Báo cáo khoa học: Knock-out of the chloroplast-encoded PSI-J subunit of photosystem I in Nicotiana tabacum PSI-J is required for efficient electron transfer and stable accumulation of photosystem I pot
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Knock-outofthechloroplast-encodedPSI-J subunit
of photosystemIinNicotiana tabacum
PSI-J isrequiredforefficientelectrontransferand stable
accumulation ofphotosystem I
Andreas Hansson
1
, Katrin Amann
2
, Agnieszka Zygadlo
1
,Jo
¨
rg Meurer
2
, Henrik V. Scheller
1
and Poul E. Jensen
1
1 Plant Biochemistry Laboratory, Department of Plant Biology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark
2 Department Biologie I, Botanik, Ludwig-Maximilians-Universita
¨
t-Mu
¨
nchen, Germany
The photosystemI (PSI) complex of higher plants con-
sists of at least 19 different polypeptides [1–3]. PSI
mediates light-driven electrontransfer from reduced
plastocyanin (Pc) inthe thylakoid lumen to oxidized
ferredoxin inthe stroma. The PSI core in higher plants
contains at least 15 different subunits named PSI-A to
PSI-L, PSI-N to PSI-P. Two subunits present in
cyanobacteria, PSI-M and PSI-X, are missing from
plants. In addition to the PSI core, higher plants con-
tain a peripheral antenna associated with PSI, also
known as light-harvesting complex I (LHCI), which is
mainly composed of four different Lhca proteins.
The major subunits of PSI, PSI-A and PSI-B, form a
heterodimer, which binds the components ofthe elec-
tron-transfer chain: the primary electron donor P700
and theelectron acceptors A
0
,A
1
and F
x
[1,4,5]. The
two remaining electron acceptors, F
A
and F
B
, are bound
to the PSI-C subunit. PSI-C is located towards the stro-
mal side of PSI and, together with PSI-D and PSI-E,
provides the docking side for soluble ferredoxin [5,6].
Keywords
antenna size; electron transport;
photosynthesis; plastocyanin kinetics;
thylakoid membrane
Correspondence
P. E. Jensen, Plant Biochemistry Laboratory,
Department of Plant Biology, Faculty of Life
Sciences, University of Copenhagen, 40
Thorvaldsensvej, DK-1871 Frederiksberg C,
Denmark
Fax: +45 35 28 33 33
Tel: +45 35 28 33 40
E-mail: peje@life.ku.dk
(Received 30 August 2006, revised 21
December 2006, accepted 31 January 2007)
doi:10.1111/j.1742-4658.2007.05722.x
The plastid-encoded psaJ gene encodes a hydrophobic low-molecular-mass
subunit ofphotosystemI (PSI) containing one transmembrane helix. Ho-
moplastomic transformants with an inactivated psaJ gene were devoid of
PSI-J protein. The mutant plants were slightly smaller and paler than wild-
type because of a 13% reduction in chlorophyll content per leaf area
caused by an % 20% reduction in PSI. The amount ofthe peripheral
antenna proteins, Lhca2 and Lhca3, was decreased to the same level as the
core subunits, but Lhca1 and Lhca4 were present in relative excess. The
functional size ofthe PSI antenna was not affected, suggesting that PSI-J
is not involved in binding of light-harvesting complex I. The specific PSI
activity, measured as NADP
+
photoreduction in vitro, revealed a 55%
reduction inelectron transport through PSI inthe mutant. No significant
difference inthe second-order rate constant forelectrontransfer from
reduced plastocyanin to oxidized P700 was observed inthe absence of PSI-
J. Instead, a large fraction of PSI was found to be inactive. Immunoblot-
ting analysis revealed a secondary loss ofthe luminal PSI-N subunitin PSI
particles devoid of PSI-J. Presumably PSI-J affects the conformation of
PSI-F, which in turn affects the binding of PSI-N. This together renders a
fraction ofthe PSI particles inactive. Thus, PSI-Jis an important subunit
that, together with PSI-F and PSI-N, isrequiredfor formation ofthe plast-
ocyanin-binding domain of PSI. PSI-Jis furthermore important for stabil-
ity or assembly ofthe PSI complex.
Abbreviations
Chl, chlorophyll; Cyt, cytochrome; LHC, light-harvesting complex; Pc, plastocyanin; PS, photosystem.
1734 FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS
In plants, the three low-molecular-mass subunits,
PSI-F, PSI-G and PSI-N, have been implicated in the
interaction between PSI and Pc [7–9]. PSI-F contains
one transmembrane helix andis exposed to both the
lumen andthe stroma: its rather large N-terminal
domain is situated inthe lumen [10], whereas the
C-terminus isin contact with PSI-E on the stromal
side [6]. The N-terminal part of PSI-F and luminal
interhelical loops of PSI-A and PSI-B form a docking
site for Pc or cytochrome (Cyt) c
6
[11–15]. In plants,
which only use Pc as an electron donor to PSI, a
longer N-terminal domain contributes to a helix–
loop–helix motif [10], which specifically enables more
efficient Pc binding and, as a result, two orders of
magnitude faster electrontransfer from Pc to P700
[16]. PSI-N is unique to eukaryotic PSI andis entirely
located inthe thylakoid lumen. However, the recently
published structural model of higher-plant PSI based
on a crystal structure at 4.4 A
˚
does not reveal the pres-
ence of PSI-N [10], and cross-linking experiments have
shown little interaction between PSI-N and other small
PSI subunits [17].
PSI-J is a hydrophobic low-molecular-mass subunit
composed of 44 amino acids with one transmembrane
helix that is located close to PSI-F [5,10]. The N-termi-
nus ofPSI-Jis located inthe stroma, andthe C-termi-
nus is located inthe lumen [6]. In cyanobacteria, PSI-J
binds three chlorophylls (Chls) andisin hydrophobic
contact with carotenoids [5], whereas in plants only
two Chl molecules are bound (Fig. 1), which has been
proposed to be important for energy transfer between
LHCI andthe PSI core [10].
