Contrasting Responses of Photosynthesis to Salt Stress
in the Glycophyte Arabidopsis and the Halophyte
Thellungiella: Role of the Plastid Terminal Oxidase
as an Alternative Electron Sink1[C][OA]
Piotr Stepien and Giles N. Johnson*
Faculty of Life Sciences, Manchester M13 9PT, United Kingdom
The effects of short-term salt stress on gas exchange and the regulation of photosynthetic electron transport were examined in
Arabidopsis (Arabidopsis thaliana) and its salt-tolerant close relative Thellungiella (Thellungiella halophila). Plants cultivated on
soil were challenged for 2 weeks with NaCl. Arabidopsis showed a much higher sensitivity to salt than Thellungiella; while
Arabidopsis plants were unable to survive exposure to greater than 150 mM salt, Thellugiella could tolerate concentrations as
high as 500 mM with only minimal effects on gas exchange. Exposure of Arabidopsis to sublethal salt concentrations resulted in
stomatal closure and inhibition of CO2 fixation. This lead to an inhibition of electron transport though photosystem II (PSII), an
increase in cyclic electron flow involving only PSI, and increased nonphotochemical quenching of chlorophyll fluorescence. In
contrast, in Thellungiella, although gas exchange was marginally inhibited by high salt and PSI was unaffected, there was a
large increase in electron flow involving PSII. This additional electron transport activity is oxygen dependent and sensitive to
the alternative oxidase inhibitor n-propyl gallate. PSII electron transport in Thellungiella showed a reduced sensitivity to
2#-iodo-6-isopropyl-3-methyl-2#,4,4#-trinitrodiphenylether, an inhibitor of the cytochrome b6f complex. At the same time, we
observed a substantial up-regulation of a protein reacting with antibodies raised against the plastid terminal oxidase. No such
up-regulation was seen in Arabidopsis. We conclude that in salt-stressed Thellungiella, plastid terminal oxidase acts as an
alternative electron sink, accounting for up to 30% of total PSII electron flow.
Salinity in soils is a major global problem and is one
that is of growing importance (Pitman and Läuchli,
2002). Research in this area has been limited by the
lack of a suitable salt-tolerant genetic model (Bressan
et al., 2001; Flowers and Colmer, 2008). A salt-tolerant
Arabidopsis (Arabidopsis thaliana) relative, Thellungiella
(Thellungiella halophila), is now promising to help in
salt stress tolerance research (Volkov et al., 2003;
Amtmann et al., 2005). Salinity tolerance is a complex
phenomenon, brought about by adaptations in a range
of physiological processes. Plants have developed a
complex defense system, including ion homeostasis,
osmolyte biosynthesis, compartmentation of toxic
ions, and reactive oxygen species (ROS) scavenging
systems (Hasegawa et al., 2000; Mittova et al., 2004;
Stepien and Klobus, 2005; Flowers and Colmer, 2008).
Of paramount importance is the process of photosyn1
This work was supported by a Marie-Curie Fellowship of the
European Commission (grant no. MEIF–CT–2006–040053 to P.S.).
* Corresponding author; e-mail [email protected].
The author responsible for the distribution of materials integral to
the findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org) is:
Giles N. Johnson ([email protected]).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[OA]
Open access articles can be viewed online without a subscription.
www.plantphysiol.org/cgi/doi/10.1104/pp.108.132407
1154
thesis that is well established as a primary target of
many forms of environmental stress, including salinity
(Garcia-Sanchez et al., 2002; Liska et al., 2004; Stepien
and Klobus, 2006).
Soil salt prevents plants from taking up water,
exposing them to drought stress. To conserve water,
they close their stomata. This simultaneously restricts
the entry of CO2 into the leaf, reducing photosynthesis.
At higher concentrations, NaCl may also directly
inhibit photosynthesis. When such inhibition occurs,
the plant is liable to suffer from oxidative stress.
Absorption of sunlight leads to ROS formation, mainly
in the chloroplast, either via photoreduction of O2 to
form superoxide (the Mehler reaction) or through the
interaction of triplet-excited chlorophyll to form singlet excited oxygen (Asada, 2000; Foyer et al., 2002).
ROS are highly reactive and can cause widespread
damage to membranes, proteins, and DNA. To prevent
such damage, there are a number of enzymatic processes in chloroplast to scavenge ROS (Asada, 2000).
These are energetically demanding, requiring the synthesis of high concentrations of antioxidants and enzymes.
An alternative strategy, placing less of a metabolic
burden on plants, would be to avoid the production of
ROS. This can be achieved by regulation of photosynthetic electron transport (Johnson, 2005). Although the
role of salt stress in inducing oxidative damage has
been widely studied (Hernàndez et al., 2001; Bor et al.,
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Salt Responses of Arabidopsis and Thellungiella
2003; Stepien and Klobus, 2005), the extent to which
regulatory processes are induced under such conditions, and the extent to which variation in their capacity
determines the degree of damage incurred by plants
exposed to salt have not been widely investigated.
