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

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
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 photosystem I (PSI) complex of higher plants con-
sists of at least 19 different polypeptides [1–3]. PSI
mediates light-driven electron transfer from reduced
plastocyanin (Pc) in the thylakoid lumen to oxidized
ferredoxin in the 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 of the elec-
tron-transfer chain: the primary electron donor P700
and the electron 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:
(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 of photosystem I (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 of the 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 of the 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 in electron transport through PSI in the mutant. No significant
difference in the second-order rate constant for electron transfer from
reduced plastocyanin to oxidized P700 was observed in the absence of PSI-
J. Instead, a large fraction of PSI was found to be inactive. Immunoblot-
ting analysis revealed a secondary loss of the luminal PSI-N subunit in 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 of the PSI particles inactive. Thus, PSI-J is an important subunit

that, together with PSI-F and PSI-N, is required for formation of the plast-
ocyanin-binding domain of PSI. PSI-J is furthermore important for stabil-
ity or assembly of the 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 and is exposed to both the
lumen and the stroma: its rather large N-terminal
domain is situated in the lumen [10], whereas the
C-terminus is in 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 electron transfer from Pc to P700
[16]. PSI-N is unique to eukaryotic PSI and is entirely
located in the 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 of PSI-J is located in the stroma, and the C-termi-
nus is located in the lumen [6]. In cyanobacteria, PSI-J
binds three chlorophylls (Chls) and is in 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 and the PSI core [10].
In cyanobacteria, PSI-J interacts with PSI-F [18]. A
psaJ knock-out in Synechocystis PCC 6803 contained
only 20% PSI-F subunit compared with wild-type [19].
The corresponding psaJ knock-out in Chlamydomonas
contained wild-type levels of PSI-F and PSI, and the
cells were able to grow photoautotrophically. A large
fraction of the mutant PSI complexes displayed slow
kinetics of electron donation from Pc or Cyt c
6
to
P700. The absence of PSI-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 electron transfer to P700
+
. One popu-
lation behaved like wild-type with fully functional
PSI-F, and the other population had characteristics

similar to a PSI-F-deficient strain [20]. It was conclu-
ded that, in 70% of the 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 for the N-terminal domain of
PSI-F required for fast electron transfer from Pc and
Cyt c
6
to PSI [20].
To determine the role of PSI-J in plants, we gener-
ated homoplastomic psaJ knock-outs in tobacco.
Transplastomic transformants were obtained and ana-
lyzed for differences in electron transport and antenna
function. In contrast with results obtained with PSI-J-
deficient Chlamydomonas, the content of PSI was
reduced by 20% and the remaining PSI had a
decreased in vitro NADP
+
-photoreduction activity. A
secondary loss of the 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 of PSI-J in higher plants; one for

assembly of the PSI core complex and the other for
integrity and stabilization of a luminal domain invol-
ving at least PSI-N and the N-terminal part of PSI-F
which is required for efficient electron transfer.
Fig. 1. Alignment of PSI-J sequences representing cyanobacteria, algae and higher plants. In total, 44 full-length PSI-J sequences were
aligned using
CLUSTAL W. In the alignment shown are the sequences from plants [Arabidopsis thaliana (ARATH) and Nicotiana 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-J of 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-out of the J subunit of PSI
FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS 1735
Results
Targeted inactivation of the tobacco chloroplast
psaJ gene
To determine the function of PSI-J in plants, we have
taken a reverse genetics approach and constructed a
knock-out allele for targeted disruption of the tobacco
psaJ (Fig. 2A). The knock-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 in the psaJ gene and subjected to
immunoblot analysis. An antibody originally raised
against electroeluted PSI-I [22] and subsequently found
to recognize both PSI-I and PSI-J [17] was used to
confirm the absence of PSI-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 of PSI-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, and the 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 of the PSI-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 of the 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 in the sense orientation.
(B) PCR confirmation that the aadA cassette
has inserted in the 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 and the right
hand side the actual Northern blot). (D)
Immunoblot analysis of PSI complexes from

