Changes of organic acid exudation and rhizosphere pH in rice plants under chromium stress

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Environmental Pollution 155 (2008) 284e289 www.elsevier.com/locate/envpol

Changes of organic acid exudation and rhizosphere pH in rice plants under chromium stress Fanrong Zeng, Song Chen, Ying Miao, Feibo Wu, Guoping Zhang* Department of Agronomy, College of Agriculture and Biotechnology, Huajiachi Campus, Zhejiang University, Hangzhou 310029, China Received 3 September 2007; received in revised form 11 November 2007; accepted 18 November 2007

Rhizosphere pH and organic acid exudation of rice roots are markedly affected by chromium level in culture solution. Abstract The effect of chromium (Cr) stress on the changes of rhizosphere pH, organic acid exudation, and Cr accumulation in plants was studied using two rice genotypes differing in grain Cr accumulation. The results showed that rhizosphere pH increased with increasing level of Cr in the culture solution and with an extended time of Cr exposure. Among the six organic acids examined in this experiment, oxalic and malic acid contents were relatively higher, and had a significant positive correlation with the rhizosphere pH, indicating that they play an important role in changing rhizosphere pH. The Cr content in roots was significantly higher than that in stems and leaves. Cr accumulation in plants was significantly and positively correlated with rhizosphere pH, and the exudation of oxalic, malic and citric acids, suggesting that an increase in rhizosphere pH, and exudation of oxalic, malic and citric acid enhances Cr accumulation in rice plants. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Chromium; Rhizosphere; Organic acid; pH; Rice (Oryza sativa L.)

1. Introduction In recent years, heavy metal contamination, as a consequence of mining, manufacture and disposal of mineral ores, metal products and waste, has become a serious problem over the world (Boominathan and Doran, 2003). The toxicity of heavy metals to plants is well documented at various levels, from reduced yield, through effects on leaf and root growth, to inhibition on enzymatic activities. Moreover, decrease in root growth is a most common effect due to the toxicity of heavy metals in trees and crops (Breckle, 1991; Godbold and Kettner, 1991; Tang et al., 2001). Rhizosphere is a dynamic micro-environment, in which water and nutrients are taken up and many substances such as sugar, organic acids, amino acids, enzymes, endogenous hormones, and some secondary metabolites are secreted constantly by plant roots. Under environmental stress, plant roots would excrete * Corresponding author. Tel.: þ86 571 8697 1115; fax: þ86 571 8697 1117. E-mail address: [email protected] (G. Zhang). 0269-7491/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.envpol.2007.11.019

more metabolites and some unknown substances (Lo´pez-Bucio et al., 2000). It has been found that organic acids are involved in heavy metal tolerance, transport and storage in plants (Godbold et al., 1984; Krotz et al., 1989; Ma et al., 2001; Mathys, 1977; Nigam et al., 2001; Tiffin, 1970; Yang et al., 1997). According to Ma and Furukawa, 2003, plant roots under Al stress might exude various organic acids, including citric and malic acids, and these organic acids could play a very important role in alleviating Al toxicity in soil. Understanding organic acid exudation and its mechanism with plants exposed to abiotic stress may provide an effective approach for illustrating fundamental aspects of plant physiology, and making new strategies of developing crop varieties with high tolerance to environmental stress, including heavy metal stress (Pellet et al., 1997). However, chromium, unlike other toxic trace metals including cadmium, lead, mercury and aluminum, has received little attention from plant scientists (Shanker et al., 2005). Up to date, studies on chromium phytotoxicity to plants are mainly focused on the inhibition of seed germination, pigment synthesis, nutrient balance, and antioxidant enzymes in plants

