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Acta Physiol Plant (2017) 39:268
DOI 10.1007/s11738-017-2565-8
ORIGINAL ARTICLE
Identification andcomparative analysis ofBrassica juncea
pathogenesis‑related genes inresponse tohormonal, biotic
andabiotic stresses
SajadAli1,2· ZahoorAhmadMir1· AnshikaTyagi1· JavaidA.Bhat3·
NarayanappaChandrashekar1,4· PradeepKumarPapolu5· SandhyaRawat1·
AnitaGrover1
Received: 11 March 2017 / Revised: 28 October 2017 / Accepted: 2 November 2017
© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2017
Further, expression of SA marker genes decreases while as
JA-responsive genes increases during drought stress. Inter-
estingly, both SA and JA signature genes were induced after
salt stress. We also found that BjPR genes displayed ABA-
independent gene expression pattern during abiotic stresses
thus providing the evidence of SA/JA cross talk. Further, in
silico analysis of the upstream regions (1.5kb) of both SA
and JA marker genes showed important cis-regulatory ele-
ments related to biotic, abiotic and hormonal stresses.
Keywords Brassica juncea· Erysiphe cruciferarum·
PR genes· Abiotic stress· Abscisic acid· Jasmonic acid·
Salicylic acid
Introduction
The genus Brassica comprises many diversified species
that provides oil, vegetables, condiments, dietary fiber, and
vitamin C (Talalay and Fahey 2001). B. juncea var. Varuna
(Indian mustard) is one of the prominent members of Bras-
sica genus, with great economic and agricultural importance
across the globe. B. juncea is an amphidiploid species with
a chromosome number of 18, derived from interspecific
crosses between, B. nigra (n=8) and B. rapa (n=10). In
India alone, mustard is cultivated around 6 million hec-
tares and is projected that by 2020, 41% of total demand for
oilseed will solely be met by this crop (Yadava and Singh
1999). Unfortunately, productivity of this crop is hampered
by a variety of biotic (mainly fungal diseases) and abiotic
(drought and salinity) stresses which lead to significant
yield losses (Mathpal etal. 2011; Goel and Singh 2015).
Improving stress tolerance in B. juncea through conventional
breeding perspective is confounded mainly due to non-avail-
ability of suitable resistant sources within the germplasm
Abstract Pathogenesis-related proteins (PRs) are the
antimicrobial proteins which are commonly used as signa-
tures of defense signaling pathways and systemic acquired
resistance. However, in Brassica juncea most of the PR pro-
teins have not been fully characterized and remains largely
enigmatic. In this study, full-length cDNA sequences of SA
(PR1, PR2, PR5) and JA (PR3, PR12 and PR13) marker
genes were isolated from B. juncea and were named as BjPR
proteins. BjPR proteins showed maximum identity with
known PR proteins of Brassica species. Further, expression
profiling of BjPR genes were investigated after hormonal,
biotic and abiotic stresses. Pre-treatment with SA and JA
stimulators downregulates each other signature genes sug-
gesting an antagonistic relationship between SA and JA in
B. juncea. After abscisic acid (ABA) treatment, SA signa-
tures were downregulated while as JA signature genes were
upregulated. During Erysiphe cruciferarum infection, SA-
and JA-dependent BjPR genes showed distinct expression
pattern both locally and systemically, thus suggesting the
activation of SA- and JA-dependent signaling pathways.
Communicated by W. Zhou.
* Anita Grover
anitagrover@hotmail.com
1 National Research Centre onPlant Biotechnology, Pusa
Campus, NewDelhi, India
2 Centre ofResearch forDevelopment, University ofKashmir,
Srinagar, India
3 Division ofGenetics, Indian Agricultural Research Institute,
NewDelhi, India
4 Division ofCrop Improvement, ICAR-Central Institute
forCotton Research, Nagpur, Maharashtra, India
5 School ofBioengineering, SRM University, Chennai, India
Acta Physiol Plant (2017) 39:268
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268 Page 2 of 15
and, therefore, genetic engineering has become imperative to
compliment the conventional breeding approach for develop-
ing stress-tolerant varieties. However, the identity and role of
potential genes or signaling cascades in B. juncea responses
to different stresses (biotic and abiotic) are largely unknown.
Therefore, it is necessary to characterize the stress-related
gene families in B. juncea to develop resistant or toler-
ant varieties for sustainable and enhanced productivity of
oilseeds.
Plants are constantly challenged by variety of stresses
which includes both biotic and abiotic stresses (Ahmad
etal. 2015). To protect themselves, plants use multidimen-
sional approaches such as morphological, biochemical and
molecular defense responses that help them to retain their
fitness or survive under such circumstances. Furthermore,
plants also combat biotic and/or abiotic stresses by syn-
thesizing small heterogeneous group of proteins like PR
proteins (Van Loon and Van Strien 1999). After first being
identified in tobacco plant infected with TMV (tobacco
mosaic virus), PR proteins have since been reported in
many plants. PR proteins are known to be induced by a
variety of biotic and abiotic stresses; hence, are gener-
ally considered to be part of multiple defense systems in
plants. Presently, PR proteins are grouped into 17 families
with diverse functions; some of these are PR1 (unknown),
PR2 (β-1, 3-glucanase), PR3 (chitinases), PR5 (thauma-
tin like), PR9 (peroxidases), PR12 (plant defensins) and
PR13 (thionins) (van Loon etal. 2006). Under non-stress
state, most of the PR genes show basal level expression,
but increases dramatically at infection site and also plays
key role in systemic acquired resistance (SAR) pathway
(Návarová etal. 2012). Generally, SAR is activated in the
distal or uninfected tissues in response to a prior (primary
or local) infection elsewhere in the plant. In plants, SAR is
an inducible immune response that offers enhanced disease
resistance against multiple pathogens (Sticher etal. 1997).
Interestingly, PR genes have been frequently used as SAR
signature genes in the model plant Arabidopsis. They are
also induced by defense signaling inducers (SA or JA) and
are widely used as molecular indicators of the activation of
these pathways (Naidoo etal. 2013). Transcript profiling
provides evidence that increased expression of PR1, PR2
and PR5 represents the activation of SA whereas increased
expression of transcripts of PR3 and PR4 (endo-chitinases)
represents the activation of JA pathway (Narusaka etal.
