Dissecting the photosystem II light-harvesting antenna
Jenny Andersson
Institutionen för Fysiologisk Botanik, Umeå Universitet
Akademisk avhandling
som med vederbörligt tillstånd av rektorsämbetet vid Umeå Universitet, för
erhållande av filosofie doktorsexamen i ämnet Växters cell- och
molekylärbiologi, offentligen kommer att försvaras fredagen den 28 februari
2003 klockan 10:00 i hörsal KB3A9, KBC, Umeå Universitet.
Fakultetsopponent är Professor Henrik Vibe Scheller, Institut for Plantebiologi,
KVL, Frederiksberg (Köpenhamn), Danmark.
Jenny Andersson (2003)
Dissecting the photosystem II light-harvesting antenna
UPSC, Department of Plant Physiology, Umeå University, SE-901 87 Umeå, Sweden
Doctoral dissertation ISBN 91-7305-387-2
Dissertation abstract: In photosynthesis, sunlight is converted into chemical energy that
is stored mainly as carbohydrates and supplies basically all life on Earth with energy.
In order to efficiently absorb the light energy, plants have developed the outer light harvesting
antenna, which is composed of ten different protein subunits (LHC) that bind chlorophyll a
and b as well as different carotenoids. In addition to the light harvesting function, the antenna
has the capacity to dissipate excess energy as heat (feedback de-excitation or qE), which is
crucial to avoid oxidative damage under conditions of high excitation pressure. Another
regulatory function in the antenna is the state transitions in which the distribution of the trimeric
LHC II between photosystem I (PS I) and II is controlled. The same ten antenna proteins are
conserved in all higher plants and based on evolutionary arguments this has led to the suggestion
that each protein has a specific function.
I have investigated the functions of individual antenna proteins of PS II (Lhcb proteins) by
antisense inhibition in the model plant Arabidopsis thaliana. Four antisense lines were obtained,
in which the target proteins were reduced, in some cases beyond detection level, in other cases
small amounts remained.
The results show that CP29 has a unique function as organising the antenna. CP26 can form
trimers that substitute for Lhcb1 and Lhcb2 in the antenna structure, but the trimers that
accumulate as a response to the lack of Lhcb1 and Lhcb2 cannot take over the LHC II function
in state transitions. It has been argued that LHC II is essential for grana stacking, but antisense
plants without Lhcb1 and Lhcb2 do form grana. Furthermore, LHC II is necessary to maintain
growth rates in very low light.
Numerous biochemical evidences have suggested that CP29 and/or CP26 were crucial for
feedback de-excitation. Analysis of two antisense lines each lacking one of these proteins
clearly shows that there is no direct involvement of either CP29 or CP26 in this process.
Investigation of the other antisense lines shows that no Lhcb protein is indispensable for qE.
A model for feedback de-excitation is presented in which PsbS plays a major role.
The positions of the minor antenna proteins in the PS II supercomplex were established by
comparisons of transmission electron micrographs of supercomplexes from the wild type and
antisense plants.
A fitness experiment was conducted where the antisense plants were grown in the field and
seed production was used to estimate the fitness of the different genotypes. Based on the
results from this experiment it is concluded that each Lhcb protein is important, because all
antisense lines show reduced fitness in the field.
Key words: antisense, Arabidopsis thaliana, chlorophyll, carotenoid, feedback de-excitation,
fitness, LHC, NPQ, photosynthesis, state transitions, xanthophyll
Dissecting the photosystem II light harvesting antenna
Jenny Andersson
Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, Sweden
Dissertation Umeå 2003
1
© Jenny Andersson, 2003
Umeå Plant Science Centre
Department of Plant Physiology
Umeå University
SE-901 87 UMEÅ
Sweden
ISBN 91-7305-387-2
Printed by nra
Front cover (Mango leaf) by Henrik Johansson and Jenny Andersson
2
List of papers
This thesis is based on the following papers, which will be referred to in the text by their
Roman numerals.
I
J. Andersson, R. G. Walters, P. Horton, and S. Jansson (2001). Antisense inhibition of
the photosynthetic antenna proteins CP29 and CP26: implications for the mechanism of
protective energy dissipation. Plant Cell, 13:1193-1204
II
A. E. Yakushevska, W. Keegstra, E. J. Boekema, J. P. Dekker, J. Andersson, S. Jansson,
A. V. Ruban and P. Horton (2003). The structure of photosystem II in Arabidopsis:
localization of the CP26 and CP29 antenna complexes. Biochemistry 42:608-613
III
J. Andersson, M. Wentworth, R. G. Walters, C. A. Howard, A. V. Ruban, P. Horton and
S. Jansson (2003). Absence of the main light-harvesting complex of photosystem II
affects photosynthetic function. Provisionally accepted for publication in Plant J.
IV
A. V. Ruban*, M. Wentworth*, A. E. Yakushevska*, J. Andersson*, P. J. Lee, W.
Keegstra, J. P. Dekker, E. J. Boekema, S. Jansson and P. Horton (*equally contributing
authors) (2003). Plants lacking the main light harvesting complex retain photosystem II
macro-organization. Nature in press
V
J. Andersson and S. Jansson (2003). Loss of Lhcb1 and Lhcb2 decreases growth in
extreme low light. Manuscript
Papers I, II and IV are reprinted by kind permission of the publishers.
Not included in this thesis: S. Jansson, J. Andersson, S.-J. Kim and G. Jackowski (2000). An
Arabidopsis thaliana protein homologous to cyanobacterial high-light-inducible proteins. Plant
Mol.Biol. 42: 345-351.
3
Table of contents
List of papers ............................................................................................................... 3
Abbreviations .............................................................................................................. 6
Introduction: photosynthesis – the antenna perspective ............................. 8
Organization of the thylakoid membrane .................................................................... 10
The light harvesting antenna and the Lhc super gene family ................................... 11
Protein composition of the photosystems and their antennae .................................... 11
LHC structure .............................................................................................................. 13
Pigment composition ................................................................................................... 15
Does PsbS bind pigments? ......................................................................................... 15
Supermolecular organization of PS II and its antenna ............................................... 16
Different LHC II trimer binding sites ............................................................................ 16
Diverse polypeptide composition in LHC II trimers ..................................................... 16
Association of monomeric Lhcb proteins (the minor antenna) .................................... 16
LHC II not directly bound to PS II/peripheral LHC II .................................................... 17
Association of LHC-related proteins ............................................................................ 17
Supercomplex arrays ................................................................................................... 18
Regulatory mechanisms ................................................................................................ 18
The destructive power of light and oxygen .................................................................. 18
Avoiding over excitation ............................................................................................... 20
Feedback de-excitation ............................................................................................... 20
State transitions ........................................................................................................... 21
Acclimation of the light harvesting antenna to different light conditions ..................... 23
Methods ...................................................................................................................... 25
Model species: Arabidopsis thaliana ........................................................................... 25
Antisense inhibition ....................................................................................................... 26
T-DNA tagged mutants ................................................................................................... 27
Chlorophyll fluorescence .............................................................................................. 27
Fv/Fm ........................................................................................................................... 27
Non-photochemical quenching (NPQ) ......................................................................... 27
Evaluation of fitness – field experiment ...................................................................... 29
PS II antenna structure – single particle analysis ....................................................... 30
4
Discussion ................................................................................................................... 31
Transgenic lines ............................................................................................................. 31
Why did Flachmann and Kühlbrandt fail? ................................................................... 33
Feedback de-excitation in the antisense plants .......................................................... 34
Speculative model for feedback de-excitation ............................................................ 35
PS II antenna organization ............................................................................................ 37
Conservation of supercomplex ultra-structure ............................................................ 37
Photosynthesis in the absence of antisense inhibited antenna proteins ................ 39
Grana ............................................................................................................................... 40
State transitions ............................................................................................................. 40
Is each Lhcb necessary? ............................................................................................... 41
Functions of the Lhcb proteins .................................................................................... 41
CP29 ............................................................................................................................ 41
CP26 ............................................................................................................................ 41
CP24 ............................................................................................................................ 42
Lhcb1/Lhcb2 ................................................................................................................ 42
Lhcb3 ........................................................................................................................... 42
Future perspectives ................................................................................................. 43
Den ljusskördande antennen hos fotosystem II ............................................. 45
Acknowledgements .................................................................................................. 48
References .................................................................................................................. 49
5
Abbreviations
Arabidopsis
ATP; ADP
DCCD
Fm, Fo, Fv
LHC/Lhc
LHC I
LHC II
Lhca/Lhca
Lhcb/Lhcb
NADPH
NPQ
P680
P700
PCR
PS I, PS II
RC
T1 (T2, T3)
TEM
T-DNA
qE
qI
qT
VDE; ZE
Arabidopsis thaliana
adenosine triphosphate; adenosine diphosphate
dicyclohexylkcarbodiimide
maximal, minimal, variable fluorescence
light harvesting complex/gene encoding light harvesting complex
light harvesting complex I (composed of Lhca1-4)
light harvesting complex II (trimers composed of Lhcb1-3 in any combination)
light-harvesting proteins of PS I/corresponding genes
light-harvesting proteins of PS II/corresponding genes
nicotine adenine dinucleotide phosphate, reduced (oxidised)
non-photochemical quenching of chlorophyll fluorescence
the photoactive chlorophyll a molecule in PS II
the photoactive chlorophyll a molecule in PS I
polymerase chain reaction
photosystem I, photosystem II
reaction centre
the first (second, third) generation from a transformed plant line
transmission electron microscopy
the transferable part of the Ti plasmid of Agrobacterium tumefaciens
feedback de-excitation = ∆pH dependent NPQ
irreversible NPQ (photoinhibition)
fluorescence reduction due to state transitions
violaxanthin de-epoxidase; zeaxanthin epoxidase
6
Aim
Photosystem II antenna proteins have been studied for more than 30 years, since light-harvesting
complex (LHC) II was first isolated by Ogawa et al. (1966) and Thornber et al. (1967), but
many questions regarding for example the organization of the antenna complexes and the
individual functions of each protein remain. Because the LHC protein family is conserved in
all higher plants it has been hypothesised that every LHC has a separate function. The first aim
of my work was to construct a set of transgenic Arabidopsis lines, each lacking one of the Lhcb
proteins. Secondly, by comparing these lines to the wild type I wanted to uncover details about
the potentially different functions of each protein, and their positions in the antenna. Specifically
we were interested in the mechanism of protective energy dissipation, that has been assumed to
take place in the PS II antenna, and of the acclimative response to low growht ligh. We also
wanted to evaluate the evolutionary argument that claims importance for each LHC.
This thesis deals mainly with the chlorophyll a/b binding photosynthetic light-harvesting antenna
proteins of PS II. It describes the results from successful antisense inhibition of five of the
proteins (CP29; CP26; CP24; Lhcb1/Lhcb2), which give insight into the function of these
proteins. The impact of the loss of these proteins on plant growth, photosynthesis and fitness
was assessed.
7
Introduction: photosynthesis – the antenna perspective
Sunlight is the primary source of energy for life on Earth as we know it. Plants, animals, insects
and bacteria are all directly or indirectly dependent on photosynthesis (except for a few
lithotrophic organisms living in extreme environments such as in deep ocean hot springs).
Photosynthetic organisms utilise light energy to synthesise macromolecules (carbohydrates,
amino acids and fatty acids) that are in turn used by other organisms as raw material and fuel
for metabolism. The first steps, by which solar energy is converted into high-energy molecules
(ATP) and reducing power (NADPH), take place in macromolecular pigment/protein complexes
embedded in the thylakoid membrane which, in plants, is situated in the chloroplast (Figures 1
and 2). The enzymes involved in carbon fixation are soluble and located in the chloroplast
stroma.
In order to convert the light energy into
chemical energy, the first step for the
photosynthetic cell is to absorb the
photons and safely trap the energy in
a more long-lived form. The most
common pigment used for energy
absorption in higher plants is
(A)
(D)
chlorophyll, with the well-known
absorption spectrum that gives our
planet its green colour (Figure 3). The
photosynthetic pigments are highly coordinated by protein complexes in the
photosynthetic reaction centres and in
Figure 1. The chloroplast. (A) Transmission electron the light-harvesting antennae. This
micrograph showing the thylakoid membrane ultrastructure, enables them to efficiently absorb light
(B) schematic drawing, (C) schematic grana stack, (D) energy and transfer it to the reaction
centres. Moreover, the light-harvesting
enlargement of grana stacks from (A).
antenna has the ability to rapidly
switch to an energy-dissipating mode, which is essential to protect the photosynthetic apparatus
from over-excitation (Horton et al., 1996; Müller et al., 2001; Niyogi, 1999). In addition to the
chlorophylls, the antenna of higher plants also include a number of carotenoids, which are
involved both in light harvesting and, perhaps more importantly, in the defence against over
excitation (Cogdell and Frank, 1987; Demmig-Adams, 1990; Havaux and Niyogi, 1999).
