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Molecular and Global Time-resolved Analysis of a psbSGene Dosage Effect on pH- and Xanthophyll Cycle-dependent Nonphotochemical Quenching in Photosystem II

2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês

10.1074/jbc.m204797200

1083-351X

Xiaoping Li, Adam M. Gilmore, Krishna Niyogi,

Light effects on plants

Photosynthetic light harvesting in plants is regulated by a pH- and xanthophyll-dependent nonphotochemical quenching process (qE) that dissipates excess absorbed light energy and requires the psbS gene product. AnArabidopsis thaliana mutant, npq4-1, lacks qE because of a deletion of the psbS gene, yet it exhibits a semidominant phenotype. Here it is shown that the semidominance is due to a psbS gene dosage effect. DiploidArabidopsis plants containing twopsbS gene copies (wild-type), one psbS gene (npq4-1/NPQ4 heterozygote), and no psbS gene (npq4-1/npq4-1 homozygote) were compared. Heterozygous plants had 56% of the wild-type psbS mRNA level, 58% of the wild-type PsbS protein level, and 60% of the wild-type level of qE. Global analysis of the chlorophyll a fluorescence lifetime distributions revealed three components in wild-type and heterozygous plants, but only a single long lifetime component innpq4-1. The short lifetime distribution associated with qE was inhibited by more than 40% in heterozygous plants compared with the wild type. Thus, the extent of qE measured as either the fractional intensities of the PSII chlorophyll afluorescence lifetime distributions or steady state intensities was stoichiometrically related to the amount of PsbS protein. Photosynthetic light harvesting in plants is regulated by a pH- and xanthophyll-dependent nonphotochemical quenching process (qE) that dissipates excess absorbed light energy and requires the psbS gene product. AnArabidopsis thaliana mutant, npq4-1, lacks qE because of a deletion of the psbS gene, yet it exhibits a semidominant phenotype. Here it is shown that the semidominance is due to a psbS gene dosage effect. DiploidArabidopsis plants containing twopsbS gene copies (wild-type), one psbS gene (npq4-1/NPQ4 heterozygote), and no psbS gene (npq4-1/npq4-1 homozygote) were compared. Heterozygous plants had 56% of the wild-type psbS mRNA level, 58% of the wild-type PsbS protein level, and 60% of the wild-type level of qE. Global analysis of the chlorophyll a fluorescence lifetime distributions revealed three components in wild-type and heterozygous plants, but only a single long lifetime component innpq4-1. The short lifetime distribution associated with qE was inhibited by more than 40% in heterozygous plants compared with the wild type. Thus, the extent of qE measured as either the fractional intensities of the PSII chlorophyll afluorescence lifetime distributions or steady state intensities was stoichiometrically related to the amount of PsbS protein. chlorophyll antheraxanthin 3-(3,4-dichlorophenyl)-1,1-dimethylurea de-epoxidation state dithiothreitol light-harvesting complex nonphotochemical quenching photosystem pH- and xanthophyll-dependent component of NPQ violaxanthin zeaxanthin high performance liquid chromatography Absorption of light in excess of photosynthetic capacity necessitates mechanisms to protect plants from photo-oxidative damage (1Niyogi K.K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 333-359Crossref PubMed Scopus (1619) Google Scholar). Overexcitation of chlorophyll (Chl)1 and overreduction of the electron transport chain can result in increased generation of reactive intermediates and harmful byproducts of photosynthesis. For example, when excitation energy in singlet-excited Chl (1Chl*) cannot be used to drive electron transport, the lifetime of 1Chl* increases, resulting in an increased yield of triplet-excited Chl (3Chl*) via intersystem crossing. 