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BIOTIC AND ABIOTIC STRESS
Physiological responses of somaclonal variants of triploidbermudagrass (Cynodon transvaalensis 3 Cynodon dactylon)to drought stress
Shaoyun Lu Æ Chuanhao Chen Æ Zhongcheng Wang ÆZhenfei Guo Æ Haihang Li
Received: 31 August 2008 / Revised: 7 November 2008 / Accepted: 16 November 2008 / Published online: 3 December 2008
� Springer-Verlag 2008
Abstract Eight somaclonal variants with enhanced
drought tolerance were isolated from regenerated plants of
triploid bermudagrass (Cynodon dactylon 9 Cynodon
transvaalensis cv., TifEagle). Three of them (10-17,
89-02, 117-08) with strong drought tolerance were
selected for investigations of physiological responses to
drought stress. Compared to the parent control, TifEagle,
the somaclonal variants had higher relative water contents
and relative growth, and lower ion leakages in the
greenhouse tests, while no difference in evapotranspira-
tional water losses and soil water contents was observed
between the variants and TifEagle. The variants also had
less leaf firing in the field tests under drought stress.
Superoxide dismutase (SOD), catalase (CAT) and ascor-
bate peroxidase (APX) activities decreased gradually in
responses to drought stress in all plants and exhibited
negative correlations with ion leakage, indicating that the
declined activities of these antioxidant enzymes were
associated with drought injury in the triploid bermuda-
grass. However, CAT activities were significantly higher
in all three variants than in TifEagle during drought
stress. Two variants, 10-17 and 89-02, also had signifi-
cantly higher APX activities than TifEagle before and
during the first 4 days of drought treatments. These two
lines also showed higher SOD activities after prolonged
drought stress. Proline, total soluble sugars and sucrose
were accumulated under drought stress in all plants and
exhibited positive correlations with ion leakage. More
proline and sugars were accumulated in TifEagle than in
the variants. The results indicated that higher activities of
the antioxidant enzymes in the variants during drought
stress are associated with their increased drought
tolerance.
Keywords Antioxidant enzymes � Drought tolerance �Somaclonal variation �Triploid bermudagrass (Cynodon dactylon 9 Cynodon
transvaalensis)
Introduction
Drought stress is a major factor limiting turfgrass growth
in semi-arid and arid regions across the world. It
becomes more important during summer months when
evapotranspiration increases. Bermudagrass is one of the
most important warm-season turfgrasses in tropical and
subtropical regions. Hybrid bermudagrass (Cynodon dacty-
lon 9 Cynodon transvaalensis) is a F1 sterile triploid
progeny (2n = 3x = 27) and is propagated vegetatively.
TifEagle is a recent-released cultivar of triploid bermuda-
grass, which produces tighter and denser turf with strong
tolerance to mowing. It is mainly used for greens on golf
courses (Hanna and Elsner 1999). Bermudagrass grows
rapidly during summer months and needs frequent irrigation
to maintain high quality turf. Due to limited water resources,
there is an increased demand on drought-tolerant cultivars
with low irrigation requirement. As sterile triploids,
Communicated by A. Feher.
S. Lu � C. Chen � Z. Wang � Z. Guo (&)
Biotechnology Laboratory for Turfgrass and Forages,
College of Life Sciences, South China Agricultural University,
510642 Guangzhou, China
e-mail: [email protected]
H. Li
College of Life Sciences, South China Normal University,
510631 Guangzhou, China
123
Plant Cell Rep (2009) 28:517–526
DOI 10.1007/s00299-008-0649-z
conventional crossing cannot be used in breeding for their
improvement. Somaclonal variation offers an alternative,
effective tool for plant breeding (Jain 2001), but limited
reports are available on turfgrass species including
bermudagrass. An efficient system for callus suspension
culture and regeneration of triploid bermudagrass cultivar
‘TifEagle’ has been established in our group, and somaclonal
variants with enhanced drought tolerance have been
observed from the regenerated plants (Lu et al. 2006). Three
salt-tolerant lines have also been identified via in vitro
selection (Lu et al. 2007).
