ARTICLE IN PRESS

Journal of Plant Physiology 163 (2006) 165—175

www.elsevier.de/jplph

Alleviation of photoinhibition in drought-stressed

wheat (Triticum aestivum) by foliar-applied
glycinebetaine
Qian-Quan Maa,b, Wei Wangb,, Yong-Hua Lib, De-Quan Lib, Qi Zoub

a
Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Baodao Xincun,
Danzhou, Hainan 571737, P.R. China
b
College of Life Science, Shandong Agricultural University, Tai’an, Shandong 271018, P.R. China

Received 11 October 2004; accepted 30 April 2005

KEYWORDS Summary
D1 protein turnover;
Effects of foliar application of 100 mmol/L glycinebetaine (GB) on PS II photochemistry
Drought stress;
in wheat (Triticum aestivum) flag leaves under drought stress combined with high
Glycinebetaine;
irradiance were investigated. The results show that GB-treated plants maintained a
Photoinhibition;
higher net photosynthetic rate during drought stress than non-GB treated plants.
Photosynthesis;
Exogenous GB can preserve the photochemical activity of PSII, for GB-treated plants
Wheat (Triticum
maintain higher maximal photochemistry efficiency of PSII (Fv/Fm) and recover more
aestivum)
rapidly from photoinhibition. In addition, GB-treated plants can maintain higher anti-
oxidative enzyme activities and suffer less oxidative stress. Our data suggest that GB
may protect the PSII complex from damage through accelerating D1 protein turnover
and maintaining anti-oxidative enzyme activities at higher level to alleviate
photodamage. Diethyldithiocarbamate as well as streptomycin treatment can impair
the protective effect of GB on PSII. In summary, GB can enhance the photoinhibition
tolerance of PSII.
& 2005 Elsevier GmbH. All rights reserved.

Abbreviations: APX; Ascorbate peroxidase; BADH; Betaine aldehyde dehydrogenase; CAT; Catalase; CDH; Choline dehydrogenase;
CMO; Choline monooxygenase; COD; Choline oxidase; DDTC; Diethyldithiocarbamate; Fm; Maximal fluorescence in dark leaves; F0;
Minimal fluorescence in dark-adapted leaves; Fv; Variable fluorescence in dark-adapted leaves; Fv/Fm; Maximal efficiency of PSII
photochemistry in the dark-adapted state; GB; glycinebetaine; Cw; Water potential; PSII; Photosystem II; ROS; Reactive oxygen
species; RWC; Relative water content; SM; Streptomycin; SOD; Superoxide dismutase
Corresponding author. Tel.: +86 538 8242656 8234; fax: +86 538 8241341.
E-mail address: wangw@sdau.edu.cn (W. Wang).

