The molecular impacts of abiotic stress factors on photosynthesis in cyanobacteria and higher plants
Dissertation
zur Erlangung des akademischen Grades des Doktors der Naturwissenschaften (Dr. rer. nat.)
an der
Universität Konstanz
Mathematisch-naturwissenschaftliche Sektion Fachbereich Biologie
Lehrstuhl für Physiologie und Biochemie der Pflanzen
vorgelegt von
Jens Nikolaus Lohscheider
Tag der mündlichen Prüfung: 20.08.2010 Referentin: Prof. Iwona Adamska
Referent: Prof. Martin Scheffner
Konstanzer Online-Publikations-System (KOPS) URN: http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-124543
URL: http://kops.ub.uni-konstanz.de/volltexte/2010/12454/
Summary
SUMMARY
During photosynthesis, the physical energy of sunlight is used for the production of biomass, consuming atmospheric CO2 and water and at the same time releasing molecular oxygen. This process is tightly regulated and its efficiency is strongly dependent on external abiotic and biotic factors influencing the status of the photosynthetic machinery, and thus, all downstream molecular processes.
Obviously, light plays a central role in this respect. Variations of light intensity and quality are frequent in most habitats. This causes the requirement of acclimation mechanisms in the photosynthetic cells in order to guarantee optimal activity of physiological processes.
Photosynthetic organisms have developed a variety of cellular and molecular mechanisms to acclimate to conditions when light is limiting as well as to avoid or repair damage caused by high light (HL) intensities. On the organismic level, plants are able to change the orientation of their leaves in order to regulate the amount of absorbed light. In plant cells, chloroplasts can move to marginal parts of the cell if light intensities are too high to shadow themselves and reduce the absorption of light energy.
At HL intensities, reactive oxygen species (ROS) are generated during photosynthesis, which lead to damage of cellular components. On the molecular level, photosynthetic organisms can prevent this photooxidative damage by adjusting the size of their light- harvesting antennae (state transition), by effective repair mechanisms of damaged cell components (e.g. D1 repair cycle), by direct detoxification of ROS via protective molecules (carotenoids, vitamins) or enzymatic radical scavenging systems (superoxide dismutases, ascorbate peroxidases and catalases), and by the induction of light stress proteins.
The family of early light-induced proteins (ELIP) has been described to be involved in protection against photooxidative damage in cyanobacteria, algae and higher plants. They presumably act as quenchers of excess light energy. Although the stress-enhanced proteins (SEP) represent the dominant members of the ELIP family in Arabidopsis thaliana, they are not well studied yet. Therefore in this study, mRNA data concerning the expression patterns of SEPs in different organs as well as during the plant’s life cycle were verified on the protein level. In order to determine the role of SEPs in photoprotection, localisation studies of these proteins inside the cell were performed for selected SEP members. The results revealed association of all investigated SEPs with photosystem II (PSII). For studies of the physiological functions of SEPs in higher plants, mutant and transgenic A. thaliana lines were identified and used in light stress experiments. While mutants with reduced amounts of SEP3a
did not show significant differences as compared to wild type plants, sep3a over-expressor mutant lines developed a circular chlorosis of the leaf rosette, in which chlorophyll fluorescence parameters were altered. This implied a function of SEPs in stabilisation of PSII and/or a role in pigment biosynthesis.
Apart from induction of ELIPs under high light (HL) conditions, the arrangement of the photosystems’ antenna is changed and photodamaged proteins (mainly the D1 protein of the PSII reaction centre) are exchanged by newly-synthesised copies. In a process called state transition, mobile parts of the PSII antenna become phosphorylated and migrate to PSI to balance energy fluxes between the two photosystems. In this study, novel light-regulated phosphorylation sites that might be involved in regulation of state transitions are described.
Moreover, nitrations at the D1 protein were discovered, which might act as degradation signal of the damaged PSII reaction centres.
Furthermore, the impact of light stress on different cyanobacteria was analysed. It could be shown that the vertical distribution of closely related Synechococcus isolates from Lake Constance is influenced by their genetically fixed mechanisms for stress protection.
While phycocyanin-rich strains isolated from the water surface were stress resistant, phycoerythrin-rich isolates from deeper water areas of the littoral zone displayed a reduction in pigment and protein concentrations after HL exposure. In contrast, analysis of the marine cyanobacterium Trichodesmium erythraeum exposed to HL and low light conditions indeed revealed a reduction of pigment concentrations and increased growth rates. This indicates effective acclimation mechanisms in Trichodesmium reflecting the environmental variations in the natural habitat.
Apart from variations in the light regime, nutrient availability strongly influences the photosynthetic capacity of cyanobacteria, algae and higher plants. Obviously, macronutrients like phosphorus, nitrogen and carbon are important for cellular biomass production and are usually the factors limiting plant growth. However, micronutrients may also be limiting because they act as important cofactors necessary for general function or regulation of proteins. In the oceans, cyanobacterial and algal growth is strongly limited by the availability of iron, which is an essential cofactor in proteins involved in photosynthetic reactions and cellular respiration as well as in the enzyme nitrogenase which is responsible for the fixation of atmospheric nitrogen. The effects of iron limitation on the marine filamentous cyanobacterium Trichodesmium were studied by showing it’s impact on photosynthesis and nitrogen fixation.
Zusammenfassung
ZUSAMMENFASSUNG
Während der Photosynthese wird die physikalische Energie der Sonnenstrahlung für die Produktion von Biomasse verwendet, wobei Wasser und atmosphärisches Kohlenstoffdioxid verbraucht werden und molekularer Sauerstoff freigesetzt wird. Dieser Prozess ist strikt reguliert, und seine Effizienz ist stark abhängig von externen biotischen und abiotischen Faktoren, welche den Status der photosynthetischen Maschinerie und dadurch auch alle nachfolgenden molekularen Prozesse beeinflussen.
Licht spielt in dieser Beziehung ganz klar eine zentrale Rolle. Fluktuationen in Lichtintensität und –qualität sind häufig in fast allen Habitaten. Deshalb benötigen photosynthetische Zellen Akklimationsmechanismen, um eine möglichst optimale Funktionsweise aller physiologischen Prozesse zu gewährleisten. Deshalb haben photosynthetische Organismen eine Reihe von zellulären und molekularen Mechanismen entwickelt, um sich an Umweltbedingungen anzupassen, bei denen Licht entweder limitierend ist oder aber im Überschuss vorhanden ist und dadurch Schäden verursacht. Auf der organismischen Ebene sind Pflanzen dazu in der Lage, die Ausrichtung ihrer Blätter zu verändern und damit die Menge an einfallender Lichtenergie zu regulieren. Zusätzlich dazu können Pflanzenzellen durch Chloroplastenbewegung entweder an die Seiten oder ins Zentrum der Zelle die einfallende Lichtmenge beeinflussen.