Drought tolerance in plants involves various physio-
logical responses, including antioxidant defenses and
osmolytes. Antioxidant defense system plays an important
role in drought tolerance in various plant species (Bowler
et al. 1992; Noctor and Foyer 1998). Antioxidant defense
system consists of several antioxidant enzymes, such as
superoxide dismutase (SOD), catalase (CAT), ascorbate
peroxidase (APX), and glutathione reductase (GR), and
non-enzymatic antioxidants, such as ascorbic acid (AsA)
and glutathione (GSH). They protect plants from the del-
eterious effects of reactive oxygen species (ROS), such as
superoxide radicals, hydrogen peroxide (H2O2), hydroxyl
radicals and singlet oxygen, which are produced at elevated
rates when plants are exposed to abiotic stress conditions
(Bowler et al. 1992; Noctor and Foyer 1998; Mittler 2002).
Higher levels of antioxidant enzymes have been found in
drought-tolerant cultivars than in drought-sensitive culti-
vars in many crops (Bowler et al. 1992; Guo et al. 2006;
Srivalli et al. 2003), as well as in turfgrass species
(DaCosta and Huang 2007). In addition, proline and sugars
are common osmolytes in plants. They accumulate in
response to salt- or drought-induced osmotic stress
(Mattioni et al. 1997; Sanchez et al. 1998). They can
function as osmoprotectors and ROS scavengers (Bohnert
et al. 1995; Noctor and Foyer 1998; Hong et al. 2000).
Osmotic adjustment has been found to be important in
drought tolerance of some turfgrass species (DaCosta and
Huang 2006a, 2006b). Previous studies in bermudagrass
have examined cultivar variations in their overall turf
performance in responses to irrigation and drought stress
(Baldwin et al. 2006; Jiang and Carrow 2007; Lu et al.
2006). Physiological mechanisms of bermudagrass toler-
ance to drought stress are not well understood (Lu et al.
2008).
The objectives of this study are based on our previous
observation on seasonal variants of triploid bermudagrass
with improved drought tolerance (Lu et al. 2006), and to
further investigate the physiological responses of antioxi-
dant enzymes and osmolytes to drought stress to gain more
insights on the possible mechanisms of the enhanced
drought tolerance in these variants.
Materials and methods
Plant growth and treatments
TifEagle and the drought-tolerant somaclonal variant lines
(Lu et al. 2006) were transplanted to 10-cm diameter
plastic pots containing a mixture of peat and perlite (3:1, v/v).
Plants were grown in a greenhouse for at least 2 months at
temperatures between 20�C and 30�C under natural light,
with routine management of daily irrigation, weekly
mowing at 1 cm height, and biweekly fertilization with
50 ml of 0.3% solution of an N–P–K fertilizer (15–15–15)
per pot. The mowing was stopped 2 weeks before the
plants were subjected to drought stress by withholding
irrigation. Four pots of plants from each line were used as
replicates in the experiment.
Determination of relative water content and ion leakage
Relative water content (RWC) and ion leakage of the leaves
were determined as described before (Lu et al. 2006). For
measurement of RWC, fresh leaves were weighed (Wf) and
immersed in water overnight until the weight of the leaves
was constant. The water-saturated leaves were weighed (WS)
and then dried for 24 h at 80�C for determinations of the dry
weight (Wd). RWC was calculated by the formula: RWC
(%) = (Wf - Wd)/(WS -Wd) 9 100. For measurement of
ion leakage, leaf samples were rinsed with distilled water and
immersed in 10 ml of distilled water for 12 h. The conduc-
tivity of the solution (R1) was measured using a conductivity
meter (Model DDS-11A, Shanghai Leici Instrument Inc.,
Shanghai, China). Samples were then heated in boiling water
for 20 min, and cooled to room temperature. The conduc-
tivity of this solution (R2) was measured. Ion leakage was
calculated as the percentage of R1 over R2.
Determination of relative growth
Relative growth was determined as described previously
(Lu et al. 2007). The plants were mowed and then sub-
jected to drought stress by withholding irrigation for
8 days. The shoots from 2.5 cm above soil surface were
harvested, dried at 105�C for 1 h, followed by 80�C for
12 h, and then weighed to obtain dry weight (DW) data.
