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Research Article
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Assessment of Osmolyte Accumulation in Heavy Metal Exposed Salvinia natans
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B. Dhir,
S.A. Nasim,
S. Samantary
and
S. Srivastava
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ABSTRACT
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Accumulation of osmolytes in terrestrial plants in response
to environmental stresses is well reported and information about aquatic plants
is limited. Present study aimed to investigate if the aquatic weed, Salvinia
natans accumulates osmolytes/compatible solutes on exposure to various heavy
metals. Plants exposed to heavy metals viz. Cd, Cu, Ni, Cr, Mn, Fe, Co, Pb and
Zn, were harvested after 48 h and various osmolytes including sucrose, mannitol,
proline, glycine betaine and polyamines were estimated using biochemical methods.
Results suggested that heavy metal stress does trigger the accumulation of osmolytes
such as sucrose, mannitol and glycine betaine. In contrast proline accumulation
was not observed. Studies of heavy metal stress on the endogenous levels of
polyamines showed presence of free polyamines, while conjugated and bound forms
were not detected. Among free polyamines, Putrescine (Put) and Spermidine (Spd)
did not show significant decrease in heavy metal exposed Salvinia except
Pb and Fe exposed plants. Spermine (Spm) content showed decline in heavy metal
exposed Salvinia. The decrease in polyamine levels indicated their possible
role in combating oxidative stress induced by heavy metals. Studies suggest
that accumulation of osmolytes under heavy metal stress might help in imparting
tolerance in Salvinia.
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Received: February 17, 2012;
Accepted: June 25, 2012;
Published: October 10, 2012
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INTRODUCTION
Accumulation of compatible organic osmolytes in plants in response to various
environmental stresses viz., drought, salinity, extreme temperatures, UV radiation
and heavy metals is well reported in literature (Serraj and
Sinclair, 2002; Ashraf and Foolad, 2007). These are
low molecular weight, highly soluble compounds. The major contribution of these
osmolytes lies in protection of plants from abiotic stresses via different mechanisms
including adjustment of cellular osmoticum, detoxification of reactive oxygen
species, maintenance of membrane integrity and stabilization of enzymes/proteins.
Apart from this, they are also known to protect cellular components from dehydration
and injury (Sharma and Dietz, 2006; Ashraf
and Foolad, 2007). These solutes include sugars (glucose, fructose, sucrose,
trehalose, raffinose), sugar alcohols, nitrogen-containing compounds such as
proline, Quaternary Amino Compounds (QACs) such as glycine betaine, alanine
betaine, proline betaine and polyamines (Mudgal et al.,
2010).
Potential of aquatic plants for accumulation of heavy metals is well studied
(Mishra and Tripathi, 2008; Peng
et al., 2008; Verma et al., 2008;
Dhir et al., 2009a; Ndimele
and Jimoh, 2011). The accumulation of toxic heavy metals in plants induces
osmotic stress that in turn may initiate synthesis of metabolites that play
an important role in metal binding, antioxidant defence and signaling (Sharma
and Dietz, 2006; Joseph and Jini, 2010; Bhat
and Khan, 2011). Accumulation of osmolytes in response to heavy metal stress
has been documented for terrestrial species but information regarding aquatic
species is lacking (Sivaci et al., 2008). Therefore,
present investigations were carried out with an aim to investigate if aquatic
plants also show accumulation of osmolytes in response to heavy metal stress.
Salvinia natans, a free-floating aquatic weed having high growth rate
and tolerance to high element concentrations was chosen for the present Previous
findings by our group proved that Salvinia natans possesses significant
potential to accumulate various heavy metals (Dhir et
al., 2008, 2009b). Therefore, further investigations
were carried out with an aim to: (1) check if Salvinia accumulate osmolytes
under heavy metal stress and (2) investigate the alterations in the level of
endogenous polyamines in heavy metal exposed Salvinia.
MATERIALS AND METHODS
Plant material and growth conditions: Plants of Salvinia natans L.
(Salviniaceae) collected from unpolluted water bodies were maintained in cemented
pots (~1 m diameter) under natural light in outdoor conditions. The temperature
ranged from 30-32°C. The solutions having metal concentration 35 mg L-1
were prepared by using metal salts Cd(NO3)2.4H2O
(Cd2+), CuSO4.5H2O (Cu2+), Ni(NO3)2.6H2O
(Ni2+), K2Cr2O7 (Cr6+),
MnCl2.4H2O (Mn2+), FeSO4.7H2O
(Fe2+), CoCl2.6H2O (Co2+), Pb(NO3)2
(Pb2+) and ZnSO4.7H2O (Zn2+).
