National Centre for Scientific Research "DEMOKRITOS", Institute of Biosciences and Applications, Patr. Grigoriou E' & 27 Neapoleos str., 153 41, Agia Paraskevi, Greece; National and Kapodistrian University of Athens, Faculty of Biology, Department of Botany, 15784 Athens, Greece.
National and Kapodistrian University of Athens, Faculty of Biology, Department of Botany, 15784 Athens, Greece.
J Plant Physiol. 2018 Mar;222:79-85. doi: 10.1016/j.jplph.2017.12.018. Epub 2018 Jan 31.
The circadian clock is crucial for the synchronization of an organism's physiology and metabolism with the geophysical time. In plants, previous work on the common bean (Phaseolus vulgaris) has identified various differing aspects of clock function compared to the widely studied Arabidopsis thaliana clock. However, transformation of legumes for the study of the circadian clock regulatory mechanisms is extremely laborious. In the present work, we describe an easy-to-follow and rapid method of preparing bean leaf protoplasts with high transformation potential and a functional circadian clock. In this system, we show that application of trichostatin A differentially changes the expression levels of several clock genes. More importantly, we investigate the effect of the clock protein PvTOC1 (Phaseolus vulgaris TIMING OF CAB EXPRESSION 1) on the activity of bean circadian promoters. We present new evidence on the function of PvTOC1 as a repressor of the promoter activity of its own gene, mediated by its conserved CCT (CONSTANS, CO-LIKE and TOC1) domain. Using our protoplast system we were able to uncover functions of the bean circadian clock and to identify an additional target of the PvTOC1clock transcription factor, not previously reported.
生物钟对于生物体的生理和代谢与地球物理时间的同步至关重要。在植物中，与广泛研究的拟南芥生物钟相比，先前对普通菜豆（Phaseolus vulgaris）的研究已经确定了生物钟功能的各种不同方面。然而，为了研究生物钟调节机制，豆科植物的转化极其繁琐。在本工作中，我们描述了一种简单易行、快速的制备具有高转化潜能和功能性生物钟的菜豆叶片原生质体的方法。在该系统中，我们表明应用曲古抑菌素 A 可差异地改变几个生物钟基因的表达水平。更重要的是，我们研究了生物钟蛋白 PvTOC1（Phaseolus vulgaris TIMING OF CAB EXPRESSION 1）对菜豆生物钟启动子活性的影响。我们提供了 PvTOC1 作为其自身基因启动子活性的抑制剂的功能的新证据，这是由其保守的 CCT（CONSTANS、CO-LIKE 和 TOC1）结构域介导的。使用我们的原生质体系统，我们能够揭示菜豆生物钟的功能，并鉴定出以前未报道的 PvTOC1 时钟转录因子的另一个靶标。
