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Laurie K. Frankel and Terry M. Bricker
Xiaoping Yi, Stefan R. Hargett, Haijun Liu,
thaliana Arabidopsisand Photoautotrophy in Assembly/StabilityPhotosystem II Complex
The PsbP Protein Is Required for
Metabolism and Bioenergetics:
doi: 10.1074/jbc.M705011200 originally published online June 29, 2007
2007, 282:24833-24841.J. Biol. Chem.
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The PsbP Protein Is Required for Photosystem II Complex
Assembly/Stability and Photoautotrophy in
Arabidopsis thaliana
*
Received for publication, June 18, 2007, and in revised form, June 29, 2007 Published, JBC Papers in Press, June 29, 2007, DOI 10.1074/jbc.M705011200
Xiaoping Yi, Stefan R. Hargett, Haijun Liu, Laurie K. Frankel, and Terry M. Bricker
1
From the Division of Biochemistry and Molecular Biology, Department of Biological Sciences, Louisiana State University,
Baton Rouge, Louisiana 70803
Interfering RNA was used to suppress the expression of the
genes At1g06680 and At2g30790 in Arabidopsis thaliana, which
encode the PsbP-1 and PsbP-2 proteins, respectively, of photo-
system II (PS II). A phenotypic series of transgenic plants was
recovered that expressed intermediate and low amounts of
PsbP. Chlorophyll fluorescence induction and Q
A
ⴚ
decay kinet-
ics analyses were performed. Decreasing amounts of expressed
PsbP protein led to the progressive loss of variable fluorescence
and a marked decrease in the fluorescence quantum yield (F
V
/
F
M
). This was primarily due to the loss of the J to I transition.
Analysis of the fast fluorescence rise kinetics indicated no sig-
nificant change in the number of PS II

centers present in the
mutants. Analysis of Q
A
ⴚ
decay kinetics in the absence of 3-(3,4-
dichlorophenyl)-1,1-dimethylurea indicated a defect in electron
transfer from Q
A
ⴚ
to Q
B
, whereas experiments performed in the
presence of this herbicide indicated that charge recombination
between Q
A
ⴚ
and the oxygen-evolving complex was seriously
retarded in the plants that expressed low amounts of the PsbP
protein. These results demonstrate that the amount of func-
tional PS II reaction centers is compromised in the plants that
exhibited intermediate and low amounts of the PsbP protein.
Plants that lacked detectable PsbP were unable to survive in the
absence of sucrose, indicating that the PsbP protein is required
for photoautotrophy. Immunological analysis of the PS II pro-
tein complement indicated that significant losses of the CP47
and D2 proteins, and intermediate losses of the CP43 and D1
proteins, occurred in the absence of the PsbP protein. This dem-
onstrates that the extrinsic protein PsbP is required for PS II
core assembly/stability.
In higher plants, algae, and cyanobacteria, at least six intrin-
sic proteins appear to be required for oxygen evolution by PS II
2
(1–3). These are CP47, CP43, the D1 and D2 proteins, and the
␣
and

