Aluminum uptake and accumulation by aluminum excluders and a

Jing Li¹, Zhengmiao Xie^1*, Zhihong Ye² and Ming H. Wong²

¹Institute for Soil and Water Resources and Environment, Zhejiang University, Hangzhou 310029, P R China; ²Institute for Natural Resources and Environmental Management, and Department of Biology, Hong Kong Baptist University, Hong Kong, P R China

*Corresponding author, email: [email protected] & [email protected]

Key words: accumulator, aluminum, excluder, pH, rhizosphere, soil

Abstract Mechanisms for Al uptake and accumulation in Al-excluders and Al-accumulators namely Camellia sinensis, Melastoma affine, Sterculia lanceolata, Ardisia crenata, Acacia formosa and Machilus thunbergii were investigated in the natural ecosystem of an abandoned tea plantation in Hong Kong. The pH values of the fresh stems and roots of 6 plant species varied from 3 to 6 and total Al concentrations in different parts of the 6 plant species ranged from 13-12810 mg/kg. It is apparent that pH of fresh plant tissues was the most important factor controlling Al uptake, translocation and accumulation in plants. The Al concentrations in plants were significantly increased with the decrease of pH. Based on the obtained results, the plants could be classified into 2 groups: Al excluders with pH around 6 and Al contents 17-151 mg/kg in leaves including S. lanceolata, A. crenata, A. formosa and M. thunbergii, and Al accumulators with pH about 3 and 4.5 and Al contents 7820-12810 mg/kg in leaves including C. sinensis and M. affine. The ratio of water-soluble Al to total Al in fresh roots was much higher in Al accumulators (0.11-0.88) than Al excluders (0.04-0.07). A similar trend was found in fresh leaves and stems, especially in the case of M. affine. The results indicated that a higher ratio of water-soluble Al to total Al in fresh roots, leaves and stems seemed to increase Al translocation rate from the soil-plant system, leading to a higher Al uptake and accumulation. pH values between rhizosphere soil and non-rhizosphere soil were statistically significant at p<0.01 (15 sites) and p<0.05 (3 sites). In general, Al accumulators such as M. affine had low pH values in plant tissues and can decrease rhizosphere pH and rendered Al more available for its uptake. Al excluders such as S. lanceolata, A. crenata, A. formosa and M. thunbergii can increase pH values in the rhizosphere soil to avoid higher Al uptake by roots. The rhizosphere soils had significantly higher organic carbon contents than non-rhizosphere soils, because of root exudation and microbial activity. It was also observed that water-soluble Al increased in the rhizosphere soils of Al excluders while pH in the corresponding rhizosphere soils also increased. In the case of M. affine, water-soluble Al in rhizosphere soil decreased with the decrease of pH in the rhizosphere soil compared to that in the non-rhizosphere soil. Two types of mechanisms for Al uptake and accumulation by Al accumulators and excluders are proposed. Al accumulators such as M. affine possess a very low tissue pH (3) and can release H⁺, i.e., net H⁺ efflux from roots to the rhizosphere soil, which lower soil pH, leading to Al more available for higher uptake. Al excluders such as S. lanceolata, A. crenata, A. formosa and M. thunbergii possess a relatively higher pH (6), have net H⁺ influx into roots, i.e., net OH^- efflux from roots and actually increase pH in the rhizosphere soil, leading to Al accumulation in the rhizosphere soil through precipitation and complexation and finally exclude Al from roots.

Introduction

Aluminum (Al) is an ubiquitous element in soil and plant ecosystem and can be a toxicant to plants, mainly depending on soil pH and plant species (Adams et al., 1999; Bernal and Clark, 1998; Delhaize et al., 1993a; Delhaize and Ryan, 1995; Hirano and Hijii, 1998; Lidon and Barreiro, 1998; Pintro et al., 1998). Aluminum can also be a stimulator to some plant species such as tea (Camellia sinensis L.) and melastoma malabathricum L. (Konishi et al., 1985; Masunaga et al., 1998; Watanabe et al., 1997). Therefore, plant species can be divided into Al accumulator and Al non-accumulator or Al excluder based on Al concentrations in their tissues. It has been proposed that plants in which growth was reduced by low and high Al applications were designated as Al-sensitive plants and Al-medium tolerant plants, respectively, while plants in which growth was not affected or stimulated by Al application were designated as Al-tolerant plants and Al-stimulated plants, respectively (Osaki et al., 1997).

