1. Introduction. 1. Introduction. 1.1 Diatoms Diatoms in General

Transcription

1 1. Introduction 1.1 Diatoms Diatoms in General Diatoms belong to the division of the Bacillariophyceae within the phylum of Heterokontophyta. They are unicellular or chain forming algae that colonize seawater and freshwater habitats in most parts of the world and they can also be found on rocks, plants or mud that are present within or at the border of water. They are responsible for about 40% of the marine primary productivity (Field et al. 1998) and make the most important part of eukaryotic phytoplankton. One of their most characteristic features is their external wall made of amorphous silicate (SiO 2 ) n H 2 O. This cell wall consists of two parts, a larger and a smaller half, that fit together like a Petri dish (Figure 1.1). The larger half is called epitheca, and the other one is called hypotheca. They can be divided into two groups, the centric and the pennate diatoms, depending on the symmetry of the cell wall (Kooistra et al. 2003). The centric diatoms are radially symmetrical and are mostly planktonic whereas the pennate diatoms are elongated with a bilateral symmetry and are living mostly in benthic areas. Figure 1.1: Structure of a diatom with hypotheca and epitheca, the raphe and the central and polar nodules. During cell division, only the hypotheca is synthesized. Due to this fact, the diatoms become more or less smaller in every generation. After reaching a minimum size, the diatoms proliferate sexually.picture from 5

2 Like in other photosynthetic eukaryotes, the photosynthetic apparatus is localized in the plastids within the cells and placed in the thylakoid membranes. In Heterokontophytes the thylakoid membranes are arranged in a typical shape, each three thylakois are stacked (lamellae) all of them are surrounded by a girdle lamella of three stacked thylakoids (van den Hoek, 1997). In addition to Chl a and β-carotene, diatoms also have specific pigments like Chl c and xanthophylls (fucoxanthin, diadinoxanthin and diatoxanthin) Evolution of Diatoms and the Theory of Endocytobiosis According to the endocytobiotic theory plastids originate from photosynthetic prokaryotes that were engulfed by non-photosynthetic eukaryotic host cells. The first speculations about the relationship of plastids and prokaryotes were published by Schimper and Mereschkowsky at the beginning of the 20 th century (Schimper 1883, Mereschkowsky 1905). Because the plastids from diatoms are surrounded by four membranes the theory of the secondary endocytobiosis was established. It is believed that the four membranes, have developed from a photosynthetic eukaryote being swallowed by a secondary eukaryotic heterotroph (Figure 1.2): the two inner membranes are supposed to be the envelope membranes of the primary plastid (Delwiche and Palmer, 1997) whereas the third membrane (counted from the inside) might be the plasmamembrane of the primary endosymbiont. The outer membrane of the plastid seems to correspond to the plasmamembrane of the secondary host cell as it was sometimes seen with ribosomes (Bouck, 1965; Gibbs 1981) and it obviously needs a food vacuole to engulf the endosymbiont. 6

3 Fig. 1.2: Putative Evolution of Diatom Plastids. After engulfing a photosynthetic eukaryotic red alga by a eukaryotic host cell, a massive gene transfer from the nucleus of the endosymbiont (N1) to the nucleus of the host cell (N2) must have occurred (bent arrow). Degradation of nucleus, mitochondria (M) and other cytosolic structures of the endosymbiont results in plastids (P) with four bounding membranes, with the outermost membrane being transformed into an ER (endoplasmic reticulum) -type membrane (Kroth and Strotmann, 1999). 1.2 Photosynthesis General Plants and algae are able to absorb and to convert light energy into chemical energy via the process of photosynthesis. This reaction can be divided into two parts: In the so called light reaction the light energy is converted into chemical energy in the form of NADPH, at the same time water (H 2 O) is oxidized and oxygen (O 2 ) is released. This process drives a thylakoidal proton gradient coupled to electron transport, which allows the production of ATP via the ATP synthase. In the second reaction, CO 2 is assimilated in the Calvin cycle, localized in the stroma of the plastids. The Calvin cycle is often called dark reaction even though it is also taking place at light. This part of the photosynthesis is not dependent on light energy. During this reaction the ATP and NADPH molecules, produced at 7