The relative growth was calculated as percentage of dry
weight of drought treatment over controls grown under
normal irrigation condition.
Measurement of evapotranspirational water loss (ET)
Plants were fully irrigated in the evening and placed in a
greenhouse overnight. Initial weight of plants plus soil
518 Plant Cell Rep (2009) 28:517–526
123
(W1) was recorded the next morning. Weight of plants plus
soil (Wn) was recorded for 8 days. ET per pot per day was
calculated as the difference of Wn-1 - Wn.
Measurement of soil water content
Soil samples were taken from each pot at different days
during drought stress, and fresh weights were determined
(Wa). The soil was then dried at 80�C for 24 h for the
determination of dry weigh (Wb). Soil water content was
calculated by the formula: (Wa - Wb)/(Wb) 9 100.
Determinations of SOD, CAT and APX activities
Leaves (0.5 g) were ground in a mortar and pestle in 5 ml
of 50 mM phosphate buffer (pH 7.8) at 4�C. The homog-
enate was centrifuged at 13,0009g for 15 min. The
supernatant was recovered for determination of SOD and
CAT activities (Zhou et al. 2005). To determine SOD
activity, 3-ml reaction solution containing 13 lM methio-
nine, 63 lM q-nitro blue tetrazolium chloride (NBT),
1.3 lM riboflavin, 50 mM phosphate buffer (pH 7.8), and
50 ll of the supernatant was incubated for 10 min under
fluorescent light (80 lmol m-2 s-1). Absorbance was
determined at 560 nm by a spectrophotometer. One unit of
SOD activity was defined as the amount of enzyme
required for inhibition of photochemical reduction of NBT
by 50%. To determine CAT activity, 3-ml reaction solution
containing 15 mM H2O2, 50 mM phosphate buffer (pH
7.0)was added with 50 ll of the supernatant, and the
decreased absorbance of H2O2 (extinction coefficient
0.0394 mM-1 cm-1) within 1 min at 240 nm was recor-
ded. One unit of CAT activity was defined as the amount of
enzyme required for catalyzing the conversion of 1 lmol
H2O2 into water per minute.
APX activity was determined as described by Zhou
et al. (2005). Leaves (0.5 g) were ground in 3 ml
extraction solution containing 50 mM phosphate buffer
(pH 7.0), 2 mM AsA and 5 mM EDTA using a mortar
and pestle at 4�C. The homogenate was centrifuged for
15 min at 13,0009g and the supernatant was used to
measure the enzyme activity. The 3-ml reaction solution
contained 50 mM phosphate buffer (pH 7.0), 0.5 mM
AsA, 0.1 mM H2O2, and 0.1 ml supernatant. APX activity
was calculated by following the decrease in absorbance of
AsA (extinction coefficient 2.8 mM-1 cm-1) in a minute
at 290 nm.
Determination of proline and sugars
Proline and sugars were determined as described previ-
ously (Lu et al. 2007). Leaves were harvested and dried at
105�C for 1 h, followed at 80�C for 12 h. The dried
samples were powdered in a mortar with a pestle, and used
for determination of proline and sugars. For measurement
of proline, 50 mg dried powder samples were extracted
with 3 ml of 80% ethanol for 1 h. After filtration, the fil-
trate was incubated in a boiling water bath to evaporate the
ethanol, and distilled water was then added to make the
final volume to 10 ml. The solution was shaken for 10 min
after addition of permutit, and then filtrated. Into 2.5 ml of
filtrate, 2.5 ml of acetic acid glacial and 2.5 ml ninhydrin
solution (2.5 g ninhydrin dissolved in 60 ml of acetic acid
glacial and 40 ml of 6 M phosphoric acid) were added. The
mixture was incubated in a boiling water bath for 1 h, and
then extracted with 2.5 ml of benzene by shaking them
vigorously for 5 min. The benzene phase was used to
determine the absorbance at 515 nm (Troll and Lindsley
1955). Proline concentration was calculated as to the
standard curve.