Bei hohen Lichtintensitäten werden während der Photosynthese Sauerstoffradikale (‚reactive oxygen species’, ROS) gebildet, deren Akkumulation zur Schädigung der meisten zellulären Komponenten führt. Auf molekularer Ebene können photosynthetisch aktive Organismen diese photooxidativen Schäden vermeiden, indem sie die Größe ihrer lichtsammelnden Antennenkomplexe anpassen (‚state transition’), durch effektive Reparaturmechanismen (z.B. den D1 Reparaturzyklus), durch direkte Entgiftung von ROS über protektive Moleküle (Carotinoide, Vitamine) oder durch enzymatische Radikalentgiftungssysteme (Superoxiddismutase, Ascorbatperoxidase und Katalase) sowie durch die Induktion von Lichtstressproteinen.
Die ‚early light stress-induced proteins’ (ELIPs) wurden in dieser Hinsicht mit dem Schutz der Photosysteme vor photooxidativen Schäden in Cyanobakterien, Algen und höheren Pflanzen in Verbindung gebracht. Wahrscheinlich helfen sie dabei, die überschüssige Lichtenergie zu löschen. Im Modellorganismus Arabidopsis thaliana sind die so genannten
‚stress-enhanced proteins’ (SEPs) die Hauptvertreter der ELIP-Familie, jedoch ist nur wenig über sie bekannt. Aus diesem Grund wurden vorhandene Daten über organ- und
altersspezifische mRNA-Expression auf Proteinebene verifiziert. Um die photoprotektive Wirkung der SEPs näher aufzuklären, muss die subzelluläre Lokalisierung geklärt werden.
Deshalb wurden Lokalisierungsstudien zu einigen Vertretern der SEPs in Wildtyppflanzen durchgeführt, welche Colokalisation mit Photosystem II (PSII) ergaben. Zur weitergehenden Analyse der physiologischen Funktion der SEPs in A. thaliana wurden Mutanten- und transgene Pflanzenlinien erzeugt und Stressexperimente unterzogen. Während Mutanten mit reduzierten Mengen an SEP3a kaum Unterschiede zu Wildtyppflanzen zeigten, traten bei SEP3a-überexprimierenden Pflanzen kreisförmige Chlorosen der Blattrosette auf, in denen die Chlorophyllfluoreszenzparameter verändert waren, was für eine Funktion in der Stabilisierung des Photosystems und/oder der Pigmentbiosynthese spricht.
Zusätzlich zur Induktion der ELIPs wird unter Starklichteinfluss die Struktur der lichtsammelnden Antennen der Photosysteme verändert und geschädigte Proteine werden durch neusynthetisierte Kopien ersetzt, vor allem das D1-Protein des PSII Reaktionszentrums.
In der so genannten ‚state transition’ kommt es zu einer pH-abhängigen Phosphorylierung mobiler Anteile der Antennen von PSII, die zum PSI diffundieren, um den Energiefluss zwischen den Photosystemen auszubalancieren. In dieser Arbeit werden neue Phosphorylierungsstellen beschrieben, die lichtabhängig reguliert sind. Zudem wurden Nitrierungen am D1-Protein entdeckt, die als neues Signal für den Abbau des geschädigten PSII-Reaktionszentrums dienen können.
Des Weiteren wurde der Einfluss von Lichtstress auf verschiedene Cyanobakterien untersucht. Es konnte gezeigt werden, dass die Verteilung nah verwandter benthischer Synechococcus-Isolate aus dem Bodensee von ihren genetisch festgelegten Schutzmechanismen beeinflusst wird. Während phycocyaninreiche Stämme von der Wasseroberfläche starklichtresistent waren, zeigte sich bereits nach kurzer Starklichtexposition ein Rückgang im Pigment- und Proteingehalt in phycoerythrinreichen Isolaten aus größeren Wassertiefen. Im Gegensatz dazu ergaben Untersuchungen des marinen Cyanobakteriums Trichodesmium erythraeum unter Stark- und Schwachlichtbedingungen zwar eine Reduktion des Pigmentgehaltes unter Starklicht, aber erhöhte Wachstumsraten.
Dies spricht für effektive Akklimationsmechanismen in Trichodesmium, die die starken Schwankungen im natürlichen Habitat widerspiegeln.
Neben den Fluktuationen in den Lichtbedingungen wird die photosynthetische Kapazität von Cyanobakterien, Algen und höheren Pflanzen sehr stark von der Nährstoffverfügbarkeit beeinflusst. Hier sind es natürlich hauptsächlich die Makronährstoffe wie Phosphat, Stickstoff und Kohlenstoff, die aufgrund ihrer Wichtigkeit für die zelluläre Biomasseproduktion das
Zusammenfassung pflanzliche Wachstum limitieren. Allerdings können auch Mikronährstoffe durch ihre Verwendung als wichtige Cofaktoren für die generelle Funktionstätitgkeit und die Regulation von Proteinen limitierend wirken. In den Ozeanen zum Beispiel wird das Wachstum von Cyanobakterien und Algen durch die geringe Konzentration an verfügbarem Eisen eingeschränkt, welches als elementarer Cofaktor vieler Proteine mit Funktionen in der Photosynthese oder der zellulären Respiration dient. Zudem benötigt das Enzym Nitrogenase Eisen, um in Cyanobakterien atmosphärischen Stickstoff fixieren zu können. Deshalb wurden die Effekte der Eisenlimitierung auf Photosynthese und Stickstofffixierung im marinen filamentösen Cyanobakterium Trichodesmium erythraeum exemplarisch untersucht.
List of publications
LIST OF PUBLICATIONS
This thesis is based on the following publications and manuscripts:
CHAPTER 1 Küpper H., Šetlík I., Seibert S., Prášil O., Šetlikova E., Strittmatter M., Levitan O., Lohscheider J., Adamska I. and Berman-Frank I. (2008).
Iron limitation in the marine cyanobacterium Trichodesmium reveals new insights into regulation of photosynthesis and nitrogen fixation.
New Phytol. 179: 784-798.
CHAPTER 2 Andresen E., Lohscheider J., Šetlikova E., Adamska I., Šimek M. and Küpper H. (2009). Acclimation of Trichodesmium erytrhraeum ISM101 to high and low irradiance analysed on the physiological and biochemical level. New Phytol. 185: 173-188.
CHAPTER 3 Lohscheider JN., Strittmatter M., Küpper H. and Adamska I. Vertical distribution of benthic freshwater cyanobacterial Synechococcus spp.
strains depends on their ability for photoprotection. (Manuscript)
CHAPTER 4 Galetskiy D., Lohscheider JN., Kononikhin AS., Popov IA., Nikolaev EN. and Adamska I. Light stress-related turnover of nitrated and phorphorylated proteins on photosynthetic complexes of Arabidopsis thaliana. (Manuscript)
CHAPTER 5 Lohscheider JN., Rojas-Stütz MC., Andersson U., Funck D., Bruderek M. and Adamska I. Stress-enhanced protein 3a in Arabidopsis thaliana is associated with light-harvesting antenna of photosystem II.
