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INTRODUCTION |
Photosystem I (PSI)1 is
a pigment-protein complex that mediates the light-driven electron
transport across the thylakoid membrane from the soluble electron
donor, plastocyanin, to the soluble electron acceptor, ferredoxin. PSI
from plants contains 13 different subunits of which three are only
found in plants, namely PSI-G, PSI-H, and PSI-N. The remaining 10 subunits are shared between cyanobacteria and plants. In addition to
the 13 subunits of PSI in a narrow sense, plants contain light
harvesting complex I (LHCI), which is composed of four different
polypeptides, Lhca1-4, that are specifically associated with PSI (1,
2). The PSI-A/B heterodimer coordinates the reaction center P700 (a
chlorophyll (Chl) a dimer) and the electron acceptors
A0 (Chl a), A1 (phylloquinone), and
FX (a [4Fe-4S] iron-sulfur cluster). The terminal
electron acceptors FA and FB are [4Fe-4S]
clusters bound to the stromal PSI-C subunit (3, 4).
The 10 common subunits are highly conserved from cyanobacteria to
plants apart from the presence of extended N- termini of PSI-D, -E, -F,
and - L from plants. The role of specific subunits in PSI has mostly
been investigated by gene knock-out studies in cyanobacteria and algae.
However, despite the sequence similarities, PSI subunits of
cyanobacteria and plants show important functional differences. For
example, the plant-specific N terminus of PSI-F plays a role in
supporting plastocyanin-mediated donation of electrons to
P700+ (5, 6). In cyanobacterial PSI, this electron transfer
mostly follows a simple second order reaction, whereas a stable
plastocyanin-PSI complex is formed in plants before electron transfer
(6). PSI-L is essential for formation of PSI trimers in cyanobacteria
(7), but plant PSI complexes are not assembled in trimers, and the function of PSI-L in plants is thus far unsolved. Finally, the N-terminal extension of the PSI-D subunit is important for the stable
binding of PSI-C (8). PSI-C is anchored to the PSI-A/B heterodimer
directly through a domain of eight amino acid residues (9, 10) and
indirectly via PSI-D (8, 11, 12). Treatment with chaotropic agents
selectively dissociates the extrinsic subunits PSI-C, -D, and -E, but a
much harsher and more prolonged treatment is required to dissociate
these subunits from plant PSI compared with cyanobacterial PSI. (8-10,
13).
The role of the three plant-specific subunits is less understood than
the role of the 10 common subunits. PSI-N is a luminal protein, and
very recent data have shown a function of this subunit in the
interaction with plastocyanin (14). The PSI-H protein is membrane
intrinsic and has a molecular mass of about 10 kDa. PSI-H can be
cross-linked with PSI-D, PSI-I, and PSI-L, and the three membrane
intrinsic subunits are positioned peripherally in the model of the
plant PSI complex with PSI-L and -I located on the outside (15, 16).
The properties of PSI-H combined with the relatively high stability of
plant PSI have led us to suggest that the role of the N terminus of
PSI-D in stability is mediated through an interaction with the PSI-H
subunit (8). Because light-harvesting chlorophyll
a/b-binding proteins are only present in plants
and not in cyanobacteria, an alternative suggestion for the function of
PSI-H has been an involvement in the interaction with LHCI (17).
However, the topological studies indicate that such a function is less
likely because PSI-H appears not to be in close contact with any LHCI
protein (16).
To investigate the role of PSI-H we transformed Arabidopsis
plants with a psaH cDNA in sense orientation under the
control of a constitutive promoter. Cosuppressed transformants with
undetectable levels of PSI-H were obtained. The down-regulated plants
were analyzed with several methods both at the biochemical and leaf level. We conclude that PSI-H is essential for efficient electron flow
in the PSI complex. Furthermore, PSI-H is required for interaction with
PSI-L, stabilization of FX, and the overall stability of the PSI complex.
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EXPERIMENTAL PROCEDURES |
Plant Material--
Arabidopsis thaliana L. cv.
