Diurnal and Developmental Changes in Energy Allocation of
Absorbed Light at PSII in Field-Grown Rice
Satoshi Ishida1, Nozomu Uebayashi1, Youshi Tazoe1, Masahiro Ikeuchi1, Koki Homma2, Fumihiko Sato1
and Tsuyoshi Endo1,*
Division of Integrated Life Sciences, Graduate School of Biostudies, Kyoto University, Kyoto, 606-8502 Japan
Laboratory of Crop Science, Graduate School of Agriculture. Kyoto University, Kyoto, 606-8502 Japan
*Corresponding author: E-mail, [email protected]; Fax, +81-75-753-6398.
(Received May 21, 2013; Accepted November 12, 2013)
2
The allocation of absorbed light energy in PSII to electron
transport and heat dissipation processes in rice grown under
waterlogged conditions was estimated with the lake model
of energy transfer. With regard to diurnal changes in energy
allocation, the peak of the energy flux to electron transport,
JPSII, occurred in the morning and the peak of the energy flux
to heat dissipation associated with non-photochemical
quenching of Chl fluorescence, JNPQ, occurred in the afternoon. With regard to seasonal changes in energy allocation,
JPSII in the rapidly growing phase was greater than that in the
ripening phase, even though the leaves of rice receive less
light in the growing phase than in the ripening period in
Japan. This seasonal decrease in JPSII was accompanied by an
increase in JNPQ. One of the reasons for the lower JPSII in the
ripening phase might be a more sever afternoon suppression
of JPSII. To estimate energy dissipation due to photoinhibition of PSII, JNPQ was divided into Jfast, which is associated
with fast-recovering NPQ mainly due to qE, and Jslow, which
is mainly due to photoinhibition. The integrated daily
energy loss by photoinhibiton was calculated to be about
3–8% of light energy absorption in PSII. Strategies for the
utilization of light energy adopted by rice are discussed. For
example, very efficient photosynthesis under non-saturating
light in the rapidly growing phase is proposed.
Keywords: Chl fluorescence Diurnal changes Energy
allocation in PSII Non-photochemical quenching Photoinhibition Rice (Oryza sativa).
Abbreviations: Fm (Fm0 , Fm00 ), maximum fluorescence
obtained by a saturating light pulse at pre-dawn (or during
daytime under light, or during daytime after a brief dark
treatment); Fo (Fo0 ), minimum fluorescence obtained under
the measuring light in the dark (or in the light); Fs, steadystate fluorescence under light; fast (Jfast), quantum yield (or
energy flux) of dissipation associated with NPQ that relaxed
rapidly in the dark; f,D (Jf,D), quantum yield (or energy flux)
of basic dissipation in PSII; NPQ (JNPQ), quantum yield (or
energy flux) of dissipation associated with NPQ; PSII (JPSII),
quantum yield (or flow rate) of electron transport in PSII;
slow (Jslow), the quantum yield (or energy flux) of dissipation associated with NPQ that relaxed slowly in the dark;
NPQ, non-photochemical quenching of Chl fluorescence;
PAR, photosynthetically active radiation; qP, fluorescence parameters that indicate the fraction of open PSII centers.
Regular Paper
1
Introduction
In plants, only some of the absorbed light energy is used for
photosynthetic electron transport in PSII, and a large portion of
the energy is lost through regulatory thermal dissipation, which
can be visualized as non-photochemical quenching (NPQ)
based on a quenching analysis of Chl fluorescence (Schreiber
et al. 1986). NPQ-associated thermal dissipation has been
shown to be an essential photoprotective mechanism of PSII.
However, it has not been clear how large a portion of light
energy absorbed in PSII is dissipated through this mechanism,
since the original parameters for non-photochemical quenching, such as qN = 1 – (Fm0 – Fo0 )/(Fm – Fo) and NPQ = (Fm –
Fm0 )/Fm0 , are not based on the quantum yield, and thus they
cannot be compared quantitatively with the quantum yield of
electron transport expressed as PSII = (Fm0 – Fs)/Fm0 (Genty
et al. 1989). Therefore, attempts have been made to simulate
the dissipation associated with NPQ on the basis of quantum
yield. The first of such attempts was reported by DemmigAdams et al. (1996) based on the puddle model of energy transfer, in which the light energy absorbed in antennae Chl is always
transferred to the same reaction centers. Later, new simulations
based on the lake model of energy transfer, in which the excitation energy of Chls can be exchanged among reaction centers,
were proposed independently by Kramer et al. (2004) and
Hendrickson et al. (2004).
