Cadmium (Cd) is the more noxious soil pollutant and its presence in the environment is essentially due to anthropogenic activities . Because of its long biological half life, Cd2+ which belongs to the group of non-essential transition metals is highly toxic. Contamination of agricultural land, and so edible plants, is essentially due to the application of Cd-containing fertilizers and sewage sludges, atmospheric deposition or geogenic origin of Cd .
Cadmium toxicity is a major factor limiting plant growth in many soils . Cd had many dangerous effects on plants, essentially the reduction of plant growth . Cadmium inhibitor effect on growth could result from photosynthesis rate reduction .nd the decline in nitrogen metabolism .6].7].8]. A great deal of research has established the ability of cadmium to induce reactive oxygen species (ROS) production in plants .9]. Cadmium stress may also impair the plasma membrane integrity by increasing lipid peroxidation . Alternatively, it could alter plasma membrane permeability essentially nitrate and other essential-nutrients uptake. Hyperaccumulators are ideal plant species used for phytoremediation of Cd contaminated soils. Tobacco plants could be considered too, as a cadmium hyperaccumulator plant . A full understanding of metal tolerance mechanisms of hyperaccumulators will facilitate enhancing their phytoremediation efficiency.
Previous work showed that tobacco plants accumulated Cd mostly in leaves. Leaf Cd content was six times more important than root Cd content. However, tobacco leaves were less affected by Cd stress than roots. In this report, we aimed at better understanding the differences in Cd partitioning between roots and different foliar stages to explain how tobacco leaves supported the high Cd level and could protect nitrogen metabolism under stress conditions. To clarify this question, this study investigated the effects of Cd accumulation on the activity of key enzymes involved in nitrogen metabolism in roots and in different foliar stages of tobacco.
2. Materials and Methods
2.1. Plant Material and Growth Conditions
Tobacco seeds (Nicotiana tabaccum, Bureley V.) were germinated on a moistured filter paper at 25˚C in the dark. The uniform seedlings were then transferred to continuously aerated nutrient solutions containing 8 mM KNO3, 2 mM Ca(NO3)2, 1 mM KH2PO4, 1 mM MgSO4, 32.9 µM Fe-K-EDTA, and micronutrients: 30 µM H3BO4, 5 µM MnSO4, 1 µM CuSO4, 1 µM ZnSO4, 1 µM (NH4)6Mo7O24. Plants were grown in a growth chamber with a photoperiod of 16 h-light (m−2s−2)/8h-darck at 26˚C and 20˚C, respectively. The relative humidity was maintained with 70% and 90% in the light and in dark respectively. After an initial growth period of thirty days, CdCl2 (10, 20, 50 and 100 µM) was added to the medium. After 7 days of heavy metal treatment, plants were assorted into shoots and roots. Roots were rapidly washed three times in 1 L of distilled water and samples were desiccated at 60˚C. The fresh and dry weights of each sample were determined before chemical analysis. Plant materials were kept at −80˚C before analysis.
2.2. Determination of Nitrate Content
Nitrate ions were extracted from dry matter with 0.5 N H2SO4 at room temperature for 48 h. Nitrate was calorimetrically determined on an automatic analyzer following diazotation of the nitrite obtained by reduction of nitrate on a cadmium column .
2.3. Determination of Cadmium Content
Cadmium content in various plant tissues was analyzed by digestion of dried samples with an acid mixture (HNO3/HClO4, 4/1 v/v). Cadmium concentrations were determined by atomic absorption spectrophotometry (Perkin-Elmer, Analyst 300).
2.4. Protein Content
Soluble protein content was quantified using Coomassie Brilliant blue . with bovine serum albumin as a protein standard.
2.5. Sugar Content
Total soluble sugars were determined according to .
