Exploration of Nonphotochemical Quenching Mechanisms in A. thaliana via Time Correlated Single Photon Counting Snapshots

As described in chapter 1, during photosynthesis, plants harvest light energy from the sun and, through a series of several steps, convert it to chemical energy to be stored for later use driving cellular processes vital to life. However, under high light conditions, often more light energy is absorbed than can be used for productive photosynthesis. Because excess absorbed energy can cause severe damage to the photosynthetic reaction center proteins, it must be dissipated harmlessly as heat in order to protect the plant. In the first step of photosynthesis, light energy is absorbed by pigment protein complexes designed especially for light harvesting, called light harvesting, or antenna complexes. Because of their location relative to reaction centers, pigment composition, and their density, most absorbed light energy passes through antenna complexes before reaching reaction centers, making them ideal sites for photoprotective quenching, or nonphotochemical quenching. Nonphotochemical quenching, or NPQ, is the reduction in chlorophyll a fluorescence yield caused by the dissipation of excess excitation energy by mechanisms other than photochemistry. Under high light conditions, NPQ switches the function of light harvesting complexes to dissipate the energy they collect as heat in order to protect the reaction centers from damage when their capacity for productive photosynthesis is overwhelmed. The induction of NPQ opens up a new relaxation pathway for electronically excited chlorophyll molecules by altering the distance of the excited chlorophyll from, and/or orientation relative to, a quencher. Neighboring chlorophylls and other xanthophyll pigments have been proposed as potential quenching molecules and as of yet, none have been ruled out and some experimental evidence exists to support each possible quencher. One way to change the distance between, and/or relative orientation of pigments within a pigment protein complex, or PPC, is a conformational change of the PPC. Previous work has demonstrated that the function of some integral biological membrane proteins can be modulated by the lipid composition in the membrane, which in turn modulates the lateral pressure profile, and thereby the protein conformation. Chapter 2 describes fluorescence lifetime measurements taken on LCHII embedded proteoliposomes with different lipid compositions. The results reveal increased quenching in the presence of the nonbilayer forming lipid MGDG, suggesting that the quenching is induced by an increase in lateral pressure in the acyl region of the membrane bilayer. LHCII is likely able to undergo a conformational change modulated by the lipid composition in the thylakoid membrane, which brings relevant pigments closer to one another to allow for the harmless dissipation of excess energy in the form of heat. In chapter 3, two xanthophyll cycles linked to NPQ, the violaxanthin cycle (VAZ cycle) and the lutein epoxide cycle (LxL cycle), are discussed. The cycling of xanthophylls affects the kinetics and extent of the photoprotective response triggered. While the VAZ cycle is ubiquitous among vascular plants and has been studied extensively, the LxL cycle is found in only about 60% of plants studied thus far and does not exist in model plants. Lauriebeth Leonelli, in the Niyogi lab, introduced the LxL cycle into Arabidopsis thaliana and functionally isolated it from the VAZ cycle. We showed an increase in dark-acclimated PSII efficiency associated with Lx accumulation. Time correlated single photon counting (TCSPC) measurements were performed to quantify the dependence of the response of NPQ to changes in light intensity on the presence and accumulation of zeaxanthin and lutein. Changes in the response of NPQ to light acclimation were observed between two successive light acclimation cycles, suggesting that xanthophyll cycles modulate the rapid component of NPQ necessary to prevent photoinhibition. Mathematical models of the response of zeaxanthin- and lutein-dependent reversible NPQ were constructed that describe the modulation. Finally, the wild-type response of NPQ was reconstructed from isolated components with a single common scaling factor, enabling deconvolution of the relative contributions of zeaxanthin- and lutein-dependent NPQ.Chapter 4 describes TCSPC measurements at several excitation and detection wavelengths to determine the location of quenching in a new mutant of Arabidopsis thaliana. In 2013 the Niyogi lab characterized a new mutant, soq1, which displayed a novel form of qI quenching dependent on the protein, SOQ1. Further chemical mutagenesis on the soq1 mutant revealed a second mutant, soq1 otk1, that displayed severe, constitutive quenching. Further characterization and TCSPC snapshot experiments taken at several excitation and detection wavelengths on the soq1 otk1 mutant suggest that the constitutive quenching observed in soq1 otk1 is likely occurring in LHCII trimers. The measured lifetimes are commensurate with lifetimes of aggregated LHCII trimers reported in the literature. Lastly, in chapter 5, the data analysis methods developed to mitigate issues such as very large data sets, low counts, and error analysis are discussed. The MatLab code is provided in an appendix at the end of the chapter