Planta (2001) 212: 808±816
In vivo temperature dependence of cyclic and pseudocyclic
electron transport in barley
Joanne E. Clarke, Giles N. Johnson
School of Biological Sciences, University of Manchester, 3.614 Stopford Building, Oxford Road, Manchester, M13 9PT, UK
Received: 6 July 2000 / Accepted: 3 August 2000
Abstract. The e€ect of temperature on the rate of
electron transfer through photosystems I and II (PSI
and PSII) was investigated in leaves of barley (Hordeum
vulgare L.). Measurements of PSI and PSII photochemistry were made in 21% O2 and in 2% O2, to limit
electron transport to O2 in the Mehler reaction. Measurements were made in the presence of saturating CO2
concentrations to suppress photorespiration. It was
observed that the O2 dependency of PSII electron
transport is highly temperature dependent. At 10 °C,
the quantum yield of PSII (FPSII) was insensitive to O2
concentration, indicating that there was no Mehler
reaction operating. At high temperatures (>25 °C) a
substantial reduction in FPSII was observed when the
O2 concentration was reduced. However, under the same
conditions, there was no e€ect of O2 concentration on
the DpH-dependent process of non-photochemical
quenching. The rate of electron transport through PSI
was also found to be independent of O2 concentration
across the temperature range. We conclude that the
Mehler reaction is not important in maintaining a
thylakoid proton gradient that is capable of controlling
PSII activity, and present evidence that cyclic electron
transport around PSI acts to maintain membrane
energisation at low temperature.
Key words: DpH ± Cyclic electron transport ±
Hordeum (electron transport) ± Mehler reaction ±
Non-photochemical quenching
Abbreviations: k ˆ rate constant for reduction of P700; NPQ ˆ
non-photochemical quenching of chlorophyll ¯uorescence; PFD ˆ
photon ¯ux density; PSI ˆ photosystem I; PSII ˆ photosystem II;
DpH ˆ pH gradient across the thylakoid membrane; FPSII ˆ
quantum eciency of PSII photochemistry; P700 ˆ primary
electron donor of photosystem I
Correspondence to: G. N. Johnson;
E-mail: [email protected]; Fax: +44-161-2753938
Introduction
The ability to tolerate temperature stress is a major
factor in determining the productivity and distribution
of plants. It is of particular relevance given present
concerns over climate change. The primary site of
damage associated with non-optimal temperatures is
the photosynthetic apparatus (Wise 1995; Yamane et al.
1998; Bukhov et al. 1999; Kratsch and Wise 2000).
Damage commonly arises when environmental stress
reduces metabolic activity, such that its capacity is
exceeded by the absorption of light by the plant. This
can result in the production of harmful radical species,
which are able to cause substantial damage to many
cellular components (for reviews, see Robinson 1988;
Asada 1999; Foyer and Noctor 2000).
In recent years, the importance of alternative routes for
electron transport in protecting the photosynthetic apparatus from stress has been the subject of considerable
debate. The reduction of molecular oxygen in the Mehler
reaction (Mehler 1951) has received particular attention.
The pseudocyclic ¯ow of electrons to oxygen in the
Mehler reaction, from either the acceptor side of PSI
(Badger 1985; Robinson 1988) or the PSII acceptor side
(Bradbury et al. 1985) could provide an important
alternative pathway for the consumption of electrons
under conditions when the rate of photosynthetic
electron transport exceeds the capacity of metabolism
(Polle 1996). This would have the e€ect of reducing the
risk of PSII photoinhibition by limiting the build-up of
reduced intermediates in the photosynthetic electron
transport chain on the PSII acceptor side (Grace and
Osmond 1995). In addition, electron ¯ow without
concomitant consumption of H+ in ATP production
would contribute to an increased pH gradient across the
thylakoid membrane. As a result, DpH-mediated photoprotective processes, in particular high-energy-state
quenching (qE), would be enhanced (Schreiber and
Neubauer 1990; Neubauer and Yamamoto 1992; Osmond and Grace 1995). However, the reduction of
molecular oxygen results in the formation of harmful
radical species (Asada and Takahashi 1987) and the
J.E. Clarke and G.N. Johnson: Cyclic and pseudocyclic electron transport in barley
resulting damage to PSI as well as PSII may far outweigh
any potential bene®ts (Wiese et al. 1998; Ott et al. 1999).
