Plant Cell Physiol. 44(8): 828–835 (2003)
JSPP © 2003
Contribution of Photosynthetic Electron Transport, Heat Dissipation, and
Recovery of Photoinactivated Photosystem II to Photoprotection at Different
Temperatures in Chenopodium album Leaves
Tsonko D. Tsonev 1, 2 and Kouki Hikosaka 1, 3
1
2
Graduate School of Life Sciences, Tohoku University, Aoba, Sendai, 980-8578 Japan
Acad. M. Popov Institute of Plant Physiology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str., Bl. 21, 1113 Sofia, Bulgaria
;
Temperature dependence of photoinhibition and photoprotective mechanisms (10–35°C) was investigated for
Chenopodium album leaves grown at 25°C under 500 mmol
quanta m–2 s–1. The fraction of active photosystem II (PSII)
was determined after photoinhibitory treatment at different temperatures in the presence and absence of lincomycin, an inhibitor of chloroplast-encoded protein synthesis.
In the absence of lincomycin, leaves were more tolerant to
photoinhibition at high (25–35°C) than at low (11–15°C)
temperatures. In the presence of lincomycin, the variation
in the tolerance to photoinactivation became relatively
small. The rate constant of photoinactivation (kpi) was stable at 25–35°C and increased by 50% with temperature
decrease from 25 to 11°C. The rate constant of recovery of
inactivated PSII (krec) was more sensitive to temperature; it
was very low at 11°C and increased by an order of magnitude at 35°C. We conclude that the recovery of photoinactivated PSII plays an essential role in photoprotection at 11–
35°C. Partitioning of light energy to various photoprotective mechanisms was further analyzed to reveal the factor
responsible for kpi. The fraction of energy utilized in photochemistry was lower at lower temperatures. Although the
fraction of heat dissipation increased with decreasing temperatures, the excess energy that is neither utilized by photochemistry nor dissipated by heat dissipation was found to
be greater at lower temperatures. The kpi value was
strongly correlated with the excess energy, suggesting that
the excess energy determines the rate of photoinactivation.
Introduction
Although light is the energy source for plant growth,
strong light can cause photoinhibition in photosynthesis.
Photosystem II (PSII), which is the major site of photoinhibition, is believed to be inactivated when absorbed energy
exceeds that consumed by photosynthetic processes (Powles
1984, Demmig-Adams and Adams 1992, Osmond 1994, Kato
et al. 2003). Plants have evolved several photoprotective mechanisms that reduce excess energy. It has been suggested that
consumption of reducing power by photosynthetic CO2 fixation (Powles 1984), photorespiration (Powles and Osmond
1979), heat dissipation via the xanthophyll cycle (DemmigAdams and Adams 1992, Gilmore 1997), the water–water
cycle (Osmond and Grace 1995, Asada 1999, Makino et al.
2002) and by cyclic electron flow around PSII (Miyake and
Yokota 2001) contribute to photoprotection of PSII. Furthermore, fast recovery of photoinactivated PSII via degradation
and re-synthesis of damaged D1 protein (Aro et al. 1993a, Aro
et al. 1993b) also contributes to maintaining the fraction of
functional PSII in leaves.
The susceptibility to photoinhibition depends on temperature. A number of studies have shown that leaves are more susceptible at low temperatures (Powles 1984, Osmond 1994,
Huner et al. 1993, Huner et al. 1998, Allen and Ort 2001). It
has been suggested that temperature dependence in recovery of
inactivated PSII is related with the higher susceptibility to photoinhibition at lower temperatures (Allen and Ort 2001). The
rate of recovery is often comparable to that of photoinactivation in PSII at normal temperatures (Wünschmann and Brand
1992, Aro et al. 1993b, Lee et al. 2001, Kato et al. 2002b), but
is very low at low temperatures (Greer et al. 1986, Aro et al.
1990a, Salonen et al. 1998), suggesting that low temperatures
accelerate photoinhibition through the suppression of the
recovery. However, it has been indicated that the susceptibility
to photoinhibition cannot be accounted for solely by a differential capacity for repair of inactivated PSII (Huner et al. 1993).
For example, the recovery rate of inactivated PSII is not correlated with the susceptibility to photoinhibition when species
with various chilling-sensitivity were compared (Öquist and
Huner 1991). Hurry and Huner (1992) also showed that, even
when recovery of inactivated PSII is inhibited, cold-hardened
Keywords: Chenopodium album — D1 protein turnover —
Excess light energy — Photoinhibition — Photoprotective
mechanisms — Temperature dependence.
Abbreviations: a, fraction of active PSII; D, fraction of the heat
dissipation; E, fraction of the excess energy; Fm, F0, Fv, maximum,
initial and variable chlorophyll fluorescence; Fm¢ maximum chlorophyll fluorescence during illumination; Fs, steady state chlorophyll
fluorescence; Fv¢ variable chlorophyll fluorescence during illumination; kpi, krec, rate constants for photoinactivation and recovery; L, fraction of the light energy that is lost in the dark; NPQ, non-photochemical quenching; PSII, Photosystem II; qP, photochemical quenching
coefficient.