subunits of cytochrome b
559
. Insertional inactivation or
deletion of the genes for these components results in the disas-
sembly of the PS II complex and the complete loss of oxygen
evolution activity (for review, see Ref. 4). Additionally, a num-
ber of low molecular mass, intrinsic membrane protein compo-
nents are associated with PS II (5–7), although the functions of
many of these proteins remain obscure. Although PS II com-
plexes containing only these intrinsic components can evolve
oxygen in vitro, they do so at low rates (⬃25–40% of control),
are extremely susceptible to photoinactivation, and require
high, nonphysiological levels of calcium and chloride for max-
imal activity (1, 3).
In higher plants and green algae, three extrinsic proteins,
with apparent molecular masses of 33, 24, and 17 kDa, are
required for high rates of oxygen evolution at physiological
inorganic cofactor concentrations. The 33-kDa component, the
PsbO protein, has been termed the manganese-stabilizing pro-
tein due to its stabilization of the manganese cluster during
exposure to low chloride concentrations or to exogenous
reductants. In vitro, the 24- and 17-kDa proteins (termed the
PsbP and PsbQ proteins, respectively) appear to modulate the
calcium and chloride requirements for efficient oxygen evolu-
tion. The precise roles of these proteins in oxygen evolution and
PS II assembly/stability in vivo, however, remain unclear. These
three extrinsic components interact with intrinsic membrane
proteins and possibly with each other to yield fully functional
oxygen-evolving complexes.
The mature PsbP protein is highly conserved (8) in higher
plants. In Arabidopsis, there are two putative genes, At1g06680
and At2g30790, which encode PsbP-1 and PsbP-2, respectively.
It should be noted that initially only PsbP-1 was observed in
Arabidopsis (9, 10) using two-dimensional IEF-SDS-PAGE.
Recently, however, PsbP-2 has been detected during two-di-
mensional difference gel electrophoresis (11). In the cyanobac-
terium Synechocystis 6803, mutants in which the homologue of
the psbP gene had been deleted exhibited reduced photoau-
totrophic growth as well as decreased water oxidation activity
under CaCl
2
-limiting conditions (7, 12), whereas in Chlamydo-
monas, a mutant which did not accumulate PsbP was deficient
in photoactivation (13).
RNAi is a post-transcriptional gene-silencing process in
which double-stranded RNA induces the degradation of
homologous mRNA sequences (14). RNAi has been success-
fully applied as a powerful gene-silencing tool in a variety of
organisms, including Caenorhabditis elegans and Drosophila
melanogaster, and in mouse oocytes. It has also become a pop-
*This work was supported by grants from the National Science Foundation
and the Dept. of Energy (to T. M. B. and L. K. F.).The costs of publication of
this article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “advertisement” in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
To whom correspondence should be addressed: Dept. of Biological Sciences
Biochemistry and Molecular Biology Section, Louisiana State University,
206 LIfe Sciences Bldg., Baton Rouge, LA 70803. Tel.: 225-578-1555; Fax:
225-578-7258; E-mail: btbric@lsu.edu.
2
The abbreviations used are: PS I and II, photosystems I and II; DCMU, 3-(3,4-
dichlorophenyl)-1,1-dimethylurea; LiDS, lithium dodecyl sulfate; Tricine,
N-tris[hydroxymethyl]methyl glycine.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 34, pp. 24833–24841, August 24, 2007
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
AUGUST 24, 2007•VOLUME 282• NUMBER 34 JOURNAL OF BIOLOGICAL CHEMISTRY 24833
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ular research methodology for investigating the physiological
functions of target genes in plants (15). With respect to PS II
membrane proteins, RNAi has been used to study the function
of the PsbO and PsbQ proteins in Arabidopsis (16, 17) as well as
the PsbP and PsbQ proteins in tobacco (18). The studies per-
formed in tobacco indicated that RNAi suppression of the PsbQ
proteins led to no observable phenotype, but RNAi suppression
of the PsbP proteins led to retardation of photoautotrophic
growth, lower quantum yield of PS II, loss of PsbQ, and an
unstable manganese cluster that disassembled in the dark.
Accumulation of the intrinsic PS II core proteins and the
extrinsic PsbO protein in PsbP-suppressed tobacco plants was
similar to that in wild type (18). Studies performed in Arabidop-
sis showed that the RNAi suppression of PsbQ led to the
impaired assembly/stability of PS II in low light growth condi-
tions, resulting in the loss of photoautotrophy (16). In our cur-
rent study, we report on the RNAi suppression of PsbP expres-
sion in Arabidopsis. Our studies differ from those performed in
tobacco (18) in that we find, in Arabidopsis, the PsbP protein
appears to be required for photoautotrophy and that, in its
absence, significant decreases in the amounts of the PS II core
proteins D2 and CP47 occur. We provide fluorescence yield
data, immunological data, and growth characterization to sup-
port the hypothesis that PsbP is required for PS II assembly/
stability and photoautotrophic growth in Arabidopsis.
MATERIALS AND METHODS
RNA Interference Construct and Transformation—The
pHANNIBAL vector (19) was used to construct an intron-spliced
hairpin RNA (RNAi construct). The sequence (⫹261 to ⫹528) of
the psbP gene (At1g06680) was chosen to suppress the expression
of both PsbP-1 and PsbP-2. This construct will be referred to as
psbP-RNAi. The primers for psbP-RNAi were 5⬘-CCGAATTCG-
AAGCCAAAGACGAACAGA-3⬘and 5⬘-CAGGTACCAGAG-
GCAGTCTCACCGA-3⬘for the sense fragment and 5⬘-AGGAT-
CCGAAGCCAAAGACGAACAGA-3⬘and 5⬘-CCATCGATCA-
GAGGCAGTCTCACCGA-3⬘for the antisense fragment of psbP.
PCR was performed on a RapidCycler (Idaho Technology, Inc.) in
thin-walled microcentrifuge tubes in 50-
l reactions containing 5
lof10⫻PCR reaction buffer, 1.5
lof50mMMgCl
2
, 1.5
lof2.5
mMdNTP mixture, 3
lof10pM/
l primer mixture, 0.25
lof5
units/
l platinum Taq polymerase High Fidelity (Invitrogen), and
25 ng of Arabidopsis genomic DNA in purified water. Cycling
parameters were a pre-denaturation step at 95 °C for 5 min fol-
lowed by 35 amplification cycles (denaturing at 94 °C for 25 s,
annealing at 55 °C for 25 s, and extension at 72 °C for 45 s) and a
final extension at 72 °C for 7 min. The amplified sequence was
cloned in both forward and reverse orientations flanking the Pdk
intron of the pHANNIBAL vector. After construction and verifi-
cation by sequencing, the expression region was excised from
pHANNIBAL with NotI and then subcloned into pART27 for
transformation of the Agrobacterium strain GV3101 by the freeze-
thaw method (20). Four-week-old Arabidopsis plants (Col-0) were
transformed by the floral dip method as described previously (21).
Harvested seeds were surface-sterilized with 50% ethanol and 0.5%
Tween 20 for 3 min, washed briefly with 95% ethanol, and then
70% ethanol for 3 min followed by washing three times with sterile
water. Seeds were spread on solid MS medium containing 0.7%
agar, 2% sucrose, 50 mg/liter kanamycin, and 400 mg/liter carbe-
nicillin and then incubated for 2 days at 4 °C in the dark. Germina-
tion and the first 10 days of growth occurred under lighted condi-
tions at 28 °C in Petri dishes, and then the plants were transplanted
into culture boxes containing solid MS medium with 2% sucrose,
50 mg/liter kanamycin, and 400 mg/liter carbenicillin at a temper-
ature of 23 °C under ⬃50
mol photons/m
2
/s continuous light. To
test for photoautotrophic growth, plants were transplanted onto
medium from which sucrose was omitted.
Screening—The presence of the RNAi construct in the kana-
mycin-resistant plant lines was confirmed by the use of PCR
with primers designed to amplify the cauliflower mosaic virus
35S promoter and the targeted gene of the introduced DNA. All
of the plants that exhibited the kanamycin-resistant phenotype
also exhibited the expected 1.0-kbp amplification product,
which was absent in the wild-type plants (data not shown). Indi-
vidual kanamycin-resistant plants were screened for the pres-
ence of the PsbP protein by “Western” blotting. One leaf was
placed in a 1.5-ml microcentrifuge tube and ground to a powder
in the presence of liquid nitrogen. After evaporation of the liq-
uid nitrogen, a protein isolation buffer (20 mMTricine-NaOH,
pH 8.4, 10 mMEDTA, 450 mMsorbitol, 0.1% bovine serum
albumin) was added, followed by the addition of LiDS-PAGE
solubilization buffer, and the samples were incubated on ice for
at least 15 min. The samples were then centrifuged at 14,000 ⫻
gfor 5 min before loading onto a 12.5–20% gradient polyacryl-
amide gel. PAGE, gel blotting, blocking, primary and secondary
antibody probing, and chemiluminescent peroxidase substrate
were used followed by exposure to x-ray film and photographic
development. An antibody directed against the mature spinach
PsbP was found to cross-react with the PsbP protein from Ara-
bidopsis and was used in these studies. Other antibodies that
were used to investigate the stability of the Photosystem II com-
plex were the kind gifts from many investigators.
Fluorescence Measurements on Leaves—Fluorescence induc-
tion was monitored with a Photon Systems Instruments FL3000
dual modulation kinetic fluorometer (commercial version of
the instrument described in Ref. 22). Both measuring and satu-
rating flashes were provided by computer-controlled photo-
diode arrays. The flash profile exhibited a square shape for low
power measuring flashes and deviated only 5% from an ideal
square shape for saturating actinic flashes. For all of the fluo-
rescence experiments, single leaves from wild-type and PsbP-
suppressed plants grown on sucrose were excised and dark-
incubated for 5 min before initiation of the experiments. In the
standard fluorescence induction experiments (Kautsky experi-
ments), data were collected in a logarithmic time series between
1 ms and 4 s after the onset of strong actinic light. In the flash
fluorescence induction experiments, the kinetics of the rapid
fluorescence rise following a single saturating flash delivered by
light-emitting diodes were examined for 50
s with 1-
s time
resolution in the presence of DCMU. Data were collected at a
frequency of 10 MHz with 12-bit resolution. The proportions of
PS II
␣
and PS II

