As relative root elongation (RRE) has been shown to be a suitable phenotypic criterion to assess Al resistance in a wide range of plant species, we used this parameter to screen tatary buckwheat cultivars for Al resistance (Fig. ). As expected, there were genotypic differences in Al resistance among these cultivars. For instance, root elongation of ‘Chuan’ was inhibited by 36 % after 24 h exposure to 25 µm AlCl3, whereas the same treatment resulted in 57 % inhibition of root elongation in ‘Liuku2’ (Fig. ). We thus termed ‘Chuan’ as an Al-resistant cultivar and ‘Liuku2’ as an Al-sensitive cultivar.
Fig. 1. Screening of eight tatary buckwheat cultivars for Al resistance. Three-day-old seedlings were subjected to a 0·5 mm CaCl2 solution (pH 4·5) containing 0 or 25 µm AlCl3 for 24 h. Root growth was measured by a ruler before and after (more ...)
Al content in the root apex was measured to test whether the mechanism of Al exclusion accounted for the genotypic differences among these tatary buckwheat cultivars. There was a large difference in Al content (mean ± s.d.) in the root apices, ranging from 0·046 ± 0·002 µg per root apex in ‘Weihei’ to 0·133 ± 0·0098 µg per root apex in the most Al-sensitive ‘Liuku2’ after exposure to 25 µm Al for 24 h (Fig. A). Al content in the root apex of the most Al-resistant ‘Chuan’ was also lower (0·052 ± 0·0079 µg per root apex; Fig. A). The Al content of the root apex correlated negatively with RRE (Fig. B), suggesting an Al exclusion mechanism is contributing to the differential Al resistance in tatary buckwheat.
Fig. 2. (A) Al content in root apex (B) and correlation between relative root elongation and root apex Al content among eight tatary buckwheat cultivars. *P < 0·05. Root apices (apical 10 mm) were cut with a razor blade after 24 h of treatment (more ...)
To determine whether the differences in Al exclusion in the root apex are due to the Al-induced secretion of organic acids, we analysed organic acids in the root exudates. Without Al treatment, a trace amount (in some cases below the lowest detection limit) of malate and citrate was detected, and oxalate was the most abundant organic acid (data not shown). Moreover, oxalate was the only organic acid whose response was dependent on the presence of Al. Al-activated oxalate exudation rates ranged from 33·4 ± 14·6 ng per root apex h−1 in ‘Lijiang’ to 98·3 ± 1·5 ng per root apex h−1 in ‘Weihei’. The Al-activated oxalate exudation rates for ‘Chuan’ and ‘Liuku2’ were 56·1 ± 2·8 and 48·8 ± 7·1 ng per root apex h−1, respectively (Fig. ). Overall, we could not establish a relationship between root Al-activated oxalate exudation and root apex Al content (Fig. ).
Fig. 3. Al-induced secretion of oxalate in eight tatary buckwheat cultivars. Three-day-old seedlings were exposed to 0·5 mm CaCl2 solution (pH 4·5) for 3 h, and root exudates were collected. Seedlings were then exposed to 0·5 mm CaCl2 (more ...)
Correlation between Al content and oxalate secretion rate of eight tatary buckwheat cultivars. Data for Al content (n = 3) and oxalate secretion rate (n = 3) were taken from Figs 2 and 3, respectively.
The lack of correlation between Al-activated oxalate exudation and Al exclusion (Al content) implies that other exclusion mechanisms might be operating. In rice, exudation of organic acids does not respond to Al stress and is not responsible for Al exclusion (Yang et al., 2008
; Famoso et al., 2010
). Interestingly, cell-wall polysaccharides play an important role in exclusion of Al from the root apex of rice (Yang et al., 2008
). Thus, whether the cell-wall polysaccharides also have a similar function in plants that show Al-induced secretion of organic acids will be of interest. As shown in Fig. A, the root apex pectin content ranged from 1·80 ± 0·39 to 3·38 ± 0·41 µg per root apex in the eight tatary buckwheat cultivars. In general, pectin content in Al-resistant cultivars was lower than that in Al-sensitive cultivars. However, a moderately Al-resistant cultivar, ‘Dianning’, had the lowest pectin content. Therefore, we could not establish a correlation between pectin content and Al content among the eight cultivars (dashed regression line in Fig. B), but a significant positive correlation could be established if ‘Dianning’ and ‘Lijiang’ were excluded (solid regression line in Fig. B). Among the cultivars, the pectin content of the most Al-sensitive ‘Liuku2’ (3·38 ± 0·41 µg per root apex) was significantly higher than that of the most Al-resistant ‘Chuan’ (2·38 ± 0·52 µg per root apex; Fig. A), indicating that pectin content could partly explain the differential ability of Al exclusion among tatary buckwheat cultivars.
Fig. 5. Pectin content in root apex of eight tatary buckwheat cultivars (A) and correlation between root apical Al contents and pectin contents (B). For pectin content determination, 3-d-old seedlings were exposed to 0·5 mM CaCl2 solution (pH 4·5) (more ...)
Several lines of evidence indicate that not only does pectin content contribute to Al accumulation in plants but so too does its degree of methylation (Eticha et al., 2005
; Yang et al., 2008
). Therefore, we determined and compared PME activity between ‘Chuan’ and ‘Liuku2’, because these two cultivars exhibited the greatest genotypic difference and pectin content difference. First, we analysed PME activity using a sensitive colorimetric assay method based on determination of the amount of methanol released from pectin by PME extracts. As shown in Fig. , PME activity was not significantly different between ‘Chuan’ and ‘Liuku2’ in the absence of Al, but Al treatment resulted in a significant increase of PME activity especially in ‘Liuku2’. Second, monoclonal antibodies (JIM5 and JIM7), which are specific for cell-wall pectin differing in the degree of methylation, were used for immunofluorescence localization of different cell-wall pectins. JIM5 stains low-methyl-ester pectins, and the intensity of JIM5 fluorescence did not differ between ‘Chuan’ and ‘Liuku2’ in the absence of Al (Fig. A, C). Fluorescence intensity was enhanced by Al treatment in the two cultivars, but was more evident in ‘Liuku2’ (Fig. B, D). JIM7 stains high-methyl-ester pectins, and the intensity of JIM7 fluorescence was not different in the two cultivars in the absence of Al (Fig. E, G). However, Al treatment resulted in a decrease of fluorescence in the two cultivars but was more evident in the Al-sensitive ‘Liuku2’ (Fig. F, H).
Fig. 6. Pectin methylesterase (PME) activity in the root apex of two tatary buckwheat cultivars. 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 excised for cell (more ...)
Fig. 7. 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 tatary buckwheat cultivars. Three-day-old seedlings of Al-resistant ‘Chuan’ (more ...)
Given the relative importance of an internal tolerance mechanism in common buckwheat, a species closely related to tatary buckwheat, we measured Al accumulation in roots and shoots of ‘Chuan’ and ‘Liuku2’. Table shows the results for roots and shoots when grown in 25 µm Al for 24 h. The Al content in the roots of the two cultivars was quite similar, although the difference was significant at P < 0·05. The Al content in the shoots of ‘Chuan’ exceeded 100 µg g−1 d. wt. This cultivar was thus also ranked as an Al-accumulator.
Al content in roots and shoots of tatary buckwheat cultivars