J. Agronomy & Crop Science 193, 357—365 (2007)  2007 The Authors Journal compilation  2007 Blackwell Verlag, Berlin ISSN 0931-2250

doi:10.1111/j.1439-037X.2007.00270.x

Mineral Toxicity Stress Department of Botany, University of Agriculture, Faisalabad 38040, Pakistan

Effects of Cadmium on Carbon and Nitrogen Assimilation in Shoots of Mungbean [Vigna radiata (L.) Wilczek] Seedlings A. Wahid, A. Ghani, I. Ali, and M. Y. Ashraf Authors addresses: Dr A. Wahid (corresponding author; e-mail: [email protected]), Dr Abdul Ghani, Department of Botany, University of Agriculture, Faisalabad 38040, Pakistan; Dr I. Ali, Botany Department, Government College University, Faisalabad, Pakistan; Dr M. Y. Ashraf, Plant Physiology Division, Nuclear Institute for Agriculture and Biology, Faisalabad, Pakistan With 5 figures and 1 table Received February 16, 2007; accepted June 8, 2007

Abstract Increased cadmium (Cd) uptake from contaminated soils damages plant metabolism. The purpose of this study was to determine Cd-induced time-related changes in some shoot growth and physiological attributes, and their interrelationships in Cd-tolerant (NM-98) and sensitive (NM-28) mungbean varieties. Shoot Cd and leaf chlorosis increased with a concomitant reduction in shoot dry weight, leaf area, relative growth rate (RGR), net assimilation rate (NAR) and relative leaf expansion rate. Reduction in transpiration rate (E) and stomatal conductance (gs) and increase in substomatal CO2 level (Ci), indicated that Cd reduced net photosynthesis (Pn) by reducing CO2 fixation by Rubisco, albeit these changes were less pronounced in NM-98. A positive correlation of chlorosis with shoot Cd, and negative relationships of chlorosis and shoot Cd with Pn revealed that Cd damages the photosynthetic apparatus in mungbean. Time course decrease in in vivo nitrate reductase activity (NRA) and an increase in soluble nitrate in NM-28 revealed that Cd markedly hampers nitrogen assimilation. Positive correlations of RGR and NAR with Pn and NRA and negative ones with chlorosis, shoot dry weight, shoot Cd and Ci in NM-98 suggested that mungbean sensitivity to Cd is due to perturbed C and N assimilation.

Key words: C- and N-assimilation — cadmium — correlations — mungbean — nitrate reductase activity

Introduction Heavy metal contamination of soils has become a worldwide problem and great environmental threat, as these metals accumulate in soils and plants in excess and enter the food chain (Kashem and Singh

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1999, Stolt et al. 2006, Jamali et al. 2007). Among others, cadmium (Cd) is the more noxious soil pollutant and its excessive discharge as a byproduct from industries is worsening the situation. It is a non-essential and highly toxic metal to plants in decreasing dry matter and seed yields. It is readily taken up and translocated in different parts, being highly mobile in the phloem (Benavides et al. 2005). A majority of plants are more sensitive during seedling stages, and this stage is quite often used to denote metal toxicity effects (Bindhu and Bera 2001, Zhang et al. 2002, Wu et al. 2006, Ghani and Wahid 2007). Reduction in growth and biomass yield with increased levels of Cd in growth media arises because of altered physiological phenomena (Demirevska-Kepova et al. 2006). The reductions in growth and biomass yield with increased levels of Cd have been primarily attributed to perturbed photosynthesis (Chugh and Sawhney 1999, Verma and Dubey 2002). Available evidences suggest that under excessive evapo-transpiration, Cd permeates the cytosol through Ca channels on the plasmalemma and hampers the cell water status (Perfus-Barbeoch et al. 2002). Moreover, acquisition of essential nutrients in appropriate amounts is important to plant growth, as these nutrients are either structural or functional components of cells. Being mobile within the plant, Cd may hamper the nutritional status of various plant parts. In this context altered nutrient status of photosynthesizing leaves is of greater consideration (Baryla et al. 2001, Ghnaya et al. 2005, 2007).

