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 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 ...

Global spectral–kinetic analysis of room temperature chlorophyll a fluorescence from light-harvesting antenna mutants of barley

“…4) compared with all the other wt and clo f104 sample conditions. Figure 1b compares Gilmore et al (1998) downturn in the level of energy dissipation when comparing the high to the medium PFD conditions. Since suitably active thylakoids were not recovered from the clo f 2 in this work, we simply cite and con¢rm other literature that documents a clear (204 0%) decrease in the maximum levels of energy dissipation in the clo f 2 mutant, and other chlorophyllb-less mutant leaves, grown under constant but moderate PFDs (Briantais 1994;Falk et al 1994;HÌrtel & Lokstein 1995).…”

On the origin of a slowly reversible fluorescence decay component in the Arabidopsis npq4 mutant

“…When red actinic light was used, the light intensities for these experiments were chosen in order to produce the same value of qL in all genotypes. When indicated, fluorescence was measured on detached leaves infiltrated with 150 mM sorbitol containing either 50 mM nigericin [26], 100 mM lincomycin [27] or 2 mM myxothiazol [28].…”

A Mechanism of Nonphotochemical Energy Dissipation, Independent from PsbS, Revealed by a Conformational Change in the Antenna Protein CP26

“…Thylakoids for fluorescence measurements were isolated from darkadapted Arabidopsis wild-type leaves as previously described (Gilmore et al, 1998) and were diluted before fluorescence measurements to a final concentration of 0.05 mg chlorophyll/mL. Buffer for F m measurements contained 0.1 M sorbitol, 5 mM MgCl 2 , 10 mM NaCl, 10 mM KCl, 10 mM Tricine, pH 7.8, 0.2% BSA, 50 mM sodium ascorbate, and 50 mM methyl viologen.…”

“…The quenching occurs by increasing the relative amplitude of the short (1 ns) fluorescence lifetime component at the expense of the long (4 ns) component found in these Lhcb (Crimi et al, 2001), corresponding to two distinct conformations of the Lhcb proteins (Moya et al, 2001). The short lifetime component conformation is correlated with the dissipative state (Gilmore et al, 1998). Interconversion of the protein conformation between these two states would regulate the efficiency of energy transfer to the PSI I reaction centers (Holt et al, 2004).…”