®

Plant Stress ©2012 Global Science Books

Role of 24-Epibrassinolide in Amelioration of High Temperature

Stress through Antioxidant Defense System in Brassica juncea L.

Sandeep Kumar1 • Geetika Sirhindi1* • Renu Bhardwaj2 • Manish Kumar1 • Priya Arora2

1 Department of Botany, Punjabi University, Patiala-147002, Pb., India

2 Department of Botanical & Environmental Sciences, G. N. D. U., Amritsar, Pb., India

Corresponding author: * [email protected]

ABSTRACT
The present work has been undertaken to study the role of different concentrations of 24-epibrassinolide (24-EBL) (10-10, 10-8 and 10-6 M)
on growth, antioxidant enzyme [catalase (CAT), ascorbate peroxidase (APOX), superoxide dismutase (SOD)] activities and total protein
content in 10-days-old seedlings of Brassica juncea L. exposed to heat stress for 5 h daily for three consecutive days. Heat treatment
lowered total protein content of B. juncea seedlings. The seedlings treated with different concentrations of 24-EBL showed better growth
and enhanced protein content than the control. Similarly, the activities of SOD, CAT, APOX, PPO, and auxinases were enhanced by the
application of different concentrations of 24-epiBL. APOX activity was enhanced maximum with 10-10 M 24-epiBL while 10-8 M was
most effective for maximum enhancement in the activity of CAT and SOD.
_____________________________________________________________________________________________________________

Keywords: brassinosteroids, Brassica juncea heat shock protein, reactive oxygen species

INTRODUCTION ling (Kumar et al. 2010) and metal stress (Sharma et al.
2010) by enhancing antioxidant defense system and make
High temperature is one of the most important environ- the plant resistant to variety of environmental stresses
mental stresses that adversely affect plant growth and deve- (Krishna 2003). One mechanism that may be involved in
lopment thereby limiting plant productivity. It causes heat resistance to many types of stresses by escalating activity of
injury, which results into degradation of proteins, lipids and enzymes involved in antioxidant defense system. Keeping
bring changes in metabolism. These changes cause break this in mind, the aim of present experiment was to study the
down of cell products and membranes etc. Plant responds effect of high temperature on seedling growth and role of
and adapt to high temperature by several physiological, bio- 24-epiBL in amelioration of high temperature stress by
chemical and molecular responses. They tolerate high tem- regulating the activity of various enzymes involved in anti-
perature through the synthesis of heat shock proteins oxidant defense system along with Polyphenol Oxidase and
(HSPs) that either directly or indirectly governs levels of auxinases in B. juncea.
osmolytes, components of cell detoxification mechanisms
and factors that regulate membrane fluidity. Levels of reac- MATERIALS AND METHODS
tive oxygen species (ROS) are immensely high under
stressed conditions (Alscher et al. 2002). There are increa- Seeds of B. juncea L. cv. ‘RCM 619’ were procured from the
sing evidences that high temperature increased the genera- Department of Plant Breeding, Punjab Agriculture University,
tion of Reactive oxygen species (ROS) such as superoxide Ludhiana, India. This is an improved variety of brassica launched
(O·2), hydrogen peroxide (H2O2), hydroxyl radicals (OH-) recently for farmers of Punjab. This is resistant certified variety
and singlet oxygen (1O2) which have greater toxicity poten- having more seed production as compare to other varieties. Seeds
tials on biomolecules and biomembranes in plants. Plants were surface sterilized with 0.01% HgCl2 and rinsed 5-6 times
have antioxidant enzymes to scavenge the increased gene- with double distilled water. The sterilized seeds were soaked for 8
ration of ROS, which includes SOD (superoxide dismutase), h in different concentrations of 24-epiBL (Sigma-Aldrich. USA)
CAT (catalase), APOX (ascorbate peroxidase), DHAR (de- (0, 10-6, 10-8 and 10-10 M). The treated seeds were propagated in
hydroascorbate reductases) and MDHAR (monodehydroas- triplicate in Petri dishes under lab conditions at 20 ± 2°C and 16-h
corbate). Plant growth regulators like auxins, gibberellins, photoperiod while 7-days old seedling were exposed to 40°C for 5
ethylene jasmonates, and salicylic acid have been reported h daily for three days. After 40°C heat shock treatment, seedlings
to play stress protective role in plants (Cao et al. 2005). were transferred to normal lab conditions. Plants were sampled on
Recent studies indicate stress ameliorative properties of the 10th day after sowing for measuring morphological parameters
brassinosteroids (BRs), which are an important group of (root length and shoot length) and the activity of different anti-
plant steroids. (Dhaubhadel et al. 2002; Sirhindi et al. 2009) oxidant enzymes.
observed that BRs play a vital role in protecting the trans-
lation machinery and inducing HSPs under thermal stress. Preparation of enzyme extract
Similarly, Kumar et al. (2010) observed in 10-day-old
H2O2-treated Brassica juncea L. seedlings, that low tem- The enzyme extract of 10-days-old seedlings was prepared by
perature causing reduction in seed germination and seedling homogenizing 1 g fresh plant material in 3 ml of 100 mM phos-
growth but treatment of H2O2 alone or in combination with phate buffer (pH 7.0) for the estimation of total protein content
BRs makes the plant more adaptable to chilling stress. BRs and antioxidant enzyme activities.
have been reported to provide protection to plant from heat
(Zhu et al. 1996), drought (Li and van Staden 1998), chil-