In cyanobacteria, PSI-J interacts with PSI-F [18]. A
psaJ knock-outin Synechocystis PCC 6803 contained
only 20% PSI-F subunit compared with wild-type [19].
The corresponding psaJ knock-outin Chlamydomonas
contained wild-type levels of PSI-F and PSI, and the
cells were able to grow photoautotrophically. A large
fraction ofthe mutant PSI complexes displayed slow
kinetics ofelectron donation from Pc or Cyt c
6
to
P700. The absence ofPSI-J did not alter the half-lives
of the different kinetic phases, but led to the formation
of two subpopulations of PSI complexes which differed
with respect to electrontransfer to P700
+
. One popu-
lation behaved like wild-type with fully functional
PSI-F, andthe other population had characteristics
similar to a PSI-F-deficient strain [20]. It was conclu-
ded that, in 70% ofthe PSI complexes lacking PSI-J,
the N-terminal domain of PSI-F is unable to provide
an efficient binding site for either Pc or Cyt c
6
and was
explained by a displacement of this domain. Thus,
PSI-J does not appear to participate directly in binding
of Pc or Cyt c
6
, but plays a role in maintaining a
precise recognition site forthe N-terminal domain of
PSI-F requiredfor fast electrontransfer from Pc and
Cyt c
6
to PSI [20].
To determine the role ofPSI-Jin plants, we gener-
ated homoplastomic psaJ knock-outs in tobacco.
Transplastomic transformants were obtained and ana-
lyzed for differences inelectron transport and antenna
function. In contrast with results obtained with PSI-J-
deficient Chlamydomonas, the content of PSI was
reduced by 20% andthe remaining PSI had a
decreased in vitro NADP
+
-photoreduction activity. A
secondary loss ofthe luminal subunit, PSI-N, was seen
when PSI complexes were analysed and kinetic analysis
revealed a large fraction of inactive PSI. Thus, we pro-
pose a dual function ofPSI-Jin higher plants; one for
assembly ofthe PSI core complex andthe other for
integrity and stabilization of a luminal domain invol-
ving at least PSI-N andthe N-terminal part of PSI-F
which isrequiredforefficientelectron transfer.
Fig. 1. Alignment ofPSI-J sequences representing cyanobacteria, algae and higher plants. In total, 44 full-length PSI-J sequences were
aligned using
CLUSTAL W. Inthe alignment shown are the sequences from plants [Arabidopsis thaliana (ARATH) andNicotiana tabacum
(TOBAC)], algae [Chlamydomonas reinhardtii (CHLRE) and Porphyra purpurea (PORPU)] and cyanobacteria [Synechcoccus elongatus (SYNEL)
and Prochlorococcus marinus (PROMA)]. Amino-acid residues involved in Chl binding [W (Trp), E (Glu) and H (His)] are indicated with green
arrows. Note that the histidine residue is only conserved in cyanobacteria, in agreement with the notion that PSI-Jof cyanobacteria is
involved in binding three Chls, whereas plant PSI-J only binds two. Amino-acid residues making contact with b-carotene [I (Ile) and R (Arg)]
are indicated with orange arrows. The underlined residues are completely conserved in plants, algae and cyanobacteria.
A. Hansson et al. Knock-outofthe J subunitof PSI
FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS 1735
Results
Targeted inactivation ofthe tobacco chloroplast
psaJ gene
To determine the function ofPSI-Jin plants, we have
taken a reverse genetics approach and constructed a
knock-out allele for targeted disruption ofthe tobacco
psaJ (Fig. 2A). Theknock-out allele was introduced
into the tobacco plastid genome by particle bombard-
ment-mediated chloroplast transformation [21].
From 10 bombarded leaf samples, 19 chloroplast
transformants were selected and verified by PCR
and DNA gel blot analysis (data not shown). Two
independent transplastomic lines were subjected to
additional rounds of regeneration on spectinomycin-
containing medium to obtain homoplastomic tissue. In
Fig. 2B, an example of PCR verification of one of the
homoplastomic psaJ knock-out lines is shown. Nor-
thern blot analysis was also performed to demonstrate
that the psaJ gene was disrupted by the insertion of
the aadA cassette (Fig. 2C). Finally, PSI particles (PSI
holocomplexes) were prepared from wild-type and
plants disrupted inthe psaJ gene and subjected to
immunoblot analysis. An antibody originally raised
against electroeluted PSI-I [22] and subsequently found
to recognize both PSI-I andPSI-J [17] was used to
confirm the absence ofPSI-J protein from the mutant
(Fig. 2D). Altogether this clearly shows that the psaJ
gene has been disrupted causing elimination of the
PSI-J protein.
Plants devoid ofPSI-J are fully viable and fertile
but display a clear phenotype
When plants lacking PSI-J were transferred to soil,
they grew photoautotrophically and were fully fertile
(Fig. 3). The original transformed lines were self-polli-
nated, andthe seeds produced were germinated
directly on soil. The resulting offspring displayed the
same characteristics as the first generation (results not
shown).
Tobacco plants lacking PSI-J were slightly smaller
than wild-type plants (Fig. 3). This was observed for
plants grown in either a growth-chamber or a green-
house and suggests that elimination ofthePSI-J pro-
tein from PSI affects the overall photosynthetic
performance.
Besides being slightly smaller than wild-type, the
psaJ knock-out plants were visibly paler. Pigment
WT
ΔJ
T
ΔJ
M
4
7
16
17
34
45
55
105
kDa
M123
564
947
831
1375
1584
2027/1904
3530
A
B
C
D
3.7 kb
1.9 kb
WT WT
68293 70823
PetG
TrnW TrnP
PsaJ Rpl33
Rps18
250-bp
ScaI
TrnP
(PsaJ) Rpl33
(ScaI/SmaI)
(PsaJ)
(HindIII/ScaI)
AadA
ΔJΔJ
Fig. 2. (A) Construction ofthe plastid trans-
formation vector. Schematic map of the
2.53-kb chloroplast genomic fragment con-
taining the psaJ gene. The aadA cassette is
inserted in a ScaI site within the coding
sequence of psaJ inthe sense orientation.
