Responses of Salvinia plant as possible bioindication to Al toxicity and its interaction with putrescine  

Mandal C. , Adak M. K.
Plant Physiology and Plant Molecular Biology Research Unit, Department of Botany, University of Kalyani, Kalyani-741235, Nadia, West Bengal, India
Author    Correspondence author
Plant Gene and Trait, 2015, Vol. 6, No. 4   doi: 10.5376/pgt.2015.06.0004
Received: 16 May, 2015    Accepted: 07 Jul., 2015    Published: 23 Jul., 2015
© 2015 BioPublisher Publishing Platform
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.
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Mandal C. and Adak M. K., 2015, Responses of Salvinia plant as possible bioindication to Al toxicity and its interaction with putrescine, Plant Gene and Trait, 6(4) 1-10 (doi: 10.5376/pgt.2015.06.0004)

Abstract

In the present experiment some of the cellular responses in relation to carbohydrate and nitrogen metabolites were analyzed under various concentration of Al salt along with polyamine application (0, 240, 360, 480 µM of Al salt [KAl (SO4)2,12H2O] and 480 µM of Al salt with 1 mM Put). Plant responded well with some significant variation under Al concentrations when carbohydrate metabolism was studied both as reserve carbohydrate as well as from enzymatic aspects. Thus, a significant decline in total carbohydrates in whole plant including reducing sugars was the feature for Salvinia plants under Al concentration. On the contrary, the metabolism of sugars were monitored in terms of invertase and sucrose phosphate synthase activity in-vitro those recorded an up regulatory trend. This was modulated by putrescine. Photosynthetic efficiencies were measured indirectly with the assay of malate dehydrogenase and phosphoenol pyruvate carboxylseactivities and recorded to be up regulated. For the nitrogen metabolizing attributes enzymes like nitrate reductase, glutamine-2-oxoglutarate amino-transferaseand glutamine synthase recorded a diminishing trend all through Al concentration. On the contrary, glutamate dehydrogenase recorded an up-regulated trend. Therefore, nitrogen metabolism is modified with Al toxicity proportionately and thereby could be used as possible bioindicator under similar condition for Salvinia plants. With this background the present paper has discussed some aspects of carbohydrate and nitrogen metabolism in Salvinia under Al toxicity. More so, the role of polyamine has also deciphered in alleviation of Al stress with interaction of those metabolites.

AbbreviationsPA: Polyamine; Put: Putrescine; DNS: di-nitro salicylic acid; DTT: dithiothrietol; EDTA: Ethylene diamine tetra acetic acid; PMSF: Phenyl-methyl-sulfonyl-fluoride; MOPS: 3-(N-morpholino) propanesulfonic acid; PEP: phospho enol pyruvate; PVP: polyvinyl pyrrolidone; PVPP: poly-vinyl-poly-pyrrolidone; SPS: sucrose phosphate synthase; MDH: malate dehydrogenase activity; PEPC: Phosphoenol pyruvate carboxylase; NR: nitrate reductase; GDH: glutamate dehydrogenase; GOGAT: glutamine-2-oxoglutarate aminotransferase; GS: glutamine synthase

Keywords
Al toxicity; Carbohydrate metabolism; Nitrogen metabolism; Polyamine; Salvinia natans L.
Responses of plants to toxic metals are reflected either in susceptibility or tolerance at physiological, cellular and molecular levels. Al, though not a heavy metal, still, is a serious hazardous element inducing phytotoxicity (Achary et al. 2008). In case of angiospermic species the toxicity is most vulnerable in roots impairing its physiological activities. However, Al behaves as a pro-oxidant inducing the oxidative stress by changing the normal cellular redox (Mandal et al. 2013a). The results have been in succession for the assessment of resistance or tolerance to Al irrespective of plant species and thus selection and potential use of such species for remediation sets the goal (Mandal et al. 2013b). Al salt is dissolved in acidic condition therefore the water bodies with low pH are the targets of primary phytotoxicity. A few topical aquatic species mostly the weeds like Eichhornia, Pistia, Iris, Acrous, Lemna etc are common angiospermic hydrophytes employed for their hyper accumulator properties of toxic metals (Singh and Ma 2006). Non angiospermic species mostly the aquatic ferns like Marsilea, Azolla and Salvinia has established their worth as hyper accumulators. The later three species are good or potential source of biological indicators for such metals (Wolff et al. 2012, Das et al. 2014, Vestena et al. 2007). In the present experiment, Salvinia is tested for its efficacy with Al tolerant under simulated water culture with varying concentrations of Al.