Received: 12 March, 2009. Accepted: 16 April, 2011.

Original Research Paper
 Plant Stress 6 (1), 55-58 ©2012 Global Science Books

A B

Fig. 1 Effect of temperature stress on (A) shoot length (cm) and (B) root length (cm) of 10-days-old seedlings of Brassica juncea L. treated with different
concentrations of 24-epibrassinolide (epiBL) (10-6, 10-8, 10-10 M). Values represent mean ± standard error (SE). * Shows significance difference at P >
0.05.

Enzyme activity M was the most effective concentration for temperature

amelioration (15.408 ± 0.99 mg g-1 FW). The activities of
SOD activity was estimated by recording the increase in absor- antioxidant enzymes (SOD, CAT, APOX) were enhanced in
bance of superoxide nitro blue tetrazolium at 540 nm following the B. juncea seedlings, treated with 24-epiBL exposed to tem-
method of Kono (1978). CAT activity was measured according to perature stress. APOX activity (Fig. 2D) was significantly
Aebi (1983). 3 ml reaction mixture containing 1.5 ml of 100 mM enhanced at 10-10 M 24-epiBL (26.66 ± 0.67 μmol U mg-1
phosphate buffer (pH 7.0) and 0.05 ml of 75 mM H2O2, 0.05 ml protein). 10-8 M 24-epiBL was the best concentration for en-
enzyme extract and distilled water was used. Reaction was started hancement of CAT (Fig. 2C) and SOD (Fig. 2B) activities,
by adding H2O2 and CAT activity was measured as decrease in 9.34 ± 0.54 and 2.13 ± 0.97 mol U mg-1 protein, respec-
absorbance at 240 nm for 1 min. Enzyme activity was computed tively in seedlings that were exposed to temperature stress.
by calculating the amount of H2O2 decomposed. Activity of APOX The activity of auxinases (Fig. 3B) was maximum at 10-6 M
was measured following the method of Nakano and Asada (1981) 24-epiBL (0.090 ± 0.032) although all the 24-epiBL treat-
by monitoring the rate of ascorbate oxidation at 290 nm. The ments showed significantly enhanced IAAO activity com-
reaction mixture contains 100 mM phosphate buffer (pH 7.0), 5 pared to control seedlings and seedling exposed to tempera-
mM ascorbate, 5 mM H2O2 and the enzyme extract. PPO activity ture stress. On the other hand, PPO activity (Fig. 3A) was
was measured using the method of Bastin and Unlaer (1972). The enhanced in all 24-epiBL treatments, significantly so at 10-8
reaction mixture contain of 60 mM phosphate buffer (pH 7.0) and M of 24-epiBL (104.1 ± 6.28 mol U mg-1 protein). Expo-
0.01 M chlorogenic acid. The reaction mixture was incubated at 30 sure to temperature stress ameliorated the antioxidant en-
± 2°C for 1 h. Absorbance was measured at 430 nm. IAAO acti- zyme system, which was further enhanced by treating the
vity was measured according to the method of Gordon and Weber seedlings with different concentrations of 24-epiBL. How-
(1951). The reaction mixture contained Salkowski reagent and ever, the concentration of 24-epiBL that could ameliorate
0.01% IAA solution. Enzyme extract was incubated at 40°C for 20 different enzymes and make the plant more resistant to tem-
min in the dark. Then mixture was cooled and absorbance was perature stress varied from enzyme to enzyme.
taken at 535 nm. Total protein content was determined following
the method of Lowry et al. (1951) DISCUSSION

