this bacterium has the ability to remediate heavy metals and
decompose various pesticides such as dibenzo-p-dioxins (Miller
et al., 2010; Liu et al., 2014). It can accumulate intracellular Zn2+
59
and reduce Cd2+ uptake, thus enhancing the expression of

cysteine-rich metallothionein proteins that bind heavy metals
(Stadtman, 1993). The LK11 strain of Sphingomonas exhibits Cd2+
bioaccumulation and translocation by regulating the amino acids
associated with oxidative stress (Khan et al., 2014). However, its
role in counteracting abiotic stresses has not yet been explored.
Large-scale inoculation of crop plants with PGPEB, especially
the strains derived from a similar host plant, can lead to crop
improvement. Application of exogenous phytohormones can aid in
increasing crop yield by enhancing plant resistance to herbivory
and lodging (Stevens et al., 2006; Manai et al., 2014). Tomato
plants, when laden with water, can quickly become susceptible to
events which drastically hinder their growth. In such cases, the
application of exogenous JA has been shown to improve resistance
to herbivory; however, less is known about the effectiveness of
PGPEB application. Therefore, further research is required for
gaining a better understanding of the synergistic effects of PGPEB
and exogenous JA on plant growth.
Sphingomonas sp. LK11 strain is known to produce indole acetic
acid and physiologically active gibberellins when grown in
nutrient broth (Khan et al., 2014). Endophytic microorganisms
producing such phytohormones have been recently identied in
many crop plants such as Solanum lycopersicum L. (Romero et al.,
2014; Yi et al., 2015). However, research related to the wild cultivar
S. pimpinellifolium is lacking. This species is native to Ecuador and
Peru, where it grows in undisturbed areas under varied environmental conditions, ranging from arid to humid and foggy
conditions at higher altitudes (Rao et al., 2013). In the present
study, we aimed to determine the effects of JA and PGPEB
application on the growth characteristics of S. pimpinellifolium
experiencing salinity stress, hypothesizing that exogenous JA and
sodium chloride (NaCl) are not detrimental to the growth of
Sphingomonas sp. LK11 and the synthesis of its essential amino
acids. The ameliorative effects of the combined application of JA
and PGPEB were also examined in the glutamate oxaloacetate
transaminase-3 (Got-3) mutant line of S. pimpinellifolium, which
shows underdeveloped growth due to low ammonia assimilation.
These experiments were performed to further elucidate whether
sole or combined applications of JA and PGPEB can promote plant
growth and development during salinity stress through endogenous phytohormonal signalling.
2. Materials and methods
2.1. Endophytic bacterial growth
The endophytic bacteria, Sphingomonas sp. LK11 (KF515708),
were isolated previously from the leaves of a medicinal plant
Tephrosia apollinea, which grows in the mountainous area (23 040
22.0000 N; 57 400 07.0000 E) of Jabal Al-Akhdar, Sultanate of Oman
(Khan et al., 2014; Halo et al., 2015). Molecular identication of the
isolate was carried out by PCR-amplication of the 16S rDNA using
universal primers 27F (50 -AGA GTT TGA TCC TGG CTC AG-30 ) and
1492R (50 -CGG CTT ACC TTG TTA CGA CTT-30 ). In oder to evaluate its
growth potential under salt-stress conditions, Sphingomonas sp.
LK11 was grown in nutrient broth containing different concentrations of NaCl (200500 mM) at 28 2 C and 200 20 rpm in an
orbital incubator shaker for 7 days. Furthermore, the growth ability
of Sphingomonas sp. LK11 against external application of JA was
assessed to analyse the toxicity of JA for bacterial growth.
Furthermore, LK11 was cultured in nutrient broth containing JA
(100 mM) alone and in combination with NaCl, and its growth was
quantied by determining biomass assimilation and cell density of
bacteria at 600 nm. The LK11 pellets were obtained by centrifugation at 13,000 rpm at 4 C for 15 min and transferred to 80 C for
subsequent protein and amino acid analysis. We used inductively
60
coupled plasma mass spectrometry (ICP-MS) to quantify the Na+ in

bacterial cells.
2.2. SDS-PAGE analysis of bacterial protein
In order to analyse the effects of JA and NaCl on protein
expressions of the endophytes, the culture suspension of LK11 in LB
broth (pH 7 0.5) supplemented with JA (100 mM) and NaCl
(500 mM) was incubated at 28 2 C. The culture broth was
centrifuged at 4 C at 10,000 rpm for 10 min. The bacterial pellets
were then rinsed twice with phosphate buffered saline by
centrifuging at 5000 rpm for 5 min. Subsequently, the pellets
were re-suspended in a buffer (10 mM DTT, and 40 mM Tris-HCl,
pH 8.5), and sonicated to rupture the cells, after which the lysate
was centrifuged at 12,000 rpm for 30 min. Protein contents were
quantied by Bradford (1976) assay using bovine serum albumin
(BSA) as the standard. The extracted proteins were loaded on 10%
polyacrylamide gel, with a reference protein ladder (Fermentas;
10170 kDa). Twenty milligrammes of bacterial biomass (control,
NaCl, JA, and combined treatment) was used for the assessment of
amino acid regulation.
2.3. Extraction and quantication of bacterial amino acids
Extraction of the amino acids was carried out by incubating
the bacterial cell suspensions with 2 mL 10% trichloroacetic acid
at 110 C with gentle agitation at 50 5 rpm for 24 h. The extract
was dehydrated, dissolved in 0.02 N HCl and subjected to
centrifugation at 4000 g at 4 C for 15 min, and the supernatant
was collected. Amino acids were quantied with an amino acid
analyser (HITACHI L-8900, Japan) attached to a HITACHI HPLC
(packed column with ion-exchanging resin; No. 2622 PF;
4.6 mm 60 mm). Buffer systems used in the mobile phase
consisted of Wako L-8500 solutions PF-1, 2, 3, 4, and RG. A
precise amount of each sample (20 mL) was injected into the
amino acid analyser. A ninhydrin reagent set (Wako Chemical Inc.,
Japan) was used to determine the amino acid composition
(expressed in nmol mg1 cells) of each sample. Three replicates
were used for each sample.
2.4. Plant growth and interaction with endophytic bacterial
Bacterial strain LK11 was cultured in LB medium at 28 2 C
and 200 rpm for 5 days, allowing the cell density to reach 6.9 109
CFU mL1. Tomato seeds of both S. pimpinellifolium LA1831 Got-3
mutant line and wild-type S. pimpinellifolium were acquired from
the Tomato Genetics Resource Center (TGRC) at the University of
California (California, USA). Soil, distilled water, and pots were
autoclaved at 121 C for 20 min. Seeds were rinsed with autoclaved
distilled water and then treated with NaOCl (5%) for 10 min. This
was followed by thorough rinsing with distilled water to eliminate
the traces of the NaOCl from seeds. Tomato seeds were grown in
plastic pots containing horticultural soil composed of perlite (11%),
cocopeat (68%), and zeolite (8%) with macro-nutrients present as:
NO3 0.205 mg g1; NH4+ 0.09 mg g1; P2O5 0.35 mg g1 and
K2O 0.1 mg g 1. In previous experiments, we observed favourable
growth responses of LK11 with both JA and NaCl stress; therefore,
100 mM JA, 120 mM NaCl, and 50 mL LK11 suspension (108 CFU
mL1) were applied to the rhizosphere region of the 2-week-old
tomato seedlings (wild-type and mutant). After seven days, tomato
plants from different treatments were harvested, immediately
frozen in liquid nitrogen, and then transferred to 80 C for
hormonal analysis. The shoot and root lengths were recorded to
examine plant growth under different treatments. Each treatment
was replicated three times, with each replicate comprising 18
plants.
2.5. Determination of glutathione

