Abstract
Ten tomato (Lycopersicon esculentum) cultivars K-25, K-21, NTS-9, Kaveri, NBR-Uday, Swarnodya, Sarvodya, NBR-Uttam, Malti, and S-22 were soaked (i.e., shotgun approach) in various concentrations of CdCl2 (0.0, 50, 100 or 150 µM) for variable durations (0, 4, 8, or 12 h) with the aim of finding out the degree of tolerance. The data obtained at 30 days after sowing indicated that all the growth and photosynthetic characteristics were decreased significantly as the concentration of cadmium increased irrespective of soaking duration. All the cultivars differ widely in their ability to tolerate the Cd stress. The variety S-22 could not survive in the presence of any Cd concentrations, the variety Sarvodya, NBR-Uttam, and Malti experienced severe damage; however, the variety Kaveri, NBR-Uday and Swarnodya were moderately affected. Moreover the variety K-25, K-21 and NTS-9 showed the maximum resistance to cadmium concentrations.
Introduction
Literature provides extensive information about the toxicity of heavy metals on the growth and development of higher plants. Although any of the known heavy metals may be toxic to plants at an elevated level, Cd2 +, Ni2 +, Zn2 +, Cu2 + and Pb2 + cause phytotoxicity in the soil (Foy et al. Citation1978). The concentration of these metals in uncontaminated soils range from 5–50 mg/kg (Schactschabel et al. Citation1984) and in plants grown in such soils, the range is between 0.4–3 mg/kg (El-Bassam Citation1978). Of the major heavy metals Cd is an ever-increasing industrial pollutant, particularly in areas associated with smelting of Zn and heavy road traffic (Ernst Citation1980). An excess of cadmium causes a number of toxic symptoms in plants such as growth retardation (Hayat et al. Citation2007; Hasan et al. Citation2008), altered stomatal action (Barcelo et al. Citation1988), it retards the biosynthesis of chlorophyll (Singh and Tewari Citation2003), decreases the activity of various enzymes (Gouia et al. Citation2003; Hayat et al. Citation2007), alters water balance (Barcelo and Poschenrieder Citation1990), and finally reduces rate of photosynthesis (Hayat et al. Citation2007). Cadmium also interferes with the uptake, transport and use of several elements (Cu, Zn, Ni, Pb and Cr) and that of water by plants (Das et al. Citation1997). Cd can be readily taken up and accumulated in vascular plants (Prasad Citation1995). Cadmium is not an essential nutrient; most plants are sensitive to cadmium in excess of minimal concentrations. However certain plants species can grow in contaminated habitats because they have developed avoidance or tolerance strategies against heavy metals (Prasad and Hagenmeyer Citation1999). Species and cultivars display marked differences for Cd tolerance in, for example, wheat (Zhang et al. Citation2002), rice (Wu et al. Citation2004; He et al. Citation2006) and pea (Metwally et al. Citation2005). When Cd enters the food chain, it causes intricate health problems. Being more readily bioavailable, hyperaccumulation of Cd causes leaf chlorosis and inhibits chlorophyll biosynthesis (Baryla et al. Citation2001; Wang and Zhou Citation2006; Lea et al. Citation2007).
Keeping all these points in mind, the present experiment was designed with the aim to find out the degree of tolerance among 10 different varieties of Lycopersicon esculentum by soaking (i.e., shotgun approach) them in varied CdCl2 concentrations for variable duration, and to find out the most sensitive and most resistant variety by assessing photosynthetic machinery.
Material and methods
The seeds of Lycopersicon esculentum varieties K-25, K-21, NTS-9, Kaveri, NBR-Uday, Swarnodya, Sarvodya, NBR-Uttam, Malti, and S-22 were purchased from Chola Beej Bhandar, Aligarh. Healthy seeds were surface sterilized with 0.01% aqueous solution of mercuric chloride followed by repeated washing with double-distilled water and were soaked in 0.0, 50, 100 or 150 µM CdCl2 for 0, 4, 8 or 12 h. This approach is known as the shotgun method (Ashraf et al. Citation2008). These treated seeds were sown in earthen pots (6 in) filled with soil and farmyard manure (6:1). Irrigation was done by using tap water as and when required. The plant samples were collected at 30 days after sowing (DAS) to assess the parameters discussed below.
Growth characteristics
The plants were uprooted and washed under running tap water. The root and shoot portion was separated and were transferred to an oven run at 80°C. These samples were weighted after 48 h to obtain their dry mass. The leaf area was calculated on a fresh mass basis.