(Manuscript)
CHAPTER 6 Lohscheider JN., Rojas-Stütz MC., Andersson U., Funck D., Bruderek M. and Adamska I. Localisation and evolutionary origin of stress- enhanced protein 3b from Arabidopsis thaliana. (Manuscript)
CHAPTER 7 Reiser V., Galetskiy D., Susnea I., Lohscheider JN., Miller-Sulger R., Przybylski M. and Adamska I. Localisation of early light-induced proteins in the thylakoid membrane. (Manuscript)
Additional manuscripts not included in this thesis:
Galetskiy D., Lohscheider JN., Kononokhin AS., Kharybin ON., Popov IA., Adamska I. and Nikolaev EN. Light stress photodynamics of chlorophyll-binding proteins in Arabidopsis thaliana thylakoid membranes revealed by high-resolution mass spectrometric studies.
(Manuskript)
Galetskiy D., Lohscheider JN., Kononikhin AS., Popov IA., Nikolaev EN and Adamska I.
Protein modifications in the photosynthetic apparatus of Arabidopsis thaliana related to light and oxidative stress. (Manuscript)
Table of contents
TABLE OF CONTENTS
SUMMARY I
ZUSAMMENFASSUNG III
LIST OF PUBLICATIONS VII
TABLE OF CONTENTS IX
GENERAL INTRODUCTION 1
Photosynthesis and the impact of abiotic stressors 1
Light stress 3
Iron limitation 3
Mechanisms of stress protection 4
Regulation of antenna size, state transitions and
changes in the PSI:PSII ratio 4
Photoprotective pigments 5
Enzymatic radical scavenging systems 5
The photosystem II repair cycle and D1 turnover 6 Induction of light stress-related proteins of the ELIP family 6
CHAPTER 1 9
Iron limitation in the marine cyanobacterium Trichodesmium reveals new insights into regulation of photosynthesis and nitrogen fixation
Summary 10
Introduction 12
Materials and Methods 15
Results 21
Discussion 34
CHAPTER 2 39 Acclimation of Trichodesmium erythraeum ISM101 to high and low
irradiance analysed on the physiological, biophysical and biochemical level
Summary 40
Introduction 41
Materials and Methods 43
Results and Discussion 48
Supplementary Data 66
CHAPTER 3 69
Vertical distribution of benthic freshwater cyanobacterial
Synechococcus spp. strains depends on their ability for photoprotection
Summary 70
Introduction 71
Materials and Methods 74
Results 77
Discussion 87
Supplementary Data 91
CHAPTER 4 93
Light stress-related turnover of nitrated and phosphorylated proteins in photosynthetic complexes of Arabidopsis thaliana
Summary 94
Introduction 95
Materials and Methods 98
Results 100
Discussion 108
Table of contents
CHAPTER 5 113
Stress-enhanced protein 3a in Arabidopsis thaliana is associated with light-harvesting antenna of photosystem II
Summary 114
Introduction 115
Materials and Methods 117
Results 122
Discussion 131
CHAPTER 6 135
Localisation and evolutionary origin of stress-enhanced protein 3b from Arabidopsis thaliana
Summary 136
Introduction 137
Materials and Methods 139
Results 143
Discussion 152
CHAPTER 7 155
Localisation of early light-induced proteins in the thylakoid membrane
Summary 156
Introduction 157
Materials and Methods 159
Results 164
Discussion 170
CONCLUDING REMARKS 173
ACKNOWLEDGEMENTS 179
AUTHOR CONTRIBUTIONS 181
REFERENCES 183
General introduction
GENERAL INTRODUCTION
Photosynthesis and the impact of abiotic stressors
Photosynthesis is one of the most important biochemical processes on earth and provided the conditions needed for the evolution of life as we know it today. During photosynthesis, organisms harvest the physical energy of solar irradiance and use it for the generation of biomass. Thylakoids are the major membrane systems in cyanobacterial cells and the chloroplasts of higher plants and algae and contain all pigment-protein complexes needed for photosynthesis (Dekker and Boekema, 2005). A schematic representation of the molecular processes during photosynthesis is shown in Fig. 1. First, light is absorbed by chlorophyll (chl) molecules of the light-harvesting complex (LHC) in plants or the phycobilisomes (PBS) in cyanobacteria that are attached to photosystem I and photosystem II (PSI and PSII, respectively) or only to PSII, respectively, and is then transferred to the reaction centre proteins where a charge separation occurs. Across the thylakoid membranes, a proton gradient is created in a multi-step process, which is finally used for ATP production while a mostly membrane-intrinsic electron transport leads to the generation of NADPH as a reduction equivalent (Dekker and Boekema, 2005). Both ATP and NADPH are then used for fixation of CO2 in sugar biosynthesis and other metabolic processes. At the same time, water is split at PSII to fill the electron gaps created by the primary excitation of chl within the PSII reaction centre, which leads to release of O2 (Barber, 2008).
Due to the complexity of the process, the light dependency and the need for multiple cofactors, photosynthesis is very susceptible to changes in environmental conditions. The availability of nutrients, light intensity and quality as well as temperature has an enormous impact on the physiology of photosynthetic organisms because of their mostly sessile way of life (Takahashi and Murata, 2008; Kehoe and Gutu, 2006; Allakhverdiev et al., 2008). Even some rather mobile representatives, like unicellular algae or cyanobacteria, usually have to adapt to variations within short periods of time when they are not able to escape unfavourable conditions (Xue et al., 2005; Nishiyama, Allakhverdiev and Murata, 2005; Latifi, Ruiz and Zhang, 2008). Therefore, all pro- and eukaryotic photosynthetically active organisms have developed numerous cellular and molecular mechanisms to prevent or reduce damage caused by abiotic stress factors.
Fig. 1 Simplified schematic representation of photosynthetic protein complexes in cyanobacteria, algae and higher plants. Dimeric photosystems II (PSII) receive light energy harvested by either soluble phycobilisomes (PBS) or membrane-internal light- harvesting complexes (monomeric or trimeric, mLHCII and tLHCII, respectively). Electron gaps are closed by water splitting and release of molecular oxygen in the oxygen-evolving complex (OEC). Electrons released from PSII reaction centre are transferred via the plastoquinone/plastoquinol pool (PQ/PQH2), the cytochrome b6/f complex (Cyt b6/f) and plastocyanin (PC) to photosystem I (PSI). In PSI, light harvested by monomeric LHCI (in plants), by the reaction centre itself or by PBS attached due to state transition (cyanobacteria) left an electron gap, which is closed by electrons delivered by PC. Finally, electrons are used to reduce NADP+ as reduction equivalent in dark reaction of photosynthesis. A proton gradient created by water splitting and influx of protons via the PQ/PQH2 and cyt b6/f drives ATP synthesis at the F-type ATPase.
