From the Department of Biology, Plant
Physiology and Molecular Biology, University of Turku,
FIN-20014 Turku, Finland and the § Department of Plant
Sciences, University of Cambridge, Downing Street,
Cambridge CB2 3EA, United Kingdom
Received for publication, February 8, 2001, and in revised form, March 14, 2001
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ABSTRACT |
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The ycf9 (orf62)
gene of the plastid genome encodes a 6.6-kDa protein (ORF62) of
thylakoid membranes. To elucidate the role of the ORF62 protein, the
coding region of the gene was disrupted with an aadA
cassette, yielding mutant plants that were nearly (more than 95%)
homoplasmic for ycf9 inactivation. The
ycf9 mutant had no altered phenotype under standard
growth conditions, but its growth rate was severely reduced under
suboptimal irradiances. On the other hand, it was less susceptible to
photodamage than the wild type. ycf9 inactivation
resulted in a clear reduction in protein amounts of CP26, the NAD(P)H
dehydrogenase complex, and the plastid terminal oxidase. Furthermore,
depletion of ORF62 led to a faster flow of electrons to photosystem I
without a change in the maximum electron transfer capacity of
photosystem II. Despite the reduction of CP26 in the mutant thylakoids,
no differences in PSII oxygen evolution rates were evident even at low
light intensities. On the other hand, the ycf9
mutant presented deficiencies in the capacity for PSII-independent
electron transport (ferredoxin-dependent cyclic electron
transport and NAD(P)H dehydrogenase-mediated plastoquinone reduction).
Altogether, it is shown that depletion of ORF62 leads to anomalies in
the photosynthetic electron transfer chain and in the regulation of
electron partitioning among the different routes of electron transport.
The plastid genome of a number of organisms has been sequenced to
completion. Comparison of the plastome sequence among lower and higher
plants has revealed the existence of highly conserved open reading
frames (1), which have been named ycfs
(hypothetical chloroplast frames
(2)). Elucidation of the function of these genes has been enormously
facilitated by the development of efficient plastid transformation
techniques in the unicellular alga Chlamydomonas reinhardtii
(3) and the higher plant tobacco (4-5). This, together with the
homologous recombination system of plastids, allows the targeted
manipulation of the plastome. Indeed, the reverse genetics approach has
been successfully used for unraveling the function of some of these
conserved open reading frames (6-9).
The ycf9 gene is co-transcribed with psbD
and psbC, encoding the photosystem II
(PSII)1 proteins D2 and CP43,
respectively (10-12). ycf9, found in all green plant
plastid genomes sequenced so far, as well as in the chromosome of
Synechocystis sp. PCC 6803, is very highly conserved (13).
It encodes a small 6.6-kDa hydrophobic protein (ORF62), which was
recently reported to co-purify with the light harvesting complex of
PSII (LHCII) (14).
Our earlier attempts to knockout the ycf9 gene in
tobacco by biolistic chloroplast transformation yielded only largely
heteroplasmic plants. Although the transformed plants did not show any
apparent phenotypic differences compared with the wild type (WT)
plants, it was suggested that the lack of homoplasmicity might reflect an important and maybe indispensable role of the protein (13). Further
purification of the mutant lines to a 95% homoplasmic state was,
however, achieved by further regeneration cycles of the heteroplasmic
tissue in spectinomycin and ultimately through the formation of seeds.
The ycf9 mutant plants have a phenotype indistinguishable from the WT under standard growth conditions, but
their growth is severely impaired in lower light intensities. On the
other hand, the ycf9 mutants are less susceptible to
photodamage than the WT plants.
We report here that inactivation of ycf9 in tobacco
plants leads to an accelerated flux of electrons to photosystem I (PSI) without a parallel change in the maximum electron transfer capacity of
PSII. Furthermore, depletion of ORF62 causes deficiencies in the
PSII-independent routes of electron transfer
(ferredoxin-dependent cyclic electron transfer and NAD(P)H
dehydrogenase-mediated plastoquinone reduction; for a review see Ref.
15) and in the terminal oxidase (PTOX) (16-17) pathway, which might be
of importance for plant growth under suboptimal light conditions.
Altogether, the results presented suggest a role for ORF62 in
regulatory processes responsible for fine-tuning of photosynthetic
electron transfer according to the prevailing environmental conditions.
Chloroplast Transformation of Tobacco and Genetic Analysis of
ycf9 Inactivation Mutants--
Inactivation of
ycf9 in tobacco (Nicotiana tabacum var.
Samsung) by chloroplast transformation was done as described before (13) by inserting an aadA cassette conferring resistance to spectinomycin 19 base pairs downstream from the start of the
ycf9 coding region. Plant regeneration was carried
out on spectinomycin (500 µg/ml), after which the plants were
transferred to soil, and seeds were collected. Seeds were germinated on
peat compost, and plants were grown at 25 °C at a photosynthetic
photon flux density of 150 µmol photons m
Total leaf DNA was isolated according to the hexadecyltrimethylammonium
bromide extraction procedure of Rogers and Bendich (18), with slight
modifications. Estimation of the proportion of transformed to
non-transformed plastome copies was done by standard DNA gel blot
analysis (19) by digesting DNA (8 µg) with HincII and
NcoI and using a DNA probe against the
ycf9 coding region. The fragment used for the
synthesis of the probe was amplified with the following pair of
primers: forward, 5'-GAC TCT TGC TTT CCA ATT G-3' (annealing with
plastome nucleotides 37596-37614), and reverse, 5'-CAA GAG ATG AGA GAA
TTA AG-3' (annealing with plastome nucleotides 37761-37781). For PCR
analysis of WT and transformed plants primers flanking the region of
aadA insertion were used: forward, 5'-TAC GAA TAA AGT GCG
AAA GG-3' (annealing with plastome nucleotides 37425-37444), and
reverse, 5'-CAT CAG GAG AAG CAA ATA CAA- 3' (annealing with plastome
nucleotides 37667-37687). The PCR program was as follows: 1 cycle
(95 °C 4 min), 30 cycles (95 °C 30 s, 56 °C 30 s,
72 °C 2 min), and 1 cycle (72 °C 10 min).
