physiological and biochemical responses resulting from cadmium and zinc accumulation in carrot...

Journal of Plant Nutrition, 33:1066–1079, 2010Copyright C© Taylor & Francis Group, LLCISSN: 0190-4167 print / 1532-4087 onlineDOI: 10.1080/01904161003729774

PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES RESULTING

FROM CADMIUM AND ZINC ACCUMULATION IN CARROT PLANTS

Rajesh Kumar Sharma,1 Madhoolika Agrawal,2

and Shashi Bhushan Agrawal2

1G. B. Pant Institute of Himalayan Environment and Development, Himachal Unit,Mohal, Kullu, Himachal Pradesh, India2Department of Botany, Banaras Hindu University, Varanasi, Uttar Pradesh, India

� The interactive effect of cadmium (Cd) and zinc (Zn) bioaccumulation on physiological andbiochemical characteristics of carrot (Daucus carota L. var Pusa Kesar) plants grown with dif-ferent levels of Cd and Zn was examined. The combined application of Cd and Zn (Cd+Zn) hadsynergistic and antagonistic effects at low and high concentrations, respectively, on Cd accumula-tions in plants, chlorophyll b, and photosynthesis and transpiration rates. Whereas both low andhigh Cd+Zn concentrations had synergistic effects on Zn accumulation, ascorbic acid, protein con-tent, total phenols, peroxidase activity, chlorophyll a, total, carotenoids and stomatal conductance.Bioaccumulation of Cd had strong and negative relationships with measured physiological andbiochemical parameters. The study further showed that Cd and Zn in combination have more toxiceffects on carrot plants as compared to their individual treatments. This study concludes that inter-active effects of Cd and Zn on test plant depend on their ratios present in plants.

Keywords: bioaccumulation, cadmium, carrot, physiological, biochemical, zinc

INTRODUCTION

Numerous urban and industrial activities such as metal finishing, paint,pigment and battery manufacturing, leather tanning, mining and uses of mu-nicipal wastewater and sludges, urban composts, pesticides and phosphatefertilizers add significant amounts of heavy metals in aquatic and terrestrialenvironments. Soils collected around zinc (Zn)/cadmium (Cd) smelter haveZn concentrations up to 99,500 mg kg−1 in addition to other heavy metalssuch as lead (Pb) (1005 to 17220 mg kg−1), copper (Cu) (2500 to 4500 mg

Received 15 July 2008; accepted 26 July 2009.Address correspondence to Dr. Rajesh Kumar Sharma, Scientist ‘C’, G. B. Pant Institute of Himalayan

Environment and Development, Himachal Unit, Mohal, Kullu-175126, Himachal Pradesh, India. E-mail:[email protected]

1066

Responses of Carrot Plants to Heavy Metals 1067

kg−1), and Cd (28–578 mg kg−1) (Reeves et al., 1996). Soils of wastewaterirrigated areas also have Cd and Zn concentrations up to 8.9 and 387.8 mgkg−1, respectively (Sharma et al., 2007).

Heavy metals are hazardous environmental pollutants that have adverseeffects on several physiological and biochemical processes of plants (Wool-house, 1983; Singh and Tewari, 2003; Khudsar et al., 2004) and on humanhealth (WHO, 1992; Jarup, 2003; Sharma and Agrawal, 2005). Zinc is anessential micronutrient but becomes hazardous at higher concentrations(>500 mg kg−1) and inhibits plants growth (Bergmann, 1992; Ali et al.,1999; Sharma et al., 2008), chlorophyll formation (Nag et al., 1984; Kayaet al., 2000) and photosynthesis and transpiration rates (Van Assche et al.,1979). Zinc also interferes with nutrient uptake (Chaney, 1993; Kaya et al.,2000) and certain enzyme activities such as nitrogenase. (Quariti et al., 1997).

Cadmium, a nonessential heavy metal, is toxic metal even at low concen-trations in the soil. Cadmium can accumulate and redistribute in differentplant parts, causing visible injuries (Reed et al., 1999), altered metabolicactivities (Chien et al., 2001; Singh and Agrawal, 2007), and reduced photo-synthesis at different growth stages (Schroder et al., 1994; Abo-Kassem et al.,1995; Skorzynska-Polit and Baszynski, 1997; Kupper et al., 2002). Singh andAgrawal (2007) reported that Cd increased lipid perioxidation in foliage ofIndian palak (Beta vulgaris L.) plants, when grown on sewage sludge amendedsoil. Earlier studies have shown that heavy metals have significant effect onthe protein metabolism, ascorbic acid and phenol contents in plants (Tamaset al., 1997; Basak et al., 2001; Ozturk et al., 2003; Singh and Agrawal, 2007).The toxicity of heavy metals to the plants varies with plant species, nature ofheavy metals and concentration in soil.