Soluble sugars and sucrose were determined by the
anthrone method (Spiro 1966). 50 mg dried powder sam-
ples were extracted with 3 ml of 80% ethanol for 30 min
and centrifuged at 13,0009g for 15 min. The pellet was
further extracted twice with 3 ml 80% ethanol. The
combined supernatant was depigmented by addition of
activated charcoal. After filtration, the solution was
brought to 10 ml. For determination of total soluble sugars,
0.1 ml of the extracts was added to 3 ml of anthrone
reagent (150 mg anthrone was dissolved in 100 ml of
7.74 M H2SO4) and heated at 90�C for 15 min. Absor-
bance at 620 nm was read and amounts of total soluble
sugars were determined according to its calibration curve
based on D-glucose. For determination of sucrose, 0.1 ml of
the extracts was mixed with 0.1 ml of 30% KOH. The
mixture was heated at 100�C for 15 min. After it was
cooled down, 3 ml of anthrone reagent was added. The
mixture was incubated at 40�C for 10 min, and its absor-
bance at 620 nm was read. Amounts of sucrose were
determined using a calibration curve of standard sucrose.
Field tests
For field tests, the stolons of the testing lines were trans-
planted to field plots at the Experimental Station of South
China Agricultural University (Guangzhou, China) in the
spring each year, where an annual rainfall of 1,683 mm and
an annual average temperature of 21.6�C were recorded.
The tests consisted of four lines (10-17, 89-02, 117-8, and
TifEagle) with three replicates in a randomized complete
block design. The size of each plot is 1 9 1 m. The
management of the grasses was at a low level, with no
mowing, no fertilization and occasional irrigation when
needed. For evaluation of drought tolerance, leaf firing was
determined by withholding irrigation for 10 days after a
full irrigation on September 13, 2007, and the experiment
Plant Cell Rep (2009) 28:517–526 519
123
was terminated when it rained. The leaf firing ratings were
visually evaluated based on percentage of the leaves that
turned yellow or brown in a plot (Carrow and Duncan
2003).
Experimental design and statistical analysis
Randomized complete block design was used for both
greenhouse and field experiments with four replicates for
the greenhouse test and three for the field test. ANOVA
was used to perform statistical analysis, and Duncan’s test
at the 0.05 probability level for analyzing significance of
differences among the means.
Results
Evaluation of drought-tolerant somaclonal variants
In our previous study, seven somaclonal variant lines were
isolated based on their tolerance to drought treatment and
characterized on their RWC and ion leakage under drought
stress (Lu et al. 2006). In this study, more plants regener-
ated from embryogenic callus and four more drought-
tolerant lines of somaclonal variants were identified (lines
10-17, 29-06, 119-09 and 188). Their RWC and ion leak-
age under drought stress were tested and the data are
presented in Fig. 1. All the four lines had higher RWCs and
lower ion leakages under drought stress in comparison to
TifEagle.
Three variant lines from the two experiments (10-17,
89-02 and 117-08) with higher RWC and lower ion leakage
were selected for further evaluation under drought stress.
Although they had similar dry weight of shoots to TifEagle
under irrigation condition, their dry weight was higher than
TifEagle after 8 days of withholding irrigation (Fig. 2a).
The calculated relative growth of TifEagle was 70.7%
(Fig. 2b), while those of the variant lines of 10-17, 89-02
and 117-08 were between 89.3 and 90.6% (Fig. 2b), indi-
cating that the variant lines had an increased drought
tolerance when compared to TifEagle. TifEagle exhibited
extreme wilting for 6 days after water withholding,
whereas the variant lines remained turgid and were greener
(Fig. 3).
Drought tolerance of these variant lines was also eval-
uated in the field. Leaf firing, which refers to leaf chlorosis
in response to progressive drought stress, provides a good
assessment of overall turfgrass drought tolerance under
field conditions (Carrow 1996). The tested plants showed
little leaf firing under well-watered conditions (data not
shown). Leaf firing increased with prolonged water with-
holding in the field for all the plants. However, the
somaclonal variant lines displayed significantly lower leaf
firing than TifEagle when evaluated at day 5 and day 10,
after withholding of irrigation (Fig. 2c).
