CONCLUDING REMARKS
Most life on earth depends on the efficient activity of oxygenic photosynthesis. Thereby, the sustainable solar energy is used for the production of biomass and generation of molecular oxygen with simultaneous consumption of atmospheric CO2 and water. Although this description sounds rather simple, photosynthesis is a tightly regulated process involving numerous multi-protein complexes as well as multiple enzymatic reactions, most of which are influenced by environmental conditions (Kruse, 2001; Gutteridge and Jordan, 2001). While light can generally be regarded beneficial for plants because it is the primary energy source driving photosynthesis, an excess of irradiation is harmful leading to the generation of ROS and damage to cellular components, primarily to pigment-binding proteins involved in photosynthesis (Kruse, 2001; Perelman et al., 2003; Huesgen, Schuhmann and Adamska, 2005; Li et al., 2009).
In this thesis, the impacts of fluctuations in light intensity as well as iron limitation on photosynthesis were investigated on a molecular level in both cyanobacteria and higher plants. It was shown that iron limitation of the marine filamentous cyanobacterium Trichodesmium erythraeum caused a down-regulation of nitrogen fixation while the photosynthetic activity was maintained (Chapter 1). Iron is one of the nutrients in oceanic habitats limiting growth of phytoplankton because its concentration is low as compared to terrestrial environments (Sunda and Huntsman, 1995; Granger and Price, 1999). Moreover, molecular nitrogen as another important nutrient has to be chemically reduced to ammonia to be biologically fixed. Cyanobacteria like Trichodesmium overcome this problem by fixing atmospheric nitrogen (Berman-Frank, Lundgren and Falkowski, 2003). However, the essential enzyme nitrogenase is highly prone to oxidation, a fact which complicates nitrogen fixation in photosynthetic organisms. Moreover, nitrogenase needs iron as a cofactor like many proteins involved in photosynthesis (Fromme, Jordan and Krauss, 2001; Ben-Shem, Frolow and Nelson, 2003; Fukuyama, 2004; Baniulis et al., 2008; Küpper et al., 2008).
Unlike other cyanobacteria, which separate nitrogen fixation from photosynthetic reactions in specialised cells, the so-called heterocysts (Adams, 2000), Trichodesmium performs a temporal separation of nitrogen fixation from oxygen evolution (Kana, 1993; Carpenter and Roenneberg, 1995; Chen et al. 1999; Berman-Frank et al., 2001a; Küpper et al., 2004). It was found that nitrogenase activity as well as growth rates were reduced under iron limiting conditions while photosynthesis was maintained. This might represent a short-term adaptation of Trichodesmium in order to survive periods with reduced availability of this metal.
Unlike iron limitation, changes in light intensity have a more direct and also more contemporary impact on photosynthetic organisms. After HL exposure, several acclimation processes take place on various physiological levels. Apart from morphological adaptations (Niklas, 2009), the repair of the damaged D1 protein of the PSII reaction centre (Huesgen, Schuhmann and Adamska, 2005 and 2009), the detoxification of ROS via enzymatic radical scavenging systems (Asada, 2000; Herbert et al., 1992), the accumulation of photoprotective pigments is very important. Carotenoids have been shown to actively reduce light stress by dissipation of excess energy as heat (Niyogi, 2000; Demmig-Adams and Adams, 2002) and direct detoxification of ROS (Tracewell et al., 2001, Wang and Hu, 2002), which are generated upon illumination with HL intensities. Higher plants and algae use the so-called xanthophyll cyclel to convert the LHC-bound carotenoid violaxanthin (diatoxanthin in brown algae) to zeaxanthin (diadinoxanthin in brown algae) by thylakoid-associated enzymes in a pH-dependent manner (Niyogi et al., 1998; Holt et al., 2005). Cyanobacteria do not possess a xanthophyll cycle but they were shown to be able to synthesise zeaxanthin and other protective carotenoids in response to light stress (Schagerl and Müller, 2006; Steiger, Schäfer and Sandmann, 2009). In this thesis, carotenoid accumulation was investigated in different cyanobacteria after exposure to HL stress (Chapters 2 and 3, respectively). It was demonstrated that carotenoid compositions and concentrations influence the vertical distribution of various benthic Synechococcus isolates that also varied in their phycobili protein composition (Chapter 3). PC-rich strains isolated close to the water surface with generally higher concentrations of carotenoids were more light stress-resistant than PE-rich strains isolated from deeper water. This reflects the protective role of e.g. zeaxanthin, keto-carotenoids and myxoxanthophyll in cyanobacteria (Schäfer, Vioque and Sandmann, 2004).