Many studies have been conducted on the single or combination ofmulti-metal influence on the uptake and bioaccumulation of heavy metalsin a wide range of plants species (Nan et al., 2002; Sharma and Agrawal,2006; Shute and Macfie, 2006; Sharma et al., 2007; Singh and Agrawal, 2007;Sharma et al., 2008). Limited data are available on the effects of multi-metalcombinations on physiological and biochemical characteristics of plants. Thedirect and indirect effects of single and or multi-metal combinations are alsonot clear. The interaction between Zn and Cd may be either antagonistic,i.e., applied Zn reduced Cd uptake in plants (Moraghan, 1993; McKennaet al., 1993) or synergistic, i.e., Zn addition increased the Cd uptake inplants (Nan et al., 2002; Sharma and Agrawal, 2006; Sharma et al., 2007).

The present study was undertaken to examine the effects of single andcombined effects of Cd and Zn on their accumulation in leaves, stems androots and consequent physiological and biochemical responses of carrot(Daucus carota L. var Pusa Kesar) plants grown under natural field con-ditions. The objectives are (1) to investigate the effects of elevated Cdand Zn, singly and jointly on their relative uptake and bioaccumulations,and consequent effects on photosynthesis and transpiration rates, stomatal

1068 R. K. Sharma et al.

conductance, photosynthetic pigments, lipid peroxidation, protein contentand antioxidative capacities of the test plant, (2) to quantify the relation-ships between Cd or Zn bioaccumulation and the resultant physiologicaland biochemical characteristics, and (3) assessment of interaction effectsby calculating interaction factor (IF) for different parameters studied usingcarrot plants.

MATERIALS AND METHODS

Measurement of Physiological Parameters

The details of experimental design, growth of plants and heavy metalsapplication amounts are described in earlier papers (Sharma and Agrawal,2006; Sharma et al., 2008). The photosynthesis and transpiration rates, andstomatal conductance were measured at 45 DAG of plant age on the sec-ond leaf from top using a Portable Photosynthetic System (LI-6200, LI-COR,INC, Lincoln, NE, USA) at ambient climatic conditions. During the mea-surements, photosynthetically active radiation (PAR) and ambient carbondioxide (CO2) concentrations were varied between 1000–1200 µmol m−2 s−1

and 320–342 g m−3, respectively. All the measurements were made between0900 to 1100 hours as maximum photosynthetic rate was observed duringthis period in field conditions.

Measurement of Biochemical Parameters

Three plants were randomly sampled from each plot for various analysesat 70 DAG. Chlorophylls and carotenoids were extracted from the leaf discusing 80% acetone and quantified according to the methods of Maclachlanand Zalik (1963) and Duxbury and Yentsch (1956), respectively. Peroxi-dase activity in leaf tissues was assessed according to Britton and Mehley(1955). Lipid peroxidation (LPO) in leaf tissues was determined in terms ofmalondialdehyde (MDA, a byproduct of lipid peroxidation) content by thio-barbituric acid (TBA) reaction as described by Heath and Packer (1968).Total soluble protein content of leaf tissue was quantified according to themethod of Lowry et al. (1951) using bovine serum albumin as standard.Total phenols and ascorbic acid contents were quantified using the methodof Bray and Thorpe (1954) and Keller and Schwager (1977), respectively.

Heavy Metal Analysis

The details of heavy metal analysis and quality controls are given inSharma and Agrawal, (2006) and Sharma et al (2008). The concentration ofheavy metals in soil, roots, stems, and leaves were calculated in term relative


changes (RC%), i.e., the percent increase/or decrease due to treatments ofheavy metals as compared to the control.

To describe the different types of interactions (additive, antagonistic orsynergistic) between Cd and Zn treatments in term of heavy metal accumula-tion, physiological and biochemical parameters, the interaction factor (IF)was calculated following formula of Sharma (2007):

IF = (A + B)C

or (A + B)/C

Where A and B are per cent individual effects of Cd and Zn, respectively, andC is percent effects of the combined application of Cd and Zn (Cd+Zn).Interactions are classified as additive (IF = 1), synergistic (IF < 1), andantagonistic (IF > 1).

Statistical Analyses

Data were subjected to analysis of variance (ANOVA) test and the sta-tistical significance of the differences between the treatment means weredetermined by using Duncan’s multiple range test (P < 0.05). Pearson cor-relation matrix was determined between heavy metal concentrations in thesoil, plant parts with the measured physiological and biochemical parame-ters. All the statistical analyses were performed using SPSS software version12 (SPSS Inc., Chicago, IL, USA).