Water status and ion leakage during drought stress
The daily evapotranspirational water loss exhibited a
gradual decline during water withholding in all the plants
tested and showed no difference between the variant lines
and TifEagle (Fig. 4a). This decline might be dependent on
the soil water content. By withholding irrigation, soil water
content decreased gradually from the initial level of 40–
44% under irrigation condition to 7–9%, 8 days into
drought stress. Soil water content was remained at the same
level for the three variants and TifEagle during the 8 days
of drought stress (Fig. 4b).
The changes of RWC and ion leakage were limited
within 2 days after withholding irrigation, as the soil was
still wet. After 2 days, all plants showed a gradual decrease
in RWC and an increase in ion leakage during drought
stress. By day 4, the three somaclonal variants started
showing significantly lower ion leakage, and they exhibited
higher RWC from day 6 onward, than their parent,
TifEagle (Fig. 5).
Antioxidant enzyme activities during drought stress
To evaluate changes of antioxidant enzymes affected by
drought stress, SOD, CAT and APX activities were
*
***
***
*10
20
30
40
50
60
70
*
*
**
***
*
0102030405060708090 A
B
*
***
***
*
0
Plant lines
Ion
leak
age
(%)
*
*
**
***
*
TE 10-17 29-06 39-03 95-08 89-02 117-08 119-09 188
TE 10-17 29-06 39-03 95-08 89-02 117-08 119-09 188
Plant lines
RW
C (
%)
Fig. 1 RWC and ion leakage of the somaclonal variant lines in
comparison to TifEagle (TE) under drought stress. RWC (a) and ion
leakage (b) were measured at day 7 after withholding irrigation. Barson the lines represent standard error of four independent measure-
ments. Asterisk symbols above the columns indicate significant
differences from TE at P B 0.05
520 Plant Cell Rep (2009) 28:517–526
123
assayed. No difference on SOD activity was observed
before drought treatment, and the enzyme activity
decreased gradually in all the lines tested during drought
stress. By day 6 and 8, both line 10-17 and 89-02 had
higher SOD than TifEagle (Fig. 6a). No difference on CAT
activity was observed between the variants and TifEagle
before drought stress, and the enzyme activity decreased
progressively during drought treatment. Significantly
higher levels of CAT activity were observed in the three
variants than in TifEagle from day 2 to 8 (Fig. 6b). The
pattern of APX activity varied from the ones of SOD and
CAT. Variants 10-17 and 89-02 had significantly higher
APX activities than TifEagle even before the drought stress
was imposed, and the levels remained higher for the first
4 days during drought stress (Fig. 6c), although the
enzyme activity decreased in all the lines under drought
stress. From day 6 onward, no difference of APX activity
was observed among the lines (Fig. 6c).
Changes of proline and soluble sugars during drought
stress
Proline is an important osmolyte and ROS scavenger in
plants. Its level was very low (\0.1 mg g-1 DW) in all the
plants tested under well-watered conditions. Its content
increased during drought treatment. More proline was
accumulated in TifEagle than in the variants in responses to
drought stress. Proline content increased to 3.4 ± 0.3
mg g-1 DW in TifEagle at day 8 after withholding irrigation,
whereas it increased to 1.8 ± 0.2, 2.2 ± 0.2 and 2.0 ±
0.2 mg g-1 DW in lines 10-17, 89-02 and 117-08,
respectively (Fig. 7a).
Soluble sugars are also osmolytes and sucrose is the
main soluble sugars in plant cells. Both accumulated higher
when the tested plants were under drought stress. Sucrose
level became significantly higher in TifEagle at day 4
during drought stress, and the total soluble sugars started to
accumulate more in TifEagle from day 6 onward (Fig. 7b,
c). At day 8, total soluble sugars and sucrose increased 190
and 220%, respectively, in TifEagle, whereas their levels
elevated only 50–82% and 30–95%, respectively, among
the variant lines (Fig. 7b, c).
