Exploration of factors integrating vitamin B1 and the circadian clock in Arabidopsis thaliana

Circadian clocks represent a benefic trait for virtually any organism subject to the environmental cycles produced by the rotation of the Earth. By anticipating these predictable cues, the molecular oscillators optimize physiological and biochemical responses, modulate growth and development, ultimately impacting the fitness of the organism. Plants are sessile organisms that depend entirely on their environment to meet their requirements, grow and reproduce. Vitamins are essential micronutrients that are indispensable to the development of all forms of life. A member of the family of vitamin B1 (thiamine) vitamers, thiamine diphosphate (TDP), performs a crucial role as coenzyme of key-enzymes involved in energetic metabolism. Although plants are able to synthesize thiamine de novo, it represents a major energetic cost and must be tightly regulated such that TDP levels are never in excess or limiting. This is exemplified by THIC, an enzyme that mediates a branch of the thiamine de novo biosynthesis pathway and that is subjected to several layers of regulation. Its expression is controlled by a major core component of the circadian clock and its transcript abundance is regulated as a function of TDP levels by a riboswitch in its own mRNA. During this project, the role of the circadian clock in integrating the levels of the vitamin B1 related compounds has been questioned. By exploiting bioinformatics resources, the extent of circadian control on thiamine metabolism has been appreciated and denotes that fluctuations in gene expression due to circadian control does not always lead to rhythmic protein abundance. It also points towards metabolite candidates that could be relaying information on TDP levels in the cell. Then, the effects on exogenous thiamine application on the circadian clock have been characterized to find a clock candidate integrating vitamin B1 levels and the mechanisms allowing such integration. Results show that thiamine could have an impact on the amplitude of clock transcription factors, and that could be due to an increase in the activity of a major clock repressor under thiamine supplementation. The alternative roles of thiamine have also been explored and preliminary results suggest that thiamine has a role in preparing plant defenses to potential pathogenic attacks. Although the knowledge on the function of thiamine derivatives is growing, little is known about its triphosphorylated form. Here, the role of thiamine triphosphate as a potential B1 levels integrator is explored. While the list of potential integrator candidates expands, the absolute necessity of proper conditions and methodologies to produce replicable results is discussed. Finally, transgenic lines expressing THIC at inappropriate times compared to wild type plants reveal the importance of THIC regulation: Plants temporally altered in their THIC expression are stunted in growth and accumulate less biomass, reporting a reduction in fitness specifically in environmental conditions of light and dark cycles. These lines could be suffering from a disrupted dialog that normally takes place between the circadian clock, the riboswitch and THIC levels