2.6. Enzyme Assays
2.6.1. Nitrate Reductase
Plant material was homogenized with 100 mM potassium phosphate buffer (pH 7.4) containing 7.5 mM cystein, 1 mM EDTA and 1.5% (w/v) casein. The homogenate was centrifuged at 30,000 g for 15 min at 4˚C. Nitrate reductase activity (NRA) was determined according to the method described by . The extract of 0.1 ml was incubated in a reaction mixture containing 0.5 ml of 100 mM potassium phosphate buffer (pH 7.4), 0.1 ml of 0.15 mM NADH, and 0.1 ml of 100 mM KNO3 at 30˚C for 30 min. The extract was incubated with 10 mM MgCl2 (for actual NRA determination) or with 15 mM EDTA (for maximum NRA determination). The reaction was stopped by 0.2 ml of 1000 mM zinc acetate. Nitrite ions were assayed after diazotation with 1 ml of 5.8 mM sulfanilamide, 1.5 N HCl, and 1 ml of 0.8 mM N-naphthyl-ethylene-diamine-dichloride.
2.6.2. Nitrite Reductase
Enzyme extracts were prepared as described above for nitrate reductase. Nitrite reductase was assayed by the method of . The extract of 0.1 mL was incubated in a solution containing 0.4 mL of 100 mM potassium phosphate buffer (pH 7.4), 0.1 mL of 15 mM sodium nitrite, 0.2 mL of 5 mM methyl viologen, 0.2 mL of 86.2 mM sodium dithionite in a 190 mM NaHCO3. The reaction was stopped by a violent agitation on vortex. Nitrite ions were assayed as described for NRA assay.
2.6.3. Glutamine Synthetase
Samples were homogenized with grinding medium containing 25 mM Tris-HCl buffer (pH 7.6), 1 mM MgCl2, 1 mM EDTA, 14 mM β-mercaptoethanol and 1% (w/v) polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 25,000 g for 30 min at 4˚C. GS activity was determined using hydroxylamine as substrate, and the formation of γ-glutamylhydroxamate (γ-GHM) was quantified with acidified ferric chloride .
2.6.4. Glutamate Dehydrogenase
GDH extraction was performed according to the method described by . Frozen samples were homogenized in a cold mortar and pestle with 100 mM Tris-HCl (pH 7.5), 14 mM 2-mercaptoethanol, and 1% (w/v) PVP. The extract was centrifuged at 12,000 g for 15 min at 4˚C. GDH activity was determined by following the absorbance changes at 340 nm.
2.7. Western-Blot Analyses
Proteins were extracted from frozen leaf material in cold extraction buffer containing 50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM MgCl2, PVP 0.5% (w/v), 2mercaptoethanol 0.1% (v/v) and 4 mM leupeptine and separated by SDS-PAGE .qual amounts of protein (25 µg) were loaded in each track. The percentage of polyacrylamide in the running gels was 12%. Proteins were electrophoretically transferred to nitrocellulose membranes for Western blot analysis. Polypeptide detection was done using polyclonal antiserum raised against Arabidopsis GS (Masclaux-Daubresse). Antibodies were raised in rabbits against the synthetic peptide AYGEGNERRLTG by Eurogentec (Seraing, Belgium) and they detected both GS1 and GS2 isoenzymes.
2.8. Statistical Analysis
The data are presented in the figures and in the tables as the average of at least six replicates per treatment and means ± confidence limits at P = 0.05 level. Each experiment was conducted in duplicate.
3.1. Growth Response to Cadmium
Cadmium treatment induced a progressive decrease of leaf and root dry weight (DW) production (Figure 1(a)). Since low Cd treatment (10 µM), the root and leaf growth was affected. At high Cd treatment (100 µM), the reduction of root DW production was more than 70%. For the different foliar stages, the growth reduction was 36%, 64% and 76% in S1, S2 and S3, respectively. S1was the oldest foliar stage, S2 and S3 were younger than S1 (Figure 1(a)). Cd belated the emergence of the fourth foliar stage in plants treated by high Cd doses (50 and 100 µM).
Figure 1. Effects of Cd treatments (0, 10, 20, 50 100 μM) for 7 days on (a) dry weight production (DW), (b) water contents and (c) Soluble protein contents. Data are means of six replicates ± CL at 0.05 levels.
The growth inhibition of tobacco seedlings was accompanied by a decrease in water contents (Figure 1(b)). At 100 µM Cd, root water content was decreased by about 45%. The leaf hydration was significantly reduced: Old leaves (S1) were less dehydrated than S3 leaves. The decrease of S1, S2 and S3 water content was respectively about 40%, 45% and 57% (Figure 1(b)).