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(Barcelo and Poschenrieder, 1997; Panda, 2003; Panda and Khan, 2003; Panda and Patra, 1997, 1998, 2000; Poschenrieder et al., 1991). Little effort was made to study the effects of chromium on the properties of rhizosphere. Moreover, chromium toxicity to plants depends on the species of this metal. Though there are main two stable oxidation states for chromium, Cr6þ and Cr3þ, Cr6þ is considered to be more toxic than Cr3þ (Panda and Patra, 1997). In addition, Cr6þ can be reduced to Cr3þ by redox reactions. It is reported that Fe2þ (Buerge and Hug, 1997; Eary and Rai, 1988), organic matter (Wittbrodt and Palmer, 1995), and reduced S (Patterson et al., 1997) can reduce Cr6þ to Cr3þ, and this redox reaction is pH dependent, increasing with a lower pH value. Therefore, it can be predicted that the reduction of Cr6þ to Cr3þ is closely related to a rhizosphere environment. When plants are exposed to Cr stress, the rhizosphere environment will be altered correspondingly, and in turn the alteration affects the form of Cr in the rhizosphere. Understanding of the complex relationship is quite imperative to unravel the mechanism of Cr toxicity and tolerance in plants. Therefore, in order to determine the response of plants to Cr toxicity and develop crop varieties with high Cr tolerance, it is necessary to determine the variation of rhizosphere properties under Cr stress. Plants have a remarkable ability to absorb, translocate and accumulate heavy metals and organic compounds from the environment. In order to maintain their charge balance, roots release protons whenever they take up more cations than anions, and take up protons when the opposite occurs (Hinsinger et al., 2003). Thereby, plant roots are responsible for substantial changes of rhizosphere pH. Root-induced changes of rhizosphere pH have a distinct impact on bioavailability of many pH-dependent nutrients and potentially toxic metals including Cd, Cr, and Hg. The decrease in the rhizosphere pH (acidification) increases the solubility of Al- or Zn-bearing compounds, leading to an enhanced uptake of Al or Zn in the plant (Calba et al., 2004; Loosemore et al., 2004). In contrast, to some extent, when rhizosphere pH is increased, the solubility of Cu in the soil can be significantly reduced (Chaignon et al., 2004). Such pH changes that occur in the rhizosphere as a consequence of root activities have a dramatic influence on the biogeochemistry of a whole range of elements, from major and minor nutrients to potentially toxic metals and other trace elements (Hinsinger et al., 2005). In this study, two rice genotypes differing in grain Cr accumulation according to a previous study (Zeng et al., 2007), Xiushui 113 and Dan K5, were planted in a hydroponic solution with four external Cr concentrations to (1) determine the variation of rhizosphere properties in rice under chromium stress; and (2) find out the relationships between Cr accumulation and rhizosphere pH and organic acid exudation, respectively.

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surface sterilized in a 2% H2O2 solution for 10 min, rinsed with tap water for 20 min, and then washed with deionized water 5 times. Seeds were then soaked in deionized water in the dark at 25  C for 2 days, germinated on a plastic net floating on deionized water at 35  C for 1 day, and then sown into sand in a controlled chamber with photoperiod of 16 h light/8 h dark and light intensity of 225  25 mmol m2 s1. The light/dark temperatures were set at 32  C / 25  C, and relative humidity was kept at 85%. After 15 days, rice seedlings with similar size were selected and transplanted into a 40 L plastic pot containing nutrient solution (48 seedlings per pot) and cultivated in the hydroponic form. The nutrient solution was prepared according to Yoshida et al. (1976) with the following salts: NH4NO3 2.9 mM, NaH2PO4 0.32 mM, K2SO4 1.0 mM, CaCl2 1.0 mM, MgSO4 $ 7H2O 1.7 mM, MnCl2 $ 4H2O 9.1  103 mM, (NH4)6MoO24 $ 4H2O 5.2  104 mM, H3BO3 1.8  102 mM, ZnSO4 $ 7H2O 1.5  104 mM, CuSO4 $ 5H2O 1.6  104 mM, FeCl3 $ 6H2O 3.6  102 mM. The pH value of culture solution was adjusted to 5.10 using 1 M HCl or NaOH solution as required. Half concentration of the nutrient solution was applied for the first 3 days and then changed to full nutrient solution for 1 week. Thereafter, different amounts of K2Cr2O7 were added to the nutrient solutions to form 4 Cr levels, i.e. 0, 10, 50 and 100 mM, respectively. Nutrient solutions were renewed every four days (solution-renewing cycle).