Colombia ecotype 0 were grown in a Percival Arabidopsis
chamber at 20 °C, 70% relative humidity, and illumination of 100 µmol m
2 s
1. Wild type plants used for
transformation were grown with a 12-h photoperiod, whereas
transformants were grown with an 8-h photoperiod to keep plants at the
vegetative state as long as possible. Transformants were germinated on
MS medium (Sigma) containing 2% sucrose, 50 mg/liter kanamycin, and
0.9% agar and transferred to soil after approximately 4 weeks.
Vector Construction and Arabidopsis Transformation--
The
psaH cDNA from Arabidopsis was obtained from
the Arabidopsis Biological Resource Center (Columbus, OH) as
est:att12892. A segment corresponding to the entire coding region was
polymerase chain reaction amplified and cloned into the
BamHI and SacI sites of the binary vector pBIN121
(18). In this way the GUS gene of pBIN121 was replaced by the
full-length psaH in sense orientation. The vector was
transformed into Agrobacterium tumefaciens strain C58 (19).
Arabidopsis was transformed by vacuum infiltration according
to Bechtold et al. (20).
Southern Blot Analysis--
Arabidopsis genomic DNA
was isolated with a Nucleon Phytopure kit (Amersham, UK). The DNA (10 µg) was digested with BamHI, and the resulting fragments
were separated by agarose gel electrophoresis and transferred onto
nylon membrane (Boehringer Mannheim). The membranes were hybridized
with a digoxigenin-labeled psaH polymerase chain reaction
fragment in DIG Easy hybridization buffer according to the DIG System
manual (Boehringer Mannheim). The membrane was washed in 0.5% SSC
containing 1% SDS at 65 °C.
Immunoblotting and Thylakoid Preparations--
All plant
extracts and fluorescence measurements were carried out with plants
grown in soil. Only fully expanded rosette leaves of comparable size
from nonflowering plants were used for these analyses. Whole leaf
extracts and thylakoids were prepared and analyzed by immunoblotting as
described previously (14). Chl a/b ratio was
determined according to Lichtenthaler (21). P700 concentration was
determined from absorption transients at 834 nm (see "Urea Treatment
of Thylakoids and Flash-induced Absorbance Measurements").
Thylakoids were solubilized by applying decylmaltoside to a final
concentration of 0.15% to a sample adjusted to a final concentration of 100 µg Chl/ml. The solubilized thylakoids were centrifuged at
300 × g for 3 × 1 min, and the white pellet of
starch was discarded.
NADP+ Photoreduction
Measurements--
NADP+ photoreduction activity was
determined as described by Kjær and Scheller (22) from the absorbance
change at 340 nm in a 0.5-ml reaction mixture containing 20 mM Tricine (pH 7.5), 7 mM MgCl2, 40 mM NaCl, 2 mM sodium ascorbate, 60 µM 2,6-dichlorophenolindophenol, 0.5 mM
NADP+, 50 nM ferredoxin:NADP+
oxidoreductase, 2 µM plastocyanin, 0.0125-3
µM ferredoxin, and solubilized thylakoids equivalent to 5 µg of Chl. Plastocyanin, ferredoxin, and ferredoxin:NADP+
oxidoreductase were purified from barley.
Urea Treatment of Thylakoids and Flash-induced Absorbance
Measurements--
Flash-induced absorbance transients at 834 nm were
determined as described previously (9, 23). The cuvette contained solubilized thylakoids (100 µg Chl/ml), 2 mM sodium
ascorbate, and 60 µM 2,6-dichlorophenolindophenol. Solid
urea was then added to a final concentration of 6.5 M, and
the cuvette was incubated at room temperature and subjected to
flash-induced absorbance measurements at different time points during
the incubation. Apart from actinic flashes, the sample was kept dark
during incubation.
