Dataset Information

Proteomic analysis of Nitric Oxide-related Post-translational modifications of PYR/PYL/RCAR receptors of ABA in Arabidopsis thaliana

ABSTRACT: 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.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Whole Body

SUBMITTER: Jose Leon

LAB HEAD: Jose Leon

PROVIDER: PXD002396 | Pride | 2015-09-09

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	121026_S_JLJL_01_01_PYL4_C.raw	Raw
	121026_S_JLJL_01_01_PYL4_C_Sequest.msf-pride.pride.mgf.gz	Mgf
	121026_S_JLJL_01_01_PYL4_C_Sequest.msf-pride.pride.mztab.gz	Mztab
	121026_S_JLJL_01_01_PYL4_C_Sequest.msf-pride.xml.gz	Xml
	121026_S_JLJL_02_01_PYL4_30M.raw	Raw

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Publications

Inactivation of PYR/PYL/RCAR ABA receptors by tyrosine nitration may enable rapid inhibition of ABA signaling by nitric oxide in plants.

Castillo Mari-Cruz MC Lozano-Juste Jorge J González-Guzmán Miguel M Rodriguez Lesia L Rodriguez Pedro L PL León José J

Science signaling 20150901 392

Abscisic acid (ABA) is a phytohormone that inhibits growth and enhances adaptation to stress in plants. ABA perception and signaling rely on its binding to receptors of the pyrabactin resistance1/PYR1-like/regulatory components of ABA receptors (PYR/PYL/RCAR) family, the subsequent inhibition of clade A type 2C protein phosphatases (PP2Cs), and the phosphorylation of ion channels and transcription factors by protein kinases of the SnRK2 family. Nitric oxide (NO) may inhibit ABA signaling because ...[more]

PMID: 26329583

Similar Datasets

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:This model is from the article: A thermodynamic switch modulates abscisic acid receptor sensitivity. Dupeux F, Santiago J, Betz K, Twycross J, Park SY, Rodriguez L, Gonzalez-Guzman M, Jensen MR, Krasnogor N, Blackledge M, Holdsworth M, Cutler SR, Rodriguez PL, Márquez JA EMBO J. [2011 Oct; Volume: 30 (Issue: 20 )] Page info: 4171-84 21847091 , Abstract: Abscisic acid (ABA) is a key hormone regulating plant growth, development and the response to biotic and abiotic stress. ABA binding to pyrabactin resistance (PYR)/PYR1-like (PYL)/Regulatory Component of Abscisic acid Receptor (RCAR) intracellular receptors promotes the formation of stable complexes with certain protein phosphatases type 2C (PP2Cs), leading to the activation of ABA signalling. The PYR/PYL/RCAR family contains 14 genes in Arabidopsis and is currently the largest plant hormone receptor family known; however, it is unclear what functional differentiation exists among receptors. Here, we identify two distinct classes of receptors, dimeric and monomeric, with different intrinsic affinities for ABA and whose differential properties are determined by the oligomeric state of their apo forms. Moreover, we find a residue in PYR1, H60, that is variable between family members and plays a key role in determining oligomeric state. In silico modelling of the ABA activation pathway reveals that monomeric receptors have a competitive advantage for binding to ABA and PP2Cs. This work illustrates how receptor oligomerization can modulate hormonal responses and more generally, the sensitivity of a ligand-dependent signalling system. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information. In summary, you are entitled to use this encoded model in absolutely any manner you deem suitable, verbatim, or with modification, alone or embedded it in a larger context, redistribute it, commercially or not, in a restricted way or not. To cite BioModels Database, please use: Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le Novère N, Laibe C (2010) BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol., 4:92.

Project description:This model is from the article: A thermodynamic switch modulates abscisic acid receptor sensitivit y. Dupeux F, Santiago J, Betz K, Twycross J, Park SY, Rodriguez L, Gonzalez- Guzman M, Jensen MR, Krasnogor N, Blackledge M, Holdsworth M, Cutler SR, Rodrigue z PL, Márquez JA EMBO J. [2011 Oct; Volume: 30 (Issue: 20 )] Page info: 4171-84 21847091 , Abstract: Abscisic acid (ABA) is a key hormone regulating plant growth, development and the response to biotic and abiotic stress. ABA binding to pyrabactin resistance (PYR )/PYR1-like (PYL)/Regulatory Component of Abscisic acid Receptor (RCAR) intracell ular receptors promotes the formation of stable complexes with certain protein ph osphatases type 2C (PP2Cs), leading to the activation of ABA signalling. The PYR/ PYL/RCAR family contains 14 genes in Arabidopsis and is currently the largest pla nt hormone receptor family known; however, it is unclear what functional differen tiation exists among receptors. Here, we identify two distinct classes of recepto rs, dimeric and monomeric, with different intrinsic affinities for ABA and whose differential properties are determined by the oligomeric state of their apo forms . Moreover, we find a residue in PYR1, H60, that is variable between family membe rs and plays a key role in determining oligomeric state. In silico modelling of t he ABA activation pathway reveals that monomeric receptors have a competitive adv antage for binding to ABA and PP2Cs. This work illustrates how receptor oligomeri zation can modulate hormonal responses and more generally, the sensitivity of a l igand-dependent signalling system. To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information. In summary, you are entitled to use this encoded model in absolutely any manner you deem suitable, verbatim, or with modification, alone or embedded it in a larger context, redistribute it, commercially or not, in a restricted way or not. To cite BioModels Database, please use: Li C, Donizelli M, Rodriguez N, Dharuri H, Endler L, Chelliah V, Li L, He E, Henry A, Stefan MI, Snoep JL, Hucka M, Le Novère N, Laibe C (2010) BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models. BMC Syst Biol., 4:92.