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ORIGINAL RESEARCH
Inoculation of ACC deaminase-producing endophytic
bacteria down-regulates ethylene-induced pathogenesis-
related signaling in red pepper (Capsicum annuum L.) under salt
stress
Aritra Roy Choudhury
1,2
| Pankaj Trivedi
2
| Jeongyun Choi
1,2
|
Munusamy Madhaiyan
3
| Jung-Ho Park
4,5
| Wonho Choi
4
|
Denver I. Walitang
1,6
| Tongmin Sa
1,7
1
Department of Environmental and Biological
Chemistry, Chungbuk National University,
Cheongju, South Korea
2
Microbiome Network and Department of
Agricultural Biology, Colorado State
University, Fort Collins, Colorado, USA
3
Singapore Institute of Food and
Biotechnology Innovation, Agency for Science,
Technology and Research (A*STAR),
Singapore, Singapore
4
Bio-Evaluation Center, Korea Research
Institute of Bioscience and Biotechnology,
Cheongju, South Korea
5
Department of Bioprocess Engineering,
University of Science and Technology of
Korea, Daejeon, South Korea
6
College of Agriculture, Fisheries and Forestry,
Romblon State University, Romblon,
Philippines
7
The Korean Academy of Science and
Technology, Seongnam, South Korea
Correspondence
Tongmin Sa, Department of Environmental
and Biological Chemistry, Chungbuk National
University, Cheongju, South Korea.
Email: tomsa@chungbuk.ac.kr
Funding information
National Research Foundation of Korea,
Grant/Award Number: 2021R1A2C1006608
Edited by B. Huang
Abstract
Pathogenesis-related (PR) signaling plays multiple roles in plant development under
abiotic and biotic stress conditions and is regulated by a plethora of plant physiologi-
cal as well as external factors. Here, our study was conducted to evaluate the role of
an ACC deaminase-producing endophytic bacteria in regulating ethylene-induced PR
signaling in red pepper plants under salt stress. We also evaluated the efficiency of
the bacteria in down-regulating the PR signaling for efficient colonization and
persistence in the plant endosphere. We used a characteristic endophyte, Methylo-
bacterium oryzae CBMB20 and its ACC deaminase knockdown mutant (acdS
). The
wild-type M. oryzae CBMB20 was able to decrease ethylene emission by 23% com-
pared to the noninoculated and acdS
M. oryzae CBMB20 inoculated plants under
salt stress. The increase in ethylene emission resulted in enhanced hydrogen peroxide
concentration, phenylalanine ammonia-lyase activity, β-1,3 glucanase activity, and
expression profiles of WRKY,CaPR1, and CaPTI1 genes that are typical salt stress and
PR signaling factors. Furthermore, the inoculation of both the bacterial strains had
shown induction of PR signaling under normal conditions during the initial inoculation
period. However, wild-type M. oryzae CBMB20 was able to down-regulate the ethyl-
ene-induced PR signaling under salt stress and enhance plant growth and stress toler-
ance. Collectively, ACC deaminase-producing endophytic bacteria down-regulate the
salt stress-mediated PR signaling in plants by regulating the stress ethylene emission
levels and this suggests a new paradigm in efficient colonization and persistence of
ACC deaminase-producing endophytic bacteria for better plant growth and
productivity.
1|INTRODUCTION
Salt stress can induce a plethora of perturbations that can prove to be
detrimental to plant physiological and biochemical processes. The typ-
ical salt-induced perturbations can be attributed to ion toxicity,
oxidative stress, and elevated ethylene emissions leading to the acti-
vation of stress response machinery (Chatterjee et al., 2017,2018,
2019; Roy Choudhury et al., 2021; Samaddar et al., 2019). However,
recent studies have also shown that abiotic stresses can induce
pathogenesis-related (PR) signaling in plants (Pacheco et al., 2013; Roy
Received: 17 January 2023 Revised: 19 March 2023 Accepted: 3 April 2023
DOI: 10.1111/ppl.13909
Physiologia Plantarum
Physiologia Plantarum. 2023;175:e13909. wileyonlinelibrary.com/journal/ppl © 2023 Scandinavian Plant Physiology Society. 1of11
https://doi.org/10.1111/ppl.13909
Choudhury et al., 2022; Song et al., 2016) that are typically induced by
biotic stress factors (Yim et al., 2013,2014). PR signaling can be acti-
vated by various effector molecules and proteins in plants during
pathogen attacks or herbivory (Ali et al., 2018). Ethylene emission and
subsequent reactive oxygen species (ROS) accumulation are major
drivers for inducing PR signaling (Dang et al., 2013; Xing et al., 2013).
These physiological responses that induce PR signaling are classic
responses for plants exposed to salt stress (Chatterjee et al., 2017,
2018,2019; Roy Choudhury et al., 2021; Samaddar et al., 2019).
PR signaling is mainly responsible for inducing host defense
responses and can be characterized as the primary immune response
mediator (Nomura et al., 2005). There are various transcription fac-
tors, genes, and enzymes that are upregulated when the plants are
exposed to biotic stress factors, such as WRKY transcription factors,
PR1 and PT1 genes, β-1,3 glucanase, and phenylalanine ammonia lyase
(PAL) (Pacheco et al., 2013; Roy Choudhury et al., 2022; Song
et al., 2016; Yim et al., 2013,2014). These effector molecules are
important for enhancing the stress tolerance of plants and recently,
tweaking PR signaling has been observed to enhance tolerance
against abiotic stresses (Ali et al., 2017; Liu et al., 2013; Seo
et al., 2008; Singh et al., 2013; Wu et al., 2016). In addition, inocula-
tion with ACC deaminase producing an endophytic microbial inoculant
has the potential to enhance the activities of PR proteins and upregu-
late gene expression profiles (Yim et al., 2013,2014). However, there
is a lack of studies that have explored the possible role of endophytic
bacterial inoculation in modulating PR signaling in plants under abiotic
stress conditions. On the other hand, a competent endophytic inocu-
lant is supposed to successfully colonize and persist in the plant endo-
sphere and prime against the host defense responses (Balmer
et al., 2015; Hardoim et al., 2008). There is a need to evaluate how
beneficial microbes are able to colonize and persist in the plant endo-
sphere and bypass the hosts' innate immune response . Also, there is
a lack of studies that concentrates on studying the efficiency of ACC
deaminase-producing bacterial inoculant on priming against host
defense responses for successful persistence in the plant endosphere.
