Plant Physiology and Biochemistry 80 (2014) 160e167

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Plant Physiology and Biochemistry

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

Research article

Amelioration of high salinity stress damage by plant growth-

promoting bacterial endophytes that contain ACC deaminase
Shimaila Ali, Trevor C. Charles, Bernard R. Glick*
Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1

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

Article history: Plant growth and productivity is negatively affected by soil salinity. However, it is predicted that plant
Received 23 October 2013 growth-promoting bacterial (PGPB) endophytes that contain 1-aminocyclopropane-1-carboxylate (ACC)
Accepted 5 April 2014 deaminase (E.C. 4.1.99.4) can facilitate plant growth and development in the presence of a number of
Available online 18 April 2014
different stresses. In present study, the ability of ACC deaminase containing PGPB endophytes Pseudo-
monas fluorescens YsS6, Pseudomonas migulae 8R6, and their ACC deaminase deficient mutants to
Keywords:
promote tomato plant growth in the absence of salt and under two different levels of salt stress (165 mM
ACC deaminase
and 185 mM) was assessed. It was evidence that wild-type bacterial endophytes (P. fluorescens YsS6 and
Agricultural biotechnology
Ethylene
P. migulae 8R6) promoted tomato plant growth significantly even in the absence of stress (salinity).
Plant growth-promoting bacterial Plants pretreated with wild-type ACC deaminase containing endophytic strains were healthier and grew
endophyte to a much larger size under high salinity stress compared to plants pretreated with the ACC deaminase
Salt stress amelioration deficient mutants or no bacterial treatment (control). The plants pretreated with ACC deaminase con-
taining bacterial endophytes exhibit higher fresh and dry biomass, higher chlorophyll contents, and a
greater number of flowers and buds than the other treatments. Since the only difference between wild-
type and mutant bacterial endophytes was ACC deaminase activity, it is concluded that this enzyme is
directly responsible for the different behavior of tomato plants in response to salt stress. The use of PGPB
endophytes with ACC deaminase activity has the potential to facilitate plant growth on land that is not
normally suitable for the majority of crops due to their high salt contents.
Ó 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction 2009; Chinnusamy et al., 2005) and plant growth-promoting bac-

Salinity of arable lands is a major problem in agriculture. It
causes a significant loss of crop productivity each year (Francois
et al., 1994). It is estimated that at least 20% of the irrigated lands
worldwide are salt affected (Pitman et al., 2002). It is necessary to
re-examine many of the existing approaches to agriculture that
presently include the use of chemical fertilizers, herbicides, fungi-
cides, and insecticides. Instead, sustainable agriculture will need to
make much greater use of both salt tolerant plants developed by
both conventional breeding and transgenic plants (for example, see
http://www.isaaa.org/inbrief/default.asp) (Ashraf and Akram,
Abbreviations: ACC, 1-Aminocyclopropane-1-carboxylate; ACCDþ, ACC deami-
nase positive; acdS, ACC deaminase gene; Amp, Ampicillin; AVG, L-a-(amino-
ethoxyvinyl)-glycine; IAA, Indoleacetic acid; Kan, Kanamycin; pBS, Plasmid
Bluescript; PGPB, Plant growth-promoting bacteria; Tc, Tetracycline; Try, Trypto-
phan; TSB, Tryptic soy broth.
* Corresponding author. Tel.: þ1 519 888 4567x32058; fax: þ1 519 746 0614.
E-mail address: glick@uwaterloo.ca (B.R. Glick).
terial endophytes (Glick, 2012).
Plant growth-promoting bacterial endophytes colonize healthy
plant tissues without causing any disease symptoms to the plants
(Bacon and Hinton, 2006). Plant growth-promoting bacterial en-
dophytes employ mechanisms similar to those used by rhizo-
spheric plant growth-promoting bacteria (PGPB). These
mechanisms include both direct and indirect, such as nitrogen
fixation (Compant et al., 2005), ammonia production (Marques
et al., 2010), solubilization of mineral phosphate (Verma et al.,
2001), siderophore production (Lodewyckx et al., 2002), and pro-
duction of plant hormones (Costacurta and Vanderleyden, 1995). In
addition, antibiotic production (Long et al., 2004), and lytic enzyme
production (Chernin et al., 2002) are two common modes of PGPB-
based biocontrol. In addition, plant growth-promoting bacterial
endophytes may promote plant growth as a consequence of
expressing the enzyme 1-aminocyclopropane-1-carboxylate (ACC)
deaminase which cleaves ACC to a-ketobutyrate and ammonia and
thereby decreases ethylene levels in host plants (Sessitsch et al.,
2005; Sun et al., 2009; Rashid et al., 2012; Penrose et al., 2001;
Glick, 2005; Glick et al., 1998).