3
Corresponding author: E-mail, [email protected]; Fax, +81-22-217-6699.
828
Temperature dependence of photoprotective processes
wheat was still more tolerant to photoinhibition than non-hardened wheat.
It has been proposed that photoinhibition is related to the
redox state of PSII (Öquist et al. 1992, Huner et al. 1993,
Huner et al. 1998). Low temperature inhibits consumption of
light energy by photosynthesis and photorespiration, and thus
absorbed light energy likely becomes excessive, presumably
leading to over-reduction in PSII. Susceptibility to photoinhibition has been shown to be correlated with the redox state of
PSII, regardless of the environmental constraints on photosynthesis caused by low temperature or light acclimation (Ögren
and Rosenqvist 1992, Öquist et al. 1992, Gray et al. 1996).
This suggests that reduction in the consumption of light energy
is responsible for higher susceptibility to photoinhibition under
lower temperatures.
Although knowledge is accumulating on photoprotection
at low temperature, it is still unclear what photoprotective process is quantitatively responsible for temperature dependence of
the susceptibility to photoinhibition. When there exist plural
photoprotective processes, a strong correlation between photoinhibitory susceptibility and activity of one process does not
necessarily mean that contribution of this process is quantitatively large. We have demonstrated that quantitative contribution of photoprotective mechanisms to photoinhibitory susceptibility varies among leaves grown under different conditions
(Kato et al. 2002b, Kato et al. 2003). We raised Chenopodium
album plants under high-light and high-nitrogen (HL-HN),
high-light and low-nitrogen (HL-LN), and low-light and highnitrogen (LL-HN) conditions. The HL plants were more tolerant to photoinhibition than the LL-HN plants. Although the HL
plants had a higher recovery rate of inactivated PSII irrespective of nitrogen availability, there was still a difference in the
rate of photoinactivation in the presence of an inhibitor of the
PSII recovery between the HL and LL plants (Kato et al.
2002b). We further estimated partitioning of light energy to
photochemistry, heat dissipation and excess energy using the
model of Demmig-Adams et al. (1996). The HL-HN plants had
the highest rate of photochemistry and the HL-LN plants had
greater heat dissipation fraction, both contributing to reduction
in excess energy. The LL-HN plants had the lower rate of photochemistry and the smaller fraction of heat dissipation, leading to the greatest excess energy (Kato et al. 2003). Although
there was no single photoprotective process that solely
explained the tolerance to photoinhibition, the rate constant of
photoinactivation without recovery was strongly correlated
with the excess energy that is neither utilized in photosynthesis
nor dissipated as heat, suggesting that the excess energy determines the rate of photoinactivation (Kato et al. 2003). These
facts suggest that the tolerance to photoinhibition results from a
combination of various photoprotective mechanisms.
The aim of the present study was to evaluate quantitative
contribution of photoprotective mechanisms to photoinhibitory
susceptibility under various temperatures. C. album plants were
grown under HL-HN conditions, and their leaves were exposed
829
Fig. 1 Temperature dependence of the maximum quantum yield of
PSII photochemistry in the dark (Fv/Fm) (a) and photosynthetic rates at
1,000 mmol quanta m–2 s–1 under ambient CO2 partial pressure (b) for
C. album leaves. Means and SE of eight replications are shown.
to various temperatures under strong light. Using the methods
of Kato et al. (Kato et al. 2002b, Kato et al. 2003), temperature
dependence of the susceptibility to photoinhibition, photosynthetic rates, the rate constant of photoinactivation and recovery, and of partitioning of absorbed light energy was determined. We discuss how temperature affects the susceptibility to
photoinhibition.
Results
C. album plants were grown at 25/20°C (day/night) under
500 mmol quanta m–2 s–1 with sufficient nutrient supply (see
Materials and Methods). Fig. 1a shows the maximal quantum
yield of photochemistry (Fv/Fm) of leaves adapted to the dark at
a given temperature. Fv/Fm was higher than 0.8 at all temperatures, suggesting that the maximal quantum yield is less sensitive to temperatures if leaves are in the dark. Fig. 1b shows the
photosynthetic rate at 1,000 mmol quanta m–2 s–1. The photosynthetic rate was highest at 25°C.
In the present study we defined photoinhibition as the
reduction of Fv/Fm as there was a linear relationship between
quantum yield of CO2 assimilation and Fv/Fm (data not shown).