Hence, this study was conducted to evaluate the efficiency of ACC
deaminase-producing endophytic bacteria in regulating ethylene-
mediated PR signaling in plants under salt stress and their ability to
prime against host defense responses for successful persistence in the
plant endosphere. To achieve these objectives, we used a well-
characterized ACC deaminase-producing Methylobacterium oryzae
CBMB20 and it's ACC deaminase knockdown (acdS
) mutant as inoc-
ulants for red pepper plants (Capsicum annum cv. Soobicho). We stud-
ied plant growth parameters, ethylene emissions, ROS accumulation,
PAL activity, β-1,3 glucanase activity, and expression profiles of candi-
date PR genes under normal and salt stress conditions. We observed
that the inoculation of both the bacterial strains had induced host
defense responses during the initial inoculation period and it sub-
verted to the level of noninoculated plants under normal conditions.
However, only the wild-type M. oryzae CBMB20 was able to down-
regulate the ethylene emission-induced PR signaling under salt stress
conditions which suggests a novel pathway in plant–bacteria interac-
tions for efficient colonization of the plant endosphere to modulate
the stress response machinery.
2|MATERIALS AND METHODS
2.1 |Plant material, growth conditions, and
bacterial inoculation
Red pepper seeds (Capsicum annuum cv. Soobicho; Syngenta seeds)
were surface sterilized as per Samaddar et al. (2019). Briefly, the seeds
were treated with 2% NaOCl followed by 70% ethanol and three
washes with sterile distilled water. The seeds were incubated in dark
at 30C for 72 h on a moist sterile filter paper for germination. The
germinated seeds were transplanted into plastic pots (dimensions:
inner diameter =9 cm; outer diameter =10 cm; depth =9 cm) con-
taining 150 g nursery soil (composition: cocopeat 49.876%, peatmoss
25%, pearlite 12%, vermiculite 7%, zeolite 6%, pyroligneous liquor
0.004%, fertilizer 0.11%, wetting agent 0.01%; Nongwoo-Bio Co.,
Ltd., Yeoju-gun; Gyeonggi-do) and grown in greenhouse conditions
with 32C/28C day/night temperature, 70% humidity, and under
natural illumination.
Methylobacterium oryzae CBMB20 (WT), acdS knockdown M. oryzae
CBMB20 (acdS
), gfp-tagged wild-type CBMB20 (gfp
+
M. oryzae
CBMB20) and gfp-tagged acdS
CBMB20 (gfp
+
acdS
M. oryzae
CBMB20) were grown in ammonium mineral salt (AMS) medium, sup-
plied with 0.5% sodium succinate as the sole carbon source at 25C.
The construction of acdS knockdown M. oryzae CBMB20, gfp
+
M. oryzae CBMB20 and gfp
+
acdS
M. oryzae CBMB20 is provided in
the supplementary material. The induction of dCas9 for acdS knock-
down was carried out by adding 0.1% cumate at an OD
600
0.4–0.5
and grown until OD
600
0.8–1. The bacterial suspension was prepared
using 0.03 M MgSO
4
and the absorbance at OD
600
was maintained at
0.8 (10
8
CFU mL
1
). Bacterial inoculation was performed at 7 days
after transplantation by inoculating 10 mL of bacterial suspension into
the rhizosphere zone. Each treatment contained three replicates and a
total of six plants were used for each of them.
2.2 |Salt stress application
The salt stress was applied to the plants after 1 day of inoculation
(8 days after transplanting). Salt stress was applied by adding 10 mL
of 150 mM NaCl into the rhizosphere zone. The control plants were
watered with tap water throughout the experimental period and
water leaching was prohibited by watering the plants below the
water-holding capacity of the soil. The plants were harvested at 2, 9,
16, and 23 days after NaCl treatment. Plant growth parameters such
as shoot length, root length, and number of leaves were recorded
while harvesting and the dry weight of the plants was measured after
drying the plants at 70C for 72 h.
2.3 |Determination of ethylene emissions
Ethylene emissions from red pepper seedlings were determined in a
completely discrete experimental setup, as mentioned in Roy Choudh-
ury et al. (2021). Bacterization of 30 red pepper seeds, each with
2of11 ROY CHOUDHURY ET AL.
Physiologia Plantarum
wild-type M. oryzae CBMB20 and acdS
M. oryzae CBMB20, were
carried out separately by soaking them in bacterial inoculum for 4 h
with gentle shaking in a sterile flask. Noninoculated seeds were trea-
ted with 0.03 M MgSO
4
. The noninoculated and bacterized seeds
were placed inside a GC bottle. Distilled water (2 mL) was added to
the seeds and allowed to grow for 7 days in a growth chamber
(DS 54 GLP; DASOL Scientific Co., Ltd.). The seedlings were treated
with 2 mL of a 150 mM NaCl solution for the salt stress set up, dis-
tilled water was used for the control setup and the seedlings were
incubated for 4 h. The accumulated ethylene in the headspace was
analyzed using gas chromatograph (dsCHROM 6200, Donam Instru-
ments Inc.) equipped with a Poropak-Q column.
2.4 |Determination of hydrogen peroxide
concentration
The hydrogen peroxide (H
2
O
2
) concentration was measured as per
the protocol mentioned in Theocharis et al. (2012). Fresh leaf and root
samples (0.5 g) were ground with liquid nitrogen, homogenized with
5 mL cold acetone in an ice bath, and the homogenate was centri-
fuged at 6000 gfor 10 min. The supernatant (1 mL) was transferred
into a fresh test tube and 5% titanium sulfate (0.1 mL) and ammonia
(0.2 mL) were added to it. The contents were centrifuged at 10 000 g
for 10 min at 4C and the supernatant was discarded. The pellet was
resuspended in 2 mM H
2
SO
4
(5 mL) and the absorbance was recorded
at 415 nm using a UV–Vis spectrophotometer (UV-1601, Shimadzu
Corporation). H
2
O
2
content was determined using a standard curve
plotted with a known concentration of H
2
O
2
.
2.5 |Determination of β-1,3-glucanase activity
The β-1,3-glucanase activity was assayed by the laminarin-
dinitrosalicylic acid method (Pan et al., 1991). Briefly, an equal volume
(62.5 μL) of 4% laminarin and plant extract were mixed and incubated
at 40C for 10 min. Dinitrosalicylic acid (DNSA) reagent (375 μL) was
added to finish the reaction and further incubated in a boiling water
bath for 5 min. The resulting mixture was diluted with 4.5 mL distilled
water and the absorbance of the colored solution was recorded at
500 nm using UV–Vis spectrophotometer (UV-1601, Shimadzu Cor-
poration). The enzyme activity was finally expressed as 1 ng of
reduced glucose min
1
mg
1
protein.
2.6 |Determination of PAL activity
PAL activity was measured following the method of Dickerson et al.
