Article pubs.acs.org/JAFC
Localization of Fluoride and Aluminum in Subcellular Fractions of Tea Leaves and Roots Hong-jian Gao,*,†,‡ Qiang Zhao,‡ Xian-chen Zhang,‡ Xiao-chun Wan,‡ and Jing-dong Mao§ †
School of Resources and Environment, Anhui Agricultural University, Hefei 230036, People’s Republic of China Key Lab of Tea Biochemistry and Biotechnology, Ministry of Education of China, Anhui Agricultural University, Hefei 230036, People’s Republic of China § Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, Virginia 23529, United States ‡
ABSTRACT: The tea plant is a fluoride (F) and aluminum (Al) hyperaccumulator. High concentrations of F and Al have always been found in tea leaves without symptoms of toxicity, which may be related to the special localization of F and Al in tea leaves. In this study, we for the first time determined the subcellular localization of F and Al in tea roots and leaves and provided evidence of the detoxification mechanisms of high concentrations of F and Al in tea plants. Results revealed that 52.3 and 71.8% of the total F accumulated in the soluble fraction of tea roots and leaves, and vacuoles contained 98.1% of the total F measured in the protoplasts of tea leaves. Cell walls contained 69.8 and 75.2% of the total Al detected in the tea roots and leaves, respectively, and 73.2% of Al sequestered in cell walls was immobilized by pectin and hemicellulose components. Meanwhile, 88.3% of the Al measured in protoplasts was stored in the vacuoles of tea leaves. Our results suggested that the subcellular distributions of F and Al in tea plants play two important roles in the detoxification of F and Al toxicities. First, most of the F and Al was sequestered in the vacuole fractions in tea leaves, which could reduce their toxicities to organelles. Second, Al can be immobilized in the pectin and hemicellulose components of cell walls, which could suppress the uptake of Al by tea roots. KEYWORDS: fluoride and aluminum, subcellular localization, detoxification, tea plants
■
INTRODUCTION Tea is one of the most popular beverages in the world. It is well-known that drinking tea is beneficial to human health because of the antimutagenic, anticarcinogenic, and antioxidative potential of tea.1 However, the tea plant is a fluoride (F) and aluminum (Al) accumulator, and their levels in tea leaves are many times higher than those in other edible plants.2 More than 90% of the F and Al in the whole tea plant accumulates in tea leaves, especially in old leaves.3,4 The ingestion of normallevel F from drinking tea has been considered to be safe and also an approach to protecting teeth against decay.3,5 However, excessive intake of F through overdrinking some special types of tea (e.g., brick tea) containing extremely high levels of F has caused fluorosis.6 In addition, it is generally believed that overconsumption of Al is harmful to health, causing kidney weakness and other diseases.7 Therefore, the effect of ingestion of F and Al through drinking tea on human health has attracted significant attention.8,9 Most of the F and Al that accumulate in tea plants is absorbed from soil solutions by roots. Fluoride ion (F−) is passively absorbed by roots and transported to young leaves,10 but excess F− forms complexes with Al3+ and is then transported to old leaves.11 Fluoride ion can also be complexed with aluminum ion (Al3+) in solution. AlFx complexes such as AlF2+, AlF2+, AlF4−, and AlF3 contribute to total soluble Al and F levels in soil solutions, particularly in acidic soils with pH values of 0.05) among F and Al concentrations in 106 protoplasts and 106 vacuoles in tea leaves.
