Special Issue – Regular Paper
Acclimation of Tobacco Leaves to High Light Intensity Drives
the Plastoquinone Oxidation System—Relationship Among
the Fraction of Open PSII Centers, Non-Photochemical
Quenching of Chl Fluorescence and the Maximum
Quantum Yield of PSII in the Dark
Chikahiro Miyake1,*, Katsumi Amako2, Naomasa Shiraishi1 and Toshio Sugimoto1
1Department
of Biological and Environmental Science, Faculty of Agriculture, Graduate School of Agricultural Science, Kobe University,
1-1 Rokkodai, Nada, Kobe, 657-8501 Japan
2Faculty of Nutrition, Kobegakuin University, Japan
Responses of the reduction–oxidation level of plastoquinone (PQ) in the photosynthetic electron transport
(PET) system of chloroplasts to growth light intensity were
evaluated in tobacco plants. Plants grown in low light
(150 µmol photons m–2 s–1) (LL plants) were exposed to a
high light intensity (1,100 µmol photons m–2 s–1) for 1 d.
Subsequently, the plants exposed to high light (LH plants)
were returned back again to the low light condition: these
plants were designated as LHL plants. Both LH and LHL
plants showed higher values of non-photochemical
quenching of Chl fluorescence (NPQ) and the fraction of
open PSII centers (qL), and lower values of the maximum
quantum yield of PSII in the dark (Fv/Fm), compared with
LL plants. The dependence of qL on the quantum yield of
PSII [Φ(PSII)] in LH and LHL plants was higher than that in
LL plants. To evaluate the effect of an increase in NPQ and
decrease in Fv/Fm on qL, we derived an equation expressing
qL in relation to both NPQ and Fv/Fm, according to the
lake model of photoexcitation of the PSII reaction center.
As a result, the heat dissipation process, shown as NPQ,
did not contribute greatly to the increase in qL. On
the other hand, decreased Fv/Fm did contribute to the
increase in qL, i.e. the enhanced oxidation of PQ under
photosynthesis-limited conditions. Thylakoid membranes
isolated from LH plants, having high qL, showed a higher
tolerance against photoinhibition of PSII, compared with
those from LL plants. We propose a ‘plastoquinone
oxidation system (POS)’, which keeps PQ in an oxidized
state by suppressing the accumulation of electrons in the
PET system in such a way as to regulate the maximum
quantum yield of PSII.
Keywords: Acclimation • Fraction of open PSII reaction
centers (qL) • High light stress • Non-photochemical
quenching (NPQ) • Photosynthesis • Plastoquinone.
Abbreviations: CEF-PSI, cyclic electron flow around PSI;
HDP, heat dissipation process; NPQ, non-photochemical
quenching of Chl fluorescence; PCO, photorespiratory
carbon oxidation; PCR, photosynthetic carbon reduction;
PFD, photon flux density; LEF, linear electron flow; LHC,
light-harvesting complex; MV, methyl viologen; PET,
photosynthetic electron transport; POS, plastoquinone
oxidation system; qL, photochemical quenching of Chl
fluorescence; PQ, plastoquinone; ROS, reactive oxygen
species; WWC, water–water cycle.
Introduction
Photon energy drives photosynthesis required for the growth
of plants. In higher plants, the photon energy absorbed by
light-harvesting Chl in both PSI and PSII of chloroplast thylakoid membranes excites each reaction center and drives
photosynthetic electron transport (PET). These photochemical reactions produce NADPH at the reducing side of PSI,
and ATP through ATP synthase. In C3 plants, these chemical
energy compounds, NADPH and ATP, drive both the photosynthetic carbon reduction (PCR) and the photorespiratory
carbon oxidation (PCO) cycles at atmospheric CO2/O2
*Corresponding author: E-mail, [email protected]; Fax, +81-78-803-5851.
Plant Cell Physiol. 50(4): 730–743 (2009) doi:10.1093/pcp/pcp032, available online at www.pcp.oxfordjournals.org
© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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730
Plant Cell Physiol. 50(4): 730–743 (2009) doi:10.1093/pcp/pcp032 © The Author 2009.
Plastoquinone oxidation system (POS)
partial pressures and function in the acquisition of carbon in
photosynthesis.
The efficiency of photosynthesis depends on the environment. For example, in plants grown in low light (LL plants),
the photon energy absorbed by their leaves is low and their
photosynthesis rates depend on the supply of the photons.
Under these conditions, NADP+ and ADP are rapidly regenerated, and the electrons flow efficiently in PET, as observed
in the high quantum yield of PSII [Φ(PSII)]. On the other
hand, in plants grown in high light (HL plants) the photon
energy absorbed by their leaves is too high compared with
the rate of photosynthesis, i.e. photosynthesis is limited by
the supply of CO2 to chloroplasts through stomata (von
Caemmerer 2000). Under these conditions, the regeneration
of both NADP+ and ADP is suppressed, and photosynthetic
efficiency is lowered, as observed in the decreased Φ(PSII).
In another example, on exposure of plants to drought conditions, stomata close. As a result, CO2 cannot reach the
carboxylation site of ribulose-1,5-bisphosphate (RuBP) on
RuBP carboxylase/oxygenase (Rubisco), and photosynthetic
efficiency decreases (Golding et al. 2004).
Accumulation of electrons in PET, as observed in the lowered Φ(PSII), causes PSII to suffer from photoinhibition,
leading to a reduction in photosynthesis (Asada 1999,
Miyake and Okamura 2003, Park et al. 1996). Under these
conditions, the photoexcited reaction center Chl P680*
cannot donate electrons to pheophytine and de-excites to
the excited triplet Chl 3P680*. 3P680* rapidly reacts with O2,
producing the reactive species of oxygen, singlet O2 (1O2). Its
accumulation triggers the inactivation of PSII through the
oxidative degradation of D1 protein or Chl by 1O2 towards
photoinhibition (Yamashita et al. 2008). Therefore, an
efficient flow of electrons in linear electron flow (LEF) is
favorable for the alleviation of PSII inactivation.
We found that tobacco high-light grown plants showed a
higher value of photochemical quenching of Chl fluorescene
(qP), compared with tobacco low-light grown plants (Miyake
et al. 2004). The qP is a parameter of Chl fluorescence and is
derived from the puddle model for the excitation of the
reaction center of PSII (Schreiber et al. 1986). In the puddle
model, the reaction center of PSII does not interact mutually
and the photochemical yield is determined by the fraction
of open PSII reaction centers multiplied by the maximum
quantum yield of PSII in the light conditions. The fraction of
open PSII centers is defined as qP. Schreiber et al. assumed
that the value of qP reflects the reduction–oxidation (redox)
level of a primary electron acceptor of PSII, QA (Schreiber
et al. 1986). The higher the value of qP becomes, the higher
the oxidized level of QA.
However, the puddle model does not hold because the
transfer of photon energy absorbed by Chl interactively
occurs among light-harvesting complex II (LHCII) antennae
(Lazár 1999). That is, the photon energy absorbed by Chl is
used competitively by a large number of reaction centers
embedded in PSII antennae: this manner of energy usage follows the lake model (Kramer et al. 2004, Baker 2008). In the
lake model, the existing state of each reaction center of PSII
does not take only two states, perfectly open or perfectly
closed, and, in this respect it differs from the puddle model.
Stochastically, the probability for the excitation of the reaction center by the photoabsorbed excited Chl a, ranges from
0 to 1, and its constant depends on the fraction of the ground
state of the reaction center in PSII: this state is defined as the
open PSII centers in the lake model (see Materials and Methods). The fraction of open PSII centers in the model is shown
as qL, a Chl fluorescence parameter, newly derived from the
lake model. For example, the electron flux in photosynthetic
LEF is limited by NADP+ regeneration and electrons accumulate in the plastoquinone (PQ) pool. Then, charge separation of the reaction center of PSII, P680, is suppressed by the
slowed down rate of electron donation from P680 to QA.
Under these conditions the fraction of open PSII centers
decreases, as observed in the lower value of qL, i.e.
qL reflects the redox level of the PQ pool.
