Journal of Plant Nutrition Excess Nickel Alters

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Journal of Plant Nutrition

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Excess Nickel Alters Growth, Metabolism, and Translocation of Certain Nutrients in Potato Rajni Shuklaa; Rajeev Gopala a Department of Botany, Lucknow University, Lucknow, India

To cite this Article Shukla, Rajni and Gopal, Rajeev(2009) 'Excess Nickel Alters Growth, Metabolism, and Translocation of

Certain Nutrients in Potato', Journal of Plant Nutrition, 32: 6, 1005 — 1014 To link to this Article: DOI: 10.1080/01904160902872800 URL: http://dx.doi.org/10.1080/01904160902872800

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Journal of Plant Nutrition, 32: 1005–1014, 2009 Copyright © Taylor & Francis Group, LLC ISSN: 0190-4167 print / 1532-4087 online DOI: 10.1080/01904160902872800

Excess Nickel Alters Growth, Metabolism, and Translocation of Certain Nutrients in Potato Rajni Shukla and Rajeev Gopal

Downloaded By: [INFLIBNET India Order] At: 09:01 25 August 2010

Department of Botany, Lucknow University, Lucknow, India

ABSTRACT To elucidate the deleterious effects of excess nickel (Ni) on potato (Solanum tuberosum) cv. ‘Chandramukhi’, plants were grown in refined sand in a complete nutrient solution for 40 days. On the 41st day, excess Ni was superimposed to potato plants at 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mM for 108 days. A set of plants with a complete nutrient solution (0.0001 mM Ni) was maintained as control for the same period. After 12 days of metal supply (d52), in addition to growth depression plants at 0.5 mM Ni developed chlorosis on young leaves initiating from the base, gradually spreading downward. With increase in age chlorosis intensified and brown necrotic areas developed irregularly on the affected lamina. Later the similar symptoms appeared at lower supply of excess Ni (>0.3 mM) but the intensity was comparatively milder. Exposure of potato plants to excess Ni show retarded growth, decreased chlorophyll concentration, concentration of zinc (Zn) and iron (Fe) (except roots in both) and activities of antioxidative heme enzymes whereas increased the concentration of Ni, phosphorus (P) (except roots) and sulfur (S) (except roots) in different plant parts of potato. Keywords: potato, Ni toxicity, chlorophyll concentration, enzyme activities

INTRODUCTION Phytotoxicity of heavy metals earlier confined to some metalliferous soils have been found commonplace because of increasing anthropogenic pressure. Bioaccumulation and entry to the food chain have turned heavy metals such as copper (Cu), cobalt (Co), zinc (Zn), lead (Pb), and nickel (Ni) as major environmental pollutants. Nickel is one of the most significant pollutants discharged by mines

Received 29 October 2007; accepted 11 May 2008. Address correspondence to Dr. Rajeev Gopal, Botany Department, Lucknow University, Lucknow 226007, U.P., India. E-mail: [email protected] 1005


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R. Shukla and R. Gopal

and smelters. These effluents have inadvertently used by farmers for irrigation. Ni is unequivocally established as an essential nutrient for plant growth (Gerendas et al., 1999; Marschner, 2002). Both inhibitory (Denkhaus and Salnikow, 2002) and stimulatory (Schicker and Caspi, 1991; Rao and Sresty, 2000) effects of Ni on plant growth have been reported. The concentration of Ni in several crop plants has been determined (Govorina et al., 2003; Robinson et al., 2003). Numerous workers (Lou et al., 1991; Kovacevic et al., 1999) have demonstrated to plants associated with high levels of nickel. Recent reviews (Kabata Pendias and Pendias, 2001; Marschner, 2002) have indicated that 0.1 to 5 mg Ni kg−1 present in the available form in the soil solution, is the critical level for a number of plant species. Leaves and stem have been found to contain more nickel than fruit and seeds. However, a major portion of nickel remained in the roots, causing retarded plant growth, and elicits perturbations in cellular metabolism (Gabbrielli et al., 1999; Seregin and Kozhevnikova, 2006). Nickel exposure to plants was found to effect variably in uptake and translocation of other macro and micronutrients. Excess nickel tends to decrease iron, copper, zinc, and manganese in some plant species as reported by Taylor and Stadt (1990) and Yang et al. (1996). Palacios et al. (1998) studied the effect of nickel nutrition in tomato plants and observed decreases in the uptake of other divalents, such as magnesium (Mg+2 ), iron (Fe+2 ), manganese (Mn+2 ), Cu+2 , and Zn+2 in both roots and shoots. At rooting medium, Ni concentration in the low range may also inhibit the uptake of other trace elements such as Fe and Cu (Rahman et al., 2005). On the other hand, Agarwala et al. (1977) observed in barley plants that excess nickel did not inhibit the uptake and translocation of iron and increase its mobility from root to top. Dube et al. (2002) associated excess nickel with higher amounts of tissue phosphorus (P) and sulfur (S) in different plant parts in Citrullus. The objective of this paper was to find out the tolerance limit of potato to nickel and the changes in growth parameter and appearance of visible toxicity symptoms. Information has also been generated on the uptake and translocation of certain essential nutrients (P, S, Fe, and Zn) by the plant, grown in refined sand at variable nickel (nickel sulfate) supply.

