NUCLEOTIDE-DEPENDENT PROCESSES IN THE THYLAKOID LUMEN OF PLANT CHLOROPLASTS

Björn Lundin

Division of Molecular Genetics

Department of Physics, Chemistry and Biology Institute of Technology Linköping University

SE-581 83 Linköping, SWEDEN

Linköping 2008

© Björn Lundin, 2008 © Cover design: Björn Lundin All rights reserved ISBN: 978-91-7393-965-2 ISSN: 0345-7524 Linköping studies in science and technology. Dissertation No. 1165 Published articles and Figure 2 have been reprinted with the permission of the respective copyright holder: Paper I © 2004, The National Academy of Sciences of the USA Paper II © 2007, Elsevier B.V. Paper III © 2007, Blackwell Publishing Ltd Paper IV © 2008, Springer This work has been supported by the Swedish Research Council and the Graduate Research School in Genomics and Bioinformatics (FGB). We thank also the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants. Printed by: LIU-TRYCK, Linköping, Sweden, 2008

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Ph. D. Thesis Thesis No. 1165

Nucleotide-Dependent Processes in the Thylakoid Lumen

of Plant Chloroplasts

Björn Lundin

Supervisor: Docent Cornelia Spetea Wiklund Division of Molecular Genetics Department of Physics, Chemistry and Biology Linköping University 581 83 Linköping, Sweden Opponent: Professor Zach Adam Institute of Plant Sciences

The Hebrew University 761 00 Rehovot, Israel

Linköping, 4 April, 2008

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“If you have no results, you haven’t worked” Anonymous

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ABSTRACT

Plants, algae and photosynthetic bacteria are able to harvest the sunlight and use its energy to

transform water and carbon dioxide to carbohydrate molecules and oxygen, both important to

sustain life on Earth. This process is called photosynthesis and is the route by which almost all

energy enters the biosphere. As most simple things in life, the process of photosynthesis is easily

explained but unfortunately not that easy to reproduce. If we could, we would be living in a much

different world with almost unlimited energy. Light energy is harvested by chlorophyll molecules,

bound to proteins in the chloroplast thylakoid membrane and drives the oxygen-evolving

complex, to extract electrons from water. Electrons are then transferred to NADPH through

photosystem II (PSII) to cytochrome b6f and photosystem I, the major photosynthetic protein

complexes. The cytochrome b6f complex also transfers protons into the lumenal space of the

thylakoid. These protons together with those from water oxidation create an electrochemical

gradient across the thylakoid membrane, which fuels the ATP synthase to produce ATP. ATP,

NADPH and carbon dioxide are used during the dark reactions to produce sugars in the

chloroplast stroma. The thylakoid lumenal space where the water oxidation occurs has until

recently been viewed as a proton sink with very few proteins. With the publication of the genome

of Arabidopsis thaliana it seems to be a much more complex compartment housing a wide variety

of biochemical processes.

ATP is a nucleotide and the major energy currency, but there are also other nucleotides

such as AMP, ADP, GMP, GDP and GTP. Chloroplast metabolism has mostly been associated

with ATP, but GTP has been shown to have a role in integration of light harversting complexes

into the thylakoid. In this work, we have demonstrated the occurrence of nucleotide-dependent

processes in the lumenal space of spinach by bringing evidence first for nucleotide (ATP)

transport across the thylakoid membrane, second for nucleotide inter-conversion (ATP to GTP)

by a nucleoside diphosphate kinase, and third the discovery that the PsbO extrinsic subunit of

PSII complex can bind and hydrolyse GTP to GDP. The active PSII complex functions as a

dimer but following light-induced damage, it is monomerised allowing for repair of its reaction

center D1 protein. PsbO is ubiquitous in all oxygenic photosynthetic organisms and together

with other extrinsic proteins stabilises the oxygen-evolving complex. We have modelled the

GTP-binding site in the PsbO structure and showed that the GTPase activity of spinach PsbO

induces changes in the protein structure, dissociation from the complex and stimulates the

degradation of the D1 protein, possibly by inducing momerisation of damaged PSII complexes.

As compared to spinach, Arabidopsis has two isoforms of PsbO, PsbO1 and PsbO2, expressed in

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a 4:1 ratio. A T-DNA insertion knockout mutant of PsbO1 showed a retarded growth rate, pale

green leaves and a decrease in the oxygen evolution while a PsbO2 knockout mutant did not

show any visual phenotype as compared to wild type. Unexpectedly, during growth under high

light conditions the turnover rate of the D1 protein was impaired in the PsbO2 knockout,

whereas it occurred faster in the PsbO1 knockout as compared to wild type. We concluded that

the PsbO1 protein mainly functions in stabilizing the oxygen evolving complex, whereas the

PsbO2 protein regulates the turnover of the D1 protein. The two PsbO proteins also differ in

their GTPase-activity (PsbO2 >> PsbO1). Although their amino acid sequences are 90%

identical, they differ in the GTP-binding region which could explain the difference in their

GTPase activity. Based on these data, we propose that the GTPase activity of PsbO(2) leads to

structural changes in interacting loops and plays a role in the initial steps of D1 turnover such as

the PSII monomerisation step.

The nucleotide-dependent processes we discovered in the thylakoid lumen raise

questions of transporters to facilitate these processes. As stated earlier, we provided biochemical

evidence of an ATP thylakoid transporter, and most recently have identified a transporter that

may be important for the export of lumenal phosphate back to the stroma. More transporters for

GDP, metal ions and others solutes have still to be identified.

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POPULÄRVETENSKAPLIG SAMMANFATTNING

Växter, alger och fotosyntetiska bakterier lärde sig för 2.5-3 biljarder år sen att absorbera

solljusets energi och använda det för att omvandla vatten och koldioxid till kolväten. Denna

process kallas för fotosyntes och är den enskilt största energibindaren på jorden. Ljusenergin

absorberas av klorofyll-molekyler bundna till proteiner lokaliserade i kloroplastens tylakoid-

membran som driver själva vattenspjälkningen. Elektroner från vattnet vandrar till NADPH

genom de stora fotosystem-komplexen; Fotosystem II, Cytochrome b6f komplexet och

Fotosystem I. Cytochrome b6f komplexet har också som funktion att låta protoner vandra till

lumen, som är lokaliserad inne i tylakoiderna. Dessa protoner tillsammans med protonerna från

vattnet skapar en protongradient över membranet, vilket fungerar som drivmedel för ATP-

syntasens ATP produktion. ATP, NADPH och koldioxid används i mörker-reaktionen för att

producera sockerarter i stroma. Lumen där själva vattenspjälkningen sker, har tills nyligen antagits

vara en proton-uppsamlare med väldigt få proteiner. Med publikationen av backtravs genom har

en mer komplex syn på detta utrymme vuxit fram.

Nukleotiden ATP, är den huvudsakliga energitransportören nukleotid, men det finns

också andra nukleotider, till exempel AMP, ADP, GMP, GDP och GTP. Kloroplastens

metabolism har för det mesta blivit kopplat till ATP, men GTP har visat sig ha en roll vid

integrationen av ljusuppsamlande komplex in i tylakoid-membranet. I detta arbete har vi

demonstrerat förekomsten av nukleotid-beroende processer i lumen; för det första genom

nukleotid (ATP) transport över membranet; för det andra via nukelotid-konvertering, ATP till

GTP, med hjälp av en Nukleosidediphosphatekinase i lumen; för det tredje, upptäckten att

proteinet PsbO, som binder in till PSII och optimerar och skyddar vattenklyvningen, kan binda

och hydrolysera GTP till GDP.

De fotosyntetiska organismerna, särskilt växter, har svårt att röra på sig och kan inte

fysiskt skydda sig från överexponering av ljus och väder. Ljus, som är fotosyntesen drivkraft, är

också en direkt fara. För starkt ljus ger skada på fotosystem-komplexen och för att skydda sig för

det har flera skyddsåtgärder ”uppfunnits”. Reaktionscenter D1 proteinet är det protein som utstår

mest skada och har en av cellens snabbaste omsättning. Det aktiva PSII komplexet, som fungerar

som en dimer, måste monomeriseras innan det skadade D1 proteinet kan bytas ut. PsbO finns

närvarande i alla fotosyntetiska organismer och stabiliserar vattenklyvnings komplexet. GTP

aktiviteten av spenats PsbO ger strukturförändringar, dissociering från PSII och stimulerar

degradering av D1 proteinet. Arabidopsis thaliana har till skillnad från spenat, två isoformer av

PsbO; PsbO1 och PsbO2.

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En knockout-mutant av PsbO1 visar ett långsammare växtsätt, ljusgröna blad och en

minskning av syrgas-produktionen. PsbO2 knockout mutanter uppvisar däremot inte några

skillnader jämfört med vildtyps plantor. Oväntat så hade PsbO2 knockout mutanten, växande

under starkt ljus nedsatt D1 reparation medan det skedde fortare i PsbO1 knockout mutanter när

man jämförde det mot vildtyp. Vi kan dra slutsatsen att PsbO1 proteinets huvudsakliga funktion

är att stabilisera syrgaskomplexet medan PsbO2 proteinet reglerar D1 proteinets reparations-

cykel. Dessutom skiljer sig Arabidopsis isoformer av PsbO i deras GTPase aktivitet (PsbO2 >>

PsbO1). Fast deras aminosyra-sekvens är 90% identisk, skiljer de sig i GTP bindande områden,

vilket skulle kunna förklara aktiviteten. Baserat på detta föreslår vi att PsbO(2)s GTPase aktivitet

leder till struktuella förändringar och spelar en viktig roll i monomeriseringsteget av PSIIs D1

protein reparations-cykel.