3Chl* can generate singlet O2(1O2*), which can directly damage pigments, proteins, and lipids in the photosynthetic apparatus. To maintain a short lifetime of 1Chl* and minimize photo-oxidative damage, a nonphotochemical quenching process, called qE, is induced in excessive light, resulting in de-excitation of 1Chl* molecules and thermal dissipation of excess absorbed light energy in the light-harvesting antenna of photosystem (PS) II (2Bjo¨rkman O. Demmig-Adams B. Schulze E.-D. Caldwell M.M. Ecophysiology of Photosynthesis. Springer, Berlin1994: 17-47Google Scholar, 3Govindjee Aust. J. Plant Physiol. 1995; 22: 131-160Crossref Google Scholar). Because it decreases the lifetime of 1Chl*, qE can be measured easily as a decrease in the maximum yield of Chl fluorescence in isolated chloroplast membranes, algal cells, or intact leaves. qE is induced by a low pH in the thylakoid lumen of chloroplasts during illumination with excessive light (reviewed in Refs. 2Bjo¨rkman O. Demmig-Adams B. Schulze E.-D. Caldwell M.M. Ecophysiology of Photosynthesis. Springer, Berlin1994: 17-47Google Scholar, 3Govindjee Aust. J. Plant Physiol. 1995; 22: 131-160Crossref Google Scholar, 4Horton P. Ruban A.V. Walters R.G. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996; 47: 655-684Crossref PubMed Scopus (1421) Google Scholar, 5Mu¨ller P., Li, X.-P. Niyogi K.K. Plant Physiol. 2001; 125: 1558-1566Crossref PubMed Scopus (2124) Google Scholar). Low pH activates the violaxanthin de-epoxidase, which converts violaxanthin, V, into antheraxanthin, A, and then zeaxanthin, Z, as part of a xanthophyll cycle (6Eskling M. Arvidsson P.-O. Åkerlund H.-E. Physiol. Plant. 1997; 100: 806-816Crossref Google Scholar, 7Hager A. Planta. 1969; 89: 224-243Crossref PubMed Scopus (227) Google Scholar, 8Yamamoto H.Y. Nakayama T.O.M. Chichester C.O. Arch. Biochem. Biophys. 1962; 97: 168-173Crossref PubMed Scopus (238) Google Scholar, 9Yamamoto H.Y. Pure Appl. Chem. 1979; 51: 639-648Crossref Scopus (323) Google Scholar). Binding of de-epoxidized xanthophylls (A and Z) and protons (H+) to undefined PSII proteins is hypothesized to result in a conformational change that effectively switches a PSII unit into a quenched state in which nonphotochemical de-excitation of 1Chl* is favored (reviewed in Refs. 10Demmig-Adams B. Gilmore A.M. Adams III., W.W. FASEB J. 1996; 10: 403-412Crossref PubMed Scopus (605) Google Scholar and11Gilmore A.M. Physiol. Plant. 1997; 99: 197-209Crossref Google Scholar). The decrease in the maximum yield of Chl fluorescence caused by qE is associated with characteristic changes in Chl fluorescence lifetime distributions that can be described by a three-state model for PSII (12Gilmore A.M. Hazlett T.L. Govindjee Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2273-2277Crossref PubMed Scopus (188) Google Scholar, 13Gilmore A.M. Shinkarev V.P. Hazlett T.L. Govindjee Biochemistry. 1998; 37: 13582-13593Crossref PubMed Scopus (123) Google Scholar, 14Gilmore A.M. Photosynth. Res. 2001; 67: 89-101Crossref PubMed Google Scholar). According to this model, without lumen acidification and with PSII traps closed, the main Chl fluorescence lifetime distribution that is broad and centered at ∼1.7–2.2 ns is referred to as the W state. Thylakoid membrane energization by a ΔpH results in a distinct conversion of the W state distribution to a shorter, usually narrower lifetime, centered at ∼1.6–1.8 ns and referred to as the X state. The W to X shift is hypothesized to be related to protein conformational changes (15Alcala J.R. Gratton E. Prendergrast F.G. Biophys. J. 1987; 51: 587-596Abstract Full Text PDF PubMed Scopus (240) Google Scholar) in the molecular environment of the protein-bound fluorophores, in this case fluorescing Chls (12Gilmore A.M. Hazlett T.L. Govindjee Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2273-2277Crossref PubMed Scopus (188) Google Scholar, 13Gilmore A.M. Shinkarev V.P. Hazlett T.L. Govindjee Biochemistry. 