http://dx.doi.org/10.1016/j.plaphy.2014.04.003
0981-9428/Ó 2014 Elsevier Masson SAS. All rights reserved.
 S. Ali et al. / Plant Physiology and Biochemistry 80 (2014) 160e167
Ethylene is an important plant growth regulator that functions
in the processes of root initiation, fruit ripening, seed germination,
flower wilting, leaf abscission, biosynthesis of other phytohor-
mones, and stress signaling (Abeles et al., 1992). Plants normally
synthesize only small amounts of ethylene; levels that typically
confer beneficial effects on plant growth and development (except
during fruit ripening when the ethylene concentration is much
higher). However, in response to various stresses, there is often a
significant rise in endogenous ethylene biosynthesis, called “stress
ethylene” (Abeles et al., 1992; Stearns and Glick, 2003). These
stresses may include flooding, drought, salinity, wounding, tem-
perature extremes, insect predation, pathogen infection, heavy
metals, organic pollutants, mechanical stress, and nutritional stress
(Stearns and Glick, 2003; Hyodo et al., 1991; Morgan and Drew,
1997). Plants typically behave similarly when they are either
exposed to an environmental stress stimulus or they are treated
with high levels of exogenous ethylene (Morgan and Drew, 1997).
Moreover, a considerable portion of the damage that occurs in
plants as a consequence of stress is due to the deleterious action of
stress ethylene and not necessarily from the direct effects of the
stress (Stearns and Glick, 2003; Van Loon and Fontaine, 1984). In
agreement with these observations, an overall accumulation of ACC
(the immediate precursor for ethylene) was observed in tomato
plant roots, xylem sap, and leaves when they were challenged with
100 mM salt stress (Albacete et al., 2008).
Considering the role of ethylene in the physiology of plants
under stress conditions, it is believed that agents that can lower the
ethylene levels in plants can be employed to facilitate plant growth
under a variety of environmental stresses. However the use of
various chemicals has a number of drawbacks, and treating plants
with high concentrations of chemicals is potentially phytotoxic and
environmentally hazardous (Abeles et al., 1992; Ali et al., 2012). In
this regard, bacterial endophytes that exhibit ACC deaminase ac-
tivity may be the best choice to help plants to keep the stress
ethylene concentration below the growth inhibitory point
following a particular stress (Glick, 1995). ACC deaminase con-
taining plant growth-promoting bacteria have been documented to
facilitate the growth of a variety of plants under high salinity
conditions. Gamalero and colleagues (Gamalero et al., 2010) re-
ported the plant growth-promoting interaction of Pseudomonas
putida UW4 and Gigaspora rosea BEG9 on the growth of cucumber
under salt stress conditions. In this experiment, 72 mM salt (NaCl)
stress was used and a significant difference in plant growth (root
and shoot fresh biomass) was observed between cucumber plants
treated with wild-type P. putida UW4, ACC deaminase mutant
bacteria and control (untreated) plants. Similarly, when canola
seeds were inoculated with ACC deaminase containing halotolerant
bacteria under high salinity (150 mM NaCl) conditions, the fresh
and dry mass of the treated plants was increased up to 47%
(Siddikee et al., 2010). When the effect of high salt (150 mM NaCl)
on the growth of red pepper seedlings in the presence or absence of
the plant growth-promoting ACC deaminase containing halotoler-
ant bacteria was evaluated, it was reported that up to 57% of the
production of stress ethylene was reduced when inoculated with
the bacterium, and the overall biomass of the inoculated plantlets
was similar to the no salt treatment control plant (Siddikee et al.,
2011). In addition, Saravanakumar and Samiyappan
(Saravanakumar and Samiyappan, 2007) reported that ACC
deaminase-containing Pseudomonas fluorescens strain TDK1
increased the vigour index of groundnut seedlings significantly
under saline (120 mM NaCl) conditions when compared with
plants inoculated with a Pseudomonas strain lacking ACC deami-
nase activity. Another group demonstrated a protective effect of
ACC deaminase containing rhizobacteria (Pseudomonas syringae,
Enterobacter aerogenes, and P. fluorescens) on the growth of maize
161
plants under high salt conditions (Nadeem et al., 2007). In a
different type of experiment, canola plants were genetically
transformed to constitutively express a bacterial ACC deaminase
gene. The transgenic canola was stressed with 0e200 mM salt
(NaCl) concentration, and the resultant data confirmed that, as a
consequence of the expression of the bacterial ACC deaminase, the
transgenic plants became resistant to the inhibitory salt concen-
trations and their fresh and dry weight and chlorophyll content
were significantly higher than the non-transformed plants
(Sergeeva et al., 2006). In addition to the above-mentioned exam-
ples, numerous reports from laboratories all over the world have
demonstrated the role of ACC deaminase-containing rhizospheric
plant growth-promoting bacteria in protecting plant growth in a
high salinity environment (Cheng et al., 2012; Yue et al., 2007; Jalili
et al., 2008; Ahmad et al., 2011; Sadrnia et al., 2011).
In this work, we have investigated the ability of two different
ACC deaminase-containing PGPB endophytes and their ACC
deaminase deficient mutants to promote the tomato plant (Sola-
num lycopersicum H 72 cv. Better Boy) growth under high salinity
stress.
2. Materials and methods
2.1. Bacterial strains and growth conditions
Two recently isolated bacterial endophytes (Rashid et al., 2012)
and their ACC deaminase minus mutants were used in this study. P.
fluorescens YsS6 and Pseudomonas migulae 8R6 were initially iso-
lated from tomato plants grown in soils collected from France and
Canada, respectively. Both strains possess ACC deaminase, promote
root elongation in canola (Brassica campestris) seedlings, produce
siderophores, synthesize indoleacetic acid (IAA) and solubilize
phosphate (Rashid et al., 2012). Briefly, the ACC deaminase deficient
mutants were constructed by the insertion of a tetracycline resis-
tance gene in the ACC deaminase gene of the bacterial endophyte
(shown in Fig. S1 and S2). It was confirmed that the ACC deaminase
deficient mutants no longer exhibit significant ACC deaminase ac-
tivity. The ACC deaminase activity of wild-type P. fluorescens YsS6
was 12.5 mmol mg1 h1 while the ACC deaminase deficient mutant
activity was 0.11 mmol mg1 h1; wild-type P. migulae 8R6 and its
ACC deaminase deficient mutant had activity levels of 10.9 and
0.03 mmol mg1 h1, respectively (Table S1). The apparent residual
enzyme activity in the two mutants (in both cases approx. 1% of
the wild-type value) is deemed to be insignificant and is within the
margin of error of this assay. In addition, the ACC deaminase defi-
cient mutants are relatively unaffected in their IAA production
when compared to the wild-type strains (Table S1).
All four bacterial strains (P. fluorescence YsS6, P. migulae 8R6 and
their ACC deaminase deficient mutants) were grown in 250 ml of
tryptic soy broth (TSB) with appropriate antibiotics (100 mg ml1
ampicillin for wild-type strains and 100 mg ml1 ampicillin and
10 mg ml1 tetracycline for the mutant strains) at 30  C for 24 h.
Overnight cultures were washed once with 0.85% NaCl and then
centrifuged at 4500 g for five minutes and then diluted with 0.85%
NaCl to an optical density of 0.25  0.02 at 600 nm (i.e. approxi-
mately 1.75  108e1.97  108 CFU/ml).
2.2. Plant material
