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Crop Science 42:466-472 (2002)
© 2002 Crop Science Society of America

TURFGRASS SCIENCE

Cytokinin Effects on Creeping Bentgrass Response to Heat Stress

II. Leaf Senescence and Antioxidant Metabolism

Xiaozhong Liua and Bingru Huang*,b

a Dep. of Botany and Microbiology, Univ. of Oklahoma, Norman, OK 73019
b Dep. of Plant Science, Rutgers University, New Brunswick, NJ 08901

* Corresponding author (huang{at}aesop.rutgers.edu)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In another study, results of growth responses to cytokinin indicate that heat stress injury in creeping bentgrass can be alleviated to some extent by injection of cytokinin to the root zone. The objective of this study was to examine whether the alleviating effects of exogenous application of a cytokinin on heat injury in creeping bentgrass (Agrostis palustris L.) involve regulation of antioxidant activities. Creeping bentgrass was exposed to three air/soil temperature regimes for 56 d in growth chambers: (i) low air and soil temperature control (20/20°C); (ii) high soil temperature (20/35°C); and (iii) high air/soil temperatures (35/35°C). Four different concentrations (0.01, 0.1, 1, and 10 µmol) of zeatin riboside (ZR) or water (control) were injected into the root zone (0 to 5 cm depth) of plants on the day before heat stress was imposed (0 d) and 14 d after. Leaf electrolyte leakage (EL) and the content of a lipid peroxidation product, malondialdehyde (MDA), increased, whereas leaf chlorophyll content and activities of superoxide dismutase (SOD) and catalase (CAT) decreased at 20/35°C or 35/35°C for ZR-untreated plants. Exogenous ZR significantly suppressed these responses under both high temperature regimes. Application of 10 µmol ZR was most effective in slowing leaf senescence and alleviating heat-induced lipid peroxidation of cell membranes, followed by 1 µmol at 35/35°C. Applying 0.1 and 0.01 µmol ZR had no effects on creeping bentgrass responses to 20/35 or 35/35°C. Our results suggested the alleviating effects of ZR at 1 and 10 µmol in heat injury to creeping bentgrass was related to the inhibition of lipid peroxidation and slowing leaf senescence.

Abbreviations: CAT, catalase • EL, electrolyte leakage • LSD, least significance difference • MDA, malonyldiadehyde • SOD, superoxide dismutase • ZR, zeatin riboside


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
GROWTH OF cool-season grasses, including creeping bentgrass, often is suppressed during summer when temperatures exceed their optimum requirement. High temperature inhibits photosynthesis (Santarius, 1975; Schreiber and Bilger, 1987; Paulsen, 1994); damages cell membranes (Blum and Ebercon, 1981; Marcum, 1998); and causes cell death (Abernethy et al., 1989). Heat injury was related to oxidative damage to cell membranes by active oxygen species, which results from the dysfunction of the active oxygen-scavenging system under various environmental stresses (Dhindsa and Matowe, 1981; Elstner, 1982; Bowler et al., 1992; Zhang and Kirkham, 1994; Liu and Huang, 2000).

Grossman and Leshem (1978) reported that exogenous cytokinin application decreased lipoxygenase activity in Pisum sativum L. foliage and retarded leaf senescence. Pauls and Thompson (1982) observed that cytokinins facilitated scavenging of active oxygen caused by ozone damage in Phaseolus vulgaris L. Benzyladenine increased activities of superoxide dismutase (SOD) and catalase (CAT) in maize (Zea mays L.) under waterlogged conditions (Liu et al., 1996). Alleviating effects of cytokinins on heat injury have also been observed in bean (P. vulgaris) (Adedipe et al., 1971; Yordanov et al., 1997); wheat (Triticum aestivum L.) (Skogqvist and Fries, 1970; Skogqvist, 1974); and other cereal crops (Kirichenko et al., 1996). Application of cytokinin to the root zone of creeping bentgrass inhibited the declines of growth and turf quality under heat stress (Liu and Huang, 2000b). However, whether the effects of cytokinin in the alleviation of heat stress in cool-season grasses involve the maintenance of oxidative scavenging ability of antioxidants is unclear.

The objective of this experiment was to determine whether cytokinin application could retard leaf senescence and improve creeping bentgrass tolerance to heat stress by regulating antioxidant activities and lipid peroxidation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Plant Materials, Treatments, and Experimental Design
The detailed experimental procedure, including plant establishment protocol and growth conditions, was described in the companion paper (Liu et al., 2002).