2.2. Measurement of rhizosphere pH The pH values of the nutrient solutions containing different Cr concentrations were measured with a PHS-25 digital display acidometer (Shanghai Precision Instruments Co., China). Measurements were made at the last day in the second (8-day) and fourth (16-day) solution-renewing cycle. Five replications were undertaken for each treatment.

2.3. Collection and measurement of organic acid To collect root exudates, the roots of 5 rice plants were washed with tap water, distilled water and deionized water 3 times respectively, and then exposed to 150 ml of 0.5 mM CaCl2 solution (pH 5.10) with corresponding Cr levels for 12 h. Thereafter, the rice roots were washed with deionized water and the collected root exudates were diluted to 500 ml with deionized water. The treatment solution were filtrated, dried in a rotary evaporator on a hot plate at 45  C and the residue was then dissolved in 25 ml of deionized water for the measurement of organic acid. The sample solution was passed through a cation exchange column (16  14 mM) filled with 5 g of Amberlite IR-120B resin (Hþ form, Shanghai Chemical Reagent Co., China), followed by an anionexchange column (16  14 mM) filled with 2 g of Dowex 1  8 resin (100e 200 mesh, format form, Shanghai Chemical Reagent Co.). The organic acids retained on anion-exchange resin were eluted by 1 M HCl, and the elute was concentrated to 10 ml. Concentrated solutions (20 ml) were injected into a Symmetry C18 column (3.9 mM i.d.  150 mM, 5 mm). The quantitative determination of organic acids was carried out using a high-performance liquid chromatography (Waters 515, Waters Corp., MA, USA). The mobile phase used was 0.01 mol l1 (NH4)2HPO4 (pH 2.5) at a flow rate of 0.8 ml min1. Detection was conducted at a wavelength of 214 nm, and the column temperature was 20  C.

2.4. Determination of chromium concentration in rice plants

2. Materials and methods

Five rice plants were washed with tap water, distilled water and deionized water 3 times respectively, and then emerged into 20 mM EDTA-Na2 for 3 h to remove the metals from plant surface. After that, the rice plants were separated into roots, stems and leaves, and dried at 80  C in an oven for 48 h. The samples were weighted, ground, dry-ashed, extracted with 1:1 HNO3, concentrated to a certain volume and finally filtered. Cr concentrations in the samples were determined by a flame atomic absorption spectrometry (AA6300, Shimadzu, Tokyo, Japan). Three replications were undertaken for each treatment.

2.1. Growth of rice seedlings

2.5. Statistical analysis

The uniform seeds of two rice genotypes, Xiushui 113 (Cr sensitive, low Cr accumulation) and Dan K5 (Cr tolerant, high Cr accumulation), were

The data were analyzed using a statistical package, SPSS version 11.50 (SPSS, Chicago, IL). A one-way variance analysis (ANOVA) was carried

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out, followed by the Tukey HSD (honestly significant difference) test. In addition, Pearson correlation coefficients were calculated to determine the relationship between Cr accumulation and rhizosphere pH and organic acid exudation.