1998; 37: 13582-13593Crossref PubMed Scopus (123) Google Scholar, 16Govindjee Van De Ven M. Preston C. Seibert M. Gratton E. Biochim. Biophys. Acta. 1990; 1015: 173-179Crossref PubMed Scopus (54) Google Scholar), caused by protonation of lumen-exposed carboxylate groups of proteins in the PSII inner antenna. The conformational changes are associated with the activation for potential structural interactions between the xanthophylls Z and A and the PSII proteins. The final step in the model is the binding of Z (or A) to convert PSII from the X state to the Y state that exhibits a short lifetime distribution typically at ∼0.3–0.5 ns (Y state) (12Gilmore A.M. Hazlett T.L. Govindjee Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2273-2277Crossref PubMed Scopus (188) Google Scholar, 13Gilmore A.M. Shinkarev V.P. Hazlett T.L. Govindjee Biochemistry. 1998; 37: 13582-13593Crossref PubMed Scopus (123) Google Scholar, 17Gilmore A.M. Hazlett T.L. Debrunner P.G. Govindjee Photochem. Photobiol. 1996; 64: 552-563Crossref PubMed Scopus (81) Google Scholar, 18Richter M. Goss R. Wagner B. Holzwarth A.R. Biochemistry. 1999; 38: 12718-12726Crossref PubMed Scopus (51) Google Scholar). The W to X to Y state conversion is typically saturated by levels of Z or A above 2–3 molecules per PSII (12Gilmore A.M. Hazlett T.L. Govindjee Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2273-2277Crossref PubMed Scopus (188) Google Scholar, 13Gilmore A.M. Shinkarev V.P. Hazlett T.L. Govindjee Biochemistry. 1998; 37: 13582-13593Crossref PubMed Scopus (123) Google Scholar, 19Bukhov N.G. Kopecky J. Pfu¨ndel E.E. Klughammer C. Heber U. Planta. 2001; 212: 739-748Crossref PubMed Scopus (43) Google Scholar, 20Gilmore A.M. Yamamoto H.Y. Photochem. Photobiol. 2001; 74: 291-302Crossref PubMed Scopus (27) Google Scholar). Hence, it was concluded that the energy dissipation in PSII exhibited saturable kinetics and may be defined by a pH-dependent binding isotherm (13Gilmore A.M. Shinkarev V.P. Hazlett T.L. Govindjee Biochemistry. 1998; 37: 13582-13593Crossref PubMed Scopus (123) Google Scholar). According to this model, and as tested in the experiments below, the energy dissipation mechanism should therefore exhibit limiting (and potentially saturating) characteristics for both the available xanthophyll substrate concentration [A+Z] and the xanthophyll binding enzyme concentration. Characterization of mutants that are defective in qE has helped to define the factors that are required for the photoprotective mechanism (1Niyogi K.K. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1999; 50: 333-359Crossref PubMed Scopus (1619) Google Scholar). Several npq mutants of Arabidopsis and the green alga Chlamydomonas reinhardtii have been identified in forward genetics screens based on video imaging of the Chl fluorescence yield (21Li X.-P. Bjo¨rkman O. Shih C. Grossman A.R. Rosenquist M. Jansson S. Niyogi K.K. Nature. 2000; 403: 391-395Crossref PubMed Scopus (1177) Google Scholar, 22Niyogi K.K. Bjo¨rkman O. Grossman A.R. Plant Cell. 1997; 9: 1369-1380Crossref PubMed Scopus (283) Google Scholar, 23Niyogi K.K. Grossman A.R. Bjo¨rkman O. Plant Cell. 1998; 10: 1121-1134Crossref PubMed Scopus (765) Google Scholar, 24Peterson R.B. Havir E.A. Planta. 2000; 210: 205-214Crossref PubMed Scopus (38) Google Scholar, 25Shikanai T. Munekage Y. Shimizu K. Endo T. Hashimoto T. Plant and Cell Physiology. 