Mechanisms for the detoxi®cation of harmful active
oxygen species are well developed in plants (for reviews,
see Asada 1999; Foyer and Noctor 2000). However, even
assuming that these reactions are able to operate with
maximal eciency and prevent any radical-associated
damage occurring, maintaining sucient concentrations
of the enzymes needed requires a signi®cant investment
of energy and resources. Therefore, minimising the
production of radicals in the ®rst place may be a
considerable advantage (Genty and Harbinson 1996).
Central to this debate is the issue of whether the
Mehler reaction has sucient capacity to play a photoprotective role under physiological conditions. A number of early in vitro studies reported insucient levels of
Mehler activity to have a signi®cant contribution to the
generation of DpH (Heber et al. 1978; Marsho et al.
1979; Ziem-Hanck and Heber 1980; Furbank et al.
1982). In these studies, however, the ascorbate-peroxidase reaction was inactive and, more recently, there have
been a number of reports indicating that O2-dependent
electron ¯ow is a major contributor to light-induced
membrane energisation in vitro (Neubauer and Schreiber 1989; Schreiber and Neubauer 1990; Schreiber et al.
1991; Neubauer and Yamamoto 1992; Hormann et al.
1993, 1994). These studies have stressed the importance
of additional electron ¯ow associated with the reduction
of H2O2 formed in the Mehler-peroxidase reaction.
Whether or not the Mehler reaction has the capacity to
make a signi®cant contribution to the protection of the
photosynthetic apparatus in vivo is unclear. Several
authors have found the capacity of the Mehler reaction
to be small, both in vitro and in vivo, and insucient to
provide protection from photoinhibition (Cornic and
Briantais 1991; Brestic et al. 1995; Epron et al. 1995;
Habash et al. 1995; Valentini et al. 1995; Wiese et al.
1998). There are reports of levels of O2-dependent
electron ¯ow capable of increasing qE in intact leaves
(Osmond and Grace 1995; Biehler and Fock 1996; Park
et al. 1996), though these often do not distinguish
between the Mehler-peroxidase pathway and photorespiration. Indeed, a regulatory role for the latter is not
unfeasible (Wu et al. 1991).
Another pathway of electron transport that has
received considerable attention is a cyclic pathway
around PSI. While it is now largely accepted that cyclic
photophosphorylation is not required for carbon assimilation, several authors have proposed that it may have a
role in the protection of the photosynthetic apparatus
(Heber and Walker 1992; Katona et al. 1992; Bukhov
et al. 1999). Cyclic ¯ow would, like the Mehler reaction,
lead to increased thylakoid acidi®cation, required for the
de-epoxidation of violaxanthin to zeaxanthin and the
non-photochemical dissipation of excess excitation energy (Horton et al. 1996). Unlike the Mehler reaction,
however, cyclic electron transport does not have the
associated problem of harmful free-radical production,
and is able to operate safely under conditions where the
detoxi®cation of radicals would be dicult, for example,
at low temperatures when these enzymatic reactions
809
would be much slowed down. Similarly, PSI cyclic
electron transport has been shown to have much greater
resistance to high temperatures than linear electron ¯ow
(Berry and Bjorkman 1980; Bukhov et al. 1999) and it
has previously been suggested that cyclic electron
transport could control PSII activity at high temperatures (Havaux et al. 1991).
Despite the amount of work that has been carried
out in this area it remains unclear to what extent these
di€erent pathways operate in vivo and to what extent
they regulate photosynthesis. In an attempt to address
this question, we have investigated the rate of electron
transport to oxygen in intact barley leaves across a
range of environmental conditions and critically examined the potential of the Mehler reaction and cyclic
electron transport to control the activity of PSII
through the generation of an increased thylakoid
proton gradient. The results presented here may help
shed some light on the processes that interact in vivo to
regulate the electron transport chain under conditions
of temperature stress.
Materials and methods
Plant material
Plants of Hordeum vulgare L. (cv. Chariot) were grown from seed
(Samuel Yates, Maccles®eld, UK) in Viking MM compost. Plants
were grown in a growth cabinet (E.J. Stiell, Glasgow, UK) on a
12 h day/12 h night cycle at a temperature of 20 °C (day) 16 °C
(night). A photon ¯ux density (PFD) of 100 lmol m)2 s)1 was
supplied by ¯uorescent strip lights supplemented by tungsten lights.