Studies so far reported using Thellungiella as a
model for salt tolerance have focused on short-term
responses to salinity, in particular examining changes
in gene expression (Inan et al., 2004; Kant et al., 2006;
Wong et al., 2006). Fewer studies have examined the
physiology of salt tolerance in this plant and none the
effects of salt on leaf physiology. Here, we describe an
investigation into the effects of salt stress on the
regulation of photosynthesis in Arabidopsis and Thellungiella. We show that these plants respond to salt
stress in highly contrasting ways. We discuss the
implications of these results for our understanding of
salt tolerance.
RESULTS
Ion Concentrations and Chlorophyll Content
Plants of Arabidopsis and Thellungiella were grown
for 4 or 6 weeks, before being exposed to a range of salt
concentrations. Exposure of Arabidopsis to NaCl concentrations higher than 150 mM resulted in plants
dying before the end of the experiment and so higher
concentrations were not used. Exposure of Thellungiella to NaCl concentrations up to 500 mM did not
result in significant mortality, in line with previous
reports (Inan et al., 2004; Taji et al., 2004). The Na+ level
determined in control leaf tissue was considerably
higher in Thellungiella than in Arabidopsis (Fig. 1A).
This difference disappeared after exposure to salinity,
due to a rapid increase in sodium concentration in
leaves of Arabidopsis. The accumulation of Na+ in
leaves of Thellungiella was much lower at external
concentrations between 0 and 150 mM NaCl. Sodium
accumulation increased sharply in Arabidopsis leaves
over the experiment, whereas leaf Na+ content in
Thellungiella increased less, even at higher external
concentrations of NaCl. The level measured after 2
weeks salt treatment in Thellungiella subjected to 250
and 500 mM NaCl was similar to that of Arabidopsis
exposed to 150 mM NaCl. The two species also differed
in their potassium accumulation. The leaf tissue K+
concentration in plants watered with salt-free medium
was found to be 30% to 40% higher in Thellungiella
(Fig. 1B). Salt treatment and sodium accumulation
resulted in a large reduction of K+ content in leaves of
Arabidopsis. In contrast, Thellungiella revealed only a
limited decline in K+ level. Even in Thellungiella
challenged with severe salinity the leaf potassium
content was no lower than that in control Arabidopsis.
Control leaves of Thellungiella had a chlorophyll
content that was around 30% higher than in Arabidopsis (Fig. 2). Exposure of Arabidopsis to salt resulted in a progressive decline in chlorophyll content.
Figure 1. Changes in leaf Na+ (A) and K+ (B) content over time in
Arabidopsis (hatched bars) and Thellungiella (dotted bars). Four-weekold Arabidopsis and 6-week-old Thellungiella were exposed to salt for
up to 2 weeks. Plants were subjected (in following sequence on graph)
to: 0, 100, and 150 mM NaCl for Arabidopsis, and 0, 100, 150, 250, and
500 mM NaCl for Thellungiella. Data represent the means 6 SE of at
least five replicates. [See online article for color version of this figure.]
The total chlorophyll measured after 10 d of salt treatment with 100 and 150 mM NaCl dropped by 32% and
48%, respectively. Exposure of Thellungiella to salt did
not result in any significant change in leaf chlorophyll
content.
Gas-Exchange Parameters
Gas exchange in Thellungiella and Arabidopsis was
measured daily for 14 d, starting at the onset of salt
treatment (Fig. 3, A and B). Under control conditions,
stomatal conductance in Thellungiella was lower than
in Arabidopsis, resulting in a lower transpiration rate
(data not shown); however, CO2 assimilation, measured under saturating light and atmospheric CO2,
was higher. Exposure of Arabidopsis to salt induced
stomatal closure, this being induced rapidly and then
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Stepien and Johnson
developing further over time. This decline was accompanied by a similar drop in CO2 assimilation. In
Thellungiella there was no rapid stomatal closure
observed, even upon exposure to 500 mM salt. By the
end of the experiment, a small decline in stomatal
conductance was seen, this resulting in only a slight
drop in photosynthesis.
Measurements of the relationship between assimilation (A) and calculated internal CO2 concentrations
(Ci) showed that this was unaffected in Thellungiella
plants exposed to a wide range of salt concentrations
(Fig. 3C). This relationship was also unchanged in
Arabidopsis subjected to 100 mM NaCl over the first 10
d of salt treatment. In Arabidopsis treated with a high
salt concentration (150 mM) the A/Ci relationship was
found to be modified. An external CO2 concentration
of 2,000 mmol m22 s21 was not sufficient to restore
carbon fixation to the control level.
Chlorophyll Fluorescence Analysis
Measurements of chlorophyll fluorescence provide
detailed information about PSII in intact leaves. In
control plants of both species the ratio Fv/Fm, a measure of the maximum quantum yield of photosynthesis, was close to 0.8 (Fig. 4A), consistent with
measurements on a wide range of unstressed higher
plants (Bjorkman and Demmig, 1987; Johnson et al.,
1993). Exposure to salt did not have any immediate
effect on Fv/Fm in either species; however, during the
development of salt stress over a 14 d period, this
parameter fell in Arabidopsis exposed to either 100 or
150 mM NaCl, consistent with a slow accumulation of
photoinhibited PSII. In Thellungiella, no change in Fv/
Fm occurred, even at the highest salt concentration.