Leaves of the tomato plants were ground to a homogenate in
50 mM Tris-HCl buffer (3 mM MgCl2, 1.0% PVP, and 1 mM EDTA; pH
7.0). The homogenate was centrifuged at 5000 g at 4 C for
15 min. The total protein content was quantied using the Bradford
assay (Bradford, 1976). In addition, reduced glutathione was
estimated by following the procedure described by Ellman (1959).
Sample extracts were obtained by grinding the fresh leaves of
tomato plants in liquid nitrogen, followed by the addition of 3 mL
5% (v/v) trichloroacetic acid to homogenate and centrifugation at
10,000 g for 15 min. The supernatant (0.1 mL) was decanted into
a tube containing 3.0 mL of 150 mM NaH2PO4 (pH 7.4). Subsequently, 500 mL of 5,50 -dithiobis(2-nitrobenzoic acid) (DTNB;
75.3 mg of DTNB dissolved in 30 mL of 100 mM phosphate buffer,
pH 6.8) was added to the suspension and incubated at 30 2 C for
5 min, and the absorbance was measured at 412 nm. The GSH
contents were estimated by comparing with the standard curve.
This experiment was repeated three times.
2.6. Extraction and quantication of endogenous phytohormones
The abscisic acid (ABA) contents of the freeze-died samples
were determined according to procedures described by Qi et al.
(1998) and Kamboj et al. (1999). ABA was extracted from the aerial
parts of plants into an extraction solution containing 95%
isopropanol, 5% glacial acetic acid and 20 ng of [()-3,5,5,7,7,7d6]-ABA. The suspension was ltered, and the ltrate was
concentrated using a rotary evaporator. The residue was suspended
in 4 mL of 1N NaOH solution and rinsed thrice with 3 mL of
methylene chloride in order to eliminate traces of lipophilic
material. After decreasing the pH of aqueous phase to 3.5 by adding
6N HCl, it was extracted through solvent-solvent extraction with
ethyl acetate (EtOAc) thrice. The EtOAc extracts were then
evaporated, and the nearly dry residue was re-suspended in
phosphate buffer solution (pH 8.0), which was passed through a
polyvinylpolypyrrolidone (PVPP) column. The eluted phosphate
buffered solution was once again partitioned three times with
EtOAc after adjusting the pH to 3.5 with 6N HCl. All three aliquots
of EtOAc extracts were pooled and evaporated using a rotary
evaporator. The residue was dissolved in dichloromethane (CH2Cl2)
and then passed through a silica cartridge (Sep-Pak; Water
Associates, Milford, Massachusetts, USA) pre-rinsed with 10 mL
of dichloromethane and 10 mL of diethyl ether: methanol (3:2, v/
v). The resulting extract was dried with N2 gas and subsequently
methylated by adding diazomethane for gas chromatographymass spectrometry (GCMS) analysis using selected ion monitoring (SIM) 6890N network GC system, and the 5973 network massselective detector; Agilent Technologies, Palo Alto, CA, USA). The
monitor responses to ions of m/z of 190 and 162 for Me-ABA, and
194 and 166 for Me-[2H6]-ABA, were obtained using Lab-Base
(ThermoQuest, Manchester, UK) data system software.
For the quantication of endogenous jasmonic acid (JA), the
optimised protocol of McCloud and Baldwin (1997) was used. The
freeze-dried stem tissues were ground to a homogenate, and 0.1 g
of the homogenate powder was suspended in a mixture of acetone
and 50 mM citric acid (70:30, v/v). Internal standard [9,10-2H2]9,10-dihydro-JA (20 ng) was also added to the suspension. The
extracts were left overnight at low temperatures to allow the
highly volatile organic solvent to evaporate but to retain the less
volatile fatty acids. The aqueous solution was ltered, and then
extracted with 10 mL diethyl ether three times. The combined
extracts were subsequently loaded on a solid-phase extraction
cartridge (500 mg of sorbent, aminopropyl), and the cartridges
were washed using 7.0 mL of trichloromethane and 2-propanol
(2:1, v/v). The exogenous JA and the relevant standard were eluted
with 10 mL of diethyl ether and acetic acid (98:2, v/v). Upon