NPQ-associated dissipation can be further divided into subcategories because NPQ has been shown to be induced by at
least three independent mechanisms: qE (energy quenching),
qT (NPQ associated with a state transition) and qI (NPQ associated with photoinhibition) (Quick and Stitt 1989). The most
Plant Cell Physiol. 55(1): 171–182 (2014) doi:10.1093/pcp/pct169, available online at www.pcp.oxfordjournals.org
! The Author 2013. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Plant Cell Physiol. 55(1): 171–182 (2014) doi:10.1093/pcp/pct169 ! The Author 2013.
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S. Ishida et al.
well studied mechanism of these dissipations is qE, which is
dependent on the presence of the PsbS subunit (Li et al.
2000), and is associated with the xanthophyll cycle (DemmigAdams 1990). Dissipation associated with qE is considered to be
an important regulatory process for photoprotection of PSII
because it is triggered by the light-induced generation of a
pH gradient across thylakoid membranes (for reviews, see
Horton et al. 1996, Müller et al. 2001). In a previous study
with rice transformants, in which psbS genes were silenced by
RNA interference (RNAi), we showed that PsbS-dependent
energy dissipation functioned in the photoprotection of PSII
by minimizing other types of more harmful dissipation pathways, but the absence of PsbSs did not enhance electron transport over a wide range of light intensities (Ishida et al. 2011).
Photoinhibition of PSII is a process that involves photodamage of the reaction center protein under strong light, and the
rate of photoinhibition is determined by the balance between
the damage and repair of damaged PSII complex through the
rapid synthesis of new reaction center protein. This damage–
repair cycle can be regarded as another important photoprotective mechanism in PSII (for recent reviews, see Nixon et al.
2010, Takahashi and Badger 2011). Energy loss associated with
qI can be quantified separately from that of qE (Kasajima et al.
2009), since both types of NPQ have been shown to relax in the
dark with different kinetics (Quick and Stitt 1989).
Although the new models for energy allocation in PSII seem
to be a good tool for deeper understanding of environmental
adaptation of photosynthesis, there is a paucity of field studies
using these models. Only a few studies have been carried out in
fruit trees (for example, see Losciale et al. 2010).
In this study, we tried to quantify diurnal energy usage and
dissipation in PSII in field-grown rice using Hendrickson’s lake
model, where absorbed light energy in PSII was allocated to
either electron transport, basal dissipation or NPQ-associated
dissipation. We divided total NPQ-associated dissipation into
NPQ with fast relaxation (corresponding to qE) and that with
slow relaxation (corresponding to qI). The contribution of qT,
which relaxes at an intermediate rate, has been reported to be
minor in higher plants (Quick and Stitt 1989).
We estimated the diurnal changes in energy allocation in
PSII in rice plants cultivated in Kyoto, Japan, from July to
September, which corresponds to the rapidly growing period
and ripening period. The integrated energy flow in PSII over an
entire day and its seasonal (developmental) changes were also
quantitatively analyzed.
Results
Dark recovery of NPQ in field-grown rice
To estimate the degree of photoinhibition during the daytime,
rice plants under full sunlight were dark adapted, then Fv/Fm
was measured (Fig. 1). To distinguish this from pre-dawn Fv/Fm,
the maximum quantum yield thus measured during daytime
was expressed as Fv00 /Fm00 . Recovery of NPQ, i.e. an increase in
172
Fig. 1 Recovery of Fv00 /Fm00 during dark treatment in rice leaves. Rice
plants were cultivated in pots for 6 weeks in a greenhouse. At noon on
a sunny day in July, plants were moved to a dark room and further
exposed to 1,100 mmol m2 s1 white light, which corresponded to
the light intensity of full sunlight in this season (see Figs. 2, 10), for
1 h to induce uniform photoinhibition among individual leaves (n = 8)
used for the measurements. The plants were then dark treated, and
Fv00 /Fm00 values were measured repeatedly.
Fv00 /Fm00 , during the dark adaptation, appeared biphasic, as
found in other plants. The first phase is generally regarded as
the recovery process of energy quenching (qE) and the slow
phase as that of photoinhibition (qI) (Quick and Stitt 1989).
From the recovery curve of NPQ, a part of NPQ, which was
found after 20 min dark adaptation, can represent slowly reversible NPQ. Therefore, in this study, we define photoinhibion
as light-induced inactivation of PSII, which can be visualized as
the remaining part of NPQ after 20 min dark adaptation.
‘Photoinhibition’ thus defined was an indicator of the balance
between light-induced inactivation of PSII centers and reconstruction of the damaged PSII complex.
Diurnal changes in photoinhibition
To estimate photoinhibition in field conditions, diurnal
changes in Fv00 /Fm00 in sunlit upright (vertical) leaves of rice
were examined in waterlogged field on July 22, 23 and 31,
and August 5 (rapidly growing phase before heading) and
September 11 and 18 (ripening phase) in 2010. The youngest
fully expanded leaves were chosen for the measurements.