0176-1617/$ - see front matter & 2005 Elsevier GmbH. All rights reserved.
doi:10.1016/j.jplph.2005.04.023
 ARTICLE IN PRESS
166
Introduction
Photoinhibition is a physiological status induced
in plants when the photons absorbed by the
photosynthetic apparatus exceed their utilization
in carbon assimilation. Usually, it is observed as a
light-induced depression of photosynthetic capa-
city (Anderson and Barber, 1996) and thus it affects
both plant growth and productivity (Aro et al.,
1993; Long et al., 1994). Photoinhibition is often
enhanced when plants are exposed to environmen-
tal stresses such as drought, high salinity, extreme
temperature and high irradiance. Drought severely
restricts the productivity and territorial distribu-
tion of plants. Under field conditions, it is generally
characterized by a combination of water shortage,
high temperature and high irradiance. Drought
stress leads to a substantial reduction in net
photosynthetic rate, due to stomatal closure,
which restricts the diffusion of CO2 into the leaf
(Cornic and Massacci, 1996), or non-stomatal
factors, such as inhibition of Rubisco or ATP
synthesis (Lawlor and Cornic, 2002). In conse-
quence, the deficiency of CO2, ATP or RUBP as well
as the inhibition of Rubisco will decrease the
oxidation of NADPH in the Calvin cycle. Conse-
quently, the primary acceptor for photosynthetic
electrons, NADP+, is not sufficiently available, and
a low irradiance is required to saturate photo-
synthesis under drought stress. Therefore, when a
high irradiance is imposed to drought-stressed
plants, susceptibility to photoinhibition may be
increased (Flexas and Medrano, 2002).
Photosystem II (PSII) is believed to play a key role
in the response of leaf photosynthesis to environ-
mental stresses (Baker, 1991). PSII is the most
vulnerable part of the photosynthetic apparatus
(Anderson and Barber, 1996). Studies suggest that
drought stress can cause a considerable depletion
of the PSII core, enhancing the degradation of CP43
as well as D1 protein and interfering with the
phosphorylation of PSII core proteins (Giardi et al.,
1996). During the reorganization process, the turn-
over of D1 protein is enhanced by drought stress to
rebuild PSII and to maintain the remaining PSII
functional. However, the lower amount of D1
protein in drought-stressed leaves suggests that
the enhanced synthesis of D1 protein cannot match
its degradation (Giardi et al., 1996).
Glycinebetaine (N,N,N-trimethyl glycine, GB) is
an amino acid derivative that is synthesized by a
large number of organisms including members of
the plant families Chenopodiaceae, Amarantha-
ceae, Gramineae, Compositae and Malvaceae
(Rhodes and Hanson, 1993; Gorham, 1996; Blunden
et al., 1999). In addition to its function in
Q.-Q. Ma et al.
osmoregulation (Wyn Jones et al., 1977), GB has
been shown to protect functional proteins, en-
zymes (e.g. Rubisco), and lipids of the photosyn-
thetic apparatus and to maintain electron flow
through thylakoid membranes (Xing and Rajashe-
kar, 1999 and references therein; Allakhverdiev et
al., 2003). Moreover, Allard et al. (1998) found that
foliar-applied GB prevented photoinhibition in
wheat (Triticum aestivum) during freezing stress.
Studies in transgenic rice (Sakamoto et al., 1998),
Arabidopsis (Alia et al., 1999) and tobacco (Holm-
ström et al., 2000), which express transgenes for
enzymes involved in GB synthesis (betaine aldehyde
dehydrogenase (BADH), choline dehydrogenase
(CDH), choline monooxygenase (CMO) or choline
oxidase (COD)), suggested that the transgenic
plants were more tolerant to photoinhibition
caused by cold, high irradiance or high salinity.
Wheat (T. aestivum) often experiences photoinhibi-
tion due to a combination of drought, high
temperature and high irradiance. It is important
to alleviate photoinhibition for maintaining abun-
dant yield of wheat. The present communication
presents the effects of foliar-applied GB on photo-
inhibition in wheat under drought stress and
discusses the possible mechanism of its protective
function.
Materials and methods
Growth conditions and treatments
Seeds of two winter wheat (T. aestivum L.)
cultivars, HF9703 (drought-tolerant) and SN215953
(drought-sensitive) (Li et al., 2003), were sown in
earthenware pots (15-L, F ¼ 25 cm) filled with
7.5 kg soil composed of loam and organic fertilizer
by the ratio in 7:3 and 500 g complete fertilizer
(NH4NO3:K3PO4:KNO3 ¼ 20:10:20) was added. Ten
seeds were sown in each pot initially, and five
uniform seedlings were left when the third leaves
were fully expanded. The plants were grown under
natural conditions and watered when required. At
anthesis, aqueous 100 mmol/L GB containing 0.1%
Tween 20 was sprayed on the leaves until runoff
twice a day for 3 days. Control plants were sprayed
with water containing 0.1% Tween 20. Drought
stress was induced through withholding watering
