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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 靳宗洛 | zh_TW |
| dc.contributor.advisor | Tsung-Luo Jinn | en |
| dc.contributor.author | 席 嵐 | zh_TW |
| dc.contributor.author | Silamparasan Dhanasekar | en |
| dc.date.accessioned | 2025-02-13T16:11:11Z | - |
| dc.date.available | 2025-02-14 | - |
| dc.date.copyright | 2025-02-13 | - |
| dc.date.issued | 2025 | - |
| dc.date.submitted | 2025-02-10 | - |
| dc.identifier.citation | Ahmed, I., Kumar, A., Bheri, M., Srivastava, A. K. & Pandey, G. K. (2023) Glutamate receptor like channels emerging players in calcium mediated signaling in plants. Int J Biol Macromol, 234, 123522.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/96375 | - |
| dc.description.abstract | 細胞膜之上鈣通透性通道在植物生長、發育以及對環境刺激的反應中有至關重要功能。阿拉伯芥有 20 種類麩胺酸受體 (GLR) 蛋白,這些蛋白在結構上與動物離子型麩胺酸受體相似,並在植物中介導 Ca²⁺的內流。鈣依賴型蛋白激酶 (CDPKs)能夠磷酸化 GLR3.7 蛋白的絲氨酸 (Ser)-860,該蛋白與 14-3-3ω 相互作用,通過調節Ca²⁺信號傳導在阿拉伯芥鹽與離層酸 (ABA) 反應有重要功能。然而,CDPK 介導的 GLR3.6 中特定 Ser 位點的磷酸化在其功能中的意義仍不明確。在本研究中,我們利用 CDPK16 與包含 GLR3.6 中 14-3-3ω 相互作用域的胜肽片段進行體外激酶實驗。結果顯示,GLR3.6 的 Ser861/862 位點對於與 14-3-3ω 的交互作用是必要的,而Ser856位點僅由CDPK16專一性磷酸化,而不會被CDPK3或CDPK34磷酸化。此外,鹽逆境會迅速下調 GLR3.6 的表現,且 glr3.6 突變體植物在正常生長條件下的根長度較野生型 Col 短,過量表現 35S::GLR3.6-YFP 植株根長度則較長。而35S::GLR3.6-S856A-YFP 植株在鹽逆境下表現出與 glr3.6 突變體相似的低敏感,顯示 Ser856 的磷酸化對於鹽逆境反應至關重要。針對 CDPK16 的研究發現,CDPK16 是一個關鍵的 Ca²⁺偵測蛋白質,影響多種生理過程。CDPK16 位於細胞iv膜和細胞核,在花與根中表現較高。在鹽逆境和 ABA處理下,其表達顯著上調,指出其可能參與逆境反應。性狀分析顯示,與 cdpk16 突變體以及野生型 Col 相比,35S::CDPK16-YFP 植株表現出增強的主根生長能力以及對鹽與 ABA 處理較低的敏感性。蛋白質-蛋白質交互作用實驗證實,CDPK16 能夠與細胞週期依賴性激酶抑制因子 (Kip相關蛋白) KRP4以及 KRP7交互作用。此外,CDPK16磷酸化 KRP4與 KRP7,促進其降解,進而以 Ca²⁺依賴的方式正調節細胞週期進程。這些發現建立了一個功能框架,揭示了 CDPK16 藉由整合 Ca²⁺ 訊號通路,藉由調節細胞週期依賴性激酶抑制因子和 GLR3.6 的活性發揮作用,調控根生長與逆境反應的機制。本研究強調 CDPK16 作為 Ca²⁺介導信號通路中的分子樞紐的重要作用,並為植物根生長與逆境防禦調控提供新的見解。 | zh_TW |
| dc.description.abstract | Calcium-permeable channels in the plasma membrane play crucial roles in plant growth, development, and response to environmental stimuli. Arabidopsis contains 20 glutamate receptor-like (GLR) proteins that share structural similarities with animal ionotropic glutamate receptors and mediate Ca2+ influx in plants. Calcium-dependent protein kinases (CDPKs) phosphorylate serine (Ser)-860 of GLR3.7 protein, which interacts with 14-3- 3ω and is essential for salt and abscisic acid (ABA) responses in Arabidopsis by modulating Ca2+ signaling. However, the functional significance of CDPK-mediated phosphorylation specific Ser residues of GLR3.6 remains unclear. In this study, we performed an in vitro kinase assay using CDPK16 and peptides containing the 14-3-3ω interacting domain of GLR3.6. Our results demonstrate that Ser861/862 of GLR3.6 are necessary for interaction with 14-3-3ω, and Ser856 of GLR3.6 is specifically phosphorylated by CDPK16 but not by CDPK3 and CDPK34. Additionally, GLR3.6 expression was rapidly downregulated by salt stress, and glr3.6 mutant plants exhibited shorter root lengths under normal growth conditions compared to Col. Conversely, 35S::GLR3.6-YFP lines displayed longer root lengths. The 35S::GLR3.6-S856A-YFP showed reduced sensitivity to salt stress, similar to glr3.6 mutant plants, indicating that Ser856 phosphorylation is critical for salt stress response. Parallel investigations revealed that CDPK16, a key Ca²⁺ sensors, influence diverse physiological processes. CDPK16 localizes to the plasma membrane and nucleus, with prominent expression in flowers and roots. Its expression is significantly upregulated in response to salt stress and ABA, suggesting its involvement in stress-responsive pathways. Phenotypic analyses showed that 35S::CDPK16-YFP plants exhibited enhanced primary root growth and reducedvi sensitivity to salt and ABA treatments compared to cdpk16 mutants and Col. Proteinprotein interaction assays confirmed