P700 oxidation and chlorophyll ¯uorescence
The redox state of P700, the primary electron donor of PSI, was
measured as an absorbance change at 830 nm using a Walz PAM 101
¯uorometer system in combination with a ED-P700DW-E emitterdetector unit (Walz, E€eltrich, Germany). Actinic light was supplied
by a Volpi Intralux 6000 lamp (Volpi, Schlieren, Sweden), which was
shuttered with a Uniblitz 6-mm electronic shutter (Vincent Associates, Rochester, N.Y., USA) and controlled by a custom-built
shutter controller. Far-red light was provided by ®ltering the light
from a Volpi Intralux 150H lamp through an RG715 far-red glass
®lter (H.V. Skan, Solihull, UK). This lamp was shuttered with a
Uniblitz 14-mm electronic shutter (Vincent Associates) under
control of a Uniblitz shutter controller. All light sources were
®ltered with Cal¯ex-X heat-re¯ecting glass ®lters (Balzers, Lichtenstein) to prevent interference with the 830-nm signal.
Chlorophyll ¯uorescence emission was measured using the same
¯uorometer but in combination with a 101-ED emitter-detector
unit. Actinic light for ¯uorescence measurements was supplied by a
Schott KL1500 lamp (Schott Fibre Optics, Doncaster, UK). Pulses
of light of saturating PFD were supplied by a Volpi Intralux 150H
lamp, shuttered as described previously.
All light sources were fed, via a ®ve-armed glass ®bre optic
bundle (Walz), into a leaf-disk chamber (LD2/2; Hansatech, King's
Lynn, UK), modi®ed to allow the light ®bres to come within 2 cm
of the leaf surface.
Transient absorbance changes on a millisecond time scale were
captured using a data acquisition board (1700 series; Keithley
Metrabyte, Taunton, Mass., USA) ®tted in a computer running
software created using the Testpoint software development package (CEC, Billerica, Mass., USA). Maximum oxidation of P700 was
810
J.E. Clarke and G.N. Johnson: Cyclic and pseudocyclic electron transport in barley
determined by applying a single 20-s pulse of far-red light of
saturating intensity to a dark-adapted leaf. The kinetics and degree
of re-reduction following a light-dark transition was measured after
25 min actinic illumination. Each signal represented an accumulation of up to 20 individual measurements made during 100-ms
closures of the shutter, performed at 10-s intervals. Exponential
curves were ®tted using the Gra®t software package, to determine
the pseudo-rate constant for the re-reduction of P700 (k) (see Ott
et al. 1999).
The relative rate of PSI electron transport was calculated as:
PSI ETR ˆ P‡
700 = P700 …total†k
where P‡
700 is estimated as the signal size following a light-dark
transition, P700(total) as the maximum signal size induced by
saturating far-red light on the dark-adapted leaf sample and k is the
rate constant for P700 re-reduction. Saturation of the far-red signal
was judged by the absence of any signal increase upon application
of a white light ¯ash.
Fluorescence signals were recorded using a chart recorder and
the following parameters recorded: the quantum yield of PSII
photochemistry (FPSII) is calculated as Fm ) Ft/F ¢m as de®ned by
Genty et al. (1989); non-photochemical quenching (NPQ) is
calculated as (Fm ) F ¢m)/F ¢m using the equation of Bilger and
Bjorkman (1991); the relative rate of electron transport through
PSII was calculated as FPSII á PFD. Quenching relaxation measurements were made using the method of Walters and Horton
(1991) and calculations made as described by Johnson et al. (1993).
Leaf-chamber temperature was controlled using a Grant cooling
water bath and monitored continuously during measurements using
a k-type thermocouple, located just below the leaf lower surface,
connected to a Kane-May KM330 thermometer (Comark, Welwyn
Garden City, UK). An atmosphere of saturating CO2 was used in all
measurements and was generated by bubbling gas through a
solution of 1 M Na2CO3/NaHCO3 (pH 9). This produced a gas
stream that was always greater than 1,200 ll l)1 CO2 as measured
using an ADC LCA2 infrared gas analyser. At 1,200 ll l)1, CO2
®xation was found to be 95% saturated at 25 °C in these plants
(data not shown). Gas was supplied from a cylinder of either
compressed air or 2% O2 /balance N2 (BOC Gases, Guildford, UK).