Soluble protein (SP) contents were gradually decreased with Cd treatment (Figure 1(c)). The highest Cd stress (100 μM) resulted in a decrease in soluble protein contents in roots (20%) and in leaves. The decrease of SP content was more important in S1 leaves (50%) and lesser in S3 and S2 leaves (25%).
3.2. , Cd and Soluble Sugar Contents
In control plants, more than 85% of total ions were accumulated in leaves (Figure 2(a)). Under increasing Cd concentration, contents were greatly decreased in both leaves and roots. At 100 µM Cd, the decrease of root content was 80% with respect to control. In leaves, the reduction of content was, respectively, 60%, 90% and 95% in S1, S2 and S3 leaves. The content of S1 leaves was the less reduced under high Cd stress (Figure 2(a)).
Cadmium ions were more accumulated in leaves than in roots. At 10 µM Cd, the leaves accumulated more than 90% of the total of cadmium absorbed by plant (Figure 2(b)). In leaves, S1 accumulated 50% of the total Cd quantity accumulated per plant. S2 and S3 accumulated respectively 30% and 3% of the total
Figure 2. Changes in contents of (a) (g/plant), (b) Cd (g/plant) and (c) soluble sugars (µmol/plant) in different part of plants under Cd treatments (0, 10, 20, 50, 100 μM) for 7 days. Data are means of six replicates ± CL at 0.05 levels
quantity of Cd accumulated in each plant (Figure 2(b)).
In control tobacco seedlings, the major quantity of soluble sugars was accumulated in leaves: 80% of the total sugar quantity accumulated per plant. Roots accumulated 6% of the whole sugar quantity accumulated per plant. Under Cd stress, the soluble sugar quantity increased in roots (+20%) and in S1 leaves (+15%). The soluble sugar quantity decreased apparently in S2 (50%) and in S3 (25%) leaves (Figure 2(c)).
3.3. Effects of CdCl2 on the Nitrogen-Assimilating Enzymes
3.3.1. Nitrate Reductase Activity
In control tobacco seedlings, Nitrate reductase (NR) activity was higher in the leaves (80%) than in the roots (Figure 3(a)). Under high Cd treatment, root NR activity decreased with 80% with respect to control. In leaves, the reduction of NR activity was respectively 56%, 60% and 40% in S1, S2 and S3. The NR activity reduction was more severe in roots. At 100 µM Cd treatment, root NR activity did not exceed 1, 8% of the whole NR activity for each plant (Figure 3(a)).
Figure 3. Effects of Cd treatments (0, 10, 20, 50, 100 µM) for 7 days on (a) Nitrate reductase activity (µmol formed. g−1FWh−1), and (b) Nitrite reductase activity (µmol reduced. g−1FWh−1) in different organs. Data are means of six replicates ± CL at 0.05 levels
3.3.2. Nitrite Reductase Activity
In leaves of control seedlings, NiR activity was more important than in roots; it represented 70% of the whole NiR activity in each plant. The NiR activity in roots was 18% of the total NiR activity per plant. The Cd addition in the culture medium caused a decrease of NiR activity in each plant organ. At 100 µM treatment, root NiR activity was more affected; it was decreased by about 35%. In leaves, the reduction of S1, S2 and S3 NiR activity were correspondingly, 11%, 15% and 20% with respect to control (Figure 3(b)). S1 NiR activity was less affected by high Cd treatment.
3.3.3. Glutamine Synthetase Activity
In the control plants, more than 80% of total GS activity was restricted in leaves. The inhibitory effect of Cd on GS activity appeared in each plant organ except for S1 GS activity. At 100 µM, GS activity decreased by 40% in roots with respect to control. Leaves from S1 GS activity increased to become 2 times more important than control. Leaves from S3 GS activity decreased by 60% with respect to the control (Figure 4(a)). Whereas, leaves from S2 GS activity was even under high Cd treatment. Western blot analysis showed that GS2 is the major isoen-
Figure 4. Effects of Cd treatments (0, 10, 20, 50, 100 µM) for 7 days on (a) GS activity (µmol γ-glutamyl hydroxamate. g−1 FW h−1) in different foliar stages and roots. Data are means of six replicates ±CL at 0.05 levels. (b)Western-Blot analysis is assayed from S1 and S3 leaves in response to 7 days of 0, 50 and 100 µM Cd grown on hydroponic medium. Total proteins are extracted from the seedlings as described in “Materials and Methods”. An aliquot of 25 µg of total protein from each sample was loaded onto each line: in Control leaves (CL), stressed leaves (SL).
zyme in tobacco leaves. Cadmium treatment reduced GS1 and GS2 protein quantity in S3 leaves. The reduction of GS protein quantity is correlated to the decrease of GS activity. This reduction may be the result of protein degradation. Cadmium could have inhibitory effect on GS expression. Cadmium stress induced GS1 and GS2 protein quantity in leaves from S1 (Figure 4(b)). So, the stimulation of GS activity corresponded to GS protein accumulation.
