Figure 1.
Reversible oxidation of HAB1 by H2O2.
A) Sensitivity of full length (1–511 aa) and phosphatase domain (171–511 aa) of HAB1 to H2O2 treatment. B) Reactivation of H2O2 treated HAB1 by reducing agents (n = 3, error bar represent s.d.).
Figure 2.
Sequence alignment of HAB1, ABI2 and ABI1.
Cysteines in the phosphatase domain of HAB1 are indicated by black boxes and asterisks. C186 and C274 are highlighted by red asterisks. The numbers on top of the alignment indicate amino acid positions within HAB1.
Figure 3.
HAB1 C186S/C274S mutant protein is resistant to inactivation by H2O2.
A) Relative phosphatase activities of HAB1 wild type and cysteine point mutant proteins to H2O2 treatment. B) H2O2 resistance of C186S and C274S single and double mutant proteins compared to wild type HAB1 (n = 3, error bar represent s.d.).
Figure 4.
Ribbon representation of the HAB1 phosphatase domain (PDB: 4LA7) with C186 and C274 presented as stick models and the remaining cysteine residues as yellow patches. The SnRK2.6 interacting site is shown in magenta and MgCl2 ions as magenta spheres (figure drawn from).
Figure 5.
H2O2 induces formation of HAB1 dimers that are catalytically compromised.
A, B, upper panels: Gel filtration profiles of HAB1 wildtype and C186S/C274S mutant protein in the presence and absence of H2O2. Wildtype His6-MBP-HAB1 elutes as monomers in the absence of H2O2 (blue chromatogram) and as a mixture of dimers (at 60 ml elution volume) and monomers (at 70 ml) upon treatment with 0.3 mM H2O2 (red chromatogram). In contrast, His6-MBP-HAB1 C186S/C274S elutes exclusively as monomers. *MW: molecular weight standards; mAU: absorbance at 280 nm×1000. Lower panels: Reducing and non-reducing SDS PAGE of gel filtration fractions of wild type and C186S/C274S HAB1 proteins, respectively. C) Phosphatase activity of HAB1 protein from the untreated monomer fraction (no H2O2), from the dimer and monomer fractions of wild type HAB1 treated with 0.3 mM H2O2, and from the monomer fraction of HAB1 C186S/C274S treated with 0.3 mM H2O2.
Figure 6.
H2O2 prevents the HAB1-mediated inhibition of SnRK2.6 kinase activity.
A) SnRK2.6 trans-phosphorylation activity at high (30∶50 molar) HAB1:SnRK2.6 ratio. B) SnRK2.6 auto- and trans-phosphorylation activity at low (30∶1000 molar) HAB1:SnRK2.6 ratio. Recombinant SnRK2.6 was incubated at the indicated concentrations with a fragment of the transcription factor ABF2 [GST-ABF2(73–120 aa)] and with 32P-γATP in the presence and absence of H2O2-treated wild type (wt) and mutant (mt; C186S/C274S) HAB1.
Figure 7.
H2O2 inhibits the direct interaction of HAB1 with SnRK2.6 and PYR1.
A, B) AlphaScreen assays showing interactions between His6-MBP-HAB1(171–511 aa) wild type, C186/274S double mutant and R505A mutant protein and biotin-SnRK2.6(11–362 aa), biotin-SnRK2.6 ABA box peptide and biotin-PYR1(9–182 aa) with 10 µM ABA in the absence or presence of 0.3 mM (A) or 1 mM H2O2 (B). Controls are HAB1 proteins in the absence of SnRK2.6 or PYR1 as well as SnR2.6 or PYR1 in the absence of HAB1 (n = 3, error bar represent s.d.). C) Non-reducing SDS PAGE of untreated wild type HAB1 (−) and HAB1 treated with 1 mM of H2O2 (+).
Figure 8.
Cartoon representation of the regulation of HAB1 by H2O2 during water stress.
See text for details.