2015). Over-expression of PR genes in different crop
systems showed enhanced disease resistance against bio-
trophic and necrotrophic pathogens (Kusajima etal. 2010;
Jiang etal. 2015). Recently, SA and JA signaling cascades
have also emerged as potential tools for improving plant
stress tolerance to abiotic stresses (Khan etal. 2012; Khan
and Khan 2014). ABA is a well-known regulator of abiotic
stresses including drought, salinity, and cold and has been
extensively studied (Shinozaki and Yamaguchi-Shinozaki
2007). In addition to abiotic stress, ABA has also gained
the importance in plant defense signaling as a positive or
a negative regulator based on the plant–pathogen interac-
tions (Cao etal. 2011). Recently, another group of plant
growth hormones such as auxin, cytokines (CKs) and gib-
berellins (GAs) have also emerged as important modula-
tors of plant defense response but their function remains
elusive (Pieterse etal. 2012). Hormonal crosstalk modu-
lates and also optimizes plant fitness to biotic and abiotic
stresses when they occur simultaneously. More studies are
needed to study the dynamic roles of these versatile small
molecules during individual or multiple stresses in plants.
PR genes are not only induced after pathogen attack but
their involvement has also been shown to combat differ-
ent abiotic stresses. In Arabidopsis, PR genes like AtPR1,
AtPR2 and AtPR5 are induced by both drought and salin-
ity stress (Seo etal. 2008; Singh etal. 2013). Transcripts
of SAR marker gene PR1 in pepper plants increased sig-
nificantly after its exposure to a variety of abiotic stresses
(Hong and Hwang 2005). In addition, transcript levels of
JA marker gene PR12 (PDF.1) was also increased dur-
ing cold stress (Archambault and Strömvik 2011). Gaudet
etal. (2003) reported that freezing increases the transcript
accumulation of antifungal (PR13) thionin genes in wheat.
Historically, known PR proteins like β-1,3-glucanase and
chitinases possess antifreeze activity and protect cell dam-
age due to cold stress (Janska etal. 2010). Recently, it
was shown that transcript levels of PR4 increased signifi-
cantly during salinity, wounding and cold stress (Kim etal.
2014). Furthermore, maize PR10 gene was also upregu-
lated after various abiotic stresses (Fountain etal. 2010).
Activation of transcription factors such as cup-shaped
cotyledon (CUC), drought-induced protein 19 (Di19) and
dehydration-responsive element binding proteins (DREB)
by abiotic stresses leads to PR gene induction (Tsutsui
etal. 2009). Availability of transcriptomic data from indi-
vidual biotic and abiotic stress experiments in both model
and crop plants has been utilized to identify mutual stress-
responsive genes (Narsai etal. 2013). In field conditions,
plants may be exposed to different stresses that may likely
occur simultaneously, a greater effort must be made to
imitate these conditions in lab studies (Mittler and Blum-
wald 2010). Considering the mutagenic natures of stress
tolerance, identification of different stress-specific genes
are required that can be transferred into crop systems
through transgenic approach to confer resistance to mul-
tiple stresses. Therefore, the major goal of this study was
to reveal the molecular mechanisms underlying BjPR gene
response to hormonal, biotic and abiotic stress. Notably,
in this work we have identified multiple stress inducible
genes in B. juncea.
Acta Physiol Plant (2017) 39:268
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Materials andmethods
Plant growth conditions andtreatments
Brassica juncea var. Varuna plants were grown in pots
containing a mixture of soil and organic manure (2:1) at
24°C under a 16-h/8-h light–dark cycle with irradiance of
100–125 μmolm−2s−1, and a relative humidity of 80%. For
cDNA library construction, B. juncea plants were sprayed
with 2mM SA (pH 7.0) and 100µΜ JA, respectively. Con-
trol plants were similarly treated with sterile distilled water
(SDW). Leaf samples were collected from control and hor-
mone-treated plants at different time points.
Construction ofB. juncea cDNA library
Total RNA was isolated from the SA-, JA- and water
(control)-treated B. juncea leaf samples using the protocol
of PureLink RNA Mini Kit (Ambion, Carlsbad, CA, USA).
B. juncea cDNA libraries were constructed from total RNA
of SA- or JA-treated leaf samples using BD SMART cDNA
library synthesis kit (Clontech Inc., USA). For cDNA ampli-
fication long-distance PCR (LD-PCR) was performed and
the reaction mixture contains 11µL first-strand cDNA,
Advantage 2 PCR buffer, dNTP Mix, CDS 1II/3′ PCR
primer (5′ATT CTA GAG GCC GAG GCG GCC GAC ATG3′,
5′ PCR primer (5′-AAG CAG TGG TAT CAA CGC AGAGT-
3′) Advantage 2 polymerase mix and deionised H2O. The
program of LD-PCR was: 72°C for 10min; 95°C for 20s
(3 cycles) and 68°C for 8min. The LD-PCR product was
treated with 2µL of proteinase K (20μgμl−1) to inactivate
DNA polymerase activity and was further purified. Purified
cDNA was digested with SfiI enzyme to generate cohesive
ends for directional cloning into λTriplEx2 vector (Clontech,
Inc., USA). cDNA was ligated into the λTriplEx2 vector and
the reaction mixture contains 0.5μl 10× ligation buffer,
0.5μl 10mM ATP, 0.5μl cDNA, 1.0μl vector (500ngμl−1),
0.5μl T4 DNA ligase and 2μl deionised H2O incubated
at 16°C overnight. Furthermore, bacterial plate culturing,
tittering the unamplified library as well as the percentage
of recombinant clones were determined according to the
protocol SMART cDNA synthesis kit. Prior to sequencing,
conversion of a recombinant λTriplEx2 to the pTriplEx2
vector was carried out into the E. coli BM25.8.
