«Tünde Tóth Thesis committee Promotor Prof. Dr H. van Amerongen Professor of Biophysics Wageningen University Co-promotors Dr G. Garab Group leader, ...»
Response of the photosynthetic
system to altered protein composition
and changes in environmental
Prof. Dr H. van Amerongen
Professor of Biophysics
Dr G. Garab
Group leader, Institute of Plant Biology
Biological Research Centre, Szeged, Hungary
Dr L. Kovács
Senior research associate, Institute of Plant Biology
Biological Research Centre, Szeged, Hungary
Prof. Dr O. van Kooten, Wageningen University Dr J.C.P. Matthijs, University of Amsterdam Dr J.P. Dekker, VU University Amsterdam Prof. Dr C. Mullineaux, Queen Mary University of London, United Kingdom This research was conducted under the auspices of the Graduate School of Experimental Plant Sciences Response of the photosynthetic system to altered protein composition and changes in environmental conditions Tünde Tóth Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Wednesday 3 September 2014 at 11 a.m. in the Aula.
Tünde Tóth Response of the photosynthetic system to altered protein composition and changes in environmental conditions, 150 pages.
PhD thesis, Wageningen University, Wageningen, NL (2014) With references, with summaries in Dutch and English ISBN 987-94-6257-050-4 “Life is bottled sunshine” Winwood Reade, 1874 Table of contents Abbreviations 2 Chapter 1 4 Introduction Chapter 2 27 Carotenoid deficiency induces structural and functional changes in the phycobilisomes and photosystems of Synechocystis PCC 6803 Chapter 3 68 The PsbW protein stabilizes the supramolecular organization of Photosystem II in higher plants Chapter 4 101 The role of light-harvesting complex II in the macro-organisation of thylakoid membranes, as revealed by circular dichroism spectroscopy in vivo Chapter 5 132 Cadmium exerts its toxic effects on photosynthesis via a cascade mechanism in the cyanobacterium, Synechocystis PCC 6803 Chapter 6 157 Genera
Introduction 4 Introduction Scope of the thesis Photosynthesis is one of the fundamental processes on Earth, which is responsible for the transformation of inorganic carbon to organic forms and it also determines to a large extent the composition of the atmosphere. Therefore, there is a large interest to understand the basic processes of photosynthesis and the organization of the photosynthetic apparatuses as well as the impact of environmental factors on their performance. Photosynthesis research represents a broad and diverse field and many aspects have been studied intensively for over more than two centuries. After many aspects have been investigated at the molecular level in vitro for several decades, there is now a renewed interest in the study of the performance of the various complexes in vivo, trying to couple the obtained knowledge of isolated complexes to the overall performance in situ.
The thylakoid membranes, where virtually all light reactions of photosynthesis take place, is a well-organised hierarchic system, but the interaction between complexes might be relatively instable and possibly change upon membrane isolation. Therefore, in this thesis we have tried to apply mostly in vivo spectroscopic (absorption and fluorescence spectroscopy/microscopy) methods to investigate the photosynthetic apparatus of various organisms under various conditions.
In this thesis we used photosynthetic organisms with modified pigmentor protein-compositions to investigate their effect on the organisation and function of the photosynthetic pigment-protein complexes. We also studied the responses of the photosynthesis apparatus to the toxic heavy metal pollutant cadmium.
Simplified model of the photosynthetic electron transport chain of oxygenic photosynthetic organisms. Abbreviations: PSI, Photosystem I; PSII, Photosystem II; PQ, plastoquinone; PC, plastocyanin; Cyt b6f, cytochrome b6f complex; ATPase, ATP synthase.
The key components of photosynthetic energy conversion system are PSI, PSII, the cytochrome b6f (Cyt b6f) and the ATP synthase (ATPase), protein complexes, which are embedded in the thylakoid membranes [1-3]. The intersystem electron transport occurs by the use of electron carriers, i.e., the lipid soluble pool of plastoquinone (PQ) molecules, the membrane-intrinsic cyt b6f protein complex and the water-soluble small plastocyanin (PC) protein, which is localised in the thylakoid lumen (Fig. 1.1). The light reactions of photosynthesis reduce NADP+ to NADPH on the stromal side, and generate a proton gradient across the membrane and a transmembrane electrical field, components of the electrochemical potential gradients for protons. This is used by the ATP synthase to convert ADP to ATP and the process known as photophosphorylation.