centers were calculated using proprietary Pho-
ton Systems Instruments software (22). In the fluorescence
decay experiments, the kinetics of the electron transfer
between Q
A
⫺
and Q
B
were examined in the absence of DCMU,
while the recombination reactions of Q
A
⫺
with PS II donor-
PsbP Required for PS II Assembly/Stability
24834 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 282•NUMBER 34• AUGUST 24, 2007
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side components were examined in the presence of DCMU. For
these experiments data were collected between 150
sto60s
following a single saturating flash. Data were analyzed using the
equations outlined previously (23). In this mathematical treat-
ment, three exponential decay components and a long-lived
(
⬎10 s) residual component were included. In the DCMU
treatment experiments, the leaves were immersed in 40
M
DCMU, 0.1% Tween 20 in water for 30 min prior to performing
the fluorescence experiments. Data were analyzed using the
Origin 6.1 software package and proprietary software provided
by Photon Systems Instruments.
Immunological Characterization of Thylakoid Proteins—For
a more in-depth analysis of the protein complement of the thy-
lakoid membranes, chloroplasts were isolated from wild-type
and four mutant plant lines that expressed low levels of PsbP.
These lines, selected from the initial screening, were RNAi-P1,
-P8, and -P11. Leaves were ground in a glass homogenizer with
a chloroplast isolation buffer (300 mMsorbitol, 5 mMMgCl
2
,5
mMEGTA, 5 mMEDTA, 20 mMHEPES/KOH, pH 8.0, 10 mM
NaHCO
3
). The homogenate was then passed through two lay-
ers of Miracloth威(Calbiochem), and the chloroplasts were pel-
leted by centrifugation at 6000 ⫻gfor 5 min. The chloroplasts
were then resuspended in a small amount of isolation buffer,
and the chlorophyll concentration was determined by the
method of Arnon (24). LiDS-PAGE was performed on a 12.5–
20% gradient gel with 3
g of chlorophyll loaded/lane. “West-
ern” blotting, blocking, and probing with primary and second-
ary antibodies were as described above. For detection of the
immobilized antibodies, a chemiluminescent substrate (Super-
Signal威West Pico chemiluminescent substrate, Pierce) was
used, and the x-ray film was exposed to the blots. After devel-
opment, the x-ray films were scanned with a UMax PowerLook
III scanner at 600-d.p.i. resolution and an 8-bit color depth.
RESULTS AND DISCUSSION
Screening of Transgenic Plants—Seeds from wild type and
from plants transformed with the psbP-RNAi construct were
distributed onto agar plates containing Murashige and Skoog
medium supplemented with 50
g/ml each of kanamycin and
carbenicillin. Although wild-type seeds could not germinate
successfully on medium containing these antibiotics, a few anti-
biotic-resistant seedlings were recovered from plants trans-
formed with the RNAi construct. When, however, seeds from
the treated plants were sown onto agar plates containing
Murashige and Skoog medium plus antibiotics plus sucrose,
many more antibiotic-resistant seedlings were recovered (data
not shown). The presence of the psbP-RNAi construct in 10
antibiotic-resistant plants was confirmed by PCR amplification
of the cauliflower mosaic virus 35S promoter region. All of the
plants that exhibited antibiotic resistance also exhibited the
1-kbp PCR amplification product, indicating the presence of
the cauliflower mosaic virus 35S promoter region of the RNAi
construct (data not shown).
To screen individual transgenic plants for the presence of the
PsbP proteins, “Western” blot analysis with a polyclonal anti-
body that recognizes the PsbP proteins was performed. The
results from a typical screening experiment are shown in Fig. 1.
In wild-type plants, two immunoreactive bands were observed
that bound the anti-psbP protein antibody. The nature of the
minor band is unclear at this time, although it may represent a
migrational variant of the PsbP-1 protein. It should be noted
that this minor band is not the PsbP-2 protein, because this
component migrates at a significantly lower apparent molecu-
lar mass (11). No immunoreactive band was detected at the
PsbP-2 location in the RNAi-suppressed plants, which we ana-
lyzed in this communication (data not shown). Most plants
transformed with the psbP-RNAi construct showed either a
partial or complete loss of the PsbP-1 band. In total, 14 plants
were screened for the presence of the PsbP protein. These
results indicated that 28% of the plants had expression levels
similar to that of wild type for the PsbP-1 protein, 14% exhibited
an intermediate level of expression, and 28% of the transgenic
plants exhibited almost complete loss of the PsbP-1 protein.
These results are consistent with the results obtained in other
RNAi studies targeting other proteins. In almost all instances,
different RNAi-containing plant lines exhibit varying degrees
of suppression of the protein targets (20), often allowing the
isolation of a graded phenotypic series of mutants deficient in
the targeted component.
Plants That Express Low Levels of PsbP Cannot Grow
Photoautotrophically—Plants containing psbP-RNAi that were
germinated on Murashige and Skoog medium plus antibiotics
plus sucrose were transplanted onto MS medium plus antibiot-
ics. Prior to transplantation, the plant that lacked PsbP-1 (Fig.
2A,plant P8) was lighter green than the wild-type plant or
plants that accumulated detectable amounts of PsbP-1 (Fig. 2A,
FIGURE 1. Immunological screening for the presence of PsbP proteins in
eleven transgenic plants. Proteins from whole leaf extracts of wild-type
(WT) and eleven transgenic plants (1through 11) were resolved by LiDS-PAGE
followed by “Western” blotting, probing with an anti-PsbP antibody, and sub-
sequent chemiluminescent detection. Individual RNAi-P plants exhibited
variable amounts of the PsbP protein.
FIGURE 2. Growth of wild type and RNAi-P transgenic plants on solid MS
medium. WT, Columbia wild type; P1,P8, and P11 represent RNAi-PsbP1, -P8,
and -P11 transgenic plant lines, respectively. A, growth on sucrose-containing
solid Murashige and Skoog medium; B, photoautotrophic growth on solid
Murashige and Skoog medium without sucrose. Please note that the medium
of the transgenic plants contained kanamycin and carbenicillin in addition to
salts.
PsbP Required for PS II Assembly/Stability
AUGUST 24, 2007•VOLUME 282• NUMBER 34 JOURNAL OF BIOLOGICAL CHEMISTRY 24835
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plants P11 and P1). After transplantation, the plant that lacked
detectable levels of PsbP-1 yellowed and died (Fig. 2B,plant
P8), whereas the other PsbP-deficient plants, which expressed
small but detectable amounts of PsbP, grew somewhat more
slowly than wild type (Fig. 2B). These results indicate that PsbP
is required for photoautotrophic growth in Arabidopsis.
Changes in Fluorescence Induction Caused by Suppressed
Expression of PsbP—Fig. 3Ashows the chlorophyll afluores-
cence rise (Kautsky curves) observed in Arabidopsis wild-
type and in the RNAi-P mutant plants. The normalized fluores-
cence yield is shown. From these curves it is possible to calculate
the quantum yield of variable fluorescence (F
V
/F
M
). F
V
, the vari-
able fluorescence, is equal to F
M
minus F
O
. For any given Kautsky
curve of dark-adapted samples, F
O
is the minimal fluorescence
observed upon onset of illumination
and F
M
is the maximal amount of
fluorescence observed. In Fig. 3A,
F
O
and F
M
correspond to the points
labeled O and P, respectively, for
wild-type. These results are summa-
rized in Table 1. Wild type exhibits a
high quantum yield, whereas the
RNAi-P mutants exhibit progres-
sively lower quantum yields (WT ⬎
P11 ⬎P1 ⬎P8). In Synechocystis
6803, a strong correlation exists (r⫽
0.94) between the quantum yield of
variable fluorescence (F
V
/F
M
) and
the PS II content (25) of wild-type
and mutant strains. Because such a
correlation has never been directly
established in either green algae or
higher plants, we view such an anal-
ysis as being only semiquantitative
when applied to the higher plant
system. With this caveat in mind,
our results indicate that there
appears to be a dramatic decrease in
the quantity of fully functional PS II
reaction centers in the phenotypic
series of PsbP-deficient plants. This
finding is fully consistent with the
results obtained by Ifuku et al. (18)
in tobacco.
Additionally, using a logarithmic
timing series, a polyphasic fluores-
cence rise exhibiting the O-J-I-P
transients was observed for the
wild-type sample (26, 27). An inspection of the curves shown in
Fig. 3Areveals several interesting features. First, the O to J tran-
sition occurs with higher fluorescence yield in the RNAi-P
mutants than in wild type. A phenotypic series is evident, with
RNAi-P11 and -P1 being least affected and RNAi-P8 being
most affected. This initial O-J transition constitutes the photo-
chemical phase of the chlorophyll afluorescence rise. This
result indicates that electron transfer from Q
A
⫺
to Q
B
is slowed
in the mutant. This would result in an increased accumulation
of Q
A
⫺
and consequently an increased fluorescence yield. This
result is similar to, but less extreme than the effects of treatment
with DCMU on the fluorescence induction curve (28). Second,
in wild type the J and I transients appeared at 3 ms and 30 ms,
respectively; however, in the RNAi-P mutants the J transient
occurred earlier (⬃2 ms), and there was a progressive loss of the
I transient throughout the phenotypic series. In wild type the J
to I transition accounted for 30% of the total fluorescence yield.
However, in the RNAi-P plants there is a progressive loss of the
J to I transition, with RNAi-P1 and -P11 being the least affected
and RNAi-P8 being the most affected. Indeed, this latter plant
exhibited essentially no J to I transition. This result may indi-
cate a defect in the oxygen-evolving complex. A similar
decrease in the magnitude of the J to I transition has been
observed in treatments that damage the oxygen-evolving com-
FIGURE 3. Chlorophyll fluorescence of wild type and PsbP-deficient plants. A, the standard fluorescence
induction rise following continuous illumination (Kautsky curves). The location of the O, J, I, and P transitions for
wild type are indicated. B, the fluorescence rise following a single saturating flash. Note different time scales for
Aand B.Cand Dillustrate the fluorescence decay following a single saturating flash in the absence and
presence of 40
MDCMU, respectively. f, wild type; ⽧, RNAi-P11; orange dot, RNAi-P1; green triangle, RNAi-P8.
n⫽2– 6, error bars,⫾1.0 S.D.; in some instances the error bars are smaller than the symbols.
TABLE 1
Fluorescence induction characteristics of wild type and the RNAi-P
mutants
Strain F
V
/F
M
PS II
␣
PS II