weigh) per 106 protoplasts and vacuole rich fractions, respectively (Table 5). Approximately 88.0% of the total Al in protoplasts was stored in the vacuole rich fractions. This result indicated that most of the F and Al was localized in the vacuole rich fractions of protoplasts after entering the cytoplasm. Distributions of Aluminum in Different Fractions of Tea Roots. The total concentration of Al in the whole cell walls was 108.1 μg/g, but the Al concentrations were reduced to 52.1 and 29.0 μg/g, respectively, after the pectin fractions and pectin and hemicellulose fractions had been removed (Table 6). This indicated that pectin and hemicellulose fractions immobilized 51.8 and 21.4% of the total Al, respectively, in the whole cell walls. The adsorption experiments revealed that the quantities of Al adsorbed on the whole cell walls, cell wall residues without pectin, and cell wall residues without pectin and hemicellulose were increased by 41.0, 6.7, and 9.4%, respectively, as the adsorption time increased from 1 to 2 h (Table 6) . At each adsorption time, the amounts of Al adsorbed on the whole cell walls were significantly larger than those on the cell wall residues without pectin and the cell wall residues without pectin and hemicellulose. The amounts of Al adsorbed on the cell wall residues without pectin were reduced by 28.0 and 54.6% compared with those on the whole cell walls after adsorption for 1 and 2 h, respectively. When both pectin and hemicellulose were removed from the cell walls, the amounts of Al adsorbed on the residual fractions were reduced by 72.5 and 82.0% 2317
dx.doi.org/10.1021/jf4038437 | J. Agric. Food Chem. 2014, 62, 2313−2319
Journal of Agricultural and Food Chemistry
■
young and old tea leaves preferentially accumulated in the cell walls and that the Al concentration in this compartment increased with time. Because cell walls are regarded as a pivotal site for metal storage in plants,40,41 the deposition of metals in cell walls is considered to be a crucial mechanism for improving heavy metal tolerance. Taylor et al.35 pointed out that the cell wall is the major site of Al accumulation, and the immobilization of Al in cell walls was one of the crucial exclusion mechanisms for Al detoxification.16 Wang et al.42 revealed that more than 85% of the Al absorbed in maize roots was localized in cell walls. In plant cell walls, pectin is a polysaccharide containing galacturonic acids with high electronegativity, which can easily chelate with Al and thus immobilize the Al in cell walls. It was found that both the contents and methylation of pectin contributed to the tolerance of maize to Al toxicity.43 These results indicate that when pectin was removed from the cell walls of tea roots, the Al concentrations were reduced by 51.8%, and the adsorption of Al in the cell wall residues also significantly declined. These findings suggested that the pectin fractions in the cell walls of tea roots may fix the Al in apoplasts and thus reduce the toxicity of Al to tea plants. The positive correlations between F and Al concentrations in tea plants have been observed, leading to the hypothesis that the uptake of F and Al were somewhat linked.44 Free fluoride ions (F−) can be chelated with aluminum ions (Al3+) in the forms of AlF2+, AlF2+, AlF3, AlF4−, etc.34 F and Al in the forms of Al−F complexes were readily taken up and transported in the xylem of tea shoots.13,45 Al−F complexes such as AlF2+ and AlF2+ were less toxic than hydroxy and Al3+ forms such as Al3+, Al(OH)2+, and Al(OH)2+.46 These findings suggested that the forms of F and Al also determined their toxicity to tea plants. F and Al complexes could be in the form of AlF2+, AlF2+, AlF3, AlF4−, etc., or their combinations. However, the exact forms of F and Al complexes in the different subcellular fractions of tea plants are still unclear. Consequently, further studies are needed to understand the chemical species of Al−F complexes in different parts of tea plants to determine the mechanisms of the tolerance of tea plants to F and Al toxicity.
■
Article
REFERENCES