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As a leguminous species, mungbean shows considerable changes in the nitrogen (N) metabolism, particularly the amino acids biosynthesis (Gouia et al. 2000, Chaffei et al. 2004, Ghnaya et al. 2005, 2007). Coordinated carbon (C) and nitrogen (N) metabolism under Cd stress is critical for normal growth. Among various enzymes nitrate reductase (NR) is a key enzyme in the conversion of nitrate to nitrite, and its sustained activity is crucial to N assimilation (Gouia et al. 2000, Ghnaya et al. 2005, 2007). Reductions in NR activity, reduced nitrogen fixation and ammonia assimilation in nodules have been reported in legumes with applied Cd (Balestrasse et al. 2004). Likewise, different aspects of photosynthesis are sensitive to increased Cd levels. Many studies witness that Cd disturbs the chloroplast metabolism by hampering light and dark reactions of photosynthesis (de Filippis and Zeigler 1993, Vassilev et al. 2005). In addition, conductance and index of stomata, transpiration and net CO2 uptake are greatly reduced with elevated Cd levels in the growth media (Bindhu and Bera 2001, Balakhnina et al. 2005). Conducting time course experiment is an effective approach to assess temporal changes in the growth and dry matter yield (Poorter 1989, Harris and Taylor 2004, Wahid 2007). Such parameters better reflect the efficiency of the plants to grow under stressful conditions (Wahid et al. 1999, Zhang et al. 2002). In view of the prediction that effects of Cd toxicity on the net assimilation capacity of mungbean are time dependent, we explored comparative changes in Cd accumulation pattern, growth, gas exchange and nitrate assimilation of shoot and possible correlations of these attributes using two differentially Cd-tolerant varieties.

Materials and Methods

Wahid et al.

alkaline pH of the soil. Physico-chemical properties of soil were: texture, sandy loam; saturation, 29%; pH, 8; ECe, 2.4 dS m)1; organic matter, 1.15%; total N, 0.52%; other nutrients (mg kg)1) total P, 6.6; K+, 160 and Ca2+, 10.3. Soil mechanical properties were determined as described by Moodie et al. (1959), pH and EC from the saturated extract of soil, while organic matter, total N and P were estimated as described by Black (1965). Calcium was determined on atomic absorption spectrophotometer (Model AAnalyst 3000; Perkin Elmer, Norwalk, CT, USA) from the soil extract. The environmental conditions were: ambient temperature 30 ⁄ 25 ± 3 C (day ⁄ night), relative humidity 55–65% and bright sunlight prevailed during the experimental duration. The experimental design was completely randomized with four replications. Plants were irrigated whenever needed to keep the soil moisture to optimum level. Half-strength Hoagland nutrient solution was applied only once to avoid nutrient depletion.

Chlorosis, shoot growth and gas exchange Plants were harvested at 2-day interval for seven times starting with day 0. Leaf chlorosis was quantified as composite value and expressed as per cent of total area. Leaf area per plant was determined of intact plant by taking the image of leaf on a paper and area determined subsequently on a leaf area metre. Whole shoot fresh weight was taken immediately after harvest, while dry weight determined after drying at 70 C for a week. Shoot water content (%) was computed as: (fresh weight ) dry weight) · 100 ⁄ fresh weight. Relative growth rate (RGR), net assimilation rate (NAR) and relative leaf expansion rate (RLER) were derived using formulae described by Hunt (1982). Net photosynthetic (Pn), and transpiration rates (E), stomatal conductance (gs) and substomatal CO2 concentrations (Ci) were determined between 9 and 10 am from second and third fully expanded leaves using infrared gas analyzer (IRGA; ADC, Hoddesdon, UK). Set of conditions for these determinations was: air flow per unit area 335–340 mmol m)1 s)1, atmospheric pressure 98.6– 99.8 kPa, photosynthetically active radiations (PAR) on leaf surface 900–1100 lmol m)2 s)1, CO2 concentration 357 lmol mol)1 and air temperature 26–28 C.