wild-type and DpsaJ plants. The panel on
the left is the stained gel, and the 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 of the J subunit of 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 in the psaJ knock-out leaves
compared with 3.25 in the 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 in the 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 of the 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 in the light by measuring
oxidation of P700 in the leaf as DA at 810 minus 860 nm
as described in Experimental procedures. The light
dependence of the 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, in the 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 in the absence of PSI-J. This usually sug-
gests that electron donation from Pc to P700
+
is affec-
ted. Comparison of the curves suggested that about
20% of the PSI has very inefficient electron donation
from Pc in the absence of PSI-J.
Table 1. Chl a and b content per leaf area, Chls per PSI reaction centre, PSI activity, and the 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-out of the J subunit of 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 in the growth chamber
under the light conditions to which the plants were
adapted. Under these conditions 1–q
P
was increased
1.7-fold in the 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 of electron flow at PSI.
The amount of PSI is reduced in the 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 of PSI-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 and the ATP synthase were present in amounts
equal or close to the amounts found in wild-type
(Fig. 5). In contrast, the amounts of the analysed sub-
units of the 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 of PSI-J and confirms the spectroscopic deter-
mination of Chl per P700 above. Together this sug-
gests that PSI-J is implicated in stable accumulation of
PSI because of a requirement for this subunit either
during assembly or subsequently for the stability of
the PSI complex.
To analyse the effect of the absent PSI-J in more
detail, immunoblot analysis of PSI particles purified
using sucrose density gradient centrifugation was also
performed (Fig. 6). This revealed that most of the sub-
units analysed were present in the complex of the
mutant in amounts similar to that found in the wild-
type. This included the PSI-F subunit, which is known
to be located next to PSI-J in the complex [5,10]. Sur-
prisingly, the only subunit that was reduced in content
was PSI-N, which was reduced to 30–40% of the 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 of the wild-type and 1.0–0.5 lg Chl of the 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 and the 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, and the blots were
incubated with antibodies as indicated.
Knock-out of the J subunit of 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 of the 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 of the Lhca proteins are present in rel-
ative excess of the 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 of the peripheral
antenna, which is because either PSI-J plays a func-
tional role in the binding ⁄ function of the 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 in the 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 of the 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 of the signals
obtained in the immunoblot analysis. (B)
Representative example of the 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, and the mutant consistently showed a 3-nm blue
shift in the far-red florescence emission peak. Excitation wave-
length was 435 nm, and the spectra were normalized to the peak
at 685 nm.

A. Hansson et al. Knock-out of the J subunit of 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% of the P700 in the 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 of PSI-J and further-
more ruling out the possibility that PSI-J is strictly
required for binding of any of the Lhca antenna
proteins.
The presence of free Lhca1 and Lhca4 in the
thylakoid membrane was verified by gentle solubiliza-
tion of the 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, and the 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 in the fractions where mainly LHCII trim-
ers and ⁄ or Lhcb monomers are normally found. How-
ever, this analysis also suggested that PSI–LHCI
complexes devoid of PSI-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 of the distribution of Lhca
proteins in the thylakoid membrane of
DpsaJ (DJ) plants. Shown is the centrifuga-
tion tubes after separation of the 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 of the J subunit of 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-J is to maintain PSI-F in the correct orienta-
tion, facilitating fast electron transfer from Pc or
Cyt c
6

to P700 [20]. A similar role for PSI-J in higher
plants is likely, and, in order to analyse this, NADP
+
photoreduction was determined using thylakoids puri-
fied from plants without PSI-J and 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 of PSI-J (Table 1). Thus, PSI devoid of
PSI-J only has 45% of the NADP
+
photoreduction
activity of the wild-type.
This result clearly suggests that PSI-J affects electron
transport. As indicated from work with green algae [20]
and the in vivo measurement of the P700 redox level in
Fig. 4, the most obvious step to be affected is the 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 in the 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, electron transfer 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 the electron transfer 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 of the 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 the electron 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 in the absence of PSI-J.
Discussion
PSI-J is a subunit of 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 of PSI-J in higher plants has so far not been
investigated. We have successfully generated transgenic
Nicotiana tabaccum plants devoid of the J subunit of
PSI and been able to investigate the role of PSI-J in
higher plants. The PSI-J-less plants were analysed with
various biochemical and physiological methods.
PSI-J is required for stable accumulation of 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), and the lower Chl a ⁄ b ratio
(Table 1). This suggests that PSI-J is implicated in sta-
bility or assembly of the 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 the PSI-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) for the 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-out of the J subunit of 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 of PSI-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 of PSI-J display certain similarities to
plants devoid of PSI-G [9,33,34]. In the absence of
PSI-G, less PSI core, a relative excess of LHCI, and a
less stable PSI is also observed. To distinguish whether
it is the stability or the assembly of the PSI complex that
is affected needs further investigation.
The reduced content of PSI was readily revealed by
the appearance of the 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 the in vitro
NADP