For all experiments mutant plants of the F1 generation were used. For
some experiments (growth at low light, examination of the thylakoid
polypeptide content, and phosphorylation status of PSII proteins) the
number of individual mutant plants screened ranged from 10 to 20, in
order to rule out segregation of the mutant phenotypes.
For examination of growth rates in lower light conditions, seeds were
germinated as described above. After 3 weeks, WT and transformed
plantlets of similar size were transferred to new pots, and the light
intensity was reduced (20 µmol photons m Protein Analysis and Enzyme Activity Assays--
Thylakoid
isolation, SDS-PAGE, and immunodetection were performed as described
earlier (20). The gels were loaded on a chlorophyll (chl) basis (if not
otherwise stated, 15 or 1 µg of chl per well for studies of the
NAD(P)H dehydrogenase (NDH) complex and PTOX or other thylakoid
membrane complex proteins, respectively). For silver staining of
polypeptides (21), thylakoid proteins were separated on
Tricine/SDS-PAGE gels (22). Polyclonal antibodies against the DE loop
of D1 and D2 were purchased from Research Genetics, Inc. Other
antibodies were kindly provided as follows: LHCB2 and CP26 by Dr. S. Jansson (Sweden), NdhH by Dr. G. Peltier (France), coupling
factor 1 by Dr. T. Hundal (Sweden), cytochrome f by Dr.
F.-A. Wollman (France), PSI by Dr. R. Barbato (Italy), and PTOX by Dr.
M. Kuntz (France).
NDH activity was solubilized from thylakoids as described (23).
Thylakoids corresponding to 40 µg of chl (~180 µg of protein) were solubilized with 2% Triton X-100 for 30 min on ice and
centrifuged for 45 min at 105,000 × g. The supernatant
was diluted 2-fold with running buffer and ran in a linear 3-10% (2%
bisacrylamide) native PAGE at 4 °C (24). NDH activity was detected
by incubating gel slices for 30 min at 30 °C in darkness in 50 mM potassium phosphate, pH 8.0, 1 mM
Na2EDTA, 0.2 mM NADH, and 0.5 mg/ml nitro blue tetrazolium.
For determination of ascorbate peroxidase (APX) activity, three leaf
discs (3 cm diameter) were ground on liquid nitrogen and 3 ml of
extraction buffer (0.1 M Tricine-KOH, pH 8.0, 1 mM dithiothreitol, 10 mM MgCl2, 50 mM KCl, 1 mM EDTA, 0.1% Triton X-100) were
added to the resulting fine powder. The extract was filtered through
Miracloth and was immediately assayed for peroxidase activity. 10, 20, or 30 µl of filtered extract were added to 1 ml of reaction buffer
(125 µM ascorbate, 0.1 mM
H2O2, 1 mM EDTA, 0.1 M
Hepes-KOH, pH 7.8), and enzyme activity was recorded as changes in
absorbance at 265 nm. Three leaves were assayed for both the WT and the mutant.
Chlorophyll a Fluorescence Measurements--
The ratio of
variable to maximum fluorescence
(Fv/Fmax) of intact leaves
was measured with a pulse amplitude modulation fluorometer (PAM 101;
Walz GmbH, Effeltrich, Germany). A non-actinic 1.6-kHz measuring beam
alone was used to measure the initial F0, and
maximum fluorescence, Fmax, was induced with a
2-s pulse of white light (5000 µmol photons
m
For measurements of fluorescence induction kinetics in
3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-poisoned thylakoids, a
500-µl thylakoid sample was used (20 µg of chl/ml in 5 mM NH4Cl, 0.33 M sorbitol, 40 mM Hepes-KOH, pH 7.6, 5 mM NaCl, 5 mM MgCl2, 1 M glycine betaine, 1 mM KH2PO4). After a dark adaptation
period of 5 min, DCMU was added (20 µM), and the sample
was illuminated (90 µmol photons m
Fluorescence emission spectra were measured at 77 K with a diode
array spectrophotometer (S2000, Ocean Optics, Dunedin, FL) equipped with a reflectance probe as described (26). Fluorescence was
excited with light below 500 nm (defined with LS500S and LS700S filters, Corion Corp., Holliston, MA, placed in front of a slide projector), and the emission was recorded between 600 and 780 nm. A
100-µl thylakoid sample (10 µg of chl/ml in 0.1 M
sorbitol, 10 mM Hepes, pH 7.4, 5 mM NaCl, and
10 mM MgCl2) was used.
Measurement of PSII and Whole Electron Transfer Chain
Activities--
PSII and whole electron transfer chain activities of
thylakoids were measured with a Hansatech oxygen electrode at 20 °C. PSII activity was measured as oxygen evolution in a reaction mixture (1 ml) consisting of 5 mM NH4Cl, 0.33 M sorbitol, 40 mM Hepes-KOH, pH 7.6, 5 mM NaCl, 5 mM MgCl2, 1 M glycine betaine, 1 mM
KH2PO4, and thylakoids equivalent to 10 µg of
chl. 2,6-Dichloro-p-benzoquinone (0.25 mM) was used as an electron acceptor. Whole electron
transfer chain activity was recorded as net oxygen consumption using
methyl viologen (MV) as an electron acceptor in 1 ml of buffer
consisting of 40 mM sodium phosphate, pH 7.4, 1 mM NaCl, 0.6 mM NaN3, 0.12 mM MV, 5 mM NH4Cl.
