Qualitative profiling of phenols and extracellular proteins induced in ...

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

Abstract

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

Özet

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 a盤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

ortaya koymaktad›r.

Anahtar kelimeler: Brassica juncea , Benzotiyadiazol, ekstra hücresel proteinler.

51

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52 Sanjay Guleria and Ashok Kumar

Introduction

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

defense response.

In this paper we demonstrate that treatment of

mustard plants cv. Varuna with BTH induce changes in

the qualitative profile of phenols and extracellular

proteins.

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,

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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

treatment.

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

(Fermentas, USA).

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

enhanced (P

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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

Control Treated

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

compounds.

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.

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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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