PXD046802-1

PXD046802 is an original dataset announced via ProteomeXchange.

Title	DIA-based phosphoproteomics identifies early phosphorylation events in response to EGTA and mannitol in Arabidopsis
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.
HostingRepository	PRIDE
AnnounceDate	2024-04-18
AnnouncementXML	Submission_2024-04-18_01:14:45.973.xml
DigitalObjectIdentifier
ReviewLevel	Peer-reviewed dataset
DatasetOrigin	Original dataset
RepositorySupport	Unsupported dataset by repository
PrimarySubmitter	Chuan-Chih Hsu
SpeciesList	scientific name: Arabidopsis thaliana (Mouse-ear cress); NCBI TaxID: 3702;
ModificationList	phosphorylated residue
Instrument	Q Exactive

Revision	Datetime	Status	ChangeLog Entry
0	2023-11-08 18:37:29	ID requested
⏵ 1	2024-04-18 01:14:46	announced
2	2024-10-22 06:36:33	announced	2024-10-22: Updated project metadata.

Dataset with its publication pending

submitter keyword: Arabidopsis, DIA,Phosphoproteomics, EGTA

Pengcheng Wang
contact affiliation	nstitute of Advanced Biotechnology and School of Life Sciences, Southern University of Science and Technology
contact email	wangpc@sustech.edu.cn
lab head
Chuan-Chih Hsu
contact affiliation	Academia Sinica
contact email	without1102@gmail.com
dataset submitter

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PRIDE project URI

• PRIDE
  1. PXD046802
     1. Label: PRIDE project
     2. Name: DIA-based phosphoproteomics identifies early phosphorylation events in response to EGTA and mannitol in Arabidopsis