Determination of the Redox State of P700--
The redox state of
P700 (reaction center chlorophyll of PSI) in isolated thylakoids was
determined from the absorbance of P700+ (oxidized P700) at
810 nm, using OD860 as a reference. Absorbance changes were monitored using an ED-P700DW unit attached to the PAM 101 fluorometer. Measurements were done under anaerobic conditions in a
temperature-regulated cuvette (25 °C) containing 0.5 ml of buffer
(50 mM Tricine, pH 7.5, 5 mM MgCl2,
6 mM glucose, 2 mM NH4Cl, 400 units/ml catalase, 50 µM ferredoxin (Fd)) (27). The mixture was thoroughly flushed with nitrogen, and 2 units of glucose oxidase and thylakoids (25 µg chl) were added. An initial
illumination period (1000 µmol photons m
For determination of the amount of oxidizable P700, leaves were kept in
darkness for 1 h on moist paper prior to measurements. Ten
separate measurements were done from each leaf, and three leaves were
used from both WT and transformed plants. The leaf was placed on a
silver plate, and the light guide of the PAM-101 fluorometer was placed
to a constant distance from the leaf (~5 mm). The measurements
consisted of a 12-s dark period (during which the system was calibrated
with the aid of the ED-P700DW unit) followed by illumination with
far-red LED (PAM-102-FR; Walz, GmbH, Effeltrich, Germany). The values
were normalized by dividing with the chl content per leaf area,
measured according to Ref. 28.
High Light Treatment of Plants--
For studies of light
tolerance of PSII, detached leaves from 6- to 8-week-old plants were
floated on a water bath and exposed to 1500 µmol photons
m Phosphorylation of PSII Core Polypeptides and LHCII--
For
determining the phosphorylation level of D1, D2, and CP43 according to
changing light conditions, leaves were detached from the plants, the
petioles were immersed in water, and the leaves were kept in darkness
overnight to induce maximal dephosphorylation of the proteins (dark).
Leaf disc samples (3 cm diameter) were thereafter transferred for
3 h to 50 (low light, LL), 150 (growth light, GL), or 850 µmol
photons m Inactivation of the Chloroplast ycf9
Gene--
Homoplasmicity for ycf9 inactivation was
assayed by DNA gel blot analysis of the F1 generation of two
independently transformed lines (G and F) regenerated at different
stages of the selection process (G3, three regeneration cycles, F5 and
F6, five and six regeneration cycles, respectively). As can be seen
from Fig. 1B (ycf9 probe), no WT copies could be detected with DNA
gel blot analysis in the mutant lines, suggesting that all of them had reached homoplasmicity. The presence of the aadA cassette in
the mutant copies was further confirmed by using a probe against the aadA sequence (Fig. 1B, aadA probe).
Importantly, this probe detected only one fragment, the expected
2650-base pair restriction fragment of the mutant plastid DNA (Fig.
1A), indicating that no additional mutations
(e.g. in the nuclear genome) had been caused by random insertion of the aadA cassette.
Since the DNA gel blot technique might fail to detect very small
amounts of non-transformed plastid DNA, we also performed PCR analysis
of the WT and transformed plants (Fig. 1C). This analysis
revealed the presence of a residual pool of WT plastid DNA copies in
the mutants which, as judged from a dilution series of template DNA,
constituted less than 5% of the total plastome population.
Chloroplast transformation of tobacco for insertional inactivation of
ycf9 yielded in our previous work only largely
heteroplasmic plants (50% of the plastid DNA molecules contained an
insert) (13). Here, we obtained nearly homoplasmic (95%)
ycf9 inactivation mutants by further regeneration
cycles on spectinomycin and ultimately by the formation of seeds.
Nevertheless, none of the mutant lines reached full homoplasmicity, a
result that might still be indicative of an essential role of the ORF62 protein.
Growth of the ycf9 Mutants Under Different Light
Conditions--
ycf9 mutants (G3, F5, and F6) did
not exhibit an altered phenotype under normal growth conditions (GL,
150 µmol photons m Thylakoid Protein Composition--
Analysis of thylakoid
polypeptide composition by silver staining of polyacrylamide gels
revealed the absence of a small polypeptide in the
ycf9 mutants (Fig.
3A), in line with a recent
report by Ruf and co-workers (14) on a ycf9
inactivation mutant. These authors concluded that this protein band,
which co-purified with isolated LHCII complexes, corresponded to ORF62,
based on the specific absence of a 6.581-kDa mass peak (the theoretical
molecular mass of ORF62) in the mutant LHCII fractions, as revealed by
matrix-assisted laser desorption ionization/time of flight
analysis.
Protein blot analysis showed that the amounts of major protein subunits
of PSII, PSI, and their antenna, as well as the cytochrome bf complex, and the ATP synthase remained unchanged
following inactivation of ycf9 (Fig. 3B).
The ycf9 inactivation mutants, however, were
specifically depleted of a 26-kDa protein (Fig. 3A), which
corresponded to the CP26 component of the inner PSII antenna (Fig.
3B).
