Different Roles of α- and β-Branch Xanthophylls in Photosystem Assembly and Photoprotection
2007; Elsevier BV; Volume: 282; Issue: 48 Linguagem: Inglês
10.1074/jbc.m704729200
ISSN1083-351X
AutoresLuca Dall’Osto, Alessia Fiore, Stefano Cazzaniga, Giovanni Giuliano, Roberto Bassi,
Tópico(s)Light effects on plants
ResumoXanthophylls (oxygenated carotenoids) are essential components of the plant photosynthetic apparatus, where they act in photosystem assembly, light harvesting, and photoprotection. Nevertheless, the specific function of individual xanthophyll species awaits complete elucidation. In this work, we analyze the photosynthetic phenotypes of two newly isolated Arabidopsis mutants in carotenoid biosynthesis containing exclusively α-branch (chy1chy2lut5) or β-branch (chy1chy2lut2) xanthophylls. Both mutants show complete lack of qE, the rapidly reversible component of nonphotochemical quenching, and high levels of photoinhibition and lipid peroxidation under photooxidative stress. Both mutants are much more photosensitive than npq1lut2, which contains high levels of viola- and neoxanthin and a higher stoichiometry of light-harvesting proteins with respect to photosystem II core complexes, suggesting that the content in light-harvesting complexes plays an important role in photoprotection. In addition, chy1chy2lut5, which has lutein as the only xanthophyll, shows unprecedented photosensitivity even in low light conditions, reduced electron transport rate, enhanced photobleaching of isolated LHCII complexes, and a selective loss of CP26 with respect to chy1chy2lut2, highlighting a specific role of β-branch xanthophylls in photoprotection and in qE mechanism. The stronger photosystem II photoinhibition of both mutants correlates with the higher rate of singlet oxygen production from thylakoids and isolated light-harvesting complexes, whereas carotenoid composition of photosystem II core complex was not influential. In depth analysis of the mutant phenotypes suggests that α-branch (lutein) and β-branch (zeaxanthin, violaxanthin, and neoxanthin) xanthophylls have distinct and complementary roles in antenna protein assembly and in the mechanisms of photoprotection. Xanthophylls (oxygenated carotenoids) are essential components of the plant photosynthetic apparatus, where they act in photosystem assembly, light harvesting, and photoprotection. Nevertheless, the specific function of individual xanthophyll species awaits complete elucidation. In this work, we analyze the photosynthetic phenotypes of two newly isolated Arabidopsis mutants in carotenoid biosynthesis containing exclusively α-branch (chy1chy2lut5) or β-branch (chy1chy2lut2) xanthophylls. Both mutants show complete lack of qE, the rapidly reversible component of nonphotochemical quenching, and high levels of photoinhibition and lipid peroxidation under photooxidative stress. Both mutants are much more photosensitive than npq1lut2, which contains high levels of viola- and neoxanthin and a higher stoichiometry of light-harvesting proteins with respect to photosystem II core complexes, suggesting that the content in light-harvesting complexes plays an important role in photoprotection. In addition, chy1chy2lut5, which has lutein as the only xanthophyll, shows unprecedented photosensitivity even in low light conditions, reduced electron transport rate, enhanced photobleaching of isolated LHCII complexes, and a selective loss of CP26 with respect to chy1chy2lut2, highlighting a specific role of β-branch xanthophylls in photoprotection and in qE mechanism. The stronger photosystem II photoinhibition of both mutants correlates with the higher rate of singlet oxygen production from thylakoids and isolated light-harvesting complexes, whereas carotenoid composition of photosystem II core complex was not influential. In depth analysis of the mutant phenotypes suggests that α-branch (lutein) and