4 light, molecules are used. The photoreactions provide the necessary energy for the organism to grow and reproduce The Photochemistry The thylakoid membranes carry the photosynthetic complexes, the photosystems I and II (PSI and PSII), the Cyt b 6 /f complex and the ATP-Synthase. The photosystems contain a light harvesting protein complex (LHC), consisting of several hundred chlorophylls (a,b,c) and accessory carotenoids, because every pigment does not absorb light of a certain wavelength (e.g. Chl a does not absorb green light). With this antenna they are able to absorb a larger spectrum of wavelengths than with just one sort of pigment molecules. The antenna pigments collect the photons and transfer the excitation energy from pigment to pigment to the second part of the photosystem, the reaction center (RC). There are several Chl a molecules in the center of PSII, also called P680, which are responsible for the first charge separation. P680 is excited by the excitation energy in P680 * and transfers an electron to a pheophytin (Chl a without magnesium). P680 * relaxes to P680, using the electrons that were produced during the oxidation of water (2H 2 O O 2 + 4e - + 4H + ). The electron is then given from the reduced Pheophytin to Quinone A (Q A ), which is bound to the reaction center protein complex. Q A transfers the electron to Quinone B (Q B ), another bound Quinone molecule. When Q B has received two electrons from Q A it detaches from the reaction center and diffuses through the hydrophobic core of the thylakoid membrane to the Cyt b 6 /f complex. The electrons are passed to a iron-sulfur protein (Rieske Protein) and then to a mobile copper protein, the Plastocyanin (Pc) which finally carries a single electron to the oxidized reaction center of Photosystem I, the P700 +, which is reduced to P700. After light driven charge separation, the electrons are transferred via several steps from P700 to a soluble ferredoxin at the stromal side of the thylakoid membrane. At last a flavoprotein, the ferredoxin-nadp + -reductase (FNR) passes the electron from ferredoxin to the final electron acceptor NADP + which is thereby reduced to NADPH that can be used in the Calvin cycle (Figure 1.3). In parallel, the ATP-Synthase is using the trans-thylakoidal proton gradient ( ph) to produce ATP. This gradient develops because of the four protons that 8

5 are released for every O 2 in the PSII and the double reduction of Q B. When the double protonated plastoquinone PQH 2 is oxidized the protons are liberated into the thylakoidal lumen and can be used by the ATP-Synthase. Figure 1.3: The Z-Scheme of the photosynthesis shows the electron transport pathway during the light reactions of the photosynthesis. Abbreviations used are (from left to the right of the diagram): Mn: manganese complex containing 4 Mn atoms; Tyr: particular tyrosine in PSII; O 2 : oxygen; H + : protons; P680: reaction center chlorophyll (Chl) in PSII; P680*: excited P680; Pheo: pheophytin molecule : Q A : quinone A; Q B : quinone B; FeS: Rieske Iron Sulfur protein; Cyt. F: Cytochrome f; Cytb6(L and H): Cytochrome b6 ; PC: plastocyanin; P700: reaction center chlorophyll of PSI; P700*: excited P700; Ao: chlorophyll a molecule); A 1: phylloquinone (Vitamin K) molecule; FX, FA, and FB: three separate Iron Sulfur Centers; FD: ferredoxin; FNR: Ferredoxin-NADP-oxido- Reductase(FNR). ATP is synthisized by the ATP synthase located in the chloroplast membranes utilizing the protons released from water and reduced plastoquinone ( 1.3 Photoprotection Many plants and algae have to cope with light stress. For example diatoms have to deal with large fluctuation of light intensity as they are immobile and are exposed to water motions, especially in coastal and estuarine areas (Figure 1.4). 9