Screening ofcDNA libraries forBjPR genes
For screening, the unamplified libraries were used. SA-
induced library was used for screening of BjPR1, BjPR2
and BjPR5 clones while as JA-induced library was used
for screening of BjPR3, BjPR12 and BjPR13 clones using
probes derived from Arabidopsis homolog PR genes. All
the BjPR clones were further sequenced and analysed
using different bioinformatic tools.
Phylogenetic andstructural analysis ofBjPR genes
Protein sequence similarity analysis of BjPR proteins
(BjPR1, BjPR2, BjPR3, BjPR5, BjPR12 and BjPR13)
were carried out using BLAST tool (http://www.ncbi.nlm.
nih.gov/blast). To elucidate the evolutionary relationship
of BjPR proteins, additional homologs of PR proteins were
retrieved from Brassica database (BRAD) and NCBI data-
bases, respectively. Phylogenetic trees of BjPR proteins
were constructed using the neighbour-joining method with
bootstrapping (1000 replicates) using MEGA.7 program.
In silico protein structure of BjPR proteins was analysed
using EXPASY software (http://www.expasy.org/). In
addition, the isoelectric point and molecular weight of
BjPR proteins were determined by Compute PI/MW tool
of Expasy. In silico subcellular localizations of BjPR pro-
teins were determined by Cell-PLoc 2.0 program (Chou
and Shen 2010).
Hormonal treatment ofB. juncea plants
For hormonal treatments, 10-day-old B. juncea plants were
sprayed with 50µM ABA, 100µM JA and 2mM SA indi-
vidually and kept separately to prevent hormonal cross
talk. Control plants were treated with sterile distilled water
(SDW) containing equal amount of solvent used for hormone
preparation. Leaf samples for RNA isolation were collected
from both hormone-treated and control plants after 1, 4 and
6h post-treatment.
Erysiphe cruciferarum infection inB. juncea plants
The pure culture of E. cruciferarum pathogen was isolated
from B. juncea-infected leaves collected from the fields of
Indian Agricultural Research Institute (IARI) New Delhi
India. It was further confirmed as E. cruciferarum by Her-
barium Cryptogamae Indiae Orientalis (H.C.I.O-ID: no.
52067) IARI, New Delhi, India. To investigate the distal
and local expression of BjPR genes after E. cruciferarum,
1-month-old B. juncea plants were infected with E. cruci-
ferarum as described by (Meur etal. 2006). For control,
plants were sprayed with SDW. The inoculated plants were
kept in a growth chamber at 100% RH and 22°C. For RNA
isolation, samples were harvested from both local (E. cruci-
ferarum infected) and distal non-infected leaves of B. juncea
plants.
Acta Physiol Plant (2017) 39:268
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268 Page 4 of 15
Abiotic stress treatments inB. juncea plants
For abiotic stress treatments, B. juncea seeds were first
treated with triton ×100 for 5min followed by treatment
with 0.1% HgCl2 for 10min. After HgCL2 treatment,
seeds of B. juncea were washed with SDW and grown on
half-strength MS medium (Murashige and Skoog 1962) in
Magenta boxes (Magenta vessel Corp, USA) at 24±2°C
under cool white florescent light (90–150µmol photons
m−2s−1) in a 16-/8-h (light/dark) photoperiod. Ten-day-
old B. juncea seedlings were aseptically transferred from
MS solid agar medium to ½ liquid MS medium and stabi-
lized for 4h before stress. Drought stress was performed
by transferring the B. juncea seedlings from MS medium
to sterile Whatman filter paper (3MM) in petri dishes,
while control plants were kept on sterile 3MM Whatman
filter paper in petri dishes supplied with ½ liquid MS.
For imposing salt stress, 10-day-old B. juncea seedlings
were transferred to ½ MS liquid media supplemented with
150mM NaCl and control plants were kept in normal
½ MS liquid media incubated at room temperature. All
control and treated plants were maintained under white
light conditions. Only leaf samples were collected from
both control and treated B. juncea seedlings at 1, 4 and
6h for RNA isolation. Three biological replicates were
used for each treatment.
cDNA synthesis andRT‑qPCR analysis
For cDNA synthesis, total RNA was extracted from
100mg leaf samples of both control and treated B. juncea
seedlings using PureLink RNA Mini Kit (Ambion Life
Technologies, USA). Purity and concentration of RNA
was measured by Nanodrop spectrophotometer (Nan-
oDrop 2000 Thermo Scientific, Wilmington, DE). First-
strand cDNA was generated from 2µg of DNase-treated
total RNA using Superscript III cDNA synthesis kit fol-
lowing the manufacturer’s protocol (Invitrogen, Canada).
Primers of BjPR genes and alpha-tubulin were designed
using Oligoanalyzer software (Table1). qRT-PCR mix-
ture contained 2µl of cDNA, 5µl of SYBR green qRT-
PCR master mix (Takara, Japan) and 0.5µl (10 picomol)
of each primer and was run at 95°C for 5min, followed
by 40 cycles of 94°C for 30s, 60°C for 30s, and 72°C
for 30s. The reactions were performed in triplicates and
repeated using three biological replicates. Housekeeping
gene, alpha-tubulin, was used in all the experiments as
an internal control and the relative expression levels of
each gene were analysed by delta CT method (Livak and
Schmittgen 2001). Fold changes with p values less than
0.05 were considered significant.
In silico analysis ofBjPR gene promoters
The upstream regions of BjPR genes were isolated from
the B. juncea by genome walking using universal genome
walker kit (Clontech, CA). 1.5kb promoter region of BjPR
genes was scanned by PLACE (Higo etal. 1999) and Plant-
CARE (Lescot etal. 2002), promoter databases for finding
biotic, abiotic stresses and hormonal responsive cis-regula-
tory elements.
Statistical analysis
For all the experiments, three biological replicates were
used. Student’s t test was used to determine significant dif-
ferences of expression data of BjPR genes between treated
and control B. juncea plants which are shown as statistically
significant (*p<0.05) or extremely significant (**p<0.01).