At all temperatures Fv/Fm decreased with illumination time in
the absence of lincomycin (Fig. 2). This reduction was mitigated with time and Fv/Fm reached in a near-equilibrium level
after about 300 min. Such an equilibration to a steady-state
level of active PSII can be expected if a first-order degradation
830
Temperature dependence of photoprotective processes
Fig. 2 Photoinactivation of PSII (indicated by relative reduction
in Fv/Fm) measured in the presence (open circles) and absence
(closed circles) of the chloroplast-encoded protein synthesis
inhibitor, lincomycin, for C. album leaves illuminated with 900–
1,000 mmol quanta m–2 s–1 at different temperatures. Fitted curves
are a = {krec + kpiexp[– (kpi + krec)t]}(kpi + krec)–1 (continuous line)
and a = exp(– kpi t) (broken line), where a is the fraction of active
PSII and t is the illumination time. See Fig. 3 for values of kpi and
krec. a–f represent results obtained at 11, 15, 20, 25, 30, and 35°C,
respectively.
is counteracted by a first-order recovery reaction during highlight treatment (Tyystjärvi et al. 1992). At higher temperatures
(25–30°C) the Fv/Fm values at 300 min decreased only by 10%
from the initial values while larger decreases were found at
lower temperatures.
Photoinactivation can be revealed only when the rate of
de novo synthesis of D1 protein is inhibited. Lincomycin was
used to inhibit the D1 protein synthesis. It is known that lincomycin exacerbates photoinhibition without causing any other
effects (Tyystjärvi et al. 1992, Aro et al. 1993b). The reduction
of Fv/Fm was thus greater in lincomycin-treated leaves than in
untreated ones. Temperature dependence of the reduction of
Fv/Fm in lincomycin-treated leaves was not remarkable, in contrast to that in untreated leaves.
We assumed that reduction in the fraction of active PSII
with illumination time follows a model of two opposing firstorder reactions with different rate constants for photoinactivation (kpi) and concurrent recovery (krec). Assuming that the rates
of photoinactivation and recovery are proportional to the concentration of active and inactivated PSII, respectively, the fraction of active PSII (a) is expressed as:
a = {krec + kpiexp[– (kpi + krec)t]}(kpi + krec)–1,
(1)
(see Tyystjärvi et al. 1992, Kato et al. 2002b), where t is illumination time (min). If lincomycin is added, krec becomes zero.
Then Eqn. 1 is simplified as:
a = exp(–kpi t).
(2)
First, kpi was determined by applying Eqn. 2 to a time-course
of Fv/Fm during illumination in the presence of lincomycin.
With the kpi value, then krec was obtained from a time-course of
Fv/Fm during illumination in the absence of lincomycin by Eqn.
1. Fitted curves of Eqn. 1 and 2 are shown in Fig. 2.
Calculated rate constants (kpi and krec) were plotted against
temperature (Fig. 3a, b). The curve of the rate constant of photoinactivation (kpi) had weak temperature dependence; it was
similar at higher temperatures (25–35°C) and increased by 50%
with temperature decline from 25 to 11°C. The rate constant of
recovery (krec) showed a strong temperature dependence; it
increased by an order of magnitude with temperature and
reached a maximum at about 30–35°C. Fig. 3c shows the fraction of active PSII at the equilibrium calculated from Eqn. 1
(t >10,000 min). At 11°C, half of PSII were inactive at the
equilibrium because the values of kpi and krec were similar to
each other. At higher temperatures, the fraction of active PSII
Temperature dependence of photoprotective processes
831
Fig. 3 Temperature dependence of the rate constants for photoinactivation, kpi (a), and recovery, krec (b), and the fraction of active PSII at
the equilibrium (c) in C. album leaves illuminated with 1,000 mmol
quanta m–2 s–1 calculated according to Tyystjärvi et al. (1992). See text
for the fraction of active PSII at the equilibrium.
Fig. 4 Temperature dependence of the quantum yield of PSII photochemistry in the light (DF/Fm.) (a), the fraction of open PSII (qP) (b),
the quantum yield of open PSII (Fv¢/Fm.) (c), and NPQ (d) in C. album
leaves at 1,000 mmol quanta m–2 s–1. Means and SE of eight replications are shown.
is greater due to higher values of krec.
Fig. 4 shows the quantum yield of photochemistry in the
light (DF/Fm¢) as the product of the fraction of open PSII (qP)
and the quantum yield of open PSII (Fv¢/Fm¢) (Genty et al.
1989). DF/Fm¢ (Fig. 4a) and qP (Fig. 4b) increased with increasing temperature at low temperatures and had stable values at
25–35°C. Fv¢/Fm¢ also tended to be higher at higher temperatures but its temperature dependence was smaller than those of
DF/Fm¢ and qP (Fig. 4c). Conversely, non-photochemical
quenching (NPQ) decreased and showed a minimum at about
30°C (Fig. 4d).
We used these data to estimate partitioning of light energy
to various pathways as a function of temperature (Fig. 5). If
PSII could completely utilize absorbed photon energy for photochemistry, the maximum quantum yield in the dark (Fv/Fm)
and in the light (Fv¢/Fm¢) should be 1.0 (Demmig-Adams et al.
1996). The difference between 1 and Fv/Fm (L) can result from
some form of energy loss in the dark. Fv/Fm – Fv¢/Fm¢ defines
the fraction of the variable heat dissipation in the light (D)
(Demmig-Adams et al. 1996). P is the fraction of photochemical electron transport defined by DF/Fm¢ (Genty et al. 1989).