(1984). The plant extract (100 μL) was mixed with 500 μL50mM
Tris HCl (pH 8.8), and 600 μL1mM
L-phenylalanine. The reaction
mixture was incubated at room temperature for 1 h and the reac-
tion termination was carried out by adding 2 N HCl. The organic
phase was extracted with 1.5 mL toluene and the absorbance of the
toluene phase was recorded at 290 nm using UV–Vis spectropho-
tometer (UV-1601, Shimadzu Corporation). Finally, the enzyme
activity was expressed as nmol trans-cinnamic acid released min-
1
mg
1
protein.
2.7 |Determination of superoxide dismutase and
catalase activities
Superoxide dismutase (SOD) and catalase (CAT) were extracted
by homogenizing ground leaf samples in 50 mM potassium phos-
phate buffer supplemented with 1% (w/v) polyvinylpyrrolidone.
The SOD activity was measured by monitoring the photochemical
reaction of nitro-blue tetrazolium at 560 nm, whereas CAT activ-
ity was measured by monitoring the ability of the enzyme extract
to convert hydrogen peroxide at 390 nm using a UV–Vis spectro-
photometer (UV-1601, Shimadzu Corporation; Roy Choudhury
et al., 2021).
2.8 |Determination of culturable bacterial
endophytes in the red pepper endosphere
The colony-forming units (CFU) of wild-type M. oryzae CBMB20-gfp
and gfp
+
acdS
M. oryzae CBMB20 were determined from surface
sterilized red pepper plants. Culturable bacterial populations were
noted after serial dilution and plating of 1 g crushed plant root or
shoot in ammonium salt media supplemented with 50 μgmL
1
kana-
mycin, incubated in 28C for 3 days.
2.9 |RNA extraction and quantitative reverse-
transcriptase PCR (qRT-PCR)
Total RNA was isolated from ground leaf tissues using the RNeasy
Plant Mini Kit (QIAGEN) following the manufacturer's protocol and
stored at 80C. cDNA was synthesized using Superscript III first
strand synthesis system (Invitrogen). The gene expression analyses
were carried out by quantitative reverse transcription (qRT)-PCR using
the CFX96 real-time system (Bio-Rad Laboratories) with the SYBR
Green master mix (iQ SYBR Green Supermix, Bio-Rad). Ubiquitin was
used as the internal control for the normalization of data. All experi-
ments were performed in triplicates with three repeats to ensure data
validity. A list of primers is available in Table S1.
2.10 |Statistical analysis
The experiments were carried out in a randomized block design.
The data were subjected to analysis of variance (ANOVA) and the
significant differences between means were determined by least sig-
nificant difference (LSD) at p< 0.05 using the SAS package, Ver-
sion 9.4.
ROY CHOUDHURY ET AL.3of11
Physiologia Plantarum
3|RESULTS AND DISCUSSION
3.1 |ACC deaminase is important for improving
plant growth parameters under salt stress
Salt stress reduces plant growth and ACC deaminase-producing
bacteria have been documented to enhance plant growth parame-
ters (Chatterjee et al., 2017; Roy Choudhury et al., 2021;
Samaddar et al., 2019). In this study, the plant growth parameters
were measured in terms of shoot length, root length, plant dry
weight, and number of leaves (Table 1). The growth parameters
had significantly decreased under salt stress across all the treat-
ments irrespective of bacterial inoculation. However, the plants
inoculated with wild-type M. oryzae CBMB20hadrecordedsignif-
icantly higher growth parameters compared to noninoculated and
acdS
M. oryzae CBMB20 inoculated plants under salt stress. The
number of leaves, shoot length, root length, and plant dry weight
were approximately 11%, 14%, 16%, and 11% higher respectively
for wild-type M. oryzae CBMB20 inoculated plants compared to
the noninoculated and acdS
bacteria inoculated plants at 23 days
after salt stress (Table 1). On the other hand, both the wild type
and acdS
M. oryzae CBMB20 inoculated red pepper plants had
shown significantly higher plant growth parameters compared to
the noninoculated plants under normal conditions. The ability of
the acdS
M. oryzae CBMB20 to enhance plant growth can be
attributed to its multifunctional PGP characteristics such as IAA
production, cytokinin production, etc. which are important for
enhancing plant growth (Madhaiyan et al., 2006,2007). This
observation can be supported by a previous report where inocu-
lation of a ACCD deletion mutant Streptomyces spp. lacking indig-
enous IAA production ability was not able to enhance plant
growth under normal and salt stress conditions (Jaemsaeng
et al., 2018). Hence, ACC deaminase might not hold much signifi-
cance under nonstress conditions; however, it can be highly
regarded for enhancing plant growth under environmental stress
conditions.
TABLE 1 Growth parameters of red pepper plants at 2, 9, 16, and 23 days after salt stress and inoculated with wild type and acdS
Methylobacterium oryzae CBMB20.
DAS Inoculation Salt conc. (mM) Number of leaves Shoot length (cm) Root length (cm) Plant dry weight (mg)
2 NI 0 5.67 ± 0.23
a
8.38 ± 0.19
b
10.59 ± 0.39
b
63.67 ± 3.91
a
WT 0 6.06 ± 0.22
a
9.47 ± 0.26
a
14.27 ± 0.37
a
68.00 ± 4.47
a
acdS
0 6.06 ± 0.28
a
9.38 ± 0.20
a
4.03 ± 0.52
a
67.00 ± 3.72
a
NI 150 5.11 ± 0.35
a
7.56 ± 0.20
b
10.46 ± 0.43
b
51.33 ± 5.80
b
WT 150 5.39 ± 0.13
a
8.91 ± 0.20
a
13.86 ± 0.85
a
60.67 ± 3.16
a
acdS
150 5.33 ± 0.23
a
8.48 ± 0.41
a
12.98 ± 1.00
a
54.67 ± 2.94
ab
9 NI 0 6.72 ± 0.36
b
9.83 ± 0.19
b
11.99 ± 0.44
b
86.00 ± 8.81
b
WT 0 7.50 ± 0.23
a
10.95 ± 0.18
a
15.02 ± 0.89
a
100.33 ± 2.15
a
acdS
0 7.44 ± 0.05
a
10.66 ± 0.14
a
14.84 ± 0.76
a
99.67 ± 3.44
a
NI 150 5.56 ± 0.40
b
8.28 ± 0.67
b
10.72 ± 0.25
c
69.67 ± 11.39
b
WT 150 6.56 ± 0.13
a
9.63 ± 0.31
a
14.20 ± 0.70
a
79.67 ± 4.88
a
acdS
150 5.72 ± 0.25
b
8.94 ± 0.03
b
12.99 ± 0.18
b
71.00 ± 5.87a
b
16 NI 0 7.39 ± 0.42
b
10.47 ± 0.71
b
13.78 ± 1.08
b
89.74 ± 4.25
b
WT 0 8.39 ± 0.57
a
11.46 ± 0.41
a
15.69 ± 0.44
a
110.56 ± 11.57
a
acdS
0 8.06 ± 0.57
ab
11.36 ± 0.52
a
15.44 ± 0.72
a
105.89 ± 5.49
a
NI 150 6.11 ± 0.30
b
9.01 ± 0.27
b
12.41 ± 0.35
c
76.16 ± 2.81
b
WT 150 6.50 ± 0.17
a
10.2 ± 0.12
a
14.49 ± 0.37
a
85.56 ± 3.20
a
acdS
150 6.22 ± 0.28
b
9.22 ± 0.57
b
13.02 ± 0.12
b
77.91 ± 1.28
b
23 NI 0 8.61 ± 0.10
a
12.01 ± 0.17
b
14.13 ± 0.49
b
111.32 ± 3.51
b
WT 0 8.72 ± 0.05
a
12.29 ± 0.11
a
17.23 ± 0.37
a
129.54 ± 6.73
a
acdS
0 8.56 ± 0.18
a
12.01 ± 0.30
a
16.75 ± 0.71
a
132.84 ± 7.46
a
NI 150 6.50 ± 0.09
b
9.32 ± 0.36
b
12.81 ± 0.75
b
84.42 ± 1.79
b
WT 150 7.33 ± 0.54
a
10.68 ± 0.51
a
15.12 ± 0.12
a
94.28 ± 3.92
a
acdS
150 6.50 ± 0.17
b
9.34 ± 0.35
b
13.10 ± 0.48
b
85.90 ± 2.43
b
Note: All data represent the mean ± SD of three replicates and the differences between means were analyzed using LSD test (p< 0.05). Different letters
indicate significant differences within the salt treatments.
