Journal of Cell and Molecular Biology 5: 51-56, 2006.
Haliç University, Printed in Turkey.
Qualitative profiling of phenols and extracellular proteins induced in
mustard (Brassica juncea) in response to benzothiadiazole treatment
Sanjay Guleria* and Ashok Kumar
CSK H.P. Agricultural University, Shivalik Agricultural Research and Extension Centre, Kangra-176 001
(H.P.) India (*author for correspondence)
Received 19 October 2005; Accepted 20 December 2005
Treatment of mustard [Brassica juncea (L.) Czern. & Coss.] cv. Varuna with benzothiadiazole (BTH) induced
changes in the qualitative profile of total soluble phenols and acid soluble extra cellular proteins. There was temporal
increase in the level of total soluble phenolics after BTH treatment and maximum content was observed 72 h after
treatment. Thin layer chromatography of an aqueous methanol extract of BTH treated leaves of mustard revealed
presence of new phenolic compounds which were not present in control. Twelve acid soluble proteins with apparent
molecular masses ranging from 13.2 – 69.5 kDa accumulated in BTH treated leaves of mustard plants. Proteins
P13.8, P33.7 and P34.5 were present in traces in control. The most prominent proteins 24 h after BTH treatment were
with apparent molecular mass of 33.0 and 33.7 kDa indicating towards their early induction, whereas, P33.0 was the
most prominent protein 48 h after treatment with BTH. It is suggested that changes in specific phenols and proteins
as a result of BTH treatment might be the useful markers of induced resistance in mustard.
Key words: Brassica juncea , benzothiadiazole, extracellular proteins
Benzotiyadiazol uygulamas›na cevap olarak hardal bitkisinde (Brassica juncea) teflvik
edilen fenol ve hücre d›fl› proteinlerin kalitatif profili
Benzotiyadiazol (BTH) ile hardal [Brassica juncea (L.) Czern. & Coss.] cv Varuna’n›n muamelesi toplam çözünür
fenoller ve asidik çözünebilir hücre d›fl› proteinlerin kalitatif profillerinde de¤ifliklikleri teflvik eder. BTH
uygulamas›ndan sonra toplam çözünebilir fenoliklerin seviyesinde geçici bir art›fl vard› ve maksimum miktar
uygulamadan 72 saat sonra gözlendi. Hardal bitkisinin BTH muameleli yapraklar›n›n sulu methanol ekstrakt›n›n ince
tabaka kromotografisinde, kontrolde mevcut olmayan yeni fenolik birleflikler aç›¤a ç›kt›. 13.2 - 69,5 kDa aras›nda
de¤iflen moleküler kütleli 12 asidik çözünebilen protein BTH ile muamele edilen hardal bitkilerinin yapraklar›ndan
topland›. P13.8, P33.7 ve P34,5 proteinleri kontrolde çok az miktarda vard›r. BTH uygulamas›ndan 24 saat sonra en
çok göze çarpan proteinler erken teflvik edilenlerin görüldü¤ü 33.0 ve 33.7 kDa l›k moleküler kütlede görüldü.
Halbuki P33.0 BTH uygulamas›ndan 48 saat sonra en çok görülen proteindi. BTH uygulamas› sonucunda spesifik
fenoller ve proteinlerde de¤ifliklikler hardal bitkisinde teflvik edilen direncin faydal› bir belirleyicisi olabilece¤ini
Anahtar kelimeler: Brassica juncea , Benzotiyadiazol, ekstra hücresel proteinler.