In all cases leaves used were removed from well-watered plants that
had been dark-adapted for at least 16 h prior to use. Two 1-cm
segments were cut at a distance of 1 cm from the tip of the leaf and
immediately placed in the leaf-disk chamber. Leaf segments from
di€erent leaves were used for each individual measurement.
Results
Oxygen and temperature dependence
of PSII quantum yield
Measurements of the quantum yield of PSII (FPSII) and
non-photochemical quenching of chlorophyll ¯uorescence (NPQ) were made over a range of temperatures, in
the presence of saturating CO2 and either ambient
(21%) or 2% O2, following a 25-min period of actinic
illumination at a PFD of 1,600 lmol m)2 s)1 (Fig. 1).
Under these non-photorespiratory conditions, any difference between the quantum yields of PSII obtained at
the two di€erent O2 concentrations can be attributed to
the reduction of molecular oxygen. At 10 °C, FPSII was
independent of O2 concentration, indicating that little or
no electron transport to O2 occurred at this temperature.
Between 15 and 25 °C, reducing the O2 concentration
led to a decrease in FPSII, suggesting that some electron
transport to O2 was occurring. It was only above 25 °C
that electron transport to O2 became substantial.
Fig. 1. Relationship between temperature and PSII quantum yield,
FPSII (A) and non-photochemical quenching, NPQ (B) in barley
leaves in the presence of ambient (open symbols) and 2% (closed
symbols) O2 at 1,600 lmol m)2 s)1. Each point represents the
mean ‹ SE of at least three measurements on separate leaves
It can be seen from Fig. 1B that, over the same range
of temperatures, the amount of NPQ was relatively
constant and was insensitive to O2 concentration. At the
highest temperatures, when the greatest amount of
Mehler reaction was observed, reducing the O2 concentration appeared, if anything, to slightly increase the
amount of NPQ. The fact that NPQ is insensitive to O2
concentration, even at temperatures where there is
apparently substantial electron transport to O2, suggests
that any Mehler reaction that is occurring is not
necessary for the generation of DpH.
In order to address the question of whether increases
in photoinhibition were in¯uencing total NPQ, the
relaxation of ¯uorescence quenching was measured.
Relaxation measurements were carried out in the presence of saturating CO2 and either 21% or 2% O2, at
three di€erent temperatures (Table 1). Non-photochemical quenching was found to consist almost entirely of
fast relaxing quenching, with a small contribution from
a slowly relaxing photoinhibitory component. The
relative contribution of the two components was independent of O2 concentration and temperature.
Sensitivity of PSI electron transport
to oxygen concentration and temperature
The relaxation of the absorbance signal at 830 nm,
during a light-dark transition, following a period of
25 min continuous actinic illumination at a PFD of
J.E. Clarke and G.N. Johnson: Cyclic and pseudocyclic electron transport in barley
811
Table 1. Values of the fast- and slow-relaxing components of NPQ (NPQf and NPQs, respectively), measured at 10, 25, and 35 °C, in the
presence of 21% and 2% O2
10 °C
NPQf
NPQs
25 °C
35 °C
21% O2
2% O2
21% O2
2% O2
21% O2
2% O2
1.859 ‹ 0.059
0.755 ‹ 0.0278
1.757 ‹ 0.125
0.618 ‹ 0.088
1.966 ‹ 0.037
0.281 ‹ 0.072
2.258 ‹ 0.087
0.276 ‹ 0.043
2.052 ‹ 0.010
0.435 ‹ 0.045
1.998 ‹ 0.198
0.576 ‹ 0.151
1,600 lmol m)2 s)1, was measured in barley leaves at a
range of temperatures, in the presence of 21% and 2%
O2. Figure 2 shows the temperature sensitivity of k, the
pseudo-rate constant for the re-reduction of P700, and of
PSI electron transport rate. The PSI electron transport
rate was calculated as the extent of P700 oxidation in the
light multiplied by k. In previous studies, PSI electron
transport rate has been estimated as the product of
reduced P700 and irradiance (Harbinson and Woodward
1987; Klughammer and Schreiber 1994). This approach
makes the assumption that the eciency of trapping by
reduced PSI centres is constant under all conditions. The
method we adopted makes no such assumption. Although our approach gave quantitatively di€erent
results, the conclusions reached were essentially the
same using both methods (data not shown). It is
apparent from Fig. 2 that both k and PSI electron
transport rate are insensitive to a decrease in the O2
concentration across the range of temperatures investi-
Fig. 2. Relationship between temperature and the pseudo-rate
constant for the reduction of P700, k (A) and the relative rate of
electron transport through PSI (B) in barley leaves in the presence of
ambient (open symbols) and 2% (closed symbols) O2 at 25 °C. Each
point represents the mean ‹ SE of at least three measurements on
separate leaves
gated. This is in clear contrast to the marked e€ect of O2
concentration on PSII quantum yield that was observed
at high temperatures (Fig. 1A).
