Toxic levels of aluminum (Al) in acid soils inhibit root growth and cause substantial reduction in yields of Al-sensitive crops. Aluminum-tolerant cultivars detoxify Al through multiple mechanisms that are currently not well understood at genetic and molecular levels. To enhance our understanding of the molecular mechanisms involved in soybean Al tolerance and toxicity, we conducted proteomic analysis of soybean roots under Al stress using a tandem combination of 2-D-DIGE, mass spectrometry, and bioinformatics tools and Al-tolerant (PI 416937) and Al-sensitive (Young) soybean genotypes at 6, 51 or 72 h of Al treatment. Comparison of the protein profile changes revealed that aluminum induced Al tolerance related proteins and enzymes in Al-tolerant PI 416937 but evoked proteins related to general stress response in Al-sensitive Young. Specifically, Al upregulated: malate dehydrogenase, enolase, malate oxidoreductase, and pyruvate dehydrogenase, in PI 416937 but not in Young. These enzymes contribute to increased synthesis of citrate, a key organic acid involved in Al detoxification. We postulate that simultaneous transgenic overexpression of several of these enzymes would be a robust genetic engineering strategy for developing Al-tolerant crops. 1. Introduction Toxic levels of aluminum (Al) in acid soils inhibit root growth and cause substantial reduction in yields of Al-sensitive crops [1, 2]. Its toxicity mechanisms include interference with nutrient and water uptake and translocation [3], disruption of calcium homeostatis [4], disruption of cytoskeleton [5, 6], callose deposition in apoplast that affects movement of substances from cell to cell [7], lipid peroxidation and reactive oxygen species production [8], and interference with cell division and elongation [9, 10]. In concert, these disorders thwart root growth and development that is typically manifested in stunted and swollen root system at the morphological level [11, 12]. Al disrupts cellular components and processes by high binding affinity to phosphate, sulfate, and carbonyl functional groups of cellular components in apoplast and symplast [11]. Perhaps as a direct and parallel evolutionary response to the nature of Al-ligand interaction, plants secret substances that possess these functional groups namely, organic acids [13], phenolics [14–16], and phosphate and polypeptides [17, 18] to bind and detoxify Al in the rhizosphere. Sequestration of Al in the rhizosphere with root secreted organic acids mainly citrate, malate, and oxalate is a common and well-documented physiological mechanism of
References
[1]
J. V. Magalhaes, “Aluminum tolerance genes are conserved between monocots and dicots,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 26, pp. 9749–9750, 2006.
[2]
S. R. Valle, J. Carrasco, D. Pinochet, and D. F. Calderini, “Grain yield, above-ground and root biomass of Al-tolerant and Al-sensitive wheat cultivars under different soil aluminum concentrations at field conditions,” Plant and Soil, vol. 318, no. 1-2, pp. 299–310, 2009.
[3]
G. R. Rout, S. Samantaray, and P. Das, “Aluminium toxicity in plants: a review,” Agronomie, vol. 21, no. 1, pp. 3–21, 2001.
[4]
Z. Rengel and W. H. Zhang, “Role of dynamics of intracellular calcium in aluminium-toxicity syndrome,” New Phytologist, vol. 159, no. 2, pp. 295–314, 2003.
[5]
E. B. Blancaflor, D. L. Jones, and S. Gilroy, “Alterations in the cytoskeleton accompany aluminum-induced growth inhibition and morphological changes in primary roots of maize,” Plant Physiology, vol. 118, no. 1, pp. 159–172, 1998.
[6]
M. Amenós, I. Corrales, C. Poschenrieder, P. Illé？, and F. Balu？ka, “Different effects of aluminum on the actin cytoskeleton and brefeldin a-sensitive vesicle recycling in root apex cells of two maize varieties differing in root elongation rate and aluminum tolerance,” Plant and Cell Physiology, vol. 50, no. 3, pp. 528–540, 2009.
[7]
K. A. Schreiner, J. Hoddinott, and G. J. Taylor, “Aluminum-induced deposition of (1,3)-β-glucans (callose) in Triticum aestivum L,” Plant and Soil, vol. 162, no. 2, pp. 273–280, 1994.
[8]
Y. Yamamoto, Y. Kobayashi, S. R. Devi, S. Rikiishi, and H. Matsumoto, “Aluminum toxicity is associated with mitochondrial dysfunction and the production of reactive oxygen species in plant cells,” Plant Physiology, vol. 128, no. 1, pp. 63–72, 2002.
[9]
W. J. Horst, “The role of the apoplast in aluminum toxicity and resistance of higher plants,” Zeitschrift für Pflanzenern？rung und Bodenkunde, vol. 158, pp. 419–428, 1995.
[10]
S. Doncheva, M. Amenós, C. Poschenrieder, and J. Barceló, “Root cell patterning: a primary target for aluminium toxicity in maize,” Journal of Experimental Botany, vol. 56, no. 414, pp. 1213–1220, 2005.
[11]
C. Poschenrieder, B. Gunsé, I. Corrales, and J. Barceló, “A glance into aluminum toxicity and resistance in plants,” Science of the Total Environment, vol. 400, no. 1–3, pp. 356–368, 2008.