Statistical analysis Preliminary studies have demonstrated that EBR treatment

improved heat tolerance of wheat leaf cells (Kulaeva et al.
One way analysis of variance (ANOVA) was performed in order to 1991), and bromegrass suspension cells (Wilen et al. 1995).
compare the mean of all measured characteristics of 24-epiBL In the present study, B. juncea seedlings showed suscepti-
(10-6, 10-8, 10-10 M) treated, control and temperature treated seed- bility to temperature stress. After temperature stress treat-
lings. Significant differences between the means were evaluated ment to seedlings, growth (e.g., shoot and root length) was
by an honestly significant difference (HSD) test. significantly inhibited. High temperature stress decreased
the germination rate and shoot length of tomato seedlings as
RESULTS reported by Singh and Shono (2005) and in barley and
radish seeds by Cavusoglu and Kabar (2007). Our previous
Better initial plant growth was observed in all 24-epiBL studies also illustrated that BR protects plants starting from
treated seeds at 20 ± 2°C than control seedlings without 24- their germination till maturity by up and down regulation of
epiBL treatment. Root length (Fig. 1B) was inhibited by various non-enzymatic and enzymatic activities at the cel-
10-6 M 24-epiBL (1.11 ± 0.89 cm) to a greater extent than lular level (Sirhindi et al. 2009). BRs also enhanced the
that of temperature stress (1.22 ± 0.33 cm) than control germination rate of seeds and the growth after direct sowing
seedlings (1.29 ± 0.88 cm). Temperature stress inhibited in submerged paddy pots at low temperature (Bajguz and
shoot length (Fig. 1A) of 10-day-old seedlings, but this was Hayat 2008). Protein synthesis was maintained in BR-
ameliorated by 24-epiBL. 10-8 M was the most effective treated leaves at 43°C at levels similar to those at 23°C
concentration for this amelioration (1.95 ± 0.95 cm). High- (Kulaeva et al. 1991), whereas in untreated leaves it de-
est total protein (Fig. 2A) content (20 ± 1.98 mg g-1 FW) creased 2.5-fold at 43°C compared to samples at the control
was observed in 10-day-old seedlings than in temperature- temperature. Sirhindi et al. (2009) and Kumar et al. (2010)
treated seedlings (13.29 ± 0.99 mg g-1 FW). 24-epiBL observed that 24-epiBL treatment helped to mitigate stress
helped the seedlings overcome temperature stress and 10-8 by regulating activities of antioxidant enzymes. High tem-

56
 24-epiBL ameliorates high temperature stress in Brassica juncea L. Kumar et al.

A
(A) B
*
* *
*
* *
* *

C D
*
*
*
*

Fig. 2 (A) Total protein (mg mg-1 FW), (B) activity of SOD (mol U mg-1 protein), (C) activity of CAT (mol U mg-1 protein), and (D) APOX (mol U mg-1
protein) of 10-days-old seedlings of Brassica juncea L. exposed to temperature stress (40°C) and different concentrations of 24-epiBL (10-6, 10-8, 10-10
M). Values represent mean ± standard error (SE). * Shows significance difference at P > 0.05.