From the above discussion, the Chl fluorescence parameter, qL, is considered to behave as qP. If qL responds to the
growth light intensity, as observed in qP in tobacco leaves
(Miyake et al. 2005b), we conclude that plants adjust the
redox level of the PQ pool. We can try to elucidate the
mechanism for the regulation of the redox level by relating
qL to non-photochemical quenching of Chl fluorescence
(NPQ) and the maximum quantum yield of PSII in the dark
(Fv/Fm). In the present study, we observed that high lightacclimated tobacco increased qL and NPQ, and decreased
Fv/Fm. We found that a decrease in Fv/Fm largely contributed
to the increase in qL. Furthermore, we demonstrated that
thylakoid membranes from the high light-acclimated
tobacco leaves showed a higher value of qL and a tolerance
against photoinhibition of PSII, compared with the thylakoid
membranes from tobacco LL plants. We call the regulation
mechanism of the redox level of the PQ pool the plastoquinone oxidation system (POS), and propose that POS
suppresses the accumulation of electrons in PET, as observed
in the increased qL of Chl fluorescence.
Results
Comparison of photosynthetic characteristics
among plants grown in low light (LL), low light to
high light (LH) and low light to high light to low
light (LHL)
Tobacco plants were grown under a light intensity of 150 µmol
photons m–2 s–1 until the fifth leaf fully expanded. These
plants were defined as LL plants. Subsequently, some LL plants
were transferred to a growth light intensity of 1,100 µmol
Plant Cell Physiol. 50(4): 730–743 (2009) doi:10.1093/pcp/pcp032 © The Author 2009.
731
C. Miyake et al.
Dependence of Φ(PSII) on the electron sink
The effects of intensity transition of growth light on the
dependency of Φ(PSII) on the activity of the electron sink
were studied. The activities of the electron sink of LL, LH
and LHL plants were regulated by changing the atmospheric
partial pressure of CO2 at 1,100 µmol photons m–2 s–1 and
2 ka Pa O2 (see Materials and Methods). The activity of the
electron sink was expressed as 4 × (A + Rd), where A is the net
CO2 assimilation rate and Rd is the day respiration rate (Fig. 1).
Under non-photorespiratory conditions, almost all electrons
produced in PSII flow to the PCR cycle (von Caemmerer
2000) and, in its stoichiometry, net fixation of 1 mol of CO2
requires four electrons. Φ(PSII) showed a positive linear relationship with the activity of the electron sink. These results
indicated that almost all electrons produced in the photooxidation of water in PSII flowed mainly to the PCR cycle
(Genty et al. 1989, von Caemmerer 2000). There were no
significant differences in the dependency of Φ(PSII) on the
activity of the electron sink, i.e. the slope of its positive linear
relationship, among LL, LH and LHL plants. These results
reflected the correspondence of the values of αII, the distribution ratio of light illuminating the leaf to PSII, in these
plants to each other.
Dependence of NPQ on Φ(PSII)
We studied the effects of intensity transition of growth light
on the dependency of NPQ on Φ(PSII). Several Φ(PSII)s were
evaluated by changing the atmospheric partial pressure of
CO2 in the measurement of the net CO2 assimilation rate
(Fig. 1). We found that the intensity of transition of growth
732
0.2
0.15
F(PSII)
photons m–2 s–1. These plants were defined as LH plants.
At 24 h after this transfer, LH plants were used in the experiments. Also 24 h after the transfer to high light intensity,
some LH plants were moved back to the growth light intensity of 150 µmol photons m–2 s–1. These plants were defined
as LHL plants. At 24 h after the transition from high light to
low light conditions, LHL plants were used for further analysis. The photosynthetic characteristics of these plants (LL,
LH and LHL plants) were comparatively analyzed (Table 1).
The light absorption efficiency (p) of a leaf, the distribution
ratio of light illuminating the leaf to PSII (αII), Chl content of
a leaf and the ratio of Chl a to Chl b (Chl a/b) were determined in LL, LH and LHL plants. We could not find any difference in such parameters among these plants. Furthermore,
there were no significant differences in the following parameters; the net CO2 assimilation rates [A(20) and A(>60) at
intercellular partial pressures of CO2 (Ci) at 20 and >60 Pa],
and the total leaf nitrogen content (N). On the other hand,
we found that the maximal quantum yields of PSII in the
dark (Fv/Fm) in both LH and LHL plants were significantly
lower than those in LL plants.
0.1
; LL-plants
; LH-plants
; LHL-plants
0.05
0
0
20
40
60
80
4 x (A + Rd) (mmol CO2
100
120
m–2 s–1)
Fig. 1 Φ(PSII) vs. 4 × (A + Rd). Measurements of Φ(PSII), A and Rd were
made simultaneously at a leaf temperature of 25°C, a PFD of 1,100 µmol
photons m–2 s–1 and 2 kPa O2. The range of 4 × (A + Rd) was generated
by applying different partial pressures of CO2 to a leaf in the analysis of
Chl fluorescence and gas exchange (see Materials and Methods).
Representative data were plotted (circles, LL plants; squares, LH plants;
diamonds, LHL plants).
light from low to high light increased NPQ (Fig. 2A). Compared with LL plants, both LH and LHL plants showed higher
NPQ values in the range of Φ(PSII). Furthermore, in all plants,
the dependency of NPQ on Φ(PSII) was the same. These
results showed that acclimation of LL plants to a high intensity of growth light required a higher activity of the heat
dissipation process (HDP) which is driven by the xanthophyll
cycle and PsbS protein (Demmig-Adams and Adams 1996,
Niyogi et al. 1998, Niyogi 1999). Also, the increased activity
of HDP was maintained after the end of high light stress, as
observed in LHL plants (Fig. 2B).
Dependence of qL on Φ(PSII)
We studied the effects of the intensity transition of growth
light on the dependency of the redox level of the PQ pool on
Φ(PSII). We derived an equation (Equation 14 in Materials
and Methods) relating a parameter of Chl fluorescence, qL,
to NPQ and Fv/Fm in order to evaluate the redox level of the
PQ pool (see Materials and Methods). In all plants, qL showed
a positive linear relationship with Φ(PSII) (Fig. 3A). These
results reflected the increase in the electron sink oxidized
PQ pool. Transition of growth light to the high intensity
caused plants to regulate the redox level of the PQ pool to
the oxidized state in LH plants, compared with LL plants
(Fig. 3A). Compared with LL plants, both LH and LHL plants
showed higher qL values. Also, the slopes of the positive
linear relationship between qL and Φ(PSII) in LH plants were
significantly higher than those in LL plants. Furthermore, the
increased qL in LH plants was maintained even after the
Plant Cell Physiol. 50(4): 730–743 (2009) doi:10.1093/pcp/pcp032 © The Author 2009.
Plastoquinone oxidation system (POS)
A
A
0.3
2.5
; LL-plants
; LH-plants
; LHL-plants
0.25
2
0.2
qL
NPQ
1.5
1
0.15
0.1
; LL-plants
; LH-plants
; LHL-plants
0.5
0.05
0
0
0
0.05
0.1
F(PSII)
0.15
0
0.2
0.05
0.1
F(PSII)
0.15
0.2
B
B
0.1
2.5
b
b
0.08
2
a
b
b
0.06
NPQ
qL
1.5
a
0.04
1
0.02
0.5
0
an
LH
L-
pl
la
-p
LH
-p
LL
LLH
Fig. 2 (A) NPQ vs. Φ(PSII). Measurements of NPQ and Φ(PSII) were
made simultaneously at a leaf temperature of 25°C, a PFD of 1,100 µmol
photons m–2 s–1 and 2 kPa O2, as described in Fig. 1. The range of
Φ(PSII) was generated by applying different partial pressures of CO2 to
a leaf in the analysis of Chl fluorescence and gas exchange (see
Materials and Methods). Representative data were plotted (circles, LL
plants; squares, LH plants; diamonds, LHL plants). (B) NPQ at about
0.05 of Φ(PSII) in leaves of tobacco plants (LL, LH and LHL plants).