Abbreviations: acdS
, ACC deaminase knockdown mutant of M. oryzae CBMB20; DAS, days after salt stress; LSD, least significant difference; NI, Non-
inoculation; WT, wild-type M. oryzae CBMB20.
4of11 ROY CHOUDHURY ET AL.
Physiologia Plantarum
3.2 |Salt stress-induced elevated levels of
ethylene in red pepper seedlings
Elevated ethylene emissions are a major response of plants under
environmental stress conditions (Chatterjee et al., 2017; Madhaiyan
et al., 2006; Roy Choudhury et al., 2021; Samaddar et al., 2019).
Similar observations were drawn in the current study, where ethyl-
ene emission was observed to be significantly higher under salt
stress across treatments irrespective of bacterial inoculation. In addi-
tion, the noninoculated and acdS
M. oryzae CBMB20 inoculated
red pepper seedlings had shown significantly higher ethylene emis-
sion levels compared to the wild-type bacteria inoculated plants
under salt stress (Figure 1). The wild-type M. oryzae CBMB20 inocu-
lation resulted in approximately 23% reduction in ethylene emis-
sions compared to noninoculated and acdS
M. oryzae CBMB20
inoculated plants. The decrease in ethylene emissions from wild-
type M. oryzae CBMB20 inoculated plants were due to its ACC
deaminase activity which enables them to scavenge the precursor
of ethylene, ACC and use it as a nitrogen source (Madhaiyan
et al., 2006).
3.3 |Salt-induced ethylene emissions enhanced
ROS accumulation in red pepper
Salt stress can exert oxidative stress on plants through an accumula-
tion of ROS (Chatterjee et al., 2017; Roy Choudhury et al., 2021;
Samaddar et al., 2019), which has been reported to be regulated by
ethylene emissions (Yao et al., 2017). In this study, the hydrogen per-
oxide concentrations of red pepper plants were significantly higher
for salt-stressed plants compared to the control conditions in both
shoot and root, irrespective of bacterial inoculation (Figure 2). Salt
stress accumulated 2.5 fold and 3 fold higher H
2
O
2
in the shoots and
roots of wild-type M. oryzae CBMB20 inoculated plants (Figure 2B, D)
compared to the plants grown under nonstress conditions. Whereas
H
2
O
2
accumulation of 3.5 fold and 4 fold higher was recorded for
noninoculated and acdS
M. oryzae CBMB20 inoculated plants at
23 days after salt stress treatment compared to the plants grown
under nonstress conditions (Figure 2B,D). The higher ROS accumula-
tion under salt stress can be attributed to ethylene emission depen-
dent membrane-bound RBOHD protein which catalyzes the reaction
for synthesis of superoxides (Yao et al., 2017). However, the wild-type
M. oryzae CBMB20 inoculation resulted in a 40.37% and 32.59%
decrease in H
2
O
2
accumulation for both shoots and roots compared
to the noninoculated and acdS
M. oryzae CBMB20 inoculated plants
at 23 days after salt stress treatment, respectively (Figure 2B, D).
These results are in agreement to a previous study where M. oryzae
CBMB20 inoculation was able to decrease hydrogen peroxide con-
centration of tomato plants under salt stress by enhancing the antixo-
dant enzyme activities (Chanratana et al., 2019). The CAT and SOD
activities were significantly increased under salt stress across all treat-
ments compared to the plants grown in nonstress conditions
(Figure 3). Furthermore, in this study, the wild-type M. oryzae
CBMB20 inoculated plants had shown a 30.29% and 55.57% increase
in CAT and SOD activities compared to the noninoculated and acdS
M. oryzae CBMB20 inoculated plants (Figure 3B,D). Hence, these
results support the potential of ACC deaminase-producing endophytic
bacteria in ameliorating the toxic effect of ROS accumulation under
salt stress.
3.4 |Salt stress-induced ethylene emissions and
ROS accumulation enhanced PR protein activities
The increase in ROS concentrations can directly up-regulate PAL
activity, which can induce chloroplast dysfunction and cell death
(Xing et al., 2013). In the present study, the PAL activity was
observed to be significantly higher in salt-stressed plants across
treatments in both shoots and roots compared to the plants grown
under nonstress conditions (Figure 4). These results are in agree-
ment with previous studies where the up-regulation of PAL was
observed under salt stress in sugar cane (Pacheco et al., 2013) and
rice (Roy Choudhury et al., 2022). The increase in PAL activity
under salt stress conditions can be attributed to plant's ability to
FIGURE 1 Ethylene emissions from red
pepper seedlings. Effect of wild type and acdSˉ
Methylobacterium oryzae CBMB20 inoculation on
ethylene emissions from red pepper seedlings
imposed with 0 and 150 mM NaCl treatments.
Different letters indicate significant differences
within the salt treatments.
