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. © 2001 Kluwer Academic Publishers. Printed in the Netherlands. 107 Cadmium mobilisation and plant availability – the impact of organic acids commonly exuded from roots Rashmi Nigam, Shalini Srivastava, Satya Prakash & M. M. Srivastava 1 Department of Chemistry, Faculty of Science, Dayalbagh Educational Institute, Dayalbagh, Agra-282005, India. 1 Corresponding 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 althoughit 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 uptakeby plants. Organicallyboundformsof Cd in the presence of different organic (carboxylic and amino) 108 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/cm 3 , CEC (cation exchange capacity ) 25.7 cmol (+)/kg, soluble ions Ca 108, Mg 432, Na 1403, K 35, Cl 2023, SO 4 624 mg/kg. Abasal 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/m 2 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 solution. 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 KNO 3 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 109 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 Conc. ratio Sand Culture Soil Culture Cd:Org. Root Shoot Fruit Root Shoot Fruit acids Citric acid 1:1 93±933±68.3±1.0 78±719±37.5±0.7 1:10 138±544±5 10.4±0.9 88±623±49.8±0.9 1:20 144±454±4 11.6±0.8 95±531±6 10.8±0.3 1:50 152±759±4 13.5±1.1 112±840±5 11.5±0.1 1:100 172±866±5 15.4±1.2 128±747±5 13.4±0.2 p 0.020 0.001 0.000 0.020 0.001 0.004 Malic acid 1:1 90±830±48.0±0.8 70±815±37.2±0.6 1:10 132±636±49.3±0.7 80±722±49.2±1.0 1:20 141±642±5 10.1±0.5 89±830±6 10.1±0.6 1:50 149±449±6 12.2±0.6 108±937±3 11.1±0.5 1:100 155±458±7 14.2±1.5 119±841±4 12.8±0.7 p 0.036 0.000 0.002 0.004 0.001 0.001 Aspartic acid 1:1 53±723±37.8±0.3 38±611±37.0±0.8 1:10 63±832±38.3±0.3 45±518±58.0±0.4 1:20 75±637±49.9±0.2 57±627±49.5±0.5 1:50 94±742±4 11.1±0.1 65±732±3 10.2±0.6 1:100 106±846±3 12.8±0.3 84±636±3 11.9±0.5 p 0.001 0.001 0.002 0.002 0.001 0.001 Glycine 1:1 49±821±27.7±0.3 35±410±26.8±0.8 1:10 60±628±48.2±0.1 41±415±48.0±0.3 1:20 69±734±49.5±0.5 53±622±59.1±0.6 1:50 91±841±3 10.6±0.4 63±728±49.9±0.3 1:100 101±646±4 11.5±1.1 80±10 35±4 10.5±0.2 p 0.002 0.001 0.001 0.020 0.000 0.001 Cd Conc. 41±618±26.1±0.9 31±39±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 110 Table 2. Source-to-plant transfer coefficients for cadmium in maize plants treated with cadmium in the presence of organic acid supplementation Conc. ratio Cd in Sand Cd in Soil Cd:Org. Citric Malic Aspartic Glycine Citric Malic Aspartic Glycine acid acid acid acid acid acid acid 0 13.0 13.0 13.0 13.0 9.2 9.2 9.2 9.2 1:1 26.9 25.6 16.8 15.5 20.7 18.4 11.2 10.4 1:10 38.5 35.7 20.7 19.2 24.2 22.2 14.2 12.8 1:20 41.9 38.6 24.4 22.5 27.4 25.8 18.7 16.8 1:50 44.9 42.0 29.4 28.5 32.7 31.2 21.4 20.2 1:100 50.7 45.4 31.8 31.7 37.7 34.6 26.4 25.3 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-boundCd, 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 Cd 2+ 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 (Ka 1 ,Ka 2 ) 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 )arein conformitywith the order obtainedin our experiments. From Table 1, Cd complexation and resulting up- take are low for amino acids as comparedto carboxylic 111 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 recentyears, 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 Cd 2+ 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, electrophoreticnature (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 organicacids 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: organicacid interactionsas a major contributorforcadmiumuptake 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. 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Sci. 7, 399–406. . 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. 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