Fluorescence Quenching Analysis--
In vivo
chlorophyll fluorescence was measured at room temperature using a
PAM-101/103 fluorometer (Walz, Effeltrich, Germany). 12-week-old plants
were dark-adapted for 30 min prior to the measurements. The initial
fluorescence in the dark-adapted state (F0) was
determined using a very dim red light modulated at 1.6 kHz. The maximal
level of fluorescence (Fm) was induced by an
800-ms pulse of saturating white light (>4000 µmol m
2
s
1). The maximum quantum yield of PSII was estimated in
dark-adapted leaves as
PSII = (Fm
F0)/Fm. Steady state
fluorescence was measured during illumination at approximately 5-10
and 100 µmol m
2 s
1 with a KL1500 halogen
lamp (Schott). During actinic illumination, the measuring light was
modulated at 100 kHz. The fluorescence was allowed to stabilize over 10 min, and maximum fluorescence yield Fm' was then
determined by giving a second 800-ms pulse. Finally, actinic light was
turned off, and minimum fluorescence in the light-adapted state
(F0') was determined. The photochemical and
nonphotochemical quenching coefficients were calculated as qP = (Fm'
F)/(Fm'
F0') and qN = 1
(Fm'
F0')/(Fm
F0) (24), where qP
indicates photochemical fluorescence quenching and
qN indicates nonphotochemical fluorescence quenching.
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RESULTS |
Cosuppression of the PSI-H Polypeptide--
A total of 26 transgenic plants (T1) with kanamycin resistance and
containing the psaH cDNA were produced. Leaf extracts of the T1 plants were analyzed by immunoblotting, but no
substantial down-regulation of PSI-H was observed in any of the plants.
The original 26 T1 plants were self-fertilized, and the
seeds were reselected on kanamycin. Five to ten T2 plants
from each T1 plant were analyzed by immunoblotting of leaf
extracts. PSI-H was undetectable in the plants designated 15.2 and 20.2 (detection level,
3% of wild type level) and was substantially
down-regulated in plants 20.5, 3.2, and 3.4 (Fig.
1A). T3 progeny of
plant 15.2 was not down-regulated, whereas 75% of the progeny of plant
20.2 lacked PSI-H (Fig. 1B). Further, 10% of the progeny of
3.4 and 5% of the 3.2 and 20.5 progeny lacked PSI-H (Fig.
1B). Progeny from the remaining T1 plants did
not show substantial down-regulation of PSI-H and were not further
analyzed.

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Fig. 1.
Identification of plants lacking PSI-H by
immunoblotting. Total leaf protein equivalent to 1.5 µg of Chl
was loaded in each lane of the gel. The blots were developed with
antibodies against PSI-H and PSI-F. A, analysis of
T2 plants 3.2 and 3.4 were progeny of T1 plant
3, 20.2-20.5 were progeny of the T1 plant 20, and 15.2 were progeny of the T1 plant 15. B, examples of
analysis of T3 progeny of the T2 plants shown
in A. Due to variation in the development of the blots, the
content of PSI-H and PSI-F in each sample should only be compared with
the wild type (WT) sample from the same blot.
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Genomic DNA of T2 progeny of T1 plants numbered
3, 15, and 20 was digested with BamHI, which cleaves at the
5' end of the inserted psaH. The blot was hybridized with a
psaH-specific probe (Fig. 2).
The blot shows that wild type plants have two copies of psaH
that are also found in all the transformants. Several Arabidopsis EST sequences are available that clearly contain
psaH sequence, and these are easily divided into two groups.
Sequence alignment shows that Arabidopsis has two expressed
psaH genes with coding sequences that are 97% identical and
that encode proteins with identical amino acid sequences (data not
shown). Southern blot analysis showed that the T2
generation of the transformants contained 2-4 copies of inserted
psaH in addition to the two wild type genes (Fig. 2).
Because of the variation in down-regulation, all plants were analyzed
by immunoblotting at the time they were used for other experiments.
However, the initial analysis of leaf extracts of small plants was
confirmed by immunoblotting of three different thylakoid preparations
of the same plants performed with an interval of 4 weeks. Thus,
cosuppression of the PSI-H subunit seemed stable throughout the life of
individual plants despite the variation between plants (data not
shown).

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Fig. 2.
Southern blot analysis of transformants
(T2). Total DNA from wild type and transformants
(designated by numbers) was digested with BamHI, which
cleaves at the 5' end of the inserted psaH clone,
electrophoresed, transferred to nylon membrane, and hybridized with a
psaH probe. T2 plants have two to four copies of
psaH in the genome in addition to the two wild type
(WT) genes. Sizes of marker DNA are shown in kb (lane
M).
