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Development of quantitative models of nonphotochemical quenching regulatory dynamics in Arabidopsis thaliana through the use of multi-period actinic light exposure measurements of chlorophyll fluorescence


In photosynthesis, solar energy is absorbed and converted into chemical energy. Chlorophyll embedded in proteins absorb light and transfer excitation energy to reaction centers where charge separation occurs. However, the solar flux incident on photosynthetic organisms is highly variable, requiring complex feedback systems to regulate the excitation pressure on reaction centers and prevent excess absorbed energy from causing damage. Upon exposure to transient high intensity light, processes to dissipate excess absorbed energy are activated. This is routinely observed upon exposure of a photosynthetic sample to actinic light as the quenching of chlorophyll fluorescence, and often broadly referred to as non-photochemical quenching (NPQ). Understanding NPQ and its regulation at a quantitative and mechanistic level is an important challenge for optimizing crop yields and design of biomimetic solar devices for energy harvesting.

The regulation of NPQ allows for photosynthetic organisms to responds to changes in light intensity that occur on multiple timescales, from as little as a few seconds due to e.g. changes in shading of a leaf, to daily oscillations due to the position of the sun in the sky, and even seasonal oscillations. Various biochemical regulators of NPQ have been identified from measurements of the chlorophyll fluorescence of model organisms upon chemical and genetic manipulation. In vascular plants such as Arabidopsis thaliana, the rapidest regulation of NPQ is triggered by a pH gradient across the thylakoid membrane (∆pH) that is mediated by the PsbS protein. ∆pH simultaneously regulates the concentration of various xanthophylls, that are thought to influence direct photochemical mechanisms of quenching via the chemical composition of pigment-protein complexes and structural aspects of pigment-protein complex fluctuations and membrane organizations. However, the various regulatory responses often depend on shared biochemical regulatory components, such as the accumulation of the carotenoid zeaxanthin or the presence of the pH-sensitive PsbS protein. This makes distinguishing contributions of different regulatory responses to the overall response a challenging problem.

The biochemical regulation of NPQ ultimately activates photochemical mechanisms of energy dissipation, where excess solar energy absorbed by chromophores is dissipated via a non-radiative process to prevent photodamage. Proposed mechanisms include the nonradiative decay of an excited state of xanthophylls, a xanthophyll radical cation formation and recombination, and chlorophyll-chlorophyll charge separation and recombination. However, the electronic states of the chromophores proposed to be involved in energy dissipation indicate that even small fluctuations in the protein environment could preference which mechanisms are most favorable for any particular chromophore and therefore suggests that the quenching process is heterogeneous and dependent on a number of factors including protein conformation and membrane organization. Therefore, understanding the mechanisms through which photosynthetic systems dissipate excess energy and regulate excitation pressure in response to variable light conditions requires extensive quantitative measurements and modeling of the photosynthetic system and energy dissipation.

Developing a comprehensive understanding of the regulation of NPQ and the underlying photochemical mechanisms of energy dissipation is an challenging question in the study of NPQ and photosynthesis. Although numerous elements of the regulatory response of NPQ have been identified, each of the elements of the regulatory response occur on various timescales that contribute to the overall response. What are the timescales of these regulatory responses? What are the mechanisms of energy dissipation and which do various regulatory processes activate? How much do these mechanisms contribute to the overall quenching response? This work describes attempts to address these questions through the use of time correlated single photon counting (TCSPC) measurements of the chlorophyll fluorescence of Arabidopsis thaliana over multiple periods of exposure to high intensity actinic light and subsequent dark recovery to inform kinetic modeling of the regulation in order to attempt to distinguish features of the regulatory processes and connect the regulatory processes to proposed photochemical mechanisms of energy dissipation in a quantitative manner. The work makes important steps toward the ability to incorporate energy dissipation into first principles models of the photosynthetic system that relate membrane scale models of energy transfer to experimental observations, and establishing predictive models of how modifications of the regulatory systems will influence the resulting quenching and yields of photosynthesis for the optimization of crop yields.

Chapter 1 contains a review discussing efforts to model energy dissipation, or quenching, in Arabidopsis thaliana and their connections to models of regulatory systems that control quenching. First, theory used to describe energy transfer and experimental data obtained to construct energy transfer models of the photosynthetic antenna system that underlie the interpretation of chlorophyll fluorescence quenching is reviewed. Second, experimental evidence leading to proposed molecular mechanisms of quenching and the implications for modeling are discussed. The initial incorporation of depictions of proposed mechanisms into quantitative energy transfer models is reviewed. Finally, the necessity of connecting energy transfer models that include molecular models of quenching mechanisms with regulatory models is discussed.

Chapter 2 discusses experimental TCSPC measurements were performed on Arabidopsis thaliana to quantify the dependence of the response of NPQ to changes in light intensity on the presence and accumulation of zeaxanthin and lutein. Measurements were performed on wild type and mutant plants deficient in one or both of the xanthophylls, as well as a transgenic line that accumulates lutein via an engineered lutein epoxide cycle. Changes in the response of NPQ to light acclimation in wild type and mutant plants were observed between two successive light acclimation cycles, suggesting that the character of the rapid and reversible response of NPQ in fully dark-acclimated plants is substantially different than in conditions plants are likely to experience due to changes in light intensity during daylight. Mathematical models of the response of zeaxanthin- and lutein-dependent reversible NPQ were constructed that accurately describe the observed differences between the light acclimation periods. Finally, the wild-type response of NPQ was reconstructed from isolated components present in mutant plants with a single common scaling factor, which enabled deconvolution of the relative contributions of zeaxanthin- and lutein- dependent NPQ.

Chapter 3 discuses measurements undertaken In order to simultaneously resolve timescales of regulatory processes operating on different timescales, but with shared biochemical regulators, TCSPC measurements were performed on several Arabidopsis thaliana mutants during periodic actinic light exposure. Over successive periods of actinic light, TCSPC measurements show distinct intra-period and inter-period dynamics and demonstrate complex roles of the biochemical regulators PsbS and zeaxanthin in both fast and slow timescale responses of NPQ. Comparison between mutant lines suggests evidence of a role of PsbS in the longer timescale quenching response not previously emphasized. Finally, a mathematical model was constructed demonstrating how short timescale, rapidly reversible quenching processes and longer timescale quenching processes combine to produce the overall quenching response.

Chapter 4 discusses aspects of analyzing and interpreting snapshot fluorescence lifetime data obtained from in vivo samples using complex actinic exposure patterns to probe and quantify aspects of the regulatory response of quenching. First, a technique involving interleaving data from measurements on separate leaves to achieve increased actinic timescale resolution is discussed, including the application of filters to remove artifacts of leaf-to-leave systematic variability that introduce high frequency oscillation in the data. Second, the application of singular value decomposition on complex data sets for validation and filtering is discussed.

Chapter 5 summarizes conclusions of this work and provides an outlook towards future steps necessary for the optimization of crop yields.

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