Indian Journal of Experimental Biology Vol. 38, August 2000, pp. 819-823

Influence of cadmium on growth and development of Vicia faba Linn.

Neelu, Manoj Kumar, Manju Tomar & A K Bhatnagar*

Environmental Biology Laboratory, Department of Botany; University of Delhi, Delhi 1 JO 007, India

Received 5 August 1999; revised 24 January 2000

Influence of cadmium (Cd) on growth and development of broad bean (V. faba) was assessed in pot cultures with cadmium iodide (CdI2) in different concentrations ranging from 15 to 500 mg per kg of soil. There was a decline in plant height and total dry weight. Root size decreased most significantly with a corresponding reduction in the frequency of root nodules. Total soluble protein in leaf, stem and root suffered a pronounced loss with increasing concentration of cadmium. Chlorophyll a was the most sensitive pigment followed by chlorophyll band carotenoids. Nitrate reductase activity too was adversely affected. Cadmium contamination induced abnormalities in stomata and trichomes.

Cadmium is regarded as one of the most toxic elements in the environment. It is widely distributed on earth, occurring as sulphide along with zinc, lead and copper ores. Soil around mines, refineries and thermal power plants, and in fields receiving discharge from a variety of industries, often shows enhanced levels of cadmium. The metal accumulates in soil, from where it is absorbed by roots of plants. It enters into the food chain and becomes toxic to various living components of the ecosystem. Cadmium is shown to cause degradation of the chloroplast inner membrane structure, affecting the rate of photosynthesis I. It also inhibits the rate of transpiration, protein synth~sis, stomatal functions2

and activities of some important enzymes3. Plant root

growth and pollen germination are adversely affected. In the present work, the impact of enhanced

cadmium in soil on growth parameters of broad bean, Viciafaba (Fabaceae) has been assessed. The plant is grown as fodder and vegetable in northern India.

Materials and Methods Seeds of broad bean, Vicia faba cv. Pusa Sumeet

were obtained from Agronomy Division, Indian Agricultural Research Institute, New Delhi and grown in pots, each with four kg of air dried soil thoroughly mixed with cadmium iodide (CdI2). The concentrations used were 15, 30, 60, 120, 250, and 500 mg CdI2 per kg of soil. Pots having soil without Cdl2 served as controls. In each pot 8 seeds were sown and after thinning 5 seedlings were retained. To study the impact of cadmium, plant height, root length and shoot length were measured with a scale in

*Correspondent author

mature, 65 days old plants. Number of root nodules per plant were counted in untreated and treated plants. For estimation of dry weight, the material was kept in oven at 80°C for 72 hr. The density of chlorophyll a, chlorophyll band carotenoids was calculated after 68 days of treatment by the formulae given by Arnon4

,5.

Soluble protein in the crude extracts of root, stem and leaf of 70 days old plants was precipitated with trichloro acetic acid (TCA) and estimated by treating aliquot with Bradford's reagent6

• Nitrate reductase activity was studied7 in root, stem and leaf of 72 days old plants. For the study of leaf epidermal traits, epidermis was separated from other leaf tissues with the help of HN03.

Results and Discussion With increase in concentration of cadmium in soil,

the growth of plants suffered correspondingly more severe decline. Plant height, dry weight, seed yield and root growth showed significant reduction. The inhibitory effect was concentration dependent (Table I). The decline in plant height was 10% at 15 mg cadmium and 58% at 250 mg cadmium in soil. There was a corresponding reduction in the seed yield and maximum reduction was 40% at 250 mg cadmium (Fig. 1). Root, stem and leaves exhibited differential effect. Maximum reduction in dry weight was observed in roots (68%) at the highest concentration of treatment (250 mg), in stems the loss was 50% and in leaves 59% (Table I) .