3.3.4. Aminating and Deaminating GDH Activities
In control plants, GDH aminating activity (NADH-GDH) was more important in roots than in leaves (Figure 5(a)). In both organs, aminating GDH activity was more important than deaminating GDH activity (NAD-GDH) (Figure 5(b)). Under Cd treatments, the aminating GDH activity was enhanced in the
Figure 5. Effects of Cd treatments (0, 10, 20, 50, 100 µM) for 7 days on (a) NADH-GDH activity (μmol NADH oxidized. g−1FWh−1) and (b) NAD-GDH activity (μmol NAD reduced. g−1FWh−1) in different foliar stages and roots. Data are means of six replicates ±CL at 0.05 levels.
roots and especially in leaves (Figure 5(a)). At 100 μM Cd, the aminating GDH activity was stimulated by 30% in the roots and more than 70% in S1 leaves, with respect to controls (Figure 5(a)). Cd stress induced a slight decrease in aminating GDH activity of young leaves (S3 and S4). In stressed seedlings, GDH deaminating activity (NAD-GDH) was stimulated in leaves. At high Cd treatment, NAD-GDH activity in young leaves (S2, S3 and S4) was at least two times higher than controls. In the roots, Cd stress had an inhibitory effect on the deaminating GDH activity which was decreased by 70% at 100 μM Cd treatment (Figure 5(b)). Cd stress caused a slight increase in deaminating GDH activity in S1 leaves (3%) with respect to control.
Under increasing Cd treatments, we observed a mean biomass decrease in leaves and in roots . This effect was mainly observed in the roots (70%), while leaves were apparently damaged only by the highest Cd concentrations. At 100 µM Cd treatment, the reduction of DW production in leaves from S1 (oldest foliar stage) was less important than the decrease in leaves from S2 and S3. As well, old leaves (S1) were, less dehydrated than younger leaves (S3) (Figure 1(b)). So, old leaves from S1 were less affected by Cd than younger leaves (S2 and S3).
Ion analysis showed that Cd stress led to an elevated decrease in contents in roots and leaves. The inhibitory effect of Cd on nitrate contents was reported in tomato seedlings .n Solanum nigrum L. .nd in rice (Huang and Xiong, 2009). The content decrease was more important in young leaves and roots. Our data showed that Cd content in leaves and roots did not follow the same trends according to Cd exposure in the same variety of tobacco (Bureley) which had been reported in Bovet et al. (2006) . Cadmium was accumulated essentially in leaves: at 100 µM treatment, leaves had 90% of Cd accumulated per plant .20]. This data confirm what had been described by .22]. At high Cd treatment, leaves from S1 accumulated more than the half of current Cd per plant, while S3 accumulated only 3%. Tobacco plant could adopt exclusive strategy of Cd ions to protect young leaves from Cd accumulation. These old leaves that accumulated the most important quantity of Cd were less affected by Cd stress compared to different foliar stages of tobacco plant (Figure 1(a); Figure 2(a) and Figure 2(b)).
The obtained decrease in nitrate contents in leaves and roots, could affect the subsequent processes involved in nitrate reduction and assimilation. In fact, regulated the NR and NiR expressions .nd activities . In tobacco seedlings, leaf NR activity was more important than root NR activity (Figure 3(a)). This elevated nitrate reduction was related to the higher leaf NR protein contents .nd a sufficient availability of light and reducing power .