In contrast, carotenoid accumulation does not seem to be a central part of the acclimation mechanisms in the marine cyanobacterium Trichodesmium (Chapter 2). After HL treatment, Trichodesmium cells showed increased growth rates as well as higher rates for nitrogen fixation. At the same time, the overall concentrations of different carotenoid classes were strongly reduced. This might reflect a preference and/or preadaptation of this organism to increased light intensities. These data demonstrate that acclimation to changes in the environment always involve multiple responses that differ between organisms.
Another photoprotective acclimation mechanism investigated in this thesis is the rearrangement of the antenna system in cyanobacteria and higher plants in response to HL illumination. The so-called state transition operates in cyanobacteria and plants in order to balance the amount of harvested light between PSII and PSI (Mullineaux, Tobin and Jones,
Concluding remarks 1997; Kruse, 2001). In higher plants, mobile parts of LHCII become phosphorylated by membrane-associated kinases leading to unstacking of the grana thylakoids and release of photodamaged PSII (Kruse, 2001). While the PSII reaction centre is repaired, phosphorylated LHCII trimers migrate to PSI and contribute to light harvesting. It was shown in the course of this study that phosphorylation as well as nitration signals are involved in this process in the plant model organism A. thaliana (Chapter 4). Apart from the known phosphorylation sites in LHCII proteins (Vener et al., 2001; Vener, 2007), it was possible to detect novel phosphorylation sites, whose levels were influenced by light intensity. These phosphorylation sites might be involved in signalling of antenna rearrangement during light stress treatment.
Moreover, nitrations were detected and could be connected to the PSII repair cycle in which the damaged D1 protein of the PSII reaction centre is cleaved and replaced by a newly-synthesised functional copy (Andersson and Aro, 2001). Antenna rearrangement was also observed in Trichodesmium cells growing under iron limited conditions (Chapter 1), where also the iron stress-induced protein A (IsiA) accumulated in response to iron starvation. IsiA was shown to be involved both in light harvesting and non-photochemical energy dissipation (Ihalainen et al., 2005) as well as reorganisation of PSI (Kouril et al., 2005). These findings underline the importance of antenna reorganisation in response to various abiotic stress conditions.
Fig. 3 Schematic representation of dynamic changes of PSI and PSII and their LHC. In low light (LL), SEPs and OHPs are tightly associated with reaction centres. After high light (HL) exposure, however, LHCII and the PSII reaction centre become phosphorylated and mobile parts of the major LHCII migrate to PSI in order to balance light harvesting between both photosystems. Light stress leads to ELIP induction and an enhancement of SEP and OHP expression. Here, ELIP family members might be involved in non-photochemical energy dissipation, pigment biosynthesis/ligation and/or reorganisation of the LHC both in PSI and PSII.
Concluding remarks Induction of ELIPs from the expanded LHC superfamily represents another important protective mechanism in photosynthetic organisms. Members of the ELIP family were discovered in all organisms performing oxygenic photosynthesis investigated so far and were reported to be related to various stress conditions (Adamska, 2001; Heddad and Adamska, 2002). ELIPs were first discovered during greening of etiolated barley and pea seedlings where they preceded the accumulation of proteins related to photosynthesis (Meyer and Kloppstech, 1984; Grimm and Kloppstech, 1987). Later, they were also found to be induced in response to light stress (Adamska and Kloppstech, 1991; Pötter and Kloppstech, 1993).
Since then it was shown that ELIPs in pea bind chl a as well as carotenoids (Adamska, Kruse and Kloppstech, 2001; Adamska et al., 1999) and are located within the LHC of PSII (Heddad et al., 2006) or PSI (Andersson, Heddad and Adamska, 2003). Due to these features, a role in photoprotection by heat dissipation of excess energy was proposed (Hutin et al., 2003). Thus, ELIPs can be regarded a connection between accumulation of protective carotenoids and antenna rearrangement. The main aim of this thesis was to investigate localisation of known and novel ELIP family members in the higher plant model organism A. thaliana. It could be demonstrated that ELIP1 and SEP2 are localised within different subpopulation of LHCII while OHP2 is associated with PSI under light stress conditions (Chapter 7) confirming and expanding previous results (Heddad et al., 2006). Additionally, localisation of two novel SEP3 paralogues was studied under ambient light conditions, also revealing association with LHCII (Chapters 5 and 6). These findings reflect the observed general localisation pattern of ELIP family members: ELIPs and SEPs associated with PSII (Heddad et al., 2006) and OHPs located within PSI (Andersson, Heddad and Adamska, 2003). Although the localisation of SEP1, SEP4 and SEP5 have not been investigated, preliminary localisation studies using sucrose density gradient centrifugation indicated association of these proteins with PSII under ambient light conditions (not shown). A schematic representation of the results is shown in Fig. 3, trying to demonstrate the dynamic changes during the acclimation of both photosystems and their LHC.