MATERIALS AND METHODS Potato (Solanum tuberosum L.) cv. ‘Chandramukhi’ plants were grown in refined sand (Agarwala and Sharma, 1976) in polyethylene containers of 10 L capacity in a glass house at an ambient temperature (25◦ –30◦ C). Each pot had a central drainage hole covered with inverted watch glass lined with glass wool. Plants were supplied with complete nutrient solution for 40 days. The composition of the complete nutrient solution (Hewitt, 1966) was: 4 mM potassium nitrate (KNO 3 ), 4 mM calcium nitrate [Ca(NO 3 ) 2 ], 2 mM


Excess Nickel in Potato

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magnesium sulfate (MgSO 4 ), 1.5 mM sodium phosphate (NaH 2 PO 4 ), 0.1 mM sodium chloride (NaCl), 100 µM Fe-ethylenediaminetetraacetic acid (EDTA), 10 µM manganese sulfate (MnSO 4 ), 30 µM boric acid (H 3 BO 3 ), 1 µM zinc sulfate (ZnSO 4 ), 1 µM copper sulfate (CuSO 4 ), 0.2 µM sodium molybdate (Na 2 MoO 4 ), 0.1 µM nickel sulfate (NiSO 4 ), and 0.1 µM cobalt sulfate (CoSO 4 ). On d 41 the pots were separated into seven lots. One lot was allowed to grow as such and was treated as control (0.0001 mM Ni), in the other six lots, nickel was superimposed at 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mM as nickel sulfate. There were three replicates in each treatment. The nutrient solution was given to the plant daily after emergence of first leaf except on weekend. On Sundays each pot was flushed with sufficient deionized water to drain out any surface contamination and root exudates. After germination each pot had an equal number of plants. The plants were maintained in culture till maturity to obtain economic yield. Apart from visible symptoms, biomass and concentration of Ni, Fe, Zn, P, and S were determined at d 108 (68 days after metal supply) in all plant parts. At d 70 (30 days after metal supply), concentration of chlorophylls a and b were measured colorimetrically in 80% (v/v) acetone (Arnon, 1949). Activities of antioxidative stress enzymes catalase and peroxidase were assayed in crude extract of fresh leaves. The activity of catalase (E.C.1.11.1.6) was measured by the method of Euler and Josephson (1927). In a 50 mL test tube, 10 mL substrate mixture containing 0.5 mM hydrogen peroxide (H 2 O 2 ) and 0.1 mM phosphate buffer pH 7.0 was taken and stabilized at 25◦ C for 5 min. The reaction was initiated by adding 1 mL suitably diluted enzyme extract to the reaction mixture and allowed to proceed for 5 min. The reaction was stopped after 5 min. by the addition of 5 mL 2 N sulfuric acid (H 2 SO 4 ) . In corresponding blanks, H 2 SO 4 was added before the addition of enzyme extract. The reaction mixture was then titrated against 0.1 N potassium permanganate (KMnO 4 ) and the amount of H 2 O 2 reduced by enzyme was determined after blank correction. The results were expressed as µmole H 2 O 2 split per mg protein. Peroxidase (EC.1.11.17) was assayed by the modified method of Luck (1963). The assay system contains 5 mL 0.1 M phosphate buffer pH 6.0, 1 mL 0.01% (v/v) H 2 O 2 , and 1 mL 0.5% p-phenylene diamine were stabilized at 25◦ C. The reaction was carried out for 5 min. by adding suitably diluted enzyme extract and was stopped by adding 2 mL of 5 N H 2 SO 4 . The blanks were run simultaneously in which H 2 SO 4 was added before the addition of enzyme extract. The oxidation of p-phenylene diamine was measured as change of color due to enzyme activity. The plant samples were harvested at d 108 for biomass, thoroughly washed with running tap water and rinsed twice with distilled water. The plant samples were blotted gently to wipe off the absorbed water and separated into different plant parts—root, stem, leaves, and inflorescence/pod. The biomass was determined after drying these fresh samples in an electric oven at 70◦ C for 48 h. This oven-dried material was digested in a nitric and perchloric acids

