Nitrogen cycle: intermediate steps NO 3 NO 2 NH 3

Transcription

1 Regulation of Photosynthesis for Nitrogen Fixation Hendrik Küpper, visit to Aberdeen, February 2014

2 Nitrogen cycle: intermediate steps NO 3 - Nitrite oxidase Nitrate reductase NO 2 - NO H 2 NOH Anammox N 2 O N 2 NH 3

3 Total equation of biological nitrogen fixation N +8H + +16MgATP+8e NH 3 + H MgADP + 16 P i

4 Organisms involved in nitrogen fixation Anabaena Trichodesmium Gloeothece Azotobacter Azolla Rhizobien

5 Mechanism of biological nitrogen fixation: nitrogenase of cyanobacteria

6 Evolution of biological nitrogen fixation in comparison to photosynthesis Berman-Frank I_Lundgren P_Falkowski P_2003_Research in Microbiology154_

7 Strategies of photosynthesis regulation for nitrogen fixation in cyanobacteria Berman-Frank I_Lundgren P_Falkowski P_2003_Research in Microbiology154_

8 Unicellular cyanobacteria

9 Regulation of photosynthesis for nitrogen fixation in unicellular cyanobacteria (II) glycogen storage begins max. photosynthetic capacity light period: carbon + energy storage down regulation of photosynthesis up regulation of photosynthesis maximum glycogen storage nitrogen fixation begins dark period: nitrogen fixation maximum nitrogen fixation maximum respiration

10 Heterocyst forming cyanobacteria vegetative Zellen Pro-Heterocyste Heterocyste From: Culture service of Instituto de Bioquímica Vegetal y Fotosíntesis, Sevilla, Spain From:El-Shehawy et al 2003 Physiol Plant 119 (1), 49-55

11 Heterocyst differentiation: distribution maps of chlorophyll fluorescence kinetic parameters Maximal fluorescence quantum yield (F m ) 25 µm 13 h 36 h 55 h 112 h Photosystem II activity (F v /F m ) Ferimazova N, Felcmanová K, Šetlíková E, Küpper H, Maldener I, Hauska G, Šedivá B, Prášil O (2013) PhotRes 116, 79-91

12 Heterocyst differentiation: changes in parameters of chlorophyll fluorescence kinetics Ferimazova N, Felcmanová K, Šetlíková E, Küpper H, Maldener I, Hauska G, Šedivá B, Prášil O (2013) PhotRes 116, 79-91

13 Trichodesmium: anoxygenic photosynthesis energizing nitrogen fixation in the same cells during the photoperiod

14 Trichodesmium Marine filamentous, non-heterocystous cyanobacteria contribution of Trichodesmium to marine N 2 fixation: 30-50% Nitrogen fixation is confined to the photoperiod and occurs simultaneously with oxygenic photosynthesis. How nitrogenase is protected from damage by photosynthetically produced O 2 has remained an enigma. Surface blooms in the Arafura Sea Colonies tuft and puff formation

15 ) Ti Trichodesmium- i bloom Trichodesmium Relative e F v/ F m Aktivitätszyklus von Trichodesmium Nitrogen fixation (n nmol C2H2 µg chl a -1 h -1 colonies: Tuft and Puff GP (µm O2 µg chl a -1 h -1 ) Dark respiration (µm µg chl a -1 h -1 O2 ) Local time (h) Berman-Frank I, Lundgren P, Chen Yi-B, Küpper H, Kolber Z, Bergman B, Falkowski P (2001) Science 294,

16 Co-Localisation of nitrogenase and PSII in Trichodesmium D1 protein (green) and Nitrogenase (rot). Big picture: Overlay, small picture: only nitrogenase (Immunostain) Berman-Frank I, Lundgren P, Chen Yi-B, Küpper H, Kolber Z, Bergman B, Falkowski P (2001) Science 294,

17 Inhibitor-Tests: Need of PSII-activity for nitrogen fixation in Trichodesmium Influence of DCMU (10 µm), ascorbic acid (100 µm), and DTT (100 µm) were tested for cultures incubated under aerobic (white columns) and anaerobic (blue columns) conditions. Changes in nitrogenase activity as measured by acetylene reduction. Berman-Frank I, Lundgren P, Chen Yi-B, Küpper H, Kolber Z, Bergman B, Falkowski P (2001) Science 294,

18 Proof of Mehler-reaction during nitrogen fixation Staining with DAB (Diaminobenzochinon) shows intracellular distribution ib ti of H 2 O 2 as brown stain in all cells Mehler-reaction: reaction: H 2 O+2O 2 --> >HO 2 2 +O 2 Berman-Frank I, Lundgren P, Chen Yi-B, Küpper H, Kolber Z, Bergman B, Falkowski P (2001) Science 294,