Values of NPQ were analyzed by one-way ANOVA (Sokal and Rohlf
1995), and a post hoc, Tukey HSD test was carried out on the ground
means. Bars denote the standard deviation. Data were averages of four
experiments (n = 4) using leaves of tobacco plants from each group.
Values with the same letter are not significantly different (P >0.05).
NPQ and Φ(PSII) in Chl fluorescence were measured simultaneously
at about 0.05 of Φ(PSII), obtained by regulating an ambient partial
pressure of CO2 at 2 kPa O2 and 1,100 µmol photons m–2 s–1.
ts
s
nt
nt
la
ts
an
pl
la
-p
LH
LL
-p
la
nt
nt
s
s
s
0
Fig. 3 (A) qL vs. Φ(PSII). Measurements of NPQ and Φ(PSII) were
made simultaneously at a leaf temperature of 25°C, a PFD of 1,100 µmol
photons m–2 s–1 and 2 kPa O2, as described in Fig. 1. The range of
Φ(PSII) was generated by applying different partial pressures of CO2 to
a leaf in the analysis of Chl fluorescence and gas exchange (see
Materials and Methods). Representative data were plotted (circles, LL
plants; squares, LH plants; diamonds, LHL plants). (B) qL at about 0.05
of Φ(PSII) in leaves of tobacco plants (LL, LH and LHL plants). Values of
qL were analyzed by one-way ANOVA (Sokal and Rohlf 1995), and a
post hoc, Tukey HSD test was carried out on the ground means. Bars
denote the standard deviation. Data were averages of four experiments
(n = 4) using leaves of tobacco plants from each group. Values with
the same letter are not significantly different (P >0.05). qL and Φ(PSII)
in Chl fluorescence were measured simultaneously at about 0.05 of
Φ(PSII) obtained by regulating an ambient partial pressure of CO2 at
2 kPa O2 and 1,100 µmol photons m–2 s–1.
Plant Cell Physiol. 50(4): 730–743 (2009) doi:10.1093/pcp/pcp032 © The Author 2009.
733
C. Miyake et al.
transition of growth light to low light, as observed in LHL
plants (Fig. 3B). These results showed that, similar to HDP,
the oxidized level of the PQ pool was maintained after the
relief of high light stress.
A
2.5
2
Effects of the PCO cycle and the water–water cycle
(WWC) on qL and NPQ in LL and HL plants
NPQ
1.5
Both the PCO cycle and the WWC function as an electron
sink (Miyake and Yokota 2000). Therefore, increases in these
activities would enhance qL. Since the electron fluxes in both
the PCO cycle and the WWC depend on the partial pressure
of O2 (Park et al. 1996, Miyake and Yokota 2000, Miyake et al.
1998, Makino et al. 2002, Miyake et al. 2006), we studied the
effects of O2 on the dependencies of both NPQ and qL on
Φ(PSII) (Fig. 4). In both LL and LH plants, an increase in the
partial pressure of O2 did not affect the dependency of either
NPQ or qL on Φ(PSII) (Fig. 4A, B). The values of Φ(PSII)
obtained at 21 kPa O2 distributed in the higher range, compared with the distribution at 2 kPa O2. These results indicated that in the presence of 21 kPa O2, the activities of both
the PCO cycle and the WWC were stimulated. Furthermore,
the effects of both the PCO cycle and the WWC on the
increase in Φ(PSII), NPQ and qL, observed in LL and LH
plants, were also observed in HL plants (Fig. 5A, B). That is,
in general, these electron sinks would contribute to the
increase in qL by enhancing Φ(PSII), in the same manner as
those obtained in the presence of 2 kPa O2.
1
0.5
0
Effects of the increase in qL on photoinhibition
We studied the effects of an increase in qL, i.e. the increase in
the oxidation level of the PQ pool, on photoinhibition of
thylakoid membranes from chloroplasts. Thylakoid membranes were prepared from the leaves of LL and LH plants.
The dependency of Φ(PSII) on the activity of LEF was
the same in thylakoid membranes from LL and LH plants
(Fig. 6A). These results were consistent with the dependency
of Φ(PSII) on the net CO2 assimilation rate under nonphotorespiratory conditions, obtained from intact leaves of
734
0
0.05
LL-plants
LH-plants
0.1
F(PSII)
0.15
0.2
0.15
0.2
B
0.3
; 2 kPa O2
; 21 kPa O2 LL-plants
; 2 kPa O2
LH-plants
; 21 kPa O2
0.25
qL
0.2
0.15
0.1
0.05
Comparison of CEF-PSI activity between LL, LH
and LHL plants
Compared with LL plants, both LH and LHL plants showed a
higher value of NPQ (Fig. 2). NPQ is induced by the formation of ∆pH across thylakoid membranes (Miyake et al.
2004). ∆pH is produced by cyclic electron flow around PSI
(CEF-PSI) (Miyake et al. 2005a). Under the limited photosynthesis conditions, the activity of CEF-PSI was analyzed comparatively (Table 1). The activity of CEF-PSI was evaluated as
Φ(PSI) at about 0.05 of Φ(PSII), where photosynthesis was
suppressed. Among LL, LH and LHL plants, there was no
significant difference in the activity of CEF-PSI. These results
suggested that the increase in qL was not due to the activity
of CEF-PSI.
; 2 kPa O2
; 21 kPa O2
; 2 kPa O2
; 21 kPa O2
0
0
0.05
0.1
F(PSII)
Fig. 4 (A) Effects of O2 on the relationship between NPQ and Φ(PSII)
in LL and LH plants. Measurements of NPQ and Φ(PSII) were made
simultaneously at a leaf temperature of 25°C and a PFD of 1,100 µmol
photons m–2 s–1 in the presence of 2 (open symbols) and 21 (filled
symbols) kPa O2, respectively. The range of Φ(PSII) was generated by
applying different partial pressures of CO2 to a leaf in the analysis of
Chl fluorescence and gas exchange (see Materials and Methods).
Representative data were plotted (circles, LL plants; squares, LH
plants). (B) Effects of O2 on the relationship between qL and Φ(PSII).
Measurements of qL and Φ(PSII) were made simultaneously at a leaf
temperature of 25°C and a PFD of 1,100 µmol photons m–2 s–1 in the
presence of 2 (open symbols) and 21 (filled symbols) kPa O2,
respectively. The range of Φ(PSII) was generated by applying different
partial pressures of CO2 to a leaf in the analysis of Chl fluorescence and
gas exchange (see Materials and Methods). Representative data were
plotted (circles, LL plants; squares, LH plants).
LL and LH plants (Fig. 1). On the other hand, the dependency of qL on Φ(PSII) in thylakoid membranes from LH
plants was higher than that from LL plants, and the values
of qL in thylakoid membranes from LH plants were higher
than those from LL plants in the range of Φ(PSII) (Fig. 6B).
Plant Cell Physiol. 50(4): 730–743 (2009) doi:10.1093/pcp/pcp032 © The Author 2009.
Plastoquinone oxidation system (POS)
A
2.5
0.5
2
0.4
1.5
1
B
0.6
qL
NPQ
3
0.3
0.2
; 2 kPa O2
0.5
0
0
0.1
0.2
0.3
0.4
; 2 kPa O2
0.1
; 21 kPa O2
0.5
; 21 kPa O2
0
0
0.1
F(PSII)
0.2
0.3
0.4
0.5
F(PSII)
Fig. 5 (A) Effects of O2 on the relationship between NPQ and Φ(PSII) in plants grown in high light (HL plants). Measurements of NPQ and
Φ(PSII) were made simultaneously at a leaf temperature of 25°C and a PFD of 1,100 µmol photons m–2 s–1 in the presence of 2 (open symbols)
and 21 (filled symbols) kPa O2, respectively. The range of Φ(PSII) was generated by applying different partial pressures of CO2 to a leaf in the
analysis of Chl fluorescence and gas exchange (see Materials and Methods). Representative data of HLplants were plotted. HL plants were grown
at the light intensity of 1,100 µmol photons m–2 s–1 and all measurements were made when the fifth to tenth leaves were fully expanded.