In control conditions, root was healthy with optimum length and weight, profuse branching, numerous root hairs and a large number of nodules per plant. However, in cadmium treated plants, roots were smaller in size with insignificant branching,

820 INDIAN J EXP BIOL, AUGUST 2000

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scanty root hairs and reduced number of root nodules. Among the plant parts, roots were most severely affected due to their direct interaction with soil particles containing cadmium, or because of the tendency of cadmium to accumulate in higher amounts in roots8

. A similar reduction in root growth has been observed in wheat9

• In broad bean at 250 mg cadmium level, 87% decrease in number of root nodule per plant was observed after 65 days of treatment. This decrease may be attributed to several factors involved in nitrogen fixation. Even low concentration of cadmium is found to drastically reduce the number of colonies of rhizobium strains and number of root hairs. Hence, the effective interaction sites between plant root hairs and rhizobium are reduced. Cadmium not only affects bacterial activity but also inhibits the activity of various enzymes involved in nitrogen fixation. The activity of enzymes such as nitrate reductase and nitrogenase was adversely affected. Thus, the reduction in nodulation may be a combined result of all these factors 10. Roots are dependent on translocation of carbohydrates from aerial parts. Cadmium disrupts translocation process through the sieve elements, resulting in accumulation of starch in leaves of cadmium treated plants 11. This may also inhibit nitrogen fixation as it is a high energy consummg process.

Concentration of Cd (mg/kg)

The reduction in plant growth could be a consequence of cadmium interference with a number

Fig. I - Effect of different concentrations of Cd on seed yield per plant.

Table I - Effect of CdI2 (15, 30, 60, 120, 250 & 500 mg Cd) on plant height, number of nodules, dry weight and stomatal index in Viciafaba

[Values are mean ± SD]

Parameters Control CdI. (mg) 15 30 60 120 250

Plant height (cm) 48.12±5.40 43.05 ±3.42 35.47±2.15 30.72± 1.87 24.02±2.21 19.75 ± 1.81 (-10.53) (-26.28) (-36.15) (-50.08) (-58.95)

Root nodules/plant 24.60±3.97 19.00±2.91 13.60±2.50 8.00 ± 1.58 5.40± 1.14 3.00 ± 0.81 (-22.76) (-44.71) (-67.48) (-78.04) (-87.80)

Stomatal index Upper Epidermis 21.60±0.14 22.2± 2.16 24.2±0.84 26.2±0.84 27.2 ±0.83 28.8 ±0.84

(+ 2.27) (+12.03) (+19.40) (+25.90) (+ 33.40) Lower Epidermis 28.8 ± 1.78 28.8±0.71 29.0± 1.00 30.2± 1.00 32.6 ±0.54 34.8 ±0.84

(+4.48) (+ 8.20) (+13.43) (+21.64) (+29.80) Dry Wt (g) Leaf 0.552 ± 0.034 0.493 ± 0.1 04 0.432±0.066 0.386 ± 0.041 0.329±0.061 0.223 ±0.390

(-10.68) (-21.70) (-30.07) (-40.38) (-59.78) Stem 0.341 ±0.041 0.320 ± 0.020 0.274 ± 0.042 0.254 ± 0.046 0.21O±0.012 0.168±0.035

(-3 .80) (-19.64) (-25.51) (-38.45) (-50.73) Root 0.155 ± 0.062 0.119 ±0.073 0.098±0.0l2 0.074±0.017 0.068 ±0.049 0.049 ±0.OO3

(-23.22) (-36.78) (-52.25) (-56.12) (-68.38)

Figures in parentheses indicate percentage increase (+) or decrease (-) over control.

NEELU et al.: INFLUENCE CADMIUM ON GROWTH AND DEVELOPMENT OF V1CIA FABA LINN. 821

of metabolic processes such as protein synthesis and photosynthesis. Growth reduction may be due to some genotoxic effects. It is reported by several workers that cadmium delays the process of cell division and cell elongation. The cycle time of cells in S phase is lengthened by 1.7 times as compared to control. Cadmium also displays other deleterious features, such as chromosomal breakage and adhesiveness, and cells with damaged DNA having a prolonged cell cycle that would lead to retarded growth l2

. A decrease in dry matter yield can result from breakdown of proteins, reduced binding capacity of amino acids to form polypeptide chains and consequently increased level of free amino acids under the impact of cadmium toxicity l3.