[12]
S. Zhou, R. Sauvé, and T. W. Thannhauser, “Proteome changes induced by aluminum stress in tomato roots,” Journal of Experimental Botany, vol. 57, pp. 4201–4213, 2009.
[13]
J. F. Ma, P. R. Ryan, and E. Delhaize, “Aluminium tolerance in plants and the complexing role of organic acids,” Trends in Plant Science, vol. 6, no. 6, pp. 273–278, 2001.
[14]
P. S. Kidd, M. Llugany, C. Poschenrieder, B. Gunsé, and J. Barceló, “The role of root exudates in aluminium resistance and silicon-induced amelioration of aluminium toxicity in three varieties of maize (Zea mays L.),” Journal of Experimental Botany, vol. 52, no. 359, pp. 1339–1352, 2001.
[15]
R. Tolrà, J. Barceló, and C. Poschenrieder, “Constitutive and aluminium-induced patterns of phenolic compounds in two maize varieties differing in aluminium tolerance,” Journal of Inorganic Biochemistry, vol. 103, no. 11, pp. 1486–1490, 2009.
[16]
D. Duressa, K. Soliman, and E. Cebert, “Protein and polyphenol profile changes in soybean roots under aluminum stress,” International Journal of Plant Physiology and Biochemistry, vol. 2, no. 3, pp. 38–45, 2010.
[17]
D. M. Pellet, L. A. Papernik, and L. V. Kochian, “Multiple aluminum-resistance mechanisms in wheat roles of root apical phosphate and malate exudation,” Plant Physiology, vol. 112, no. 2, pp. 591–597, 1996.
[18]
L. V. Kochian, O. A. Hoekenga, and M. A. Pi？eros, “How do crop plants tolerate acid soils? Mechanisms of aluminum tolerance and phosphorous efficiency,” Annual Review of Plant Biology, vol. 55, pp. 459–493, 2004.
[19]
C. M. Bianchi-Hall, T. E. Carter, M. A. Bailey et al., “Aluminum tolerance associated with quantitative trait loci derived from soybean PI 416937 in hydroponics,” Crop Science, vol. 40, no. 2, pp. 538–545, 2000.
[20]
Z. M. Yang, M. Sivaguru, W. J. Horst, and H. Matsumoto, “Aluminium tolerance is achieved by exudation of citric acid from roots of soybean (Glycine max),” Physiologia Plantarum, vol. 110, no. 1, pp. 72–77, 2000.
[21]
I. R. Silva, T. J. Smyth, C. D. Raper, T. E. Carter, and T. W. Rufty, “Differential aluminum tolerance in soybean: an evaluation of the role of organic acids,” Physiologia Plantarum, vol. 112, no. 2, pp. 200–210, 2001.
[22]
Q. Yang, Y. Wang, J. Zhang, W. Shi, C. Qian, and X. Peng, “Identification of aluminum-responsive proteins in rice roots by a proteomic approach: cysteine synthase as a key player in Al response,” Proteomics, vol. 7, no. 5, pp. 737–749, 2007.
[23]
S. Zhou, R. Sauve, and T. W. Thannhauser, “Aluminum induced proteome changes in tomato cotyledons,” Plant Signaling and Behavior, vol. 4, no. 8, pp. 769–772, 2009.
[24]
Y. Zhen, J. L. Qi, S. S. Wang et al., “Comparative proteome analysis of differentially expressed proteins induced by Al toxicity in soybean,” Physiologia Plantarum, vol. 131, no. 4, pp. 542–554, 2007.
[25]
W. J. Hurkman and C. K. Tanaka, “Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis,” Plant Physiology, vol. 81, pp. 802–806, 1986.
[26]
M. M. Bradford, “A rapid and sensitive method for rapid quantification of micrograquantities of protein utilizing the principle of protein-dye binding,” Anals of Biochemistry, vol. 72, pp. 248–254, 1976.
[27]
N. L. Houston, M. Hajduch, and J. J. Thelen, “Quantitative proteomics of seed filling in castor: comparison with soybean and rapeseed reveals differences between photosynthetic and nonphotosynthetic seed metabolism,” Plant Physiology, vol. 151, no. 2, pp. 857–868, 2009.
[28]
A. Giannakoula, M. Moustakas, T. Syros, and T. Yupsanis, “Aluminum stress induces up-regulation of an efficient antioxidant system in the Al-tolerant maize line but not in the Al-sensitive line,” Environmental and Experimental Botany, vol. 67, no. 3, pp. 487–494, 2010.
[29]
I. R. Silva, T. J. Smyth, D. F. Moxley, T. E. Carter, N. S. Allen, and T. W. Rufty, “Aluminum accumulation at nuclei of cells in the root tip. Fluorescence detection using lumogallion and confocal laser scanning microscopy,” Plant Physiology, vol. 123, no. 2, pp. 543–552, 2000.
[30]
C. Cobbett and P. Goldsbrough, “Phytochelatins and metallothioneins: roles in heavy metal detoxification and homeostasis,” Annual Review of Plant Biology, vol. 53, pp. 159–182, 2002.