(B) Effects of O2 on the relationship between qL and Φ(PSII) in HL plants. Measurements of qL and Φ(PSII) were made simultaneously at a leaf
temperature of 25°C and a PFD of 1,100 µmol photons m–2 s–1 in the presence of 2 (open symbols) and 21 (filled symbols) kPa O2, respectively.
The range of Φ(PSII) was generated by applying different partial pressures of CO2 to a leaf in the analysis of Chl fluorescence and gas exchange
(see Materials and Methods). Representative data of HL plants were plotted. HL plants were grown at the light intensity of 1,100 µmol photons
m–2 s–1 and all measurements were made when the fifth to tenth leaves were fully expanded.
These results were also consistent with those from their
intact leaves (Fig. 3A).
Next, we compared the photoinhibition of thylakoid
membranes from LL and LH plants. We set the activity of
LEF, the electron sink activity, of thylakoid membranes to
the same value in LL and LH plants. Both thylakoid membranes showed an initial activity of LEF of about 30 µmol O2
[(mg Chl)]–1 h–1 before light treatments, by regulating the
concentration of the electron acceptor, methyl viologen
(MV) (Miyake and Okamura 2003).
The activity of LEF decreased as the duration of light
treatment increased (Fig. 6C). After 30 min, thylakoid membranes in LL plants largely lost LEF activity, compared with
LH plants. The activity of LEF in LL plants decreased to about
30% of its initial activity. On the other hand, the activity of
LEF in LH plants decreased to about 70% of its initial activity.
Similar to the activity of LEF, the activity of PSII also decreased
during the light treatments (Fig. 6D). The magnitude of the
decrease in the activity of PSII in LL and LH plants was similar
to the decrease in activity of LEF in LL and LH plants. On the
other hand, the activity of PSI in LL and LH plants was not
affected by the light treatment (Fig. 6E). These results
showed that the decrease in the activity of LEF with light
treatment was due to the loss of the activity of PSII (i.e. photoinhibition of PSII), and was remarkable when qL was low
and the PQ pool was reduced as in LL plants, compared with
LH plants.
Discussion
In the present study, we analyzed the response of photosynthetic characteristics of LL plants to high light conditions.
After the transition of light intensity from low to high, the
quantum yield of PSII in the dark (Fv/Fm) decreased significantly
in LH and LHL plants, compared with LL plants (Table 1).
Furthermore, the value of NPQ increased significantly in LH
plants, compared with LL plants (Fig. 2). The value of qL of
Chl fluorescence also increased significantly in LH plants
compared with LL plants (Fig. 3). The increases in both NPQ
and qL of Chl fluorescence and the decrease in Fv/Fm were
maintained even after the transition of light intensity back
from high to low, as observed in LHL plants. These results
indicate that plants regulate the redox level of the PQ pool
in response to the growth light intensity. To elucidate its
mechanism of regulation, a parameter of the redox level of
the PQ pool, qL, was derived from the lake model, in the
excitation of PSII reaction centers, where the parameter qL
was related to NPQ and Fv/Fm, as described below.
We discussed the effect of the NPQ on qL, using Equation 14
(see Materials and Methods). On exposure to high intensity
growth light conditions, plants increased the capacity of
the HDP process, as observed in the higher value of
NPQ (Demmig et al. 1988, Demmig and Adams 1994,
Demmig et al. 1996, Verhoeven et al. 1997, Verhoeven
et al. 1998, Verhoeven et al. 1999). Under such conditions,
Plant Cell Physiol. 50(4): 730–743 (2009) doi:10.1093/pcp/pcp032 © The Author 2009.
735
C. Miyake et al.
Table 1 Effects of growth light intensity on p, αII, Chl, Chl a/b,
A(20), A(>60), Φ(PSI) and Fv/Fm in tobacco leaves
Parameter
LL plants
LH plants
LHL plants
Growth PFD (µmol
photons m–2 s–1)
150
150→1,100
150→1, 100→150
N (mmol mol–1)
96a
100a
103a
(n = 4)
(8)
(7)
(8)
p
0.850a
0.843a
0.844a
(n = 6)
(0.011)
(0.014)
(0.010)
αII
0.43a
0.40a
0.41a
(n = 4)
(0.02)
(0.05)
(0.04)
Chl (mmol m–2)
0.58a
0.55a
0.52a
(n = 4)
(0.04)
(0.06)
(0.05)
Chl a/b
3.20a
3.31a
3.28a
(n = 4)
(0.12)
(0.09)
(0.11)
A(20) (µmol CO2
m–2 s–1)
13a
14a
14a
(n = 4)
(3)
(2)
(3)
A(>60) (µmol CO2
m–2 s–1)
28a
27a
28a
(n = 4)
(2)
(2)
(3)
Φ(PSI) at about 0.05 of
Φ(PSII)
0.13a
0.15a
0.15a
(n = 4)
(0.02)
(0.03)
(0.03)
Fv/Fm
0.818a
0.706b
0.710b
(n = 6)
(0.011)
(0.013)
(0.008)
These effects were analyzed by one-way ANOVA (Sokal and Rohlf 1995). For data
presented in the table, a post hoc, Tukey HSD test was carried out on the grouped
means. Abbreviations (LL, LH and LHLplants) are explained in the text. Figures in
parentheses represent the SD. Data are the averages of 4–6 experiments (n =4 –6)
using leaves of tobacco plants from each group. Within the same experiment,
values followed by the same letter are not significantly different (P >0.05).
both the pool size of the xanthophyll cycle and the amount
of PsbS protein, both of which function in the induction of
the HDP process, increased and contributed to the dissipation of excess light energy against photosynthesis as heat
(Li et al. 2002a, Li et al. 2002b, Li et al. 2004, Dall’Ostro et al.
2005). The HDP process is expected to decrease the
efficiency of photoexcitation of the reaction center of PSII
and to suppress the influx of electrons from H2O to the PET.
That is, the HDP process would contribute to the oxidation
of the PQ pool under conditions limiting photosynthesis. In
general, an increase in NPQ of leaves of plants grown under
abiotic stress conditions, such as high light, drought and
high temperature, was compared from the aspects of dependency of NPQ on either photon flux density (PFD) or CO2
partial pressure (Miyake et al. 2004, Golding et al. 2004,
Miyake et al. 2005a). However, the value of NPQ depends on
736
the activity of the electron sink (Miyake et al. 2005b). Therefore, a comparison of NPQ should be based on the same
activity of the electron sink. In Fig. 2, at the same activity of
the electron sink, Φ(PSII), for example at Φ(PSII) = 0.1, NPQ
and qL in both LH and LHL plants were 2.20 and 0.16, respectively. On the other hand, these values in LL plants were 1.75
and 0.073, respectively. The ratios of NPQ of both LH and
LHL plants to that in LL plants were 1.26-fold higher. From
this value, the ratios of qL were expected to be 1.16-fold
higher, estimated from Equation 14. However, in fact, the
values of qL in both LH and LHL plants were 2.21-fold higher
than in LL plants. That is, we found that the increase in qL in
high light-acclimated plants cannot be accounted for only
by an increase in NPQ. These results are consistent with
those in Yamamoto et al. (2006). Transplastomic plants
overexpressing ferredoxin in chloroplasts showed a higher
activity of CEF-PSI, compared with wild-type plants, and the
increased activity of CEF-PSI contributed to enhanced HDP.
However, we could not find any increase in qP (Yamamoto
et al. 2006). From these results, we concluded that only an
increase in NPQ could not enhance qL, i.e. the oxidation of
the PQ pool.
From Equation 14, we recognized that the decrease in
maximal quantum efficiency of PSII photochemistry [Fv/Fm =
Φ(PSII) in the dark] contributes to the increase in qL. In fact,
both LH and LHL plants had lower values of Fv/Fm compared
with LL plants (Table 1). These decreased values of Fv/Fm in
both LH and LHL plants increased their qL values (Fig. 2). As
described above, at a Φ(PSII) of 0.1, increases in NPQ in both
LH and LHL plants accounted for only 16% of the enhanced
qL. On the other hand, decreased values of Fv/Fm in both LH
and LHL plants, 0.706 and 0.710, accounted for about 90% of
the enhanced qL.
