A Model for the Involvement of HAB1, SWI3B, and a Putative Plant SWI/SNF Complex in the Regulation of Plant Transcriptional Response to ABA on the Chromatin Template.
HAB1 is a negative regulator of ABA signaling that interacts with SWI3B, which is a positive regulator of ABA signaling. SWI3B must play a key role as a core subunit of an unidentified SWI/SNF complex, which is predicted to regulate nucleosomal structure in response to ABA. In the absence of ABA, HAB1 is localized in the vicinity of the RAB18 and RD29B promoters and negatively regulates the expression of these genes. ABA inhibits HAB1 and releases its inhibitory effect on a putative SWI/SNF complex. [See online article for color version of this figure.]

The Presence of HAB1 in the Vicinity of the ABA-Responsive RD29B and RAB18 Promoters Is Abolished by ABA.
(A) Immunoprecipitated samples (IP anti-HA) were subjected to immunoblot analysis (W anti-HA-HRP).
(B) to (D) ChIP assays of the RD29B, RAB18, and ACT8 promoters in hab1-1 or hab1-1∷ProHAB1-HAB1-dHA plants. Genomic DNA fragments that coimmunoprecipitated with HAB1-dHA were analyzed by RT-qPCR using primers of the RD29B (B), RAB18 (C), and ACT8 (D) promoters. Results are presented as ratios of the amount of DNA immunoprecipitated from HAB1-dHA samples to that from the hab1-1 control (value set to equal 1). The positions of the ABA-responsive elements (triangles) and TATA boxes (diamonds) in the sequences of the different promoters are indicated, as well as the primers used for RT-qPCR analysis. The numbering refers to the ATG translation start codon, and the beginnings of the open reading frames are indicated by arrows.

HAB1 and SWI3B Interact in a Yeast Two-Hybrid Assay.
Interaction was determined by growth assay on medium lacking His and adenine. Dilutions (10⁻¹, 10⁻², and 10⁻³) of saturated cultures were spotted onto the plates.
(A) Top, interaction assay with ΔNHAB1 as bait (fused to the GBD) and either full-length or different deletions of SWI3B as putative preys (fused to the GAD). Schemes of SWI3B domains and the different protein deletions are shown. Deletions C1 and C2 lacked C-terminal amino acid residues 346 to 469 and 221 to 469, respectively. Deletion N1 lacked N-terminal amino acid residues 1 to 220. GAD-SWIRM and GAD-ZZ comprised amino acid residues 1 to 140 and 134 to 220, respectively. Middle, interaction assay with SWI3A, SWI3B, SWI3C, and SWI3D as putative preys. Bottom, interaction assay with ΔNPP2CA, ΔNABI1, and ΔNABI2 as baits and SWI3B as prey.
(B) Protein phosphatase activity of MBP-HAB1, MBP-ΔNHAB1, and MBP-G246D ΔNhab1 fusion proteins. Values are averages ± se from three independent experiments.
(C) Interaction assay with ΔNHAB1 and G246D ΔNhab1 as baits and SWI3B as prey.

HAB1 Localizes at Both Cytosol and Nucleus.
(A) Subcellular localization of HAB1 and SWI3B in Agrobacterium-infiltrated tobacco leaves. Epifluorescence and bright-field images of epidermal leaf cells infiltrated with a mixture of Agrobacterium suspensions harboring the indicated constructs and the silencing suppressor p19. BiFC-induced bZIP63 dimerization served to identify the nuclei of tobacco epidermal cells (asterisks). The expression of the proteins is demonstrated by immunodetection with anti-GFP for HAB1-GFP and SWI3B-GFP (center, between panels). Treatment with 50 μM ABA for 1 h (+ABA) did not change the subcellular localization of both HAB1-GFP and SWI3B-GFP.
(B) Biochemical fractionation of HAB1-dHA (full-length protein marked with asterisks). Plant material was obtained from the hab1-1∷ProHAB1-HAB1-dHA transgenic line after mock treatment or treatment with 50 μM ABA for 1 h. The soluble cytosolic fraction (S), nuclei/organelles wash fraction (W), nuclear insoluble fraction (Ni), and nuclear soluble fraction (Ns) were analyzed using anti-HA, anti-histone 3 (H3), and anti–ribulose-1,5-bis-phosphate carboxylase/oxygenase (RBC) antibodies.
(C) Relative amount of HAB1-dHA in the soluble cytosolic (S) and nuclear (N) fractions after mock treatment or treatment with 50 μM ABA for 1 h.