Results
Isolation, phylogenetic andstructural analysis ofBjPR
genes fromB. juncea
PR gene families constitute a large and important group of
defense proteins in plants. In this study, two cDNA libraries
(SA and JA cDNA library) were constructed from B. jun-
cea plants after SA and JA treatment. The cDNA sequences
of BjPR1, BjPR2 and BjPR5 were isolated from SA cDNA
library while as BjPR3, PR12 and PR13 were isolated from
JA cDNA library using probes derived from A. thaliana
homologous PR gene families. The sizes of cDNA sequences
of BjPR genes range between 137 and 1041 nucleotides. A
homology search against the NCBI database was carried out
to confirm whether the obtained sequences from B. juncea
Table 1 List of primers used in this study
Gene Primer
RT-PR1 F-5′ GAA CAC GTG CAA TGG AGA ATG 3′
R-5′ CCA TTG TTA CAC CTC GCT TTG 3′
RT-PR2 F-5′ CGT CTC TCT ACA ATT CGC TCTG 3′
R-5′ CGA TAT TGG CGT CGA ATA GGT 3′
RT-PR3 F-5′ AAG ACC AGG TTC TTG CCT TC 3′
R-5′ TCC GGT ACA CTC CCT ACT ATTC 3′
RT-PR5 F-5′ GCA GAA CAA TTG CCC TTA CAC 3′
R-5′ R-GCG CCT GGA TTC AGT TGA TA
RT-PR12 F-5′ CAA TGG TGA AAG CGC AGA AG3′
R-5′ AGG TTG ATG CAC TGG TTC TT 3′
RTPR13 F-5′ GAG AAG CAA TGG CAG GTT CTA 3′
R-5′ CGC ACT CCG TGT TGT AGT T 3′
Alpha-tubulin F-5′ GCC TCG TCT CTC AGG TTA TTTC 3′
R-5′ TGA AGT GGA TTC TTG GGT ATGG 3′
Acta Physiol Plant (2017) 39:268
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Page 5 of 15 268
encoded PR gene family. BLASTP analysis revealed that the
deduced amino acids of BjPR genes were closely related to
PR proteins of Brassicaceae family, respectively. The phy-
logenetic relationship of BjPR1 protein with its homologs
from other plants revealed that they were closely related to
PR1 proteins of B. rapa, B. napus, B. nigra and Schren-
kiella parvula (Fig.1a). Similarly, BjPR2, BjPR3, BjPR5
and BjPR12 clustered within the clade containing their
respective homologs from B. napus, B. oleracea and B. rapa
(Fig.1b, e). Phylogenetic analysis also showed that BjPR13
protein was closely related to PR13 proteins of B. napus and
B. oleracea and Arabidopsis (Fig.1f). Phylogenetic analysis
of BjPR proteins with other PR homologs of different plant
species suggests that they may share a common ancestor.
The accession numbers, molecular weight, isoelectric point,
predicted coding sequences, protein size and in silico subcel-
lular localisation of BjPR1, BjPR2, BjPR3, BjPR5, BjPR12
and BjPR13 proteins are mentioned in Table2.
Expression profiling ofBjPR genes afterhormonal
treatments reveals unique interactions
Hormonal regulations of PR genes are well documented in
model plants but not in crop plants. This study investigates
the effect of various phytohormones (ABA, SA and JA) on
the expression of BjPR genes. Here, after ABA treatment,
distinct expression pattern of BjPR genes was observed.
The transcript levels of PR1, PR2 and PR5 significantly
decreased after ABA treatment as compared to control.
In contrast to SA, JA-responsive genes PR3 (17.33-fold)
and PR13 (5.12-fold) were significantly increased at 6h
after ABA treatment while PR12 was marginally induced
(Fig.2a). As expected, the higher accumulation of PR3 and
PR13 further supports previous findings of synergistic rela-
tion between JA and ABA. After SA treatment the expres-
sion of PR1, PR2, and PR5 increases at 1h and reaches a
maximum at 6h of post-treatment. In contrast, to JA signa-
ture genes PR3, PR12 and PR13 were not induced by SA
treatment (Fig.2b). Following the JA treatment, PR3, PR12
and PR13 exhibited increased transcript accumulation at
1h and reached maximum at 6h. SA signatures (PR1, PR2
and PR5) were not induced after JA treatment in B. juncea
(Fig.2c).
Disease progression anddefense expression (local
andsystemic) ofBjPR genes
Erysiphe cruciferarum is one of the important pathogens
of rapeseed mustard crops. However, the molecular mech-
anism of powdery mildew and Brassica pathosystem as
well as disease resistance is not fully understood. In this
study, we isolate and identified this obligate ascomycete
from naturally infected B. juncea plants. Typical powdery
mildew symptoms like white, star-shaped colonies of myce-
lia were visually observed with naked eye on the naturally
infected leaves of B. juncea (Fig.3a). Further, microscopic
observations showed ovoid to cylindrical hyaline conidia
(70–115×8–10μm) of E. cruciferarum (Fig.3b). The
appearance of spores of our isolate (H.C.I.O-ID: no. 52067)
was similar to previous records of E. cruciferarum HUST-
WUH1 (GenBank no. KP730001) isolated from infected
rapeseed plants by Alkooranee etal. (2015). We further
investigated the disease progression in healthy B. juncea
leaves after E. cruciferarum inoculation. Our results revealed
that after E. cruciferarum inoculation, spores germinated on
30-day-old B. juncea leaf surface and produced the same dis-
ease symptoms thus satisfying Koch’s postulates (Fig.3c).
However, no symptoms were seen on uninfected leaves of B.
juncea (Fig.3d). These results further reveal that B. juncea
is highly susceptible to powdery mildew disease.
An overview of typical SAR in distal leaves after infec-
tion is shown in (Fig.3e). To investigate the transcriptional
changes of BjPR genes in local (inoculated) and distal (non-
inoculated) leaves, B. juncea plants were infected with E.
cruciferarum. Based on the results transcript levels of SA
signature genes (BjPR1, BjPR2, and BjPR5) increased sig-
nificantly both locally as well as systematically after fungal
infection. However, among SA-dependent genes the tran-
script levels of BjPR1 (55.48-fold in local and 30.33-fold in
distal tissues) gene was comparatively higher than BjPR2
(25.29-fold in local and 20.87-fold in distal tissues) and
BjPR5 (7.67-fold in local and 6.45-fold in distal tissues)
after infection. In contrast to JA signatures, BjPR3 (15.67-
fold) gene was significantly induced in local-infected leaves
while very low transcript levels of BjPR12 and BjPR13 were
detected in local or distal tissues after infection (Fig.3f). In
B. juncea, SAR was accompanied by a systemic accumula-
tion of transcripts of most of the PR genes which further
reveals that role of PR genes in SAR is associated with acti-
vation of these genes.