(B)
(C)
When light hits a chlorophyll molecule in the light-harvesting antenna, absorption of the energy
of the photon brings one of the unpaired electrons (Π-electrons) in the conjugated porphyrin
8
STROMA
LUMEN
Buchanan et al., 2000, ASPB, printed with permission
Figure 2. The photosynthetic electron transport chain. Excitation of photosystem II (PS
II) by light (hυ) causes charge separation in the reaction centre where the primary radical
pair (P680 + Pheo - ) is formed. P680 + withdraws one electron from a tyrosine residue in the D1
protein which in turn is re-reduced by electrons from the manganese cluster which oxidises
water and release protons (H + ) and O 2 into the lumen. Pheo - reduces a bound quinone (Q A )
which passes the electron on to a second quinone (Q B ) to form a semiquinol (Q B - ). In a second
turn, Q B is fully reduced to quinol and acquires two H + from the stroma and diffuses from its
binding site as plastoquinol (PQH 2 ). PQH 2 is oxidised in the Q-cycle by the cytochrome b 6 f
complex (Cyt b 6 f) that reduces plastocyanin (PC) and release protons into the lumen. In
photosystem I (PS I) light absorption leads to charge separation between P700 and the primary
electron acceptor A 0 (a chlorophyll). The electron is passed on via phylloquinone (A 1 ) and a
number of Fe-S centres (F X , F A and F B ) to the soluble Fe-S protein ferredoxin (Fdx). The FdxNADP + reductase (FNR) reduces NADP + to NADPH with electrons from Fdx and a H + from the
stroma. P700 + is re-reduced with electrons from PC. The translocation of H + from the stroma
to the lumen generates a proton motive force that drives phosphorylation of ADP to ATP by
the ATP synthase (CF 0 CF 1 ). As a summary, solar energy is used to oxidise water to protons,
electrons and molecular oxygen. The electrons are converted into reducing power in the form
of NADPH. The H + from water oxidation and the Q-cycle are used to synthesise the high
energy molecule ATP. The next part of photosynthesis is the consumption of NADPH and ATP
for the assimilation of CO 2 into carbohydrates in the Calvin-Benson cycle (not shown).
9
Absorption
Violet Blue Green Yellow Orange
Red
ring to an excited energy state
(provided the photon is of an
Chlorophyll a
appropriate wavelength, see Figure
Chlorophyll b
3). Chlorophyll has two major
Carotenoid
Thylakoid
absorption bands in the visible region,
one in the blue and one in the red part
of the spectrum. Absorption of red
wavelengths excites the electron to the
first excited level, Qy. Absorption of
blue wavelengths excites the electron
400
500
600
700
to a higher energy level, the Soret
Wavelength (nm)
transition, which rapidly relaxes to the
Figure 3. Absorption spectra of pigments and
Qy level dissipating the spare energy
pigment-protein complexes. Absorption spectra of
as heat. The excitation is passed on
chlorophyll a, chlorophyll b, and carotenoid in nonpolar
to neighbouring chlorophyll by
solvents, and a thylakoid preparation where
resonance transfer with a high
chlorophylls and carotenoids are protein bound.
efficiency, and this is repeated until
the excitation reaches the reaction centre. In the reaction centre, charge separation occurs
resulting in the transfer of electrons, rather than the transfer of excitation energy, and this is the
beginning of the photosynthetic electron transport chain. The chemical environment around
the pigment, in this case amino acid residues in the apoprotein that binds the pigment, influences
the energy level of the excited states. This is demonstrated in Figure 3 by the differences
between the absorption spectra of isolated pigments in solution and the thylakoid membrane
preparation. The excitation energy of chlorophyll a is lower than that of chlorophyll b and in
the antenna, the excitation energy of pigment molecules decreases slightly with decreasing
distance to the reaction centre. This directs the energy towards the reaction centre, regardless
where in the antenna the photon was absorbed. However, the energy difference is small enough
to allow substantial probability for the excitation to travel in the opposite direction, away from
the reaction centre (Schatz et al., 1988).
Organization of the thylakoid membrane
In most higher plants and some green algae, the thylakoid membrane is organized into appressed
domains called grana stacks that are interconnected via stroma exposed thylakoids (Figure 1).
Grana stacking is dynamic. Rozak et al. (2002) show that the size and number of grana stacks
change within minutes of transition between different light conditions. The distribution of
photosynthetic complexes in the thylakoid membrane shows a high degree of lateral
heterogeneity (Figure 1) which was observed already in 1980 (Andersson and Anderson, 1980).
PS II is mainly localised to the appressed membranes (PS II α) and grana margins (PS II β)
while PS I is mainly localized to the stroma thylakoids and grana margins. The ATPase show
10
the same distribution pattern as PS I, and the cytochrome b6f complex is found in all membrane
fractions.
There are several suggestions to explain the occurrence of lateral heterogeneity. Separation of
the photosystems in different membrane domains prevent energy drain from PS II to PS I
(Anderson, 1999). PS I contains some chlorophylls (the so called red chlorophylls) with lower
energy excited state than any of the chlorophylls in PS II. Hence, if the antenna systems of both
photosystems were connected, the excitation energy would end up in PS I. Also, energy trapping
by PS II is three fold slower than in PS I (Anderson, 1999) which further increases the need for
separation of the photosystems. Another advantage of stacked membranes is that it allows
close packing of pigment proteins (Anderson, 1999), which is important in light limited
conditions where a large antenna system is necessary. It has been observed that low light grown
plants have more grana than high light grown plants. Horton (1999) suggests that grana allow
interactions to occur between PS II and the PS II antenna both within a thylakoid and between
adjacent membranes in the grana stack. This is hypothesised to prevent aggregation of the
antenna complexes which could induce a highly dissipative state due to high chlorophyll
concentration (Horton et al., 1991). Examination of the structures of the two photosystems
gives a steric explanation for their distribution (Allen and Forsberg, 2001). PS I has large
stroma exposed subunits which would not fit into the narrow space between adjacent thylakoids
in a granum while PS II protrudes only 10 Å at the stroma side of the membrane (Zouni et al.,
2001). Also, PS I donates electrons at the stroma side of the membrane to the soluble ferredoxin,
which may not have free access to grana stacks while PS II donates electrons to plastoquinone
which diffuses in the membrane.
The light harvesting antenna and the Lhc super gene family
Protein composition of the photosystems and their antennae
PS II is a complex structure composed of more than 20 protein subunits (Barber et al., 1997;
Hankamer et al., 1997a; Wollman et al., 1999), including the reaction centre (RC; the D1 and
D2 proteins with cofactors and the cytochrome b-559) that perform the primary charge
separation, the oxygen evolving complex that splits water, the core and outer antennae, that
enhances light harvesting and provides most of the excitation energy that powers the other
reactions. RC, the oxygen evolving complex and the core antenna form the PS II core complex,
which is dimeric in vivo (Wollman et al., 1999) review.
All higher plants investigated so far, have light harvesting antennae composed of the same set
of light harvesting proteins. The PS II core antenna consists of chloroplast-encoded polypeptides
(CP43 and CP47) binding chlorophyll a and β-carotene. In PS I, the core antenna is located in
the same polypeptides as the reaction centre, PSI-A and PSI-B, which are also chloroplast
11
encoded and bind chlorophyll a and β-carotene. The outer antenna consists of ten different
chlorophyll a/b and carotenoid binding (LHC) proteins encoded by a nuclear gene family
(Dunsmuir, 1985; Jansson, 1994; Jansson, 1999). The outer antenna of PS I is referred to as
LHC I and is composed of hetero dimers of Lhca1 and Lhca4 (LHCI-730) and hetero or homo
dimers of Lhca2 and Lhca3 (LHCI-680; Jansson et al., 1996; Schmid et al., 1997). In PS II the
outer antenna is composed of the monomeric, minor LHCs CP29, CP26 and CP24 and the
trimeric LHC II, which is composed of Lhcb1, Lhcb2 and Lhcb3 (described more thoroughly
below). LHC II also associates with PS I and the distribution of LHC II may contribute to
balance the excitation level between the two photosystems (Allen, 1992) and is regulated by
the so called state transitions (see below). Electron microscopy indicates that LHC I binds to
one side of PS I, and LHC II to the other (Boekema et al., 2001), notably to the PSA-H subunit
(Lunde et al., 2000).
Tabell 1 The members of the Lhc super-gene family of Arabidopsis thaliana
Gene
Protein
TAIR
EST clone GenBank mRNA Size MSH Ref.
Lhca1
LHCI-730
At3g54890 93I7T7
M85150
15
197
3
1
LHCI-680
At3g61470 32F4T7
AF134120 15
213
3
2
Lhca2
Lhca3
LHCI-680
At1g61520 40G8Y7
U01103
30
232
3
3
Lhca4
LHCI-730
At3g47470 91O23T7
M63931
15
199
3
4
?
At1g45474 177O9T7
AF134121 1
211
?
2
Lhca5
?
At1g19150 E1H6T7
U03395
1
220
?
4
Lhca6
Lhcb1.1 Lhcb1
At1g29920 36H7T7
X03907
5
232
3
5
Lhcb1.2 Lhcb1
At1g29910 98N12T7
X03908
5
232
3
5
Lhcb1.3 Lhcb1
At1g29930 138O13T7 X03909
80
232
3
5
Lhcb1.4 Lhcb1
At2g34430 39E1T7
X64459
25
231
3
6
At2g34420 35F5T7
X64460
40
232
3
6
Lhcb1.5 Lhcb1
At2g05100 31F8T7
AF134122 6 (+1) 228
3
2
Lhcb2.1 Lhcb2
Lhcb2.2 Lhcb2
At2g05070 167L10T7 AF134123 8
228
3
2
Lhcb2.3 Lhcb2
At3g27690 227K7T7
AF134125 1
228
3
2
Lhcb3
At5g54270 98K5T7
AF134126 10
223
3
2
Lhcb3
At5g01530 103O22T7 X71878
20
258
3
7
Lhcb4.1 CP29
Lhcb4.2 CP29
At3g08940 20D3T7
AF134127 15
256
3
2
At2g40100 149G3T7
AF134128 1
244
3
2
Lhcb4.3 CP29
CP26
At4g10340 37A1T7
AF134129 30
243
3
2
Lhcb5
Lhcb6
CP24
At1g15820 23A1T7
AF134130 20
211
3
2
PsbS
PsbS
At1g44575 137M5T7
AF134131 15
205
4
2
ELIP1
At3g22840 127N22T7 U89014
4
149
3
2
Lil1.1
Lil1.2
ELIP2
At4g14690 VCVCD09 AF134132 1
151
3
2
HLIP
At5g02120 105P6T7
AF054617 1
69
1
2
Lil2
?
At4g17600 114M20T7 AF134133 2
?
?
2
Lil3.1
Lil3.2
?
At5g47110 187G12T7 1
?
?
2
Lil4
SEP1
At4g34190 235A5T7
AF133716 1
103
2
8
SEP2
At2g21970 212I19T7
AF133717 1
181
2
8
Lil5
Fe-chelatase At2g30390 122F6T7
Y13156
1
?
1
9
FC
Gene: the common name of the gene; Protein: the common name of the protein; TAIR:
accession number in the Arabidopsis genomic database; EST clone: accession number to
an EST clone; GenBank: accession number to the full length coding region; mRNA: the
number of EST’s found (Jansson, 1999); Size: the size of the deduced mature protein in
amino acid residues. 1 Jensen et al., 1992; 2 Jansson et al., 1999; 3 Wang et al., 1994; 4
Zhang et al., 1991; 5 Leutweiler et al., 1986; 6 McGrath et al., 1992; 7 Green et al., 1993; 8
Heddad et al., 2000; 9 Chow et al., 1998.
12
(A)
LHC structure
The atomic structure of Lhcb1/2 has been
determined (Figure 4) based on a threedimensional map at 3.4 Å resolution, obtained
by electron microscopy on two-dimensional
crystals of a mixture of Lhcb1 and Lhcb2
(Kühlbrandt et al., 1994). The structure of CP26
(Green and Kühlbrandt, 1995) and CP29
(Sandonà et al., 1998) were modelled with the
Lhcb1/2 map as a template and it may be
assumed that all LHCs share the same basic
features of that structure, due to the high
conservation of amino acid sequence in
membrane spanning regions (Figure 5; Pichersky
et al., 1991. The structure comprises three
membrane-spanning α-helices in which the first
and third helix are held together by ion pairs,
and a short, amphiphatic α-helix at the lumenal
side of the membrane (not present in CP24).
2
3
1
4
(B)
Figure 4. LHC protein structure. (A) The protein structure
of an LHC II monomer indicating the three membrane
spanning helices (1-3) and the amphiphatic helix (4) (cf Figure
5). The location of the two central carotenoids and the twelve
known chlorophylls are shown. (B) Trimeric LHC II.