Heading occurred on August 16. We chose south-facing upright
leaves, which represented the major portion of actively photosynthesizing leaves, for the measurements. Typical diurnal
changes in Fv00 /Fm00 are shown in Fig. 2, and the data for
other days are shown in Supplementary Fig. S1. Values of
Fv00 /Fm00 decreased by 6–10% at around noon. On most days,
the peak of photoinhibition corresponded well with the peak of
photosynthetically active radiation (PAR). Significant photoinhibition occurred even when the maximum light intensity at
noon was <600 mmol m2 s1, which was typically observed on
July 31. Another important observation is that, during the
ripening period (September 18), pre-dawn values of Fv/Fm
Plant Cell Physiol. 55(1): 171–182 (2014) doi:10.1093/pcp/pct169 ! The Author 2013.
PSII energy allocation in field-grown rice
Fig. 3 Typical fluctuations in PSII, NPQ and f,D, on a sunny day
(September 11). Fm0 and Fs values were measured repeatedly every
2.5 min from dawn to sunset in an upright leaf.
Fig. 2 Diurnal changes in photoinhibition on days with various light
conditions (July 22, July 31 and September 18). Fv00 /Fm00 values (blue
symbol) were measured every hour (n = 5). Upright leaves were covered with aluminum foil for 20 min before the measurement of fluorescence. PAR (orange symbol) was measured for the upright leaves
every 2.5 min, and the values were averaged every hour before the
measurement of Fv00 /Fm00 .
were <0.8. This may suggest the irreversible photodamage
of PSII.
Energy allocation in PSII simulated by the
lake model
A model for energy partitioning in PSII was proposed based on
the lake model of energy exchange among PSII centers
(Hendrickson et al. 2004, Kramer et al. 2004). Recently,
Kasajima et al. (2009) demonstrated that the models proposed
by Kramer et al. (2004) and that by Hendrickson et al. (2004)
are, in fact, different expressions of the same model.
They categorized total dissipation (1 – PSII) according to its
origin, i.e. basal intrinsic decay of excited Chl (NO in the
Kramer model or f,D in the Hendrickson model) and lightdependent regulative dissipation (NPQ), which includes qEand qI-dependent dissipation. In this study we adopted the
model of Hendrickson et al. because this model dose not require measurement of the Fo0 value, which is rather difficult to
measure in field conditions.
Using this model, diurnal changes in energy allocation in PSII
were monitored in south-facing upright leaves of rice. From
pre-dawn to after sunset, Fs was measured under ambient
light in the field, and Fm0 induced by a saturating light pulse
was measured every 2.5 min (for details, see the Materials and
methods). The diurnal changes in energy allocation were measured for 23 d in growing and ripening phases, including the
same days as the Fv00 /Fm00 measurements shown above.
Typical diurnal changes in quantum yields for electron
transport and dissipation (September 11) are shown in Fig. 3.
A significant fluctuation in PAR occurred at around noon
(10:00–14:00 h) on this day (see Supplementary Fig. S2).
PSII responded very rapidly to the sudden fluctuation in
PAR, and this change in PSII was accompanied by an inverse
change in NPQ. In contrast, f,D did not fluctuate.
The energy flux (mmol m2 s1) in PSII can be estimated
from the quantum yield of each pathway and the PAR
(mmol m2 s1). For example, energy flux to electron transport
JPSII, which is also designated as the electron transport rate
(ETR), was calculated by multiplying the estimated energy absorption in PSII (= PAR 0.84 0.5) and the quantum yield
PSII. This estimation is based on the assumption that 84% of
irradiated light energy is absorbed in leaves, and that the light
energy is equally distributed to PSII and PSI.
Diurnal changes in the energy flux of absorbed light in
PSII, to electron transport JPSII, basic dissipation Jf,D and NPQassociated dissipation JNPQ, on July 22 and September 18
are shown in Fig. 4 (data for other days are shown in
Supplementary Fig. S2). The peak of the energy flux to
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S. Ishida et al.
in Fig. 5A, and data for other days are shown in Supplementary
Fig. S3A. On most days, PSII in the morning was higher than
that in the afternoon. Interestingly, the afternoon decrease in
PSII was more evident in the ripening period than in the
growing phase. It should also be noted that the afternoon decrease in JPSII persisted until the late afternoon when PAR was
very low.
The relationship between PAR and NPQ (Fig. 5B;
Supplementary Fig. S3B) confirmed that NPQ in the afternoon was higher than that in the morning. No clear changes
were seen between f,D in the morning and in the afternoon
(Supplementary Fig. S3C).
