Figure 4.

The PYR1 His60Pro mutation leads to increased affinity for ABA. Typical thermograms resulting from isothermal titration calorimetry experiments with wt PYR1 (A) and PYR1^H60P (B) (the concentration of PYR1 is 8 times higher than that of PYR1^H60P). The estimated Kds for ABA binding are indicated. The increase in affinity and the change in enthalpy, endothermic for PYR1 and exothermic PYR^H60P can be appreciated. (C) In vitro OST1 kinase activity assays in the presence of ΔNHAB1, wt PYR1 and the mutant PYR1^H60P. The experiment shows an enhanced recovery of OST1 activity in the presence of PYR1^H60P at low ABA concentrations, as compared with wt PYR1. The top panel shows a representative autoradiogram from a protein kinase assay. The bottom panel shows the quantification of the activity based on relative intensity of bands corresponding to OST1 phosphorylation products, using a phosphoimager Image Gauge V.4.0. Quantification of ΔCABF2 was also performed obtaining similar results. Standard error measurements are shown (n=3).