The xanthophyll cycle-dependent dissipation of excitation energy in higher plants is one of the most important regulatory and photoprotective mechanisms in photosynthesis. Using parallel timeresolved and pulse-amplitude modulation fluorometry, we studied the influence of the intrathylakoid pH and the xanthophyll cycle carotenoids on the PSII chlorophyll (Chl) a fluorescence yield in thylakoids of Arabidopsis, spinach, and barley. Increases in concentrations of dithiothreitol in thylakoids, which have a trans-thylakoid membrane pH gradient and are known to have decreased conversion of violaxanthin (V) to zeaxanthin (Z), lead to (1) decreases in the fractional intensity of the ∼0.5 ns Chl a fluorescence lifetime (τ) distribution component and simultaneous increases in a 1.6-1.8 ns fluorescence component and (2) increases in the maximal fluorescence intensity. These effects disappear when the pH gradient is eliminated by the addition of nigericin. To quantitatively explain these results, we present a new mathematical model that describes the simultaneous effects of the chloroplast trans-thylakoid membrane pH gradient and xanthophyll cycle pigments on the PSII Chl a fluorescence τ distributions and intensity. The model assumes that (1) there exists a specific binding site for Z (or antheraxanthin, A) among or in an inner antenna complex (primarily CP29), (2) this binding site is activated by a low intrathylakoid pH (pK ≈4.5) that increases the affinity for Z (or A), (3) about one Z or A molecule binds to the activated site, and (4) this binding effectively "switches" the fluorescence τ distribution of the PSII unit to a state with a decreased fluorescence τ and emission intensity (a "dimmer switch" concept). This binding is suggested to cause the formation of an exciton trap with a rapid intrinsic rate constant of heat dissipation. Statistical analysis of the data yields an equilibrium association constant, K a , that ranges from 0.7 to 3.4 per PSII for the protonated/activated binding site for Z (or A). The model explains (1) the relative fraction of the ∼0.5 ns fluorescence component as a function of both Z and A concentration and intrathylakoid pH, (2) Interest in the molecular mechanisms used by higher plants to adapt and acclimate to light levels in excess of that used in photosynthesis has recently increased. One possible photoprotective mechanism involves xanthophyll cycledependent thermal dissipation of excess absorbed light energy in the light-harvesting complexes of photosystem II (PSII) (1-8). Light harvesting in the PSII antenna and the xanthophyll cycle-dependent heat dissipation mechanism are related to the structure and organization of the PSII pigmentproteins. The PSII holochrome is composed of (a) the PSII core that includes chlorophyll proteins CP43 and CP47 and reaction center proteins D1, D2, and cytochrome b 559 , (b) the minor inner antenna, labeled as CP24, CP26, and CP29, and (c) the major peripheral antenna complex that includes trimeric assemblies of the light-harvesting complex ...
A mnode-locked laser was used to measure fluorescence decay down to 80 picoseconds. Measurements on the fluorescence of methylene blue quenchled with potassium iodide demonstrate the effectiveness of the method. Fluorescence decay times of chlorophyll b (3.87 +/- 0.05 nianoseconds) and c-phycocyanin (1.14 +/- 0.01 nanoseconds) in vitro and chlorophyll a in the green alga Chlorella pyrenoidosa (1.14 to 1.6 nanoseconds) compare well with some of the existing data.
Chl -chlorophyll; Fm -maximum Chl a fluorescence; Fo -initial (minimal) Chl a fluorescence; Fv (= Fm -Fo) -variable Chl a fluorescence; I-step -Chl a fluorescence at ~ 30 ms; J-step -Chl a fluorescence at ~ 2 ms; OJIP curve -the 'fast' phase of the fluorescence transient ['O' is for the initial fluorescence (at ~ zero time), 'P' is for peak, and 'J' and 'I' are inflection points between 'O' and 'P']; PQ -plastoquinone; QA and QB -the first and second plastoquinone electron acceptor of PSII. Acknowledgments: We thank Zuzana Benedikty (of Photon Systems Instruments, The Czech Republic) for the generous gift of FluorPen to one of us (G. Govindjee). We are grateful to Roland Valcke for his suggestions to relate our observations to those on fluorescence imaging, which led to substantial improvement of our paper. We are highly thankful to Hartmut Lichtenthaler, the one who we are honoring here, for reading our paper, and for making crucial suggestions, before its submission. We are also grateful to the two anonymous reviewers for their very helpful comments. Conflict of interest: The authors declare that they have no conflict of interest.
Note a: See Allakhverdiev et al. (2016) for an article honoring the lifetime achievements of George C. Papageorgiou. Note b: One of us (Govindjee) is very proud to remember George Papageorgiou to be his first PhD student, whose 1968 PhD thesis in Biophysics, at the University of Illinois at Urbana-Champaign (UIUC), was extraordinarily unique, for that time; it was on 'Fluorescence induction in Chlorella pyrenoidosa and Anacystis nidulans and its relation to photophosphorylation' (available at: https://www.life. illinois.edu/govindjee/theses.html); it is equally unique that together with George, Govindjee recently paid a tribute to the father of biophysics, the prophet of photosynthesis, at UIUC, Eugene Rabinowitch, who had been on George's PhD committee (see Govindjee et al. 2019). † Lazar and Stirbet had equal contribution.
Abstract. –The absorption and fluorescence characteristics of subchloroplast particles highly enriched in P700 (1 P700 to 10–15 chlorophyll a molecules) and of two artificial systems are presented here. The fluorescence characteristics measured were excitation and emission spectra, and the degree of polarization of fluorescence. The model systems studied were chlorophyll a and pheophytin a in a polystyrene matrix, and a colloidal mixture of these two pigments with bovine serum albumin. Effects of 0.5% sodium dodecyl sulfate on the optical properties of the P700‐enriched particles are also described. The importance of pigment‐pigment and pigment‐protein interactions in determining the fluorescence properties of these particles are discussed. The possible role of pheophytin in these preparations needs further investigation.
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