The parameter FPSII provides an estimate of PSII
quantum efficiency (Genty et al., 1989). With increasing salt stress and decreasing CO2 fixation, PSII elec-
Figure 2. The effect of salt treatment on the total leaf chlorophyll
content in Arabidopsis (white bars) and Thellungiella (black bars). Fourweek-old Arabidopsis and 6-week-old Thellungiella were exposed to:
0, 100, 150, 250, and 500 mM NaCl. Leaves were collected 10 d after
initiating salt treatment to determine chlorophyll concentration. Data
represent the means 6 SE of at least five replicates.
Figure 3. Gas exchange in Arabidopsis (closed symbols) and Thellungiella (open symbols) exposed to: 0 (diamonds), 100 (squares), 150
(triangles), 250 (circles), and 500 (stars) mM NaCl. Leaves were exposed
to the white actinic light (PFD 850 mmol m22 s21) for 40 min at 25°C in
the presence of 370 mL L21 CO2. Stomatal conductance, gs (A), and
CO2 assimilation rate, A (B), were measured. C, CO2 assimilation rate
as a function of internal CO2 concentration determined 10 d after
initiating salt treatment. Symbols as above. Data points represent the
means 6 SE of at least five replicates. [See online article for color
version of this figure.]
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Salt Responses of Arabidopsis and Thellungiella
suming excess reducing power (Cornic and Briantais,
1990; Tourneux and Peltier, 1995). In salt-treated Thellungiella, FPSII did not fall below the control under
any treatment. For plants irrigated with 100 or 150 mM
NaCl, there was little change in FPSII. In plants
exposed to the highest salt concentrations, a considerable increase in FPSII was observed, with this increase
developing progressively through the experiment.
It is widely observed that plants are able to protect
themselves from high light by increasing the dissipation of light energy as heat, measured as nonphotochemical quenching of chlorophyll fluorescence
(NPQ). Increasing salinity resulted in a substantial
increase in NPQ in Arabidopsis, while in Thellungiella, NPQ remained close to control levels at all salt
concentrations (Fig. 4C). The increase in Arabidopsis
might result from changes in one of at least two
processes: protective high-energy-state quenching or
photoinhibition. These processes can be partially distinguished by the kinetics with which they relax following the end of illumination (Maxwell and Johnson,
2000). Measurements of the fast and slow relaxing
components of quenching showed that the majority of
quenching relaxed rapidly in the dark (NPQf), indicating that it was high-energy-state quenching (Fig. 5).
However, a part of the quenching was more persistent
(NPQs), suggesting the occurrence of photoinhibition
in leaves of Arabidopsis due to high NaCl. Both forms
of quenching increased in response to salt treatment,
each contributing to a similar extent to the overall
increase. The increase in total NPQ in Thellungiella
was small and was mainly due to an increase in NPQf.
PSI Photochemistry
In addition to chlorophyll fluorescence, simultaneous measurements were made of the redox state
Figure 4. Changes in the chlorophyll fluorescence parameters in
Arabidopsis (closed symbols) and Thellungiella (open symbols) exposed to: 0 (diamonds), 100 (squares), 150 (triangles), 250 (circles), and
500 (stars) mM NaCl. The leaves of plants dark adapted overnight were
illuminated with a 1.2-s pulse of white saturating light (PFD 8,000 mmol
m22 s21) at 25°C in the presence of 370 mL L21 CO2 to estimate the
maximal fluorescence. Following this, the actinic light (PFD 850 mmol
m22 s21) was switched on and the plant was left for 40 min to reach a
steady state. The maximum quantum efficiency (A), photochemical
efficiency (B), and NPQ (C) were calculated. Data points represent the
means 6 SE of at least five replicates. [See online article for color
version of this figure.]
tron transport was inhibited in Arabidopsis, as indicated by the fall in FPSII; however, this inhibition was
less marked than that of CO2 assimilation (Fig. 4B),
probably reflecting a role of photorespiration in con-
Figure 5. Fast- and slow-relaxing components of NPQ (NPQf and
NPQs, respectively) in the leaves of Arabidopsis and Thellungiella
subjected to: 0 and 150, and 0 and 500 mM NaCl, respectively.
Measurements were carried out 14 d after initiating salt treatment at
25°C in the presence of 370 mL L21 CO2. Leaves were illuminated with
850 mmol m22 s21 actinic light. Data points represent the means 6 SE of
at least three replicates.
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Stepien and Johnson
and turnover of the PSI primary electron donor, P700.
With increasing NaCl and time of treatment, P700
became more oxidized in Arabidopsis, rising at maximum of 72% oxidized (Fig. 6A). No significant changes
in P700 redox state were seen in Thellungiella subjected
to salt. The conductance of the electron transport chain
(gETC) was also estimated (Fig. 6B). This parameter
provides information about the extent to which the
electron transport chain is being regulated (Golding
and Johnson, 2003). In Arabidopsis, there was a progressive decline in gETC in response to salt, implying a
down-regulation of the electron transport chain. In
Thellungiella no change in gETC was observed.
Golding and Johnson (2003) observed that the proportion of PSI reaction centers that were active (i.e.
where P700 could be oxidized by light and then
rapidly rereduced in darkness) increased in plants of
barley (Hordeum vulgare) exposed to drought stress.
This was suggested to represent the activation of a
pool of PSI centers involved in cyclic electron flow.