In addition, the mutants had clearly reduced amounts of the NdhH
polypeptide, a subunit of the NDH complex (Fig. 3B). The NDH
complex utilizes stromal NAD(P)H for reduction of plastoquinone (PQ)
and therefore has been hypothesized to be involved in PSII-independent electron flow around PSI (30). Since to this point all the mutant lines
exhibited the same properties, we chose one of them, F6, for most of
the further experiments.
Importantly, immunodetection of the recently discovered PTOX
(16-17, 31-32) revealed a considerable reduction in the content of
the enzyme in the thylakoids of the ycf9 mutant (Fig.
3B).
Electron Transfer Properties--
To confirm the depletion of NDH
complexes, the enzyme in its active state was solubilized with Triton
X-100 from thylakoids of WT and F6, subjected to native PAGE, and the
in-gel enzyme activity was determined. This assay showed a clear
decline (3-fold) in NDH activity in the thylakoids of the mutant (Fig.
3C).
To investigate whether the other route of PSII-independent electron
transfer (recycling of electrons from reduced Fd back to PQ) was
likewise affected in the mutant, we determined P700+
reduction rates in thylakoid samples in
the presence of DCMU and reduced Fd (Table I and Fig.
4A). DCMU blocks the flow of electrons from PSII, and therefore P700+ is mainly reduced
through Fd-dependent cyclic electron flow around PSI (the
NDH-mediated pathway does not function in isolated thylakoids in the
absence of added NAD(P)H, and PSI recombination reactions do not
interfere in the presence of Fd, see Ref. 27). Under such conditions
the reduction rate of P700+ was clearly slower in the
mutant than in the WT (t1/2 0.34 and 0.23 s,
respectively, Table I and Fig. 4A), pointing to a
disturbance in the cyclic flow of electrons from Fd to PQ in the
ycf9 mutant.
Thylakoids were also illuminated in the absence of DCMU, when PSII is
the main source of electrons for reducing P700+. Under such
conditions, the ycf9 mutant showed a faster
P700+ reduction than the WT (t1/2
0.09 s versus 0.16 s, Table I; Fig.
4B), suggesting an increased flow of electrons to
P700+ in the absence of ORF62.
Since a simple explanation for the faster P700+ reduction
of the mutant would be a decreased amount of functional PSI reaction centers (RC), the functionality of PSI was also examined. The amount of
oxidizable P700 was determined by measuring the maximum change in
OD810 obtained by illuminating intact dark-adapted leaves with far-red light. This assay showed (WT, 0.25 ± 0.01, F6,
0.23 ± 0.01, arbitrary units) that there were no differences in
the amount of functional PSI complexes between the WT and the mutant.
PSII oxygen evolution rates were measured from thylakoids of GL- and
LL-grown plants under a range of light intensities (Table II). PSII oxygen evolution was similar in
WT and F6, suggesting that the accelerated post-illumination reduction
of P700+ (Fig. 4B) was not caused by a more
efficient PSII-dependent PQ reduction in the mutant.
Importantly, no differences in PSII activities between the WT and F6
were observed either when the measurements were done with
non-saturating light intensities. Instead, the oxygen evolution rates
of WT and F6 decreased in parallel upon lowering of light intensities,
arguing against differences in energy transfer properties from the
antenna to the PSII RC. Most importantly, despite the fact that the
maximum electron transfer capacity of PSII was similar in F6 and WT,
the rate of whole chain electron transfer was faster in the mutant
(Table II), indicating an accelerated flow of electrons to PSI.
Size and Function of the LHCII Antenna--
Recently, it was
suggested that the ycf9 mutants are deficient in the
transfer of energy to the PSII RC due to absence of the CP26 minor
antenna protein (14). This suboptimal antenna function, in turn, was
proposed to be the cause for reduced growth under limiting light
conditions. We determined the amount of CP26 in the plants grown at 20 µmol photons m
Possible differences in the efficiency of energy transfer from the
LHCII antenna to PSII were examined by measuring fluorescence induction
kinetics in the presence of DCMU in WT and F6 thylakoids from plants
grown in both GL and LL. It must be noted that in the presence of DCMU
the kinetics of fluorescence rise is determined by antenna size and not
by other components downstream of PSII. As can be seen in Fig.
5B, no differences were found in the kinetics of
fluorescence rise between WT and F6 thylakoids. The faster fluorescence
rise in both WT and F6 thylakoids from LL-grown plants as compared with
GL-grown plants indicates that an acclimation to LL by an increase in
the PSII antenna size occurs similarly in the WT and the
ycf9 mutant.
77 K fluorescence emission spectra from isolated thylakoids of
WT and F6 (Fig. 6) revealed no
differences in the positions or relative heights of the PSII (CP43 and
CP47 at 685 and 695 nm, respectively) or PSI (735 nm) emission peaks.
Thus, the mutant did not appear to differ from the WT in the amounts of
the photosystems and their antennas. The difference in the relative
heights of PSII and PSI peaks between GL and LL plants was obvious for
both the WT and F6 (Fig. 6) and reflects acclimation of the
photosynthetic machinery to the prevailing growth irradiance, shade and
low light plants having a lower PSII/PSI ratio (33).
High Light Tolerance and Phosphorylation of Thylakoid
Proteins--
Susceptibility of the mutant to photoinhibition of PSII
was investigated by recording changes in
Fv/Fmax (variable/maximum fluorescence) during exposure of plants to high light. As can be seen
in Fig. 7A, photoinhibition of
WT tobacco proceeded more rapidly than that of F6. In order to rule out
factors involved in the PSII repair cycle (e.g. D1 repair
rate), plastid translation was inhibited with lincomycin during a 16-h
dark adaptation period, and the control (in water) and the
lincomycin-treated plants were subsequently exposed to high light (Fig.