De nukleotidberoende processer vi visat ger upphov till fler frågor om transportörer

över tylakoid-membranet, som kan transportera in och ut nödvändiga ämnen för processerna. Vi

har visat förekomsten av en ATP thylakoid transportör och vi har nyligen identifierat en

fosfattransportör som kan ha en viktig roll för regulationen av Pi innehållet i lumen. Andra

transportörer för till exempel GDP, metall joner och andra lösliga ämnen finns fortfarande kvar

att upptäcka.

ORIGINAL PUBLICATIONS

This thesis is based on the following publications, which will be referred to in the text by their

Roman numerals (I-V).

I. Spetea, C., Hundal, T., Lundin, B., Heddad, M., and Andersson, B. Multiple

evidence for nucleotide metabolism in the chloroplast thylakoid lumen. Proc. Natl. Acad.

Sci. USA 101, 1409-1414 (2004).

II. Lundin, B*., Thuswaldner, S*., Shutova, T., Eshaghi, S., Samuelsson, G,. Barber,

J., Andersson, B., and Spetea, C. Subsequent events to GTP binding by the plant

PsbO protein: structural changes, GTP hydrolysis and dissociation from the photosystem

II complex. Biochim. Biophys. Acta (Bioenerg.) 1767, 500-508 (2007).

*These authors equally contributed to this work.

III. Lundin B., Hansson, M., Schoefs, B., Vener, A. V., and Spetea, C. Arabidopsis

PsbO2 protein regulates dephosphorylation and turnover of the photosystem II reaction

centre D1 protein. Plant J. 49, 528–539 (2007).

IV. Lundin, B., Thuswaldner, S., and Spetea, C. Arabidopsis PsbOs differ in their GTPase

activity. In: Photosynthesis: Energy from the Sun (Allen, J.F. Ed) Springer publisher, chapter 5,

773-775 (2008).

V. Ruiz Pavón, L., Lundh, F., Lundin, B., Mishra, A., Persson, B. L., and Spetea, C.

Arabidopsis ANTR1 is a thylakoid Na+-dependent phosphate transporter -functional

characterization in Escherichia coli. J.Biol. Chem. revised manuscript submitted 25.02.2008.

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OTHER PUBLICATIONS

Spetea, C., Ruiz Pavón, L., Thuswaldner, S., Lundin, B., Lundh, F., Persson, B. L.,

Schoefs, B., and Adamska, I., Screening for solute transporters in plant photosynthetic

membranes. In: Photosynthesis: Energy from the Sun (Allen, J.F. Ed) Springer publisher, chapter

4, 1075-1078 (2008).

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ABBREVIATIONS

ATP adenosine 5’-triphosphate (energy carrier produced during photosynthesis) ADP adenosine 5’-diphosphate CP chlorophyll-binding protein CD circular diochrism Cyt b6f cytochrome b6f Chl chlorophyll D1 32 kDa D1 subunit of the photosystem II reaction center DNA deoxyribonucleic acid (where genes are coded) GTP guanosine 5’-triphosphate kDa kilo Dalton (weight unit for proteins) LC liquid chromatography LHC light-harvesting complex MALDI matrix-assisted laser desorption ionization MS mass spectrometry m/z mass over charge ratio (x-axis in MS spectrum) NADPH nicotinamide adenine dinucleotide phosphate, reduced form (reducing

potential produced in photosynthesis) NDPK nucleoside diphosphate kinase OEC oxygen-evolving complex Pi Inorganic phosphate Psb(O,P,Q,R) 33, 23, 16, 10 kDa extrinsic subunits of the photosystem II complex psbo(1,2) knockout mutants of PsbO(1,2) PSI photosystem I PSII photosystem II PQ plastoquinone RC reaction center SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis TAAC thylakoid ATP/ADP carrier TOF time of flight (detector in MS) WT wild type

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TABLE OF CONTENTS

Abstract ............................................................................................................................................. VII

Populärvetenskaplig sammanfattning ............................................................................................ IX

Original Publications .......................................................................................................................XI

Other Publications .......................................................................................................................... XII

Abbreviations .................................................................................................................................. XIII

1. Introduction .......................................................................................................... 1

1.1. Photosynthesis: an overview ..................................................................................................... 1

1.2. Photosynthetic energy conversion ........................................................................................... 3

1.3. Photosynthetic complexes ......................................................................................................... 5 2. Functional genomics in plants .............................................................................. 9

2.1. Prediction studies ........................................................................................................................ 9

2.2. Photosynthetic preparations .................................................................................................... 10

2.3. Localisation studies .................................................................................................................... 11

2.4. Structural analyses ...................................................................................................................... 13

2.5. Phenotypic analyses ................................................................................................................... 14

3. Present investigation ............................................................................................ 16

3.1. Aim of this study ........................................................................................................................ 16

3.2. Working model ........................................................................................................................... 17

3.3. Nucleotide-dependent processes in the thylakoid lumen .................................................... 18

3.4. Plant PsbO as a GTPase ........................................................................................................... 20

3.5. ATP and phosphate thylakoid transporters ........................................................................... 27

4. Conclusions .......................................................................................................... 29

Acknowledgements ......................................................................................................................... 31

References ........................................................................................................................................ 33

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CHAPTER 1

1. Introduction

1.1. Photosynthesis: an overview

Plants, algae and photosynthetic bacteria are able to harvest sunlight and use its energy to

transform water and carbon dioxide to carbohydrate molecules and oxygen, both important to

sustain life on Earth. This process is called photosynthesis, and has evolved 2.5 to 3 billion years

ago from being dependent on substrates such as H2S, NH3 and Fe2+, to oxidising H2O

(McFadden, 1999; Olson, 2006) . It is the route by which almost all energy enters the biosphere.

Sunlight energy is harvested by chlorophyll molecules bound to hydrophobic proteins, located in

the chloroplast thylakoid membrane and drives the oxygen-evolving complex, where the water-

splitting reaction occurs (Lichtententhaler, 1987; Olson, 2006; Barber, 2007). Electrons are then

transferred to NADPH through photosystem II (PSII) via the cytochrome b6f complex (Cyt b6f)

and finally to photosystem I (PSI), the major photosynthetic protein complexes together with the

ATP synthase. The Cyt b6f complex also transfers protons into the lumenal space of the

thylakoid, in conjugation with the electron transport. These protons together with those from

water oxidation generate a proton gradient across the thylakoid membrane, which fuels the ATP

synthase to produce ATP. ATP, NADPH and carbon dioxide are then used in the dark reactions

to produce sugars in the chloroplast stroma. The thylakoid lumenal space where the water

oxidation occurs has until recently been viewed as a proton sink with very few proteins. With the

publication of the genome sequence of Arabidopsis thaliana (AGI, 2000) and proteomics works

(Peltier et al., 2002; Kieselbach and Schröder, 2003) it seems to be a much more complex

compartment housing a wide variety of biochemical processes.

1.1.1. Chloroplast

The chloroplast is the photosynthetic organelle in plants and green algae. It is surrounded by a

double membrane (outer and inner envelope), and contains the soluble stroma and thylakoids

(figure 1). The chloroplast, similarly to mitochondria, has its own genome, and thus the ability to

synthesise essential proteins for the photosynthetic machinery. However, a significant number of

chloroplast proteins are nuclear-encoded. For example, 10-15% of the nuclear gene products are

predicted to be targeted to the chloroplast (Peltier et al., 2002). Proteins that are targeted to the

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chloroplast have an N-terminal chloroplast transit peptide (cTP), which is processed when the

precursor protein is translocated across the chloroplast inner envelope. Proteins then have two

options; either remain in the stroma or move further into the thylakoid membrane or lumen.

Proteins in the thylakoid lumen are targeted and transported via an additional transit peptide, the

lumenal transit peptide (lTP), which is located directly after the cTP. Soluble lumenal proteins

with lTPs are translocated by at least two different pathways: Sec pathway (ATP-dependent) and

twin-arginine translocation (tat) pathway (pH-dependent) (Robinson et al., 2001; Peltier et al.,

2002).

Figure 1. Schematic illustration of the chloroplast membrane and soluble compartments.

1.1.2. Thylakoid

The thylakoid membrane in plants can be divided into two different structures, namely grana and

stroma lammelae (Menke, 1962; Menke, 1990). The grana regions usually cluster together into

grana stacks, made up from repeating units that contain paired layers. These grana stacks can be

further divided into surface-exposed, core and margin regions (Nelson and Yocum, 2006). The

grana stacks are interconnected by the stroma lammelae (figure 1). Recently, the three

dimensional structure of the plant thylakoid membrane has been revealed using electron

tomography (Shimoni et al., 2005). In this structure the top layer bends slightly upwards and

fuses with the layer above it and the other layer bends downwards and fuses with the layer below

it. This proceeds with a counter clockwise rotation of 25° around the axis (figure 1). The

thylakoid harbours the four photosynthetic complexes, PSII, Cyt b6f, PSI and ATP synthase

driving the light reactions. These complexes are located differentially in the thylakoid membrane.