Hyper accumulators species can alter their physiological activities for absorption of a particular metal. On relationship with metal absorption and the changes in activities of the those species can pose the concept of bio-indication. In most of the cases the metal induced changes in redox is related to generation of reactive oxygen species (ROS) and hyper accumulators are well tuned with moderate the oxidative damages of the biomolecules. Salvinia may be sited as a potential hyper accumulator species of Al with its possible metabolic profiling. Al induced water stress and its inherent oxidative exposure has been found to be adjusted by plants potentials to tolerate the metals (Mandal et al. 2013a). Still, the other metabolic paths which constitute the cellular integrity like carbohydrate and nitrogen metabolism have not been dealt earlier with those. Maintenance of total carbohydrates reserve its synthesis and allocation in plant parts is a possible clue for indirect reflection of photosynthetic potential (Norwood et al. 2000). Thus, the different carbohydrates fractions like reducing and non reducing sugars enzymatic machineries (anabolic: like sucrose phosphate synthase, catabolic: invertase, organic acid synthesizer: malate dehydrogenase, phospho enol pyruvae carboxylase) are the most crucial for establishing plant status for carbohydrate metabolism under metal stress. On the other hand nitrogen metabolism under metal contaminated soil also reflects the efficiency of plants to metabolized inorganic nitrogen in to most predominant biomolecules (protein, nucleic acid and other polymeric moieties) (Masclaux-Daubresse et al. 2010). It is quite in agreement those nitrogen metabolizing enzymes are two states in contributing of inorganic nitrogen in to organic moieties: direct conversion of nitrogen salt into reduced form (by NR and allied enzymes) and allocation of reduced nitrogen into various intermediatory moieties (by the enzymes GDH, GS, GOGAT). In angiospermic plants the nitrogen metabolism has unraveled the paths for metal mediated stress and its possible recovery for plant’s sustenance even in contaminated water bodies (Gao et al. 2013; Masclaux-Daubresse et al. 2010). Still, for non angiospermic plants, there recorded hardly any information to support the nitrogen metabolism in sustenance as well as bio accumulation of concern metal.At the cellular level plants can modulate their responses in perception of some elicitors moieties to metal toxicity. Those elicitors include growth regulators, allelochemicals, secondary metabolites, brassino steroids, polyaminess, nitric oxide, jasmonic acid etc. Amongst those, polyamines (PAs) have been successfully evident a potential modulator for metal induced cellular responses in the plants. Thus in the present experiment we hypothesized that Salvinia plant may also undergo some sort of changes in regards to carbohydrate and nitrogen metabolism under Al contaminated water bodies and thus may set as a possible bioindicator under Al contaminated water bodies. Moreover, those cellular responses might be modulated with application of PAs to unravel the details of carbohydrate and nitrogen metabolism to recover in plants under Al toxicity. With this the present paper documented some aspects of carbohydrate and nitrogen metabolism under influence of Al salt in simulated condition and its possible interaction with PAs.