Tomato (S. lycopersicum H 72 cv. Better Boy) seeds, purchased
from Stokes Seeds Ltd. (Thorold, ON, Canada), were surface steril-
ized by a 1 min treatment with 70% ethanol, 10 min with 1%
commercial bleach, and with several washes of sterile milli-Q wa-
ter. The seeds were then inoculated with bacterial cell suspensions
(O.D.600 0.25). The control seeds were treated with sterile Milli-Q
162 S. Ali et al. / Plant Physiology and Biochemistry 80 (2014) 160e167
water only. All of the treatments were incubated at room temper-
ature for 1.5 h.
2.3. Pot experimental set-up
Four tomato seeds (per 9.5 cm  11 cm plastic pot) pre-
inoculated either with wild-type bacteria, mutant bacteria, or no
bacteria (control) were planted in wet peat-based Sunshine4Ô mix
aggregate (w180 g dry weight) general-purpose plant growth
medium (Premier Horticulture, St. Catharines, Ontario, Canada).
The ingredients of the growth medium include Canadian
sphagnum peat moss, coarse perlite, and a starter nutrient charge
in a ratio of 14:03:22. After the seeds had been placed on the soil, a
one ml bacterial suspension was poured on the top of each seed,
and then the seeds were covered with the soil. All the plants were
arranged randomly in the Department of Biology green house
benches and grown from mid-March through August with no fer-
tilizer applications. A group of 24 pots (96 plants) was set for each
treatment, and the entire experiment was repeated two times. For
the first three weeks of the experiment, the pots were watered with
tap water in late morning of every other day. After three weeks,
plants were watered either with 165 mM salt, 185 mM salt, or no
salt (0 mM) the same time every other day. All the plants were
watered enough to ensure drainage to avoid salt build up in the
pots. At week 7, the plants were transferred to larger pots
(25 cm  30 cm) containing the same plant growth medium. After
week 11, plants were collected and fresh biomass was measured
and recorded. Then, the plants were dried in an oven at 85  C until
there was no further decrease in the weight; the dried shoot
biomass was also recorded.
2.4. Sodium content measurements
For the quantification of sodium content in the plant, 1 g of dried
plant material was digested in 20 ml of 1 M nitric acid (HNO3),
incubated at 70  C for 4 h, and then cooled at room temperature for
1 h (Cheng et al., 2007). The contents of the tubes were centrifuged
at 10,050 g for 10 min. An aliquot of 100 ml of the supernatant was
diluted to 1 ml with Milli-Q water. The sodium concentration was
measured using graphite furnace atomic absorption spectroscopy
(VarianSpectrAA 880 spectrophotometer; Varian, Palo Alto, Cali-
fornia, USA). The sodium concentration in the plant tissue was
calculated using a standard curve between 50 and 2000 mM of
sodium.
2.5. Chlorophyll measurements
One g of youngest fully expanded fresh leaves was treated with
5 ml of N,N-dimethylformamide in a small glass bottle with a tight
cap. The tube was incubated at 4  C in the dark for 48 h. The
absorbance of the resulting solution was read at 663 nm and
645 nm. The amount of total chlorophyll was calculated by using
the following formula:
Total chlorophyll ¼ ð20:2  A645 þ 8:02  A663 Þ  1=2
2.6. Presence of bacterial endophytes in plant tissue
Different parts of the plant (roots, stems, leaves, fruits) were
investigated for the presence of bacterial endophytes. Surface
sterilized plant tissue was homogenized in an alcohol-flamed
sterile mortar and pestle in 3x Ringer’s solution. The plant ho-
mogenate was incubated at room temperature for 1 h in an orbital
shaker. A series of serial dilutions was plated onto TSB agar plates,
and the plates were incubated at 30  C for 24e48 h. CFUs from each
plate were counted and 3 representative colonies from each plate
were selected for strain screening purposes.
2.7. Gnotobiotic root elongation assay
The ability of bacterial endophytes and their ACC deaminase
mutants to promote the growth of canola roots was carried out and
monitored as described by Penrose and Glick (Penrose et al., 2001).
Briefly, all test strains were grown under gnotobiotic conditions
and then diluted with 0.03 M MgSO4 to an OD of 0.15  0.02 at
600 nm. Canola seeds were surface sterilized by a 1 min treatment
with 70% ethanol, 10 min with 1% commercial bleach, and several
washes with sterile milli-Q water. The seeds were then treated with
the test strains (1 h incubation at room temperature) and growth
pouches were inoculated manually. As negative controls, surface
sterilized seeds were treated with 0.03 M MgSO4. Observations
were made on day five. A two-way ANOVA was performed to
evaluate plant growth-promotion by bacterial endophytes (if any)
at different levels of salt treatment; this test was also used to
analyze any endophyte and salinity interaction that affected plant
growth. Significant differences between groups were analyzed by
Tukey’s post-test for each test strain and compared with controls.
For each bar, the values represented by the same lower-case letter
are not significantly different at P < 0.05.
3. Results
The bacterial endophytes P. fluorescens YsS6 (YsS6WT),
P. migulae 8R6 (8R6WT), and their corresponding ACC deaminase
deficient mutants (YsS6M and 8R6M) were tested for their capacity
to facilitate plant growth under salt stress. Bacterial treatments
were given at the seed level whereas seeds treated with 0.03 M
MgSO4 only served as a control. Seeds treated with the wild-type
bacterial endophytes exhibited higher growth than the control-
treated plants while ACC deaminase deficient mutant-inoculated
plants displayed significantly lower growth than the ones that
were inoculated with the wild-type strains (Fig. 1a and b, Table 1),
and are more like the control (Fig. 2). Wild-type strain-inoculated
plants tended to prevent salt built-up in plant tissues, and more salt
was deposited per gram of dry biomass in the plants inoculated
with the mutant strains (Fig. 1c, Table 1). As a consequence of plant
growth-promotion, the chlorophyll content of plants inoculated
with wild-type bacterial endophytes was higher than plants inoc-
ulated with either the mutant strain or non-inoculated (control)
plants (Fig. 1d, Table 1). In addition, plants treated with the wild-
type strains showed early flowering and fruiting, and the
numbers of the flowers and buds were greater than plants treated
with either the mutants or the control (Table 2). On the other hand,
when these plants were stressed with either 165 mM or 185 mM
salt, the general effect of the bacterial endophytes on the growth of
the tomato plants was similar (Fig. 2). Wild-type strain-inoculated
plants significantly protected plants from salt stress. Furthermore,
when the control plants and mutant strain-inoculated plants were
watered with 165 mM salt, at week 11 their fresh and dry weight
was dramatically reduced (Fig. 3a and b, Table 1). Wild-type strains
of bacterial endophytes provided protection against salt stress by
limiting the build-up of salt in the plants therefore plants survived
longer. Fig. 3c shows that the wild-type strain-inoculated plants
contained a lower level of salt than the mutant-inoculated or non-
inoculated plants. Similarly, the chlorophyll content was also
different among the differently treated plants with the plant that
had been treated with the wild-type bacteria having the highest
chlorophyll content (Fig. 3d, Table 1). When the salt concentration
was increased to 185 mM, although the sodium content of the
 S. Ali et al. / Plant Physiology and Biochemistry 80 (2014) 160e167 163