‘Penncross’ was exposed to three constant day/night temperature regimes for 56 d: (i) control: optimum air and soil temperatures (20/20°C); (ii) high soil temperature with optimum air temperature (20/35°C); and (iii) high air and soil temperatures (35/35°C). For the 20/35°C regime, air temperature was controlled at 20°C by the temperature controller in the growth chamber, and soil temperature was controlled at 35°C by circulating warm water around PVC tubes placed vertically in water baths using an immersion circulating heater (Fisher Scientific Inc., Pittsburgh, PA). In the 20/20°C and 35/35°C regimes, both air and soil temperatures were controlled by the temperature controller in the growth chambers. Relative humidity in the growth chamber was approximately 70% at 20/20°C and 85% at 20/35°C and 35/35°C. On the day before high temperature treatments were imposed (0 d) and 14 d after treatments, 50 ml of water or 0.01, 0.1, 1.0, and 10 µmol ZR were injected into the 0–5 cm layer of root zone in each tube with hypodermic syringes.

The experimental design was a completely randomized split-plot design with temperature as the main plot and ZR concentration as the subplot in four replicates for each measurement. Each temperature treatment was replicated four times in four different growth chambers and water baths. The ZR treatments were arranged randomly in each temperature treatment. Effects of temperature, ZR concentration, and their interaction were determined by analysis of variance according to the general linear model procedure of Statistical Analysis System (SAS Inc., Cary, NC). Differences between treatment means were separated by the least significance difference (LSD) test at the 0.05 level.

Measurements
Leaves were harvested weekly with an electric clipper for analyses of leaf chlorophyll content and electrolyte leakage and biweekly for measurement of antioxidant enzyme activities and content of malonydialdehyde (MDA). Chlorophyll was extracted by soaking 0.1-g sample of leaves in 8 mL of dimethyl sulfoxide for 72 h. Absorbance of chlorophyll extractants was measured at 663 nm and 645 nm with a spectrophotometer (Spectronic instruments, Rochester, NY). Cell membrane permeability was estimated by measuring electrolyte leakage (EL) using the method described in the companion paper (Liu and Huang, 2002b) but with a 0.1-g sample of leaves.

For preparation of crude enzyme extracts, a 0.5-g sample of fresh leaves was ground in 8 mL of 50 mM cool phosphate buffer (pH 7.0) containing polyvinylpyrrolidone plus white quartz sand in an ice bath. The homogenate was centrifuged at 15,000 x g for 20 min at 4°C. The supernatant was used for the assays of enzyme activity and MDA contents.

Activity of SOD was determined by measuring its ability to inhibit the photoreduction of nitro blue tetrazolium (NBT) according to the methods of Giannopolitis and Ries (1975). The reaction solution (3 mL) contained 50 µmol NBT, 1.3 µmol riboflavin, 13 mmol methionine, 75 nmol EDTA, 50 mmol phosphate buffer (pH 7.8) and 20 to 50 µL enzyme extract. The reaction solution was irradiated under a bank of fluorescent lights at 75 µmol m-2 s-1 for 15 min. The absorbance at 560 nm was read against the blank (nonirradiated reaction solution) with a spectrophotometer (Spectronic Instruments, Rochester, NY). One unit of SOD activity was defined as the amount of enzyme that inhibited 50% of NBT photoreduction.

Activities of CAT were measured by the method of Chance and Maehly (1955) with modification. The CAT reaction solution (3 mL) contained 50 mmol phosphate buffer (pH 7.0), 15 mmol H2O2, and 0.1 mL enzyme extract. The reaction was initiated by adding the enzyme extract. The decline at abosorbance in 240 nm was scanned automatically with the spectrophotometer. One unit of CAT activity was defined as the absorbance change by 0.01 per min at 240 nm.

Enzyme activities were expressed on a per unit protein basis. Protein concentration of the crude extract was measured by the method of Bradford (1976). A 0.1-mL aliquot of crude extract was added to 5 mL of Bright Blue G-250 solution, and absorbance at 595 nm was read between 10 and 60 min.

The lipid peroxidation level was determined in terms of MDA content by the method of Dhindsa et al. (1981) with modification. To 2 ml of enzyme solution, 1 ml 20% trichloroacetic acid containing 0.5% of thiobarbituric acid was added. The mixture was heated in a water bath at 95°C for 30 min, cooled at 22°C, and centrifuged at 10,000 x g for 10 minutes. Absorbance of the supernatant was read at 532 nm, and the nonspecific absorbance at 600 nm was subtracted. The MDA content was calculated by the extinction coefficient of 155 mM-1 cm-1 (Heath and Packer, 1968).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Electrolyte Leakage
Electrolyte leakage increased significantly above the control level (20/20°C) for plants treated with or without ZR, beginning at 7 d under high soil temperature (20/35°C) (Fig. 1A, B) and high air/soil temperatures (35/35°C) (Fig. 1C, D). The increase in EL was more pronounced at 35/35°C than at 20/35°C, particularly for untreated plants. At the end of the experimental period (56 d), EL was 2 to 3 times above the control level at 20/35°C and 3 to 4 times greater at 35/35°C, depending on ZR concentrations.



