Li Yixing, Xu Meng, Wang Ning, Li Youguo
Guangxi Experiment Centre of Science and Technology, Guangxi University, Nanning 530004, People's Republic of China; College of Animal Science and Technology, Guangxi University, Nanning 530004, People's Republic of China.
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, People's Republic of China.
PLoS One. 2015 Oct 13;10(10):e0139964. doi: 10.1371/journal.pone.0139964. eCollection 2015.
Leghemoglobins (Lbs) play an important role in legumes-rhizobia symbiosis. Lbs bind O2 and protect nitrogenase activity from damage by O2 in nodules, therefore, they are regarded as a marker of active nitrogen fixation in nodules. Additionally, Lbs are involved in the nitric oxide (NO) signaling pathway, acting as a NO scavenger during nodule development and nitrogen fixation. However, regulators responsible for Lb expression and modulation of Lb activity have not been characterized. In our previous work, a Jasmonate-Zim-domain (JAZ) protein interacting with a Lb (AsB2510) in Astragalus sinicus was identified and designated AsJAZ1. In this study, the interaction between AsJAZ1 and AsB2510 was verified using a yeast two-hybrid system and in vitro Glutathione S-transferase (GST) pull-down assays, resulting in identification of the interaction domain as a TIFY (previously known as zinc-finger protein expressed in inflorescence meristem, ZIM) domain. TIFY domain is named after the most conserved amino acids within the domain. Bimolecular fluorescence complementation (BiFC) was used to confirm the interaction between AsJAZ1 and AsB2510 in tobacco cells, demonstrating that AsJAZ1-AsB2510 interaction was localized to the cell membrane and cytoplasm. Furthermore, the expression patterns and the symbiotic phenotypes of AsJAZ1 were investigated. Knockdown of AsJAZ1 expression via RNA interference led to decreased number of nodules, abnormal development of bacteroids, accumulation of poly-x-hydroxybutyrate (PHB) and loss of nitrogenase activity. Taken together, our results suggest that AsJAZ1 interacts with AsB2510 and participates in nodule development and nitrogen fixation. Our results provide novel insights into the functions of Lbs or JAZ proteins during legume-rhizobia symbiosis.
豆血红蛋白（Lbs）在豆科植物与根瘤菌的共生关系中发挥着重要作用。Lbs结合氧气，并保护固氮酶的活性不受根瘤中氧气的损害，因此，它们被视为根瘤中活跃固氮的一个标志。此外，Lbs参与一氧化氮（NO）信号通路，在根瘤发育和固氮过程中作为NO清除剂发挥作用。然而，负责Lb表达和Lb活性调节的调控因子尚未得到鉴定。在我们之前的工作中，鉴定出一种与紫云英中的一种Lb（AsB2510）相互作用的茉莉酸-Zim结构域（JAZ）蛋白，并将其命名为AsJAZ1。在本研究中，使用酵母双杂交系统和体外谷胱甘肽S-转移酶（GST）下拉试验验证了AsJAZ1与AsB2510之间的相互作用，确定相互作用结构域为TIFY（以前称为在花序分生组织中表达的锌指蛋白，ZIM）结构域。TIFY结构域以该结构域中最保守的氨基酸命名。采用双分子荧光互补（BiFC）技术在烟草细胞中证实了AsJAZ1与AsB2510之间的相互作用，表明AsJAZ1-AsB2510相互作用定位于细胞膜和细胞质。此外，还研究了AsJAZ1的表达模式和共生表型。通过RNA干扰敲低AsJAZ1的表达导致根瘤数量减少、类菌体发育异常、聚-β-羟基丁酸酯（PHB）积累以及固氮酶活性丧失。综上所述，我们的结果表明AsJAZ1与AsB2510相互作用并参与根瘤发育和固氮。我们的结果为Lbs或JAZ蛋白在豆科植物与根瘤菌共生过程中的功能提供了新的见解。