Buchanan et al., 2000, ASPB,
printed with permission
Proteins with sequence similarity to the LHCs
In addition to the antenna proteins, there are several other proteins, with more or less obscure
functions but with sequence similarity to the LHCs, that are grouped together in the Lhc super
gene family (Table 1; Jansson, 1999). PsbS has recently been shown to play a major role in
protective energy dissipation (Li et al., 2000). The ELIPs are transiently induced during greening
of etiolated plants (Meyer, 1984), during desiccation (Bartels 1992) and high light stress
(Adamska et al., 1992), and are speculated to be involved in high light protection. HLIP has
one membrane spanning helix and share its highest sequence similarity with the cyanobacterial
HLIP (Dolganov et al., 1995), and its expression is up-regulated in high light (Jansson et al.,
2000). Lil3.1 and Lil3.2 have been found in EST databases (Jansson, 1999) and in the
Arabidopsis genome, but the corresponding proteins are unknown. Lil4 and Lil5, encoding
13
14
Transit peptide
Helix 2 (C)
Helix 4 (D)
EAEDL–––––––––––––––––––LYPGG–SFDPLGLAT–––––––––DPEAFAELKVKELKNGRLAMFSMFGFFVQAIVTGKG–––––––PIENLADHLADPVNNNAWAFATNFVPGK
EGLDP–––––––––––––––––––LYPGG–AFDPLNLAE–––––––––DPEAFSELKVKELKNGRLAMFSMFGFFVQAIVTGKG–––––––PIENLFDHLADPVANNAWSYATNFVPGN
EGND––––––––––––––––––––LYPGGQYFDPLGLAD–––––––––DPVTFAELKVKEIKNGRLAMFSMFGFFVQAIVTGKG–––––––PLENLLDHLDNPVANNAWAFATKFAPGA
R–––––––––––––––––––––––LYPGGKFFDPLGLAA–––––––––DPEKTAQLQLAEIKHARLAMVAFLGFAVQAAATGKG–––––––PLNNWATHLSDPLHTTIIDTFSSS
DK––––––––––––––––––––––LHPGG–PFDPLGLAK–––––––––DPEQGALLKVKEIKNGRLAMFAMLGFFIQAYVTGEG–––––––PVENLAKHLSDPFGNNLLTVIAGTAERAPTL
SQSVEWATPWSKTAENFANYTGDQGYPGGRFFDPLGLAGKNRDGVYEPDFEKLERLKLAEIKHSRLAMVAMLIFYFEAGQ–GKTPLGALGL
Helix 3 (A)
–––––––––––––––––––––––––PETFARNRELEVIHSRWAMLGALGCVFPELLARNGVKFG–EAVWFKAGSQIFSDGGLDYLGNPSLVHAQSILAIWATQVILMGAVEGYRVAGNGPLG
–––––––––––––––––––––––––PETFAKNRELEVIHSRWAMLGALGCTFPEILSKNGVKFG–EAVWFKAGSQIFSEGGLDYLGNPNLIHAQSILAIWAVQVVLMGFIEGYRIGG–GPLG
–––––––––––––––––––––––––PEAFAKNRALEVIHGRWAMLGAFGCITPEVLQKWVRVDFKEPVWFKAGSQIFSEGGLDYLGNPNLVHAQSILAVLGFQVILMGLVEGFRINGLDGVG
LAGDVIGTRTEAADAKSTPFQPYSEVFGIQRFRECELIHGRWAMLATLGALSVEWLTGVT––––––––WQDAGKVELVDGS–SYLGQPLPF–––SISTLIWIEVLVIGYIEFQRNAELDSEK
–––––––––––––––––––––––––PENFAKYQAFELIHARWAMLGAAGFIIPEALNKYGANCGPEAVWFKTGALLLDGNTLNYFGKNIPI–––NLVLAVVAEVVLLGGAEYYRITNGLDFE
–––––––––––––––––––––––––PAFLKWYREAELIHGRWAMAAVLGIFVGQAWSG––––––––VAWFEAGAQPDAIAPF––––––––––––SFGSLLGTQLLLMGWVESKRWVDFFNPD
Helix 1 (B)
MAASTMALSSPAFAGKAVKLSPAASEVLGSGRVTMRKTVAKPKGPSGSPWYGSDRVKYL––GPFS–GESPSYLTGEFPGDYGWDTAGLSAD––––––––––––––––––
MATSAIQQSSFAGQTALKPSNELLRKVGVSGGGRVTMRRTVKS–––TPQSIWYGPDRPKYL––GPFS–ENTPSYLTGEYPGDYGWDTAGLSAD––––––––––––––––––
MASTFTSSSSVLTPTTFLGQTKASSFNPLRDVVSLGSPKYTM––––––––G––NDLWYGPDRVKYL––GPFS–VQTPSYLTGEFPGDYGWDTAGLSAD––––––––––––––––––
MAATSAAAAAASSIMGTRVAPGIHPGSGRFTAVFGFGKKKAAPKKSAKKTVTTD–RPLWYPGAIS–––––––––––––PDWLDGSLVGDYGFDPFGLGKPAEYLQFDIDSLDQNLAKN
MASLGVSEMLGTPLNFRAVSRSSAPLASSPSTFKTVALFSKKKPAPAKSKAVSETSDELAKWYGPDRRIFLPDGLLDRSEIPEYLNGEVAGDYGYDPFGLGKK––––––––––––––––––
MAMAVSGAVLSGLGSSFLTGGKRGATALASGVGTGAQRVGRKTLIVAAAAAQPKKSWIPAVKGGGN–––––––FLDPEWLDGSLPGDFGFDPLGLGKD––––––––––––––––––
267
265
265
290
280
258
184
182
182
214
197
158
88
87
85
104
103
91
indicate the amino acids known to be involved in trimer formation in LHC II (Hobe et al., 1995 and Kuttkat et al., 1996).
putative cleavage site. Bold letters indicate the known chlorophyll ligands in LHC II (Kühlbrandt et al., 1994). Triangles above the alignment
1994). The helices are sometimes denoted with the letters A-D which is also shown. The N-terminal transit peptide is boxed according to the
thereafter manually adjusted. Four boxes indicate the membrane spanning (1-3) and amphiphatic (4) helices (according to Kühlbrandt et al.,
as judged from the occurrence in the EST database, cf Table 1) of each of the six Lhcb proteins were aligned with the ClustalW program and
Figure 5. Alignment of the Arabidopsis Lhcb proteins. The deduced amino acid sequences of one representative (the most highly expressed
Lhcb1.3
Lhcb2.1
Lhcb3
Lhcb4.1
Lhcb5
Lhcb6
Lhcb1.3
Lhcb2.1
Lhcb3
Lhcb4.1
Lhcb5
Lhcb6
Lhcb1.3
Lhcb2.1
Lhcb3
Lhcb4.1
Lhcb5
Lhcb6
SEP1 and SEP2 respectively, are expressed in response to stress, but the functions are unknown
(Heddad and Adamska, 2000). Finally, one of the two Arabidopsis ferrochelatases is included
in the family, due to LHC sequence similarity in a region believed to form a membrane spanning
helix (Jansson, 1999).
Pigment composition
It is estimated that PS II, including the LHC antenna, binds 150-200 chlorophyll molecules, of
which approximately 40 associate with the PS II core. From these numbers it is concluded that
70-80 % of the PS II light energy absorption occurs in the LHC antenna. All LHC proteins bind
both chlorophyll a and b but the ratio between them varies and in addition to chlorophyll, the
LHC proteins bind various carotenoids (Table 2).
Table 2 Pigment binding stochiometry of the light-harvesting proteins
Protein
Chl a
Chl b
Chl a/b
Lutein
a
nLHC II
7
5
1.4
1.68
a
nLHC II
6.6
5.0
1.32
1.9
nLHC IIa
1.4
1.8
nCP29
6.8
2.0
3.40
0.9
rCP29
6
2
3.0
0.89
nCP26
7.5
3.0
2.5
1.2
nCP24
2.7
2.3
1.2
0.53
rLhca1
5.47
1.57
3.48
1
rLhca1
4.0
1.81
rLhca2
4.99
2.19
2.28
1
rLhca3
4.63
0.76
6.14
1
rLhca4
5.56
2.15
2.59
1
rLhca4
2.3
1.5
Average pigment binding of the Lhcb complexes
Violax.
0.32
0.2
0.2
1.2
0.64
0.9
0.47
0.17
1.05
0.13
0.14
0.13
0.5
Neox.
1.05
1.0
1
0.6
0.47
1.0
<0.01
0.20
0.12
0.07
0.16
0.09
0
Protein
LHC II trimer
CP29
CP26
CP24
Violax.
1
1
1
0 or 1
Neox.
3
0 or 1
1
0
Chl a
21
7
7
3
Chl b
15
2
3
2
Lutein
6
1
1
0 or 1
β-car
0
0
0
0
0
0
0
0.03
0.03
0.09
0.03
-
Ref.
1
2
3
2
4
2
5
6
7
6
6
6
7
a
per monomer; n – native protein; r – recombinant protein reconstituted with pigments
1 Remelli et al., (1999); 2 Ruban et al., (1999); 3 Croce et al., (1999); 4 Clinque et al.,
(2000); 5 Pagano et al., (1998); 6 Schmid et al., (2002); 7 Croce et al., (2002)
Does PsbS bind pigments?
The data regarding the pigment binding properties of PsbS are ambiguous. Funk et al. (1994
and 1995) showed that it does bind both chlorophyll (mainly chlorophyll a) and carotenoids
(including violaxanthin). On the other hand, Dominici et al. (2002) found no pigments on the
native protein and also showed that PsbS could be reconstituted without pigments under
conditions where LHC proteins recruit pigments. Dominici et al. conclude that either PsbS is
not a pigment binding protein, or its pigment binding mechanism is very different from the
LHC proteins.
15
Supermolecular organization of PS II and its antenna
Different LHC II trimer binding sites
When thylakoid membranes are solubilized with
n-dodecyl-α,D-maltoside, purified on a gel
filtration column or by sucrose gradient
centrifugation and subjected to transmission
electron microscopy (TEM), supercomplexes
with outer antennae of varying size can be
obtained (Boekema et al., 1995). Three different
binding sites for LHC II trimers have been defined
and are named after their apparent binding
strength to the PS II core: strongly (S), moderately
Figure 6. Hypothetical supercomplex. Four
LHC II binding sites per PS II have been found. (M) and loosely (L) bound (Boekema et al., 1998;
In this figure all of them are shown, although such
1999a; 1999b). In each supercomplex six binding
a supercomplex has never been observed.
(Boekema et al., 1999b; printed with permission.) possibilities exists for LHC II, two S, two M and
two L sites (Figure 6), although it needs to be
pointed out that a supercomplex with a full set of six LHC II has never been observed.
Diverse polypeptide composition in LHC II trimers
LHC II itself consists of three distinct proteins (Lhcb1, Lhcb2 and Lhcb3), which in different
combinations form the trimers. Lhcb1 and Lhcb2 are encoded by multi-gene families in most
species (see Table 1 for Arabidopsis). Several different trimeric complexes have been
characterised in terms of their polypeptide composition in maize (De Luca et al., 1999) carnation
(Jackowski and Jansson, 1998), Arabidopsis (Jackowski et al., 2001) and spinach (Jackowski
and Pielucha, 2001). S-LHC II trimers are composed of Lhcb1 and Lhcb2 in a 2:1 ratio
(Hankamer et al., 1997b), and M-LHC II trimers of Lhcb1 and Lhcb3 in a 2:1 ratio (Boekema
et al., 1999b). Lhcb1/Lhcb2/Lhcb3 heterotrimers have also been observed (Jackowski et al.,
2001).
Association of monomeric Lhcb proteins (the minor antenna)
Several methods have been used to assign the specific locations of the minor antenna proteins
in relation to the core complex and LHC II. Cross linking studies in Marchantia polymorpha
(Harrer et al., 1998), which is similar to higher plants in terms of PS II, was used to identify the
masses seen on TEM and yielded the tentative model shown in Figure 6. In Paper II we provide
evidence based on analysis of antisense plants lacking CP26 or CP29, which show that this
model is correct. CP29 is most intimately associated with the core complex and seems to be
essential for the stability of the supercomplex, since it is present in all preparations (Boekema
16
et al., 1999b) and supercomplexes cannot be isolated from antisense plants lacking this protein
(Paper II, see also the Discussion section). CP29 is located in direct contact with CP47 of the
inner antenna. CP24 is, as shown previously, believed to be in contact with CP29, but more
peripheral from the core complex. CP24 seems loosely attached to the supercomplex, and for
stable association, M-LHC II seems necessary (Boekema et al., 1999b). CP26 is located in
contact with CP43 in the “corner” of the supercomplex. In order for CP26 to be stable within
the supercomplex, binding of S-LHC II is required (Boekema et al., 1999b).
LHC II not directly bound to PS II/peripheral LHC II
The supercomplex described above has six putative LHC II binding sites, although it seems
exceptional that all of them are filled. In Arabidopsis, the PS II dimer is associated with no
more than four LHC II (Yakushevska et al., 2001).
On the other hand, biochemical analyses indicate
a stochiometry of up to eight LHC II per core
dimer (Dainese and Bassi, 1991; Jansson, 1994;
Peter and Thornber, 1991). This leaves a large
proportion of LHC II unaccounted for. A
peripheral population of LHC II exist, that is not
as closely associated with PS II. Several studies
show that this subpopulation can be
phosphorylated, and participates in the state
transition process (further discussed below). Peter
and Thornber (1991) saw a multimeric complex
of LHC II that was not directly associated with
Figure 7. Heptameric LHC II. A complex PS II, and more recently (Dekker et al., 1999)
of seven LHC II trimers (that is 21 protein observed structures composed of seven LHC II
s u b u n i t s ) . T h r e e o f t h e t r i m e r s a r e trimers that did not interact with PS II (Figure
indicated with tripods. (Dekker et al., 1999) 7). Furthermore, (Boekema et al., 2000) observed
LHC II in membrane domains deficient in PS II.
