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Sumoylation: a regulatory mechanism for circadian clock function

Final Report Summary - MOLECULAR CLOCK (Sumoylation: a regulatory mechanism for circadian clock function)

The main objective of the project MOLECULAR CLOCK was the characterization of clock protein sumoylation as a regulatory mechanism for clock function. We have focused our efforts on the characterization of photoreceptor sumoylation, and more specifically on the CRYPTOCHROME 2 (CRY2) protein, that is a key factor integrating light signals into the clock, as well as contributing to the regulation of other important developmental processes such as photoperiodic flowering and hypocotyl elongation. This project aimed to be part of the solution for the current need to increase crop productivity in order to alleviate the food needs of the increasing world population. The understanding of how plants perceive environmental changes and how they adapt their responses to the dynamic surrounding is crucial for the development of more productive and sustainable crop management systems.
A lot of information was available at the beginning of this project regarding the transcriptional regulation of diverse developmental processes including circadian clock function, flowering time determination and control of cell elongation and growth. However, recent studies have shown that factors controlling circadian clock function, as well as other regulatory networks, are tightly regulated not only at the transcriptional level but also translational and post-translational. Post-translational modifications of proteins often regulate their stability, their 3D conformation or their subcellular localization, having a profound impact on protein active pools in the cell. Examples of post-translational modifications are the addition of phosphate groups (phosphorylation), ubiquitin (ubiquitination) or sumo (sumoylation) to the target proteins. The consequences of those modifications are diverse and it often exists a crosstalk between different post-translational modifications that will lead to the fine-tuning of protein abundance and activity at each particular moment.
In this project we have analysed the post-translational modification of CRY2, an important factor in circadian synchronization in plants. CRY2 is blue light photoreceptor that is stable in the dark but it is rapidly degraded upon exposure to blue light. CRY2 protein is phosphorylated in blue light and then ubiquitinated. This modification targets CRY2 for degradation by the proteasome. We have unveiled a new post-translational modification that affects CRY2 protein: sumoylation. We have analysed the effects of CRY2 sumoylation on its activity and its biological relevance.
At the start of the project, we predicted by in silico analysis that CRY2 could be sumoylated in several sites. We used a semi in vivo technique (transformation of Arabidopsis protoplasts) to show that CRY2 was indeed sumoylated and more specifically that the protein was polysumoylated with at least two target sites along the protein sequence. We mutagenize the putative lysine residues where the sumoylation could take place and identify a main sumoylation site in the C-terminal domain of CRY2. However, other sites are present as well in the protein and we did not manage to identify the target lysine residues in the N-terminal domain of the CRY2 protein. However, the identification of sumoylation as a new post-translational modification affecting CRY2, together with the identification of one of the major target residues for this modification, represents a major milestone in our project.
The next step was to characterize the biological relevance of CRY2 sumoylation. Our first approach was to check whether a CRY2 mutant version affecting the identified sumoylated residue, thereafter named CRY2_S2, would affect the ability of the protein to bind its known cofactor flavin adenine dinucleotide (FAD). FAD is necessary for CRY2 to perceive blue light. To rule out that the mutation affected FAD binding, causing a “blind” version of CRY2, we produced and purified in vitro CRY2 protein wild type and S2, and compare their absorption spectra in darkness and after blue light exposure. These experiments showed that the both WT and S2 CRY2 bind FAD in a similar manner. We conclude at this point that sumoylation does not affect the ability of CRY2 to bind its cofactor FAD. Another important question was to find out whether sumoylation of CRY2 altered its subcellular localization. In Arabidopsis, CRY2 protein is located in the nucleus. We transformed Arabidopsis protoplasts with a construct over-expressing a GFP_CRY2 fusion protein. We could confirm that both WT and S2 mutant proteins were located in the nucleus. This set of experiments rule out the possibility that sumoylation changed subcellular localisation of CRY2. We then explored the possibility that sumoylation altered the stability of the protein by chaging the ratio between phosphorylated and non-phosphorilated CRY2 protein upon exposure to blue light. We confirmed that this ratio was increased in S2 CRY2 proteins compared to WT CRY2. Furthermore, we have analysed the relation between sumoylation and ubiquitination and established that CRY2_S2 protein suffers a faster protein turnover upon exposure to blue light compared to CRY2_WT. This stability difference suggests that sumoylation is involved in the fine-tuning of CRY2 activity during light perception.
In the final section of the project, we have characterized the biological consequences of CRY2 sumoylation. We analysed the responses in which CRY2 plays an important role: circadian clock synchronization, flowering time and hypocotyl elongation. I did not find significant differences in circadian clock synchronization in free running experiments between plants expressing WT or S2 mutated forms of CRY2, under the control of its own promoter, in a cry2 mutant brackground (pCRY2::CRY2:_WT cry2 and pCRY2::CRY2_S2 cry2). I did find slight differences in hypocotyl elongation at very low intensities of blue light. This would suggest that sumoylation is important for CRY2 function in hypocotyl elongation. Regarding flowering time determination, we proved that both construct pCRY2::CRY2:_WT cry2 and pCRY2::CRY2_S2 cry2 complemented the late flowering phenotype of the cry2 mutant. However, slight differences among these lines could be observed. Unfortunately, there was a substantial variability from experiment to experiment, indicating that several other factors besides light input, are contributing to the fine-tuning of CRY2 activity. We observed that pCRY2::CRY2:_WT cry2 plants complemented the cry2 mutation and flower at the same time as Col0 plants; whereas only a percentage of the pCRY2::CRY2_S2 cry2 plants did complement the mutation. The rest of the plants resemble cry2 mutant regarding the flowering response. Another interesting observation was made on certain pCRY2::CRY2_S2 cry2 plants where inflorescences present phyllotaxis defects and floral buds present defects at anthesis.
These results are very relevant because they give an insight in the crosstalk between different posttranslational modifications and the regulation of light perception and photoreceptor function. A better understanding of the developmental processes that govern plant shape and adaptability is very valuable and entails a substantial advance in the field. Light input signal is one of the signals that have a greater impact on plant growth and development. If we want to design crops adapted to the environmental changes, achieving a better crop management system, it is of central importance to have detail information of the regulation of the key factors of the corresponding gene/protein network. As CRY2 represents a key factor integrating signals from the environment, it is clear that the data unveiled during this project will be useful to have a global understanding of the influence of environmental signal on plant development. Moreover, the Arabidopsis CRY2 protein has been used since some years as the bases for the generation of optogenetic tools to control gene expression trough light exposure in different biological systems. The novel information regarding CRY2 regulation provided here might help to better design this tools for a precise fine-tuning of gene expression in those systems.

The paper attached is under preparation and is confidential