Circadian-regulated dynamics of translation in Arabidopsis thaliana

Completed under a Cotutelle arrangement between the University of Melbourne and University of Potsdam© 2021 Michael Kien Yin TingCircadian clocks are an endogenous timing mechanism that allows an organism to fine tune diverse cellular processes in anticipation of external stimuli. To date, most plant circadian studies have focussed on transcriptional control, so our knowledge at the level of mRNA translation is very limited. Thus, the overall goal of this thesis was to close this gap in knowledge by monitoring translational dynamics in Arabidopsis thaliana (Arabidopsis) over a diel and circadian cycle in order to uncover translational regulation imposed by the circadian clock. To achieve these goals, the ribosome profiling method was first optimized for 2-week old Arabidopsis seedlings. Quality control assessment revealed that this optimized method generates translatomic data that exceeds the sequencing depth of currently published plant Ribo-seq studies. This protocol was therefore applied to generate a diel and circadian translatome with 2 h temporal resolution. Data integration with complementary transcriptomes revealed widespread translational regulation. Most notably, many genes were identified that are not rhythmic at the level of transcript abundance, but maintained rhythms at the level of translation output. These translational unique-cyclers are phased at select times of the day, revealing that select transcripts involved in coordinated biological processes are preferentially translated by ribosomes. Such translational preference is observed at subjective dawn for genes involved in the general translation machinery, and at subjective dusk for genes involved in protein catabolism. Thus, it is reported here that the translational machinery itself appears to be under translational control by the plant circadian clock. Finally, an Agrobacterium-mediated seedling transformation approach was tailored for circadian studies as an independent method for defining the molecular mechanisms of regulatory elements that were identified from the ribosome profiling data. Using luciferase reporters, the promoter activity of core clocks genes following this “transient” transformation system was reported to be highly comparable to equivalent stable transgenic approaches. Furthermore, transient transformation of reporter constructs into clock mutants reproduced published circadian defects. Together, this verified the robustness and reliability of this approach. Subsequently, the transient transformation system was used to assess the functionality of candidate upstream open reading frames (uORFs) found in the leader sequence of core clock genes. The results suggest that CCA1, GI and PRR7 contain uORFs that may contribute to rhythm phase. In summary, this thesis describes widespread translational regulation in a diel and circadian context, and implicates uORFs as potential translational regulators of the core circadian oscillator