β-branch (zeaxanthin, violaxanthin, and neoxanthin) xanthophylls have distinct and complementary roles in antenna protein assembly and in the mechanisms of photoprotection. Carotenoids are a group of C40 terpenoid compounds with a wide distribution in several biological taxa, ranging from archaea to bacteria, fungi, algae, and higher plants. Xanthophylls form a subgroup of oxygenated carotenoids, whose importance in the oxygenic photosynthesis is well known. Xanthophylls play essential roles in higher plant photosynthesis, as components of the photosynthetic apparatus of the chloroplast. In higher plants, β-carotene binds to reaction center subunits of both photosystem I (PSI) 4The abbreviations used are: PSI and PSII, photosystems I and II, respectively; 1O2, singlet oxygen; ABA, abscisic acid; α- and β-DM, n-dodecyl-α-d-maltoside and n-dodecyl-β-d-maltoside, respectively; Chl a and b, chlorophyll a and b, respectively; 3Chl*, triplet excited state of chlorophyll; ETR, electron transport rate; Lhca and Lhcb, light-harvesting complexes of photosystems I and II, respectively; LHCI, antenna complex of photosystem I; LHCII, major light-harvesting complex of PSII; Lute, lutein; MDA, malondialdehyde; Neo, neoxanthin; NPQ, nonphotochemical quenching; qE, ΔpH-dependent component of NPQ; qI, photoinhibition quenching; qP, photochemical quenching; ROS, reactive oxygen species; SOSG, singlet oxygen sensor green; Viola, violaxanthin; WT, wild type; Zea, zeaxanthin; HPLC, high pressure liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. and II (PSII), whereas xanthophylls are both accessory pigments and structural elements of light-harvesting complexes (Lhcs). Together with β-carotene, they act both as chromophores, absorbing light energy that is used in photosynthetic electron transport, and as photoprotectants of the photosynthetic apparatus from excess light and from the reactive oxygen species (ROS) that are generated during oxygenic photosynthesis. A remarkable characteristic of higher plant xanthophylls is that they show very similar spectral properties in the visible region. This evidence is apparently incoherent with the high conservation of their relative abundance across a range of plant taxa, which suggests that each xanthophyll species serves a specific role. Xanthophyll biosynthesis in plants is divided into two distinct branches; hydroxylation of α-carotene gives rise to lutein (Lute), the most abundant xanthophyll in leaves (Fig. 1), whereas hydroxylation of β-carotene gives rise to zeaxanthin (Zea). In normal light conditions, zeaxanthin is epoxidized into antheraxanthin and violaxanthin (Viola) (Fig. 1), whereas in excess light, de-epoxidation prevails, leading to the accumulation of Zea (1Yamamoto H.Y. Nakayama T.O. Chichester C.O. Arch. Biochem. Biophys. 1962; 97: 168-173Crossref PubMed Scopus (238) Google Scholar, 2Demmig-Adams B. Adams W.W. Ann. Rev. Plant Physiol. Plant Mol. Biol. 1992; 43: 599-626Crossref Scopus (2081) Google Scholar). LHCII, the major light-harvesting complex of photosystem II, binds Lute, Viola, and neoxanthin (Neo) (Fig. 1) (3Liu Z. Yan H. Wang K. Kuang T. Zhang J. Gui L. An X. Chang W. Nature. 2004; 428: 287-292Crossref PubMed Scopus (1391) Google Scholar). The minor complexes CP24, CP26, and CP29 bind the same pigments and, in excess light, Zea (4Bassi R. Pineau B. Dainese P. Marquardt J. Eur. J. Biochem. 1993; 212: 297-303Crossref PubMed Scopus (350) Google Scholar, 5Morosinotto T. Caffarri S. Dall'Osto L. Bassi R. Physiol. 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Neo has a role in the protection of photosynthetic apparatus by promoting the scavenging of superoxide anions, a ROS formed in the Mehler reaction (20Dall'Osto L. Cazzaniga S. North H. MArion-Poll A. Bassi R. Plant Cell. 2007; 19: 1048-1064Crossref PubMed Scopus (150) Google Scholar). The function of Viola is more controversial; it has been suggested to be involved both in the reversion of NPQ (21Gilmore A.M. Mohanty N. Yamamoto H.Y. FEBS Lett. 1994; 350: 271-274Crossref PubMed Scopus (66) Google Scholar) and in chlorophyll (Chl) triplet quenching (22Peterman E.J. Gradinaru C.C. Calkoen F. Borst J.C. van Grondelle R. Van Amerongen H. Biochemistry. 