Subjecting Arabidopsis plants to salt stress induced a
considerable increase in the proportion of active PSI at
either salt concentration used (Fig. 6C). In Thellungiella, regardless of salt treatment, the active PSI pool
was unaltered. Interestingly, although the proportion
of P700 oxidized was measured to be similar in control
plants of both species, the proportion of the active
P700 in the same plants was notably higher for
Thellungiella.
Providing P700 is .20% oxidized, PSI turnover can
be estimated from the reduction kinetics of P700+,
following transition from actinic light to dark (Clarke
and Johnson, 2001; Golding et al., 2005). The electron
transport rate through PSI (PSI ETR) estimated in
Arabidopsis increased by up to 23% in response to salt
treatment (Fig. 6D), in spite of the greatly inhibited
rate of PSII electron transport. This implies an increase
in cyclic electron flow around PSI in Arabidopsis
exposed to salt stress. In salt-treated Thellungiella,
PSI ETR was unaltered.
Electron Transport to Oxygen under Salt Stress
Reducing potential produced by photosynthetic
electron transport can be consumed by a number of
alternative pathways. Of particular note are reactions
involving oxygen. Two major oxygen-using pathways
have been described: photorespiration and the Mehler
reaction. To evaluate the importance of these pathways, the oxygen dependence of electron transport
was examined. Arabidopsis and Thellungiella were
exposed to a range of irradiances in the presence of
2,000 mL L21 CO2 and either 21% or 2% O2. In control
plants of both species, PSII ETR, calculated as the
product of PSII photochemical efficiency (FPSII) and
photon flux density (PFD), exhibited a standard light
response reaching a maximum at around 600 mmol m22
s21 of light (Fig. 7, A and B). Subjecting control plants
to low (2%) oxygen resulted in a decrease of PSII ETR
at saturating irradiances. NPQ was unaffected by low
O2 at all irradiances (Fig. 7, C and D).
Simultaneous measurements of the redox state and
turnover of the PSI primary electron donor revealed
slight effects of oxygen concentration in control plants.
With increasing irradiance, P700 became progressively
more oxidized in both species (Fig. 7, E and F).
Although the proportion of P700 oxidized was insensitive to oxygen in control plants, the conductance of
the electron transport chain (gETC) decreased and the
PSI ETR was lowered by a similar amount (Fig. 7, G–J).
In Arabidopsis exposed to 150 mM salt, maximum
PSII ETR at high CO2 was lower than in control plants,
Figure 6. Changes in PSI parameters in
leaves of Arabidopsis (closed symbols) and
Thellungiella (open symbols) exposed to: 0
(diamonds), 100 (squares), 150 (triangles),
250 (circles), and 500 (stars) mM NaCl. The
redox state of the PSI primary donor (A),
the conductance of electron transport
chain, gETC (B), the proportion of active
PSI centers, P700Act (C), and the PSI ETR
(D) were estimated as described in “Materials and Methods.” Measurements were
carried out on the same leaves used in
Figure 4. Data points represent the
means 6 SE of at least five replicates. [See
online article for color version of this
figure.]
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Salt Responses of Arabidopsis and Thellungiella
Figure 7. Oxygen dependence of electron transport:
PSII ETR (A and B), NPQ (C and D), P700+ (E and F),
gETC (G and H), and PSI ETR (I and J), measured in
leaves of Arabidopsis and Thellungiella subjected to
NaCl (circles): 150 mM for Arabidopsis and 250 mM
for Thellungiella. Control plants (squares) were maintained in a NaCl-free soil. The measurements were
carried out 10 d after initiating salt treatment under
saturating CO2 (2000 mL L21), at 25°C and under 21%
(closed symbols) or 2% (open symbols) oxygen. Data
points represent the means 6 SE of at least five
replicates. [See online article for color version of
this figure.]
consistent with observations at ambient CO2. As in the
control, electron transport through PSII fell slightly in
response to low O2 (Fig. 7A). In contrast, salt treatment
resulted in an increase in PSII ETR in Thellungiella
relative to plants not exposed to salt; however, this
increase in electron transport was entirely abolished
when the O2 concentration was lowered (Fig. 7B). In
salt-treated Arabidopsis, lowering the O2 concentration
resulted in a decrease in NPQ (Fig. 7C). In Thellungiella,
NPQ was insensitive to O2 concentration (Fig. 7D).
The proportion of P700 oxidized in salt-stressed
Arabidopsis was significantly higher under low O2
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Stepien and Johnson
conditions (Fig. 7E). This, in spite of the small reduction in gETC, resulted in the electron transport through
PSI being maintained at the same level (Fig. 7I). In
contrast, PSI ETR in Thellungiella subjected to NaCl,
like the control, decreased at low O2 (Fig. 7J). This
resulted from an increase in P700 oxidation and a drop
in gETC.
