Introduction

Environmental factors, such as heat, cold, drought, salinity and heavy metals result in a massive loss of crop yield all over the world. Although metals are required as structural and catalytic components of enzymatic proteins involved in various physiological processes, they can still be toxic to a plant if present at supraoptimal concentrations (Clemens 2001). Zinc is typically the second most abundant transition metal in organisms after iron (Fe). Mean soil Zn concentrations of 50 and 66 μg total Zn g−1 soil are typical for mineral and organic soils, respectively, with most agricultural soils containing 10–300 μg Zn g−1 (Alloway 1995; Barber 1995). The levels of Zn in the plant material are low and generally in the range of 10–100 mg kg−1 of dry matter. Large parts of agricultural soil are contaminated with zinc by natural and anthropogenic activities including mining and industrial processes and agricultural practices, such as use of fertilizers containing various heavy metals (Staker and Cummings 1941; Lee and Page 1967; Ross 1994; Singla-Pareek et al. 2006; Dudka et al. 1996). The pollution of soil by zinc has been a major environmental concern (Zarcinas et al. 2004). Among heavy metals, zinc is an important element for both plants and animals. It plays an important role in several plant metabolic processes and it activates enzymes and is involved in protein synthesis and in carbohydrate, nucleic acid and lipid metabolism. It is the only metal represented in all six enzyme classes (oxidoreductases, transferases, hydrolases, lyases, isomerases, ligases) (Broadley et al. 2007). In catalytic sites, Zn is directly involved in the catalytic function of the enzyme (e.g. carbonic anhydrases); histidine (His) is the primary amino acid and Zn2+ is complexed with water and any three S, N or O donors. In co-catalytic sites, Zn2+ can be used for catalytic, regulatory and structural functions (e.g. superoxide dismutases (SODs), purple acid phosphatases, metallo-β-lactamases). It forms complexes with DNA and RNA and affects the stability of these compounds (Collins 1981; Pahlsson 1989). When heavy metals accumulated in excess in plant tissues, these components cause alterations in various physiological processes, such as transpiration, photosynthesis and photosynthetic electron transport, biosynthesis of chlorophyll as well as cell membrane integrity (Richardson et al. 1993). In particular, zinc excess has been reported to have a negative effect on mineral nutrition and enzyme activities related to metabolism of plants (Van Assche and Clijsters 1986; Chaoui et al. 1997). To counteract this metabolic dysfunction caused by heavy metal stress, higher plants employ defense strategies. Generally, plant cells develop a mechanism by which the metal ion, entering the cytosol of the cell, is immediately complexed and inactivated. This process is mediated by phytochelatins (Zenk 1996). For their protection, plant cells are also equipped with oxygen radical detoxifying enzymes, such as catalase, peroxidase, ascorbate peroxidase (APX) and SOD (Asada 1987; Foyer and Harbinson 1994). The key role of SOD in the protection against harmful oxidative reaction resulting from metal stress has been reported previously (Monk et al. 1989). Cultivation of sugarcane adjacent to industries and metal polluted fills and with application of municipal wastes and indiscriminate use of phosphatic fertilizers and pesticides may enhance the problem of zinc toxicity in sugarcane crop. Reports are available on the effect of Zn deficiency to excess on sugarcane plant (Chatterjee et al. 1998; Bowen 1981). The objective of present study was to investigate the effects of high concentrations of Zn in growing medium on growth and metabolism of sugarcane (Saccharum spp.). The aim of study was to observe the instant effect of Zn stress on root growth and mitotic efficiency using very high doses of Zn (65.0 and 130 mg l−1 Zn) and its further impact on shoot growth, lipid peroxidation, oxidative stress and nutrient transport in different parts of sugarcane plant.

Materials and methods

Single bud setts of sugarcane (Saccharum spp.) cultivar CoLk 8102 were planted in 10 L polyethylene pots filled with refined sand at three levels of zinc viz., 0.065 (control), 65.0 and 130 mg l−1 Zn (excess) as zinc sulfate, respectively. Graded level of zinc was supplied as zinc sulfate as described earlier (Bonnet et al. 2000; Sagardoy et al. 2009). Initially, five setts were planted in each pot. After root sampling for cytological analysis, three plants were maintained in each pot. These pots were kept under net house conditions at natural photoperiod of 12–13 h and ambient temperature (day temperature 28–31°C; night temperature 15.6–19.7°C).