1997; 36: 12208-12215Crossref PubMed Scopus (106) Google Scholar). A previous report showed that the overaccumulation of Viola increases photoprotection in vivo (23Davison P.A. Hunter C.N. Horton P. Nature. 2002; 418: 203-206Crossref PubMed Scopus (309) Google Scholar). Furthermore, cooperative effects on photoprotection by different xanthophyll species have been suggested by early experiments using recombinant Lhc proteins reconstituted with different xanthophylls (24Croce R. Weiss S. Bassi R. J. Biol. Chem. 1999; 274: 29613-29623Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). These data suggest that different xanthophyll species may have distinct roles in photoprotection. Nevertheless, the role of β-β-xanthophylls (in particular, Neo and Viola) in the photosynthetic process is not completely understood. The recent elucidation of the higher plant β-ring hydroxylation (25Kim J. DellaPenna D. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3474-3479Crossref PubMed Scopus (198) Google Scholar, 26Fiore A. Dall'Osto L. Fraser P.D. Bassi R. Giuliano G. FEBS Lett. 2006; 580: 4718-4722Crossref PubMed Scopus (57) Google Scholar) showed that two different classes of enzymes are involved in hydroxylation of the ϵ- and β-ionone rings of carotenes: ferredoxin-dependent di-iron oxygenases (CHY1 and CHY2), active in β-ring hydroxylation, and cytochromes P450: LUT1, required for ϵ-ring hydroxylation, and LUT5, showing in vivo a major hydroxylation activity on the β-ring of α-carotene as well as minor activity on the β-rings of β-carotene. Plant mutants are now available, specifically lacking xanthophylls in the β-β-branch (Neo, Viola, and Zea) and useful for the analysis of xanthophyll function in photosynthesis. In this work, we compare the photosystem structure and photoprotection properties of two triple mutants containing low levels of xanthophylls but mutually exclusive in composition; chy1chy2lut5 contains only Lute, whereas chy1chy2lut2 contains only β-β-xanthophylls (Neo, Viola, and Zea). We find that Lute alone is unable to provide sufficient levels of photoprotection even in moderate light, thus conferring to the chy1chy2lut5 mutant the highest sensitivity to photooxidation ever described in a xanthophyll mutant. Plant Material—T-DNA insertion mutants were identified in the Syngenta and Salk collections. Individual mutants were crossed, and F1 seeds were grown and self-fertilized to obtain the F2 generation. The genotype of the F2 individuals was checked by PCR using gene-specific primers and T-DNA primers (26Fiore A. Dall'Osto L. Fraser P.D. Bassi R. Giuliano G. FEBS Lett. 2006; 580: 4718-4722Crossref PubMed Scopus (57) Google Scholar). All mutants were in the Columbia background and were grown at 22 °C under a photoperiod of 16 h of light (45 μmol m−2 s−1) and 8 h of darkness. In Vivo Fluorescence and NPQ Measurements—Nonphotochemical quenching of chlorophyll fluorescence, qP (photochemical quenching) and PSII yield (ΦPSII) was measured on whole leaves at room temperature (22 °C) with a PAM 101 fluorimeter (Walz, Effeltrich, Germany). Minimum fluorescence (F0) was measured with a 0.15 μmol m−2 s−1 beam, maximum fluorescence (Fm) was determined with a 0.6-s light pulse (4500 μmol m−2 s−1), and white continuous light (1200 μmol m−2 s−1) was supplied by a KL1500 halogen lamp (Schott, Mainz, Germany). NPQ, qP, and relative electron transport rate (ETR) were calculated according to the equation (27Van Kooten O. Snel J.F.H. Photosynth. Res. 1990; 25: 147-150Crossref PubMed Scopus (2072) Google Scholar), NPQ = (Fm - F′m)/F′m, qP = (Fm - Fs)/(Fm - Fo), rel ETR =ΦPSII·PAR, where Fm is the maximum Chl fluorescence from dark-adapted leaves, F′m is the maximum Chl fluorescence under actinic light exposition, Fs is the stationary fluorescence during illumination, and PAR is the photosynthetic active radiations (white light, measured as μmol m−2 s−1). Calculation of the ΔpH-dependent component of chlorophyll fluorescence quenching (qE) was performed as described previously (28Walters R.G. Horton P. Planta. 