In vivo bioluminescence based studies of the circadian clock in Arabidopsis
All organisms from basic single-cell organisms through to complex multicellular organisms, including humans, are subject to a 24-hour cycle due to the rotation of the earth. Organisms have evolved a specific biological clock system, often called the Circadian Clock to synchronise with this cycle. Since the circadian clock has been found to play a key role in many important biological functions, research into the underlying regulatory processes are becoming of increasing interest.
Example of bioluminescence timelapse of Arabidopsis to investigate the circadian clock.
The biological clock system can be divided into three functional parts:
Input system: reflects the changes of the external environment. For example for plants, light and temperature are the most important input parameters.
The second part is called the central oscillator which can be defined as the biological circadian system that maintains the biological stability.
Output system: acts by regulating the function of downstream genes.
Professor Liu (Liu Lab, Shanghai Institutes for Biological Sciences, CAS, Shanghai), focuses on the study of the mechanism of light regulated transcription, and light regulated development using Arabidopsis as a model organism. They also work on the molecular mechanism of light regulated transcription and circadian clock regulated transcription.
In order to perform research in this area, Professor Liu’s group have built a complete bioluminescence detection system that enables the precise control of light and temperature levels, this is shown in figure 1. In this system, the output signal is measured using the bioluminescence signal from specifically labelling with Luc protein in Arabidopsis grown in a 24-well plate. A high sensitivity camera equipped with a commercial lens is then used to record the image.
Figure 1: The Bioluminescence detection system used by the Liu Lab features precise control of light and temperature and high performance CCD imaging camera.
An example image for two 24-well plates is shown in figure 2(a). The whole bioluminescence image of the Arabidopsis in the two sets of 24-well plates was captured simultaneously. The average of the signal was used to determine the expression of the specific protein of interest. This experiment was carried out at 1 hour intervals over the course of 6 days. The circadian rhythm curve generated from this is shown in figure 2(b).
Figure 2: (a) typical bioluminescence image of Arabidopsis imaged in two sets of 24-well plate;(b) typical circadian rhythm curve
Due to the very low signal levels that are produced and the long duration of the experiments the detector choice is critcal to peforming such studies effectively. When compared against the traditional leaf movement analysis method, there are two advantages (data available in reference 5):
The first is that the signal was more accurate and the period, phase and amplitude in circadian rhythm curves can be computed with higher accuracy. These parameters are very important to determine growth, metabolism and phenotypical analysis of the biological system.
The second advantage is achieving as large a field of view as possible because it is better to analyse as many plants as possible for better accuracy of data accuracy in a short time-frame.
The iKon-M CCD camera was found to be the ideal solution for this work. The iKon-M features a high sensivity sensor with 95% QE. Very low dark current means that there is a very low noise floor. This combination is important since it means that the signals can be detected against the background noise over the long timescales these experiments require. The 13 mm x 13 mm sensor size of the iKon-M is perfectly matched to capturing two sets of 24-well plate in high resolution making for efficent collection of experimental data.
"The iKon-M CCD camera helps us obtain data more accurately and more conveniently. We can also do other important scientific research using protein interaction based BiFC and BiLC methods."
- Libang Ma, Liu Lab
All data courtesy of the Liu Lab. Find out more about the exciting research at the Liu Lab here.
Fei Wang, Yongshun Gao, Yawen Liu, Xin Zhang, Xingxing Gu, Dingbang Ma, Zhiwei Zhao, Zhenjiang Yuan, Hongwei Xue, Hongtao Liu*.(2019) BES1 regulated BEE1 controls photoperiodic flowering downstream of blue light signaling pathway in Arabidopsis. New Phytologist.
Qian Zhang, Zhi Xie, Rui Zhang, Peng Xu, Hongtao Liu, Hongquan Yang, Monika S. Doblin, Antony Bacic, and Laigeng Lia*.(2018) Blue Light Regulates Secondary Cell Wall Thickening via MYC2/MYC4 Activation of the NST1-Directed Transcriptional Network in Arabidopsis. The Plant Cell.
Liang T, Mei S, Shi C, Yang Y, Peng Y, Ma L, Wang F, Li X, Huang X, Yin Y, Liu H*. (2018) UVR8 interacts with BES1 and BIM1 to regulate transcription and photomorphogenesis inArabidopsis. Developmental Cell. 44:1-12
Yang Y, Liang T, Zhang L, Shao K, Gu X, Shang R, Shi N, Li X, Zhang P, Liu H*.(2018) UVR8 interacts with WRKY36 to regulate HY5 transcription and hypocotyl elongation in Arabidopsis. Nature Plants. doi: https://doi.org/10.1038/s41477-017-0099-0
Xu Li, Dingbang Ma,Sheen X. Lu,Xinyi Hu,a Rongfeng Huang, Tong Liang, Tongda Xu, Elaine M. Tobin and Hongtao Liu*. (2016) Blue Light- and Low Temperature-Regulated COR27 and COR28 Play Roles in the Arabidopsis Circadian Clock ,The Plant Cell, 28: 2755–2769, DOI: https://doi.org/10.1105/tpc.16.00354
Date: September 2019
Author: Wang Kun and The Liu Lab, Shanghai Institutes for Biological Sciences, CAS, Shanghai