DataSheet_1_Calcium-dependent protein kinase CDPK16 phosphorylates serine-856 of glutamate receptor-like GLR3.6 protein leading to salt-responsive root growth in Arabidopsis#.pdf

Calcium-permeable channels in the plasma membrane play vital roles in plant growth, development, and response to environmental stimuli. Arabidopsis possesses 20 glutamate receptor-like proteins that share similarities with animal ionotropic glutamate receptors and mediate Ca2+ influx in plants. Calcium-dependent protein kinases (CDPKs) phosphorylate serine (Ser)-860 of glutamate receptor-like (GLR)3.7 protein, which interacts with 14-3-3ω and plays an essential role in salt and abscisic acid response in Arabidopsis by modulating Ca2+ signaling. However, the significance of CDPK- mediated phosphorylation status of Ser residues of GLR3.6 with regard to the functioning of GLR3.6 remains to be elucidated. In this study, we performed an in vitro kinase assay using CDPK16 and peptides containing the 14-3-3ω interacting domain of GLR3.6. We showed that Ser861/862 of GLR3.6 are required for the interaction with 14-3-3ω and that Ser856 of GLR3.6 is specifically phosphorylated by CDPK16 but not by CDPK3 and CDPK34. In addition, the expression of GLR3.6 was quickly downregulated by salt stress, and plants of glr3.6 mutants and GLR3.6-overexpression lines presented shorter and longer root lengths, respectively, under normal growth conditions than Col. Overexpression of the GLR3.6-Ser856 to Ala mutation resulted in a less sensitive phenotype in response to salt stress similar to glr3.6. Our results indicated that the Ser861/862 residues of GLR3.6 are required for interaction with 14-3-3ω. Additionally, the phosphorylation status of Ser856 residue of GLR3.6, which is mediated specifically by CDPK16, regulates root growth in normal and salt stress and conditions.

质膜上的钙通透性离子通道在植物生长、发育以及环境胁迫响应中发挥至关重要的作用。拟南芥（Arabidopsis）拥有20种谷氨酸受体类蛋白（glutamate receptor-like proteins, GLR），这类蛋白与动物离子型谷氨酸受体具有序列相似性，可在植物中介导钙离子内流。钙依赖性蛋白激酶（Calcium-dependent protein kinases, CDPKs）可磷酸化谷氨酸受体类蛋白GLR3.7的丝氨酸（Ser）860残基，该位点磷酸化后可与14-3-3ω蛋白互作，并通过调控钙信号通路在拟南芥的盐胁迫与脱落酸响应中发挥核心作用。然而，GLR3.6的丝氨酸残基被CDPK介导的磷酸化状态对GLR3.6功能的重要性仍有待阐明。本研究采用CDPK16与包含GLR3.6的14-3-3ω互作结构域的肽段开展了体外激酶活性测定。实验结果证实，GLR3.6的Ser861/862残基是其与14-3-3ω蛋白互作所必需的；GLR3.6的Ser856残基可被CDPK16特异性磷酸化，而CDPK3与CDPK34则无此磷酸化活性。此外，GLR3.6的表达会被盐胁迫快速下调；在正常生长条件下，glr3.6突变体与GLR3.6过表达株系的根长分别较野生型Col更短与更长。将GLR3.6的Ser856突变为丙氨酸（Ala）并过表达该突变体，可使植株呈现出与glr3.6突变体类似的盐胁迫不敏感表型。本研究结果表明，GLR3.6的Ser861/862残基是其与14-3-3ω蛋白互作所必需的；此外，由CDPK16特异性介导的GLR3.6的Ser856残基磷酸化状态，可调控正常及盐胁迫条件下的植株根生长。