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.