The dimeric PSII complex is located in the grana, PSI and the ATP synthase are located in the

stroma lammelae and the surface-exposed grana, whereas Cyt b6f is evenly distributed across the

two thylakoid structures (Anderson, 1980; Allen and Forsberg, 2001; Anderson, 2002). However

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other reports suggest that Cyt b6f, which transports electrons between PSII and PSI, might

mostly be localised in the grana margins (Allen and Forsberg, 2001).

1.1.3. Lumen

The thylakoid lumenal space is located inside the thylakoid and is continuous between the grana

and stroma lammelae, as revealed in the recent 3-D structure (Shimoni et al., 2005). It has been

predicted to contain around 200 proteins, although no more than 80 proteins have been

identified using proteomics (Peltier et al., 2002; Schubert et al., 2002). There is also tremendous

evidence for various biochemical activities in the lumen, such as chaperones (Schlicher and Soll,

1996), immunophilins (Fulgosi et al., 1998), carbonic anhydrases (Park et al., 1999), violaxanthin

deepoxidases (Hieber et al., 2000), peroxidases (Kieselbach et al., 2000) and proteases (Adam,

2001). This growing complex view of the thylakoid lumen also raises the question of nucleotide-

dependent processes in this chloroplast compartment.

1.2. Photosynthetic energy conversion

Photosynthesis is a unique and complex process converting sunlight into chemical energy. This

process can be divided into two sets of reactions, the light and the dark reactions. In the light-

driven reactions, light energy is used by chlorophyll bound to proteins located in the chloroplast

thylakoid membrane, to drive the oxygen-evolving complex to form ATP from ADP, reduce

electron carrier molecules, such as NADP+ to NADPH, and produce oxygen as a by-product.

The dark reactions, called so because they proceed without the need for light, represent the

second part of photosynthesis, where the energy of ATP and reducing power of NADPH are

used for carbon dioxide fixation into sugars (Barber, 2007).

An overall equation of the oxygenic photosynthetic reactions is the following:

CO2 + H2O + light -> (CH2O)n + O2

The photosynthetic pigments such as chlorophylls, carotenoids and phycobilins are

embedded in the thylakoid membrane in units called photosystems (PS) (Lichtententhaler, 1987).

One photosystem can contain 250-750 pigment molecules (Dunahay et al., 1984; Neale and

Priscu, 1995), and consist of two subcomplexes, an antenna protein complex (LHC) and a

reaction centre (RC) complex, that capture the light at different wavelengths. There are several

types of chlorophyll, among which chlorophyll a occurs in all photosynthetic eukaryotes and

cyanobacteria. Chlorophyll b is found in vascular plants, bryophytes, green algae and euglenoids,

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and functions as an accessory pigment to broaden the spectrum of the light that can be absorbed.

When a chlorophyll b, molecule absorbs the light, the energy is transferred to chlorophyll a which

leads the energy to the oxygen-evolving complex (OEC) (Mathews and Holde, 1990; Raven et al.,

1992).

When the light energy is absorbed by the pigments, the energy is transferred until it

reaches the reaction center, which has one special pair of chlorophyll molecules. When one of

these two chlorophylls absorbs the light energy, it looses one electron which is then transferred

to a primary acceptor molecule, pheophytin (Pheo) (Raven et al., 1992).

There are two different photosystems in algae and plants, called photosystem II and I,

having their own reaction center molecules, P680 and P700, with maximum absorption at 680

and 700 nm, respectively. In both photosystems, the primary step driven by the light energy is a

charge separation from where one electron is provided to the electron transfer chain (figure 2)

(Baker et al., 2007).

The water-splitting reaction takes place on the lumenal side of PSII complex, and is

performed by the OEC complex, composed of four Mn2+ and one Ca2+ ion (figure 2). Water is

split into protons, oxygen and electrons. A tyrosine residue (Yz), positioned on the D1 protein,

functions as an electron transfer intermediate between P680 and the OEC complex. It reduces by

electron transfer P680 to its ground state, while itself is reduced by an electron from the splitting

Figure 2. A schematic representation of the electron and proton transfer chain and of the photosynthetic

subunits involved.

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of water (Baker et al., 2007).

Electrons are transferred via redox components through PSII to the Cyt b6f and PS I,

where the second type of charge separation occurs, involving P700 (figure 2). Electrons are

transferred to the primary acceptor Chl A0, then to PhQ A1, FeSx, FeSA and FeSB, from where

they leave PSI and reduce ferredoxin (Fd). The reduced ferredoxin reduces NADP+, via

ferredoxin-NADP+ oxidoreductase to NADPH (Merchant and Sawaya, 2005). The Cytochrome

b6f complex also transfers protons into the lumenal space of the thylakoid. The proton gradient

across the thylakoid membrane drives the ATP synthase. ATP together with NADPH are used

during carbon dioxide fixation (Mathews and Holde, 1990; Raven et al., 1992) (Baker et al., 2007).

For details of the light reactions, see the recent review by (Merchant and Sawaya, 2005).

Photosynthetic organisms, especially plants, have a hard time to cope with the

environment. They are literally rooted to the ground and cannot with ease move from their spot.

Sunlight, which drives photosynthesis, can be damaging to the photosynthetic machinery and

especially when the plants are exposed to high radiation. To overcome this problem, they have

developed several protection systems; from turning of leaves to extensive repair systems and

movement of the LHCII between PSII and PSI.

1.3. Photosynthetic complexes The four photosynthetic complexes are the PSII complex, Cyt b6f complex, the PSI complex and

the ATP synthase. Among them, the PSII complex has been the main focus of research in the

present investigation. There is overwhelming structural and functional information about each of

these complexes, for recent reviews see (Dekker and Boekema, 2005; Nelson and Yocum, 2006).

1.3.1. Photosystem II

Photosystem II (PSII), a multi-subunit membrane–protein complex, uses light energy to perform

the most thermodynamically demanding and unique reaction of photosynthesis, namely the

oxidation of water to molecular oxygen and reducing equivalents. X-ray crystal structures

obtained for the PSII complex isolated from Synechococcus elongatus, Thermosynechococcus vulcanus and

Thermosynechococcus elongatus, under various resolutions (3.8 to 3.0) (Zouni et al., 2001; Kamiya and

Shen, 2003; Ferreira et al., 2004; Loll et al., 2005) (figure 3). The PSII dimer has a 105 Å of depth,

205 Å of length and 110 Å of width at 3.5 Å resolution, and a molecular mass of 650 kDa

(Ferreira et al., 2004). Each monomer contains more than 20 different proteins where 16 are

intrinsic and four are extrinsic (Hankamer et al., 1997; De Las Rivas et al., 2004). Based on these,

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a homologous refined structure of the LHCII-PSII supercomplex of higher plants was recently

presented (figure 3) (Nield and Barber, 2006).

Figure 3. Homology model of a plant LHCII-PSII supercomplex based upon the X-ray crystal structures of the

LHCII-PSII complex from Synechococcus elongatus, of the plant PsbP and PsbQ proteins. Taken from Nield and

Barber (2006) Biochim. Biophys. Acta 1757:353-361, with kind permission from Professor Jim Barber (Imperial

College, London).

The assembly of PSII subunits of plants is regulated by the nucleus, but the core of the integral

PSII subunits are encoded by the chloroplast genome. These include D1, D2, CP43 and CP47

proteins. For a review, see (Kessler and Schnell, 2006; Suorsa and Aro, 2007; Suorsa M, 2007).

The reaction centre of PSII consists mostly of two homologous proteins named D1 and

D2, which bind cofactors for the electron transport. Closely associated to the D1 and D2

proteins are two chlorophyll-binding proteins called CP43 and CP47 (Barber, 2006). The OEC

complex is located on the lumenal side of the PSII complex (Seibert et al., 1987). In plants and

green algae, there are four extrinsic proteins, PsbO, PsbP, PsbQ and PsbR associated with the

OEC (Seidler, 1996; De Las Rivas et al., 2007). Cyanobacteria have PsbU and PsbV, as well as

PsbO as OEC extrinsic proteins, although there is evidence that they may also have homologs of

PsbP and PsbQ, associated with their PSII complexes (Thornton et al., 2004).

The water splitting in the RC of PSII has its side effects: the production of single

oxygen and of highly oxidising redox components, which may cause damage to the PSII, and

especially to its reaction centre D1 subunit. To survive, the D1 protein undergoes an extensive

repair cycle, including degradation and replacement by a newly synthesised protein (Aro et al.,

1993).

There are two mechanisms of D1 degradation, a donor and an acceptor-side inhibition.

The acceptor-side (Aro et al., 1993) occurs under high light and is due to an over reduction of the

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PQ pool. This in turn leads to the formation of P680 triplet state that can cause the formation of

singlet oxygen. The single oxygen, produced in the vicinity of the D1 protein, leads to an

excessive damage and the need for repair (Aro et al., 1993). The donor-side inhibition occurs

when the donor-side of PSII can not keep up with the withdrawal of electrons from P680.

Leading to long lived P680+ and the oxidised tyrosine electron donor Yz+, which causes an

inactivation of the PSII electron transport (Aro et al., 1993).

