Supplementary Figure 1:

1. Supplementary Figure 1: b PYL5 Marker PYL4 PYL6 PYR1 PYL1 PYL2 PYL3 a None PYL2 PYL3 PYL4 PYL5 PYL6 PYR1 PYL1 c apo-PYL2 ABA-PYL2 apo-PYL1 ABA-PYL2 -HAB1 None PYL2 PYL3 PYL4 PYL5 PYL6 PYR1 PYL1 d HAB1 Mg2+ PYL2 pre- post- gelfiltration column None PYL2 PYL3 PYL4 PYL5 PYL6 PYR1 PYL1

2. Supplementary Figure 2: N N a C C gate gate latch latch b C N N C

3. Supplementary Figure 3: a K64 K64 N173 N173 E98 E98 E147 E147 b c latch + gate ABA-PYL2 apo-PYL2 ABA * ABA PYL2 G246D Mg2+ Mg2+ HAB1/PP2C HAB1/PP2C

4. Supplementary Figure 4: a 680 nm 520-620 nm Singlet Oxygen b Streptavidin -Coated Donor Bead Ni-coated Acceptor Bead PYL2-H6 b-HAB1 IC50 = 250nM + untagged PYL2 competitor PYL2 680 nm Singlet Oxygen X no signal Streptavidin Ni-coated Acceptor Bead b-HAB1 PYL2 PYL2-H6

5. Supplementary Figure 5: ligand binding pocket gate relative luciferase activity latch

6. Supplementary Figure 6: a PYR1 residue number gate latch c b d