ROY CHOUDHURY ET AL.5of11
Physiologia Plantarum
catalyze the synthesis of secondary metabolites, which is important
for stabilizing ROS (Gao et al., 2011). Furthermore, the PAL activity
was observed to be significantly lower for the wild-type M. oryzae
CBMB20 inoculated salt-stressed plants at 2, 9, 16, and 23 days
after stress treatment (Figure 4B, D). The inoculation of wild-type
M. oryzae CBMB20 had recorded a 28.63% and 27.27% decrease in
PAL activities of shoots and roots at 23 days after salt stress treat-
ment compared to the noninoculated and acdS
M. oryzae CBMB20
inoculated plants, respectively (Figure 4B,D). This can be attributed
to the reduction in ROS accumulation due to a decrease in ethylene
emissions and enhanced antioxidant enzyme activities by ACC
deaminase-producing endophytic bacteria.
Salt stress also significantly enhanced β-1,3 glucanase activity
compared to the plants grown in control conditions (Figure 5). The
increase in β-1,3 glucanase activity are observed to be regulated by
ethylene emissions (Felix & Meins, 1987), as well as being attributed
to the expression of wounding-related genes under salt stress that
are typical in enhancing β-1,3 glucanase activity (Choudhury
et al., 2010; Dombrowski, 2003). Moreover, the ability of M. oryzae
CBMB20 to produce ACC deaminase and reduce the salt-stress-
mediated ethylene emission levels, resulted in a significant decrease
in β-1,3 glucanase activity compared to noninoculated and acdS
M. oryzae CBMB20-inoculated red pepper plants in both shoots and
roots under salt stress at 9, 16, and 23 days after stress treatment
(Figure 5B,D). The wild-type M. oryzae CBMB20 inoculation showed
a 21.8% and 36.6% decrease in β-1,3 glucanase activity in shoots
and roots of red pepper plants at 23 days after stress treatment
compared to the noninoculated and acdS
M. oryzae
CBMB20-inoculated plants, respectively (Figure 5B, D). The
decrease in PR protein activities during inoculation can be attributed
to the ability of ACC deaminase-producing endophytic bacteria in
reducing stress susceptibility by lowering ethylene emission levels
and in turn resulting in the modulation of ethylene-induced down-
stream signaling.
FIGURE 2 Reactive oxygen species concentration of red pepper plants under salt stress. Effect of wild type and acdSˉMethylobacterium
oryzae CBMB20 inoculation on hydrogen peroxide concentration in shoot (A, B) and root (C, D) of red pepper plants imposed with 0 and 150 mM
NaCl treatments at 2, 9, 16, and 23 days after stress treatment. All data represent the mean ± SD of three replicates and the differences between
means were analyzed using least significant difference (LSD) test ( p< 0.05).
FIGURE 3 Antioxidant enzyme
activities of red pepper plants. Effect of
wild type and acdSˉMethylobacterium
oryzae CBMB20 inoculation on
catalase (A, B) and superoxide
dismutase (C, D) activities of red
pepper plants imposed with 0 and
150 mM NaCl treatments at 2, 9,
16, and 23 days after stress treatment.
All data represent the mean ± SD of
three replicates and the differences
between means were analyzed using
LSD test (p< 0.05).
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Physiologia Plantarum
3.5 |PR genes are regulated by salt-induced
ethylene emission levels
The expression profiles of PR genes were characterized to evaluate
whether salt stress can induce their expression. WRKY gene expres-
sion profiles were significantly higher in the shoots and roots of
salt-stressed plants compared to the plants grown under control
conditions (Figure 6). These observations are in agreement with a
previous study where the high-throughput sequencing of RNAs
extracted from Capsicum annuum leaves showed higher expression
of WRKY genes under salt stress (Song et al., 2016). Also, WRKY
gene expressions and ethylene emissions have been observed to be
positively correlated under biotic stress conditions (Dang
et al., 2014), which might be a causation of the increase in WRKY
expressions upon enhanced salt stress-induced ethylene emissions.
Salt stress also significantly enhanced the expression of PR1 expres-
sion profiles in red pepper leaves compared to the plants grown in
control conditions (Figure 7A, B). Another study showed similar
FIGURE 4 Phenylalanine ammonia-lyase (PAL) activity of red pepper plants. Effect of wild type and acdSˉMethylobacterium oryzae CBMB20
inoculation on PAL activity in shoot (A, B) and root (C, D) of red pepper plants imposed with 0 and 150 mM NaCl treatments at 2, 9, 16, and
23 days after stress treatment. All data represent the mean ± SD of three replicates and the differences between means were analyzed using
least significant difference (LSD) test (p< 0.05).
FIGURE 5 β-1,3 glucanase activity of red pepper plants. Effect of wild type and acdSˉMethylobacterium oryzae CBMB20 inoculation on β-1,3
glucanase activity in shoot (A, B) and root (C, D) of red pepper plants imposed with 0 and 150 mM NaCl treatments at 2, 9, 16, and 23 days after
stress treatment. All data represent the mean ± SD of three replicates and the differences between means were analyzed using least significant
difference (LSD) test (p< 0.05).
ROY CHOUDHURY ET AL.7of11
Physiologia Plantarum
trends in increased PR1 expression profiles in cucumber plants
imposed with salt stress (Chojak-Ko´
zniewska et al., 2017). A typical
root localized ethylene response factor in red pepper, the CaPTI1
expression profile, was also significantly higher under salt stress
compared to the plants grown in normal conditions (Figure 7C, D).
Moreover, the wild-type M. oryzae CBMB20-inoculated plants had
significantly lower WRKY expression profiles at 16 and 23 days after
stress treatment in shoots and roots at 9, 16, and 23 days after
stress treatment compared to the noninoculated and acdS
M. oryzae CBMB20 inoculated plants under salt stress (Figure 6B,D).
Similarly, the shoot CaPR1 and root CaPTI1 expression profiles were
significantly lower for wild-type M. oryzae CBMB20-inoculated
plants compared to noninoculated and acdS
M. oryzae CBMB20
inoculated plants under salt stress (Figure 7B,D). Overall, the inocu-
lation of wild-type M. oryzae CBMB20 showed an approximate
range of 1.8–3folddecreaseinWRKY, CaPR1,andCaPTI1 gene
expression profiles compared to the noninoculated and acdS
M. oryzae CBMB20-inoculated red pepper plants under salt stress.
FIGURE 6 Gene expression profile of WRKY transcription factor of red pepper plants. Effect of wild type and acdSˉMethylobacterium oryzae
CBMB20 inoculation on WRKY gene expression profiles in shoot (A, B) and root (C, D) of red pepper plants imposed with 0 and 150 mM NaCl
treatments at 2, 9, 16, and 23 days after stress treatment. All data represent the mean ± SD of three replicates and the differences between
means were analyzed using least significant difference (LSD) test ( p< 0.05).