Association of LHC-related proteins
There is not much information of the locations of the other members of the LHC family that are
assumed to belong to PS II. Supercomplexes containing PsbS has been isolated (Eshaghi et al.,
1999), which indicates that this protein associates closely with the PS II core. However, in the
M. polymorpha cross linking study mentioned above, supercomplex preparations were depleted
in PsbS compared to grana membranes (Harrer et al., 1998). Moreover, supercomplexes isolated
from a mutant (npq4-1) that does not synthesise PsbS appear identical on TEM (Boekema,
unpublished), that is, no apparent mass is missing. This indicates that PsbS is not located close
to the reaction centre or the inner antenna of PS II.
17
Supercomplex arrays
In freeze-etch and freeze-fracture studies several authors have observed crystalline areas in the
thylakoid membrane. Boekema et al. (2000) isolated paired grana membranes free of stroma
membranes and grana margins from spinach. On electron micrographs they found that most,
but not all, membranes contained semi-crystalline domains that appeared as rows spaced by
26.3 nm and were shown to consist of supercomplex arrays (for an example see Figure 4 in
Paper II). The repeating unit was suggested to be C2S2M supercomplexes, possibly intermixed
with some C2S2M2 supercomplexes causing some disorder of the crystal lattice. There was
space for one CP24 per PS II dimer in this type of lattice. Some membranes (~1%) had rows
spaced by 23 nm and were concluded to consist of arrays of C2S2 supercomplexes. Furthermore,
it was found that PS II centres in one membrane domain frequently faced a domain containing
exclusively LHC II in the adjacent membrane, and it is suggested that energy transfer may
occur between adjacent membranes. In a similar study in Arabidopsis (Yakushevska et al.,
2001), the arrays were spaced by 25.6 nm and were concluded to consist of repetitions of
C2S2M2. In Papers II and IV (see also Discussion), it is shown that antisense plants lacking
certain antenna protein complexes assemble in lattices with some differences from the wild
type.
It should be noted that all PS II does not exist in ordered lattices. There is a high degree of
heterogeneity in antenna size and composition within one membrane.
Regulatory mechanisms
Under conditions when light energy is present in excess, which means that all energy that is
absorbed cannot be converted into electron transport and proton translocation, several
detrimental processes may take place (Niyogi, 1999). This occurs for example at increased
irradiances, at decreased temperatures or under insufficient CO2 concentrations, which may be
induced by stomata closure upon water deficiency. Figure 8 shows the natural variations in
light intensity a normal summer day, when clouds and trees occasionally shade the sun. In
order to compete successfully with other plants, as much energy as possible must go into
photosynthesis, without unnecessary loss, and surplus energy must quickly be dissipated during
periods of over excitation. The light-harvesting antenna has the dual capacity for efficient light
capture and energy dissipation and this is strictly regulated to avoid destruction by over excitation
and still maximise energy transfer during more favourable conditions.
The destructive power of light and oxygen
Singlet oxygen (1O2), superoxide radicals (O2-•), hydrogen peroxide (H2O2) and hydroxyl radicals
(•OH), collectively called reactive oxygen species, are chemically aggressive molecules that
can cause oxidative damage to proteins, pigments and membrane lipids. In addition, the PS II
18
1200
PAR (µmol photons m-2s-1)
1000
Sunny
800
600
400
Shady
200
0
0
2
4
6
8
10
12
14
16
18
20
22
24
Time (h)
F i g u r e 8 . Va r i a t i o n s i n n a t u r a l s u n l i g h t . T h e i n t e n s i t y o f t h e s u n l i g h t w a s
recorded during two consecutive 24 h periods at the two locations where our
field experiment is conducted. The sunny location is not shaded by any vegetation
while in the shady location, the sunlight is filtered through trees and bushes.
primary electron donor itself in its oxidised form, P680+, is a powerful oxidising agent (Anderson
et al., 1998). 1O2 may be formed by interaction of molecular oxygen, which is a triplet in the
ground state, with triplet chlorophyll (3Chl), which can be formed via recombination of P680+
and Pheo- that may occur when the forward electron transport is blocked, or by intersystem
crossing from singlet excited chlorophyll. Another pathway of oxidative damage may occur at
very low light intensities. When the time between the first and second charge separation event
is long, QA- or QB- may recombine with the PS II donor side causing the formation of reactive
oxygen species (Keren et al., 1997).
O2-• is formed in the Mehler reaction, in which PS I uses O2 as the terminal electron acceptor,
for example when NADP+ is not available. O2-• can reduce metal ions, such as Fe3+ and Cu2+,
which in turn may react with H2O2 and produce •OH. O2-• is converted to water via H2O2 by
superoxide dismutatse and ascorbate peroxidase in the water-water cycle (Asada, 1999).
Carotenoids protect against 1O2 in several ways (Cogdell and Frank, 1987). Firstly carotenoids
prevent 1O2 formation by accepting energy from 3Chl and dissipate it as heat. Secondly by
“scavenging” where carotenoids interact directly with 1O2 and dissipate the energy as heat.
Carotenoids are also involved in the regulation of feedback de-excitation which is discussed
throughout this thesis, which prevents the formation of 3Chl by allowing 1Chl to relax via heat
dissipation.
19
Avoiding over excitation
In addition to the protection against reactive oxygen, plants may prevent excitation damage by
mechanisms that decrease the excitation pressure. Several regulatory mechanisms have
developed to ensure a proper balance between the amount of excitation energy that reaches the
RC, and the amount of energy that may be utilised. There are two ways to lower the excitation
pressure: either decrease the amount of energy that comes in or increase the amount of energy
that goes out. In order to decrease the amount of light intercepted by the antenna, many plant
species respond physiologically by adjusting the angle of the leaf towards the sun and relocating
the chloroplasts. Plants may also synthesise antocyanins, water-soluble pigments that act as a
sunscreen, filtering out wavelengths otherwise absorbed by chlorophyll b (Gould et al., 1995).
The long term response by the antenna to high light is to reduce its size (Anderson, 1986;
Bailey et al., 2001; Mäenpää and Andersson, 1989). Alternatively, plants can increase the
energy usage, either by increased electron transport rate and turnover of ATP and NADPH, or
by dissipation of excess energy by the phenomenon known as the qE type of non-photochemical
quenching (NPQ; Horton et al., 1996; Niyogi, 1999), which we now prefer to call feedback deexcitation, in which excitation energy is dissipated as heat. Ways to sustain a high electron
transport rate during excess light may involve terminal electron acceptors other than CO2, for
example O2 in the Mehler reaction, O2 in photorespiration or N and S in amino acid metabolism.
Feedback de-excitation
When the enzymatic reactions that consume ATP cannot keep pace with the light reactions, the
trans-thylakoid ∆pH increases because protons that are transported into lumen along with
electron transport cannot be released through the ATP synthase in the absence of ADP. The low
lumenal pH activates Violaxanthin de-epoxidase (VDE; Rockholm and Yamamoto, 1996), which
converts violaxanthin via anteraxanthin to zeaxanthin in the so-called xanthophyll cycle (Figure
9; Demmig-Adams, 1990), and leads to protonation of several antenna polypeptides. Zeaxanthin
formation and antenna protonation trigger the feed back de-excitation (qE), thus preventing
over excitation and its destructive consequences. The Arabidopsis npq1 mutant that is deficient
in VDE and hence has a non-functioning xanthophyll cycle is incapable of inducing qE (Niyogi
et al., 1998). It has also been shown that the PsbS protein is essential for this process, because
the Arabidopsis npq4 mutant lacking PsbS has no feed back de-excitation (Li et al., 2000). The
mechanism of this kind of energy dissipation is not clear, but a tentative model will be presented
in the Discussion.
Violaxanthin has 9 conjugated carbon double bonds whereas zeaxanthin has 11. This led to the
hypothesis that violaxanthin had a higher S1 state that could not take energy from singlet excited
chlorophyll, but zeaxanthin, with its larger conjugated system would have an S1 state low
enough to quench chlorophyll excitation (Frank et al., 1994). Although it is tempting to believe
20
β-Carotene
β-hydroxylase
Zeaxanthin
High light
VDE
ZE
Antheraxanthin
VDE
Low light
that zeaxanthin acts as a direct
dissipater of chlorophyll excitation,
while violaxanthin would not have this
capacity, it is not likely to be the case.
Determination of the energy level of
the S 1 state for violaxanthin and
zeaxanthin shows that both are lower
than the Qy state of chlorophyll a
(Polívka et al., 1999). This indicates
no difference in their capacity for
quenching the singlet excited state of
chlorophyll a.
ZE
Violaxanthin
Another explanation is that zeaxanthin
Figure 9. The xanthophyll cycle. Violaxanthin is
might bring about a conformational
synthesised from β-carotene by β-hydroxylase and
change that induces dissipation (Crofts
zeaxanthin epoxidase (ZE). Under conditions that lead
and Yerkes, 1994; Horton et al., 1996).
to lumen acidification (indicated to the left by ”High
The shapes of violaxanthin and
light”), violaxanthin de-epoxidase (VDE) is activated and
zeaxanthin are different and may
converts violaxanthin to antheraxanthin, which has one
induce different conformations of the
epoxide group, and zeaxanthin, which has no epoxide.
protein to which the pigment bind. The
When the lumen pH raises (indicated to the right by
end groups of the zeaxanthin molecule
”Low light”), VDE is deactivated and ZE convert
are in the plane with the polyene chain
zeaxanthin and antheraxanthin back to violaxanthin.
whereas the epoxides in violaxanthin
twist the end groups out of the plane (Ruban et al., 1998b). Evidence supporting the modulating
role of zeaxanthin comes from in vitro experiments using the non-native xanthophyll auraxanthin
(Ruban et al., 1998b). Auraxantin is similar in size to zeaxanthin, but has the shape of
violaxanthin and a smaller system of conjugated carbon double bonds (7 double bonds). Ruban
et al. show that auraxanthin has the capacity to induce quenching in isolated LHC II similarly
to zeaxanthin. This strongly indicates that it is the molecular structure of zeaxanthin that is
important for qE, and not the energy level of the S1 state.
State transitions
Several studies have shown the presence of two pools of LHC II, which are often referred to as
the inner (or tightly bound) and the peripheral pools (Larsson and Andersson, 1985; Larsson et
al., 1987b). The inner pool appears to be more tightly associated with PS II (perhaps
corresponding to S, M and L-LHC II). Peripheral LHC II is more loosely associated with PS II
and participates in the state transitions (Larsson et al., 1987b). Peripheral LHC II may correspond
to the trimers not associated directly with PS II, as described above.
21
PS I and PS II have different absorption spectra, with PS I being red-shifted compared to PS II
and even capable of using far-red wavelengths. These discrepancies mean that different light
qualities excite the two photosystems unequally. In order for photosynthesis to function
optimally, electron transfer between PS II and PS I must be coordinated, which requires
regulation of the excitation balance (Allen, 1992). Under conditions that excites PS I more, the
mobile pool of LHC II transfer energy to PS II (state 1). Light that excite PS II more, leads to
the migration of LHC II to PS I (state 2). One can speculate that state 2 can be advantageous in
other conditions as well. For example when the cellular need for ATP is increased, cyclic
electron transport around PS I may benefit from increased PS I absorption.
A widely accepted model for the regulation of state transitions is based on phosphorylation of
LHC II, which leads to migration from PS II and attachment to PS I (Allen, 1992). In vivo
experiment (Andrews et al., 1993) using light enriched in either PS I or PS II wavelengths
showed phosphorylation of LHC II in PS II light. Peripheral LHC II is phosphorylated by an
LHC II kinase that is activated under conditions that cause a reduced plastoquinone to remain
bound to the Qo site of reduced cytochrome b6f complex (Vener et al., 1997), that is for example
when PS I is less excited than PS II. However, regulation of the LHC II kinase is more complex
(Hou et al., 2002). (Rintamäki et al., 2000) suggest that the kinase can be inactivated by
thioredoxin in high light.
The mechanism that cause the migration has been explained as electrostatic repulsions between
the negatively charged phosphate groups or to depend on molecular recognition between the
altered conformation of phosphorylated LHC II and PS I. However, other factors besides
phosphorylation are likely to be necessary to induce LHC II migration. Antisense plants lacking
the PSA-H subunit of PS I (Lunde et al., 2000), that are deficient in state transitions, show a
high level of LHC II phosphorylation, although LHC II remains to excite PS II, showing that
phosphorylation itself does not cause LHC II to migrate away from PS II.
Nevertheless, antisense plants with reduced activity of a kinase that is shown to phosphorylate
LHC II in vitro, has reduced capacity for state transitions (Snyders and Kohorn, 2001), and a
Chlamydomonas state transition mutant was shown to lack LHC II phosphorylation
(Fleischmann et al., 1999; Kruse et al., 1999), indicating that phosphorylation is a prerequisite
for state transitions.
Phosphorylation of LHC II in vivo has been shown to be most pronounced in low light (half the
growth light intensity) and very low in high light (Pursiheimo et al., 1998; Rintamäki et al.,
1997). This may be explained by the emphasised need for ATP in very low light, where the
plant does not assimilate carbon so reducing power (NADPH) is not needed, but steady state
cellular activities require ATP. Cyclic electron transport generates a pH gradient that drives
22
ATP formation without NADP+ reduction. Since PS I, but not PS II, is involved in this process,
it is favourable to induce state 2. In addition to the migration of LHC II, state transitions are
accompanied with a redistribution of the cytochrome b6f complex (Vallon et al., 1991) with a
larger proportion of cytochrome b6f in the stroma-exposed thylakoids (where PS I is located)
in state 2. This also enhances the capacity for cyclic electron transport. However, there are no
direct evidences for enhanced cyclic electron transport in low light. Besides state transitions,
phosphorylation of LHC II may have other functions such as regulating the structure of LHC II
(Nilsson et al., 1997; Zer et al., 2002).