that CDPK16 interacts with cyclin-dependent kinase (CDK) inhibitors KRP4 (Kip-related protein) and KRP7. Furthermore, CDPK16 phosphorylates KRP4 and KRP7, targeting them for degradation to regulate cell cycle progression in a Ca²⁺-dependent manner. These findings establish a functional framework in which CDPK16 integrates Ca²⁺ signaling pathways to regulate root growth and stress responses by modulating the activities of both CDK inhibitors and GLR3.6. This study highlights the central role of CDPK16 as a molecular hub in Ca²⁺ mediated signaling pathways, providing novel insights into the regulation of root growth and stress resilience in plants. | en |
| dc.description.provenance | Submitted by admin ntu (admin@lib.ntu.edu.tw) on 2025-02-13T16:11:11Z No. of bitstreams: 0 | en |
| dc.description.provenance | Made available in DSpace on 2025-02-13T16:11:11Z (GMT). No. of bitstreams: 0 | en |
| dc.description.tableofcontents | Acknowledgments ii
摘要 iii Abstract v List of Tables xii List of Figures xii List of Suppelementary Figures xiv Abbreviations xv Introduction 1 Environmental stresses 1 Abiotic stresses 1 Responses of plants to salt stress 1 Phytohormones 2 Abscisic acid (ABA) 3 Root growth and development 3 Architecture of primary roots 3 14-3-3 Proteins 5 Glutamate receptors (GLRs) 5 Glutamate-like receptors in plants 6 Arabidopsis glutamate receptors (AtGLR) 7 Cell cycle 7 Cell cycle regulation 8 Cyclin-dependent kinases (CDKs) 8 Kip-related proteins (KRPs) 9 Degradation of KRPs 9 Role of calcium signaling in cell cycle regulation 10 Calcium signaling in plants 10 Calcium‐dependent protein kinases (CDPKs) 11 Structure and function 11 Distribution and diversity 11 CDPKs in ABA and salt stress signaling 12 Role of CDPK16 in Arabidopsis 13 Motivations and objectives 13 Materials and Methods 16 Plant materials 16 Growth conditions 16 Generation of overexpression and gene silencing (RNAi) lines 17 Arabidopsis transformation by floral dipping 18 Isolation of genomic DNA for genotyping 19 RNA extraction and gene expression analysis 19 Primary root growth assay 20 Histochemical β-glucuronidase (GUS) staining 20 Isolation of Arabidopsis leaf protoplasts 21 In planta phosphorylation assay 22 Bimolecular fluorescence complementation (BiFC) assay 22 Plasmid construct for BiFC analysis 22 BiFC analysis using Arabidopsis mesophyll protoplasts 23 BiFC analysis in Nicotiana benthamiana 23 Luciferase complementation imaging (LCI) assay 24 Site-directed mutagenesis 24 Construction and designing of recombinant proteins 25 Expression and purification of 6×His- and GST-tagged proteins 26 In vitro kinase assay 27 Liquid chromatography−tandem mass spectrometry (LC−MS/MS) analysis 28 Pull-down assay and western blotting 29 Cell-free protein degradation assay 29 Statistical analysis 29 Primers and accession numbers 30 Chapter 1- 31 Calcium-dependent protein kinase CDPK16 phosphorylates serine-856 of glutamate receptor-like GLR3.6 protein leading to salt-responsive root growth in Arabidopsis. 31 Results 32 Sequence similarity of GLR3.6 and GLR3.7 32 Ser856 residue of GLR3.6 is phosphorylated by CDPK16 but not by CDPK3 or CDPK34 34 Protein-protein interaction between GLR3.6 and 14-3-3ω 34 Negative regulation of GLR3.6 in response to salt stress 36 Overexpression of the GLR3.6-S856A mutant shows a less salt-responsive primary root growth phenotype 36 Discussion 38 14-3-3 scaffold proteins and their role in protein-protein interactions 38 Calcium signaling in plant growth and development 38 Consensus phosphorylation motifs of CDPKs and GLR3.6 40 Phosphorylation of GLR3.6 by CDPK16 and 14-3-3 binding 40 Role of GLR3.6 and its mutants in salt stress response 41 Interaction between GLR3.6 and other GLR proteins 43 Chapter 1. Perspective and future work 43 Chapter 2- 46 