RESULTS AND DISCUSSION

The present study showed that Zn application in combination with Cdhas increased the Cd concentrations in all plant parts as compared to the in-dividual treatments of both low and high Cd concentration (Table 1). Earlierstudies have shown that applications of Zn reduced Cd uptake significantlyin a wide range of plants (Moraghan, 1993; McKenna et al., 1993). In thepresent study, the uptake of Cd decreased significantly with increasing con-centrations of Cd + Zn, whereas the uptake of Cd increased with increasingconcentrations of Cd only (Table 1). This result reveals that Zn at low con-centrations induces Cd uptake, whereas inhibits at higher concentrations.However, Zn uptake increased significantly with increasing concentrationsof Cd + Zn as well as Zn alone (Table 1). Interactive effects of Cd and Znin terms of interaction factor (IF) on their uptake and bioaccumulation indifferent plant parts were observed either synergistic or antagonistic depend-ing on the Cd and Zn combinations (Table 1). The results further showedthat Zn, when applied with Cd, affects Cd uptake and its accumulation inroots, stems and leaves synergistically and antagonistically, respectively at lowand high concentrations (Table 1). In roots Zn accumulation was affected


TABLE 1 Relative changes (± RC%) in Cd and Zn accumulation in soil and different parts of carrotplants, grown in soil elevated with low and high Cd (10 and Zn 100 mg/l, respectively), Zn (100 and Zn300 mg/l, respectively), singly and in combination

Cd Zn

Heavy metals Concentrations Soil Root Stem Leaf Soil Root Stem Leaf

Cd Low 25.9c 51.8c 44.9c 102.9c 1.5d 1.7c 17.1b −11.9f

Zn Low 18.6d 15.8e 8.2e 17.5e 25.5c 48.2b 18.6b 20.1c

Cd + Zn Low 9.6e 77.1a 78.6a 132.9a 35.0b 48.3b 17.6b 10.1d

IF <1 <1 <1 >1 >1 <1Cd High 96.3b 63.1b 64.8b 108.8b 0.3d 2.8c 8.9bc −15.7f

Zn High 12.6e 22.4d 17.9d 45.3d 52.7a 59.3a 38.4a 32.3b

Cd + Zn High 140.9a 65.4b 64.8b 118.3b 55.7a 64.9a 40.6a 42.8a

IF >1 >1 >1 <1 >1 <1

IF = Interaction factor, Values followed by different letters are significantly different from each otherat P < 0.05 (Duncan’s multiple range test).

antagonistically and synergistically by Cd + Zn at the low and high concen-trations, respectively. The results further showed that Zn accumulation instems was affected antagonistically and in leaves, synergistically, respectivelyby the Cd + Zn at low and high concentrations (Table 1). This may beascribed to variation in translocation patterns of Zn from roots to stems orfrom stems to leaves.

An oxidative stress in different plants caused by heavy metals is oftendemonstrated by the accumulation of malondialdehyde (MDA), a byprod-uct of membrane peroxidation. The MDA content (an index of lipid per-oxidation) increased significantly with increasing metal application rates,singly and in combination as compared to the control plants (Figure 1).Enhancement of LPO was reported upon Cd and Zn treatments in bean(Phaseolus vulgaris L.) plants (Chaoui et al., 1997) and in Indian palak (Betavulgaris) by heavy metal presence in sewage sludge (Singh and Agrawal,2007). The increment in LPO levels may be ascribed to peroxidation of es-sential membrane lipids due to generation of more reactive oxygen species(ROS) under heavy metals stresses (Lin and Kao, 2000). Protonation ofsuper oxide radical (·O2

−) can produce the hydroperoxyl radicals (∗OH,H2O2), which convert fatty acids to toxic lipid peroxides that destroy biolog-ical membranes (Shah et al., 2000). In this study, the MDA content in leaftissues of carrot plants increased significantly when supplemented with Cd+ Zn as compared to their individual treatments (Figure 1). This may bedue to a higher accumulation of Cd and Zn in leaf tissues. The data showedthat LPO had a positive and significant relationship with Cd accumulation inleaf and insignificant relationship with Zn accumulation (Table 2). The LPOwas synergistically and antagonistically affected by low and high concentra-tions of Cd + Zn, respectively. These results suggest that increasing levels ofendogenous Cd and Zn indirectly led to the production of more free


Treatments

C ontro l Low H igh

LP

O (

nM

/ml)

0

5

10

15

20

25

30

CdZnCd+Zn

e

d

e

b

c

d

aIF > 1 (low)

IF < 1 (high)

FIGURE 1 The effects of low and high concentrations of Cd (10 and 100 µg mL−1, respectively) and Zn(100 and 300 µg mL−1, respectively) on lipid peroxidation (LPO) in the leaves of carrot (Daucus carota)plants. Bars represent mean ± S.E.; n = 3. Different letters are significantly different from each other atP ≤ 0.05. (IF = Interaction factor).

radicals, resulting in an increased in membrane LPO in leaf tissues of carrotplants.