(B) PCR confirmation that the aadA cassette
has inserted inthe psaJ gene. M, marker;
1, total DNA from transgenic plant as tem-
plate; 2, plasmid DNA used to transform the
plants as template; 3, total DNA from wild-
type tobacco as template. (C) Northern blot
showing that there is no wild-type-sized
psaJ mRNA (as a loading control the left
hand side shows the stained andthe right
hand side the actual Northern blot). (D)
Immunoblot analysis of PSI complexes from
wild-type and DpsaJ plants. The panel on
the left isthe stained gel, andthe panel on
the right is an immunoblot using an antibody
directed against a mixture of PSI-I and
PSI-J. The arrow indicates PSI-J.
Knock-out ofthe J subunitof PSI A. Hansson et al.
1736 FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS
extraction of leaf discs using boiling ethanol and spec-
trophotometric quantification showed a 13% reduction
in the content of Chl per leaf area compared with
wild-type (Table 1). Estimated from the leaf extracts,
the Chl a ⁄ b ratio was 2.95 inthe psaJ knock-out leaves
compared with 3.25 inthe wild-type leaves. This differ-
ence was caused by a bigger decrease in Chl a (15%
less) and a smaller decrease in Chl b (6% less) in the
mutant (Table 1). Similar measurements on several
independent preparations of thylakoids also revealed a
lower Chl a ⁄ b ratio inthe mutant, although the abso-
lute numbers were different. The reduced Chl a ⁄ b ratio
suggests that plants without PSI-J either have less of
the core complexes or increased content ofthe Chl b
containing peripheral antenna.
To monitor the photosynthetic electron flow through
PSI during steady-state photosynthesis in vivo, we esti-
mated the redox state of P700 inthe light by measuring
oxidation of P700 inthe leaf as DA at 810 minus 860 nm
as described in Experimental procedures. The light
dependence ofthe P700 oxidation ratio (DA ⁄ DA
max
)
was examined, and, in both the wild-type and DPSI-J
plants, P700 oxidation was almost linearly related to
increasing light intensity. However, inthe DPSI-J plants
the redox state of P700 was higher than wild-type at all
light intensities (Fig. 4). This means that P700 stays
more oxidized inthe absence of PSI-J. This usually sug-
gests that electron donation from Pc to P700
+
is affec-
ted. Comparison ofthe curves suggested that about
20% ofthe PSI has very inefficient electron donation
from Pc inthe absence of PSI-J.
Table 1. Chl a and b content per leaf area, Chls per PSI reaction centre, PSI activity, andthe plastoquinone redox state under different light
conditions.
Wild-type n DPSI-J n
Chl (lg ⁄ cm
2
) Leaf 19.1 ± 2.1 6 16.6 ± 1.0* 6
Chl a ⁄ b Leaf 3.25 ± 0.3 6 2.95 ± 0.1* 6
Chl a (lg) Leaf 14.6 ± 1.8 6 12.4 ± 0.7* 6
Chl b (lg) Leaf 4.5 ± 0.3 6 4.2 ± 0.3 6
Chl ⁄ P700 Thylakoids 435 ± 17 3 531 ± 32* 3
NADP
+
photoreduction
a
Thylakoids 24.8 ± 2.0 3 11.1 ± 1.0*** 3
[lmol NADP
+
Æs
)1
Æ(lmol P700)
)1
]
1–q
P
Growth chamber ⁄ growth light 0.024 ± 0.003 3 0.04 ± 0.01* 5
1–q
P
Greenhouse ⁄ cloudy and rainy 0.013 2 0.019 2
1–q
P
Greenhouse ⁄ sunny, no clouds 0.028 2 0.065 2
a
Mean of three independent thylakoid preparations. *P<0.05; ***P<0.001.
WT
ΔPsaJ
Fig. 3. Phenotype of homoplastomic DpsaJ plants grown under
growth chamber conditions. Note that the DpsaJ plant is slightly
smaller and paler than the wild-type plant.
Li
g
ht intensity (μE)
0 100 200 300 400
oitar noitadixo
007P
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
WT
ΔJ
Fig. 4. P700 oxidation state in leaves of wild-type and DpsaJ plants.
Light response of P700 oxidation ratio (DA ⁄ DA
max
) in leaves of
wild-type (WT) and DPSI-J plants (DJ). All data points are
mean ± SD (n ¼ 3), but in some cases the error bars are covered
by the marker.
A. Hansson et al. Knock-outofthe J subunitof PSI
FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS 1737
The PSII excitation pressure (estimated as 1–q
P
) was
subsequently measured in vivo inthe growth chamber
under the light conditions to which the plants were
adapted. Under these conditions 1–q
P
was increased
1.7-fold inthe plants lacking PSI-J (Table 1), indica-
ting that the PSII excitation pressure was significantly
increased as the result of a more reduced plastoqui-
none pool. Measuring 1–q
P
under greenhouse condi-
tions on either a cloudy or a sunny day confirmed the
higher excitation pressure in plants without PSI-J,
especially under conditions where the plants have to
cope with higher light intensities (Table 1). This is in
agreement with a restriction ofelectron flow at PSI.
The amount of PSI is reduced inthe absence
of PSI-J
To analyze the content of PSI further, the amount of
P700 was determined in solubilized thylakoids using
flash-induced absorption changes in P700 at 834 nm.
The number of Chls per P700 reaction centre was esti-
mated to be 435 ± 17 for wild-type and 531 ± 32 for
thylakoids from the PSI-J-less plants (Table 1). Similar
values were obtained using chemical oxidation and
reduction of P700 (data not shown). This clearly indi-
cates an % 20% reduction in P700 in plants lacking
PSI-J.
