Journal of Plant Physiology & Pathology ISSN: 2329-955X

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Research Article, J Plant Physiol Pathol Vol: 2 Issue: 3

Effects of Exogenous Spermidine on Cell Wall Composition and Carbohydrate Metabolism of Marsilea Plants under Cadmium Stress

Kingsuk Das1, Chiranjib Mandal1, Nirmalya Ghosh1, Sidhartha Banerjee1, Narottam Dey2 and Malay Kumar Adak1*
1Plant Physiology and Plant Molecular Biology Research Unit, Department of Botany, University of Kalyani, Kalyani, WB, India
2Department of Biotechnology, Visva Bharati, Santiniketan, WB, India
Corresponding author : Malay Kumar Adak
Department of Botany, University of Kalyani, Kalyani– 74 1235 (W.B.), India
Tel: 033-2582-8282, 09432418218
E-mail: [email protected]
Received: May 26, 2014 Accepted: July 16, 2014 Published: July 21, 2014
Citation: Das K, Mandal C, Ghosh N, Banerjee S, Dey N, et al. (2014) Effects of Exogenous Spermidine on Cell Wall Composition and Carbohydrate Metabolism of Marsilea Plants under Cadmium Stress. J Plant Physiol Pathol 2:3. doi:10.4172/2329-955X.1000127


Effects of Exogenous Spermidine on Cell Wall Composition and Carbohydrate Metabolism of Marsilea Plants under Cadmium Stress

In an experiment to detect the cellular changes of carbohydrate content and its related enzymatic activities, a study was undertaken with Marsilea minuta L., an aquatic fern species in simulated condition of cadmium (Cd) toxicity. From the varying doses of Cd (0, 50, 100 and 200 μM) and supplemented with spermidine (2 mM), it revealed that plants were suffered from accumulation of total carbohydrate in a dose-dependent manner under Cd stress. Maximum depletion of carbohydrate content was 58% with respect to control which was retrieved by 1.42 fold with spermidine application. In a similar manner, plants were also affected with starch, total reducing sugar content by 42% and 63.04% respectively over the control. The fall in both starch and total reducing sugar were retrieved by plants by 1.32 fold and 1.52 fold, respectively.