A synergistic effect of Cd and Zn both at low and high concentrations wasobserved for protein content, ascorbic acid, peroxidase activity, chlorophyll

TABLE 2 Correlation of resultant physiological and biochemical parameters with Cd or Znbioaccumulation in soil and plant parts

Cd Zn

Parameters Soil Root Stem Leaf Soil Root Stem Leaf

Photosynthesis −0.84∗∗ −0.76∗∗ −0.76∗∗ −0.76∗∗ −0.45∗ −0.31NS −0.52∗ −0.29NS

Transpiration −0.54∗ −0.90∗∗ −0.88∗∗ −0.92∗∗ −0.42NS −0.31NS −0.47∗ −0.16NS

Stomatal conductance −0.57∗∗ −0.92∗∗ −0.90∗∗ −0.92∗∗ −0.35NS −0.23NS −0.40NS −0.12NS

Chlorophyll a −0.73∗∗ −0.81∗∗ −0.84∗∗ −0.79∗∗ −0.45∗ −0.36NS −0.38NS −0.30NS

Chlorophyll b −0.71∗∗ −0.90∗∗ −0.89∗∗ −0.91∗∗ −0.45∗ −0.34NS −0.49∗ −0.23NS

Carotenoids −0.76∗∗ −0.78∗∗ −0.76∗∗ −0.79∗∗ −0.53∗ −0.43NS −0.59∗∗ −0.40NS

Total chlorophyll −0.74∗∗ −0.87∗∗ −0.88∗∗ −0.85∗∗ −0.46∗ −0.36NS −0.43∗ −0.28NS

Ascorbic acid 0.53∗ 0.92∗∗ 0.92∗∗ 0.92∗∗ 0.47∗ 0.39NS 0.44∗ 0.24NS

Total Phenols 0.61∗∗ 0.81∗∗ 0.80∗∗ 0.80∗∗ 0.36NS 0.28NS 0.33NS 0.20NS

Lipid peroxidation 0.71∗∗ 0.73∗∗ 0.74∗∗ 0.71∗∗ 0.52∗ 0.45∗ 0.46∗ 0.42NS

Total soluble proteins −0.07NS −0.66∗∗ −0.61∗∗ −0.64∗∗ 0.03NS −0.08NS 0.04NS 0.15NS

Peroxidase activity 0.56∗∗ 0.89∗∗ 0.89∗∗ 0.91∗∗ 0.35NS 0.29NS 0.35NS 0.15NS

∗∗Correlation is significant at the 0.01 level.∗Correlation is significant at the 0.05 level.NSCorrelation is not significant.


mg

/g f

resh

leaf

0.0

0.5

1.0

1.5

2.0

Treatments

Control Low High

mg

/g d

ry weig

ht

0

2

4

6

8

10

12

14

mg

/g fresh

leaf

0

5

10

15

20

25

Treatments

Control Low High

µM p

ur.

form

ed/ m

in/m

g f

resh

leaf

0

5

10

15

e

d

e

b

cd

a

db

d

ab c

a dc d

ab

c

a

aa

bcb

d

IF < 1 (low)IF < 1 (high)

IF < 1 (low)IF > 1(high)



d

)b()a(

)d()c(

CdZnCd+Zn

FIGURE 2 The effects of low and high concentrations of Cd (10 and 100 mg/l, respectively) and Zn(100 and 300 mg/l, respectively) on the (a) ascorbic acid, (b) total phenols, (c) peroxidase activity and(d) protein content in the leaves of carrot (Daucus carota) plants. Bars represent mean ± S.E.; n = 3.Different letters on bars in each figure are significantly different from each other at P ≤ 0.05 (Duncan‘smultiple range test). (IF = Interaction factor).

a, total, carotenoid content and stomatal conductance (Table 2; Figures 2–4).The Cd + Zn treatment also affected total phenol content, chlorophyll b,photosynthesis, and transpiration rates synergistically and antagonistically attheir low and high concentrations, respectively (Figures 2–4). This might bedue to direct relationships of these parameters with the accumulation of Cdand Zn in different parts of carrot plants.

In this study, the ascorbic acid content was found to be higher in planttreated with Cd and Zn in combination as compared to the control and theirsingle treatments (Figure 2a). Ozturk et al. (2003) found that ascorbic acid indurum wheat (Triticum turgidum L.) decreased under Cd stress as comparedto the control plants. Total phenol content increased with increasing heavy


metal application rates both singly as well as in combination. The percentincrease in total phenol content was recorded in plants treated with Cd +Zn as compared to the either Cd or Zn or control plants (Figure 2b). To-tal peroxidase activity, however, increased significantly with both the plantage and increasing heavy metal application rates, singly as well as in com-bination as compared to the control plants (Figure 2c). Singh and Tewari(2003) have reported a 163% increase in total peroxidase activity of mustard(Brassica juncea) plants treated with high Cd as compared to the controlplants. In the present study, total peroxidase activity increased by 57% at70 DAG of plant age in high Cd (100 µg mL−1) treatments. The increasein total peroxidase activity is due to increase in the levels of free radicalsdue to heavy metals stresses (Singh and Tewari, 2003; Singh and Agrawal,2007).

