High Light Response of the Thylakoid Proteome in Arabidopsis Wild Type and the Ascorbate-Deficient Mutant vtc2-2. A Comparative Proteomics Study1[W]

The thylakoid proteome of chloroplasts contains multiple proteins involved in antioxidative defense, protein folding, and repair. To understand this functional protein network, we analyzed the quantitative response of the thylakoid-associated proteome of Arabidopsis (Arabidopsis thaliana) wild type and the ascorbate-deficient mutant vtc2-2 after transition to high light (HL; 1,000 μmol photons m−2 s−1). The soluble thylakoid proteomes of wild type and vtc2-2 were compared after 0, 1, 3, and 5 d of HL using two-dimensional gels with three independent experiments, followed by a multivariant statistical analysis and tandem mass spectrometry. After 5 d of HL, both wild-type and vtc2-2 plants accumulated anthocyanins, increased their total ascorbate content, and lost 10% of photosystem II efficiency, but showed no bleaching. Anthocyanin and total ascorbate concentrations in vtc2-2 were respectively 34% and 20% of wild type, potentially leading to enhanced oxidative stress in vtc2-2. Forty-five protein spots significantly changed as a consequence of genotype, light treatment, or both. Independent confirmation was obtained from western blots. The most significant response was the up-regulation of thylakoid YCF37 likely involved in photosystem I assembly, and specific fibrillins, a flavin reductase-like protein, and an aldolase, each located in thylakoid-associated plastoglobules. Fe-superoxide dismutase was down-regulated in vtc2-2, while Cu,Zn-superoxide dismutase was up-regulated. vtc2-2 also showed a systematic up-regulation of a steroid dehydrogenase-like protein. A number of other stress-related proteins, several thylakoid proteases, and lumenal isomerases did not change, while PsbS increased in wild type upon light stress. These findings are discussed in terms of plastid metabolism and oxidative stress defense, and emphasize that understanding of the chloroplast stress-response network must include the enzymatic role of plastoglobules.

[1]  J. Whitelegge,et al.  The Chloroplast Grana Proteome Defined by Intact Mass Measurements from Liquid Chromatography Mass Spectrometry* , 2002, Molecular & Cellular Proteomics.

[2]  S. Prat,et al.  Leaf C40.4: a carotenoid-associated protein involved in the modulation of photosynthetic efficiency? , 1999, The Plant journal : for cell and molecular biology.

[3]  W. Sakamoto,et al.  Coordinated Regulation and Complex Formation of YELLOW VARIEGATED1 and YELLOW VARIEGATED2, Chloroplastic FtsH Metalloproteases Involved in the Repair Cycle of Photosystem II in Arabidopsis Thylakoid Membranes Article, publication date, and citation information can be found at www.plantcell.org/cgi/d , 2003, The Plant Cell Online.

[4]  A. Wilde,et al.  Characterization of the cyanobacterial ycf37: mutation decreases the photosystem I content. , 2001, The Biochemical journal.

[5]  R. Mittler,et al.  Reactive oxygen gene network of plants. , 2004, Trends in plant science.

[6]  G. Ben-Ari,et al.  Expression in Multigene Families. Analysis of Chloroplast and Mitochondrial Proteases1 , 2004, Plant Physiology.

[7]  K. Niyogi,et al.  Zeaxanthin Deficiency Enhances the High Light Sensitivity of an Ascorbate-Deficient Mutant of Arabidopsis1 , 2003, Plant Physiology.

[8]  K. Niyogi,et al.  Ascorbate-Deficient Mutants of Arabidopsis Grow in High Light Despite Chronic Photooxidative Stress1 , 2004, Plant Physiology.

[9]  B. Haas,et al.  Proteome Map of the Chloroplast Lumen of Arabidopsis thaliana * , 2002, The Journal of Biological Chemistry.

[10]  D. Voytas,et al.  Mutations in the Arabidopsis VAR2 locus cause leaf variegation due to the loss of a chloroplast FtsH protease. , 2000, The Plant journal : for cell and molecular biology.

[11]  B. Pogson,et al.  Global Changes in Gene Expression in Response to High Light in Arabidopsis1,212 , 2002, Plant Physiology.