19 ) Diurnal cycle of activity: Distribution of F 0 values ntical cells) lls or group ps of neighb bouring ide Number of objects (ce low F 0 normal bi bright "very bright" type I = bright type II Non-diazotrophic period Diazotrophic period F 0 Küpper H, Ferimazova F, Setlík I, Berman-Frank I (2004) Plant Physiology 135,

20 Diurnal cycle of activity: correlation between bright cells, pigment content and nitrogenase activity e activity reduction) ) -1.min -1 ) Nitrogenase ml ethylene. (ml culture) N ((m % Brigh ht cells Chl conce entration (µm M) Nitrogenase activity % bright cells Chl : : :00 15:00 18:00 21:00 00:00 control 5% oxygen 50% oxygen Küpper H, Ferimazova F, Setlík I, Berman-Frank I (2004) Plant Physiology 135,

21 Küpper H, Ferimazova F, Setlík I, Berman-Frank I (2004) Plant Physiology 135,

22 Hypothesis about the regulation of photosynthesis for nitrogen fixation in Trichodesmium Licht initiates photosynthesis Reduction of the PQ pool Induction of a state 2 --> state 1-transition cells have a high F 0 ( bright cells type I ) due to surplus phycobiliproteins Energy and reduction equivalent for CO 2 - assimilation Stimulation of pseudocyclic electron transport High respiration rates in early light period Oxygen consumption exceeds oxygen production: Opportunity for nitrogen fixation Carbohydrates are consumed Respiration decreases, transition to the normal F 0 inactive or quenching state Intracellular oxygen rises Nitrogenase becomes inhibited, no further nitrogen fixation until the following day, cells return to "normal F 0 active" state

23 Deconvolution of spectrally resolved in vivo fluorescence kinetics shows reversible coupling of individual phycobiliproteins Basic dark-adapted fluorescence yield F 0 Non-photochemical changes in fluorescence yield quenching normal F 0 active bright I =diazotrophic Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787,

24 Sequence of uncoupling events in a bright g II cell Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787,

25 Reversible coupling of individual phycobiliproteins......as a basis for diazotrophic photosynthesis Küpper H, Andresen E, Wiegert S, Šimek M, Leitenmaier B, Šetlík I (2009) Biochim. Biophys. Acta (Bioenergetics) 1787,

26 Iron limitation: rescue of photosynthetic components......by sacrificing nitrogenase PsaC new PE isoform Küpper H, Šetlík I, Seibert S, Prášil O, Šetlikova E, Strittmatter M, Levitan O, Lohscheider J, Adamska I, Berman-Frank I (2008) New Phytologist 179,

27 Conclusions For Trichodesmium, photosystem II activity is essential for providing energy for nitrogen fixation. Regulation of PSII activity for nitrogen fixation is achieved mainly by quickly reversible (un)coupling of individual phycobiliproteins. The nitrogen fixing activity state is characterised by a particularly large PSIIassociated antenna, which is achieved mainly by coupling of additional units of phycourobilin (PUB) isoforms. Therefore, acclimation to light limitation ( low light stress ) involves enhanced synthesis mainly of phycourobilin, which h is then mainly coupled to PSII. Synthesis and levels of other photosynthetic components decrease. Because of their vital importance, when adverse conditions require a choice, Trichodesmium in contrast to other cyanobacteria does not sacrifice its phycobilisomes, but rather its nitrogenase. Stress leads to expression of alternative phycobiliprotein isoforms

28 Current and former lab members involved in this project Elisa Andresen, Barbara Leitenmaier, Qiyan Wang-Müller, Sven Seibert, Verena Rösch, Christian Lang Collaborators Iwona Adamska, Jens Naila Ferimazova, Ivan Šetlík, Eva Lohscheider, Martina Šetlíkova, Ondrej Prašil: Photosynthesis Strittmatter: Universität Research Centre, Institute of Konstanz, Germany Microbiology, Třeboň, Czech Republic Ilana Berman-Frank: Bar Ilan University, Israel Pernilla Lundgren, Birgitta Yi-Bu Chen, Zbigniew Kolber, Paul Bergman: Stockholm University Falkowski: Sweden Rutgers University, U.S.A Main sources of external funding Deutsche Forschungsgemeinschaft NATO Science for Peace programme Ministry of Education of the Czech Republic

29 All slides of my lectures can be downloaded from my workgroup homepage Department of Biology Workgroups Küpper lab, or directly and on the ILIAS website