Arabidopsis thaliana
Dmitry Galetskiy1,2, Jens N. Lohscheider1, Alexey S. Kononikhin2,3, Igor A. Popov2,3, Eugene N. Nikolaev2,3 and Iwona Adamska1
1Department of Physiology and Plant Biochemistry, University of Konstanz, DE-78457 Konstanz, Germany
2Institute of Biochemical Physics and the 3Institute for Energy Problems of Chemical Physics, Russian Academy of Sciences, 119334 Moscow, Russian Federation
Running head: Light stress-related protein turnover
Address correspondence to: Iwona Adamska, Department of Physiology and Plant Biochemistry, University of Konstanz, Universitätsstr. 10, DE-78457 Konstanz, Germany.
Tel: +49 7531 88 2561; Fax: +49 7531 88 3042; E-mail: Iwona.Adamska@uni-konstanz.de
Abbreviations: BN-PAGE, blue-native polyacrylamide gel electrophoresis; DM, N-dodecyl-ß-D-maltoside; PSI and PSII, photosystem I and II, respectively.
SUMMARY
The photosynthetic pigments that capture light energy in higher plants are bound to various proteins of photosystem I (PSI) and II (PSII) and their light-harvesting antenna (LHC) forming photosynthetically active PSI-LHCI and PSII-LHCII complexes. Reactive oxygen and nitrogen species (ROS and RNS, respectively) generated by oxygenic photosynthesis may affect directly and indirectly turnover of photosynthetic complexes, de novo synthesis and proteolytic degradation of the damaged proteins. Protein modifications by nitration as well as by phosphorylation are key indicators for the regulation of redox signalling and quality control mediated by ROS and RNS. Using a label-free mass spectrometric approach we investigated dynamic changes in the distribution of nitrated and phosphorylated proteins within photosynthetic complexes in the thylakoid membrane of Arabidopsis thaliana leaves adapted to low light and subsequently exposed to light stress. Photosynthetic complexes were isolated using blue-native polyacrylamid gel electrophoresis (BN-PAGE) and subjected to tryptic digestion followed by liquid chromatography/tandem mass spectrometry (LC-MS/MS). Nitrated proteins undergo down regulation in PSI-LHCI and PSII-LHCII complexes and their supercomplexes and up regulation in the damaged subcomplexes under increased light illumination, suggesting the role of RNS and protein nitration in inactivation, aggregation and degradation of photosynthetically active complexes. Especially Tyr-262 nitration in the electron acceptor binding pocket of the PSBA reaction center protein leads to in vivo disassembly of PSII-LHCII dimers. Light-dependent regulation of the turnover of nitrated proteins and rearrangement of photosynthetic complexes by reversible phosphorylation is shown here as an interplay between the phosphorylation and nitration levels in PSII-LHCII monomers, dimers and supercomplexes.
Stress-related modifications and turnover of thylakoid proteins
INTRODUCTION
The photosynthetic pigments are bound to various proteins located within photosystem I (PSI) and II (PSII). At least two types of chlorophyll (chl)-binding proteins can be distinguished in higher plants: those that bind only chl a and those that bind both, chl a and b. The plastid-encoded chl a-binding proteins are represented by PSI and PSII reaction centre heterodimers, PSAA/PSAB and PSBA/PSBD (also called D1/D2 proteins), respectively, and the PSII core antenna proteins PSBB and PSBC (also called CP47 and CP43, respectively). The nuclear-encoded chl a/b-binding proteins (CAB) form light-harvesting antenna complexes around PSI (LHCI) and PSII (LHCII) reaction centres. Four major LHCA1-4 proteins build the LHCI complex in A. thaliana (Jansson, 1999). Two other proteins, LHCA5 and LHCA6, are additional PSI antenna subunits based on sequence similarity (Ganeteg et al., 2004; Klimmek et al., 2006). The crystal structure of pea PSI-LHCI complex revealed that LHCI consists of two separate dimers (LHCA1/LHCA4 and LHCA2 /LHCA3) that are arranged in a half-moon-shaped belt bound to one side of PSI (Ben-Shem, Frolow and Nelson, 2003). Six LHCII proteins are organized in monomeric/trimeric major (LHCB1-3 and their isoforms) and monomeric minor (LHCB4-6 and their isoforms, also called CP29, CP26 and CP24, respectively) antenna systems (Jansson, 1999; Jansson, 2006). The major antenna is either tightly associated with the PSII core complex or forms a loosely bound mobile pool located peripheral to the PSII core (Jansson, 2006). This pool can reversibly dissociate from PSII and associate with PSI during state transition (Allen, 2003; Rochaix, 2007; Kargul and Barber, 2008). Recently, two rarely expressed LHCB7 and LHCB8 were also reported (Klimmek et al., 2006).