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(10:1) according to the method of Piper (1942). The diluted samples were analyzed for phosphorus colorimetrically as phosphomolybdic blue complex (Wallace, 1951), sulfur turbidimetrically (Chesnin and Yien, 1951) whereas the concentration of Ni, Fe, and Zn were estimated by atomic absorption spectrophotometry (AAS 4141). Soluble protein (Lowry et al., 1951) was also estimated in the enzyme extract to express specific activity of enzymes. The entire data presented in the Table and Figures were analyzed statistically and tested for Least Significant Difference at P = 0.05 (Panse and Sukhatme, 1985).

RESULTS AND DISCUSSION At d 52 (12 days after metal supply), the potato plants at 0.5 mM Ni were depressed in growth. In due course, the growth of plants became depressed at other levels of Ni, i.e., at 0.4 and 0.3 mM Ni. The branches were short, leaves were less in number and small with reduced lamina. At d 66 (26 days after metal supply), the young leaves of the affected plants developed diffused chlorosis initiating from the base. Gradually chlorosis intensified and brown necrotic areas developed irregularly on the affected (chlorotic) lamina. These leaves turned bleached with necrosis and as a result leaves appeared distorted with broken shrunken edges. Some of them turned inward. Almost all young leaves were affected. These symptoms were less acute at 0.2 and 0.1 mM Ni supply. The depressed growth of plants at higher levels of Ni (>0.3 mM) coincides with the results on green gram (Pandey and Pathak, 2006) and wheat (Dube et al., 2002). Depression in growth with stunted shoot and root observed in potato was a common feature of nickel excess and has been earlier reported by many workers (Palacios et al., 1998; Nagy and Proctor, 2001).The affected young leaves showed severe iron deficiency type symptoms as has earlier seen in barley (Agarwala et al., 1977). At d 108 compared to the biomass of potato at control level the dry weight of potato decreased with an increase in nickel supply (Table 1). The depression in biomass was very marked (Lou et al., 1991; Baccouch et al., 2001; Kukier and Chaney, 2004) might be due to accumulation of nickel in different part and reduction in that of carbohydrate and protein contents in leaves (Marschner, 2002). The weight of tubers were also affected and decreased drastically as a result of excess nickel (Table 1). The concentration of nickel in various parts of potato at control level was very low and compared to this, its concentration increased in all parts of potato with an increase in nickel supply (Figure 1). Compared to this, concentration of nickel in various parts at variable nickel was lowest in tubers. Nickel mobility from root to top is: root > leaves > stem > tubers. The accumulation of nickel in roots at all levels of nickel was quite high and resemble with the results


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Table 1 Effect of excess nickel on dry matter, tuber dry weight at d108 (68 days after metal supply) and chlorophyll concentration of potato at d 70 (30 days after metal supply) mM Ni supply

0.0001

0.05

0.1

0.2

0.3

0.4

0.5

LSD (P = 0.05)

Dry matter: g/plant 20.3

15.6

14.5

9.5

7.6

7.1

6.4

3.2

2.8

1.04

0.265

0.147

0.13

0.082

0.028

0.02

0.347

0.175

0.11


Tuber dry weight: g/plant 10.8

6.9

5.8

5.6

3.4

3.1

Chlorophyll: mg/g fresh weight a: 1.132

1.016

0.834

0.815

0.451

0.370

0.333

0.306

1.584

1.386

1.168

1.121

0.580 b: 0.220 Total: 0.800

observed by other workers (Kovacevic et al., 1999; Baccouch et al., 2001; Syshchykov and Hryshko, 2003; Licina et al., 2007). Excess nickel significantly restricted the translocation of iron from root to shoot. At d 108 (68 days after metal supply), compared to the values of iron obtained at various parts of potato, its concentration decreased in all parts except roots with an increase in nickel supply (Figure 1). In roots there was an increase in iron concentration with an increase in nickel supply. This might suggest that nickel like other heavy metal can displace several ions from physiologically important binding sites and can thus decrease the uptake and mode of other heavy metal including iron. Interference of heavy metal like nickel with iron in plant metabolism is known to induce iron deficiency (van Assche and Clijsters, 1990; Yang et al., 1996; Palacios et al., 1998). The concentration of phosphorus increased in all parts (except roots) with an increase in nickel supply (Table 2). The increased concentration of phosphorus in present study is not in consonance with the results on Helianthus and Hyptis reported for nickel toxicity (Vasanthapillay et al., 1987). It might be suggested that excess nickel disturbed the phosphorus metabolism by creating a hindrance in the mobility of these nutrient to those organ from where they are needed. The disturbance in phosphorus and iron contents due to excess nickel affect the carbohydrate and nitrogen metabolism and this might be responsible for depressed growth and lowered biomass in excess nickel.

