BRAZILIAN JOURNAL OF PLANT PHYSIOLOGY

The official journal of the SHORT COMMUNICATION

Brazilian Society of Plant Physiology

28-Homobrassinolide alleviates oxidative stress in salt-

treated maize (Zea mays L.) plants
Nitika Arora, Renu Bhardwaj*, Priyanka Sharma and Hardesh K. Arora

Department of Botanical & Environmental Sciences, Guru Nanak Dev University, Amritsar 143005 (Punjab), India.
*Corresponding author: renubhardwaj82@gmail.com
Received: 03 May 2008; Returned for revision: 18 June 2008; Accepted: 30 June 2008

The present investigation was undertaken to study the effects of 28-homobrassinolide on the activities of antioxidative
enzymes such as superoxide dismutase (EC 1.15.1.1), guaiacol peroxidase (EC 1.11.1.7), catalase (EC 1.11.1.6), glutathione
reductase (EC 1.6.4.2) and ascorbate peroxidase (EC 1.11.1.11), as well as protein and malondialdehyde concentrations in 30-
d-old plants of Zea mays L. grown under salt stress. The seeds were soaked in 28-homobrassinolide solutions (0, 10-8, 10-6 and
10-4 mM) for 12 h and then sown in the field in a randomized block layout. The blocks were salinised with NaCl at
concentrations of 0, 25, 50 and 75 mM. The activities of antioxidative enzymes and protein concentration increased in 28-
homobrassinolide-treated plants. Despite the enhancement of enzyme activities under salt stress alone, lipid peroxidation
increased and protein concentration decreased. However, pre-sowing treatments of 28-homobrassinolide further enhanced
the activities of antioxidative enzymes in addition to lowering lipid peroxidation and increasing protein concentration, thus
suggesting that 28-homobrassinolide can alleviate oxidative stress in salt-treated maize plants.
Key words: antioxidative enzymes, brassinosteroids, lipid peroxidation, maize, salt stress

28-Homobrassinolídeo reduz o estresse oxidativo em plantas de milho (Zea mays L.) tratadas com sal: Objetivou-se estudar
os efeitos do 28-homobrassinolídeo sobre as atividades de enzimas antioxidantes [dismutase do superóxido (EC 1.15.1.1),
peroxidase do guaiacol (EC 1.11.1.7), catalase (EC 1.11.1.6), redutase da glutationa (EC 1.6.4.2) e peroxidase do ascorbato (EC
1.11.1.11)] e concentrações de proteínas e aldeído malônico em plantas de milho com 30 d de idade, cultivadas sob estresse
salino. As sementes foram embebidas em soluções de 28-homobrassinolídeo (0, 10-8, 10-6 and 10-4 mM) por 12 h e, então,
semeadas no campo, seguindo-se um desenho experimental de blocos ao acaso. Os blocos foram salinizados com NaCl a
concentrações de 0, 25, 50 e 75 mM. As atividades das enzimas antioxidantes e a concentração protéica aumentaram nas
plantas tratadas com 28-homobrassinolídeo. A despeito do aumento das atividades das enzimas sob estresse salino
isoladamente, a peroxidação lipídica aumentou e a concentração protéica reduziu-se. Todavia, tratamentos de pré-emergência
com 28-homobrassinolídeo promoveram aumentos adicionais nas atividades das enzimas antioxidantes, além de acarretar
decréscimos na peroxidação lipídica e aumentos na concentração protéica, sugerindo, portanto, que o 28-homobrassinolídeo
pode aliviar o estresse oxidativo em plantas de milho sob estresse salino
Palavras-chave: brassinosteróides, enzimas antioxidantes, estresse salino, milho, peroxidação lipídica

Salt stress is one of the major abiotic stresses faced superoxide radical, hydrogen peroxide, hydroxyl radical
by plants, which adversely affect their productivity. and alkoxyl radical. These ROS produced in the cell are
Many crop plants such as barley, maize and rice, are often detoxified by both non-enzymatic and enzymatic
subject to salinity stress (Sairam and Tyagi, 2004). antioxidant systems. The enzymatic antioxidative system
Salinity also leads to oxidative stress in plants due to the consists of several enzymes such as guaiacol peroxidase
production of reactive oxygen species (ROS) such as the (POD), catalase (CAT), superoxide dismutase (SOD),

