Understanding and engineering photoresponses in Arabidopsis thaliana

Light is a remarkably versatile and precise tool, the prevalent nature of which has caused it to become a common stimulus in biological processes. Along with metabolic signals derived from photosynthesis, plants have evolved a suite of five known families of photoreceptor proteins which make up photoperception array of the organism. These photoreceptors are the red and far-red light sensitive phytochromes, the blue light sensitive cryptochromes, phototropins and zeitlupe family and the UV-B receptor UVR8. The role of phototropins in the maintenance of circadian rhythms in the chloroplast has been recently identified, suggesting the potential for further involvement of phototropins in the circadian system of Arabidopsis. This study shows that in mutant plants lacking functional phototropins, the lack of phototropin function has little effect upon circadian rhythms of luciferase bioluminescence but is sufficient to induce loss of robustness in rhythms of chlorophyll fluorescence, indicating that phototropins are most likely indirectly involved in the regulation of circadian rhythms via the chloroplast and not directly involved in the regulation of the nuclear circadian clock. No known plant photoreceptors have a peak of absorbance within the green range of the photosynthetically active spectrum. While plants are capable of developing to maturity in the absence of green light, this study examines some of the subtle ways in which plants respond to green light. Via observation of hypocotyl elongation in seedlings lacking specific photoreceptor functions we show that phytochromes and cryptochromes are required to induce de-etiolation in seedlings grown under green light. Additionally, luciferase bioluminescence imaging of mutants lacking cryptochrome function is used to show that cryptochromes are required to maintain circadian rhythms in Arabidopsis under certain green light spectra, although the presence of blue wavelengths within the spectra of many green lights are sufficient to mask this response. Finally, the subtlety of green light responses in plants has provided a non-invasive input for an optogenetic construct which allows for light controlled manipulation of gene expression in planta. In this study, we present the design and engineering of a green light sensitive optogenetic system for use within plants based upon a cyanobacterial photoreceptor. This prototype system is functional in transiently transformed Nicotiana benthamiana leaves but displays significant leakiness and requires further development before its uses can be developed upon. Additionally further development is required in order to better engineer the system for the production of stable transgenic lines in Arabidopsis. The studies presented here seek to define and explore the roles of phototropins and green light within the circadian system and to design and engineer a green light sensitive optogenetic system for use in plants.