52 Sanjay Guleria and Ashok Kumar
Plants have several lines of defense against invading
pathogens including preformed barriers and induced
responses. The later include a rapid production of
reactive oxygen species, synthesis of phenolics and
production of large amount of pathogenesis related
(PR) proteins (Kombrink et al., 1993). Van Loon
(1985) defined them as proteins which have low
molecular weights, are extremely soluble in acids,
have low isoelectric points (pI), are resistant to
photolytic digestion, are secreted into the intercellular
fluid and have no apparent enzymatic activities. PRproteins
have been shown to possess antimicrobial
activity (Brunner et al., 1995). Their synthesis occurs
after pathogen attack at a faster rate in incompatible
than compatible interaction (Kombrink and Somssich,
1997). Apart from this, they also accumulate in
response to treatment with abiotic elicitors such as
salicylic acid (SA), benzo(1,2,3)-thiadiazole-7carbothioic
acid S-methyl ester (BTH) and 2,6dichloroisonicotinic
acid (INA) (Cohen, 1994;
Vernooij et al., 1995; Gorlach et al., 1996; Spletzer
and Enyedi, 1999). The expression of genes encoding
basic PR-1 from barley and acidic PR-1 from maize
has also been shown to be induced after plant
treatment with INAor BTH respectively (Kogel et al.,
1994; Morris et al., 1998). Two groups of PR-proteins,
namely chitinases and β-1, 3-glucanases were found to
have enzymatic functions (Legrand et al., 1987;
Kauffmann et al., 1987). A role for these hydrolytic
enzymes in plant defense response against fungal
pathogens is suggested as they degrade chitin and β-1,
3-glucan, major structural polysaccharides in the cell
walls of many fungi (Mauch et al., 1988).
Induced synthesis of phenolic compounds is
associated with host-pathogen interaction and specific
phenolics have been implicated in host resistance
(Glazener, 1982; Hammerschmidt et al., 1982; Friend
et al., 1973). Ferulic and p-coumaric acids in bound
form have been found to be involved in the resistance
of wheat leaves to Puccinia recondita f. sp. tritici
(Southerton and Deverall, 1990).
Benzothiadiazole has been developed by Novartis
for the protection of crop plants from potential
pathogens by inducing systemic acquired resistance
(SAR) in the host plant. SAR refers to a distinct signal
transduction pathway that plays an important role in
the ability of plants to defend themselves against
pathogens (Hunt and Ryals, 1996). Kaur and Kolte
(2001) showed that foliar treatment of mustard plants
by BTH protects them against staghead phase of
white rust (Albugo candida) by activating plants own
In this paper we demonstrate that treatment of
mustard plants cv. Varuna with BTH induce changes in
the qualitative profile of phenols and extracellular
Materials and methods
Seeds of mustard cv. Varuna were surface sterilized
with two changes of sterile distilled water before
sowing in 800 ml (16 cm height) pots containing a
medium of soil:sand:vermi compost (2:1:1 v/v/v).
Plants were maintained under controlled conditions
and irrigation was applied in the pots every third day.
For studying the qualitative profile of phenols 4-weekold
plants were sprayed with 1.428 mM aqueous BTH
solution. The leaves sprayed with water alone served
as control. Level of total soluble phenols was
determined in second and third leaves harvested 24,
48, 72 and 96 h after elicitor treatment. Qualitative
profile of phenols was determined by sampling the
second and third leaf of 4-week-old mustard plants 72
h after elicitor treatment. For the determination of
qualitative protein profile, second and third leaves of
4-week-old plants were treated with elicitor (BTH)
solution. The elicitor was applied in sterile aqueous
solution (0.095 mM) through the petioles for 12 h and
then the leaves were placed in sterile distilled water in
petri plates. The analogous treatment with water
served as control. The intercellular fluid (IF) was
isolated 24 and 48 h after treatment.
Quantification of total soluble phenolics
The method of Swain and Hillis (1959) was used for
the extraction and quantification of total soluble
phenolics. Fresh leaves (1 g) were extracted in 80%
methanol for 90 min at 80 °C. The extract was
centrifuged at 14,000 g for 15 min, and 100 μl of the
extract was diluted to 1 ml with water and mixed with
0.5 ml of 2.0 M Folin-Ciocalteu’s reagent and 0.5 ml
of 1 M Na 2CO 3. After 1h, absorbance of the sample
solution was measured at 725 nm using T 11 7
spectrophotometer (Systronics, India). Concentration
of total soluble phenolics in the extract was calculated
from a standard curve prepared with gallic acid. The
data were analyzed using t-test (Gupta and Kapoor,
1993). Values in Fig.1 are the means and the bars
indicate the standard deviations.