evaporation of the solvents, the residue was esteried with excess
diazomethane and its volume was brought to 50 mL with
dichloromethane, and the samples were analysed with GCMS
(6890N network GC system and the 5973 network mass selective
detector; Agilent Technologies, Palo Alto, CA, USA) in the selected
ion mode. The ion fragment was monitored at m/z = 83 amu
corresponding to the base peaks of JA and [9,10-2H2]-9,10-dihydroJA; the amount of endogenous JA was estimated from the peak
areas compared with the respective standards.
SA was quantied according to the method described by Seskar
et al. (1998) and Enyedi et al. (1992). Freeze-dried plant samples
were homogenised, and 0.1 g of the homogenate powder was
extracted sequentially with 90 and 100% methanol by centrifuging
at 10,000 g at 4 C. The resulting pellets were resuspended in
2.5 mL of 5% trichloroacetic acid, and the suspension was
partitioned
with
ethyl
acetate/cyclopentane/isopropanol
(49.5:49.5:1, v/v). The upper layer, free of SA, was decanted to a
new vial, and the residues were dried with N2 gas and resuspended in 1 mL of 70% methanol. High-performance liquid
chromatography (HPLC) analyses were carried out using a
Shimadzu uorescence detector (Shimadzu RF-10AXL, excitation
and emission detected at 305 nm and 365 nm, respectively;
Shimadzu, Japan). A C18 reverse-phase HPLC column (HP hypersil
ODS; particle size, 5 mm; pore size, 120- water; Supplementary
Table 1) with a ow rate of approximately 1.0 mL min1 was used
for the analysis.
2.7. Statistical analyses
All experiments were conducted in triplicates and each
treatment consisted of a set of three replicates, with each replicate
comprising 18 plants. The data is presented as mean standard
deviation (SD). Two-way analysis of variance (ANOVA) of the mean
values was performed to determine the signicance level (*p
< 0.05, ** p < 0.01, *** p < 0.001, ns = not signicant) among the
bacterial, JA, and NaCl treatments in comparison with control. In
addition, for each treatment, Duncans multiple range test was
performed using statistical analysis software SAS, version 9.1 (SAS
Inc., Cary, NC, USA). Graphical presentation was carried out with
GraphPad Prism, version 5.0 (GraphPad Software Inc., San Diego,
California, USA).
3. Results
3.1. Synergism of PGPEB during salinity and JA applications
Sphingomonas sp. LK11 was grown initially in media of various
saline concentrations to examine its survival rates under saline
stress. Repeated experimentation demonstrated that the endophytic bacterium maintained a sustainable growth in saline
conditions of up to 500 mM. The growth rate of LK11 was
signicantly high with only JA treatment, whereas it was
signicantly low under high saline conditions (Fig. 1). The
concentrations of bacteria in the presence and absence of JA
application were 12.49 108 and 12.32 108 cells mL1, respectively. The sole application of NaCl reduced the growth of LK11
cells, decreasing their concentration to 10.97 108 cells mL1.
However, the growth of LK11 cells was found to be signicantly
higher (12.14 108 cells mL1) with JA application under NaCl
treatment (Fig. 1), suggesting that JA was benecial for LK11
growth under high NaCl conditions. This was supported by the
quantity of Na+ in the bacterial growth media. ICP-MS analysis of
bacterial cells showed that Na+ accumulation was signicantly
higher in bacteria growing under the combined treatment of JA and
NaCl (LK + JA + NaCl) than those growing under the sole treatment
61
of NaCl (LK11 + NaCl) and without NaCl (LK11 control; Fig. 1).
Another reason might be that Na+ gets reduced through a reaction
with the JA-methyl group or, alternatively, Na+ might convert the
JA-ester group to carboxylic acid or alcohol, thus, allowing steady
growth (Fig. 1). However, further research on the bacterial
microenvironment is required to understand the mechanisms of
the effects of NaCl and JA on microbial growth.
Furthermore, Fig. 1 shows that JA might counteract the negative
effects of Na+ ions on LK11 cells. The concentrations of proline, an
amino acid that is used to understand stress induction in living
organisms, were signicantly reduced by JA treatments with or
without NaCl as compared to the sole treatment of NaCl (Fig. 1). To
further understand the adverse effects of NaCl and benecial
effects of JA on protein expression, we carried out an SDS-PAGE
analysis of extracted proteins of differently treated bacterial cells.
The SDS-PAGE analysis showed signicantly high expression of
proteins in the LK11 + NaCl treatment as compared to the LK11 +
JA + NaCl treatment (Fig. 1), with the least protein expression in
untreated LK11 cells. We elucidated the detailed prole of amino
acids and quantied them by examining protein expression
(Supplementary information 2).
The reduction of protein expression in LK11 cells exposed to JA
and NaCl, as shown by the SDS-PAGE analyses, was an intriguing
observation warranting further quantication and understanding
of the regulation of essential amino acids. Accordingly, the amino
acids present in the LK11 cells exposed to JA and NaClasparagine
(Asp), leucine (Leu), arginine (Arg), threonine (Thr), serine (Ser),
phenylalanine (Phe), tyrosine (Tyr), alanine (Ala), cysteine (Cys),
valine (Val), methionine (Met), isoleucine (Ile), glutamine (Glu),
lysine (Lys), histidine (His), and proline (Pro)were quantied
using an amino acid analyser and compared to those present in the
control LK11 cells. The concentration of bacterial cell-wall amino
acid, Ala with NH3+ group was found to be signicantly high in LK11
cells treated with NaCl (NaCl + LK11; Supplementary information
2). The concentration of Gly, Leu, Asp, and Val, was moderately
high, whereas that of Ser, Cys, Met, Tyr, Phe, Lys, and His was
signicantly low during LK11 growth (Supplementary information
2). As compared to the control LK11 cells, a two-fold increase was
observed in the amino acid contents of the LK11 cells growing
under NaCl stress. No signicant difference was observed in the
contents of Ser, Cys, Met, Tyr, and His between the control and
treated bacterial cells. A similar trend was observed in the
JA + NaCl + LK11 treatment; however, the contents of some amino
acids (Asp, Glu, Gly, Ala, Leu, NH3, and Arg) were signicantly lower
than the LK11 + NaCl treatment (Supplementary information 2).
This is also in agreement with the results of the SDS-PAGE analyses,
in which relatively lower levels of protein expression were
detected for the LK11 + JA + NaCl treatment.
Some of the novel species of the bacterial genera, such as
Bacillus and Sphingomonas, possess specialised sets of genes either
in their circular chromosomes or plasmids to counteract high ionic
imbalance caused by inorganic chemical transport of metals or, in
the present case, Na+/Cl ions (Liu et al., 2014). Sphingomonas spp.
comprise gene sets that help them in surviving and sustaining their
growth prociently in harsh living conditions. LK11 genome
possesses genes to protect it from cellular ionic imbalances. The
genome comprising genes encoding glutathione peroxidases
(AV944_12175), i.e. three genes encoding glutathione S-transferase
(AV944_12110, AV944_13280, AV944_05350), a gene encoding
glutathione-disulde reductase (AV944_15970), and two genes
encoding superoxide dismutases (AV944_13570, AV944_06030).
Furthermore, the LK11 genome has ve genes encoding various
kinds of catalases and eight genes encoding peroxidases. Besides,
three genes encoding peroxiredoxin and two genes encoding
glutaredoxin were identied in the LK11 genome (unpublished
data). These ndings suggest that LK11 possess a strong network of
62
Fig. 1. Interactive dynamics of Sphingomonas sp. LK11 and exogenous jasmonic acid (JA; 100 mM) under high sodium chloride (NaCl; 500 mM) treatment. (a) Showing the
bacterial endophyte growth during various treatments. (b) Sodium (Na+) concentration was recorded on ICP-MS from the treated bacterial cells. (c) Effect of different
treatments on stress-responsive proline amino acid. (d) SDS-PAGE with Coomassie Brilliant blue (10%) was carried out on the extracted proteins from different treatments and
a Fermentas protein ladder (10170 kDa) was used as reference. The scatter plot represents the means values of three replicates and standard deviation. Different letter(s)
indicate signicant differences (P < 0.05) determined by Duncans multiple range test. (For interpretation of the references to colour in this gure legend, the reader is referred
to the web version of this article.)
genetic makeup, which could counteract the negative effects of

