Biology Department, Concordia University, 7141 Sherbrooke W. Montreal (Quebec) H4B 1R6, Canada.
Biology Department, Concordia University, 7141 Sherbrooke W. Montreal (Quebec) H4B 1R6, Canada.
J Plant Physiol. 2022 Dec;279:153841. doi: 10.1016/j.jplph.2022.153841. Epub 2022 Oct 14.
The investigation of the caleosin CLO7 in relation to heterotrimeric G-protein signalling in Arabidopsis showed that the gene plays a role in seed germination and embryo viability. The caleosin CLO7 belongs to a multi-gene family of calcium-binding proteins which are characterized by single EF-hand motifs. Other members of the caleosin gene family have been shown to affect transpiration and seed germination as well as play a role in both abiotic and biotic stress responses. The proteins are associated with lipid droplets/oil bodies and some members of the gene family have been shown to have peroxygenase activity. Members of the gene family have also been shown to interact with the α subunit of the heterotrimeric G protein complex. In this study, we further expand on the diversity of physiological responses in which members of this gene family play regulatory roles. Utilizing BiFC and Y2H protein-protein interaction assays, CLO7 is identified as an interactor of the heterotrimeric G protein α subunit, GPA1. The full-length CLO7 is shown to interact with both the wild-type GPA1 and its constitutively active form, GPA1, at the plasma membrane. Point mutations to critical amino acids for calcium binding in the EF-hand of CLO7 indicate that the interaction with GPA1 is calcium-dependent and that the interaction with GPA1 is enhanced by calcium. Protein-protein interaction assays also show that CLO7 interacts with Pirin1, a member of the cupin gene superfamily and a known downstream effector of GPA1, and this interaction is calcium-dependent. The N-terminal portion of CLO7 is responsible for these interactions. GFP-tagged CLO7 protein localizes to the endoplasmic reticulum (ER) and to lipid bodies. Characterization of the clo7 mutant line has shown that CLO7 is implicated in the abscisic acid (ABA) and mannitol-mediated inhibition of seed germination, with the clo7 mutant displaying higher germination rates in response to osmotic stress and ABA hormone treatment. These results provide insight into the role of CLO7 in seed germination in response to abiotic stress as well as its interaction with GPA1 and Pirin1. CLO7 also plays a role in embryo viability with the clo7gpa1 double mutant displaying embryo lethality, and therefore the double mutant cannot be recovered.
钙结合蛋白 CLO7 与拟南芥异三聚体 G 蛋白信号转导的关系研究表明，该基因在种子萌发和胚胎活力中起作用。CLO7 钙结合蛋白属于钙结合蛋白的多基因家族，其特征是单个 EF 手模体。该基因家族的其他成员已被证明会影响蒸腾和种子萌发，并在非生物和生物胁迫反应中发挥作用。这些蛋白质与脂质滴/油体相关，并且该基因家族的一些成员已被证明具有过氧化物酶活性。该基因家族的成员还与异三聚体 G 蛋白复合物的α亚基相互作用。在这项研究中，我们进一步扩展了该基因家族成员发挥调节作用的生理反应的多样性。利用 BiFC 和 Y2H 蛋白-蛋白相互作用测定法，鉴定 CLO7 是异三聚体 G 蛋白α亚基 GPA1 的相互作用蛋白。全长 CLO7 被证明与野生型 GPA1 及其组成型激活形式 GPA1 在质膜上相互作用。CLO7 EF 手模体中关键氨基酸的点突变表明，与 GPA1 的相互作用依赖于钙，并且钙依赖性增强了与 GPA1 的相互作用。蛋白-蛋白相互作用测定法还表明，CLO7 与 Pirin1 相互作用，Pirin1 是 cupin 基因超家族的成员，也是 GPA1 的已知下游效应物，这种相互作用依赖于钙。CLO7 的 N 端部分负责这些相互作用。GFP 标记的 CLO7 蛋白定位于内质网（ER）和脂质体。CLO7 突变体的特征表明，CLO7 参与脱落酸（ABA）和甘露醇介导的种子萌发抑制，CLO7 突变体在对渗透胁迫和 ABA 激素处理的反应中表现出更高的萌发率。这些结果提供了 CLO7 在响应非生物胁迫的种子萌发中的作用的深入了解，以及它与 GPA1 和 Pirin1 的相互作用。CLO7 还在胚胎活力中发挥作用，clo7gpa1 双突变体显示胚胎致死性，因此双突变体无法恢复。