after GB application. Thus, the plants were divided
into four groups: (1) WW ¼ well-watered and non-
GB treated, (2) DS ¼ drought stressed and non-GB
treated, (3) GB+WW ¼ GB treated and well wa-
tered, and (4) GB+DS ¼ GB treated and drought
stressed.
 ARTICLE IN PRESS
Glycinebetaine alleviates photoinhibition in wheat
Water status measurements
The degree of drought stress was assessed by leaf
water potential (Cw) and relative water content
(RWC) at predawn. Cw was measured with a HR-
33 T dew point micro voltmeter (Wescor InC.,
Logan, UT, USA) after equilibration in the chamber
for 2 h. RWC was estimated using the following
formula: RWC(%) ¼ [(FW-DW)/(TW-DW)]
100,
where FW ¼ immediate weight of freshly collected
leaves, TW ¼ weight of leaves after rehydration for
24 h at 4 1C in the dark and DW ¼ weight of the
same leaves after drying at 80 1C for 48 h (Bajji et
al., 2001). Water status was measured in penulti-
mate leaves. No differences in Cw and RWC were
found between the penultimate leaves and the flag
leaves of the same plants in a pre-experiment.
Other measurements were conducted on flag leaves
of the same plant.
Plants with RWC decreased by 0%, 10%, 18% and
25% were chosen to represent non (T0), mild (T1),
moderate (T2) and severe (T3) drought-stressed
plants, respectively (Hsiao, 1973).
Photoinhibitory treatment and recovery
Plants from the different groups that suffered
different levels of drought stress were transferred
to a growth chamber. Photoinhibitory treatment
was performed by using high-pressure sodium lamps
at 2571C. The irradiance reaching the upper side
of the leaves was approximately 1600 mmol/m2/s.
The light was filtered through a 10 cm-deep running
water bath to avoid overheating the leaf surface.
Photosynthetic activity and chlorophyll fluores-
cence were measured at 12:00 A.M. to 1:00 P.M.,
2 h after the plants were transferred into the
growth chamber.
For the recovery experiments, severe drought-
stressed plants were kept in the growth chamber
for 4 h to get a more photoinhibited status. After
Fv/Fm was measured, the plants were allowed to
recover at an irradiance of 507mmol/m2/s, during
which course Fv/Fm was measured every 30 min.
Photosynthetic activity and chlorophyll
fluorescence measurements
During the photoinhibitory treatment, gas ex-
change analysis was made using an open system
Infrared Gas Analyzer (CIRAS-I, PP systems, UK).
Net CO2 assimilation rate was determined at
360 ppm CO2, 80% relative humidity, and 21% O2.
Fv/Fm parameters of wheat flag leaves were
measured with a portable fluorometer (FMS-2,
167
Hansatech, UK). The minimal fluorescence (F0)
was determined with a weak modulated light that
was low enough not to induce any significant
variable fluorescence. A 0.8 s saturating pulse light
at 8000 mmol/m2/s was applied to the dark-adapted
leaves to obtain the maximal fluorescence (Fm).
Variable chlorophyll fluorescence (Fv) was calcu-
lated as Fv ¼ FmF0.
In vitro experiments and inhibitors feeding
The flag leaves of well-watered wheat plants
that had been pretreated with or without GB were
cut at dusk, and the cut end of the blade sheath
was immersed in 1 mmol/L diethyldithiocarbamate
(DDTC), an inhibitor of SOD (Jiao and Ji, 2001), or
3 mmol/L streptomycin (SM), an inhibitor of D1
protein synthesis (Darkó et al., 2000), or in
deionized water (controls). The inhibitors were
absorbed by the leaves under low irradiance
(50 mmol/ m2/s) for 12 h at 2571C. Then the
detached leaves were cut into two parts. One part
was used for the measurement of RWC and the
other for Fv/Fm measurement. Both parts were
placed on tissue paper and dehydrated to different
RWC under an irradiance of 300 mmol/m2/s. Then
the irradiance was turned to 1600 mmol/m2/s for
30 min and Fv/Fm was measured after 20 min dark
adaptation.
GB determination
Flag leaves from each group were excised and
cleaned with wet tissue. Samples (0.5 g FW) for
each replicate were stored in liquid nitrogen.
Glycinebetaine determination was done by HPLC
(Shimadzu-LC-6A, Japan) using a modified proce-
dure after Chen et al. (2000). Briefly, samples were
extracted in methanol at 4 1C for 24 h. Extracts
were then partitioned in a mixture of methanol
extract:chloroform:water (ultra pure) (10:5:6).
The upper aqueous phase was concentrated to
dryness using a rotor evaporator and dissolved in
ultrapure water. The samples were then passed
through columns of Amberlite CG250 cation and
Dowex1-X2 anion exchange resins. GB was eluted
with 4 mol/L of NH4OH and concentrated to
dryness. The dried eluate was dissolved in metha-
nol and filtered through a 0.25 mm membrane
before subjection to the HPLC system. A Spherisorb
10SCX column (4.6
250 mm) was used and the
mobile phase was NH4H2PO4. GB concentration was
detected by a UV detector at 195 nm, and
quantification was done by comparing the peak
 ARTICLE IN PRESS
168 Q.-Q. Ma et al.