The composition and methods of preparing complete nutrient solution have been described earlier (Agarwala et al. 1985). Nutrient solution was supplied daily except on Sundays when each pot was flushed with pure water to remove deleterious substances from the rooting medium. There were three replications in each treatment.

For cytological analysis, 1–1.5 cm long root tips from germinating setts were fixed directly in Carnoy’s 6:3:1 fluid (6 alcohol:3 chloroform:1 glacial acetic acid) as well as after pretreatment with 1:1 v/v aqueous solution of saturated para-dichlorobenzene and 0.002 M 8-hydroxyquinoline for 3–3.5 h (Srivastava 1995). The fixed root was transferred in 70% alcohol and stored at low temperature (10–12°C). The root tips were hydrolyzed in 1 N HCl for 30 min at 60°C, washed thoroughly and stained in 2% aceto-orcein for 5–6 h. The stained root tips were squashed on a glass slide in a drop of 45% glacial acetic acid. Well spread plates from at least 20 meristems per replication were scored for each treatment for various types of cytological effects. Mitotic index (MI) was calculated using the following formula:

$$ {\text{MI}} = \left\{ {A/\left( {A + B} \right)} \right\} \times 100 $$

where A is the number of dividing cells (metaphase and anaphase) and B the number of non-dividing cells.

Visible symptoms of zinc toxicity were observed at 30 days after planting (DAP). To determine growth parameters, viz. leaf area, length, plant height, root number and length, fresh weight, dry weight and Ca, Fe, Mn, Cu and Zn content in different plant parts, sugarcane plants were harvested and sampled. All plant samples were thoroughly washed with tap water, rinsed with distilled water and oven-dried to constant weight at 85°C as described by Chatterjee et al. (1998). Dried plant material was digested in HNO3: HClO4 (10:1) mixture. Fe, Mn, Cu and Zn contents were estimated in clear digest by atomic absorption spectrophotometer and expressed as μg g−1 dry weight. Phosphorus was determined colorimetrically in clear digest by method of Fiske and Subbarow (1925).

Fresh last transverse mark leaves were used for various biochemical estimations. Chlorophyll a, b and carotenoids contents were determined in fresh leaves by the method of Arnon (1949). To measure hydrogen peroxide (H2O2) content, finely chopped fresh leaves were homogenized in 50 mM phosphate buffer solution (pH 6.8). The homogenate was centrifuged at 6,000g for 25 min and 2 ml supernatant was mixed with 1 ml of 0.1% titanium chloride (Aldrich) in 20% (v/v) H2SO4 (sulfuric acid) and the mixture was centrifuged at 6,000g for 5 min. The intensity of yellow color of the supernatant was measured at 410 nm. The amount of H2O2 was calculated from the extinction coefficient, 0.28 µM−1 cm−1 (Lin and Kao 2001).

To determine the malondialdehyde (MDA) content, finely chopped fresh leaves were homogenized in 5% trichloroacetic acid solution. The homogenate was centrifuged at 15,000g for 10 min and 2 ml supernatant was added to 2 ml 0.5% thiobarbituric acid (TBA) in 20% TCA. The mixture was incubated at 90°C for 20 min. The samples were centrifuged at 10,000g for 5 min, and the absorbance of the supernatant was read at 532 nm and corrected for non-specific turbidity by subtracting the value at 600 nm. The amount of MDA–TBA complex (red pigment) was calculated using the extinction coefficient 155 mM−1 cm−1 (Cakmak and Horst 1991).

