9

1. Introduction

Temperature is a limiting element for distribution of 
plants worldwide (Sakai and Larcher, 1987). Cold season 
turfgrasses such as Festuca in temperate regions have a 
good adaptability to low temperature, but under severe 
winter conditions may suffer considerable damage (Lev-
itt, 1980). Although fescues are cultivated in transition 
zones, warm season turfgrasses such as bermudagrass are 
preferred (Carrow, 1994). Plant responses to cold stress 
and subsequent adaptation occurs at physiological and 
biochemical levels, as well as cellular and molecular ex-
tents (Gulzar et al., 2011). Harsh low temperatures re-
sult in oxidative stress and a change in proline and sugar 
content of the cells. The vital means for interaction of 
plants to these stresses is a balance between antioxidant 
enzymes and reactive oxygen species (ROS). The damag-
ing ROS responsible for oxidative stress consists of free 
radicals: superoxide (O

2
.-), hydroxyl (OH.), hydroperoxyl 

(HO
2

.) and other molecules such as hydrogen peroxide 
(H

2
O

2
) and singlet oxygen (1O

2
) (Gill and Tuteja, 2010). 

The precise role of antioxidant enzymes which give tol-
erance to cold stress in turfgrasses has not yet been in-

vestigated, but their relieving effect to other oxidative 
stresses has been reported by other researchers (Jiang 
et al., 2005). Rogers et al. (1975) examined the proline 
amount of Zoysia japonica Steud. ‘Meyer’ during the 
months from October to March and found that there is 
an increase in proline from October to December. It has 
been shown that for the period of adaptation to cold, SOD 
and CAT activity in Agrostis stolonifera L., Poa pratensis 
L. and Lolium perenne L. significantly increased (Sarkar 
and Bhowmik, 2009).

The main objective of the present study was to investi-
gate the effects of low temperature stress on biochemical 
and physiological responses of tall fescue and common 
bermudagrass. To the best of our knowledge, this is the 
first report on how these turfgrasses counter cold stress. 

2. Materials and Methods

Plant materials and experimental conditions
The experiment was conducted in a growth chamber 

(Gallenkamp, Germany) at the Department of Horticultur-
al Sciences at the College of Agriculture, Shiraz University 
(29°36’ N and 52°32’ E, elevation 1810 m). Seeds of com-
mon bermudagrass (Cynodon dactylon [L.] Pers. ‘Califor-
nia origin’) and tall fescue (Festuca arundinacea Schreb. 
‘Starlett’) were cultivated in 5 L plastic pots containing a 

Biochemical and physiological adjustments in common 
Bermudagrass (Cynodon dactylon [L.] Pers.) and tall 

Fescue (Festuca arundinacea Schreb.) under low
temperature stress

R. Manuchehri, H. Salehi(1), A. Jowkar
Department of Horticultural Science, College of Agriculture, Shiraz University, Shiraz, Iran.

Key words: Antioxidant enzymes activity, common Bermudagrass, low temperature stress, tall fescue, Turfgrasses.

Abstract: Low temperature is a restrictive factor for turfgrass growth and development in temperate regions. A study was 
conducted with the purpose of examining the physiological and antioxidant response of two turf species, Festuca arun-
dinacea Schreb. ‘Starlett’ and Cynodon dactylon [L.] Pers. ‘California Origin’ to cold stress in a growth chamber at the 
College of Agriculture, Shiraz University. Five temperatures (25, 15, 7.5, 0 and -7.5°C) in four replicates were examined 
in a completely randomized design experiment. It was revealed that under low temperature stress, soluble sugar contents, 
proline, malondialdehyde (MDA) and hydrogen peroxide (H

2
O

2
) were increased in both turfgrasses. Antioxidant enzyme 

activity, particularly catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1), was increased as a result 
of temperature reduction from 25°C to 0°C. Tall fescue is thought to be better adapted to cold stress than common bermu-
dagrass due to higher soluble sugar contents, proline, malondialdehyde and antioxidant enzyme activity. The results show 
that scavenging enzymes have a direct effect in cold season tolerance of turfgrass and improve the defense mechanism of 
plants, but their exact role merits further investigation.

