Cadmium mobilisation and plant availability the impact of organic acids

Cd ở dạng di động và dạng dễ hấp phụ và các tác động của hợp chất hữu cơ

Plant and Soil 230: 107–113, 2001.

Cadmium mobilisation and plant availability – the impact of organic acids commonly exuded from roots

Rashmi Nigam, Shalini Srivastava, Satya Prakash & M M Srivastava1

Department of Chemistry, Faculty of Science, Dayalbagh Educational Institute, Dayalbagh, Agra-282005, India.

1Corresponding author∗

Received 11 July 2000 Accepted in revised form 14 November 2000

Key words: root exudates, organic acids, complexation, cadmium solubilisation, maize plants

Abstract

The present work highlights metal-organic acid interactions with special reference to their plant availability Pot experiments were conducted to investigate the effect of various organic (carboxylic and amino) acids on the uptake

and translocation of root-absorbed Cd by maize (Zea mays) plants grown in sand and soil culture Statistically

significant increases in Cd accumulation from Cd-treated plants in the presence of increasing concentrations of organic acids, suggest the existence of Cd-organic acid interactions in the soil-plant system In order to support the above hypothesis of formation of organically bound Cd, separate experiments were performed to synthesize and estimate its various forms viz cationic, anionic and neutral The chemical nature of the organically bound forms was ascertained by electrophoretic experiments Amino acids have been found to be less effective in the mobilisation of cadmium compared to carboxylic acids The results are discussed on the basis of the potential of organic acids to form complexes with Cd

Introduction

Concern over the possible health and ecosystem

ef-fects of heavy metals in soils and accumulation in

plants has increased in recent years Among the

vari-ous toxic metals, Cd is of particular concern, because

although it is not an essential element (Kabata-Pendias

and Pendias, 1992), it is readily absorbed and

ac-cumulated in plants, thus increasing the potential

for contamination of the food chain (Galal-Gorchev,

1993) Its long-distance translocation as a metal ion is

limited, probably due to the binding of cations to

ex-change sites located in the xylem cell walls (White et

al., 1981; Wolterbeek et al., 1984) The possible

form-ation of metal chelates or complexes in soils, however,

may result in easy availability of soil Cd and effective

transport of Cd-organic complexes in plants (Senden

and Wolterbeek, 1990; Tiffin, 1970, 1972)

Root exudates released into the rhizosphere have

been implicated in several mechanisms for altering

∗ FAX No.: +0562 281226

the level of soluble ions and molecules within the rhizosphere (Cataldo et al., 1988) Included among the various root exudates are organic acids which are negatively charged anions under a wide range of soil conditions, allowing them to react strongly with metal ions in both the soil aqueous and solid phases (Jones and Darrah, 1994; Jones et al., 1996) The interactions

of organic acids with metals in the soil-plant system are important for solubilising/binding the metals from highly insoluble mineral phases in the soil and have become an area of sustained research

In continuation of our work on mobilisation of metals and their plant availability (Srivastava et al.,

1999 a, b, c), the present communication describes the impact of some organic acids on the uptake and trans-location of root-absorbed Cd in various parts of maize plants grown in sand and soil culture The comparative studies in soil and quartz sand (inert matrix) are expec-ted to highlight the role of organic acids in modifying the chemical nature of Cd supplied and its subsequent uptake by plants Organically bound forms of Cd in the presence of different organic (carboxylic and amino)