In general, a decrease in Fv/Fm is considered to reflect photoinhibition of PSII (Niyogi 2000, Hikosaka et al. 2004). Plants
exposed to high intensity light showed a decrease in Fv/Fm,
and these decreased values correlated with the decrease in
net CO2 assimilation rate (Niyogi 2000, Hikosaka et al. 2004).
However, we did not observe any decreases in CO2 assimilation rate at a Ci of 20 Pa and >60 Pa in the range of Fv/Fm
obtained in the present work, i.e. we propose that plants
regulate the activity of PSII in the direction of lowering the
maximal quantum efficiency of PSII photochemistry. Its
decreased efficiency would contribute to the protection of
PSII from photoinhibition. In fact, we showed that thylakoid
membranes from LH plants had a tolerance against photoinhibition of PSII, compared with those from LL plants (Fig. 6).
From these results, we propose that plants have a mechanism to keep the PQ pool in the oxidized state, i.e. the POS,
and the POS regulates the accumulation of electrons in the
PET system by lowering the maximal quantum efficiency of
PSII photochemistry (Fig. 7). Under low light conditions, the
level of reduction of the PQ pool is low, which means less
Plant Cell Physiol. 50(4): 730–743 (2009) doi:10.1093/pcp/pcp032 © The Author 2009.
Plastoquinone oxidation system (POS)
A
0.2
B
0.08
; LL-plants
; LH-plants
; LL-plants
; LH-plants
0.07
0.06
0.15
qL
F(PSII)
0.05
0.1
0.04
0.03
0.02
0.05
0.01
0
0
0
10
20
30
40
0
50
0.05
C
la
-p
LL
400
la
-p
350
LH
b
300
a
0.15
D
400
s
nt
Activity of PSII
(mmol O2 (mg Chl)–1h)–1
Activity of Linear Electron Flow
(mmol O2 (mg Chl)–1h)–1
500
s
nt
0.1
F(PSII)
V (O2) (mmol O2 (mg Chl)–1 h–1
b
200
a
100
la
-p
LL
s
nt
s
nt
la
-p
LH
b
300
a
b
250
200
150
a
100
50
0
0
0
15
30
Time for Light Treatment (min)
0
15
30
Time for Light Treatment (min)
E
800
Activity of PSI
(mmol O2 (mg Chl)–1h)–1
700
la
-p
LL
s
nt
s
nt
la
-p
LH
600
500
400
300
200
100
0
0
15
30
Time for Light Treatment (min)
Fig. 6 Light treatment of thylakoid membranes and photoinhibition. (A) Φ(PSII) was plotted against the activity of linear electron flow (LEF)
[V(O2)] in thylakoid membranes from both LL (open circles) and LH (filled circles) plants. LEF was evaluated as the methyl viologen- (MV)
dependent O2 uptake rate: a range of rates was obtained by changing the concentration of MV in the reaction mixture (Miyake and Yokota
2001). The reaction mixture (2 ml) contained 50 mM potassium phosphate buffer (pH 7.5), 10 mM NaCl, 2 mM MgCl2, 400 mM sucrose, 0.67 µM
nigericin, 0.1 mM KCN, MV (0–0.5 µM) and thylakoid membranes (14 µg of Chl). Φ(PSII) and V[O2] were determined simultaneously at the PFD
of 200 µmol photon m–2 s–1 (red light, ≥640 nm). (B) qL in thylakoid membranes from both LL (open circles) and LH (filled circles) plants was
Plant Cell Physiol. 50(4): 730–743 (2009) doi:10.1093/pcp/pcp032 © The Author 2009.
737
C. Miyake et al.
Low-Light
High-Light
High-Light
Stressed!
Mitigated!
Acclimation
Transition
Red.
Red.
Red.
PQ
Reduced
Oxidized
Ox.
Ox.
Ox.
POS
PCO-Cycle
PCO-Cycle
WWC
WWC
HDP
HDP
Stress mitigation mechanism
Fig. 7 Mitigation mechanism of plants against high light stress. Under low light conditions, the reduction level of the plastoquinone (PQ) pool is
low, which means less accumulation of electrons in the photosynthetic electron transport (PET) system. In this situation, the probability of
production of reactive oxygen species (ROS) is negligible, resulting in low oxidative stress on plants. When plants are exposed to high light
intensity, i.e. transition from low light conditions to high light, the electron sink such as the photosynthetic carbon reduction (PCR) cycle limits
photosynthesis, resulting in the accumulation of electrons in the PET system, as observed in the increased reduction level of the PQ pool. Under
these conditions, the mitigation mechanism of plants against oxidative stress functions to suppress the enhanced production of ROS.
Photorespiratory carbon oxidation (PCO cycle) and the water–water cycle (WWC) function as an electron sink (shown as purple) to reduce the
reduction level of the PQ pool. Furthermore, the xanthophyll cycle functions as a heat dissipation process (HDP, shown as green) to leak excess
photon energy against photosynthesis and to contribute to the suppression of the over-reduction of the PQ pool. Furthermore, we propose a
new mitigation mechanism against high light stress in the acclimation of plants, where the oxidation–reduction level of the PQ pool is regulated
to the oxidized state from the reduced state, through lowering the maximum quantum efficiency of PSII photochemistry. We named this new
mechanism the plastoquinone oxidation system (POS), shown in red. POS suppresses the accumulation of electrons in the PET system.
Fig. 6 (caption continued)
estimated from Equation 14, and plotted against Φ(PSII). (C) Light treatments of thylakoid membranes from both LL and LH plants were
conducted. The reaction mixture was the same as in (A), except that KCN was omitted and 0.25 µM MV giving about 30 µmol O2[(mg Chl)]–1 h–1
of LEF, 200 U of Mn-superoxide dismutase and 1,000 U of catalase were present. Superoxide dismutase and catalase were added to the reaction
mixture to prevent the oxidative attack on thylakoid membranes by O2– and H2O2 photoproduced via the reaction of photoreduced MV with
O2 at PSI. Light treatments were conducted (Miyake and Okamura 2003). At the indicated time of light treatment, the activities of LEF of
thylakoid membranes were assayed in the presence of 0.67 µM nigericin, 0.1 mM KCN and 0.1 mM MV at 1,600 µmol photons m–2 s–1 (white
light) (LL plants, unshaded; LH plants, shaded). The light-dependent O2 uptake rate was measured as the activity of LEF. Data were the average
(n = 3) of three experiments using the present preparation of thylakoid membranes, and vertical bars represent the standard deviation of
measurement. Within the same time after light treatments, different letters are significantly different [P <0.05, Student's t-test (Sokal and Rohlf
1995)]. (D) Light treatments were the same as in (C). At the indicated time, the activities of PSII of thylakoid membranes were assayed in the
presence of both 0.5 mM phenyl-p-benzoquinone and 0.5 µM 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone at 1,600 µmol photons m–2 s–1
(white light) (LL plants, unshaded; LH plants, shaded). The light-dependent evolution rate of O2 was measured as the activity of PSII. Within the
same time after light treatments, different letters are significantly different [P <0.05, Student's t-test (Sokal and Rohlf 1995)]. (E) Light treatments
were the same as in C. At the indicated time of light treatment, the activities of PSI of thylakoid membranes were assayed in the presence of
0.1 mM KCN, 10 µM DCMU, 500 µM dichlorophenolindophenol, 2 mM ascorbate, 0.67 µM nigericin and 0.1 mM MV at 1,600 µmol photons
m–2 s–1 (white light) (LL plants, unshaded; LH plants, shaded). The light-dependent uptake rate of O2 was measured as the activity of PSI.
738
Plant Cell Physiol. 50(4): 730–743 (2009) doi:10.1093/pcp/pcp032 © The Author 2009.