The D1 repair cycle starts with phosphorylation at its N-terminus by the STN8 kinase,

activated by the reduction of the PQ pool; second, the oxidative damage to the D1 protein due to

highly reactive singlet oxygen; third, the monomerisation of PSII and detachment of CP43, PsbP

and PsbQ; fourth, migration of the momomerised PSII complex to the stroma lamellae; fifth,

dephosporylation of the D1 protein; sixth, multi-step degradation: first, by a primary cleavage by

DegP2 resulting in a 23 kDa N-terminus and 10 kDa C-terminal fragments followed by a

secondary cleavage by FtsH; seventh, a cotranslation insertion of a new D1 protein; eight, PSII

monomer migration to grana and dimerisation of the PSII complex; ninth, activation of the PSII

dimer to a functional state. (Adir et al., 1990; Aro et al., 1993; Spetea et al., 1999; Lindahl et al.,

2000; Haußühl et al., 2001; Zaltsman et al., 2005; Kapri-Pardes et al., 2007).

1.3.1.1. Extrinsic proteins

The 33, 23, 16 and 10 kDa extrinsic proteins of PSII (PsbO, PsbP, PsbQ and PsbR) are

associated on the lumenal side of thylakoid membrane (figure 3). They were thought to be

directly involved in oxygen evolution since the removal of the 23 and 16 kDa subunits led to a

partial inactivation of the oxygen evolution (Seidler, 1996). The PsbO subunit, also called the

manganese stabilising protein, is conserved in all known oxygenic photosynthetic organisms,

showing a 40–50% sequence homology between cyanobacteria and higher plants. PsbO

optimizes the ionic environment, provides a polar channel for protons/water, and stabilises the

metal cluster (Seidler, 1996; De Las Rivas and Barber, 2004; Murray and Barber, 2007).

PsbP together with PsbQ have been thought to be responsible of Ca+ and Cl- binding

(Seidler, 1996), but a recent paper states that PsbP is required to maintain the active Mn2+, Ca-, Cl-

cluster in vivo, and that PsbQ is not necessary for either PSII function or growth (Ifuku et al.,

2005).

In the present investigation, the focus has been on the PsbO protein. A review by (De

Las Rivas et al., 2007) goes through the other extrinsic proteins in more detail.

7

1.3.2. Cytochrome b6f (Cyt b6f)

The oxidation of plastoquinol occurs by an integral membrane protein complex, Cyt b6f. It

functions as a plastoquinone-plastocyanin oxidoreductase, transferring electrons from

plastoquinol to plastocyanin, a copper-containing protein. This electron transfer is accompanied

by the translocation of protons across the membrane, this creates a proton gradient across the

thylakoid membrane, which drives the ATP synthesis in the stroma (Cramer et al., 2006).

1.3.3. Photosystem I (PSI)

The plant photosystem I (PSI) complex, is a monomer as compared to cyanobacterial PSI which

is a trimer as to PSII which is a dimer, reviewed in (Melkozernov et al., 2006). PSI accepts

electrons from the cyt b6f complex via plastocyanin. PSI reaction centre Chl absorbs light with a

maximum wavelength of 700 nm (P700). The electrons are transported through a chain of redox

cofactors to the ferredoxin-NADP+ complex, completing the electron flow from H2O to

NADP+. PSI has a peripheral LHC (LHCI), but can share the LHCII with PSII. Under changing

light conditions (state transitions) LHCII detaches itself from PSII and connects to PSI (Allen

and Forsberg, 2001)

1.3.4. ATP synthase

There are three types of ATPases, P, F and V–type, where the F-type is located in the inner

membrane of mitochondria and in the thylakoid membrane of chloroplasts. The sequence of

thylakoid ATP synthase is termed CF1CF0 complex. By analogy with mitochondrial ATPase

which transfers protons into the matrix, the thylakoid ATP synthase is driven by the H+

emigration from the lumenal space to the stroma (Mathews and Holde, 1990). The mechanism of

the proton-driven ATP synthesis can be described in three steps; first, translocation of protons

carried out by F0; second, catalysis of formation of the phosphoanhydride bond of ATP carried

out by F1; third, coupling of the dissipation of the proton gradient with ATP synthesis (Mathews

and Holde, 1990).

8

CHAPTER 2

2. Functional genomics in plants

Identification and functional characterisation of proteins from organisms with sequenced

genomes (eg. Arabidopsis, (AGI, 2000)) involves several steps in a functional genomic approach.

The prediction provides information about the sequence of the protein of interest, in which

species it can be found, the putative function and where it can be localised. Also, by using

bioinformatics, protein candidates for a certain biochemical activity can be found. Then,

localisation- and characterisation studies in vivo and in vitro provide the experimental validation of

the predictions. An important part of the characterisation is represented by phenotypic studies of

knockout mutants for the gene candidate.

2.1. Prediction studies There are numerous databases, servers and tools that can be accessed over the Internet or used as

a stand alone program; a few of them and routine analyses are listed below.

2.1.1. Databases and tools

Below, I briefly describe the databases and tools that I have used in the present investigation.

NCBI (http://www.ncbi.nlm.nih.gov/) was established in 1988 as a National Resource for

Molecular Biology Information, creates public databases, conducts research in computational

biology, develops software tools for analysing genome data, and disseminates biomedical

information (NCBI).

ExPASy (http://www.expasy.ch/) is a server dedicated for the analysis of protein sequences and

structures. Swiss-Prot is the most popular and used database available at this server (Gasteiger et

al., 2003).

BLAST is a widely used tool at several servers (NCBI, ExPASy), and searches for homologies to

sequences in the same organism and other species.

ClustalW is a tool found at EBI, used to align sequences and to identify conserved regions.

9

TargetP (http://www.cbs.dtu.dk/services/TargetP/) predicts the sub-cellular location of

eukaryotic proteins. The location assignment is based on the predicted presence of any of the N-

terminal presequences: chloroplast transit peptide (cTP), mitochondrial targeting peptide (mTP)

or secretory pathway signal peptide (SP) (Nielsen et al., 1997; Olof Emanuelsson, 2000).

PyMOL (http://pymol.sourceforge.net/) is a PDB viewer program, based upon an opensource

project, and visualises protein crystal structures saved in a pdf format (PyMOL, 2006).

CAVER (http://loschmidt.chemi.muni.cz/caver/index.php) calculates tunnels leading from

cavities inside static or dynamic protein or protein complexes. It does this by calculating the least

costing path, by giving numerical numbers corresponding to the resistance of closely located

molecules. It can be used both as a plugin for the PyMOL program or as an online application

(Petřek et al., 2006; Damborský et al., 2007).

Signal (http://signal.salk.edu/) is used to search for availability of public collection of Arabidopsis

knockout mutants and the insert location (Alonso et al., 2003).

Geneinvestigator (https://www.genevestigator.ethz.ch/) is a reference expression database and

meta-analysis system. It allows to study the expression and regulation of genes in a broad variety

of contexts by summarizing information from hundreds of microarray experiments into easily

interpretable results (Zimmermann et al., 2004; Zimmermann et al., 2005).

2.2. Photosynthetic preparations

For the main parts of this thesis, Arabidopsis thaliana has been used as plant m

aterial (papers I, III and IV). Spinach was used in the earlier stages of this work (papers I and II).

Arabidopsis is a small flowering plant that was discovered by Johannes Thal already in the

sixteenth century. It is favoured by scientists for its small genome which was fully sequenced in

2000. The genome has a total of 125 Mbp with 25,498 genes, encoding proteins belonging to

11,000 families (AGI, 2000).

Isolation of chloroplasts, thylakoids and various PSII components are described in the attached

papers. Here I provide a summarised flow scheme (figure 4); first, the leaves are homogenised in

a mixer; second, chloroplasts are isolated through centrifugation and washing steps; third,

thylakoids are extracted by the disruption of the chloroplast envelope using osmotic shock

followed by differential centrifugation; forth, using thylakoids, various preparations

10

Figure 4. Flow scheme for preparation of various chloroplast sub-fractions.

can then be obtained: LHCII-PSII supercomplexes, PSII membranes (BBY), PSII cores and the

soluble lumen content, by using detergents or mechanical pressure combined with various

centrifugation steps. There are major differences between the different types of preparations. The

PSII membranes are fragments from the grana regions enriched in LHCII-PSII dimeric

complexes, and containing all of the OECs extrinsic proteins. PSII cores are PSII monomers

largely devoid of LHCII as well of all extrinsic proteins except for PsbO.

2.3. Localisation studies To localise your protein of interest, there are many standard techniques; those used in the present

investigation are listed below.

2.3.1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE

Gel electrophoresis is a technique where proteins are separated in a solid matrix using an electric

field. The rate of the migration is based on the protein net charge, size, electric field, and also on

11

the composition of the mediums (Westermeier, 2001). The most common used variant is SDS-

PAGE, a discontinuous system originally described by Laemmli (Laemmli, 1970). SDS is an

anionic detergent, which binds strongly to and denatures proteins. SDS removes the intrinsic

charge of the protein, and a constant negative net charge per unit is obtained. When run under

the same conditions on a polyacrylamide gel, different sized proteins will travel at various speed,

and therefore be separated on the gel.

2.3.2 Immuno detection

The protein of interest can be probed with antibodies raised against a homologous protein from

another organism or against a specific peptide from this protein. The protein, that can be in an

isolated form or associated to a complex, can be detected by Western blotting or fished out using

immonoprecipitation.