Chlorophyll and photosynthetic characteristics
The SPAD chlorophyll in the fresh leaf samples was measured by a Minolta chlorophyll meter (SPAD-502; Konica Minolta Sensing Inc. Japan), whereas the water use efficiency, internal CO2 concentration, stomatal conductance, transpiration rate and photosynthetic rate were measured by the LI-COR 6400 photosynthesis system (LI-COR, Lincoln, NE, USA). Gas exchange in the LI-COR 6400 is measured in an open-mode design. The atmospheric conditions during measurement were photosynthetically active radiation (PAR), 1016±6 µmolm−2 s−1, relative humidity 60±3%, atmospheric temperature 22±1°C and atmospheric CO2, 360 µmol mol−1. The ratio of atmospheric CO2 to intercellular CO2 concentration was constant.
Estimation of carbonic anhydrase (CA) activity
The activity of CA was determined following the procedure described by Dwivedi and Randhawa (Citation1974). The leaf samples were cut into small pieces and suspended in cystein hydrochloride solution. The samples were incubated at 4°C for 20 min. The pieces were blotted on the filter paper and transferred to the test tubes, containing phosphate buffer (pH 6.8) followed by the addition of alkaline bicarbonate solution and bromothymol blue indicator. The test tube was incubated at 5°C for 20 min. The reaction mixture was titrated against 0.05N HCl after addition of 0.2 ml of methyl red indicator.
Statistical analysis
The experiment was conducted according to simple randomized block design. A total of ten replicates for each treatment was taken. Treatment means were compared by analysis of variance using SPSS (SPSS, Chicago, IL, USA). Least Significance Difference (LSD) was calculated at the 5% level of probability.
Results
Root dry mass
The root dry mass exhibited a linear decrease as the soaking duration was increased from 4–12 hours (). The decrease in the root dry mass was also proportionate to the concentration (50, 100 or 150 µM) of the metal. Among the concentrations, 150 µM was the most toxic at all the treatments duration (4, 8 or 12 h). Soaking the seeds in 50 µM of Cd for 4 h had a negligible effect on the root dry mass of all the varieties, whereas soaking of seeds for 8 or 12 h duration in 50 µM of Cd significantly decreased the root dry mass. The other two concentrations (100 or 150) of Cd proved highly toxic irrespective of the duration of soaking. The varieties K-25, K-21 and NTS-9 were the least affected among all the varieties.
Table 1. Effect of pre-sowing seed soaking treatment of cadmium chloride (CdCl2) for 4, 8 or 12 h on the root dry mass (g) in 10 varieties of tomato (Lycopersicon esculentum Mill.) at 30 days after sowing.
Shoot dry mass
The data in show that the degree of toxicity varied with different varieties of tomato. Among the varieties, S-22 was too sensitive to survive any of the Cd concentrations. The varieties NBR-Uttam and Malti survived in all the Cd concentration supplied for 4 h. However, these two varieties could not germinate when the seeds were pre-treated with 150 µM or 100 µM CdCl2 for 8/12 h duration. The varieties Kaveri, NBR-Uday, Swarnodya and Sarvodya were moderately affected by Cd in 4 or 8 h. The longest duration of soaking was highly toxic for these varieties, which inhibited their shoot dry mass at 150 µM concentration. The varieties K-25, K-21 and NTS-9 were comparatively resistant to Cd treatments. The order of toxicity followed the trend 12 h > 8>4 h.
Table 2. Effect of pre-sowing seed soaking treatment of cadmium chloride (CdCl2) for 4, 8 or 12 h on the shoot dry mass (g) in 10 varieties of tomato (Lycopersicon esculentum Mill.) at 30 days after sowing.
Leaf area
The data in show that pre-sowing seed soaking caused a significant decrease in the leaf area of the resulting plants. As the concentration of the metal and the duration of soaking increased the degree of inhibition also increased. The variety Malti showed the maximum inhibition, whereas, decrease in K-25 was minimum. The varieties NBR-Uttam and Malti could not germinate when treated with 150 µM of Cd for 8 or 12 h. The variety S-22 was completely perished in response to any of the Cd concentration and the soaking duration. Cd exerted a moderate toxicity on Kaveri, NBR-Uday and Swarnodya.
Table 3. Effect of pre-sowing seed soaking treatment of cadmium chloride (CdCl2) for 4, 8 or 12 h on the leaf area (cm2) in 10 varieties of tomato (Lycopersicon esculentum Mill.) at 30 days after sowing.