1999; 40: 1134-1142Crossref PubMed Scopus (80) Google Scholar). Besides providing genetic evidence for the necessary roles of the ΔpH and de-epoxidized xanthophylls in qE (22Niyogi K.K. Bjo¨rkman O. Grossman A.R. Plant Cell. 1997; 9: 1369-1380Crossref PubMed Scopus (283) Google Scholar, 23Niyogi K.K. Grossman A.R. Bjo¨rkman O. Plant Cell. 1998; 10: 1121-1134Crossref PubMed Scopus (765) Google Scholar, 26Munekage Y. Takeda S. Endo T. Jahns P. Hashimoto T. Shikanai T. Plant J. 2001; 28: 351-359Crossref PubMed Scopus (81) Google Scholar), mutant analysis has recently identified the PSII subunit PsbS as a critical component of the qE mechanism (21Li X.-P. Bjo¨rkman O. Shih C. Grossman A.R. Rosenquist M. Jansson S. Niyogi K.K. Nature. 2000; 403: 391-395Crossref PubMed Scopus (1177) Google Scholar, 27Peterson R.B. Havir E.A. Planta. 2001; 214: 142-152Crossref PubMed Scopus (32) Google Scholar). PsbS is a member of the light-harvesting complex (LHC) protein superfamily (28Kim S. Sandusky P. Bowlby N.R. Aebersold R. Green B.R. Vlahakis S. Yocum C.F. Pichersky E. FEBS Lett. 1992; 314: 67-71Crossref PubMed Scopus (78) Google Scholar, 29Wedel N. Klein R. Ljungberg U. Andersson B. Herrmann R.G. FEBS Lett. 1992; 314: 61-66Crossref PubMed Scopus (76) Google Scholar). PsbS has been reported to bind substoichiometric levels of V (30Funk C. Schro¨der W.P. Napiwotzki A. Tjus S.E. Renger G. Andersson B. Biochemistry. 1995; 34: 11133-11141Crossref PubMed Scopus (111) Google Scholar), although recent attempts to purify or reconstitute the protein with bound pigments were unsuccessful (31Dominici P. Caffarri S. Armenante F. Ceoldo S. Crimi M. Bassi R. J. Biol. Chem. 2002; 277: 22750-22758Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). In thenpq4-1 mutant of Arabidopsis, the nuclear gene encoding PsbS has been completely deleted, resulting in the absence of PsbS protein and a severe defect in qE (21Li X.-P. Bjo¨rkman O. Shih C. Grossman A.R. Rosenquist M. Jansson S. Niyogi K.K. Nature. 2000; 403: 391-395Crossref PubMed Scopus (1177) Google Scholar). In contrast to other mutants in which the accumulation of de-epoxidized xanthophylls is affected, the npq4-1 mutant exhibits xanthophyll cycle conversion and generation of the thylakoid ΔpH that are indistinguishable from that of the wild type. Loss-of-function mutations like the npq4-1 deletion usually behave as recessive mutations in genetic crosses, yet npq4-1 is semidominant: heterozygous F1 plants resulting from a cross betweennpq4-1 and the wild type have a phenotype that is intermediate between those of the parents. This observation suggested that the dosage of the psbS gene somehow affects the level of qE (21Li X.-P. Bjo¨rkman O. Shih C. Grossman A.R. Rosenquist M. Jansson S. Niyogi K.K. Nature. 2000; 403: 391-395Crossref PubMed Scopus (1177) Google Scholar). In this study, the effect of psbS gene dosage was investigated systematically by comparing psbS mRNA levels, PsbS protein levels, extent of qE, and global analysis of the PSII Chl fluorescence lifetime distributions. Materials in the investigation included the homozygous npq4-1mutant plants (no psbS gene copies), heterozygous F1 plants (one psbS gene copy), and homozygous wild-type plants (twopsbS gene copies). The targeted global analysis employed the measured xanthophyll concentrations, lumen pH, and multifrequency phase and modulation data to simulate the changes in the fractional intensity changes in the PSII components corresponding to the W, X, and Y states (13Gilmore A.M. Shinkarev V.P. Hazlett T.L. Govindjee Biochemistry. 1998; 37: 13582-13593Crossref PubMed Scopus (123) Google Scholar, 15Alcala J.R. Gratton E. Prendergrast F.G. Biophys. J. 1987; 51: 587-596Abstract Full Text PDF PubMed Scopus (240) Google Scholar, 32Beechem J.M. Knutson J.R. Ross J.B. Turner B.W. Brand L. Biochemistry. 