High aluminum resistance in buckwheat. I. Al-induced specific secretion of oxalic acid from root tips

Abstract

High Al resistance in buckwheat (Fagopyrum esculentum Moench. cv Jianxi) has been suggested to be associated with both internal and external detoxification mechanisms. In this study the characteristics of the external detoxification mechanism, Al-induced secretion of oxalic acid, were investigated. Eleven days of P depletion failed to induce secretion of oxalic acid. Exposure to 50 &mgr;M LaCl3 also did not induce the secretion of oxalic acid, suggesting that this secretion is a specific response to Al stress. Secretion of oxalic acid was maintained for 8 h by a 3-h pulse treatment with 150 &mgr;M Al. A nondestructive method was developed to determine the site of the secretion along the root. Oxalic acid was found to be secreted in the region 0 to 10 mm from the root tip. Experiments using excised roots also showed that secretion was located on the root tip. Four kinds of anion-channel inhibitors showed different effects on Al-induced secretion of oxalic acid: 10 &mgr;M anthracene-9-carboxylic acid and 4, 4'-diisothiocyanatostilbene-2,2'-disulfonate had no effect, niflumic acid stimulated the secretion 4-fold, and phenylglyoxal inhibited the secretion by 50%. Root elongation in buckwheat was not inhibited by 25 &mgr;M Al or 10 &mgr;M phenylglyoxal alone but was inhibited by 40% in the presence of Al and phenylglyoxal, confirming that secretion of oxalic acid is associated with Al resistance.

Figure 1

Effect of Al on root elongation in buckwheat and wheat. Three-day-old seedlings were exposed to 0.5 mm CaCl₂ solution, pH 4.5, containing no Al (white bars) or 25 μm AlCl₃ (black bars) for 16 h. Error bars represent ±sd (n = 10).

Figure 2

Effect of P deficiency followed by Al treatment on the secretion of oxalic acid by buckwheat roots. Ten-day-old seedlings were grown in nutrient solution devoid of P, and root exudates were collected every other day in 0.5 mm CaCl₂ solution at pH 4.5 for 6 h. On d 12 and 14 after P deficiency, the roots were exposed to 50 μm Al solution, and the root exudates were collected for 6 h. After passage of the root exudates through a cation-exchange resin column followed by an anion-exchange resin column, the anionic fraction was eluted using 1 m HCl and concentrated. Organic acids were analyzed by HPLC. Error bars represent ±sd (n = 3).

Figure 3

Effect of La³⁺ and Al³⁺ on the secretion of oxalic acid by buckwheat roots. Seedlings were exposed to 0.5 mm CaCl₂ solution, pH 4.5, containing 50 μm AlCl₃, 50 μm LaCl₃, or both. After 6 h the root exudates were collected and organic acids were analyzed as described in Figure 1. Error bars represent ±sd (n = 3).

Figure 4

Effect of a 3-h pulse of 150 μm Al on the secretion of oxalic acid (□). Seedlings were exposed to 0.5 mm CaCl₂ solution, pH 4.5, containing 150 μm AlCl₃ for 3 h and subsequently to 0.5 mm CaCl₂ solution, pH 4.5, without Al. Root exudates were collected during the periods 0 to 3, 3 to 7 , 7 to 11, and 27 to 31 h. For comparison, exudates of roots continuously exposed to 150 μm Al (○) or 0 μm (⋄) were also collected at the same interval. Organic acids were analyzed as described in Figure 1. Error bars represent ±sd (n = 3).

Figure 5

Al-induced secretion of oxalic acid from a different section of buckwheat roots. Excised root segments 0 to 5 and 5 to 10 mm from root tips were incubated in 0.5 mm CaCl₂ solution, pH 4.5, containing 0 or 150 μm AlCl₃ after washing. After 3 h the root exudates were collected. Soluble oxalic acid in the 0- to 5- and the 5- to 10-mm root segments were extracted with distilled water at 55°C. Organic acids were analyzed as described in Figure 1. Shown are the oxalic acid content in roots (white bars), oxalic acid excreted by the roots not treated with Al (black bars), and oxalic acid secreted by roots treated with Al (shaded bars). Error bars represent ±sd (n = 3).

Figure 6

Location of secretion of oxalic acid (OX) along buckwheat roots. Fifteen roots exposed to 0 or 150 μm Al for 3 h were placed on the filter paper. After 8 h the amount of oxalic acid was assayed by the method described in Methods. For quantification, 5 μL of a 1- to 4-mm oxalic acid solution was spotted onto the filter paper and assayed by the same procedures as intact roots (top).

Figure 7

Effect of anion-channel inhibitors on Al-induced secretion of oxalic acid. Seedlings were exposed to 0.5 mm CaCl₂ solution, pH 4.5, containing 50 μm Al in the presence or absence of each inhibitor (10 μm). After 6 h root exudates were collected, and organic acids were analyzed as described in Figure 1. Error bars represent ±sd (n = 3).

Figure 8

Combined effect of Al and PG on root elongation in buckwheat. Seedlings were exposed to 0.5 mm CaCl₂ solution, pH 4.5, containing no Al (−Al), 25 μm Al (+Al), 10 μm PG, or 25 μm Al plus 10 μm PG for 16 h. Error bars represent ±sd (n = 10).

Figure 9

Effect of different molar ratios of Al to oxalic acid on the root elongation of corn. The Al concentration was 20 μm in 0.1 mm CaCl₂ solution. Seedlings were exposed to different treatment solutions for 22 h. Error bars represent ±sd (n = 6).