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

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。

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.