Keywords: Marsilea minuta; Cadmium; Carbohydrate metabolism; Amylase


Marsilea minuta; Cadmium; Carbohydrate metabolism; Amylase


It is the ever increasing threats from environmental pollution that make the plants unsuitable to grow and develop properly. Out of environmental pollution, the metal toxicity in soil might be the most serious for crop plants. Starting from seed germination to grain/fruit development, plant’s survival undergoes hampered in various ways both from anthropogenic as well as natural activities. Cadmium (Cd), a divalent cation of such heavy metal, contaminates the soil and leads to various stress responses in plants with various visible symptoms of toxicity. Overall, these include growth inhibition, damages of roots, perturbations of cellular metabolic pathways and finally drastic fall in yield. Cd accumulation in crop species becomes an integral part of human solid food chain. Besides, water and its improper use for irrigation emerges as a potential source of Cd pollution. Thus, it intoxicates both the soil-water environment and aquatic plants/crop species. In this way, a substantial amount of aquatic bodies, particularly, those are characterized by lowland with stagnation of water for prolonged period. Toxicity of Cd is found in these aquatic plants, mostly in roots which are based on an impairment of phosphate uptake system. Cd behaves as a distant phosphate analogue and enters through phosphate transports trailing the phosphate concentration lower in cytosol [1]. In the cytosolic fractions, Cd undergoes competition with phosphate and it depletes adequate phosphate to form ATP from ADP and thereby it depletes the energy conservation in tissues [2].
Carbohydrate biosynthesis and its accumulation is the direct reflection of plant’s inherent ability to exercise photosynthetic efficiency and its concomitant allocation to dry matter. Therefore, any situation shifting plants from its optimum growth might be due to Cd interference with the carbohydrate metabolism. A number of studies have revealed the adaptation of plants for altered metabolism to allocate more sugars into maintenance respiration [3] .These may cause the diversion of photosynthetic pools into incompatible solutes into compatible solutes [4]. In the present experiment, Marsilea plants are analyzed for its few aspects of carbohydrate metabolism with reference to Cd toxicity. This has gone more insight when spermidine (Spd), a polyamine, was found to minimize the effect of Cd toxicity as well as to moderate the activities of carbohydrate metabolism- related enzymes. In fern species the efficacy of Spd in moderation of metabolic aspects has also been evident [5]. In fact, the level of Spd that was detected in the subcellular fractions has put its relevance to shield the cellular membrane from ROS induced damages in many plants. Another level of Cd toxicity adhered to ROS sensitivity is based on development of some sort of anoxic or hypoxic condition [6]. The accumulation of Cd though not a typical redox metal but blocks some channel proteins [7] or some transporters on root membrane based solute transport. This gets more aggravated when metal induced perturbances of osmotic potential are made to lower the water potential of tissues. Thereby, it is a conjoint effort down regulating the osmotic turgidity of plants and thus plants become prone to hypoxic. The sustenance of respiratory activity is another adaptive feature to realize the hypoxic stress and thus the over expression of some genes are resulted [8,9].
Taking all these together, the present work hypothesizes that carbohydrate metabolism may be adhering the tolerance and hyper accumulation of heavy metal in Marsilea plant. This may give impetus to the cellular responses of bimolecular organization that was modulated with Spd. This happens to be a preliminary report that a water fern like Marsilea could also respond to polyamine similar to higher plants in mode of action for alleviation of Cd stress. In general, non-angiospermic plants have been less studied in depth with cadmium toxicity along with related metabolic pathways. In brief, this study illustrates the few cellular responses in respect to carbohydrate metabolism that could be granted the possible biomarkers under Cd toxicity in aquatic environment.