For enzyme assay, fresh leaf samples were ground in a mortar with liquid nitrogen and extracted (2 ml 0.2 g FW) in 50 mM sodium phosphate buffer, pH 7.0 containing 2 mM EDTA and 5 mM β-ME. The homogenate was centrifuged at 10,000g for 10 min at 4°C. The supernatant was dispensed into aliquots for further analyses. The SOD activity was assayed in reaction mixture containing 50 mM phosphate buffer (pH 7.8), 50 mM Na2CO3, 75 µM nitroblue tetrazolium (NBT), 13 mM methionine, and 2 µM riboflavin in a 3.0 ml volume. The reduction of NBT proceeded by placing the tubes under 500 μM photons m−2 s−1 of white light. The reaction was terminated after 8 min by removing the tubes from the light source. Enzyme activity was measured by monitoring the inhibition of NBT photo-reduction at 560 nm. One unit of SOD activity was defined as the amount of the enzyme that inhibited the reduction in NBT by 50%. Specific activity has been defined as units per mg protein (Beauchamp and Fridovich 1971). Catalase activity was determined spectrophotometrically by following the decline in A 240 as H2O2 (ε = 39.4 mM−1 cm−1) was consumed (Aebi 1984). The 3 ml reaction mixture contained 50 mM sodium phosphate buffer, pH 7.0, 10 mM H2O2 and enzyme extract. For peroxidase assay, the reaction mixture containing 5 ml 0.1 M phosphate buffer, pH 6.0, 1 ml 0.01% H2O2, 1 ml 0.5% p-phenylenediamine and leaf extract (0.1 ml) was incubated for 5 min at 25°C (Luck 1963). The color developed was measured at 485 nm, and the peroxidase activity was expressed as change in OD mg−1 protein. Soluble protein was determined in extracts prepared for enzyme assays according to Lowry et al. (1951).

Statistical analysis

Treatment means were separated at P = 0.05 by the critical difference (CD) as described by Panse and Sukhatme (1985).

Results

Plants supplied at higher doses of Zn (65 and 130 mg l−1 Zn) exhibited growth depression and dark green leaves; the effects were more marked at 130 mg l−1 Zn supply. Root number and length decreased with an increase in Zn supply; decrease was maximum at 130 mg l−1 Zn supply. Reduction in leaf attributes viz., leaf area, leaf length, width and perimeter and plant height occurred due to high Zn levels (Table 1). Fresh and dry weight of different plant parts reduced due to high Zn supply; reduction was found in all plant parts (Table 1). The highest reduction was observed in leaf weight and lowest in roots.

Table 1 Effect of Zn on growth attributes of sugarcane plants
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The mean mitotic index of sugarcane genotype showed a reduction in 40.86 and 88.22% in 65.0 and 130 mg l−1 Zn treated setts when compared with control (Table 2). The decline of mitotic index in response to high dosages of zinc indicated the inhibitory effect of excess zinc on mitotic cell division of somatic cells. Analysis of different stages of cell division affected by high doses of Zn (130 mg l−1) revealed chromosome aberrations in 66.66% cells at metaphase and 57.77% cells at anaphase stages when compared with 3.38 and 3.63% aberrant cells at both the stages in control treatment (0.065 mg l−1 Zn). The presence versus absence of chromosomal aberrations, e.g. chromosome clumping, stickiness, cytomixis, grouping, and lagging etc. and their extent was recorded in all treatments (Table 3).

Table 2 Effect of Zn on mitotic efficiency of root tip cells of sugarcane plants
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Table 3 Effect of Zn on somatic chromosomal aberrations at metaphase and anaphase in root tips of sugarcane plants
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In the present study, the addition of zinc in the culture medium led to a decrease in the concentration of Fe, copper, and phosphorus in the leaves (Fig. 1; Table 4). High levels of Zn decreased total phosphorus in leaves (20–28%) and increased it in root tissues (31–50%) (Table 4). Zn concentration increased in different plant parts with an increase in Zn supply; its concentration was the highest in roots (277 µg g−1 dry weight), and the lowest in leaves (60.9 µg g−1 dry weight) which was slightly higher than control plants (Fig. 1). High levels of Zn in the nutrient medium depressed the concentration of Fe; depression was found highest in root tissues. When compared with control, Cu content decreased in all plant parts except in stalk due to higher levels of Zn in the growing medium. At higher level, copper concentration in stalk tissues increased from 15.9 μg g−1 dry weight (0.065 mg l−1 Zn) to 31.2 μg g−1 dry weight (130 mg l−1 Zn). Although Mn concentration increased in roots with an increase in zinc supply and it decreased slightly in leaves and stalk due to high Zn supply (Fig. 1). When comparing the nutrient contents on whole plant basis, zinc and Mn contents increased, while Fe and Cu decreased due to high zinc in the growing medium. In present study, when plants were submitted to higher levels of Zn, increase in chlorophyll and carotenoids contents was observed (Table 5).