1995; 197: 306-312PubMed Google Scholar). Variable fluorescence was induced in leaf discs, infiltrated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (30 μm), sorbitol (150 mm), with a green light of 7 μmol m−2 s−1 produced by a light-emitting diode. The time corresponding to two-thirds of the fluorescence rise (T⅔)was taken as a measure of the functional antenna size of PSII (29Malkin S. Armond P.A. Mooney H.A. Fork D.C. Plant Physiol. 1981; 67: 570-579Crossref PubMed Google Scholar). In 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated leaves, the rate of fluorescence rise depends on light intensity and functional antenna size of PSII. Thus, keeping constant the saturating flash intensity, PSII with higher functional antenna size will reduce more rapidly all of the available QA pool and will have a lower T⅔ of fluorescence rise. For measurements of the PSII repair process, whole plants were illuminated at room temperature for 1.5 h to induce 50-60% photoinhibition of PSII (photon flux density of 1800 μmol m−2 s−1 for WT plants, 700 μmol m−2 s−1 for chy1chy2lut5 plants), and restoration of the Fv/Fm ratios was subsequently followed at irradiances of 30 μmol m−2 s−1 at room temperature (30Aro E.M. McCaffery S. Anderson J.M. Plant Physiol. 1994; 104: 1033-1041Crossref PubMed Scopus (111) Google Scholar). Pigment Analysis—Pigments were extracted from dark-adapted leaf discs; samples were frozen in liquid nitrogen and ground in 85% acetone buffered with Na2CO3, and then the supernatant of each sample was recovered after centrifugation (15 min at 15,000 × g, 4 °C); separation and quantification of pigments were performed by HPLC (31Gilmore A.M. Yamamoto H.Y. Plant Physiol. 1991; 96: 635-643Crossref PubMed Scopus (218) Google Scholar) and by fitting of the spectrum of the acetone extract with spectra of individual pigments (32Croce R. Canino g. Ros F. Bassi R. Biochemistry. 2002; 41: 7334-7343Crossref PubMed Scopus (161) Google Scholar). Thylakoid Isolation and Thylakoid Protein Preparation—Unstacked thylakoids were isolated from leaves as previously described (33Casazza A.P. Tarantino D. Soave C. Photosynth. Res. 2001; 68: 175-180Crossref PubMed Scopus (61) Google Scholar). Membranes corresponding to 500 μg of chlorophylls were washed with 5 mm EDTA and then solubilized in 1 ml of 0.6% α-DM) 10 mm HEPES, pH 7.5. Solubilized samples were then fractionated by ultracentrifugation in a 0.1-1 m sucrose gradient containing 0.06% α-DM, 10 mm HEPES, pH 7.5 (22 h at 280,000 × g, 4 °C). Gel Electrophoresis and Immunoblotting—SDS-PAGE analysis was performed with the Tris-Tricine buffer system as previously described (34Schägger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10505) Google Scholar). For immunotitration, thylakoid samples corresponding to 0.5, 1, 2, and 4 μg of chlorophylls were loaded for each sample and electroblotted on nitrocellulose membranes. Filters were incubated with antibodies raised against Lhcb1, Lhcb2, Lhcb3, CP29 (Lhcb4), CP26 (Lhcb5), CP24 (Lhcb6), or CP47 (PsbB) and were detected with alkaline phosphatase-conjugated antibody, according to Ref. 35Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar. Signal amplitude was quantified using the GelPro 3.2 software (Bio-Rad). Electron Microscopy—Intact leaf fragments from wild-type and mutant 3-week-old leaves were fixed, embedded, and observed in thin section as previously described (36Sbarbati A. Merigo F. Benati D. Tizzano M. Bernardi P. Osculati F. Chem. Senses. 