Plastid Terminal Oxidase as a Sink for Electrons
Results in Figure 7 strongly indicate that the additional turnover of PSII seen in salt-treated leaves is due
to electron transport to oxygen. Given that sensitivity
to oxygen is seen at high CO2, we can exclude a
contribution of photorespiration to this effect. Photoreduction of oxygen may occur at the acceptor side of
PSI, via the Mehler reaction; however, the absence of a
sensitivity of PSI parameters to oxygen tends to speak
against this. It has been observed that higher plant
chloroplasts contain a putative quinone-oxygen oxidoreductase, the plastid terminal oxidase (PTOX) or
IMMUTANS protein. To determine whether the PTOX
plays a role in electron transport from PSII to oxygen,
measurements of PSII ETR were repeated in control
and salt-treated leaves vacuum infiltrated with either
water or a solution of the PTOX inhibitor n-propyl
gallate (nPG; 3,4,5-trihydroxy-benzoic acid-n-propyl
ester; Josse et al., 2003). In Arabidopsis, PSII ETR was
insensitive to nPG, regardless of whether plants had
been exposed to salt treatment (Fig. 8A). This was also
the case in control Thellungiella. In Thellungiella
exposed to 250 mM NaCl, PSII ETR was sensitive to
nPG. PSII ETR, measured 10 d after initiating salt
treatment, was reduced by 35% in leaves infiltrated
with to 1 mM nPG, falling to the control level.
The effect of nPG suggests the activity of a PTOX
protein in Thellungiella leaves; however, it does not
rule out a contribution of the Mehler reaction to
electron transport. To measure electron transport to
oxygen in the absence of that reaction, leaves were
infiltrated with the cytochrome b6f inhibitor 2#iodo-6isopropyl-3-methyl-2#,4,4#-trinitrodiphenylether (DNPINT), a specific inhibitor of the Qo-binding site (Trebst
et al., 1978). In Arabidopsis, this almost completely
blocked PSII electron transport, regardless of salt
treatment (Fig. 8B). In control Thellungiella leaves,
DNP-INT also largely blocked PSII electron transport,
although a significant residual level of FPSII remained.
In salt-treated Thellungiella leaves, DNP-INT only partially inhibited electron transport. Lowering O2 further
inhibited electron transport. The extent of DNP-INTinsensitive, oxygen-sensitive electron transport was
close to that of nPG-sensitive electron transport in the
same leaves.
Immunoblot analyses of thylakoid membrane extracts using antibodies raised against the PTOX from
Arabidopsis revealed the presence of a 40-kD band in
both Arabidopsis and Thellungiella leaves (Fig. 9). The
band detected in control plants, loaded on the basis of
equal protein content, was more prominent in Thel-
Figure 8. The effect of nPG (A) and DNP-INT (B) on PSII photochemical
efficiency measured in the leaves of Arabidopsis and Thellungiella
subjected to: 0 and 100, and 0 and 250 mM NaCl, respectively.
Measurements were carried out 10 d after initiating salt treatment at
25°C in the presence of 370 mL L21 CO2. Leaves were illuminated with
850 mmol m22 s21 red light. Leaves were vacuum infiltrated with water
(white bars) or with 1 mM nPG (A) or 35 mM DNP-INT (B) in the
presence of 21% (gray bars) and 2% (black bars) oxygen. Data points
represent the means 6 SE of at least five replicates.
lungiella than in Arabidopsis. In Arabidopsis, subjecting plants to salt did not result in any change in the
estimated PTOX content. Thellungiella responded to
250 mM NaCl by increasing polypeptide content nearly
4-fold, relative to the plants maintained in a NaCl-free
medium.
DISCUSSION
A considerable effort has been made, over many
years, to understand the fundamental basis of salttolerance physiology. A number of studies have examined the responses of Arabidopsis to salt (Zhu, 2001).
This has the advantage of being a well-studied model
system with a wealth of molecular and genetic information; however, as is illustrated here, its usefulness is
limited by the fact that it is a glycophyte. Thellungiella
in contrast has a high degree of salt tolerance, as well as
tolerance of other environmental stresses, but, as a close
Arabidopsis relative, shares a high degree of homology
and is amenable to many of the same experimental
approaches. In particular, a comparative approach
studying both species has the potential to identify key
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Salt Responses of Arabidopsis and Thellungiella
Figure 9. The effect of salt treatment on PTOX protein expression in the
leaves of Arabidopsis and Thellungiella subjected to: 0 and 100, and 0
and 250 mM NaCl, respectively. Leaves from control and salt-treated
plants were collected 10 d after initiating salt treatment for immunodetection after SDS-PAGE, separation of 35 mg protein from the
thylakoid membrane samples, and electrophoretic transfer to nitrocellulose membrane. Immunoblot was quantified by the optical densitometry. Data points represent the means 6 SE of six blots from three
separate membrane preparations. Insert shows typical bands from an
original blot, loaded on an equal protein basis.
characteristics that might be transferred between species to enhance stress tolerance.
Exposure of Arabidopsis to salt resulted in substantial Na+ uptake (Fig. 1) and loss of chlorophyll (Fig. 2)
and induced stomatal closure (Fig. 3A). The resulting
limitation on CO2 entry and any direct toxic effect
of salt accumulation resulted in inhibition of CO2
assimilation. Consistent with previous findings
(Allakhverdiev et al., 2000; Stepien and Klobus, 2005;
Stepien and Klobus, 2006), there were indications that
the highest salinity caused nonstomatal limitation of
photosynthesis. High CO2 (2,000 mL L21), the highest
experimentally available in our gas-exchange system,
failed to reverse the inhibition of assimilation, although internal Ci is liable to be overestimated under
these conditions.
The inhibition of assimilation in salt-stressed Arabidopsis is accompanied by a decrease in electron transport through PSII, indicated by the decline in FPSII,
and cumulative damage to PSII, indicated by the
progressive drop in Fv/Fm. This was not, however,
mirrored in the responses of PSI electron transport.