Relationship between (PSII and Fv00 /Fm00
Fig. 4 Diurnal changes in energy allocation based on the model proposed by Hendrickson et al. (2004) in the rapidly growing phase (July
22) and ripening phase (September 18). PAR, Fm0 and Fs values were
measured repeatedly every 2.5–5 min from dawn to sunset in an upright leaf. JPSII, JNPQ and Jf,D were the rates of energy use or dissipation
calculated from the quantum yield for each process PSII, NPQ, and
f,D , respectively. For example, JPSII = PAR 0.5 0.84 PSII.
The relationship between PSII and photoinhibition as estimated by Fv00 /Fm00 was examined (Fig. 6; Supplementary
Fig. S4). A linear relationship between PSII and Fv00 /Fm00 was
found. It should be noted that in July, the leaves accepted light
in non-saturating ranges, while in September the leaves accepted saturating light at around noon, as pointed out in the
previous section (see Fig. 4). The very low PSII in September
was due to strong light. A minor decrease in Fv00 /Fm00 resulted in
a significant decrease in PSII in September, compared with July.
For example, when Fv00 /Fm00 was decreased to 0.74 under moderate sunlight, a leaf in July showed a PSII of 0.46, while in
September it showed a PSII of 0.34. There seemed to be no
clear difference in the relationship of Fv00 /Fm00 to PSII between
morning and afternoon.
Electron transport in photoinhibited leaves
electron transport JPSII appeared in the morning and gradually
decreased, which might coincide with the afternoon suppression of CO2 assimilation in rice reported by Ishihara and Saitoh
(1987). The peak of the energy flux to basic dissipation Jf,D
corresponded well with the peak of PAR, and the peak of the
energy flux to NPQ-associated dissipation JNPQ occurred in the
afternoon. Therefore, JPSII seems to be inversely correlated
with JNPQ.
The upright leaves received more sunlight in the ripening
phase (September 18) than in the growing phase (July 22) since
the altitude of the sun at noon in Kyoto decreased from 75 to
55 (see Table 1 for the integrated PAR over a whole day). In
July, leaves accepted light in non-saturating ranges
(<800 mmol m2 s1) even at around noon because the angle
of incidence was small, while in September the leaves accepted
more light (>1,100 mmol m2 s1).
In spite of more light acceptance, a larger portion of the
absorbed light energy seems to be dissipated as heat via an
NPQ-associated mechanism in September.
174
The above finding that PSII was linearly correlated with
Fv00 /Fm00 suggests that photoinhibition may regulate PSII.
To evaluate this possibility, young leaves of rice cultivated in
a growth chamber were exposed to strong light to induce
photoinhibition, and then PSII was measured (Fig. 7). Since
the photoinhibited state seemed to be stably maintained
during dark treatment from 20 to 80 min as shown in Fig. 1,
we could estimate changes in PSII caused by photoinhibition.
Photoinhibition that was comparable with the most severe
photoinhibition under natural light in our field conditions
(see Fig. 2) resulted in a decrease in PSII over low and moderate light ranges, while PSII under saturating light was at the
same level as that in the control (before light stress). The
decrease in PSII was evident under a light intensity
<600 mmol m2 s1. Thus, once photoinhibition occurred,
leaves would keep showing a low PSII when sunlight was
filtered through thin or heavy clouds (approximately
200–600 mmol m2 s1).
Relationship between PAR and quantum yields for
energy usage and dissipation
Calculation of quantum yield for the dissipation
associated with photoinhibition ((slow) and its
contribution to total dissipation
The relationship between PAR and PSII in the rapidly growing
phase (July 23) and the ripening phase (September 18) is shown
There were two minor components of NPQ besides qE: qI,
which is associated with photoinhibition; and qT, which is
Plant Cell Physiol. 55(1): 171–182 (2014) doi:10.1093/pcp/pct169 ! The Author 2013.
PSII energy allocation in field-grown rice
Table 1 Developmental changes in integrated daily energy flows (integrated daily quantum yields) in PSII
DPSII (ratio)
Df,D (ratio)
Dslow (ratio)
Dfast (ratio)
Daily integrated
amount of PAR
(mol quanta m2)
July 22
0.46
0.28
0.07
0.19
17.7
July 23
0.50
0.30
0.04
0.16
12.7
44
July 24
0.50
0.26
0.06
0.17
10.9
51
Jul 31
0.48
0.29
0.03
0.20
11
55
August 5
0.55
0.25
0.04
0.16
12.6
87
September 7
0.33
0.29
0.08
0.30
10.7
91
September 11
0.30
0.25
0.06
0.39
17.6
98
September 18
0.26
0.22
0.04
0.48
26.2
Days after
transplanting
Date
42
43
The values are expressed relative to total light energy absorbed in PSII (PAR 0.84 0.5).
Fig. 5 The relationship of PAR vs. PSII (A) and NPQ (B) in the growing phase (July 23) and ripening phase (September 18). Data in the morning
are shown by blue symbols and those in the afternoon by red symbols. PAR and quantum yield for each process shown as in Figs. 2 and 3 were
averaged every hour.
associated with a state transition (Quick and Stitt 1989). NPQ
associated with photoinhibition that relaxes slowly in the dark
can be distinguished from rapidly relaxing NPQs due to qE
and qT.