acids, have been estimated in separate experiments

using ion exchange chromatography Electrophoretic

experiments have also been conducted to determine

the chemical nature of organically bound Cd

Materials and methods

Pot experiments under laboratory conditions were

per-formed using maize (Zea mays) plants grown for 60

d in sand and soil (2.5 kg) using plastic containers

Quartz sand was used after prescribed washings

(He-witt, 1966) Plants grown in sand were irrigated with

a complete nutrient solution (Hoagland and Arnon,

1950) Various parameters of soil samples were

char-acterised by the Nuclear Research Laboratory (NRL),

Indian Agricultural Research Institute (IARI), New

Delhi The soil used in the experiment has the

fol-lowing characteristics: sandy loam inceptisol, pH 7.4,

EC 0.23 ds/m, organic carbon 0.8 g/kg, total Cd

0.012 µmol/L, bulk density 1.25 g/cm3, CEC (cation

exchange capacity ) 25.7 cmol (+)/kg, soluble ions

Ca 108, Mg 432, Na 1403, K 35, Cl 2023, SO4

624 mg/kg A basal dose of N:P:K (60:20:18 mg /kg of

soil ) was initially supplied The plants were irrigated

with distilled water as and when required

A carrier solution of cadmium nitrate was tagged

with Cd-115 m radiotracer.Treatments started by

ap-plying to the surface of growing media of

60-day-old plants a single pulse addition of a solution (200

mL) containing radiolabelled Cd (44 µm) with

vary-ing amount of different organic acids: citric acid

(23,237,474,1189,2379 µm), malic acid (37, 373,

746, 1865, 3731µM), aspartic acid (37, 375, 750,

1878, 3756 µm) and glycine (66, 666, 1333, 3333,

6666 µm) in order to obtain Cd: organic acid

ra-tios (1:1,1:10,1:20,1:50 and1: 100 w/w) These are the

predominant acids released by maize plants in root

exudates (Mench and Martin, 1991) The pH of the

solution was finally adjusted to 5.5 with 0.1 N HCl

Cd-115m was obtained from Board of Radiation and

Isotope Technology (BRIT), BARC, Mumbai Plants

were grown with these treatments for 10 d Natural

light (diurnal cycle of 14 h) was supplemented with

Phillips Fluorescent tubes (40 W) and Toshiba lamps

(15 W) providing an irradiation of approximately 600

W/m2 at the plant tops Plants were harvested and

washed thoroughly with tap water, followed by pH 4

water and distilled water They were then cut into root,

shoot and edible parts and packed into plastic vials for

oven drying (50◦C) to obtain dry matter yields The

pH of the final washings were tested to ensure that no detectable acidity was left

Accurately weighed amounts of plant material were counted over a planar NaI (TI) detector coupled

to a 4 K MCA ( Canberra Accuspec Card with PC-AT 386) The counting geometry was pre calibrated for ef-ficiency with known amount of Cd-115m activity from the 0.934 MeV photopeak area The activity of Cd-115m was calculated and reported as Cd in different parts of plant per gram of dry weight

Source-to-plant transfer coefficients (SPT) for Cd with increasing organic acid supplementation in both sand and soil medium were calculated by dividing the

Cd concentration in the plants (DW) by Cd concentra-tion in feeding soluconcentra-tion

A quantity of Cd (100 µm) was taken in

Er-lenmeyer flasks and radiolabelled with Cd-115m

tracer Organic acids; Carboxylic acids: Citric acid and Oxalic acid: Amino acids: Aspartic acid and

Glutamic acid were added separately in the ratio (1:20 w/w).Ionic strength was maintained by adding KNO3 solution (20 mM) After adjusting to pH 6, the solution was shaken for 16 h and the supernatant was removed after centrifuging for 45 min The supernatant solu-tion, representing the organically bound form, was subjected to chemical speciation Based on the inher-ent capability of the combination of radiotracer and ion-exchange resins, i.e Amberlite XAD (neutral), Dowex-50 (cationic) and Dowex-1 (anionic) (Batley, 1991), the percentage of neutral, cationic and anionic forms of the organically bound Cd was estimated The quantification (%) of different forms of organically bound Cd was calculated by difference, using the pro-cedure of Deb et al.(1976) Electrophoretic radioassay was also carried out to ascertain the existence of or-ganically bound Cd in neutral, anionic and cationic forms