State transitions cause a small decrease in PS II fluorescence, qT, because the antenna connected
to PS II is smaller in state 2 than in state 1, leading to a decrease in absorbed energy in state 2.
However, the state transition mechanism is not a high light protection, as for example shown
by the low phosphorylation levels of LHC II in high light, but function as a modulation of the
relative efficiencies of PS I and PS II.
Acclimation of the light harvesting antenna to different light conditions
In contrast to animal development, which is predominantly genetically determined, plant
development is to a significant extent governed by environmental factors. Being sessile, plants
need to be able to respond and acclimate to a broad spectrum of growth climates and stress
factors that vary at timescales from seasons to seconds. Light has a profound impact at most
levels of plant existence, for example leaf size and thickness, stem elongation, mesophyll
structure, the number of chloroplasts per cell, the onset of flowering and senescence, as well as
net biomass production. Both light quality, intensity and diurnal rhythm are important and are
perceived through various signal transduction pathways mediated by for example phytochrome
(Neff et al., 2000), cryptochromes (Cashmore et al., 1999) and chloroplast redox potential
(Pfannschmidt, 2003).
A comparison of the antenna protein composition in plants from the same species grown in
high or low light intensity show that the main difference lies in the amount of the subunits of
the peripheral LHC II namely Lhcb1 and Lhcb2 (Anderson, 1986; Bailey et al., 2001; Larsson
et al., 1987a), which increase with decreasing intensity of growth light. In addition, plants
grown in extremely low light increase the amounts of CP26 and Lhca4 (Bailey et al., 2001),
but not to the same extent as LHC II. These discrepancies in protein composition are regulated
via altered gene expression. Lhc transcription is induced by phytochromes and cryptochromes
in a circadian mode (Anderson and Kay, 1995; Hamazoto et al., 1997; Mazzella et al., 2001).
The transcription rate is modulated by signals from the chloroplast but the source and nature of
these signals are poorly described (Surpin et al., 2002). Some evidence indicates that the redox
state of the PQ pool regulates Lhc expression. Manipulation of the redox state by DCMU that
oxidises PQ (mimics low excitation pressure) and DBMIB that reduces PQ (mimics high
excitation pressure) have been used to investigate this. In Dunaliella tertiolecta the expression
23
of Lhc is high in the presence of DCMU, and low in the presence of DBMIB (Escoubas et al.,
1995). The cue-1 (cab under-expressed) mutant, which is deficient in the chloroplast envelope
phosphoenolpyruvate/phosphate translocator is hypothesised to under-express Lhc as a
consequence of a small PQ pool (Streatfield et al., 1999) - the synthesis of PQ is dependent on
the shikimate and isopentenyl pathways, to which phosphoenolpyruvate is a substrate.
Chlorophyll intermediates have been suggested to act as signals from the chloroplast and it
was recently shown that accumulation of Mg-protoporphyrin IX, represses Lhc expression
along with a range of other nucleus encoded photosynthesis genes (Strand et al., 2003). Other
hypotheses involve reactive oxygen species in the regulation of nuclear photosynthesis genes
(Mullineaux and Karpinski, 2002; Rodermel, 2001).
The difference in antenna size and composition influences the chlorophyll content of the leaf
both quantitatively and qualitatively. In high light the chlorophyll content per leaf area is higher
and because the outer antenna is smaller the chlorophyll a/b ratio is higher (chlorophyll b is
only present in the outer antenna).
Plants growing in high light plants invest energy into synthesising more of the electron transport
chain subunits and enzymes involved in carbon assimilation. These are rate limiting steps in
saturating light that determine the maximum photosynthetic rate (Pmax), hence Pmax is higher for
plants grown at high light. Surprisingly, however, it may be difficult to detect differences in the
quantum yield of O2 evolution or CO2 uptake in limiting light (Anderson, 1986; Lee et al.,
1999).
24
Methods
I would like to elaborate on some of the methods that were used, because they were important
to the work and may be less well known to the reader.
Model species: Arabidopsis thaliana
All work presented in this thesis was done in the model plant Arabidopsis thaliana (Figure 10).
Arabidopsis is naturally found over a large area on earth, and different ecotypes have developed
(Figure 10A). A few of them are in use in laboratories over the world, for example Columbia
(which I used), Landsberg erecta and Wassilevskija. This annual plant has an extraordinarily
short generation time. Depending on growth conditions (mainly day length) it can be as short
as six weeks from seed to seed. For photosynthesis experiments, wide leaves and large biomass
are beneficial which may be achieved by a short day length (8 h) and moderate light intensity
(150 µmol photons m-2 s-1), and under these conditions generation time increases to at least 10
weeks. Arabidopsis is self fertile, often pollinating already before the flower opens, but may be
cross-pollinated by hand. Seeds are small, facilitating screening, and keeping, many thousands
of them. Of superior importance for my work was the possibility to transform Arabidopsis with
exogenous DNA, which is easily done by routine protocols using Agrobacterium tumefaciens.
Figure
10.
(A)
Arabidopsis
thaliana. In (A) the
distribution
of
different ecotypes is
shown.
For
my
e x p e r i m e n ts , t h e
plant
material
usually looked as in
(B) which shows a
seven weeks old
plant grown at 150
µmol photons m -2 s -1
in 8 h photoperiod,
(B)
(C)
23/18°C day/night
temperature. (C)
shows a flowering
plant
and
an
enlargement of the
f l o w e r s..............
25
Recently the complete genome of Arabidopsis (Columbia) was sequenced (Arabidopsis Genome
Initiative, 2000). One significant reason for the choice of Arabidopsis to be fully sequenced
was that the genome size is the smallest of all known plant genomes, 7x107 bp, which can be
compared to the largest known plant genome, Fritillaria, 1x1011 bp, and the human genome,
3x109 bp (Ferl and Paul, 2000). The small size is due to little non-gene DNA, but also that
many proteins are encoded by singe copy genes. An advantage given by the compact genome
is that it is feasible to saturate the genome with insertion inactivation tags. Determination of
the DNA flanking the tag, gives, together with the complete genome sequence, the exact position
of the insert and the potentially inactivated gene. In addition to the genomic sequence, a large
number of expressed sequence tags (ESTs) have been obtained from different tissues and
different growth and stress conditions, which gives information about expressed genes.
Knowledge of gene sequences, families and expression patterns gives the opportunity to
construct antisense and over-expression lines.
Antisense inhibition
Mutants and transgenic organisms are useful tools to understand biological processes on a
molecular level. For a long time, scientists have gone from phenotype to genotype: an aberrant
individual was discovered, it was found out what was wrong in it and after tedious crossings
and marker analyses the mutated gene could be identified. When gene sequence information
started to build up, many genes with unknown function became known. This raised numerous
questions about the structure and function of the gene products. With the possibility to create
individuals that differ from the wild type only in one known gene, it is often possible to draw
some conclusions about the gene function. In bacteria, it is very easy to disrupt gene function
by introducing a deletion or insertion via homologous recombination. However, this is not
possible in plants, since they lack an efficient homologous recombination process. Instead, a
new copy of the gene of interest, in antisense direction, is inserted into the genome. The antisense
gene will be transcribed and diminish, and sometimes abolish, translation of the original gene.
The gene function may also be disrupted by co-suppression, in which a sense copy of the gene
of interest is introduced. For the integration into the genome there are a few methods in use, for
example bombarding plant material with DNA coated particles, or Agrobacterium tumefaciens
mediated transformation.
Antisense inhibition functions by a mechanism in which double stranded RNA (dsRNA)
molecules silence similar genes (for example Hannon, 2002; Hutvagner and Zamore, 2002). In
this process, dsRNA is cleaved into ~22 bp fragments (Bernstein et al., 2001) that are
incorporated into an enzyme complex and used as a template for the degradation of mRNAs
that contain the 22 nt sequence in question. This suggests that efficient antisense inhibition of
heterologous genes requires absolute sequence identity in a ~22 nt sequence.
26
T-DNA tagged mutants
If the gene sequence is known, another way to retrieve a plant with disrupted gene expression
is to screen for a favourable T-DNA insertion among the numerous collections of randomly
mutated Arabidopsis plants that are available. Either, the screen is performed with gene specific
primers that are designed in a way that they generate a PCR product if one gene specific primer
is used in combination with a T-DNA specific primer on a template of genomic DNA provided
that a T-DNA is inserted into the gene of interest. A PCR fragment is not produced if there is no
T-DNA in or close to the gene. Another way is to determine the genomic DNA sequence flanking
the insert. In the case of Arabidopsis, where the entire genome has been sequenced, this will
tell exactly in which gene the insert is found.
Chlorophyll fluorescence
When a chlorophyll molecule is excited, it has four ways to return to the ground state. 1)
transfer of the energy to another chlorophyll molecule, 2) charge separation in the reaction
centre leading to electron transport (photochemistry), 3) dissipation of the absorbed energy as
heat, or 4) fluorescence, in which the energy is emitted as light, which is possible to detect and
quantify. Since all the excitation energy in a leaf will meet one of these four fates, it is possible
to deduce a lot of information about photochemistry and quenching from the amount of
fluorescence that is emitted when known amounts of light is applied to a leaf (or to a thylakoid
or chloroplast suspension) (Maxwell and Johnson, 2000) .
Fv/Fm
A commonly used parameter is Fv/Fm, which is the ratio between the variable and the maximum
fluorescence that reflects the optimal photochemical efficiency of PS II. This is recorded on
dark-adapted leaves in order to avoid any effects of energy dissipation mechanisms, and the
light intensity given to induce maximum fluorescence should be applied as a short (<1s)
saturating (that is reducing all PS II centres) pulse.
Non-photochemical quenching (NPQ)
A dark-adapted leaf is neither ready to do photochemistry or energy dissipation, therefore a
high amount of fluorescence is obtained when light is applied to such a leaf. When photosynthesis
starts, which occurs within a few minutes upon light exposure and reflects the light dependent
activation of carbon metabolism enzymes, the amount of fluorescence decreases because more
of the excitation energy can be used in photochemistry. The decrease in fluorescence that is
caused by photochemistry is termed photochemical quenching. In addition to the induction of
photosynthesis, the antenna begins to dissipate energy, especially in excess light, which also
decreases the amount of fluorescence. This is commonly denoted non-photochemical quenching
of chlorophyll fluorescence, or non-photochemical quenching (NPQ), or sometimes just
27
Fm.......
.......Fmr
.......Fm’
.......Ft
Fo.......
Fo’.......
1 min
SP
MB
AL on
AL off
Figure 11. Fluorescence trace. This figure shows a chlorophyll fluorescence experiment performed
on an attached leaf. At MB the measuring beam is turned on and the Fo level is recorded as
indicated. At SP a short (~800 ms) pulse of saturating light intensity is given and the Fm level is
recorded as indicated. At AL on the actinic light, which drives photosynthesis, is turned on. Saturating
pulses are given (not indicated) and give the Fm’ value, as indicated. When the actinic light is
turned off (AL off) far red light is given to oxidise the electron transport chain and obtain the minimal
fluorescence ”in the light”, F o ’. After relaxation in the dark, F m r is recorded.
Table 3 Commonly used fluorescence parameters (See also Figure 11)
Quantum yield of PSII
(Fm´-Ft)/Fm´)
φPSII
qP
Proportion of open PSII
(Fm´-Ft)/(Fm´-Fo´)
Fv/Fm
Maximum quantum yield of PSII
(Fm-Fo)/Fm
NPQ
Non-photochemical quenching
(Fm-Fm´)/Fm´
NPQS
Slowly relaxing NPQ
(Fm- Fmr)/ Fmr
r
NPQF (qE) Rapidly relaxing NPQ (NPQ-NPQS)
(Fm/Fm´)-(Fm/Fm )
quenching. Non-photochemical quenching may be split up into at least three separate
components:
NPQ = qT + qE + qI
qT is the reduction in fluorescence due to detachment of LHC II from PS II in the State Transition
process. qI is the sustained reduction in quenching that is caused by non-functional PS II and
28
is sometimes referred to as photo-inhibition. The major part of NPQ is qE. This is induced by
the trans-thylakoid ∆pH and is sometimes referred to as the ∆pH dependent, or energy dependent,
or reversible, quenching. Quenching is a rather confusing term, relating to the detection method,
and not to the physiological cause and consequences of the energy dissipation. We have suggested
a new term for this phenomenon: feedback de-excitation, that reflects the fact that excitation
energy is released as a feedback response to the high trans-thylakoid ∆pH, which in turn indicates
high excitation pressure. Furthermore, it points out that the important parameter is de-excitation
of chlorophyll, not increase in the quenching of chlorophyll fluorescence, which is just observed
when performing a particular kind of experiment.
Fluorescence is measured with a weak measuring beam without and with light supporting
photosynthesis (actinic light), and saturating pulses are applied at certain intervals to obtain
the maximum fluorescence. Figure 11 shows a typical fluorescence trace and important variables
are indicated. In Table 3 the calculations of some important fluorescence parameters are
explained.