7B). The difference in Fv/Fmax values between the WT
and F6 was also evident in the lincomycin-treated plants, indicating
that the slower photoinhibition kinetics of the mutant was not due to a
more efficient PSII repair machinery.
Next, the phosphorylation of PSII core polypeptides was examined, the
level of which has been shown to respond to the redox state of the PQ
pool (34-35). For this purpose, dark-adapted leaf discs of both WT and
mutant plants were exposed to different light intensities, and changes
in the phosphorylation level of PSII core polypeptides were determined.
Fig. 8 shows that phosphorylation of PSII
core polypeptides increased with increasing light intensities in the
ycf9 mutant, indicating that the kinase was active
and responded to oscillations in the PQ redox state in a similar manner as in the WT. However, in F6 the phosphorylation level of these proteins was consistently lower than in the WT, suggesting a greater oxidation of PQ in the mutant under all light conditions
studied.
Ascorbate Peroxidase Activity--
Results from measurements of
the rate of P700+ reduction (Fig. 4B and Table
I) and whole chain electron transfer (Table II) indicated a faster flux
of electrons to PSI. This was not transduced, however, into a more
rapid carbon fixation, as can be deduced from the similar growth rate
of the WT and F6 under standard conditions (Fig. 2A). It is
known that in the presence of excess reductant (reduced Fd), the
electron transport system may divert electrons to molecular oxygen
instead of NADP+ (Mehler reaction) (36), at a rate
proportional to the concentration of reduced Fd (37). Diversion of
electrons to oxygen leads to a rapid formation of superoxide radicals,
which have to be metabolized by the detoxification system of
chloroplasts. In this system superoxide dismutase and APX work in chain
to ultimately convert superoxide into water and monodehydroascorbate
(38).
We determined APX activities from total leaf extracts and found that
ascorbate degradation was faster in the mutant than in the WT (5.3 ± 0.5 and 3.9 ± 0.1 mmol of ascorbate oxidized
h Depletion of ORF62 Leads to Anomalies in Photosynthetic Electron
Transfer--
Comparison of the electron transfer properties of
thylakoids of WT and the ycf9 inactivation mutant
revealed a higher rate of whole chain electron transfer in the latter
(Table II). This was not accompanied, however, by an increase in the
electron transfer capacity of PSII, which was similar in WT and F6
under a wide range of measuring light intensities (Table II). Based on
this and the finding that the amount of functional PSI RCs remained unchanged in the mutant, we conclude that the function of some intermediate component of the electron transfer chain between PSII and
PSI is modified upon severe depletion of ORF62. In line with this
conclusion is the finding that the post-illumination reduction of
P700+ was faster in the thylakoids of the mutant in the
absence of DCMU (Fig. 4B). It is important to emphasize that
these results are not caused by artifacts generated during measurements
in vitro, since a similar difference with respect to the WT
has been obtained for cyanobacterial ycf9
inactivation mutants using intact cells instead of isolated thylakoids
in the experiments (unpublished results).
Considering the accelerated whole chain electron transfer in F6, the
next question is how the faster linear electron flow is utilized in the
mutant. In chloroplasts there are three main sinks for PSI electrons as
follows: (i) the Calvin-Benson cycle, (ii) cyclic electron flow around
PSI, and (iii) the Mehler reaction. Similar growth rates of WT and F6
in normal light (Fig. 2A) indicate that the accelerated flow
of electrons to PSI is not transduced into a faster carbon fixation in
the Calvin-Benson cycle. Neither are electrons recycled back from Fd to
PQ, as can be deduced from the deficiency in Fd-dependent
cyclic electron transfer in F6 (Fig. 4A). Another possible
route for the return of electrons to PQ is the NDH complex. This
complex reduces PQ using stromal NAD(P)H as a substrate (40) and has
been hypothesized to be involved both in the electron flow around PSI
in the light and in a putative respiratory chain in the chloroplast
("chlororespiration," see Refs. 17, 30, 41, and 42) resembling the
one occurring in cyanobacteria. However, the reduction in NdhH
protein levels (Fig. 3B) and the decline in NDH activity
(Fig. 3C) of the mutant argue against this alternative.
Instead, by taking into account the increased APX activity of the
mutant as an indicator of increased O2 reduction through the Mehler reaction, it seems that electrons leak from the system and
react with O2 to form superoxide. This harmful species is then metabolized by the concerted action of superoxide dismutase and
APX (Mehler-peroxidase reaction; see Ref. 38). Stimulation of APX
activity has been reported in maize leaves, in which excess reductant
was produced due to restrictions in CO2 assimilation upon
chilling (43).
Considering all the different steps of photosynthetic electron
transport affected in the ycf9 mutant, one putative
component whose function might be modified upon depletion of ORF62 is
the cytochrome bf complex. Oxidation of plastoquinol by the
cytochrome bf complex is considered to be the rate-limiting
step in photosynthetic electron transport (44-45), and thus, it might
be hypothesized that this control step is altered in plants nearly
deprived of ORF62. Furthermore, it has been reported that conditions
favoring cyclic electron flow induce migration of the cytochrome
bf complex from the grana to the stroma-exposed membranes
(46), thereby raising the possibility that this migration could be
involved in the triggering of cyclic electron flow. The deficiencies in cyclic electron transport of the ycf9 mutant might
then be related to a modification of the function or migration of the
cytochrome bf complex. Finally, the cytochrome bf
complex is distributed over the grana appressions and the
stroma-exposed thylakoids (47), and accordingly, it is conceivable that
ORF62 could interact with the cytochrome bf complex in the
grana. Based on results by Ruf et al. (14), ORF62
co-purifies with the LHCII antenna, and therefore it is expected to be
located mainly in the grana stacks. We have previously reported the
localization of ORF62 in the stroma-exposed membranes on the basis of
the specific immunodetection of a 6.5-kDa protein in this thylakoid
fraction (13). However, testing of the antiserum with the homoplasmic
mutant plants revealed that our earlier conclusion on the location of
ORF62 did not hold.