Calcium-dependent protein kinase CDPK16 mediates salt stress and ABA responses in Arabidopsis root growth by phosphorylating cyclin-dependent kinase inhibitory proteins 46 Results 47 Expression pattern of CDPK16 in Arabidopsis tissues 47 Loss of CDPK16 function impairs root growth in Arabidopsis 47 Kinase activity and EF-motifs are essential for CDPK16-mediated root development and subcellular localization 48 CDPK16 expression is regulated by salt stress and ABA 50 CDPK16 modulates root growth under salt stress and ABA 50 CDPK16 interacts with cell cycle-related proteins in BiFC asay in tobacco cells 51 CDPK16 interacts with KRP4 and KRP7 in LCI 52 Autophosphorylation of CDPK16 and the role of structural domains 53 CDPK16 phosphorylates KRP4 and KRP7 in a Ca2+-dependent manner 54 Identification of phosphorylation sites on KRP4 and KRP7 targeted by CDPK16 55 Validation of CDPK16-targeted phosphorylation sites on KRP4 and KRP7 using LC-MS/MS data profile 56 CDPK16-mediated phosphorylation of KRP4 and KRP7 in Arabidopsis protoplasts 57 Functional roles of KRP4 and KRP7 in Arabidopsis root growth 58 KRP4 and KRP7 regulate root growth responses to salt stress and ABA 59 CDPK16 promotes KRP4 and KRP7 degradation 60 Discussion 61 Functional dynamics of CDPK16 in response to Ca²⁺ signaling 61 CDPK16 expression and its role in root development 62 Autophosphorylation and substrate specificity of CDPK16 63 Phosphorylation dynamics of KRPs, insights into CDPK16 substrate specificity 64 CDPK16 in salt stress and ABA signaling 65 CDPK16 regulation of root growth via KRP phosphorylation 66 Proposed model for CDPK16 in root growth and stress response 67 Perspective and future work 68 Tables 70 Figures 80 References 133 Appendix 152 | - |
| dc.language.iso | en | - |
| dc.subject | abscisic acid | zh_TW |
| dc.subject | stress responses | zh_TW |
| dc.subject | root growth | zh_TW |
| dc.subject | phosphorylation | zh_TW |
| dc.subject | kip-related proteins | zh_TW |
| dc.subject | glutamate receptor-like | zh_TW |
| dc.subject | CDK inhibitors | zh_TW |
| dc.subject | CDPKs | zh_TW |
| dc.subject | Ca²⁺ signaling | zh_TW |
| dc.subject | Arabidopsis | zh_TW |
| dc.subject | 14-3-3ω | zh_TW |
| dc.subject | stress responses | en |
| dc.subject | 14-3-3ω | en |
| dc.subject | abscisic acid | en |
| dc.subject | Arabidopsis | en |
| dc.subject | Ca²⁺ signaling | en |
| dc.subject | CDPKs | en |
| dc.subject | CDK inhibitors | en |
| dc.subject | glutamate receptor-like | en |
| dc.subject | kip-related proteins | en |
| dc.subject | phosphorylation | en |
| dc.subject | root growth | en |
| dc.title | 逆境下 CDPK16 與類麩胺酸受體 GLR3.6 調控阿拉伯芥 根系發育之研究 | zh_TW |
| dc.title | Study of CDPK16 and GLR3.6 in Regulating Root Growth and Stress Response in Arabidopsis | en |
| dc.type | Thesis | - |
| dc.date.schoolyear | 113-1 | - |
| dc.description.degree | 博士 | - |
| dc.contributor.oralexamcommittee | 謝明勳;葉國楨;楊健志;鄭秋萍;謝旭亮 | zh_TW |
| dc.contributor.oralexamcommittee | Ming-Hsiun Hsieh;Kuo-Chen Yeh;Chien-Chih Yang;Chiu-Ping Cheng;Hsu-Liang Hsieh | en |
| dc.subject.keyword | 14-3-3ω,abscisic acid,Arabidopsis,Ca²⁺ signaling,CDPKs,CDK inhibitors,glutamate receptor-like,kip-related proteins,phosphorylation,root growth,stress responses, | zh_TW |
| dc.subject.keyword | 14-3-3ω,abscisic acid,Arabidopsis,Ca²⁺ signaling,CDPKs,CDK inhibitors,glutamate receptor-like,kip-related proteins,phosphorylation,root growth,stress responses, | en |
| dc.relation.page | 160 | - |
| dc.identifier.doi | 10.6342/NTU202500616 | - |
| dc.rights.note | 同意授權(全球公開) | - |
| dc.date.accepted | 2025-02-11 | - |
| dc.contributor.author-college | 生命科學院 | - |
| dc.contributor.author-dept | 植物科學研究所 | - |
| dc.date.embargo-lift | 2025-02-14 | - |
| 顯示於系所單位: | 植物科學研究所 | |
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