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Plants Lacking PSI-H Grow Poorly on Agar Medium and Have Diminished
Nonphotochemical Quenching--
Growth and development of plants
lacking PSI-H were similar to wild type plants when grown in soil. In
contrast, the plants clearly showed a decreased growth rate compared
with wild type if kept on agar medium (Fig.
3). The difference becomes apparent about
4 weeks after germination.

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Fig. 3.
Phenotype of plants lacking PSI-H (20.2)
compared with wild type (WT). Plants (6-7 weeks
old) were grown on Murashige and Skoog (MS) medium containing 2%
sucrose, at 20 °C and 70% humidity for an 8-h photoperiod at 100 µmol photons m 2 s 1.
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The fluorescence quenching parameters: maximal quantum yield of PSII
photochemistry (
PSII), photochemical quenching
(qP), and nonphotochemical quenching (qN) were
determined at two different light intensities (Table
I).
PSII was slightly but
significantly decreased in plants lacking PSI-N (t test,
p < 0.05). No significant change in qP,
which reflects the steady state redox level of quinone QA
in PSII and thus reflects the redox state of the plastoquinone pool,
was observed in plants lacking PSI-H. However, qN, which largely reflects energy-dependent quenching due to the
proton gradient across the thylakoid membrane was decreased to about 60% of wild type levels.
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Table I
Fluorescence quenching coefficients and quantum yield of PSII
photochemistry
The values are the means ± S.E. based on independent measurements
of 7-14 plants in each group.
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PSI fluorescence emission spectra determined on leaves at 77 K were
identical in plants lacking and containing PSI-H (25). Furthermore, the
functional antenna size of PSI is unaffected by the lack of PSI-H as
evidenced by measurements of P700 absorption changes in leaves
illuminated with varying intensity of far red light (25).
Absence of PSI-H Affects Binding of PSI-L to the PSI
Complex--
The Chl/P700 ratio (±S.E., n = 7-14))
was 696 ± 32 in wild type thylakoids and 606 ± 17 in
thylakoids from plants devoid of PSI-H. These numbers are significantly
different (t test, p < 0.05) and indicate
that plants devoid of PSI-H compensate for the deficiency by increasing
the PSI/PSII ratio. To analyze the polypeptide composition of a PSI
complex lacking PSI-H, the isolated thylakoids were subjected to
immunoblotting. The blots were probed with antibodies directed against
the subunits that are close to PSI-H, i.e. PSI-D, -L, and -I
(15, 16) as well as with antibodies against PSI-F and -J (Fig.
4). Plants lacking PSI-H had a decreased content of PSI-L (<50%), whereas PSI-D, -I and -J were present at
normal levels. Some plants appeared to have an increased content of
PSI-D (Fig. 4), but this was not consistently observed and was not
correlated with the content of PSI-H. Similar blots were incubated with
antibodies directed against: PSI-C, -A/B, and -N and Lhca1, and no
differences between wild type and plants without PSI-H were
observed.

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Fig. 4.
Immunoblot analysis of accumulation of PSI
subunits in wild type and plants lacking PSI-H. Thylakoids were
prepared from different plants lacking PSI-H (20.2.2, 20.5.9, 3.2.9, and 3.4.17), and wild type (WT) and samples of protein
equivalent to 0.5 µg of Chl were loaded on SDS gels. The blots were
incubated with antibodies against PSI-D, -F, -H, -I/J, and -L. The
PSI-I/J antibody recognizes both PSI-I (upper band) and
PSI-J (lower band).
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Electron Transfer Rate Is Decreased in the Absence of
PSI-H--
To analyze the importance of PSI-H for electron transfer,
NADP+ photoreduction was determined. At standard assay
conditions of 3 µM ferredoxin, NADP+
reduction of thylakoids from plants devoid of PSI-H was only 61% of
wild type NADP+ reduction (Fig.
