Research Article

Polyamine mediated anti-oxidation activity facilitates of Marsilea minuta L. under Cd toxicity  

K. Das , Malay Kumar Adak
1 Department of Botany, Serampore College, Serampore, Hooghly, West Bengal, 712201, India
2 Plant Physiology and Plant Molecular Biology Research Unit, Department of Botany, University of Kalyani, Nadia, West Bengal, 741235, India
Author    Correspondence author
Plant Gene and Trait, 2015, Vol. 6, No. 8   doi: 10.5376/pgt.2015.06.0008
Received: 02 Nov., 2015    Accepted: 14 Dec., 2015    Published: 24 Dec., 2015
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This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:
Das K. and Adak M.K., 2015, Polyamine mediated anti-oxidation activity facilitates of Marsilea minuta L. under Cd toxicity, Plant Gene and Trait, 6(8): 1-13 (doi: 10.5376/pgt.2015.06.0008)
Abstract

The Marsilea minuta (L) plant was evaluated on the basis of their anti-oxidation path in vitro under Cd mediated metal stress. The plants were grown under varying concentrations of Cd stress and their metal acquisition with concomitant oxidative stress. The different anti-oxidation assay systems were analyzed with regards to putrescine as polyamine interaction and their possible modulation. Thus, Cd treated plants were recorded ABTS and DPPH free radical scavenging activities in terms of 1.75 and 1.59 fold under maximum concentration (200 µM) respectively as compared to control. The spermidine could moderate the activity by 7.53% and 24.91% respectively against the same Cd concentration. On the contrary, a down regulatory trend with a fall in 44.01% activity suggested the chelation efficiency of metal against highest Cd concentration. The other free radical quenching attributes like HOCl- and OH- showed the defense of plants under oxidative stress with records of 1.94 and 2.63 fold over control. In both the cases, spermidine was the moderator by curtailing the activities with 10.70% and 3.18%. Finally, H2O2, the peroxide crossed the limit of tolerance in tissues with an wider variation by 1.45 fold activity and that was again moderated by spermidine by 10.54%. The different interfaces of anti-oxidations in aquatic fern species was discussed in details taking Cd as pro-oxidant and its possible dealing with polyamine.

Keywords
Anti-oxidation; Pro-oxidants; Metal stress; Polyamines; Fern
 Introduction
The development of byproducts in plant metabolism is an important concern to interfere the normal growth and development. Out of those byproducts, the free radicals are the most serious moieties to interfere almost every physiological process in plant tissues. Free radicals in general have the chemical configurations integrally associated with an unpaired electron spinning around the nucleus. The origin of free radicals is predominantly based on snatching of electrons from electron transport system in sub-cellular fractions (mostly mitochondria and chloroplast) into molecular oxygen in order to attain stability.

It is the cellular homeostasis which maintains a steady balance between reactive oxygen species (ROS) production and its lysis. However, under abiotic stresses, the imbalance between the overproduction of ROS and its lysis is the causative effect to damage the cellular integrity (Gill and Tuteja, 2010). The latter is mostly focused to macromolecules including proteins, lipids, nucleic acids and other conjoint moieties for cellular functional status (Fedoroff, 2006). Antioxidants are the most effective path to be exercised by the plants in quenching of excess energy of ROS (Sharma et al., 2012). Along with antioxidants, the enzymatic antioxidative cascades synergistically run in plant system to make a balance between production and lysis of ROS.

A number of free or conjugant anti-oxidants are characterized with species variation as well as in expression. Of those, the most frequent is ascorbic acid in complementation with glutathione. In Haliwell-Asada pathway, ascorbic acid and glutathione are the predominant electron donors for accomplishing the enzymatic conversion of different ROS (Bailly et al., 2000). Thus, in enzymatic cascades, a sequential lysis of superoxides (O2-) results into the final product water (H2O) via hydrogen peroxide (H2O2). Still, a few intermediate or byproducts of these reaction sequences are established as equally harmful for plants. Those include hydroxyl radical (OH-), hypochlorous acid (HOCl-), nitrous oxide (NO-) etc. These products are more over-expressed in plants where there is an excess accumulation of metals mostly heavy and transitional in address (Dey et al., 2012). Thus, copper (Cu2+), iron (Fe2+), zinc (Zn2+) along with cadmium (Cd2+) have been reported in many plant species like Xanthium (Nazir et al., 2015), Arabidopsis (Roschzttardtz et al., 2013), sunflower plants (Moraghan et al., 1993), Lemna (Ducic and Polle, 2015) etc. to over-express the ROS generation.

Marsilea minuta L. (family-Marsiliaceae), an aquatic pteridophyte is regarded as wild weed. In special reference, this fern has the immense potential to accommodate metals in excess quantities (Das et al., 2013). Even the heavy and toxic metals are also accommodated in abundance for this species with concomitant expression of antioxidative pathways. The distribution of this species is mostly tropical to subtropical lowland, marshy environment with relatively higher humidity. This environment allows mostly exercising the photo-respiratory pathways leading the possibilities for ROS accumulation (Das et al., 2014). Thereby, this species might be expressing the antioxidative pathways with diverse phenolic conjugants either to lysis the ROS or quench the excess energy in oxidative damages (Mandal et al., 2013). The latter is facilitated with different the category antoxidants moieties. The development of antioxidants and its interplay in contaminated environment alerted this species more suitable for hyper-accumulation of heavy metals. The physiological changes in accordance with heavy metal accumulation have been well documented earlier (Minocha et al., 2014).