Materials and Methods

Marsilea minuta L. was collected from the industrial belts of Kalyani, Nadia. The plants were thoroughly washed and were grown for 7 days in Hoagland’s solution. Thereafter, plants were treated with various concentrations (0, 50, 100 and 200 μM) of cadmium chloride (CdCl2) in the same solution in different sets. Another set was done with 2 mM Spd with 200 μM of CdCl2 solution. The plants were then grown for 7 days in growth chamber (37±1°C), 85% relative humidity and 14 h light (irradiance 72-80 μM/m2/s) and 10 h in dark. The nutrient solution was changed at every two alternate day. On completion of the incubation period, the plants were sampled and frozen in liquid nitrogen following storage at -80°C for further biochemical assays.
The total carbohydrate and starch content of the sample were estimated following by the method of Yoshida et al. [10]. For each of the estimation, plant samples were crushed with liquid nitrogen and ground to a fine powder. The powdered sample was extracted with 80 % ethanol (v/v). The extract so obtained was used for the analysis of total carbohydrate content by adding ice cold 2% (v/v) anthrone reagent. The absorbance was recorded at 630 nm using a spectrophotometer Cecil (CE 7200). After extraction of total carbohydrate, the residue left was dried and extracted using 52% perchloric acid for the analysis of starch content using anthrone reagent. The absorbance was read at 630 nm. Using the standard curve of glucose, the total carbohydrate content and starch content was estimated and expressed as mg/g FW.
Cellulose content of plant sample was estimated following the procedure of Thimmaiah [11]. The dried powder of the sample was extracted with acetic nitric reagent (150 ml of 80% acetic acid and 15 ml of conc. nitric acid). The residue so obtained was diluted and the cellulose content was analyzed by adding ice cold anthrone reagent. The absorbance was observed at 630 nm. The amount of cellulose was calculated from the standard curve of glucose and expressed as mg cellulose/g DW.
Hemicellulose content of the sample was estimated adopting the procedure of Loomis and Shull [12]. The dried powder of the sample was extracted in hot 80% ethanol (v/v). The sugar free residue so obtained, was hydrolyzed using 1(N) HCl and was treated with ice cold anthrone reagent. The absorbance was taken at 630 nm. The amount of sugar was calculated from the standard curve of glucose. The hemicellulose content was obtained by multiplying the amount of sugar by 0.9 as suggested by Loomis and Shull. Hemicellulose content was expressed as mg hemicellulose/g DW.
Estimation of total reducing sugar was done according to procedure of Chow et al. [13]. 100 mg of powdered sample was treated with 80% ethanol and evaporated in water bath at 80°C. The sugar was estimated by adding DNSA reagent followed by boiling in water bath. The total reducing sugar content was analyzed by adding Rochelle’s salt. The absorbance was monitored at 630 nm. Reducing sugar content was expressed as μg/g FW.
The assay of soluble and cell wall bound invertase ( were based on the method of Hubbard et al. [14]. 1 g of plant sample was crushed in liquid nitrogen and the powdered sample was centrifuged at 10,000 rpm. For soluble invertase assay, properly diluted enzyme extract obtained from plants was reacted with 1.5 ml of assay mixture containing sodium citrate buffer (pH 3.8), 200 mM sucrose at 37°C. The reaction mixture was incubated for 90 min. The reaction was terminated with 1.5 M NaOH (pH 6.5) at 100°C for 30 min. Absorbance was read at 510 nm. For cell wall bound invertase estimation, the pellet was taken after centrifugation. The pellet was washed thrice and dissolved in 150 μl of sodium acetate buffer (pH 5.2). After that, the process followed as that of soluble invertase. Using the standard curve of glucose, invertase activity was expressed as μg/min/g FW.
The assay of α-amylase (EC and β-amylase (EC were done following the procedure of Peter Bernfield [15]. For α-amylase estimation, the plant sample was extracted using ice cold calcium chloride (10mM) solution and centrifuged at 54,000×g at 4°C for 20 min. The supernatant, so obtained, was used as the enzyme source and incubated with equal volume of starch solution at 27ºC for 15 min. The reaction was terminated using DNSA reagent and the absorbance was recorded at 560 nm. For β-amylase estimation, the plant sample was defatted by acetone. The acetone defatted sample was extracted with 66mM phosphate buffer (pH 7.0) containing 0.5M NaCl. The extract was centrifuged at 20,000 rpm for 15 min. The supernatant was used as source of β-amylase and followed the procedure as above of α-amylase. Using the standard curve of maltose, both of the amylase activity was calculated and expressed as μg/min/g FW.
Alcohol dehydrogenase (ADH, EC assay was done according to Fukao et al. [16]. 50 mg of powdered sample was homogenized in ice cold extraction buffer (100 mM Tris-HCl, pH 9.0, 20 mM MgCl2, 0.1% (v/v) 2-mercaptoethanol), centrifuged at 20,000 g for 20 min at 4°C. The supernatant was used for enzyme activity. The assay mixture contained 50 mM Tris-HCl, pH 9, 1 mM NADH and 50 μl enzyme extract. Ethanol added to initiate the reaction. The changes in absorbance were observed after every 60 s were recorded at 340 nm. The ADH activity was expressed as μMol NADH oxidized/ min/mg protein.
Assay of malate dehydrogenase (MDH, EC was done by the method of Kumar et al. [17]. 1 g of plant sample was homogenized in 50 mM Tris-HCl, pH 9.0, 50 mM MgCl2, 5 mM 2-mercaptoethanol and 1 mM EDTA. The supernatant was used as enzyme source and was added with 5 μM oxaloacetic acid, 10 μM MgCl2, 0.4 μM NADH and 0.1 M Tris-HCl. The decrease in absorbance was observed after every 60 s were recorded at 340 nm. The ADH activity was expressed as μMol NADH oxidized/min/mg protein.
All observations were recorded with three replications (n=3) and data were presented as mean value of ± SE. The statistical was analysis was performed by one-way ANOVA analysis by Duncan’s test taking p ≤ 0.05.