It was shown with elip1/elip2 double knock-out mutants in A. thaliana that a loss of both proteins did not result in a significant impairment of photoprotection (Rossini et al., 2006). Therefore, it was further hypothesised that their function might be related to pigment biosynthesis (Tzvetkova-Chevolleau et al., 2007). The latter function is likely for SEPs and OHPs because of their expression under ambient light conditions (Adamska, 2001; Chapters 5 and 6) and the increased need for newly-synthesised pigments under light stress conditions (Heddad and Adamska, 2000; Adamska, 2001). However, both SEPs and OHPs, contain only
one membrane-spanning α-helix with the conserved chl-binding motif (Adamska, 2001) of which two are needed to coordinate chl molecules (Green and Kühlbrand, 1995). Therefore, they might act as homo- or heterodimers or in association with not yet identified interaction partners. The phenotypes of mutant plants with a T-DNA insertion in one of the two OHP genes of A. thaliana imply dimerisation of OHP1 and OHP2 because a lack of one protein also resulted in a loss of the second (Rojas-Stütz, 2008). In contrast, it was suggested for highly similar SEP3 paralogues from A. thaliana localised in PSII that they rather have redundant function(s) (Chapter 6). Although this suggestion does not exclude interaction and/or cooperation of the SEP3 proteins, it indicates a higher degree of functional independence of each paralogue as compared to OHPs.
In this thesis, selected short-term responses to light stress and iron limitation were studied in higher plants and cyanobacteria. It can be summarised that the general mechanisms of stress protection are similar in all investigated organisms. However, individual differences could be described with an impact on ecological niche occupation. The dynamic changes in LHC and PBS composition and function analysed here seem to be an important factor during abiotic and biotic stress responses. In future experiments, localisation and function of ELIP family members will be investigated in more detail by physiological analysis of transgenic mutant lines that have already been identified for several SEPs in A. thaliana. Moreover, the pigment-binding capacity of ELIP family members will be tested and the search for interaction partners will be initiated. These measures might then help to further elucidate the complex mechanisms involved in acclimation of photosynthetic organisms to stress conditions.
Acknowledgements
ACKNOWLEDGEMENTS
I would like to thank
• Iwona Adamska for encouragement, enabling conference participations, freedom of work and support during the whole time I could stay in her lab. Thank you for your patience, your confidence and for just being Iwona!
• Prof. Martin Scheffner for being the second referee/examiner and Prof. Ralf Oelmüller for being third examiner
• the many co-workers, who helped to accomplish this thesis, especially Martina Strittmatter for sharing ups and downs, Hendrik Küpper for support, helpful discussions, productivity and delicious Czech bear and Dmitry Galetskyi for persistence
• the ELIP people, especially Marc Rojas-Stütz and Ulrica Andersson for teamwork and discussions and Verena Reiser for being a nice flatmate
• Dr. Dietmar Funck for general and special help with nasty nucleic acids, for Arabidopsis advice, critical questioning and partly revision of the thesis
• all past and present members of the Adamska group for the pleasant working atmosphere and numerous delicious cakes and barbeques. I want to specially thank Gudrun Müller, Ulrike Mogg and Karlo Gasparic for thorough revision of this thesis and help with layout and Regina Grimm for lab management and sarcastic comments
• the gardeners in the greenhouse, especially Claudia Martin for taking care of my plants
• all former and present students directly or indirectly involved in this thesis, especially Clemens Jäger for his great effort in establishing methods and demonstrating stamina, as well as Anne Kummer, Barbara Weber and Michael Bruderek for gathering all the little details and for their frustration tolerance
• all friends, who listened to problems and supported me, especially Sonja Erath
I am deeply grateful to my family, especially my parents Anne and Eberhard Lohscheider for enabling my course of studies and for their confidence and to my grandmother Berta Lohscheider for extra financial support and interest in my work.
I want to thank Marion Schneider for love, support and many beautiful moments throughout the major period of this thesis.