title: Quantitative analysis of the effects of intrathylakoid pH and xanthophyll cycle pigments on chlorophyll a fluorescence lifetime distributions and intensity in thylakoids.
abstract: 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 time-resolved 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 approximately 0.5 ns Chl a fluorescence lifetime (tau) 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 tau 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 approximately 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 tau distribution of the PSII unit to a state with a decreased fluorescence tau 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, Ka, 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 approximately 0.5 ns fluorescence component as a function of both Z and A concentration and intrathylakoid pH, (2) the dependence of the ratio of F'm/Fm on the fraction of the 0.5 ns fluorescence tau component (where F'm and Fm are maximal fluorescence intensities in the presence and the absence of a pH gradient), and (3) the dependence of the ratio of F'm/Fm on the concentration of Z and A and the intrathylakoid pH.
authors.affiliation: Photobioenergetics Group, Australian National University Research School of Biological Sciences, Canberra, ACT. gov@uiuc.edu
authors.forename: A M
authors.initials: AM
authors.lastname: Gilmore
authors.forename: V P
authors.initials: VP
authors.lastname: Shinkarev
authors.forename: T L
authors.initials: TL
authors.lastname: Hazlett
authors.forename: G
authors.initials: G
authors.lastname: Govindjee
mesh_terms: D002338:Carotenoids / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D002734:Chlorophyll / Q000737:chemistry* / Q000378:metabolism; D000077194:Chlorophyll A; D002736:Chloroplasts / Q000737:chemistry* / Q000378:metabolism; D005454:Fluorescence Polarization; D001467:Hordeum; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000737:chemistry* / Q000378:metabolism; D014975:Lutein / Q000737:chemistry*; D010777:Photochemistry; D010860:Pigments, Biological / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D018724:Spinacia oleracea; D024341:Xanthophylls; D065146:Zeaxanthins; D019207:beta Carotene / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism
chemical_list: D010860:Pigments, Biological; D024341:Xanthophylls; D065146:Zeaxanthins; D019207:beta Carotene; C031140:antheraxanthin; D002734:Chlorophyll; D002338:Carotenoids; D014975:Lutein; D000077194:Chlorophyll A