1983; 22: 6054-6058Crossref Scopus (114) Google Scholar, 33Beechem J.M. Methods Enzymol. 1992; 210: 37-54Crossref PubMed Scopus (336) Google Scholar). The implications of the results for the concentration-dependent function of the PsbS protein in the pH- and xanthophyll-dependent control of qE are discussed. A. thalianaplants (Col-0 background) were grown in a growth chamber with a short-day photoperiod of 10 h light (150 μmol photons m−2 s−1, 22–23 °C) and 14 h dark (19–20 °C). Plants between the ages of 5 and 7 weeks, prior to bolting, were used for all experiments except for genomic DNA extraction and crosses. Heterozygous plants (npq4-1/NPQ4; hereafter referred to as F1) were generated by crossing wild-type plants (NPQ4/NPQ4) with homozygous npq4-1 mutant plants (npq4-1/npq4-1; hereafter referred to asnpq4-1). Wild-type plants have two copies of thepsbS gene, the F1 plants have only one copy of thepsbS gene, and npq4–1 plants do not have anypsbS gene. For RNA extraction and immunoblot analysis, leaves were harvested 3 h into the light period, frozen immediately in liquid nitrogen, and then stored at −80 °C. Chloroplast thylakoid membrane isolations, including grinding, resuspension, and reaction buffers and sample preparation protocols for the lifetime measurements were described previously (13Gilmore A.M. Shinkarev V.P. Hazlett T.L. Govindjee Biochemistry. 1998; 37: 13582-13593Crossref PubMed Scopus (123) Google Scholar, 14Gilmore A.M. Photosynth. Res. 2001; 67: 89-101Crossref PubMed Google Scholar). Genomic DNA was extracted from young flower buds (34Shure M. Wessler S. Federoff N. Cell. 1983; 35: 225-233Abstract Full Text PDF PubMed Scopus (637) Google Scholar), and RNA was extracted from leaves (35Bugos R.C. Yamamoto H.Y. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6320-6325Crossref PubMed Scopus (126) Google Scholar). For DNA gels, genomic DNA was digested with XbaI. A 0.9-kb fragment of thepsbS cDNA (EST clone 135M5T7) was amplified by PCR with the primers KN118 (5′-TCCTTCTCTCATCCTCAGAAA-3′) and KN119 (5′-CAACATGAAGAGAAGGTCACA-3′) and used as a hybridization probe for both DNA and RNA blots. DNA gel blot analysis was conducted using the AlkPhos Direct Kit detected with the CDP-Starchemiluminescent method (Amersham Biosciences). RNA gel blot analysis was done using the DIG-High Prime DNA Labeling and Detection Kit (Roche Molecular Biochemicals). Thylakoids used in the Chl fluorescence lifetime experiments described below were frozen in liquid nitrogen, stored at −80 °C, and then dissolved in solubilization buffer (250 mm Tris-HCl, pH 6.8, 3.5% SDS, 10% (v/v) glycerol, 1m urea, 10% (v/v) β-mercaptoethanol). SDS-PAGE was conducted under denaturing conditions with a precast 10–20% acrylamide gel with Tris-glycine buffer (NOVEX, San Diego). A protein sample containing 1 nmol of Chl was loaded in each lane. The polyclonal antibody recognizing PsbS was generated (Sigma-Genosys) in rabbits against an Arabidopsis PsbS peptide, which is between the second and third transmembrane helices at the stromal side of the native protein. The sequence used to generate the antibody was CGDRGKFVDDPPTG. The anti-D1 antibody was kindly provided by Prof. Anastasios Melis (University of California, Berkeley). Proteins were detected using an enhanced chemiluminescent Western blotting kit (Amersham Biosciences). PsbS levels were normalized to D1 protein on the same blot. The Chl fluorescence data for Fig. 2 were obtained from attached rosette leaves using a commercial fluorometer (FMS2, Hansatech, King's Lynn, UK). After an overnight dark period, standard Chl fluorescence parameters (36van Kooten O. Snel J.F.H. Photosynth. Res. 1990; 25: 147-150Crossref PubMed Scopus (2072) Google