3. Results 3.1. Change of rhizosphere pH The effects of Cr level, exposure time on rhizosphere pH of the two rice genotypes are shown in Table 1. In general, rhizosphere pH increased with the increased Cr level in the nutrient solution. When plants were exposed to relatively low Cr levels, no significant increases in rhizosphere pH were found. However, rhizosphere pH showed significant increases when the Cr level in nutrient solution was 50 mM or above. Moreover, rhizosphere pH tended to be higher with the plant growth after culture solution was renewed, and the plants exposed to Cr stress showed more rapid pH increase. In addition, there was a significant difference between the two rice genotypes in the response of rhizosphere pH to Cr stress, with Xiushui 113 having higher pH in low Cr level (50 mM or less) and lower pH values in high Cr level (100 mM) than Dan K5. 3.2. Change of organic acid exudation in rice root The effects of Cr stress on organic acid exudation are presented in Table 2. Among organic acids measured in this study, oxalic and malic acid contents were much higher than the other four organic acids. Cr stress markedly increased oxalic acid exudation, irrespective of rice genotypes and the time of Cr exposure. The malic acid exudation in rice roots varied greatly with the time of Cr exposure and rice genotype. At the 8th day of Cr exposure, malic acid exudation showed a decrease in the treatment of 10 mM Cr relative to that of the control (without Cr addition), and an increase in the treatment of 50 mM Cr; and then again showed decline in the treatment of 100 mM Cr. Both Xiushui 113 and Dan K5 had the maximum malic acid exudation in the treatment of 50 mM Cr. When the Table 1 Effects of different Cr levels on rhizosphere pH value of two rice genotypes Genotype

Xiushui 113

Dan K5

Cr treatment (mM)

pH value 8 days of Cr exposure

16 days of Cr exposure

0 10 50 100 0 10 50 100

3.78 3.85 4.91 5.23 3.72 3.74 4.78 5.35

4.05 4.36 5.28 5.67 4.21 4.22 4.99 5.71

0.09 0.07 Sa

0.04 0.03 S

Genotype (LSD0.05) Cr level (LSD0.05) Interaction a

S: significant at 95% probability level.

time of Cr exposure was prolonged to 16 days, malic acid exudation in Dan K5 showed a massive increase under 100 mM Cr, while that in Xiushui 113 still decreased under 100 mM Cr compared to other Cr concentrations. There was little exudation for both lactic and acetic acids, irrespective of Cr level and exposure time. Similar to malic acid, citric acid exudation was dependent on the time of Cr exposure and rice genotypes. At the 8th day of Cr exposure, citric acid exudation of the two genotypes showed an increase with increased Cr level up to 50 mM, and declined when the level of Cr increased to 100 mM. In contrast, at the 16th day of Cr exposure, both genotypes had the maximum citric acid exudation under 100 mM Cr. Significant differences were found for succinic acid exudation among Cr levels and between two genotypes. At the 8th day of Cr exposure, Dan K5 showed the highest succinic acid exudation under 50 mM and as the exposure time extended, Dan K5 under 100 mM stress had the highest succinic acid exudation. 3.3. Cr concentration and accumulation in rice plant Cr concentration and accumulation in rice plants are shown in Table 3. The total Cr concentration in the seedlings of both genotypes increased with increased Cr level in the nutrient solution. However, Xiushui 113 did not show a further increase when the level of Cr was increased from 50 mM to 100 mM. Cr concentration in the roots was much higher than that in the stem and leaf, indicating that the root is the main part for Cr accumulation in a rice plant. Cr accumulation per plant also increased with increased Cr level in the nutrient solution. Again, Xiushui 113 had the maximum Cr accumulation in the treatment of 50 mM Cr, which may be attributed to highest Cr concentration in roots and greater root weight in the treatment. 3.4. Relationships between Cr accumulation, pH value and organic acid exudation Pearson correlation coefficients were calculated between Cr accumulation, rhizosphere pH and organic acid exudation (Table 4). Cr accumulation showed significantly synergistic relationships with rhizosphere pH and oxalic, malic and citric acid exudation, respectively, indicating that the increase of rhizosphere pH and oxalic, malic and citric acid exudation would cause more Cr accumulation in rice plants. Significant correlations were also detected between rhizosphere pH and oxalic, malic and citric acid exudation, suggesting that these three organic acids exudation might also play an important role in the variation of rhizosphere pH. No significant correlations were found between Cr accumulation and lactic, acetic or succinic acid. 4. Discussion The variation of rhizosphere pH has been observed in many researches (Foy et al., 1965; Hinsinger et al., 2005; Pellet et al., 1997). In the present experiment, the rhizosphere pH

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Table 2 Effects of different Cr levels on organic acid contents in root exudation of two rice genotypes Days of Cr exposure