Salvinia has been established earlier as a potential bio-accumulator for toxic metals (Dhir et al. 2012). The physiological responses for such bioaccumulation in Salvinia for Al have registered both at sequestering as well as elevation of antioxidation metabolism (Mandal et al. 2013b). On the other hand, a plant could modify its predominant metabolic domains in reversible way with the application of PA and could mitigate the stress (Mandal et al. 2014). Carbohydrate and nitrogen metabolism are of those predominant area that are to be dealt with Al tolerance in relation to bioaccumulation of the metal. In aquatic species few reports are also available where modification of different metabolic pools could supersede the normal metabolic rate when exposed to Al toxicity (Mandal et al. 2013a; 2013b). Photosynthetic rate of plant and the effect on it with metal toxicity could be measured by analyzing the carbohydrate metabolism and related enzymes. With this background, the different carbohydrate reactions with some invitro enzymes activities were assayed under Al toxicity with supplementation of Put. With this back ground an analysis of those metabolic profiles may add some insights in possible bio indication for Al phytotoxicity in Salvinia plant. In addition, the interactive responses with polyamine application would probe another possibility in recovery of metal induced changes or impairment of metabolic activities.Collectively, there might pose a scope for Salvinia plant to be treated as suitable hyper accumulator for the water bodies with Al toxicity.
1 Plant material
Salvinia natans (Linn.) is a free-floating aquatic fern, belonging to the family of Salviniaceae, class Pteridopsida and division Pteridophyta, was chosen as the experimental material for the present experiment.
1.1 Culture of the plant
Plants were collected from wet land and washed with deionized water to remove adhering salts/debris. Thereafter, it was kept in de-ionized water to leach out absorbed salts, if any. After completion, plants were transferred to 1/4 X Murashige and Skoog medium for seven days for acclimatization (Murashige and Skoog 1962). Thereafter, plants were treated with varying concentrations 0, 240, 360, 480 µM of Al salt [KAl (SO4)2,12H2O] and 480 µM of Al salt with 1 mM Put adjusted to pH 4.5 (as mentioned by Giannakoula et al. 2008; 2010; Parker et al. 1995) and kept in a poly house for 7 days under the ambient condition: 37±10C of temperature, 75-85% of relative humidity and photoperiod of 14-10 h light and dark. All the experiment was replicated thrice for each concentration of Al treatment. After completion of treatments, plants were harvested, immediately freezed in liquid nitrogen and preserved in -700C for further biochemical analysis.
1.2 Carbohydrate metabolism
Estimation of total carbohydrate
100 mg of whole plant sample was hydrolyzed by 5 ml of 2.5 N HCl for 2½ h and followed by neutralization with sodium carbonate (Na2CO3) until effervescence ceases. After completion, the final volume was made up to 100 ml by adding double distilled water. Mixed vigorously and it was centrifuged at 12,500 × g for 15 min followed by supernatant collection in a clean test tube. 1 ml of supernatant was gently mixed with 4 ml of anthrone reagent, incubated in a boiling water bath for 10 min followed by cooled it immediately. The absorbance was recorded at 630 nm. Total carbohydrate is obtained from the standard curve made by glucose and expressed as mg g-1 FW (Yoshida et al. 1976).
1.2.1 Determination of reducing sugar
100 mg of fresh whole plant sample was homogenized thoroughly in liquid nitrogen and extracted with 80% hot ethanol. Followed by 10 ml distilled water was added to this to dissolve the sugar with gentle mixing. 1 ml of sample is mixed with 3 ml of DNS reagent and kept at boiling water bath for 5 min. Thereafter, 1 ml of 40% Rochelle salt solution added to this when the content of the test tube were still warm. After cooling, the color intensity was measured at 510 nm and value has been determined from the standard curve made by glucose and expressed as mg g-1 FW (Adak et al. 2011).
1.2.2 Assay of invertase activity (EC 3.2.1.26)
The assay of invertase activity was performed based on the method of Adak et al. (2011) with slight modification. The sample was homogenized in liquid nitrogen and enzyme was extracted in 20 mM sodium acetate buffer. The aliquots were centrifuged at 10,000 × g for 12 min at 4°C. The supernatant was collected and reacted with 2 ml of assay mixture composed of citrate buffer (pH 3.8), sucrose (200 mM-1 L-1) at 30°C of incubation for 30 min. The reaction was terminated with NaOH in boiling water bath for 15 min. From the standard curve of glucose; invertase activity was calculated and expressed as µg min-1 g-1 FW.