The pH of all the solutions ranged between 4.5-5.0. Distilled water was taken
as control. Five plants of Salvinia (each having five nodes with two
leaves at each node) were floated in each tub having 750 mL of metal solution.
All the measurements were carried out after 48 h of metal exposure.
Sucrose estimation: Sugars were extracted by overnight submersion of
dried plant material in 80% (v/v) ethanol at 25°C with periodic shaking.
Sucrose content was determined first by degrading reactive sugars present in
0.1 mL extracts with 0.1 mL 5.4 N KOH at 97°C for 10 min. Three mL of freshly
prepared anthrone reagent were then added to the cooled reaction product and
the mixture was heated at 97°C for 5 min, cooled and absorbance was read
at 620 nm (El-Shihaby et al., 2002). Sucrose
content was expressed as μg g-1 fresh wt.
Estimation of mannitol: Leaves were ground in liquid nitrogen, lyophilized
overnight and stored at -25°C until used. The lyophilized powder was placed
in a capped 1.5 mL Eppendorf tube and 1 mL of hot (80°C) distilled water
was added. The tube was heated at 80°C for 30 min and then cooled and centrifuged
at 12000 g for 15 min twice. Plant extracts were deionized by passage through
cationic and anionic resins.
The 0.1 mL of biological extract was dispensed in microcentrifuge tube and
0.5 mL of 0.5 M formate (pH 3.0) was added. To this solution 0.3 mL of 5 mM
sodium periodate (reagent 1) was added. The contents were vortexed and left
at room temperature for 15 sec and 0.3 mL of a solution containing 0.1 M acetylacetone,
2 M ammonium acetate and 0.02 M sodium thiosulfate (reagent 2), were added.
The tube was closed and heated in boiling water for 2 min and cooled under running
tap water and the absorbance at 412 nm was measured (Sanchez,
1998.). The mannitol content was expressed in nM g-1 fresh wt.
Proline estimation: Proline was measured in leaf tissues following method
described by Bates et al. (1973). The optical density
was measured at 520 nm and proline content was expressed as μg g-1
fresh wt.
Glycine betaine estimation: Glycine betaine was estimated in leaf tissues
following modified protocol of Desingh and Kanagaraj (2007).
The absorbance was measured at 365 nm and glycine betaine content was expressed
as μg g-1 fresh weight.
Estimation of polyamines: Polyamines (free, conjugated and bound forms)
present in leaf tissue were quantified according to modified protocol of Flores
and Galston (1982). The polyamines were extracted in perchloric acid. Supernatant
was used for estimation of free polyamines, while supernatant treated with HCl
was used for estimation of conjugated polyamines. The pellet was dissolved in
1 N NaOH was used for estimation of bound polyamines. Two hundred microliter
each of supernatent (untreated), supernatent hydrolysate and pellet hydrolysate
were taken separately. Two hundred microliter of saturated Na2CO3
and 400 μL of dansyl chloride (5 mg mL-1 in acetone) were added
to each tube, vortexed and incubated at 25°C in dark (overnight). 300 μL
of proline (100 mg mL-1) was added to dansylation mixture and samples
were incubated in dark for 30 min at room temperature. To extract polyamines,
50 μL benzene was added to dansylation mixture, vortexed and allow to stand
till the layers separate. Equal volumes of polyamine solutions and standards
were applied and resolved by Thin-Layer Chromatography (TLC) with cyclohexane
and ethylacetate in a ratio of 5:4 (v/v) as the solvent. The polyamines were
located with a UV detector, polyamine-containing silica spots were collected
and eluted with 4 mL of ethyl acetate. The fluorescence of these solutions was
measured with a UV-fluorescence spectrophotometer (Hitachi, F-2000) at 359 nm
(excitation) and 495 nm (emission) wavelengths, respectively (Chang
et al., 1999).
Statistical analysis: Analysis of Variance (ANOVA) for all measured
variables was performed by using software new MSTAT-C (version 2.1). The level
of significance was measured using Duncans Multiple Range Test (DMRT)
taking p≤0.05 as significant.