2004; 29: 683-692Crossref PubMed Scopus (44) Google Scholar). Spectroscopy—Spectra were obtained using samples in 10 mm HEPES, pH 7.5, 0.06% α-DM, 0.2 m sucrose. Absorption measurements were performed using an Aminco DW-2000 spectrophotometer (SLM Instruments, Rochester, NY) at room temperature. Fluorescence emission spectra were measured at room temperature using a Jobin-Yivon Fluoromax-3 spectrofluorimeter at room temperature. Measure of ΔpH—The kinetics of ΔpH formation across the thylakoid membrane were measured using the method of 9-aminoacridine fluorescence quenching, as previously described (37Evron Y. McCarty R.E. Plant Physiol. 2000; 124: 407-414Crossref PubMed Scopus (26) Google Scholar). Reaction buffer contained 50 mm Tricine, pH 8.0, 50 mm NaCl, 2 μm 9-aminoacridine. The chlorophyll concentration in the reaction buffer was adjusted to 10 μg/ml. Determination of the Sensitivity to Photooxidative Stress—Photooxidative stress was induced in detached leaves by a strong light treatment at low temperature. Detached leaves on wet filter paper were exposed to high light (1000 μmol m−2 s−1 for 5 h) in a growth chamber at low temperature (10 °C) and then immediately frozen in liquid nitrogen. Photoxidative stress was assessed by measuring malondialdehyde (MDA) formation, as indirect quantification of lipid peroxidation. MDA is a reactive, low molecular weight aldehyde derived from radical attack of polyunsaturated fatty acids; in our measurement, leaf MDA levels were stabilized through the formation of a colored, thiobarbituric acid adduct. The MDA-(thiobarbituric acid adduct)2 complex was separated from other thiobarbituric acid adducts and quantified by HPLC as previously described (38Havaux M. Eymery F. Porfirova S. Rey P. Dormann P. Plant Cell. 2005; 17: 3451-3469Crossref PubMed Scopus (404) Google Scholar). Measurements of Singlet Oxygen Production—Measurements of singlet oxygen (1O2) production either from thylakoids and purified pigment-protein complexes were performed with singlet oxygen sensor green (SOSG; Molecular Probes, Eugene). SOSG is a fluorescent probe highly selective for 1O2 that increases its 530 nm emission band in the presence of this ROS (39Flors C. Fryer M.J. Waring J. Reeder B. Bechtold U. Mullineaux P.M. Nonell S. Wilson M.T. Baker N.R. J. Exp. Bot. 2006; 57: 1725-1734Crossref PubMed Scopus (377) Google Scholar). Thylakoids were resuspended in a reaction buffer (0.33 m sorbitol, 10 mm NaCl, 10 mm KCl, 5 mm MgCl2, 10 mm Hepes, pH 7.5, 30 mm ascorbate, 100 μm methyl viologen, 2 μm SOSG) at a final Chl concentration of 20 μg/ml and kept under continuous stirring. Pigment-protein complexes were harvested from sucrose gradient and diluted in a reaction buffer (10 mm Hepes, pH 7.5, 0.06% α-DM, 2 μm SOSG) to the same absorption area in the wavelength range 600-750 nm (around 2.2 μg Chls/ml). Thylakoids and isolated complexes were illuminated with red light (λ > 600 nm) for 5 min; fluorescence yields of SOSG (λex 480 nm, λem 530 nm) were determined before and after light treatment in order to quantify 1O2-dependent fluorescence increase. Pigment Composition—Wild type and single and triple mutants chy1chy2lut5 and chy1chy2lut2, described by Fiore et al. (26Fiore A. Dall'Osto L. Fraser P.D. Bassi R. Giuliano G. FEBS Lett. 2006; 580: 4718-4722Crossref PubMed Scopus (57) Google Scholar), were grown in low light conditions (45 μmol m−2 s−1) for 3 weeks. None of the single or double mutants displayed a visible phenotype, whereas the triple mutants chy1chy2lut2 and chy1chy2lut5 showed, respectively, paler leaves and a highly retarded growth. Since Viola and Neo are precursors of the plant growth regulator abscisic acid (ABA) (40Milborrow B.V. J. Exp. Bot. 2001; 52: 1145-1164Crossref PubMed Google Scholar), the lack of β-β-xanthophylls in