Adv. Hort. Sci., 2014 28(1): 9-13

(1) Corresponding author: hsalehi@shirazu.ac.ir

Received for publication 6 December 2013
Accepted for publication 18 March 2014

Short note



10

mixture of 1:2 (v/v) of loamy soil/decomposed farmyard 
manure. Irrigation was carried out on a daily schedule. Es-
tablished turfs were clipped from 3 cm above ground by 
a hand mower and were transferred to the growth cham-
bers prior to the application of treatments. All treatments 
received a constant light intensity of 3000 Lux, relative 
humidity of 65±5% and a 12 h photoperiod. Low tempera-
tures were maintained at 25, 15, 7.5, 0 and -7.5°C for 48 h.

Experimental design and data analysis
Experiment factors were arranged in a completely 

randomized design with four replications. Data were an-
alyzed using SAS software (ver. 9.1.3) and means were 
compared using the least significant difference (LSD) 
test at p<0.05.

Reducing sugars and proline content
Phenol-sulfuric acid reactions were used to determine 

the reducing sugar content. Shoot samples were oven dried 
at 60°C for 48 h and then ground to a fine powder using an 
electric mill. Samples (0.2 g) were diluted with 80% etha-
nol and centrifuged at 13500 rpm. Supernatant was further 
diluted to 25 ml by 80% ethanol. Then, 1 ml of extract was 
mixed with 1 ml of 5% phenol. Five ml of concentrated 
sulfuric acid were added to tubes and immediately stirred. 
Light absorption was measured by a spectrophotometer 
(Biochrome, UK) at 490 nm wavelength (Dubois et al., 
1956). Proline was determined according to the method 
used by Bates et al. (1973) using a spectrophotometer at 
520 nm wavelength.

Measurement of antioxidant enzyme activity
To extract antioxidant enzymes, fresh leaf samples (0.5 

g) were collected and ground to a fine powder in a mortar by 
adding liquid nitrogen and then homogenized with an ice cold 
enzyme extraction buffer containing 0.5% polyvinylpyrrol-
idone (PVP), 3 mM EDTA, and 0.1 M potassium phosphate 
buffer (pH=7.5). The extracted samples were centrifuged for 
10 min at 13500 rpm and 2-4°C and stored on ice until used. 
The resulting supernatants were used for enzyme analysis. 
CAT activity was determined according to the procedure used 
by Dhindsa et al. (1981) and SOD activity was determined as 
described by Beauchamp and Fridovich (1971).

Malondialdehyde (MDA)
As for H

2
O

2
, 0.25 g of leaf samples were ground in a 

mortar containing 5 ml TCA (0.1%). Leaf extracts were 
centrifuged at 10000 rpm for 5 min. Supernatants (250 µl) 
were mixed with 1 ml MDA solution containing 20% TCA 
and 0.5% thiobarbituric acid. The mixtures were warmed at 
95°C for 30 min and then immediately cooled on ice. Sam-
ple tubes were centrifuged at 10000 rpm for 10 min. Ab-
sorption of light was measured by a spectrophotometer at 
532 nm wavelength according to Heath and Packer (1969).

Hydrogen peroxide (H
2
O

2
)

Leaf samples (0.25 g) were ground in a mortar contain-
ing 5 ml trichloroacetic acid (TCA) (0.1%). Extracts were 

centrifuged at 10000 rpm for 5 min. Supernatants (250 µl) 
were mixed with 250 µl phosphate buffer (100 mM) and 
500 µl potassium iodide (1 M). Absorption of light was 
measured by a spectrophotometer at 390 nm wavelength 
according to Alexieva et al. (2001).