surface areas with those obtained with pure Statistical analysis

glycinebetaine standards.
Enzyme extraction and assays
Wheat flag leaves (1 g FW) were excised and
ground with a pestle in an ice-cold mortar with
8 mL 0.05 mol/L Na2HPO4/NaH2PO4 (pH 7.8) buffer
containing 1 mmol/L EDTA-Na2 and 2% (W/V) poly-
vinylpolypyrrolidone. The homogenates were fil-
tered through four layers of cheesecloth and then
centrifuged at 4 1C for 15 min at 15,000 g. The
supernatants were collected and the activities of
superoxide dismutase (SOD, EC 1.15.1.1), catalase
(CAT, EC 1.11.1.6) and ascorbate peroxidase (APX,
EC 1.11.1.11) were determined according to Alia
et al. (1999).
Each pot (five plants) was considered as one
replicate. The data are presented with standard error
(SE) of at least three replicates (pots). For no obvious
difference was found between the WW and GB+WW
treatments, we omitted the data from the GB+WW
group (except the inhibitor feeding experiment).
Results
GB accumulation and water status
With increasing drought stress, GB concentration
increased in HF9703 whether it had been pre-
treated with GB or not (Fig. 1A). In SN215953,
it first increased and then declined (Fig. 1B).

Fig. 1. Effects of foliar application of 100 mmol/L GB on GB concentration (A,B), relative water content (RWC) (C,D)
and water potential (Cw,)(E,F) in wheat flag leaves under drought stress. Filled circle (K) ¼ well-watered plants; Open
circle (J) ¼ drought-stressed plants without GB pretreatment; filled triangle (m) ¼ drought-stressed plants pretreated
with GB. Values are average of five replicates7SE.
 ARTICLE IN PRESS
Glycinebetaine alleviates photoinhibition in wheat 169

GB-treated plants always kept a higher GB level
than non-GB treated controls in both cultivars (Fig.
1A and B). However, no obvious difference in water
status was found between the GB-treated and non-
GB treated plants. Exogenous GB only improved the
RWC and Cw slightly in leaves of SN215953 at the
beginning of drought stress (Fig. 1).
stomatal conductance decreased continuously
(Fig. 2A and B) while the intercellular CO2
concentration increased (Fig. 2C and D). GB-
treated plants always maintained a higher stomatal
conductance than non-GB treated plants in both
wheat cultivars during the whole drought stress
course (Fig. 2).

Gas exchange Chlorophyll fluorescence and PSII recovery

Net photosynthesis rate was reduced in non-GB
and GB-treated plants by drought stress in both
cultivars. GB application was able to reduce these
decreases (Fig. 2E and F). Stomatal conductance
(Fig. 2A and B) and intercellular CO2 concentration
decreased simultaneously (Fig. 2C and D) under
mild drought stress. With increasing drought stress,
Mild drought stress (T1) had no effect on the
efficiency of PSII (Fv/Fm) in leaves of HF9703 and
only caused a slight decrease in that of SN215953.
Moderate (T2) and severe (T3) drought stresses
combined with high irradiance (1600 mmol/m2/s)
led to a decrease in Fv/Fm (Fig. 3E and F). The
decrease in Fv/Fm resulted from the increase in F0

Fig. 2. Effects of foliar application of 100 mmol/L GB on stomatal conductance (A,B), intercellular CO2 concentration
(C,D) and net photosynthesis rate (E,F) in wheat flag leaves under drought stress and high irradiance (1600 mmol/m2/s).
T0, T1, T2 and T3 represent the plants under non-, mild-, moderate- and severe-drought stress, respectively. Filled
circle (K) ¼ well-watered plants; open circle (J) ¼ drought-stressed plants without GB pretreatment; filled triangle
(m) ¼ drought-stressed plants pretreated with GB. Values are average of five replicates7SE.
170 Q.-Q. Ma et al.

Fig. 3. Effects of foliar application of 100 mmol/L GB on Fo (A,B), Fm (C,D) and Fv/Fm (E,F) in wheat flag leaves under
drought stress. Filled circle (K) ¼ well-watered plants; open circle (J) ¼ drought-stressed plants without GB
pretreatment; filled triangle (.) ¼ drought-stressed plants pretreated with GB. Values are average of five
replicates7SE.