chy1chy2lut5 and the concomitant reduction in ABA content could be in principle a cause for some of the chy1chy2lut5 phenotypes. However, chy1chy2lut5 plants sprayed daily with ABA did not show a visible phenotypic reversion (data not shown). This result is in agreement with a recent report showing that ABA deficiency is not responsible for increased photooxidation of thylakoid membranes (20Dall'Osto L. Cazzaniga S. North H. MArion-Poll A. Bassi R. Plant Cell. 2007; 19: 1048-1064Crossref PubMed Scopus (150) Google Scholar). The lut2 and lut5 mutants were included in this characterization, respectively, as lutein-less and increased α-carotene internal controls, whereas mutants npq1 (16Niyogi K.K. Grossman A.R. Björkman O. Plant Cell. 1998; 10: 1121-1134Crossref PubMed Scopus (765) Google Scholar) and npq1lut2 (7Niyogi K.K. Shih C. Chow W.S. Pogson B.J. DellaPenna D. Bjorkman O. Photosynth. Res. 2001; 67: 139-145Crossref PubMed Google Scholar, 41Gilmore A.M. Yamamoto H.Y. Photochem. Photobiol. 2001; 74: 291-302Crossref PubMed Scopus (27) Google Scholar) were used as a reference for increased photosensitivity (7Niyogi K.K. Shih C. Chow W.S. Pogson B.J. DellaPenna D. Bjorkman O. Photosynth. Res. 2001; 67: 139-145Crossref PubMed Google Scholar, 18Havaux M. Niyogi K.K. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8762-8767Crossref PubMed Scopus (568) Google Scholar). The steps affected by the various mutations are described in Fig. 1A, whereas Fig. 1B and Table 1 show the leaf pigment composition of the mutants analyzed (see additional results in supplemental Table a1 for a complete description of leaf pigment composition of all genotypes used in this characterization).TABLE 1Pigment content and photosynthetic parameters Fv/Fm and T⅔ of dark-adapted leaf tissue from wild type and mutant genotypes Parameters were obtained from measurements on the same samples described in the legend to Fig. 1B. The time of the fluorescence rise (T⅔) was measured in 3-(3,4-dichlorophenyl)-1,1-dimethylurea-infiltrated leaves and was taken as a measure of the functional antenna size of photosystem II. Data are expressed as means ± S.D., n = 3. Car, carotenoid; Xant, xanthophyll.Chl a/bChl/CarChl/XantChl/cm2Fv/FmT⅔msWT2.9 ± 0.23.4 ± 0.15.4 ± 0.119.1 ± 0.70.79 ± 0.01338 ± 54lut53.0 ± 0.23.5 ± 0.27.0 ± 0.2aValues significantly different from the wild type (p < 0.05)18.7 ± 1.70.80 ± 0.01463 ± 60aValues significantly different from the wild type (p < 0.05)chy1chy2lut53.9 ± 0.3aValues significantly different from the wild type (p < 0.05)3.6 ± 0.113.5 ± 2.0aValues significantly different from the wild type (p < 0.05)12.6 ± 1.7aValues significantly different from the wild type (p < 0.05)0.68 ± 0.02aValues significantly different from the wild type (p < 0.05)592 ± 87aValues significantly different from the wild type (p < 0.05)lut23.5 ± 0.23.5 ± 0.16.6 ± 0.117.9 ± 0.80.81 ± 0.02416 ± 95chy1chy2lut24.4 ± 0.3bValues significantly different from lut2 (p < 0.05)3.3 ± 0.1bValues significantly different from lut2 (p < 0.05)12.5 ± 0.7bValues significantly different from lut2 (p < 0.05)10.3 ± 0.3bValues significantly different from lut2 (p < 0.05)0.77 ± 0.03bValues significantly different from lut2 (p < 0.05)909 ± 151bValues significantly different from lut2 (p < 0.05)a Values significantly different from the wild type (p < 0.05)b Values significantly different from lut2 (p < 0.05) Open table in a new tab At 45 μmol m−2 s−1, the triple mutants had Fv/Fm ratios (the maximal photochemical yield of PSII) decreased with respect to WT (Table 1); chy1chy2lut5 scored a value of 0.68 versus 0.77 for chy1chy2lut2. In these conditions, lut2npq1, the most light-sensitive xanthophyll mutant so far described (7Niyogi K.K. Shih C. Chow W.S. Pogson B.J. DellaPenna D. Bjorkman O. Photosynth. Res. 2001; 67: 139-145Crossref PubMed Google Scholar), had an Fv/Fm ratio of 0.8, the same as WT (data