3. Results

With a decrease of temperature from 25°C to -7.5°C, 
reducing sugars increased considerably, with tall fes-
cue showing a greater increase than common bermudag-
rass. The highest soluble sugar content in tall fescue was 
formed at -7.5°C and the highest reducing sugar content 
produced in common bermudagrass was detected at 0°C 
(Table 1). There was no significant difference between the 
turfgrasses for proline content. The highest proline content 
was observed at 0°C and the lowest proline content was 
seen at 25°C. The interaction of temperature and turf spe-
cies showed that tall fescue at 25°C had the lowest proline 
content, while tall fescue at -7.5°C had the highest (Table 
1). It was found that as temperature decreased from 25°C 
to 7.5°C, CAT activity increased.

The greatest CAT activity was observed in tall fescue 
at 7.5°C and the least was seen in bermudagrass at -7.5°C 
(Table 1). Comparison of the means showed that SOD ac-
tivity in tall fescue is greater, but not significantly differ-
ent from common bermudagrass. Maximum SOD activity 
in bermudagrass was detected at 0°C, while the minimum 
was found at 25°C in tall fescue. As the temperature di-
minished from 25°C to -7.5°C, MDA amassed continuous-
ly in the plants. MDA accumulated significantly more in 
common bermudagrass with the highest amount built up at 
-7.5°C (Table 2). H

2
O

2
 increased in plants as the tempera-

ture lowered to 0°C. The most H
2
O

2
 was produced in com-

mon bermudagrass at 0°C, whilst the lowest was observed 
in tall fescue at 25°C (Table 2).

4. Discussion and Conclusions

As the temperature decreased from 25°C to -7.5°C, 
soluble sugars and proline content increased, which tall 
fescue had higher amounts at -7.5°C (Table 1). A similar 
behavior was found in saltgrass (Distichlis spicata L.), 
centipedegrass (Eremochloa ophiuroides [Munro]), an-
nual bluegrass (Poa annua L.) and buffalograss (Boutel-
oua dactyloides [Nutt.]) (Fry, 1993; Shahba et al., 2003). 
Generally, one of the first reactions by these plants to 
counter the chilling stress of winter is a buildup of sugar 
(Fry, 1993; Ball et al., 2002), whilst amino acids help 
adapt the plants to low temperature (Guy, 1990). Proline 
and reducing sugars serve as cryoprotectants through in-
creasing the concentration of cell content and reducing 
the water potential (Ball et al., 2002). Comparable results 
were observed in zoysiagrass (Zoysia japonica Steud.) 
and annual bluegrass (Dionne et al., 2001).



11

The increase in CAT and SOD activity found in this 
study is assumed to protect the cells from oxidative dam-
age caused by cold stress as seen in other plants (Matsu-
mura et al., 2002; Larkindale and Huang, 2004; Jiang et 
al., 2011). SOD converts superoxide (O

2
.-) to H

2
O

2
 and 

CAT detoxifies the latter to water and oxygen (Fuchs et 
al., 1997; Polidoros and Scandalios, 1999). Antioxidant 
enzymes help maintain cell homeostasis under severe low 
temperatures by scavenging as well as signaling, although 
their definite function should be further elucidated (Polle, 
1997). Higher antioxidant enzyme activity in tall fescue 
could be attributed to better cold tolerance compared to 
common bermudagrass. MDA and H

2
O

2 
increase in the 

turfgrasses in this research is dependent on the cold stress 
received (Table 2). MDA and H

2
O

2
 are produced by lipid 

peroxidation of plants under chilling stress (Leshem, 1987; 

Wise and Naylor, 1987). These two sensitive indicators are 
considered to point toward the extent of low temperature 
stress and damage inflicted to the plant (Xu et al., 2006). 
Greater amounts of these two substances in common ber-
mudagrass compared to tall fescue could be interpreted as 
a greater sensitivity to and injury from low temperatures 
(Table 2), which is consistent with the reports in Manila 
grass (Zoysia matrella L.) (Wang et al., 2009).

