Cell wall polysaccharides are specifically involved in the exclusion of aluminum from the rice root apex

Abstract

Rice (Oryza sativa) is the most aluminum (Al)-resistant crop species among the small-grain cereals, but the mechanisms responsible for this trait are still unclear. Using two rice cultivars differing in Al resistance, rice sp. japonica 'Nipponbare' (an Al-resistant cultivar) and rice sp. indica 'Zhefu802' (an Al-sensitive cultivar), it was found that Al content in the root apex (0-10 mm) was significantly lower in Al-resistant 'Nipponbare' than in sensitive 'Zhefu802', with more of the Al localized to cell walls in 'Zhefu802', indicating that an Al exclusion mechanism is operating in 'Nipponbare'. However, neither organic acid efflux nor changes in rhizosphere pH appear to be responsible for the Al exclusion. Interestingly, cell wall polysaccharides (pectin, hemicellulose 1, and hemicellulose 2) in the root apex were found to be significantly higher in 'Zhefu802' than in 'Nipponbare' in the absence of Al, and Al exposure increased root apex hemicellulose content more significantly in 'Zhefu802'. Root tip cell wall pectin methylesterase (PME) activity was constitutively higher in 'Zhefu802' than in 'Nipponbare', although Al treatment resulted in increased PME activity in both cultivars. Immunolocalization of pectins showed a higher proportion of demethylated pectins in 'Zhefu802', indicating a higher proportion of free pectic acid residues in the cell walls of 'Zhefu802' root tips. Al adsorption and desorption kinetics of root tip cell walls also indicated that more Al was adsorbed and bound Al was retained more tightly in 'Zhefu802', which was consistent with Al content, PME activity, and pectin demethylesterification results. These responses were specific to Al compared with other metals (CdCl(2), LaCl(3), and CuCl(2)), and the ability of the cell wall to adsorb these metals was also not related to levels of cell wall pectins. All of these results suggest that cell wall polysaccharides may play an important role in excluding Al specifically from the rice root apex.

Figure 1.

Different Al resistance and Al content in rice ‘Nipponbare’ and ‘Zhufu802’. Three-day-old seedlings were exposed for 24 h to 0.5 mm CaCl₂ solution (pH 4.5) containing 0, 25, 50, or 100 μm Al.Al/root elongation without Al × 100 and the root length was measured before and after treatment. Error bars represent ±sd (n = 10). After treatments, root apices were cut for Al extraction or for cell wall extraction. Al in root apices (B) or cell wall fraction (C) was extracted by 2 m HCl for 24 h and determined by ICP-AES. Error bars represent ±sd (n = 3). *, Differences between the cultivars at P < 0.05.

Figure 2.

Al-induced secretion of citrate in rice ‘Zhefu802’ and ‘Nipponbare’. Six-week-old seedlings were exposed to a 0.5 mm CaCl₂ solution (pH 4.5) containing 0 or 25 μm Al. Root exudates were collected after 24-h exposure and citrate was analyzed by HPLC. Error bars represent ±sd (n = 3).

Figure 3.

Effect of Al on root elongation in pH-buffered solution. Three-day-old seedlings were exposed to 0.5 mm CaCl₂ solution containing 0 or 25 μm Al with or without 2.5 mm Homo-PIPES buffer (pH 4.5). Root length was measured in both buffered and unbuffered conditions with a ruler before and after treatments (24 h). White bars represent the RRE between buffered and unbuffered control conditions; black bars represent the RRE between control and Al treatment both under unbuffered conditions; hatched bars represent RRE between control and Al treatment both under buffered conditions. Error bars represent ±sd (n = 10).

Figure 4.

Uronic acid content of cell wall fractions extracted from the rice root apex of ‘Zhefu802’ and ‘Nipponbare’. Three-day-old seedlings were exposed to 0.5 mm CaCl₂ solution containing 0 or 25 μm Al for 24 h. Root apices were cut and cell wall polysaccharides were fractionated into pectin (A), HC1 (B), and HC2 (C) for uronic acid content measurement. Error bars represent ±sd (n = 3). Bars with different letters are significantly different at P < 0.05.

Figure 5.

PME activity in the root apex of rice. Three-day-old seedlings were exposed to 0.5 mm CaCl₂ solution (pH 4.5) containing 0 or 25 μm Al for 24 h. Root apices were cut for cell wall and PME extraction. PME activity was determined colorimetrically. Data are means ±sd (n = 3). Bars with different letters are significantly different at P < 0.05.

Figure 6.

Immunolocalization of low-methyl-ester pectin (JIM5 epitope; A–D) and high-methyl-ester pectin (JIM7 epitope; E–H) in root cross-sections of two rice cultivars. Three-day-old seedlings of Al-sensitive ‘Zhefu802’ (A, B, E, F) or Al-resistant ‘Nipponbare’ (C, D, G, H) were subjected to 0.5 mm CaCl₂ solution with (B, D, F, H) or without (A, C, E, G) 25 μm Al for 24 h. Root sections were taken from 1 to 3 mm behind the apex. Scale bar = 50 μm for all images.

Figure 7.

Adsorption (A) and desorption (B) kinetics of Al in the root cell wall of rice cultivars ‘Zhefu802’ and ‘Nipponbare’. Cell wall materials were extracted as described in “Materials and Methods.” Cell wall materials (10 mg) were placed in a 2-mL column and kinetics were conducted as previously described (Zheng et al., 2004).

Figure 8.

Effect of other cations on root elongation of rice ‘Zhefu802’ and ‘Nipponbare’, and the adsorption ability of cell wall materials extracted from the root apex of rice ‘Zhefu802’ and ‘Nipponbare’ to metals. A, Three-day-old seedlings were exposed to 0.5 mm CaCl₂ solution (pH 4.5) containing 0, 25 μm CdCl₂, 10 μm LaCl₂, or 0.5 μm CuCl₂ for 24 h. Root length was measured before and after treatments. Error bars represent ±sd (n = 10). B, Cell wall materials (3 mg) were suspended with 1.5 mL of 2 μm CdCl₂, 2 μm LaCl₂, or 2 μm CuCl₂ for 1 h. Metal content in suspension solution before or after adsorption was determined by ICP-AES. Error bars represent ±sd (n = 3). *, Differences between cultivars at P < 0.05.