perature stress often causes membrane damage, decrease with 10-8 and 10-10 M 24-epiBL showing significant dif-
hydrolytic enzyme activities and increases the lipid peroxi- ferences in APOX activity compared to control seedlings.
dation level. It may stimulate the formation of ROS, such as The results shown in the present study proved that 24-
OH-, H2O2, and O2.- radicals. Among ROS, superoxide radi- epiBL enhances APOX activity at a very dilute concentra-
cal (O2.-) is dismutated by SOD in H2O2 and is further sca- tion. Bhardwaj et al. (2007) reported that exogenous treat-
venged by CAT and various peroxides. APOX and GR also ment is effective in stressful rather than in optimal con-
play a key role by reducing H2O2 to water through the As- ditions. The effect of 24-epiBL and MH5 (polyhydroxylated
corbate-glutathione cycle (Noctor and Foyer 1998). The spirostanic analogue of brassinosteroids) was analyzed by
level of CAT, SOD and APOX were increased by the ap- Mazorra et al. (2002) on CAT, APOX and SOD activity in
plication of 24-epiBL to overcome the stress generated by tomato leaf discs at 25-40°C; BRs altered the activity of
high temperature and to boost the resistance capacity of B. these enzymes, suggesting a possible role of 24-epiBL and
juncea plants. Various studies indicated that 24-epiBL treat- MH5 in the reduction of cell damage produced by heat
ment enhanced the antioxidant enzyme activities under dif- stress. Although PPO is not considered a component of the
ferent abiotic stresses (Dhaudhadel et al. 2002; Bhardwaj et antioxidant defense system, it plays an important role to
al. 2007; Arora et al. 2008; Sharma et al. 2010). The pre- control oxidative stresses. The activity of PPO was in-
sent investigation also revealed the induction of oxidative creased under stress (Zhukova et al. 1996). PPO activity
stress and ROS production during temperature stress treat- was enhanced by the application of 24-epiBL in the present
ment in B. juncea seedlings. Reports are also available indi- study. Darbyshire (1971) reported that the activity of IAA
cating that a mild increase in temperature enhanced the Oxidase increased under water stress in pea plants. The pre-
growth of plant, but when this temperature exceeded a per- sent work also shows an increase in IAA Oxidase activity
missible limit, it increased the production of ROS. The under the influence of 24-epiBL in seedlings subjected to
effects of high temperature stress in BR-treated and -untrea- temperature stress.
ted Brassica napus and tomato seedlings were examined at In conclusion, the present study reveals that temperature
the expression level of total protein synthesis, which in- is an important environmental factor responsible for normal
creased in BR-treated seedlings (Dhaubhadel et al. 1999). functioning of plant growth and metabolism but an elevated
In the present investigation total protein content increased degree of temperature beyond a certain limit cause growth
quantitatively in 24-epiBL-treated and untreated seedlings. inhibition and damages the cell and ultimately leads to
In the present study SOD and CAT activity were enhanced necrosis. 24-epiBL showed stress-ameliorative properties in
in temperature- and BR-treated seedlings. 10-8 M 24-epiBL B. juncea seedlings exposed to temperature stress by im-
was best in ameliorating temperature stress. APOX activity proving seedling growth and enhancing protein content and
was the same in temperature-treated and control seedlings. activities of CAT, SOD, APOX, PPO, IAA Oxidase. This
However, this activity was enhanced when seedlings ex- study culminates to the role of BR as an anti-stress agent for
posed to temperature stress were treated with 24-epiBL, protecting plants exposed to various stresses although ex-

57
 Plant Stress 6 (1), 55-58 ©2012 Global Science Books

A B *
* * * * *
*

Fig. 3 (A) Activity of IAAO (mol U mg-1 protein), (B) activity of PPO (mol U mg-1 protein) of 10-days-old seedlings of Brassica juncea L. exposed to
temperature stress (40°C) and different concentrations of 24-epiBL (10-6, 10-8, 10-10 M). Values represent mean ± standard error (SE). * Shows
significance difference at P > 0.05.