Overall, cold stress produces large amounts of ROS 
which causes oxidative damage to plants through vast 
destruction of proteins, carbohydrates, lipids, cellular 
membranes, DNA and major decline of ATP reserve, and 
finally cell death (Dionne et al., 2001; Gill and Tuteja, 
2010). Since ROS has multifunctional roles, it is es-
sential for the cells to control the level of ROS tightly 
to avoid any oxidative injury and not to eliminate them 

Table 1 -  Effects of cold stress on biochemical changes [reducing sugar, proline content, catalase (CAT) and superoxide dismutase (SOD) activity] 
in the two turfgrasses used in this study

Turfgrass
Temperature (°C)

Mean
-7.5 0 +7.5 +15 +25

Reducing sugar (mg·g-¹ d.w.)

Tall fescue 176.4±60.1 a 160.8±16.3 ab 130.2±27.3 bcd 114.8±13.2 cd 104.3±22.4 dz 137.4 A

Bermudagrass 121.9±11.5 bcd 158.9±21.0 abc 140.9±18.7 a-d 117.3±19.8 bcd 96.5±14.7 d 127.1 A

Mean 149.2 A 159.8 A 135.5 AB 116.1 BC 100.4 C

Proline content (µg·g-¹ d.w.)

Tall fescue 35.8±3.7 a 35.1±3.4 a 27.2±2.7 b 13.7±3.1 cd 9.2±0.9 d 24.2 A

Bermudagrass 26.4±1.7 b 31.8±1.5 ab 27.3±5.1 b 17.3±4.5 c 12.7±1.3 cd 23.1 A

Mean 31.1 A 33.5 A 27.2 B 15.5 C 11.0 D

CAT (U g·g-¹ d.w.)

Tall fescue 36.3±5.4 c 46.7±6.1 ab 52.8±9.8 a 37.5±3.6 c 32.8±5.7 c 41.2 A

Bermudagrass 31.5±4.5 c 39.5±4.4 bc 47.5±2.9 ab 41.5±3.9 bc 36.9±5.8 c 39.3 A

Mean 35.4 C 43.1 B 50.1 A 39.5 BC 34.8 C

SOD (U g·g-¹ d.w.)

Tall fescue 179.6±17.6 abc 161.6±33.1 a-d 186.6±30.5 ab 126.0±19.0 de 106.6±17.0 e 152.1 A

Bermudagrass 127.3±23.1 cde 193.3±25.1 a 145.0±42.7 a-e 137.6±58.6 b-e 120.6±11.7 de 144.8 A

Mean 153.5 AB 177.5 A 165.8 AB 131.8 BC 113.6 C

z In each variable, data followed by the same letters±SD (small letters for interactions and capital letters for means) are not significantly different 
at 5% level of probability using LSD test.

Table 2 -  Effects of cold stress on malondialdehyde (MDA) and hydrogen peroxide (H
2
O

2
) in the two turfgrasses used in this study

Turfgrass
Temperature (°C)

Mean
-7.5 0 +7.5 +15 +25

MDA (µmol g-1 f.w.)

Tall fescue 7.5±1.6 cd 7.8±1.3 bc 6.8±0.8 cde 4.4±0.7 fg 3.9±0.7 gz 6.1 B

Bermudagrass 11.6±0.7 a 9.5±0.7 b 7.7±0.8 c 5.9±1.0 def 5.1±0.7 efg 8.0 A

Mean 9.6 A 8.6 A 7.3 B 5.1 C 4.5 C

H
2
O

2
 (µmol g-1 f.w.)

Tall fescue 5.1±0.9 a 4.7±0.7 a 3.5±0.6 b 2.9±0.4 b 2.7±0.4 b 3.8 B

Bermudagrass 5.0±0.3 a 5.3±0.4 a 4.9±0.7 a 3.5±0.5 b 3.1±0.7 b 4.3 A

Mean 5.0 A 5.0 A 4.2 B 3.2 C 2.9 C

(z)  In each variable, data followed by the same letter ± sd (small letters for interactions and capital letters for means) are not significantly different 
at 5% level of probability using LSD test.