The fraction of absorbed light neither going into photochemical electron transport nor heat dissipation defines excess energy
(E) and is given by 1 – L – D – P (Demmig-Adams et al. 1996,
Kato et al. 2003). The fraction of energy consumed by photochemistry decreased by a half as temperature decreases from 35
to 10°C (Fig. 5). The fraction of heat dissipation increased in a
counteracting manner, which partly contributed to decrease in
the excess energy. However, the decrease in photochemistry
was larger than that of heat dissipation, resulting in doubling of
excess energy as temperature decreases from 35°C to 10°C. To
test the hypothesis that the rate of photoinactivation depends on
the excess light energy we plotted the rate constant of photoinactivation (kpi) against excess light energy (Fig. 6). Excess light
832
Temperature dependence of photoprotective processes
Fig. 5 Temperature dependence of the fractions of absorbed light in
PSII that is lost in the dark (L), that is dissipated as heat (D), that is
utilized in photochemistry (P) in C. album leaves at 1,000 mmol quanta
m–2 s–1. The fraction of absorbed light neither going into P, D, nor L
defines excess (E). Means and SE of eight replications are shown.
energy gave a strong linear relationship with the kpi value.
Discussion
As it has been known in various plant species, strong temperature dependence was found in the susceptibility to photoinhibition in C. album leaves (Fig. 2). The fraction of active PSII
at the equilibrium was found to be lowest (49%) at 11°C while
it was about 90% at 25–35°C (Fig. 3c). However, when the
repair of D1 protein is blocked, the photoinactivation was
found at all temperatures in a range of 11–35°C. Although the
rate constant of photoinactivation (kpi) was found to be temperature dependent, the difference was only 50% (Fig. 3a). On the
other hand, the rate constant of recovery (krec) was highly sensitive to temperature. The value of krec was similar to that of kpi at
11°C, leading to 49% of the fraction of active PSII at the equilibrium (Fig. 3c) and increased by an order of magnitude from
11 to 35°C (Fig. 3b). To evaluate their quantative contribution
to photoprotection, we conducted a simple sensitivity analysis.
Leaves photoinhibited at 25°C had the kpi and krec values of
0.00159 and 0.0134 min–1, respectively, resulting in the fraction of active PSII of 89% at the equilibrium. When we replace
the kpi value by 0.00236, which was observed at 11°C, the fraction of active PSII is calculated to be 85%. When we similarly
replace the krec value by 0.00226, which was observed at 11°C,
the fraction greatly decreases to 60%. Thus the temperature
dependence of recovery of inactivated PSII was most responsible for the susceptibility to photoinhibition.
Inactivated PSII is recovered in the following processes:
inactivated PSII complex is transferred from the appressed
region to the non-appressed region of thylakoid membranes
Fig. 6 Relationship between the rate constant of photoinactivation
(kpi) and the excess energy (E). Data from Fig. 3 and 5 are used.
and then is repaired after degradation and de novo synthesis of
D1 protein (Aro et al. 1993a). It was shown that low temperature markedly reduces the rate of enzymatic PSII repair processes and a decrease in membrane fluidity limits the migration
of inactive PSII complexes between stroma and grana thylakoids (Aro et al. 1990b, Baroli and Melis 1998, Salonen et al.
1998). Nishiyama et al. (2001) suggested that oxidative stress
inhibits the repair of inactivated PSII. These factors might
cause the large decrease in krec at lower temperatures.
Although temperature dependence of kpi was smaller than
that of krec, temperature had a significant effect on the rate of
photoinactivation: kpi increased by 50% as temperature declines
from 25 to 11°C (Fig. 3a). Decrease in the quantum yield of
PSII photochemistry in the light (DF/Fm¢) was partly related to
the increase in the rate of photoinactivation at low temperatures (Fig. 4). The reducing equivalents produced by photochemistry are consumed by photosynthetic CO2 fixation, photorespiration, the PSII cyclic electron transport, and the water–
water cycle. Temperature dependence of the photosynthetic
rate, which had a maximum at 25°C, was slightly different
from that of DF/Fm¢. In C3 plants, inhibition of photosynthetic
rates at moderately high temperature is usually attributed to an
increase in the ratio of oxygenation/carboxylation activities in
ribulose-1,5-bisphosphate carboxylase (RuBPCase). As temperature increases, the ratio of dissolved O2/CO2 and the specificity of RuBPCase for O2 increases, thus favoring oxygenase activity (Sage and Sharkey 1987, Crafts-Brandner and
Salvucci 2002). Therefore, at higher temperatures photorespiration is suggested to have larger contribution to photoprotection. On the other hand, low temperatures suppress the activity
of many photosynthetic processes (Berry and Björkman 1980).