Experimental details Experiments were conducted during February–March, 2004 and 2005 to determine the changes in absolute and derived growth, Cd accumulation pattern, leaf gas exchange capacity nitrate reductase activity (NRA) and soluble nitrate (SN) levels of two differentially Cd-tolerant mungbean [Vigna radiata (L.) Wilczek] varieties. Seeds were sown in plastic pots containing 5 kg soil and kept in a wire-house under bright sunlight. Three days after emergence, the seedlings were thinned to five per pot. One set of 7-day-old seedlings was applied with 12 mg Cd kg)1 soil using CdCl2Æ2.5H2O (Cd stress), while control plants received no Cd (control). This Cd level was selected after preliminary experiments under a range of Cd levels (1–25 mg Cd kg)1 soil) in view of the

Shoot Cd concentration For the determination of shoot Cd, 0.5 g of dried and ground using high speed mill (Foss Tecator AB, Hoganas, Sweden) plant tissue was digested in 5 ml mixture of nitric acid and perchloric acid (3 : 1 ratio) on a heating block in a fume hood by gradually raising the temperature to 250 C for 2–3 h or until the sample became clear. The digested samples were diluted to 50 ml with distilled water and filtered. Determination of Cd content was carried out on the above mentioned atomic absorption spectrophotometer. The sample values were compared with the standard series prepared from 1000 mg l)1 stock solution in appropriate concentrations.

Cd Induced Disruption in C and N Metabolism in Bean

Shoot dry weight (mg plant–1)

In vivo NRA was determined as described by Radin et al. (1975). Leaf segments (0.5 g) were transferred to 5 ml of 0.1 m phosphate buffer (pH 7) containing 0.02 m KNO3 and the tubes were incubated in dark at 32 C for 1 h. After incubation, 1 ml of solution was mixed with 0.5 ml of sulphanilamide, immediately shaken and 0.5 ml of N-1-naphthylethyalene diamine diheydrachloride added. After 20 min the absorbance of the coloured complex was read at 542 nm on a spectrophotometer (Hitachi, Model U-2001, Tokyo, Japan) against standard as blank containing all of the above reagents except leaves. For SN estimation, according to the method described by Kowalenco and Lowe (1973), 0.1% stock solution of chromotropic acid disodium salt (CTA) was prepared by dissolving 0.247 g in 100 ml of reagent grade H2SO4. A 0.01% working solution of CTA was prepared by diluting 10 ml with concentrated H2SO4. An aliquote (3 ml) of the sample was mixed with 7 ml of the working solution. Absorbance of yellow CTA–NO3) coloured complex was taken at 430 nm on the spectrophotometer. Final quantities were computed by comparison with a standard NO3) curve.

400

Results Absolute and derived growth attributes Shoot dry weight, although similar in both the varieties under control, indicated greater a reduction in NM-28 (sensitive) than NM-98 (tolerant) under Cd stress, leading to significant difference in varieties, Cd treatments and harvest days together with significant interactions of day · treatments and treatments · varieties (Fig. 1). Time course changes in shoot water content were similar in both varieties, although Cd stress reduced it significantly (Fig. 1). Showing a greater leaf area under control, NM-28 exhibited a steeper decline in this attribute under Cd stress (Fig. 1), thus showing significant differences in varieties, treatments and harvest days together with a significant interaction of day · treatments and treatments · varieties (Fig. 1). Although RGR decreased both under control and Cd stress, the effect of applied Cd in declining

300

200

100

100

Statistical analysis

80 60 V = P > 0.05 C = P < 0.01 D = P > 0.05 V x C = P > 0.05 V x D = P ? 0.05 C x D = P ? 0.05 V x C x D = P > 0.05

40 20 0 40

Leaf area (cm2 plant–1)

In the absence of any remarkable differences in various growth and physiological characteristics for both the years, the data were averaged to perform anova using COSTAT software (COHORT, Monterey, CA, USA), and to find significance and interactions of mungbean varieties, Cd treatments, harvest days and their interactions. Correlations coefficients (r) was determined to find possible relationships of various attributes separately for both the varieties.