To investigate this by an independent method and
also to analyze whether the absence ofPSI-J caused
changes in photosynthetic complexes, we performed
immunoblot analysis of thylakoid proteins using a
variety of antibodies directed against subunits of the
PSI, PSII and ATP synthase complexes (Fig. 5). The
gels were loaded with proteins corresponding to equal
amounts of Chl. This analysis showed that subunits of
PSII andthe ATP synthase were present in amounts
equal or close to the amounts found in wild-type
(Fig. 5). In contrast, the amounts ofthe analysed sub-
units ofthe PSI core were consistently reduced by 15–
25% compared with the wild-type (Fig. 5A). This
shows that there are fewer PSI core complexes in the
absence ofPSI-Jand confirms the spectroscopic deter-
mination of Chl per P700 above. Together this sug-
gests that PSI-Jis implicated instableaccumulation of
PSI because of a requirement for this subunit either
during assembly or subsequently forthe stability of
the PSI complex.
To analyse the effect ofthe absent PSI-Jin more
detail, immunoblot analysis of PSI particles purified
using sucrose density gradient centrifugation was also
performed (Fig. 6). This revealed that most ofthe sub-
units analysed were present inthe complex of the
mutant in amounts similar to that found inthe wild-
type. This included the PSI-F subunit, which is known
to be located next to PSI-Jinthe complex [5,10]. Sur-
prisingly, the only subunit that was reduced in content
was PSI-N, which was reduced to 30–40% ofthe wild-
type level.
Fig. 5. Immunoblot analysis of proteins in thylakoids prepared from
DpsaJ and wild-type plants. (A) Content of a range of PSI core pro-
teins and ATP synthase (CF
1
-b). Thylakoids were prepared from
leaves from two to four different wild-type or DpsaJ plants. A dilu-
tion series containing protein corresponding to 1.0, 0.5, and
0.25 lg Chl ofthe wild-type and 1.0–0.5 lg Chl ofthe mutant was
separated by SDS ⁄ PAGE, blotted and analyzed with the antibodies
indicated. Wild-type (WT) and DpsaJ dilutions were run side by
side, and, for each antibody, the resulting signal was quantified
using the LabWorks software as described in Experimental proce-
dures. Quantification was performed on two independent prepara-
tions of both wild-type and DpsaJ thylakoids. (B) Content of light-
harvesting Chl a ⁄ b proteins of PSI. Thylakoid proteins were separ-
ated as above andthe blots were incubated with antibodies as indi-
cated. The Lhca2 antibody also detects Lhcb4 (CP29). (C) Content
of light-harvesting Chl a ⁄ b proteins of PSII and PSII core proteins.
Thylakoid proteins were separated as above, andthe blots were
incubated with antibodies as indicated.
Knock-out ofthe J subunitof PSI A. Hansson et al.
1738 FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS
PSI-J is not involved in binding LHCI
The four Lhca proteins, which constitute the major
part ofthe peripheral antenna of PSI (LHCI), were
not reduced to the same extent as the core subunits.
Lhca1 and Lhca4 were present in near wild-type
amounts, and Lhca2 and Lhca3 were reduced by
15–25% compared with wild-type (Fig. 5B). This indi-
cates that some ofthe Lhca proteins are present in rel-
ative excess ofthe PSI core complexes.
The antenna properties were further analysed by
fluorescence emission measurements at low tempera-
ture. Fluorescence emission spectra between 650 and
800 nm during excitation at 435 nm at 77 K using
intact leaves of wild-type plants and plants devoid of
PSI-J are shown in Fig. 7. The spectra revealed that,
in the absence of PSI-J, there is a 2–3 nm blue shift in
the far-red emission originating from PSI–LHCI. The
blue shift suggests a perturbation ofthe peripheral
antenna, which is because either PSI-J plays a func-
tional role inthe binding ⁄ function ofthe LHCI
antenna or free Lhca complexes are present in the
membrane. However, low-temperature fluorescence
emission measurements on PSI–LHCI particles
enriched using sucrose density gradient centrifugation
as shown in Fig. 8 did not display the 2–3 nm blueshift
(data not shown), indicating that the blue shift is
caused by excess free Lhca complexes inthe thylakoid
membrane.
This was further supported by estimation of the
functional antenna size of PSI using light-induced
P700 absorption changes at 810 nm after very gentle
solubilization ofthe thylakoid membrane using digito-
nin as described in Experimental procedures. We have
WT ΔJ
B
PSI-D
PSI-E
PSI-F
PSI-K
PSI-H
PSI-L
PSI-J
PSI-C
PSI-N
% of WT)(ecnadnu
baevitaleR
0
20
40
60
80
100
Δ
J
A
Fig. 6. Immunoblot analysis of proteins in
PSI particles prepared from DpsaJ and wild-
type plants. (A) Quantification ofthe signals
obtained inthe immunoblot analysis. (B)
Representative example ofthe signals with
the various PSI antibodies.
Emission wavelen
g
th (nm)
660 680 700 720 740 760 780
ecnecseroulf evi
taleR
0.0
0.5
1.0
1.5
2.0
WT
ΔJ
Fig. 7. Low-temperature fluorescence emission. Shown are the
spectra of intact leaves from a wild-type plant (WT) and a DpsaJ
plant (DJ). Leaves from several individual plants of both genotypes
were measured, andthe mutant consistently showed a 3-nm blue
shift inthe far-red florescence emission peak. Excitation wave-
length was 435 nm, andthe spectra were normalized to the peak
at 685 nm.
A. Hansson et al. Knock-outofthe J subunitof PSI
FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS 1739
previously used this method to successfully detect
changes in PSI antenna caused by association with
LHCII during state transitions [23] or genetic elimin-
ation of individual Lhca proteins in Arabidopsis [24].