1.2.3 Assay of sucrose phosphate synthase (SPS, EC 2.4.1.13)
SPS activity was assayed according to Saman et al. (1995). Plant sample (whole plant) was homogenized in liquid nitrogen and enzyme was extracted in 100 mM MOPS buffer (pH 7.5) and centrifuged at 14,000 × g for 20 min. Enzyme extract was collected and followed by 2 ml of reaction mixture containing 50 mM L-1 MOPS (pH 7.9), 10 mM UDP-glucose, 10 mM L-1 fructose-6-phosphate, 40 mM L-1 glucose-6-P, 15 mM L-1 MgCl2, 2.5 mM L-1 DTT for 10 min at 25°C. The reaction was terminated by adding 30% KOH in the reaction mixture, kept in a boiling water bath for 10 min. The mixture was treated with ice cold anthrone reagent for 30 min and the absorbance was recorded at 620 nm. The enzyme activity was expressed as µM sucrose produced min-1 g-1 FW.
1.2.4 Assay of malate dehydrogenase activity (MDH, EC 1.1.1.37)
MDH activity was assayed according to Ritambhara et al. (2000). 0.2 g of whole plant sample was taken and homogenized in liquid nitrogen and extracted in 5 ml of 100 mM Tris–HCl buffer (pH 7.8) containing 20 mM MgCl2, 1 mM DTT, 1 mM EDTA and 1 mM PMSF followed by centrifuged at 20,000 × gfor 15 min at 4°C. The supernatant was collected and assayed in a reaction mixture containing 100 mM Tris–HCl buffer (pH 7.8), 20 mM MgCl2, 0.1 mM NADH, 1 mM EDTA, 0.5 mM oxaloacetate. The reaction was initiated at 25°C with the addition of the enzyme extract and oxidation of NADH was recorded at 340 nm. Enzyme specific activity was expressed as nmol NADH oxidized S-1 mg-1 protein.
1.2.5 Phosphoenol pyruvate carboxilase (PEPC, EC 4.1.1.31)
PEPC activity was accomplished according to Idrees et al. (2010). 1 g of leaves sample was homogenized in liquid nitrogen and enzyme was extracted in 3 ml of 25 mM phosphate buffer containing 1 mM EDTA, 25 mM NaF, 5 mM malate, 5 mM thiourea, 5% PVPP, 10 mM MgCl2, followed by centrifugation at 15,000 × g for 30 min at 4°C. Supernatant was collected and mixed with 3 ml of reaction mixture containing 50 mM Tris-HCl (pH 8), 2.5 mM PEP, 5 mM KHCO3, 5 mM MgCl2, 15% (v/v) glycerol, 0.15 mM NADH and 5 IU of malate dehydrogenase (MDH). The mixture was kept at 30°C for coupling reaction and absorption was read at 350 nm. Enzyme activity was expressed as International Units (IU) mg-1 protein.
1.3 Nitrogen metabolism
Assay of nitrate reductase (NR, EC 1.6.6.1)
NR was assayed according to Botrel et al. (1996). 1 g of whole plant sample was taken and homogenized in liquid nitrogen. Then the sample was extracted with ice cold 0.1 M potassium phosphate buffer (pH 7.6) followed by centrifugation at 12,000 × g for 15 min. 0.2 ml of supernatant was reacted in an assay mixture with 0.2 ml of 0.1 M potassium nitrate, 0.4 ml of 2 mM NADH dissolved in 0.1 M potassium phosphate buffer (pH 7.6) and kept in incubation for 15 min at 30ºC. The reaction was terminated by the rapid addition of 1 ml of 1% sulphanilamide followed by 1 ml of 0.2% napthyl-ethylene-diamine reagent and again incubated for 30 min. The absorbance was recorded at 540 nm. NR activity was expressed as µM min-1 g-1 FW.
