Dataset Information

DIA-Based Phosphoproteomics Identifies Early Phosphorylation Events in Response to EGTA and Mannitol in Arabidopsis.

ABSTRACT: Osmotic stress significantly hampers plant growth and crop yields, emphasizing the need for a thorough comprehension of the underlying molecular responses. Previous research has demonstrated that osmotic stress rapidly induces calcium influx and signaling, along with the activation of a specific subset of protein kinases, notably the Raf-like protein (RAF)-sucrose nonfermenting-1-related protein kinase 2 (SnRK2) kinase cascades within minutes. However, the intricate interplay between calcium signaling and the activation of RAF-SnRK2 kinase cascades remains elusive. Here, in this study, we discovered that Raf-like protein (RAF) kinases undergo hyperphosphorylation in response to osmotic shocks. Intriguingly, treatment with the calcium chelator EGTA robustly activates RAF-SnRK2 cascades, mirroring the effects of osmotic treatment. Utilizing high-throughput data-independent acquisition-based phosphoproteomics, we unveiled the global impact of EGTA on protein phosphorylation. Beyond the activation of RAFs and SnRK2s, EGTA treatment also activates mitogen-activated protein kinase cascades, Calcium-dependent protein kinases, and receptor-like protein kinases, etc. Through overlapping assays, we identified potential roles of mitogen-activated protein kinase kinase kinase kinases and receptor-like protein kinases in the osmotic stress-induced activation of RAF-SnRK2 cascades. Our findings illuminate the regulation of phosphorylation and cellular events by Ca²⁺ signaling, offering insights into the (exocellular) Ca²⁺ deprivation during early hyperosmolality sensing and signaling.

SUBMITTER: Sang T

PROVIDER: S-EPMC11325057 | biostudies-literature | 2024 Jun

REPOSITORIES: biostudies-literature

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DIA-Based Phosphoproteomics Identifies Early Phosphorylation Events in Response to EGTA and Mannitol in Arabidopsis.

Sang Tian T Chen Chin-Wen CW Lin Zhen Z Ma Yu Y Du Yanyan Y Lin Pei-Yi PY Hadisurya Marco M Zhu Jian-Kang JK Lang Zhaobo Z Tao W Andy WA Hsu Chuan-Chih CC Wang Pengcheng P

Molecular & cellular proteomics : MCP 20240618 8

Osmotic stress significantly hampers plant growth and crop yields, emphasizing the need for a thorough comprehension of the underlying molecular responses. Previous research has demonstrated that osmotic stress rapidly induces calcium influx and signaling, along with the activation of a specific subset of protein kinases, notably the Raf-like protein (RAF)-sucrose nonfermenting-1-related protein kinase 2 (SnRK2) kinase cascades within minutes. However, the intricate interplay between calcium sig ...[more]

PMID: 38901673

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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:BackgroundProtein phosphorylation regulated by plant hormone is involved in the coordination of fundamental plant development. Brassinosteroids (BRs), a group of phytohormones, regulated phosphorylation dynamics remains to be delineated in plants. In this study, we performed a mass spectrometry (MS)-based phosphoproteomics to conduct a global and dynamic phosphoproteome profiling across five time points of BR treatment in the period between 5 min and 12 h. MS coupling with phosphopeptide enrichment techniques has become the powerful tool for profiling protein phosphorylation. However, MS-based methods tend to have data consistency and coverage issues. To address these issues, bioinformatics approaches were used to complement the non-detected proteins and recover the dynamics of phosphorylation events.ResultsA total of 1104 unique phosphorylated peptides from 739 unique phosphoproteins were identified. The time-dependent gene ontology (GO) analysis shows the transition of biological processes from signaling transduction to morphogenesis and stress response. The protein-protein interaction analysis found that most of identified phosphoproteins have strongly connections with known BR signaling components. The analysis by using Motif-X was performed to identify 15 enriched motifs, 11 of which correspond to 6 known kinase families. To uncover the dynamic activities of kinases, the enriched motifs were combined with phosphorylation profiles and revealed that the substrates of casein kinase 2 and mitogen-activated protein kinase were significantly phosphorylated and dephosphorylated at initial time of BR treatment, respectively. The time-dependent kinase-substrate interaction networks were constructed and showed many substrates are the downstream of other signals, such as auxin and ABA signaling. While comparing BR responsive phosphoproteome and gene expression data, we found most of phosphorylation changes were not led by gene expression changes. Our results suggested many downstream proteins of BR signaling are induced by phosphorylation via various kinases, not through transcriptional regulation.ConclusionsThrough a large-scale dynamic profile of phosphoproteome coupled with bioinformatics, a complicated kinase-centered network related to BR-regulated growth was deciphered. The phosphoproteins and phosphosites identified in our study provide a useful dataset for revealing signaling networks of BR regulation, and also expanded our knowledge of protein phosphorylation modification in plants as well as further deal to solve the plant growth problems.

Dataset Information

DIA-Based Phosphoproteomics Identifies Early Phosphorylation Events in Response to EGTA and Mannitol in Arabidopsis.

Publications

DIA-Based Phosphoproteomics Identifies Early Phosphorylation Events in Response to EGTA and Mannitol in Arabidopsis.

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