tensive studies are still needed on various aspects related to Kulaeva ON, Burkhanova EA, Fedina AB, Khokhlova VA, Bokebayeva GA,
stress and the role of BRs in regulating them. Vorbrodthm HM, Adam G (1991) Effect of brassinosteroids on protein syn-
thesis and plant –cell ultra structure under stress conditions. In: Cutler HG,
Yokota T, Adam G (Eds.) Brassinosteroids: Chemistry, Bioactivity and Ap-
ACKNOWLEDGEMENTS
plications, American Chemical Society Symposium Series 474, American
Chemical Society, Washington DC, pp 141-157
The financial assistance provided by UGC (University Grants Kumar M, Sirhindi G, Bhardwaj R, Kumar S, Jain G (2010) Effect of exo-
Commission) is duly acknowledged. genous H2O2 on antioxidant enzymes of Brassica juncea L. seedlings in rela-
tion to 24-epibrassinolide under chilling stress. Indian Journal of Biochemis-
REFERENCES try and Biophysics 47 (6), 378-382
Li L, van Staden J (1998) Effect of plant growth regulators on the antioxidant
Aebi H (1983) Catalase. In: Bergmeyer HU (Ed) Methods of Enzymatic Analy- system in seedlings of two maize cultivars subjected to water stress. Plant
sis, Verlag Chemie, Weinhan, pp 673-684 Growth Regulation 25, 81-87
Alscher RG, Erturk N, Health LS (2002) Role of superoxide dismutases Lowry OH, Rosenbrough NJ, Farr AL, Randall RL (1951) Protein measure-
(SODs) in controlling oxidative stress in plants. Journal of Experimental ment with folin-phenol reagent. The Journal of Biological Chemistry 193,
Botany 53, 1331-1341 265-275
Arora N, Bhardwaj R, Sharma P, Arora HK (2008) Effect of 28-homo- Mazorra LM, Núñez M, Hechavarria M, Coll F, Sánchez-Blanco MJ (2002)
brassinolide on growth, lipid peroxidation and antioxidative enzyme acti- Influence of brassinosteroids on antioxidant enzymes activity in tomato under
vities in seedlings Zea mays L. under salinity stress. Acta Physiologia Plan- different temperatures. Biologia Plantarum 45 (4), 593-596
tarum 30, 833-839 Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate spe-
Bajguz A, Hayat S (2008) Effects of Brassinosteroids on the plant responses to cific peroxidase in spinach chloroplasts. Plant and Cell Physiology 22, 867-
environmental stresses. Plant Physiology and Biochemistry 47, 1-8 880
Bastin M, Unleur O (1972) Effect of actinomycin–D on the formation of en- Noctor G, Foyer CH (1998) Ascorbate and glutathione: Keeping active oxygen
zymes in Jerusalem artichoke tuber slices. Planta 102, 357-361 under control. Annual Review of Plant Physiology and Plant Molecular Biol-
Bhardwaj R, Arora N, Sharma P, Arora HK (2007) Effect of 28-homobras- ogy 49, 249-279
sinolide on seedling growth, lipid peroxidation and antioxidative enzyme Ozdemir F, Bor M, Demiral T, Turkan I (2004) Effects of 24-epiBL on seed
activities under nickel stress in seedlings of Zea mays L. Asian Journal of germination, seedling growth, lipid peroxidation, proline content and antioxi-
Plant Sciences 6, 765-772 dative system of rice (Oryza sativa L.) under salinity stress. Plant Growth
Cao S-Q, Xu Q-T, Cao Y-J, Qian K, An K, Zhu Y, Hu B-Z, Zhao H-F, Kuai Regulation 42, 203-211
B (2005) Loss of function mutations in DET2 gene lead to an enhanced resis- Sharma I, Pati PK, Bhardwaj R (2010) Regulation of growth and antioxidant
tance to oxidative stress in Arabidopsis. Physiologia Plantarum 123, 57-66 enzyme activities by 28-homobrassinolide in seedlings of Raphanus sativus L.
Cavusoglu K, Kabar K (2007) Comparative effects on some plant growth under cadmium stress Indian Journal of Biochemistry and Biophysics 47,
regulator on the germination of barley and radish seeds under high tempera- 172-177
ture stress. EurAsian Journal of BioSciences 1, 1-10 Singh I, Shono M (2005) Physiological and molecular effects of 24-epibras-
Darbyshire B (1971) The effect of water stress on indoleacetic acid oxidase in sinolide, a brassinosteroid on thermotolerance of tomato. Plant Growth Regu-
pea plants. Plant Physiology 47, 65-67 lation 47, 111-119
Dhaubhadel S, Browning KS, Gallie DR, Krishna P (2002) Brassinosteroid Sirhindi G, Kumar S, Bhardwaj R, Kumar M (2009) Effects of 24-epibras-
functions to protect the transnational machinery and heat shock protein syn- sinolide and 28-homobrassinolide on the growth and antioxidant enzyme
thesis following thermal stress. Plant Journal 29, 681-691 activities in the seedlings of Brassica juncea L. Physiology and Molecular
Dhaubhadel S, Chaudhary S, Dobinson KF, Krishna P (1999) Treatment Biology of Plants 15 (4), 335-341
with 24-epibrassinolide (a brassinosteroid) increase the basic thermotolerance Wilen RW, Sacco M, Gusta LV, Krishna P (1995) Effect of 24-epibrassinolide
of Brassica napus and tomato seedlings. Plant Molecular Biology 40, 333- on freezing and thermotolerence bromegrass (Bromus inermis) cell cultures.
342 Physiologia Plantarum 95, 195-202
Gordon SA, Weber RP (1951) The effect of X-radiation on indoleacetic acid Zhu C, Zeng GW, Liu F (1996) Effect of epibrassinolide on the heat sock tol-
and auxin levels in the plant. American Journal of Botany 37, 678 (Abstract) erance and antioxidant metabolism in cucumber seedling. Journal of Zheji-
Kono Y (1978) Generation of superoxide radical during auto oxidation of ang Agricultural University 22, 284-288
hydroxylamine and an assay for superoxide dismutase. Archives of Bioche- Zhukova LA, Voskresenskaya OL, Grosheva NP (1996) The effect of stand
mistry and Biophysics 186, 189-195 density on morphological and physiological features of Calendula officinalis
Krishna P (2003) Brassinosteroids-mediated stress responses. Journal of L. development. Ekologiya 2, 104-110
Plant Growth Regulation 22, 289-297

58