Evaluation of fitness – field experiment
Most experiments using transgenic or mutant plants deficient in photosynthetic proteins have
failed to detect a phenotype and have led to the conclusion that the plants grow as the wild type
(Paper I; Haldrup et al., 1999; Varotto et al., 2002). This contrasts with the view that evolution
will expel proteins that are not important for the fitness of the plant. However, there are great
discrepancies between the natural climate and the growth conditions we provide plants with in
the growth chamber. First of all, weather changes rapidly over significant magnitudes and
varies over the seasons whereas in the growth chamber light, temperature, water availability
and humidity are nearly ideal at all times. In nature the plants also have to fight biotic stresses
in form of fungal, viral and bacterial infections as well as grazers. In addition, nutrient supplies
are frequently limiting growth in the field, forcing the plants to use alternative metabolic
pathways.
In order to evaluate the potential impact of fitness from antisense inhibition of antenna proteins,
we have grown plants in the field, and used seed production as a measure of fitness (Külheim
et al., 2002). As the first, and so far only, group in Europe, we obtained a permit from the
Swedish ministry of agriculture (Jordbruksverket, Dnr 22-2151/01 and 22-2623/02) to grow
transgenic Arabidopsis in the field, with certain precautions taken. The different genotypes
were grown in bottom-less pots that were randomized in trays filled with soil. After germination
in a growth chamber, trays were dug down in the ground to allow free diffusion of water and
nutrients. No artificial watering or fertilization occurred. Once, during a severe infestation
with the Diamond back moth (Plutella xylostella) that threatened to kill of the entire population,
plants were treated with a pyrethrum compound. The only significant difference from natural
29
growth was the absence of competition between
individuals. At harvest, the number of siliques
per plant was determined. Three siliques from
each individual were opened and the seeds were
counted and weighed. From the average number
of seeds per silique and the number of siliques
from the same individual, the total number of
seeds per individual was calculated.
For an annual plant the total seed yield represents
its potential success to propagate to the next
generation. Provided that the seed dispersal and
germination frequency are the same between the
genotypes, seed number can be used as an
indicator of fitness.
PS II antenna structure – single particle
analysis
There are difficulties to obtain detailed structure
information about large membrane protein
Figure 12. Field experiment. This figure shows
the shady (top left) and sunny (bottom) complexes. Crystal structures, needed for nearlocations where the field experiment was atomic resolution, are only possible to obtain with
conducted. The different genotypes were colour homogeneous material. PS II is inherently
coded and randomised in trays that were dug heterogenic with a diverse protein composition
down in the soil. In the shady spot, spreading and variable antenna size, depending for example
of pollen and seeds were prevented by covering on the chloroplast’s location in the leaf and the
a glasshouse frame with insect net. Most of the photosystem’s location in the thylakoid
glass was removed to let in rain and avoid membrane. An alternative approach has to be
unnatural temperatures. In the sunny spot, taken. Grana membrane preparations may be
similar precautions were taken by building a gently solubilized to generate individual
small cage of insect net around the plants. All supercomplexes that can be subjected to
soil from the site was collected and destroyed. transmission electron microscopy. The resolution
in individual micrographs is low, but if many
hundreds of similar particles are averaged, some
details may be obtained. Yet another approach makes use of natural near crystalline areas of
the thylakoid membrane, which contains only PS II. In these membrane areas PS II develop
regular rows that can be treated as crystals and give detailed information on the antenna
composition.
30
Discussion
Transgenic lines
In order to investigate the functions of individual PS II light harvesting proteins we chose the
approach of reverse genetics. Antisense transformants were obtained for Lhcb1/Lhcb2, CP29,
CP26 and CP24. In addition, T-DNA tagged mutant collections were screened for insert into
any of the single gene encoded proteins (Lhcb3, CP26 and CP24). At a later stage, individuals
carrying a disruptive insertion in Lhcb5 lacking CP26 were identified. The different lines are
summarised in Table 4.
Table 4. Summary of PS II light harvesting antenna transformants
Type of
Lines
transformant
Affected transcript(s)
asLhcb2
antisense
Lhcb1.1; Lhcb1.2; Lhcb1.3; Lhcb1.4;
Lhcb1.5; Lhcb2.1; Lhcb2.2; Lhcb2.3
asLhcb4
antisense
Lhcb4.1; Lhcb4.2
asLhcb5
antisense
Lhcb5
salkLhcb5 T-DNA insert
asLhcb6
antisense
Lhcb6
Effect on protein
level
Lhcb1~100%
Lhcb2~100%
CP29~90%
CP26~100%
CP24~100%
Antisense constructs were made in the Agrobacterium tumefaciens binary vector pSJ10 (Ganeteg
et al., 2001), against CP29, CP26, CP24 and Lhcb2 using complete cDNA sequences of Lhcb4.1,
Lhcb5, Lhcb6 and Lhcb2.1, respectively. Major features of the transferable part of the vector
(T-DNA) are a cloning cassette for insertion of the antisense gene, driven by the constitutive
Cauliflower Mosaic Virus-35S promoter, and a kanamycin selectable marker (Figure 13). Plants
(Arabidopsis thaliana, ecotype Columbia) were transformed using in planta vacuum infiltration
(Ganeteg et al., 2001). This method transforms the inflorescence (Ye et al., 1999) and yields
heterozygous seeds carrying, most frequently, one or two insertion sites composed of an array
Lhc cDNA in
antisense direction
BL
bla
nos polyA
CaMV 35S
promoter
BR
NPT II
Figure 13. Antisense construct. The transferable part (T-DNA) of the Agrobacterium
tumefaciens vector pSJ10, that was used in the construction of the antisense plants described
in this thesis, has a cloning cassette that allows insertion of a DNA fragment which expression
will be driven by the Cauliflower mosaic virus (CaMV) 35S promoter. In all constructs used
here, a full length cDNA was inserted in the antisense direction. Other features of the pSJ10
T-DNA are the polyA signal, the NPT II gene that gives kanamycin resistance in plants, and
the bla gene that gives ampicillin resistance in bacteria. BL and BR symbolises the left and
right border sequences necessary for insertion into the plant genome......................................
31
(A)
(B)
Figure 14. Primary transformants. Several hundred seeds of primary transformants (T1) were
surface sterilised and spread on kanamycin plates (A). Kanamycin resistant seedlings are large
and green (for example the one the arrow points at). Many primary transformants were recovered
from each transformation and planted in soil. In (B) eight randomly selected T1 thylakoid
preparations, and two wild type samples in the outer lanes, were separated on SDS-PAGE and
subjected to immunoblotting using an antibody against the potentially antisense inhibited protein,
in this case CP26. All of them had basically nothing left of the antisense target protein.
EcoRI
WT
2
3
XbaI
14
WT
2
3
14
Figure 15. DNA blot analysis. In order to
investigate if three individuals had the antisense
gene inserted at different positions in the genome,
a DNA gel blot was run. DNA samples from the
wild type (WT) and three different T1 plants (CP29
antisense lines 2, 3 and 14) were cut with EcoRI
and XbaI. Detection with a radiolabeled PCR
fragment of the relevant cDNA (Lhcb4) yields a
different banding pattern showing that the insertion
site is different in the three individuals....................
of T-DNA copies (Feldmann, 1991). In most
cases, several tens of kanamycin resistant
seedlings (Figure 14A) of the first transformed
(T1) generation were isolated, and screened for
reduced protein levels (Figure 14B). A potential
problem when using the antisense method is that
the site of insertion into the genome cannot be
controlled. If the antisense construct is
accidentally inserted into another gene,
disruption of that gene may cause a phenotype
that will interact with the antisense induced
phenotype. To circumvent this problem, we
have used more than one antisense line that were
shown to be independent by the generation of
different restriction fragment patterns in DNA
gel blot analyses (Figure 15). Consistency
between lines ensures that the observed
phenotype is indeed caused by the intended
antisense effect, and not by random insertion
inactivation. We found that the antisense effects
with our constructs were extraordinarily high.
32
In all cases we were able to obtain at least one individual with close to nothing left of the
targeted protein (Papers I and III; Ganeteg et al., 2001). In the case of asLhcb2, both Lhcb1
and Lhcb2 were affected both on RNA and protein level (Paper III). In asLhcb5 only CP26
was affected and in asLhcb4 CP29 was nearly absent and in addition CP24 protein levels were
decreased, but the mRNA was still present (Paper I). In asLhcb6 only CP24 was affected.
Several collections of T-DNA tagged mutants were screened, both manually (PCR based
screening of pooled DNA; Campisi et al., 1999; McKinney et al., 1995; Sussman et al., 2000)
and by searching sequence databases of insert/genome junctions (SALK Institute Genomic
Analysis Laboratory and Syngenta Arabidopsis Insertion Library). One line was obtained from
the SALK collection carrying the disruptive insert in the Lhcb5 gene. The insert is located in
the 3’ end of the coding region (the Cterminal of the polypeptide), so nearly
1
2
3
4
5
6
WT
30
the entire reading frame is conserved.
However, protein gel blotting shows that
15
this line has no detectable amount of
CP26 (Figure16). It is possible that the
Figure 16. Immunoblot analysis of six putative
insert corrupts the polyA signal which
Lhcb5 knockout plants. Thylakoids from wild
would lead to an instable mRNA that is
type (WT) and six putative Lhcb5 knockouts (1rapidly degraded. Other possibilities are
6) were analysed with the CP26 antibody. Plants
number 1 and 5 had no detectable level of the
that the insert leads to a very long open
protein. Plants 2-4 and 6 have lower levels than
reading frame that cannot be transcribed
the wild type and may be heterozygous. Two size
or a polypeptide that cannot be imported
markers, in kDa, are shown to the left...............
into the chloroplast.
Antisense has the advantage that proteins encoded by gene families, such as CP29, Lhcb1 and
Lhcb2, may be abolished, provided they share some sequence homology. Insertion inactivation,
on the other hand, has the advantage that the insertion site is known and the knocked out gene
is likely to be constantly deactivated. They are also useful for complementation studies. We
have crossed antisense lines in order to obtain multiple antenna protein deficiencies, but found
that the antisense effect decreased in the offspring. Maybe that it is due to that the vector used
for all antisense constructs was the same. We believe that it will be possible to cross our antisense
lines with the T-DNA insertion mutants. Alternatively, the T-DNA knock-out may be transformed
with the antisense construct.
Why did Flachmann and Kühlbrandt fail?
There have been several attempts to obtain a plant lacking Lhcb1 and Lhcb2 proteins, of which
one was published. In 1995 Flachmann and Kühlbrandt (Flachmann and Kühlbrandt, 1995)
reported an antisense experiment in Nicotiana tabacum, in which the levels of Lhcb1 and
33
Lhcb2 transcripts were severely decreased (although not zero) from all six known genes, but
protein levels were as the wild type. No differences in chlorophyll content, photosynthesis
rates or fluorescence were detected. I compared their 509 bp antisense construct from the N.
tabacum Lhcb1.2 gene (CAB-21), with the other four N. tabacum Lhcb1 genes and the single
N. tabacum Lhcb2 gene. Apparently, the Lhcb2 gene did not share any continuous sequence
more that 17 nt in length with exact nucleotide identity with the antisense gene, which indicates
that this gene might not be affected by the antisense construct. However, Flachmann and
Kühlbrandt did see a decrease in transcription also from this gene (although some mRNA was
retained), so an additional explanation is required. They hypothesise that Lhcb protein synthesis
is post transcriptionally regulated, and that normal protein levels may accumulate from the
small amount of mRNA present. In addition to this explanation, one should consider the very
likely possibility of additional Lhcb1 and Lhcb2 genes in tobacco, from which nothing is known
about their expression pattern in the antisense lines.
Feedback de-excitation in the antisense plants
Several lines of evidence have pointed towards CP29 and/or CP26 being either the site, or the
sensor(s) for feedback de-excitation (qE). Their positions between the core antenna and LHC
II have led to the hypothesis that they function as a gate for the excitation before it would reach
the sensitive reaction centre (Bassi et al., 1993). CP29 and CP26 have been shown to have
protonation sites sensitive to DCCD, and have been suggested to be the sensor(s) for the lowered
lumenal pH (Walters et al., 1994; 1996). DCCD is a chemical agent that interacts with protonation
sites in a hydrophobic environment and has empirically been shown to inhibit qE, and CP29
has been shown to bind DCCD. Formation of feedback de-excitation goes along with deepoxidation of violaxanthin to zeaxanthin in the xanthophyll cycle, and CP29 and CP26 each
bind one molecule of these pigments (Bassi et al., 1993). Furthermore, their in vitro quenching
characteristics seemed to mimic qE (Ruban et al., 1996; 1998a).
However, when we analysed the antisense plants lacking either CP29 or CP26, only small
effects on NPQ was observed (Paper I)*. Most importantly, the capacity for feedback deexcitation in high light grown plants were the same for both antisense lines and the wild type,
which show that maximum qE does not depend on CP29 or CP26. It may be argued that these
two proteins have redundant functions and loss of one is compensated for by the other. This
cannot be conclusively ruled out yet. However, we could not observe any increase in the protein
* Five months after these results were obtained, parallel work showed that PsbS was
likely to be the most important PS II protein in qE (Li et al. 2000). However, the idea
of CP29 and/or CP26 as the quenching site(s) remained in the research community
for some time, and everyone is not yet convinced of the opposite......................................