Thin layer chromatography
Thin layer chromatography was used for the
qualitative profiling of phenolic compounds in the
leaves of Brassica juncea cv. Varuna using the
methods of Harborne (1973). Leaves were extracted in
Figure 1. Total soluble phenolic content in the leaves of
Brassica juncea cv. Varuna 24, 48, 72 and 96 h after BTH
80% methanol and the extracts concentrated to the
aqueous phase using a vacuum rotary evaporator.
Phenolics were then partitioned into ethyl acetate (2
x equal volume). A single spot of extract (25 µl) was
applied onto silica gel TLC plates (250 µm) and the
plates developed in n- butanol-acetic acid-water
(BAW, 4:1:5, v/v/v). Response to ultra-violet light and
diagnostic spray reagents was determined.
Extraction of intercellular fluids
The procedure adopted was a simplified form of the in
vacuo infiltration method described by Parent and
Asselin (1984). Leaves were submerged in a 32
mM/84 mM phosphate/citrate buffer pH 2.8
containing 1% β-mercaptoethanol (20 ml g -1 fresh
weight) and vacuum infiltrated for 10 min. The leaves
were dried on the filter paper, rolled into cylindrical
bundle and placed in a 5 ml syringe inside a centrifuge
tube. The intercellular fluid was obtained by
centrifuging the tissue at 1000 g for 10 min at room
temperature. The protein content of the intercellular
fluid was determined as µg BSA equivalents using
Bradford (1976) method.
SDS-polyacrylamide gel electrophoresis
The proteins were separated on the gels according to
Läemmli (1970) with the following concentrations
used in the stacking /separation gels-5/12%
acrylamide :N,N’-methylenebisacrylamide (30:0.8);
125/375 mM Tris-HCl pH 8.8; 0.1% (w/v) SDS;
0.05/0.01% (v/v) ammoniumpersulphate. The running
buffer was 25 mM Tris, 190 mM glycine, and 0.1%
(w/v) SDS, pH 8.3. Protein in the intercellular fluid
was precipitated with nine volumes of ice cold
acetone. After 16 h at 4 o C, the precipitates were
collected by centrifugation at 5000 g for 20 min at
4 o C. The pellets were suspended in sample buffer
(0.0625 M Tris-HCl pH 6.8, 2% SDS, 5% βmercaptoethanol,
10% glycerol and 0.002%
brompohenol blue), heated to 95 o C for 3 min, spun
rapidly , and loaded (max. 20 µg protein per slot) onto
80 x 70 x 0.1 mm slab gels. The current was
maintained at 10 mA per gel until the samples had
passed the stacking gel then increased to 20 mA and
run for approximately 2 h. The gel was stained and
fixed for 1h in 40% MeOH, 10% acetic acid and 0.1%
Coomassie brilliant blue G-250. Overnight destaining
was carried out in 10% MeOH and 7.5% acetic acid.
Relative molecular weight of the proteins was
determined using protein molecular weight marker
Results and discussion
Treatment of mustard with BTH 53
The resistance-inducing activity of BTH probably
relies on the activation of a natural defense pathway
called systemic acquired resistance ( Sticher et al.,
1997; Lucas, 1999) Treatment of Brassica juncea cv.
Varuna with BTH caused temporal changes in
concentration of soluble phenolics and accumulation
of acid soluble proteins. It is evident from the Fig.1
that total soluble phenolic content is significantly
54 Sanjay Guleria and Ashok Kumar
Table 1. Thin layer chromatography of extracts from leaves of Brassica juncea cv. Varuna 72 h after treatment with BTH.