High light (HL) intensity is a major stress factor encountered by oxygenic phototrophs, which often leads to over-excitation of the photosynthetic apparatus and production of reactive oxygen species (ROS), such as singlet oxygen, superoxide, hydrogen peroxide, and hydroxyl radicals (Nield, Redding and Hippler, 2004; Asada, 2006; Popísil, 2009). It was shown that HL illumination leads to accelerated turnover of proteins in photosynthetic complexes, e.g. a rapid degradation of photodamaged proteins followed by their replacement by newly-synthesised functional copies (Aro et al., 2005). The D1 and D2 proteins of the PSII reaction centre are the most susceptible to irreversible damage by generated ROS, because they bind all cofactors involved in primary and secondary electron flow (Yamamoto, 2001;
Yamamoto et al., 2008).
Adaptation of the photosynthetic apparatus to excess light might take place at morphological, anatomical and subcellular levels and occurs on a time scale of seconds to hours (short-term adaptation) or days and seasons (long-term adaptation). Phosphorylation of threonine residues in LHCBII and PSII proteins is dynamically regulated according to light intensity and is proposed to be connected to light acclimation (Tikkanen et al., 2010).
According to the state transition theory, the N-terminal threonine residues of the most abundant PSII antenna polypeptides LHCB1 and LHCB2 become phosphorylated by thylakoid-bound STN7 and TAK kinases (Kargul and Barber, 2008; Tikkanen et al., 2006), the phosphorylated LHCB1 and LHCB2 trimers dissociate from PSII and bind to the subunit H of PSI in order to maintain an optimal excitation balance between both photosystems Chuartzman et al., 2008). Other reports suggest a more subtle mechanism for the regulation of energy distribution between PSII and PSI by LHCII and PSII core phosphorylation regulated by STN7 and STN8 kinases (Tikkanen et al., 2010; Tikkanen et al., 2008a). The PSII core protein phosphorylation was also shown to control PSII turnover (Tikkanen et al., 2008b).
Posttranslational modification of proteins by nitration is an important biomarker for pathological states (Beal, 2002; Dalle-Donne et al., 2005; Møller, Jensen and Hansson, 2007;
Wormuth et al., 2007). An increase of protein tyrosine nitration has been reported to be an early defense mechanism of plants during biotic stress (Chaki et al., 2009a,b; Corpas et al., 2009). The protein tyrosine nitration can modify the conformation and structure of proteins, the catalytic activity of enzymes and its susceptibility to proteolysis, and therefore nitrotyrosine is a marker of nitrosative stress in plants under biotic conditions (Corpas, del Río and Barroso, 2007; Corpas et al., 2009). The protein tyrosine nitration is suggested to be mediated by peroxynitrite (Ischiropoulos, 2003; Radi, 2004), which is produced by a rapid reaction between superoxide radicals and nitric oxide (Kissner et al., 1997). It was proposed that peroxynitrite formed in chloroplasts (Radi, 2009) inhibits oxygen evolution within PSII membranes under continuous illumination acting at the electron acceptor site (Gonzáles-Pérez et al., 2008).
Recently, large scale mass spectrometry-based studies of the chloroplast proteome have not only shown a large number of identified proteins, but also localisation data, protein accumulation levels and post-translational modifications (Sun, Emanuelsson and van Wijk, 2004; Zybailov et al., 2008; Plöscher et al., 2009). Protein phosphorylation is one of the most studied modifications in chloroplasts, and especially in their photosynthetic membranes (Aro, Rokka and Vener, 2004; Sugiyama et al., 2008; Reiland et al., 2009). By contrast, information about protein nitration in plants is rare. There are several studies showing protein
Stress-related modifications and turnover of thylakoid proteins nitration under certain stress conditions but almost no information exists about the identity of nitrated proteins in plants.
Here we combine separation of native pigment-protein complexes from A. thaliana thylakoid membranes using blue-native polyacrylamide gel electrophoresis (BN-PAGE, (Wittig, Braun and Schägger, 2006; Reisinger and Eichacker, 2007, 2008) with high-resolution liquid chromatography/mass spectrometry (LC-MS/MS) for determination of HL-induced changes in phosphorylation and nitration levels in photosynthetic complexes. The method described here does not contain any enrichment, precipitation or chemical modification steps, and therefore is especially suitable for analysis of protein modifications at the physiologically relevant level. Determination of modified proteins based on highly accurate ion chromatograms of corresponding tryptic peptides is shown here to be an efficient tool for discovery of light stress-related protein turnover.