Therefore, we calculated the energy allocation associated
with qI according to Kasajima et al. (2009) with a minor
modification:
00
20 min dark 1=Fm Fs
slow ¼ 1=Fm
The fraction of the total absorption of light energy at midday
that is allocated to qI-associated dissipation (slow) is about
3–8% (Fig. 8). The energy allocated to qI-associated dissipation
was significant in the morning when PAR rapidly increased.
Integrated electron transport and dissipation over
an entire day
The total daily usage and loss of absorbed light energy in PSII
can be calculated by integrating the energy flux of each process
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S. Ishida et al.
Fig. 7 The light curve of PSII in photoinhibited leaves. Rice plants
were cultivated in a growth chamber at 28 C in 80% relative humidty
under a 300 mmol2 s1 quanta fluorescent lamp. The fifth or sixth
leaves of 4-week-old plants were used. First, Fv00 /Fm00 was measured in
leaves that had been dark treated for 20 min. Then PSII was measured
under increasing light intensity in a stepwise manner (each step for
3 min). For high light treatment, plants were illuminated with light of
2,000 mmol m2 s1 quanta from sodium lamps for 20 min. Then the
plants were dark treated for 20 min, after which Fv00 /Fm00 and PSII was
measured again under increasing light intensity in a stepwise manner.
Values are the averages and SDs of three samples.
Fig. 6 The relationship between PSII and Fv00 /Fm00 in the growing
phase (July 23) and ripening phase (September 18). Data in the morning are shown by blue symbols and those in the afternoon by red
symbols. Values of PSII measured every 2.5 min were averaged every
hour.
over an entire day. The seasonal changes in the daily energy flux
in the year 2010 are shown in Fig. 9, and the relative contribution of each process is shown in Table 1. As the season proceeded from July to September, when the altitude of the sun
gradually decreased, the integrated PAR to the upright leaves
increased accordingly. In July, when rice plants were in the
rapidly growing phase (early reproductive growth phase), the
rate of PSII in the integrated light absorption in PSII was about
50% (Table 1), meaning that about half of the absorbed light
energy was used for electron transport. In September, in the
ripening phase, the integrated daily use of energy for electron
transport decreased to 30%. This seasonal (developmental) decrease in energy use for electron transport was compensated by
an increase in NPQ-associated dissipation.
To examine the total fraction of photoinhibition-associated
dissipation in absorbed light energy in PSII, NPQ was divided
into fast and slow (Table 1). The daily quantum yield
of energy dissipation associated with photoinhibition represented by slow was 3–8% of the total light energy absorbed
in PSII.
176
Seasonal changes in the relationship between
light intensity and JPSII
The relationship between light intensity and JPSII on the days of
measurement during the season of 2010 is shown in Fig. 10.
In the growing phase, JPSII increased steadily up to about
1,000 mmol m2 s1, while it was saturated at about
600–700 mmol m2 s1 in the ripening phase. The energy
usage for electron transport was especially limited in the afternoon in the ripening phase.
On most days, PAR in the growing phase was less than
the level of saturation (1,000 mmol m2 s1) even at noon
(see Figs. 4, 5A), which suggests a constant shortage of light
in this period. In contrast, light intensity was excessive in the
ripening phase.
There was a sudden decease in JPSII in early September that
corresponded to about 20 d after heading. A significant metabolic shift toward ripening may have occurred at this time.
Discussion
Response of PSII energy allocation upon
fluctuating light in field conditions
Upon rapid fluctuation of PAR, the change in PSII was very
closely associated with inverse changes in NPQ, as shown in
Fig. 3. This means that the dissipation mechanism associated
with NPQ (most probably with qE) could very efficiently
Plant Cell Physiol. 55(1): 171–182 (2014) doi:10.1093/pcp/pct169 ! The Author 2013.
PSII energy allocation in field-grown rice
Fig. 8 Diurnal changes in energy allocation in PSII and the contribution of photoinhibition-associated thermal dissipation. The quantum yields of
each process were averaged every hour. Photoinhibition-associated thermal dissipation was calculated as shown in the text.
discharge excess light energy, which is not used for electron
transport, even when rapid fluctuations in PAR occurred
under field conditions.
In contrast, f,D was kept constant even under fluctuating
PAR. The origin of f,D has been suggested to be the constitutive deactivation of excited Chl through thermal and radiative
dissipation (Hendrickson et al. 2004, Kramer et al. 2004).
Afternoon decrease in energy flux to
electron transport
A midday and afternoon decrease in photosynthetic CO2
assimilation has been reported in a variety of plant species
(Schulze et al. 1974, Tenhunen et al. 1984, Küppers et al.