ionic stress, in current case, caused by Na+/Cl ions. Microbes
possessing such genes could be an interesting alternative to
mitigate salinity stress in economically important crops.
3.2. Plant-Microbe (tomato-LK11) interaction
In order to better understand the above-mentioned benecial
effects of PGPEB, we applied LK11 bacteria to the wild-type (S.
pimpinellifolium) and mutant Got-3 tomato. Got-3 mutant line
exhibits stunted growth due to its inability to produce high
glutamate oxaloacetate transaminase (Got), which is involved in a
major assimilation pathway for ammonia in most of the higher
plants and synthesis of glutamate and aspartate (Appels and
Haaker, 1991). Lack of such events in plant cells can devoid them of
the mechanisms used for resisting stressful conditions. Under
salinity stress, PGPEB (Sphingomonas sp. LK11) showed high
survival potential, which in turn might help increase the stresstolerance of tomato plants. Out results showed that, in case of
wild-type tomato plants, the sole application of JA and LK11 to the
root zones signicantly (P < 0.0001) increased the shoot length
compared to the untreated controls (Fig. 2). However, the shoot
length of plants treated solely with LK11 was relatively lower than
that of plants treated solely with JA (Fig. 2). In contrast, the
combined application of LK11 and JA signicantly increased the
shoot length compared to all other treatments. In case of Got-3

mutant tomato line, we observed similar patterns as those for the
wild-type tomato cultivar (Fig. 2); both LK11 and LK11 + JA
treatments considerably increased the growth of tomato plants,
but the sole application of JA showed only minor ameliorative
effects on Got-3 plants.
The root development was also signicantly enhanced by the
application of LK11 and JA in wild-type plants under no stress. Our
results showed that the application of LK11, JA, and LK11 + JA
signicantly increased the root length by 28.8, 26.2, and 29.1%,
respectively, as compared to the untreated control (Fig. 2). In case
of Got-3 plants, the application of LK11 cells alone and in
combination with JA signicantly increased the root length.
However, the increase in root length was slightly lower with the
combined JA + LK11 treatment as compared to the sole LK11
treatment. Contrarily, the sole application of JA showed reduced
root lengths, which were almost similar to control treatment in
Got-3 plants.
Variable effects were observed for wild-type and Got-3 plants in
response to the salinity stress coupled with JA and PGPEB
applications. Sole NaCl treatment drastically affected the growth
of both kinds of plants. The shoot growth was signicantly low
among all treatments. The wild-type plants exhibited a relatively
lower level of sensitivity to salinity stress and showed somewhat
stronger plant growth attributes than the Got-3 plants (Fig. 2).
63
Fig. 2. Synergistic effects of Sphingomonas sp. LK11 and jasmonic acid (JA) on the shoot growth (a) and phenotype (b) of the wild-type and Got-3 tomato plants with or without
NaCl treatment. The plant images are representative of 18 plants per treatment. Bars represent the means and standard deviation of three replicates. Different letter(s)
indicate signicant differences (P < 0.05) determined by Duncans multiple range test.
Surprisingly, the growth of wild-type tomato plants was found to

be signicantly reduced with the sole JA treatment as compared to
the other treatments under salinity stress. At the same time, the
sole LK11 and the combined LK11 and JA treatments effectively
counteracted the NaCl-induced salinity stress as compared to the
sole JA treatment and untreated controls. In the mutant Got-3 line,
however, distinct applications of JA and LK11 signicantly
increased the growth attributes of tomato plants as compared to
the combined JA + LK11 + NaCl treatment (Fig. 2; Table 1), with leaf
curling and chlorosis occurring more commonly in the combined
treatment. For wild type tomato plants, leaf primordia displayed
well-differentiated lobes and glandular hairs; however, these
attributes were poorly differentiated in Got-3 plants, for which leaf
curling was also signicantly more frequent in them (Fig. 2).
Similar negative effects were observed on the root lengths of
the wild-type and Got-3 plants during salinity stress. The results
showed that the sole LK11 and combined LK11 + JA treatment
signicantly increased the root length of the wild-type plants
under salinity stress as compared to the sole JA treatment and
untreated control. The sole application of JA signicantly reduced
the root growth under salinity stress (Fig. 2). In case of Got-3
plants, salinity stress adversely affected the root growth and
development. However, these negative effects were ameliorated by