Another alternative is that ORF62 interacts with a component
independent of the cytochrome bf complex. In line with this
is the effect of ycf9 inactivation on the NDH complex
(Fig. 3, B and C), cyclic electron flow (Fig.
4B, Table I) and PTOX (Fig. 3B), all affecting
directly the PQ pool. The presence in thylakoid membranes of a terminal
oxidase (PTOX) that catalyzes oxidation of plastoquinol and reduction
of oxygen to water was recently reported (16-17), but so far the role
of this enzyme in photosynthesis is unknown. It is considered that
electron flow from plastoquinol to O2 is marginal in normal
light conditions (17), and indeed decreased PTOX amounts (Fig.
3B) did not affect the growth of the ycf9
mutant under optimal light conditions (Fig. 2). However, one could
argue that PTOX might have a still unknown role, which might affect the
overall photosynthetic rate and be of importance for acclimation to
suboptimal light intensities.
ycf9 Mutants Tolerate High Light Better Than the Wild
Type--
A faster flow of electrons to PSI without changes in the
maximum electron transfer capacity of PSII can be expected to result in
oxidation of the PQ pool. This, in combination with a stimulation of
the scavenger system of thylakoids, might be beneficial under high
light stress (48-49). Our photoinhibition experiments showed that
indeed the ycf9 mutant was less susceptible to
photoinhibition than the WT (Fig. 7A). Importantly, this
capacity to cope better with higher irradiances was not due to a more
efficient PSII repair machinery since the differences in high light
tolerance remained after pretreatment of leaves with lincomycin (Fig.
7B). In addition to the photoinhibition experiments, the
lower phosphorylation level of PSII proteins in the
ycf9 mutant (Fig. 8) is also in good agreement with a
considerable oxidation of PQ, since the level of PSII phosphorylation
is known to respond to PQ reduction (34, 35). Notably, PSII
phosphorylation in the mutant was lower also in darkness (Fig. 8). In
the dark there is predicted to be no pumping of electrons through the
NDH complex in the mutant, and thus PQ remains fully oxidized. In the
case of the WT, however, one could expect that the observed residual
PSII phosphorylation results from NDH activity.
Reduced Growth of ycf9 Mutants in Low Light Is Not Due to
Deficiencies in Antenna Function--
The ycf9
inactivation mutants presented a WT-like phenotype under standard
growth conditions (Fig. 2A). However, a clear difference in
size became apparent when the plants were grown under low light intensities (Fig. 2B). Recently, it was proposed that the
reduced growth rate of the tobacco ycf9 mutants in LL
was due to inefficient energy transfer to the PSII RC, caused by the
lack of the CP26 antenna protein (14). Our results, however, do not
support this view, although the CP26 protein amounts are indeed
strongly reduced in the mutant under normal light conditions (Fig. 3,
A and B). In contrast, under LL, where the
slow-growth phenotype becomes apparent, CP26 does clearly accumulate in
the mutant (Figs. 2 and 5A). Moreover, the similar kinetics
of fluorescence induction (Fig. 5B) rules out the
possibility of deficiencies in energy transduction to the RC. Further
evidence comes from experiments with CP26 antisense
Arabidopsis plants (50). These plants do not exhibit reduced
growth in LL and have wild type PSII activities under a wide range of
measuring light intensities. Moreover, ycf9 mutants
of Synechocystis likewise show slower growth rates than the
WT under low irradiances,2 despite the fact that the
light-harvesting system of cyanobacteria, the phycobilisome antenna,
differs considerably from its eukaryotic counterpart and has no
subunits homologous to CP26.
Adaptation of plants to low light intensities is accompanied by
rearrangements in the photosynthetic machinery, which result in a
reduction of the PSII/PSI ratio (33). This is considered to set the
photosynthetic apparatus for cyclic electron flow in order to adjust
the ATP/NADPH ratio (51-52). 77 K fluorescence emission spectra
showed that the ycf9 mutant was able to adjust the
PSII/PSI ratio in LL in a similar manner as the WT (Fig. 6). Nevertheless, its growth in LL was clearly retarded compared with the
WT (Fig. 2). Cyanobacterial psaE (essential for
Fd-dependent cyclic electron flow) and ndhF (NDH
subunit) mutants (53-54) have been reported to exhibit decreased
growth under low light conditions. This has been interpreted as a
failure to produce sufficient ATP in such conditions due to
deficiencies in either of the two PSII-independent electron transfer
pathways. It is therefore conceivable that the defects detected in both
the NDH-mediated (Fig. 3, B and C) and the
Fd-dependent electron transfer routes (Fig. 4A,
Table I) in the ycf9 mutant are at least partly
responsible for the impairment of growth under limiting light conditions.
