PYRABACTIN RESISTANCE1-LIKE8 plays an important role for the regulation of abscisic acid signaling in root

Abstract

Abscisic acid (ABA) signaling plays a critical role in regulating root growth and root system architecture. ABA-mediated growth promotion and root tropic response under water stress are key responses for plant survival under limiting water conditions. In this work, we have explored the role of Arabidopsis (Arabidopsis thaliana) PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS for root ABA signaling. As a result, we discovered that PYL8 plays a nonredundant role for the regulation of root ABA sensitivity. Unexpectedly, given the multigenic nature and partial functional redundancy observed in the PYR/PYL family, the single pyl8 mutant showed reduced sensitivity to ABA-mediated root growth inhibition. This effect was due to the lack of PYL8-mediated inhibition of several clade A phosphatases type 2C (PP2Cs), since PYL8 interacted in vivo with at least five PP2Cs, namely HYPERSENSITIVE TO ABA1 (HAB1), HAB2, ABA-INSENSITIVE1 (ABI1), ABI2, and PP2CA/ABA-HYPERSENSITIVE GERMINATION3 as revealed by tandem affinity purification and mass spectrometry proteomic approaches. We also discovered that PYR/PYL receptors and clade A PP2Cs are crucial for the hydrotropic response that takes place to guide root growth far from regions with low water potential. Thus, an ABA-hypersensitive pp2c quadruple mutant showed enhanced hydrotropism, whereas an ABA-insensitive sextuple pyr/pyl mutant showed reduced hydrotropic response, indicating that ABA-dependent inhibition of PP2Cs by PYR/PYLs is required for the proper perception of a moisture gradient.

Figure 1.

PYL8 plays a nonredundant role in root sensitivity to ABA. A, Quantification of ABA-mediated root growth inhibition of pyr/pyl mutants compared with the wild type. Data are averages ± se from three independent experiments (n = 15 each). *P < 0.01 (Student’s t test) with respect to the wild type in the same experimental condition. B, Seedling establishment of pyr/pyl mutants compared with Col and Landsberg erecta (La-er) wild types in medium supplemented with 0.5 μm ABA at 3, 5, 7, and 10 d after sowing. Data show the percentage of seeds that germinated and developed green cotyledons. Values are averages ± se for three independent experiments (100 seeds each). *P < 0.01 (Student’s t test) with respect to the wild type in the same experimental condition. C, ABA-insensitive phenotypes of pyl8-1 and pyl8-2 alleles compared with the Col wild type. Photographs show representative seedlings 10 d after the transfer of 4-d-old seedlings from Murashige and Skoog medium (MS) to plates lacking or supplemented with 10 μm ABA. D, Complementation of the pyl8-1 allele by introduction of a 35S:3HA-PYL8 transgene (pyl8-1 complemented). The photograph shows representative seedlings 12 d after the transfer of 4-d-old seedlings from Murashige and Skoog medium to plates supplemented with 20 μm ABA. [See online article for color version of this figure.]

Figure 2.

GUS expression driven by ProPYR1:GUS, ProPYL1:GUS, ProPYL2:GUS, ProPYL4:GUS, ProPYL5:GUS, ProPYL6:GUS, ProPYL7:GUS, ProPYL8:GUS, and ProPYL9:GUS genes in the apical root. A, GUS expression visualized using modified PS-PI staining and confocal laser scanning microscopy. B, Quantification of GUS activity in 15-d-old roots using 4-methylumbelliferyl β-d-glucuronide as a substrate. RFU, Relative fluorescence units. C, Immunoblot analysis of protein extracts from 15-d-old roots using anti-GUS antibody. Ponceau staining from a 43-kD protein is shown as a loading control. D, Magnification of the apical root from ProPYL8:GUS lines that were stained as described in A. E, GUS expression driven by ProPYL1:GUS, ProPYL4:GUS, and ProPYL8:GUS genes in columella cells. GUS staining was observed in the absence of subsequent PS-PI staining.

Figure 3.

Quantification of ABA-mediated inhibition of the root growth of pyr/pyl mutants compared with the wild type. Data are averages ± se from three independent experiments (n = 15 each). The letters denote significant differences among the different genetic backgrounds (P < 0.05, Fisher’s lsd tests). Primary root lengths of 15 plants per genotype (three independent experiments) were measured after 8 d in medium lacking or supplemented with 10, 20, or 50 μm ABA. The 145, 148, 1458, and 12458 mutants contain the pyr1-1 allele; the 114, 1124, 11458, and 112458 mutants contain both pyr1-1 and pyl1 alleles. The rest of the abbreviations reflect the corresponding pyl number.

Figure 4.

Genetic and biochemical interaction of PYL8 with clade A PP2Cs. A, The reduced sensitivity of pyl8 to ABA-mediated inhibition of root growth is abrogated by knocking out clade A PP2Cs. The quantification of ABA-mediated root growth inhibition of the indicated genotypes compared with the wild type is shown. Data are averages ± se from three independent experiments (n = 15 each). *P < 0.01 (Student’s t test) with respect to the wild type in the same growth conditions. Photographs show representative seedlings 10 d after the transfer of 4-d-old seedlings to Murashige and Skoog plates (MS) lacking or supplemented with 5 µm ABA. Bars = 1 cm. B, Clade A PP2Cs interact in vivo with PYL8 in the presence of ABA. GS-PYL8 and PYL8-GS interact with clade A PP2Cs expressed in Arabidopsis cell suspension cultures. The SDS-PAGE analysis shows the zone where HAB1/HAB2 (top two bands) and ABI1/ABI2/PP2CA (other bands) were recovered as interacting partners of PYL8 when extracts and TAP purification buffers were supplemented with 50 μm ABA. C, Quantification of significantly (P > 95%) matched peptides of clade A PP2Cs recovered in independent TAP experiments using either GS-PYL8 or PYL8-GS as bait. ABA supplementation (50 μm; +ABA) dramatically increased the recovery of clade A PP2Cs compared with samples lacking ABA supplementation (−ABA). Detailed results of the peptides identified by MS analyses are provided as Supplemental Table S1. [See online article for color version of this figure.]

Figure 5.

Enhanced hydrotropic response of the pp2c quadruple mutant and reduced hydrotropic response of the pyr/pyl sextuple mutant. A, ABA-hypersensitive phenotype of the hab1-1abi1-2pp2ca-1abi2-2 quadruple mutant, abbreviated as Qabi2-2, compared with the Col wild type. Photographs show representative seedlings 10 d (left) or 20 d (right) after the transfer of 4-d-old seedlings to Murashige and Skoog plates lacking or supplemented with 10 µm ABA. B, ABA-hypersensitive root growth inhibition of the Qabi2-2 mutant compared with the Col wild type. C, Enhanced hydrotropic response of the Qabi2-2 mutant compared with the wild type. D, Reduced hydrotropic response of the pyr/pyl sextuple mutant compared with the wild type. C and D show hydrotropism assays with 7-d-old Arabidopsis seedlings. Data represent measures of the root curvature angle taken 14 h after the transfer of 7-d-old seedlings to split agar plates containing 0.4 m sorbitol in the region with low water potential. Values are averages from three independent experiments ± se (n = 42 each). *P < 0.05 (Student’s t test) when comparing data from each genotype and the wild type in the same assay conditions. E and F, Photographs show the experiments described in C and D, respectively, at 3 d after the transfer of 7-d-old seedlings to split agar plates containing 0.4 m sorbitol. The arrows mark the limit between Murashige and Skoog medium and medium supplemented with 0.4 m sorbitol. [See online article for color version of this figure.]