Braz. J. Plant Physiol., 20(2):153-157, 2008

154 ARORA et al.

ascorbate peroxidase (APX), and glutathione reductase grown under field conditions during the normal crop-
(GR) (Asada and Takahashi, 1987; Arora et al., 2002). A growing season (June-August) with a supplementary
number of plant hormones such as ethylene, abscisic water supply (sprinkler irrigation). After 30 d from
acid, salicylic acid and steroids are involved in the sowing, leaves of the maize plants were harvested from
regulation of the plant antioxidative enzymatic system the second whorl from the top for the study of various
(Cao et al., 2005). biochemical parameters.
Brassinosteroids (BRs) are hydroxylated derivatives For biochemical assays, 1 g of shoot tissue was
of cholestane, which play an essential role in plant homogenized in 3 mL of 100 mM potassium phosphate
growth and development by influencing various buffer (pH 7.0). The homogenate was centrifuged at 4oC
physiological responses (Bajguz and Tretyn, 2003). One for 20 min at 15,000 g. The supernatant was used for
of the most important roles of BRs is their ability to confer assays of antioxidative enzymes and protein
resistance to plants against various abiotic/biotic concentration. Each treatment consisted of three
stresses such as heat, drought, heavy metals, infection, replicates. The activity of SOD was determined by
pesticides, salt and even viruses (Kagale et al., 2007). monitoring its ability to inhibit photochemical reduction
Under the influence of this group of plant steroids, of nitrobluetetrazolium at 540 nm (Kono, 1978), and CAT
Özdemir et al. (2004) and Nunez et al. (2003) observed that activity was determined by following the initial rate of
resistance to stresses involves regulation of disappearance of H 2 O 2 at 240 nm (Aebi, 1974). The
antioxidative enzyme activities. However until now, no activities of POD, APX and GR were measured according
data have been documented on salt stress management to Putter (1974), Nakano and Asada (1981) and Carlberg
by BRs in maize plants. The objective of the present and Mannervik (1975), respectively. Protein
study was therefore to investigate the influence of 28- concentration was determined following the method of
homobrassinolide (HBL) on activities of antioxidative Lowry et al. (1951). Lipid peroxidation was determined as
enzymes, lipid peroxidation and protein content of maize the concentration of malondialdehyde (MDA) in a shoot
plants under salt (NaCl) stress. extract prepared in 0.1% trichloroacetic acid, using the
A field experiment was conducted to study the effects thiobarbituric acid reaction as described by Heath and
of seed-presowing treatment of HBL on biochemical Packer (1968). The data were analyzed statistically by
parameters of maize (Zea mays L. var. Partap-1) plants using one-way analysis of variance (ANOVA); means
grown under salt stress. Seeds were surface sterilised were compared by Tukey’s HSD (Honestly Significant
with 0.05% mercuric chloride for 5 min followed by Differences) test and differences with P values ≤ 0.05
repeated rinses in sterile distilled water. Seeds were were considered significant (Bailey, 1995). Data are
soaked for 12 h in aqueous solution of different presented as the mean ± SE.
concentrations of HBL. The stock solution (1 mM) of HBL The studies conducted on biochemical parameters of
(Sigma Aldrich, New Delhi, India) was prepared in DMSO salt-stressed maize plants indicated significant effects of
and further serial dilutions were made with double- HBL treatments (Table 1). Activities of antioxidative
distilled water to prepare different concentrations of HBL enzymes (SOD, POD, CAT, APX and GR) were enhanced
(0, 10-8, 10-6 and 10-4 mM). The field area was divided into in maize plants raised from seeds pre-treated with HBL
randomised blocks that were salinised with NaCl. For alone, with the 10 -6 mM concentration being the most
salinisation of each block, soil was removed to a depth of effective since it produced the highest HSD (Honestly
30 cm. The soil was weighed and salt mixed with this soil Significant Differences) value (Table 1). The activities of
to obtain a final concentration of salt in the soil of 0, 25, 50 these enzymes also increased in response to salt
and 75 mM. The treated soil was then uniformly additions with further enhancement in plants pre-treated
distributed in the respective blocks. One salinised block with HBL and grown in salinised soil (Table 2). Maximum
was separated from another by a border of ca. 20 cm long increase in SOD activity (17.47 U min-1 mg-1 protein) was
and 15 cm in height to avoid cross-contamination by salt observed in plants treated with HBL at 10 -8 mM and
between blocks. The field soil consisted of clay, sand and grown under 25 mM NaCl as compared to untreated
manure in the ratio of 2:1:1, respectively. The plants were plants grown under 25 mM NaCl only (11.01 U min-1 mg-1

