photosynthesis and nitrogen fixation
Hendrik Küpper1, 2, *, Ivan Šetlík2, 3, Sven Seibert1, Ondrej Prášil2, 3, Eva Šetlikova2, 3, Martina Strittmatter1, Orly Levitan4, Jens Lohscheider1, Iwona Adamska1 and Ilana Berman-Frank4
1 Universität Konstanz; Mathematisch-Naturwissenschaftliche Sektion; Fachbereich Biologie;
D-78457 Konstanz; Germany
2 University of South Bohemia, Faculty of Biological Sciences and Institute of Physical Biology, Branišovská 31, CZ-370 05 České Budejovice, Czech Republic
3 Academy of Sciences of the Czech Republic, Institute of Microbiology, Dept. of Autotrophic Microorganisms, Opatovický mlýn, CZ-37981 Třeboň, Czech Republic
4 Bar Ilan University, Mina and Everard Goodman Faculty of Life Sciences, Ramat Gan, 52900, Israel
* To whom correspondence should be addressed. E-mail: Hendrik.Kuepper@uni-konstanz.de, Tel.: (++49)-7531-884112, Fax.: (++49)-7531-884533
SUMMARY
• Since iron limitation is a main limiting factor of ocean productivity, we investigated its effects on interactions between photosynthesis and nitrogen fixation in the marine non-heterocystous diazotrophic cyanobacterium Trichodesmium IMS101.
• We used biophysical methods such as fluorescence kinetic microscopy, FRR fluorimetry, in vivo and in vitro spectroscopy of pigment composition, we measured nitrogenase activity and abundance of key proteins.
• Fe-limitation caused fast down-regulation of nitrogenase activity and protein levels. In contrast, abundance of Fe-requiring PSI components remained constant. Total levels of phycobiliproteins remained unchanged according to single-cell in vivo spectra.
However, the regular 16 kDa phycoerythrin band decreased and finally disappeared 16-20 d after initiation of Fe-limitation, concomitant with the accumulation of a 20 kDa protein crossreacting with the phycoerythrin antibody. Concurrently, nitrogenase expression and activity increased. Fe-limitation dampened the daily cycle of PSII activity characteristic for diazotrophic Trichodesmium cells. Further, it increased the number and prolonged the time period of occurrence of cells with elevated basic fluorescence F0. Additionally, it increased the effective cross-section of PSII, probably due to enhanced coupling of phycobilisomes to PSII, and led to up-regulation of the iron stress protein IsiA.
• Trichodesmium survives short-term iron limitation by selectively downregulating nitrogen fixation while maintaining but re-arranging the photosynthetic apparatus.
Key words: chlorophyll fluorescence imaging, in vivo spectroscopy, iron limitation stress, nitrogen fixation, photosynthesis, state transitions, Trichodesmium
Iron limitation in Trichodesmium
Abbreviations
Chl = Chlorophyll
FKM = Microscope for two-dimensional (imaging) measurements of fluorescence kinetics FRRf = Fast repetition rate fluorimeter
F0 = Basic fluorescence yield of a dark-adapted sample, fluorescence in non-actinic measuring light
Fm = Maximum fluorescence yield of a dark-adapted sample
Fm’ = Maximum fluorescence yield of a sample during the exposure to actinic light, i.e.
diminished by non-photochemical quenching
Fm’’ = Maximum fluorescence yield of a fully light-adapted sample at the end of the actinic light period of the measurement, diminished by non-photochemical quenching
Ft’ = Fluorescence under actinic irradiance immediately before the measurement of Fm’ Fv = Variable fluorescence; Fv = Fm-F0, i.e. response to a supersaturating flash in the
dark-adapted state of PS II.
Fv/Fm = “Maximal efficiency of dark-adapted PS II”. In this study, activity of PS II was measured by the variable fluorescence (Fv, see above) without the usual normalisation to Fm (e.g. Maxwell and Johnson, 2000) because the latter value would be influenced by the fluorescence emitted by the uncoupled antenna that leads to the elevated F0, (see results and Küpper et al., 2004 for details).
Fqp = “photochemical quenching” was measured as the difference between Fm’ and Ft’, i.e. the response to a supersaturating flash during actinic light exposure. This was done without normalisation, for the same reason as described for Fv vs. Fv/Fm above.
Fqnp = “Nonphotochemical quenching” was measured as the difference between the dark-adapted Fm and the light-adapted Fm’, i.e. Fqnp = Fm - Fm’ without the usual normalisation to Fm’ for the same reason as described for Fv vs. Fv/Fm above.
LED = light emitting diode
PBS = phosphate buffered saline, buffer used for extracting phycobiliproteins
INTRODUCTION
Biological fixation of atmospheric nitrogen is performed by certain cyanobacteria when bioavailable forms of nitrogen (nitrate and ammonia) are in short supply. The nitrogen-fixing enzyme, nitrogenase, is irreversibly inactivated when exposed to molecular oxygen (reviewed by Postgate, 1998). Therefore, nitrogen-fixing (diazotrophic) cyanobacteria must prevent nitrogenase from being damaged by oxygenic photosynthesis (reviewed by Gallon, 1992, 2001; Bergman et al., 1997; Berman-Frank, Lundgren and Falkowski, 2003). Most diazotrophic cyanobacteria achieve this by separating photosynthesis and nitrogen fixation either spatially by differentiating highly specialised cells called heterocysts, or temporally by fixing nitrogen at night (usually found in unicellular diazotrophic cyanobacteria). In contrast, filamentous non-heterocystous marine cyanobacteria of the genus Trichodesmium execute both processes during the light period without irreversible differentiation of specialised cells.