␣
/

%
Wild type 0.78 ⫾0.01
a
56 ⫾645⫾6 1.2
RNAi-P11 0.68 ⫾0.04
b
48 ⫾252⫾2 0.9
RNAi-P1 0.61 ⫾0.03
b
55 ⫾33 45 ⫾33 1.2
RNAi-P8 0.42 ⫾0.15
b
43 ⫾29 56 ⫾29 0.8
a
Values given are means ⫾1.0 S.D., n⫽3–6.
b
p⬍0.05 as compared with wild type using Student’s t-test.
PsbP Required for PS II Assembly/Stability
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plex, such as mild Tris or heat treatment (29), and in plants that
possess only the defective PsbO-2 protein in Arabidopsis (30).
Alternatively, the absence of the J to I transition may indicate a
defect in the ability to form the Q
A
⫺
Q
B
⫺
state (27). It should be
noted that these two explanations are not mutually exclusive,
and both could, in principal, contribute to the loss of the J to I
transition in the mutants. In both wild type and the RNAi-P
mutants, the fluorescence signal started to rise at 30–40 ms,
with all strains reaching the P level in 200–300 ms. Overall, the
total fluorescence yield of the thermal phase (J to P transition) is
progressively reduced (wild type ⬎P11 ⫽P1 ⬎P8) in the
RNAi-P mutants.
Flash Fluorescence Induction—Fig. 3Bshows the flash-in-
duced fluorescence induction at a 1-
s time resolution in the
absence of DCMU. The observed fluorescence rise is the result
of the re-reduction by the oxygen-evolving complex of Y
Z
,
which is in equilibrium with the primary electron donor P
680
⫹
(40). The shape of this fluorescence rise contains information
bearing on the amount of PS II
␣
and PS II

reaction centers
present in the sample. Exponential fluorescence rise kinetics
indicate that the antennae of individual PS II centers are not
coupled (i.e. a characteristic of PS II

centers), while sigmoidal
fluorescence rise kinetics indicate a high degree of interconnec-
tivity between the antennae of PS II centers (i.e. a characteristic
of PS II
␣
centers) (41). This is true under conditions of both
continuous relatively weak light illumination (slow fluores-
cence induction) and under single saturating flash conditions
(fast fluorescence induction) as demonstrated by Nedbal et al.
(22). This experiment allows the determination of the relative
proportions of PS II
␣
and PS II

reaction centers present (22)
(Table 1). In addition to the differences noted above, PS II

centers have a smaller antenna size, are enriched in chlorophyll
a, and are depleted of the light-harvesting chlorophyll proteins.
They appear to be principally located in the stromal thylakoid
membranes (31) and at the grana margins (32). Additionally, PS
II

centers appear to be defective in their ability to transfer
electrons from Q
A
⫺
to Q
B
(33). In wild type, we find the ratio of
PS II
␣
to PS II

to be 1.2. This value is similar to those obtained
in other studies (34–36). In the PsbP mutants we find that the
PS II
␣
to PS II

ratio is slightly decreased, although none of the
values observed for the mutants was statistically significant at
the p⫽0.05 level. The trend toward a slightly higher propor-
tion of PS II

centers in the RNAi-P mutants may indicate a
slightly increased rate of metabolic turnover of PS II in the
mutant, as conditions that increase photoinactivation also lead
to increases in the amount of PS II

centers observed (33). This
is in marked contrast to a mutant that contains only the PsbO-2
protein. In this mutant, loss of PsbO-1 leads to a dramatic
increase in the number of PS II