[31]
S. Saka, W. Aouacheri, and C. Abdennour, “The capacity of glutathione reductase in cell protection from the toxic effect of heated oils,” Biochimie, vol. 84, no. 7, pp. 661–665, 2002.
[32]
M. Sivaguru, F. Balu？ka, D. Volkmann, H. H. Felle, and W. J. Horst, “Impacts of aluminum on the cytoskeleton of the maize root apex. Short-term effects on the distal part of the transition zone,” Plant Physiology, vol. 119, no. 3, pp. 1073–1082, 1999.
[33]
S. Grabski and M. Schindler, “Aluminum induces rigor within the actin network of soybean cells,” Plant Physiology, vol. 108, no. 3, pp. 897–901, 1995.
[34]
M. Sivaguru, S. Pike, W. Gassmann, and T. I. Baskin, “Aluminum rapidly depolymerizes cortical microtubules and depolarizes the plasma membrane: evidence that these responses are mediated by a glutamate receptor,” Plant and Cell Physiology, vol. 44, no. 7, pp. 667–675, 2003.
[35]
R. Cruz-Ortega, J. C. Cushman, and J. D. Ownby, “cDNA clones encoding 1,3-β-glucanase and a fimbrin-like cytoskeletal protein are induced by Al toxicity in wheat roots,” Plant Physiology, vol. 114, no. 4, pp. 1453–1460, 1997.
[36]
L. Kadouri, A. Hubert, Y. Rotenberg et al., “Cancer risks in carriers of the BRCA1/2 Ashkenazi founder mutations,” Journal of Medical Genetics, vol. 44, no. 7, pp. 467–471, 2007.
[37]
T. Watanabe, M. Osaki, H. Yano, and I. Rao, “Internal mechanisms of plant adaptation to aluminum toxicity and phosphorus starvation in three tropical forages,” Journal of Plant Nutrition, vol. 29, no. 7, pp. 1243–1255, 2006.
[38]
X. F. Li, J. F. Ma, and H. Matsumoto, “Pattern of aluminum-induced secretion of organic acids differs between rye and wheat,” Plant Physiology, vol. 123, no. 4, pp. 1537–1543, 2000.
[39]
J. F. De Carvalho Gon？alves, J. Cambraia, P. R. Mosquim, and E. F. Araújo, “Aluminum effect on organic acid production and accumulation in sorghum,” Journal of Plant Nutrition, vol. 28, no. 3, pp. 507–520, 2005.
[40]
R. D. Hamel and V. D. Appanna, “Modulation of TCA cycle enzymes and aluminum stress in Pseudomonas fluorescens,” Journal of Inorganic Biochemistry, vol. 87, no. 1, pp. 1–8, 2001.
[41]
M. Tesfaye, S. J. Temple, D. L. Allan, C. P. Vance, and D. A. Samac, “Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum,” Plant Physiology, vol. 127, no. 4, pp. 1836–1844, 2001.
[42]
V. M. Anoop, U. Basu, M. T. McCammon, L. McAlister-Henn, and G. J. Taylor, “Modulation of citrate metabolism alters aluminum tolerance in yeast and transgenic canola overexpressing a mitochondrial citrate synthase,” Plant Physiology, vol. 132, no. 4, pp. 2205–2217, 2003.
[43]
P. Barone, D. Rosellini, P. LaFayette, J. Bouton, F. Veronesi, and W. Parrott, “Bacterial citrate synthase expression and soil aluminum tolerance in transgenic alfalfa,” Plant Cell Reports, vol. 27, no. 5, pp. 893–901, 2008.
[44]
H. H. Begum, M. Osaki, T. Watanabe, and T. Shinano, “Mechanisms of aluminum tolerance in phosphoenolpyruvate carboxylase transgenic rice,” Journal of Plant Nutrition, vol. 32, no. 1, pp. 84–96, 2009.
[45]
V. M. Anoop, U. Basu, M. T. McCammon, L. McAlister-Henn, and G. J. Taylor, “Modulation of citrate metabolism alters aluminum tolerance in yeast and transgenic canola overexpressing a mitochondrial citrate synthase,” Plant Physiology, vol. 132, no. 4, pp. 2205–2217, 2003.
[46]
M. A. Pi？eros, J. V. Magalhaes, V. M. Carvalho Alves, and L. V. Kochian, “The physiology and biophysics of an aluminum tolerance mechanism based on root citrate exudation in maize,” Plant Physiology, vol. 129, no. 3, pp. 1194–1206, 2002.
[47]
M. Sivaguru, B. Ezaki, Z. H. He et al., “Aluminum-induced gene expression and protein localization of a cell wall-associated receptor kinase in Arabidopsis,” Plant Physiology, vol. 132, no. 4, pp. 2256–2266, 2003.
[48]
W. A. Carlezon, R. S. Duman, and E. J. Nestler, “The many faces of CREB,” Trends in Neurosciences, vol. 28, no. 8, pp. 436–445, 2005.
[49]
W. Wang, B. Vinocur, O. Shoseyov, and A. Altman, “Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response,” Trends in Plant Science, vol. 9, no. 5, pp. 244–252, 2004.