R f values Visible UV a Folin b Van-HCl c
0.09 0.09 - - db -
0.25 0.25 - - db -
0.38 0.38 - db db -
0.52 0.52 - - db -
0.64 0.64 y-br - b -
- 0.76 - b b p-y
- 0.89 - b b-grey -
0.93 0.93 y db b -
a Colour reaction of the phenolic after treatment: y, yellow; br, brown; b, blue; db, dark blue; p, pink, b Folin-Ciocalteu reagent (2
M), c Vanillin-HCl (1 g Vanillin in 10 ml concentrated HCl).
with water (control) or BTH were run on the TLC
plates which contained fluorescent indicator and plates
observed under short wave UV (254 nm), separated
compounds appear as dark spots. When plates were
sprayed with Folin-Ciocalteu reagent, blue to blue
gray bands indicated the presence of at least eight
phenolic compounds in BTH treated and six in control
(Table 1). Of these, two of R F values 0.64 and 0.93
were yellow-brown and yellow in the visible. One
major band of high R F (R F = 0.64) changed to blue
immediately after spraying with Folin-reagent, a
characteristic of phenols which are derivatives of
catechol. A major band of high R F (R F = 0.76) which
was observed only in BTH treated leaves, showed pink
yellow colour after spraying with vanillin-HCl. Bands
corresponding to R F values 0.76 and 0.89 were only
observed in the extract from BTH treated leaves and
were absent in the control. Induced synthesis of
phenolic compounds is a common feature of host
pathogen interaction or treatment with certain
chemicals (elicitors) and specific phenolics have been
implicated in host resistance. Southerton and Deverall
(1990) reported that ferulic and p-coumaric acids were
involved in the resistance of wheat leaves to Puccinia
recondite f. sp. tritici. Similarly ferulic and p-coumaric
acids in corn leaves infected with Colletotrichum
g r a m i n i c o l a were inhibitory to spore germination
(Nicholson et al.,1989). Differences in phenolic
profile of Phytophthora cinnamomi resistant and
susceptible Eucalyptus cultivars have been reported
(Cahill and McComb, 1992). In our study of the types
of phenolics found in BTH treated and control leaves
of mustard differences in phenolics were evident.
There was a change in number and type of phenolics
after BTH treatment and accumulation of new
A typical separation of acid soluble proteins
accumulated in the intercellular spaces of BTH treated
mustard leaves obtained by SDS-PAGE is shown in
Fig.2. Intercellular fluid isolated from control leaves
contained 15 μg acid soluble proteins per 1g fresh
Figure 2. Accumulation of PR proteins in the intercellular
space of mustard leaves treated with 0.095 mM BTH. The
elicitor was applied through the petiole by placing the leaf in
BTH solution for 12 h and then in water. In the control
experiment (48c) leaves were treated with water instead of
BTH solution. 20 μg acid soluble proteins extracted from
intercellular spaces after vacuum infiltration were subjected
to SDS-polyacrylamide gel electrophoresis: gel stained with
Coomassie brilliant blue G-250.
weight infiltrated water treated leaves after 48 h. The
amount of acid soluble protein in the IF extracted from
BTH treated leaves was 50 μg and 75 μg per 1g fresh
weight infiltrated BTH treated leaves respectively
after 24 and 48 h after elicitor treatment. The numbers
on right hand side of Fig.2 indicate the apparent
molecular mass of each protein band.
The induction of PR-proteins has been reported in
several crop plants after treatment with biotic and
abiotic elicitors (Kagale et al., 2004; Reiss and
Bryngelsson, 1996; Silué et al., 2002). In total, 12 acid
soluble proteins, with molecular masses ranging from
13.2-69.5 kDa accumulated in BTH treated leaves of
mustard plants with P18.6, P26.8, P33.0 and P33.7
accumulating to the greatest extent. The protein P55.1
was present only in the control and its level reduced
after BTH treatment. Proteins P13.8, P33.7 and P34.5
were present in traces in control. The most prominent
proteins 24 h after BTH treatment were with apparent
molecular mass of 33.0 and 33.7 kDa indicating
towards their early induction, whereas, P33.0 was the
most prominent protein 48 h after treatment with BTH.
The expression of genes encoding acid soluble PRprotein
(PR-1) from maize has also been shown to be
induced after treatment with BTH (Morris et al.,
1998). Similarly, Sauerborn et al. (2002) reported
accumulation of PR-proteins in sunflower (Helianthus
annuus) treated with BTH.
From above study it may be concluded that BTH
induces changes in qualitative profile of total soluble
phenols and extracellular proteins in mustard by
activation of natural defense pathway and changes in
specific phenols and proteins might be the useful
markers of induced resistance in mustard.
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