RESULTS AND DISCUSSION
Sucrose and mannitol levels: Overproduction of compatible organic solutes
is one of the most common stress responses in plants (Serraj
and Sinclair, 2002). Exposure to heavy metal stress induced a substantial
increase in osmolyte content (Table 1). Sucrose levels recorded
enhancement in plants exposed to heavy metals, though the response varied for
each metal. The increase was significant in Cr, Cd and Zn exposed Salvinia.
Cadmium (352), Cr (317)and Zn (293) exposed plants showed ~3 fold increase,
while Ni (268), Pb(250), Fe(239), Mn(202), Co(214) and Cu(184) exposed plants
showed ~2 fold enhancement in sucrose level in comparison to control (108).
Table 1: |
Alterations in various solutes measured after 48 h of metal
exposure |
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Values are Mean±SEM of three independent experiments
with three replicates each, Different letters in a row are significantly
different at p≤0.05 |
Sucrose functions as an osmoprotectant under stress conditions and has been
postulated to possess ROS-scavenging capacity (Ende and
Valluru, 2008). The increase in sucrose content can be correlated to its
reduced utilization during stress and/or block in its transport rather than
overproduction (El-Shihaby et al., 2002). Heavy
metal induced increase in soluble sugars particularly sucrose has been reported
(Alaoui-Sosse et al., 2004; Rosa
et al., 2009).
Mannitol, a sugar alcohol plays an important role in storage of carbon and
energy, regulation of coenzymes, osmoregulation and free radical scavenging
(Prabhavathi and Rajam, 2007). Heavy metal exposed Salvinia
showed increase in mannitol content except Cu, Pb and Mn exposed plants where
no change was observed. Plants exposed to Co (796) and Ni (547) showed ~4 and
~2.5 fold increase, while Cd, Cr (471, 443) exposed plants showed ~2 fold enhancement
in mannitol level in comparison to control (208). The increase in mannitol content
suggests its possible role as a free radical scavenger thus preventing cells
from lipid peroxidation (Sickler et al., 2007).
Glycine betaine and proline levels: Glycine betaine levels increased
in plants exposed to metal stress except for Cr and Mn, where no change was
observed. The increase was more significant in Cu, Fe and Co exposed plants.
Glycine betaine levels increased by ~1.9-1.6 fold in Cu, Fe and Co (342, 322,
293) exposed plants, while ~1.2 to 1.3 fold increase was noted in Pb, Zn, Ni
and Cd (209, 219, 222, 237) exposed plants in comparison to control (179). Similar
response in terms of glycine betaine accumulation under environmental stress
has been reported both in terrestrial and aquatic plant species such as
Spartina and Phragmites (Zhu et al.,
2003; Al-Garni, 2006; Islam
et al., 2009). Glycine betaine is shown to confer tolerance in Cd
exposed tobacco by protecting cellular components and increasing activities
of antioxidant enzymes (Islam et al., 2009).
Glycine betaine (GB), a quaternary ammonium compound, plays a vital role in
protection of enzyme like RuBisCo, maintenance of membrane integrity and osmotic
potential. All these roles thus protect photosynthetic efficiency (Chen
and Murata, 2002; Shirasawa et al., 2006;
Kattab, 2007).
Proline, an amino acid is reported to play role in osmotic adjustment and maintenance
of cellular integrity (e.g. membranes and proteins) via scavenging free radicals,
maintaining cellular redox potential and NADP+/NADPH ratios (Sharma
and Dietz, 2006; Ashraf and Foolad, 2007). Salvinia
exposed to heavy metal stress showed decline in proline accumulation. Decline
was more significant in plants exposed to Zn, Fe and Ni. Proline levels noted
decline of ~3 fold in plants exposed to Ni, Fe, Zn, (25, 24, 22) while exposure
to Cd and Co (34, 30) led to ~2 fold decrease in comparison to control (Table
1). This is in accordance with earlier studies where aquatic species viz.
Ceratophyllum, Wolffia, Hydrilla and Lemna polyrhiza
exhibited decline in proline levels in response to Cd stress (Dhir
et al., 2004; John et al., 2008).
Similar response of decrease in proline content has been documented for Cu exposed
Spirodela polyrrhiza (Xing et al., 2010).