+
measurements and the in 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-J in higher plants are sug-
gested to be important for the energy transfer between
LHCI and the 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 required for 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 of the two Chl molecules bound to
PSI-J causes inefficient transfer of excitation energy
from the peripheral antenna to the core.
In vitro the absence of PSI-J affects the stability of the
PSI–LHCI complex. The results of the 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 in the 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 is required for efficient electron transfer
PSI-J affects the electron transport through PSI. Meas-
ured as in vitro NADP
+
photoreduction activity, a
55% decrease in the steady-state electron transport in
the absence of PSI-J was observed. The kinetic analysis
of the reaction between Pc and P700 did not reveal
any significant difference in the 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 of the 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 in the 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% of the 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 electron transfer in
the absence of PSI-J and has been suggested to be due
to a stabilizing effect of PSI-J on PSI-F [20]. Similar to
this, we observe a 20% decrease in the amplitude of
the second-order component of electron transfer with
plant thylakoids devoid of PSI-J. Thus, in plants, there
is also an increased proportion of PSI complexes that

are incompetent for efficient electron 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 of the luminal PSI-N to PSI
was affected in the absence of PSI-J (Fig. 6). This loss
of PSI-N is probably due to increased sensitivity to
detergent during preparation of the PSI particles, but,
despite this, it strongly suggests a perturbation of the
luminal side of PSI involving PSI-F and PSI-N. The
absence of PSI-J might affect the conformation of
PSI-F, which, in turn, changes the binding of PSI-N.
PSI-F provides part of the Pc-binding site in plants
[16], and it is known that the depletion of PSI-F by
antisense suppression of the 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 for the efficient interaction
Knock-out of the J subunit of 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% in the absence of PSI-N [7].
The increase in the pool of inactive PSI observed in
plants devoid of PSI-J is 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 in the absence of PSI-J
renders a fraction of the PSI complexes inactive.
The in vivo measurements of the P700 redox level
indicate that P700 in the 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 the in vivo experiment is in 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 in the absence of PSI-J in vivo.
However, the 20% increase in the pool of inactive
PSI complexes in the absence of PSI-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 electron transfer from Pc to P700 is unaffected.
However, the release of oxidized Pc has been shown to
limit electron transfer between the cytochrome b
6
f
complex and PSI in vivo [35], and the absence of PSI-J
may affect the k
off

value, so that oxidized Pc stays lon-
ger in the active site and thereby blocks efficient
exchange with reduced Pc. Alternatively, the changed
conformation of PSI-F in the absence of PSI-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 and efficient electron
transfer to ferredoxin [6], and, from the structures, it is
known that PSI-E is in contact with the C-terminus of
the PSI-F subunit [5]. Changes in binding or amounts
of any of the stromal subunits of PSI were not detec-
ted in our immunoblot analysis; however, a subtle
change in arrangement of the subunits is still possible.
In conclusion, PSI-J is needed for stable accumulation
of the PSI core complex and proper electron transfer.
Despite the location of PSI-J close to the rim of the 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 in the absence of PSI-J,
resulting in an increased pool of inactive PSI.
Experimental procedures
Vector construction, chloroplast transformation,
and plant material
The region of the tobacco chloroplast genome containing
700 bp upstream and downstream of the 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 in the 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 and the 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 and the 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-out of the J subunit of PSI
FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS 1743
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 directed against
subunits of the various thylakoid membrane complexes as
indicated in the Figure legends. An antibody originally raised

against electroeluted PSI-I [22] but subsequently found to
recognize both PSI-I and PSI-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, IL, USA) according to the
instructions of the manufacturer. The chemiluminescent
signal produced was recorded digitally using a cooled CCD
camera with the AC1 AutoChemi System (Ultra-Violet
Products Ltd, Cambridge, UK). The exposure time was set
to 5 min, with accumulative snapshots at 30 s intervals.
Signal intensity was quantified using the labworks analysis
software (Ultra-Violet Products Ltd).
Fluorescence measurements
Fluorescence emission spectra were recorded at 77 K on
intact leaves from dark-adapted plants or PSI particles
using a bifurcated light guide connected to a spectrofluo-
rimeter (Photon Technology International, Lawrenceville,
NJ, USA). The excitation light had a wavelength of
435 nm, and emission was detected from 650 to 800 nm.
Standard fluorescence parameters
The Q
A
redox state (1–q
P
), PSII quantum yield (F
PSII
),
nonphotochemical quenching (NPQ) under growth light
conditions were performed as in [33], and the parameters
for standard fluorescence were calculated using the follow-

ing equations:
1 À q
P
¼ðF
s
À F
0
0
Þ=ðF
0
m
À F
0
0
Þ
U
PSII
¼ðF
0
m
À F
s
Þ=F
0
m
;U
PSII
ðdark-adaptedÞ¼ðF
m
À F