centers present (30).
Analysis of the Q
A
⫺
Decay Kinetics in Leaves of PsbP-deficient
Plants—Additional information concerning the electron trans-
fer characteristics on both the reducing and oxidizing side of
the photosystem was obtained by the examination of Q
A
⫺
reoxidation kinetics in either the absence (Fig. 3C) or presence
of DCMU (Fig. 3D), respectively. In the absence of DCMU, the
fluorescence decay after a single saturating flash can be resolved
into three exponential components and a residual component
with a
⬎10 s (23). The fastest (and dominant) exponential
decay component that we observed for wild type is related to
the electron transfer from Q
A
⫺
to Q
B
(280
s, 67%) (Table 2).
The middle exponential decay component (7.3 ms, 18%) is asso-
ciated with electron transfer from Q
A
⫺
to Q
B
in reaction cen-
ters that have to bind plastoquinone to the Q
B
site before Q
A
⫺
oxidation can occur. The slowest decay component (1.6 s, 7%) is
related to a charge recombination reaction in which the reoxi-
dation of Q
A
⫺
occurs with donor-side components. Finally, a
residual fraction of the fluorescence yield (around 5%) is very
long-lived and may result from the equilibrium between Q
A
⫺
and Q
B
(37). For the RNAi-P mutants, the time constant for the
fast phase increased significantly throughout the phenotypic
series while the amplitude of the fast component decreased.
This indicates that electron transport from Q
A
⫺
to Q
B
is slowed
in the mutant. The time constant for the middle exponential
decay component and its amplitude were very similar in wild
type and the RNAi-P mutants. Additionally, the time constant
for the slow phase significantly decreased throughout the phe-
notypic series while its amplitude increased. Finally, the resid-
ual amplitude (for the decay component(s) with
⬎10 s) also
increased significantly, perhaps indicating a change in the
Q
A
⫺
Q
B
equilibrium in favor of Q
A
⫺
. Overall, these results
indicate that the principal modification on the reducing side
of the photosystem observed in the mutants was a significant
slowing of electron transfer between Q
A
⫺
and Q
B
and an
increased rate of charge recombination between Q
A
⫺
and
oxidizing side component(s).
TABLE 2
Q
A
ⴚ
reoxidation kinetics of wild type and RNAi-P mutants in the absence and presence of DCMU
Strain Fast phase Middle phase Slow phase Residual
Amplitude
Amplitude
Amplitude
ms %ms% s % %
⫺DCMU
a
Wild type 0.28 ⫾0.02
b
67.5 ⫾1.4 7.3 ⫾3.7 18 ⫾2.0 1.57 ⫾0.39 7.4 ⫾1.4 5.0 ⫾1.0
RNAi-P11 0.28 ⫾0.05 62.8 ⫾4.2 6.2 ⫾3.9 18.9 ⫾1.6 1.28 ⫾0.87 10.5 ⫾2.7 5.9 ⫾0.8
RNAi-P1 0.45 ⫾0.09
c
52.8 ⫾3.2
c
7.9 ⫾3.7 21.8 ⫾2.4 0.93 ⫾0.23
c
13.1 ⫾1.3
c
10.2 ⫾1.2
c
RNAi-P8 0.47 ⫾0.05
c
46.0 ⫾5.5
c
7.0 ⫾2.8 21.1 ⫾2.3 0.85 ⫾0.29
c
18.9 ⫾3.6
c
11.2 ⫾0.6
c
⫹DCMU
a
Wild type 156 ⫾32 18.9 ⫾6.7 612 ⫾126 48.9 ⫾4.5 2.41 ⫾0.57 30.5 ⫾9.3 1.1 ⫾1.3
RNAi-P11 2.8 ⫾0.8
c
1.9 ⫾0.9
c
330 ⫾39
c
42.8 ⫾8.2 1.50 ⫾0.23 53.4 ⫾5.9
c
1.8 ⫾1.5
RNAi-P1 20.6 ⫾34.8
c
3.7 ⫾1.8
c
537 ⫾164 29.6 ⫾7.8
c
2.81 ⫾1.15 47.1 ⫾4.7
c
19.0 ⫾8.0
c
RNAi-P8 2.8 ⫾1.0
c
8.0 ⫾7.1 437 ⫾53 24.3 ⫾6.6
c
3.98 ⫾0.28
c
30.2 ⫾16.5 36.6 ⫾2.5
c
a
The data were fit to a model containing three exponential decaying components plus a residual long-lived (
⬎10 s) component (23). The curve fits in the absence of DCMU
exhibited average X
2
/DoF values between 0.00017 and 0.00022 for the different strains, whereas in the presence of DCMU the average X
2
/DoF values were between 0.00001
and 0.00059.
b
Values given are means ⫾1.0 S.D., n⫽2–6.
c
p⬍0.05 as compared with wild type using Student’s t-test.
PsbP Required for PS II Assembly/Stability
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In the presence of DCMU, which prevents electron transfer
from Q
A
⫺
to Q
B
, the decay of fluorescence following a saturat-
ing flash is dominated by charge recombination between Q
A
⫺
and the oxidizing-side components of the photosystem. Fig. 3D
illustrates the fluorescence decay kinetics of wild type and the
RNAi-P mutants in the presence of 40
MDCMU. The
observed fluorescence decay curves were fit to the same model
described above (38). Significant alterations are observed in the
mutants. It should be noted that alternative models containing
two exponential decay components and a hyperbolic compo-
nent (38), or only two exponential decay components, failed to
adequately fit the data. Table 2 shows the kinetic parameters
obtained for the fluorescence decay in the presence of DCMU.
The fastest decaying component observed in wild type exhib-
ited a time constant of 156 ms and is attributed to a fraction of
PS II reaction centers that lack a functional manganese cluster
(39), in which Q
A
⫺
recombines with oxidized Y
Z
. The slowest
decay component observed for wild type exhibited a time con-
stant of 2.4 s and is attributed to charge recombination between
Q
A
⫺
and the S
2
and, possibly, the S
3
states (40). The origin of
the intermediate decay component observed in wild type (612
ms) is unclear, although it may represent a sub-fraction of reac-
tion centers in which the charge recombination rate between
Q
A
⫺
and the S
2
state is 4- to 5-fold faster than the 2.4-s com-
ponent (23). Finally, a small proportion (⬃1%) of wild-type
reaction centers exhibited a time constant of ⬎10 s. It should be
noted that, in the presence of DCMU, which displaces Q
B
,no
equilibrium between Q
A
⫺
and Q
B
exists. In this case the resid-
ual amplitude is the result of slow charge recombination
between Q
A
⫺
and oxidizing-side components. In the mutant
RNAi-P8, the slowest decay component was observed to have a
time constant of nearly 4 s. This result indicates that the S
2
and