1.3.1 Assay of glutamate dehydrogenase (GDH, EC 1.4.1.2)
The GDH activity was assayed according to Loyola-Vargas and Jimenez (1984). 1 g of fresh plant sample was homogenized in two volumes (w/v) of extraction buffer containing 50 mM Tris-HCl (pH 8), 1 mM calcium chloride (CaCl2) 5 mM β-mercaptoethanol, and 5% polyvinyl pyrrolidone (PVP). The homogenate was filtered through 4 layers of cheese cloth and then centrifuged at 12,000 × g for 30 min. The supernatant was collected for the determination of enzyme activity. The whole procedure was carried out under cold condition. The amination reaction was carried out at 30°C in 100 mM Tris-HCl (pH 7) containing, 0.16 mM NADH, 10 mM 2-oxoglutarate, 100 mM NH4Cl and 4 mM calcium chloride. The oxoglutarate dependent oxidation of NADH was recorded at 340 nm. Enzyme units were expressed as NADH oxidized min-1 mg-1 protein.
1.3.2 Glutamine-2-oxoglutarate aminotransferase (GOGAT, EC 1.4.1.13)
1 g of fresh plant sample was ground in liquid nitrogen and extracted in 50 mM phosphate buffer (pH 7.8), containing 1 mM EDTA, 2 mM 2-oxoglutarate, 1 mM PMSF, 1 mM DTT and 0.1% PVPP. The homogenate was then filtered through 4 layers of muslin cloth and centrifuged at 15,000 × g for 20 min at 4°C. The GOGAT activity was assayed within 2 h of enzyme extraction by monitoring NAD(P)H oxidation according to Esposito et al. (2005). The assay mixture consisted of 50 mM Tris-HCl (pH 7.5), 5 mM 2-oxoglutarate, 5 mM glutamine, 0.25 mM NAD (P)H. The reaction was initiated by immediate adding of glutamine following the enzyme preparation and incubated for 15 min. The absorption was read at 340 nm and the activity was expressed as nM Glu mg-1 protein min-1.
1.3.3 Assay of glutamine synthatase (GS, EC 6.3.1.2)
1 g fresh plant sample was homogenized in liquid nitrogen and extracted with 0,1 M Tris–HCl buffer (pH 8) containing 20 mM MgCl2, 1 mM β-mercaptoethanol (2-ME) and 0.05% TRITON X-100 according to Kwinta and Cal (2005). Followed by the slurry was centrifuged at 10,000 × g for 20 min at cold condition. The supernatant was added in an assay mixture containing 0.2 M of L-glutamine, 20 mM sodium arsenate, 50 mM hydroxylamine, 3 mM MnCl2, 1 mM ADP. The activity of glutamine synthetase was assayed following spectrophotometric method and enzyme activity was defined in μM of 4-glutamyl hydroxamate formed min-1 g-1 dry weight (DW).
1.4 Statistical analysis
All the observations were recorded with three replications (n = 3) and data were expressed as mean ± SE. The statistical analysis was performed with SPSS software (SPSS Inc., version 10.0) by one-way ANOVA analysis, taking P≤0.05 and P≤0.01 as significant (Gomez and Gomez 1984).
2 Result
2.1 Determination of total carbohydrate
A significant subdued status of total carbohydrate was observed in Al treated plants as compared to control. The decline of total carbohydrate had shown a consistent manner with the Al doses. Thus, we recorded a significant (p≤0.05) depletion of total carbohydrate with 8.69%, 16.15%, 26.93% at 240, 360, 480 μM of Al concentration respectively as compared to control. Put has been able revert the carbohydrate accumulation and acted as a modulator. The total carbohydrate was retrieved by 1.3 fold as compared to highest concentration of the Al exposure (480 μM) (Figure 1A).


Figure 1 Determination of total carbohydrate content (a) and reducing sugar content (b) of the Salvinia plants grown under varying concentration 0 (control), 240, 360, 480μM of Al and 480μM of Al supplemented with 1mM Put (480μM+1mM Put). The values are plotted from means (±SE) of replication (n=3), Bars showing different letters indicate significant differences according to Duncan’s test at (p≤0.05)

2.2 Determination of reducing sugar
In continuation of carbohydrate status we have also quantified the reducing sugar from the Salvinia plant under various Al concentrations (0, 240, 360, 480 and 480 μM of Al supplemented with 1mM Put). We found a consistent significant (p≤0.05) decline in reducing sugar activity according to concentration gradient of Al and those recorded 11.2%, 29.5%, 34.2% respectively at 240, 360, 480 μM of Al concentration as compared to control. With theapplication of PA, Salvinia plant had responded well with Put and that was increased by 1.3 fold than highest concentration of Al (480μM) (Figure 1B).