Foliar protein content declined significantly in plants under single andcombined treatments of Cd and Zn (Figure 2d). The decrease in proteincontent may also be ascribed to generation of ROS under heavy metal stresscausing fragmentation of protein structure. In contrast, protein content in-creased in linseed under Cd and Zn treatments (Chakravarty and Srivastava,1997). Chaoui et al. (1997) did not find any effect of Cd and Zn treatmentson soluble protein content in mung bean (Phaseolus vulgaris) plants. Thedecrease in protein content in the leaves of palak (Beta vulgaris) plants wasfound by Singh et al. (2008), grown in soil amended with fly ash high inheavy metals.

Application of Cd has significantly decreased the contents of chlorophylla, b, total and carotenoid (Figure 3). The greater effects of Cd and Zn werefound on chlorophyll b that increased chlorophyll a/b ratio. The highersensitivity of chlorophyll b to Cd could be due to either greater degrada-tion of chlorophyll b or reduced conversion of chlorophyll a to chlorophyllb (Singh and Tewari, 2003). A number of studies have shown that Cd re-duced significantly the photosynthetic pigments in plants either directlyor indirectly (Singh and Tewari, 2003; Stobert et al., 1985). The results ofpresent study further showed that Cd accumulation in soil and in plant partshave significant and negative relationships with chlorophyll a, b, total andcarotenoids, whereas, Zn accumulation in soil and stem showed only negativerelationships (Table 2). The reduction in chlorophyll content in leaf mayalso be attributed to interaction of Cd or Zn with the functional –SH groupof chlorophyll synthesizing enzymes during the various steps of chlorophyllbiosynthesis (Stobert et al., 1985; Padmaja et al., 1990; Boddi et al., 1995;Horvath et al., 1996). The reduction in chlorophyll content and physio-logical activity due to heavy metals stress was also ascribed to disruption oflight harvest complex (LHC) through inhibition of LHC protein synthesisat transcription level (Horvath et al., 1996).

In the present study, combined treatments of Cd and Zn decreasedchlorophyll a, b and total significantly as compared to their individual


mg

/g d

ry w

eig

ht

0.0

0.5

1.0

1.5

2.0

Treatments

Control Low High

mg

/g d

ry w

eig

ht

0.0

0.5

1.0

1.5

mg

/g d

ry weig

ht

0.0

0.5

1.0

1.5

2.0

Treatments

Control Low High

mg

/g d

ry weig

ht

0.0

0.5

1.0

1.5

a b a

d

c

b

e

a

b

a

d cb

d

aba

c c b

d

a

b

a

dc

b

e


IF < 1 (low)IF > 1 (high)



(a) (b)

(c) (d)

CdZnCd+Zn

FIGURE 3 The effects of low and high concentrations of Cd (10 and 100 mg/l, respectively) and Zn(100 and 300 mg/l, respectively) on photosynthetic pigments (a) chlorophyall a, (b) total chlorophyall,(c) chlorophyll b and (d) carotenoids in the leaves of carrot (Daucus carota) plants. Bars representmean ± S.E.; n = 3. Different letters on bars in each figure are significantly different from each other atp ≤ 0.05. (IF = Interaction factor).

treatments and control plants (Figure 3). The intense decrease in pigmentcontents under combined treatment of Cd and Zn was ascribed to moreaccumulation of Cd and Zn (Table 1). The result suggests that Cd and Znhave a synergistic effect on photosynthetic pigments. The reduction of totalchlorophyll was accompanied by carotenoid content reduction during thepresent study. However, an increase in carotenoid content has been reportedin plants (Kenneth et al., 2000).

Zn at low (100 µg mL−1) concentration did not significantly affectphotosynthesis, transpiration and stomatal conductance (P > 0.05). How-ever, other treatments of Cd and Zn significantly reduced photosynthesis,


µMol

CO

2/m2 /s

0

5

1 0

1 5

2 0

2 5

3 0M

ol H

2O/m

2 /s

0 .0

0 .1

0 .2

T re a tm e n ts

C o n tro l L o w H ig h

cm/s

0

2

4

6

8

1 0

1 2

1 4

1 6

C dZ nC d + Z n

ab

a

cd

e

ab

a

cb

b

c

c

a

c

a

dc

b

d

IF < 1 ( lo w )IF > 1 (h ig h )

IF < 1 ( lo w )IF > 1 (h ig h )

IF < 1 ( lo w )IF < 1 (h ig h )

(a )

(b )

(c )

