Analysis of non-photochemical quenching in photosystem II and assessment of plant water loss based on chlorophyll fluorescence measurements
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Magnitude measurement of chlorophyll a fluorescence (ChlF) involves challenges, and dynamic responses to variable excitations may offer an alternative. ChlF was measured during strong actinic light by using a pseudo-random binary sequence as a time-variant multiple frequency illumination excitation. The responses were observed in the time domain but were primarily analyzed in the frequency domain in terms of amplitude gain variations. The excitation amplitude was varied, and moisture loss was used to induce changes in the plant samples for further analysis. The results show that when nonphotochemical quenching (NPQ) activities start, the amplitude of ChlF responses vary, making the ChlF responses to illumination excitations nonlinear and nonstationary. NPQ influences the ChlF responses in low frequencies, most notably below 0.03 rad/s. The low frequency gain is linearly correlated with NPQ and can thus be used as a reference to compensate for the variations in ChlF measurements. The high-frequency amplitude gain showed a stronger correlation with moisture loss after correction with the low-frequency gain. This work demonstrates the usefulness of dynamic characteristics in broadening the applications of ChlF measurements in plant analysis and offers a way to mitigate variabilities in ChlF measurements during strong actinic illumination. Photochemical reactions were analyzed and modeled to observe the photoenergy regulation mechanisms in PSII from measured ChlF kinetics. Two pH-driven mechanisms were revealed: One is the PsbS-mediated NPQ of activated antennae (A*), as commonly understood, and the other is a disruption of energy transfer from A* to P680. Representing the latter with a conformational change resulting from complex formation of zeaxanthin and lutein with antennae proved necessary and closely described measured ChlF from initially dark-adapted state to light-adapted state of both Spinacia oleracea and Arabidopsis thaliana. The two mechanisms also correctly represented the differences in measured NPQ between A. thaliana wild type and koLhcb6 mutant (no CP24 monomer antenna). Analysis of the reactions indicates that zeaxanthin and lutein lead to slow protracted reductions in P680* and ChlF via the second mechanism. Protonation of PsbS plays a major role via the first mechanism in responding to fast changes in illumination. The research provides important insights into the mechanisms of photoenergy regulation and a validated kinetic model of photochemical reactions in PSII. ChlF in light-adapted state is a convenient indicator of plant status but heavily depends on NPQ. In this research, we demonstrated that measured NPQ varies significantly among replicate samples and is by itself not a consistent indicator of relative water content (RWC) of plant leaves. Using NPQ as a scaler in ChlF-based photochemical efficiency measures resulted in an NPQ-adjusted expression of ChlF characteristics, which turned out to be PSII PQ (photochemical quenching), and an NPQ-scaled PSII photochemical efficiency (Y(II)') as improved indicators of RWC. The results were experimentally demonstrated with Arabidopsis thaliana and then validated with varied illumination intensities and Spinacia oleracea. Further analysis of NPQ-related ChlF variables revealed a robust linear correlation between steady-state ChlF (Fs) and maximum fluorescence (Fm') across different RWC status and also uncovered the use of a major time constant as a substitute to account for the effect of NPQ. This research shows that ChlF characteristics depend on NPQ during water loss and PSII efficiency measures adjusted with NPQ effects provide enhanced effectiveness in indicating plant water status.
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Ph. D.