Introduction1.1.2 Photosynthetic model organisms The cyanobacterial Synechocystis PCC 6803 strain and the higher plant Arabidopsis thaliana used in this thesis are commonly applied model organisms in photosynthesis research. Their entire genomes have been sequenced [4, 5] and extensive mutant collections are available for these organisms.
Cyanobacteria are ancient prokaryotes with a relatively small genome size (3.6 Mbp)  and low degree of complexity. Due to their relative simplicity cyanobacteria are commonly used model organisms to study basic photosynthetic processes. In the presence of adequate carbon sources some of the cyanobacterial strains, including Synechocystis PCC 6803, are able to grow heterotrophically without operating photosynthesis . This property makes Synechocystis PCC 6803 strain suitable to study several photosynthesis-related mutations which otherwise would be lethal [7, 8].
Higher plants possess a bigger genome size, with more complex organisation and combined physiological responses. One of the most widely studied model plants is Arabidopsis thaliana (Arabidopsis). Its relatively small genome (125 Mbp) makes it advantageous to work with Arabidopsis, but the results obtained on these plants can be also relevant for crops.
1.2 Photosynthetic pigments In photosynthetic organisms pigments are responsible for the absorption of light. They are positioned close together in the pigment-protein complexes to provide efficient energy transfer. The different photosynthetic taxa possess different pigment species owing to evolutionary adaptation to various environmental conditions. In this section the pigments occurring in cyanobacteria and higher plants are described in details.
Molecular structure of chlorophyll a, chlorophyll b (A) and phycocyanobilin (B) pigments 7
Chapter 11.2.1 Chlorophylls Chlorophylls are cyclic tetrapyrrole molecules, and more precisely, belong to the group of reduced porphyrins. Their porphyrin ring contains a magnesium atom in the centre and is associated with a phytol tail consisting of four isoprene units. All photosynthetic organisms contain chlorophyll-type pigments, but their fine structure is characteristic for the taxa . Cyanobacteria contain only chlorophyll a (Chl a), while higher plants also contain chlorophyll b (Chl b) (Fig. 1.2). These two types of chlorophylls are different only in one functional group, which is a methyl group in Chl a and a formyl group in Chl b. Inspite of their structural similarity, Chl a and Chl b have distinct spectral properties. The chlorophylls show most absorption in the near-UV region, the Soret (By and Bx) bands, and in the near-IR, the Qy and Qx bands. Chl a absorbance peaks are found in the ~ 350-450 nm and ~ 650-700 nm regions and Chl b around ~ 400nm and ~ 625-675 nm (Fig. 1.3).
There are some special chlorophylls in photosynthetic systems. In photosystem I complexes a special Chl a stereoisomer is present, the so called Chl a’, which forms one half of the special P700 Chl pair. The pheophytins are metal-free chlorophylls, where the magnesium is replaced by hydrogens. Two pheophytin molecules are present in photosystem II where they have important function in the process of charge separation. On the other hand the pheophytin can also be a product of chlorophyll degradation.
Fig 1.3. The absorption spectra of various photosynthetic pigments: β-carotene (orange), chlorophylls a (light green) and b (dark green), dissolved in non-polar solvents. Phycocyanin is a protein containing phycocyanobilin pigments covalently bound to the peptide, it was measured in aqueous buffer (cyan). Red curve, represents the solar spectrum incident on the surface of Earth.
Introduction1.2.2 Bilins Bilins are open-chain tetrapyrrole molecules. They are present in the phycobilisome antennae of cyanobacteria, red algae, glaucophytes and some cryptophytes. They are unique in a way that they are covalently bound to the proteins via thioether bounds. The most common bilins are phycocyanobilin and phycoerytrobilin (Fig. 1.3). In Synechocystis PCC 6803 strain, used in this thesis, phycocyanobilin is present only (Fig. 1.2), but exists in two spectrally different forms depending on the phycobiliprotein to which it is bound.
Phycocyanobilin in allophycocyanin has more red-shifted absorption and fluorescence spectra than in phycocyanin.