FIGURE 7 Gene expression profiles of shoot CaPR1 and root CaPTI1 of red pepper plants. Effect of wild type and acdSˉMethylobacterium
oryzae CBMB20 inoculation on CaPR1 gene expression profiles in shoot (A, B) and CaPTI1 gene expression profiles in root (C, D) of red pepper
plants imposed with 0 and 150 mM NaCl treatments at 2, 9, 16, and 23 days after stress treatment. All data represent the mean ± SD of three
replicates and the differences between means were analyzed using least significant difference (LSD) test ( p< 0.05).
8of11 ROY CHOUDHURY ET AL.
Physiologia Plantarum
The down-regulation of PR genes upon inoculation of ACC-
producing endophytic bacteria can be attributed to its ability in
decreasing ethylene emission levels and enhancing the stress toler-
ance of red pepper plants. These results are validated by previous
studies which established that WRKY,CaPR1,andCaPTI1 expression
profiles are modulated by the ethylene emission pathway under
stress conditions (Dang et al., 2013,2014;Huangetal.,2004;Jin
et al., 2016;Xuetal.,2007). Hence, the ACC deaminase activity of
M. oryzae resulting in decreasing the salt stress-mediated ethylene
emission levels is a characteristic trait in reducing the expression
profiles of these particular PR genes in planta.
3.6 |Beneficial bacteria can induce host defense
responses during initial inoculation period
Host defense responses are generally up-regulated during patho-
gen infection (Yim et al., 2013,2014). However, bacterial endo-
phytes need to be competent to colonize and persist in the plant
endosphere and it is also noteworthy that the colonization of bac-
terial endophytes can induce host defense responses (Compant
et al., 2005; Hardoim et al., 2008). In this study, both the wild
type and acdS
M. oryzae CBMB20-inoculated plants showed sig-
nificantly higher hydrogen peroxide concentrations in red pepper
plants grown under control conditions during the early inoculation
period (Figure 2A, C). Similar observations were also incurred for
PAL (Figure 4A, C)andβ-1,3 glucanase activities (Figure 5A,C)
where the bacteria-inoculated plants were observed to have sig-
nificantly higher enzyme activities compared to the noninoculated
plants grown under control conditions. These results are in agree-
ment with a previous study where the inoculation of Burkholderia
sp. enhanced the host defense responses by an accumulation of
polyphenol in grapevines (Compant et al., 2005). Similar observa-
tions were recorded for the expression profiles of WRKY in shoots
and roots (Figure 6A, C), shoot CaPR1 (Figure 7A)androotCaPTI1
(Figure 7C) expression profiles. The activation of these genes by
red pepper plants during the early stages of inoculation can be
attributed to the colonization of non-native bacterial endophytes
and the eventual decrease can be a potential response of bacteria
to successfully prime and sustain in the endosphere. In addition,
these are not dependent on the jasmonate biosynthesis pathway;
rather, they are tightly regulated by ethylene levels in plants
which minimize the restriction of colonization by bacterial endo-
phytes (Iniguez et al., 2005; Miché et al., 2006). Also, the CFU
count of wild type and acdS
M. oryzae CBMB20 in the endo-
sphere of shoot and root compartments of red pepper plants were
invariant when plants were grown under control conditions
(Figure 8A,C). These results are in line with another study, where
the CFU count of wild type and ACC deaminase knockout mutant
of Pseudomonas putida did not show any difference in the Arabi-
dopsis thaliana endosphere (Ravanbakhsh et al., 2019). Both the
wild type and the mutant bacterial strains were able to persist in
the endosphere of red pepper plants which validates the compe-
tence of M. oryzae CBMB20 as an effective endophytic inoculant.
On the other hand, the wild-type M. oryzae CBMB20 CFU count
was significantly higher under salt-stress conditions compared to
the CFU count of acdS
M. oryzae CBMB20 (Figure 8B,D). These
results might infer that the ability of ACC deaminase-producing
bacteria in utilizing the ethylene precursor ACC under environ-
mental stress conditions can enhance a mutualistic relationship
between the plant and the endophyte (Hardoim et al., 2008;
Pieterse et al., 2014).
FIGURE 8 Persistence of bacterial inoculant in the red pepper endosphere. Colony forming units of wild type and acdSˉMethylobacterium
oryzae CBMB20 in shoot (A, B) and root (C, D) endosphere of red pepper plants imposed with 0 and 150 mM NaCl treatments at 2, 9, 16, and
23 days after stress treatment. All data represent the mean ± SD of three replicates and the differences between means were analyzed using
least significant difference (LSD) test (p< 0.05).
ROY CHOUDHURY ET AL.9of11
Physiologia Plantarum
4|CONCLUSIONS
ACC deaminase is important for enhancing plant growth under salt-
stress conditions and holds minimal significance under normal envi-
ronmental conditions. On the other hand, salt stress can also induce
the activation of ethylene emission-mediated PR signaling in plants
and the inoculation of ACC deaminase-producing endophytic bacteria
has the ability to down-regulate PR signaling by modulating stress-
induced ethylene emissions. Also, plants inoculated with non-native
bacterial endophytes can induce PR signaling during the early stages
of inoculation but the beneficial effect as well as the competence of
the inoculant can subvert the host defense responses and successfully
persist in the plant endosphere. Overall, this study extrapolates the
reasons behind the efficiency of M. oryzae CBMB20 in alleviating vari-
ous environmental stresses and their beneficial interaction with multi-
ple plant hosts.
AUTHOR CONTRIBUTIONS
Aritra Roy Choudhury and Tongmin Sa conceptualized the study. Ari-
tra Roy Choudhury designed the experimental setup. Aritra Roy
Choudhury, Jeongyun Choi and Wonho Choi performed the experi-
ments. Jung-Ho Park provided the resources. Aritra Roy Choudhury
and Pankaj Trivedi did the data analysis and writing of the original
draft. Aritra Roy Choudhury, Pankaj Trivedi, Munusamy Madhaiyan,
Jung-Ho Park, and Denver I. Walitang wrote, critically reviewed, and
edited the work. Tongmin Sa was responsible for the supervision and
funding acquisition.
ACKNOWLEDGMENTS
We would like to thank Prof. Tobias J. Erb and Dr. Dae-Hee Lee for
providing the required plasmids for this study. This study was sup-
ported by the Basic Research Program through the National Research
Foundation (NRF) funded by the Ministry of Education, Science and
Technology (2021R1A2C1006608), Republic of Korea.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
ORCID
Aritra Roy Choudhury https://orcid.org/0000-0002-8373-1348
Pankaj Trivedi https://orcid.org/0000-0003-0173-2804
Jeongyun Choi https://orcid.org/0000-0001-8650-1735
Tongmin Sa https://orcid.org/0000-0001-7444-5852
REFERENCES
Ali, S., Ganai, B.A., Kamili, A.N., Bhat, A.A., Mir, Z.A., Bhat, J.A. et al. (2018)
Pathogenesis-related proteins and peptides as promising tools for
engineering plants with multiple stress tolerance. Microbiological
Research, 212, 29–37.