the sole application of LK11 and up to some extent by JA, but the
combined application of both JA and LK11 did not rescue the root
growth in Got-3 plants.
3.3. Regulation of glutathione by JA and LK11 treatments
Glutathione, a non-protein thiol common in plants, regulates
stress responses and maintains intracellular redox state by
reacting with reactive oxygen species (ROS). The presence of
glutathione may ameliorate plant growth and enhance salt
tolerance in plants (Chen et al., 2012; Csiszr et al., 2011).
Concentrations of glutathione were found to be signicantly
higher (P < 0.0001) in the combined JA + LK11 treatment under
salinity stress as compared to the other treatments (Fig. 3). The
application of JA alone also resulted in high concentrations of
glutathione in wild-type tomato plants. However, JA + NaCl, sole
NaCl, and LK11 + JA treatments showed signicantly lower
glutathione contents in wild-type tomato plants.
For Got-3 plants, glutathione concentrations were signicantly
higher in the sole LK11 and combined LK11 + NaCl treatments as
compared to the other treatments and the control. JA treatment, on
Table 1
Interaction effects of LK11, JA, and NaCl on the growth of normal and Got-3 tomato cultivars.
Parameters
(LK11 JA NaCl)
Sum of square
Degrees of freedom
Mean square
Variation (%; interaction wild Got-3)
P value
Signicance
Shoot length
Root length
Glutathione
Abscisic acid
Salicylic acid
Jasmonic acid
669.9
442.74
6145
952866
42180
6145
32
32
6
6
6
6
20.94
11.2
1024
158811
7030
1024
0.7651
0.43
18.26
71.55
44.9
18.29
P < 0.0001
P < 0.0001
P < 0.0001
P < 0.0001
P < 0.0001
P < 0.0001
***
***
Highly signicant.
***
***
***
***
***
64
Fig. 3. Regulation of glutathione in wild-type (a) and Got-3 (b) tomato plants after
inoculation with Sphingomonas sp. LK11 and application of exogenous jasmonic acid
(JA) with or without NaCl treatment. Bars represent the means and standard
deviation of three replicates. Different letter(s) indicate signicant differences
(P < 0.05) determined by Duncans multiple range test.
the other hand, did not effectively activate the synthesis of

glutathione; however, JA in combination with LK11 increased the
content of glutathione in Got-3 plants under normal growth
conditions while a reverse of this effect was observed for the same
treatment under salinity stress conditions (Fig. 3; Table 1). Overall,
the synthesis of glutathione was signicantly (P < 0.0001) higher
in wild-type plants under different treatment conditions than in
Got-3 plants.
3.4. Effects of LK11 and JA treatment on ABA signalling
Endogenous phytohormones (ABA and JA) were quantied in all
treatments through deuterated internal standards during extraction and GCMS/SIM analysis. In wild-type tomato plants,
signicantly high amounts of endogenous ABA were observed in
response to NaCl treatment followed by LK11, JA + NaCl, and
LK11 + JA + NaCl treatments as compared to the untreated control
plants (Fig. 4). The sole JA and combined JA + LK11 treatments
showed almost similar levels of ABA accumulation. These results
suggested that the sole and combined applications of LK11 and JA
reduced the synthesis of ABA under saline conditions.
In Got-3 plants, ABA accumulation was signicant as compared
to the untreated control and sole NaCl treated plants (Fig. 4). LK11
application resulted in an increase in ABA synthesis, but this was
signicantly lower than that in the control and sole NaCl treatment
(Fig. 5). However, the accumulation of ABA with the sole treatment
of JA was signicantly higher than that with the combined
LK11 + JA treatment. In addition, ABA accumulation was signicantly lower in JA + NaCl and LK11 + JA + NaCl combined treatments
as compared to the NaCl treatment. In conclusion, ABA regulation
in response to various treatments was signicantly higher in Got-3
plants than the wild-type plants. However, when the bacterial
Fig. 4. Regulation of the endogenous stress hormone Abscisic acid (ABA) following
application of Sphingomonas sp. LK11 and exogenous jasmonic acid (JA) with or
without NaCl treatment in wild-type and Got-3 tomato plants. Bars represent the
means and standard deviation of three replicates. Different letter(s) indicate
signicant differences (P < 0.05) determined by Duncans multiple range test.
inoculation and exogenous JA and NaCl treatments were applied,

ABA biosynthesis increased in wild-type (Fig. 4; Table 1).
3.5. SA contents during LK11 and NaCl treatments
SA is known to counteract the negative impacts of abiotic stress
and plays a role in the induced systemic resistance against PGPR
(Caarls et al., 2015). In wild-type tomato plants, SA accumulation
was found to be signicantly higher with JA + NaCl and LK11 + JA
combined treatment than with the sole JA treatment. However, the
sole LK11 and combined treatments exhibited signicantly lower
SA concentrations (Fig. 5). In Got-3 tomato plants, the SA content
was signicantly higher in plants inoculated with only LK11 under
salinity stress, followed by untreated control plants. However, the
sole NaCl treatment resulted in signicantly lower amount of SA
synthesis in Got-3 plants. The combined treatment of LK11 + JA +
NaCl led to signicantly high SA synthesis in Got-3 plants, whereas
the sole LK11 and the combined LK11 + JA treatments resulted in
almost similar levels of SA synthesis (Fig. 5; Table 1). These results
suggested that in wild-type plants, SA biosynthesis was activated
under salinity stress, whereas in Got-3 plants inoculated with LK11
and JA, SA biosynthesis was signicantly higher than the untreated
control plants under salinity stress.
3.6. Endogenous JA regulation by LK11 and salinity treatments
Endogenous JA was also quantied in wild-type and Got-3
tomato plants using GCMS/SIM and deuterated standards. The
results showed that JA synthesis was signicantly increased in
Fig. 5. Inuence of Sphingomonas sp. LK11 and exogenous jasmonic acid (JA) on the
endogenous stress hormones Salicylic acid (SA) with or without NaCl treatment in
normal and Got-3 tomato plants. Bars represent the means and standard deviation
of three replicates. Different letter(s) indicate signicant differences (P < 0.05)
determined by Duncans multiple range test.
both the sole JA and the combined LK11 + JA + NaCl treatments,

whereas the sole LK11 inoculation generated relatively less JA than
did the sole JA and combined treatments (Fig. 6). However, JA levels
in the wild-type plants were signicantly lower with the sole LK11
inoculation and combined LK11 + JA treatments as compared to the
untreated control plants. An irregular response of endogenous JA
was observed in response to the various treatments in wild-type
plants (Fig. 6). In contrast, and as with glutathione, JA biosynthesis
was substantially inhibited in Got-3 plants; JA synthesis was
signicantly lower in Got-3 plants than that in the wild-type
plants. Concentrations of JA were similar in control and LK11inoculated plants, but declined appreciably in plants with sole JA
and combined LK11 + JA treatments with or without NaCl (Fig. 6;
Table 1). The downward regulatory shift continued in the
combined LK11 + NaCl and LK11 + JA + NaCl treatments, with which
the concentrations of JA were signicantly lower than those with
the other treatments.
4. Discussion
Salinity imposes a spectrum of stresses on crop plants by
affecting their normal physiological, morphological, and biochemical functions (Shrivastava and Kumar, 2015). As a result, all
developmental process from seed germination till reproductive
stages are hampered due to the inadequate uptake of nutrients,
osmotic stress that triggers cell death, and oxidative stress that
impairs normal cell functions (Shrivastava and Kumar, 2015). The
ultimate approach is to develop an efcient and cost-effective
solution that could be widely practised without any constraint.
65
Fig. 6. Sphingomonas sp. LK11 and jasmonic acid (JA) application to the tomato
plants and regulation of endogenous stress hormones jasmonic acid (SA) with or
without NaCl treatment in normal and Got-3 tomato plants. Bars represent the
means and standard deviation of three replicates. Different letter(s) indicate
signicant differences (P < 0.05) determined by Duncans multiple range test.
One such environmentally friendly promising solution is the use of