1.2.3 Carotenoids Carotenoids (Cars) are the most wide-spread pigments in nature. Cars are tetraterpenes comprising eight isoprene units. They contain double bonds in a conjugated system, which is responsible for the colour and rod-shape of the molecules. Carotenes and xanthophylls form the two main classes of Cars.
Carotenes, such as β-carotene, are pure hydrocarbon molecules containing a cyclohexene ring (ionone ring) at one or both ends. They are extremely hydrophobic, non-polar molecules. Xanthophylls carry one or more oxygencontaining functional groups, generally attached to their terminal ionone rings (e.g., echinenone, zeaxanthin). In Cars glycosides, a sugar group is attached to the molecule at one side instead of the ionone ring (e.g., myxol-dimethylfucoside) (Fig. 1.4 A). In photosynthetic organisms, carotenoids are typically localised in protein complexes and lipid membranes and their location is highly influenced by the polarity of Cars and direct environment. Both photosynthetic reaction centre core complexes (PSI and PSII) contain Cars and this is also the case for light-harvesting complexes of higher plants (see section 1. 3). Cars have various functions in photosynthetic organisms . They can serve as accessory light-harvesting antenna pigments by absorbing in the blue-green region of the solar spectrum (Fig. 1.3) . Cars have also protective roles exerted by quenching of singlet or triplet excited state Chl upon excess light conditions and by directly scavenging the singlet excited state oxygen [11, 12]. Cars also decrease the susceptibility of lipid-membranes to oxidative degradation . In lipid bilayers the Car molecules seem to significantly influence the membrane fluidity and form penetration barrier of small molecules, including oxygen .
9 Chapter 1
Figure 1.4. Chemical structure of three carotenoid molecules (A) adapted from Domonkos et al. (2013), and a simplified scheme of the biosythesis pathways of the most abundant carotenoids of Synechocystis PCC 6803 (B). White colour represents the non-polar parts of the molecule and red colour, the polar regions. Relative amounts are shown in parantheses. CrtB, phytoene synthase;
CrtP, phytoene desaturase; CrtQ, ζ-carotene desaturase; CrtH, cis-trans carotene isomerase; CrtR, carotene β-hydroxylase; CrtO, carotene β-ketolase.
In Synechocystis the main Car forms are as follows: β-carotene (~26% of total carotenoid content), echinenone (~18%), zeaxanthin (~14%) and myxoxanthophyl (~36%) (present as myxol 2’-dimethyl-fucoside) . The biosynthesis of Cars was recently reviewed by Domonkos and co-workers .
Figure 1.4 B shows the key steps of the synthesis and the catalysing enzymes.
The first step of Car synthesis is the condensation of two geranylgeranyldiphosphates by the phytoene synthase (CrtB) enzyme . The produced cisphytoene is transformed to all-trans-lycopene via multiple steps, which include desaturation and isomerisation. The desaturation steps are catalysed by phytoene desaturase (CrtP) and ζ-carotene desaturase (CrtQ). Until now only one cis-trans carotene isomerase (CrtH) has been characterized from cyanobacteria.
Masamoto and co-workers  demonstrated that the presence of the CrtH enzyme or light is essential for the isomerisation steps, and thus for the production of all-trans-lycopene. Subsequent steps require the presence of cyclases. Formation of one ionone ring results in the production of γ-carotene, which is a branching point of the synthesis. γ-carotene can be used to synthetize myxoxanthophyll, which, in Synechocystis, is myxol 2’-dimethyl-fucoside .
γ-carotene is also used for the production of β-carotene by the formation of the 10
Introductionionone ring on the other side of the molecule. β-carotene is an intermediate product for echinenone and zeaxanthin synthesis. The final form of the xanthophylls require the presence of carotene β-hydroxylase (CrtR) and carotene β-ketolase (CrtO) .
1.3 Photosynthetic pigment-protein complexes
1.3.1 Photosynthetic reaction centres Photosynthetic reaction centres (RCs) are special pigment-protein complexes where light induces electron transfer reactions, which results in charge separation across the membrane. The basic structure of the photosystems is evolutionary highly conserved and rather similar for cyanobacteria and higher plants [1, 3].