Yakir Esther, Hilman Dror, Kron Ido, Hassidim Miriam, Melamed-Book Naomi, Green Rachel M
Department of Plant and Environmental Sciences, The Silberman Institute for Life Sciences, The Hebrew University, Givat Ram, Jerusalem 91904, Israel.
Plant Physiol. 2009 Jun;150(2):844-57. doi: 10.1104/pp.109.137414. Epub 2009 Apr 1.
As an adaptation to life in a world with predictable daily changes, most eukaryotes and some prokaryotes have endogenous circadian (approximately 24 h) clocks. In plants, the circadian clock regulates a diverse range of cellular and physiological events from gene expression and protein phosphorylation to cellular calcium oscillations, hypocotyl growth, leaf movements, and photoperiod-dependent flowering. In Arabidopsis (Arabidopsis thaliana), as in other model organisms, such as Drosophila (Drosophila melanogaster) and mice, circadian rhythms are generated by molecular oscillators that consist of interlocking feedback loops involving a number of elements. CIRCADIAN CLOCK ASSOCIATED1 (CCA1) and LATE ELONGATED HYPOCOTYLS (LHY) are closely related single myb transcription factors that have been identified as key elements in the Arabidopsis oscillator. Research in other model organisms has shown that posttranslational regulation of oscillator components plays a critical role in the generation of the approximately 24-h cycles. To examine the role of posttranslational regulation of CCA1 and LHY in the Arabidopsis oscillator, we generated transgenic plants with tagged CCA1 and LHY under the control of their own promoters. We have shown that these tagged proteins are functional and can restore normal circadian rhythms to CCA1- and LHY-null plants. Using the tagged proteins, we demonstrate that CCA1 can form both homodimers and heterodimers with LHY. Furthermore, we also show that CCA1 is localized to the nucleus in vivo and that there is no significant delay between the translation of CCA1 and its translocation to the nucleus. We discuss our findings in the context of the functioning of the Arabidopsis oscillator.
作为对生活在一个具有可预测每日变化的世界的适应，大多数真核生物和一些原核生物都有内源性昼夜节律（约24小时）时钟。在植物中，昼夜节律时钟调节从基因表达、蛋白质磷酸化到细胞钙振荡、下胚轴生长、叶片运动以及光周期依赖的开花等多种细胞和生理事件。在拟南芥（Arabidopsis thaliana）中，与其他模式生物如果蝇（Drosophila melanogaster）和小鼠一样，昼夜节律由分子振荡器产生，这些振荡器由涉及多个元件的连锁反馈环组成。昼夜节律相关1（CIRCADIAN CLOCK ASSOCIATED1，CCA1）和晚伸长下胚轴（LATE ELONGATED HYPOCOTYLS，LHY）是密切相关的单个MYB转录因子，已被确定为拟南芥振荡器中的关键元件。对其他模式生物的研究表明，振荡器组件的翻译后调节在大约24小时周期的产生中起关键作用。为了研究CCA1和LHY的翻译后调节在拟南芥振荡器中的作用，我们构建了在其自身启动子控制下带有标签的CCA1和LHY的转基因植物。我们已经表明，这些带标签的蛋白质具有功能，并且可以将正常的昼夜节律恢复到CCA1和LHY缺失的植物中。使用带标签的蛋白质，我们证明CCA1可以与LHY形成同二聚体和异二聚体。此外，我们还表明CCA1在体内定位于细胞核，并且在CCA1翻译与其转运到细胞核之间没有明显延迟。我们在拟南芥振荡器功能的背景下讨论我们的发现。