At 35 °C, PSI electron transport rate showed a
marked decline. Since k was also drastically reduced at
this temperature, this is likely to re¯ect a direct e€ect of
the super-optimal temperature on the ultrastructural
stability of the photosynthetic apparatus.
E€ect of temperature and oxygen concentration
on PFD dependency of PSII electron transport
In order to understand in more detail the e€ects of
temperature on electron transport we measured the
relationship between PFD and the quantum yield of
PSII photochemistry, PSII electron transport rate and
non-photochemical quenching, in the presence of saturating CO2 and ambient or 2% O2, at 10, 25 and 35 °C
(Fig. 3). At 10 °C, FPSII was found to be largely
insensitive to O2 concentration across the range of
PFDs investigated (Fig. 3A), con®rming the absence
of any electron transport to O2. At this temperature,
PSII electron transport rate was saturated at a PFD
of 250 lmol m)2 s)1 (Fig. 3D). Non-photochemical
quenching was also largely insensitive to O2 concentration (Fig. 3G).
When measurements were made at 25 °C, FPSII
again appeared to be largely insensitive to O2 concentration, though a slight decrease in FPSII was observed
upon lowering of O2 concentration at PFDs saturating
for electron transport (Fig. 3B). This di€erence was
re¯ected more clearly in the rates of PSII electron
transport (Fig. 3H) observed at PFDs greater than
750 lmol m)2 s)1. This suggests that at moderate temperatures there is some electron transport to O2 at
saturating light intensities. Under the same conditions,
however, there was no noticeable increase in the amount
of NPQ (Fig. 3E). The data in Fig. 3E also suggest that
at 1,600 lmol m)2 s)1 the capacity of NPQ may be
saturated. This could provide an explanation for the
insensitivity of NPQ to temperature seen in Fig. 1B.
It was only when measurements were conducted at
the relatively high temperature of 35 °C that substantial
amounts of electron transport to O2 were observed
(Fig. 3C, I). At this temperature, reducing the O2
concentration to 2% resulted in a clear decrease both
in FPSII and the rate of PSII electron transport at all
PFDs greater than 250 lmol m)2 s)1. The electron ¯ow
to O2 does not seem to result in an increased DpH, as
indicated by the amount of non-photochemical quench-
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J.E. Clarke and G.N. Johnson: Cyclic and pseudocyclic electron transport in barley
Fig. 3. Relationship between
PFD and PSII quantum yield,
FPSII, (A±C), non-photochemical quenching of chlorophyll
¯uorescence, NPQ (D±F) and
the relative rate of electron
transport through PSII (G±I), in
barley leaves, in the presence of
ambient (open symbols) and 2%
(closed symbols) O2 at 10 °C and
35 °C respectively. Each point
represents the mean ‹ SE of at
least three measurements on
separate leaves
ing. In fact, NPQ was found to be greater when the O2
concentration was low. At all three temperatures, NPQ
continued to rise with increasing PFD long after the
PSII electron transport rate had saturated. It seems
reasonable that the additional electron transport and
associated proton pumping that would be required to
generate this increase in NPQ could be supplied by the
cyclic transport of electrons around PSI.
E€ect of temperature and oxygen concentration
on PFD dependency of PSI electron transport
Figure 4 shows the PFD dependency of the pseudo-rate
constant for the re-reduction of P700 (k) and PSI electron
transport rate in the presence of saturating CO2 and
either 21% or 2% O2 at three temperatures. At all three
temperatures, k remained unchanged with increasing
PFD. Both k and the rate of PSI electron transport were
insensitive to O2 concentration under all conditions. It is
clear that at each temperature the rate of PSI electron
transport continued to rise with increasing PFD, long
after PSII electron transport rate had become saturated
(compare Fig. 3). This is consistent with an increase in
cyclic electron transport occurring at higher PFDs.