[12]  C. Mullineaux,et al.  A Critical Role for the Var2 FtsH Homologue of Arabidopsis thaliana in the Photosystem II Repair Cycle in Vivo * , 2002, The Journal of Biological Chemistry.

[13]  M. M. Bradford.  A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. , 1976, Analytical biochemistry.

[14]  G. Friso,et al.  In-Depth Analysis of the Thylakoid Membrane Proteome of Arabidopsis thaliana Chloroplasts: New Proteins, New Functions, and a Plastid Proteome Database On-line version contains Web-only data. , 2004, The Plant Cell Online.

[15]  G. Csucs,et al.  Tocopherol Cyclase (VTE1) Localization and Vitamin E Accumulation in Chloroplast Plastoglobule Lipoprotein Particles* , 2006, Journal of Biological Chemistry.

[16]  B. Buchanan,et al.  Redox regulation: a broadening horizon. , 2005, Annual review of plant biology.

[17]  K. V. van Wijk.  Plastid proteomics. , 2004, Plant physiology and biochemistry : PPB.

[18]  K. Niyogi,et al.  Ascorbate Deficiency Can Limit Violaxanthin De-Epoxidase Activity in Vivo1 , 2002, Plant Physiology.

[19]  P. Mullineaux,et al.  Signal transduction in response to excess light: getting out of the chloroplast. , 2002, Current opinion in plant biology.

[20]  D. Inzé,et al.  Genome-Wide Analysis of Hydrogen Peroxide-Regulated Gene Expression in Arabidopsis Reveals a High Light-Induced Transcriptional Cluster Involved in Anthocyanin Biosynthesis1[w] , 2005, Plant Physiology.

[21]  M. Kuntz,et al.  Accumulation of plastid lipid-associated proteins (fibrillin/CDSP34) upon oxidative stress, ageing and biotic stress in Solanaceae and in response to drought in other species. , 2001, Journal of experimental botany.

[22]  K. Niyogi.  Safety valves for photosynthesis. , 2000, Current opinion in plant biology.

[23]  F. Wollman,et al.  A chloroplast-targeted heat shock protein 70 (HSP70) contributes to the photoprotection and repair of photosystem II during and after photoinhibition. , 1999, The Plant cell.

[24]  Karen Schlauch,et al.  Cytosolic Ascorbate Peroxidase 1 Is a Central Component of the Reactive Oxygen Gene Network of Arabidopsisw⃞ , 2005, The Plant Cell Online.

[25]  W. Sakamoto.  Protein degradation machineries in plastids. , 2006, Annual review of plant biology.

[26]  G. Pastori,et al.  Effects of leaf ascorbate content on defense and photosynthesis gene expression in Arabidopsis thaliana. , 2003, Antioxidants & redox signaling.

[27]  H. Scheller,et al.  Photoinhibition of photosystem I at chilling temperature and subsequent recovery in Arabidopsis thaliana. , 2004, Plant & cell physiology.

[28]  R. Last,et al.  Arabidopsis Map-Based Cloning in the Post-Genome Era , 2002, Plant Physiology.

[29]  W. Sakamoto,et al.  The VAR1 locus of Arabidopsis encodes a chloroplastic FtsH and is responsible for leaf variegation in the mutant alleles , 2002, Genes to cells : devoted to molecular & cellular mechanisms.

[30]  R. Herrmann,et al.  Identification and Characterization of SppA, a Novel Light-inducible Chloroplast Protease Complex Associated with Thylakoid Membranes* , 2001, The Journal of Biological Chemistry.

[31]  K. V. van Wijk,et al.  New Functions of the Thylakoid Membrane Proteome of Arabidopsis thaliana Revealed by a Simple, Fast, and Versatile Fractionation Strategy* , 2004, Journal of Biological Chemistry.

[32]  P. Schürmann,et al.  PLANT THIOREDOXIN SYSTEMS REVISITED. , 2000, Annual review of plant physiology and plant molecular biology.

[33]  Peter Roepstorff,et al.  Central Functions of the Lumenal and Peripheral Thylakoid Proteome of Arabidopsis Determined by Experimentation and Genome-Wide Prediction Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010304. , 2002, The Plant Cell Online.