Braz. J. Plant Physiol., 20(2):153-157, 2008

 28-HOMOBRASSINOLIDE ALLEVIATES OXIDATIVE STRESS IN SALT-TREATED MAIZE 155

protein). The activity of POD was maximum in plants Pre-sowing treatment of HBL significantly improved
grown under 75 mM NaCl (12.27 mmol GDHP min-1 mg-1 plant tolerance to saline conditions by enhancing the
protein) when compared with the other salt treatments activities of antioxidative enzymes and protein
without HBL application. Seed-presowing treatment of concentration. Generally, salt stress impairs plant growth
HBL to salt-stressed plants further enhanced POD by affecting water absorption and other biochemical
activity that was maximum (13.99 mmol GDHP min-1 mg-1 processes such as increases in activity of antioxidative
protein) in plants raised from seeds pre-treated with HBL enzymes and antioxidants of the Asada-Halliwell
at 10-8 mM concentration and grown under 25 mM NaCl. pathway (Chen et al., 1997; Sairam and Srivastava, 2002).
Similarly maximum CAT activity in plants treated with salt In this pathway, the superoxide radical suffers
only was observed at 75 mM NaCl (9.13 mol H2O2 min-1 mg-1 dismutation by SOD into H2O2 which in turn is scavenged
protein). Maximum enhancement in CAT (11.30 mol H2O2 by CAT and various peroxidases. Both APX and GR also
min -1 mg-1 protein) and APX (13.95 mmol ascorbate min -1 play a key role by reducing H2O2 to water through the
mg-1 protein) activities were found in plants treated with ascorbate-glutathione cycle (Noctor and Foyer, 1998). 28-
HBL at 10-8 mM and grown under 50 mM of NaCl. Similarly Homobrassinolide may confer tolerance to salt stress by
GR activity was maximum (3.71 mmol NADPH min-1 mg-1 increasing the activities of antioxidative enzymes and/or
protein) in plants raised from 10-4 mM HBL-pre-treated by reducing the uptake of salts as indicated by previous
seeds and grown under 50 mM NaCl (Tables 1 and 2). studies where HBL reduced the uptake of heavy metals
Plants raised from seeds pre-treated with HBL alone and activated the antioxidative enzymes of Oryza sativa,
showed an increase in soluble protein concentration in Brassica juncea and Zea mays plants (Özdemir et al.,
comparison with untreated seedlings, 10-6 mM of HBL 2004; Bhardwaj et al., 2007; Sharma and Bhardwaj, 2007;
being the most effective (Table 1). On the other hand, Arora et al., 2008). In addition, Zhang et al. (2007)
protein concentration decreased with increasing salt observed that brassinoloide treatment of Medicago
concentration (Table 2). Plants treated with HBL at 10-6 sativa increased the germination percentage, fresh
mM and grown under 25 mM NaCl, led to maximum weight and activities of antioxidative enzymes (POD,
enhancement in protein concentration (30.78 g kg-1 FW) SOD and CAT).
relative to control plants grown under the same salt Brassinosteroids are found to affect the transcription
concentration (27.30 g kg-1 FW). The concentration of and translation processes of specific genes related to
MDA increased in salt-stressed plants. However HBL stress-tolerance (Kagale et al., 2007). As membrane
application decreased the MDA levels, especially in destruction results from ROS-induced oxidative damage,
plants treated with HBL at 10-6 mM and grown under 50 which ultimately increases the MDA content, the HBL
mM NaCl (4.66 mmol kg-1 FW) relative to HBL-untreated treatments may be involved in scavenging ROS more
plants raised under 50 mM NaCl (6.74 mmol kg -1 FW) effectively in the plants. Our observations are in
(Tables 1 and 2). agreement with the results of Özdemir et al. (2004), who

Table 1. Effect of HBL on protein and malondialdehyde (MDA) concentrations, and specific activities of antioxidative
enzymes [superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX) and
glutathione reductase (GR)] of 30-d-old maize plants. n = 3 ± SE. Asterisks indicate significance at P ≤ 0.05 [Inside
bracket is HSD (Honestly Significant Differences) value].