Scholar) were measured before, during, and after illumination with 1000 μmol of photons m−2 s−1 for 6 min. NPQ was calculated as (Fm-Fm′)/Fm′. The measurements were performed on six different plants of each genotype. The Chl fluorescence data for Fig. 3A were obtained from intact detached leaf samples inside a light-tight, temperature-controlled cuvette with a PAM Chl fluorometer (37Schreiber U. Schliwa U. Bilger W. Photosynth. Res. 1986; 10: 51-62Crossref PubMed Scopus (2396) Google Scholar) equipped with the ED101-BL emitter detector unit (Heinz-Walz, Effeltrich, Germany). The fluorescence was excited by the blue excitation LED (440 nm) and measured with a 10-nm bandpass emission filter centered at 685 nm. The analog PAM signal was digitized and recorded at a sampling rate of 3 Hz through the auxiliary channel of an SLM 8100 spectrofluorimeter (Spectronic Inc., Rochester, NY). After dark-adapting the plant material for at least 60 min, the intensity of the open PSII traps (Fo) was determined for 30 s with the weak 1.6-kHz measuring beam (<0.25 μmol of photons m−2s−1). A 2-s pulse of high intensity white light (10,000 μmol of photons m−2 s−1, Walz DT-Cyan filter) was delivered simultaneously with switching the PAM measuring beam from 1.6 to 100 kHz to determine the maximal fluorescence level (Fm) with all PSII traps closed. A train of identical high intensity pulses ensued at a rate of 1 pulse every 100 s for the duration of the experiment to determine the maximal fluorescence level under the conditions of thylakoid membrane energization (Fm′). Immediately following the initial Fo and Fm determination, the leaf samples were exposed to a continuous white actinic light (750 μmol of photons m−2s−1, Walz DT-Cyan filter) for 300 s at a temperature of 25 °C with the PAM measuring beam at 100 kHz. The temperature was attenuated to −2 °C over the next 300 s upon which time actinic light was extinguished, and the PAM measuring beam was switched to 1.6 kHz. The Fm′ was determined every 100 s for 600 s upon which time the temperature was ramped back up to 25 °C over a 300-s interval. The Fm′ (or Fm) determinations (depending on the interpretation of dark-sustained thylakoid membrane lumen acidification) continued for an additional 600 s. The determination of the PSII Chl afluorescence intensity and lifetimes followed the eight-step protocol defined by Gilmore et al. (13Gilmore A.M. Shinkarev V.P. Hazlett T.L. Govindjee Biochemistry. 1998; 37: 13582-13593Crossref PubMed Scopus (123) Google Scholar). The only notable exception was that the lifetimes were measured with an ISS K2-004 lifetime spectrofluorimeter (ISS Inc., Urbana, IL) via a quartz fiber optic probe using the emission/excitation wavelength specifications defined by Gilmore & Yamamoto (20Gilmore A.M. Yamamoto H.Y. Photochem. Photobiol. 2001; 74: 291-302Crossref PubMed Scopus (27) Google Scholar). In addition to the reaction buffer, other chemical additions included 7.5 μmtotal Chl, 30 mm sodium ascorbate, 0.3 mm ATP, and 35 μm methylviologen in 3-ml quartz cuvettes for all samples. When necessary, DCMU was added to 10 μmand nigericin to 2 μm. The thylakoids were treated in the same temperature-controlled cuvette described for the leaf experiments above except that the actinic light intensity was 500 μmol of photons m−2 s−1 for 10 min at 25 °C followed by addition of dithiothreitol (DTT) to a final concentration of 5 mm and continued actinic illumination for 10 min while the reaction temperature was lowered to 0 °C. The level of violaxanthin de-epoxidation was controlled by addition of a subsaturating level of DTT (0–1 mm) prior to actinic illumination. Pigment measurements from leaves were performed by HPLC according to Mu¨ller-Mouleá et al. (38Mu¨ller-Mouleá P. Conklin P.L. Niyogi K.K. Plant Physiol. 