Journal of Advanced Research ( IF 11.4 ) Pub Date : 2022-08-06 , DOI: 10.1016/j.jare.2022.08.001 Peng Xu 1 , Yingxin Zhang 2 , Xiaoxia Wen 2 , Qinqin Yang 2 , Ling Liu 2 , Shulei Hao 2 , Jiaxin Li 2 , Zhaozhong Wu 2 , Liaqat Shah 2 , Amir Sohail 2 , Qunen Liu 2 , Lianping Sun 2 , Yongbo Hong 2 , Daibo Chen 2 , Xihong Shen 2 , Xiaodeng Zhan 2 , Shihua Cheng 2 , Liyong Cao 3 , Weixun Wu 2
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Introduction
Circadian clocks coordinate internal physiology and external environmental factors to regulate cereals flowering, which is critical for reproductive growth and optimal yield determination.
Objectives
In this study, we aimed to confirm the role of OsLUX in flowering time regulation in rice. Further research illustrates how the OsELF4s–OsELF3-1–OsLUX complex directly regulates flowering-related genes to mediate rice heading.
Methods
We identified a circadian gene OsLUX by the MutMap method. The transcription levels of flowering-related genes were evaluated in WT and oslux mutants. OsLUX forms OsEC (OsELF4s–OsELF3-1–OsLUX) complex were supported by yeast two-hybrid, pull down, BiFC, and luciferase complementation assays (LCA). The EMSA, Chip-qPCR, luciferase luminescence images, and relative LUC activity assays were performed to examine the targeted regulation of flowering genes by the OsEC (OsELF4s–OsELF3-1–OsLUX) complex.
Results
The circadian gene OsLUX encodes an MYB family transcription factor that functions as a vital circadian clock regulator and controls rice heading. Defect in OsLUX causes an extremely late heading phenotype under natural long-day and short-day conditions, and the function was further confirmed through genetic complementation, overexpression, and CRISPR/Cas9 knockout. OsLUX forms the OsEC (OsELF4s–OsELF3-1–OsLUX) complex by recruiting OsELF3-1 and OsELF4s, which were required to regulate rice heading. OsELF3-1 contributes to the translocation of OsLUX to the nucleus, and a compromised flowering phenotype results upon mutation of any component of the OsEC complex. The OsEC complex directly represses Hd1 and Ghd7 expression via binding to their promoter's LBS (LUX binding site) element.
Conclusion
Our findings show that the circadian gene OsLUX regulates rice heading by directly regulating rhythm oscillation and core flowering-time-related genes. We uncovered a mechanism by which the OsEC target suppresses the expression of Hd1 and Ghd7 directly to modulate photoperiodic flowering in rice. The OsEC (OsELF4s–OsELF3-1–OsLUX)–Hd1/Ghd7 regulatory module provides the genetic targets for crop improvement.
中文翻译：
时钟成分 OsLUX 通过募集 OsELF3-1 和 OsELF4s 抑制 Hd1 和 Ghd7 来调控水稻抽穗
介绍
生物钟协调内部生理和外部环境因素来调节谷物开花，这对于生殖生长和最佳产量决定至关重要。
目标
在这项研究中，我们旨在确认OsLUX在水稻开花时间调节中的作用。进一步的研究阐明了 OsELF4s–OsELF3-1–OsLUX 复合物如何直接调控开花相关基因以介导水稻抽穗。
方法
我们通过 MutMap 方法鉴定了一个昼夜节律基因OsLUX 。在 WT 和oslux突变体中评估了开花相关基因的转录水平。OsLUX 形成 OsEC (OsELF4s–OsELF3-1–OsLUX) 复合物得到酵母双杂交、pull down、BiFC 和荧光素酶互补测定 (LCA) 的支持。进行 EMSA、Chip-qPCR、荧光素酶发光图像和相对 LUC 活性测定以检查 OsEC (OsELF4s–OsELF3-1–OsLUX) 复合物对开花基因的靶向调控。
结果
昼夜节律基因OsLUX编码一个 MYB 家族转录因子，作为重要的生物钟调节器并控制水稻抽穗。OsLUX的缺陷导致在自然长日照和短日照条件下极晚抽穗的表型，并且通过基因互补、过表达和 CRISPR/Cas9 敲除进一步证实了该功能。OsLUX 通过募集调控水稻抽穗所需的 OsELF3-1 和 OsELF4s 形成 OsEC (OsELF4s–OsELF3-1–OsLUX) 复合体。OsELF3-1 有助于 OsLUX 向细胞核的易位，并且在 OsEC 复合体的任何成分发生突变时导致受损的开花表型。OsEC 复合体直接抑制Hd1和Ghd7通过与其启动子的 LBS（LUX 结合位点）元素结合来表达。
结论
我们的研究结果表明，昼夜节律基因OsLUX通过直接调节节律振荡和核心开花时间相关基因来调节水稻抽穗。我们发现了 OsEC 靶标直接抑制Hd1和Ghd7的表达以调节水稻光周期开花的机制。OsEC (OsELF4s–OsELF3-1–OsLUX)– Hd1 / Ghd7调控模块为作物改良提供了遗传目标。
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