Discussion
It is clear from the results in Fig. 1 that light-dependent
oxygen uptake is highly temperature dependent. Notably, we found that it does not operate in this species at
low temperature. At 10 °C, both FPSII and the rate of
PSI electron transport were found to be insensitive to O2
concentration across a range of light intensities. This is
in contrast to the ®ndings of Fryer et al. (1998) working
on maize, who presented evidence for the occurrence of
the Mehler reaction when plants were exposed to chilling
temperatures. This discrepancy might be explained by
the fact that barley is a chilling-tolerant species, whereas
the chilling sensitivity of maize is well documented. At
chilling temperatures, chilling-sensitive and chillingtolerant plants have been shown to respond di€erently
in terms of the phase behaviour of their thylakoid lipids
and inhibition of membrane-associated processes
(Hodgson et al. 1987; Hodgson and Raison 1991). It is
possible that the ability to restrict the ¯ow of electrons
to oxygen, thus preventing the production of harmful
radicals at temperatures when the enzymatic reactions
required for their detoxi®cation will be much slowed, is
what gives tolerant species such as barley the ability to
withstand chilling.
J.E. Clarke and G.N. Johnson: Cyclic and pseudocyclic electron transport in barley
813
Fig. 4. Relationship between
PFD and the pseudo-rate constant for the reduction of P700, k
(A±C) and the relative rate of
electron transport through PSI
(D±F) in barley leaves in the
presence of ambient (open symbols) and 2% (closed symbols) O2
at 10, 25 and 35 °C respectively.
Each point represents the
mean ‹ SE of at least three
measurements on separate leaves
It seems likely that, at low temperatures, photosynthesis in barley is limited by the physical properties of
the electron transport chain itself. This has also been
suggested by Ott et al. (1999) working with the plant
Silene dioica. In particular, the di€usion of plastoquinone within the membrane and plastocyanin in the intrathylakoid space are likely to be highly temperature
sensitive. Thus, at 10 °C, the PSII electron transport rate
was found to be saturated at all PFDs greater than
250 lmol m)2 s)1 (Fig. 3G). Under such conditions,
when the capacity of the Calvin cycle is also likely to be
limited, the cyclic transport of electrons around PSI
would be favoured. Indeed, measurement of the rate of
PSI electron transport at 10 °C showed that this
continues to rise with increasing PFD well beyond the
point at which FPSII had become saturated. This
provides evidence of a cyclic pathway of electron ¯ow
operating around PSI at high light intensities. The fastrelaxing component of NPQ, high-energy-state quenching (NPQf), is a pH-dependent process; a low intrathylakoid pH is required for the de-epoxidation of
violaxanthin to zeaxanthin, which is believed to be
involved in the non-radiative dissipation of excess
excitation energy in PSII. Therefore, NPQf can be used
to indicate relative changes in the DpH across the
thylakoid membrane. It should be noted, however, that
there is a non-linear relationship between NPQ and DpH
(Noctor and Horton 1990) and measurements of NPQ
can, therefore, provide only an indication of the DpH.
The light intensity range over which PSII electron
transport is saturated and PSI electron transport is
rising corresponds to that over which most NPQ is
formed. This points to an essential role of cyclic electron
transport in generating the DpH required to induce
protective non-photochemical quenching.
At moderate and high temperatures the quantum
yield of PSII photochemistry was found to be greater in
ambient than in 2% O2, indicating that electron transport to O2 is occurring. This was not, however, re¯ected
in measurements of PSI electron transport rate. One
explanation might be that oxygen was being directly
reduced by the acceptor side of PSII. This has previously
been suggested by Bradbury et al. (1985) and, in the face
of the data we have obtained, this is perhaps the most
straightforward explanation. Alternatively, the apparent
lack of sensitivity of PSI electron transport rate to O2
observed could be due to a compensatory increase in
cyclic electron transport when the pathway of electron
transfer to O2 is removed. Distinguishing between these
two possible alternatives would be dicult to accomplish in vivo. Interestingly, in vitro it has been demonstrated that cyclic electron transport may be suppressed
by Mehler-peroxidase activity (Hormann et al. 1994).