It has been well documented that many plant species exhibit significant genetic variability in their ability to resist Al toxicity (Delhaize and Ryan, 1995; Paul et al., 1998, and refs. therein). Plants can deal with Al toxicity through two strategies: exclusion from the root apex or development of the ability to tolerate Al once it enters the plant symplasm. In addition, plant-soil relationship concerning Al uptake is very complex, especially in rhizosphere soil-root surface ecosystem. There have been a number of studies related to the relationships of Al availability in rhizosphere soils and plant root surface. Aluminium concentration in the rhizosphere soil was increased due to pH decrease in rhizosphere soil as a result of acidification from NH₄-N uptake compared to NO₃-N uptake by plants (Calba et al., 1999; Gohoonia, 1993; Ruan et al., 2000; Schier and Mcquattie, 1999; Watanabe et al., 1998). On the contrary, Al concentration in the rhizosphere soil was decreased due to pH increase by plants in the rhizosphere soil (Cuenca et al., 1990; Marion and Ursula, 1999). Aluminium accumulator plants can avoid Al toxicity by binding with detoxifying complexes especially the organic complexes resulted from root exudation ( Delhaize et al., 1993b; Diatloff et al., 1998; Heim et al., 1999; Marscher, 1991; Miyasake et al., 1991; Pellet et al., 1995; Ryan et al., 1995).

However, the above results on exclusion-accumulation mechanisms were based on hydroponics or pot experiments conducted in controlled conditions. Up to now, these seem to be a lack of direct evidence related to Al-resistance mechanisms, especially in the natural ecosystem in which soil-plant interaction is more complex. The tea plantation at the peak of Lantau Island, Hong Kong provides a unique site for this study. The tea plantation had been intensively cultivated for more than 40 years, but was disused for over 10 years. Due to the lack of management, other plant species invaded the tea plantation, leading to a new plant-soil ecosystem in which Al accumulators and Al excluders coexist. The objective of this research was to investigate the mechanisms for Al uptake and accumulation in Al-excluders and Al-accumulators in relation to pH difference in plants and pH change in the rhizosphere soils in the natural ecosystem.

Materials and methods

Descriptions of study site

The tea plantation occupied about 420 hectares and located at a high elevation (440-579 m) in the Lantau Peak of Lantau Island. The area is always full of mist and high humidity and the annual rainfall is about 2400 mm. The tea plantation had been intensively farmed for over forty years and was disused for more than ten years. Due to the lack of management, the different plant species including aluminum excluders and aluminum accumulators invaded the abandoned tea plantation. The soil is red yellow podzolic soil (clayey loam) with pH ranging from 3.84 to 4.90.

Based on the ecological survey *, several plant species namely Camellia sinensis, Melastoma affine, Sterculia lanceolata, Ardisia crenata, Acacia formosa and Machilus thunbergii, were selected to compare their Al uptake and accumulation. There have been more than 10 plants present both within and around the borders of the plantation. The selected 6 plants had varied Al contents and higher abundance and coverage in the area.

Sampling of soil and plant

To study Al compartmentation in different species, samples of leaves, stems and roots were taken and washed carefully by deionised water and then dried at 105⁰C and ground to pass through a 0.15 mm sieve and stored in plastic bottles for further analyses. For each species, three random sites were chosen for sampling.

Rhizosphere soil samples and non-rhizosphere (bulk) soil samples were collected at the same sites where plant samples were collected. The rhizosphere soil (< 2 mm from root surface) was obtained by gently shaking the plant roots, so that the soil that had been attached to the roots was collected. The non-rhizosphere soil or bulk soil (5 mm-10 mm from root surface) was also collected.