1986, Huc et al. 1994, Zotz et al. 1995, Pathre et al. 1998,
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S. Ishida et al.
Fig. 9 Seasonal changes in daily energy use and loss in PSII. Integrated
absorbed light energy (PAR), electron transport (PSII) and thermal
dissipations (NPQ, f,D) over a whole day were calculated from PAR,
JPSII, JNPQ and Jf,D shown in Fig. 3 and Supplementary Fig. S2. The first
and last measurements were performed on July 21 and October 6,
respectively.
Ishida et al. 1999), including rice in a paddy field (Ishihara and
Saitoh 1987). In most cases, stomatal closure due to a decrease
in water potential has been shown to be one of the main reasons for this decrease. The afternoon decrease in CO2 assimilation in rice has also been reported to be closely associated with
a decrease in CO2 supply (Ishihara and Saitoh 1987). However,
the afternoon decrease in PSII observed here was most evident
in the late afternoon, i.e. under lower light, when CO2 uptake
was probably not the rate-limiting factor for overall photosynthetic processes. Therefore, we have to assume that some other
factors, besides the decrease in CO2 supply that can limit PSII
in the afternoon, are involved. The inverse relationship between
PSII and NPQ might mean that NPQ is the factor that restricts PSII. However, previous studies showed that the level of
NPQ did not affect PSII over a wide range of light intensities
(Ishida et al. 2011). Therefore, qE seems not to have the potential to down-regulate PSII. Another factor of NPQ, photoinhibition (qI), can regulate PSII (see Fig. 7). However, under field
conditions, the peak of photoinhibition corresponded well to
Fig. 10 Relationship between light intensity and JPSII on every measuring day from July 21 (shown as 7/21 in the explanatory notes) to October 6
(10/6). For each morning or afternoon, all data for JPSII in the indicated ranges of PAR were collected and averaged.
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PSII energy allocation in field-grown rice
the peak of PAR (see Fig. 2; Supplementary Fig. S1), which
shows that photoinhibiton was not the main factor that
induced the afternoon decrease in PSII.
Rather, further analysis of Chl fluorescence in this study
indicated that the PSII was closely correlated with qP, an indicator of the acceptor-side oxidation (Supplementary Fig. S5).
Therefore, the low PSII in the afternoon was always associated
with high acceptor-side reduction. This suggests that rhw electron transport chain including PSI, or the carbon fixation cycle
(including CO2 supply) can be the rate-limiting factor that
induces the afternoon decrease in PSII.
Relationship between electron transport and
photoinhibition
High Fv00 /Fm00 indicated that only a small proportion of PSII was
photoinhibited. Therefore, we initially expected that Fv00 /Fm00
might remain high under non-saturating light, and might drop
steeply under saturating light while PSII decreased steadily as
light intensity increased. However, an unexpected linear correlation was found between these two parameters (see Fig. 6),
suggesting that photoinhibition regulated PSII over a wide
range of non-saturating light intensities (for details, see Fig. 8
and corresponding text).
The cause and effect relationship between PSII and Fv00 /Fm00
can be deduced from the results shown in Fig. 7. In photoinhibited leaves, the lower Fv00 /Fm00 leads to a decrease in PSII
under low light. Under saturating light, where the rate-limiting
step shifts from light absorption to CO2 fixation, no such restriction is found. Thus, photoinhibition could largely affect
PSII under low light but not under high light. Similar observations have been reported for wheat (Hurry et al. 1992) and
Arabidopsis (Russell et al. 1995). However, some reports
showed that photoinhibitied leaves had lower PSII even
under high light in physiological conditions (for example,
Hikosaka et al. 2004), which can happen when the electron
transport rather than CO2 fixation limits the rate of photosynthesis even under high light. Alternatively, lower PSII under
high light in photoinhibited leaves might be explained by the
fact that photoinhibited leaves can induce more photoinhibtion under high light than non-photoinhibited leaves (see
Chow et al. 2005). Therefore, whether or not photoinhibtion
limits PSII under high light may vary depending on the plant
species and also on physiological and environmental conditions, such as temperature and humidity, as well as the intensity
of irradiance. Further, the rates of photodegradation and repair
of the PSII complex might be independently changed according
to environmental conditions (Sundby et al. 1993).
In natural weather, light conditions fluctuate very frequently, but Fv00 /Fm00 values are very slow to fluctuate; thus,
the restriction of PSII by a lowered Fv00 /Fm00 may occur frequently. Hikosaka et al. (2004) also showed in Chenopodium
album that photoinhibition at any level of light intensity
decreased the photosynthetic rate, causing a significant reduction in daily carbon gain.