Plastoquinone oxidation system (POS)
accumulation of electrons in the PET system. In this situation, the probability of producing reactive oxygen species
(ROS) is negligible, resulting in the low oxidative stress on
plants. When plants are exposed to high light intensity, in
the transition from low light conditions to high light, the
electron sink such as the PCR cycle limits photosynthesis,
resulting in the accumulation of electrons in the PET system,
as observed in the increased level of reduction of the PQ
pool. Under these conditions, the mitigation mechanism of
plants against oxidative stress functions to suppress the
enhanced production of ROS. The PCO cycle and the WWC
function as an electron sink to reduce the level of reduction
of the PQ pool. Furthermore, the xanthophyll cycle functions as an HDP to leak photon energy that is excess to photosynthesis and to contribute to the suppression of the
over-reduction of the PQ pool. Furthermore, we propose a
new mitigation mechanism against high light stress in the
acclimation of plants, whereby the oxidation–reduction
level of the PQ pool is regulated to the oxidized state from
the reduced state, through lowering the maximum quantum efficiency of PSII photochemistry. We named this new
mechanism the plastoquinone oxidation system (POS). The
POS suppresses the accumulation of electrons in the PET
system. We intend to clarify the detailed mechanism of POS
in future publications.
Materials and Methods
Plant growth conditions
Tobacco plants (Nicotiana tabacum cv Xanthi) were grown
from seeds under standard air-equilibrated conditions with
16 h/8 h day–night cycles at 25 and 22°C, respectively, and
50–60% relative humidity. PFDs were adjusted to 150 (low
light treatment) and 1,100 µmol photons m–2 s–1 (high light
treatment). Seedlings were kept in 0.5 dm3 pots containing
commercial peat-based compost, and were watered daily.
Plants were fertilized with 1,000-fold diluted Hyponex
8–12–6 (Hyponex Japan, Osaka, Japan) three times a week.
All measurements described below were made 4 weeks after
sowing. These plants were also used in the transition experiments of growth conditions, where plants grown under low
light intensity were exposed to high light intensity, and vice
versa, with 16 h/8 h day–night cycles.
CO2 fixation, Chl fluorescence and P700+ absorbance
For measurements of photosynthetic parameters and collection of leaves, tobacco plants were transferred to a dark
room 4 h after the start of the light period. After incubation
of tobacco in the dark room for about 60 min, CO2 fixation
(gas exchange) and Chl fluorescence were measured simultaneously. P700+ absorbance was measured sequentially
after Chl fluorescence measurement. All measurements
were repeated at least three times using three different
plants. The measurements of the leaf attached to the plant
were done over a leaf area of 6 cm2. The basal system of gas
exchange was adopted as previously detailed by Miyake and
Yokota (2000), except that LI-6400 (Li-Cor, Lincoln, NE, USA)
was used as the IRGA (infrared gas analyzer). Leaf temperature was adjusted to 25.0 ± 0.5°C. The mixture of gases was
saturated with water vapor at 16 ± 0.1°C, which corresponded to 1.825 kPa. Irradiance was provided by a halogen
lamp (KL-1500; Walz, Effeltrich, Germany) to the leaf chamber through the glass fiberoptics that were linked to a PAM
Chl fluorometer, as described below.
Chl fluorescence was measured with the PAM Chl fluorometer through the same fiberoptics. The steady-state fluorescence yield (Fs) was monitored continuously and a
1,000 ms pulse of saturating light was supplied at intervals of
60 s to determine maximum variable fluorescence (Fm′). The
relative Φ(PSII) at steady state was defined as (Fm′ – Fs)/Fm′, as
proposed by Genty et al. (1989). NPQ and qP of Chl fluorescence were calculated as (Fm/Fm′ – 1), according to Bilger and
Björkman (1994) and as (Fm′ – Fs)/(Fm′ – Fo′), according to
Oxborough and Baker (1997), respectively. qL was determined as described in the Discussion.
The absorbance of P700+ was measured with the same
PAM Chl fluorometer by exchanging the Chl fluorescence
detector unit for a ED-P700DW-E emiter–detector unit
(Walz, Effeltrich, Germany) (Miyake et al. 2004). The amplitude of full P700 oxidation was measured in the dark for
each leaf before the illumination was started. In darkness,
P700 is in its reduced sate, and full oxidation of P700,
[P700]total, was achieved by illumination with far-red
light (>700 nm), which excites only PSI. The oxidation of
P700, [P700+], was monitored by the change in the A810–860.
During illumination, the same amount of oxidizable P700
should be available, unless the PSI electron acceptors are
already in their reduced sate and cannot accept more electrons. During illumination, the fraction of reduced [reduced
P700] or PSI acceptor (A–) is determined by short saturating
light pulses, which give full oxidation of P700, followed by a
‘dark pulse’, which yields fully reduced P700. The difference
between the P700 amplitude in the light and the far-redinduced amplitude determined in the dark-adapted leaf
must be attributed to A–. Φ(PSI) was calculated as described
by Klughammer and Schreiber (1994), Φ(PSI) = [reduced
P700]/[P700]total.
The value of the distribution ratio of light illuminating the
leaf to PSII (αII) was determined under non-photorespiratory
conditions, where electron flux in PSII [Je(PSII)] was expressed
from the stoichiometry of the Calvin cycle, as follows. Je(PSII)
= αII×Φ(PSII) × PFD = 4 × (A + Rd), where A was the net
CO2 assimilation rate and Rd was the day respiration rate
(Genty et al. 1989, Miyake and Yokota 2000, Ruuska
et al. 2000, von Caemmerer 2000, Makino et al. 2002, Miyake
et al. 2004). Rd was estimated from curves of A vs. Ci obtained
Plant Cell Physiol. 50(4): 730–743 (2009) doi:10.1093/pcp/pcp032 © The Author 2009.
739
C. Miyake et al.
at various PFDs, as described by Brooks and Farquhar (1985).
We obtained the constant value of αII under any light intensity, and Ca at 2 kPa O2 (Miyake et al. 2004).
The ratio of light absorbed by chloroplasts in tobacco
leaves, p, was determined with an LI-1800 spectroradiometer
and the 1800-12S integrating sphere attachment (Li-Cor
Inc.). For each leaf, both a reference scan and a sample scan
of reflectance or transmittance were made from 400 to
700 nm at 1 nm intervals. The sample scan was divided by its
corresponding reference scan, and integrated over the
wavelength range to obtain the average reflectance or transmittance (Chen and Cheng 2003). The p was calculated as:
1 – reflectance – transmittance.
Derivation of the equation relating qL to both NPQ
and Fv/Fm
To elucidate the mechanism for regulation of the redox level
of the PQ pool, we derived the equation relating qL to both
NPQ and Fv/Fm. The qL is a parameter that shows the fraction of open PSII reaction centers and reflects the redox level
of QA, a primary electron acceptor of PSII, i.e. the redox level
of the PQ pool (Kramer et al. 2004). Kramer et al. (2004)
assumed the lake model in the derivation of qL. In this model,
excitons photoproduced in PSII pigment beds flow to reaction centers competitively: these centers exist in an open
state. Also, the efficiency of photochemical reaction of the
open reaction centers in PSII depends on the ratio of QA,
which is in an oxidized state (Baker 2008). The value of qL is
proportional to the efficiency of the photochemical reaction
of PSII reaction centers, and is different from the value of qP
used previously (Baker 2008). Thus, qL is a superior parameter for the evaluation of the redox level of the PQ pool (Baker
2008). The parameter qL is expressed in Equation 1 (Kramer
et al. 2004, Baker 2008).
β depends on both the intensity of measuring light and the
sensitivity of the instrument for detecting Chl fluorescence.
The magnitude of kp is determined by two parameters: an
‘intrinsic’ rate constant (kpi) for capture of exciton energy by
the reaction centers and the fraction of ‘open’ reaction centers (qL) (Kramer et al. 2004). Then,
(3)
(4)
At Fs in the dark (Fo), minimal fluorescence from darkadapted leaves, qL is 1 and kNPQ is zero. Then,
(5)
At Fm, maximal fluorescence from dark-adapted leaves, qL
and kNPQ are zero. Then,
(6)
At Fm′, maximal fluorescence from light-adapted leaves, qL is
zero. Then,
(7)
Then, NPQ is expressed as follows:
(8)
(1)
where Fm′ is the maximal fluorescence from a light-adapted
leaf; Fs is the fluorescence emission from a light-adapted leaf;
Fo′ is the minimal fluorescence from a light-adapted leaf; and
qP is the photochemical quenching (Baker 2008).