Many authors have indicated that electron transport in the thylakoid membranes is strongly temperature dependent (Armond
et al. 1978, Kirschbaum and Farquhar 1984, Laisk and Oja
Temperature dependence of photoprotective processes
1994, Hikosaka et al. 1999). Thus the electron transport may
mainly explain temperature dependence of DF/Fm¢ at lower
temperatures.
In contrast to DF/Fm¢, NPQ and 1 – Fv¢/Fm¢ were found to
be higher at lower temperatures (Fig. 4). This suggests that
leaves could dissipate more energy as heat at lower temperatures. However, according to the model of Demmig-Adams et
al. (1996), the fraction of energy dissipated as heat increased
only by 10% from 25 to 10°C, which was smaller than the
decrease in the fraction of energy consumed in photochemistry
(30%) (Fig. 5). Thus, although heat dissipation contributed to
photoprotection at low temperatures, its contribution was not
sufficient to fully compensate for the reduction in the electron
transport activity.
The excess energy (E) that is neither consumed by photochemistry nor dissipated as heat increased with decreased
temperature (Fig. 5). kpi was positively correlated with E, suggesting that the excess energy determines the rate of photoinactivation. This is consistent with our previous finding that
kpi is proportional to E irrespective of photoinhibitory irradiance and growth conditions (Kato et al. 2003). E can also be
expressed as (1–qP) ´ Fv¢/Fm¢ (Demmig-Adams et al. 1996).
This is the excitation energy absorbed by closed PSII, which
was not dissipated by heat. Accumulation of excitation energy
in closed PSII may generate long-lived excited states of Chl
(3Chl*) and singlet excited oxygen (1O2). 1O2 is generated in
the PSII reaction centre by interaction of the ground state
oxygen (3O2) with the 3Chl* produced in the recombination
reaction of reduced pheophytin with P680+ (Macpherson et al.
1993). 1O2 generated within the protein matrix of the reaction centre brings about specific damage (Aro et al. 1993a,
Andersson and Barber 1996). Thus the increased amount of
excess energy may lead to proportional increase in the production of 1O2.
It should be noted that the kpi-E relationship was a linear
function from the origin in the study of Kato et al. (2003),
while it had a positive Y-intercept in the present study (Fig. 6),
suggesting that the effect of the excess energy on photoinactivation is relatively weak at lower temperatures. This may be
explained partly by the re-activation of PSII without turnover
of D1 protein. It is known that recovery after low temperature
photoinhibition exhibits a rapid initial phase which seems to be
independent of protein synthesis (Hurry and Huner 1992,
Leitsch et al. 1994). This rapid recovery has been attributed to
epoxidation of zeaxanthin via the xanthophyll cycle in leaves
of several plant species (Thiele et al. 1996, Thiele et al. 1998,
Jahns and Miehe 1996, Xu et al. 2000). Huner et al. (1993)
speculated that a population of inactivated PSII can revert rapidly to active PSII at low temperatures through conformational
changes. In the present study, recovery of inactivated PSII
through processes other than protein synthesis was ignored. If
the population of recovered PSII through such processes is
large at low temperatures, the rate of photoinactivation might
be underestimated.
833
We can summarize the quantitative contribution of photoprotective mechanisms at different temperatures as follows.
The energy utilized by photochemistry, which may be eventually consumed either by photosynthesis, photorespiration, the
water–water cycle, and the PSII cyclic flow, decreased as temperature reduces. The energy dissipated as heat partly increased
with decreased temperature in a counteracting manner to the
decrease in photochemistry, but the increase was insufficient to
compensate for the reduction in photochemistry. As a result,
the excess energy increased with decreased temperature, accelerating the rate constant of photoinactivation by 50%. The rate
constant of recovery of inactivated PSII had stronger temperature dependence than the rate constant of photoinactivation,
indicating that the rate of recovery was the factor most responsible for temperature dependence of the susceptibility to photoinhibition. One may consider that the present result contradicts previous results suggesting that reduction in consumption
of light energy, especially by photosynthesis, is responsible for
higher susceptibility to photoinhibition (Öquist et al. 1992,
Huner et al. 1993, Huner et al. 1998, Gray et al. 1996). However, these previous authors did not necessarily evaluate quantitative contribution of photoprotective mechanisms. Consider
a situation where two leaves have similar rate constants of
recovery to each other but have different photosynthetic capacities, leading to different susceptibilities to photoinhibition.
There will be a correlation between the susceptibility and photosynthetic capacity irrespective of contribution of the recovery. We stress that quantitative evaluation is indispensable to
clarify how tolerance to photoinhibition is determined, because
it results from a combination of various photoprotective mechanisms.
Materials and Methods
C. album L. is a broad-leaved summer annual, which colonizes
disturbed habitats. Leaves of C. album do not exhibit apparent chloroplast movement (Oguchi et al. 2003). Plants were grown in a phytotron
(14/10 h photoperiod, 25/20°C day/night temperature, 80% relative air
humidity). Metal halide lamps (DR400/T; Toshiba, Tokyo, Japan)
served as a light source. Two-week-old seedlings germinated in sand
were transplanted to pots (1.5 liter volume) filled with sand and grown
at a photosynthetic photon flux density of 500 mmol quanta m–2 s–1.
