Effect of Heavy Metals and salt on biochemical changes of Sorghum
bicolor (L.)
Rekha Yadav, Sumita Kacchwava, SL Kothari and Usha Jain
Department of Botany and Biotechnology,
University of Rajasthan, JLN Marg Jaipur
Email :- rekhayadavbiotech@gmail.com
ABSTRACT
Experiments were carried out in order to investigate the deleterious effects of different
concentrations of heavy metals and salt in combination on sorghum bicolor seedling and activity
of lipid peroxidation (Malondialdehyde, MDA content), peroxidase (PRX) and its isozyme, were
also studied for different time intervals. Seedlings of Sorghum bicolor (csv-17) were grown
through Hydroponics system containing Hoagland solution under optimum condition of 70%
humidity and temperature (28ºC). MDA contents and activity of peroxidase were determined in
the leaves after 7 days of induction under salinity and heavy metal stress. In salinity (200mM)
application during 24 hrs MDA contents increased and in remaining time intervals MDA
contents decreased significantly. The activity of peroxidase increased significantly in salinity
(200 mM) conditions with corresponding control and in different time durations (24, 48 and 96
hrs. ) The activity of peroxidase significantly increased in first two time durations (24 and 48
hrs.) and significantly decreased in remaining last time duration ( 120 hrs.). Native PAGE, we
observed stressed, isoform band B3 (L3) and B4 (L5) were newly expressed. Further it is
assumed that there is a correlation between physiological roles of these enzymes in stress
tolerance mechanism.
Key words; - Peroxidase, Abiotic stress, Hydroponics system, Isozyme, Instat software and
Enzyme activity.
INTRODUCTION
Sorghum bicolor is of main interest in research all over world after accepting it as a model plant.
It has been ranked fifth in the world and second in united state as an important crop plant [2].
Sorghum, an African grass related to sugar cane and maize family is especially important in dry
regions such as northeast Africa and the southern plains of the United States due to their drought
tolerance. The Saccharine plants are characterized by C4 photosynthesis mechanism, comprising
biochemical and morphological specializations that increase net carbon assimilation at high
temperatures [1].
Environmental stress factors like drought, temperature, high salinity and heavy metals are the
major constraint that limits plant growth and productivity, by disturbing the intracellular water
balance. Usually, in fields or on agricultural land, unlike in a laboratory or even a greenhouse
environment, plants are subjected to a manifold array of stress factor. [3]. However, most of the
studies have been devoted to assess the physiological response of plants in a single stress
environment like salinity [4,5 6) drought and heavy metals [7.8].
Many physiological factors might be involved for causing drought or heat stress injuries to
Sorghum bicolor. In some species, drought or heat injury induces oxidative stress, resulting from
the production and accumulation of toxic oxygen species such as superoxide radicals, hydrogen
peroxide (H2O2), and hydroxyl radicals (OH·) [9,10,11]. However, the function of the
scavenging enzyme systems can be interrupted by drought or heat stresses, which results in
increase in lipid peroxidation and consequent membrane damage [12,13,14,15]. Identification of
abiotic stress tolerant lines to design a breeding programs aim at tolerance for stress
environments is a great challenge because of defining the suitable criterion associated with the
stress and the complexity of the inheritance to stress environments as well.
To best of our knowledge there are scanty report on combination stress including salt and Heavy
metals in our present manuscript. We studied synergetic effect of salt and heavy metals with
increasing time intervals to evaluate biochemical changes in Sorghum bicolor.
MATERIAL AND METHODS
Seeds of Sorghum bicolor (csv-17) were grown hydroponically in plastic container with 2 L of
Hoagland solution, having 4 boxes for seedling cultivation. Surface sterilized seeds were
germinated in culture chamber with optimum condition (28ºC) and 70% humidity on an average.
After 7 days of germination of seedling in combination stresses (Salinity and Heavy metal) were
imposed, with the gap of 24 hrs time intervals (24, 48 72, 96, 120 and 144)
Enzyme extractions and assays: 0.3 g of leaves were frozen in liquid nitrogen and then ground
in
6
mL
solution
containing
50
mM
phosphate
buffer
(pH
7.0),
1%
(w/v)
polyvinylpolypyrrolidone, and 0.2 mM ascorbic acid (ASA). The homogenate was centrifuged at
15000 g for 30 min, and supernatant was collected and used for enzyme assays.