(1) Yamamoto, T.; Juneja, L. R.; Kim, M. Chemistry and application of green tea; CRC Press Inc.: Boca Raton, FL, 1997. (2) Xie, Z. M.; Ye, Z. H.; Wong, M. H. Distribution characteristics of fluoride and aluminum in soil profiles of an abandoned tea plantation and their uptake by six woody species. Environ. Int. 2001, 26, 341−346. (3) Simpson, A.; Shaw, L.; Smith, A. J. The bio-availability of fluoride from black tea. J. Dent. 2001, 29, 15−21. (4) Guo, B. Y.; Cheng, Q. K. Chelating capability of tea components with metal ion. J. Tea Sci. 1991, 11, 139−144. (5) Gao, H. J.; Jin, Y. Q.; Wei, J. L. Health risk assessment of fluoride in drinking water from Anhui province in China. Environ. Monit. Assess. 2013, 185, 3687−3695. (6) Cao, J.; Zhao, Y.; Liu, J. Brick tea consumption as the cause of dental fluorosis among children from Mongol, Kazak and Yugu populations in China. Food Chem. Toxicol. 1997, 35, 827−833. (7) McLachlan, D. R. C. Aluminum and the risk for Alzheimer’s disease. Environmetrics 1995, 6, 233−275. (8) Cao, J.; Zhao, Y.; Liu, J.; Xirao, L. D.; Danzeng, S. B.; Daji, D. W.; Yan, Y. Brick tea fluoride as a main source of adult fluorosis. Food Chem. Toxicol. 2003, 41, 535−542. (9) Erdemoǧlu, S. B.; Pyrzyniska, K.; Gücȩ r, Ş. Speciation of aluminum in tea infusion by ion-exchange resins and flame AAS detection. Anal. Chim. Acta 2000, 411, 81−89. (10) Gao, H. J.; Zhang, Z. Z.; Wan, X. C. Influences of charcoal and bamboo charcoal amendment on soil fluoride fractions and bioaccumulation of fluoride in tea plants. Environ. Geochem. Health 2012, 34, 551−562. (11) Karak, T.; Bhagat, R. M. Trace elements in tea leaves, made tea and tea infusion: A review. Food Res. Int. 2010, 43, 2234−2252. (12) Wenzel, W. W.; Blum, W. E. H. Fluorine speciation and mobility in F-contaminated soils. Soil Sci. 1992, 153, 357−364. (13) Nagata, T.; Haytsu, M.; Kosuge, N. Aluminum kinetics in the tea plant using 27Al-NMR and 19F-NMR. Phytochemsity 1993, 32, 771−775. (14) Ding, R. X.; Huang, X. Biogeochemical cycle of aluminum and fluorine in tea garden soil system and its relationship to soil acidification. Turang Xuebao 1991, 28, 229−236. (15) Matsumoto, H.; Hirasawa, E.; Morimura, E.; Takahashi, E. Localization of aluminum in tea leaves. Plant Cell Physiol. 1976, 17, 627−631. (16) Taylor, G. J. Current views of the aluminum stress response: The physiological basis of tolerance. Curr. Top. Plant Biochem. Physiol. 1991, 10, 57−93. (17) Zheng, S. J.; Yang, J. L.; He, Y. F.; Yu, X. H.; Zhang, L.; You, J. F.; Shen, R. F.; Matsumoto, H. Immobilization of aluminum with phosphorus in roots is associated with high aluminum resistance in buckwheat. Plant Physiol. 2005, 138, 297−303. (18) Morita, A.; Horie, H.; Fujii, Y.; Takatsu, S.; Watanabe, N.; Yagi, A.; Yokota, H. Chemical forms of aluminum in xylem sap of tea plants (Camellia sinensis L.). Phytochemistry 2004, 65, 2775−2780. (19) Shen, R. F.; Ma, J. F.; Kyo, M.; Iwashita, T. Compartmentation of aluminum in leaves of an accumulator, Fagopyrum esculentum Moench. Planta 2002, 215, 394−398. (20) Hall, J. L. Cellular mechanisms for heavy metal detoxification and tolerance. J. Exp. Bot. 2002, 53, 1−11. (21) Kü p per, H.; Zhao, F. J.; McGrath, S. P. Cellular compartmentation of zinc in leaves of the hyperaccumulator Thlaspi caerulescens. Plant Physiol. 1999, 119, 305−311. (22) Weigel, H. J.; Jäger, H. J. Subcellular distribution and chemical form of cadmium in bean plants. Plant Physiol. 1980, 65, 480−482. (23) Yoo, S. D.; Cho, Y. H.; Sheen, J. Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nat. Protoc. 2007, 2, 1565−1572. (24) Cooking, E. C. A method for the isolation of plant protoplasts and vacuoles. Nature 1960, 4771, 962−963. (25) Wagner, G. J.; Siegelman, H. W. Large-scale isolation of intact vacuoles and isolation of chloroplasts from protoplasts of mature plant tissues. Science 1975, 190, 1298−1299.
AUTHOR INFORMATION
Corresponding Author
*School of Resources and Environment, Anhui Agricultural University, 130 W. Changjiang Rd., Hefei 230036, People’s Republic of China. E-mail:
[email protected]. Telephone: +86-551-65867447. Fax: +86-551-65786316. Funding
This work was supported by the National Natural Science Foundation of China (41071158 and 31272254), the Science Foundation for Distinguished Young Scholars of Anhui Province (1408085J01), and the earmarked fund for Modern Agro-industry Technology Research System by the Chinese Ministry of Agriculture (nycytx-26). Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank Hai-sheng Cao and Xun-gang Gu at Anhui Agricultural University for analyzing Al and F in tea plants using ion chromatography. 2318
dx.doi.org/10.1021/jf4038437 | J. Agric. Food Chem. 2014, 62, 2313−2319
Journal of Agricultural and Food Chemistry
Article
by Avena sativa and Lycopersicon esculentum. Plant Soil 1997, 192, 81− 93.