Treatments of Protein (g kg-1 MDA (mmol SOD (U min-1 POD (mmol CAT (mol APX (mmol GR (mmol
HBL FW) kg-1 FW) mg-1 protein) GDHP min-1 H2O2 min-1 ascorbate min-1 NADPH min-1
mg-1) mg-1) mg-1) mg-1)
0 28.38±1.86 6.06±0.16 9.02±0.85 8.79±0.22 4.06±0.09 5.67±0.23 1.75±0.16
10-8 mM 32.04±0.87 5.50±0.23 10.37±0.54 9.83±0.27 5.71±0.60 8.45±0.49 2.07±0.13
10-6 mM 33.54±0.54 4.67±0.20 12.93±0.54 9.99±0.46 6.32±0.46 8.90±0.15 2.50±0.08
10-4 mM 33.33±0.75 5.87±0.19 10.59±0.51 9.61±0.22 5.68±0.49 8.45±0.32 2.44±0.22
F-ratio 4.46 (5.11)* 9.50 (0.90)* 12.84 (2.15)* 2.92 (1.41) 4.48 (2.07)* 20.60 (1.47)* 4.8 (0.72)*

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156 ARORA et al.

Table 2. Effect of HBL on protein and malondialdehyde (MDA) concentrations, and specific activities of antioxidative
enzymes [superoxide dismutase (SOD), guaiacol peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX) and
glutathione reductase (GR)] of 30-d old salt-stressed maize plants. Statistics as in Table 1.

Treatments Protein (g kg-1 MDA (mmol SOD (U min-1 POD (mmol CAT (mol APX (mmol GR (mmol
FW) kg-1 FW) mg-1) GDHP min-1 H2O2 min-1 ascorbate NADPH min-1
mg-1) mg-1) min-1 mg-1) mg-1)
NaCl (25 mM) ± 1.24
27.30± ± 0.13
6.54± ± 0.26
11.01± ± 0.58
8.96± 5.24± ± 0.33 7.85±± 0.35 1.99±± 0.06
NaCl (25 mM) + 28.64±1.35 5.91±0.16 17.47±1.22 13.99±0.26 9.17±0.85 10.41±0.40 2.18±0.05
HBL (10-8 mM)
NaCl (25 mM) + 30.78±2.59 5.23±0.22 11.73±0.71 8.99±0.74 6.25±0.85 7.93±0.72 2.31±0.10
HBL (10-6 mM)
NaCl (25 mM) + 28.00±1.83 6.33±0.42 16.47±1.14 13.60±1.65 8.05±0.06 12.10±0.43 2.03±0.07
HBL(10-4 mM)
F-ratio 0.67 (8.30) 4.96 (1.17)* 12.67 (4.16)* 8.52 (4.35)* 8.08 (2.81)* 16.96 (2.26)* 4.31 (0.32)*
NaCl (50 mM) 21.38±± 1.39 6.74±± 0.16 12.03±± 1.48 11.96±± 1.43 8.82±± 0.64 ± 0.38
9.82± 2.170.13
NaCl (50 mM) + 24.09±1.18 5.44±0.49 14.76±0.25 10.77±0.49 11.3±0.57 13.95±1.15 2.46±0.2
HBL (10-8 mM)
NaCl (50 mM) + 30.38±1.15 4.66±0.30 12.24±0.22 13.28±0.49 10.66±0.27 10.04±0.52 2.91±0.50
HBL (10-6 mM)
NaCl (50 mM) + 22.77±0.67 6.38±0.37 16.19±1.10 11.95±0.23 9.39±0.57 10.57±0.51 3.71±0.27
HBL (10-4 mM)
F-ratio 12.31 (5.12)* 6.97 (1.61)* 4.61 (4.25)* 1.60 (3.66) 4.51 (2.42)* 7.38 (3.21)* 4.61 (1.41)*
NaCl (75 mM) ± 0.81
20.5± 8.08±± 0.58 13.37±± 0.84 12.27±± 0.35 9.13±
± 0.54 10.24±± 0.42 3.48±±0.50
NaCl (75 mM) + 28.04±1.72 6.98±0.33 13.82±0.57 13.44±0.56 11.00±1.08 10.87±0.48 3.65±0.13
HBL (10-8 mM)
NaCl (75 mM) + 25.39±2.25 7.49±0.32 14.73±0.62 12.49±0.42 9.31±1.47 10.86±0.67 3.52±0.33
HBL (10-6 mM)
NaCl (75 mM) + 24.48±0.41 6.40±0.28 13.45±1.42 12.35±1.81 9.15±0.49 11.49±0.52 3.57±0.37
HBL (10-4 mM)
F-ratio 4.37 (6.76)* 3.25 (1.79) 0.45 (4.20) 0.30 (4.48)* 0.84 (4.45) 0.91 (2.41) 0.04 (1.16)

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