THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 276, No. 23, Issue of June 8, pp. 20795–20802, 2001
Printed in U.S.A.
Abnormal Regulation of Photosynthetic Electron Transport in a
Chloroplast ycf9 Inactivation Mutant*
Received for publication, February 8, 2001, and in revised form, March 14, 2001
Published, JBC Papers in Press, March 20, 2001, DOI 10.1074/jbc.M101255200
Elena Baena-González‡, John C. Gray§, Esa Tyystjärvi‡, Eva-Mari Aro‡¶, and Pirkko Mäenpää‡
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
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 dehydrogenasemediated 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).
* 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. This 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, FIN20014 Turku, Finland. Tel.: 358-2-333-5931; Fax: 358-2-333-5549; Email: [email protected].
This paper is available on line at http://www.jbc.org
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.
EXPERIMENTAL PROCEDURES
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
1
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)-1,1dimethylurea; MV, methyl viologen; P700, reaction center chlorophyll
of PSI; P700⫹, oxidized P700; Fd, ferredoxin; PQ, plastoquinone; RC,
reaction center; ␮E, ␮mol photons m⫺2 s⫺1; PCR, polymerase chain
reaction; PAGE, polyacrylamide gel electrophoresis; LL, low light; GL,
growth light.
20795
20796
Inactivation of ycf9 Alters Photosynthetic Electron Transport
coding region. Plant regeneration was carried out on spectinomycin
(500 ␮g/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 ␮mol
photons m⫺2 s⫺1 with a 16-h light/8-h dark rhythm.
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 ␮g) 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 ␮mol photons m⫺2 s⫺1).
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 ␮g 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 ␮g of chl (⬃180 ␮g 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 ␮l of
filtered extract were added to 1 ml of reaction buffer (125 ␮M 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 ␮mol photons m⫺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).
For measurements of fluorescence induction kinetics in 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-poisoned thylakoids, a 500-␮l thylakoid sample was used (20 ␮g 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 ␮M), and the sample was illuminated (90 ␮mol photons
m⫺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).
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-␮l thylakoid
sample (10 ␮g 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 ␮g 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 ␮M ferredoxin (Fd)) (27). The mixture was thoroughly flushed with nitrogen, and
2 units of glucose oxidase and thylakoids (25 ␮g chl) were added. An
initial illumination period (1000 ␮mol photons m⫺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 ␮M)
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.
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 ␮mol photons m⫺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 ␮mol 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.
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 ␮mol photons m⫺2 s⫺1
(high light, HL). Thylakoid samples corresponding to 1 ␮g 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
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
Inactivation of ycf9 Alters Photosynthetic Electron Transport
20797
FIG. 2. WT and ycf9 mutant tobacco plants grown under standard and low light intensities. A, plants grown at 150 ␮mol photons
⫺2 ⫺1
m s for 6 weeks. B, plants grown at 20 ␮mol photons m⫺2 s⫺1 for 6
weeks.
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 ␮g) 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.
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 ␮mol photons m⫺2 s⫺1, Fig. 2A). However, when the light intensity in the
growth chamber was lowered to 20 ␮mol photons m⫺2 s⫺1 (LL),
the growth of the mutants was clearly retarded as compared
with the WT (Fig. 2B).
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 26kDa 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
20798
Inactivation of ycf9 Alters Photosynthetic Electron Transport
TABLE I
Rate constants and corresponding half-times of the fast phase of
P700⫹ reduction in WT and F6 thylakoids
P700⫹ reduction was measured in the presence of 50 ␮M Fd by
monitoring changes in the absorbance of P700⫹ at 810 nm in the dark
after light-induced reduction of Fd. The rate constants were calculated
by fitting the average curve of 30 repetitions into the sum of two
exponentials (only the faster component is shown, which represented
57– 89% of the reduction of P700⫹).
Rate constant
WT
F6
WT DCMU
F6 DCMU
t1⁄2
s⫺1
s
4.2 ⫾ 0.36
7.5 ⫾ 0.92
3.0 ⫾ 0.12
2.0 ⫾ 0.15
0.16
0.09
0.23
0.34
FIG. 3. Analysis of thylakoid membrane proteins from WT and
ycf9 mutants (G3, F5, and F6). Silver-stained PAGE (A) (2 ␮g chl/
lane; the asterisks denote protein bands missing in the mutant lines)
and immunoblots (B) (1 ␮g chl per lane, except for NdhH and PTOX, 15
␮g) 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 ␮g of chl (⬃180 ␮g 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.
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.
FIG. 4. Dark re-reduction of P700ⴙ 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 ␮M 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.
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
Inactivation of ycf9 Alters Photosynthetic Electron Transport
20799
TABLE II
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 ␮E (GL) or 20 ␮E (LL)
Activities were measured at saturating light and at 1000, 500, 100, or 25 ␮E. Values are given in ␮mol of O2 evolved (PSII measurements) or
consumed (WC measurements) per mg chl per h. The numbers in parentheses are the percentage values with regard to the maximum activity of
the sample at saturating light.
WT GL
F6 GL
WT LL
F6 LL
PSII,
saturating
PSII, 1000 ␮E
PSII, 500 ␮E
PSII, 100 ␮E
PSII, 25 ␮E
Whole chain,
saturating
582 (100%)
561 (100%)
384 (100%)
392 (100%)
478 (82%)
448 (80%)
345 (90%)
323 (82%)
297 (51%)
272 (48%)
241 (63%)
211 (53%)
94 (16%)
86 (15%)
82 (21%)
81 (21%)
27 (5%)
26 (5%)
25 (6%)
22 (5%)
278
329
86
109
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 ␮mol photons m⫺2 s⫺1 (LL) and found
that, in contrast to the situation in growth light (150 ␮mol
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).
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 lincomy-
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 ␮mol
photons m⫺2 s⫺1 (GL) and at 20 ␮mol 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 ␮M
DCMU. Each curve is an average of three independent measurements.
cin 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, darkadapted 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
20800
Inactivation of ycf9 Alters Photosynthetic Electron Transport
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 ␮mol photons m⫺2 s⫺1 (high light, HL). The gel was loaded on
a chl basis (1 ␮g 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).
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.
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⫺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
FIG. 7. Photoinhibition kinetics of WT and ycf9 mutant (F6)
plants. A, photoinhibition kinetics during a 3-h exposure to 1500 ␮mol
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 ␮mol 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).
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
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
Inactivation of ycf9 Alters Photosynthetic Electron Transport
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 stromaexposed 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
20801
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
20802
Inactivation of ycf9 Alters Photosynthetic Electron Transport
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 PSIIindependent 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 finetuning 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.
Acknowledgments—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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