Transcriptional profiling ofBjPR genes duringabiotic
stresses
Plants are very often exposed to drought stress which stim-
ulates the accumulation of various stress signaling mol-
ecules especially ABA which in turn regulates a number
of genes. This study reveals the effect of drought stress on
biotic-responsive genes in B. juncea. Compared to control,
transcript levels of SA marker genes PR1 (3.16-fold), PR2
(10.85-fold) and PR5 (4.63-fold) showed upregulation at 1h
but downregulates at 4 and 6h after drought stress. On the
other hand, PR3 (17.93-fold) and PR13 (9.06-fold) showed
a significant increase at 4h post-treatment. However, the JA
marker gene PR12 (2.83-fold) was marginally induced when
compared to control by drought stress in B. juncea (Fig.4a).
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Acta Physiol Plant (2017) 39:268
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A few reports have explored the effect of salt stress on
PR genes (Seo etal. 2008). To examine whether salinity
regulates the expression of PR genes in B. juncea, we treated
plants with 150mM NaCl and expression analysis were car-
ried out at different time points. In this study, significant
upregulation of PR1 (4.92-fold), PR2 (13.36-fold) and PR5
(6.58-fold) was observed at 4h after salt treatment in B.
juncea. On the other hand, PR3 (26.49), PR12 (2.6-fold)
and PR13 (9.25-fold) showed maximum expression at 6h
(Fig.4b). These observations suggest that salt stress modu-
lates key immune genes in B. juncea largely through SA-/
JA-dependent signaling pathways.
We have summarized the detailed expression analysis
data of BjPR genes in B. juncea after hormonal, biotic and
abiotic stress which clearly show the kinetics of gene expres-
sion (upregulation or downregulation) during these stresses
(Table3).
In silico analysis ofSA‑ andJA‑dependent BjPR gene
promoters reveals cis‑elements responsive tomultiple
stresses
To further investigate the regulation aspect of BjPR genes
in response to hormonal, biotic and abiotic stress, we ana-
lysed the upstream sequences of BjPR genes to identify the
cis-elements involved in multiple stresses. In silico analy-
sis of both SA and JA marker BjPR genes showed many
biotic stress-related cis-regulatory elements such as TC-rich
repeats (ATT TTC ), SARE (SA-responsive element) or (JA-
responsive element) (TGACG) motifs, W BOX [(T) TGAC
(C/T)] and GT1GMSCAM4 motif (GAA AAA ). Many
potential abiotic-responsive elements such as ABREs motif
(ACGT) for ABA-dependent expression, DREs motif (TAC
CGA CAT) for ABA-independent expression during salt
and drought stress, (LTRE) motif TGG/ACC GAC for low-
temperature response, MYB motif (TAA CTG ) for drought
stress, MYC motif CAT GTG , CAC ATG ) for early response
to drought and ABA induction, Wbox (TTGAC, TGACT)
for the activation of defense and wounding-related genes,
GT1 motif (GAA AAA , GGT TAA ) for pathogen and salt
response and HSE (CNNGAANNTTCNNG) involved in
heat stress were also found in single or multiple copies in
BjPR gene promoters (Fig.5). The presence of these cis-
regulatory elements in the upstream regions of B. juncea
PR genes reveals that they might be regulated by multiple
stresses.
Discussion
Plants are very often exposed to multiple stresses resulting
in substantial agricultural losses. Global climate change will
possibly increase the emergence of virulent strains of phy-
topathogens with broad host range. Therefore, understanding
the mechanisms underlying plant resistance or tolerance to
above stresses will help us to genetically engineer crops for
multiple stress tolerance. Plant responses to multiple stresses
are generally complicated process and involve a network of
genes and signaling cascades. However, the regulation and
molecular function of most of these genes or signaling cas-
cades to these stresses are largely unknown. Therefore, we
carried out transcriptional analysis of one of the important
stress-related gene families (PRs) in B. juncea after multi-
ple stresses. We identified the BjPR genes in B. juncea to
understand Brassica immune response and their signaling
pathways. Based on BLAST algorithms, domain prediction
and phylogenetic analysis, we found that BjPR genes share
similar protein sequence identities as well as conserved
domains of known PR proteins from other crucifers.
Phytohormones are essential not only for plant growth
and development but also play a vital role in the stress tol-
erance (Wani etal. 2016). Plants respond to stress through
a multifaceted group of signaling cascades which are
mainly regulated by small molecules called hormones such
as ABA, ET, JA and SA that interact synergistically and/or
antagonistically to each other. PR genes are generally con-
sidered as the molecular indicators of SA and JA defense
signaling pathways in model plants. Recently, their role in
various abiotic stresses has gained importance to study hor-
monal cross talk (Khan and Khan 2013). In Arabidopsis,
SA and JA exert antagonistic interactions with each other
(Van-der-Does etal. 2013); however, there are reports that
these pathways also act synergistically (Lazniewska etal.