plant growth-promoting microbes and phytohormones, which
exert their benecial effects via direct and indirect mechanisms.
Among these plant growth-promoting microbes, endophytic
bacteria have been identied as having an opportunistic role
and function in regulating host plant growth (Brader et al., 2014;
Dar et al., 2015; Hardoim et al., 2015). These bacteria, along with
their co-evolved fungal counterparts, offer a unique means of
nutrient and metabolite transport that helps in maintaining and
supporting host plant growth under both normal and extreme
environmental conditions, such as excessive salinity, drought, and
heat (Khan et al., 2015). This potential has been attributed to the
bacterial production of various physiologically active metabolites,
including hormones. Here, we examined the synergism between
the plant growth-promoting endophytic bacteria (PGPEB) Sphingomonas sp. LK11, which produces gibberellins (GA4; 2.97 ng mL1;
Khan et al., 2014) and grows well in saline environments (Halo
et al., 2015), and exogenously applied JA on tomato plants under
salinity stress.
Various studies have reported the salinity stress tolerance
ability of plant growth-promoting bacteria (PGPB) belonging to
different genera and characterised them by growing in media
amended with variable NaCl concentrations (Nadeem et al., 2016;
Natarajan et al., 2016). During exposure to salinity stress, these
PGPB not only sustain their growth but also exhibit certain plant
growth-promoting traits like production of phytohormones and
siderophores (Nadeem et al., 2016). In the presence of JA, the
growth of Sphingomonas sp. LK11 was found to be signicantly high
along with an increase in Na+ accumulation and a decrease in the
proline levels (Fig. 1), suggesting a synergism between endophytic
bacteria and phytohormones. However, the addition of NaCl to the
66
bacterial growth medium resulted in a signicantly higher proline

accumulation and reduced Na+ levels, suggesting the activation of
bacterial stress mitigation assembly, which either converts NaCl
into a stable product or safeguards the bacteria from the negative
effects of NaCl attack. Besides, proline accumulation is accompanied by the activation of Glu and Asp, which are used to replenish
the cellular pool of the glutamate pathway and act as osmoprotectants (Zaprasis et al., 2014).
The SDS-PAGE prole clearly showed signicantly high protein
expressions in Sphingomonas sp. LK11 with NaCl treatment as
compared to the bacteria growing under sole JA treatment (Fig. 1).
This was further veried by the increased levels of amino acids
detected among different treatments (Fig. 2). High contents of Ala,
Glu, Leu, and Asp in Sphingomonas sp. LK11 growing in the presence
of NaCl suggested the increased activation of these cell-wall
peptidoglycan amino acids in order to maintain cellular integrity
(Schleifer and Kandler, 1972; Turner et al., 2013). Increased
activation of the proteogenic amino acids (Glu, Asp, and Arg)
was observed in LK11 + NaCl treatment as compared to the sole
LK11 and combined treatments. Thus, the viability and structure of
bacterial cells were not disturbed as a result of NaCl stress. Utilizing
PGPEB under NaCl stress can be benecial as they counteract the
negative impacts of NaCl by regulating their own stress-tolerance
apparatus. Such regulatory networking of endophytes with crop
plants can extend their ameliorative responses to the plants
growing in the same niche, as previously shown by Hoper et al.
(2006), Tsuzuki et al. (2011), and Brader et al. (2014). In addition,
the presence of peroxiredoxin-, glutathione S-transferase-, and
glutaredoxin-related genes in LK11 cells suggests a higher ability of
this bacterial strain to counteract NaCl-induced salinity stress,
which could be used to mitigate the salinity stress of crop plants.
Although several studies have shown the advantages of using
plant growth-promoting rhizobacteria as a commercial inoculant
for crops, the use of PGPEB has not been thoroughly investigated.
Some studies have clearly demonstrated that PGPEB improve plant
growth traits in various crops, including tomato (Bordiec et al.,
2011; Khan et al., 2014; Romero et al., 2014; Halo et al., 2015; Yi
et al., 2015). In the current study, Sphingomonas sp. LK11
inoculation to wild-type and Got-3 tomato cultivars signicantly
increased plant growth attributes (Fig. 2), which is consistent with
the results of our previous work on the same strain (Khan et al.,
2014). The inoculation of Sphingomonas sp. LK11 also helped in
counteracting salinity stress in both wild-type and Got-3 tomato
plants.
In the current study, we used S. pimpinellifolium, which has been
a source of useful genes for many other important horticultural
traits (Mahuad et al., 2013) for understanding mechanisms of salt
tolerance. S. pimpinellifolium Got-3 is a glutamine oxoglutarate
aminotransferase (or glutamate synthase) mutant line that
exhibits stunted growth and limited responses to salinity. The
enzyme glutamate synthase catalyses the synthesis of two
glutamate molecules by transferring the amide group of glutamine
to 2-oxoglutarate. One glutamate serves as a substrate for the
glutamine synthetase, and the other glutamate is used for amino
acid metabolism (Appels and Haaker, 1991; Ivan, 2014). This cycle
represents the major assimilation pathway for ammonia in most of
the higher plants. Using physiologically variant plant strains is an
effective way of understanding stress responses as well as effects of
exogenous growth regulators and endophytic bacteria in plants
(Bacon et al., 2015). The presence of glutathione in plants is
essential for scavenging reactive oxygen species and electrophilic
compounds during oxidative stress (Chen et al., 2012); thus, the
accumulation of glutathione suggests regulation of oxidative stress
through PGPEB inoculation in plants (Nazar et al., 2015). Our
results demonstrated that PGPEB inoculation signicantly increased the growth of Got-3 mutant cultivars both in the presence
and absence of salinity stress (Fig. 3). In addition, LK11 also helped
in increasing glutathione synthesis in Got-3 plants under salinity
stress (Fig. 4), a response that is considered to be a global switch to
activate stress tolerance in Arabidopsis thaliana (Cheng et al., 2015).
In addition, glutathione regulates biotic and abiotic stress (see
review Gill et al., 2013; Diaz-Vivancos et al., 2015) and plays an
essential role in photosynthetic regulation of plants. Increased
glutathione levels were observed in LK11-treated wild-type/
mutant plants under salinity, indicating a relatively high ROS
scavenging potential, which in turn improved their shoot/root
growth as well as photosynthesis. Similarly Fatma et al. (2014)
suggested that glutathione levels could improve plant growth and
photosynthesis during salinity stress. The whole genomic sequence of LK11 (unpublished data) also revealed the presence of a
set of genes that help in counteracting stress induced through ionic
imbalances in bacterial cells by activating glutathione pathways.
Pietrini et al. (2003) suggested that the elevated levels of cytosolic
glutathione increase photosynthesis and activities of related
enzymes during heavy metal-induced ionic imbalance in Phragmites australis.
The application of exogenous JA proved to be signicantly
ameliorative to the growth of wild-type as well as Got-3 tomato
plants growing under salinity stress, but varying responses were
observed in both plants. The growth of wild-type plants treated
with JA + LK11 under saline conditions was enhanced, but the
opposite was observed for Got-3 plants (Fig. 3). Previous studies
have shown that the endogenous JA plays a role in plant responses
to abiotic and biotic stresses as well as in plant growth and
development (Wasternack et al., 2006; Dar et al., 2015; Kazan,
2015), and mitigates induced osmotic stress and extends stressresistance in various crop plants such as wheat (Qiu et al., 2014;
Shan et al., 2015), grey mangrove (Yan et al., 2015), grapevine
(Bidabadi et al., 2013), tomato (Mayak et al., 2004), and soy
(Bidabadi et al., 2013; Xia et al., 2015). However, in some plants,
higher levels of JA might act as a growth suppressant as its
application to the root zone might be harmful to some mycorrhizae
as well as root growth. Our results showed reduced root length in
wild-type and Got-3 tomato cultivars during normal and saline
conditions. Snchez-Romera et al. (2016) showed that JA positively
counteracts plant stress by demonstrating that JA application
increases root hydraulic activity during mycorrhizae symbiosis.
However, if JA concentration increases, it might inhibit plant
growth. In addition, some specic mutant is unable to decide
whether to defend itself from stress or benet from the microbial
association. As Got-3 plants were decient in glutathione, a potent
scavenger for reactive oxygen species during stress conditions in
plants, it was presumed that LK11 cells triggered their week
apparatus to counteract salinity stress, whereas JA could not
participate in combination. This might be true in the case of some
previous studies conducted by Kumari and Sudhakar (2003) and
Heinrich et al. (2013). A similar study by Yan et al. (2015) suggested
that the application of relatively high concentrations of JA could
inhibit glutathione synthesis as compared to the lower concentrations. The stunted growth of plants with the combined JA + NaCl
treatment might be due to changes in their signalling responses
under conditions of high salinity; however, these responses were
activated in plants treated with PGPEB. This is analogous to the
ndings of Heinrich et al. (2013), who showed that JA antagonises
the biosynthesis of gibberellins and reduces shoot growth;
however, this could be improved by treating plants with exogenous
gibberellic acid (GA3). The inhibitory effect of JA at high
concentrations (100 mM and 250 mM) was also indicated by the
reduction in growth and fresh and dry weights of groundnut
seedlings, an important leguminous crop (Kumari and Sudhakar,
2003). It has been suggested that JA diverts carbon and nitrogen
resources toward secondary metabolite development instead of
growth; however, further transcriptomic investigation is needed to