The energy loss due to photoinhibition in a natural environment has been rather difficult to estimate. However, our
estimation of integrated energy use and losses over a whole
day can now show how much energy is lost due to photoinhibition. Although the apparent fraction of photoinhibitiondependent loss was minor and comprised only about 3–8%
of the total absorption of light energy in PSII (Table 1), the
consequent decrease in JPSII can result in growth retardation,
since a considerable portion of the energy fixed by photosynthesis is used for respiration and only the remaining energy is
used for growth. We also have to consider that some portion of
the energy allocated to PSII is used for the repair of damaged
PSII complex, since a rapid damage–repair cycle especially on a
sunny day might consume considerable energy. While previous
studies on photoinhibition have focused on the damage to PSII
under saturating light, the present study under field conditions
demonstrates that more attention should be paid to the relevance of effects of photoinhibition under low light or fluctuating light in the field.
Nature of energy dissipation associated
with photoinhibition
Kasajima et al. (2009) divided NPQ into fast and slow. The
value of NPQ represents energy dissipation induced by light
illumination. Thus, slow is categorized as the portion of thermal dissipation in PSII. While the molecular nature of slow is
not clear, it is reasonable to assume that photoinhibitionassociated disconnection between antennae and PSII centers
may induce the additional de-excitation of Chl in antennae.
Therefore, in this study, we used slow as an indicator of
energy dissipation associated with photoinhibition. However,
Johnson and Ruban (2010) demonstrated the presence of a
slowly relaxing component of qE, which could not be distinguished from qI in Arabidopsis. Therefore, we have to consider
that our definition of photoinhibition adopted here might
include energy dissipation associated with the slowly relaxing
component of qE.
In addition to the light-induced decrease in Fv00 /Fm00 , we
should consider the physiological role of the irreversible
decrease in pre-dawn Fv/Fm in the late ripening period (see
Fig. 2). This seasonal decrease in Fv/Fm can probably reduce
overall JPSII, as in the case of the light-induced decrease in
Fv00 /Fm00 associated with photoinhibition within the narrow definition. When this seasonal decrease in Fv/Fm was considered as
a form of photoinhibition, the total contribution of photoinhibition in the energy dissipation processes in autumn leaves
would be greater than the calculated values shown in
Table 1. In this sense, the photoprotective role of reduced Fv/
Fm in autumn should be reconsidered. The molecular nature of
the decrease in pre-dawn Fv/Fm in autumn is not clear.
However, it may be explained in part by the senescenceassociated disorder of the PSII complex and the degradation
of Chl (Holloway et al. 1983). Alternatively, sustained NPQ,
which is associated with high zeaxanathin + anthrexanthin
Plant Cell Physiol. 55(1): 171–182 (2014) doi:10.1093/pcp/pct169 ! The Author 2013.
179
S. Ishida et al.
contents, typically observed in photoinhibited or overwintering
evergreens (Demmig-Adams et al. 1998, Ebbert et al. 2001),
might occur in rice.
Seasonal changes in energy use
It should be noted that seasonal changes in JPSII shown here
were based on the assumption that the light absorption value
of 0.84 was not changed from July to September. In this study
we used the constant light absorption value because (i) the
absolute value of Fm, which can be a rough indicator of PSII
density, was not changed: 1.00 ± 0.15 in August (n = 7) and
1.04 ± 0.13 in September (n = 6); and (ii) Fv/Fm was stable
until September 25, after which it drastically decreased (not
shown). However, the contents and the composition of pigments including Chls and carotenoids as well as the thickness
and the structure of the leaf blade vary depending on the
growth stage. In addition, leaf senescence starts after heading
in rice plants. Therefore, these aspects of changes in leaf quality
should be more carefully considered in future studies.
Values of PSII and total energy use for JPSII were decreased in
the ripening period compared with the growing phase (see
Fig. 4). This is partly because upright leaves accept more sunlight in autumn. However, the main cause might be the decrease in CO2 fixation activity in autumn, as discussed above.
The decrease in energy use for JPSII was compensated by
an increase in NPQ-associated dissipation, most probably qEassociated dissipation. Photosynthetic products in rice plants
are stored in the leaf sheaths and the basal part of culms in the
early reproductive stage, and the stored carbohydrates are
transferred to panicles in the ripening stage (Togari et al.
1954, Murayama et al. 1955). As a result, the net daily assimilation of CO2 has been reported to be maximal at the early
reproductive growth phase and then gradually deceases on a
leaf area basis (Jiang et al. 1988) as well as a whole-plant basis
(Saitoh et al. 1990). Therefore, the higher JPSII in the early
reproductive stage may be explained by the greater photosynthetic sink capacity.