6 MacIntyre et al., 2000 Figure 1.4: Fluctuations of the light intensity as a function of time in estuarine (coastal) and oceanic areas, submitted to a low and high turbulence of water, respectively. PAR: Photosynthetic Active radiations. (MacIntyre et al., 2000) A consequence from light stress is photoinhibition, which is a result of an unbalance between the absorption of the light energy and its use. Exposure to high light can be harmful for photosynthesis, generating photo-oxidative damages within the chloroplasts (Falkowsky and Raven, 1997). To survive high light exposure and light fluctuations, plants and algae developed regulatory mechanisms, which are rapidly induced within the photosynthetic apparatus and allow the dissipation of excess energy. To regulate photosynthesis during very rapid changes of light intensity, the mechanism of non-photochemical quenching of fluorescence (NPQ), by which excess energy is turned into heat, is very important (Holt et al., 2004). This process is controlled by the ph over the thylakoid membrane, by the enzymatic conversion of xanthophylls (xanthophylls cycle, Figure 1.6) and at least by one special polypeptide (PsbS in higher plants). In Diatoms the capacity of NPQ can be very large (2 to 5 times the values in higher plants, see Figure 1.5) and the kinetics are very rapid. It has already been shown that this high capacity to dissipate excess energy is very effective in protecting the photosynthesis activity (Lavaud et al., 2002). 10

7 Fluorescence (a. u.) Fm 8 6 Fo 4 2 Synechococcus NPQ = 1.5 NPQ Fm' Figure 1.5: Non-photochemical quenching (NPQ = (Fm-Fm )/Fm ) in cells of P. tricornutum, leaves of A. thaliana and the cyanobacterium Synechococcus PCC6803 induced by a saturating actinic white light intensity (2000 µmol m -2 s -1 ). P saturating pulse; AL arrow up, light on; arrow down, light off. F 0, minimal level of fluorescence emission in dark-adapted cells and leaves; Fm, maximal level of fluorescence in illuminated cells and leaves. Data from Ruban et al and Cadoret et al Hence, diatoms are able to preserve their photosyntheitic productivity in a highly variable light environment (Falkowski et al. 1997). The high NPQ capacity is due to the high amount of the xanthophylls diadinoxanthin/diatoxanthin (DD/DT), the special pigments of the diatoms (Lavaud et al. 2003). Low light, darkness Excess light Figure 1.6: The xanthophyll cycle in diatoms. The diadinoxanthin (DD) is deepoxidized into diatoxanthin (DT) via the diadinoxanthindeepoxidase (DDE) under high light exposure. Under low light or darkness DT is epoxidized back to DD (Pfündel et al., 1994) 11

8 The regulation of the extent and the kinetics of NPQ is dependent on the irradiance as well as the light regime the cells are acclimated to (Lavaud et al. 2002). Under high light acclimation, the synthesis of DT and the relative amount of DD/DT is increased. The more DD/DT is present in the cells, the better they can cope with light stress conditions because, amongst other reasons, they can develop a very high capacity for NPQ. The xanthophylls are bound to the LHC subunits which are composed of several highly homologous proteins encoded by a multigene family (fucoxanthin chlorophyll proteins, Fcp) (Bhaya and Grossman, 1993). These proteins are closely related to the Chlorophyll a/b binding proteins (Cab proteins) as described in the following chapter High light induced proteins (Hlips) and Photoprotection The Hlips are members of the Elip (early light induced protein) protein family, which can be separated into three groups, differing in their number of transmembrane helices: the three-helices Elips and related proteins, the two-helix Seps (stress enhanced proteins) also called Lils (light-harvesting-like) and the one-helix Hlips (high light induced proteins), Scps (small Cab-like proteins) and Ohps (one-helix proteins) (Adamska 2001) (Figure 1.7). Proteins of the Elip family were first described by Kloppstech and coworkers (Meyer and Kloppstech, 1984; Grimm and Kloppstech 1987). The Elip family consists of stress proteins with low molecular weight that are located in the thylakoid membranes of pro- and eukaryotes. They are related to the light-harvesting antenna proteins of higher plants and green algae. The first Helix of all Elip family members is highly conserved in its amino acid composition and contains an Elip consensus motif. 12