Results and Discussion

As heavy metal, Cd has diverse impacts on plants mainly to reduce growth and development. In addition, Spd has also been documented to recover the Cd induced retardation of plant’s metabolism, however, it’s exogenous application mostly for angiospermic crops (Figures 1a-1d). Therefore, the Cd induced changes of plant’s metabolism and its interaction with Spd ought to be dealt with some non-angiospermic plants. Thus, in the present experiment, Marsilea plant which is an aquatic pteridophyte has been observed with this objective. We have noticed a significant decline in total carbohydrate in linear manner with Cd concentrations s. The maximum decline in carbohydrate content was about 58% at 200 μM of Cd over control (Figure 1a). The Spd had retrieved the total carbohydrate by 1.42 fold when applied at the concentration of 2 mM. Almost a similar trend was recorded for total reducing sugar under Cd interaction which had the value of 63.04% decrease. With the application of Spd, it was recovered by 1.52 fold when compared with highest concentration of Cd (Figure 1e). For both the cases, the features for down-regulation of total carbohydrate and reducing sugar are indicative of the fact for suppression of photosynthetic carbon assimilation and its concomitant allocation of carbohydrate fraction. Similar interpretation has also been found in heavy metal affected carbohydrate status in plants [18]. The impairment of carbon metabolism can limit the source sink relationship in plants under metal stress. So, plants have to use the storage carbohydrates for its mobilization [19]. Thus the Marsilea plants tended to accumulate more cellulose and hemicellulose content on their leaf tissues with the maximum values of 3.84 fold for cellulose and 4.66 fold for hemicelluloses at highest Cd concentration over control (Figures 1c and 1d). However, throughout the Cd exposure, plants recorded a steady rise of storage carbohydrates significantly. Some hyper accumulating species are characterized by the deposition of metals in the apoplastic spaces, cell wall and intercellular spaces. The mechanical rigidity of the cell wall is offered by furnishing the more accumulation of structural carbohydrates (like cellulose and hemicellulose) [20]. This is also characterized by other depositing materials for lignifications process in higher plants under abiotic stresses like water deficit, salinity, oxidative exposures [21]. Our result showed that t he lignifications process is accomplished for Cd tolerance. Moreover, the Marsilea plants may not be excluded for adoption of such strategy to overcome the metal stress. However, previous studies reported that aquatic plant species, may not be maintaining more rigidity on their roots and stem, still, the depositions of structural carbohydrates in excess may circumvent their mechanical rigidity [22]. The Cd induced production of complex carbohydrates like hemicelluloses has been established in some aquatic plant species like Lemna and Pistia [23]. On the other hand, in the present study, the enzymatic machineries are also imperative to justify as referred to soluble invertase, cell wall bound invertase, α-amylase, β-amylase and malate dehydrogenase (Figures 2a-2f). Acid invertase activity was demarcated by both soluble and wall bound. From the result it is revealed that the activity of both the enzyme of invertase recorded declining trend through Cd concentrations. The range of fall in activities recorded 51.27% and 42.07% at highest concentration of Cd compared to control in soluble and wall bound invertase activity. Sucrose is regarded as the most stable photosynthates which is also the easily transportable form amongst sugars. Lysis of sucrose into reducing sugars is behaves as readily source of respiratory substrate. Invertase is the main enzyme which under low pH activates the hydrolysis of sucrose into hexose monomers. Under Cd stress, the declining rate of this enzyme may be indicative of the fact of plant’s less demand for respiratory substrate [24]. Marsilea plant also behaved similarly to have an impairment in enzyme activity. The activity of this enzyme particularly which are wall bound and tonoplast seated are more important to non-cellular sucrose hydrolysis and thus provides the osmolyes under metal stress.
Figure 1: Effects of Cd concentration (0, 50, 100, 200 μM of Cd salt) and 200 μM of Cd salt with 2 mM Spermidine (200 μM+2 mM Spd) on content of (a) total carbohydrate, (b) starch, (c) cellulose, (d) hemicelluloses, (e) total reducing sugar. The data represented as mean value of observations (n=3) ± SE and put by the vertical bars in each bar. Statistical differences (p ≤ 0.05) have been compared with student’s t test. Different letters indicate significant differences and similar letters indicate insignificant.