Ali, S., Mir, Z.A., Tyagi, A., Bhat, J.A., Chandrashekar, N., Papolu, P.K. et al.
(2017) Identification and comparative analysis of Brassica juncea
pathogenesis-related genes in response to hormonal, biotic and abiotic
stresses. Acta Physiologiae Plantarum, 39, 1–15.
Balmer, A., Pastor, V., Gamir, J., Flors, V. & Mauch-Mani, B. (2015) The
‘prime-ome’: towards a holistic approach to priming. Trends in Plant
Science, 20, 443–452.
Chojak-Ko´
zniewska, J., Linkiewicz, A., Sowa, S., Radzioch, M.A. &
Ku´
zniak, E. (2017) Interactive effects of salt stress and Pseudomonas
syringae pv. Lachrymans infection in cucumber: involvement of antioxi-
dant enzymes, abscisic acid and salicylic acid. Environmental and Experi-
mental Botany, 136, 9–20.
Chanratana, M., Joe, M.M., Choudhury, A.R., Anandham, R.,
Krishnamoorthy, R., Kim, K. et al. (2019) Physiological response of
tomato plant to chitosan-immobilized aggregated Methylobacterium
oryzae CBMB20 inoculation under salinity stress. 3 Biotech,9,1–13.
Chatterjee, P., Samaddar, S., Anandham, R., Kang, Y., Kim, K.,
Selvakumar, G. et al. (2017) Beneficial soil bacterium Pseudomonas fre-
deriksbergensis OS261 augments salt tolerance and promotes red pep-
per plant growth. Frontiers in Plant Science, 8, 705.
Chatterjee, P., Samaddar, S., Niinemets, Ü. & Sa, T.M. (2018) Brevibacter-
ium linens RS16 confers salt tolerance to Oryza sativa genotypes by
regulating antioxidant defense and H+ATPase activity. Microbiological
Research, 215, 89–101.
Chatterjee, P., Kanagendran, A., Samaddar, S., Pazouki, L., Sa, T.M. &
Niinemets, Ü. (2019) Methylobacterium oryzae CBMB20 influences
photosynthetic traits, volatile emission and ethylene metabolism in
Oryza sativa genotypes grown in salt stress conditions. Planta, 249,
1903–1919.
Choudhury, S.R., Roy, S., Singh, S.K. & Sengupta, D.N. (2010) Molecular
characterization and differential expression of β-1, 3-glucanase during
ripening in banana fruit in response to ethylene, auxin, ABA, wound-
ing, cold and light–dark cycles. Plant Cell Reports, 29, 813–828.
Compant, S., Reiter, B., Sessitsch, A., Nowak, J., Clément, C. & Barka, E.A.
(2005) Endophytic colonization of Vitis vinifera L. by plant growth-pro-
moting bacterium Burkholderia sp. strain PsJN. Applied and Environmen-
tal Microbiology, 71, 1685–1693.
Dang, F.F., Wang, Y.N., Yu, L.U., Eulgem, T., Lai, Y.A.N., Liu, Z.Q. et al.
(2013) CaWRKY40, a WRKY protein of pepper, plays an important
role in the regulation of tolerance to heat stress and resistance to Ral-
stonia solanacearum infection. Plant, Cell and Environment, 36,
757–774.
Dang, F., Wang, Y., She, J., Lei, Y., Liu, Z., Eulgem, T. et al. (2014) Overex-
pression of CaWRKY27, a subgroup IIe WRKY transcription factor of
Capsicum annuum, positively regulates tobacco resistance to Ralstonia
solanacearum infection. Physiologia Plantarum, 150, 397–411.
Dickerson, D.P., Pascholati, S.F., Hagerman, A.E., Butler, L.G. &
Nicholson, R.L. (1984) Phenylalanine ammonia-lyase and hydroxycin-
namate: CoA ligase in maize mesocotyls inoculated with Helminthos-
porium maydis or Helminthosporium carbonum.Physiological Plant
Pathology, 25, 111–123.
Dombrowski, J.E. (2003) Salt stress activation of wound-related genes in
tomato plants. Plant Physiology, 132, 2098–2107.
Felix, G. & Meins, F. (1987) Ethylene regulation of β-1, 3-glucanase in
tobacco. Planta, 172, 386–392.
Gao, L., Yan, X., Li, X., Guo, G., Hu, Y., Ma, W. et al. (2011) Proteome analy-
sis of wheat leaf under salt stress by two-dimensional difference gel
electrophoresis (2D-DIGE). Phytochemistry, 72, 1180–1191.
Hardoim, P.R., van Overbeek, L.S. & van Elsas, J.D. (2008) Properties of
bacterial endophytes and their proposed role in plant growth. Trends in
Microbiology, 16, 463–471.
Huang, Z., Zhang, Z., Zhang, X., Zhang, H., Huang, D. & Huang, R. (2004)
Tomato TERF1 modulates ethylene response and enhances osmotic
stress tolerance by activating expression of downstream genes. FEBS
Letters, 573, 110–116.
Iniguez, A.L., Dong, Y., Carter, H.D., Ahmer, B.M., Stone, J.M. &
Triplett, E.W. (2005) Regulation of enteric endophytic bacterial
colonization by plant defenses. Molecular Plant-Microbe Interactions,
18, 169–178.
10 of 11 ROY CHOUDHURY ET AL.
Physiologia Plantarum
Jaemsaeng, R., Jantasuriyarat, C. & Thamchaipenet, A. (2018) Molecular
interaction of 1-aminocyclopropane-1-carboxylate deaminase
(ACCD)-producing endophytic Streptomyces sp. GMKU 336 towards
salt-stress resistance of Oryza sativa L. cv. KDML105. Scientific
Reports,8,1–15.
Jin, J.H., Zhang, H.X., Tan, J.Y., Yan, M.J., Li, D.W., Khan, A. et al. (2016) A
new ethylene-responsive factor CaPTI1 gene of pepper (Capsicum
annuum L.) involved in the regulation of defense response to Phy-
tophthora capsici. Frontiers. Plant Science, 6, 1217.
Liu, W.X., Zhang, F.C., Zhang, W.Z., Song, L.F., Wu, W.H. & Chen, Y.F.
(2013) Arabidopsis Di19 functions as a transcription factor and modu-
lates PR1,PR2, and PR5 expression in response to drought stress.
Molecular Plant, 6, 1487–1502.
Madhaiyan, M., Poonguzhali, S., Ryu, J. & Sa, T. (2006) Regulation of ethyl-
ene levels in canola (Brassica campestris) by 1-aminocyclopropane-
1-carboxylate deaminase-containing Methylobacterium fujisawaense.