It has previously been suggested that electron ¯ow to
O2 provides an important means of regulating the
activity of PSII through the generation of an increased
proton gradient across the thylakoid membrane (see
Schreiber et al. 1991). We found that electron transport
to O2 did not result in a measurable increase in nonphotochemical quenching. On the contrary, at higher
temperatures NPQ was increased when O2 concentration
was lowered. This would seem to argue against a role for
the Mehler reaction in the protection of the photosynthetic apparatus through the control of PSII activity. It
is important to note that all the data presented here were
made in an atmosphere of saturating CO2. Under these
conditions, the oxygenase activity of Rubisco is largely
inhibited and so O2-dependent electron ¯ow is almost
entirely restricted to the Mehler reaction. Photorespiration may itself make a signi®cant contribution to the
814
J.E. Clarke and G.N. Johnson: Cyclic and pseudocyclic electron transport in barley
regulation of PSII activity in vivo, particularly under
conditions where the internal concentration of CO2 is
reduced, for example during drought stress (Wu et al.
1991). The relative importance of the Mehler reaction in
many previous studies, which have simply considered
O2-dependent electron transport is, therefore, not always clear.
Evidence of cyclic electron transport was found at
moderate and high as well as low temperatures. Comparative measurements of PSI and PSII electron transport rates at both 25 °C and 35 °C again showed that
the rate of PSI electron transport continued to increase
with PFD after that of PSII had saturated (Fig 3H, I;
Fig. 4E, F). Non-photochemical quenching was similarly observed to increase at PFDs where linear electron
transport was saturated. In line with the ®ndings of
previous studies (Wu et al. 1991; Heber and Walker
1992; Heber et al. 1992; Katona et al. 1992), this points
to a clear role for cyclic electron transport in controlling
PSII activity when the light energy absorbed by the plant
exceeds the capacity of carbon metabolism to utilise it.
Such conditions were found to occur either when PFDs
were very high or at low temperatures when PFDs that
would not normally be considered excessive became
super-saturating.
Comparing Figs. 1 and 2, it is notable that PSII
electron transport has a very di€erent temperature
dependence to PSI electron transport rate. The PSI
electron transport rate approximates to a Q10 ˆ 2
relationship, with the rate of electron transport doubling
between 10 and 20 °C (Fig. 2). By contrast, the Q10 for
PSII electron transport is nearer 3 (Fig. 1). From this we
conclude that the contribution of cyclic electron transport to the ¯ux through PSI must rise with falling
temperature. This di€erence may relate to the di€erence
in sensitivity to temperature of plastocyanin and plastoquinol di€usion, with the former occurring in an
aqueous medium and the latter in the lipid phase. This
leads us to suggest a model (Fig. 5) for the temperature
sensitivity of cyclic and linear electron transport in
barley that points to a central role of cyclic electron
transport in generating DpH at low temperature.
We conclude that coupled cyclic electron transport is
more important in the DpH-dependent control than
electron transport to O2 in the Mehler reaction. Instead,
the excessive amounts of O2 uptake that apparently
occur when high temperature and PFD are combined,
might better be viewed as an unavoidable and probably
detrimental symptom of the stress conditions, rather
than as a photoprotective mechanism. Although such
electron transport may protect PSII from damage (Park
et al. 1996), the negative e€ects of oxidative stress are
likely to outweigh this bene®t. However, it is not
unfeasible that a low rate of Mehler reaction, possibly
in combination with photorespiration, is necessary for
the poising of the electron transport chain so as to
permit coupled cyclic electron transport to occur under
conditions where the electron transport chain would
otherwise be over-reduced. Such a role has been envisaged previously (Wu et al. 1991; Kobayashi and Heber
1994).
Fig. 5. Model for the response of linear and cyclic electron transport
at ambient and chilling temperatures. At ambient temperatures, cyclic
electron transport is the major pathway for electron ¯ow. At low
temperatures, plastoquinone di€usion through the membrane is
inhibited, causing linear electron transport to slow faster than cyclic,
leading to an increase in the ratio of cyclic to linear electron transport.
cyt b6f Cytochrome b6f complex; PC plastocyanin; PQ plastoquinone
J.E.C. was in receipt of a Biotechnology and Biological Sciences
Research Council studentship. G.N.J. would like to acknowledge
support from the Natural Environment Research Council and the
Royal Society.
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