There are several reports dealing with abnormalities of stomatal structure caused oy air pollution l4

, but the effect of soil pollution on stomatal structure needs to be investigated. In the present work the stomata were observed to be adversely affected by enhanced cadmium in soil. Stomatal index of leaf showed a rise in cadmium treated plants. Maximum increase was observed at 250 mg level (Table 1). The increase in

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Concentration of Cd (mg/kg)

Fig. 2 - Effect of different concentrations of Cd on chlorophyll density of 68 days old plants.

stomatal frequency was found to be due to reduction in the size of guard cells because of inhibition of cell elongation. It is possible that the reduced efficiency of such stomata is partly responsible for the decrease in rate of photosynthesis. Cadmium is known to enhance st<?matal resistance to gaseous exchangel5

• The increase in stomatal frequency is supposed to be an ecological adaptation to sustain adequate rate of gaseous exchange necessary for growth and development. In cadmium treated plants, fused stomata and trichomes were observed on leaf epidermal surface. This indicates a more extensive impact on metabolic processes occurring during ontogeny of leaf and differentiation of stomata. Normally stomatal and trichome meristemoids maintain a certain distance from each other on account of their inhibitory influence or nutritional competition among themI6

•17

• However, in treated plants such a balance seems to be disturbed.

One of the most deleterious impact of heavy metals on plants can be observed in terms of reduction in chlorophyll contentl8

. In Vi cia faba too there was a gradual decline in chlorophyll content with increasing levels of cadmium in soil (Fig. 2) . Chlorophyll a,

2500 I m LEAF 0 STEM un ROOT I ~ cD -,... N 0

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Fig. 3 - Effect of different concentrations of Cd on soluble protein in 70 days old plants.

822 INDIAN J EXP BIOL, AUGUST 2000

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Fig. 4 - Effect of different concentrations of Cd on nitrate reductase activity in 72 days old plants.

chlorophyll band carotenoids show a decrease of 42%, 380/0 and 370/0 respectively at 250 mg cadmium. Chlorophyll a is most adversely affected among the three photosynthetic pigments. The reduction of chlorophyll pigments is attributed to enhanced activity of chlorophyllase enzyme l9 and interference of pigment metabolism20. Cadmium can also alter chlorophyll biosynthesis by' inhibiting protochloro­phyllide reductase responsible for chlorophyll synthesis and photosynthetic electron transport by inhibiting water splitting enzyme located at the oxidising site of PS_II21 .22.

Total soluble protein shows a reduction of 37% in roots, 360/0 in stem and 320/0 in leaves at 250 mg cadmium (Fig. 3). Under cadmium stress the activity of catabolic enzymes like proteases increases which leads to degradation of protein23. The binding of cadmium with free amino acids inhibits the initiation process of translation or interferes with peptide chain elongation process. This drastically affects the enzyme synthesis and leads to reduction in protein content24.

Nitrate reductase, a significant enzyme of nitrogen metabolism, is also adversely affected. There is a gradual decline in nitrate reductase activity in

different plant parts. It shows a decline of 560/0, 620/0 and 740/0 in leaf, stem and root respectively at 250 mg of cadmium (Fig. 4). Nitrate reductase is a molybedoflavo protein with Mo as a cofactor required for transfer of electron from NADH2 to nitrate. The source of NADH2 is either Kreb's cycle or light reactions of photosynthesis. Cadmium is known to cause distortion of the structure of mitochondria and chloroplasts which results in the impaired generation of NADH2

25. On the other hand, it also inhibits the absorption of N03 -, which acts as inducer for nitrate reductase, in cadmium treated plants. The inadequate supply of N03- and NADH2 is responsible for decline in the enzymatic activities26.

The present work suggests that growth and yield of broad bean are adversely affected in soil with cadmium pollution. There is a significant decline in biomass, seed yield and protein content. At 500 mg Cd/kg the plants succumbed before flowering. This indicates that broad bean is quite sensitive to cadmium pollution. This plant shows less resistance towards cadmium in comparison to other leguminous crops such as pea27, mung bean28 and soybean29.

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