Trichodesmium is abundant and forms widespread blooms, thousands of kilometres-wide over the subtropical and tropical oceans, and contributes a larger fraction to the total marine nitrogen fixation than any other organism (Capone et al., 1997, 2005; Westberry and Siegel, 2006).
In Trichodesmium, active photosynthetic components (such as PS I and PS II complexes, Rubisco, carboxysomes) are found in all cells, even those harbouring nitrogenase (Janson, Carpenter and Bergman, 1994; Fredriksson and Bergman, 1997; Fredriksson et al.
1998; Berman-Frank et al. 2001a). During nitrogen fixation, nitrogenase is expressed in a fraction of the cells that are often arranged consecutively along the trichome (Lin et al. 1998;
Berman-Frank et al. 2001a). Protection against oxygen in Trichodesmium is a complex interaction between reversible spatial and temporal segregation of photosynthesis, respiration and nitrogen fixation (Kana, 1993; Carpenter and Roenneberg, 1995; Chen et al. 1999;
Berman-Frank et al. 2001a; Küpper et al., 2004). Earlier research demonstrated light and photosystem II (PS II) dependent (DCMU sensitive) oxygen consumption by the Mehler reaction (Kana, 1993; Berman-Frank et al., 2001, Milligan et al. 2007) as well as an unusually high dark respiration rate (Kana, 1993; Carpenter and Roenneberg, 1995) suggesting that oxygen-consuming mechanisms protect nitrogenase. Measurements of chlorophyll (Chl) fluorescence kinetics yield comprehensive information about the regulation of photosynthesis in cyanobacteria (reviewed e.g. by Campbell et al., 1998).
Investigations of Chl fluorescence kinetics in cyanobacteria have led to the discovery and better understanding of daily activity cycles in Cyanothece sp. (Meunier, Colón-Lopez
Iron limitation in Trichodesmium and Sherman, 1997, 1998; review by Sherman, Meunier and Colón-Lopez, 1998), Synechococcus sp. (Behrenfeld and Kolber, 1999) and Plectonema boryanum (Misra and Mahajan, 2000). Fluorescence kinetics of cyanobacteria differ in several ways from those of green plants and algae, mainly due to the presence of phycobilisomes in cyanobacteria instead of the LHC II in Chlorophyta (Campbell et al., 1998). In particular, "state transitions"
between associations of the phycobilisomes with PS II ("state I") or with PS I ("state II") profoundly change many parameters of the fluorescence kinetics. In two previous studies, we utilised Chl fluorescence kinetic microscopy (FKM: Küpper et al., 2000) to resolve the spatial and temporal patterns of photosynthetic activity in Trichodesmium in relation to nitrogen-fixation (Berman-Frank et al. 2001a; Küpper et al., 2004). Thus, we showed that Trichodesmium trichomes have a homogeneous high activity of PS II during most of the day, and perform a reversible partial differentiation of cells for the period of nitrogen fixation. This partial differentiation involves a decline in oxygen production by enhancing Mehler reaction correlated to a reversible change in PS II activity (Berman-Frank et al. 2001a; Küpper et al., 2004, Milligan et al. 2007). Chl fluorescence kinetic microscopy revealed that during the period of high nitrogen fixation some cells had a much higher basic Chl fluorescence yield (F0) than all the cells outside the diazotrophic period; these cells have been termed “bright zones/cells” (Berman-Frank et al. 2001a). Rapid reversible switches between fluorescence levels were observed, which indicated that the elevated F0 of the bright cells originates from reversible uncoupling of PS II antenna proteins from the PS II reaction centre (Küpper et al., 2004). Two physiologically distinct types of bright cells were observed (Küpper et al., 2004).
Bright I with about double F0 compared to the normal F0 in the non-diazotrophic state had a high PS II activity and were correlated with nitrogen fixation. Type II bright cells, in contrast, had more than three times the normal F0, exhibited hardly any PS II activity measurable by variable fluorescence and were not related to nitrogen fixation, but to stress. In addition to the two high fluorescence states, cells were observed to reversibly enter a low-fluorescence state.
Biological nitrogen fixation is also controlled by the bioavailable iron, which is limiting in many regions of the world's oceans (reviewed e.g. by Morel and Price, 2003). The bioavailable iron concentration in the oceans is still a matter of debate, due to the fact that not only free iron (at the surface down to <0.1 nM, Morel and Price, 2003), but also the much more abundant (high nM range) bound and colloidal iron is often bioavailable, which was recently investigated also with Trichodesmium (Wang and Dei, 2003). Amongst the organisms most susceptible to iron limitation are the photosynthetic diazotrophs, due to their high requirement for iron in nitrogenase (19 Fe per nitrogenase), in addition to the
Fe-containing proteins of the photosynthetic units. Thus, diazotrophs have higher intracellular iron quotas than non-diazotrophic phytoplankton (Raven, Evans and Korb, 1999; Kustka et al., 2003; Tuit, Waterbury and Ravizzaz, 2004). Availability of iron influences N2 fixation in cyanobacteria by its direct effect on Fe-rich protein synthesis of nitrogenase, and by effects on photosynthesis, growth, and global productivity (Paerl, Crocker and Prufert, 1987, Rueter, Ohki and Fujita, 1990; Falkowski, 1997; Berman-Frank et al., 2001b; Fu and Bell, 2003).
Thus we expect that the availability of iron would further influence the regulation of photosynthesis for nitrogen fixation in Trichodesmium.
In the present study, we used newly available techniques to extend our understanding of Trichodesmium's response to iron limitation. Biophysical measurements were combined with assays of nitrogenase activity, Chl and carotenoid composition, Western blot analysis of protein expression, measurements of C, N, and phosphate content. These investigations were done in Trichodesmium cultures grown in chemostats either at the normal or continuously reduced iron concentration.
Iron limitation in Trichodesmium