General introduction
Light stress
In general, photosynthesis is affected by most abiotic stress factors but it is obvious that especially light intensity and quality play a very important role (Dietzel, Bräutigam and Pfannschmidt, 2008). When the energy of the collected light exceeds the capacity of the photosystems for photosynthetic electron transport (PET), photoinactivation occurs as the first phase of light stress (Andersson and Aro, 2001). During photoinactivation, all components of the PET remain reduced leading to deceleration of photosynthetic reactions. Photoinactivation can be overcome by up-regulation of the Calvin cycle and cyclic electron flow between PSI and the cytochrome b6/f complex (cyt b6/f) (Rumeau, Peltier and Cournac, 2007). After continuous illumination with high light (HL) intensities, reversible photoinhibition occurs when proteins of the PET are severely damaged by reactive oxygen species (ROS) that are generated by excited states of chl molecules (chl triplett, 3chl) (Shao et al., 2008; Latifi, Ruiz and Zhang, 2009). Here, the D1 protein of the PSII reaction centre is the main target of photoinhibition because it binds most cofactors necessary for electron transport within PSII (Yamamoto, 2001). As long as the cellular repair mechanisms are able to cope with the oxidative damages caused by HL illumination, the organism can survive. However, if the rate of photooxidative damage exceeds the capacities of the cellular repair mechanisms, irreversible photoinhibition occurs leading to oxidation of cellular components like proteins, pigments and lipids. This will finally lead to photobleaching and death of the cell (Mittler, 2002).
Iron limitation
Apart from light energy, micronutrient availability influences the photosynthetic performance due to the need for many trace metals and vitamins as enzymatic cofactors. For photosynthetic organisms, iron as a micronutrient plays an important role (Pilon et al., 2006). It is typically associated with sulphur ions in iron-sulphur clusters acting as cofactors e.g. in the cyt b6/f (Baniulis et al., 2008), in the reaction centre of PSI (Fromme, Jordan and Krauss, 2001; Ben- Shem, Frolow and Nelson, 2003), in ferredoxins (Fd) (Fukuyama, 2004) and in the enzyme nitrogenase of nitrogen-fixing diazotrophic cyanobacteria (Küpper et al., 2008).
However, iron limitation is usually most pronounced in the oceans where the concentration of bioavailable iron is very low as compared to terrestrial ecosystems (Sunda and Huntsman, 1995; Granger and Price, 1999). Iron deficiency leads to so called iron chlorosis due to the need for this trace metal in the biosynthesis of chl (Moseley et al., 2002;
Varotto et al., 2002; Pilon et al., 2006). It has been shown that marine photosynthetic
organisms as well as terrestrial plants living under iron-limited conditions developed different strategies to manage the problems resulting from iron deprivation. For example, land plants and green algae are able to increase availability of iron by chemical reduction of external iron (Guerinot and Yi, 1994; Eckhardtand Buckhout, 1998). Moreover, cyanobacteria, algae and plants are able to increase the cellular iron concentration by excretion of low-molecular mass molecules, so-called siderophores (Guerinot and Yi, 1994; Wilhelm, Maxwell and Trick, 1996; Eckhardtand Buckhout, 1998).
Mechanisms of stress protection
Regulation of antenna size, state transitions and changes in the PSI:PSII ratio
One very effective mechanism to regulate the amount of energy harvested by the protein complexes of the thylakoid membranes is the adjustment of antenna size according to the irradiated light intensities (Kruse, 2001). This can be achieved by two separate approaches:First of all, the overall amount of antennae surrounding the two photosystems can be generally reduced as a response to prolonged illumination or increased when light intensity is limiting (Anderson, Chow and Park, 1995). Moreover, it is possible to transfer a mobile fraction of the antenna of PSII to PSI under HL conditions to reduce the energetic pressure on the PSII reaction centre, the main target for light stress damage. Under ambient light conditions, the antenna of PSII in higher plants and algae is composed of monomeric LHC proteins surrounding a PSII reaction centre dimer. In this so called state I, the monomeric antenna is surrounded by groups of LHC trimers composed of LHCB1-3 (Kargul and Barber, 2008). Upon HL exposure, these trimeric antennae become phosphorylated in a light- dependant manner leading to relaxation of the grana stacks and release of PSII into the stroma thylakoids (Kruse, 2001). At the same time, the phosphorylated LHC populations diffuse and associate with PSI (state II). Due to the lack of membrane-located LHC and the existence of PBS, the mode of state transition differs in cyanobacteria as compared to higher plants and algae. Besides a plant-like mechanism of state transition (transfer of PBS from PSII to PSI;
Mullineaux, Tobin and Jones, 1997), various mechanisms have been proposed including redistribution of the energy absorbed by PBSs between PSII and PSI (McConnell et al., 2002).
Another mode of adjusting the amount of energy collected in the thylakoid membranes of cyanobacteria, algae and higher plants is the regulation of the PSI:PSII ratio (Hihara and Sonoike, 2001). Cyanobacteria and plants are able to increase the amount of PSII in a process
General introduction regulated by the reduction state of the cyt b6/f to reduce the number of photons absorbed by a single photosystem (Allen and Pfannschmidt, 2000).
Photoprotective pigments
Pigments play a central role in photosynthesis because they are the primary receptors for the light energy absorbed in the pigment-protein complexes of the photosynthetic membranes (Green and Durnford, 1996). While chl acts as a primary photosynthetic pigment, a large number of carotenoids serve as accessory or secondary photosynthetic pigments. Lutein and violaxanthin for example are common in higher plants and algae and help to extend the range of light available for photosynthesis but other carotenoids are able to reduce the amount of energy collected by the photosystems (Demmig-Adams, Gilmore and Adams, 1996).
In a mechanism called xanthophyll cycle, higher plants and algae are able to enzymatically convert the accessory xanthophyll violaxanthin in a pH-dependent manner into zeaxanthin (Baroli and Niyogi, 2000). Unlike violaxanthin, zeaxanthin cannot transfer the absorbed light energy to neighbouring chl molecules but is capable of re-emitting the absorbed energy as heat, a process also known as non-photochemical quenching (NPQ) (Baroli and Niyogi, 2000). Cyanobacteria lack the xanthophyll cycle but are able to induce the biosynthesis of the photoprotective xanthophyll zeaxanthin upon exposure to HL intensities (Schagerl and Müller, 2006).