We related qL to both NPQ and Fv/Fm, as described below.
Photoexcited Chl a (Chl a*) in LHCII de-excites to the ground
state with loss of its energy through the following five processes: fluorescence emission with the rate constant (kf);
photochemical reaction with the rate constant (kp); nonradiative decay with the rate constant (kd); intersystem
crossing to form triplets with the rate constant (kisc); and a
heat dissipation process observed as NPQ with the rate constant (kNPQ). Then, Fs is expressed as,
and, maximal quantum efficiency of PSII photochemistry
[Fv/Fm = Φ(PSII) in the dark] is expressed as follows:
(9)
From Equation 9,
(10)
From Equation 8,
(2)
740
Plant Cell Physiol. 50(4): 730–743 (2009) doi:10.1093/pcp/pcp032 © The Author 2009.
(11)
Plastoquinone oxidation system (POS)
Quantum yield of photochemical energy conversion in PSII,
equivalent to Φ(PSII) in the light, is expressed as follows.
(12)
From Equations 11 and 12,
thylakoid membranes were suspended in the same medium
and centrifuged again under the same conditions. The pellet
was suspended in the reaction mixture, which consisted of
50 mM potassium phosphate buffer (pH 7.5), 10 mM NaCl,
2 mM MgCl2, 0.1 mM KCN and 400 mM sucrose, and used
for experiments as the preparation of thylakoid membranes.
Concentrations of Chl were determined as described by
Arnon (1949).
Measurement of oxygen uptake
(13)
Finally, we can get the equation relating qL to both Fv/Fm
and NPQ, from Equations 10 and 13, as follows:
(14)
In contrast to Equation 1, we can evaluate qL without the
concept Fo′. Furthermore, we can relate both NPQ and Fv/Fm
to qL, i.e. qL is proportional to the factor (NPQ + 1). In the
present study, we calculated qL using Equation 14.
Measurements of leaf nitrogen and Chl
Total leaf nitrogen was determined on the same leaves as
used for the gas-exchange studies (Makino et al. 1988). After
the photosynthetic measurements, the leaf was quickly
cut off and its fresh weight and leaf area were measured;
then the leaf was immediately homogenized in 50 mM Naphosphate buffer (pH 7.5) containing 10 mM dithiothreitol
(DTT) and 12.5% (v/v) glycerol at a ratio of leaf to buffer
of 1 : 7 (g : ml) using a chilled mortar and pestle with
acid-washed quartz sand (0.30 g). Total Chl was determined in this homogenate (Makino et al. 1992). A 100 µl
aliquot of this homogenate was weighed and subjected
to Kjeldahl digestion. Total leaf nitrogen was determined
with the SuperKjel 1200/1250 System (ACTAC, Tokyo,
Japan).
Isolation of thylakoid membranes from tobacco
chloroplasts
Intact chloroplasts were isolated from tobacco leaves of
both LL and LH plants, and purified by Percoll density gradient centrifugation, as described previously (Asada et al.
1990). Isolated chloroplasts were subjected to osmotic shock
by 10-fold dilution with 50 mM potassium phosphate buffer
(pH 7.5), 10 mM NaCl and 2 mM MgCl2, and then the mixture was centrifuged at 5,000×g for 10 min. The sedimented
Uptake of O2 and Chl fluorescence were measured simultaneously. Oxygen uptake was monitored with an oxygen electrode (Hansatech, King's Lynn, UK). After incubation in
darkness for 5 min under air-equilibrated conditions, the
reaction mixture (2 ml) was illuminated with an iodine lamp
at the indicated light intensity at 25°C.
Measurements of Chl fluorescence in thylakoid
membranes
The Chl fluorescence originating from PSII in thylakoid
membranes was measured with a Chl fluorometer (PAM101; Walz, Effeltrich, Germany). The steady-state fluorescence yield (Fs) was monitored at a light intensity of 200 µmol
photon m–2 s–1, as indicated, and 1,000 ms pulses of saturating light (PFD, 5,000 µmol m–2 s–1) were supplied at intervals
of about 60 s for the determination of maximum variable
fluorescence (Fm′). The protocol for measurements of Chl
fluorescence was similar to that described by Genty
et al. (1989).
Acknowledgments
The authors are grateful to Professor Amane Makino
(Tohoku University) for stimulating discussions at Kobe
University, and thank Emeritus Profesor Kozi Asada (Kyoto
University) for his encouragement throughout the course
of this research.
References
Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts.
Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24: 1–15.
Asada, K. (1999) The water–water cycle in chloroplasts: scavenging of
active oxygens and dissipation of excess photons. Annu. Rev. Plant
Physiol. Plant Mol. Biol. 50: 601–639.
Asada, K., Neubauer, C., Heber, U. and Schreiber, U. (1990) Methyl
viologen-dependent cyclic electron transport in spinach chloroplasts
in the absence of oxygen. Plant Cell Physiol. 31: 557–564.
Baker, N.R. (2008) Chlorophyll fluorescence: a probe of photosynthesis
in vivo. Annu. Rev. Plant Biol. 59: 89–113.
Bilger, W. and Björkman, O. (1994) Relationship among violaxanthin
deepoxidation, thylakoid membranes conformation, nonphotochemical chlorophyll fluorescence quenching in leaves of
cotton (Gossypium hirsutum L.). Planta 193: 238–246.
Plant Cell Physiol. 50(4): 730–743 (2009) doi:10.1093/pcp/pcp032 © The Author 2009.
741
C. Miyake et al.
Brooks, A. and Farquhar, G.D. (1985) Effect of temperature on the CO2/
O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase
and the rate of respiration in the light. Planta 165: 397–406.
Chen, L.-S. and Cheng, L. (2003) Both xanthophyll cycle-dependent
thermal dissipation and the antioxidant system are up-regulated in
grape (Vitis labrusca L. cv. Concord) leaves in response to N
limitation. J. Exp. Bot. 54: 2165–2175.
Dall’Osto, L., Caffarri, S. and Bassi, R. (2005) A mechanism of
nonphotochemical energy dissipation, independent from PsbS,
revealed by a conformational change in the antenna protein CP26.
Plant Cell 17: 1217–1232.
Demmig, B., Winter, K., Krüger, A. and Czygan, F.C. (1988) Zeaxanthin
and the heat dissipation of excess light energy in Nerium oleander
exposed to a combination of high light and water stress. Plant
Physiol. 87: 17–24.
Demmig-Adams, B. and Adams, W.W., III, (1994) Capacity for energy
dissipation in the pigment bed in leaves with different xanthophyll
cycle pools. Aust. J. Plant Physiol. 21: 575–588.
Demmig-Adams, B. and Adams, W.W., III, (1996) Xanthophyll cycle
and light stress in nature: uniform response to excess direct sunlight
among higher plant species. Planta 198: 460–470.
Demmig, B., Adams, W.W., III, Barker, D.H., Logan, B.A.,
Bowling, D.R. and Verhoeven, A.S. (1996) Using chlorophyll
fluorescence to assess the fraction of absorbed light allocated to
thermal dissipation of excess excitation. Physiol. Plant. 98:
253–264.
Genty, B., Briantais, J.-M. and Baker, N.R. (1989) The relationship
between the quantum yield of photosynthetic electron transport
and quenching of chlorophyll fluorescence. Biochim. Biophys. Acta
990: 87–92.
Golding, A.J., Finazzi, G. and Johnson, G.N. (2004) Reduction of the
thylakoid electron transport chain by stromal reductants—evidence
for activation of cyclic electron transport upon dark adaptation or
under drought. Planta 220: 356–365.
Hikosaka, K., Kato, M.C. and Hirose, T. (2004) Photosynthetic rates and
partitioning of absorbed light energy in photoinhibited leaves.
Physiol. Plant. 121: 699–708.