2.3 In-vitro assay of acid invertase
As mentioned that Salvinia plant had maintained a declining trend of reducing sugar content with increasing concentration of Al, hence, there must be some changes in some enzymatic reactions. Therefore, the significant (p≤0.05) changes in activity of acid invertase recorded 1.3 fold 1.4 fold 1.7 fold higher over control under 240, 360, 480 μM of Al concentration respectively. On the other hand, application of Put reduced the activity by 24.8% compared to highest concentration of Al (Figure 2A).
2.4 Sucrose phosphate synthase (SPS)
We performed the experiment for in-vitro analysis of sucrose synthesis by SPS activity from Salvinia plant under Al toxicity. It recorded a stepper up regulation of activity by 1.3 fold, 1.4 fold, 1.7 fold under 240, 360, 480 μM of Al concentration respectively than control, Whereas, Put down regulated the activity by 20.8% as compared to highest concentration of Al (480 μM) (Figure 2B).
2.5 Malate dehydrogenase (MDH)
A significant (p≤0.05) increase in activity was observed in the order of 1.2 fold, 1.4 fold, 1.6 fold at 240, 360, 480 μM of Al concentration respectively over control.On the other hand, while plant was fed with Put, MDH activity was subdued by 32.4% over highest concentration of Al (480 μM) (Figure 2C).


Figure 2 Assay of acid invertase activity (a) SPS activity (b) MDH activity (c) and PEPC activity (d) in Salvinia grown under varying concentration 0 (control), 240, 360, 480μM of Al and 480μM of Al supplemented with 1mM Put (480μM+1mM Put). The values are plotted from means (±SE) of replication (n=3), Bars showing different letters indicate significant differences according to Duncan’s test at (p≤0.05)

2.6 Phosphoenolpyruvate carboxylase (PEPC)
The Al sensitivity was effected on PEPC activities and its concomitant responses showed a significant (p≤0.05) up regulation by 1.1 fold, 1.2 fold, 1.4 fold higher at 240, 360, 480 μM of Al concentration respectively over control. On the contrary, Put down regulated the activities by 20.1% as compared to highest concentration of Al (Figure 2D).
2.7 Nitrate Reductase (NR)
NR is the most studied enzyme for nitrogen metabolism and in the present study it showed that the significant (p≤0.05) reduction in activity by 13.3%, 22.6%, 35.8% under varying Al concentration (0, 240, 360, 480 μM) as compared to control. As stated earlier, Put application had overcome the detrimental effects of Al toxicity on the NR enzyme. Thus, the retrieval of NR activity was 1.3 fold over 480 µM of Al (Figure 3A).
2.8 Glutamate dehydrogenase (GDH)
GDH activity showed a dose dependent response behavior of the enzyme activity and recorded 1.2 fold, 1.3 fold, 1.4 fold higher at 240, 360, 480 μM of Al concentration respectively than control. The activity of GDH was down regulated by 20.7% with the application of Put (Figure 3B).


Figure 3 Assay of NR activity (a) GDH activity (b) GOGAT activity (c) and GS activity (d) in Salvinia grown under varying concentration 0 (control), 240, 360, 480μM of Al and 480μM of Al supplemented with 1mM Put (480μM+1mM Put). The values are plotted from means (±SE) of replication (n=3), Bars showing different letters indicate significant differences according to Duncan’s test at (p≤0.05)

2.9 Glutamate oxoglutarate amino transferase (GOGAT)
The changes in GOGAT activity under Al treatments was significant (p≤0.05). The NADHdependent GOGAT activity was subdued by 15.4%, 31.6%, 38.2% under 240, 360, 480 μM of Al concentration respectively over control. Put was highly active to retrieve the activity by 1.2 fold as compared to the highest concentration of Al (480 μM) (Figure 3C).