(26) Granstedt, R. C.; Huffaker, R. C. Identification of the leaf vacuole as a major nitrate storage pool. Plant Physiol. 1982, 70, 410− 413. (27) Boller, T.; Kende, H. Hydrolytic enzymes in the central vacuole of plant cells. Plant Physiol. 1979, 63, 1123−1132. (28) Leonard, R. T.; Hansen, D.; Hodges, T. K. Membrane-bound adenosine triphosphatase activities of oat roots. Plant Physiol. 1973, 51, 749−754. (29) Hiiusler, E.; Petersen, M.; Alfermann, A. W. Isolation of protoplasts and vacuoles from cell suspension cultures of Coleus blumei Benth. Plant Cell Rep. 1993, 12, 510−512. (30) Zhong, H. L.; Lauchli, A. Changes of cell wall composition and polymer size in primary roots of cotton seedlings under high salinity. J. Exp. Bot. 1993, 44, 773−778. (31) Mort, A. J.; Moerschbacher, B. M.; Pierce, M. L.; Maness, N. O. Problems encountered during the extraction, purification and chromatography of pectin fragments, and some solution to them. Carbohydr. Res. 1991, 215, 219−277. (32) Zhang, X. C.; Gao, H. J.; Zhang, Z. Z.; Wan, X. C. Influences of different ion channel inhibitors on the absorption of fluoride in tea plants (Camellia sinesis L.). Plant Growth Regul. 2013, 69, 99−106. (33) Martinoia, E.; Heck, U.; Wiemken, A. Vacuoles as storage compartments for nitrate in barley leaves. Nature 1981, 289, 292−293. (34) Xie, Z. L.; Chen, Z.; Sun, W. T.; Guo, X. J.; Yin, B.; Wang, J. H. Distribution of aluminum and fluoride in tea plant and soil of tea garden in central and southwest China. Chinese Geographical Science 2007, 17, 376−382. (35) Taylor, G. J.; McDonald-Stephens, J. L.; Hunter, D. B.; Bertsch, P. M.; Elmore, D.; Rengel, Z.; Reid, R. J. Direct measurement of aluminum uptake and distribution in single cells of Chara corallina. Plant Physiol. 2000, 123, 987−996. (36) Morita, A.; Yanagisawa, O.; Takatsu, S.; Maeda, S.; Hiradate, S. Mechanism for the detoxification of aluminum in roots of tea plant (Camellia sinensis (L.) Kuntze). Phytochemistry 2008, 69, 147−153. (37) Heim, A.; Luster, J.; Brunner, I.; Frey, B.; Frossard, E. Effects of aluminum treatment on Norway spruce roots: Aluminum binding forms, element distribution, and release of organic substances. Plant Soil 1999, 216, 103−115. (38) Tolra, R.; Vogel-Mikus, K.; Hajiboland, R.; Kump, P.; Pongrac, P.; Kaulich, B.; Gianoncelli, A.; Babin, V.; Barcelo, J.; Regvar, M.; Poschenrieder, C. Localization of aluminum in tea (Camellia sinensis L.) leaves using low energy X-ray fluorescence spectro-microscopy. J. Plant Res. 2011, 124, 165−172. (39) Carr, H. P.; Lombi, E.; Kupper, H.; McGrath, S. P.; Wong, M. H. Accumulation and distribution of aluminum and other elements in tea (Camellia sinensis L.) leaves. Agronomie 2003, 23, 705−710. (40) Lozano-Rodriguez, E.; Hemandz, L. E.; Bonay, P.; Carpena-rui, R. O. Distribution of cadmium in root tissues of maize and pea plants: Physiological disturbances. J. Exp. Bot. 1997, 306, 123−128. (41) Carrier, P.; Baryla, A.; Havaux, M. Cadmium distribution and microlocalization in oilseed rape (Brassica napus) after long-term growth on cadmium-contaminated soil. Planta 2003, 216, 939−950. (42) Wang, Y.; Stass, A.; Horst, W. J. Apoplastic binding of aluminum is involved in silicon-induced amelioration of aluminum toxicity in maize. Plant Physiol. 2004, 136, 3762−3770. (43) Eticha, D.; Stass, A.; Horst, W. J. Cell wall pectin and its degree of methylation in the maize root-apex: Significance for genotypic differences in aluminum resistance. Plant, Cell Environ. 2005, 28, 1410−1420. (44) Ruan, J. Y.; Ma, L. F.; Shi, Y. Z.; Han, W. Y. Uptake of fluoride by tea plant (Camellia sinensis L.) and the impact of aluminum. J. Sci. Food Agric. 2003, 83, 1342−1348. (45) Yumata, H.; Hattori, T. Investigation of the relationship between aluminum and fluorine in plants (Part 1). Relationship between aluminum and fluorine in tea leaves. J. Sci. Soil Manure 1977, 48, 253−261. (46) Stevens, D. P.; McLaughlin, M. J.; Alston, A. M. Phytotoxicity of aluminum-fluoride complexes and their uptake from solution culture 2319
dx.doi.org/10.1021/jf4038437 | J. Agric. Food Chem. 2014, 62, 2313−2319