Analytical methods

All soil samples were air-dried for several days and subsequently ground to pass through a mesh sieve with a pore size of 0.15 mm. The following soil properties were tested: texture (Hydrometer Method), pH (pH meter, soil : distilled water = 1:2.5 w/v), moisture (dried at 105^oC until a constant weight), total organic carbon (TOC) (modified Walkey and Black Method) according to the methods described in Sparks et al (Sparks et al, 1996). All plant samples were dried at 105^oC for 24 h and ground to pass through a 0.15 mm sieve. The total Al (digested with conc. hydrochloric acid-conc. nitric acid using a microwave digester, model: MARS, CEM Corporation, USA) contents in plants and water soluble Al in soil were measured using high-purity standard solution (EPA, USA) by means of an inductively coupled plasma (ICP)-Emission Spectroscopy (model: Perkin Elmer in USA).

pH determination of fresh shoots and roots

In order to measure pH in fresh stems and roots of the plant species, two types of pH test paper (Macherey-NAGEL, Art.-Nr. 92110 and Advantec, Toyo Roshi Kaisha Ltd., made in Japan) were used. The stems or roots were cut open and a piece of pH test paper was inserted between the opened stems or roots before they were closed and pressed tightly. After the juice from the sap and vacuole wet the test paper, the pH values were obtained by comparing the standard colors.

Determination of water-soluble Al in fresh leaves, stems and roots

In order to study the translocation of Al in plant species, water-soluble Al in fresh leaves, stems and roots were determined. The fresh leaves, stems and roots were washed carefully with deionised water and air-dried until there was no free water attached on the sample surface. Half of each sample was tested for moisture (105^oC until constant weight). While the other half (2.00 g) of each sample was cut to about 1 mm², and extracted with 25 ml deionized water (pH was equal to that of each fresh stems, respectively) by shaking for 2 h at 20^oC and Al in filtered supernatants was determined. One gram of each sample (roots) was extracted with 50 ml deionized water (pH was equal to that of each fresh root, respectively) by shaking for 10 h at 20^oC and Al in filtered supernatants was determined.

Determination of water-soluble Al in rhizosphere and non-rhizosphere soil

Samples (2.00 g of each fresh rhizosphere or non-rhizosphere soil sample) were equilibrated with 25 ml deionized water and shaken for 30 min. at 20^oC, centrifuged (4000 rpm) for 5 min. The supernatants were acidified by adding 1 ml conc. nitric acid and stored in plastic bottles for testing Al. Soil moisture was also tested (105^oC until constant weight).

*Hilda P. Carr, 2000. The accumulation of Aluminium and its Effect on the Uptake and Distribution of Ca, Mg, K, Mn, Fe, Cu and Zn in C. Sinensis (L.). Ph. D. Dissertations, Hong Kong Baptist University.

Results

pH in fresh shoots and roots

The pH of the fresh stems and roots of 6 plant species and total Al concentration (based on dry weight) in different parts of the 6 plant species are listed in Table 1. It is apparent that pH of fresh plant tissues was the most important factor controlling Al uptake, translocation and accumulation in plants. The Al concentrations in plants were significantly increased with the decrease of pH. Based on the results, the plants could be classified into 2 groups: Al excluder with pH around 6 including Sterculia lanceolata, Ardisia crenata, Acacia formosa and Machilus thunbergii, and Al accumulator with pH about 3 and 4.5 including Camellia sinensis, Melastoma affine.

Water-soluble Al in fresh leaves, stems and roots in Al accumulators and excluders

The ratio of water-soluble Al to total Al in fresh roots was much higher in Al accumulators (0.11-0.88) than Al excluders (0.04-0.07) (Table 2). A similar trend was found in fresh leaves and stems, especially in the case of M. affine (Table 2), although there were other factors such as water-soluble Al in soil and chemical composition in plants. The results indicated that higher ratios of water-soluble Al to total Al in fresh roots, leaves and stems seemed to increase Al translocation rate from soil-plant system, leading to higher Al uptake and accumulation. And vice versa.

Plant induced pH change in rhizosphere soil

pH was found to be statistically significant at 1% p<0.01 (15 sites) and p<0.05 (3 sites) between rhizosphere soil and non-rhizosphere soil and related to plant species (Table 4). M. affine induced pH decrease in the rhizosphere soil compared to non-rhizosphere soil, because of the lowest pH 3 (Table 1) of its roots, which is much lower than pH of non-rhizosphere soil. While S. lanceolata, A. Crenata, A. formosa, M. thunbergii and M. sinensis induced pH increase in the rhizosphere soil, mainly due to higher pH in plant roots (Table 1). Basically, Al accumulator such as M. affine decreased rhizosphere pH and rendered Al more available for its uptake. Al excluders such as S. lanceolata, A. Crenata, A. formosa, and M. thunbergii increased pH values in the rhizosphere soil to avoid higher Al uptake by roots.