FIGURE 4 The effects of low and high concentrations of Cd (10 and 100 mg/l, respectively) and Zn(100 and 300 mg/l, respectively) on (a) photosynthesis, (b) transpiration and (c) stomatal conductancein the leaves of carrot (Daucus carota) plants. Bars represent mean ± S.E.; n = 3. Different letters on barsin each figure are significantly different from each other at P ≤ 0.05 (Duncan‘s multiple range test) (IF =Interaction factor).


transpiration and stomatal conductance (Figure 4–c). As compared to thecontrol, photosynthesis reduced by 19 and 53%, respectively at low and highdoses of Cd, 31% at high dose of Zn and 35 and 65%, respectively, at low andhigh doses of Cd + Zn. This result suggests that combined treatments of Cdand Zn have a larger negative effect on photosynthesis than the individualtreatments. These changes in physiological parameters may be due to higheraccumulation of Cd and Zn in carrot plants under combined treatment ofCd and Zn than their individual treatments (Table 1). The rate of photo-synthesis showed a negative relationship with Cd in soil and in the roots,stems, and leaves of carrot plants and also with the Zn concentrations in thesoil and stem (Table 2). The accumulation of Cd and Zn may interfere withvarious enzymes of photosynthetic pathways.

In the present study, reduction in photosynthesis can be directly cor-related to decline in chlorophyll content and stomatal conductance. Inhi-bition of photosynthesis at higher Cd concentration was ascribed to theincrease in stomatal and mesophyll resistances to CO2 uptake (Lamoreauxand Chaney, 1978). In the present study, the transpiration rate also de-creased significantly at the single and combined treatments of Cd and Zn.(Figure 4). This reduction may be ascribed to the heavy metal induced rootdamage, interference of heavy metals with stomatal regulation and xylemobstruction (Lamoreaux and Chaney, 1978). It has been suggested thatheavy metal stress causes reduction in the transpiration rate due to its influ-ence on water flow through root as well as stomatal aperture (Hatch et al.,1988).

CONCLUSION

The present investigation clearly showed that Zn at low concentrationhas no detrimental effects on the examined parameters of carrot plants.The other treatments significantly reduced the photosynthesis and transpi-ration rates, stomatal conductance and chlorophyll a, b total, carotenoids,and protein contents. However, increments were observed in ascorbic acid,total phenols, peroxides activity and lipid peroxidation. The results furthershowed the synergistic effects of Cd + Zn both at low and high concentrationson Zn accumulation in leaf, ascorbic acid and protein contents, peroxidaseactivity, chlorophyll a, total, carotenoid contents and stomatal conductance.Cd accumulations in root, stem and leaf, chlorophyll b, photosynthesis andtranspiration rates responded synergistically and antagonistically under lowand high Cd + Zn concentrations, respectively. The component-wise ac-cumulations of Cd showed strong relationships with studied parameters ascompared to Zn accumulation. The study concludes that the interactive ef-fects of Cd and Zn on carrot plants depends on the ratios of Cd and Znpresent in the plants.


ACKNOWLEDGMENTS

The staff of the Department of Botany and Farm Incharge, Institute ofAgricultural Science BHU, Varanasi, India are gratefully acknowledged forproviding the laboratory facilities and field for conducting the experiment.Rajesh Kumar Sharma is also thankful to the Department for InternationalDevelopment, United Kingdom and Council of Scientific and IndustrialResearch, New Delhi, India for providing financial support.

REFERENCES

Abo-Kassem, E., A. Sharaf-el-din, J. Rozema, and E. A. Foda. 1995. Synergistic effect of cadmium, NaClon the growth, photosynthesis and iron content on wheat plants. Biologia Plantarum 37: 241–249.

Ali, G., P. S. Srivastava, and M. Iqbal. 1999. Morphogenic and biochemical responses of Bacopa monnieracultures to zinc toxicity. Plant Science 139: 1–7.

Basak, M., M. Sharma, and U. Chakraborty. 2001. Biochemical responses of Camellia sinensis (L.) O.Kuntze to heavy metal stress. Journal of Environmental Biology 22: 37–41.

Bergmann, W. 1992. Nutritional Disorders of Plants. Jena: Gustav Fisher Verlag.Boddi, B., A. R. Oravecz, and E. Lehoczki. 1995. Effects of cadmium on organization and photoreduction

of protochlorophyllide in dark-grown leaves and etioplast inner membrane preparations of wheat.Photosynthetica 3: 411–420.

Bray, H. C., and W. Y. Thorpe. 1954. Analysis of phenolic compounds of interest in metabolism. In:Methods of Biochemical Analysis, ed. D. Click, pp. 27–52. New York: InterScience.

Britton, C., and A. C. Mehley. 1955. Assay of catalase and peroxides. In: Methods in Enzymology, eds. S. P.Colowick, and N. O. Kaplan, Vol. II, p. 764. New York: Academic Press.