9 Figure 1.7: Protein structure of the Elip family members. The predicted secondary structure allows the division into three groups: The Elips and related proteins with three transmembrane helices, the two-helix Seps (also called Lils) and the one-helix Hlips, Scps and Ohps. The transmembrane helices I and III are highly conserved, while the helix II is polymorphic and differs between these proteins (Adamska 2001) The relation of the Elips to Cab (chlorophyll a/b-binding proteins) and Fcps (fucoxanthin-chlorophyll -binding proteins) has been deduced from their sequence similarities. The main difference between them is that Cab proteins are structural components of PSI and PSII, whereas the expression of Elips is only induced under special physiological conditions. In general, Elips are expressed under light stress, dehydrative stress and morphogenesis and are also thought to play a role in the regulation of NPQ in cyanobacteria and green algae (Adamska 2001). Another relative of the Elips is the PsbS protein of higher plants which is essential for the NPQ process. It differs from the Elips, Cabs and Fcps in a fourth transmembrane helix, but the sequences of the single helices showed a clear relation to the other Cab protein family members. One striking difference was that the expression of PsbS is not dependent on light and was stable even in the absence of pigments (Funk, 1999). Li et al. showed that an NPQ deficient mutant of Arabidopsis thaliana mutant had a single nuclear mutation in the PsbS gene (Li et al., 2000). The Hlips were first described in the cyanobacterium Synechocystis PCC 6803 by He et al., They showed that all Hlips were overexpressed in high light and that Hlip mutants were not able to cope with high light conditions. Hence the Hlips are critical for the surviving of Synechocystis. More precisely it has been shown in 13

10 cyanobacteria that Hlips could be involved in the NPQ mechanism (Havaux et al., 2003). Additionally, in the green alga Dunaliella sp. an Elip like protein (Cbr) plays a role in the regulation of both the xanthophyll cycle and the NPQ mechanism (Braun et al., 1996). 1.4 Aim of the Project Members from the Elip family have been found in the genome of P. tricornutum (Gruber and Lavaud, unpublished). They consist of 2 Hlips, 1 Sep, 1 Elip and 2 putative Elips. Nothing is known regarding the regulation of their synthesis, localisation in the photosynthetic apparatus and the physiological role of these proteins in diatoms. Therefore the purpose of this work is to start some investigations on the Elip family of diatoms. One of the most striking features of the diatom NPQ is the absence of a PsbS protein (Armbrust et al., 2004). It is likely that as in Chlamydomonas (Elrad, 2002) another antenna-related protein plays a similar role. Thus one of our working hypothesis is that one (or some) of the members of the Elip family might be involved in the NPQ mechanism in diatoms. In the recent years, cells of P. tricornutum, which show a high capacity for high light acclimation and NPQ, were grown and characterized (Lavaud 2002). These high NPQ cells show an increase in the amount of DD/DT in specific subfractions of the LHC antenna as well as an increase/presence of specific polypeptides (Lavaud 2003, unpublished). The localisation of these polypeptides within the LHC antenna would help to identify them. The first purpose of this work has been to set-up in Konstanz the necessary methods to pursue the investigations on these cells, like the purification of the photosynthetic complexes by gel filtration and the characterization of their pigment and polypeptide contents. In parallel, some tools were set up to gain some information on the localization of two members of the Elip family: Sep and Hlip2. The first step was to verify the targeting of these polypeptides to the plastid. Second, the production of specific antibodies for future investigations on the precise localization of Hlips within the LHC antenna system was started. 14