2.10 Glutamine synthatase (GS)
In our present experiment, GS shows opposite trend to that of GDH and we recorded a significant (p≤0.05) reduction in activity by 6.5%, 19.5%, 37.5% under 240, 360, 480 μM of Al concentration respectively over control. Interesting to note that Put was an effective elicitor to regain the GS activity by 1.2 fold as compared to highest concentration of Al (480 μM) (Figure 3D).
3 Discussion
Plants initially recorded a linear decrease in total carbohydrate and reducing sugar. This is not any exception of the plant with metal toxicity that undergoes loss of photosynthetic carbohydrate biosynthesis. Total carbohydrates, reducing sugars and their concomitant metabolic fate under any sort of abiotic stress opine the possibility for the suppression of photosynthetic rate. It also describes the allocation of reduced carbon into polymeric complex moieties like starch or cellulose in plant tissue. Thus, with the view of photosynthetic performances under Al toxicity the behaviour of Salvinia plant was analyzed in terms of few enzymatic assays. Of those PEPC is an enzyme that catalyzes the conversion of PEP to OAA which is then subsequently decarboxylated into significant amount of pyruvate. PEPC, however, is regarded as the key enzyme for C4 photosynthesis, still, its non photosynthetic form as found in cytosol of C3 plants (Sangwan et al. 1992). Salvinia being a typical C3 plant was characterized in the present experiment with an increased activity of PEPC. This result bears the conformity with a same under water stress condition as reported by trend Idrees et al. (2010). The over expression of PEPC activity in an increasing order as a function of Al concentration undoubtedly suggests the involvement of more carbon flux in addition to normal photosynthetic reactions. However, based on the present observations, it could be speculative for the facts that plants are prone to some sorts of metabolic constraints under Al toxicity. The later was illustrated in regulation of sugar metabolism. Thus, in the present study, the activities of enzymes like invertase and SPS were measured. For the first enzyme, which is sugar hydrolyzing in nature, is more focused for its increasing tendency all through the Al concentrations. However, it has been down regulated with the application of Put. Over accumulation of metal, invariably among plant species alters the energy yielding pathways that keep the plants vulnerable for sustenance. Rapid replenishment of reducing sugars, the substrate for respiratory energy may be corroborated with the up regulation of invertase activity as documented in the present case. In fact, sucrose accumulation and its physiological function are more attributed as osmolytes also besides being a readily transportable moiety. However, it plays in balance of osmoregulation which under goes perturbance due to over accumulation of Al (Talbott and Zeiger 1998). Therefore, Salvinia plant with its enhanced activities of invertase may be more corroborated for supplementation of energy yielding metabolism under such conditions. Accumulation of various fractions of soluble sugars were evident as reliable physiological markers for plants subjected to metal stress, with reference to tolerant genotypes (Arbona et al. 2013). With regard to another enzyme for carbohydrate metabolism, the activity of the SPS followed the same trend as that of invertase throughout Al concentration. The linear increase in SPS activity was significantly subdued when Put was applied along with highest concentration of Al. This may unravel the possibility that synthesis of sucrose was a factor to compensate depressed photosynthetic activity even under Al toxicity. In earlier works, metal toxicity and its concomitant effects on photosynthetic carbon fixation following sucrose synthesis has thoroughly been clarified. PA (as Put in the present case) may be related to retrieval of sugar metabolism mostly due to maintenances of photosynthetic apparatus at the cellular level. The later deals with mostly the stability of chloroplast membrane and its integrally bound protein for carboxylationand other downstream reaction are maintained by application of PA as documented in metal toxicity (Gill and Tuteza 2010). However, for fern species, the impetus or PA metabolism in regulation to photosynthetic reaction leading sucrose synthesis and its allocation to other carbohydrate fraction demand similar mechanism of resistance to Al as that of higher plants. Under depleted osmotic potential the cellular turgidity could be compensated by over expression of polyamine content (Dhir et al. 2012). One of the key regulatory enzyme involved in TCA cycle, is MDH. Keto acid pathways are a major source of energy yielding mechanism in plants and some of the enzymes plays regulatory role in keto acid metabolism. Malic acid accumulation and its consequent metabolism give a special protection to plant for metal tolerance. Under increasing concentration of Al, the conversion of malic acid into OAA by MDH is hypothesized for dual roles: the rapid supplementation of OAA as substrate to different amino acid biosynthesis through amination reaction and the utilization of malic acid as a chelating substance with metals (Ma 2000). With regard to the later, specificity for Al toxicity by a tolerant species is characterized with production of significant amount of malic acid forming ligand with cations (Yang et al. 2011). Therefore, Salvinia plants an aquatic C3 species has shown an extensive growth in some cases when absorb a higher concentration of metallic pollutants. In that case plants have the possibility to over express the keto acid pool to be utilize in more energy yielding path ways or/and sequestering the metal with different chelate compound (Muthukumaran and Rao 2014; Ritambhara et al. 2000).