5). The analyzed thylakoids contained
only intact PSI complexes with respect to the electron acceptor
composition as shown by flash photolysis (see Fig. 6A;
0 h of urea treatment). Therefore, the observed decrease in
NADP+ reduction of PSI lacking PSI-H must be explained by a
corresponding decrease in forward electron transfer rate. As the
ferredoxin concentration became rate-limiting for the measured
NADP+ reduction at a ferredoxin concentration below 1 µM, the difference between thylakoids without PSI-H and
wild type disappeared (Fig. 5). Thus, the second order rate constant
for ferredoxin reduction appears to be unchanged in PSI lacking PSI-H.

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Fig. 5.
NADP+ photoreducing activity of
PSI with and without PSI-H. Thylakoid preparations from plants
lacking PSI-H ( ) (20.2.2, 20.2.17, 20.5.9, 3.4.17, and 3.2.9) and
wild type ( ) were analyzed. The samples were supplied with 2 µM plastocyanin, 0.05 µM
ferredoxin:NADP+ oxidoreductase, 0-3 µM
ferredoxin, and thylakoids corresponding to 5 µg of Chl. The
error bars correspond to S.E. (n = 5).
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PSI Is Unstable in the Absence of PSI-H--
To investigate the
stability of the PSI complexes, thylakoids were treated with 6.5 M urea for up to 4 h, and the change in electron
acceptor composition of the PSI complexes was followed by flash-induced
absorption changes at 834 nm. In an intact PSI complex carrying all
electron acceptors from P700 to [FA/FB], the
time constant for charge recombination between
[FA/FB]
and P700+
is >30 ms. When PSI-C carrying [FA/FB] is
dissociated by urea treatment, the charge recombination between
FX
and P700+ of 1 ms is observed.
Finally, when FX is destroyed the observed charge
recombination from A1
to
P700+ takes place much faster than 1 ms. No accurate
determination of the time constant of the fast reaction (approximately
50 µs) was attempted in these experiments. After a saturating flash, the recorded absorbance changes of P700 can be resolved into a sum of
exponential decays with the characteristic time constants. For a
discussion of recombination rates see Refs. 26 and 27.
Both PSI complexes containing and lacking PSI-H were intact before the
addition of urea as evidenced by the observation that the 30-ms phase
was the single component of the absorbance decay (Fig.
6A). Urea was then added to
the samples to a final concentration of 6.5 M, resulting in
a slow dissociation of the PSI complex. After 1 h of urea
treatment, the 1-ms phase was visible in the wild type PSI complex and
the 50-µs phase in the complex without PSI-H (Fig. 6B).
The 50-µs signal increased in PSI without PSI-H to become the major
component after 2 and 4 h of urea treatment (Fig. 6, C
and D). The curve traces for the wild type PSI showed increasing amounts of the FX
to
P700+ 1-ms phase. However, faster recombinations indicating
the destruction of FX were not observed (Fig. 6,
B, C, and D).

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Fig. 6.
Stability of PSI in the presence of 6.5 M urea. Thylakoid preparations from three plants
lacking PSI-H and three wild type (WT) plants were analyzed
by flash-induced absorption transients at 834 nm during incubation with
urea. Charge recombination between electron acceptors in the PSI
complex (FA/FB ,
FX , and
A1 ) and P700+
was recorded. Before urea treatment, charge recombination is single
phased consisting exclusively of the
FA/FB to P700+ charge
recombination of >30 ms. After addition of urea, charge recombination
becomes bi- and triphasic, consisting of recombinations between
different electron acceptors and P700. A-D, the
traces shown are examples of one wild type and one
PSI-H-less sample. The duration of incubation is indicated in the
figure. E, average extent of the 30-ms ( , ) and 1-ms
( , ) recombination in wild type ( , ) and PSI-H-less ( ,
) samples during urea incubation.
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Urea treatments were carried out with three samples of thylakoids from
each type of plant. The absorption decay components calculated for each
time point were averaged and plotted as a function of time (Fig.