Fig. 1
figure 1

Effect of Zn on nutrient accumulation in sugarcane plants

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Table 4 Effect of Zn on total phosphorus content in sugarcane
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Table 5 Effect of Zn on chlorophyll and carotenoids contents in sugarcane leaves
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In the present study, induction of oxidative stress by zinc toxicity was studied by measuring H2O2 content. The H2O2 level was found to be increased (37% at 130 mg l−1 Zn) upon exposure to higher levels, thereby documenting that plants experience oxidative stress when exposed to higher levels of zinc (Table 6). Further, to analyze the damage caused by enhanced levels of H2O2, lipid peroxidation in terms of production of MDA was measured. MDA levels were found to increase by 43% when exposed to 130 mg l−1 zinc, whereas its level increased by 14% at 65 mg l−1 zinc supply (Table 6).

Table 6 Effect of Zn on H2O2 and MDA contents and activity of antioxidative enzymes in sugarcane leaves
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Three enzymes of the cellular antioxidative system involved in H2O2 metabolism were studied: SOD, which catalyses the disproportionation of O2 − radicals into H2O2 and molecular oxygen, catalase and peroxidase, enzymes that scavenge H2O2. Total SOD activity was measured in leaves of plants submitted to different levels of Zn. It was observed that activity of this enzyme was enhanced with increasing concentrations of the heavy metal; 130.0 mg l−1 Zn showed the highest activity (Table 6). By application of 130 mg l−1 Zn in the culture medium, the leaf catalase and peroxidase activity increased by more than 50% of the initial activity. At other concentration (65 mg l−1 Zn), the increase was less evident (Table 6).

Discussion

In the present study, relatively higher amounts of zinc than those reported earlier related to zinc toxicity (Chatterjee et al. 1998) have been used to impose heavy metal stress. It is observed that a very high concentration of zinc is required to cause immediate stress to the roots and leaves of sugarcane plant. To study mitotic efficiency of root tips, nutrient solution with graded doses of zinc was supplied immediately after planting. Sugarcane plants were watered daily with nutrient solution except Sunday to observe phytotoxicity (Chatterjee et al. 1998). At very high concentration of Zn (130 mg l−1), the growth of sugarcane plants reduced as compared to the control and other level of zinc supply (65 mg l−1). Similar to sugarcane, other plants showed stunted growth and reduced plant height under high zinc conditions (Singla-Pareek et al. 2006; Boawn and Rasmussen 1971; Chaney 1993; Sagardoy et al. 2009). Excess Zn causes changes in root growth and morphology in Datura (Vaillant et al. 2005). In radish, higher content of leaf Zn (from 36 to 1,013 mg kg−1 DW) is associated with a 50% yield reduction (Davies 1993).

A decrease in the mitotic index indicates that excess zinc interferes in the normal sequences of mitosis and such a reduction in mitotic activity could be due to the inhibition of DNA synthesis (Beu et al. 1976). This, in turn affected the early growth of settlings. The wide spectrum of cytotoxic effects induced by different dosages of Zn included spindle inhibition and concomitant multipolarity, chromosome stickiness resulting in chromosome clumping, laggards due to delayed anaphases, c-mitotis, and bi/multi nucleate cells and such aberrations were more pronounced at 130 mg l−1dose of Zn; such chromosome irregulations can affect the vigor, fertility, yield, or competitive ability of the exposed plants (Beu et al. 1976). Many cytological studies have been carried out to detect the harmful effects of various heavy metals on sugarcane and other plants (Jain et al. 2000; Nandi 1985; Lerda 1992). The inhibition of spindle formation has been shown to lead to severe abnormalities, such as stickiness, unequal distribution, multipolar anaphase, chromosomal bridges, and laggards in plants (Amer and Ali 1968; Badr 1983).