V = P < 0.01 C = P < 0.01 D = P < 0.01 V x C = P < 0.01 V x D = P > 0.05 C x D = P < 0.01

0

Shoot water content (%)

In vivo nitrate reductase activity and soluble nitrate concentration

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V = P < 0.05 C = P < 0.01 D = P < 0.01 V x C = P < 0.05 V x D = P > 0.05 C x D = P < 0.01 V x C x D = P > 0.05

32 24 16 8 0 2 4 6

8 10 12

NM-28

2 4 6

8 10 12

NM-98

Days after treatment application Fig. 1: Time course changes in the shoots dry weight, water content and leaf area of differentially Cd-tolerant mungbean varieties grown in the absence (s) or presence () of Cd. In this and subsequent figures, subsets are statistical analysis of data (V, varieties; C, Cd levels and D, harvest days)

RGR was steeper in NM-28, in a steady state in NM-98 and approached control plants towards the end. Varieties, harvest days and Cd levels indicated significant differences, together with significant interactions of varieties · days and varieties ·

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treatments for RGR (Fig. 2). Like RGR, NAR also declined under control and Cd treatments, nonetheless the effect of Cd was well pronounced. Of the two varieties, NM-28 displayed about twofold greater decline in NAR than NM-98 over the experimental period. These changes led to significant differences in the varieties, Cd levels and harvest days and significant interactions of these factors (Fig. 2). For RLER, Cd levels and

RGR (mg mg–1 day–1)

0.08 V = P < 0.01 C = P < 0.01 D = P < 0.01 V x C = P < 0.05 V x D = P > 0.05 C x D = P < 0.01 V x C x D = P > 0.05

0.06

0.04

0.02

harvest days, but not the varieties, showed significant differences. Although with no significant difference in the varieties, applied Cd produced a time course decrease in RLER (Fig. 2). Shoot-Cd concentration and leaf chlorosis Varieties, Cd levels and harvest days revealed significant differences with an interaction of these factors. Shoot-Cd concentration, detected in trace amounts under control condition in both the varieties, indicated a time-dependent accumulation at 12 mg kg)1 Cd level, but with substantial difference between the varieties. Comparison of varieties revealed about 1.5-fold greater Cd concentration in NM-28 than NM-98 (Fig. 3). Leaf chlorosis was nominal in control plants of both the varieties. However, applied Cd produced this symptom in both varieties, being substantially

0.00 50 Cd (mg kg dry weight–1)

NAR (mg cm–2 day–1)

0.6

0.4

V = P < 0.05 C = P < 0.01 D = P < 0.01 V x C = P < 0.01 V x D = P < 0.01 C x D = P < 0.01 V x C x D = P < 0.01

0.2

0

40 30 20 10 0 15

Chlorosis (% of total leaf area)

0.06 RLER (cm2 cm–2 day–1)

V = P < 0.01 C = P < 0.01 D = P < 0.01 V x C = P < 0.01 V x D = P < 0.01 C x D = P < 0.01 V x C x D = P < 0.01

V = P > 0.05 C = P < 0.01 D = P < 0.01 V x C = P > 0.05 V x D = P > 0.05 C x D = P > 0.05 V x C x D = P > 0.05

0.05 0.04 0.03 0.02 0.01

12 9 6 3 0 2 4 6

0

2

4 6

8 10 12

NM-28

2

4 6

8 10 12

NM-98

8 10 12

NM-28

2

4 6

8 10 12

NM-98

Days after treatment application

Days after treatment application Fig. 2: Time course changes in some derived growth parameters of differentially Cd-tolerant mungbean varieties grown in the absence (s) or presence () of Cd

Fig. 3: Time course changes in the shoot-Cd concentration and leaf chlorosis of differentially Cd-tolerant mungbean varieties grown in the absence (s) or presence () of Cd

