Verena Reiser1, Dmitry Galetskiy2, Iuliana Susnea2, Jens N. Lohscheider1, Roswitha Miller-Sulger1, Michael Przybylski2 and Iwona Adamska1*
1 Department of Biology, Physiology and Plant Biochemistry, University of Konstanz, DE-78457 Konstanz, Germany
2 Department of Chemistry, Analytical Chemistry and Biopolymer Structure Analysis, University of Konstanz, DE-78457 Konstanz, Germany.
Corresponding author: Iwona Adamska, Department of Biology, University of Konstanz, Universitätsstraße 10, D-78457 Konstanz, Germany
Phone: +49 7531 88 2561, Fax: +49 7531 88 3042; E.mail: iwona.adamska@uni-konstanz.de
Abbreviations: Chlorophyll a/b-binding protein, CAB; Chlorophyll, chl; Early light-induced protein, ELIP; Light-harvesting complex, LHC; Mass spectrometry, MS; One-helix protein, OHP; Photosystem I and photosystem II, PSI and PSII, respectively; Stress-enhanced protein, SEP.
SUMMARY
The reaction centres of photosystem I (PSI) and photosystem II (PSII) in higher plants and algae are surrounded by light-harvesting complexes (LHCI and LHCII, respectively). Under light stress conditions, the LHC undergo dynamic changes including a migration of a loosely associated LHCII pool between PSI and PSII due to a process called state transition as well as accumulation of proteins from early light-induced protein (ELIP) family. This family consists of three-helix ELIPs, two-helix stress-enhanced proteins (SEPs) and one-helix proteins (OHPs). It was shown that ELIP1 and ELIP2 are associated with LHCII, while the related one-helix protein 2 (OHP2) is localised within LHCI in Arabidopsis thaliana. Nothing is known about the localisation of SEPs. In this study, localisation in subpopulations of LHCII and LHCI were investigated in high light-stressed plants using sucrose density gradient centrifugation in combination with gel filtration. Fractions with maximal abundance of ELIP/SEP or OHP were analysed by 2 dimensional gel electrophoresis and mass spectrometry. It was found that ELIP1 and SEP2 accumulate in different subpopulation of LHCII, while OHP2 is localised within the LHCI antenna. Co-localisation with fibrillins might indicate a role of ELIPs in the turnover of pigments during light stress.
Localisation of ELIPs in the thylakoid membrane
INTRODUCTION
The antenna of photosystem II (PSII) is composed of the core pigment-protein complexes, CP47 and CP43, and the light-harvesting complex II (LHCII) (Green and Durnford, 1996).
The latter is divided into minor monomeric antenna composed of LHCB4 (CP29), LHCB5 (CP26) and LHCB6 (CP24) and the major antenna composed of LHCB1-3. The major antenna is either tightly associated with the PSII core complex or forms an outer pool at more distant locations relative to PSII that is loosely bound (Bassi and Dainese, 1992; Drepper et al., 1993; Vink et al., 2004; Consoli et al., 2005). This pool can reversibly dissociate/associate with PSII during the process of state transition (Allen and Forsberg, 2001;
Haldrup et al., 2001; Wollmann, 2001). Recent crystallisation of spinach (Spinacia oleracea) LHCII revealed that the basic structural and functional unit of the major antenna is the trimer (Liu et al., 2004). These trimers are composed of mixtures of LHCB1-3 proteins and bind approximately 60% of the PSII chlorophyll (chl) (Peter and Thornber, 1991). Each PSII reaction centre is associated with two to fourtrimers depending on plant material and light conditions. TheLHCII antenna is associated with a dimeric reaction centre core(C2) to give rise to a highly conserved structural unit, theC2S2 LHCII-PSII supercomplex, consisting of two copies each of CP26, CP29, and two LHCII trimers (Dekker and Boekema, 2005).