Klughammer, C. and Schreiber, U. (1994) An improved method, using
saturating light pulses, for the determination of photosystem I
quantum yield via P700+-absorbance change at 830 nm. Planta 192:
261–268.
Kramer, D.M., Johnson, G., Kiirats, O. and Edwards, G.E. (2004) New
fluorescence parameters for determination of QA redox state and
excitation energy fluxes. Photosynth. Res. 79: 209–218.
Lazar, D. (1999) Chlorophyll a fluorescence induction. Biochim. Biophys.
Acta 1412: 1–28.
Li, X.P., Gilmore, A.M., Caffarri, S., Bassi, R., Golan, T., Kramer, D., et al.
(2004) Regulation of photosynthetic light harvesting involves
intrathylakoid lumen pH sensing by the PsbS protein. J. Biol. Chem.
279: 22866–22874.
Li, X.P., Gilmore, A.M. and Niyogi, K.K. (2002a) Molecular and global
time-resolved analysis of a psbS gene dosage effect on pH- and
xanthophylls cycle-dependent non-photochemical quenching in
photosystem II. J. Biol. Chem. 277: 33590–33597.
Li, X.P., Müller-Moulè, P., Gilmore, A.M. and Niyogi, K.K. (2002b) PsbSdependent enhancement of feedback de-excitation protects
photosystem II from photoinhibition. Proc. Natl Acad. Sci. USA 99:
15222–15227.
742
Makino, A., Mae, T. and Ohira, K. (1988) Differences between wheat
and rice in the enzymatic properties of ribulose-1,5-bisphosphate
carboxylase/oxygenase and the relationship to photosynthetic gas
exchange. Planta 174: 30–38.
Makino, A., Miyake, C. and Yokota, A. (2002) Physiological functions of
the water–water cycle (Mehler reaction) and the cyclic electron
flow around PSI in rice leaves. Plant Cell Physiol. 43: 1017–1026.
Makino, A., Sakashita, H., Hidema, J., Mae, T., Ojima, K. and Osmond, B.
(1992) Distinctive responses of ribulose-1,5-bisphosphate
carboxylase and carbonic anhydrase in wheat leaves to nitrogen
nutrition and possible relationships to CO2-transfer resistance. Plant
Physiol. 100: 1737–17473.
Miyake, C., Miyata, M., Shinzaki, Y. and Tomizawa, K. (2005a) CO2
response of cyclic electron flow around PSI (CEF-PSI) in tobacco
leaves—relative electron fluxes through PSI and PSII determine the
magnitude of non-photochemical quenching (NPQ) of Chl
fluorescence. Plant Cell Physiol. 46: 629–637.
Miyake, C., Horiguchi, S., Makino, A., Shinzaki, Y., Yamamoto, H. and
Tomizawa, K.I. (2005b) Effects of light intensity on cyclic electron
flow around PSI and its relationship to non-photochemical
quenching of Chl fluorescence in tobacco leaves. Plant Cell Physiol.
46: 1819–1830.
Miyake, C. and Okamura, M. (2003) Cyclic electron flow within PSII
protects PSII from its photoinhibition in thylakoid membranes from
spinach chloroplasts. Plant Cell Physiol. 44: 457–462.
Miyake, C., Schreiber, U., Hormann, H., Sano, S., and Asada, K. (1998)
The FAD-enzyme monodedhydroascorbate radical reductase
mediates photoproduction of superoxide radicals in spinach
thylakoid membranes. Plant Cell Physiol. 39: 821–829.
Miyake, C., Shinzaki, Y., Miyata, M., and Tomizawa, K. (2004)
Enhancement of cyclic electron flow around PSI at high light and its
contribution to the induction of non-photochemical quenching
(NPQ) of Chl fluorescence in intact leaves of tobacco plants. Plant
Cell Physiol. 45: 1426–1433.
Miyake, C., Shinzaki, Y., Nishioka, M., Horiguchi, S., and Tomizawa, K.I.
(2006) Photoinactivation of ascorbate peroxidase in isolated
tobacco chloroplasts: Galdieria partita APX maintains the electron
flux through the water–water cycle in transplastomic tobacco
plants. Plant Cell Physiol. 47: 200–210.
Miyake, C. and Yokota, A. (2000) Determination of the rate of
photoreduction of O2 in the water–water cycle in watermelon
leaves and enhancement of the rate by limitation of photosynthesis.
Plant Cell Physiol. 41: 335–343.
Miyake, C. and Yokota, A. (2001) Cyclic flow of electrons within PSII in
thylakoid membranes. Plant Cell Physiol. 42: 508–515.
Niyogi, K.K. (1999) Photoprotection revisited: genetic and molecular approaches. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50:
333–359.
Niyogi, K.K. (2000) Safety valves for photosynthesis. Curr. Opin. Plant
Biol. 3: 455–460.
Niyogi, K.K., Grossman, A.R. and Björkman, O. (1998) Arabidopsis
mutants define a central role for the xanthophylls cycle in the
regulation of photosynthetic energy conversion. Plant Cell 10:
1121–1134.
Oxborough, K. and Baker, N.R. (1997) Resolving chlorophyll a
fluorescence images of photosynthetic efficiency into photochemical
and non-photochemical components—calculation of qP and Fv′/Fm′
without measuring Fo′. Photosynth. Res. 54: 135–142.
Plant Cell Physiol. 50(4): 730–743 (2009) doi:10.1093/pcp/pcp032 © The Author 2009.
Plastoquinone oxidation system (POS)
Park, Y.-I.I., Chow, W.S., Osmond, C.B. and Anderson, J.M. (1996)
Electron transport to oxygen mitigates against the photoinactivation
of photosystem II in vivo. Photosynth. Res. 50: 23–32.
Ruuska, S.A., Badger, M.R., Andrews, T.J. and von Caemmerer, S. (2000)
Photosynthetic electron sinks in transgenic tobacco with reduced
amounts of Rubisco: little evidence for significant Mehler reaction.
J. Exp. Bot. 51: 357–368.
Schreiber, U., Schliwa, U. and Bilger, W. (1986) Continuous recording of
photochemical and non-photochemical chlorophyll fluorescence
with a new type of modulation fluorometer. Photosynth. Res. 10:
51–62.
Sokal, R.R., Rohlf, F.J., (1995) Biometry. 3rd edn. Freeman, New York.
Verhoeven, A.S., Adams, W.W., III and Demmig-Adams, B. (1998) Two
forms of sustained xanthophyll cycle-dependent energy dissipation
in overwintering Euonymus kiautschovicus. Plant Cell Environ. 21:
893–903.
Verhoeven, A.S., Adams, W.W., III and Demmig-Adams, B. (1999) The
xanthophyll cycle and acclimation of Pinus ponderosa and Malva
neglecta to winter stress. Oecologia 118: 277–287.
Verhoeven, A.S., Demmig-Adams, B. and Adams, W.W., III, (1997)
Enhanced employment of the xanthophyll cycle and thermal energy
dissipation in spinach exposed to high light and N stress. Plant
Physiol. 113: 817–824.
von Caemmerer, S., (2000) Biochemical Models of Leaf Photosynthesis.
CSIRO Publishing, Collingwood, Australia.
Yamamoto, H., Kato, H., Shinzaki, Y., Horiguchi, S., Shikanai, T., Hase, T.,
et al. (2006) Ferredoxin limits cyclic electron flow around PSI (CEFPSI) in higher plants—stimulation of CEF-PSI enhances nonphotochemical quenching of Chl fluorescence in transplastomic
tobacco. Plant Cell Physiol. 47: 1355–1371.
Yamashita, A., Nijo, N., Pospisil, P., Morita, N., Takenaka, D., Aminaka, R.,
et al. (2008) Quality control of photosystem II. Reactive oxygen
species are responsible for the damage to photosystem II under
moderate heat stress. J. Biol. Chem. 283: 28380–28391.
(Received December 29, 2008; Accepted February 23, 2009)
Plant Cell Physiol. 50(4): 730–743 (2009) doi:10.1093/pcp/pcp032 © The Author 2009.
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