0
Þ=F
m
NPQ ¼ðF
m
À F
0
m
Þ=F
0
m
:
P700 oxidation state in wild-type and DPSI-J
leaves
The redox level was estimated from absorption changes at
810–860 nm determined with a PAM 101–103 chlorophyll
fluorimeter (Walz, Effeltrich, Germany) connected to a
dual-wavelength emitter-detector unit, ED 700 DW as des-
cribed by Klughammer & Schreiber [41]. The dual-wave-
length emitter-detector system detects strictly differential
absorbance changes (810 nm minus 860 nm) and is selective
for absorbance changes caused by P700 [42]. Oxidized P700
(DA
max
) was recorded during far-red light illumination. The
level of oxidized P700 in the leaf (DA) was determined dur-
ing white light illumination (25–800 lmol photons Æ m
)2
Æs
)1

).
NADP
+
photoreduction measurements
and Chl ⁄ P700
The NADP
+
photoreduction activity of PSI was deter-
mined from DA
340
as described by Naver et al. [43] using
thylakoids equivalent to 5 lg Chl. Thylakoids were solubi-
lized in 0.1% N-dodecyl-b-d-maltoside before the measure-
ment.
The total P700 content was determined from the ferricya-
nide-oxidized minus ascorbate-reduced difference spectrum
using an absorption coefficient of 64 000 m
)1
Æcm
)1
at
700 nm. The thylakoids were solubilized with 0.2% Triton
X-100, and the measurements were repeated 3–5 times on
several independent thylakoid preparations.
For spectroscopic determination of the 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

.
Antenna size of PSI
Functional PSI antenna size was determined from light-
induced P700 absorption changes at 810 nm using the
dual-wavelength emitter-detector unit, ED-P700DW-E, con-
nected via a PAM 101 fluorimeter to a Tektronix TDS420
oscilloscope using thylakoids equivalent to 33 lg Chl as
outlined in [23]. Thylakoids were solubilized in 0.01% dig-
itonin before the measurement. For each sample, four
traces were averaged, and the measurements were repeated
five times on several independent thylakoid preparations.
The absorption curves were fitted with single-exponential
functions, and relative antenna sizes (percentage of wild-
type) were calculated from the half-times (t
1 ⁄ 2
) with the
assumption that all Chls functionally connected to a reac-
tion centre contribute equally to P700 oxidation in the
monitored millisecond time scale.
Kinetic measurements
Flash-induced P700 absorption decay was measured at
834 nm, as described previously [9]. The saturating actinic
pulse (532 nm, 6 ns) was produced by a Nd:YAG laser.
Thylakoids (30 lg Chl) were dissolved in a final volume of
Knock-out of the J subunit of PSI A. Hansson et al.
1744 FEBS Journal 274 (2007) 1734–1746 ª 2007 The Authors Journal compilation ª 2007 FEBS
300 lL20mm Tricine (pH 7.5), 40 mm NaCl, 8 mm
MgCl
2
, 0.15% dodecyl-b-d-maltoside, 2 mm sodium ascor-

bate, 6 lm 2,6-dichlorophenolindophenol and 100 lm
Methyl Viologen. The solution was centrifuged once for
10 s at 200 g using an Eppendorf 5417 R, rotor F45-30-11
to remove starch. The sample (300 lL) was transferred to a
cuvette with 1 cm path length, and Pc was added to 5 or
25 lm. A diode laser provided the measuring beam, which
was detected using a photodiode. The signal was passed via
a preamplifier (Tektronix ADA400A) to an oscilloscope. A
total of 32 absorbance transients were collected with 4 s
interval and averaged for each decay curve. The recorded
absorbance changes were resolved into two exponential
decay components using a Levenberg–Marquardt nonlinear
regression procedure.
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) and
the EU (Contract No. HPRN-CT-2002-00248) are
gratefully acknowledged.
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