solve these underlying mechanisms involving the interactions of
endogenous phytohormones like ABA, JA, and SA.
Endogenous phytohormones are essential pre-requisites for
improving stress tolerance in plants. Previous studies have shown
that endogenous ABA synthesis increases two fold in plants
exposed to salinity or drought stress conditions (Larkindale and
Huang, 2005). However, a converse trend was observed when plant
growth-promoting fungus (Paecilomyces formosus LHL10) was
inoculated in plants under salinity and drought (Khan et al.,
2012, 2014). Relatively lower ABA concentrations have been related
to the attenuated stomatal closing ability (Aliniaeifard and
Meeteren, 2013), reduced stomatal sensitivity to closing signals
of fully developed leaves for a lower transpiration rate during
growth (Giday et al., 2014), and better osmotic adjustment to
salinity driving a more efcient water uptake (Aroca et al., 2006;
Yoshida et al., 2015). In current study, we observed a relatively
lower ABA content in wild-type plants than in Got-3 mutants
(Fig. 5). In Got-3 control plants with signicantly high ABA
contents, PGPEB and JA treatments with or with salinity stress
helped to regulate its synthesis. Signicantly low levels of ABA in
PGPEB under salinity stress suggested better stress management in
wild-type plants, but not in Got-3 plants, which showed
signicantly low growth with combined treatment. Treatment
with exogenous JA alone or in combination with PGPEB produced
low levels of endogenous ABA, but plants treated solely with JA
under salinity showed higher levels of ABA. Endogenous ABA tends
to accumulate in plants under stress conditions, which compromises plant growth and development, but exogenous plant growth
regulators, such as PGPEB or JA, tend to overcome such responses
by adopting either ABA-dependent or independent regimes
(Rodriguez et al., 1998; Roychoudhury et al., 2013).
A signicant increase in the endogenous JA levels in wild-type
plants in the absence of external stress is consistent with the
previously reported experiments involving even low levels of
exogenous JA application (Menzel et al., 2014). For instance,
spraying lima beans with low concentrations of exogenous JA
increased their endogenous JA levels and induced plant defence
responses. A signicant decrease in the endogenous JA levels of
wild-type plants treated with LK11 and LK11 + JA as compared to
the untreated control and sole JA treatments (Fig. 3) might indicate
a priming effecta well-known characteristic of endophytic
microorganisms. This might be the reason for the opposite
behaviour observed under salt-stress conditions, and extremely
high levels of endogenous JA accumulated in LK11 and LK11 + JA
treated plants. Furthermore, decreasing levels of the endogenous
JA might be indicative of delayed senescence and prolonged
lifespan under normal conditions or of enhanced response
mechanisms to salinity stress in plants treated with LK11 and
LK11 + JA. This was supported by the studies of Larkindale and
Huang (2005) and Conn et al. (2008), which showed that a marked
decrease in ABA contents induced the same priming effect of
endophytic actinobacteria by down-regulating systemic acquired
resistance (SAR) and jasmonic acid/ethylene (JA/ET) pathways
under normal conditions in Arabidopsis thaliana. However, defensive genes were highly expressed in the same inoculated plants
when challenged with pathogens. The behaviour of JA is quite
interesting as it gets down-regulated under normal conditions, but
hyper-accumulates under salinity stress in wild-type tomato
plants treated with LK11 + JA. Before further discussion, it is worth
mentioning that this sort of mechanism can only be ascribed to the
association of JA with LK11. Correlating the plant growth
promotion data of LK11-infected wild-type plants with or without
salinity stress in the presence of JA indicates the stress-mitigating
effect of LK11 bacteria. Under normal conditions, LK11 bacteria
might serve to neutralise the detrimental effects on plant growth
67
by ne-tuning the amount of JA to optimal levels, which is reected