possibly the S
3
states are more stable in this mutant than in wild
type. Little change was observed for the time constant of the
intermediate decay component for the RNAi-P mutants,
although the amplitude generally decreased throughout the
phenotypic series. The amplitude of the residual decay compo-
nent (
⬎10 s) increased dramatically throughout the pheno-
typic series, becoming the dominant decay component in
RNAi-P8. This result indicates that a major disruption in the
water-oxidizing complex has occurred in the RNAi-P mutants
and that charge recombination with Q
A
⫺
and oxidizing-side
components is dramatically slowed. A large difference was also
observed for the rapidly decaying component. The time con-
stant for this decay component decreased from 156 ms in wild
type to 3 ms in RNAi-P8, and its amplitude dropped from 18.9%
to 8%. The origin of these changes is unclear at this time; how-
ever, a similar change was observed in an Arabidopsis mutant
that lacked the PsbO-1 protein and exhibited similar dramatic
changes in a variety of fluorescence induction and decay
parameters (30).
In an earlier study, the effects of PsbP and PsbQ depletion on
the kinetic properties of isolated PS II membrane preparations
were reported (54). In this study, the extrinsic proteins were
removed from spinach membranes by treatment with 2 MNaCl,
which elicited changes in both the oxidizing side and reducing
side of the photosystem. These investigators observed a gradual
loss in the coupling between the oxygen-evolving complex and
Y
Z
䡠, which occurred in the light and which could be reversed by
the addition of CaCl
2
or reconstitution with the PsbP and PsbQ
proteins. They also observed changes that could not be reversed
by these additions. These include a slowing of the S
3
f(S
4
)f
S
0
transition, changes in the Kok parameters and loss of the
period two oscillations associated with the two electron gating
mechanism for quinone reduction. The authors attributed
these changes to irreversible alterations in PS II engendered by
the high NaCl concentrations used in their studies. At this time
we cannot comment on their observed alterations in the rate of
the S state transition or changes in the Kok parameters. How-
ever, our observation that the loss of PsbP (and, consequently,
PsbQ, see below) leads to alterations in Q
A
⫺
to Q
B
electron
transfer may indicate that their earlier observation was not an
artifact. Overall, the results that we have obtained in vivo are
consistent with the in vitro results reported in Ref. 54.
Suppressed Expression of PsbP Leads to Alterations of the
Thylakoid Protein Complement—To determine whether
decreased expression of PsbP led to a loss of other PS II com-
ponents, immunological analysis using chemiluminescent
detection was performed. The relative amounts of selected PS II
and control proteins present were analyzed in chloroplast prep-
arations of wild-type Arabidopsis and the transgenic plants in
the phenotypic series (Fig. 4). Four proteins that are present in
the intrinsic core of PS II (CP47, CP43, D1, and D2) and the
three extrinsic proteins (PsbO, PsbP, and PsbQ) were exam-
ined. In this particular gel, the PsbP protein was not detected in
the RNAi-P1 and P8 lanes. In other immunoquantification
experiments, when compared with wild-type plants, RNAi-P11
exhibited 5–10% of the PsbP protein while RNAi-P1 exhibited
barely detectable, and RNAi-P8 exhibited no detectable
amounts of this protein. The expression of the extrinsic PsbO
and PsbQ extrinsic proteins was also assessed. In wild-type and
PsbP-deficient plants, two immunoreactive bands were
observed that bound the anti-PsbO antibody. These were pre-
viously identified to be PsbO-1 and PsbO-2 (17). The PsbO
components were only modestly affected by the loss of PsbP. A
markedly different pattern was observed for the PsbQ protein,
because PsbP-deficient plants exhibited a complete loss of this
extrinsic component. Even in the RNAi-P11 plant, in which
PsbP was only partially suppressed, PsbQ could not be detected.
This result is consistent with the earlier tobacco study (18). It is
unclear at this time whether the loss of the PsbQ protein in
Arabidopsis was due to decreased synthesis or increased degra-
dation of this component due to weak binding to PSII. It should
be noted, however, that large pools of unassembled mature
PsbQ protein can exist in the thylakoid lumen without being
degraded either in the presence (41, 42) or in the absence (43,
44) of assembled and functional PS II reaction centers. Conse-
quently, it is possible that the loss of the PsbQ protein may be
the direct result of the loss of expression of PsbP and not a
consequence of the reduced levels of PS II reaction centers
resulting from decreased PsbP expression. Indeed, the RNAi-
P11 plant exhibits a complete loss of the PsbQ protein even
though the amount of PS II reaction centers is near normal. The
previous studies performed in tobacco indicated that RNAi
suppression of the PsbP protein led to a dramatic decrease in
PsbQ, whereas accumulation of PS II core proteins and PsbO
PsbP Required for PS II Assembly/Stability
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was similar to that of wild type (18). An earlier report on a
PsbP-deficient Chlamydomonas mutant showed that it synthe-
sized and accumulated the PSII core proteins and extrinsic
PsbO and PsbQ proteins with no visible differences from wild
type (43, 45).
Two of the intrinsic PS II components examined, D2 and
CP47, also exhibited significant losses upon the loss of PsbP
expression, whereas the D1 and CP43 proteins were more mod-