Measurements of PSI electron transport have been the
subject of some controversy recently (see Johnson,
2005 for discussion). We have based our estimates on
the kinetics of rereduction of P700+ following a light to
dark transition (Clarke and Johnson, 2001); however,
using estimates based on the proportion of centers that
are reduced but can be oxidized (P700Act-P700+; see
Klughammer and Schreiber, 1994), we reach the same
conclusions. PSI turnover does not drop under salt
stress in line with assimilation and PSII turnover and
indeed we argue that it rises under certain experimental conditions. From this we conclude that cyclic
electron flow around PSI increases under salt stress.
This is in line with studies of the responses of a
number of plant species to a variety of stresses, including for example studies of high light, chilling, and
low CO2 or drought stress (Clarke and Johnson, 2001;
Golding and Johnson, 2003; Miyake et al., 2005a,
2005b). The increase in cyclic electron transport was
accompanied by an increase in NPQ. NPQ is made up
of both reversible, DpH-dependent quenching, which
protects PSII from light damage, and irreversible NPQ,
which reflects photoinhibition. Although we did not
attempt to resolve fast and slowly relaxing across all
experimental conditions, measurements under selected conditions showed that the additional quenching resulted from both these processes increasing
(Fig. 5).
The inhibition of linear electron flow in Arabidopsis
under salt stress was accompanied by a down-regulation
of electron flow through the cytochrome b6f complex,
as indicated by the decline in the conductance of the
electron transport chain, gETC. A similar response has
been seen previously in response to low CO2 or
drought (Harbinson, 1994; Golding and Johnson,
2003). This down-regulation has recently been shown
to occur in response to changes in the redox state of the
NADP/NADPH pool and serves to limit oxidative
stress (Hald et al., 2008).
Overall, the response of Arabidopsis to salt stress is
very much in line with that expected from the responses of a variety of other species to a variety of
stresses. Down-regulation of linear electron transport
limits oxidative stress and increased cyclic flow enhances photoprotective energy dissipation.
In contrast, the response of the halophyte Thellungiella was rather different. Our results confirmed the
salt-tolerant nature of Thellungiella (Inan et al., 2004;
Taji et al., 2004). Plants grew rapidly at moderate
salinity and survived exposure to concentrations of up
to 500 mM NaCl without significant mortality. In terms
of the responses of photosynthesis, even the highest
salt concentration used did not represent a substantial
stress. The chlorophyll content of leaves did not drop
significantly in response to salt (Fig. 2) and both
stomatal conductance and assimilation at atmospheric
CO2 concentrations were maintained (Fig. 3).
Given the ability of Thellungiella to maintain stomatal conductance and assimilation even under severe
salt stress, it might be expected that the photosynthetic
apparatus would show little or no response to salt.
This is however not the case. Even at the highest salt
concentration, there is no sign that the photosynthetic
apparatus is stressed, there is no decline in Fv/Fm,
which would indicate photoinhibition, and no increase
in NPQ (Fig. 4). There was, however, a substantial
effect of salt treatment on PSII photochemistry. With
prolonged exposure to salt, the quantum efficiency of
PSII, FPSII, increased.
An increase in FPSII might be explained in a number of ways. Most simply, this could reflect saltinduced changes in PSII concentration and/or antenna
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Stepien and Johnson
complex size. If the concentration or antenna size of
PSII decreased, then the efficiency of each reaction
center would have to increase to maintain the same
overall electron flux. However, given that there is no
significant change in either chlorophyll content (Fig. 2)
or chlorophyll a/b ratio (data not shown), this is
unlikely to play a major role. The fact that there is no
concomitant increase in the CO2 assimilation rate
measured in the same plants, strongly suggests that
there must be an alternative pathway for use of electrons coming from PSII in Thellungiella exposed to
severe salinity. A number of pathways in addition to
CO2 fixation are known to derive their reducing potential from the photosynthetic electron transport,
including nitrogen and sulfur metabolism, and the
demand for these certainly might increase in Thellungiella under salt stress to meet the needs, for example,
for Pro accumulation as a compatible solute. However,
the increase in FPSII we observe is seen only at high
light—at growth light intensities there was no significant change in FPSII. This implies that it is not simply
an increase in the competition from an alternative
electron sink that has increased but the overall capacity of that alternative sink. Crucially, the additional
electron transport through PSII was sensitive to low O2
concentrations (Fig. 7B). This indicates that it is oxygen
acting as the additional sink for electrons.
There are a number of different ways in which
oxygen might act as a sink for reducing equivalents
from PSII. The most widely known is photorespiration, when the carbon-fixing enzyme Rubisco reacts
ribulose bisphosphate with oxygen rather than CO2.
