Operationally Defined Apoplastic and Symplastic Aluminum Fractions in Root Tips of Aluminum-Intoxicated Wheat

Abstract

Knowledge of the mechanistic basis of differential aluminum (Al) tolerance depends, in part, on an improved ability to quantify Al located in the apoplastic and symplastic compartments of the root apex. Using root tips excised from seedlings of an Al-tolerant wheat cultivar (Triticum aestivum L. cv Yecora Rojo) grown in Al solutions for 2 d, we established an operationally defined apoplastic Al fraction determined with six sequential 30-min washes using 5 mM CaC12 (pH 4.3). Soluble symplastic Al was eluted by freezing root tips to rupture cell membranes and performing four additional 30-min CaC12 washes, and a residual fraction was determined via digestion of root tips with HNO3. The three fractions were then determined in Yecora Rojo and a sensitive wheat cultivar (Tyler) grown at 18, 55, or 140 Mm total solution Al (AIT). When grown at equal AlT, Tyler contained more Al than Yecora Rojo in all fractions, but both total Al and fractional distribution were similar in the two cultivars grown at AIT levels effecting a 50% reduction in root growth. Residual Al was consistently 50 to 70% of the total, and its location was elucidated by staining root tips with the fluorophore morin and examining them using fluorescence and confocal laser scanning microscopy. Wall-associated Al was only observed in tips prior to any washing, and the residual fraction was manifested as distinct staining of the cytoplasm and nucleus but not of the apoplastic space. Accordingly, the residual fraction was allocated to the symplastic compartment for both cultivars, and recalculated apoplastic Al was consistently approximately 30 to 40% of the total. Distributions of Al in the two cultivars did not support a symplastic detoxification hypothesis, but the role of cytoplasmic exclusion remains unsettled.Root lesions caused by Al toxicity can cause disruption of membrane structure and function, disruption of DNA synthesis and mitosis, cell wall rigidification and reductions in cell elongation, and/or disturbance in mineral assimilation and metabolism, and these postulated toxicity mechanisms have recently been reviewed by Taylor (29). Despite vast quantities of published research, however, the principal physiological mechanism(s) of Al rhizotoxicity remains unresolved, and it remains unclear which are primary dysfunctions and which should more properly be considered secondary effects. There are broad, genetically determined differences in Al tolerance between plant species and genotypes (8). Intraspecfic differences in responses to Al may provide clues to mechanisms of toxicity and aid in plant breeding for superior Al tolerance (8).In general, theories concerning mechanisms of differential tolerance may be divided into three categories (3, 30): (a) the primary lesion of Al rhizotoxicity is cytoplasmic, and differential tolerance is the result of variation in the ability of a plant to detoxify or tolerate Al within the symplast; (b) the primary lesion is cytoplasmic, and differential tolerance is a result of genotypic variation in abili...

Direct Evaluation of the Ca2+-Displacement Hypothesis for Al Toxicity

“…Third, we have demonstrated that the accumulation of A1 in the apices of AI-tolerant roots was significantly less than in AI-sensitive genotypes (Table 111). This agrees with previous studies by Polle et al (1978), Tice et al (1992), Rincón and Gonzales (1992), and Delhaize et al (1993a), but in the present study we included severa1 improvements. First, we used near-isogenic lines of wheat, which allowed us to be confident that any differences observed between the tolerant and sensitive genotypes were related to the Alfl locus, which confers AI tolerance in these lines (Delhaize et al, 1993a).…”

“…Most that are available indicate an overwhelming accumulation of A1 in the apoplasm (Clarkson, 1967;Clarkson and Sanderson, 1969;Huett and Menary, 1979;Zhang and Taylor, 1990). Tice et al (1992) cautioned that many of the earlier studies used solutions that might have encouraged precipitation of AI in the cell wall and led to an overestimation of the contribution from the apoplasm. In their own study with wheat roots they concluded that most of the A1 accumulated over 2 d was located in the symplasm.…”

“…The compositions of the treatments (Table I) were kept simple to minimize the potential for precipitation of A1 in the cell wall (Tice et al, 1992). Furthermore, the concentrations of CaC1, and AlC1, were varied so that the activity of the free Ca2+ ions was maintained near 200 FM and, where present, the activity of the trivalent A1 cation A13+ was maintained near 1.4 p~.…”

“…These investigations often require reliable estimates of AI uptake into the symplasm, but the capacity for the cell wall to accumulate high concentrations of cations makes the resolution of apoplasmic and symplasmic fractions difficult (Dainty and Hope, 1959; Zhang and Taylor, 1990; Reid and Smith, 1992). Some research indicates that A1 can readily cross the plasma membrane (Lazof et al, 1994) and that a large proportion of the A1 in roots resides in the symplasm (Tice et al, 1992; see Taylor, …”

The Physiology and Biophysics of an Aluminum Tolerance Mechanism Based on Root Citrate Exudation in Maize

“…However, some plants have evolved Al tolerance mechanisms that enable them to grow in Al-toxic, acid soil environments (for review, see Kochian, 1995;Ma et al, 2001). Earlier work in this field showed that Al tolerance in wheat (Triticum aestivum) was associated with a reduced accumulation of Al in the root apex, but not the mature root (see, for example, Rincon and Gonzales, 1992;Tice et al, 1992; Delhaize et al, 1993a). Delhaize et al (1993aDelhaize et al ( , 1993b subsequently provided compelling evidence for the existence of an Al tolerance mechanism based on root tip Al exclusion that involved Al-activated release of malate from the root apex of an Al-tolerant near-isogenic line of wheat.…”