estly affected (Fig. 4). This result differs substantially from the
earlier tobacco study (18) in which PsbP-suppressed plants
showed a wild type-like accumulation of all of the PS II core
proteins examined. It should be noted that, in our study, small
amounts of accumulated PsbP (for instance, in RNAi-P11) sup-
ported near wild-type levels of the CP47 and D2 proteins. In the
tobacco study, the authors state that small amounts (⬃5% of
wild type) of the PsbP proteins were present (18). This small
amount, approximately equivalent to plant RNAi-P11 in our
study, appears sufficient to maintain the number of assembled
PS II reaction centers in near normal amounts. This assertion is
supported by the near wild-type fluorescence characteristics
exhibited by RNAi-P11. In plant RNAi-P8 we were not able to
detect any PsbP protein. Under these conditions the loss of the
D2 and CP47 components, and consequently fully assembled
PS II core complexes, is exacerbated. It is clear that the absence
of the PsbP protein leads to a profound alteration in the PS II
protein complement. These results indicate that the absence of the
PsbP protein leads to the loss of the assembly/stability of PS II.
In addition to these PS II proteins, three control proteins
were also examined. As expected, the amount of cytochrome f,
a component of the cytochrome b
6
/fcomplex, was not affected
by decreased expression of the PsbP protein. The PsaB protein,
a core component of PS I, was somewhat decreased in the
RNAi-P1 and P8 plants. It should be noted that this could not be
the result of a general loss of thylakoid membranes, because
such a situation would also lead to a loss of the cytochrome b
6
/f
complex. The mechanism for this phenomenon is not well
established but is similar to the effects observed in other studies
in which the steady-state amounts of PS I reaction centers were
decreased by loss of PS II components (46). Similar results were
obtained for the nuclear mutant hcf136, which is deficient in the
HCF136 protein and does not assemble PS II reaction centers.
The loss of PS II reaction centers apparently led to decreased
accumulation of PS I but not the cytochrome b
6
/fcomplex.
These plants also accumulate lower steady-state amounts of
RuBp carboxylase.
Earlier, it had been (41, 42) demonstrated that there was a
large pool of unassembled PS II extrinsic proteins comprising
20–50% of the total extrinsic proteins present in the thylakoid
lumen and that these proteins were competent in binding to the
photosystem. They suggested that this unbound pool partici-
pated in the maintenance of homeostasis with respect to turn-
over and assembly of PS II. Our observation that a lower level of
PsbP in PsbP-deficient plants led to the loss of PS II reaction
center proteins suggests that maintenance of this unassembled
pool is essential for the normal expression levels of PS II reaction
center proteins, supporting the hypothesis of Hashimoto et al.
(42).
In an earlier study, we examined the effect of RNAi suppres-
sion of the expression of the PsbO-1 and PsbO-2 proteins in
Arabidopsis (17). In that study, we determined that loss of these
PsbO proteins also led to a marked decrease in the number of
functional PS II reaction centers and a loss of photoautotrophic
growth. Because, at least in vitro, it appears that the PsbO pro-
tein is required for binding of the PsbP component (47– 49), it is
formally possible that the detrimental effects we observed for
the PsbO protein were, in fact, due to the loss of the association
of PsbP with the photosystem. Several observations, however,
appear to indicate that this is not the case. First, the loss of PsbO
leads to a marked decrease in the amounts of the CP43 intrinsic
component (17). In contrast, RNAi suppression of PsbP led to a
substantial loss of the CP47 component with the CP43 protein
FIGURE 4. Immunological analysis of selected thylakoid membrane pro-
teins of wild-type and RNAi-P transgenic plants. Wild-type (WT) plants and
RNAi-P plants (P11,P1, and P8) were examined by LiDS-PAGE followed by
“Western” blotting, probing with various antibodies, and subsequent chemi-
luminescence detection. Individual proteins are labeled to the right. All
immunoblots for a given protein were performed on the same gel; the order,
however, was rearranged to reflect the phenotypic series. Please note that,
although on this gel no PsbP protein is visible in the P1 lane, the protein is
detected on gels that were overloaded. No PsbP protein was ever detected in
the P8 plant.
PsbP Required for PS II Assembly/Stability
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being modestly affected. These differential patterns of protein
loss in plants lacking PsbO and PsbP may indicate different
roles for these components in the stability/assembly of the pho-
tosystem. Second, in the case of the phenotypic series resulting
from RNAi suppression of PsbO expression, incremental loss of
the PsbO protein was paralleled by the incremental loss of PS II
functionality and PS II assembly/stability (17). In the case of the
phenotypic series resulting from RNAi suppression of PsbP,
which we report here, even very small amounts of PsbP could
support near normal levels of PS II function and accumulation
(for example, the phenotype of RNAi-P11). In this regard, it is
interesting to note that, in cyanobacteria, a PsbP homologue
is necessary to maintain fully functional PS II even though it is
present in substoichiometric amounts (50). Finally, we have
recently demonstrated that the Arabidopsis mutant psbo1,
which lacks the major PsbO isoform PsbO-1, accumulates large
quantities of PS II