The standard hydroponic solution as described by Hikosaka et al.
(1994) (12 mM N) was fed at 1 liter per pot. The solution was continually aerated and was renewed every week. Fully expanded young
leaves (4–5) of 6-week-old plants were sampled. Leaves were cut with
a sharp razor in water at a petiole length of 3±0.5 cm, such treatment
having been observed not to affect photosynthetic characteristics (Kato
et al. 2002a). Four independent experiments were done.
The first experiment was performed to study the role of chloroplast-encoded protein turnover in the susceptibility to photoinhibition
at different temperatures (11, 15, 20, 25, 30, and 35°C). The petioles
were soaked in water or lincomycin solution (0.7 mM) and the leaf
laminae were exposed to 20 mmol quanta m–2 s–1 for 2 h at 25°C. The
leaves were placed in a temperature-controlled chamber with saturating humidity and illuminated through a glass window. Light (900–
1,000 mmol quanta m–2 s–1) was provided by metalhalide lamps. Air
834
Temperature dependence of photoprotective processes
was passed through soda lime to remove CO2 and mixed with 100%
(v/v) CO2 using mass-flow controllers (RK-150; Kojima, Utsunomiya,
Japan). Then the air was passed through water whose temperature was
regulated to adjust air humidity. The concentration of CO2 in the entering air was adjusted such that the concentration in the outlet air was at
360 mmol mol–1. Leaf temperature was measured with a copper-constantan thermocouple. Ten leaves were placed in the chamber and samples were taken every 40 or 60 min. The samples were kept in the dark
for 40 min at the corresponding temperature and later initial fluorescence (F0) and maximum fluorescence after a saturating pulse of light
(Fm) were measured using a chlorophyll fluorescence measuring
device (PAM-2000, Walz, Effeltrich, Germany). The fraction of active
PSII was evaluated as Fv/Fm = (Fm – Fo)/Fm.
In the second experiment simultaneous measurements of the rate
of leaf net gas exchange and chlorophyll fluorescence were done using
a portable photosynthesis measurement system with fluorescence
measuring supplement (Li-6400, LiCor, Lincoln, NE, U.S.A.) to assess
the different fates for absorbed light energy. Intact leaves were used.
After 40 min adaptation of a plant to the corresponding temperature
and to dark, the initial fluorescence (Fo) and maximum fluorescence
after a saturating pulse of light (Fm) were measured. Later the light
(1,000 mmol quanta m–2 s–1, 10% blue light) was switched on. About
30 min were necessary for the photosynthetic rate to reach steady state.
Then it was registered as well as the actual fluorescence level, Fs. To
obtain Fm¢, the leaf was exposed to a saturating flash during illumination. To determine the minimal level of fluorescence during illumination (Fo¢), far-red light was turned on to rapidly deoxidize the PSII
centres. The quantum yield of open PSII was determined by Fv¢/Fm¢
(Genty et al. 1989). The photochemical quenching coefficient (qP) was
calculated as (Fm¢ – Fs)/(Fm¢ – Fo¢) (Harbinson et al. 1989, Schreiber et
al. 1994). The quantum yield of PSII photochemistry was calculated as
DF/Fm¢=(Fm¢ – Fs)/Fm¢ (Genty et al. 1989). The level of energy dissipation activity was estimated from the non-photochemical quenching of
Fm¢ as Fm/Fm¢ – 1 (NPQ, see Bilger and Björkman 1990).
Acknowledgment
We thank R. Oguchi for his technical support to the study. This
work was supported in part by fellowship No. 02561 from Japan Society for Promotion of Science to TDT and by grants from the Ministry
of Education, Science, Sports and Culture of Japan to KH.
References
Allen, D.J. and Ort, D.R. (2001) Impacts of chilling temperatures on photosynthesis in warm-climate plants. Trends Plant Sci. 6: 36–42.
Andersson, B. and Barber, J. (1996) Mechanisms of photodamage and protein
degradation during photoinhibition of photosystem II. In Photosynthesis and
the Environment. Edited by Baker, N.R. pp. 101–121. Kluwer Academic Publishers, Dordrecht.
Armond, P.A., Schreiber, U.S. and Björkman, O. (1978) Photosynthetic acclimation to temperature in the desert shrub, Larrea divaricata. II. Light-harvesting
efficiency and electron transport. Plant Physiol. 61: 411–415.
Aro, E.M., Hundal, T., Carlberg, I. and Andersson, B. (1990a) In vitro studies
on light-induced inhibition of photosystem II and D1-protein degradation at
low temperatures. Biochim. Biophys Acta 1019: 269–275.
Aro, E.M., McCaffery, S. and Anderson, J.M. (1993b) Photoinhibition and D1
protein degradation in peas acclimated to different growth irradiances. Plant
Physiol. 103: 835–843.