Changes of water-soluble Al and organic carbon in rhizosphere soils

Organic carbon increased significantly in the rhizosphere soils compared with non-rhizosphere soils, ranging from 26.85 to 65.40 g/kg (Table 3), because of root exudation and microbial activity. It was observed that water-soluble Al increased in the rhizosphere soils of S. lanceolata, A. Crenata, A. formosa, M. thunbergii and M. sinensis while pH in the corresponding rhizosphere soils also increased. In the case of M. affine, water-soluble Al decreased with pH decrease in the rhizosphere soil compared to that in the non-rhizosphere soil.

Discussion

Aluminum uptake, translocation and accumulation are controlled by pH inherent in plants

Aluminum solubility increases substantially with the decrease of pH in solution when pH < 5. Around pH 6, the Al solubility is lowest and then gradually increases when pH > 6.5, because of soluble Al(OH)^-₄ formation in solution. When pH in solution is < 4.5, the predominant ion is Al³⁺, followed by Al(OH)²⁺ and Al(OH)⁺. It is quite reasonable to conclude that lower pH (< 4.5) in media (such as plants and soils) is a highway for Al translocation as pH decreases by one unit, the Al³⁺ concentration will increase by one thousand times. Therefore, It has been well established that in acid soils (pH <5.5) the phytotoxic species Al³⁺ is solubilized to levels that inhibit root growth and crop yield (Hirano and Hijii, 1998; Larsen et al., 1998, and refs. therein).

However little is known how some plant species exhibit significant genetic ability to resist Al toxicity, when available Al concentration is very high. The pH values in fresh roots/stems of tested plant species were not usually detected in most of the studies (e.g. Adams et al., 2000; Bernal et al., 1998; Cuenca et al., 1990; Schier and Mcquattie, 1999; Watanabe et al., 1998). The present results (Table 1 and Table 2) suggest that Al uptake, translocation and accumulation are controlled by pH inherent in roots and stems and by ratio of water-soluble Al to total Al concentration in different plant species. Al accumulators such as C. sinensis and M. affine, have very low pH (3, 4.5) in roots and shoots (Table 1) and relatively higher ratio of H₂O-Al to total Al in the roots, stems and leaves (Table 2). This lower pH (< 4.5) in plant media (H₂O) is a highway for Al³⁺ translocation from soil to roots and then to stems and leaves. Therefore Al accumulator has pH highway for Al³⁺ migation. On the contrary, Al excluders such as Sterculia lanceolata, Ardisia crenata, Acacia formosa and Machilus thunbergii, have higher pH (about 6) in roots and stems and relatively lower ratio of H₂O-Al to total Al in the roots, stems and leaves. This high pH functions as pH barrier to Al³⁺ translocation from soil to roots and then to other plant parts.

Aluminum depletion or increment in rhizosphere soils as related to intrinsic pH in roots and pH change in rhizosphere

Aluminium increment in the rhizosphere soils was found due to pH decrease in rhizosphere (Adams et al., 1999; Bernal and Clark, 1998; Delhaize et al., 1993a; Delhaize and Ryan, 1995; Hirano and Hijii, 1998; Marion and Ursula, 1999), although most of the studies were based on hydroponics and pot experiments in greenhouse. In the present investigation, on natural ecosystem, a depletion of Al in the rhizosphere soil of M. affine was first evident while pH in the rhizosphere decreased (Table 3). It seems unreasonable from the point of view of Al chemistry. The Al depletion in rhizosphere of high accumulator (or hyperaccumulator), such as Melastoma with pH 3 itself, had resulted from strong Al absorption by M. affine and Al concentration gradient and lower diffusion rate than needed. On the other hand, Al concentrations in the rhizosphere soils of Al excluders with pH about 6 themselves, such as Sterculia lanceolata, Ardisia crenata, Acacia formosa and Machilus thunbergii,increased even if pH in the rhizosphere also increased (Table 3).