Correlation analysis
The data on osmolytes and antioxidant enzymes in the
three variants and TifEagle were analyzed for their corre-
lations with ion leakage. The activities of SOD, CAT, and
APX showed negative correlations with ion leakage during
drought stress (R2 = 0.72, 0.79, 0.62, respectively), indi-
cating that the decreased antioxidant enzyme activities
were associated with the drought-induced injury in ber-
mudagrass (Fig. 8a–c). The accumulation of proline, total
sugars, and sucrose during drought stress were positively
correlated with elevation of ion leakage (Fig. 8d, e)
(R2 = 0.82, 0.87, 0.87, respectively). Similarly, the anti-
oxidant enzyme activities and osmolyte contents were
negatively correlated with RWC during drought stress
(data not shown).
Discussion
Somaclonal variation provides good opportunities to
modify traits of plants randomly. A number of somaclonal
a
a aaaa
b
0.2
0.4
0.6
0.8
1 Control Drought
0
20
40
60
80
100
d
cc
b
bc
b
a
0
10
20
30
40 5 d 10 d
A
B
C
a
0TE 10-17 89-02 117-08
TE 10-17 89-02 117-08
Plant lines
Plant lines
TE 10-17 89-02 117-08
Plant lines
Dry
wei
ght (
g)
Rel
ativ
e gr
owth
(%
)
bc
Lea
f fi
ring
(%
)
Fig. 2 Dry weight of shoots (a), the relative growth (b) and leaf
firing (c) of the somaclonal variant lines in comparison to TifEagle
(TE) under drought stress. The dry weights of shoots were obtained
after plants were withheld irrigation for 8 days as drought treatments
or irrigated daily as controls. The relative growth was calculated as
the percentage of dry weight of drought treatments over that of
controls. Leaf firing was measured at day 5 and 10 after withholding
irrigation in the field. Different letters indicate statistically difference
among plant lines and treatments at P B 0.05
Plant Cell Rep (2009) 28:517–526 521
123
variants have been released as cultivars in a few crops,
including banana, a vegetatively propagated crop (Jain
2001). A group of sugarcane somaclones derived from
callus tissues have been selected for 10 years for eyespot
disease resistance in fields, and the superiority of two so-
maclones, one resistant and one tolerant to eyespot disease,
was confirmed (Ramos Leal et al. 1996). Our previous
studies, together with a report by other scientists, revealed
that a wide range of variations in leaf length, leaf width,
and internode length among the regenerated plants can be
recovered from bermudagrass cv. TifEagle (Lu et al. 2003,
2006; Goldman et al. 2004). The study reported here sug-
gests somaclonal variation with improved drought
tolerance was also identified. All these indicate that so-
maclonal variation offers an effective tool for breeding in
triploid bermudagrass.
Eight somaclonal variants with higher RWC and lower
ion leakage under drought stress were selected in the
Fig. 3 Plant responses of
somaclonal variant lines to
drought stress in comparison to
TifEagle (TE). The photos were
taken at day 6 under regular
irrigation (a) or no irrigation (b)
00 2 4 6 8
10
20
30
-1)
TE
89-02 117-08
A
0
10
20
30
40
Soil
wat
er (
%)
B
40
Days after drought stress
0 2 4 6 8
Days after drought stress
ET
(g
pot
10-17
50
Fig. 4 Evapotranspirational water losses (ET, a) and soil water
contents (b) of the somaclonal variant lines in comparison to TifEagle
(TE) under drought stress. Asterisk symbols in the figures indicate
significant difference between the somaclonal variant lines and TE at
P B 0.05 at a given day of treatment
**
20
40
60
80
100
Days after drought stress
TE 10-17
89-02
***
0
20
40
60
80TE 10-17
89-02
A
B
**
00 2 4 6 8
Days after drought stress
0 2 4 6 8
RW
C (
%)
117-08
***Io
n le
akag
e (%
)
117-08
Fig. 5 Effects of drought stress on RWC (a) and ion leakage (b) of
the variant lines in comparison to TifEagle (TE). Data are the means
of four independent measurements with standard errors. There are no
significant differences of RWC and ion leakage among the variant
lines. Asterisk symbols indicate significant differences between TE
and the variants at P B 0.05
522 Plant Cell Rep (2009) 28:517–526
123
present study. RWC reflects water status of plants, while
ion leakage implicates the injury of plasmalemma. Higher
RWC and lower ion leakage were maintained in the vari-
ants than in TifEagle under drought stress, indicating that
the variants had an increased drought resistance. Among
them, three variants (lines 10-17, 89-02, 117-08) had sig-
nificant higher relative growth than TifEagle under drought
stress. Their enhanced drought tolerance was also demon-
strated by leaf firing ratings in the field test.
