3.2 Al-tolerant Tibetan wild barley XZ29 differs from Dayton in response to Al
We then attempted to figure out the roles of evolution of P metabolism and P transport for Al tolerance using barley and other plant species in different experiments. In the short-term field trials, XZ29 and Dayton showed greater tolerance to acid soil than XZ9 and Franklin (Figure 3a). In hydroponic experiments with 5 μM AlCl3, XZ29 and Dayton also showed greater Al tolerance than XZ9 (Figure 3b). In long-term hydroponic experiments with full nutrition and 0, 15 μM or 60 μM AlCl3, XZ29 and Dayton are consistently more tolerant than XZ9 (Figures S6). Tissue analysis following these treatments showed that the Al concentration in roots of XZ9 was approximately 1.5-fold greater than in XZ29 or Dayton (Figure 3c).
One of the major established mechanisms for Al tolerance in cultivated barley is the citrate efflux from the root apices via the HvAACT1 transporter (Furukawa et al., 2007). Therefore, we measured the Al-dependent citrate efflux from excised root tips of XZ29, XZ9 and Dayton and found that Dayton was the only cultivar to show an Al-activated efflux of citrate (Figure 3d). Furthermore, the constitutive expression of HvAACT1 in Dayton roots was four-fold greater than both of the two Tibetan wild barley accessions and the expression of HvACCT1 was unaffected by Al treatment in all genotypes (Figure 3e). The higher expression of HvAACT1 in Dayton is known to be caused by a 1 kb insertion upstream of the HvAACT1coding region and a PCR marker is available to detect the present or absence of this mutation (Fujii et al., 2012). Our results indicated that the 1-kb insertion was present in Dayton, as expected, but not in XZ29 or XZ9 (Figure 3f). This same marker was used to test another 108 Tibetan wild barley accessions with differing levels of Al tolerance. Surprisingly, none of these accessions possessed the 1 kb insertion upstream of HvAACT1 (Table S5). Thus, there is likely to be another mechanism controlling Al tolerance in X29.