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greater in NM-28 (13%) than NM-98 (9%) at the final harvest day (Fig. 3). Pn (µmol m–2 s–1)

9

V = P < 0.01 C = P < 0.01 D = P < 0.01 V x C = P < 0.01 V x D = P < 0.01 C x D = P < 0.01 V x C x D = P > 0.05

6 3

E (mmol m–2 s–1)

3

2

V = P > 0.01 C = P < 0.01 D = P < 0.01 V x C = P > 0.01 V x D = P > 0.01 C x D = P > 0.01 V x C x D = P > 0.05

1

0 0.35 gs (mol m-2 s-1)

Soluble nitrate and nitrate reductase activity The SN concentration did not change much in control shoots, but indicated a time course increase under Cd stress. This led to significant differences in varieties, Cd levels and harvest days with significant interactions of these factors (Fig. 5). The NRA indicated slight fluctuations in control shoots but exhibited a time course decline under Cd stress. NM98 exhibited an initial declined but a subsequent improvement in NRA, whilst NM-28 indicated a consistent decline. These changes caused significant differences in varieties, Cd levels and harvest days along with significant interactions of days · treatments and treatment · varieties (Fig. 5).

12

0

0.28 0.21

V = P > 0.05 C = P < 0.01 D = P < 0.05 V x C = P > 0.05 V x D = P > 0.05 C x D = P < 0.05 V x C x D = P > 0.05

0.14 0.07 0 350

Ci (µmol m1–1)

Gas exchange parameters The Pn was similar in the varieties under control, but applied Cd produced a greater reduction in this character in NM-28 as compared with NM-98 over the experimental period. These changes produced significant differences in varieties, Cd treatments and harvest days, and significant interactions of these factors (Fig. 4). Both E and gs although reduced linearly because of Cd stress were not different in the varieties. Moreover, no interaction of varieties, treatments and days was evident, except Cd stress · days interaction for gs (Fig. 4). The level of Ci significantly increased in both varieties under Cd stress over the experimental period, and showed an interaction of varieties · Cd stress (Fig. 4).

15

280 210 V = P < 0.01 C = P < 0.01 D = P < 0.05 V x C = P < 0.01 V x D = P > 0.05 C x D = P > 0.01 V x C x D = P > 0.05

140 70 0

2

Correlations Responses of both varieties for changes in absolute growth parameters (shoot dry weight and leaf area) were similar in control plants (data not shown). However, under Cd-stress RGR and NAR, showing no relationship with gas exchange attributes and NRA in NM-28, were closely related in NM98, although chlorosis and Ci were negatively, and Pn, E and gs were positively correlated. NRA showing no correlation with RGR and NAR in NM-28 exhibited a positive relationship in NM-98 (Table 1). RLER, not correlated with chlorosis, shoot Cd, Ci and SN in NM-28, was negatively related with these attributes in NM-98. Relationships of shoot dry weight, shoot Cd and leaf

4

6

8 10 12

NM -28

2

4

6

8 10 12

NM -98

Days after treatment application

Fig. 4: Time course changes in some gas exchange parameters of differentially Cd-tolerant mungbean varieties grown in the absence (s) or presence () of Cd

chlorosis with gas exchange attributes, NRA and SN although showed a similar trend, but were tighter in NM-98 (Table 1).

Discussion Studies showing time course changes in growth and physiological attributes and their relationships with

NRA (µmol NO2 h-1 g-1 fresh weight)

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Wahid et al.