The functional PSI antenna size was expressed by the
t
1 ⁄ 2
value which is defined as the time it takes to
oxidize 50% ofthe P700 inthe sample and was esti-
mated at three different intensities of actinic light. At
all three light intensities, there was no significant dif-
ference in t
1 ⁄ 2
in the samples lacking PSI-J compared
with the values obtained with wild-type samples
(Table 2), suggesting that the PSI antenna size is
unaffected by the elimination ofPSI-Jand further-
more ruling out the possibility that PSI-Jis strictly
required for binding of any ofthe Lhca antenna
proteins.
The presence of free Lhca1 and Lhca4 in the
thylakoid membrane was verified by gentle solubiliza-
tion ofthe various thylakoid membrane complexes
using dodecyl-b-d-maltoside and subsequent separation
of the complexes using sucrose density gradient centrif-
ugation. After separation, the gradients were harvested
in 0.5-mL fractions, andthe individual fractions were
analysed by gel electrophoresis and immunoblotting
using antibodies against the four Lhca proteins and
the PSI-F subunit (Fig. 8). This revealed that signifi-
cant amounts of free Lhca1 and Lhca4 proteins indeed
were found inthe fractions where mainly LHCII trim-
ers and ⁄ or Lhcb monomers are normally found. How-
ever, this analysis also suggested that PSI–LHCI
complexes devoid ofPSI-J are slightly more sensitive
to the detergent treatment, as some free Lhca2 and
Lhca3 proteins were also detected.
Table 2. Measurements of antenna size using time course of P700
photo-oxidation in solubilized thylakoid preparations from wild-type
and DPSI-J plants. lE, l moles photonÆm
)2
Æs
)1
.
lE
t
1 ⁄ 2
(ms)
Wild-type n DPSI-J n
20 104.5 ± 3.4 3 102.6 ± 12.8 4
33 66.3 ± 4.6 3 61.7 ± 7.7 4
58 38.0 ± 1.2 3 37.5 ± 3.9 4
12345678910111213141516171819202122232425
wt
ΔJ
PSI-LHCI
PSII-core
LHCII trimers
and monomers
wt Lhca1
wt Lhca2
wt Lhca3
wt Lhca4
ΔJ Lhca2
ΔJ Lhca3
ΔJ Lhca4
ΔJ Lhca1
wt PsaF
ΔJ PsaF
Fig. 8. Analysis ofthe distribution of Lhca
proteins inthe thylakoid membrane of
DpsaJ (DJ) plants. Shown isthe centrifuga-
tion tubes after separation ofthe solubilized
membrane complexes in a sucrose density
gradient (top panels) and an immunoblot
analysis using the four Lhca antibodies and
a PSI-F antibody on individual fractions har-
vested from the sucrose density gradient
fraction (bottom part).
Knock-out ofthe J subunitof PSI A. Hansson et al.
1740 FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS
PSI-J is important for proper electron transfer
On the basis of work with mutants of Chlamydomonas
lacking PSI-J, it has been proposed that the function
of PSI-Jis to maintain PSI-F inthe correct orienta-
tion, facilitating fast electrontransfer from Pc or
Cyt c
6
to P700 [20]. A similar role forPSI-Jin higher
plants is likely, and, in order to analyse this, NADP
+
photoreduction was determined using thylakoids puri-
fied from plants without PSI-Jand wild-type plants. In
our standard assay with 2 lm Pc, an activity of
24.8 ± 2.0 lmol NADPHÆs
)1
Æ(lmol P700)
)1
was
obtained with thylakoids from wild-type and
11.1 ± 1.0 lmol NADPHÆ s
)1
Æ(lmol P700)
)1
with thyl-
akoids devoid ofPSI-J (Table 1). Thus, PSI devoid of
PSI-J only has 45% ofthe NADP
+
photoreduction
activity ofthe wild-type.
This result clearly suggests that PSI-J affects electron
transport. As indicated from work with green algae [20]
and thein vivo measurement ofthe P700 redox level in
Fig. 4, the most obvious step to be affected isthe elec-
tron transfer from Pc to P700. To investigate the kinetics
of the Pc–P700 interaction, flash-induced P700 absorp-
tion transients were determined by following the absorp-
tion at 834 nm inthe presence of Pc. Flash excitation of
PSI results in a very rapid absorption increase at 834 nm
caused by photo-oxidation of P700 to P700
+
, followed
by a slower absorption decrease due to reduction of
P700
+
by Pc. The reaction between Pc and P700 is a
multistep reaction, which can be divided into three major
steps: binding of Pc to P700, electrontransfer within a
complex between Pc and P700, and release of oxidized
Pc from the complex between Pc and P700. The absorp-
tion decrease at 834 nm can be modelled as the sum of
three exponential decays discerned as a fast phase corres-
ponding to theelectrontransfer between preformed Pc–
PSI complexes, an intermediate phase corresponding to
the bimolecular reaction between Pc in solution and PSI,
and a slow phase corresponding to inactive PSI and a
contribution from absorption of oxidized Pc at 834 nm
[25–27]. For analysis of wild-type and mutants lacking
PSI-J, Pc concentrations of 5 an 25 lm were used, and
the first 20 ls ofthe data were ignored. With 5 and
25 lm Pc, the fast reduction of P700
+
by Pc bound to
PSI before photo-oxidation is negligible. Therefore, good
fits to the experimental data could be obtained using a
sum of two exponential decays. The results show that
there is no difference between wild-type and mutant in
the apparent second-order rate constants (Table 3), sug-
gesting that PSI-J does not affect theelectron transfer
from Pc to PSI directly. However, the amplitude of the
intermediate phase is 80% in wild-type and only 63% in
the PSI-J-less samples, indicating that the absence of
PSI-J results in % 20% more inactive PSI compared with
wild-type. Thus, the observed decrease in NADP
+
pho-
toreduction can, at least in part, be explained by a larger
fraction of inactive PSI inthe absence of PSI-J.