Lipid Peroxidation Assay: Lipid peroxidation was determined by measuring the amount of
malondialdehyde (MDA) produced by the thiobarbituric acid reaction as described by [16]. The
enzyme extract was mixed with the same volume of a 0.5% (w/v) thiobarbituric acid solution
containing 20 % (w/v) tricholoroacetic acid. The mixture was heated at 95CO for 30 min and
then quickly cooled in an ice bath. The mixture was centrifuged at 3,000 g for 5 min and the
absorbance of the supernatant measured at 532 and 600 nm. MDA concentrations were
calculated by means of an extinction coefficient of 156 mM-1 cm-1: MDA (μmol/g fresh wt.) =
[(A532 - A600)/156] x 103 x dilution factor [17].
Peroxidase (PRX) assay: Total peroxidase activity towards abiotic stresses Salinity and Heat
stress was determined as describe [18] in a reaction mixture (0.4 ml) containing 100mM
phosphate buffer (pH 7.0), o.1 μM EDTA, 5 Mm guaiacol, 15mM H2O2 and 100 μl enzyme
extract .The initiation of reaction by addition of the enzyme extract and the increase of
absorbance were recorded at 470 nm for every 15 second up to 5 min. The enzyme activity
quantified by the amount of tetraguaiacol formed per second per mg.
Statistical analysis: All the data were analyses by the help of Instat software. The data were
analyzed as the mean ± SEM of number of observations. Comparisons of means were carried
out using analysis of variance (ANOVA) followed by Bonferroni’s test to compare means
between the different treatment groups. Differences were considered significant at p≤0.05 unless
otherwise stated in the text. Data point in the figures represents the means ± SEM and significant
effect at p≤0.05 at least three independent replicate per cultivars of abiotic stress treatments.
Native polyacrylamide gel electrophoresis (PAGE) and PRX extraction: Leaf material was
collected from plant of stress treatment (Salinity and Heavy metal). Triplicate samples of leaf
tissues were right away frozen and ground in liquid N2 and stored at -80oC until used. PRX was
extracted from leaf tissues using the extraction methods described [19]. Ground leaf tissues (0.1
g) were homogenized at 4oC in 0.6 ml extraction buffer [0.1 M potassium phosphate pH 7.5, 30
mM boric acid, 50 mM L-ascorbic acid, 17 mM sodium metabisulfite, 16 mM dithiocarbamic
acid, 1 mM EDTA and 4% (w/v) PVP-40 and final pH was readjusted to 7.5 with NaOH].
Homogenates were centrifuged at 15 000 rpm for 20 min and supernatant was used for
electrophoresis. Native PAGE was performed with a PROTEAN III vertical electrophoresis unit
(Bio-Rad, Hercules, Calif.) peroxidase, respectively, as described [20, 21] Five percent stacking
gels and 10% separating gels were prepared for both systems. For each sample 20 μl of crude
extract was loaded to the gel. Electrophoresis was performed at 20 mA for 30 min, followed by
40 mA for 3 hrs. Gels were stained for peroxidase using the established protocol. [22].
MALDI-TOF
RESULTS
a.
Lipid peroxidation assay
It was observed that with increasing time intervals the MDA content also increased
significantly with corresponding control.
Fig. 1 Lipid peroxidation, expressed by the content of malondialdehyde (MDA) in leaves of Sorghum bicolor in
salt and heavy metal combinely stress treatment condition. Plants were grown under control combined stress
conditions; values represent the mean and the bars indicate standard deviation.
b.
Peroxidase assay
It was observed that with increasing time intervals the peroxidase content also increased
significantly with corresponding control.
.
Fig. 2 Effect on peroxidase activity by salt and heavy metal combinely stress treatment of Sorghum bicolor
leaves. Differences between values with matching symbol notations within each column are not statistically
significant at 5% level of probability
c.
Native PAGE (Heavy metal lead (Pb)+ cadmium (Cd)+ Salt stress)
C Control 24
L1 Stress 24
L4 Stress 96
L2 Stress 48
L5 Stress 120
L3 Stress 72
L6 Stress 144
Sorghum bicolor plants were cultivated in the presence of abiotic stresses (Salinity and heavy
metal stress combinely) and also as control without any stress was used for comparison of MDA
contents and enzyme activity assay as described earlier. Cultivation of Sorghum bicolor plants
under abiotic stress conditions leads to reduced growth of leaves in both stress condition and also
chlorosis in salinity stress relative to the control plants. The delay in growth was visible in plants
after both stress treatments. In the above figures in L3 and L5 dominant isoform (P1 and P2 )
were found to be expressed. In all the lanes B3 isoform were newly expressed.