Reduction in biomass production is general response of higher plants to heavy metal toxicity (Ouariti et al. 1997). Inhibition of both cell elongation and division by heavy metals could explain the decline in biomass production (Arduini et al. 1994; Hewitt 1983). In addition, it has been shown that growth of the upper plant parts is more sensitive to heavy metals, despite their low metal content compared with roots, with the hypothesis that roots could play an important role in the retention of metals by preventing an excess of toxic accumulation in the shoots. In the same way, roots of higher plants were considered as a barrier against heavy metal translocation to the top parts (Wallace and Romney 1977), reflecting a potential tolerance mechanism operating in the root cell.

Similar to sugarcane, highest zinc content in roots was reported in Arabidopsis (Verret et al. 2004). Importantly, in this study, the leaves remained practically free from excess zinc contents. Excess Zn is known to interfere with Fe, P, Mg and Mn uptake (Carroll and Loneragan 1968; Boawn and Rasmussen 1971; Foy et al. 1978; Chaney 1993; Bowen 1981; Hewitt 1983; Broadley et al. 2007). Excess zinc may cause iron deficiency in some plants (Bonnet et al. 2000; Hewitt 1983). Changes in nutrient contents in different plant parts of sugarcane plants under excess Zn conditions indicate poor root growth due to zinc accumulation in root tissues (Broadley et al. 2007).

Higher contents of photosynthetic pigments in leaf tissues of sugarcane at excess zinc levels may be caused probably by lower fresh mass of these plant leaves and higher amounts of pigments per one unit of mass (Hewitt 1983). However, Sagardoy et al. (2009) observed reduced chlorophyll and carotenoids concentrations in sugar beet leaves in response to excess zinc, when the pigments content were expressed per leaf area. In addition to this, sugar beet leaves showed very high zinc concentration (230–260 µg g−1 dry weight) when compared with sugarcane leaves (60.9 µg g−1 dry weight). In most cases, excess Zn generates reactive oxygen species and displaces other metals from active sites in proteins and induces chlorosis in young leaves (Marschner 1995). Other common zinc toxicity effects, include decrease in water and changes in the P and Mg concentrations in plant tissues (Marschner 1995). In the present study, reduced water content of leaf tissues may be resulted in higher chlorophyll and carotenoids contents per unit weight in high zinc treatment.

Several biotic and abiotic factors impose oxidative stress onto the living system has been observed previously (Weckx and Clijsters 1997). MDA is one of low molecular weight end products formed via decomposition of certain primary and secondary lipid peroxidation products; MDA enhancement is lipid peroxidation and oxidative stress indicator. In the present study, sugarcane plants undergo lipid peroxidation, thereby, affecting membrane integrity of leaves under high level of zinc. Toxic oxygen species, generated by the presence of heavy metal in leaves were evident by the increased lipid peroxidation in bean seedlings (Chaoui et al. 1997; Somashekaraiah et al., 1992), sunflower (Gallego et al. 1996) and maize and pea plants (Lozano-Rodriguez et al. 1997). Therefore, phytotoxicity of zinc in sugarcane is established by enhancement of free radical and lipid peroxidation.

Apparently, an increase in the capacity of distinct antioxidant enzymes in sugarcane seems to be a rather general response to phytotoxic doses of several heavy metals (Van Assche and Clijsters 1986; Jain et al. 2008). The main reason of enzyme activity increase was oxidative stress induced by heavy metals (Saffar et al. 2009; Van Assche and Clijsters 1986).

The results indicated reduction in plant growth, leaf expansion, root growth, mitotic efficiency, enhanced chromosomal aberrations and reduced concentration of Fe, Cu and P in sugarcane due to excess Zn in the growing medium. The presence of chromosomal aberrations clearly indicates towards the cytotoxic effects of high doses of zinc. A probable reason might be the disturbance in the synthesis of proteins and nucleic acids resulting in a change in nucleoprotein configuration of chromosomes, which in turn affects the growth attributes of sugarcane. Higher H2O2, MDA contents and increased activity of SOD, catalase and peroxidase enzymes in sugarcane leaves indicating oxidative damage by zinc toxicity.