6E). The sum of the wild type components was adjusted to
100%, and the phases of the components of the samples lacking PSI-H
were normalized according to this. The 30-ms component
(FA/FB
to P700+) of
the wild type decreased with the duration of urea treatment and was
replaced by the 1-ms phase (Fig. 6E). However, in the absence of PSI-H, the 30-ms component decreased about twice as fast as
in the wild type. Thus, PSI-C with FA/FB is
released approximately twice as fast from PSI when PSI-H is absent
compared with the release of PSI-C in the wild type samples. The 1-ms
component in the samples lacking PSI-H replaced the 30-ms phase for a
short period but was then replaced by the 50-µs phase
(A1
to P700+).
This fast component was essentially absent in the wild type. Thus,
FX is much less stable in the absence of PSI-H. After
4 h of urea treatment the sum of the three decay components in the absence of PSI-H was only 21% of the wild type. The remaining 79%
represents urea-degraded PSI complex where no P700 absorption changes
could be detected with the time resolution of the measurement. Thus,
prolonged urea treatment of PSI lacking PSI-H leads to inactivation of
electron acceptors earlier than FX or possibly damage to
P700 itself.
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DISCUSSION |
The cosuppression strategy in Arabidopsis was
successful and yielded plants without detectable PSI-H. This results
has enabled us to investigate the function of the PSI-H subunit
in vivo as well as in vitro. Although the
approach was successful, the frequency of substantial down-regulation
in the transformed plants was low, and no plants lacking PSI-H were
obtained in the T1 generation. It has previously been
reported that down-regulation of LHCII in an antisense approach was not
observed, although the mRNA level was reduced to extremely low
levels (28). On the other hand, down-regulation of the PSI-N subunit
was very efficient in a similar cosuppression and antisense approach
(14), and the successful down-regulation of the LHCI subunit Lhca4 by
an antisense approach was recently reported (29). Possibly, the
difficulty in obtaining down-regulation of PSI-H is related to the
presence of two expressed copies of psaH in
Arabidopsis. In contrast, PSI-N is expressed from a single
gene. Gene knock-out by homologous recombination is an excellent way of
studying the role of PSI subunits in cyanobacteria. However, in plants
this technique is not yet straightforward. The present results show
that PSI subunits can be efficiently down-regulated in plants by
cosuppression. Thus, this approach can be very useful in dissecting the
role of individual components of PSI.
During growth on sterile medium, plants without PSI-H showed pronounced
stunting of growth and yellowing of leaves (Fig. 3). Surprisingly,
however, no difference in visual appearance of the plants was seen when
pants were grown in soil. Only plants grown in soil were used for
biochemical and physiological studies.
Forward Electron Transport Is Decreased in Plants Lacking
PSI-H--
Steady state electron transport is clearly decreased in PSI
lacking PSI-H. The decrease does not appear to be due to heterogeneity of PSI in the absence of PSI-H because there is no difference at low
ferredoxin concentration. The interaction between ferredoxin and PSI is
kinetically complex (12, 30). Ferredoxin forms a complex with PSI in
both the reduced and oxidized form. Intracomplex electron transfer is
heterogeneous and takes place with several different time constants. At
ferredoxin concentrations below the dissociation constant for the
PSI-ferredoxin complex, the limiting factor for electron transfer is
the second order rate constant. Thus, this rate constant appears to be
unaltered in PSI lacking PSI-H. At higher ferredoxin concentration,
intracomplex electron transfer may become limiting, and the lower rate
in the absence of PSI-H suggests that intracomplex electron transfer is
affected. This may be due to different rate constants of transfer or to different relative contributions of the different time constants. The
situation in PSI lacking PSI-H may resemble the situation in
cyanobacterial PSI where the rate of ferredoxin reduction is much lower
than in plant PSI, at least in a large fraction of complexes (30). In
principle, the difference at high ferredoxin concentration could also
be due to another limiting step in electron transfer. However, we find
this unlikely. Forward electron transfer from P700 to
FA/FB is very fast compared with the rates of
NADP+ reduction, and the interaction between plastocyanin
and PSI appears to be largely governed by PSI-F and PSI-N.
Nevertheless, further detailed investigations of different steps of
electron transport will be necessary to determine the precise mechanism
by which PSI-H affects electron transport. A likely explanation is that lack of PSI-H perturbs the binding of PSI-D and PSI-C, causing a less
productive binding of ferredoxin.