Many of the stress injuries to plants, including drought,
are associated with oxidative damages at cellular level
(Bowler et al. 1992; Srivalli et al. 2003). In present study,
the activities of SOD, CAT, and APX decreased in response
to drought stress and showed a significant correlation to ion
leakage, suggesting that the drought injury on triploid ber-
mudagrass is associated with declined antioxidant enzyme
activities. The association of drought injury with the
decrease of antioxidant enzyme activities has been observed
in the cool-season turfgrasses (Jiang and Huang 2001;
DaCosta and Huang 2007). In bermudagrass, such associ-
ations are more obvious in CAT and APX activities. CAT
activity was maintained higher than in TifEagle throughout
the drought stress process in all the three variant lines,
suggesting that CAT may play an important role in drought
tolerance in bermudagrass. Variants 10-17 and 89-02 also
had higher APX activities than TifEagle when plants were
not under drought stress and during early stage of drought
**
00 2 4 6 8
1000
2000
3000
4000
Days after drought stress
0 2 4 6 8
Days after drought stress
0 2 4 6 8
Days after drought stress
-1 D
W)
TE 10-17
89-02
* **
0
20
40
60
80
100
120
APX
(U
g -1
TE 10-17
89-02
* * **
0
2000
4000
6000
-1 D
W)
TE 10-17
89-02 117-08
A
B
C
**
SOD
(U
g
117-08
* **
DW
)
117-08
* * **
8000
CA
T (
U g
Fig. 6 Effects of drought stress on activities of SOD (a), CAT (b)
and APX (c) of the somaclonal variant lines in comparison to
TifEagle (TE). Data are the means of four independent measurements
with standard errors. There are no significant differences of enzyme
activities among the variant lines. Asterisk symbols indicate signif-
icant differences between TE and the variants at P B 0.05
* *
0
20
40
60
80
100
120
-1 D
W)
TE 10-17
89-02 117-08
**
*
00 2 4 6 8
1
2
3
4
Pro
line
(mg
g -1
TE 10-17
89-02 117-08
A
B
C
* **0
20
40
60
80
100
120
-1 D
W)
TE 10-17
89-02 117-08
* *
Sug
ars
(mg
g
**
*
Days after drought stress
0 2 4 6 8
Days after drought stress
0 2 4 6 8
Days after drought stress
DW
)
* **Suc
rose
(m
g g
Fig. 7 Effects of drought stress on contents of proline (a), total
soluble sugars (b) and sucrose (c) in the somaclonal variant lines in
comparison to TifEagle (TE). Data are the means of four independent
measurements with standard errors. There are no significant differ-
ences of proline, total soluble sugars and sucrose among the variant
lines. Asterisk symbols indicate significant differences between TE
and the variants at P B 0.05
Plant Cell Rep (2009) 28:517–526 523
123
stress, which may suggest its role in protecting plants from
drought-induced injury, may differ from CAT. Higher
activities of antioxidant enzymes in drought-tolerant culti-
vars of rice and wheat have been reported (Lascano et al.
2001; Guo et al. 2006). Higher activities of SOD, CAT, and
APX were also observed in the cold-tolerant bermudagrass
cultivar ‘Riviera’ in comparison to the cold-sensitive cul-
tivar ‘Princess-77’ during cold acclimation (Zhang et al.
2006) and in bentgrass resistance to drought stress (DaCosta
and Huang 2007). Put together, it seems the coordinated
changes of antioxidant enzymes which may be an important
part of a mechanism to protect plants from stress injuries in
grass species.