Aro, E.M., Tyystjärvi, E. and Nurmi, A. (1990b) Temperature-dependent
changes in photosystem II heterogeneity of attached leaves under high light.
Physiol. Plant. 79: 585–592.
Aro, E.M., Virgin, I. and Andersson, B. (1993a) Photoinhibition of photosystem
II – Inactivation, protein damage and turnover. Biochim. Biophys Acta 1143:
113–134.
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.
Baroli, I. and Melis, A. (1998) Photoinhibitory damage is modulated by the rate
of photosynthesis and by the photosystem II light-harvesting chlorophyll
antenna size. Planta 205: 288–296.
Berry, J.A. and Björkman, O. (1980) Photosynthetic response and adaptation to
temperature in higher plants. Annu. Rev. Plant Physiol. 31: 491–543.
Bilger, W. and Björkman, O. (1990) Role of the xanthophyll cycle in photoprotection elucidated by measurements of light-induced absorbance changes, fluorescence and photosynthesis in leaves of Hedra canariensis. Photosynth.
Res. 25: 173–186.
Crafts-Brandner, S.J. and Salvucci, M.E. (2002) Sensitivity of photosynthesis in
a C4 plant, maize, to heat stress. Plant Physiol. 129: 1773–1780.
Demmig-Adams, B. and Adams, W.W. III (1992) Photoprotection and other
responses of plants to high light stress. Annu. Rev. Plant Physiol. Plant Mol.
Biol. 43: 599–626.
Demmig-Adams, B., Adams, W.W. III, Baker, 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.
Gilmore, A.M. (1997) Mechanistic aspects of xanthophyll cycle-dependent photoprotection in higher plant chloroplasts and leaves. Physiol. Plant. 99: 197–
209.
Gray, R.G., Savith, L.V., Ivanov, A.G. and Huner, N.P.A. (1996) Photosystem II
excitation pressure and development of resistance to photoinhibition. Plant
Physiol. 110: 61–71.
Greer, D.H., Berry, J.A. and Björkman, O. (1986) Photoinhibition of photosynthesis in intact bean leaves: Role of light and temperature and requirement of
chloroplast-protein synthesis during recovery. Planta 168: 253–260.
Harbinson, J., Genty, B. and Baker, N.R. (1989) Relationship between the quantum efficiencies of photosystems I and II in pea leaves. Plant Physiol. 90:
1029–1034
Hikosaka, K., Murakami, A. and Hirose, T. (1999) Balancing carboxylation and
regeneration of ribulose-1,5-bisphosphate in leaf photosynthesis: temperature
acclimation of an evergreen tree, Quercus myrsinaefolia. Plant Cell Environ.
22: 841–849.
Hikosaka, K., Terashima, I. and Katoh, S. (1994) Effects of leaf age, nitrogen
nutrition and photon flux density on the distribution of nitrogen among leaves
of a vine (Ipomoea tricolor Cav.) grown horizontally to avoid mutual shading of leaves. Oecologia 97: 451–457.
Huner, N.P.A., Öquist, G., Hurry, V.M., Krol, M., Falk, S. and Griffith, M.
(1993) Photosynthesis, photoinhibition at low temperature acclimation in cold
tolerant plants. Photosynth. Res. 37: 19–39.
Huner, N.P.A., Öquist, G. and Sarhan, F. (1998) Energy balance and acclimation
to light and cold. Trends Plant Sci. 3: 224–230.
Hurry, V.M. and Huner, N.P.A. (1992) Effect of cold hardening on sensitivity of
winter and spring wheat leaves to short-term photoinhibition and recovery of
photosynthesis. Plant Physiol. 100: 1283–1290.
Jahns, P. and Miehe, B. (1996) Kinetic correlation of recovery from photoinhibition and zeaxanthin epoxidation. Planta 198: 202–210.
Kato, M.C., Hikosaka, K. and Hirose, T. (2002a) Leaf discs floated on water are
different from intact leaves in photosynthesis and photoinhibition. Photosynth. Res. 72: 65–70.
Kato, M.C., Hikosaka, K. and Hirose, T. (2002b) Photoinactivation and recovery
of photosystem II in Chenopodium album leaves grown at different levels of
irradiance and nitrogen availability. Funct. Plant Biol. 29: 787–795.
Kato, M.C., Hikosaka, K., Hirotsu, N., Makino, A. and Hirose, T. (2003) The
excess light energy that is neither utilized in photosynthesis nor dissipated by
photoprotective mechanisms determines the rate of photoinactivation in Photosystem II. Plant Cell Physiol. 44: 318–325.
Kirschbaum, M.U.F. and Farquhar, G.D. (1984) Temperature dependence of
whole-leaf photosynthesis in Eucalyptus pauciflora Sieb. ex Spreng. Aust. J.
Plant Physiol. 11: 519–538.
Laisk, A. and Oja, V. (1994) Range of photosynthetic control of postillumination P700+ reduction rate in sunflower leaves. Photosynth. Res. 39: 39–50.