In addition, Marsilea plant exhibited a significant up-regulation of different anti-oxidative enzymes to moderate oxidative damages under heavy metal like Cd (Dey et al., 2012). On the other hand, plants’ antioxidation pathways are also in tune with some elicitor molecules for its modulation. A number of citations have documented the utility of chemical elicitors to change the anti-oxidative activities under metal stress. In higher plants, and even in non-flowering pteridophytic species, records, however, in skimpy show the variations in elicitors influencing the oxidative stress (Assuncao et al., 2003). Among the elicitors, the polyamines, polycationic, variably aminated, saturated straight chain moieties have also been effective in amelioration of oxidative damages (Kumar et al., 2013). With this background, the Marsilea plant is hypotheticated to vary the anti-oxidation system in terms of free radical scavenging activities and with polyamine application. Thus, in present experiment, the Marsilea plant’s extract under different concentrations of Cd, has been evaluated for a few free radical scavenging activities in vitro with reference to polyamine application. It is expected that polyamine can modulate the biosynthesis and functions of different non-enzymatic antioxidants of the plant. Expectedly, this could possibly be utilized in amelioration of metal stress in relation to oxidative damages.

2 Result
2.1 Total free radical scavenging activity by DPPH assay
From the leaves of Marsilea plant under varying concentrations of Cd, the plant extracts were made and homogenized in ethanol and the extract was applied for DPPH radical scavenging assay in vitro with the presentation of IC50 values as 0.306 1 mg/mL, 0.245 mg/mL, 0.228 mg/mL, 0.191 9 mg/mL and 0.255 6 mg/mL under 0, 50, 100 and 200 µM of Cd and 200 µM of Cd with 2 mM Spd respectively (Table 1). In addition to this, it is also notable that the variations in concentrations (within the range of 0.1 mg/mL, 0.2 mg/mL and 0.3 mg/mL) for each Cd treated plant extract, there recorded a similar increasing trend (Figure 1). Therefore, it is obvious that percent of inhibition of DPPH radical scavenging method is dose-dependent. It is also important to note that plants have modulated the generation of free radicals under influence of Spd, a significant decrease in DPPH radical scavenging assay was recorded for Spd treated sample as compared to 200 µM of Cd (Figure 2). Therefore, the changes in DPPH radical scavenging assay was in the order of 1.249 fold, 1.339 fold and 1.595 fold as compared to control plant (0 µM of Cd). In addition to this, the decline in DPPH assay was detected by 24.91 % with Spd treatment (Figure 3). In percentage of inhibition treated in the order of table of IC50 values. It is very much clear that percent inhibition and IC50 are inversely proportional.
 
   
Figure 1 DPPH radical scavenging activity of Marsilea plant extract grown under varying concentrations: 0 (control), 50, 100, 200 µM of Cd and 200 µM of Cd supplemented with 2 mM Spd. Extract from each cases was diluted with varying concentrations of 0.1 mg/mL, 0.2 mg/mL,0.3 mg/mL. X-axis indicates different concentrations of cadmium and Y-axis indicates inhibition percentage of Marsilea plant.
 
   
Figure 2 Standardization of butylated hydroxyl toluene (BHT) in different concentrations served as positive control compound. X-axis indicates different concentrations of positive control and Y-axis indicates inhibition percentage of positive control.
 
   
Figure 3 DPPH radical scavenging activity of Marsilea plant extract under varying treatments: 0 (control), 50, 100, 200 µM of Cd and 200 µM of Cd supplemented with 2 mM Spd and BHT as positive control. Each extract was made with 100 µg/mL. The values are plotted from means (±SE) of replication (n=3). Symbols with different letters indicate significant differences according to Duncan’s test at (p<0.05).
 
 
Table 1 IC50 values of different free radical scavenging assays of Marsilea leaf extract treated under different Cd concentrations. (Values are mean (±SE) (n=3))
 
2.2 Total free radical scavenging activity by ABTS method
Another mode of assay in total antioxidation activity in plants is the exercise of ABTS chemicals to scavenge the free radicals. This is interesting to note that Marsilea plant had scored its better antioxidation activities in relation to Cd toxicity in a dose dependant manner. The percent inhibition with reference to BHT as positive control showed a linear rise according to its increasing trend of concentrations. With this, the BHT could also be standardized as an effective and reliable positive control (Figure 5). Thus the percent inhibition taking the leaf extract from different Cd treatment as well as Spd supplementation is depicted in Figure 6. Marsilea plant with this scavenging activity was detected by the absorbance at 414 nm which declined as the ABTS radical is eliminated. The free radical scavenging assay was done with the reference standard taking BHT as positive control. The profile in percent inhibition showed a linear increase as taken the m