Polyamines: Polyamines including spermidine (Spd), spermine (Spm) and
their obligate precursor, putrescine (Put), are polybasic aliphatic amines that
influence variety of growth and development processes in plants (Liu
et al., 2007). They play role in wide range of basic cellular regulatory
processes including DNA replication, transcription, translation, cell division,
modulation of enzyme activities, cellular cation anion balance and membrane
stability due to their potent binding ability to negatively charged macromolecules
and membranes. They function as signalling molecules, antioxidants and act as
second messengers (Verma and Mishra, 2005). Though, specific
role of polyamines in plants under metal stress is not yet known but there is
a strong possibility that they can effectively stabilize and protect the membrane
systems against the toxic effects of metal ions particularly the redox active
metals. Their role of metal chelators has been postulated (Sharma
and Dietz, 2006; Groppa et al., 2007; Gill
and Tuteja, 2010). Being polycationic in nature, they bind to negatively
charged groups in the cell membrane. Spermine, in particular has been noted
to acts as a free radical scavenger and is capable of quenching chemically generated
singlet oxygen (Groppa et al., 2007; Wen
et al., 2010).
Table 2: |
Alterations in level of free polyamines observed after 48
h of metal exposure. |
 |
Values are Mean±SEM of three independent experiments
with three replicates each, Different letters in a column are significantly
different at p≤0.05 |
Spermine has been suggested to play a protective role against the oxidative
damage produced by metals. Moreover, role of other polyamines in reducing ROS
formation by inhibiting NADPH oxidase activity has been reported (Wen
et al., 2010). Role of spermidine as a stress-protecting compound
and stress-signaling regulator has been noted (Kasukabe
et al., 2004).
Polyamines have been considered to act as antioxidants and reduce oxidative
damage produced by metals. Salvinia showed accumulation of free polyamines,
while conjugated and bound polyamines were not detected (Table
2). Among free polyamines, putrescine and spermidine was present in higher
amount, while spermine accumulated to a lesser extent. Polyamine levels showed
a variation in response to each metal exposure. Putrescine levels did not show
any significant difference in comparison to control. A slight increase was noted
in Co and Cd exposed Salvinia, while no difference was observed in Ni
and Zn exposed plants in comparison to control. In contrast, decline in putrescine
levels was noted in plants exposed to other metals (Cr, Cu, Pb, Mn and Fe).
Spermidine levels showed increase in Cd, Cr, Co and Zn exposed plants, though
increase was significant in Cd (27) exposed Salvinia (Table
2). In contrast, a decline was noted in plants exposed to Ni, Pb, Cu, Mn
and Fe. Spermine levels showed a general decline in plants exposed to metal
stress. Stress conditions can affect polyamine metabolism in different manner
that includes an increase or decrease of endogenous polyamines (Liu
et al., 2007). Increase in free putrescine and spermidine levels
noted in Cd and Co exposed Salvinia could be due to enhanced de novo
synthesis resulting from increased activity of enzymes involved in putrescine
biosynthesis [Arginine Decarboxylase (ADC) or Ornithine Decarboxylase (ODC)]
or reduced degradation, although the exact mechanism remains unclear (Liu
et al., 2007). Similar increase in putrescine and decrease in spermine
has been observed in wheat, Hydrocharis dubia exposed to Cd, Ni (Groppa
et al., 2007; Zhao et al., 2008).
Ni treatment significantly increased the putrescine (Put) level and lowered
spermidine (Spd) and spermine (Spm) levels, thereby significantly reducing the
ratio of free (Spd+ Spm)/Put in leaves, which has been considered as the signal
under stress (Zhao et al., 2008). Significantly
increase in free putrescine (Put) level and alterations in other PAs levels
under Pb treatment has been noted in Potamogeton crispus (Xu
et al., 2011). In contrast, heavy metal induced decrease in putrescine
and spermine level has also been reported in plant species (Groppa
et al., 2007; Zhao et al., 2008).
CONCLUSIONS
The present investigations revealed that Salvinia produced osmolytes
that helped in combating osmotic stress induced by heavy metals, though the
response varied from metal to metal. In summary, Salvinia possesses effective
metabolic machinery that is capable of overcoming the osmotic stress induced
by heavy metals. This is evident from enhanced accumulation of metabolites viz.
glycine betaine, mannitol and sucrose. Decline in the level of metabolites such
as proline and polyamines may not suggest a direct role but they might contribute
in imparting tolerance in Salvinia.
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