2010). To further investigate the hypothesis of synergistic
or antagonistic relationship in B. juncea, expression profil-
ing of SA and JA signature genes were studied after hormo-
nal treatment. This work also aimed to find the signatures
of SA and JA signaling in B. juncea. Upon SA treatment,
transcripts of PR1, PR2, PR5 increases dramatically while
as PR3, PR12 and PR13 decreases which were similar to
the findings observed in Arabidopsis (Seo etal. 2008). In
contrast, JA upregulates PR3, PR12 and PR13 but down-
regulates SA marker genes PR1, PR2 and PR5; thus, our
results are consistent with previous reports observed in
Arabidopsis (Thomma etal. 1998). Our findings suggest
Fig. 1 Phylogenetic relationship of BjPR proteins of B. juncea with
other PR proteins from plant species. A. thaliana (At), B. napus (Bn),
B. nigra (Bni), B. oleracea (Bo), B. rapa (Br), Camelina sativa (Cs),
Capsella rubella (Cr), Leavenworthia alabamica (La), Oryza sativa
(Os), Raphanus sativus (Rs), Sisymbrium irio (Si), Schrenkiella par-
vula (Sp), Thellungiella halophila (Th), Thellungiella salsuginea
(Ts), Vitis Vinifera (Vv). a Phylogenetic analysis of BjPR1, b BjPR2,
c BjPR3, d BjPR5, e BjPR12 and f BjPR13 protein sequences with its
homologs from other plant species obtained from Brassica and NCBI
databases. The phylogenetic trees of BjPR proteins were constructed
by neighbour-joining method using MEGA7.0 software with 1000
bootstrap
◂
Acta Physiol Plant (2017) 39:268
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268 Page 8 of 15
that the known SA/JA antagonistic relationship in the
model plant Arabidopsis was also observed in B. juncea
during early hours of hormonal treatments. Furthermore,
BjPR genes studied in this work can be used as suitable
markers or molecular indicators for SA and MeJA signaling
pathways in B. juncea.
Table 2 BjPR proteins along with their accession numbers, molecular weight (M.wt.), isoelectric point (PI), CDS and protein length, and sub-
cellular localization
Protein Accession numbers M. wt. (kDa) PI CDS length (bp) Protein length
(a.a)
Subcellular
localization
BjPR1 (unknown) ABC94641 17.53 7.07 661 161 Vacuole
Bj-PR2 (β,1-3 glucanase) ABC94639 38.08 9.13 1041 346 Vacuole
BjPR3 (chitinase) ABQ57389 17.03 4.82 468 155 Vacuole
BjPR5 (thaumatin) ABX10753 11.65 5.98 341 114 Cell wall
Cytoplasm
BjPR12 (defensin) AHB85724 8.93 8.47 243 80 Vacuole
BjPR13 (thionin) ABO71662 4.44 4.10 137 45 Unknown
Fig. 2 Expression analysis
of BjPR genes after hormonal
treatments. 10-day-old B. jun-
cea plants were treated with a
SA (1mM), b JA (100µM) and
c ABA (50µM). Leaf samples
were harvested at different
time points for RNA isolation.
Control plants for each treat-
ment were treated with sterile
distilled water containing equal
amount of solvent used for hor-
mone preparation. SE for each
bar is shown. Treatment bars
marked by an asterisk are sig-
nificantly greater than untreated
(controls) (p<0.05)
Acta Physiol Plant (2017) 39:268
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Page 9 of 15 268
Generally, ABA not only plays a central role in abiotic
stresses, but also plays a critical role in plant–pathogen
interactions (De-Torres-Zabala etal. 2007; Fan etal. 2009;
Lim etal. 2015). To further understand the role of ABA in
B. juncea defense response, we studied the expression of
key immune genes (BjPR genes) after ABA treatment. Our
results revealed that ABA downregulates the expression of
SA marker genes PR1, PR2 and PR5 in B. juncea at all time
points which suggest that ABA and SA interact antagonisti-
cally in B. juncea. There are reports which have shown that
ABA paralyzes the plant defense responses by suppressing
the SA pathway, thereby acting as negative regulator of
SA-mediated immunity (De Vleesschauwer etal. 2013). In
addition, many reports have shown ABA suppresses the SA
pathway which fails to establish SAR both in Arabidopsis as
well as in tobacco plants (Yasuda etal. 2008; Kusajima etal.
2010). Our results are in agreement with the above find-
ings that ABA inhibits the expression of SAR marker genes
PR1, PR2 and PR5 in B. juncea, therefore, might increase
susceptibility to biotrophic pathogens. A significant finding
of our study was that SA marker genes were downregulated
by ABA but showed upregulation during various abiotic
stresses. These results further provide the affirmation of
the participation of other signaling pathways like SA/JA or
the occurrence of interrelated pathways which could trigger
the expression of these genes in ABA-independent man-
ner. Interestingly, we observed the synergistic interaction
between ABA and JA in B. juncea as JA marker genes PR3
and PR13 were significantly induced. Similar expression
pattern of PR3 and PR13 genes was reported in Arabidopsis
and rice after ABA treatment (Yazaki etal. 2003; Seo etal.
2008). Furthermore, JA signature genes were also induced
by various abiotic stresses; therefore, there induction would
be either JA or ABA dependent in B. juncea. In Arabidop-
sis, exogenous application of ABA increases disease resist-
ance to necrotrophic pathogens Alternaria brassicicola and
Pythium irregulare (Adie etal. 2007). Therefore, our results
suggest that induction of JA marker genes by ABA might
have a role in combating biotic and abiotic stresses in B.
juncea.
Brassica juncea crop production is adversely affected by
biotic stresses. Among them fungal diseases are rated as the
most important factor for significant yield losses with no
proven source of disease resistance. One of the important
biotrophic fungal pathogens of B. juncea and other cruci-
fers is E. cruciferarum which causes severe damage and
Fig. 3 In planta infection of B. juncea with E. cruciferarum and
expression profiling of BjPR genes in local (inoculated) and dis-
tal (non inoculated) leaves. a B. juncea-infected leaf with E. crucif-
erarum in IARI fields, bar=2 cm. Pure culture of E. cruciferarum
(H.C.I.O-ID: no. 52067) was isolated from above-infected leaf of B.
juncea and used as inoculum. b Microscopic identification of E. cru-
ciferarum fungus (Conidia under 40X microscope), bar= 40µm. c
E. cruciferarum-inoculated B. juncea plants after seven days of post-
inoculation, bar=0.36mm. d Uninfected or control B. juncea plants,
bar=2cm. e Schematic diagram of SAR in plants. After infection,
plants show PAMP-triggered immunity (PTI) and effector-triggered
immunity (ETI), in the infected leaves, bar=2cm. f Transcriptional
profiling of the BjPR1, BjPR2, BjPR3, BjPR5, BjPR12 and BjPR13
genes were investigated in the uninfected (control= C) and E. cru-
ciferarum-infected leaves. The expression levels of BjPR genes in
control seedlings were normalized to a value of 1. SE for each bar
is shown. The α-tubulin gene was used as an internal control. A sig-
nificant difference (p< 0.05) between control and treated samples is
denoted by an asterisk above the bar
Acta Physiol Plant (2017) 39:268
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268 Page 10 of 15
yield losses. The defense response of the B. juncea to this
pathogen has not been fully deciphered at molecular level.