(Fig. 3A and B) and the decrease in Fm (Fig. 3C and

D). GB application was able to reduce the decrease
in Fv/Fm. For example, severe drought stress
decreased Fv/Fm to 0.65 in non-GB treated
HF9703, but to only 0.73 in GB-treated plants
(Fig. 3E).
In the recovery experiments, GB-treated plants
recovered more rapidly and recovered to a
higher level than non-GB-treated ones. For exam-
ple, when Fv/Fm in both non-GB and GB-treated
HF9703 leaves were photoinhibited to about
Fig. 4. Effects of exogenous GB on the recovery of PSII
0.41, it recovered to 0.72 within 4 h under low from photoinhibition. Severe drought-stressed wheat
irradiance (50 mmol/m2/s) in GB-treated leaves, plants that had been treated with (K) or without (J)
but it only recovered to 0.62 in non-GB- GB were put under a high irradiance (1600 mmol/m2/s)
treated leaves within the same time (Fig. 4, left). for 4 h and then recovered at 50 mmol/m2/s. Values are
Similar trends were observed in SN21593 (Fig. 4, average of three independent experiments with five
right). leaves each.
 ARTICLE IN PRESS
Glycinebetaine alleviates photoinhibition in wheat 171

Anti-oxidative enzyme activities Effects of DDTC and SM on GB application

Drought stress induced different changes in the
anti-oxidative enzyme activities. SOD activity was
increased by mild and moderate drought stresses,
but began to decrease when severe drought stress
was imposed to both wheat cultivars (Fig. 5A and
B). APX activity in both cultivars (Fig. 5C and D) and
CAT activity in HF9703 (Fig. 5E) increased at the
onset of drought stress and then declined with
increased drought stress. CAT activity in SN215953
kept declining from the beginning of the drought
stress (Fig. 5F).
GB application increased the activity of SOD
under moderate (T2) and severe (T3) drought stress
(Fig. 5A and B) and that of APX under all the levels
of drought stress (Fig. 5C and D) in both cultivars,
but had no effect on CAT activity (Fig. 5E and F).
To explore the mechanism of the protective
effect of GB on the PSII complex, we examined
the effects of GB on SOD activity and D1 protein
turnover in detached wheat leaves using inhibitors
of SOD (DDTC) and D1 protein synthesis (SM).
GB treatment could slightly improve the RWC in
detached flag leaves of both wheat cultivars
(Fig. 6). Although slight decreases in RWC were
observed during adaptation under an irradiance of
1600 mmol/m2/s, leaves from non-GB and GB-
treated plants still had similar RWC (data not
shown).
SM treatment caused decrease in Fv/Fm in both,
well-watered or drought-stressed, plants with or
without GB pretreatment (Fig. 7). A similar effect
on Fv/Fm was observed with DDTC (Fig. 8).