Genotype

Cr level (mM)

Organic acid concentration (mg plant1) Oxalic

Malic

Lactic

Acetic

Citric

Succinic

8

Xiushui 113

0 10 50 100 0 10 50 100

0.14 0.22 0.91 2.00 0.21 0.27 1.06 1.54 0.27 0.19 Sb

0.14 0.11 0.20 0.01 0.53 0.14 1.10 0.06 0.02 0.01 S

NDa ND 0.06 0.03 0.06 0.05 0.01 0.03 0.01 0.01 NSb

ND 0.04 0.02 0.04 ND ND 0.03 0.03 0.01 0.01 S

0.11 0.13 0.17 0.05 0.03 0.03 0.11 0.05 0.01 0.01 S

ND ND 0.04 0.11 0.16 0.10 0.43 0.12 0.05 0.04 S

0 10 50 100 0 10 50 100

0.14 0.22 1.47 1.28 0.28 0.23 1.49 1.87 0.39 0.27 NS

0.11 0.38 0.20 0.14 0.56 0.20 0.03 2.94 0.02 0.02 S

ND 0.09 0.02 0.03 0.16 0.03 ND 0.21 0.07 0.05 S

ND 0.03 ND ND 0.03 0.04 0.04 0.02 0.03 0.03 NS

0.02 0.02 0.06 0.07 0.04 0.03 0.05 0.18 0.02 0.01 S

0.04 0.13 0.15 0.15 0.13 0.04 0.16 0.98 0.04 0.03 S

Dan K5

Genotype LSD0.05 Cr level LSD0.05 Interaction 16

Xiushui 113

Dan K5

Genotype LSD0.05 Cr level LSD0.05 Interaction a b

ND, not detected. S and NS: significant and not significant at 95% probability level.

showed a significant increase with increased Cr level in the nutrient solution. There are several possible explanations for the increase of rhizosphere pH. As reported by Song et al. (2004), the rhizosphere pH is formed from (1) the absorption of cation and anion being out of balance; (2) CO2 being generated by rhizosphere breath; and (3) the secretion of organic acids, Hþ, and other chemical components from the roots. Tao et al. (2003) suggested that root-induced changes in rhizosphere pH occurred primarily as a consequence of differential rates in the uptake of cations and anions by plants. Nye (1981) reported that Al3þ stress can also lead to the release of HCO 3 from the roots into the rhizosphere to maintain electrical Table 3 Cr concentration and accumulation of the two rice cultivars under different Cr level Genotype

Cr level Cr concentration (mg g1) (mM) Root Stem Leaf

Accumulation (mg plant1)

Xiushui 113

0 10 50 100 0 10 50 100

Dan K5

Genotype (LSD0.05) Cr level (LSD0.05) Interaction a

22.29 629.51 2449.46 2285.42 22.92 599.24 973.10 2718.70

9.64 12.37 37.71 120.46 7.34 9.62 37.99 122.61

9.50 11.63 37.82 75.35 8.86 7.59 38.88 85.20

157.40 1062.58 4254.41 2829.85 140.74 1203.04 2349.83 4434.07

118.78 83.99 Sa

56.05 39.63 NSa

9.75 6.89 NS

161.52 114.21 S

S, significant; NS, not significant.