The only explanation is due to net H⁺ influx into the plant roots and organic acid release from roots (Degenhardt et al., 1998; Larsen et al., 1998). It is possible for net H⁺ influx from the rhizosphere to Al excluders with pH 6. The results obtained in the present study on natural ecosystem can support a new mechanism proposed by Degenhaedt et al. (1998) for Al uptake on plant root surface, in which an Al-inducible increased H⁺ influx at the root tip was found, resulting in a higher rhizosphere pH. Miyasaka et al. (1989) have also found that Al-resistant wheat cv Atlas maintained a slightly higher pH (approximately 0.15 pH unit) at root tip compared with the Al-sensitive cv Scout.

Why water-soluble Al increased with pH increase in the rhizosphere?

Why water soluble Al increased with pH increase in the rhizosphere? It is because of higher organic matter content in the rhizosphere soils compared with the non-rhizosphere soils. In natural forest ecosystem, very high organic matter exists in soil (Table 3), while experiments done before were based on hydroponics with no addition of organic matter or pot experiments with very lower organic matter in red soil, for example, 9.8 g/kg (Ruan et al., 2000). The results (Adams et al., 2000) indicated when pH < 5.0, Al solubility was controlled by complexation of Al with organic matter in soil. Paw et al. (Paw et al., 2000) evaluated the mechanisms controlling the concentrations of free Al³⁺ and total Al in the soil solution of three podzolic soils and concluded that in the E and most of the B1 horizon solutions, Al activity was controlled by equilibrium with soluble organic acids. It was found > 85% of the total Al was organically bound (Paw and Lundstrom, 2000; Zysset et al., 1999). Several studies suggested that solution concentrations of Al in organic surface soils were controlled by complexation with organic matter and organic matter solubility was greatest at high pH (Helene et al., 1999). Geottlein et al. (Geottlein et al., 1999) studied the Al chemistry at the acid forest soil root interface and found that while the concentration of nutrients, especially Ca²⁺ and Mg²⁺, decreased in the vicinity of growing roots the concentrations of Al³⁺ significantly increased. Therefore, in the present work, it is possible that water-soluble Al increased with the increase of pH in the rhizosphere soil compared to that in the non-rhizosphere soil mainly due to higher organic matter, which makes soluble organically bound Al complexation increased when pH relatively increased.

Two types of mechanisms for Al uptake and accumulation by Al accumulators and excluders

Based on the results from references cited in this paper and the results obtained in the present work, two types of mechanisms for Al uptake and accumulation by Al accumulators and excluders are proposed more clearly. Al higher accumulators such as M. affine with very low pH (3) itself can release H⁺ (or positively-charged organic acids/matter), i.e., net H⁺ efflux from roots to the rhizosphere soil and make rhizosphere soil pH decrease, leading to Al more availability for its higher Al absorption. Watanabe et al. (1998) found that roots of Malastoma malabathricum could release H⁺ in culture solution. Al excluders such as Sterculia lanceolata, Ardisia crenata, Acacia formosa and Machilus thunbergii with relatively higher pH (6) in themselves have net H⁺ influx into roots (or release of negatively-charged organic acids/matter), i.e., net OH^- efflux from the roots and actually increase pH in the rhizosphere soil, leading to Al accumulation in the rhizosphere soil mainly through precipitation and complexation and finally exclude Al from the roots. Thus Al tolerants can be from either Al accumulators or Al excluders. And what is more, pH inherent in roots and stems is the determining factor when plant species are classified into Al excluders and Al accumulators.

Acknowledgements

This research was conducted in the Department of Biology, Hong Kong Baptist University, sponsored by the Croucher Foundation Visiting Fellowship. The financial support from the Research Grants Council of the University Grants Committee of Hong Kong, and the China National 973 Project (No.G19990118) funded by National Natural Science Foundation of China is gratefully acknowledged. The authors thank Bob Poon and Martin Fung for their technical assistance.

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Table 1. The pH and Al concentrations (mg/kg, DW) in fresh tissue of stems and roots of six different plant species. *Same letters in the same column indicate no significant difference at p < 0.01 level (n=3), according to Duncan‘s Multiple Range Test (SAS). **Old leaves.