Fig. 1. The effect of bacterial endophytes on the tomato plants growth. The effect of the bacterial endophytes P. fluorescens YsS6 wild type, P. fluorescens YsS6 ACC deaminase
deficient mutant, P. migulae 8R6 wild type, and P. migulae 8R6 ACC deaminase deficient mutant P. migulae 8R6 on shoot fresh weight (Panel a), shoot dry weight (Panel b), sodium
content (Panel c), and chlorophyll content (Panel d) of the tomato plants. Seeds treated with 0.03 M MgSO4 served as a control (C). A group of 24 pots (96 plants) was set for each
treatment, and the entire experiment was repeated two times. A two-way ANOVA was performed. Significant differences between groups were analyzed by Tukey’s post-test for
each test strain and compared with controls. For each bar, the values represented by the same lower-case letter are not significantly different at P < 0.05. All the measurements were
taken following 11 weeks of growth. Error bars indicate 1 standard error of the mean.

plants inoculated with the wild-type strains also increased (Fig. 4c,
Table 1), the overall fresh shoot weight for that treatment was not
significantly changed (Fig. 4a, Table 1) when compared with the
165 mM salt treatment plants (Fig. 2). Among the bacterial endo-
phytes, wild-type P. migulae 8R6 remediated the salt stress more
efficiently and provided better protection than the wild-type strain
of P. fluorescens YsS6.
The presence of bacterial endophytes in different plant tissue
was also investigated. Plant tissues were homogenized and then
checked for the presence of bacteria in the surface sterilized plant
tissues. Plants from the seeds that were inoculated with a bacterial
cell suspension (either wild-type or mutant) carry bacteria in their
internal tissues. Both strains and their respective ACC deaminase
deficient mutants colonized plant stem and root tissues with oc-
casional colonization of leaf tissues (Table 3). Interestingly, no ev-
idence for the presence of these bacteria was found in the tomato
fruit (Table 3). The bacteria were recognized and verified by their
ability to synthesize ACC deaminase, produce IAA, demonstrate
characteristic salt tolerance, and antibiotic resistance.
4. Discussion
High salinity in agricultural land is a worldwide problem. High
salt concentrations are plant growth inhibitory in many crops