Upright leaves of the growing phase accepted less light than
those in the ripening period, even though the maximum JPSII in
the growing phase was higher than that in the ripening phase
(see Figs. 4, 5A). As a result, the leaves in the growing phase
always performed photosynthesis under non-saturating light
(see Fig. 10). When crop photosynthesis is considered, while
upright leaves accept less light, the leaf area that can accept
direct sunlight is greater when the sun is high above the horizon
(Duncan 1971). Under saturating light, a large portion of the
light energy is lost as JNPQ, and thus the efficiency of energy
usage (PSII) is greater under non-saturating light than under
saturating light. Therefore, even from the limited data based on
single-point measurement adopted in this study, we can suppose that ‘non-light-saturated photosynthesis’ in the growing
phase results in the very efficient use of light energy, which
might maximize total CO2 fixation in the whole plant.
The higher PAR and lower PSII in autumn (see
Table 1) suggest that autumn leaves are subjected to severe
180
photo-oxidative stress. However, no clear increase in slow was
observed in autumn (see Fig. 8 and Table 1). This probably
means that photoprotective mechanisms, such as NPQ induction and the water–water cycle that scavenges active oxygen
species (Asada 1999), functioned well to cope with the severe
photo-oxidative stress in autumn. We need further experiments with P700 redox measurement and a gas exchange
chamber in combination with Chl fluorescence analysis to
estimate the contribution of the water–water cycle as a photoprotection mechanism.
Materials and Methods
Plants
Japonica rice cv. Nipponbare (Oryza sativa L.) was cultivated in
a paddy field on the experimental farm of the Graduate School
of Agriculture of Kyoto University, Japan. The seeds were sown
on April 30, 2009 and May 10, 2010, respectively, and the
1-month-old seedlings were transplanted in a paddy field at a
plant density of 22.2 plants m2 in 2 m2 plots. Fertilizer was
applied at 5–5–5 g m2 of N–P2O5–K2O before transplantation. The field was continuously waterlogged throughout
the experiment. The measurements of Chl fluorescence were
done during the vegetative growth phase, which could
be divided into two phases; the rapidly growing phase before
heading (August 16 in 2010) and the ripening phase after
heading.
Chl fluorescence
Chl fluorescence parameters were measured using a PAM2000
Chl fluorometer (Waltz). The leaf clip of the fluorometer was
set on the surface of a south-facing upright (vertical) leaf at a
position located >50 mm away from the top. The leaf clip was
fixed throughout the whole day; thus, the fluorescence parameters shown below were obtained from a single point of a leaf.
PAR was measured with a light sensor positioned on the leaf
clip. The minimum fluorescence at open PSII centers was determined by measuring light. It was confirmed by the application
of far-red light that the intensity of the measuring light was
sufficiently weak not to induce any photochemical increase in
Fo level fluorescence. After an application of a 1 s pulse of
saturating white light (3,000 mmol m2 s1 white light) to estimate the precise Fv/Fm (maximum fluorescence yield at closed
PSII centers at pre-dawn), the measuring light intensity was
increased by changing the light interval from 0.6 to 20 kHz to
stabilize the level of the steady-state Chl fluorescence (Fs).
During daytime, Fs was continuously monitored under ambient
light. Fm and Fm0 (maximum fluorescence yield during the daytime under light) were measured every 2.5 min by the application of the saturating light pulse.
For the measurement of Fm00 (maximum fluorescence yield
during the daytime in dark-treated leaves), a section of leaf was
dark treated for 20 min before the application of the saturating
pulse. Fm00 was measured every hour.
Plant Cell Physiol. 55(1): 171–182 (2014) doi:10.1093/pcp/pct169 ! The Author 2013.
PSII energy allocation in field-grown rice
The quantum yield of photochemistry in PSII during steadystate photosynthesis (PSII) was calculated as (Fm0 –Fs)/Fm0 .
NPQ was calculated as (Fm – Fm0 )/Fm0 . The quantum yields of
basal dissipation and NPQ-associated dissipation were calculated as f,D = Fs/Fm and NPQ = 1 – (PSII + f,D), respectively,
according to Hendrickson et al. (2004). Fo0 was calculated as
Fo/(Fv/Fm + Fo/Fm0 ) according to Oxborough and Baker (1998),
from Fm and Fo values measured before dawn and Fm0 . qP was
calculated as (Fm0 – Fs)/(Fm0 – Fo0 ). Fm0 , Fs, light intensity and
temperature in the field were measured every 2.5 min from predawn to after sunset.
The rates of electron transport JPSII, JNPQ, and basal dissipation Jf,D were calculated by multiplying the estimated energy
absorption in PSII (= PAR 0.84 0.5) and the quantum
efficiencies, PSII, NPQ and f,D, respectively.
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by the Ministry of Agriculture and
Fishery of Japan [the project ‘Functional analysis of genes relevant to agriculturally important traits in rice genome’].
Disclosures
The authors have no conflicts of interest to declare.
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