50 40 30 V = P < 0.01 C = P < 0.01 D = P < 0.01 V x C = P < 0.01 V x D = P > 0.05 C x D = P < 0.01 V x C x D = P > 0.05

20 10 0

NO3 (µmol g-1 dry weight)

1000

V = P < 0.01 C = P < 0.01 D = P < 0.01 V x C = P < 0.01 V x D = P > 0.05 C x D = P < 0.01 V x C x D = P < 0.05

750

500

250

0 2

4

6

8 10 12

NM-28

2

4

6

8 10 12

NM-98

Days after treatment application Fig. 5: Time course changes in the nitrate reductase activity (NRA) and soluble nitrate concentration of differentially Cd-tolerant mungbean varieties grown in the absence (s) or presence () of Cd

accumulation of Cd in the plant parts are scarce. The available information suggests that plant exposure to Cd stress leads to reduced N-assimilation because of influence on NR in pea (Chugh and Sawhney 1999), chlorosis in Brassica napus and mungbean (Baryla et al. 2001, Ghani and Wahid 2007) and oxidative damage on various plants (Chaoui et al. 1997, Smeets et al. 2005, Demirevska-Kepova et al. 2006). This study revealed that NM-98 (tolerant) excelled NM-28 (sensitive) by exhibiting greater shoot dry weight and available photosynthetic area (Fig. 1) and derived growth attributes (Fig. 2). Contrarily, leaf chlorosis and shoot Cd were substantially lower in NM-98, while NM-28 manifested about 1.5 and 2 times greater values of these parameters respectively (Fig. 3), suggesting that Cd accumulation in shoot is a noxious factor in reducing growth. However, varieties did not differ for shoot water content

under applied Cd, which is contrary to earlier report (Perfus-Barbeoch et al. 2002). This further revealed that root hydraulic conductivity was minimally affected in mungbean because of Cd stress. The study of time-dependent changes and their interrelationships have been regarded as functional approaches to appraise the ability of plants to withstand stressful conditions (Poorter 1989, Wahid et al. 1999, Zhang et al. 2002, Wahid 2007). From no prominent changes in RLER and very prominent ones in RGR and NAR (Fig. 2), it appeared that applied Cd reduced the net assimilation capacity of mungbean to utilize available resources from soil and atmosphere for dry matter accumulation. Thus, both attributes can be taken as important criteria for Cd tolerance. Data further suggest that at early growth stage, the changes in growth, leaf chlorosis and shoot-Cd accumulation as manifested by NM-98 are important for better growth in Cd-affected soils. As evident from the above (Figs. 1 and 2), reduced net assimilation capacity is a Cd-sensitivity response in mungbean. As a leguminous species, carbon and nitrogen assimilation are crucial to mungbean because of amino acids biosynthesis (Gouia et al. 2000, Chaffei et al. 2004, Ghnaya et al. 2007). Therefore, plants maintaining coordination between C and N metabolism under stressful conditions are desirable. Various aspects of photosynthesis including gas exchange, photochemical and biochemical processes in chloroplast are first targets of Cd toxicity (Verma and Dubey 2002, Mobin and Khan 2007). Determination of leaf gas exchange parameter revealed that a greater time-dependent reduction in gs and E and an increase in Ci (Fig. 4) indicated a diminished Rubisco activity (Vassilev et al. 2005), which appeared to be mainly responsible for reduced Pn. Similarly, Cd is toxic to NRA, a key enzyme early in N assimilation and amino acid biosynthesis (Gouia et al. 2000, Chaffei et al. 2004). Changes in the in vivo NRA and the level of SN in this study revealed a 1.5-fold greater reduction in NRA of the sensitive variety, which was accompanied with a concomitant rise in the level of SN (Fig. 5). From comparative changes in Pn, NRA and SN, it appears that better net assimilation by leaves of tolerant variety is due to sustained activities of carbon and nitrogen assimilation pathway enzymes in mungbean. Earlier studies on various plant species revealed enhanced metal ions accumulation in various plant

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Table 1: Interrelationships (correlation coefficient, r) of absolute and derived growth parameters, shoot-Cd concentration, shoot dry weight, leaf chlorosis, Pn, NRA and soluble nitrate content of both the mungbean varieties at 12 mg kg)1 level of Cd X-variable Relative growth rate