Table 1
Plants inoculated with P. fluorescens YsS6 wild-type (YsS6WT), P. fluorescens YsS6 ACC deaminase deficient mutant (YsS6M), P. migulae 8R6 wild-type 8R6WT), and P. migulae
8R6 ACC deaminase deficient mutant (8R6M) in the presence of no salt, 165 mM salt, or 185 mM salt. Each value represents the mean value. Data was collected on week 11 after
seed sowing. Two e way ANOVA was performed followed by TUKEYS post-test for each treatment. For each number in a column, values represented by the same lower-case
letters are not significantly different at P < 0.05. Table key: FW, Fresh Weight; DW, Dry weight; Na, Sodium contents; and Chll, Chlorophyll contents.a

Treatment No salt 165 mM salt 185 mM salt

FW DW Na Chl. FW DW Na Chl. FW DW Na Chl.

a a a a a a a a a a a
Control 7.22 1.03 18.2 136.1 1.85 0.35 76.8 26.6 1.24 0.25 82.0 17.4a
8R6WT 12.8b 2.0b 15.4c 176.1b 8.97b 1.21b 63.8c 140c 8.89c 1.09c 69.3b 76.8c
8R6M 8.87a 1.34a 20.6b 148.4a 1.90a 0.38a 80.1a 22.6a 1.82a 0.37a 81.1a 17.4a
YsS6WT 10.7b 1.75b 14.1c 178.6b 7.40b 0.88b 69.2b 69.5b 7.0b 0.64b 72.9b 36.9b
YsS6M 7.03a 1.15a 18.2a 141.9a 1.87a 0.37a 78.1a 28.9a 1.40a 0.11a 80.7a 14.1a
a
Plants inoculated with P. fluorescens YsS6 wild-type (YsS6WT), P. fluorescens YsS6 ACC deaminase deficient mutant (YsS6M), P. migulae 8R6 wild-type 8R6WT), and
P. migulae 8R6 ACC deaminase deficient mutant (8R6M) in the presence of no salt, 165 mM salt, or 185 mM salt. Each value represents the mean value. Data was collected on
week 11 after seed sowing. Two e way ANOVA was performed followed by TUKEYS post-test for each treatment. For each number in a column, values represented by the same
lower-case letters are not significantly different at P < 0.05. Table key: FW, Fresh Weight; DW, Dry weight; Na, Sodium contents; and Chll, Chlorophyll contents.
164 S. Ali et al. / Plant Physiology and Biochemistry 80 (2014) 160e167

Fig. 2. Effect of bacterial endophytes on the growth of tomato plants in the presence and absence of salt. “A” represents control. “B” represents P. migulae 8R6 wild type. “C”
represents P. migulae 8R6 ACC deaminase deficient mutant. “D” represents P. fluorescens YsS6 wild type. “E” represents P. fluorescens YsS6 ACC deaminase deficient mutant. Bacterial
treatments were given at seed levels and salt treatment was started at 3rd week of seed sowing. Pictures were taken at the end of week 13 of seed sowing.