The data represent the mean of three replicates

of four plants per pot Statistical analyses were per-formed using SPSS/PC+M software package Testing for non-normal data distributions was computed by Mann Whitney (independent) U test comparing indi-vidual means Correlation coefficients were used to relate concentration in root and aerial parts to various organic acid treatments

Results

Table 1 shows the accumulation of Cd in various plant

parts supplied with 44 µm of Cd in the presence

Table 1 Plant tissue concentrations of cadmium (µg/g dry weight) in maize plants

supplied with cadmium (44 µm) in the presence of varying concentrations of organic

acids

acids

Citric acid

Malic acid

Aspartic acid

Glycine

Cd Conc 41 ±6 18 ±2 6.1 ±0.9 31 ±3 9 ±0.5 6.2 ±0.3 without

Org acid (control exp.) Values ±SD

of increasing concentrations of various organic acids

The distribution of Cd in various parts of maize plant

shows the following order: root > shoot > fruit.

Cd added with organic acids (1:1, 1:10, 1:20, 1:50

and 1:100 w/w) resulted in statistically significant

in-creases in Cd accumulation in root and aerial parts of

the plant in both the sand and soil cultures (p≤ 0.03)

Relatively higher increases in Cd accumulation were observed in plants grown in sand (Mann Whitney

U-test p≤ 0.03)

The effect of organic acid amendments on the Cd enrichment from the Cd treatment has been calculated

in terms of the source-to-plant transfer (SPT) coeffi-cient (Table 2) Experiments showed that the affinity

Table 2 Source-to-plant transfer coefficients for cadmium in maize plants treated with cadmium in

the presence of organic acid supplementation

Cd:Org Citric Malic Aspartic Glycine Citric Malic Aspartic Glycine

of organic acids for complexation with Cd was (Mann

Whitney U-test): citric > malic > aspartic≈ glycine

To support the hypothesis of the formation of

organically-bound Cd, separate experiments were

per-formed to synthesize organically-bound Cd [Cd- citric

acid, Cd-malic acid and Cd-aspartic acid] and are

depicted in Figure 1

Discussion

The distribution of Cd in the plant tissues (root, shoot

and fruit) indicated that 70–80% of the Cd was

re-tained in roots and only a small proportion, was

trans-located to aerial parts Jarvis et al (1976) also reported

that more than 70% of supplied Cd was incorporated

in roots of Zea mays and other plants Increasing

con-centrations of organic acids increased plant uptake of

Cd, with a similar trend of distribution ratio as

ob-tained in control experiments where no organic acid

was provided

An increase in Cd uptake from the Cd treatments

with increasing supplementation of organic acids may

be ascribed to the interaction of Cd with organic

ligands leading to the formation of mobile

organically-bound Cd Peterson and Alloway (1979) have also

recorded that organically complexed Cd was more

readily translocated than similar amounts of the ionic

form.Organic acids such as citric,malic,oxalic,aspartic

and glutamic acids, have been reported as being

po-tential metal chelators (Nakayama, 1981; Naidu and

Harter, 1998)

Higher uptake of Cd from the treatment of Cd

with organic acids occurred in plants grown in sand

as compared to soil (Table 1) Quartz sand being

in-ert in nature, does not have a sorption tendency for

Cd, therefore, provides a better site for Cd- organic

acid complexation Moreover, the slower degradation

of the organic complex of the cadmium is accepted

in the sand medium On the contrary, soil has greater capacity to adsorb Cd2+ ions (Haghiri, 1974; Miller

et al., 1976) and thus reduces the extent of Cd or-ganic acid complexation, resulting in a lower plant availability There was an increasing trend in SPT val-ues of Cd with increasing concentration of organic acids provides further support to the demonstration of the existence of Cd-organic acid interaction (Table 2) SPT coefficients for Cd uptake when no organic acid was provided, are considered as a reference standard