2002; 128: 970-977Crossref PubMed Scopus (198) Google Scholar). For each condition, measurements were done on six individual plants of each genotype. Thylakoid samples were immediately frozen at 77 K following the experiments and stored at −80 °C until HPLC analysis according to the method of Gilmore and Yamamoto (39Gilmore A.M. Yamamoto H.Y. J. Chromatogr. 1991; 543: 137-145Crossref Scopus (412) Google Scholar). As outlined in previous studies (12Gilmore A.M. Hazlett T.L. Govindjee Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2273-2277Crossref PubMed Scopus (188) Google Scholar, 13Gilmore A.M. Shinkarev V.P. Hazlett T.L. Govindjee Biochemistry. 1998; 37: 13582-13593Crossref PubMed Scopus (123) Google Scholar, 14Gilmore A.M. Photosynth. Res. 2001; 67: 89-101Crossref PubMed Google Scholar, 40Gilmore A.M. Hazlett T.L. Debrunner P.G. Govindjee Photosynth. Res. 1996; 48: 171-187Crossref PubMed Scopus (88) Google Scholar), we analyzed the fluorescence lifetime distributions assuming three major distinct PSII Chl fluorescence lifetime states that correspond to the following structural model assumptions. 1) W is a PSII unit containing unprotonated unknown CP protein(s) incapable of binding the Z or A molecules. 2) X is a PSII unit containing the protonated unknown CP protein(s) capable of, but not actively binding a Z or A molecule; and finally 3) Y is a PSII unit with protonated unknown CP protein(s) and with a bound Z or A molecule. In simplest terms conversion of the protonation of the unknown CP protein is defined with a simple Henderson-Hasselbach titration (defined by a pKavalue). Also the concentration dependence of the binding of Z and A to the protonated unknown CP protein is defined as an equilibrium association (defined by a Ka value). In SchemeFS1 the total sum of all three PSII states is normalized and defined as [W] + [X] + [Y] = 1 wherepKa = pH − log[W]/[X] andKa = [X]/[Y]·[A + Z]. The following balanced analytical Equations Equation 1, Equation 2, Equation 3 defined as, [W]=1/(1+10pKa­pH(1+Ka[A+Z]))Equation 1 [X]=10pKa­pH/(1+10pKa­pH(1+Ka[A+Z]))Equation 2 [Y]=Ka[A+Z]10pKa­pH/(1+10pKa­pH(1+Ka[A+Z]))Equation 3 were derived from the Scheme FS1 with the assistance of Dr. V. P. Shinkarev of the University of Illinois at Urbana-Champaign Center for Biophysics and Computational Biology (11Gilmore A.M. Physiol. Plant. 1997; 99: 197-209Crossref Google Scholar, 13Gilmore A.M. Shinkarev V.P. Hazlett T.L. Govindjee Biochemistry. 1998; 37: 13582-13593Crossref PubMed Scopus (123) Google Scholar, 41Gilmore A.M. Govindjee Singhal G.S. Renger G. Sopory S.K. Irrgang K.-D. Govindjee Concepts in photobiology: Photosynthesis and Photomorphogenesis. Narosa Publishing House, New Delhi, India1999: 513-548Google Scholar). Equations Equation 1, Equation 2, Equation 3 were used to calculate the pKa, [W], [X], and [Y] given the measured constant level of the ATPase-induced thylakoid lumen pH and the variable [A + Z]/[V + A + Z] levels. The integral fractions of the [W] + [X] + [Y] parameters were normalized to unity by employing the area form of the Lorentzian distribution equations. The lumen pH was estimated using the 9-aminoacridine technique (42Schuldiner S. Rottenberg H. Avron M. Eur. J. Biochem. 1972; 25: 64-70Crossref PubMed Scopus (529) Google Scholar), and the levels of [A + Z]/[V + A + Z] were varied with DTT concentrations ranging from 0 to 1 mm, essentially as described by Gilmore et al. (13Gilmore A.M. Shinkarev V.P. Hazlett T.L. Govindjee Biochemistry. 