(Cheng et al., 2007; Cuartero and Fernández-Muñoz, 1998; Mayak
et al., 2004). Tomato is a dicotyledonous, ethylene and salt sensi-
tive important food crop; it has been previously documented that
when tomato plants are exposed to high levels of salt, their
ethylene production is increased (Mayak et al., 2004). ACC
deaminase-containing plant growth-promoting rhizospheric bac-
teria have been reported to facilitate plant growth under salt stress
by reducing the ethylene level that is produced as a consequence of
abiotic stress (i.e. stress ethylene) (Cheng et al., 2007; Mayak et al.,
2004). In previous studies, 1) mainly rhizospheric plant growth-
promoting bacteria were employed, 2) plants were grown and
observed for shorter time periods, and 3) plants were stressed with
relatively low salt concentrations. In the present study, two ACC
deaminase containing plant growth-promoting bacterial endo-
phytes P. fluorescens YsS6, P. migulae 8R6 and their ACC deaminase
deficient mutants were used. ACC deaminase is a key enzyme,
produced by bacteria, which helps in ameliorating plant stress
under a variety of abiotic and biotic stress conditions (Glick, 2005,
1995; Glick et al., 1998). In this study, when tomato seeds were
treated with the plant growth-promoting ACC deaminase positive
bacterial endophytes, the emerging plant was primed to deal with
the given salt stress more effectively and provided better protection
because of the presence of the “pre colonized-bacterial endo-
phytes” in various parts of the plant. Even in the absence of any
stress (no salt added), tomato seeds treated with the wild-type
bacterial endophytes exhibited dramatically better growth when
compared to the control plants (no bacterial treatment) (Fig. 1a, b
and 2, Table 1). To prove that ACC deaminase is the main modu-
lator of plant growth, the knockout mutants were constructed. It
was shown that plants those were pre-inoculated with wild-type
endophytes significantly promoted plant growth under gnotobi-
otic conditions than the corresponding ACC deaminase deficient

Table 2
Plants inoculated with P. fluorescens YsS6 wild-type (YsS6WT), P. fluorescens YsS6 ACC deaminase deficient mutant (YsS6M), P. migulae 8R6 wild-type 8R6WT), and P. migulae
8R6 ACC deaminase deficient mutant (8R6M) in the presence of no salt, 165 mM salt, or 185 mM salt. Each value is the mean value of four individual plants. Data was collected
on week 13 after seed sowing. One e way ANOVA was performed followed by TUKEYS post-test for each treatment. For each number in a column, values represented by the
same lower-case letters are not significantly different at P < 0.05.a

Treatment No salt 165 mM salt 185 mM salt

Flower Bud Fruit Flower Bud Fruit Flower Bud Fruit

a a a a a a a a
Control 5.0 3.5 0.5 0.5 0 0 0 0 0a
8R6WT 7.5b 2.5a 1a 1.5b 2.5b 0.25b 3.5b 2b 0.25b
8R6M 4.5a 4.5a 1a 0.5a 1a 0a 0.5a 2b 0a
YsS6WT 8.5b 7b 2.5b 3b 4b 0.5b 2.5b 4c 0.25b
YsS6M 3.0a 4.5a 0.5a 1a 0.5a 0a 0a 0a 0a
a
Plants inoculated with P. fluorescens YsS6 wild-type (YsS6WT), P. fluorescens YsS6 ACC deaminase deficient mutant (YsS6M), P. migulae 8R6 wild-type 8R6WT), and
P. migulae 8R6 ACC deaminase deficient mutant (8R6M) in the presence of no salt, 165 mM salt, or 185 mM salt. Each value is the mean value of four individual plants. Data was
collected on week 13 after seed sowing. One e way ANOVA was performed followed by TUKEYS post-test for each treatment. For each number in a column, values represented
by the same lower-case letters are not significantly different at P < 0.05.
 S. Ali et al. / Plant Physiology and Biochemistry 80 (2014) 160e167 165