Plants Lacking PSI-H Accumulate More PSI--
The plants devoid of
PSI-H have compensated by synthesizing more PSI as evidenced by the
15% lower Chl/P700 ratio. This compensation is sufficient under
optimal conditions, where lack of PSI-H had little impact on plant
growth. The lower efficiency of PSI might be expected to lead to
overreduction of the electron transport components connecting PSI and
PSII. However, again the compensation seems sufficient because no
significant increase in qP was observed. Furthermore, the
size of the functional PSI antenna is identical in plants with and
without PSI-H, at least in state 1 (25). Plants lacking PSI-H show less
nonphotochemical quenching of fluorescence, which may indicate a
smaller transthylakoidal proton gradient. A smaller proton gradient
could result from less overall electron transport. However, because the
plants do not exhibit differences in growth, we do not think that this
is likely. Alternatively, the less efficient reduction of ferredoxin
may lead to altered redox levels in the stroma with a resulting
decrease in cyclic electron transport and therefore a decrease in
proton pumping. Finally, it may be imagined that lack of PSI-H lead to
altered permeability of the membrane for protons.
The lower scope for PSI activity and the lower nonphotochemical
quenching in the absence of PSI-H may have little significance under
optimal and constant growth conditions. However, under photoinhibitory conditions it may be predicted that plants lacking PSI-H would suffer
more severely from overreduction of plastoquinone. Other stress
condition such as low light intensity or conditions where the demand
for ATP is increased may also be expected to lead to more severely
affected plants.
PSI-H Stabilizes PSI--
A substantial decrease in stability of
PSI was observed in thylakoids from plants without PSI-H. The relative
instability resembles the situation in cyanobacteria, where urea has a
much faster and stronger effect on PSI than in plants. We have
hypothesized that PSI-H interacts with the N-terminal extension of
PSI-D which is important for the high stability of plant PSI (8). The
lower stability of the PSI complex lacking PSI-H is therefore in good agreement with the hypothesis. Future experiments with in
vitro reconstitution in the presence or absence of PSI-H should
allow us to test the hypothesis further and investigate the interaction of PSI-H with PSI-D and other subunits in detail. The instability of
FX and earlier acceptors was a surprising result because
PSI-H is unlikely to be directly involved in coordinating electron
acceptors. Possibly, lack of PSI-H leads to a general instability of
the PSI complex in the presence of urea and a progressive
disintegration of the entire complex. Although the instability of PSI
was easily observed in the in vitro experiments, no severe
disintegration of the PSI complex appears to take place during normal
growth because all electron acceptors were functional in the thylakoids lacking PSI-H. However, the partial lack of PSI-L suggests a lower stability of the complex also under in vivo conditions.
Possibly, more severe disintegration of PSI could occur under certain
stress conditions, e.g. heat stress. Because PSI-L content
is decreased, it can be speculated whether the role of PSI-H in
electron transport is mediated through PSI-L. We find this unlikely
because PSI-L is relatively far removed from the site of interaction of
the soluble electron transfer proteins (4). However, experiments with
plants lacking only PSI-L will be required to address this issue.
Conclusion--
In summary, PSI-H has been shown to be not
important for LHCI interaction with PSI (25) but to be essential for
efficient electron transfer of PSI and for stability of the PSI
complex. The cyanobacterial PSI complex is dissociated eight times
faster in the monomeric than in the trimeric form as shown by urea
treatment of monomers and trimers (4, 31). Plant PSI is a monomer that is more stable upon urea treatment than the trimeric cyanobacterial PSI
(8). The peripheral antenna in cyanobacteria consists of extrinsic
phycobiliproteins. In contrast, plants have adopted a different antenna
consisting of membrane intrinsic Chl a/b-binding proteins, i.e. LHCI and LHCII. It can be speculated that the
trimeric structure of PSI was abandoned as LHCI became associated with PSI in plants, and this resulted in a need for stabilizing the now
monomeric PSI complex. PSI-H may have evolved simultaneously, fulfilling the role as a stabilizing factor.