Characterization of Thylakoid Immunophilins in Arabidopsis

Author(s): Garcia, Veder Joel | Advisor(s): Luan, Sheng | Abstract: The FK506-binding proteins (FKBPs) and cyclophilins (CYPs), collectively called immunophilins, were originally discovered as cellular receptors for immunosuppressive drugs FK506 and cyclosporine A. A common feature of immunophilins is their peptidyl-prolyl cis/trans isomerase (PPIase) activity that often functions in protein folding. These proteins exist in a wide range of organisms from bacteria to animals and plants. The abundance and diversity of immunophilins identified to date underlie the functional versatility of this protein family. In plants, immunophilins are present in various cellular compartments and, in particular, they constitute a significant portion of the chloroplast proteome. Recent studies suggest that chloroplast immunophilins play important roles in assembly and maintenance of photosynthetic complexes. However, the majority of chloroplast immunophilins await detailed functional characterization. As a first step to investigate plant chloroplast immunophilins, an evolutionary analysis was performed on sequences derived from eight representative species across a large evolutionary time-scale; from algae to higher plants. Multiple blast searches in these genomes resulted in identification of fifty CYPs and seventy-three FKBPs. Sequence analysis and phylogenetic tree reconstruction indicated that chloroplast immunophilin proteins are largely conserved from green algae to higher plants. Reverse genetics was employed in Arabidopsis, coupled with molecular and biochemical analyses, to examine the function of chloroplast immunophilins. Among them, CYP37 plays a critical role in response to high light stress. A cyp37 mutant displayed defect in anthocyanin accumulation under high light. Consistently, cyp37 accumulated higher levels of reactive oxygen species as compared to wild-type plants. Moreover, leaf chlorophyll fluorescence analysis indicated a higher electron transport rate in the mutant. However, thylakoid membrane protein composition was not significantly altered. Further yeast two-hybrid assays identified cyt f and PsbM as CYP37 interactors, suggesting that CYP37 may be involved in regulation of cytb6f complex or photosystem II (PSII). Taken together, these results suggest CYP37 plays a role in defense mechanisms under high light stress conditions. Functional redundancy appears to be a major feature among chloroplast immunophilins. Data collected herein suggest that AtCYP28 might be functionally redundant with other immunophilins as plants lacking CYP28 do not exhibit any phenotypic change under a number of stress conditions. To further address the potential functional redundancy of immunophilins, we generated double mutants between four FKBP single mutants (AtFKBP16-3, 16-4, 17-1, and 20-2). Among the double mutants produced, only the fkbp20-2/fkbp16-3 showed a significant phenotypic change under normal growth conditions. Plants lacking these two FKBPs were stunted in growth and their photosynthetic efficiency was compromised. FKBP20-2 interacted with CP43 and FKBP16-3 interacted with CP47 in a yeast two-hybrid assay. As both CYP43 and CP47 are subunits of the PSII complex, our data suggest that FKBP20-2 and FKBP16-3 may work together in the assembly/maintenance of PSII. In conclusion, the work presented in this thesis supports the hypothesis that chloroplast immunophilins have distinct and overlapping functions. They serve as critical regulators in photosynthesis-related processes including the assembly of photosynthetic complexes and adaptation to various light intensities in the natural environment.