34
level of CP26 in CP29 antisense lines or vice versa, which may argue against a compensatory
response between these two proteins. The fact that the maximum level of NPQ was not affected
in any of the lines, clearly points at no direct involvement of either of these proteins in feedback
de-excitation. By crossing CP29 antisense plants with CP26 T-DNA knockouts, plants lacking
both proteins are likely to be found. Such plants would finally settle this question.
In asLhcb2, lacking both Lhcb1 and Lhcb2, the capacity for feedback de-excitation was reduced,
but its induction kinetics was similar to the wild type. The maximum photosynthesis rate was
un-affected and the xanthophyll cycle was operational, indicating that the trans-membrane
proton gradient is normal and is not the cause of lowered qE. In addition, NPQ was recorded in
light intensities as high as 10 000 µmol photons m-2s-1 and it was found that NPQ was saturated
at the same light intensity in the antisense lines as in the wild type which shows that it is a true
decrease in feedback de-excitation, and not simply a shift of the induction to higher light
intensity due to decreased energy absorption. This decrease may occur due to fewer dissipation
sites or decreased connectivity between the antenna and the dissipation site. We have some
evidence that the protein level of PsbS was decreased, when expressed per PS II, in asLhcb2
(Paper III). It is known that the capacity for feedback de-excitation is related to the amount of
PsbS (Li et al., 2000; Li et al., 2002a) and that its expression increases with growth light
intensity (Külheim et al., Submitted manuscript). Perhaps the asLhcb2 plants with their small
light harvesting antenna experience a low light response which leads to down regulation of the
expression of PsbS. Another possibility is that the lack of Lhcb1 and Lhcb2 impair the energy
transfer between the antenna and the dissipating species (possibly PsbS). A third alternative is
that all antenna proteins contribute to energy dissipation and that loss of a part of the antenna
leads to a proportional decrease in dissipation capacity. It would be interesting to cross asLhcb2
lines with the PsbS over-expressing line in order to distinguish between these alternatives.
Speculative model for feedback de-excitation
The mechanism that actually dissipates the excitation is obscure. Concentrated solutions of
chlorophyll quench fluorescence efficiently (Beddard and Porter, 1976) and altered chlorophyll
interactions have been suggested as the mechanism for qE (Crofts and Yerkes, 1994). It has
been shown in vitro that aggregation of antenna proteins (both LHC II and the minor ones;
Horton et al., 1991; Ruban et al., 1994 and 1997; Walters and Horton, 1999) leads to fluorescence
quenching, perhaps by allowing close interaction between pigments. Disruption of the aggregates
(by addition of detergents) causes a fluorescent state of the proteins. In vitro studies also show
that addition of violaxanthin inhibits whereas zeaxanthin promotes the formation of aggregates
(Ruban et al., 1997). Although we do not believe this is the exact mechanism behind qE, it
shows that there is a thin line between energy transfer and heat dissipation in the chlorophyll
dense antenna. Minor conformational changes may be sufficient to promote close chlorophyll
interactions that bring about heat dissipation pathways for de-excitation of 1Chl (Crofts and
35
Yerkes, 1994; Horton et al., 1996). It is now known that the PS II antenna alone cannot perform
feedback de-excitation and qE is absent in the npq4 mutant where all Lhcb proteins are present
in wild type amounts. There is also no indication of an obligatory interaction of any of the
Lhcb proteins. We have ruled out every Lhcb protein because the antisense plants lacking
CP29, CP26 and CP24, respectively, all perform qE to the same maximum level as the wild
type. The plants without Lhcb1/Lhcb2 have a reduced maximum level, although it still has a
significant amount of qE. In order to rule out Lhcb3, barley chlorina mutants (chlorina126 and
134
) lacking Lhcb3 and LHC I-730 (Bossmann et al., 1997) were examined and found to have
extensive NPQ.
Solid evidence point at a direct involvement of PsbS in feed back de-excitation. 1) The capacity
for qE is proportional to the amount of PsbS (Li et al., 2000; 2002a). 2) It has been shown to
bind DCCD (Dominici et al., 2002) and putatively protonatable amino acid residues that are
essential for PsbS function have been identified (Li et al., 2002b). 3) The change in leaf
absorbance at 535 nm (∆A535) that has been observed to accompany the onset of qE (Ruban et
al., 1993), and has been hypothesised to stem from a conformational change leading to the
formation of a quencher, was demonstrated to be PsbS dependent (Ruban et al., 2002). Ruban
(2002) also concluded that only 1-2 zeaxanthin molecules are needed to induce quenching.
It is apparently straightforward to isolate PsbS without pigments, but rather difficult to obtain
a pigment-protein complex (Dominici et al., 2002; Funk et al., 1994; 1995). However, the data
presented in (Funk et al., 1994) are convincing. Furthermore, PsbS prepared from spinach was
shown to bind zeaxanthin when it was reconstituted in the presence of this pigment (AspinallO’Dea et al., 2002), which also induced the characteristic 535 nm absorbance change. I believe
PsbS does bind both xanthophyll cycle pigments and chlorophyll. All chlorophyll binding sites
in LHC proteins are not conserved in PsbS, but the function of the chlorophylls in the LHC
proteins are radically different, and their binding and coordination are likely to be distinct.
According to my view, PsbS is the site of feedback de-excitation. Protonation of the protein
stimulates exchange of violaxanthin for zeaxanthin which induces a conformation change that
bring chlorophyll molecules close enough to form new excitation levels that allow relaxation
of excited states via heat dissipation. PsbS may accept energy from the other antenna proteins
and when it is activated by zeaxanthin the excitation energy is dissipated as heat. It is likely
that PsbS can interact with the PS II antenna at multiple sites. Evidence for this idea comes
from the wide variation in the amount of PsbS per PS II that have been observed in mutants
without this protein, wild type plants grown in various light conditions and transgenic plants
that over-express PsbS. Furthermore, qE in the PS II antenna deficient lines does not indicate
any specific interaction site for the heat dissipating species.
36
PS II antenna organization
In order to determine the PS II subunit organization,
cross-linking studies has been used to obtain nearest
neighbour information (Harrer et al., 1998).
Transmission electron microscopy has yielded a
contour map of the PS II supercomplex, including
tentative positions of the antenna proteins (Boekema
et al., 1999b). However, the minor antenna proteins
are similar in size and shape and cannot be
distinguished in the map, although the model shown
in Figure 6 assigns certain masses to particular
proteins. The antisense plants provided an excellent
tool to test the model.
Figure 17. Organisation of the PS II antenna. The dimeric core complex is shown in white.
Minor antenna proteins are light grey. LHC II trimers are dark grey. 1 = Lhcb1, 2 = Lhcb2, 3 =
Lhcb3. The rotational orientation of LHC II is not intended to be accurate. The exact position of
the Lhcb3 subunit is not known, although it is located in the same trimer where it is drawn.................
Supercomplexes prepared from the CP26 antisense plants have a different shape compared to
wild type supercomplexes (Paper II). A mass corresponding to a minor antenna is missing from
two diametric corners of the structure. It is most likely that the difference is due to the lack of
CP26 and establishes the position of CP26. In the model the same sites were assigned to CP26.
Having located CP26, the positions of the other two minor antenna complexes are given.
Interestingly, it was not possible to isolate supercomplexes from the CP29 antisense line (Paper
II). Apparently this protein is essential for supercomplex stability, and no other LHC could
compensate for this function. CP29 may have the unique function of interacting both with the
core antenna and anchoring LHC II and CP24 to the complex.
Figure 17 shows the organization of the PS II antenna in the wild type. In Figures 18-20 the
putative antennas of the antisense plants lacking the minor antenna proteins are drawn.
Conservation of supercomplex ultra-structure
asLhcb2 supercomplexes, lacking Lhcb1 and Lhcb2, were expected to confirm the lack of
LHC II trimers. However, the shape of the supercomplex was similar to the wild type, although
slightly smaller (Paper IV). We had previously found an extensive increase in CP26 and Lhca4
in these plants (Paper III). It became apparent that CP26 were able to form trimers that were
incorporated into the antenna complex in the positions usually occupied by LHC II. In Figure
37
Figure 18. Organisation of the PS II antenna
in asLhcb5. According to TEM (Paper II) the only
difference from the wild type antenna is the lack
of the CP26 protein. No rearrangements appear
to occur. Labelling as in Figure 17.......................
Figure 19. Disorganisation of the PS II
antenna in asLhcb4. No supercomplexes were
detected in asLhcb4 (Paper II). According to
immunoblot analysis (Paper I) the protein levels
of CP26 and the LHC II subunits are present in
wild type amounts whereas the CP24 level is
decreased. The question is how the antenna is
arranged. Are LHC II, CP26 and CP24
completely disconnected or are they located at
their normal positions although not securely
bound? Labelling as in Figure 17................
Figure 20. Tentative organisation of the PS II
antenna in asLhcb6. asLhcb6 has wild type
amounts of all Lhcb proteins except CP24 which is
not detectable. Although this plant has not been
subject to TEM, it is likely that the antenna is
organised as shown in this figure. Supercomplexes
without CP24 have been observed in wild type plants
(Boekema et al., 1999b). Labelling as in Figure 17.....
38
Figure 21. Conserved antenna organisation
in asLhcb2. According to TEM (Paper IV) the
antenna ultrastructure is conserved. These
plants have highly increased amounts of CP26
which is assembled into trimers that resemble
LHC II. It is not known whether the Lhcb3
subunit, that is present at least in wild type
amounts, form homotrimers, or if is trimerises
together with CP26. Labelling as in Figure 17.
21, the hypothetical organization of the antenna in this plant is shown. It is interesting to note
that amino acids in the N terminal loop that are regarded as important for LHC II trimerisation
(Hobe et al., 1995) are conserved between Lhcb1, Lhcb2, Lhcb3 and CP26, but not in CP29 or
CP24 (Figure 6). However, one amino acid residue in the C terminal domain (W222) that is
also important for LHC II trimer formation (Kuttkat et al., 1996) is not conserved in CP26. It
appears as if the supercomplex integrity is important and the minor antenna protein CP26 is
recruited to maintain this structure.
Photosynthesis in the absence of antisense inhibited antenna proteins
70-80% of the photosynthetic light energy is absorbed by the LHC proteins, and only a small
amount of energy is absorbed directly by the reaction centres and the core antenna. This gives
the impression that the outer antenna is very important for efficient light harvesting and
photosynthetic quantum yield in limiting light.
We measured the rates of photosynthetic O2 evolution on all antisense lines grown at 150 µmol
photons m-2 s-1 and compared to the wild type. Surprisingly, none of the antisense lines performed
strikingly worse than the wild type neither in terms of limiting light quantum yield nor saturating
light maximum photosynthesis. In fact, asLhcb2 had a higher quantum yield and asLhcb5 had
a higher maximum photosynthesis whereas asLhcb4 had a slightly lower maximum
photosynthesis. High and low light grown plants have previously been shown to have similar
quantum yields (Anderson, 1986; Lee et al., 1999). However, when interpreting these results
one should be aware that the light in the growth chamber always differs from the light source
used in the O2 measurements. For our measurements we used a LED light source which gives
a spectrum that excites PS II more than PS I. This leads to a PS I limited photosynthesis in low
light. In saturating light photosynthesis is not, by definition, limited by energy absorption,
instead the enzymatic reactions in CO2 fixation and the inter-photosystem electron transport
39
chain, especially the cytochrome b6f complex, is regarded as the limiting factors. Hence, it is
not surprising that antenna protein deficiency has little effect on maximum photosynthetic
rates. It should also be noted that none of the antisense lines had impaired growth rates in
standard climate chamber conditions (150 µmol photons m-2 s-1, 8 h photoperiod, 23/18°C day/
night temperature), so it may be expected that photosynthesis functions normally under these
conditions.
We also measured CO2 uptake. In this case a white light source was used and we were indeed
able to detect a small decrease in quantum yield for the Lhcb2 antisense line compared to the
wild type (Paper III). Although the difference was very small the discrepancy between the
different methods demonstrates the difficulty of accurate photosynthesis rate determinations.
Grana
Although investigations of plants with decreased antenna size due to various mutations and
extreme growth regimes have shown that grana stacking can be present even with a minimum
of Lhcb proteins, the idea of LHC II as the driving force for stacking remains (e.g. Allen and
Forsberg, 2001; Rozak et al., 2002). asLhcb2 that specifically lack Lhcb1 and Lhcb2 still form
grana, although we have not done any thorough quantification it appears to be similar to the
wild type in this respect. This shows that LHC II is not a prerequisite for stacking.
State transitions
asLhcb2 plants had no state transitions showing that the “CP26 trimers” or the Lhcb3 protein
could not take over this function (Paper III). CP26 does not have the phosphorylation site
present in LHC II. Moreover, it is believed that it is the peripheral LHC II pool that participates
in state transitions, and this pool may be completely absent in these plant lines.
The increase in Lhca4 in asLhcb2 indicates that LHC II is indeed important for PS I. In vivo
studies of LHC II phosphorylation have found that it is only present in light intensities well
below growth light, which suggests that LHC II would only transiently associate with PS I. In
contrast, asLhcb2 grown in constant moderate light intensity (150 µmol photons m-2 s-1, Paper
III) increase the amount of an LHC I protein, which could mean that PS I senses a decrease in
antenna size which initiates an increase in Lhca4.
40
Is each Lhcb necessary?