SPAD chlorophyll
The plants grown from the seeds soaked in different concentrations of Cd for various durations possessed the lower SPAD value than the unstressed controls (). The highest concentration (150 µM) was the most toxic, irrespective of the duration of soaking. The varieties K-25, K-21 and NTS-9 showed a slight resistance to 150 µM of Cd. The response was closely followed by 100 µM concentration. The SPAD value in Kaveri, NBR-Uday and Swarnodya was less affected by the cadmium concentrations given for 4 or 8 h, whereas, Sarvodya, NBR-Uttam and Malti exhibited a severe loss in SPAD value, irrespective of the concentration/duration of the soaking treatment.
Table 4. Effect of pre-sowing seed soaking treatment of cadmium chloride (CdCl2) for 4, 8 or 12 h on the SPAD chlorophyll value in 10 varieties of tomato (Lycopersicon esculentum Mill.) at 30 days after sowing.
Water use efficiency (WUE) and transpiration rate
The WUE and transpiration rate in all the varieties gradually decreased as the concentrations of Cd and the treatment durations increased ( and ). K-25, K-21 and NTS-9 exhibited the minimum decrease in response to Cd, irrespective of its treatment duration. The higher concentrations (100 and 150 µM) caused a substantial decline in WUE and transpiration in all the varieties.
Table 5. Effect of pre-sowing seed soaking treatment of cadmium chloride (CdCl2) for 4, 8 or 12 h on the water use efficiency (WUE) in 10 varieties of tomato (Lycopersicon esculentum Mill.) at 30 days after sowing.
Table 6. Effect of pre-sowing seed soaking treatment of cadmium chloride (CdCl2) for 4, 8 or 12 h on the transpiration rate (mol m−2 s−1) in 10 varieties of tomato (Lycopersicon esculentum Mill.) at 30 days after sowing.
Photosynthetic parameters
The photosynthetic parameters (stomatal conductance, internal CO2 concentration and net photosynthetic rate) exhibited a similar response to the treatments (Tables ). These parameters showed a linear decrease as the concentration of the metal was increased in the pre-sowing treatment. The lowest concentration (50 µM) of the metal was the least toxic, irrespective of the duration of its application, for all the varieties. Sarvodya, NBR-Uttam and Malti were highly sensitive to the metal. The higher concentrations (100 and 150 µM) of Cd were extremely toxic for these varieties. K-25, K-21 and NTS-9 showed a significant resistance to the Cd concentrations.
Table 7. Effect of pre-sowing seed soaking treatment of cadmium chloride (CdCl2) for 4, 8 or 12 h on the stomatal conductance (mol m−2 s−1) in 10 varieties of tomato (Lycopersicon esculentum Mill.) at 30 days after sowing.
Table 8. Effect of pre-sowing seed soaking treatment of cadmium chloride (CdCl2) for 4, 8 or 12 h on the intercellular CO2 concentration (mg/l) in 10 varieties of tomato (Lycopersicon esculentum Mill.) at 30 days after sowing.
Table 9. Effect of pre-sowing seed soaking treatment of cadmium chloride (CdCl2) for 4, 8 or 12 h on the photosynthesis rate (µmCO2 m−2s−1) in 10 varieties of tomato (Lycopersicon esculentum Mill.) at 30 days after sowing.
Leaf carbonic anhydrase (CA) activity
The activity of CA varied significantly with respect to Cd concentration, soaking durations and the varieties (). The lowest concentration (50 µM) was less toxic for all the varieties. The other two concentrations (100 and 150 µM) showed the magnitude of toxicity, which varied from variety to variety. Among the varieties K-25, K-21 and NTS-9 were comparatively resistant in terms of the CA activity, whereas, Kaveri, NBR-Uday and Swarnodya were neither too resistant nor too sensitive. However, Sarvodya, NBR-Uttam and Malti were highly sensitive.
Table 10. Effect of pre-sowing seed soaking treatment of cadmium chloride (CdCl2) for 4, 8 or 12 h on the CA activity (mol CO2 kg−1 (F.M.) s−1) in 10 varieties of tomato (Lycopersicon esculentum Mill.) at 30 days after sowing.