Fig. 3. The effect of the bacterial endophytes on the growth of tomato plant in the presence of 165 mM salt. The effect of the bacterial endophytes P. fluorescens YsS6 wild type,
P. fluorescens YsS6 ACC deaminase deficient mutant, P. migulae 8R6 wild type, and P. migulae 8R6 ACC deaminase deficient mutant on the fresh shoot mass (Panel a), dry shoot mass
(Panel b), sodium content (Panel c), and chlorophyll content (Panel d) of the tomato plants in the presence of 165 mM salt: Seeds treated with 0.03 M MgSO4, (no salt, no bacteria)
served as control (1st bar). For the test strains, strains were grown and diluted with 0.03 M MgSO4 to achieve an OD of 0.25  0.02 at 600 nm. Surface sterilized seeds were
inoculated with the test strains (1 h incubation at room temperature) and sown in pots manually. Salt treatment was started at 3rd week of seed sowing. A group of 24 pots (96
plants) was set for each treatment, and the entire experiment was repeated two times. A two-way ANOVA was performed. Significant differences between groups were analyzed by
Tukey’s post-test for each test strain and compared with controls. For each bar, the values represented by the same lower-case letter are not significantly different at P < 0.05. All the
measurements were taken following 11 weeks of growth. Error bars indicate 1 standard error of the mean.

mutants (Fig. S3). It was previously observed that when tomato
plants were pre-treated with the ACC deaminase containing the
rhizospheric plant growth-promoting bacterium Achromobacter
piechaudii ARV8, and grown under high salt conditions (up to
172 mM), although the stress-mediated ethylene production by the
tomato plants was significantly reduced, when salt was added the
sodium ion concentration of plant shoots was statistically the same
in the presence and absence of the bacterium (Mayak et al., 2004).
In those experiments, since the added wild-type bacterium pro-
moted plant growth, the sodium content (i.e. concentration times
biomass) was actually increased. Interestingly, when the effect of
salt (150 mM) on the growth of canola in the presence of an ACC
deaminase containing plant growth-promoting rhizospheric bac-
terium P. putida UW4 was evaluated, the results were somewhat
different (Cheng et al., 2007). The wild-type rhizospheric bacterium
tends to facilitate the accumulation of much higher concentrations
of sodium in the shoots than the untreated plant or its ACC
deaminase mutant does. However, in both reports, the biomass of
the wild-type treated plants was significantly increased (Cheng
et al., 2007; Mayak et al., 2004). It seems that different plant
growth-promoting bacteria affect plants differently under (salt)
stress conditions. Rhizospheric binding bacteria (such as P. putida
UW4) allow the salt to accumulate and presumably partition into
the vacuole (away from the cytosolic metabolism so that it cannot
affect plant growth) but facilitate plant growth by keeping the
stress ethylene level low with ACC deaminase activity. On the other
hand, plant growth-promoting bacterial endophytes limit the
concentration of sodium in plant shoots (Figs. 1c, 3c and 4c,
Table 1). The mechanism by which the bacterium does this, and
whether this effect is plant specific, is unknown.
In the treatment of tomato plants with 185 mM salt, plants
behaved in a similar way to what was observed at the lower level of
salt, although the condition of the control plants and the ACC
deaminase mutant inoculated plants were worse than the 165 mM
salt treatment (Fig. 2). The plants, either inoculated with the ACC
deaminase deficient mutants or no bacterial inoculation, were
completely dead before week 11. However, wild-type strains of
bacterial endophytes P. fluorescens YsS6 and P. migulae 8R6 pro-
tected tomato plants against salt stress and facilitated their growth
under these harsh conditions (Fig. 4, Table 1).
Leaf chlorophyll concentration is an important indicator of salt
tolerance and responses to increasing salinity (Percival et al., 2003).
Increasing the salt concentration from 0 to 185 mM had the effect of
progressively decreasing the chlorophyll contents in both plants
pretreated with wild-type bacterial endophytes, their ACC deami-
nase mutants, and control tomato plants while at the same time
increasing the amount of sodium in the plant, with the largest
changes occurring at 185 mM salt. However, it was observed that
inoculation with wild-type bacterial endophytes also significantly
increased the total chlorophyll contents of tomato plants compared
with their ACC deaminase mutants and control (Figs. 1d, 3d and 4d,
Table 1). Increased ethylene levels by salt stress can cause senes-
cence (Arshad and Frankenberger, 2002), but in the presence of ACC
deaminase-containing bacterial endophytes P. fluorescence YsS6 or
P. migulae 8R6, ethylene synthesis was significantly suppressed so
that the chlorophyll decay was decreased. Numerous others
166 S. Ali et al. / Plant Physiology and Biochemistry 80 (2014) 160e167

Fig. 4. The effect of the bacterial endophytes on the growth of tomato plant in the presence of 185 mM salt. The effect of the bacterial endophytes P. fluorescens YsS6 wild type,
P. fluorescens YsS6 ACC deaminase deficient mutant, P. migulae 8R6 wild type, and P. migulae 8R6 ACC deaminase deficient mutant P. migulae 8R6 on the fresh shoot mass (Panel a),
dry shoot mass (Panel b), sodium content (Panel c), and chlorophyll content (Panel d) of the tomato plants in the presence of 185 mM salt: Seeds treated with 0.03 M MgSO4 (no salt,
no bacteria) served as control (1st bar). For the test strains, strains were grown and diluted with 0.03 M MgSO4 to achieve an OD of 0.25  0.02 at 600 nm. Surface sterilized seeds
were treated with the test strains (1 h incubation at room temperature) and sown in pots manually. Salt treatment was started at the 3rd week of seed sowing. A group of 24 pots
(96 plants) was set for each treatment, and the entire experiment was repeated two times. A two-way ANOVA was performed. Significant differences between groups were analyzed
by Tukey’s post-test for each test strain and compared with controls. For each bar, the values represented by the same lower-case letter are not significantly different at P < 0.05. All
the measurements were taken following 11 weeks of growth. Error bars indicate 1 standard error of the mean.