Discussion
PSI-J is a subunitof PSI in almost all photosynthetic
organisms studied so far. However, the unicellular
cyanobacterium, Gleobacter violaceus PCC 7421,
appears to have a PSI without PSI-J [28,29]. The func-
tion ofPSI-Jin higher plants has so far not been
investigated. We have successfully generated transgenic
Nicotiana tabaccum plants devoid ofthe J subunit of
PSI and been able to investigate the role ofPSI-J in
higher plants. The PSI-J-less plants were analysed with
various biochemical and physiological methods.
PSI-J isrequiredforstableaccumulationof PSI
In the absence of PSI-J, the steady-state accumulation
of PSI is reduced by % 20%, as evidenced by the esti-
mates of Chl ⁄ P700, the immunoblotting analysis of
thylakoid proteins (Fig. 5), andthe lower Chl a ⁄ b ratio
(Table 1). This suggests that PSI-Jis implicated in sta-
bility or assembly ofthe PSI complex in tobacco. This
is in contrast with results reported for Chlamydomonas
lacking PSI-J, where it was concluded that steady-state
accumulation of PSI does not require thePSI-J sub-
unit [20]. Differences between higher plants and green
algae with respect to PSI stability and function have
also been reported after removal of PSI-F, which in
Arabidopsis resulted in severe destabilization of PSI
and especially loss of stromal subunits such as PSI-C,
PSI-D and PSI-E [8]. In contrast, a deletion of PSI-F
Table 3. Apparent second-order rate constant (k) forthe reduction of P700
+
by plastocyanin. The rate constants were obtained from a
curve-fitting analysis of flash-induced absorption transients recorded at 834 nm in samples of dodecyl-b-
D-maltoside-solubilized thylakoids.
Wild-type n DPSI-J n
k (
M
)1
Æs
)1
) 1.75 · 10
8
± 2.17 · 10
7
10 1.97 · 10
8
± 4.76 · 10
7
8
Percentage of amplitudes relative
to the total amplitude
80 ± 5 10 63 ± 9 8
A. Hansson et al. Knock-outofthe J subunitof PSI
FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS 1741
in Chlamydomonas did not affect the stability of the
PSI complex [11,20].
Transgenic Arabidopsis plants without PSI-N, PSI-H,
PSI-K and PSI-L compensate for a poorly functioning
PSI by making 15–20% more PSI [7,30–32]. Apparently,
the plants devoid ofPSI-J cannot compensate in a sim-
ilar way, which again suggests that PSI-J affects the sta-
bility or assembly in a different way from the absence of
PSI-N, PSI-H, PSI-K and PSI-L. In some aspects,
plants devoid ofPSI-J display certain similarities to
plants devoid of PSI-G [9,33,34]. Inthe absence of
PSI-G, less PSI core, a relative excess of LHCI, and a
less stable PSI is also observed. To distinguish whether
it isthe stability or the assembly ofthe PSI complex that
is affected needs further investigation.
The reduced content of PSI was readily revealed by
the appearance ofthe transgenic tobacco plants, which
were slightly smaller and paler than wild-type. Plants
devoid of PSI-G or PSI-K have been reported to be
reduced in mean size [34], and plants devoid of PSI-G
have a 40% reduction in content of PSI [33] and also
a slightly lighter pigmentation [34]. Thus, there is good
correlation between the amount of PSI, plant size, and
pigmentation, although one would not expect a 20%
reduction in PSI to affect the growth to the extent seen
for the tobacco plants without PSI-J. However, com-
bined with a less efficient PSI, as both thein vitro
NADP
+
measurements andthein vivo estimations of
the PSII excitation pressure indicate, the observed
growth phenotype is explainable.
PSI-J is not necessary for binding of the
peripheral light-harvesting antenna
The two Chls bound to PSI-Jin higher plants are sug-
gested to be important forthe energy transfer between
LHCI andthe PSI core [10]. However, the functional
PSI antenna size is unaffected by the elimination of
PSI-J from the PSI complex (Table 2). Thus, PSI-J
is not requiredfor binding or the function of the
peripheral antenna, or at least the PSI that is formed
is unaffected by the missing PSI-J. The measurements
of the functional antenna size using P700 oxidation
rates do not allow enough time resolution to tell whe-
ther the absence ofthe two Chl molecules bound to
PSI-J causes inefficient transferof excitation energy
from the peripheral antenna to the core.
In vitro the absence ofPSI-J affects the stability of the
PSI–LHCI complex. The results ofthe fractionation of
mildly solubilized thylakoid membrane complexes as pre-
sented in Fig. 8 indicate that some Lhca proteins, mainly
Lhca1 and Lhca4 are present in relative excess compared
with the core subunits, as also indicated inthe immuno-
blot analysis on nonsolubilized thylakoids (Fig. 5) and
the 77 K fluorescence emission measurements on
detached leaves (Fig. 7). Alternatively, the solubilization
with detergent affects the PSI-J-deficient complexes more
than the wild-type complexes, and thereby more of the
Lhca proteins are released from the complex.