ORF62, a Possible Role in the Regulation of Photosynthetic Electron
Transfer--
Photosynthetic organisms have a capacity to regulate
photosynthetic electron transfer in order to respond efficiently to
changes in environmental conditions. Adjustments in the function of the photosynthetic machinery are required to coordinate the synthesis of
ATP and NADPH with the rate of their consumption, and thereby to
optimize the use of excitation energy and to avoid damage to the
photosystems (for reviews see Refs. 33, 55, and 56). Optimization of
the use of limited energy and adjustment of the synthesis of
metabolites to their rate of use in the Calvin cycle is achieved in
part by a redistribution of energy between PSII-dependent and PSII-independent electron transfer routes (15, 57, 58). Despite
intense study, it is not known how the balance between the activities
of the photosystems is established and what is the molecular mechanism
that triggers the shift between the different modes of electron transfer.
On the basis of our results, ORF62 is involved in this fine-tuning of
photosynthesis. In plants deprived of ORF62, the partitioning between
PSII-dependent and PSII-independent electron flow is
impaired. Furthermore, without affecting the maximum electron transfer
capacity of PSII, depletion of ORF62 leads to a faster flow of
electrons to PSI, probably resulting in the stimulation of
O2 reduction in the Mehler reaction. It is tempting to
propose a model in which ORF62 could be part of an "electron gate"
which, according to the physiological needs of the cell, would divert
electrons to the different electron transport pathways. Accurate
assignment of ORF62 to a specific thylakoid protein complex will be
essential for development of hypothesis of how this might be
accomplished at the molecular level. Obviously, further work is needed
for unraveling the complex regulatory networks that coordinate the function of the thylakoid protein complexes and the exact molecular role of ORF62 in these processes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2
s
1 with a 16-h light/8-h dark rhythm.
2
s
1).
2 s
1). Each
Fv/Fmax measurement was
preceded by a 1-h dark adaptation period. The fluorometer was
controlled, and the data were digitized with the FIP software (QA-Data
OY, Turku, Finland).
2
s
1) through a Corning 4-96 and a Balzers K-1
filter (center wavelength 450 nm). The fluorescence signal was recorded
with a home-built fluorometer at 685 nm (25).
2
s
1) of 30 s was applied to reduce Fd.
After that, the samples were kept in darkness for 10 s, during
which DCMU was added (final concentration of 10 µM)
through a small hole on the side of the cuvette. Thereafter, 30 cycles
of actinic light (1.2 s) and darkness (8.8 s) were applied, and the
average post-illumination change in the P700+ signal of the
30 repetitions was resolved into the sum of two exponentials. The
results reported consider only the fast component of the signal.
2 s
1 for 3 h.
Small leaf discs (2 cm diameter) were cut from the leaves at specific
time points for fluorescence measurements. For some experiments the
leaves were detached, and the petioles were immersed either in water
(control) or in 2 mM lincomycin for 16 h in darkness to inhibit plastid translation. The leaves were thereafter exposed to
850 µmol photons m
2
s
1 for 1 h.
Fv/Fmax was determined prior
to the beginning of the light treatment to ensure that the lincomycin
treatment in darkness did not affect the photochemical efficiency of
PSII.
2 s
1 (high
light, HL). Thylakoid samples corresponding to 1 µg of chl per lane
were ran in SDS-PAGE, and thylakoid phosphoproteins were immunodetected
with a rabbit polyclonal phosphothreonine antibody (New England
Biolabs) as described (29).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (23K):
[in a new window]
Fig. 1.
Construction of plastid
ycf9 inactivation mutants. A,
restriction map of the plasmids used for chloroplast transformation and
the corresponding wild type (WT) region. The hybridization
probe is indicated as a solid bar under the
ycf9 gene. B, DNA gel blot analysis of WT
tobacco and three mutant lines using the probe indicated in
A (ycf9 probe) or a probe against the
aadA cassette (aadA probe). G and
F are two independently transformed lines; the
numbers designate the number of regeneration rounds of each
line. Total DNA (8 µg) was cut with NcoI and
HincII. All the three lines had the aadA cassette
inserted in the opposite orientation to the ycf9
gene. C, PCR analysis of the WT and transformed plants with
primers flanking the region of insertion of the aadA
cassette. bp, base pair.
2
s
1, Fig.
2A). However, when the light
intensity in the growth chamber was lowered to 20 µmol photons
m
2 s
1 (LL), the
growth of the mutants was clearly retarded as compared with the WT
(Fig. 2B).
View larger version (55K):
[in a new window]
Fig. 2.
WT and ycf9 mutant
tobacco plants grown under standard and low light intensities.
A, plants grown at 150 µmol photons
m 2 s
1 for 6 weeks.
B, plants grown at 20 µmol photons
m
2 s
1 for 6 weeks.
View larger version (56K):
[in a new window]
Fig. 3.
Analysis of thylakoid membrane proteins from
WT and ycf9 mutants (G3, F5, and F6).
Silver-stained PAGE (A) (2 µg chl/lane; the
asterisks denote protein bands missing in the mutant lines)
and immunoblots (B) (1 µg chl per lane, except for
NdhH and PTOX, 15 µg) demonstrate the protein composition of
thylakoid membranes. G and F are two
independently transformed lines; the numbers designate the number of
regeneration cycles of each line. C, zymogram comparing NDH
activities in WT and F6. NDH activity was solubilized from thylakoids
corresponding to 40 µg of chl (~180 µg of protein). Activity
assays were done after native PAGE as described under "Experimental
Procedures." Equal loading of the samples in the zymogram is shown by
immunoblotting with a PSI antibody.
Rate constants and corresponding half-times of the fast phase of
P700+ reduction in WT and F6 thylakoids
View larger version (13K):
[in a new window]
Fig. 4.
Dark re-reduction of P700+ in
isolated thylakoids of WT and ycf9 inactivation
mutant (F6) in anaerobic conditions in the presence of Fd.