Under metal stress, not only the balance of carbohydrate, but also the nitrogen compounds could be reflectant for sensitivity of plants under metal toxicity (Wang et al. 2008; Debouba et al. 2007). After absorption of mineral nitrogen it remains inter convertible in transformation of amino acid and keto acid. GDH converts oxoglutarate into glutamate in a reversible way. GDH is attributed for rapid elimination of free ammonia through incorporation in amino acids. It serves for two purposes: to avoid ammonium toxicity and secondarily, making provision of reduced nitrogenous forms. The later are most suitable for maintaining the source-sink relationship. Salvinia plants in the present experiment recorded with a linear increase in GDH activity with Al concentration and thereafter moderated with Put. Our data is in agreement with elevated GDH functioning in response to others studies (Labboun et al. 2009), where there is a clear report that a substantial rate of glutamine dependent respiration is based on GDH reaction. The depletion of GDH activity could also be correlated from the flow of reduced carbon limitation as well as it’s starvation under metal stress. Put has raised the provision for such carbon flow in adequate manner for the physiological functioning of GDH (Awasthi et al. 2013). In higher plants the glutamine happens to be one of the key regulatory moieties for nitrogen balance in terms of its fate on reversible reaction into keto acids. Therefore, GS also happens to be a regulatory enzyme in conjugation with GOGAT for generation of glutamate and amino acids (Bao et al. 2014). Amines are major source of organic nitrogen in plants and glutamine synthatase is the key enzyme where glutamic acid is converted to glutamine through the aminative reduction. Our findings are in confirmation with others where GS and GOGAT showed a significant down regulation in context to Al concentration. It is concluded that ammonium would be very much toxic in plant tissues unless it is properly sequester incorporated in keto acids. However, our result supported that the GDH induction of ammonium incorporation may not be at par with the biosynthesis of glutamine before it is converted into glutamic acids. Thereby, GS-GOGAT synchronization may not have marked for any significant influences in glutamines compensation for Salvinia plants. Still, Put had recovered the efficiency of plants to biosynthesize glutamic acid and glutamine when supplied with Al doses. PA has also intrinsic effects on root membrane potential to increase the permeability for nitrate / nitrite accumulation even under depleted concentration in contaminated soil (Sangwan et al. 2014). This could be illustrated in Salvinia plant also where the regulation of GOGAT and GS activity had accounted the possible ammonium accumulation in Al stressed plants as reported earlier (Panda et al. 2009).
Conclusively our results clearly indicated that the Salvinia plants suffered and showed some distinct variation in carbohydrate and nitrogen metabolism when encountered with various concentration of Al toxicity. This probably is reflection of the fact of interlinking of organic acids and its corresponding assimilation into amino acids by ammonium metabolism. Therefore, the serious constrain for Al toxicity in plants could be justified with metabolic prospect and those parameter could reliably serve as a signal for bioindication of Al toxicity.
Acknowledgments
This work is financially supported by Department of Science and Technology, DST-PURSE programme, Govt. of India, New Delhi activated to University of Kalyani.
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