| Abstract: | Despite the rhizotoxicity of aluminum (Al) being identified over 100 years ago, there is still no consensus regarding the mechanisms whereby root elongation rate is initially reduced in the approximately 40% of arable soils worldwide that are acidic. We used high-resolution kinematic analyses, molecular biology, rheology, and advanced imaging techniques to examine soybean (Glycine max) roots exposed to Al. Using this multidisciplinary approach, we have conclusively shown that the primary lesion of Al is apoplastic. In particular, it was found that 75 µmAl reduced root growth after only 5 min (or 30 min at 30 µmAl), with Al being toxic by binding to the walls of outer cells, which directly inhibited their loosening in the elongation zone. An alteration in the biosynthesis and distribution of ethylene and auxin was a second, slower effect, causing both a transient decrease in the rate of cell elongation after 1.5 h but also a longer term gradual reduction in the length of the elongation zone. These findings show the importance of focusing on traits related to cell wall composition as well as mechanisms involved in wall loosening to overcome the deleterious effects of soluble Al.Acid soils, in which soluble aluminum (Al) is elevated, comprise approximately 4 billion ha of the global ice-free land or approximately 40% of the world’s total arable land (Eswaran et al., 1997). Although it has been known for over a century that Al decreases plant root growth, the underlying reasons for its toxic effects remain elusive. In a highly cited review of literature, Horst et al. (2010) stated that the “mechanism of Al-induced inhibition of root elongation is still not well understood, and it is a matter of debate whether the primary lesions of Al toxicity are apoplastic or symplastic.” For example, in the symplast, Al has been reported to cause interference with DNA synthesis and mitosis, disrupt the function of the Golgi apparatus, damage membrane integrity, and inhibit mitochondrial functions. In the apoplast, Al may rigidify the cell wall (prevent loosening), inhibit cell wall enzymes, such as expansin, and cause cell rupturing (for review, see Rengel, 1997; Horst et al., 2010). The identification of numerous processes influenced by Al (such as those listed above) has occurred for a number of reasons. First, it is almost certain that Al does, indeed, have multiple mechanisms by which it reduces growth in both the short and long term. Second, there has been a lack of studies that have related the changes observed in these processes to the actual underlying changes in root elongation rate (RER). Thus, there typically has been no clear separation of the primary and secondary toxic effects of Al. Although some studies have examined the speed with which Al reduces RER, these studies have generally (1) been at comparatively coarse time steps and (2) not taken the additional step of relating these changes in RER to the underlying mechanism of toxicity (for example, see Llugany et al., 1995; Parker, 1995; Kidd et al., 2001; and Blamey et al., 2004).In this study, we used kinematic analyses as the basis for elucidating the underlying mechanisms by which Al exerts toxic effects on the growth of soybean (Glycine max) roots. First, after exposure to Al, we captured digital images every 0.5 to 1 min so as to calculate changes in overall RER with a resolution of 5 to 10 min. Second, we examined whether these changes in overall RER resulted from either or both (1) changes in the length of the root elongation zone (LEZ) or (2) changes in elemental elongation rate (EER), which is defined as the change in length per unit length of a small portion of tissue (Silk, 1992), as a measure of the rate at which individual cells elongate. Based upon these data, it seemed that Al is toxic by at least three separate but interrelated mechanisms. To provide additional information on these mechanisms, we: (1) used kinematic analyses to investigate the effects of aminoethoxyvinyl-Gly (AVG), an ethylene synthesis inhibitor; (2) examined changes in auxin distribution and movement in the root apex using a highly active synthetic auxin-response element (referred to as DR5) with a minimal promoter-GUS reporter gene (DR5::GUS); (3) investigated rapid changes in the mechanical properties of root cell walls using creep analysis; and (4) used synchrotron-based low-energy x-ray fluorescence spectromicroscopy (LEXRF) and high-resolution secondary ion mass spectroscopy (NanoSIMS) to examine the spatial distribution of Al on cellular and subcellular levels in roots exposed to Al for only 30 min. This integrated approach has allowed us to identify the sequence of processes whereby Al reduces the growth of soybean roots in the short term. |