After Cd exposure of tobacco seedlings, NR activity induced a significant decrease .23].hich was more pronounced in the roots than in leaves (Figure 3(a)). In leaves, NR activity reduction was more pronounced in S2 leaves. The NR activity decrease in leaves from S2 was associated to a severe decrease in nitrate content (Figure 2(b)). The nitrate content plays a direct role for NR protein production and activation. Soluble sugar content decreased visibly in leaves from S2 under Cd stress. This reduction of sugar content could cause NR activity decrease in S2 leaves. Klein et al. 2000 .eported that low sugar repressed NR gene expression that affected NR protein quantity and NR activity . Thereafter, the Cd-induced inhibition of NR activity in the leaves may result from the low nitrate availability at the enzyme reduction site. The decrease of NR activity in Cd-treated plants could also reflect an increase in the enzyme breakdown induced by toxic oxygen species generated during stress treatment. Indeed free radicals could cause the breakdown of proteins directly by oxidative reaction or indirectly by increasing proteolytic activity. The NiR activity was less affected by Cd stress than the NR activity. This higher NiR activity in the leaves and the roots, disable the toxic accumulation of nitrite ions .
With increasing Cd concentration, NiR activity was slightly decreased in the leaves and mainly in the roots (Figure 3(b)). The ammonium produced by NiR was then incorporated into an organic form primarily by the GS enzyme. The presence of Cd in the nutrient solution caused a significant decrease in GS activity in roots (Figure 4(a)). At 100 µM Cd treatment, GS activity increased in S1 leaves, to become 2 times more important than control.
GS activity induction is correlated to the stimulation of GS1 and GS2 protein accumulation (Figure 4(b)). This result was reported previously in tomato seedlings . The GS activity induction and the cytosolic GS isoforme (GS1) protein increase were probably related to the induction of GLN transcripts. While in S3 leaves, cadmium treatment reduced GS1 and GS2 protein quantity. The reduction of GS protein quantity is correlated to GS activity decrease. Cadmium stress affected the nitrogen enzyme activity by enzyme protein alterations.
At the same time as, Cd stress was found to increase the aminating GDH activity in S1 leaves and roots, even at high Cd concentrations (100 µM) (Figure 5(a)). Conformingly to the increase in S1 GS activity, aminating GDH activity was stimulated to provide GS activity with Glutamate. This increase of aminating GDH was reported by many authors . Aminating GDH activity seems to be also involved in the ammonium detoxification under stress conditions .
Cd stress caused a clear increase in deaminating GDH (NAD-GDH) activity in young leaves. Deaminating GDH activity became 2 times more important than control. This increase in deaminating GDH activity could provide young leaves with carbohydrates. While an important deaminating GDH activity decrease was noted in roots (70%). In S1 leaves, the NAD-GDH activity reduction was insignificant (3%) with respect to control (Figure 5(b)).
We noted that Cd was accumulated in a lessening gradient from basal to apical leaves. This lessening gradient of Cd accumulation was accompanied with a Cd tolerance gradient in the same direction. Thus the response difference of tobacco leaves to the cadmium could be bound either to the leaf mature or/and the contact with important Cd quantities. Although, more work is needed at the molecular level for further information towards the subcellular accumulation of Cd in young and old leaves, phytochelatins accumulation and the Cd effects on protein and gene expression of nitrogen metabolism enzymes. Although foliar Cd accumulation, roots were more affected by Cd stress. The lower sensitivity of S1 leaves to Cd could be related, at least in part, to a lesser reduction of nitrate reduction and ammonium assimilation, concomitantly with a high increase in aminating GDH activity under Cd stress and an ability of S1 leaves to accumulate this metal in non-active form. Tobacco plant could be considered as a hyperaccumulator plant used to clean up soil contaminated with cadmium. Ultimately, the large accumulation of Cd in leaves invited tobacco manufactories using leaves for cigarette production, to strictly make sure that exploited soils are not contaminated by Cd or other heavy metals. The reduction of cadmium content can reduce health hazards to smokers by selection of young leaves rather than old leaves and control of pH soil that have an effect on Cd uptake. S1 leaves are a target organ to verify an eventual soil contamination per cadmium.
CL confidence limit
Chl a chlorophyll a
Chl b chlorophyll b
DW dry weight
Fd-GOGAT ferredoxine glutamate synthase
GDH glutamate dehydrogenase
GS glutamine synthetase
NADH-GOGAT NADH glutamate synthase
NR nitrate reductase
NiR nitrite reductase
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