A, re-reduction of P700+ in WT and F6 in the
presence of 10 µM DCMU. B, re-reduction of
P700+ in WT and F6 in the absence of DCMU.
P700+ reduction was measured by monitoring changes in the
absorbance at 810 nm in the dark after light-induced reduction of Fd.
Each curve is an average of 30 repetitions.
Electron transfer activities (PSII, electron transfer from H2O
to dichloro-p-benzoquinone; whole chain, electron transfer from
H2O to MV) of thylakoids from wild type (WT) and ycf9
knockout plants (F6) grown at 150 µE (GL) or 20 µE (LL)
2
s
1 (LL) and found that, in contrast to the
situation in growth light (150 µmol photons
m
2 s
1, GL), the
ycf9 mutants were able to accumulate CP26 under low light intensities (Fig. 5A),
where the slow-growth phenotype becomes apparent (Fig. 2).
Interestingly, the amount of CP26 was also increased in LL in the WT,
in a similar manner as the phosphorylation level of LHCII polypeptides
(Fig. 5A).
View larger version (22K):
[in a new window]
Fig. 5.
Analysis of antenna function in thylakoids of
WT and ycf9 mutant (F6) plants.
A, comparison of CP26 amounts and LHCII phosphorylation
levels in WT and F6 from plants grown at 150 µmol photons
m 2 s
1 (GL) and at
20 µmol photons m
2
s
1 (LL). Upper panels,
silver-stained PAGE and immunoblot with a CP26 antibody. Lower
panels, immunoblots with a phosphothreonine antibody recognizing
the phosphorylated form of LHCII (P-LHCII) and with a LHCB2
antibody (LHCII). B, fluorescence induction
kinetics of WT and F6 plants grown in GL and LL conditions. Chlorophyll
a fluorescence was measured at 685 nm from isolated
thylakoids in the presence of 20 µM DCMU. Each
curve is an average of three independent measurements.
View larger version (21K):
[in a new window]
Fig. 6.
Chlorophyll fluorescence emission spectra of
WT and ycf9 mutant (F6) at 77 K. The upper and lower
panels correspond to thylakoids of plants grown at standard (GL)
and LL conditions, respectively.
View larger version (12K):
[in a new window]
Fig. 7.
Photoinhibition kinetics of WT and
ycf9 mutant (F6) plants. A,
photoinhibition kinetics during a 3-h exposure to 1500 µmol photons
m 2 s
1. Each point
represents the mean of two independent experiments (3 leaves measured
in each experiment). Standard errors are drawn if bigger than the
symbols. B, changes in
Fv/Fmax induced by 1-h
treatment at 850 µmol photons m
2
s
1. Before the light treatment, the petioles
were kept immersed either in water (WT, F6) or in a 2 mM lincomycin solution (WT+, F6+) overnight in
the dark. Open bars,
Fv/Fmax before exposure to
high light; solid bars,
Fv/Fmax after 1 h of
high light treatment. Each bar is the mean of two independent
experiments (3 leaves measured in each experiment).
View larger version (55K):
[in a new window]
Fig. 8.
Phosphorylation level of PSII core
polypeptides in thylakoids isolated from differentially light-treated
WT and ycf9 mutant (F6) leaves. Thylakoids
were isolated from dark-adapted leaves (D), and from leaf
discs illuminated for 3 h at 50 (LL), 150 (GL), and 850 µmol
photons m 2 s
1 (high
light, HL). The gel was loaded on a chl basis (1 µg per
lane). Immunodetection of the phosphoproteins (P-D1, P-D2,
and P-CP43) was performed with a phosphothreonine antibody
(upper panel), and immunodetection of D1 (as a control of
equal loading) was performed with a D1-specific polyclonal antibody
(lower panel).
1 (mg chl)
1,
respectively). Chloroplast APX constitutes around 80% of the total APX
content of the cell (39), and therefore measurement of APX activity
from total leaf extracts mainly reflects the activity of the
chloroplastic enzyme.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
---|
We thank Dr. S. Jansson, Dr. G. Peltier, Dr. T. Hundal, Dr. F.-A. Wollman, and Dr. R. Barbato for the generous gift of antibodies. We thank Dr. M. Kuntz and E.-M. Josse for help with the PTOX immunoblots. We also thank M. Keränen for valuable help with biophysical measurements and computer analysis and Dr. S. Jansson and Dr. H. V. Scheller for helpful discussions.
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FOOTNOTES |
---|
* This work was supported by grants from the Academy of Finland (to E.-M. A.), the Emil Aaltonen foundation (to E. B.-G.), The Royal Society, London, UK, and Robinson College, Cambridge, UK (to P. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Plant Physiology and Molecular Biology, Dept. of Biology, University of Turku, FIN-20014 Turku, Finland. Tel.: 358-2-333-5931; Fax: 358-2-333-5549; E-mail: evaaro@utu.fi.
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M101255200
2 ?. Ulas et al., manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
PSII, photosystem
II;
LHC, light harvesting complex;
WT, wild type;
PSI, photosystem I;
PTOX, plastid terminal oxidase;
chl, chlorophyll;
NDH, NAD(P)H-dehydrogenase complex;
APX, ascorbate peroxidase;
DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea;
MV, methyl viologen;
P700, reaction center chlorophyll of PSI;
P700+, oxidized P700;
Fd, ferredoxin;
PQ, plastoquinone;
RC, reaction center;
µE, µmol
photons m2 s
1;
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel electrophoresis;
LL, low light;
GL, growth light.
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