Plants generally involve LAR and SAR immune responses to
counterpart pathogen attack (Fu and Dong 2013). Both these
resistance pathways are associated with activation of array of
genes like PR genes. Till date, limited studies have been car-
ried out on the defense mechanisms and signaling pathways
involved in LAR or SAR in B. juncea after pathogen attack.
Therefore, we studied the transcriptional regulation of BjPR
genes in local and distal tissues of B. juncea in response
to E. cruciferarum pathogen. Among PR genes, PR1 has
been universally known as molecular indicator of induced
plant immune system and exhibits antifungal activity (Zhu
etal. 2012). Similarly, during our study on the interaction of
B. juncea and E. cruciferarum pathogen, PR1 was strongly
upregulated to a greater level both locally and systematically
and can be used as SAR marker in B. juncea. Interestingly,
higher accumulation of PR2 gene was also seen in B. juncea
leaves after E. cruciferarum infection. Generally, transcript
levels of PR2 genes are comparatively low in healthy or non-
infected plants but increases dramatically after biotrophic or
necrotophic fungal pathogen attack, thus implying its role in
disease resistance (Cheong etal. 2000; Shi etal. 2006; Zhu
etal. 2013). Another important member of the SA-dependent
Fig. 4 Relative expression
analysis of BjPR genes in B.
juncea after abiotic stresses at
different time points. Expres-
sion profiling of BjPR genes in
drought (a), salt (b) and control
(C=untreated) leaves of B.
juncea at various time points.
The expression levels of BjPR
genes in control seedlings were
normalized to a value of 1.
Expression of BjPR1, BjPR2,
BjPR3, BjPR5, BjPR12 and
BjPR13 at 1, 4 and 6h after
different stresses are represented
in different colours and control
(C) is represented with black
colour bar. The relative expres-
sion levels of BjPR genes are
compared with that of a control
alpha-tubulin gene. The data are
the mean±SE of three biologi-
cal replicates. SE for each bar is
shown. A significant difference
(p<0.05) between control and
treated samples is denoted by an
asterisk above the bar
Table 3 Differential gene
expression profiling of BjPR
genes in response to hormonal,
biotic and abiotic stress
treatments
‘+’ to ‘+++’ strong upregulation, ‘+−’ low expression ‘−’ weak to ‘−−’ strong down-regulation
Gene name SA JA ABA E. cruciferarum Drought Salt
Local Distal
BjPR1 +++ −− − +++ ++ + +
BjPR2 ++ −− − ++ ++ + +
BjPR3 −− +++ ++ ++ − ++ ++
BjPR5 ++ −− − + + + +
BjPR12 −− +++ + +− +− + −
BjPR13 −− ++ + +− +− + +
Acta Physiol Plant (2017) 39:268
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Page 11 of 15 268
PR gene family is PR5 or thaumatin-like genes which have
been reported to be induced by diverse pathogens. Our
results also showed that PR5 gene of B. juncea was induced
by fungal pathogen both in local and distal leaves. Among
SA marker genes, transcript level of PR1 gene was relatively
higher than PR2 and PR5 after infection. On the onset of
SAR, SA is transported from the infected leaf to the distal
leaves, which leads to the activation of SAR downstream
genes such as PR1, PR2 and PR5 in the pathogen-free tissues
(Dempse and Klessig 2012). Therefore, these genes might
play crucial part in LAR and SAR in B. juncea. Interestingly,
among JA signature genes, PR3 (chitinase) was significantly
induced by biotrophic pathogen (E. cruciferarum) but the
expression was restricted to local-infected tissues. Previous
study has also shown that powdery mildew increases tran-
script levels of PR3 gene in grapevine (Jacobs etal. 1999).
Plant defensins (PR12) and thionins (PR13) are known be
strongly induced by fungal pathogens (Kong etal. 2005). In
this study, we found less induction of BjPR12 and BjPR13
after E. cruciferarum infection in B. juncea. It is generally
well established, with some exceptions, that SA pathway
provides resistance to biotrophic pathogens while as JA/ET
pathways show resistance to necrotrophs and herbivorous
pests, respectively (Glazebrook 2005). Earlier reports in
A. thaliana suggested that many of the powdery mildew-
regulated genes are unlikely to be directed by SA signaling,
but are also regulated by other signals like hydrogen per-
oxide, ET, JA or by fungal elicitors (Chandran etal. 2009).
Therefore, our results suggests that E. cruciferarum medi-
ated expression of BjPR3, BjPR12 and BjP13 genes is SA
independent, as they were downregulated by SA. Altogether,
these results revealed that increased pathogen inducible
expression of BjPR genes might directly contribute to dis-
ease resistance because most of the PR genes isolated from
different plants have shown antifungal activity invitro and
enhanced resistance to pathogenic fungi when constitutively
overexpressed in planta.