PSI-J isrequiredforefficientelectron transfer
PSI-J affects theelectron transport through PSI. Meas-
ured as in vitro NADP
+
photoreduction activity, a
55% decrease inthe steady-state electron transport in
the absence ofPSI-J was observed. The kinetic analysis
of the reaction between Pc and P700 did not reveal
any significant difference inthe second-order rate con-
stant between wild-type and PSI-J-deficient plants that
can explain the observed decrease in PSI activity. The
kinetic parameters ofthe reaction between Pc and
P700 was also found to be unaffected when PSI from
DPSI-J and wild-type Chlamydomonas was analysed
[20], and it therefore seems that PSI-J does not partici-
pate directly inthe binding of Pc in either plants or
green algae. In Chlamydomonas, the amplitude of the
PSI-F-dependent second-order kinetics was 76% and
42% ofthe total amplitude with wild-type and PSI-J-
deficient thylakoid membranes, respectively [20], which
correspond to a 45% decrease. This decrease is
thought to be caused by an increased proportion of
PSI complexes incompetent for fast electrontransfer in
the absence ofPSI-Jand has been suggested to be due
to a stabilizing effect ofPSI-J on PSI-F [20]. Similar to
this, we observe a 20% decrease inthe amplitude of
the second-order component ofelectrontransfer with
plant thylakoids devoid of PSI-J. Thus, in plants, there
is also an increased proportion of PSI complexes that
are incompetent forefficientelectron transfer. Interest-
ingly, the immunoblotting analysis of PSI particles
purified using sucrose density gradient centrifugation
after solubilization with dodecyl-b-d-maltoside clearly
suggested that binding ofthe luminal PSI-N to PSI
was affected inthe absence ofPSI-J (Fig. 6). This loss
of PSI-N is probably due to increased sensitivity to
detergent during preparation ofthe PSI particles, but,
despite this, it strongly suggests a perturbation of the
luminal side of PSI involving PSI-F and PSI-N. The
absence ofPSI-J might affect the conformation of
PSI-F, which, in turn, changes the binding of PSI-N.
PSI-F provides part ofthe Pc-binding site in plants
[16], and it is known that the depletion of PSI-F by
antisense suppression ofthe corresponding gene leads
to a secondary loss of PSI-N [8], indicating an interac-
tion between these two subunits. PSI-N has further
been shown to be necessary fortheefficient interaction
Knock-out ofthe J subunitof PSI A. Hansson et al.
1742 FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS
with Pc, as the second-order rate constant was reduced
by 40% inthe absence of PSI-N [7].
The increase inthe pool of inactive PSI observed in
plants devoid ofPSI-Jis not caused by the absence of
PSI-N because mutants lacking PSI-N clearly have a
changed second-order rate constant for Pc–P700 inter-
action but not an increased proportion of inactive PSI
complexes [7]. Furthermore, the immunoblotting ana-
lysis of thylakoid proteins (Fig. 5) clearly indicates that
PSI-N is present in amounts similar to the other PSI
core subunits. Instead it seems plausible that the chan-
ged conformation of PSI-F inthe absence of PSI-J
renders a fraction ofthe PSI complexes inactive.
The in vivo measurements ofthe P700 redox level
indicate that P700 inthe DPSI-J plants constantly
stays more oxidized, which is usually caused by a limi-
tation of electron-transfer activities on the donor or lu-
minal side of PSI. The 20% permanently oxidized PSI
estimated from thein vivo experiment isin excellent
agreement with the 20% inactive PSI determined with
the flash excitation. At the same time, the plastoqui-
none pool is more reduced, as indicated by the
increased PSII excitation pressure. These observations
are consistent with a greater pool of inactive PSI cen-
tres inthe absence ofPSI-Jin vivo.
However, the 20% increase inthe pool of inactive
PSI complexes inthe absence ofPSI-J does not explain
the dramatic reduction in PSI activity measured by
NADP
+
photoreduction activity. The kinetic analysis
clearly indicates that the second-order rate constant
for electrontransfer from Pc to P700 is unaffected.
However, the release of oxidized Pc has been shown to
limit electrontransfer between the cytochrome b
6
f
complex and PSI in vivo [35], andthe absence of PSI-J
may affect the k
off
value, so that oxidized Pc stays lon-
ger inthe active site and thereby blocks efficient
exchange with reduced Pc. Alternatively, the changed
conformation of PSI-F inthe absence ofPSI-J could
affect proper functioning of stromal subunits in con-
tact with PSI-F, such as PSI-E or PSI-D. These sub-
units are involved in docking andefficient electron
transfer to ferredoxin [6], and, from the structures, it is
known that PSI-E isin contact with the C-terminus of
the PSI-F subunit [5]. Changes in binding or amounts
of any ofthe stromal subunits of PSI were not detec-
ted in our immunoblot analysis; however, a subtle
change in arrangement ofthe subunits is still possible.
In conclusion, PSI-Jis needed forstable accumulation
of the PSI core complex and proper electron transfer.
Despite the location ofPSI-J close to the rim ofthe core
complex facing LHCI, it is not needed for correct inter-
action with the peripheral antenna complexes. Clearly
the luminal side of PSI is perturbed, probably because
of destabilization of PSI-F inthe absence of PSI-J,
resulting in an increased pool of inactive PSI.
Experimental procedures
Vector construction, chloroplast transformation,
and plant material
The region ofthe tobacco chloroplast genome containing
700 bp upstream and downstream ofthe psaJ reading
frame was amplified using PCR. The 1535-bp fragment was
ligated into the SacI and BamHI sites of pUC19. The psaJ
knock-out allele was created by digestion of this construct
with ScaI, and a chimeric aadA gene conferring resistance
to aminoglycoside antibiotics [21] was inserted into this
ScaI site to disrupt psaJ and to facilitate selection of
chloroplast transformants. ScaI causes disruption of the
132-bp psaJ coding region after nucleotide 38. A plasmid
clone carrying the aadA gene inthe same orientation as
psaJ yielded the transformation vector pPsaJ (Fig. 2).
Chloroplasts of N. tabaccum cv. Petit Havanna were
transformed by particle bombardment of leaves [21]. Selec-
tion and culture of transformed material as well as assess-
ment of plastome segregation andthe homoplastomic state
were performed essentially as described by De Santis-
Maciossek et al. [36] and Swiatek et al. [37]. Essentially, 10
leaves were used for particle bombardment, and 19 antibi-
otic resistant transformants were selected. The material was
maintained on agar-solidified MS medium [38] containing
2% sucrose, and grown in 12 h dark ⁄ light cycles at 25 °C
and 20 l mol photonsÆm
)2
Æs
)1
and, under selective condi-
tions, 500 lgÆmL
)1
spectinomycin. For thylakoid isolation
and physiological measurements, wild-type and transformed
plants (originating from two independent transplastomic
lines) were planted in compost and kept in growth chamber
conditions in 8 h light and 120–140 lmol photonsÆm
)2
Æs
)1
.