DISCUSSION
Over the last ten years the development of a number of the functional tools allowed us to dissect
many of the fundamental questions associated with stress tolerance which included MDA
contents and antioxidant enzyme activity mechanism to determine the efficacy of these
approaches in the field of crop plants.
Lipid peroxidation
The accumulation of MDA often is used as an indicator of lipid peroxidation [23]. The results
indicated that membrane lipid peroxidation occurred from the malfunction of the scavenging
system, which could lead to damage to main cellular components [24].
Combination stress of salt and heavy metal
It has been demonstrated that salinity induces oxidative stress in plant tissues, and lipid
peroxidation has frequently been used as an indicator of oxidative stress when plants are
subjected to salinity. This has been shown for Morus alba [25]., Beta vulgaris [26]., Oryza sativa
and Gossypium hirsutum [27]. The data of present study also agree with this idea initially for 24
hrs of salt stress but disagreed in 48 and 96 of salt stress hrs, mainly because decrements were
found in lipid peroxidation saline treatments when compared with corresponding control in
plants of Sorghum bicolor genotype (Fig.1) suggesting that lipid peroxidation cannot be regarded
as the universal marker for salt tolerance in all abiotic stress condition species. However, lipid
peroxidation is not the only oxidative stress damage, because reactive oxygen species (ROS)
may also damage macromolecules such as DNA and proteins [28]. To overcome the effects of
salinity-induced oxidative stress, plants make use of a complex antioxidant system. Relatively
higher activities of ROS-scavenging enzymes have been reported in tolerant genotypes
suggesting that the antioxidant system plays an important role in plant tolerance against
environmental stresses.
Peroxidase activity
Combination stress of salt and heavy metal
Our enzyme activity data (Fig. 3) under combinely stress strongly suggests the antioxidant
enzyme activity of peroxidase increases that might be cooperating to plants for survival in
nature. Anyhow, a higher degree of protection against oxidative damage should require a fast
removal of H2O2 by other scavenging systems, thus minimizing H2O2 toxicity and the
formation of the highly toxic hydroxyl radicals [34] Since APX and GR are key enzymes of the
ascorbate-glutathione cycle [29], this pathway could be a potential mechanism for sorghum
acclimation or adaptation to salt stress. Even though in some species salt tolerance was
associated with increases in both APX and GR activities [30,31], the same was observed here.
The increases observed in peroxidaae (PX) activity for the salt-tolerant genotype (Fig. 1) The
intercellular level of H2O2 produced under stress conditions is regulated by peroxidases.
Ascorbate peroxidases (APX) can scavenge H2O2 that is inaccessible for catalase because of
their high affinity for H2O2 and their presence in different subcellular locations (32)
Antioxidant activities are known to increase in a variety of environmental stresses like soil sa
linity, drought, extremes of temperature and heavy metals. Physiologically these stresses cause
oxidative damage to plants either directly or indirectly [33,34]. In fact, oxidative stress hazards
are due to the production of reactive oxygen species which include superoxide radical (O2 +),
hydroxyl radical (OH+) and hydrogen peroxide (H2O2). Reactive oxygen species (ROS) products
in turn cause damage to the biomolecules by peroxidation, electrophilic substitution reaction,
reduction of membrane lipids, proteins, chloroplast pigments, enzymes, nucleic acids, etc. [35].
Native PAGE analysis
As the product of the gene expression, isozyme could be a biochemical criterion to know the
tolerance of plant to stress and detect the inheritance and variance at the molecular level. In
recent years, many studies had shown that isozymes of plant growth or induce some desirable
effects. Some people found the similar results [36,37,38], some new peroxidase isozyme bands
induced by the treatments of high salt (200mM) concentration may be corrected with salt
compliance. In their study, data from native PAGE indicated in both salinity and heat
isoperoxidases with different intensities (Figure 3), since data indicated a linear relationship
between band intensities and the duration of the stress treatment, it is correlated to salt and heat
stress-acclimations of Sorghum bicolor leaf tissues. Regarding the temperature stress, one basic
isoperoxidase band was correlated with lignification and recovery of cell membrane damage
under heat stress in strawberry leaf [39]. Recently, there are some reports on expression of acidic
peroxidase bands with different band intensities which are responsible for tolerance to
temperature stress [40]. In our present investigation on combination stress (heavy metal and salt
stress ) we observed some novel isoforms, these might be due to combine stress as their
mechanism is still not clear as there are no previous reports available on these parameters.
ACKNOWLEDGEMENT
The authors thank to Head, Department of Botany for granting me the permission of facilities
and Seminal Applied Sciences, Jaipur, Rajasthan, India for technical support and facility.
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