Generally, PR genes are universally known as marker
genes of plant defense responses. However, few reports have
shown the activation of PR genes by various abiotic stresses
and have gained importance. Abiotic stress factors (salinity,
heat and drought) possess huge threat to modern agriculture
and decreases yields in most of the major crops. B. jun-
cea is greatly affected by drought stress causing significant
yield losses (Chauhan etal. 2007; Khan etal. 2017; Raza
etal. 2017). Plants exposed to drought stress have shown
Fig. 5 In silico analysis of PR gene promoters of B. juncea. Promoter
cis-elements of SA (BjPR, BjPR2, BjPR5) and JA signature (BjPR3,
BjPR12 and BjPR13) genes in response to biotic, abiotic and hormo-
nal stresses are shown in different shapes and colours along with their
respective positions from the start codon ATG
Acta Physiol Plant (2017) 39:268
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268 Page 12 of 15
to modulate plant defense response which further leads to
susceptibility or disease resistance. To further investigate
how drought stress regulates defense responses in B. juncea,
we examined the expression profiling of SA and JA signa-
ture genes after drought stress. Our findings showed that
SA-responsive genes increased at 1h and decline at later
time points whereas transcript levels of JA-responsive genes
were relatively higher after 1h of drought stress. But if we
look at the timing of induction of PR genes by SA and JA,
it is different from drought stress but ABA induced genes
to follow the same pattern. It seems that ABA is involved
in regulating the expression of PR genes during drought
stress. In addition, decreased expression of SA marker genes
like PR1, PR2 and PR5 might be due to high accumula-
tion of ABA which may suppress SA-responsive genes in
B. juncea during drought stress. Similar reports were also
observed in Arabidopsis where high accumulation of ABA
due to drought stress or exogenous treatment antagonizes
the SA signaling pathway (De-Torres-Zabala etal. 2007).
Our findings suggest the occurrence of so-called crosstalk
between biotic and abiotic stresses in B. juncea. Interest-
ingly, drought-induced expression of JA signature genes
may provide disease resistance to pathogens if both stresses
occur simultaneously in B. juncea, as there are reports which
have shown that drought stress reduces fungal biomass in
tomato plants during gray mold disease caused by B. cinerea
and also prevents powdery mildew infection (Oidium neoly-
copersici) (Achuo etal. 2006). On the other hand, it has
been shown that pathogens also improve plant tolerance to
drought stress which might be due to the activation of PR
genes.
Nowadays, interest for studying salinity stress is rising
rapidly because it is not only inhibiting plant growth but
also interferes with other plant responses to environmen-
tal stimuli such as disease response. Recent, studies have
also reported that salinity stress severely impairs B. juncea
productivity and, therefore, molecular biology intervention
is essential for the betterment of sustainable mustard culti-
vation particularly in northwestern agroclimatic region of
India (Yousuf etal. 2016). It has been demonstrated that SA
and JA defense hormones play important role in combat-
ing salt tolerance in many plants (Khan and Khan 2014).
However, the molecular mechanism underlying how SA
and JA combating salt tolerance in plants is poorly under-
stood. Therefore, we evaluated the expression analysis of
BjPR genes which are known as molecular indicators of SA
and JA signaling pathways in B. juncea after salt stress. We
observed the salt-mediated expression of both SA- and JA-
dependent PR genes in B. juncea. However, the expression
of BjPR3 and BjPR2 was relatively higher than other PR
genes. These results provide the evidence that SA- and JA-
mediated salt tolerance in plants could be because of the
coordinated expression of PR genes or other SA/JA pathway
genes. Moreover, earlier studies have also shown the positive
impact of salt stress on disease resistance to B. cinerea and
Oidium neolycopersici in tomato (Achuo etal. 2006). Here,
we found that the universally known antifungal genes such
as PR2 (glucanase), PR3 (chitinase) and PR13 (thionin) were
induced by salt stress that may lead to disease resistance
against fungal pathogens. Together, these results indicate
that PR genes are not only induced by pathogens but also by
salinity stress in B. juncea.
To further investigate the stress-related expression of PR
genes in B. juncea, 1.5kb promoter regions were scanned
for cis-elements involved in multiple stresses. Based on in
silico analysis, BjPR promoters showed many stress-related
cis-acting regulatory elements and were present in single or
multiple copies. Among biotic stress-related cis-regulatory
elements are TC-rich repeats (ATT TTC ), SARE (TCA GAA
GAGG, TCA TCT TCTT), JA (TGACG) motifs, W BOX [(T)
TGAC (C/T)], GT1GMSCAM4 motif (GAA AAA ) found in
BjPR gene promoters which further confirms the fact that
BjPR genes might play an important role in biotic stress.
Activation of abiotic stress-related genes usually occurs
either by ABA-dependent pathway which is conferred by the
presence of single or multiple copies of ABREs motifs, or
independently which possess DRE motifs (TAC CGA CAT)
to which different groups of DREBPs bind (Roychoudhury
etal. 2013). MYB and MYC motifs have also been known
to regulate genes during abiotic stresses. In addition, LTRE
a low-temperature-responsive element motif is commonly
found in cold-responsive genes (Brown etal. 2001). A well-
known pathogen-related motif W box mediates abiotic stress
responses in plants to wounding, oxidative stress, drought,
heat, cold and salinity by binding various WRKY transcrip-
tion factors (TFs). The presence of SA- and JA-responsive
motifs in stress-related genes is known to increase stress
tolerance to wide range of stresses. These motifs were also
found in B. juncea PR genes in single or multiple copies that
further confirm the fact that these genes might play a role
in abiotic stress.
Conclusion
This is the first report of a comparative analysis of B. juncea
PR genes after hormonal, biotic and abiotic stresses. Our
results showed that E. cruciferarum induces both LAR and
SAR pathways in B. juncea which will help us to select the
potential candidate gene like PR1, PR2 and PR3 for devel-
oping disease-resistant plants. This study also revealed that
besides ABA, SA and JA are also involved in abiotic stress
signaling in B. juncea and there is a hormonal crosstalk. In
this work, SA marker genes were downregulated by ABA but
showed upregulation during abiotic stresses which further
provides the evidence that SA could trigger the expression
Acta Physiol Plant (2017) 39:268
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Page 13 of 15 268
of these genes in ABA-independent manner. These identified
PR genes can serve as potential candidates for developing
transgenic crops resistant to multiple stresses which are the
theme of future research in plant genetic engineering and
molecular breeding.
Author contribution statement AG conceived and
designed research. SA has performed all the experiments
and wrote the manuscript. ZAM and PKP contributed to
data analysis. AT has contributed to bioinformatic analysis.
NC and SR contributed to RNA isolation. AG contributed
to manuscript proofreading. All authors read and approved
the manuscript.
Acknowledgements We gratefully acknowledge the Project Director,
National Research Centre on Plant Biotechnology, Pusa Campus, New
Delhi, for providing all the facilities required to complete this work.
Compliance with ethical standards
Conflict of interest The authors declare that there is no conflict of
interest.
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