Plant species Al in leaves Al in stems Al in roots pH in stems pH in roots Camellia sinensis 7820-8136** 906-949 1363-2082 4.5 B* 4.5 C Melastoma affine 9099-12810 3499-3633 2072-3826 3 C 3 D Sterculia lanceolata 17-22 13-38 575-803 6 A 5.7 B Ardisia crenata 42-97 33-168 633-672 6 A 6 A Acacia formosa 55-64 23-43 452-765 6 A 6 A Machilus thunbergii 44-151 30-185 1217-1457 6 A 6 A

Table 2. Water-soluble Al and total Al (mg/kg, DW) in fresh leaves, stems and roots of six plant species. *Ratio of H₂O-Al to Total-Al in plants; ** Means±Standard deviation (n=4) and same letters in each column indicate no significant difference at p < 0.05, according to Duncan‘s Multiple Range Test. ***BDL--Below detection limit

Plant species Part of plant H₂O-Al

in plant Total-Al

in plant Ratio* H₂O-Al

in soil Camellia sinensis New leaves 72±2 f ** 928±12 g 0.08 27±0.4 b Old leaves 443±3 d 7978±130 b 0.06 Roots 191±2 e 1723±21 e 0.11 Melastoma affine Leaves 3605±45 a 10955±103 a 0.33 26±0.5 b Stems 1323±20 c 3566±55 c 0.37 Roots 2593±21 b 2949±45 d 0.88 Sterculia lanceolata Leaves 5.3±0.5 i 20±4.5 i 0.27 25±0.1 b Stems 3.9±0.5 i 25±5.5 i 0.16 Roots 47±3 fgh 689±6 h 0.07 Ardisia crenata Leaves 1.4±0.3 i 70±6 i 0.02 25±0.4 b Stems BDL*** i 101±3.5 i ~0 Roots 23±1.6 ghi 653±9 h 0.04 Acacia formosa Leaves BDL i 55±7.5 i ~0 28±0.9 b Stems 0.5±0.4 I 33±8.5 i 0.02 Roots 27±0.7 ghi 609±6 h 0.04 Machilus thunbergii Leaves 16±0.4 hi 98±3.5 i 0.16 35±0.3 a Stems 15±1.4 hi 108±7 i 0.14 Roots 60±1.6 fg 1337±13 f 0.04

Table 3. Differences of pH, water soluble Al (mg/kg) and organic carbon (g/kg) between rhizosphere soil and non-rhizosphere soil. *R--rhizosphere, N--non-rhizosphere; **Lower case is significantly different p < 0.05 and upper case p < 0.01 between rhizosphere soil and non-rhizosphere soil at the same site, according to Duncan‘s Multiple Range Test (SAS).

Species Sites pH H₂O-Al Organic carbon R* N* R N R N C. sinensis 1 4.68 A** 4.33 B 33.18 a** 35.88 a 30.78 A 27.82 B 2 4.16 A 3.92 B 18.97 b 26.44 a 46.70 A 44.09 B 3 4.54 A 4.49 B 20.78 b 38.95 a 41.53 A 38.71B M. affine 1 3.96 A 4.06 B 15.78 b 20.41 a 30.93 A 27.79 B 2 4.00 A 4.19 B 14.62 b 23.15 a 39.77 A 36.68 B 3 3.98 A 4.09 B 13.60 b 19.69 a 35.94 a 35.15 a S. lanceolata 1 4.29 A 4.13 B 23.42 a 15.84 b 50.41 A 42.56 B 2 4.30 a 4.25 b 30.53 a 28.11 b 42.73 a 39.18 b 3 4.44 a 4.33 b 49.54 a 31.29 b 44.58 A 36.69 B A. crenata 1 4.50 A 4.24 B 29.03 a 28.26 a 43.71 A 39.71 B 2 4.54 A 4.25 B 28.99 a 24.34 b 47.88 A 40.11 B 3 4.54 A 4.34 B 28.51 a 24.03 b 46.32 A 38.98 B A. formosa 1 4.26 A 3.92 B 13.20 a 11.40 a 45.62 A 42.70 B 2 4.21 A 3.84 B 45.53 a 13.68 b 65.40 A 50.53 B 3 4.38 A 3.90 B 40.33 a 15.13 b 53.33 a 45.76 a M. thunbergii 1 4.57 A 4.21 B 37.32 a 21.39 b 35.52 A 26.85 B 2 4.90 A 4.64 B 35.18 a 33.81 a 58.59 A 53.51 B 3 4.87 A 4.67 B 34.00 a 32.83 b 57.47 A 52.32 B