State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, College of Life Sciences, Northwest A & F University, Yangling, Shaanxi, 712100, China.
State Key Laboratory of Soil Erosion and Dryland Farming on the Loess Plateau, College of Life Sciences, Northwest A & F University, Yangling, Shaanxi, 712100, China.
Biochem Biophys Res Commun. 2019 Oct 22;518(4):719-725. doi: 10.1016/j.bbrc.2019.08.118. Epub 2019 Aug 28.
Reactive oxygen species (ROS) play important roles in plant growth, development, responses to abiotic and biotic stresses. Hypersensitive response (HR)-like cell death is often associated with excess ROS. However, how a calcium-dependent protein kinase (CPK) modulates this process remains elusive in rapeseed (Brassica napus L.). In the present study, we identified and characterized CPK6L from rapeseed as a novel regulator of ROS and cell death. The subcellular localization of BnaCPK6L was investigated through GFP and was found to be located at the endoplasmic reticulum membrane. Overexpression of the constitutively active BnaCPK6LCA resulted in significant accumulation of ROS and HR-like cell death than the full-length. A quantitative RT-PCR survey identified that the expression levels of a few ROS, cell death and defense-related marker genes were up-regulated upon BnaCPK6LCA expression. Mating-based split ubiquitin system (mbSUS) screening revealed that BnaCPK6L interacted with BnaRBOHD (Respiratory Burst Oxidase Homolog D), which was validated by bimolecular fluorescence complementation (BiFC). An in vitro phosphorylation assay indicated that BnaCPK6L phosphorylated BnaRBOHD. Lastly, we also found that three 2C type protein phosphatases (PP2Cs) interacted with BnaCPK6L. Taken together, this study indicates that BnaCPK6L plays an important role in ROS and HR-like cell death through interacting with and phosphorylating RBOHD.
活性氧（ROS）在植物生长、发育以及对非生物和生物胁迫的响应中发挥着重要作用。超敏反应（HR）样细胞死亡通常与过量的 ROS 有关。然而，钙依赖蛋白激酶（CPK）如何调节这一过程在油菜（Brassica napus L.）中仍不清楚。在本研究中，我们从油菜中鉴定并表征了 CPK6L，它是 ROS 和细胞死亡的一种新型调节因子。通过 GFP 进行亚细胞定位研究，发现 BnaCPK6L 位于内质网膜上。组成型激活的 BnaCPK6LCA 的过表达导致 ROS 和 HR 样细胞死亡的显著积累，超过全长。定量 RT-PCR 调查发现，BnaCPK6LCA 表达后，一些 ROS、细胞死亡和防御相关标记基因的表达水平上调。基于交配的分裂泛素系统（mbSUS）筛选显示，BnaCPK6L 与 BnaRBOHD（呼吸爆发氧化酶同源物 D）相互作用，这通过双分子荧光互补（BiFC）得到了验证。体外磷酸化实验表明，BnaCPK6L 磷酸化 BnaRBOHD。最后，我们还发现三种 2C 型蛋白磷酸酶（PP2Cs）与 BnaCPK6L 相互作用。综上所述，这项研究表明，BnaCPK6L 通过与 RBOHD 相互作用和磷酸化 RBOHD，在 ROS 和 HR 样细胞死亡中发挥重要作用。