12

entirely (Sharma et al., 2012). It is concluded that both 
turfgrasses increase reducing sugars, proline, CAT, SOD, 
MDA and H

2
O

2
 in response to lower temperatures, but 

tall fescue has a better defense mechanism than common 
bermudagrass and is more tolerant to cold stress. The 
results show that scavenging enzymes have a direct ef-
fect in cold season tolerance of turfgrass and improve the 
defense mechanism of plants, but their exact role merits 
further investigation.

Acknowledgements

The authors wish to thank the administration of the 
Students’ Scientific Association of Shiraz University for 
their financial support.

References

ALEXIEVA V., SERGIEV I., MAPELLI S., KARANOV E., 
2001 - The effect of drought and ultraviolet radiation on 
growth and stress markers in pea and wheat. - Plant Cell and 
Environment, 24: 1337-1344.

BALL S., QIAN Y.L., STUSHNOFF C., 2002 - Soluble carbo-
hydrates in two Buffalograss cultivars with contrasting freez-
ing tolerance. - J. Amer. Soc. Hortic. Sci., 127(1): 45-49.

BATES L.S., WALDERN R.P., TEAVE I.D., 1973 - Rapid de-
termination of free proline for water stress standies. - Plant 
and Soil, 39: 107-205.

BEAUCHAMP C., FRIDOVICH I., 1971 - Superoxide dis-
mutases: improved assays and an assay predictable to acryl-
amide gels. - Analytical Bioch., 44: 276-287.

CARROW R., 1994 - Zoysiagrass cultivar performance as af-
fected by traffic and N-level. - Agronomy Abstracts, ASA, 
Madison, WI, USA, p. 183.

DHINDSA R.S., PLUMB-DHINDSA P., THORPE T.A., 1981 
- Leaf senescence: correlated with increased levels of mem-
brane permeability and lipid peroxidation, and decreased 
levels of superoxide dismutase and catalase. - J. Exp. Bot., 
32(1): 93-101.

DIONNE J., CASTONGUAY Y., NADEAU P., DESJARDINS 
Y., 2001 - Freezing Tolerance and carbohydrate changes 
during cold acclimation of green-type annual bluegrass (Poa 
annua. L) ecotypes.- Crop Science, 41: 443-451.

DUBOIS M., GILLES K.A., HAMILTON J.K., REBERS P.A., 
SMITH F., 1956 - Colorimetric method for determination of 
suger and related substances. - Analytical Bioch., 28: 350-356.

FRY J.D., 1993 - Freezing tolerance and carbohydrate content 
of low-temperature-acclimated and nonacclimated centipe-
degrass. - Crop Science, 33: 1051-1055.

FUCHS D., BITTERLICH G.B., WEDE I., WACHTER H., 
1997 - Reactive oxygen and apoptosis, pp. 139-167. - In: 
SCANDALIOS J.G. (eds.) Oxidative stress and the molecu-
lar biology of antioxidant defenses. - Cold Spring Harbor 
Laboratory Press, Cold Spring Harbor, NY, USA, pp. 890.

GILL S.S., TUTEJA N., 2010 - Reactive oxygen species and 
antioxidant machinery in abiotic stress tolerance in crop 

plants. - Plant Physiology and Biochemistry, 48: 909-930.
GULZAR S.S., SHABIR H.W., WASIM H., SINGH N.B., 2011 

- Engineering cold stress tolerance in crop plants. - Current 
Genomics, 12: 30-43.

GUY C.L., 1990 - Molecular mechanism of cold acclimation, pp. 
35-61. - In: KATTERMAN F. (eds.) Environmental injury to 
plants. - Academic Press, San Diego, CA, USA, pp. 302.

HEATH R.L., PACKER L., 1969 - Photoperoxidation in iso-
lated chloroplast; Kinetics and stoichiometry of fatty acid 
peroxidation. - Arch. Bioch. Biophy., 125: 189-198.

JIANG X., SONG Y., XI X., GUO B., MA K., WANG Z., LI B., 
ZHANG D., AN X., ZHANG Z., 2011 - Physiological and 
biochemical responses to low temperature stress in hybrid 
clones of Populususs uriensis Kom.× P. deltoides Bartr. - Af-
rican J. Biotech., 10: 19011-19024.