BiFC Visualization and Coimmunoprecipitation Experiments Show Interaction between HAB1 and SWI3B in the Nucleus of Tobacco Leaves.
(A) Introduction of the G246D substitution into HAB1 abolishes the interaction with SWI3B. Epifluorescence and bright-field images of epidermal leaf cells infiltrated with a mixture of Agrobacterium suspensions harboring constructs HAB1-HA-YFP^C/SWI3BN-myc-YFP^N (panel 1) or G246D-HA-YFP^C/SWI3BN-myc-YFP^N (panel 2) and the silencing suppressor p19. The bar corresponds to 75 μm. Green color corresponds to YFP, whereas red color is generated by chlorophyll fluorescence.
(B) Coimmunoprecipitation assay demonstrates the interaction between HAB1 and SWI3B in planta. Protein extracts obtained from tobacco leaves infiltrated with Agrobacterium suspensions harboring constructs HAB1-HA-YFP^C/SWI3BN-myc-YFP^N (lanes 1) or G246D-HA-YFP^C/SWI3BN-myc-YFP^N (lanes 2) were analyzed using anti-HA or anti-c-myc antibodies. Input levels of epitope-tagged proteins in crude protein extracts (20 μg of total protein) were analyzed by immunoblotting. Immunoprecipitated epitope HA–tagged proteins were probed with anti-c-myc antibodies to detect coimmunoprecipitation of SWI3BN-myc-YFP^N with HAB1-HA-YFP^C.
(C) BiFC assays show the interaction of ABI1, ABI2, and PP2CA with SWI3B in the nucleus of tobacco leaves. Cells were infiltrated with a mixture of Agrobacterium suspensions harboring constructs SWI3BN-HA-YFP^C/YFP^N-ABI1 (panel 3), SWI3BN-HA-YFP^C/YFP^N-ABI2 (panel 4), or SWI3BN-HA-YFP^C/YFP^N-PP2CA (panel 5) and the silencing suppressor p19.
(D) Protein gel blot analysis demonstrates the expression of SWI3BN-HA-YFP^C and the corresponding YFP^N-PP2Cs (asterisks). Protein extracts obtained from tobacco leaves infiltrated with Agrobacterium suspensions harboring the silencing suppressor p19 and constructs SWI3BN-HA-YFP^C/YFP^N-ABI1 (lane 3), SWI3BN-HA-YFP^C/YFP^N-ABI2 (lane 4), SWI3BN-HA-YFP^C/YFP^N-PP2CA (lane 5), or p19 alone (lane p19) were analyzed using anti-HA or anti-GFP^N antibodies.

swi3b Mutants Show Reduced Sensitivity to ABA-Mediated Inhibition of Germination and Growth.
(A) T-DNA insertions in the swi3b-1 and swi3b-2 alleles and localization of ethyl methanesulfonate–induced mutations in swi3b-3 and swi3b-4 alleles. The numbering begins at the ATG translation start codon. The gray boxes represent exons. The SANT domain is spotted within the third exon.
(B) ABA effects on germination in the progeny of swi3b-1 and swi3b-2 heterozygous plants. The percentage of seeds that germinated and developed green cotyledons in the presence of the indicated concentrations of ABA is shown. Values are averages ± sd for three independent experiments (n = 200 seeds per experiment). * P < 0.01 (Student's t test) when comparing data from each genotype and the wild type in the same assay conditions.
(C) Reduced sensitivity of swi3b-1 and swi3b-2 heterozygous (+/−) seedlings to ABA-mediated growth inhibition. Fresh weight was measured in 12-d-old seedlings grown in MS medium either lacking or containing 10 μM ABA. Values are averages ± sd for three independent experiments (n = 20 seedlings per experiment). Representative seedlings of Col (wild type [wt]), swi3b-1 +/− (1), and swi3b-2 +/− (2) were removed from medium lacking or containing 10 μM ABA and rearranged on agar plates (at right).
(D) Reduced sensitivity to ABA-mediated inhibition of seed germination in swi3b-3 and swi3b-4 mutants compared with Col-er105. The percentage of seeds that showed radicle emergence at 96 h after seed stratification is shown. Values are averages ± sd for three independent experiments (n = 200 seeds per experiment). Asterisks are as described for (B).
(E) Reduced sensitivity to ABA-mediated inhibition of early growth in the swi3b-3 mutant compared with Col-er105 in medium supplemented with 0.8 μM ABA. The photograph was taken at 18 d after sowing.
(F) Reduced expression of ABA-inducible genes in swi3b-3 compared with Col-er105. Values are expression levels reached in the mutant with respect to Col-er105 (value 1) as determined by RT-qPCR analyses. Expression of gene markers was analyzed in 7-d-old seedlings grown in medium supplemented with 0.3 μM ABA. Values are averages ± sd for three independent experiments (n = 30 to 40 seedlings per experiment).
(G) The swi3b-3 phenotype is epistatic to hab1-1. The percentage of seeds that showed radicle emergence at 96 h after seed stratification is shown. Values are averages ± sd for three independent experiments (n = 200 seeds per experiment). * P < 0.01 (Student's t test) when comparing data from swi3b-3 and the wild type, or hab1-1swi3b-3 and hab1-1, in the same assay conditions.
(H) Reduced sensitivity to ABA-mediated inhibition of early growth in swi3b-3 and the hab1-1swi3b-3 double mutant. Photographs were taken at 7 d (MS) and 11 d (0.5 μM ABA) after sowing. As in (C), plants were removed from growth medium and rearranged on plates for photography.
[See online article for color version of this figure.]