not shown). These mutants showed a strong increase in chlorophyll a/b ratios as well as a reduced Chl content per leaf area with respect to WT and lut2 (chy1chy2lut2 (45% reduction) and chy1chy2lut5 (35% reduction) (Table 1)) and a severely reduced xanthophyll content with respect to WT and lut2 plants, the main leaf carotenoids being α- and/or β-carotene (Fig. 1B). β-Carotene normally binds photosynthetic reaction centers and Lhca, whereas α-carotene binds the same sites as β-carotene and is found in shade-adapted plants (42Krause G.H. Koroleva O.Y. Dalling J.W. Winter K. Plant Cell Env. 2001; 24: 1345-1352Crossref Scopus (113) Google Scholar, 43Krause G.H. Grube E. Koroleva O.Y. Barth C. Winter K. Funct. Plant Biol. 2004; 31: 743-756Crossref PubMed Scopus (41) Google Scholar). In this case, its accumulation is induced by a knock-out mutation in the LUT5 gene, which encodes a cytochrome P450 carotenoid hydroxylase (25Kim J. DellaPenna D. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3474-3479Crossref PubMed Scopus (198) Google Scholar, 26Fiore A. Dall'Osto L. Fraser P.D. Bassi R. Giuliano G. FEBS Lett. 2006; 580: 4718-4722Crossref PubMed Scopus (57) Google Scholar). Both lut2 and lut5 mutations induced a slight increase in the chlorophyll/xanthophyll ratio, which led to a slight decrease in functional antenna size (Table 1). Chlorophyll/xanthophyll ratio was essentially the same in both triple mutants. Still they show distinct compositions of the xanthophyll fraction; Lute is the only oxygenated carotenoid in chy1chy2lut5, whereas chy1chy2lut2 leaves contain Neo, antheraxanthin, Viola, and Zea in approximately equal amounts. lut2 and lut5 had similar functional antenna size, measured from kinetics of fluorescence rise in 3-(3,4-dichlorophenyl)-1,1-dimethylurea, whereas chy1chy2lut5 antenna size was 57% with respect to WT, and chy1chy2lut2 antenna size was 45% with respect to lut2, consistent with their lower Chl b content (Table 1). Chloroplast Ultrastructure and Pigment-Protein Complex Composition—Carotenoids are important ligand and structural elements of several photosynthetic subunits; thus, changes in thylakoid pigment composition can result in structural modification of photosynthetic membranes as well as in changes of the relative amount of pigment-protein complexes. Electron microscopy analysis was performed to verify if the thylakoid structure was changed as an effect of the different carotenoid composition (Fig. 2A). The mutants showed a membrane organization very similar to that of wild-type chloroplasts. All genotypes formed well defined grana, containing ∼9.1 ± 3.3 (WT, lut5), 8.9 ± 2.5 (chy1chy2lut5), 10.1 ± 3.6 (lut2), 6.9 ± 2.7 (chy1chy2lut2) stacks. Statistical analysis revealed that only the chloroplasts from chy1chy2lut2 plants formed grana with a number of stacks significantly lower with respect to all other genotypes (Student's t test, p < 0.05, n > 15). One striking observation was the difference in the number of osmiophilic globules found; whereas WT and lut2 showed few of these globular structures (5 ± 3 and 4 ± 2 per chloroplast, respectively), the two triple mutants showed a strongly increased incidence of these structures: 14 ± 5 and 18 ± 6, respectively, for chy1chy2lut5 and chy1chy2lut2. Moreover, these structures were also larger in the latter genotypes. Chlorophyll proteins from wild type, lut2, lut5, chy1chy2lut5, and chy1chy2lut2 were fractionated by sucrose gradient ultracentrifugation (Fig. 2B). The amount of LHCII trimers was found to be roughly proportional to the Lute/chlorophyll ratio, in agreement with previous results (44Lokstein H. Tian L. Polle J.E. DellaPenna D. Biochim. Biophys. Acta. 2002; 1553: 309-319Crossref PubMed Scopus (145) Google Scholar). The chy1chy2lut5 pattern was very similar to that of WT, with abundant LHCII trimers and
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