in the signicant increase of plant growth compared to the other
treatments. It might also be true that the resultant high
concentrations of endogenous JA in treated plants were relocated
to the roots, which might induce resistance against necrotrophic
pathogens or herbivores (Sato et al., 2009; Kazan 2015). Moreover,
in the same treatment under salinity stress, LK11 might not have
induced the down-regulation of endogenous JA, but instead
increased JA levels for use as a defence against salinity stress, as
shown by the enhanced phenotypic results. Garcia-Abellan et al.
(2015) reported that the mutant tomato res (restored cell structure
by salinity) was capable of producing high levels of endogenous JA
that gets accumulated in the roots and inhibits plant growth, like
Got-3 plants, when treated with exogenous JA. Owing to the high
levels of JA in the roots of res, it showed marked growth inhibition
as compared to the wild-type plant. However, when both the res
mutant and wild-type plants were exposed to extremely high
salinity levels (200 mM), the opposite effect was observed, with
signicant increase in the growth characteristics, such as shoot
weight, root weight, chlorophyll content, and uorescence, of the
res mutant. However, in our study, growth characteristics of the
Got-3 mutants were unresponsive to JA application under normal
and salinity stress conditions.
In the presence of JA, SA induces plant defences against
biotrophic pathogens and extends plant tolerance during salinity
stress (Brodersen et al., 2006; Caarls et al., 2015). Exogenous SA
plays an essential role in ameliorating plant physiology during
salinity; it increases plant biomass, photosynthesis, cellular
membrane integrity, and salinity stress tolerance (Stevens et al.,
2006; Miura and Tada, 2014). During salinity stress, an increase in
the level of endogenous SA was accompanied by the stomatal
closure due to reactive oxygen species attack (Yang et al., 2004;
Miura and Tada, 2014). This is in agreement with our results which
showed that signicantly high SA was synthesised in response to
salinity and LK11 application; however, exogenous JA triggered it
under normal and stress conditions. These applications have
further enhanced the levels of endogenous SA in the glutathioneimpaired Got-3 mutants, which were unable to tackle the ROS
attack and hence recruited a higher amount of SA. Miura and Tada
(2014) also shared a similar opinion in their recent review. On the
other hand, JA and ABA counteract the negative impacts of
herbivore attack on plants (Larkindale and Huang, 2005; Vos et al.,
2015). An antagonistic relationship between SA and JA has been
reported (Kazan and Manners, 2008; Kazan, 2015). It can be
assumed that mitogen-activated protein kinase 4 (MAPK 4)
mediates the antagonistic effect (Brodersen et al., 2006) of the
exogenous JA on the endogenous SA content of wild tomato under
normal and salinity stress conditions, and vice versa for the mutant
Got-3 plants. This factor could be an underlying cause for the
susceptibility of Got-3 plants to salinity stress and consequent
reduction in plant growth. These results support the ndings of
Wang et al. (2001), who examined endogenous hormonal
regulation in the native wetland species Iris hexagona under salt
stress. They found that the up-regulation of endogenous JA and
ABA combined with the down-regulation of SA and indole-3 acetic
acid (IAA) enhanced adaptation to salinity stress. The upregulation of endogenous JA in response to salinity stress can be
further inferred from the results of Pedranzani et al. (2003), who
observed relatively high levels of endogenous JA in salt-tolerant
tomato cultivars in comparison to salt-susceptible cultivars.
Moreover, the accumulation of JA, especially in roots, was
considered to be important for salinity stress resistance. In
conclusion, the current study shows that PGPEB promulgate
benecial impacts to plant growth during salinity stress. The
application of JA in high concentrations can compromise the plant
growth (as shown by Heinrich et al., 2013), but the application of
68
LK11 bacteria positively inuence the growth attributes of tomato

plants under salinity stress. Our ndings indicate that the
combination of PGPEB and JA is not essentially harmful to plants;
however, both can reprogramme crop plant responses to overcome
the adverse effects of salinity stress.
Acknowledgements
This work was nancially supported by the National Research
Foundation of Korea (NRF), Ministry of Science, ICT and FuturePlanning (MSIP), Republic of Korea through a Basic-Science
Research Program (2014R1A1A2A10058022). The author wishes
to thank Ihsan Ullah for his help with the initial experiment.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
envexpbot.2016.09.009.
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Contents lists available at ScienceDirect

Environmental and Experimental Botany

Plant growth-promoting endophyte Sphingomonas sp. LK11 alleviates

cell-division impairment, and membrane degeneration (Munns

under various environmental stress conditions, including salinity

and reduce Cd2+ uptake, thus enhancing the expression of

coupled plasma mass spectrometry (ICP-MS) to quantify the Na+ in

2.5. Determination of glutathione

with 10 mL of diethyl ether and acetic acid (98:2, v/v). Upon

genetic makeup, which could counteract the negative effects of

shoot length compared to all other treatments. In case of Got-3

Surprisingly, the growth of wild-type tomato plants was found to

development. However, these negative effects were ameliorated by

Variation (%; interaction wild  Got-3)

the other hand, did not effectively activate the synthesis of

inoculation and exogenous JA and NaCl treatments were applied,

both the sole JA and the combined LK11 + JA + NaCl treatments,

One such environmentally friendly promising solution is the use of

bacterial growth medium resulted in a signicantly higher proline

growth; however, further transcriptomic investigation is needed to

by ne-tuning the amount of JA to optimal levels, which is reected

LK11 bacteria positively inuence the growth attributes of tomato

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Variation (%; interaction wild Got-3)