This occurs in competition with CO2 fixation and can
be suppressed when leaves are supplied with high
CO2. In our experiments however the oxygen sensitivity of PSII was also seen at high CO2 concentrations,
so photorespiration cannot explain the data. Photoreduction of oxygen can occur at the acceptor side of PSI
to produce superoxide—the Mehler reaction. The protective potential of this reaction has been widely
discussed. It will provide a sink for reductant, taking
electrons away from the electron transport chain and
so tend to protect PSII from photoinhibition. It is also
believed to generate a DpH across the thylakoid membrane, supporting protective, NPQ of energy (Asada,
2000; Chen et al., 2004); however, it is liable to produce
ROS. These are tightly controlled by coupling the
Mehler reaction to detoxifying processes (Asada,
2000), but will still impose a metabolic burden on the
plant. Also, given that the primary reaction involved is
the essentially spontaneous, uncatalyzed reaction between iron sulfur centers and molecular oxygen, it is
not clear how the capacity of this would increase. The
Mehler reaction is limited by control of electron flux
through the cytochrome b6f complex (Hald et al.,
2008); however, there is no indication that this is
altered in Thellungiella under salt stress.
The Mehler reaction involves consumption of reducing potential after PSI. Therefore, this should be
reflected in an increased PSI turnover, in addition to
the enhanced PSII photochemistry observed. This we
do not observe. The PSI ETR (Fig. 6D) remained
unaltered in response to salt. Furthermore, PSI electron transport was seen to be relatively insensitive to
altering oxygen concentration; indeed, PSI turnover
tended to increase at low oxygen. This speaks against
the Mehler reaction being involved in acting as a sink
for electrons from PSII, although a contribution of this
pathway cannot be totally excluded.
Various authors have discussed the possibility of
oxygen acting as an electron sink prior to PSI. Reduction of oxygen might occur at PSII itself, either directly
at the QB site or via cytochrome b559 (Cleland and
Grace, 1999; Bondarava et al., 2003). It has also been
discussed that oxygen might oxidize plastoquinol
directly, although the extent to which this can occur
spontaneously is uncertain (Khorobrykh and Ivanov,
2002). The reaction with PQH2 can however occur in
an enzyme-catalyzed reaction in the chloroplast, mediated by the PTOX (Aluru et al., 2006). The PTOX
protein was first detected as the protein responsible for
the variegated or bleached phenotype of various mutants (immutans or ghost mutants; Carol et al., 1999;
Josse et al., 2000). PTOX shares sequence similarity
with the alternative oxidase in the mitochondrion and
is able to divert the electron flow from PSII via PQ to
O2, producing water rather than superoxide (Josse
et al., 2003). The variegated phenotype seen in the
immutans mutants arises because PTOX participates in
carotenoid biosynthesis. It is also suggested to participate in a pathway of chlororespiration, together with
an NADPH plastoquinone oxidoreductase complex,
ndh (Cournac et al., 2002; Joet et al., 2002; Aluru et al.,
2006). The exact role of chlororespiration remains
obscure; however, it is suggested to function in regulating cyclic electron flow around PSI (Joet et al., 2002).
In Thellugiella, we observe a substantial up-regulation
of PTOX protein in response to salt stress (Fig. 9). In
Arabidopsis, PTOX is only ever expressed at very low
concentrations, approximately 1% of PSII, and is therefore thought unlikely to ever act as a significant
electron sink (Lennon et al., 2003). Based on immunoblot analysis, it seems possible that even in control
conditions, the PTOX content of Thellungiella leaves is
substantially higher and this is increased significantly
in response to salt stress (Fig. 9). Furthermore, in salttreated leaves of Thellungiella, electron transport was
observed to be sensitive to the known PTOX inhibitor
nPG. No such sensitivity was seen in either control
Thellungiella or control or salt-treated Arabidopsis. In
salt-treated Thellungiella, in the presence of the cytochrome b6f inhibitor DNP-INT, there was a substantial
residual electron transport activity, indicated by FPSII
measurements, which was sensitive to oxygen (Fig. 8).
These observations provide strong evidence that
PTOX is acting as the additional electron sink in salttreated Thellungiella.
High expression of PTOX has been observed previously. The montane plant Ranunculus glacialis acclimated to high-light and low-temperature conditions
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Salt Responses of Arabidopsis and Thellungiella
was demonstrated to possess an alternative electron
sink and to contain high PTOX protein abundance
(Streb et al., 2005). These authors suggested this to
serve as a mechanism to consume electrons; however,
apart from protein expression, no practical test for the
PTOX activity was shown. This lead Rosso et al. (2006)
to question whether PTOX was in fact the electron sink
in R. glacialis. They examined plants of Arabidopsis in
which the native PTOX was overexpressed and were
unable to detect any effect on the redox poising of the
PSII acceptor pool or any protection from photoinhibition. Rosso et al. (2006) did not however demonstrate
that the overexpressed PTOX was catalytically active.
Joet et al. (2002) examined the effect of overexpressing
Arabidopsis PTOX in tobacco (Nicotiana tabacum)
leaves and were able to detect activity; however, the
rate of turnover shown was low, and no data was
presented to indicate that PTOX significantly affected
steady-state electron transport.
The contradictions in the above studies might be
explained in a number of ways. Diffusion of plastoquinol in the thylakoid membrane is heavily restricted,
due to the high protein concentration (Kirchhoff et al.,
2008). So, to act as an efficient electron sink, PTOX
would probably need to be colocated with PSII. In
spinach (Spinacia oleracea), PTOX is localized to the
stromal lamellae (Lennon et al., 2003) whereas PSII is
concentrated in the granal stacks (Albertsson, 2001).