Accumulation of proline and sugars in response to
drought stress has been observed in many plants. Osmotic
adjustments for pea and bentgrass have been shown to be
associated with accumulation of soluble sugars and proline
during drought stress (Sanchez et al. 1998; DaCosta and
Huang 2006a). Both the variants and TifEagle accumulated
proline and sugars under drought stress, but less proline
and sugars were accumulated in the variants than in
TifEagle. The induced proline in both the variants and
TifEagle may function in osmotic adjustment, ROS scav-
enging and protein stabilization (Ramanjulu and Bartels
2002) for well adaptation of drought stress. However,
contradictory results have been reported on the relationship
between proline and sugar accumulations and stress toler-
ance in response to drought stress in different plants. Some
investigators observed that more proline and sugars
accumulated in drought-tolerant cultivars than in drought-
sensitive cultivars (Mali and Mehta 1977; Sivaramakrish-
nan et al. 1988; Kerepesi and Galiba 2000), while other
studies show that less proline is accumulated in drought-
tolerant cultivars than in drought-sensitive cultivars under
drought stress in many crops, such as barley (Hanson
et al. 1979), maize (Ilahi and Dorffling 1982), sorghum
(Premachandra et al. 1995), and cassava (Sundaresan and
Sudhakaran 1995). Ibarra-Caraliero et al. (1988) observed
no correlation between proline accumulation and the
variety or population of maize. They suggested that proline
accumulation caused by drought stress was not an indica-
tion of drought stress resistance, but a symptom of drought
stress. In turfgrasses a similar pattern has been reported that
salt-tolerant species had less proline accumulated com-
pared to the salt-sensitive species under salt stress
(Marcum 1999; Qian et al. 2001). Less proline accumu-
lated in the in vitro selected salt-tolerant triploid
bermudagrass lines than their parent TifEagle under salt
stress (Lu et al. 2007). As the accumulation of proline and
sugars appeared to coincide with the increased ion leakage
R21000
2000
3000
4000
Ion leakage
SOD
A
R2
0
2000
4000
6000
8000
CA
T
B
R2 = 0.62*
0
50
100
150A
PXC
R2 = 0.82**
0
1
2
3
4D
2 = 0.87**
0
20
40
60
80
100E
R2 = 0.87**
0
20
40
60
80
= 0.72**
00 10 20 30 40 50 60
Ion leakage0 10 20 30 40 50 60
Ion leakage0 10 20 30 40 50 60
Ion leakage0 10 20 30 40 50 60
Ion leakage0 10 20 30 40 50 60
Ion leakage0 10 20 30 40 50 60
= 0.79**
Prol
ine
R
Tot
al s
ugar
s
Sucr
ose
F
Fig. 8 Correlations of ion
leakage with other parameters in
the variant lines and Tiffeagle
under drought stress. a SOD,
b CAT, c APX, d proline, e total
sugars, f sucrose. Asterisksymbols mean significant at
P B 0.05, and double asteriskmeans significant at P B 0.01
524 Plant Cell Rep (2009) 28:517–526
123
under drought stress in TifEagle and the variants, more
accumulation of proline and sugars in TifEagle than the
variants may be resulted from the more drought-caused
injury in TifEagle. More rapidly and significantly declined
RWC and increased ion leakage in TifEagle resulted in
larger amounts of accumulation of proline and sugars
compared to the variants under drought stress.
In summary, eight somaclonal variants have been
selected from plants regenerated from the long-time sus-
pension-cultured calli of TifEagle. Investigations on three
somaclonal variants, in comparison to TifEagle, indicated
that the declined activities of SOD, CAT, and APX were
associated with the drought injury in the triploid bermu-
dagrass, while the higher levels of CAT and APX in the
variants during drought stress are associated with their
increased drought tolerance. The accumulated proline and
sugars were associated with the drought injury in the
triploid bermudagrass.
Acknowledgments This work was funded by grants from the
National Basic Research Programs of China (2007CB108905), the
Natural Science Foundation of China (30371014, 30671478) and
Guangdong Provincial Natural Science Foundation (06025818).
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