Dataset Information

Rebuilding core abscisic acid signaling pathways of Arabidopsis in yeast

ABSTRACT: The signaling pathway of the phytohormone abscisic acid (ABA) regulates responses towards abiotic stress such as drought and high osmotic conditions. The multitude of functionally redundant components involved in ABA signaling, poses a major challenge for elucidating the largely unresolved response selectivity. We decided rebuilding single linear ABA signaling pathways in yeast for combinatoric permutation of ABA receptors and coreceptors, as well as the response-mediating SnRK2 protein kinases and their targeted transcription factors to drive luciferase expression in a heterologous host. We show that SnRK2s differ in the regulation by ABA receptor complexes, affect ABA responsiveness of the pathway, and differ in their transactivation activity but have similar preferences for ABA-responsive transcription factors. SnRK2s thought to act ABA-independently and known to be activated under osmotic stress in plants were regulated by ABA receptor complexes in yeast and competed with ABA receptor components in an ABA-dependent manner in plant tissue. The study reveals the suitability of the yeast system for analysis of ABA signaling factors and allowing the future dissection of ligand-receptor specificities in a functional response pathway. The analysis provides new insights into SnRK2 regulation indicating that four SnRK2 members of the osmotic stress response are tightly embedded into the ABA signaling pathway.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast) Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Cell Culture

SUBMITTER: Julia Mergner

LAB HEAD: Bernhard Kuster

PROVIDER: PXD013669 | Pride | 2019-07-30

REPOSITORIES: Pride

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Rebuilding core abscisic acid signaling pathways of Arabidopsis in yeast.

Ruschhaupt Moritz M Mergner Julia J Mucha Stefanie S Papacek Michael M Doch Isabel I Tischer Stefanie V SV Hemmler Daniel D Chiasson David D Edel Kai H KH Kudla Jörg J Schmitt-Kopplin Philippe P Kuster Bernhard B Grill Erwin E

The EMBO journal 20190801 17

The phytohormone abscisic acid (ABA) regulates plant responses to abiotic stress, such as drought and high osmotic conditions. The multitude of functionally redundant components involved in ABA signaling poses a major challenge for elucidating individual contributions to the response selectivity and sensitivity of the pathway. Here, we reconstructed single ABA signaling pathways in yeast for combinatorial analysis of ABA receptors and coreceptors, downstream-acting SnRK2 protein kinases, and tra ...[more]

PMID: 31368592

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Project description:The rapid activation of RAF-SnRK2 cascades in response to hyperosmolarity is a crucial event in plants' adaptation to osmotic stress, yet the underlying mechanisms have remained elusive. In this study, we reveal that the calcium chelator EGTA, but not other calcium modulators, can potently activate both RAFs and SnRK2s. We demonstrate that EGTA induces the phosphorylation of RAF24, serving as an indicator of rapid RAF activation. Furthermore, we show that EGTA treatment results in increased phosphorylation of Ser171 and Ser175 in SnRK2.6, known markers of SnRK2 activation. In-gel kinase assays confirmed the activation of RAFs and SnRK2s. These findings collectively highlight that exogenous EGTA application mimics mannitol treatment, effectively triggering the activation of both RAF and SnRK2 subfamily kinases. Utilizing a DIA-based phosphoproteomics approach, we profiled global phosphorylation changes in response to EGTA and mannitol treatments. We identified 22,991 phosphorylated peptides corresponding to 6,515 phosphosites on 1,790 proteins. Notably, 5,067 sites were classified as class I phosphorylation sites, indicating high confidence in their localization. Principal Component Analysis confirmed the distinct phosphoproteomic profiles of the three conditions, with excellent reproducibility among biological replicates. EGTA treatment induced substantial changes in global phosphorylation, with 3,987 phosphosites increased and 964 decreased, signifying a profound impact on protein phosphorylation. The activation of RAFs and SnRK2s was supported by significant phosphorylation changes in these kinases. Additionally, EGTA-induced phosphoproteins included downstream effectors of RAF-SnRK2 cascades. GO analysis revealed distinct functional categories for EGTA and mannitol-responsive phosphoproteins, shedding light on the divergent roles of these treatments. Our study provides novel insights into the role of EGTA in the activation of RAF-SnRK2 cascades and its broader impact on the plant phosphoproteome, offering a valuable resource for understanding the downstream events related to exocellular calcium depletion in plants. It also highlights the potential involvement of MAP4Ks and resistosome-related kinases in osmotic stress signaling. Overall, our research enhances our understanding of osmotic stress response mechanisms and the power of DIA-based phosphoproteomics in unraveling complex phosphorylation events in plants.

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.

Dataset Information

Rebuilding core abscisic acid signaling pathways of Arabidopsis in yeast

Publications

Rebuilding core abscisic acid signaling pathways of Arabidopsis in yeast.

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