Net assimilation rate

Relative leaf expansion rate

Shoot-Cd concentration

Shoot dry weight

Chlorosis

Y-variable

NM-28

NM-98

Chlorosis Shoot Cd Net rate of photosynthesis Transpiration rate Stomatal conductance Substomatal CO2 level Nitrate reductase activity Soluble nitrate concentration Chlorosis Shoot Cd Net rate of photosynthesis Transpiration rate Stomatal conductance Substomatal CO2 level Nitrate reductase activity Soluble nitrate concentration Chlorosis Shoot Cd Net rate of photosynthesis Transpiration rate Stomatal conductance Substomatal CO2 level Nitrate reductase activity Soluble nitrate concentration Chlorosis Net rate of photosynthesis Transpiration rate Stomatal conductance Substomatal CO2 level Nitrate reductase activity Soluble nitrate concentration Chlorosis Net rate of photosynthesis Transpiration rate Stomatal conductance Substomatal CO2 level Nitrate reductase activity Soluble nitrate concentration Net rate of photosynthesis Transpiration rate Stomatal conductance Substomatal CO2 level Nitrate reductase activity Soluble nitrate concentration

)0.921** )0.875* 0.723 ns 0.756 ns 0.808 ns )0.805 ns 0.808 ns )0.936** )0.896* )0.845* 0.697 ns 0.721 ns 0.803 ns )0.808 ns 0.781 ns )0.915* )0.759 ns )0.801 ns 0.946** 0.920** 0.825* )0.744 ns 0.874* )0.686 ns 0.992** )0.883** )0.909** )0.995** 0.981** )0.967** 0.969** 0.989** )0.870* )0.897** )0.992** 0.990** )0.958** 0.967** )0.846* )0.883* )0.977** 0.974** )0.946** 0.992**

)0.970** )0.980** 0.970** 0.831* 0.837* )0.966** 0.971** )0.948** )0.955** )0.980** 0.941** 0.803 ns 0.815* )0.947** 0.941** )0.929** )0.915** )0.930** 0.973** 0.753 ns 0.746 ns )0.901* 0.950** )0.873* 0.997** )0.956** )0.907** )0.862* 0.997** )0.981** )0.991** )0.998** )0.935** )0.942** )0.867* 0.998** )0.969** 0.998** )0.939** )0.935** ).855* 0.997** )0.968** 0.993**

Significant at **P £ 0.01, *P £ 0.05 and not significant (ns) at P ‡ 0.05.

parts in time course manner, which led to appearance of foliar toxicity effects, perturbed growth and metabolic activities (Chaoui et al. 1997, Harris and Taylor 2004, Stolt et al. 2006, Ghani and Wahid 2007). Both the varieties in this study indicated a time course accumulation of Cd

in the shoot and an enhanced leaf chlorosis, with a concomitant decline in absolute and derived growth, gas exchange parameters and nitrate assimilation (Figs. 1–3). To substantiate the validity of these findings, changes in derived growth attributes were correlated with shoot-Cd concen-

364

tration, leaf chlorosis, gas exchange parameters, NRA and SN separately of both the varieties at 12 mg kg)1 Cd level (Table 1). Although the trend of changes was similar in both the varieties, most prominent of these relationships were those of RGR and NAR with Pn, Ci and NRA, not present in NM-28, were tighter in NM-98 (Table 1). From tighter relationships of leaf chlorosis with shoot Cd and dry matter yield and negative one with Pn in sensitive variety under Cd stress, it emerged that damage to photosynthetic apparatus because of gradual Cd accumulation in shoot is a specific sensitivity response in mungbean. These findings strongly supported the prediction that Cd had a great effect on the carbon and nitrate assimilation efficiency in utilizing the available resources for growth. In conclusion, Cd perturbs mungbean growth primarily by reducing the net assimilation capacity of shoot mainly by damaging the photosynthetic apparatus and disrupting the coordination between C and N metabolism. Therefore, capacity of shoot to display sustained and coordinated C and N metabolism is important for sustained net assimilation capacity and the growth of mungbean in the soils where Cd is relatively more abundant.

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