While the accumulation of zeaxanthin prevents the formation of ROS generated during HL stress, carotenoids are also capable of detoxifying radicals in later phases of the stress response. Many carotenoids like e.g. β-carotene covalently bind ROS. By resonance transfer of electrons, the ROS are converted into O2 and released while excess energy is emitted as heat (Tracewell et al., 2001, Wang and Hu, 2002).
Enzymatic radical scavenging systems
Besides the detoxification of ROS by carotenoids, enzymatic radical scavenging systems are important factors in protection against oxidative stress. ROS may be produced at PSII, PSI and cyt b6/f when PET is impaired (Krieger-Liszkay, 2005). In higher plants and algae, the superoxide dismutases (SOD) catalyse the detoxification of the superoxide radical (O2-) to hydrogen peroxide (H2O2) (Asada, 2000; Herbert et al., 1992). Since H2O2 is toxic itself, it can be converted into water and molecular oxygen by the enzyme catalase (CAT) (Wilhelm, 1999) or can be subjected to the glutathione-ascorbate cycle. In this case, the enzyme ascorbate peroxidase (APX) reduces H2O2 by oxidising ascorbate, which is subsequently
regenerated via reduced glutathione (GSH) (Asada, 2000). GSH levels are then restored by oxidation of NADPH generated at PSI (Karpinki et al., 1997).
Although this is true for higher plants, the comparable mechanisms in cyanobacteria are only poorly understood. However, SOD (Herbert et al., 1992) as well as catalase-like enzymes (Obinger et al., 1999; Perelman et al., 2003) have been described in cyanobacteria speaking in favour of a similar function in these organisms.
The photosystem II repair cycle and D1 turnover
The D1 protein is a main target of photooxidation during HL stress because it binds almost all cofactors necessary for electron transport within PSII (Barber and Andersson, 1992; Aro, Virgin and Andersson, 1993). Therefore, the photodamaged D1 protein needs to be quickly and efficiently replaced after exposure to photooxidative conditions in order to prevent further damage to surrounding molecules. For both cyanobacteria and higher plants, a conformational change of D1 has been proposed (Andersson and Aro, 2001) prior to proteolytic degradation by stromal/cytosolic proteases after light stress exposure (Andersson and Aro, 2001; Haußühl, Andersson and Adamska, 2001, Silva et al., 2002). The family of Deg/Htr proteases identified in cyanobacteria as well as in higher plants seems to be of special importance (Huesgen, Schuhmann and Adamska, 2005 and 2009) although the specific mechanisms underlying the D1 replacement are not fully understood.
Induction of light stress-related proteins of the ELIP family
Photosynthesis is driven by the absorption of sunlight. This requires the presence of structures which allow the collection of solar energy. The photosystems of plants, algae and cyanobacteria contain a variety of proteins that are able to bind pigments in order to enable harvesting of sunlight as well as photosynthetic charge separation and finally the generation of ATP, NADPH, molecular oxygen and biomass (Montané and Kloppstech, 2000). These proteins can be divided into two major groups: The chl a-binding proteins and the chl a/b- binding proteins (CAB) (Montané and Kloppstech, 2000). The first group comprises the reaction centre proteins of the two photosystems and the core antenna proteins of PSII as well as proteins with similar structure such as the IsiA protein that is induced under iron limitation and light stress in cyanobacteria (Kouril et al., 2005; Murray, Duncan and Barber, 2006). The second group contains the typical light-harvesting proteins of higher plants and algae and a small subgroup of structurally related proteins that is involved in stress responses, the so- called early light-induced proteins (ELIP) (Fig. 2) (Adamska, 2001; Heddad and Adamska,
General introduction 2002). First ELIPs were discovered during the greening of etiolated pea and barley seedlings where they transiently accumulated before the induction of the LHC proteins (Meyer and Kloppstech, 1984; Grimm and Kloppstech, 1987). Later it was found that ELIPs are also induced in response to light stress (Adamska and Kloppstech, 1991; Pötter and Kloppstech, 1993). Their predicted secondary structure with three membrane-spanning helices, of which helix I and III are conserved, resembles that of the LHC proteins (Adamska, 2001). After genome-wide searches, two groups of related proteins could be identified containing different amounts of transmembrane helices but sharing the so-called ELIP consensus motive embedded in these helices (Adamska, 2001). The stress-enhanced proteins (SEPs) (Fig. 2) or light harvesting-like proteins (LIL) contain two membrane helices, of which helix I is conserved and helix II is polymorphic, and are present in all photosynthetic eukaryotes investigated so far (Heddad and Adamska, 2002). The third group of ELIP proteins was found in cyanobacteria, algae and higher plants and is called one-helix proteins (OHPs) (Fig. 2) (Jansson et al., 2000; Andersson, Heddad and Adamska, 2003), small CAB-like proteins (Scp) (Funk and Vermaas, 1999) or HL-induced proteins (Hlip) (Dolganov, Bhaya and Grossman, 1995). These proteins contain a single conserved membrane helix. Another LHC- and ELIP-related protein is the PSBS protein of PSII in plants and algae with four membrane- spanning helices, of which helices I and III are conserved (Fig. 2) (Funk et al., 1994; Niyogi et al., 2005).
All members of the ELIP family have been shown to be related to stress. Thereby, the classical ELIPs are only detected in response to light stress conditions, while SEPs and OHPs are constitutively expressed at ambient light conditions and up-regulated under light stress conditions (Adamska, 2001).
Fig. 2 Membrane topology and gene accession numbers of early light stress-induced proteins (ELIPs) in Arabidopsis thaliana. According to the number of polymorphic and conserved membrane-spanning helices, four groups of these proteins can be distinguished: the subunit S of photosystem II (PSBS), the classical ELIPs, the stress-enhanced proteins (SEP) and the one-helix proteins (OHP).
Iron limitation in Trichodesmium
CHAPTER 1
Iron limitation in the marine cyanobacterium Trichodesmium reveals new insights into regulation of 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
MATERIALS AND METHODS
Culture media and culture conditions
Cultures of Trichodesmium IMS101 were grown in YBCHK medium, with the following composition: 420 mM NaCl, 10 mM KCl, 20 mM MgCl2, 10 mM CaCl2, 25 mM MgSO4, 2.5 mM NaHCO3, 464 µM H3BO3, 780 µM KBr, 50 µM KH2PO4, 68 µM NaF, 25 µM LiCl, 2 µM RbCl, 1 µM FeNa-EDTA, 450 nM NaIO3, 80 nM Na2MoO4, 20 nM MnCl2, 7 nM ZnSO4, 7 nM NiSO4, 2.5 nM CoCl2, 1 nM CuSO4, dissolved in redistilled water. The 1:1 ratio of Fe3+ and EDTA in our experiments may have caused some formation of (invisible) iron hydroxide precipitates, but this neither affected the controls (as judged by their healthy physiology and growth) nor the iron limitation. The latter was shown by the decreased and finally ceasing growth under these conditions. Pre-cultivation was in batch cultures in glass tubes of 3 cm inner diameter and 250 ml volume. Altogether, we carried out three experiments with step-down type iron removal in batch cultures and two experiments with gradual iron removal in chemostat cultures. Since the chemostat cultures and gradual iron removal are closer to the natural situation, all data presented here are taken from these experiments. The step-down batch culture experiments yielded very similar results, however, in terms of filament fragmentation, in vivo single cell and acetone extract absorption spectra, and maintenance of the photosynthetic apparatus.
