Transient activity assays were performed in Arabidopsis mesophyll protoplasts from Columbia wild-type or SnRK2.2/2.3/2.621 as described previously (http://genetics.mgh.harvard.edu/sheenweb)28 (link). Transfected protoplasts were incubated for 5 h in light in the presence of 0 (open bars) or 5 μM (+) ABA, and then used for measuring LUC and GUS activities as described previously28 (link). Yeast two-hybrid and triple-hybrid assays, co-immunoprecipitation and BiFC assays were similar to those described previously13 . Purification of GST-HAB1, His-PYR1 and His PYR1P88S was carried out as described previously12 (link). GST, GST-ABI1, GST-ABF2 fragment, MBP and MBP-SnRK2.6 constructs were transformed into E. coli Rosetta cells (Novagen) and the recombinant proteins isolated by affinity purification. Purification of MBP-ABI1, MBP-ABI2, His-PYL8, His-PYL5 and His-SnRK2.6 was as described previously13 . In-gel kinase assays were performed as described previously20 (link) with the modification that 300 μg protein was loaded for samples without ABA treatment. For germination assays, seeds were plated on MS medium containing 3% sucrose. In each experiment, at least 50 seeds per genotype were stratified at 4 °C for 3 d, and the presence of green cotyledons was scored after 6-day incubation at 23 °C.