[34]  C. Foyer,et al.  Low ascorbic acid in the vtc-1 mutant of Arabidopsis is associated with decreased growth and intracellular redistribution of the antioxidant system. , 2001, Plant physiology.

[35]  G Peltier,et al.  Over-expression of a pepper plastid lipid-associated protein in tobacco leads to changes in plastid ultrastructure and plant development upon stress. , 2000, The Plant journal : for cell and molecular biology.

[36]  K. Niyogi,et al.  Two P-Type ATPases Are Required for Copper Delivery in Arabidopsis thaliana Chloroplasts , 2005, The Plant Cell Online.

[37]  H. Hirt,et al.  Reactive oxygen species: metabolism, oxidative stress, and signal transduction. , 2004, Annual review of plant biology.

[38]  G Peltier,et al.  Molecular characterization of CDSP 34, a chloroplastic protein induced by water deficit in Solanum tuberosum L. plants, and regulation of CDSP 34 expression by ABA and high illumination. , 1998, The Plant journal : for cell and molecular biology.

[39]  O. Björkman,et al.  Nigericin insensitive post-illumination reduction in fluorescence yield in Dunaliella tertiolecta (chlorophyte) , 1996, Photosynthesis Research.

[40]  G. Pastori,et al.  Leaf Vitamin C Contents Modulate Plant Defense Transcripts and Regulate Genes That Control Development through Hormone Signaling Online version contains Web-only data. Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010538. , 2003, The Plant Cell Online.

[41]  G. Blobel,et al.  Identification of proteins associated with plastoglobules isolated from pea (Pisum sativum L.) chloroplasts , 1999, Planta.

[42]  R. Last,et al.  Identification of ascorbic acid-deficient Arabidopsis thaliana mutants. , 2000, Genetics.

[43]  K. V. van Wijk,et al.  Protein Profiling of Plastoglobules in Chloroplasts and Chromoplasts. A Surprising Site for Differential Accumulation of Metabolic Enzymes1[W] , 2006, Plant Physiology.

[44]  R. Wrolstad,et al.  Characterization and Measurement of Anthocyanins by UV‐Visible Spectroscopy , 2001 .

[45]  A. Wilde,et al.  Analysis of photosynthetic complexes from a cyanobacterial ycf37 mutant. , 2006, Biochimica et biophysica acta.

[46]  H. Scheller,et al.  Photoinhibition of photosystem I , 2005, Planta.

[47]  Graham R Fleming,et al.  Toward an understanding of the mechanism of nonphotochemical quenching in green plants. , 2004, Biochemistry.

[48]  Stefan Jansson,et al.  A pigment-binding protein essential for regulation of photosynthetic light harvesting , 2000, Nature.

[49]  N. Shah,et al.  Temporal evolution of the Arabidopsis oxidative stress response , 2005, Plant Molecular Biology.

[50]  R. Bassi,et al.  Lhc proteins and the regulation of photosynthetic light harvesting function by xanthophylls , 2004, Photosynthesis Research.

[51]  K. Dietz,et al.  Redox regulation: an introduction. , 2004, Physiologia plantarum.

[52]  T. Oniki,et al.  Regulation of Peroxidase-Dependent Oxidation of Phenolics in the Apoplast of Spinach Leaves by Ascorbate , 1992 .

[53]  S. Kikuchi,et al.  Levels of active oxygen species are controlled by ascorbic acid and anthocyanin in Arabidopsis. , 2003, Journal of agricultural and food chemistry.

[54]  G. Friso,et al.  Proteomics of the Chloroplast: Systematic Identification and Targeting Analysis of Lumenal and Peripheral Thylakoid Proteins , 2000, Plant Cell.

[55]  G. Pastori,et al.  Common Components, Networks, and Pathways of Cross-Tolerance to Stress. The Central Role of “Redox” and Abscisic Acid-Mediated Controls1 , 2002, Plant Physiology.

[56]  C. Foyer,et al.  Redox Homeostasis and Antioxidant Signaling: A Metabolic Interface between Stress Perception and Physiological Responses , 2005, The Plant Cell Online.

[57]  C. Schofield,et al.  Structure and mechanism of anthocyanidin synthase from Arabidopsis thaliana. , 2002, Structure.