2.3.3. Radioactive labelling

Radioactive assays are used to give experimental proof of protein function. The methods use

radioactively labelled compounds such as 14C, 32P, relevant for the function of the protein. The

radio-labelled protein is detected on a phosphorimage of SDS-gels. The radio-labelled nucleotides

are detected by phosphorimage of a thin layer chromatographic plate. The amount of

radioactivity is determined by liquid scintillation spectrometry.

2.3.4. Organelle import

Organelle import is a classic method for localisation studies. The protein of interest is synthesized

in vitro in a radiolabelled precursor form, containing a targeting peptide at its N-terminus that

directs the protein for example into the chloroplast. The protein can also be synthesised as a

fusion construct with a recognition label at the C-terminus, such as a green fluorescent protein

(GFP) tag, which can be visualised in a fluorescence microscope.

2.3.5. Mass spectrometry

The protein of interest can be enzymatically cleaved and extracted from a protein mixture, SDS-

gel or membrane and the obtained peptides can be analysed without breaking the covalent

structure of these molecules by mass spectrometry (MS) techniques. In the matrix-assisted laser

desorption ionization (MALDI) technique (Tanaka et al., 1988), and further developed in the

time-of-flight (TOF), ionized peptides are accelerated in an electric field and enter a flight tube.

During the flight in this tube, different molecules are separated according to their mass to charge

12

ratio (m/z) and reach the detector at different times. The masses of the peptides are then

analysed and searched in various databases. MS techniques can also be used for quantitative

applications, for example to compare the levels of a certain protein in knockouts mutants and

wild type plants (Edvardsson et al., 2007).

2.4. Structural analyses

There are four different aspects of the structure of a protein. Its primary structure is the amino

acid sequence of the polypeptide chain. The secondary structure (sub structure) shows the α-

helix, β-sheet and random coils distribution in the protein. The tertiary structure shows how the

protein is folded in space. The quaternary structure shows how proteins are folded together in

complexes. Different techniques listed below have been used to investigate protein structure in

this thesis (Mathews and Holde, 1990).

2.4.1 Circular dichroism

Circular dichroism (CD) is a technique to characterise the secondary structure of proteins. The

CD spectra are generated based on the optical spectra of the polypeptide chain. When a protein

is exposed to circulary polarised light the different structures of the protein give characteristic

bands, reflecting the electronic excitation energies. Secondary structure elements such as the α-

helix, β-sheet and random coil all give specific bands. This analysis can therefore lead to the

determination of the secondary structure of a protein. CD can also be used to detect

conformational changes induced with substrates (Bulheller et al., 2007).

2.4.2. Intrinsic fluorescence

Intrinsic fluorescence of the aromatic amino acids tryptophan, tyrosine and phenylalanine is used

to monitor structural changes in proteins. Among them, tryptophan has the highest fluorescence

and quantum yield. The tryptophan fluorescence is used as a tool to determine if the residue is

buried in the protein core or exposed on the surface, and thus to investigate changes in the

tertiary structure of a protein during folding and substrate binding. For reviews, see (Cioni and

Strambini, 2002; Bulheller et al., 2007).

13

14

2.5. Phenotypic analyses

2.5.1. Plant fitness

The overall fitness of knockout mutants has been compared to that of wild type (WT) plants

during growth under normal (120 µmol photons m-2 s-1) and under high light (1000 µmol photons

m-2 s-1).

2.5.2. Leaf weight

The weight of leaves is in direct correlation with how large they are. Comparing the leaf weight in

plants under various growth conditions, as in paper III, may provide indications for an impaired

function. The reduced growth of a knockout mutant points to impaired functions due to the

absence of an important protein.

2.5.3. Chlorophyll a/b ratio

To determine total chlorophyll Chl (a+b) concentration and Chl a/b ratio of a photosynthetic

preparation, samples are measured in a spectrophotometer at specific wavelengths, 663 and 645

nm, corresponding to maximum absorption by chlorophyll a and chlorophyll b, respectively. The

equation of Arnon (Arnon, 1949) was previously routinely used, but recently the equation of

Porra (based upon a modification of Arnon) has gained popularity. A ratio of around 3 is typical

for thylakoids, around 2 for isolated PSII membranes and greater-than or equal to 4 for PSII

cores (Porra, 2002).

2.5.4. Chlorophyll content

The chlorophyll content of a photosynthetic tissue gives indications about the rate of senescence

as well as the efficiency of photosynthesis. For comparing young to mature and senescent tissues,

a ratio of the chlorophyll content per mass of tissue is calculated.

2.5.5. Oxygen evolution

Molecular oxygen produced during water oxidation by OEC of PSII, can be used as an indicator

how well the photosynthetic apparatus performs. It can be measured by using an oxygraph with a

Clark-type electrode. By using saturated light and electron acceptors, which withdraw electrons

from different parts of the photosynthetic machinery, we can locate the irregularity in the

electron transfer chain.

2.5.6. Chlorophyll fluorescence

The light energy that is absorbed by the leaf may take three directions; it can be dissipated as

heat, drive photosynthesis or can be emitted as fluorescent light. The emission of fluorescent

light from the leaf is called chlorophyll fluorescence. Although the emitted light is low (only 1-2%

of the total light absorbed), it can be measured using a fluorometer. The three directions are

always in conjugation with each other, if one goes up the others go down so that the energy

always is the same as the light energy coming in. By measuring the yield of chlorophyll

fluorescence, information about the other directions can be gained, as reviewed in (Maxwell and

Johnson, 2000).

Once PSII has absorbed light and QA has accepted one electron, it is not able to accept

another one until it has passed the first electron to the next electron carrier QB. During this time,

the reaction centre is called “closed”. In darkness the reaction centers are open and by exposing

them to light they will gradually be closed. An increase in the ratio between closed and open

reaction centers leads to an increase in the yield of chlorophyll fluorescence.

15

CHAPTER 3

3. Present Investigation

3.1. Aim of this study

The lumenal space, of the thylakoid was known in the past to harbour just a few proteins, but

recent biochemical and proteomic reports point to a much more active role for this compartment

in photosynthetic regulation. This new and complex view of the lumenal space raises several

questions of the chloroplast function and regulation. One of these questions concerns the

presence of nucleotides, nucleotide-binding proteins and nucleotide regulation in the thylakoid

lumen of plant chloroplasts, which was a general aim of this work.

This chapter summarises the findings of the papers I to V. A short description of the aim of each

paper is listed below.

Paper I: To identify nucleotide-dependent reactions in the thylakoid lumen of spinach

chloroplasts and an ATP transporter in thylakoid membrane.

Paper II: To characterise the GTP-binding properties and GTPase activity of spinach

PsbO.

Paper III: To study the roles of Arabidopsis PsbO isoforms.

Paper IV: To investigate if Arabidopsis PsbO isoforms differ in their GTPase activity.

Paper V: To identify and functionally characterise a phosphate transporter in the

Arabidopsis thylakoid membrane.

16

3.2. Working model

Figure 5. Working model during the present investigation

1. ATP import and ADP export across the thylakoid membrane, via the thylakoid

ATP/ADP carrier, TAAC (Paper I)

2. Intra-conversion of ATP to GTP, by transfer of the γ-phosphate from ATP to GDP via

Nucleoside diphosphate kinase, NDPK3 (Paper I)

3. GTP binding and hydrolysis by the PSII extrinsic PsbO protein (Papers I, II and IV)

4. Following light-induced damage to PSII, the PsbO1 protein of Arabidopsis thaliana is

exchanged for the PsbO2 protein. The GTPase activity of the PsbO2 protein (PsbO in

spinach), initiates the monomerisation of the PSII dimer as an initial step of D1 turnover

(Papers III and IV)

5. Lumenal Pi is exported to the stroma via the thylakoid anion transporter 1, ANTR1

(Paper V)

17

3.3. Nucleotide-dependent processes in the chloroplast

The outer and inner envelope of the chloroplast host several nucleotide-dependent processes and

have the function to transport and exchange proteins, solutes and metabolites between the

cytosol and the stroma. Most chlorophyll proteins are nuclear-encoded and need to be imported

through these membranes from the cytosol. Two protein complexes are responsible for the

protein import, namely the translocon at the outer envelope membrane of chloroplasts, Toc, and

the translocon at the inner envelope membrane of chloroplasts, the Tic apparatus. Toc binds the

preprotein, coming from the cytosol, in the presence of ATP and GTP and the preproteins

transit peptide associates with the Tic complex and forms a Toc and Tic supercomplex, reviewed

in (Chen and Schnell, 1999; Spetea and Thuswaldner, 2008). The insertion of the protein from

the Tic complex is made by the help of hydrolysis of stromal ATP. In addition to protein import,

the inner envelope holds other nucleotide-dependent processes such as lipid translocation and

transporters (Lu et al., 2007; Spetea and Thuswaldner, 2008).

Inside the chloroplast are nucleotides, such as ATP, produced by the ATP synthase on

the stromal side of the thylakoid membrane. These are involved not just in the photosynthetic

dark reactions but also play an important roll in energy-dependent processes such as

phosphorylation, transport, degradation and folding of proteins (Spetea and Thuswaldner, 2008).