Further antenna complexes associate with this supercomplex to give rise to C2S2M2
supercomplexes,which contain in addition two copies of CP24 and two additional LHCII trimers. In the grana membranes, supercomplexes form various megacomplexes, sometimes in higher-order semi crystalline arrays (Boekema et al., 2000).
Past reports revealed that 20 different chl a/b-binding (CAB) proteins are encoded by the genome of Arabidopsis thaliana (Jansson, 1999). Based on the three-dimensional (3D) structure determined at 3.4 Å (Kühlbrandt, Wang and Fujiyoshi, 1994) and 2.72 Å (Liu et al., 2004) resolution for one CAB member from higher plants, it was proposed that all CAB proteins in higher plants and green algae have three transmembrane α-helices, where helices I and III are evolutionarily related to each other and kept together by ion pairs formed by charged residues.
Several distant relatives of the CAB proteins with conserved chl-binding residues and a transient expression pattern related to various stress conditions have been described from higher plants, algae and cyanobacteria (Adamska, 2001). These distant relatives include: a four-helix PSBS protein of PSII (Funk, 2001) and a family of proteins called early light-induced proteins (ELIPs) (Montané and Kloppstech, 2000; Adamska, 2001). The ELIP family
consists of three-helix ELIPs (Grimm, Kruse and Kloppstech, 1989), two-helix stress-enhanced proteins (SEPs) (Heddad and Adamska, 2000) and one-helix proteins (OHPs) (Jansson et al., 2000; Andersson, Heddad and Adamska, 2003), called high light-induced proteins (Hlips) (Dolganov, Bhaya and Grossman, 1995) or small chl a/b-binding-like proteins (Scps) (Funk and Vermaas, 1999) in prokaryotic organisms. While transcription of ELIP genes and protein accumulation is only induced under light stress conditions, SEPs and OHPs are present also in the absence of light stress but their transcript and protein levels increased during illumination with high intensity light (Heddad and Adamska, 2000;
Andersson, Heddad and Adamska, 2003). The accumulation of ELIPs increased almost linearly with increasing light intensity (Adamska, Scheel and Kloppstech, 1991; Pötter and Kloppstech, 1993) and correlated with the degree of photoinactivation and photodamage of PSII reaction centres (Heddad et al., 2006). It was reported that other stress conditions trigger a transient induction of these proteins in various plant species (Adamska, 2001).
It was demonstrated that ELIP1 and ELIP2 in A. thaliana are located within the LHCII (Heddad et al., 2006) while OHP2 was found in PSI (Andersson, Heddad and Adamska, 2003). Recently, it was reported that Hlips in cyanobacteria are associated with the monomeric photodamaged PSII (Yao et al., 2007).
Isolation of ELIP from light-stressed pea leaves and analysis of pigments confirmed the theoretical expectation that this protein binds chl. Chl a and also lutein were detected in the purified ELIP fraction (Adamska et al., 1999). However, very unusual pigment composition and pigment-binding characteristics were reported for isolated ELIPs, such as a weak excitonic coupling between chl a molecules and an extremely high lutein content as compared with other CAB proteins (Adamska et al., 1999). Based on these features it was proposed that ELIPs are not involved in light harvesting but play a protective role within the thylakoids under light stress conditions either by transiently binding free chl molecules and preventing the formation of free radicals and/or by acting as sinks for excitation energy (Montané and Kloppstech, 2000; Adamska, 2001). A similar photoprotective function was demonstrated for the PSBS protein (Li et al., 2000).
Here, we investigated the localisation of different ELIP family members within the thylakoid membrane. We could confirm that OHP2 is located in PSI. SEP2 and ELIP1 were found to be located in different subcomplexes of LHCII. SEP2 was mainly associated with LHCB proteins from the outer trimeric antenna, whereas ELIP1 was found associated to the inner monomeric LHCII. In all of the ELIP subcomplexes we also found another stress-induced protein group, fibrillins.
Localisation of ELIPs in the thylakoid membrane