Poor correlation (non-significant at p< 0.03)

between the dry matter yield and organic acids ad-dition indicates that the treatments imposed have no toxic effects The non-toxic behaviour in spite of Cd accumulation in the plants, may be ascribed to the fact that organic ligands not only enhance the solu-bility of the trace metals, but also reduce their toxicity

to plants (Sposito, 1985) The free trace metal ions are reported to be more toxic compared with organic-ally complexed molecules (Xue Dongsen et al., 1995) However, a slight decrease was observed in dry matter yield of plants grown in sand culture amended with the highest concentration of citric acid ( Cd: Citric acid; 1:100)

The metal-solubilising ability of the organic acids

is parallel to their metal binding ability (Mench and Martin, 1991) which in turn is correlated with their dissociation constants The dissociation con-stants (Ka1, Ka2) for citric acid (7.10× 10−4,1.68×

10−4), malic acid (3.9× 10−4, 7.8× 10−6), aspartic acid (1.38× 10−4) and glycine (1.67× 10−10) are in conformity with the order obtained in our experiments From Table 1, Cd complexation and resulting up-take are low for amino acids as compared to carboxylic

Figure 1 % of formation of organically bound form of Cd with citric, malic and aspartic acids.

acids Carboxylic acids, particularly citric and malic

acids, can bind divalent cations strongly and form

stable complexes (Cieslinski et al., 1998; Senden and

Wolterbeek, 1990) Furthermore, the efficiency of

cit-ric acid towards metal complexation over malic acid

has been previously reported (White et al., 1981) It is

also suggested that proteinaceous amino acids released

into the rhizosphere do not play a major role in

mobil-ising metals from the soil (Jones et al., 1994) Costa

(1997) has indicated that amino acids effect

complex-ation to lesser extent than carboxylic acids because of

their limited nutrient mobilisation capacity

In recent years, it has been emphasised that

consid-eration of total metal concentration does not provide

the real picture of bioaccumulation It needs

inform-ation regarding various physicochemical forms of the

metals Research attention has been focussed on the

formation of organically bound forms of the metals

virtually responsible for their uptake by plants by in

vivo and in vitro experiments Any resulting complex

of a metal and a ligand (organic acid) might have a

net charge that can be negative, positive or neutral

Cataldo et al (1988) complexed the whole exudates

of soyabean plants with the Cd2+in vitro and reported

the existence of anionic and cationic forms of

organic-ally bound Cd However, the electrophoretic shape of

the anionic component suggests that it is near neutral

in charge

We have conducted separate experiments on

syn-thesis, electrophoretic nature (Figure 2) and estimation

of various forms of organically bound Cd (Figure 1) Results clearly show the existence of all the three forms – neutral, anionic and cationic species of or-ganically bound Cd However, the cationic form has been found to be predominant in each case Our ob-servations are supported by the Donnan dialysis quan-tification (Cox et al., 1984) showing that about 84% of the Cd present in the plant extract was in the cationic form which is more labile The affinity of organic acids under study to form cationic complex is found to be as follows:

citric > malic > aspartic

Conclusions

Plant uptake of Cd from the Cd treatments with in-creasing concentration of organic acids seems to be the resultant of the interactions of Cd with organic lig-ands resulting in the formation of mobile organically bound Cd The experiments highlight Cd: organic acid interactions as a major contributor for cadmium uptake

by plants than similar amounts of the ionic form The extent of Cd complexation and its resultant uptake is less for amino acids as compared to carboxylic acids Attempts on chemical speciation of organically bound forms existing in neutral, anionic and cationic nature provide support to the above facts

Figure 2 Plot of counts vs migration (cm) on the electrophoretic

strip for Cd (a) citric acid (b) malic acid.

Acknowledgements

The authors are grateful to Prof P.S Satsangi,

Dir-ector, Dayalbagh Educational Institute, Agra for

providing necessary facilities Financial support given

by Board of Studies in Nuclear Science (BRNS),

Department of Atomic Energy (DAE) is gratefully

acknowledged Thanks are due to Project Director,

Nuclear Research Laboratory, Indian Agricultural

Re-search Institute, New Delhi for help in getting soil

sample analysed

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