In the case of asLhcb2-12 we detected a significant decrease in growth in low light (50 µmol
photons m-2 s-1, Paper V) but for the other antisense lines no such decrease was observed. It has
been argued that each LHC protein has a function in the antenna, based on the conservation of
the entire LHC family in all higher plants. Since we could not see a clear phenotype in the
growth chamber, we decided to grow the plants in the field where they would meet a wide
spectrum of environmental conditions, and the flexibility of their metabolism would be tested
(Ganeteg, Andersson, Külheim and Jansson, manuscript in preparation). In order to evaluate
each genotype’s fitness, we determined the number of siliques and seeds per silique for at least
2x40 individuals of each genotype grown either in a sunny or shady spot. Regardless which
Lhcb protein that was taken away, it led to a decrease in fitness. The cause of the reduction is
likely to be different in each case, and has to be further investigated in controlled growth
chamber experiments.
Functions of the Lhcb proteins
The field experiment shows that every Lhcb protein is necessary. The main question is in what
way? From the characterization of the antisense plants described in this thesis, the following
may be concluded about individual light-harvesting proteins.
CP29
- is not a prerequisite for feedback de-excitation, in contrast to what was proposed on the basis
of biochemical analyses. Antisense plants with very low levels of CP29 do induce qE to a level
comparable with the wild type.
- is important in antenna organization and appears to be connecting LHC II to the core antenna.
PS II supercomplexes are absent, or at least very unstable, in the antisense line. It is likely that
the observed reduction in fitness is a consequence of the disorganization of the antenna that is
caused by the loss of CP29.
CP26
- is also dispensable for feedback de-excitation, concluded on the same basis as CP29.
- confers flexibility to antenna composition. It can form trimers that resemble LHC II, and
conserve supercomplex structure in the absence of Lhcb1 and Lhcb2.
CP26 may be important for the relation between PS II units. Perhaps the connectivity between
the antenna systems is impaired when this protein is absent.
41
CP24
- depend on CP29 for stability.
- is important for overall fitness in the field, but further studies are required to understand the
reason.
Lhcb1/Lhcb2
- are important for growth in low light in the growth chamber, and the compensatory increase
in CP26 and Lhca4 does not completely make up for the loss of these proteins in asLhcb2
plants.
- are essential for the state transition process, and the CP26 trimers that assemble in the absence
of Lhcb1 and Lhcb2 cannot take over this function.
- are dispensable for the formation of grana stacks.
The observed decrease in fitness may be contributed in part to the lack of state transitions and
in part to the decrease in antenna size leading to a decrease in light limited photosynthesis.
Lhcb3
- has previously been shown to form trimers with Lhcb1 and/or Lhcb2. It is also present in
asLhcb2 which lack these proteins where it forms trimers either by itself, or with CP26. Many
question remains about the localization and function of Lhcb3 in the antisense plants as well as
in the wild type.
42
Future perspectives
There are still many questions that the transgenic plants described here may help to answer.
For example, it would be interesting to...
Isolate LHC II trimers from low light grown wild type plants, which are known to have increased
amounts of CP26, to determine the protein composition. Does CP26 participate in LHC II
trimer formation also in wild type under certain (perhaps extreme shade) conditions?
Exchange W222 in recombinant Lhcb1 and/or Lhcb2 for lysine (which is the amino acid in
corresponding position in CP26) and investigate trimerisation characteristics.
Investigate PS I antenna structure in the asLhcb2 plants. Where does the extra Lhca4 bind?
Lhca4 is also known to increase extensively in low light conditions. Does the PS I antenna of
wild type plants grown in low light resemble the PS I antenna of asLhcb2 plants?
Cross asLhcb2 with an insertion inactivation mutant lacking Lhcb3 to obtain plants lacking all
proteins that are known to form LHC II. Will trimers form with only CP26? Is supercomplex
ultra-structure preserved? Are these plants able to compensate for the loss of light harvesting
function even in growth chamber conditions?
Cross asLhcb2 with SALK-Lhcb5 to obtain plants lacking the major constituents of LHC II in
wild type plants, as well as CP26 that compensate for the Lhcb1/Lhcb2 loss in asLhcb2 plants.
Do yet other antenna proteins have the potential to be recruited as LHC II trimer constituents?
What happens to the thylakoid ultra-structure?
Cross asLhcb2 with PsbS over-expresser. What happens to the NPQ capacity? If NPQ is the
same as for asLhcb2 it may be concluded that LHC II has a direct function in feedback deexcitation either as connecting the antenna to the quencher or (less likely) itself dissipating
energy. If NPQ is higher, it is likely that the decrease in feedback de-excitation in asLhcb2 was
a low light response leading to a down regulation of quenching capacity (the amount of PsbS).
It would also be informative to grow asLhcb2 in very high light in order to determine the
maximum capacity for qE.
43
Do more fitness experiments in other conditions: extreme shade, shade from vegetation or
“abiotic” shade, different light qualities (filtered sunlight), wind exposed, dry, well watered,
climate chamber, climate chamber with rotating shade, climate chamber with rotating shade
from leaves, climate chamber in constant shade from leaves. These experiments would pinpoint
under which environmental factor each antenna protein is most important.
Investigate the dynamics of grana stacking in asLhcb2. Will there be rapid changes along with
alterations in the light quality and quantity. Perhaps these fast changes depend on state transitions
(or more specifically on relocation of LHC II) and will be absent in plants lacking this process.
Explore the signal transduction pathway leading to the increase in CP26 and Lhca4 in asLhcb2.
Is this similar to a low light response (these proteins are also up-regulated in low light), and if
so, how is this sensed?
Transform the CP26 knockout line with site directed mutagenised Lhcb5 sequences. This enables
the in vivo investigation of for example the pigment binding sites, protein interaction sites,
function of the DCCD sensitive amino acids and amino acid relevant for protein stability.
44
Den ljusskördande antennen hos fotosystem II
Solljuset är den primära energikällan för praktiskt taget allt liv på jorden. Fotosyntetiserande
organismer (till exempel växter, mossor, alger och cyanobakterier) omvandlar solenergin till
kemiskt bunden energi, främst i form av kolhydrater, som i sin tur används av icke
fotosyntetiserande organismer (till exempel människor och andra djur) som energikälla och
byggmaterial för cellerna. De flesta känner till att vi är beroende av växterna för att de
producerar syret vi andas, men det är även så att all vår föda har sitt ursprung i fotosyntesen.
I växter sker fotosyntesen i kloroplasten (Figur 1). Den första delen, när solenergin binds i
cellens energitransportörer ATP och NADPH, utförs av stora komplex bestående av massor
av olika proteiner som sitter bundna i tylakoidmembranet (Figur 2). Den andra delen, när
luftens koldioxid omvandlas till kolhydrater, utförs av enzymer som förbrukar ATP och
NADPH.
Det pigment som används för att fånga in solenergin är främst klorofyll. Den gröna färg som
dominerar vår planet är en följd av att klorofyll absorberar blått och rött ljus (Figur 3),
medan den gröna delen av ljusspektret reflekteras tillbaka. För att effektivt absorbera
solljuset har växterna utvecklat en ljusskördande antenn (light-harvesting antenna) som
består av tio olika proteiner som binder klorofyll och några andra pigment (Tabell 1 och 2).
Antennen är mycket effektiv på att samla in solljus och skicka energin vidare till de två
fotosystemen (PS I och PS II; Figur 2) som utför de första stegen i fotosyntesreaktionen.
Solen är en mycket kraftfull energikälla, och det är inte alltid växten hinner med att förbruka
all energi. Figur 8 visar hur ljusstyrkan varierar under en vanlig sommardag med växlande
molnighet. Den ena kurvan visar en helt öppen plats, den andra är i skuggan av träd och
buskar. När för mycket energi absorberas av klorofyll, så kan skador uppstå på grund av att
det bildas olika typer av syreradikaler som är mycket aggressiva och kan förstöra proteiner
och membranlipider. Eftersom det inte går att stänga av solen, och växten inte kan flytta på
sig, så har det utvecklats en mekanism som gör det möjligt att spilla bort överskottsenergi i
form av värme. Det har varit mycket oklart hur detta går till, men man börjar nu förstå
ungefär vad som händer.
Jag har utforskat den ljusskördande antennen hos fotosystem II. Den utgörs av sex olika
proteiner varav de första hittades redan på 1960-talet. Trots att de har varit några av de allra
mest undersökta växtproteinerna (speciellt av biokemister) så var mycket okänt när jag
inledde mina forskarstudier. Man visste till exempel inte hur de var placerade i förhållande
till varandra eller varför det fanns så många olika typer av antennproteiner. Man trodde sig
45
dock veta att två av dem, CP29 och CP26, var mycket viktiga för bortspillandet av
överskottsenergi.
En vanlig metod inom växtforskningen för att ta reda på hur ett protein fungerar, är att ta
fram en växt som saknar det proteinet. Genom att jämföra den förändrade växten med
ursprungsväxten (vildtypen) så kan man utifrån skillnaderna dra en del slutsatser om vilka
funktioner proteinet är inblandat i. För att få en växt som saknar just det protein man är
intresserad av, så kan man använda sig av en metod som kallas antisens. Lite förenklat så går
det praktiskt till så att man tar hjälp av en jordbakterie (Agrobacterium tumefaciens) för att
sätta in en bak-och-framvänd gen (antisensgen) i någon av växtens kromosomer.
Antisensgenen gör så att originalgenens budbärar-RNA bryts ned, och inget protein kan
bildas.
Den vanligaste växten inom molekylärbiologin idag är det lilla ogräset backtrav,
Arabidopsis thaliana (Figur 10, syns även på Figur 12). Det är en oansenlig växt med många
fördelar för forskarna. Bland annat så är den ringa storleken bra när man vill odla många
individer, fröna är också mycket små så det går att spara tusentals av dem. Backtrav har även
ett ovanligt litet genom, det vill säga den samlade längden på alla växtens gener, vilket har
lett till att man har kunnat läsa av hela genomsekvensen. En stor fördel är också att det är
möjligt att transformera backtrav (sätta in nya gener). Denna lilla växt har i sig inget värde
exempelvis i jordbruket, men de grundläggande funktionerna hos alla växter (och i många
fall hos alla levande organismer) är i regel väldigt lika. Det man lär sig av backtrav är alltså
ofta giltigt för växter i allmänhet.
Jag har använt antisensmetoden för att ta fram fyra olika växter som saknar ett eller två
antennproteiner var. Genom att studera dessa har vi kunnat visa följande:
De olika antennproteinerna sitter runt PS II såsom visas i Figur 17 där CP29 spelar en
nyckelroll genom att samla de andra komponenterna omkring sig.
LHC II-trimeren (mörkgrå i Figur 17) är mycket viktig för att upprätthålla
tillväxthastigheten när växten odlas i mycket svagt ljus. Däremot verkar den inte spela lika
stor roll för hur tylakoidmembranet veckas som man tidigare trott.
CP26 kan agera ställföreträdande LHC II i de växter som inte har proteinerna som normalt
utgör LHC II.
CP29 och CP26 utför inte bortspillning av överskottsenergi trots att åratal av biokemiska
experiment har visat på det. Vi har visat att i själva verket är inte något av antennproteinerna
46
direkt inblandat in den processen vilket många har varit övertygade om (och vissa är det
fortfarande).
I ett fältförsök, där vi odlade alla antisensväxterna utomhus (vilket kräver tillstånd av
Jordbruksverket eftersom det här är transgena växter), kunde vi visa att alla antennproteiner
är viktiga för att växten ska vara konkurrenskraftig gentemot sina artfränder. De växter som
saknade något antennprotein gjorde färre frön än vildtypsplantorna.
Det är fortfarande många obesvarade frågor kvar som antenn-antisensväxterna kan hjälpa till
att lösa. Till exempel skulle de kunna användas för att öka förståelsen för hur det fungerar
när växten känner av att den befinner sig i skugga och behöver göra en större ljusantenn. De
kommer också att användas i den fortsatta forskningen om hur detaljerna i bortspillande av
överskottsenergi ser ut.
47
Acknowledgements
First of all I would like to express my gratitude to each and every one working at FysBot.
You are a nice group of people and I will miss you. In particular the PhD students, both past
and present, have always been a friendly gang, disproving the myth of rival scientists.
I am thankful to all collaborators and co-authors, especially Peter Horton, Sasha Ruban and
Robin Walters.
I wish to mention ”the group”: Ulrika, Carsten, Johanna, Rupali, Andreas, Frank, Oskar,
Roberth, and all temporary members that have come and gone over the years. The Friday
breakfasts are memorable, as well as the traditional summer barbeque and (perhaps new
born tradition) mid winter star gazing.
Personal thanks to
... my supervisor, Stefan Jansson, for gathering a great working group, and for a successful
project. It is a fact that I would not be here today without your tireless belief in my
capability.
... my reference group, Göran Samuelsson and Vaughan Hurry, for your support over the
years and for valuable comments during the writing process.
... my co-supervisor, Petter Gustafsson, for just being extremely positive.
... Ulrika Ganeteg, my room mate and fellow antenna researcher, for introducing me to the
practices of LHC study, for being a good working partner and friend.
... Tomtebo Odlingsområde, for providing me space to practice some real plant science.
... Henrik, you know why.
Finally I want to say that I have had a good time living in Umeå (although the concept of
spring in completely on its head). Moving here was a big step that I am glad I took.
48
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