Temperature dependence of photoprotective processes
Lee, H.-Y., Hong, Y.-N. and Chow, W.S. (2001) Photoinactivation of PSII complexes and photoprotection by non-functional neighbours in Capsicum
annuum L. leaves. Planta 212: 332–342.
Leitsch, J., Schnettger, B., Critchley, C. and Krause, G.H. (1994) Two mechanisms of recovery from photoinhibition in vivo: reactivation of photosystem
II related and unrelated to D1-protein turnover. Planta 194: 15–21.
Macpherson, A.N., Telfer, A., Barber, J. and Truscott, T.G. (1993) Direct detection of singlet oxygen from isolated PSII reaction centers. Biochim. Biophys
Acta 1143: 301–309.
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.
Miyake, C. and Yokota, A. (2001) Cyclic flow of electrons within PSII in thylakoid membranes. Plant Cell Physiol. 42: 508–515.
Nishiyama, Y., Yamamoto, H., Allakhverdiev, S.I., Inaba, M., Yokota, A. and
Murata, N. (2001) Oxydative stress inhibits the repair of photodamage to the
photosynthetic machinery. EMBO J. 20: 5587–5594.
Ögren, E. and Rosenqvist, E. (1992) On the significance of photoinhibition of
photosynthesis in the field and its generality among species. Photosynth. Res.
33: 63–71.
Oguchi, R., Hikosaka, K. and Hirose, T. (2003) Does the photosynthetic lightacclimation need change in leaf anatomy? Plant Cell Environ. 26: 505–512.
Öquist, G., Chow, W.S. and Anderson, J.M. (1992) Photoinhibition of photosynthesis represents a mechanism for long-term regulation of photosystem II.
Planta 186: 450–460.
Öquist, G. and Huner, N.P.A. (1991) Effects of cold acclimation on the susceptibility of photosynthesis to photoinhibition in Scots pine and in winter and
spring cereals: A fluorescence analysis. Funct. Ecol. 5: 91–100.
Osmond, C.B. (1994) What is photoinhibition? Some insights from comparisons of shade and sun plants. In Photoinhibition of Photosynthesis: From
Molecular Mechanisms to the Field. Edited by Baker, N.R. and Bowyer, J.R.
pp. 1–24. BIOS Scientific Publishers, Oxford.
Osmond, C.B. and Grace, S.C. (1995) Perspectives on photoinhibition and photorespiration in the field: Quintessential inefficiencies of the light and dark
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reactions of photosynthesis? J. Exp. Bot. 46: 1351–1362.
Powles, S.B. (1984) Photoinhibition of photosynthesis induced by visible light.
Annu. Rev. Plant Physiol. 35: 15–44.
Powles, S.B. and Osmond, C.B. (1979) Photoinhibition of intact attached leaves
of C3 plants illuminated in the absence of both carbon dioxide and photorespiration. Plant Physiol. 64: 982–988.
Sage, R.F. and Sharkey, T.D. (1987) The effect of temperature on the occurrence of oxygen and carbon dioxide insensitive photosynthesis in field grown
plants. Plant Physiol. 84: 658–664.
Salonen, M., Aro, E.-M. and Rintamäki, E. (1998) Reversible phosphorylation
and turnover of the D1 protein under various redox states of photosystem II
induced by low temperature photoinhibition. Photosynth. Res. 58: 143–151.
Schreiber, U., Bilger, W. and Neubauer, C. (1994) Chlorophyll fluorescence as a
nonintrusive indicator for rapid assessment of in vivo photosynthesis. In Ecophysiology of Photosynthesis. Edited by Schulze, E.D. and Caldwell, M.M.
pp. 49–70. Springer-Verlag, Berlin.
Thiele, A., Krause, G.H. and Winter, K. (1998) In situ study of photoinhibition
of photosynthesis and xanthophyll cycle activity in plants growing in natural
gaps of the tropical forest. Aust. J. Plant Physiol. 25: 189–195.
Thiele, A., Schirwiz, K., Winter, K. and Krause, G.H. (1996) Increased xanthophyll cycle activity and reduced D1 protein inactivation related to photoinhibition in two plant systems acclimated to excess light. Plant Sci. 115: 237–
250.
Tyystjärvi, E., Ali-Yrkkö, K., Kettunen, R. and Aro, E.-M. (1992) Slow degradation of the D1 Protein is related to the susceptibility of low-light-grown
pumpkin plants to photoinhibition. Plant Physiol. 100: 1310–1317.
Wünschmann, G. and Brand, J.J. (1992) Rapid turnover of a component required
for photosynthesis explains temperature dependence and kinetics of photoinhibition in a cyanobacterium, Synechococcus 6301. Planta 186: 426–433.
Xu, C.C., Kuang, R., Li, L. and Lee, C.H. (2000) D1 protein turnover and carotene synthesis in relation to zeaxanthin epoxidation in rice leaves during
recovery from low temperature photoinhibition. Aust. J. Plant Physiol. 27:
239–244.
(Received April 14, 2003; Accepted June 5, 2003)