Figure 2: Effects of Cd concentration (0, 50, 100, 200 μM of Cd salt) and 200 μM of Cd salt with 2 mM Spermidine (200 μM+2 mM Spd) on activities of (a) alcohol dehydrogenase (ADH), (b) soluble invertase, (c) cell wall bound invertase, (d) α-amylase (e) β-amylase and (f) malate dehydrogenase (MDH). The data represented as mean value of observations (n=3) ± SE and put by the vertical bars in each bar. Statistical differences (p ≤ 0.05) have been compared with student’s t test. Different letters indicate significant differences and similar letters indicate insignificant.
Marsilea plant is also found with a steady decline of reducing sugar being maximum under 200 μM of Cd with 63.04% value against control (Figure 1e). The direct correlation between invertase activity and total reducing sugar is imperative to demand a steady state complementary of carbohydrate profile. The rapid consumption of reducing sugar and its fall in cellular concentration may be correlated with invertase activity.
Activity of ADH recorded serial increase over the course of Cd concentrations (Figure 2a). The linear increase of ADH activity peaked at 200 μM with 1.83 fold increase over the control. By default, when plant’s major metabolic thrust are hindered by abiotic stresses like heavy metals, the recovery of energy currency, reducing equivalents like NADH/NADPH are limited. Plants have to divert their normal mode of metabolically energy yielding pathways like respiratory fluxes into anaerobic mode [25]. ADH is the enzyme that circumvents the key regulatory steps for anaerobic respiration. Besides angiosperms or higher crop plants, the reports in anaerobic pathways are less explored in non-angiospermic species like fern. Therefore, from our results, the support for plant’s sustenance under heavy metal accumulation is also accomplished from the nature of activities of ADH [26]. Moreover, the supplementation with Spd has undoubtedly been proven for replenishment of NADH/NADPH and other energy equivalents.
In addition to respiratory enzymes in plants it is found that, there recorded a significant decline in MDH activity under Cd stress. In general, utilizing sucrose and starch as principal respiratory substrate, plants represent a steady metabolic flexibility. These undergo, however, some perturbances, particularly, with reference to malate metabolism. Malate is served as pre-dominant carboxylic acid which often undergoes decarboxylation to supplement the carbon replenishment under abiotic stresses [27]. On the other hand, the flexibility of malate could often be used as a osmolyes for stomatal regulation, particularly, under water stress. Therefore, the down regulation of malate metabolism as evident from its MDH activity is reflecting on the fact that Marsilea plants were under osmotic deficits with Cd accumulation (Figure 2f). MDH is the enzyme which is dually beneficial by releasing CO2 as well as generation of NADH/NADPH+H+. The latter is more circumvented for donation of reducing equivalents in plants. The subdued effects of MDH activity under Cd stress has commonly encountered in many crop plants as effective biomarkers so resemble in Marsilea plant also [28]. The recovery of MDH activity by Spd thus suggests the resumption of normal metabolic flux of carbon through glycolytic pathway.
Marsilea plant in the present experiment also characterized the starch hydrolytic activity with respect to Cd interference. Starch content decreased significantly with increase in Cd doses and least decrease in 200 μM and it was 42% with compared to control. Retrieval by 2 mM Spd was 1.32 fold when it was applied on highest concentration (Figure 1b). A distinguishing trend for α-amylase and β-amylase activities recorded with decreasing trend through the course of Cd concentration. From the comparative analysis, a significant higher value of α-amylase activity that trail the β-amylase by 3.28 fold when compared at 200 μM of Cd against control (0 μM Cd). It is interesting to note that Spd had hardly any significant effect to retrieve both of the activities, however, by 1.13 fold and 1.23 fold for α and β amylase respectively (Figures 2d and 2e). It could be speculated that starch, the non-structural but storage carbohydrate is behaved as major source of energy yielding moieties [29]. So, Cd and its interference might have impair the photosynthetic carbon reactions in such a way that respiratory substrate would be only the stored starch [30]. Therefore, decline in amylase activity may be beneficial, if plant could sustain its cellular rigidity for storage carbohydrate.


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