Protein phosphorylation on the stromal side of the thylakoid membrane has an important role in

how photosynthetic organisms cope with the environment. Excess light to PSII relative to PSI

leads to the reduction of the PQ pool (Vener et al., 1997) and the phosphorylation of the light

harvesting complex signals for a reversible association of LHCII between PSII and PSI, called

state transition (Zito et al., 1999; Bellafiore et al., 2005). During high light stress, PSII core

proteins, especially the PSII D1 protein that harbours most of the redox components get

phosphorylated, dephosphorylated and degraded (Aro et al., 1993). The turnover rate of the D1

protein is the fastest known in chloroplast, with a half life of 30 minutes. The degradation step

following light-induced damage has been shown to be nucleotide dependent (Spetea et al., 1999;

Spetea et al., 2000).

While the stromal side of the thylakoid membrane has several nucleotide-dependent

processes involved in the regulation of photosynthetic light reactions, there have been no reports

on the presence of nucleotides in the thylakoid lumen before the initiation of the present

investigation.

It has earlier been reported that the presence of GTP bound to the thylakoid membrane

enhances the D1 degradation rate (Spetea et al., 1999; Spetea et al., 2000) In paper I, we have

18

attempted to find GTP-binding proteins in the thylakoid membrane with a role in D1 protein

degradation (figure 5).

For this purpose, we incubated dark-controlled and pre-illuminated thylakoid

membranes with [α-32P]8-N3GTP, which upon UV exposure binds covalently to polypeptides

containing GTP-binding sites. Two radioactive bands of 33 and 36.5 kDa could be detected. The

band of 33 kDa was detected mainly in the preilluminated thylakoids and to a lower extent in the

dark sample. When isolated PSII cores were incubated with [α-32P]8-N3GTP, the 33 kDa protein

was detected, but not the 36.5 kDa protein. The labelling of the 33 kDa band was inhibited by

DCMU, which points to an involvement in the electron transport. The photolabelled 33 kDa was

washed away by alkaline TRIS and crossreacted with an antibody against the OEC33 (PsbO)

protein. Upon incubation of thylakoid membranes with [γ-32P]8-N3ATP, only the 36.5 kDa was

detected and stronger than compared with [α-32P]8-N3GTP, pointing to a higher affinity for ATP

than GTP. Thylakoid subfractionation showed that the 36.5 kDa was mainly located to the

stroma-exposed membranes.

Nucleoside diphosphate kinases (NDPK) catalyses the transfer of the γ-phosphate of a

triphosphate nucleoside to a diphosphate nucleoside by a ping-pong mechanism. In plants several

isoforms have been purified from cytosol (NDPK1) and chloroplast (NDPK2, 3) and

mitochondria (mtNDPK) (Spetea and Thuswaldner, 2008). To test for NDPK activity, thylakoid

lumen from spinach was incubated with [γ-32P]ATP in the presence and absence of externally

added GDP. Radioactive GTP was detected only in the presence of GDP, and when incubated

with [γ-32P]GTP radioactive ATP was detected. The Km for ADP was four-fold higher than for

GDP, indicating a preference of lumenal NDPK for GDP. Bioinformatic searches indicated

NDPK3 as a candidate for lumenal NDPK. Western blotting and immunoprecipitation with a

peptide-specific antibody as well as chloroplast import studies validated the lumenal location of

NDPK3.

Nucleotide metabolism and GTP binding to PsbO pointed to existence of nucleotide

transport across the thylakoid membrane (figure 5, steps 1-3). To test if externally added ATP

could be transported across the thylakoid membrane from the stroma to the lumen, intact

thylakoid membranes were incubated with [γ-32P]ATP in darkness and under illumination. The

thylakoids were washed and reisolated and tested for the presence of [γ-32P]GTP. As shown in

paper I, radioactive GTP could be formed by the help of the lumenal NDPK3. When spinach

thylakoids were immunoblotted with an antibody against the ADP/ATP carrier (AAC) protein

from bovine mitochondria a major band of 36.5 kDa was detected. As detected earlier a

corresponding band of 36.5 kDa was detected to bind ATP using [α-32P]8-N3GTP and [γ-32P]8-

19

N3ATP. To verify that the 36.5 kDa nucleotide-binding protein was responsible for the transport,

thylakoids were immunoprecipitated with the AAC antibody or preimmune serum. The AAC

antibody reduced the NDPK mediated formation of GTP to 20% as compared with the

preimmune serums 72%. This gives support to the fact that the 36.5 kDa band correspond to the

nucleotide transporter homologous to the bovine AAC.

3.4. Plant PsbO as a GTPase

3.4.1. GTP-binding proteins

Small GTP-binding proteins (GTPases) have a

molecular mass of 20-40 kDa and constitute a

superfamily with more than 100 members. Small

GTPases can be classified in five families: Ras, Rho,

Rab, Sar1/Arf and Ran (Takai et al., 2001). The Ras

GTPases are key regulators for several events such as

differentiation, vesicle transport, nuclear assembly and

signalling pathways, as reviewed in (Lunquist, 2006;

Spetea and Thuswaldner, 2008). GTPase change from

an active to an inactive state by binding a GTP molecule followed by hydrolysis to GDP (figure

6). The rate-limiting step of the GDP/GTP exchange reaction is the dissociation of GDP from

the GDP bound form (Takai et al., 2001). Proteins regulating this event are known as GTPase

activating proteins (GAP) which promote the inactive state and GTP exchange factors (GEFs)

which facilitate the exchange of GDP for GTP, and thus favours the active state (Yang, 2002;

Siderovski and Willard, 2005; Spetea and Thuswaldner, 2008). The GDP dissociation inhibitors

(GDIs) reviewed in (DerMardirossian and Bokoch, 2005; Siderovski and Willard, 2005) are also

regulators of GTPases. They have three functional activities; first, they inhibit the dissociation of

GDP, thus holding the GTPase in its inactive form; second, GDIs can inhibit GTP hydrolysis;

third, they can maintain GTPases in a soluble phase and thus preventing it to bind to the

membrane.

Figure 6. GTP binding protein in its

active form turns inactive by hydrolysing

GTP to GDP.

Sequence analyses of small GTPases have revealed that GDP/GTP-binding proteins

have well-conserved binding motifs. The phosphate binding loop (P-loop or G1) consists of a

glycine rich sequence followed by a lysine and serine or threonine. (GX4GKS/T, PROSITE

PS00017). This P-loop may interact directly with one of the α/β-phosphates of the bound

GTP/GDP (Saraste et al., 1990; Rensland et al., 1995). The binding regions called G2 and G3

20

interact with Mg2+ and γ-phosphate, respectively. G2 consists of an aspartic acid followed by two

amino acids and one glycine (DX2G), whereas G3 consists of a conserved Threonine (Bourne et

al., 1991; Leipe et al., 2002). A fourth conserved region (G4), involved in the binding of the

guanine ring, consists of hydrophobic and polar amino acids followed by (N/T)KX(D/E)

(Bourne et al., 1991; Leipe et al., 2002).

In paper I, we discovered that the 33 kDa subunit (PsbO) of the PSII complex in

spinach could bind GTP. We further characterised spinach and Arabidopsis PsbOs as GTPases in

paper II and IV.

3.4.2. GTP-binding regions of PsbO

PsbO is exposed to the lumenal side of the PSII where it is responsible for the stability of the Mn

cluster of the OEC complex. Plant PsbOs structural information, have been gathered through

various biophysical approaches (Xu et al., 1994; Svensson et al., 1996; Shutova et al., 1997).

Cyanobacterial PsbO has two regions, a head domain that interacts with PSII and a β-barrel core

(Ferreira et al., 2004). In figure 7, the PsbO protein structure of the homology spinach PsbO and

Thermosynechococcus elongatus is shown, where the colours (red, blue, magenta) correspond

to the GTP-binding motifs of GTP in spinach; G2-G3, G1 and G4 respectively. In the structure

homology model of spinach PsbO based upon the X-ray crystal structure of cyanobacterial PSII

(Ferreira et al., 2004), we have predicted two loops, as the Switch I and II, corresponding to the

regions flanking G2 and G3 motifs, namely the long β1- β2 and the short β2- β3 (paper II).

Switch I, interacts with CP47 on the lumenal surface of the other PSII monomer constituting the

dimeric complex (De Las Rivas J, 2004). A function for Switch II has so far not been reported,

however it can be speculated to interact with another lumenal protein. In figure 4, of paper II, we

show a proposed location/orientation of the GTP molecule in the PsbO structure, namely inside

the PsbO β-barrel domain. The surface of PsbO is rich in basic and acid residues, thus making it

a hydrophilic protein, while the central part of the β-barrel is not hollow but full of bulky

hydrophobic residues (De Las Rivas J, 2004). To search for a potential pocket inside the PsbO

protein, we have used the CAVER program for finding a tunnel originating from the G2 domain.

This program has been recently used to identify water and proton channels leading from the Mn

cluster (Murray and Barber, 2007).