Planta, 22, 268–278.
Madhaiyan, M., Poonguzhali, S. & Sa, T. (2007) Metal tolerating methylo-
trophic bacteria reduces nickel and cadmium toxicity and promotes
plant growth of tomato (Lycopersicon esculentum L.). Chemosphere, 69,
220–228.
Miché, L., Battistoni, F., Gemmer, S., Belghazi, M. & Reinhold-Hurek, B.
(2006) Upregulation of jasmonate-inducible defense proteins and dif-
ferential colonization of roots of Oryza sativa cultivars with the endo-
phyte Azoarcus sp. Molecular Plant-Microbe Interactions, 19, 502–511.
Nomura, K., Melotto, M. & He, S.Y. (2005) Suppression of host defense in
compatible plant–pseudomonas syringae interactions. Current Opinion
in Plant Biology, 8(4), 361–368.
Pacheco, C.M., Pestana-Calsa, M.C., Gozzo, F.C., Mansur, C., Nogueira, R.
J., Menossi, M. et al. (2013) Differentially delayed root proteome
responses to salt stress in sugar cane varieties. Journal of Proteome
Research, 12, 5681–5695.
Pan, S.Q., Ye, X.S. & Ku
c, J. (1991) Association of β-1, 3-glucanase activity
and isoform pattern with systemic resistance to blue mould in tobacco
induced by stem injection with Peronospora tabacina or leaf inoculation
with tobacco mosaic virus. Physiological and Molecular Plant Pathology,
39, 25–39.
Pieterse, C.M., Zamioudis, C., Berendsen, R.L., Weller, D.M., van Wee, S.C.
M. & Bakker, P.A. (2014) Induced systemic resistance by beneficial
microbes. Annual Review of Phytopathology, 52, 347–375.
Ravanbakhsh, M., Kowalchuk, G.A. & Jousset, A. (2019) Root-associated
microorganisms reprogram plant life history along the growth–stress
resistance tradeoff. The ISME Journal, 13, 3093–3101.
Roy Choudhury, A., Choi, J., Walitang, D.I., Trivedi, P., Lee, Y. & Sa, T.
(2021) ACC deaminase and indole acetic acid producing endophytic
bacterial co-inoculation improves physiological traits of red pepper
(Capsicum annum L.) under salt stress. Journal of Plant Physiology, 267,
153544.
Roy Choudhury, A., Roy, S.K., Trivedi, P., Choi, J., Cho, K., Yun, S.H. et al.
(2022) Label-free proteomics approach reveals candidate proteins in
rice (Oryza sativa L.) important for ACC deaminase producing bacteria-
mediated tolerance against salt stress. Environmental Microbiology, 24,
3612–3624.
Samaddar, S., Chatterjee, P., Choudhury, A.R., Ahmed, S. & Sa, T. (2019)
Interactions between Pseudomonas spp. and their role in improving the
red pepper plant growth under salinity stress. Microbiological Research,
219, 66–73.
Seo, P.J., Lee, A.K., Xiang, F. & Park, C.M. (2008) Molecular and functional
profiling of Arabidopsis pathogenesis-related genes: insights into their
roles in salt response of seed germination. Plant and Cell Physiology,
49, 334–344.
Singh, N.K., Kumar, K.R.R., Kumar, D., Shukla, P. & Kirti, P.B. (2013) Char-
acterization of a pathogen induced thaumatin-like protein gene AdTLP
from Arachis diogoi, a wild peanut. PLoS One, 8, e83963.
Song, H., Wang, P., Hou, L., Zhao, S., Zhao, C., Xia, H. et al. (2016) Global
analysis of WRKY genes and their response to dehydration and salt
stress in soybean. Frontiers in Plant Science,7,9.
Theocharis, A., Bordiec, S., Feranadea, O., Paquis, S., Dhondt-Cordelier, S.,
Baillieul, F. et al. (2012) Burkholderia phytofirmans PsJN primes Vitis
vinifera L. and confers a better tolerance to low nonfreezing tempera-
tures. Molecular Plant-Microbe Interactions, 25, 241–249.
Wu, J., Kim, S.G., Kang, K.Y., Kim, J.G., Park, S.R., Gupta, R. et al. (2016) Over-
expression of a pathogenesis-related protein 10 enhances biotic and abi-
otic stress tolerance in rice. The Plant Pathology Journal, 32, 552–562.
Xing, F., Li, Z., Sun, A. & Xing, D. (2013) Reactive oxygen species promote
chloroplast dysfunction and salicylic acid accumulation in fumonisin
B1-induced cell death. FEBS Letters, 587, 2164–2172.
Xu, Z.S., Xia, L.Q., Chen, M., Cheng, X.G., Zhang, R.Y., Li, L.C. et al. (2007)
Isolation and molecular characterization of the Triticum aestivum
L. ethylene-responsive factor 1 (TaERF1) that increases multiple stress
tolerance. Plant Molecular Biology, 65, 719–732.
Yao, Y., He, R.J., Xie, Q.L., Zhao, X.H., Deng, X.M., He, J.B. et al. (2017)
ETHYLENE RESPONSE FACTOR 74 (ERF74) plays an essential role in
controlling a respiratory burst oxidase homolog D (RbohD)-dependent
mechanism in response to different stresses in Arabidopsis. New Phy-
tologist, 213, 1667–1681.
Yim, W.J., Kim, K.Y., Lee, Y.W., Sundaram, S.P., Lee, Y. & Sa, T.M. (2014)
Real time expression of ACC oxidase and PR-protein genes mediated
by Methylobacterium spp. in tomato plants challenged with Xanthomo-
nas campestris pv. Vesicatoria.Journal of Plant Physiology, 171, 1064–
1075.
Yim, W., Seshadri, S., Kim, K., Lee, G. & Sa, T. (2013) Ethylene emission
and PR protein synthesis in ACC deaminase producing Methylobacter-
ium spp. inoculated tomato plants (Lycopersicon esculentum mill.) chal-
lenged with Ralstonia solanacearum under greenhouse conditions.
Plant Physiology and Biochemistry, 67, 95–104.
SUPPORTING INFORMATION
Additional supporting information can be found online in the Support-
ing Information section at the end of this article.
How to cite this article: Roy Choudhury, A., Trivedi, P., Choi,
J., Madhaiyan, M., Park, J.-H., Choi, W. et al. (2023)
Inoculation of ACC deaminase-producing endophytic bacteria
down-regulates ethylene-induced pathogenesis-related
signaling in red pepper (Capsicum annuum L.) under salt stress.
Physiologia Plantarum, 175(2), e13909. Available from: https://
doi.org/10.1111/ppl.13909
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