reaction centers (30) exhibiting a PS II
␣
to
PS II

ratio of 0.3. In PsbP-suppressed plants, which we have
examined in this communication, no significant change in the
PS II
␣
to PS II

ratio was observed. In light of these observa-
tions, we conclude that the detrimental effects observed on PS
II function and assembly/stability observed upon RNAi sup-
pression of the PsbO protein are not due directly to the con-
comitant loss of PsbP from the photosystem.
It should be noted that the pattern of PS II core protein loss in
both the RNAi-suppressed PsbP plants and the RNAi-sup-
pressed PsbO plants (17) is quite interesting. Structural studies
from cyanobacteria (51, 52) indicate that the D1 protein asso-
ciates closely with CP43 and that the D2 protein associates
closely with CP47. Additionally, the cyanobacterial PsbO pro-
tein appears to interact primarily with CP43 (although CP47
also contains binding determinants for PsbO (53)). Although
there are certainly differences between cyanobacteria and
higher plant PS II (8), these structural studies on cyanobacteria
provide an initial framework for our understanding of the
structure of the higher plant photosystem. Our observation,
that loss of PsbO in higher plants leads primarily to the loss of
CP43 (17) while the loss of PsbP (this study) leads primarily to
the loss of CP47, may indicate specific roles for these two
extrinsic proteins in maintaining the assembly/stability charac-
teristics of these intrinsic membrane components.
CONCLUSIONS
Our results show that the use of RNAi methodology has
proven a useful tool in probing the effects of a reduction of PsbP
in Arabidopsis. Decreased amounts of PsbP led to a loss of pho-
toautotrophic growth, the progressive loss of variable fluores-
cence and a marked decrease in the fluorescence quantum yield
(F
V
/F
M
), fewer functional PS II reaction centers, and a signifi-
cant loss of PS II reaction center components, with D2 and
CP47 being strongly affected. We conclude that the PsbP pro-
tein is required for PS II core complex assembly/stability and
photoautotrophic growth in Arabidopsis.
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PsbP Required for PS II Assembly/Stability
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