In this work we have found four potential tunnels in the homolgous PsbO structure

from spinach and five potential tunnels in the structure from Thermosynechococcus elongatus. Only

one of them exited on the lumenal side of both of the PsbO proteins (figure 7), between the

loops of β3-β4, β4-β5 and β6-β7 and inside the loop of β4-β5, respectively. The average radius of

21

Figure 7. (A) Prediction of GTP-binding

site in the homology structural model of

the spinach PsbO. The G1, G2, G3 and

G4 as detected in blue, red and magenta,

respectively. SwI and II correspond to the

predicted Switch I and Switch II. The

tunnel is indicated as a grey density

region and exits through the lumenal exit

of the β-barrel, between the loops of β3-

β4, β4-β5 and β6-β7. (B) Overlay of the

predicted GTP-binding site of spinach

PsbO in Thermosynechococcus

elongatus 3-D structure. Tunnel is

indicated as a grey density region and

exits inside the loop of β4-β5.

these tunnels was 2 Å and the lumenal exiting tunnel had the least cost of the five. The lumenal

tunnel structure/cavity matches with prediction of the GTP-binding site in the PsbO structure.

The tunnel inside the spinach PsbO has a different exit and is much less restricted than to the

corresponding tunnel of Thermosynechococcus. The N-terminus tail of PsbO could function as a lid

and control the binding or dissociation of GTP/GDP. Beside the location of the GTP-binding

site inside the β-barrel, close to the lumenal exit of the protein, it is important to emphasise that

conformational changes in the Switch I (and II) loops upon GDP/GTP exchange may weaken

the interaction between PSII monomers (figure 5).

3.4.3. Mg GTP induces structural changes in PsbO

To study the interaction of GTP and PsbO, the secondary structure was investigated by far-UV

CD spectroscopy upon the addition of GTP to the spinach protein in solution. Figure 2 in paper

II shows that GTP induces changes in the PsbO structure in the presence of Mg2+ while without

the metal ion no structural changes are observed. Eukaryotic PsbOs contain a single tryptophan

22

23

residue Trp-241 in the mature form of spinach PsbO (figure 8), which intrinsic fluorescence is

used to monitor structural changes in its surroundings. The differential spectra with or without

MgGTP showed that the tryptophan fluorescence was affected and shifted to a shorter

wavelength, indicating that this amino acid becomes more buried inside the core of the protein.

3.4.4. GTPase activity of PsbO in its isolated and associated form

GTP hydrolysis experiments of the PsbO protein were conducted in its isolated form or

associated to PSII in different PSII preparations such as PSII membranes, NaCl-washed PSII

membranes and PSII cores. The GTPase activity was measured in darkness at pH 6.0 and 7.4,

corresponding respectively to the physiological pH under in vivo light and dark conditions in the

lumen. Only PSII membranes and isolated PsbO proteins showed significant difference between

the different pH conditions. By calculating the amount of PsbO per mol PsbO in the various

PSII preparations we could give a better comparison of the GTPase activity, see figure 3C in

paper II.

The results show that the PsbO protein has a low intrinsic GTPase activity, which is

enhanced when associated to a PSII dimer. This point to the fact that the PsbO as a GTPase

requires a certain conformational change, which is best achieved when bound to the dimeric

form of PSII. The implication of this finding is a potential role of this activity in the PSII

monomerisation step of D1 turnover (figure 5).

3.4.5. GTP stimulates the light-induced release of the PsbO protein and D1 degradation

Previously, it was reported that GTP enhanced the degradation of the D1 protein in photo-

inhibited thylakoid membranes as well as PSII complexes (Spetea et al., 1999; Spetea et al., 2000).

In paper II we show that GTP stimulates the light-induced release of PsbO protein under similar

experimental conditions as those inducing D1 degradation.

Following GTP hydrolysis, the inactive GDP form of the protein may be released from

the membrane. It has previously been known that the PsbO protein dissociates from the PSII

complex upon light exposure (Hundal et al., 1990). In paper II, we have investigated potential

roles of GTP in the mechanism of PsbOs dissociation from the PSII complex (figure 5, step 3-5).

In figure 4A of paper II we show the effect of GTP on PsbO in dark and light-exposed NaCl-

washed PSII membranes at pH 6.0 and 7.4. At pH 6.0 the PsbO release was stimulated by GTP

in both dark and light exposed membranes, while at pH 7.4 the release was only stimulated in

light-exposed membranes.

Figure 8. Prediction and location of GTP-binding domains in the structure of the PsbO protein, as shown in

paper II. PsbO sequences from the following organisms were aligned using ClustalW software: Spinacia

oleracea (spiol), Arabidopsis thaliana (Arath), Chlamydomonas reinhardtii (Chlam) and from

Thermosynechococcus elongatus (Synel). Black boxes mark the changes in the amino sequence between

PsbO1 and PsbO2 in Arabidopsis thaliana. The blue background marks the G1 domain, red the G2-G3 domains,

magenta the G4 domain. The yellow background flanking G2-G3 domain indicates Switch I (Sw I) and Switch II

(Sw II) regions. The eukaryotic tryptophan residue, located in the C-terminus of PsbO is indicated in green.

Figure 4B from paper II shows a decrease in oxygen evolution, upon light exposure and

addition of GTP to LHCII-PSII supercomplexes, which have PSII in its dimeric form that

retains all OEC proteins and have a high level of GTPase activity. The effect occurs upon

exposure to various light conditions, from darkness, low, growth to highlight. Western blot of the

supernatant and pellet of the membranes from the LHCII-PSII supercomplexes, showed an

enhanced release of PsbO and corresponding degradation of the D1 protein at enhancing light

intensities. The most remarkable effect of GTP was on degradation of D1 protein at 200 µmol

photons m-2 s-1, corresponding to the largest effect of GTP on the release of PsbO.

24

3.4.6. Different roles of PsbOs in Arabidopsis thaliana

Paper I and II provided strong evidence for new roles for PsbO in addition to stabilising the

OEC. As stated in chapter 1.3.1.1., Arabidopsis thaliana has two PsbO isoforms. In this work

(Papers III and IV), using T-DNA insertion knockout mutants, we attempted to study their roles

in PSII and determine their GTPase activity.

Previously, a PsbO1 lacking mutant, that has a single point mutation, was shown to

have retarded growth, light green leaves and a lower PSII content (Murakami et al., 2002; 2005).

Two knockout mutants of each of the psbO genes were obtained from the SALK

Institute; mutant lacking PsbO1 (SALK 093396), called psbo1 with an insertion in the 3´-UTR

region, and a mutant lacking PsbO2 (SALK 024770) called psbo2, with an insertion in one of the

exons. The psbo1 showed similar phenotype as Murakami´s knockout mutant, while the psbo2 had

dark green leaves slightly elongated (Paper III).

Immuno detection of PSII proteins (CP43, D1, D2, Lhcb2 and PsbP) showed an overall

reduction (75%) in psbo1 and increased (125%) in psbo2 as compared with wild type (WT), while

PSI content did not significantly differ among mutants and wild type. This would explain their

visual difference seen between the mutants and wild type plants. To analyse the photosynthetic

performance, we measured the oxygen evolution in thylakoids from psbo1 and psbo2. The

obtained values, 70% and 145% respectively to WT, matched their PSII content, while activity

from LHCII-PSII supercomplexes did not significantly vary. This indicates that both of the PsbO

proteins can stabilise the OEC and support the oxygen evolution but in various degrees under

stress light conditions. Phenotypic-response due to high light stress of plants, showed that the

average leaf weight was reduced with the most severe effect on psbo2 under extended high light,

pointing to a critical role for PsbO2 protein in PSII D1 repair cycle. To confirm this, we

measured PSII activity and D1 degradation in thylakoids subjected to light stress, see figure 5 in

paper III. The important finding that the D1 degradation was impaired in psbo2, implied that

PsbO2 may be essential for the initial steps of D1 degradation during high light stress. As

described by (Aro et al., 2005), reversible phosphorylation of PSII proteins regulates the

functional stability of PSII complexes, and that dephosphorylation of D1 protein is a prerequisite

for its degradation (Rintamaki et al., 1996). Interestingly, the in vivo phosphorylation levels of the

PSII D1 and D2 protein in the psbo1 were significantly lower (60%) compared to that of WT and

psbo2 knockout. This was confirmed by mass spectrometry. The endogenous level of protein

phosphorylation is the result of phosphorylation/dephosphorylation reactions. To investigate the

mechanism behind the lower phosophorylation levels in psbo1, we studied in vitro phosphorylation

25

and dephosphorylation, see figure 7 and 8 from paper III. We could conclude that psbo1 had a

faster D1 dephosphorylation and degradation in thylakoids as compared to wild type and psbo2.

3.4.7. Arabidopsis PsbOs differ in their GTPase activity

In paper IV, we have investigated the GTPase activity of Arabidopsis PsbOs in the corresponding

knockout mutants, psbo1 and psbo2. As in paper II, isolated PSII membranes and NaCl washed

PSII membranes were incubated with [α-32P]GTP in darkness at pH 6.0. We calculated the

amount of hydrolysed GTP to GDP expressed first per mg chl and then per mol protein, see

figure 1 in paper IV. The PsbO2 protein in psbo1 showed a three fold higher activity as compared

to the PsbO1 protein in psbo2.

Following GTP hydrolysis, the inactive GDP-form may be released from the membrane

(figure 5, step 4-5). Previously, (Hundal et al., 1990), showed that PsbO dissociates from its

docking site upon photoinactivation of the PSII electron transport. Figure 2 paper IV, show the

release of Arabidopsis PsbOs from illuminated PSII membranes under various light conditions

and in the presence with or without GTP. PsbO2 protein is more efficiently released in the psbo1

compared to WT and the PsbO1 protein in psbo2.