In the step-down experiments, iron was removed by filtering Trichodesmium filaments onto GF/F filters (Whatman), washing them with iron-free medium, and resuspending them in iron-free medium in the glass culture tubes. As in such step-down experiments the cells died within 5 to 6 days, to simulate more natural conditions in the later experiments shown in the results we used chemostat cultures and slow decrease of iron concentration instead. From 3 weeks before the experiments and throughout the experiments, the cultures were grown in 4 l flat glass chemostats at a flow rate of 0.5 to 1 l·d-1 that maintained the Fe-replete cultures at an OD750 of 0.05. At the start of the experiments, the medium flowing into the low-iron chemostats was exchanged by a medium containing no Fe-EDTA, so that the iron in the chemostat was gradually diluted out. The decrease of the iron concentration was stopped after two weeks by adding 50 nM Fe-EDTA into the medium pumped into the low-iron chemostats. The chemostat experiments were executed twice, each replicate with one Fe- replete and one Fe-limited culture. The preliminary step-down batch culture experiments were carried out three times, again each replicate with one Fe-replete and one Fe-limited culture.
Both the tubes and the chemostats were aerated with air. The cultures were maintained at 12:12 h Light/Dark cycle (light on 9:00 to 21:00 local time) and 27°C/25°C day/night temperature. The photon flux density during the light period followed a sinusoidal cycle simulating natural conditions, with a peak intensity of about 300 µmol·m-2·s-1 supplied by OSRAM® Dulux L 55W/12-950 (Osram, www.osram.com) fluorescent tubes.
Sampling times
Samples for measurements of nitrogen fixation, pigment quantification, FKM analyses and FRRf measurements were taken five times per day to resolve the changes in the daily activity pattern of Trichodesmium: 8:15 (just before the onset of the light period), 11:00 (at the beginning of pigment synthesis in the morning), 14:15 (in the middle of the light period and peak of nitrogen fixation), 17:00 (in the decline of nitrogen fixation), and 21:15 (directly after the end of the light period). Samples requiring very large amounts of culture were taken only three times per day (8:15, 14:15, 21:15). This applied to the samples for Western blots and C/N/P analyses.
Fluorescence kinetic measurements on a single-cell level
Photosynthetic performance was analysed on a single-cell level using a fluorescence kinetic microscope (FKM) as originally described by Küpper et al. (2000), in the updated and extended version described by Küpper et al. (2007), produced by Photon Systems Instruments (Brno, Czech Republic, www.psi.cz). The most important new features of the version of the FKM described by Küpper et al. (2007) are the possibility to excite and detect fluorescence kinetics by various wavelengths accessible by computer-controlled filter wheels, and the recording of spectrally resolved in addition to the spatially resolved (imaging) fluorescence and absorption kinetics due to the addition of a high-sensitivity fibre-optic spectrometer.
Preparation of samples for FKM measurements
Samples were prepared as previously described (Küpper et al., 2004) with few modifications.
Embedding of the living Trichodesmium filaments was done with 0.75% SeaKem Gold agarose (Cambrex BioScience Rockland, Inc.; www.cambrex.com), which yields a gel strength of >1000 g·cm-2 at this concentration. This high gel strength helped to reduce the movement of Trichodesmium filaments during the measurement. Temperature controlled (27°C) air saturated medium was pumped through the measuring chamber was with
Iron limitation in Trichodesmium 25 ml·min-1. For each time point of the daily activity cycle, fresh samples were prepared from the cultures in the chemostats.
FKM measurement parameters
All FKM measurements lasted 300 s. In the third second, a 600 ms pulse of saturating light (about 4000 µmol·m-2·s-1) was given for measurement of Fm. This was followed by 90 s of darkness, after which F0 was measured for 5 s (measuring light irradiance 5 µmol·m-2·s-1, tested to be non-actinic for Trichodesmium). Then, 100 s of actinic light (about 1000 µmol·m-2·s-1) were applied to analyse the Kautsky induction, and finally 100 s of measurement with no actinic light were used to measure dark relaxation. For the analysis of photochemical and non-photochemical quenching, further 600 ms saturating pulses were applied during the actinic light exposure and in the relaxation period (4 and 3, respectively).
The excitation light in the range of 410 to 510 nm almost excited Chl and the short- wavelength form of phycoerythrin (phycourobilin = “CU phycoerythrin”) judged by the optical properties of Trichodesmium (Subramaniam et al., 1999). The measurements were performed with an automatic subtraction of background signals and a maximum time resolution of 40 ms. A lower time resolution was applied for the slower kinetics. Each image of the resulting fluorescence kinetic records had a resolution of 512x512 pixels at 4096 grey values (12 bits). The FKM was used also as a regular epifluorescence and bright-field microscope to observe the cultures and measure the length of the filaments. The fluorescence kinetic measurements were analysed as described in detail in Küpper et al. (2000, 2004, 2007) using the FluorCam6 software from Photon Systems Instruments.
Integrative (population-level) biophysical measurements
Fast repetition rate measurements of Chl fluorescence kinetics
Fast repetition rate fluorometry (FRRf) by the “FIRe” system (Satlantic instruments, Halifax, Nova Scotia) was used to assess the effective cross-section of the PS II-associated antenna according to Strasser, Srivastava and Tsimilli-Michael (1999), the reoxidation rates of QA-, and photosystem connectivity (Kolber, Prašil and Falkowski, 1998). Samples were measured without pre-concentration. Fluorescence excitation was by blue (Chl a and phycoruobilin exciting) LEDs (Ex: 450±30 nm), and emission was detected using a > 678 nm long pass filter combination.
Low temperature spectroscopy
77K fluorescence emission spectra were recorded by a laboratory-built portable spectrofluorometer. Sample was prepared by filtering Trichodesmium cells onto a GF/F filter (Whatman), mounting this into a filter holder, freezing it in liquid nitrogen, and immersing this sample into a custom-designed dewar at liquid nitrogen temperature. Spectra were recorded using the Avantes USB-2000 fibre optic spectrometer and the SpectraWin software (Avantes Corp., The Netherlands). Excitation was performed by a white LED with filters transmitting between 450 to 550 nm, i.e. exciting Chl, phycourobilin and phycoerythrin. The fluorescence signal was recorded after a 590 nm long pass filter.