Discussion
Cadmium is strongly phytotoxic and causes a physiological drought in plants that may result in plant death. Earlier reports showed that plants were sensitive to increased Cd levels (Chaoui et al. Citation1997), and major effects were reductions in shoot and root growth and their dry mass (Rout et al. Citation1999). Roots being the first target of Cd toxicity, exhibit considerable changes at histological and molecular levels (Hernandez and Cooke Citation1997; Suzuki Citation2005). Despite substantial varietal differences, symptoms of Cd toxicity in gradual reductions in the dry weight of roots () and shoots () as well as reducing the available leaf area () are common. The changes in plant growth attributes as a result of Cd stress were because of decreased water use efficiency (). Such effects may result from the reduced uptake of essential nutrients and toxicity of metal ions accumulated in various plant parts (Zhang et al. Citation2002; Adhikari et al. Citation2006). The extent of changes in growth attributes revealed the existence of great varietal differences for Cd tolerance. The effects of Cd damage to all the varieties were largely similar to the deficiency of essential nutrients including K, Mg, Mn and Fe (Epstein and Bloom Citation2005; Ghnaya et al. Citation2007). This is plausible in view of the fact that all these elements are either structurally or functionally involved in chlorophyll biosynthesis and its activity. Hence, loss and/or reduced biosynthesis of chlorophyll in response to Cd stress are the determinants of growth in all the varieties of tomato. All the varieties differ widely in their ability to tolerate the Cd stress. The variety S-22 could not survive in the presence of any of Cd concentrations, Sarvodya, NBR-Uttam, and Malti experience the severe damage, however, Kaveri, NBR-Uday and Swarnodya were moderately affected. Moreover, K-25, K-21 and NTS-9 showed the maximum resistance to cadmium concentrations. Such reductions are assignable to hampered rate of photosynthesis () and partitioning of photoassimilates to seed during filling because of Cd (Bindhu and Bera Citation2001; Balakhnina et al. Citation2005; Wahid et al. Citation2007). An additional reason that may simplify the cause behind this damage may be the loss of cellular turgor due to the physiological drought generated by Cd, since cell expansion depend on cell wall potential, thus developing cell walls expand less and cell size will be smaller, this reduced cell size affects the growth pattern of the whole plant and results in a reduced carbon gain due to smaller leaves which lead to reduced photosynthesis (). The observed decrease in the net photosynthesis rate under Cd stress () may be due to the inhibition in the activity of phochlorophyllide reductase (Stobart et al. Citation1985) possibly by activating the sulphydryl site of the reductase protein (Ernst Citation1980) which may lead to the decrease in SPAD value (). Decreased concentrations of chlorophyll pigments that may lead to chlorosis of leaves may be an outcome of reduced synthesis and/or enhanced oxidative degradation of these pigments by imposed oxidative stress. Inhibition of ALA dehydratase in Cd stressed plants as reported by Noriega et al. (Citation2007) could have led to decreased synthesis of chlorophyll. This enzyme catalyzes a rate limiting step of sequential pathway of haem and chlorophyll synthesis from its precursor ALA (Marschner Citation1995). A similar decrease in chlorophyll due to Cd has also been reported in Brassica juncea (Hayat et al. Citation2007) and in Cicer arietinum (Hasan et al. Citation2008). Moreover photosynthesis is also sensitive to disturbance in gas exchange through stomata. Cd stimulate stomatal closure (Sandalio et al. Citation2001), as a result, leaf transpiration rate was significantly decreased (). However, the effects of Cd on transpiration rate are complex and apparently depend on metal concentration, plant species and period of treatment (Sandalio et al. Citation2001). Cd also produced disturbance in the water balance by changing the water use efficiency () probably by inhibiting the absorption and translocation of water (Barcelo et al. Citation1988). The water movement in the plant could be affected by a reduction in the size and number of xylem vessels imposed by the metal toxicity, and also by alterations of hormone balance (Poschenrieder and Barcelo Citation1999).
Carbonic anhydrase (CA) is the enzyme that catalyzed the reversible inter-conversion of CO2 and HCO3 –. Its activity is largely determined by the photon flux density, concentration of CO2, the availability of Zn (Tiwari et al. Citation2005), and the genetic expression (Kim et al. Citation1994). The stress generated by cadmium decreases the partial pressure of CO2 in the stroma by inducing the stomatal closure (Barcelo and Poschenrieder Citation1990) and thus resulting in a loss in the activity of CA (). Similar observations have also been reported in Phaseolus vulgaris (Siedlecka et al. Citation1997) Brassica juncea (Hayat et al. Citation2007) and Cicer arietinum (Hasan et al. Citation2008) when exposed to Cd.
Conclusion
It is concluded from the present observation that shotgun approach can be used as an effective method to assess the tolerant and sensitive varieties of tomato.
Acknowledgements
The authors are thankful to the anonymous reviewers for their valuable suggestions. This work was funded by University Grants Commission [Project No 32-403/ 2006 (SR)], New Delhi, India.
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