Dataset Information

Structural insights into the abscisic acid stereospecificity by the ABA receptors PYR/PYL/RCAR.

ABSTRACT: The phytohormone abscisic acid ((+)-ABA) plays a key role in many processes. The biological and biochemical activities of unnatural (-)-ABA have been extensively investigated since 1960s. However, the recognition mechanism by which only a few members among PYR/PYL/RCAR (PYLs) family can bind (-)-ABA remains largely unknown. Here we systematically characterized the affinity of PYLs binding to the (-)-ABA and reported the crystal structures of apo-PYL5, PYL3-(-)-ABA and PYL9-(+)-ABA. PYL5 showed the strongest binding affinity with (-)-ABA among all the PYLs. PYL9 is a stringently exclusive (+)-ABA receptor with interchangeable disulfide bonds shared by a subclass of PYLs. PYL3 is a dual receptor to both ABA enantiomers. The binding orientation and pocket of (-)-ABA in PYLs are obviously different from those of (+)-ABA. Steric hindrance and hydrophobic interaction are the two key factors in determining the stereospecificity of PYLs binding to (-)-ABA, which is further confirmed by gain-of-function and loss-of-function mutagenesis. Our results provide novel insights of the bioactivity of ABA enantiomers onto PYLs, and shed light on designing the selective ABA receptors agonists.

SUBMITTER: Zhang X

PROVIDER: S-EPMC3699650 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Structural insights into the abscisic acid stereospecificity by the ABA receptors PYR/PYL/RCAR.

Zhang Xingliang X Jiang Lun L Wang Guoqiang G Yu Lin L Zhang Qi Q Xin Qi Q Wu Wei W Gong Zhizhong Z Chen Zhongzhou Z

PloS one 20130702 7

The phytohormone abscisic acid ((+)-ABA) plays a key role in many processes. The biological and biochemical activities of unnatural (-)-ABA have been extensively investigated since 1960s. However, the recognition mechanism by which only a few members among PYR/PYL/RCAR (PYLs) family can bind (-)-ABA remains largely unknown. Here we systematically characterized the affinity of PYLs binding to the (-)-ABA and reported the crystal structures of apo-PYL5, PYL3-(-)-ABA and PYL9-(+)-ABA. PYL5 showed t ...[more]

PMID: 23844015

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Project description:Abscisic acid (ABA) is a key hormone for plant growth, development and stress adaptation. Perception of ABA through four types of receptors has been reported. We show here that impairment of ABA perception through the PYR/PYL/RCAR branch reduces vegetative growth and seed production, and leads to a severely open stomata and dramatic ABA insensitive phenotype, even though other branches for ABA perception remain functional. Arabidopsis sextuple mutant impaired in 6 PYR/PYL receptors, namely PYR1, PYL1, PYL2, PYL4, PYL5 and PYL8, was able to germinate and grow even on 100 mM ABA. Whole-rosette stomatal conductance (Gst) measurements revealed that leaf transpiration in the sextuple pyr/pyl mutant was higher than in the ABA-deficient aba3-1 or ABA-insensitive snrk2.6 mutants. The gradually increasing Gst values of plants lacking three, four, five and six PYR/PYLs indicate quantitative regulation of stomatal aperture by this family of receptors. The sextuple mutant lacked ABA-mediated activation of SnRK2s and ABA-responsive gene expression was dramatically impaired as was reported in snrk2.2/2.3/2.6. In summary, these results show that ABA perception by PYR/PYLs plays a major role to regulate seed germination and establishment, basal ABA signaling required for vegetative and reproductive growth, stomatal aperture and transcriptional response to the hormone. Four biological replicates were generated for the 3 genotypes, pyr1pyl1pyl2pyl4pyl5pyl8 sextuple mutant, snrk2.2/2.3/2.6 triple mutant, and Col-0. All the samples were treated with ABA for 3 hours before harvesting. The four Col-0 samples were mix to create an unique reference Col-0 sample. Two comparasion were made, fisrt one, PYR/PYL sextuple mutant versus Col-0 reference sample and second one, SnRK2 triple mutant versus Col-0 reference sample. In each comparasion four biological replicates were made. Replicas number 1 and 2 were labeled with Cy5 for the mutant sample and Cy3 for the Col-0 reference sample, while the other two replicas,#3 and #4, were reversed-labeled.

Project description:ABA signaling relies on the perception of the hormone by 14 different members of the PYR/PYL/RCAR family of ABA receptors. Upon ABA binding, receptors become active in inhibiting a family of type 2C protein phosphatases that usually function as negative regulators on protein kinases of the SnRK2 family. Previous physiological and genetic data supporting a negative role exerted by nitric oxide (NO) on ABA signaling have been reported. Our preliminary data suggested that negative regulation exerted by NO on ABA signaling may involve direct effects of NO on ABA receptors. Here we explore whether the effects of NO on ABA signaling are due to post-translational modifications (PTMs) on ABA receptors. Two main PTMs are functionally related to NO, the S-nitrosylation of C residues and the nitration of Y residues and they have been studied in this work. Because our data suggest that NO can also modulate the stability of the receptor proteins, we have also analyzed another PTM, the ubiquitination of K residues. A double approach based on in vitro nitration of recombinant PYR/PYL/RCAR proteins as well as in planta expression of HA-tagged versions of the receptors, followed by immunopurification strategies based on anti-3-nitroY-coated magnetic beads, and LC-MS/MS analyses of the immunopurified proteins under non-reducing conditions allowed us to identify peptides containing nitroY (mass shifts of +45), S-nitrosylated C (mass shifts of +29), and K-GG tags resulting from the trypsin digestion of ubiquitin tails (mass shift of + 114). Our data suggest that S-nitrosylation of cysteines from PYR/PYL receptors occur in planta but has not effect on receptor function as tested in vitro. By contrast, the nitration of several Y residues of PYR/PYL receptors completely inactivates them, promotes also their polyubiquitination and marks them for subsequent proteasomal degradation, thus representing a rapid mechanism for lowering ABA sensitivity when required in the plant.

Dataset Information

Structural insights into the abscisic acid stereospecificity by the ABA receptors PYR/PYL/RCAR.

Publications

Structural insights into the abscisic acid stereospecificity by the ABA receptors PYR/PYL/RCAR.

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