This would make it difficult for PTOX to act as a
significant direct electron sink from PSII. It may be,
therefore, that the targeting of PTOX within the thylakoid membrane is different in stress-tolerant plants
such as Thellungiella and R. glacialis. Alternatively,
additional regulation of PTOX may be required. The
mitochondrial alternative oxidase possesses a regulatory disulphide that is not conserved in the chloroplast
homolog (Berthold and Stenmark, 2003); however,
some alternative form of regulation can be envisaged.
In conclusion, we have shown substantial differences in the responses of the photosynthetic apparatus
of Arabidopsis and Thellungiella to salt stress. In
particular, Thellungiella induces an alternative pathway for electron transport that may protect the leaf
under stress. This pathway is absent in Arabidopsis;
however, a better understanding of how this pathway
operates in Thellungiella may open the possibility of
engineering this into more stress-sensitive plants.
MATERIALS AND METHODS
Plant Material and NaCl Treatment
Seeds of Arabidopsis (Arabidopsis thaliana; ecotype Columbia 0) and
Thellungiella (Thellungiella halophila; ecotype Shandong wild type) were
stratified at 4°C for 5 d and then germinated in a controlled-environment
cabinet (E.J. Stiell) in an 8-h photoperiod (photosynthetic photon flux of 120
mmol m22 s21 provided from cool-white fluorescent bulb), at 23°C/15°C (day/
night). A short day length was used to delay flowering in Arabidopsis. Oneweek-old seedlings were transferred to 7.5-cm pots filled with Viking MM
peat-based compost. Four-week-old Arabidopsis and 6-week-old Thellungiella
plants, similar in size, were irrigated with 0, 50, 100, 150, 250, and 500 mM NaCl
for up to 14 d.
Determination of Na+ and K+ Content
Leaves of control and treated plants were harvested and washed with
deionized water. The leaf samples were dried at 105°C for 1 h, subsequently at
60°C for 48 h, and then weighed for determination of dry weight. Lyophilized
leaves were milled to powder for mineral nutrient analyses. Powdered samples
(0.5 g) were then extracted with 10 mL of HNO3 for 60 min at 95°C. The resulting
solutions were filtered through Whatman filter paper, diluted appropriately,
and analyzed for Na+ and K+. Cation concentrations were determined with a
UNICAM 929 atomic absorption spectrophotometer (Unicam Ltd.).
Chlorophyll Measurements
Leaves from control and salt-treated plants were collected, weighed fresh,
washed in distilled water, and extracted in 80% (v/v) acetone. Chlorophyll
content was measured according to Porra et al. (1989).
Photosynthetic Parameters
Photosynthetic parameters were measured as described previously (Golding and Johnson, 2003; Hald et al., 2008). Briefly, gas exchange was monitored
using a CIRAS1 infrared gas analyzer (PP Systems). Leaves illuminated for 40
min, to achieve steady-state conditions. Leaf temperature (25°C 6 1°C) and
CO2 concentration within the chamber were controlled by the gas analyzer.
Chlorophyll fluorescence emission was measured using a PAM-101 chlorophyll fluorimeter (Walz). Fluorescence parameters were calculated as described by Maxwell and Johnson (2000). The redox state of the PSI primary
donor, P700, was determined using a Walz PAM 101 fluorometer in combination with an ED-P700DW-E emitter-detector unit (Walz). Light was provided by Volpi Intralux lamps, except for measurements in Figure 7 that used
a Luxeon LXHL-PD09 LED (Phillips-Lumiled) in a laboratory-built lamp.
Calibration of maximum P700 signal size was conducted using a high-power
LED array (LED 735-66-60 Roithner Laser). Two percent and 21% O2 gas were
supplied by mixing compressed oxygen and nitrogen from cylinders (BOC
Gases) using an MKS controller (MKS Instruments Inc.).
Measurements of PTOX Activity and Protein
To estimate the contribution of the PTOX to overall PSII electron transport,
the leaves of control and salt-treated plants were vacuum infiltrated with
either water or with 1 mM nPG (3,4,5-trihydroxy-benzoic acid-n-propyl ester;
Sigma) or 35 mM DNP-INT (kindly provided by Dr. Anja Krieger-Liszkay, CESaclay, France).
For immunoblot analysis, thylakoids were isolated as described by Cerovic
and Plesnicar (1984). Thylakoid proteins were extracted from membranes in
125 mM TRIS-HCl, pH 6.8, 20% glycerol, 4% (w/v) SDS, 5% (v/v) b-mercaptoethanol, 0.1% (w/v) bromphenol blue. Protein concentration was estimated
using a Bio-Rad protein assay kit (Bio-Rad Laboratories). Immunoblotting was
carried out as described by Mudd et al. (2008). Polyclonal antibodies against
PTOX were kindly provided by Dr. M. Kuntz (Université Joseph Fourier,
Grenoble, France).
ACKNOWLEDGMENTS
We would like to thank to Dr. M. Kuntz for kindly providing us with
polyclonal antibodies against PTOX and Dr. Anja Krieger-Liszkay for DNPINT. We would like to thanks Drs. Simon Hald, Rachel Webster, and
Panagiotis Madesis for help and advice with experimental procedures, and
Mr. John Simpson for help building instrumentation.
Received November 10, 2008; accepted November 26, 2008; published
December 3, 2008.
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