Chakravarty, B., and S. Srivastava. 1997. Effect of cadmium and zinc interaction on metal uptake andregeneration of tolerant plants in linseed. Agricultural Ecosystem and Environment 61: 45–50.

Chaney, R. L 1993. Zinc phytotoxicity. In: Zinc in Soil and Plants, ed. A. D. Robson, pp. 135–150. Dordrecht,the Netherlands: Kluwer Academic Publishers.

Chaoui, A., S. Mazhoudi, M. H. Ghorbal, and E. Ferjani. 1997. Cd and Zn induction of lipid peroxidationand effect on antioxidant enzymes activities in bean (Phaseolus vulgaris L.). Plant Science 127: 139–147.

Chien, H. F., J. W. Wang, C. C. Lin, and C. H. Kao. 2001. Cadmium toxicity of rice leaves is mediatedthrough lipid peroxidation. Plant Growth and Regulation 33: 205–213.

Duxbury, A. C., and C. S. Yentsch. 1956. Plankton pigment monographs. Journal of Marine Research 15:91–101.

Hatch, D. J., L. H. P. Jones, and R. G. Burau. 1988. The effect of pH on the uptake of Cd by four plantspecies grown in flowing solution culture. Plant and Soil 105: 212–226.

Heath, R. L., and L. Packer. 1968. Phytoperoxidation in isolated chloroplast. I. Kinetics and stoichiometryof fatty acid peroxidation. Archives of Biochemistry and Biophysics 125: 189–198.

Horvath, V. J. G., M. Droppa, A. Oravecz, V. I. Raskin, and J. B. Marder. 1996. Formation of the photo-synthetic apparatus during greening of cadmium poisoned barley leaves. Planta 199: 238–243.

Jarup, L. 2003. Hazards of heavy metal contamination. British Medical Bulletin 68: 167–182.Kaya, C., D. Higgs, and A. Burton. 2000. Plant growth, phosphorus nutrition and acid phosphatase

enzyme activity in three tomato cultivars grown hydroponically at different zinc concentrations.Journal of Plant Nutrition 23: 569–579.

Keller, T., and H. Schwager. 1977. Air pollution and ascorbic acid. European Journal of Forest Pathology 7:338–350.

Kenneth, E., K. E. Pallet, and I. Young. 2000. In: Carotenoids Antioxidants in Higher Plants, ed. R. G. Alscherand J. L. Hess, pp. 60–81. Boca Raton, FL: CRC Press.

Khudsar, T., M. Iqbal, and R. K. Sairam. 2004. Zinc-induced changes in morpho-physiological AndBiochemical Parameters. In Artemisia Annua. Biologia Plantarum 48: 255–260.

Kupper, H., I. Setlık, M. Spiller, F.C. Kupper, and O. Prasil. 2002. Heavy metal-induced inhibition ofphotosynthesis: Targets of in vivo heavy metal chlorophyll formation. Journal of Phycology 38: 429–441.


Lamoreaux, R. J., and W. R. Chaney. 1978. The effect of cadmium on net photosynthesis, transpirationand dark respiration of excised silver maple leaves. Physiologia Plantarum 43: 231–236.

Lin, L. C., and C. H. Kao. 2000. Effect of NaCl stress on H2O2 metabolism in rice leaves. Plant Growth andRegulation 30: 151–155.

Lowry, O. H., F. Rosenbrough, and R. J. Randall. 1951. Protein measurement with folin phenol reagent.Journal of Biological Chemistry 193: 265–275.

Maclachlan, S., and S. Zalik. 1963. Plastid structure, chlorophyll concentration and free amino acidcomposition of a chlorophyll mutant on Barley. Canadian Journal of Botany 41: 1053–1062.

McKenna, I. M., R. L. Chaney, and F. M. Williams. 1993. Effects of Cd and Zn interactions on theaccumulation and tissue distribution of Zn and Cd in lettuce and spinach. Environmental Pollution79: 113–120.

Moraghan, J. T. 1993. Accumulation of Cd and selected elements in flax seed grown on calcareous soil.Plant and Soil 96: 154–164.

Nag, P., A. K. Paul, and S. Mukherji. 1984. Toxic action of zinc on growth and enzyme activities of rice(Oryza sativa L.) seedlings. Environmental Pollution 36: 45–59.

Nan, Z., J. Li, A. Zhang, and G. Cheng. 2002. Cadmium and zinc interaction and their transfer in soil–cropsystem under actual field conditions. Science of the Total Environment 285: 187–195.

Ozturk, L., S. Eker, and F. Ozkutlu. 2003. Effect of cadmium on growth and concentrations of cadmium,ascorbic acid and sulphydryl groups in durum wheat cultivars. Turkish Journal of Agricultural Forestry27: 161–168.

