Gene expression in the stem of an Arabidopsis thaliana seedling made visible through differently colored fluorescent proteins. Image credit: Fernan Federici & Jim Haseloff (CC BY 4.0)
Plants have an internal time keeper known as the circadian clock that operates in 24-hour cycles to coordinate the plants behaviors with the environment. The clock is made of many different proteins and plants carefully control when they make and destroy these proteins to regulate the cycle.
Inside plant cells, enzymes known as E3 ubiquitin ligases determine which proteins are destroyed by labelling target proteins with a small tag. Plants have hundreds of different E3 ubiquitin ligases, leading to overlaps in the roles the different enzymes play. These overlaps make it difficult to identify the specific E3 ubiquitin ligases that are involved in a particular process. As a result, only few E3 ubiquitin ligases implicated in the circadian clock have been identified so far.
A small weed known as Arabidopsis is often used in research studies because it grows quickly and the genes can be easily manipulated. Here, Feke et al. set out to develop a new tool to identify the specific E3 ubiquitin ligases involved in regulating the circadian clock in Arabidopsis.
The team created a library of hundreds of Arabidopsis plants producing different decoy E3 ubiquitin ligases that retained their ability to bind to target proteins but were unable to degrade them. Nearly a quarter of the E3 ligases found in Arabidopsis were represented in this library. The decoy enzymes protected the target proteins from being degraded by the normal E3 ubiquitin ligases, resulting in the library plants having presumably higher levels of these target proteins compared to normal Arabidopsis plants. By tracking circadian rhythms in these plants, the team was able to identify the individual E3 ligases that control the circadian clock.
The experiments revealed several E3 ligases that may regulate the circadian clock, including two enzymes called MAC3A and MAC3B. Further experiments demonstrated that MAC3A and MAC3B have similar roles in regulating the circadian clock and can compensate for the absence of the other.
The library of Arabidopsis plants generated by Feke et al. is now available for other researchers to use in their studies. In the future this approach could be adapted to make similar libraries for crops and other plants that have even more E3 ligase enzymes than Arabidopsis.
