Development of Time-Resolved Spectroscopic Tools to Study the Dynamics of Photoprotective Quenching in Plants

Author(s): Sylak-Glassman, Emily Jane | Advisor(s): Fleming, Graham R | Abstract: Plants experience rapid fluctuations in light intensity due to intermittent shading. While under low light conditions, efficient absorption of light is important, under high light conditions, this efficient light harvesting can result in excess energy that leads to photodamage. To prevent this photodamage, plants have a set of mechanisms called non-photochemical quenching (NPQ) that allow the plant to dissipate the excess energy harmlessly as heat. While NPQ has been studied for decades, there is still much we do not know about how NPQ is triggered, the changes in the photosynthetic apparatus that occur during NPQ, and the photophysical mechanisms that quench excess energy. NPQ is difficult to study both because of the large number of feedback loops and processes that contribute to NPQ and because the photosynthetic apparatus changes as NPQ turns on and off. This thesis describes the development of new time-resolved spectroscopic tools to help elucidate the proteins and processes important in NPQ. One of the techniques developed is a tool to measure the chlorophyll fluorescence lifetimes of whole leaves as they acclimate to changing light conditions. Chlorophyll is the main pigment in photosynthetic organisms, and its excited state relaxation dynamics reveal information about the energy transfer network in plants. The chlorophyll fluorescence lifetime gives additional information about quenching than the most commonly used probe of NPQ, the chlorophyll fluorescence yield. This tool is able to examine both the nanosecond-timescale and seconds- to minutes-timescale of quenching in vivo.This thesis also describes the application of the fluorescence lifetime technique to study different types of NPQ. Energy-dependent quenching (qE) is the largest and fastest component of NPQ. By comparing the shape of the fluorescence decay curves for plants with and without PsbS, a key protein in qE, it is possible to demonstrate that the presence of the protein PsbS changes only the amount of qE and the speed of qE turn-on and turn-off, not the type of quenching. This suggests that PsbS catalyzes qE. In contrast, the presence of the enzyme violaxanthin de-epoxidase and the carotenoid zeaxanthin do seem to affect the type of quenching. The fluorescence lifetime technique is also applied to study a new family of mutants that lack the protein SOQ1. Here, the fluorescence lifetime measurements confirm that the lack of SOQ1 confers extra quenching as opposed to extra photobleaching. Furthermore, the technique is able to show that SOQ1 reduces the timescale of long-term quenching that normally turns on in approximately 45 minutes by a factor of two. Additionally, a new mutant in the soq1 family shows the highest quenching of any mutant previously studied, and may be a model organism to study energy transfer when Photosystem II reaction centers are open. The development of another tool, a super-resolution stimulated emission depletion (STED) fluorescence lifetime imaging (FLIM) microscope, is also discussed. The goal of this tool was to be able to image the thylakoid membrane of plants using chlorophyll fluorescence. Chlorophyll from different protein environments would be distinguished by their fluorescence lifetime, and the STED technique would provide the resolution needed to distinguish the different parts of the thylakoid membrane. However, different types of STED-based imaging were unable to achieve super-resolution with chlorophyll.Time-resolved measurements of chlorophyll fluorescence offer a way to sensitively probe the dynamics of quenching in plants. In the future, the fluorescence lifetime technique can be applied to other processes and components of NPQ. In addition, the lessons learned in the development of both the fluorescence lifetime technique and the microscopy technique can be used to inform the development of other tools.