Fig. 5. Effects of foliar application of 100 mmol/L GB on anti-oxidative enzyme activities in wheat flag leaves under
drought stress. Filled circle (K) ¼ well-watered plants; open circle (J) ¼ drought-stressed plants without GB
pretreatment; filled triangle (.) ¼ drought-stressed plants pretreated with GB. Values are average of five
replicates7SE.
 ARTICLE IN PRESS
172
Fig. 6. Effect of dehydration on leaf relative water
content (RWC) in detached wheat flag leaves. Leaves that
had been (K) or not been (J) pretreated with GB were
dehydrated under 300 mmol/m2/s at 2571C. Values are
average of three independent experiments with five
leaves each time7SE.
Fig. 7. Effects of SM on Fv/Fm in detached wheat flag
leaves that had been pretreated with or without GB
during dehydration. Filled circle (K) and open circle (J)
represent the non-GB pretreated leaves been treated
without or with SM respectively; Filled triangle (.) and
open triangle (&) represent the GB pretreated leaves
been treated without or with SM, respectively. Values are
average of three independent experiments with five
leaves each time7SE.
Fig. 8. Effects of DDTC on Fv/Fm in detached wheat flag
leaves that had been pretreated with or without GB
during dehydration. Filled circle (K) and open circle (J)
represent the non-GB pretreated leaves been treated
without or with DDTC, respectively; Filled triangle (.)
and open triangle (&) represent the GB-treated leaves
been treated without or with DDTC, respectively. Values
are average of three independent experiments with five
leaves each time7SE.
Q.-Q. Ma et al.
Discussion
Many plants are able to accumulate GB and other
compatible solutes in response to environmental
stress. This is regarded as one of the protective
mechanisms allowing plants to acclimate to un-
favorable environment (Rhodes and Hanson, 1993;
Hare et al., 1998; Bohnert and Shen, 1999;
Sakamoto and Murata, 2002). Wheat is such a
natural GB accumulator, and exogenously applied
GB increases the GB concentration in cells (Mäkela
et al., 1996; Rajasekaran et al., 1997; Allard et al.,
1998). The increased GB concentration in GB
pretreated plants observed here (Fig. 1A and B)
suggests that foliar-applied GB can be absorbed by
wheat under drought stress. However, different
from Xing and Rajashekar (1999), exogenous GB had
little effect on the water status of flag leaves under
drought stress in wheat (Fig. 1).
Several authors have reported that exogenous GB
is able to offset the decrease in photosynthesis in a
variety of plants under various stresses (Rajasekar-
an et al., 1997; Mäkelä et al., 1998, 1999; Xing and
Rajashekar, 1999). The present experiment finds
that exogenous GB is able to decrease the reduc-
tion in net photosynthesis rate in wheat leaves
subjected to drought stress, which may be ascribed
to increased stomatal conductance (Fig. 2A and B),
see also Mäkelä et al. (1998, 1999). GB may
maintain the photosynthetic capacity not only
through increasing stomatal conductance but also
by maintaining chloroplast ultrastructure (Rajase-
karan et al., 1997; Mäkelä et al., 2000) and Rubisco
activity (Incharoensakdi et al., 1986; Nomura et
al., 1998; Mäkelä et al., 2000) under drought or salt
stress.
The reduction in photosynthetic activity under
drought stress can be due to or lead to photo-
inhibition. PSII is believed to play a key role in the
response of leaf photosynthesis to environmental
perturbations (Baker, 1991; Anderson and Barber,
1996). The strong protective effect of GB on
structure and function of the oxygen-evolving
complex of PSII against different environmental
stresses has been well established in vitro (Mame-
dov et al., 1991, 1993; Papageorgiou et al., 1991;
Papageorgiou and Murata, 1995; Allakhverdiev et
al., 2003). Fv/Fm is usually used as a sensitive
indicator of plant photosynthetic performance
(Maxwell and Johnson, 2000) and represents a
measure of the functional status of the oxygen-
evolving complex. The increased Fv/Fm values in
GB-treated plants under photoinhibitory conditions
(Fig. 3E and F) indicate that the PSII complex is
better suited to withstand photoinduced inactiva-
tion, suggesting that exogenous GB is able to
 ARTICLE IN PRESS
Glycinebetaine alleviates photoinhibition in wheat
alleviate photoinhibition in wheat leaves caused by
drought stress combined with high irradiance
(1600 mmol/m2/s). Similarly, Allard et al. (1998)
found that exogenous GB prevents photoinhibition
in wheat caused by freezing stress. In addition,
transgenic Arabidopsis (Alia et al., 1999), tobacco
(Holmström et al., 2000), Zea mays (Quan et al.,
2004), and Brassica juncea (Prasad and Pardha
Saradhi, 2004) expressing genes for COD, CDH/
BADH, CDH or COD, respectively, also maintain
higher Fv/Fm values than wild-type plants when
subjected to photoinhibition caused by high irradi-
ance combined with salt or low-temperature stress.
How can GB enhance the tolerance of the
photosynthetic machinery to photoinhibition under
various stresses? It is well accepted that the extent
of photo-induced inactivation of the PSII complex in
vivo depends on the balance between the photo-
induced inactivation (which is mainly due to
damage and degradation of the D1 protein) and
the repair of the PSII complex (Alia et al., 1999).
Alia et al. (1999) and Prasad and Pardha Saradhi