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R. Shukla and R. Gopal Iron concentration

Nickel concentration

Leaves Stem Root Tube r

Leaves Stem Root Tube r 300

µg g dry weight

200

-1

400

200

0 0.0001

100

0 0.0001 0.001

0.01

0.1

0.001

0.01

0.1

1

1

Peroxidas e

Catalase

160

6

120

Change in OD

m moles H 2O2 decomposed


-1

µg g dry weigaht

600

80

4

2

40

0 0.0001

0.001

0.01

mM Ni

0.1

1

0 0.0001

0.001

0.01

0.1

1

mM Ni

Figure 1. Excess nickel and concentration of nickel and iron in different plant parts at d108 (68 days after metal supply) and specific activities of heme enzymes catalase and peroxidase at d 70 (30 days after metal supply). Vertical lines represent LSD (P = 0.05).

The increased concentration of sulfur in leaves of potato (Table 2) might be possible that excess nickel blocked the pathway of sulfur content. This altered sulfur content in turn might be responsible for less available sulfur for different biomolecule to be utilized in various metabolic pathways as has been suggested for excess nickel (Gopal et al., 2001). The zinc concentration decreased in all parts (except roots) of potato with an increase in nickel supply (Table 2). The increased zinc concentration in roots with an increase in nickel supply might be suggested that excess nickel check the mobility of zinc to move the upper as well as lower part of the plant like tuber. The results are not in consonance with the result of Piccini and Malavolta (1992) in bean. Robinson et al., (2003) reported a decrease in zinc concentration in excess nickel condition.


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Table 2 Effect of excess nickel on sulphur, phosphorus and zinc concentration of potato in different plant parts at d108 (68 days after metal supply) mM Ni supply

Plant parts 0.0001

0.05

0.1

0.2

0.3

0.4

0.5

LSD (P = 0.05)

0.97 0.52 0.38 0.53

0.22 0.06 0.11 0.18

0.57 0.42 0.43 0.57

0.06 0.11 0.09 0.12


Sulfur concentration: % dry matter Leaves Stem Root Tuber

0.29 0.27 0.64 0.28

0.23 0.36 0.53 0.30

0.25 0.35 0.58 0.38

0.43 0.44 0.40 0.36

0.57 0.42 0.63 0.48

0.86 0.54 0.54 0.64

Phosphorus concentration: % dry matter Leaves Stem Root Tuber

0.43 0.36 0.59 0.16

0.41 0.36 0.58 0.18

0.49 0.39 0.51 0.28

0.56 0.41 0.54 0.22

0.54 0.43 0.51 0.23

0.55 0.42 0.46 0.45

Zinc concentration: µg g−1 dry matter Leaves Stem Root Tuber

36.7 34.1 26.4 45.2

34.7 26.7 24.1 39.7

24.2 23.8 30.9 33.6

27.3 24.1 44.2 34.2

25.1 18.7 41.2 36.2

21.8 22.3 48.2 29.8

23.6 19.8 52.6 37.2

1.09 0.98 2.67 1.13

The concentration of chlorophyll a and b decreased (Table 1) in potato leaves is similar to several other reports (Molas, 2002; Gajewska and Sklodowska, 2007). The decrease in chlorophyll content might be due to low availability of iron for incorporation in protochlorophyllide, one of the intermediate in chlorophyll synthesis. However nickel is known to inactivate photosystem (PSII) activity without affecting PS-mediated electron flow (Molas, 2002). It has been suggested that uptake of toxic amounts of nickel by plant lead to quantitative changes in the structure of photosynthetic apparatus of plants (K¨upper and Kroneck, 2007). The decrease in photosynthesis is also responsible for reduced biomass of crop plants in such condition. The decrease in CAT and POX activity (Figure 1) in turn increase the H 2 O 2 concentration creating oxidative stress enhancing the inactivation of CAT preventing synthesis of new enzyme (Dat et al., 1998). The decrease activity of CAT and POX might be due to less iron availability for incorporation into the protein moiety of the enzyme (van Assche and Clijsters, 1990). The low activity of POX in excess nickel treated plant might be result in to peroxidative damages of the thylakoid membrane.

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ACKNOWLEDGMENT The authors are grateful to Prof. C. Chatterjee for valuable suggestions during the course of experiment.


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