neutrality. Miyasaka et al. (1989) reported that increase of pH was probably related to rhizosphere Ca2þ concentration. Cr stress caused strong inhibition of both Hþ and Kþ uptake, thus resulting in increase of pH (Zaccheo et al., 1985). The decrease in ATPase activity leads to a decrease in proton extrusion, causing the change of rhizosphere pH (Astolfi et al., 2003). In this experiment, the significant correlations between rhizosphere pH and oxalic, malic and citric acid exudation (Table 4) indicated that the increase of rhizosphere pH would strongly stimulate the exudation of these organic acids from rice roots. Organic acids in plant root exudates are directly involved in a number of soil processes, such as plant metabolism, metal detoxification, and nutrient solubilization during plant growth (Jorge and Arruda, 1997). Their quantitative role in these processes, however, has been difficult to establish due to the influence of many interdependent factors, including solid phase sorption/desorption reactions, metal complexation reactions, leaching, and microbial degradation (Morita et al., 2004). The current results showed that the exudation of organic acids was changeable in response to Cr stress in different levels. Either the amount or the species of organic acids exuded from rice roots under Cr stress increased with the Cr level in the nutrient solution. Among the six organic acids detected in the experiment, oxalic acid and malic acid played the most important role in the variation of organic acid exudation in the rhizosphere. These organic acids could be important in alleviating phytotoxicity induced by Cr stress. There is little or no conclusive evidence about whether these acids play a protective role and what level of organic acid content is sufficient for providing

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Table 4 Correlative coefficients among Cr accumulation, organic acid secretion and rhizosphere pH

pH Oxalic Malic Lactic Acetic Citric Succinic

Cr accumulation

pH

Oxalic

Malic

Lactic

Acetic

Citric

0.912** 0.916** 0.497* 0.142 0.267 0.751** 0.397

0.921** 0.462* 0.168 0.320 0.767** 0.379

0.474* 0.143 0.173 0.752** 0.426*

0.828** 0.062 0.887** 0.867**

0.268 0.608** 0.555**

0.119 0.185

0.774**

*Significant and **highly significant at 95% and 99% probability level, respectively.

direct protection against Cr toxicity in the rhizosphere. Further research is required for illustration of these issues. In this study, Cr concentrations in plant tissues of the two rice genotypes increased with increased Cr levels, although Xiushui 113 showed the maximum root Cr concentration under 50 mM level. Cr concentration in rice root was much higher than that in the stem and leaf, indicating that most Cr absorbed from the nutrient solution would remain in the roots with only a small proportion of them being remobilized and translocated into above-ground plant parts. The possible reason for the high accumulation of Cr in roots could be attributed to the immobilization of Cr in the vacuoles of the root cells or that the Cr6þ in cells is probably readily reduced to Cr3þ, which is retained in the root cortex cells (Shanker et al., 2004). The higher vascular plants do not contain Cr6þ-reducing enzymes, although these enzymes have been widely found in bacteria and fungi (Horitsu et al., 1987; Cervantes et al., 2001). In the present experiment, a significant correlation was found between Cr accumulation and rhizosphere pH. Moreover, both variation of rhizosphere pH and Cr accumulation in rice plants increased significantly with Cr level in nutrient solution. Acknowledgment The project was supported by Zhejiang Natural Science Foundations (Z304104, R306202) and the Key Project of Chinese Ministry of Education (No. 106096). References Astolfi, S., Zuchi, S., Chiani, A., Passera, C., 2003. In vivo and in vitro effects of cadmium on Hþ-ATPase activity of plasma membrane vesicles from oat (Avena sativa L.) roots. Journal of Plant Physiology 160, 387e393. Barcelo, J., Poschenrieder, C., 1997. Chromium in plants. In: Carati, S., Tottarelli, F., Seqmi, P. (Eds.), Chromium Environment Issue. Francotangati Press, Milan, pp. 101e129. Boominathan, R., Doran, P.M., 2003. Organic acid complexation, heavy metal distribution and the effect of ATPase inhibition in hairy roots of hyperaccumulator plant species. Journal of Biotechnology 101, 131e146. Breckle, S.W., 1991. Growth under stress: heavy metals. In: Waisel, Y., Eshel, A., Kafkafi, U. (Eds.), Plant Root: The Hidden Half. Marcel Dekker, New York, pp. 351e373. Buerge, I.J., Hug, S.J., 1997. Kinetics and pH dependence of Cr6þ reduction by Fe2þ. Environmental Science and Technology 31, 1426e1432. Calba, H., Cazevieille, P., The´, C., Poss, R., Jaillard, B., 2004. The dynamics of protons, aluminium and calcium in the rhizosphere of maize cultivated

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