JIANG Y.W., CARROW R.N., DUNCAN R.R., 2005 - Physio-
logical acclimation of seashore paspalum and bermudagrass 
to low light. - Scientia Hort., 105: 101-115.

LARKINDALE J., HUANG B., 2004 - Thermotolerance and 
antioxidant systems in Agrostis stolonifera: Involvement of 
salicylic acid, abscisic acid, calcium, hydrogen peroxide, 
and ethylene. - J. Plant Physiol., 161(4): 405-413.

LESHEM Y.Y., 1987 - Membrane phospholipids catabolism and 
Ca2+ activity in control of senescence. - Plant Physiology, 
69: 551-559.

LEVITT J., 1980 - Responses of plants to environmental stress. 
Volume 1. Chilling, Freezing, and High Temperature Stress. 
- Academic Press, New York, pp. 497.

MATSUMURA T., TABAYASHI N., KAMAGATA Y., SOUMA 
C., SARUYAMA H., 2002 - Wheat catalase expressed in 
transgenic rice can improve tolerance against low tempera-
ture stress. - Plant Physiology, 116: 317-327.

POLIDOROS N.A., SCANDALIOS, J.G., 1999 - Role of hydro-
gen peroxide and different classes of antioxidants in the regu-
lation of catalase and glutathione S-transferase gene expres-
sion in maize (Zea mays L.). - Plant Physiology, 106: 112-120.

POLLE A., 1997 - Defense against photooxidative damage in 
plants, pp. 623-666. - In: SCANDALIOS J.G. (eds.) Oxida-
tive stress and the molecular biology of antioxidant defenses. 
Cold Spring Harbor Laboratory Press. Plainview, NY, USA, 
pp. 890.

ROGERS R.A., DUNN J.H., NELSON C.J., 1975 - Cold hard-
ening and carbohydrate composition of Meyer zoysia. - 
Agronomy Journal, 67: 836-838. 

SAKAI A., LARCHER W., 1987 - Frost survival of plants: Re-
sponse and adaptation to freezing stress. - Springer-Verlag, 
Berlin.

SARKAR D., BHOWMIK P. C., 2009 - Cold acclimation re-
sponses of three cool-season turfgrasses and the role of pro-
line-associated pentose phosphate pathway.- Journal of the 
American Society for Horticultural Science, 134: 210-220.

SHAHBA M.A., QIAN Y.L., HUGHES H.G., KOSKI A.J., 
CHRISTENSEN D., 2003 - Relationship of carbohydrates 
and cold hardinesss in six saltgrass accessions. - Crop Sci-
ence, 43: 2148-2153.

SHARMA P., JHA A.B., DUBEY R.S., PESSARAKLI M., 
2012 - Reactive oxygen species, oxidative damage, and anti-
oxidative defense mechanism in plants under stressful condi-
tions. - Journal of Botany, pp. 26. doi: 10.1155/2012/217037.

WANG Y., YANG Z.M., ZHANG Q.F., LI J.L., 2009 - Enhanced 



13

chilling tolerance in Zoysia matrella by pre-treatment with 
salicylic acid, calcium chloride, hydrogen peroxide or 6-ben-
zylaminopurine. - Biologia Plantarum, 53: 179-182.

WISE R.R., NAYLOR A.W., 1987 - Chilling-enhanced photo-
oxidation, Evidence for the role of singlet oxygen and super-
oxide in the breakdown of pigments and endogenous antioxi-

dants. - Plant Physiology, 83: 278-282.
XU S., LI J.L., ZHANG X.Q., WEI H., CUI L.J., 2006 - Effects 

of heat acclimation pretreatment on changes of membrane 
lipid peroxidation, antioxidant metabolites, and ultrastruc-
ture of chloroplasts in two cool-season turf grass species un-
der heat stress. - Environ. Exp. Bot., 56: 274-285.