Padmaja, K., D. D. K. Prasad, and A. R. K. Prasad. 1990. Inhibition of chlorophyll synthesis in Phaseolusvulgaris L. seedlings by cadmium acetate. Photosynthetica 24: 399–405.

Quariti, O., H. Gouia, and M. H. Ghorbal. 1997. Responses of bean and tomato plants to cad-mium: Growth, mineral nutrition and nitrate reduction. Plant Physiology and Biochemistry 35: 347–354.

Reed, R. L., M. A. Sanderson, V. G. Allen, and A. G. Matches. 1999. Growth and Cd accumulation inselected switch cultivars. Communication in Soil Science and Plant Analysis 30: 2655–2667.

Reeves, R. D., A. J. M. Baker, and R. R. Brooks. 1996. Abnormal accumulation of trace metals by plants.Mining Environment and Management 3: 4–8.

Schroder, W. P., J. B. Arellano, T. Bittner, M. Baron, H. J. Eckert, and G. Renger. 1994. Flyash-inducedabsorption spectroscopy studies of copper interaction with photosystem II in higher plants. Journalof Biological Chemistry 269: 32865–32870.

Shah, K., R. G. Kuwar, S. Verma, and R. S. Dubey. 2000. Effect of cadmium on lipid peroxidation,super oxide and activities of antioxidant enzymes in growing rice seedlings. Plant Science 161: 1135–1144.

Sharma, R. K. 2007. Heavy metal contamination of vegetables in and around Varanasi city. Ph.D thesis,Banaras Hindu University, Varanasi, India.

Sharma, R. K., and M. Agrawal. 2005. Biological effects of heavy metals: an overview. Journal of Environ-mental Biology 26: 301–313.

Sharma, R. K., and M. Agrawal. 2006. Effects of single and combined treatment of Cd and Zn on carrotplants: Uptake and bioaccumulation. Journal of Plant Nutrition 29: 1791–1804.

Sharma, R. K., M. Agrawal, and S. B. Agrawal. 2008. Interactive effects of Cd and Zn on carrot plants:Growth and biomass bioaccumulation. Journal of Plant Nutrition 31: 1–17.

Sharma, R. K., M. Agrawal, and F. M. Marshall. 2007. Heavy metals contamination of soil and vegetablesin suburban areas of Varanasi, India. Ecotoxicology and Environmental Safety 66: 258–266.

Shute, T., and S. M. Macfie. 2006. Cadmium and zinc accumulation in soybean: A threat to food safety?Science of the Total. Environment 371: 63–73.

Singh, R. P., and M. Agrawal. 2007. Effects of sewage sludge amendment on heavy metal accumulationand consequent responses of Beta vulgaris plants. Chemosphere 67: 2229–2240.

Singh, A., R. K. Sharma, and S. B. Agrawal. 2008. Effects of fly ash incorporation on heavy metal accumu-lation, growth and yield responses of Beta vulgaris plants. Bioresource Technology 99: 7200–7207.

Singh, P. K., and S. K. Tewari. 2003. Cadmium toxicity induced changes in plant water relations andoxidative metabolism of Brassica juncea L. plants. Journal of Environmental Biology 24: 107–117.

Skorzynska-Polit, E., and T. Baszynski. 1997. Differences in sensitivity of the photosynthetic apparatus inCd-stressed runner bean plants in relation to their age. Plant Science 128: 11–21.

Stobert, A. R., W. T. Griffiths, A. J. Bukhari, and R. P. Shewood. 1985. Effect of Cd2+ on biosynthesis ofchlorophyll in leaves of barley. Physiologia Plantarum 63: 293–298.


Tamas, L., J. Huttoa, and Z. Zigova. 1997. Accumulation of stress proteins in intracellular spaces of barleyleaves induced by biotic and abiotic factors. Biologia Plantarum 39: 387–394.

Van Assche, F., H. Clijsters, and R. Marcelle. 1979. Photosynthesis in Phaseolus vulgaris L. as influenced bysupra-optimal zinc nutrition. In: Photosynthesis and Plant Development, eds. R. Marcelle, H. Clijsters,and M. Van Poucke, pp. 175–184. The Hague, the Netherlands: Dr. W Junk.

WHO. 1992. Cadmium. Environmental Health Criteria, Vol. 134, Geneva: WHO.Woolhouse, H. W. 1983. Toxicity and tolerance in the response of plants to metals. In: Encyclopedia of

Plant Physiology, eds. O. L. Lange, P. S. Nobel, C. B. Osmond, and H. Ziegler, Vol. 12C, pp. 246–300.Berlin: Springer-Verlag.

Copyright of Journal of Plant Nutrition is the property of Taylor & Francis Ltd and its content may not be

copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written

permission. However, users may print, download, or email articles for individual use.

physiological and biochemical responses resulting from cadmium and zinc accumulation in carrot...

Documents