In plants, the spatiotemporal expression of circadian oscillators provides adaptive advantages in diverse species. However, the molecular basis of circadian clock in soybean is not known. In this study, we used soybean hairy roots expression system to monitor endogenous circadian rhythms and the sensitivity of circadian clock to environmental stimuli. We discovered in experiments with constant light and temperature conditions that the promoters of clock genes GmLCLb2 and GmPRR9b1 drive a self-sustained, robust oscillation of about 24-h in soybean hairy roots. Moreover, we demonstrate that circadian clock is entrainable by ambient light/dark or temperature cycles. Specifically, we show that light and cold temperature pulses can induce phase shifts of circadian rhythm, and we found that the magnitude and direction of phase responses depends on the specific time of these two zeitgeber stimuli. We obtained a quadruple mutant lacking the soybean gene GmLCLa1, LCLa2, LCLb1, and LCLb2 using CRISPR, and found that loss-of-function of these four GmLCL orthologs leads to an extreme short-period circadian rhythm and late-flowering phenotype in transgenic soybean. Our study establishes that the morning-phased GmLCLs genes act constitutively to maintain circadian rhythmicity and demonstrates that their absence delays the transition from vegetative growth to reproductive development.

中文翻译：

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大豆发育中的光和温度可昼夜节律生物钟。

在植物中，昼夜节律振荡器的时空表达在不同物种中提供了适应性优势。但是，尚不清楚大豆中生物钟的分子基础。在这项研究中，我们使用大豆毛状根表达系统监测内源性昼夜节律和昼夜节律对环境刺激的敏感性。我们在恒定的光照和温度条件下的实验中发现，时钟基因GmLCLb2和GmPRR9b1的启动子在大豆毛状根中驱动约24小时的自我维持的强劲振荡。此外，我们证明了昼夜节律可被环境光/暗或温度循环所夹带。具体而言，我们表明，冷热温度脉冲会引起昼夜节律的相移，我们发现相位响应的大小和方向取决于这两个Zeitgeber刺激的特定时间。我们使用CRISPR技术获得了一个缺少大豆基因GmLCLa1，LCLa2，LCLb1和LCLb2的四重突变体，发现这四个GmLCL直系同源基因的功能丧失导致转基因大豆的极端短周期昼夜节律和晚花表型。我们的研究表明，早晨阶段的GmLCLs基因可组成性地维持昼夜节律，并证明它们的缺失延迟了从营养生长到生殖发育的过渡。并且发现这四个GmLCL直系同源物的功能丧失导致转基因大豆的极端短周期昼夜节律和迟花表型。我们的研究表明，早晨阶段的GmLCLs基因可组成性地维持昼夜节律，并证明它们的缺失延迟了从营养生长到生殖发育的过渡。并且发现这四个GmLCL直系同源物的功能丧失导致转基因大豆的极端短周期昼夜节律和迟花表型。我们的研究表明，早晨阶段的GmLCLs基因可组成性地维持昼夜节律，并证明它们的缺失延迟了从营养生长到生殖发育的过渡。