(Nadeem et al., 2007; Sergeeva et al., 2006; Glick et al., 1997; directly by both IAA and ACC deaminase. Notwithstanding minor
Marcelis and Van Hooijdonk, 1999)have also reported this effect changes in IAA synthesis (a 7.5e17.5% decrease or a 3.1% increase) in
of PGPB that contain ACC deaminase. the mutants, we believe that the decrease in plant growth pro-
Moreover, the IAA production by the ACC deaminase deficient motion that is observed under stressful conditions comes primarily
mutant of P. migulae 8R6 was decreased 7.5e17.5% as compared to from the absence of ACC deaminase in the mutant strains. Based on
its wild-type counterpart (Table S1). However, in case of the ACC unpublished observations in our laboratory, changes in the bacte-
deaminase deficient mutant of P. fluorescens YsS6, the IAA pro- rial IAA concentration of this magnitude are unlikely to have any
duction was decreased by 3.3% in the absence of the IAA precursor significant effect on plant growth promotion. Thus, when tested the
tryptophan, but was increased by 3.1% when the mutant strain was endophytic plant growth-promoting bacterium Burkholderia phy-
supplemented with exogenous tryptophan (Table S1). As pointed tofirmans PsJN, a complemented mutant strain (in the gene for ACC
out previously (Patten and Glick, 2002), plant growth is promoted deaminase) that produces double the wild-type level of ACC
deaminase and one sixth the amount of IAA synthesized by the
wild-type strain yields identical sized canola seedling root lengths
Table 3 (Sun et al., 2009). One important caveat here is that in practice, the
Endophytic bacterial populations in various parts of the tomato plants treated with measured ability of the bacterium to synthesize IAA reflects con-
P. fluorescens YsS6 wild-type (YsS6WT), P. fluorescens YsS6 ACC deaminase deficient
ditions in liquid culture in the presence of high levels of added
mutant (YsS6M), P. migulae 8R6 wild-type (8R6WT), P. migulae 8R6 ACC deaminase
deficient mutant (8R6M), and control (no bacterial treatment).a tryptophan while in planta free tryptophan levels are likely orders
of magnitude lower. In addition, there is no significant differences
Treatment CFU/g of fresh weight
found in the colonization abilities between wild-type and their
Root Stem Leaf Fruit respective ACC deaminase deficient mutant stains (Table 3).
3 3
8R6WT 7.1  10 2.8  10 0 0
8R6M 6.5  103 2.5  103 2 0 5. Conclusion
YsS6WT 1.3  103 4.8  103 0 0
YsS6M 0.9  103 5.0  103 0 0
Control 18 23 1 0 Bacterial endophytes are plants’ natural companions and can
a
generally be isolated from the interior of various plant tissues. Since
Endophytic bacterial populations in various parts of the tomato plants treated
with P. fluorescens YsS6 wild-type (YsS6WT), P. fluorescens YsS6 ACC deaminase
bacterial endophytes are better adapted to various host plants, they
deficient mutant (YsS6M), P. migulae 8R6 wild-type (8R6WT), P. migulae 8R6 ACC may be more beneficial in facilitating plant growth than their
deaminase deficient mutant (8R6M), and control (no bacterial treatment). counterpart, rhizospheric-binding bacteria. Moreover, being
 S. Ali et al. / Plant Physiology and Biochemistry 80 (2014) 160e167
natural flora of almost all plants, there may be less regulatory
limitation to their deliberate release into the environment. The
current study proved that ACC deaminase containing bacterial
endophytes increase the resistance of tomato plants to salt and help
them to grow under high salt stress conditions.
Author’s contribution
SA carried out the experiments, conducted the analysis, and
drafted the manuscript. BRG and TCC participated in the design of
the study and drafted the manuscript. All authors have read and
approved the final manuscript.
Acknowledgments
We are very thankful to Lynn Hoyles for taking care of plants in
the green house and to Dr. James McGeer and Katherine Chan of
Wilfrid Laurier University, Waterloo, Ontario for their help with
atomic absorption spectrometry.
This work was supported by grants from the Natural Sciences
and Engineering Research Council of Canada to B.R.G. and T.C.C.
S.A. is the recipient of scholarship from the Higher Education
Commission (H.E.C.), Pakistan.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.plaphy.2014.04.003.
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