(2004) found that the enhanced tolerance is due to
the accelerated recovery of the PSII from a
photoinactivated state by GB. Similar results were
obtained in a study on double transgenic lines of
tobacco expressing CDH and BADH (Holmström et
al., 2000). The present study shows the GB-treated
plants recover more rapidly than controls when
subjected to photoinhibitory conditions (Fig. 4).
This is confirmed by the finding that the protective
role of GB is impaired when the leaves are treated
with SM, an inhibitor of D1 protein (Fig. 7). The
present study, together with previous reports,
suggests that both, endogenous as well as exogen-
ous, GB are able to enhance photoinhibition
tolerance of plants by accelerating the recovery
of PSII from photoinactivated state.
An inevitable consequence of drought stress is
the increased production of reactive oxygen spe-
cies (ROS) in the chloroplasts (Smirnoff, 1993). ROS
cause the degradation of D1 protein if not
scavenged effectively (Asada, 1999; Prasad and
Pardha Saradhi, 2004 and reference therein), a
process known as photooxidation. Plants are en-
dowed with an array of enzymes such as SOD, APX
and CAT and some small ROS-scavenging molecules
such as glutathione, ascorbic acid, a-tocopherol
and carotenoids to cope with ROS (Mittler, 2002).
During drought stress, these defense mechanisms
might be induced to maintain the equilibrium
between the formation and detoxification of ROS.
However, as shown here and by others (Bartoli et
al., 1999), these enzymes can usually not match
the increasing production of ROS because of the
decrease in the anti-oxidative enzyme activities
173
under severe drought stress (Fig. 5). There are few
experiments on the effect of GB on anti-oxidative
enzymes, and the results are inconsistent. Quan et
al. (2004) reported that there is no significant
difference in SOD activity between wild type and
transgenic betA maize under chilling conditions.
Alia et al. (1999) and Prasad and Pardha Saradhi
(2004) reported that transgenic plants have higher
anti-oxidative activities than the wild-type plants.
More recently, Demiral and Türkan (2004) found
that GB protects rice seedlings from salinity-
induced oxidative stress. Our results suggest that
GB-treated plants exhibit increased SOD and APX
activities (Fig. 5), indicating a more effective
scavenging of ROS. We suggest this is one of the
mechanisms by which GB protects PSII, for SOD is
believed to play an important role in the protection
of the photosynthetic apparatus against photoinhi-
bition (Jiao and Ji, 2001). Application of DDTC, an
inhibitor of SOD, reverses the protective effect of
GB on PSII (Fig. 8).
The direct protective role of GB on PSII, either
through enhancing D1 protein turnover or through
alleviating oxidative stress may have additional
functions. Under drought stress, the excess light
energy absorbed by the antenna pigments must be
dissipated safely to avoid severe photoinhibition
and photooxidation. Energy dissipation relying on
D1 protein turnover is a very important mechanism
(Russell et al., 1995); the enhanced D1 protein
turnover by GB may dissipate more excess light
energy. Moreover, SOD and APX are two of the main
enzymes involved in the water–water cycle (Asada,
1999), which is regarded as one of the safe
dissipation pathways of excess photon energy under
environmental stress (Asada, 1999). The higher
anti-oxidative enzyme activity in GB-treated plants
may suggest a more effective operation of the
water–water cycle and, thus, more excess energy
may be dissipated. However, it is unclear as to how
GB maintains the anti-oxidative enzyme activities.
It may be related to its molecular features. GB is an
amphoteric compound and can interact with both
hydrophilic and hydrophobic domains of macromo-
lecules (Sakamoto and Murata, 2002) to stabilize
the quaternary structure and maintain their func-
tions. Furthermore, the role of GB in accelerating
D1 protein turnover and its possible involvement in
the xanthophylls cycle needs to be determined.
It should be mentioned that the protective effect
of GB under drought stress conditions seems not to
be directly linked to the water status since plants
of similar water have been used in the present
study. Thus, the differences observed between the
non-GB and GB treated plants are caused mainly, if
not entirely, by the application of GB.
 ARTICLE IN PRESS
174
In summary, the present study shows that foliar
application of 100 mmol/L GB is able to decrease
the susceptibility of PSII in wheat leaves to
photoinhibition caused by drought stress combined
with high irradiance; this is mainly through accel-
erating the turnover of D1 protein in PSII and
through maintaining the anti-oxidative enzyme
activities to avoid or mitigate photooxidation. GB
is able to preserve both structural and functional
stability of PSII under photoinhibition.
Acknowledgments
This research was supported by the state key
basic research and development plan (G
1998010100) from the Ministry of Science and
Technology of the Peoples Republic of China; by
the Fund of Shandong Agricultural University; and
by the Natural Science Foundation of Shandong
province. We are grateful to Prof. Hui-Yuan Gao,
Shandong Agricultural University for his critical
reading of the manuscript.
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