Arabidopsis PsbO1 protein has a structural role in the stabilisation of the OEC complex

while the PsbO2 protein regulates the PSII turnover. The reasons for their differential roles are

not known. PsbO1 and PsbO2 differ in 11 amino acids, of which two amino acids are located in

the targeting sequence. Previously, (Murakami et al., 2005) showed that mutation of three amino

acids between PsbO1 and PsbO2 in the C-terminal region, could affect oxygen evolution. Six out

of the remaining nine are in or close by the GTP-binding domain, and only one is not conserved.

The change from an Ala131 (PsbO1) -> Thr (PsbO2 as well as spinach PsbO), i.e., non polar to

polar amino acid change in the G1 domain, could explain the change of the GTPase activity

between psbo1 and psbo2.

Most striking is that the putative GTP-binding motifs are completely absent or only

partially conserved in Chlamydamonas (eukaryotic unicellular, green alga) and Synechococcus

(prokaryotic, cyanobacterium) PsbO, allowing us to suggest that GTP-binding to PsbO is a plant-

specific feature, as is the GTP-dependent D1 protein degradation (Spetea et al., 1999; paper II)

26

27

3.5. ATP and phosphate thylakoid transporters

3.5.1. Thylakoid ATP transporter

In paper I, we provide evidence of nucleotide transport in the chloroplast thylakoid membrane,

by using the lumenal NDPK activity, which can form GTP using externally added ATP. Immuno

blotting and transport inhibition, using an antibody against a bovine ATP/ADP carrier indicated

that the responsible 36.5 kDa protein in spinach is a homologous of the bovine ADP/ATP

carrier.

This protein responsible for the activity described in paper I has recently been identified

as the product of the At5g01500 gene and functionally characterised as a thylakoid ATP/ADP

carrier (TAAC) in Arabidopsis (figure 5, step 1) (Thuswaldner et al., 2007).

3.5.2. Thylakoid phosphate transporter

An active nucleotide metabolism in the thylakoid lumen, reported in paper I, implies the

existence of additional transporters (figure 9) than TAAC, such as those recycling inorganic

phosphate (Pi) to the soluble stroma (figure 5, step 5). Among the transporters in the chloroplast

envelope there are several translocators for Pi, which all functions as antiport systems using Pi or

Figure 9. An updated overview of transporters related to nucleotide metabolism in chloroplasts. Modified from

Spetea and Thuswaldner (2008), with kind permission from Cornelia Spetea (Linköping University).

phosphorylated C3- and C6-compounds as counter substrates (Flugge, 1999; Rausch and Bucher,

2002).

In Arabidopsis thaliana there are six genes encoding for anion transporters (ANTR1-6)

homologous to the membrane Na+-dependent phosphate transporter NaPi-1 (Werner et al.,

1998; Reimer and Edwards, 2004). ANTR1-3 have been predicted as chloroplast proteins (Roth

et al., 2004). ANTR2 has been characterised as an envelope protein by proteomics and immuno-

detection (Rolland et al., 2003; Roth et al., 2004). ANTR1 has been localised, by transient

expression of green fluorescent protein fusion construct, to the chloroplast (Roth et al., 2004).

In paper V we show, using two different peptide specific antibodies that the Arabidopsis

ANTR1 is a thylakoid membrane protein. The recombinant expressed ANTR1 facilitates Na+-

dependent Pi transport into Escherichia coli (E-coli).

Structural analyses of the 512 amino acids of the ANTR1 protein, show a high score for

a putative transit peptide; the theoretical mass for its processed form is 51 kDa and shows an

80% similarity with the ANTR2 protein. NaPi-1 transporter have been predicted to have 12

trans-membrane domains (Werner et al., 1998; Reimer and Edwards, 2004). Prediction of

ANTR1 topology at AREMEMNON showed 8-12 putative trans-membrane domains. See figure

1A-B in paper V. The ANTR1 protein has a 50% similarity to the rabbit NaPi-1 transporter and

60% to VGLUT2 transporters.

Recently VGLUTs have been shown to transport glutamate as well as Pi by two

independent mechanisms (Juge et al., 2006). To verify if ANTR1 also transports glutamate, an

uptake study of L-[3,4-3H]glutamate in E-coli, figure 4, 6A and table 1 in paper V, showed that

glutamate can bind to but is not transported by ANTR1. To further establish a physiological role

of ANTR1 in chloroplast Pi metabolism, phenotypic analyses by using knockout mutants are

required.

28

CHAPTER 4

4. Conclusion

Paper I

• The thylakoid lumen of spinach and Arabidopsis contains NDPK3, which converts ATP

and GDP to ADP and GTP.

• Spinach PsbO can bind GTP

• ATP transport occurs across the spinach thylakoid membrane into the lumen via a

protein homologous to the bovine ADP/ATP carrier.

Paper II

• Four GTP-binding motifs have been identified in spinach PsbO

• MgGTP induces specific changes in the structure of the spinach PsbO protein

• Spinach PsbO has a low intrinsic GTPase activity, which is enhanced when it is

associated with the PSII complex in its dimeric form

Paper III

• Both Arabidopsis PsbO proteins are able to support oxygen evolution

• PsbO1 can not substitute for PsbO2 in D1 protein turnover under high light stress

Paper IV

• Between the two PsbO proteins (PsbO1 and PsbO2) in Arabidosis, PsbO2 is the main

GTPase

Paper V

• Arabidopsis ANTR1 is a membrane transporter, located in the thylakoid membrane

transporting phosphate out of the lumen into the stroma.

29

The findings of this work, as summarised above, bring multiple evidence for nucleotide

dependent processes in the thylakoid lumen and relevant transporters in the thylakoid membrane.

Against old paradigms, this work provides a new and complex view of this subcellular

compartment, displaying an active nucleotide metabolism with roles in the PSII D1 turnover as

well as other crucial thylakoid-associated processes, yet to be identified and characterised.

30

31

ACKNOWLEDGMENTS

I have mainly two persons to thank for this thesis; first my main supervisor Docent Cornelia Spetea Wiklund, that has been very patient with me and taught me almost everything I know about science. It has been very nice that you have leant your ear for the troubles I have had over the years; second I want to thank my wife Mia, without you my life would not be as rich and this thesis would never have been written! I would like to thank my co-authors: Torill Hundal, Mounia Heddad, Iwona Adamska, Bertil Andersson, Maria Hansson, Benoít Schoefs, Alexander V. Vener, Sophie Thuswaldner, Tatiana Shutova, Said Eshaghi, Göran Samuelsson, James Barber, Lorena Ruiz Pavón, Fredrik Lundh, Arti Mishra, Bengt L. Persson and Cornelia Spetea I also want to express my gratitude to the others in the plant group! Professor Alexander V Vener, who is my second supervisor and have given me many valuable advices over the years. Masha who not only helped me in the lab but also with the repair of my flute. Lorena “Rose of the jungle”. What can I say: I am going to miss our chats and your laughter! Rikard “Rikardo” (Prego), I am really going to miss you! Thank you for the time and all the help! Björn the second, you got to keep the name Björn at a high standard. Hanna, wow a girl. I am sorry that we have not been able to spend so much time together. Patrik “the new guy”, good luck with your PhD and Lan “China doll”, I hope your dreams will come true! A special thank to Inger Carlberg for the kilos of spinach that you nicely gave to us. I especially want to thank the ex-members of our group Anna, Maria, Sophie, Julia, Markus, Alexey, Arti and Georgios. Big hugs to Anna and Maria that helped me in the lab when I was so lost! I want to thank the floor 13 at IBK - Cell Biology and give big bear hugs to: Lotta, Johan, Eva, Emelie, Thomas, Anders, Ingmar, Anna L, Anna G, Cecilia “Cilla”, Cecelia M, Kerstin, Monica, Elisavet, Maria D, Anette, Ingrid, Åke, Cecilia, Maggan, Anita S, Anna-Lotta, Annelie and Anita L and all the others! I loved the coffee breaks we have had, and the coffee cakes on Thursdays ☺. Thank you Eva and Emelie for the conversations on not just the weather or the lab but also on computer games and names. I also warmly want to thank all the people at the Cell Biology/IKE that have been very nice to me and helpful of lending materials and explaining procedures. I want to thank all the secretaries, lab personal, our everyday helper Håkan and the boys at MTA that keep the equipment working. Of course, I want to thank my “old” family; my parents Bo and Inger, that have really given me support and given me many, many things and helped me through many of the worries of life; my “little” sister Maria and her husband Erik and their two young ones Alicia and Jonathan. I also want to thank my “new” family from my wife´s side that have warmly welcome me without

32

thought; Henrik, Birgitta, Susanna and Janke thank you for everything! Then I want to thank Erik, Sarah, Christina, Kalle, Bo and Birgitta! I want to thank all of my friends! Lina, Tobias, Jonathan, Per, Martin, Mohit, FeiFei, Feng, Sun Bo, Joakim, Leif, Lisa and Roland. I give thanks to the scholarships that I have got, without them many of the trips to conferences would not have taken place. Thank you Dennis and the others at LIU-TRYCK, for the help with printing this thesis. Thank you all that have helped me, talked to me and pushed me, you know who you are! Thank you from all my heart! Björn

This work has been supported by the Swedish Research Council and the Graduate Research School in Genomics and Bioinformatics (FGB). We thank also the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants.

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