Decipher interactions between TOR and DYRK, and evaluate their role in the control of growth and lipid synthesis in plants and algae – TOR-DYRKcontrol

First, we performed genetic (including genome editing), pharmacological, and biochemical analyses to characterize the relationship between TOR kinases and certain DYRK kinases. We confirmed that DYRK YAK1 is a target of TOR in the model plant Arabidopsis and that its phosphorylation is maintained in a hyperactive TOR mutant. In order to identify targets of YAK1, we screened chemical mutants that reverse the phenotype of plants overexpressing YAK1 and identified the causal mutations by positional cloning. This genetic approach leads us directly to the function of potential targets of YAK1 in relation to the different roles of this kinase. In the microalga Chlamydomonas, we focused on a DYRK kinase involved in the accumulation of reserves (triacylglycerols and starch). We developed a strategy to globally analyze proteins produced in the mutant compared to the wild type using proteomics to identify DYRKP targets. The functions and targets identified were characterized using biochemical, cellular, and metabolomic approaches.

This work enabled us to identify two potential targets of YAK1 in the model plant Arabidopsis: one that regulates proliferation and the other that regulates metabolism. In the microalgae Chlamydomonas, the proteomic approach enabled us to identify a major role for this kinase in regulating proliferation via cell wall synthesis and in metabolism. In addition, we participated in the development of genome editing approaches in plants. These results open up prospects for applications in biotechnology and agriculture and have led us to establish new collaborations at the European level.

This project has resulted in international scientific publications, including four research articles and two review articles on the role of TOR and DYRK kinases in regulating growth and metabolism in plants and algae. In addition, other articles are in preparation. Numerous oral presentations have been given and several conferences have been co-organized. Finally, several outreach activities aimed at the general public have been undertaken (conferences, YouTube, Science Festival, festivals, etc.).

Microalgae and plant leaves are promising sources of fatty acids and triacylglycerol (TAG) for alternative energies or for green chemistry. A major biological bottleneck is the inverse correlation between proliferation and oil accumulation, which compromises productivity. Therefore it is imperative to take an integrated approach and investigate potential signaling pathways that regulates the balance between proliferation and TAG accumulation. Increasing evidence from our work and others indicates that the TOR (Target Of Rapamycin) pathway is essential for regulating growth and TAG accumulation in response to nutrient availability in both plants and algae. Moreover, recent independent genetic screens from two partners of this project and other groups suggest that members of the small family (3 to 5 members) of DYRK (dual-specificity tyrosine-phosphorylation-regulated kinases) could be essential effectors of TOR-dependent regulation of proliferation and lipid accumulation in plant and algae. First, TOR was shown to control cell growth and proliferation in Arabidopsis by phosphorylating DYRK kinase YAK1 which is a growth repressor acting downstream of TOR. Second, two DYRKs, TAR1 and DYRKP were reported to regulate the accumulation of reserve compounds (starch and oil) in the green algae Chlamydomonas.
Our central hypothesis is that interactions between DYRK and TOR coordinate lipid accumulation and cell growth in response to environmental cues (e.g., nutrient, light). This will be addressed in parallel in plant and algal models Arabidopsis and Chlamydomonas where large numbers of genetic and molecular tools and mutants are available. This project is organized in three work-packages (WPs) and built on key preliminary results. WP1 will use state of the art biochemistry methods to identify DYRKs that are phosphorylated in a TOR-dependent manner and are therefore acting downstream of TOR. WP2 is dedicated to the functional relationship between TOR and DYRK kinases. Mutants in DYRKs, TOR, and their doubles mutant will be generated and phenotypes in regards to lipid, protein and starch content relative to biomass, with a particular focus on lipids (Heliobiotec lipidomic platform). Recently developed genome editing methods using CAS9 variant will be used to generate some of these mutants, particularly Chlamydomonas mutants carrying new point mutations in the TOR gene that we have identified in Arabidopsis and modulate TOR activity. Finally, during WP3, we will develop genetic screens of suppressors of dyrk mutants, in order to identify effectors of DYRK functions related to proliferation and TAG accumulation.
The TOR-DYRKcontrol project brings together three partners with complementary expertise in TOR signaling, biochemistry, lipid metabolism, genetics, genome editing and plant and algal biology. The use of both plant and algae should shed light on the evolutionary aspects of the TOR regulation, and bridging gaps on the lack of knowledge across different evolutionary lineages. This project will advance our knowledge in the understanding of synergy between TOR pathway, cell growth and carbon storage, and allow the further use of this knowledge to create algal/plant prototypes for improved lipid production. Therefore, this project addresses two urgent societal issues, energy shortage and global warming. It should contribute to the emergency of a greener economy, replacing fossil fuels by renewable sources for the production of lipids for food, transportation and chemical industry, while lowering impact of CO2 overproduction on global warming.