Isolation of thylakoid membranes and PSI
particles from tobacco
Leaves from 6–8-week-old plants were used for isolation of
thylakoids as described previously [7]. PSI particles were iso-
lated from thylakoids after solubilization with dodecyl-b-d-
maltoside and sucrose density gradient ultracentrifugation as
described in [31]. Chl content andthe Chl a ⁄ b ratio were
determined in 80% acetone as described previously [39]. The
samples were frozen in liquid nitrogen and stored at )80 °C.
RNA gel blot analysis
Northern blot analysis of total leaf RNA was performed
using DNA probes and was carried out as described by
Meurer et al. [40]. A
33
P-labelled DNA fragment corres-
ponding to the psaJ gene was used as probe.
A. Hansson et al. Knock-outofthe J subunitof PSI
FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS 1743
[...]... antibodies directed against subunits ofthe various thylakoid membrane complexes as indicated inthe Figure legends An antibody originally raised against electroeluted PSI -I [22] but subsequently found to recognize both PSI -I andPSI-J [17] was used to detect PSI-J Primary antibodies were detected using a chemiluminescent detection system (Immun-Star, Bio-Rad, Herlev, Denmark; Super-Signal, Pierce, Rockford,... plastocyanin to photosystemIof Chlamydomonas reinhardtii requires PsaF Biochemistry 36, 6343–6349 Hippler M, Drepper F, Haehnel W & Rochaix J-D (1998) The N-terminal domain of PsaF: precise recognition site for binding and fast electrontransfer from cytochrome c (6) and plastocyanin to photosystemIof Chlamydomonas reinhardtii Proc Natl Acad Sci USA 95, 7339–7344 Hippler M, Rimbault B & Takahashi Y (2002)... Mimuro M (2004) Unique constitution ofphotosystemI with a novel subunitinthe cyanobacterium Gloeobacter violaceus PCC 7421 FEBS Lett 578, 275–279 30 Naver H, Haldrup A & Scheller HV (1999) Cosuppression ofphotosystemIsubunit PSI-H in Arabidopsis thaliana J Biol Chem 274, 10784–10789 31 Jensen PE, Gilpin M, Knoetzel J & Scheller HV (2000) The PSI-K subunitofphotosystemIis involved inthe interaction... resolved into two exponential decay components using a Levenberg–Marquardt nonlinear regression procedure Knock-outofthe J subunitof PSI 9 10 11 12 Acknowledgements We wish to thank Ingrid Duschanek and Elli Gerick for excellent technical assistance, and Steen Malmmose for assistance with growing plants The Danish National Research Foundation, the Danish Veterinary and Agricultural Research Council (23-03-0105)... (2000) Downregulation ofthe PSI-F subunitofPhotosystemIin 14 16 17 18 19 20 21 Arabidopsis thaliana The PSI-F subunitis essential for photoautotrophic growth and antenna function J Biol Chem 275, 31211–31218 Zygadlo A, Jensen PE, Leister D & Scheller HV (2005) PhotosystemI lacking the PSI-G subunit has higher affinity for plastocyanin andis less stable Biochim Biophys Acta 1708, 154–163 Ben-Shem... absence ofthe PSI-G subunit J Biol Chem 277, 2798–2803 1746 34 Varotto C, Pesaresi P, Jahns P, Lessnick A, Tizzano M, Schiavon F, Salamini F & Leister D (2002) Single and double knockouts ofthe genes forphotosystemI subunits G, K, and H of Arabidopsis Effects on photosystemI composition, photosynthetic electron flow, and state transitions Plant Physiol 129, 616–624 35 Finazzi G, Sommer F & Hippler... at 700 nm The thylakoids were solubilized with 0.2% Triton X-100, andthe measurements were repeated 3–5 times on several independent thylakoid preparations For spectroscopic determination ofthe amount of P700, the maximal flash-induced P700 absorption was determined by supplying a series of saturating flashes as outlined below (under Kinetic measurements) and using an e at 834 nm for P700 of 5 mm)1... (2004) Light-harvesting complex II binds to several small subunits ofphotosystemI J Biol Chem 279, 3180–3187 24 Klimmek F, Ganeteg U, Ihalainen JA, van Roon H, Jensen PE, Dekker JP, Scheller HV & Jansson S (2005) The structure of higher plant LHCI In vivo characterization and structural interdependence ofthe Lhca proteins Biochemistry 44, 3065–3073 25 Bottin H & Mathis P (1985) Interaction of plastocyanin... Chlorophylls and caroteinoids: pigments of photosynthetic biomembranes Methods Enzymol 148, 350–382 40 Meurer J, Meierhoff K & Westhoff P (1996) Isolation of high-chlorophyll-fluorescence mutants of Arabidopsis thaliana and their characterisation by spectroscopy, immunoblotting and Northern hybridization Planta 198, 385–396 41 Klughammer C & Schreiber U (1994) An improved method,using saturating light pulses, for. . .Knock-out ofthe J subunitof PSI A Hansson et al Chl content per leaf area Total leaf Chls were extracted by boiling leaf disks in 95% ethanol for 30 min After cooling to room temperature and volume adjustment, the Chl content and Chl a ⁄ b ratio was determined in 95% ethanol as described [39] Immunoblotting Immunoblotting analysis was performed essentially as described previously [31] using antibodies . explained by a larger
fraction of inactive PSI in the absence of PSI-J.
Discussion
PSI-J is a subunit of PSI in almost all photosynthetic
organisms studied. Knock-out of the chloroplast-encoded PSI-J subunit
of photosystem I in Nicotiana tabacum
PSI-J is required for efficient electron transfer and stable
accumulation