Abstract

The plant blue light photoreceptor proteins Flavin binding Kelch F-box protein-1 (FKF1) and LOV Kelch protein-2 (LKP2) play an important role in maintaining circadian rhythmicity and photo-periodic flowering responses. These proteins along with light sensitive Light Oxygen Voltage (LOV) domain contain F-box and Kelch repeat domains. Therefore, these proteins control blue light mediated degradation of various protein targets to regulate circadian rhythmicity, growth, and photo-periodic flowering. Blue-light excitation of these proteins leads to formation of a cysteinyl-flavin adduct, which then decays depending upon various factors. Therefore, spectral studies were conducted to measure the kinetics of light–dark recovery. Size Exclusion Chromatography (SEC) was performed to determine its oligomeric state. Attempts to crystallize these proteins to understand the signal transduction landscape were unsuccessful. Although crystal structure information of Zeitlupe a protein belonging to the same family provided by Ashutosh Pudasaini gave useful insights about potential signaling mechanisms that could apply to FKF1 and LKP2. These results collectively aid to develop a model of LOV domain function We also studied ENV1 a blue light photoreceptor protein from the industrially important fungus Trichoderma reseei ENV1 is responsible for blue-light mediated regulation of gene transcription, growth, reproduction and stress responses along with another blue light photo receptor Blue light receptor-1 (BLR1). Previous studies of a homologous protein VVD that regulates circadian rhythmicity in fungus Neurospora crassa provides useful insight into blue-light dependent signaling mechanisms. Biophysical characterization of ENV1 using techniques such as size-exclusion chromatography, multi-angle light scattering, and X-Ray crystallography shows that it controls gene transcription through competitive hetero- and homo-dimer formation. Further, it was observed that ENV1 can respond to oxidative stress conditions in addition to blue light sensitivity. Integration of blue light signaling, and oxidative stress responses were confirmed by in-vivo studies. Thus, ENV1 has evolved from its homologous protein VVD to incorporate oxidative stress sensing. Sequence alignment results indicate that it can also serve as a model to understand physiology of pathogenic fungus that affect crops. Thus, key findings of this research will aid in understanding signal transduction pathways and development of plant and fungal variants with desirable traits. Further, since these proteins are sensitive to blue light, they can be utilized for opto-genetic applications.

Degree Date

Spring 5-19-2018

Document Type

Thesis

Department

Chemistry

Advisor

Brian D. Zoltowski

Subject Categories

Biochemistry

Number of Pages

201

Format

.pdf

Creative Commons License

Creative Commons Attribution-Noncommercial 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License

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