Measurement of nitrogen fixation
Nitrogenase activity was assayed by the acetylene reduction method. Samples of 10 ml volume were taken from all three cultures at five time points throughout the daily cycle.
Sample were incubated in gas-tight glass vials, in which 10 ml air (20% of total air volume) were exchanged for 10 ml acetylene, for 1 h under the same light and temperature conditions as the cultures. Afterwards, 5 ml of the gas phase in the vials was taken out and analysed by gas chromatography on an FID-GC (310 GC, SRI Instruments, CA). Control injections+analyses were done with vessels containing pure YBCHK medium without cells in order to check for possible acetylene turnover not connected to Trichodesmium and for possible ethylene contaminations in the air and/or acetylene.
Analysis of protein, Chl and carotenoid composition
FKM measurement of single-cell absorption spectra
In vivo single-cell absorption spectra were recorded in the FKM using the white LED for transmittant light as the light source, as described in detail in Küpper et al. (2007).
Analysis of Chl, carotenoids and phycobilisomes in extracts
Chlorophyll and carotenoids were extracted with 100% acetone after filtrating 80 ml of culture on 25 mm GF/F glass fibre filters (Whatman), freezing the filters in liquid nitrogen and then lyophilising them. After 1 day of extraction at 4°C in the dark, absorbance spectra from 350 nm to 750 nm were recorded in 0.2 nm intervals at an optical bandwidth (slit) of
Iron limitation in Trichodesmium 1 nm. From these spectra, carotenoids and Chl were analysed using the Gauss-Peak-Spectra method according to Küpper et al. (2000) and Küpper, Seibert and Parameswaran (2007).
Phycobiliproteins were extracted after completion of the acetone extraction described above, which along with the pigments removed the membranes from the cells. This pre- treatment made the phycobilisomes easily accessible for extraction by incubation with phosphate buffered saline (PBS = 40 g·l-1 NaCl; 1 g·l-1 KCl; 7.2 g·l-1 Na2HPO4; 1.2 g·l-1 KH2PO4) for 1 day at 4°C in the dark. Spectra of these extracts were recorded in the same way as those of the acetone extracts.
Western blot analysis of protein expression
Samples were harvested by filtration on GF/F filters (Whatman, www.whatman.com), frozen in liquid nitrogen, and stored at -20°C until analysis. Total proteins were extracted from collected samples by grinding the frozen cells in liquid nitrogen and heating of the cell suspension in preheated (60°C) extraction buffer composed of 125 mM Tris/HCl, pH 6.8, 4%
(w/v) SDS, 200 µM phenylmethylsulfonyl fluoride (PMSF) (dissolved in 100% ethanol) and 100 mM dithiothreitol (DTT) for 10 min at 100°C after short vortexing. Cell debris was collected by centrifugation twice for 15 min at 25,000 x g at room temperature. Proteins from the collected supernatant were precipitated by addition of 4 volumes of 100% cold acetone during the incubation at -20°C for approximately 2 h. The proteins were collected by centrifugation for 15 min at 25,000 x g and 4°C and the pellets were washed with 70% (v/v) ethanol. The protein pellets were dried and resuspended in 1:100 diluted extraction buffer.
The amount of protein was determined using the RC DC Protein Assay kit (BioRad, www.biorad.com) with optional precipitation step to improve accuracy as recommended in the manufacturer’s manuals. Absorption was measured at 750 nm.
For Western blotting routinely 6 µg of total protein extract were loaded onto 10% or 15% Laemmli gels (Laemmli, 1970) prior to their transfer to Hybond-P membranes (Amersham Biosciences, Uppsala, Sweden) using a semi-dry blotting system (Biometra) as described (Towbin, Staehlin and Gordon, 1979). Primary antibodies against D1 protein from PS II (α-PsbA), nitrogenase (α-NifH) and PsaC from PS I (α-PsaC) were purchased from AgriSera, (Vännäs, Sweden), against the B-phycoerythrin (α-B-PE) from Rockland Immunochemicals (Gilbertsville, USA) and used in the dilutions recommended by suppliers.
Blots were developed using horseradish peroxidase-coupled IgG (Sigma-Aldrich, Hamburg, Germany) followed by chemiluminescence detection (ECL, Amersham Bioscience, Uppsala
Sweden) or via alkaline phosphatase-coupled IgG (Sigma-Aldrich, Hamburg, Germany) followed by NBT/BCIP colorimetric reaction (Roche Diagnostics GmbH).
For quantification of signals in Western blots, membranes and X-ray films were scanned and signals of interest were quantified by densitometry scanning. Although the chemistry behind any detection on Western blots is not a linear function of protein abundance, the resulting numbers clearly do provide an indication of increase/decrease trends.
Analysis of C/N/P ratios
Samples were harvested by filtration on pre-combusted (450°C, 4 h) and acid-washed GF/F filters (Whatman). After filtration the samples were lyophilised, and analysed for C/N and for P. The C/N analysis was analyzed on an elemental soil analyzer using a thermoconductivity detector (NC 2110, ThermoQuest). P analysis was performed using a modified version of standard protocols for particulate P (Protocol from the Hawaii Institute of Marine Biology, Analytical Services laboratory at the University of Hawaii). The method relies on the release of organically-bound phosphorus compounds as orthophosphate, by high temperature and pressure combustion. The released orthophosphate reacted with a mixed reagent containing sulphuric acid, molybdic acid and trivalent antimony to form phosphomolybdic acid. The solution was reduced to a blue molybdenum complex by the ascorbic acid in the mixture, which was then measured spectrophotometrically (880 nm).
Iron limitation in Trichodesmium
RESULTS
Growth parameters: cell number and filament length, nitrogen fixation, C/N/P ratios.
Cultures were initiated at an OD750 ~ 0.01 as in our earlier studies, and we then let the cultures adjust their growth rate to the dilution rate of the chemostats, which was about 0.18 d-1. For the Fe-replete culture, this equilibrium was reached at an OD750 of 0.5 to 0.6 (Fig. 1b). The filaments of the Fe-replete culture exhibited the phenotype characteristic for healthy Trichodesmium, i.e. they were up to several mm long (Fig. 1a1). After one week of iron limitation, growth gradually slowed down and finally stopped (Fig. 1b), and the filaments fragmented and shortened (Fig. 1a2). Under transmitted light microscopy, no other morphological differences were observed between the Fe-limited and Fe-replete cultures (Fig. 1a3,1a4).
Fig. 1. Morphology and growth of the Fe-replete and the iron-limited cultures.
a) Morphology of Trichodesmium filaments in the Fe-replete and Fe-limited cultures as observed by brightfield microscopy in the FKM. Left: 5x objective; Right: 40x; Top: Fe- replete; Bottom: Fe-limited culture 18 days after the induction of iron limitation, at about 50 nM Fe(III)-EDTA. b) Cell density, measured as OD750, of the Fe-limited cultures expressed in % of the Fe-replete cultures. The points represent average and standard error of two independent replicate experiments for both conditions.