1998; 37: 13582-13593Crossref PubMed Scopus (123) Google Scholar). The measured values for [A+Z] and the lumen pH = 5.18 ± 0.01 were fixed. The free parameters included the pKa andKa values and all the width and lifetime mode parameters of the Lorentzian distributions. The linking scheme assumed all Lorentzian mode and width parameters corresponding to each given PSII state represented mean linked values. The heart of the global target scheme reduced to a minimum of nine free parameters, including three modes plus three widths for the entire data set plus twoKa values for the two samples types with measurable [PsbS], namely, the wild type and F1. The pKawas assumed to be linked between the wild type and F1. In thenpq4-1 samples lacking PsbS, the fraction of the [W] state was normalized as unity for the final solution since there was little or no observed pH- or xanthophyll-dependent change in the fluorescence lifetime integrals, averages, or intensities. Two other minor modes were also included in the fits for all three materials in addition to the three major modes directly related to the model. One was a narrow distribution (∼50-ps wide) with the mode and width parameters linked for all samples and free amplitudes which accounted for rapid decays ( 5 ns) distribution included all free floating parameters and accounted for decay variations attributed to instrumental tuning and decay signals from Chls dissociated from proteins and other impurities and long-lived background components; the variation in this component was also possibly related to natural variation in the longer lifetime tail of the Lorentzian distributions of the W state attributed to heterogeneity of the Chl protein environment (40Gilmore A.M. Hazlett T.L. Debrunner P.G. Govindjee Photosynth. Res. 1996; 48: 171-187Crossref PubMed Scopus (88) Google Scholar). The global analysis program was written with Visual Basic for Applications 97 for use in Microsoft Windows NT4.0 (32 bit) and Excel 97, and it utilized a Large Scale General Reduced Gradient (LSGRG) minimization engine developed by Frontline Systems Inc. (Incline Village, NV). The search engine is capable of solving, in Excel 97 with a Pentium III computer with 500 Mb RAM and an 800 MHz processor, all 26 test problems prescribed by the National Institute of Standards and Technology (NIST) website for Nonlinear Regression (www.itl.nist.gov/div898/strd/nls/nls_info.shtml) with 10 digit precision for the sum of squares parameter. The global reduced chi-square equation (15Alcala J.R. Gratton E. Prendergrast F.G. Biophys. J. 1987; 51: 587-596Abstract Full Text PDF PubMed Scopus (240) Google Scholar, 43Bevington P.R. Data reduction and error analysis for the physical sciences. McGraw-Hill, Inc., New York1969Google Scholar) was minimized assuming the following uncertainties for the phase angle shift (φ) ςp = 0.025° and the demodulation ratio (M) ςm = 0.005. The final goodness of fit was judged by nonlinear regression of the φ and M to calculate an r2 regression coefficient and F-test. The standard errors for all global model parameters and regression coefficients were calculated from the diagonal elements of the inverted variance-covariance matrix (44Draper N.R. Smith H. Applied regression analysis. 3rd Ed. John Wiley and Sons, Inc., New York1998Crossref Scopus (15868) Google Scholar). The randomness and normality of the residual error distribution were further judged by calculation of a second chi square statistic (45Straume M. Johnson M.L. Methods Enzymol. 1992; 210: 87-105Crossref PubMed Scopus (111) Google Scholar) describing the fit of the binned histogram of the residual errors to a Gaussian distribution as described by Gilmore et al.(46Gilmore A.M. Itoh S. Govindjee Phil. Trans. R. Soc. Lond. B. 2000; 355: 1371-1384Crossref PubMed Scopus (35) Google Scholar). To investigate the effect of psbS gene dosage at a molecular level, the amounts of psbS DNA and mRNA and PsbS p