The Fe-replete culture with 1 µM total Fe in the medium showed high rates of nitrogen fixation. The maximum rate per volume of culture was reached in the late afternoon (Fig. 2a).
Normalisation to Chl (= to compensate for the growth of the culture) shifted the maximum nitrogenase activity to the middle of the light period (Fig. 2b) due to Chl synthesis in the light period. Iron limitation very quickly inhibited nitrogen fixation. Nine days after starting to dilute out the iron in the chemostat, when the iron concentration declined to around 200 nM (total Fe content of the medium calculated from initial concentration and dilution rate), the nitrogen fixation per volume of culture was reduced to about 25% of the Fe-replete culture (Fig. 2a) or 50% when normalised to Chl (Fig. 2b). Three days later, when Fe in the medium was reduced to ~ 100 nM, nitrogen fixation of the Fe-limited culture declined to ~ 10% of the Fe-replete culture, and became almost undetectable 16 days after induction of iron limitation.
Although the biomass of the Fe-limited culture continued declining, 20 days after induction of Fe-limitation nitrogenase activity (normalised to Chl (Fig. 2b) or OD750 (not shown)) increased again almost reaching rates measured in the Fe-replete culture. Nevertheless, the Fe-limited cultures died three weeks after induction of iron limitation (Fig. 1b).
Iron limitation in Trichodesmium
Fig. 2. Nitrogen fixation in Fe-replete and Fe-limited cultures. The Fe-replete cultures showed the typical daily trends; the data points shown represent the average and standard error of four measurements of two independent experiments. a) Nitrogen fixation measured as nmol ethylene·h-1·(ml culture)-1. b) Nitrogen fixation measured as nmol ethylene·h-1·(µmol Chl)-1. Elemental analysis of carbon, nitrogen and phosphorus reflected the decreased nitrogen fixation with increasing C:N and a decrease in the N:P ratio (Fig. 3). While the N:P ratio declined continuously until 12 days after induction of Fe-limitation, the C:N remained steady at about 130% of the control value (C:N ~ 10.2; Fig. 3b) after 9 days of Fe-limitation. This was caused by a slower decrease of the C:P ratio compared to the N:P ratio at the beginning of the experiment. From twelve days after induction of Fe-limitation, both parameters (i.e.
N:P, C:P) decreased at about the same rate. At the end of the experiment, N:P was about 28%
of the control (N:P ~ 9) and C:P was about 39% of the control (C:P ~ 93; Fig. 3).
Fig. 3. Effect of iron limitation on C:N:P ratios.
a) C:N:P ratios averaged over the whole experiment for the two Fe-replete cultures and between 9-20 days after start of iron limitation for the two Fe-limited cultures. Thus, the means and standard errors shown are from 28 replicate samples for the Fe-replete and of 34 samples for the Fe-limited cultures. b) Development of C:N:P ratios during progressing Fe- limitation. For the day 0 level, again all values of the Fe-replete cultures were averages (i.e.
n=28), for all further time points all samples taken at that day were averaged (n = 1-5).
Changes of protein and pigment composition
FKM measurement of single-cell absorption spectra
Measuring absorption spectra of individual living cells in the FKM allows for direct monitoring of cellular pigment changes without any artefact-prone sample preparation. Iron limitation led to a slight increase in the total content of Chl (peaks at 680 nm and 435 nm minus the reference point at 750 nm) and carotenoids (shoulder at 465 nm) per cell (Fig. 4a).
Iron limitation in Trichodesmium Phycoerythrin content (peaks at 542 nm and 568 nm minus the valley at 593 nm) remained constant. Phycourobilin content per cell, measured from the peak amplitude at 495 nm to the local minimum before, was higher in the Fe-limited cultures compared to the Fe-replete cultures (Fig. 4a).
Analysis of chlorophyll, carotenoids and phycobiliproteins in extracts
Partial separation of pigments into an acetone extract for Chl and carotenoids and a phosphate buffered saline (PBS) extract for phycobiliproteins, combined with narrower absorption peaks in solution, allowed for a quantitative analysis of Chl and carotenoids. Moreover, the PBS extract enabled better assessment of relative changes between phycobiliproteins due to the removal of overlapping absorption of Chl and carotenoids.
The acetone extracts revealed a slow decrease of the Chl/carotenoid ratio until one day before the end of the experiments, and a steep decrease of this ratio on the last day of the experiment (Fig. 4b). The most dramatic change induced by Fe-limitation, however, was the appearance of high concentrations of keto-carotenoids (mainly echinenone), which were only a very minor component in Fe-replete cultures (Fig. 4b).
Since no direct normalisation per cell is possible for extracts, we used the observation from the in vivo spectra (see above) showing constant phycoerythrin content throughout the experiment, and normalised the PBS extract spectra to this peak. Spectra of PBS extracts differed from the single-cell in vivo spectra mainly in two ways. First, while in the in vivo spectra phycourobilin increased only slightly (Fig. 4a), in the PBS extracts a peak at about 504 nm, close to the normal absorption of phycourobilin (497 nm), dominated the spectra 18 and 20 days after the start of iron limitation (Fig. 4c). Additionally, the PBS extract spectra showed that the phycocyanin (peak at 620 nm) decreased under iron limitation (Fig. 4c).
Phycocyanin could not be analysed in the single-cell absorption spectra due to overlapping of the phycocyanin peak with the side peak of the red absorbance band (Qy band) of Chl a.
Fig. 4. Effect of iron limitation on pigment composition. a) Single-cell in vivo absorption spectroscopy of Trichodesmium filaments. Each graph represents the average of all single-cell spectra measured on all five time points of the daily activity cycle, and the average of both replicate experiments. For the absorption spectrum of the Fe-replete cells, additionally all days of the measurement were averaged. Background was corrected only by setting OD750 to zero. No normalization was done. b) Analysis of Chl and carotenoid composition in acetone extracts. Averages and standard errors of all samples measured between 12 and 20 days after start of iron limitation, altogether six samples from two independent experiments for each data point shown. Values for “chlorophylls” are the sum of Chl a and all Chl a degradation products (chlorophyllide a, pheophytin a, and oxidation products). c) Changes in phycobilisome composition of Trichodesmium under iron limitation analysed by absorption spectra of extracts in phosphate buffered saline. Each spectrum shown represents the average of 5 or 10 samples taken at the 5 different time points of the daily cycle. Since the single cell in vivo spectra, where artefacts of extraction can be excluded, showed constant phycoerythrin per cell (see Fig. 4a), the spectra shown here are normalised to the phycoerythrin peak of the Fe-replete cultures.