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  		Fig. 1. Cell membrane integrity expressed as EL in leaves of creeping bentgrass as affected by applications of exogenous zeatin riboside (ZR) at 0 d (A, C) and 14 d (B, D) of high soil temperature (20/35°C, A, B) and high air/soil temperatures (35/35°C, C, D). Vertical bars indicate LSD values (P = 0.05) for temperature and ZR concentration comparisons at a given day.

 
Application of ZR at 0.1, 1, and 10 µmol at either 0 or 14 d reduced EL, beginning at 21 d of 20/35°C and 35/35°C. Applying 10 µmol of ZR at 0 or 14 d of heat stress was most effective in reducing EL at both 20/35°C and 35/35°C.

Chlorophyll Content
Chlorophyll content decreased significantly below the control level for untreated plants after 14 d of 20/35°C (Fig. 2A, B) and 7 d of 35/35°C (Fig. 2C, D).



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  		Fig. 2. Leaf chlorophyll content of creeping bentgrass as affected by exogenous applications of zeatin riboside (ZR) at 0 d (A, C) and 14 d (B, D) of high soil temperature (20/35°C, A, B) and high air/soil temperatures (35/35°C, C, D). Vertical bars indicate LSD values (P = 0.05) for temperature and ZR concentration comparisons at a given day.

 
Applying 10 and 1 µmol ZR at 0 d of heat stress significantly increased chlorophyll at 14 d of 20/35°C and 35/35°C compared with plants without ZR treatment. Applying 10 and 1 µmol ZR at 14 d of heat stress significantly increased chlorophyll content after 21 d of either 20/35°C or 35/35°C. Applications of 0.1 and 0.01 µmol ZR at 0 or 14 d of heat stress did not affect chlorophyll content under either high temperature regime.

Application of ZR at 0 d of 20/35°C (Fig. 2A, C) helped maintain higher chlorophyll content during the first 14 d of heat stress compared with plants not receiving ZR until 14 d (Fig. 2B, D).

Antioxidant Activity
Activity of SOD in untreated plants decreased significantly below the control level, beginning at 14 d of 20/35°C (Fig. 3A, B) and 35/35°C (Fig. 3C, D).



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  		Fig. 3. Superoxide dismutase activities in leaves of creeping bentgrass as affected by exogenous applications of zeatin riboside (ZR) at 0 d (A, C) and 14 d (B, D) of high soil temperature (20/35°C, A, B) and high air/soil temperatures (35/35°C, C, D). Vertical bars indicate LSD values (P = 0.05) for temperature and ZR concentration comparisons at a given day.

 
Applying 10 µmol ZR at 0 d or 14 d of heat stress helped maintain SOD activity (Fig. 3A, B) at the control level within the first 28 d of 20/35°C. SOD activity in plants treated with 1 and 0.1 µmol ZR at 0 or 14 d of heat stress were higher than that in untreated heat-stressed plants. Applying 0.01 µmol ZR at 0 or 14 d under either high temperature regime had no effects on SOD activity.

Significant declines in CAT activities were observed at 14 d of 20/35°C (Fig. 4A, B) and 35/35°C (Fig. 4C, D) for plants with or without ZR treatment.



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  		Fig. 4. Catalase activities in leaves of creeping bentgrass as affected by exogenous applications of zeatin riboside (ZR) at 0 d (A, C) and 14 d (B, D) of high soil temperature (20/35°C, A, B) and high air/soil temperatures (35/35°C, C, D). Vertical bars indicate LSD values (P = 0.05) for temperature and ZR concentration comparisons at a given day.

 
Plants treated with 10 and 1 µmol ZR at 0 or 14 d of heat stress had significantly higher CAT activities than untreated plants under both high temperature regimes. Applying 0.1 and 0.01 µmol ZR at 0 or 14 d of heat stress had no significant effect at 20/35°C but increased CAT activity after 56 d at 35/35°C.

Malonyldiadehyde Content
The MDA contents increased significantly at both 20/35°C (Fig. 5A, B) and 35/35°C (Fig. 5C, D) for plants with or without ZR treatment.



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  		Fig. 5. Malonyldidehyde (MDA) contents in leaves of creeping bentgrass as affected by exogenous applications of zeatin riboside (ZR) at 0 d (A, C) and 14 d (B, D) of high soil temperature (20/35°C, A, B) and high air/soil temperatures (35/35°C, C, D). Vertical bars indicate LSD values (P = 0.05) for temperature and ZR concentration comparisons at a given day.

 
Applying ZR at both 0 and 14 d of heat stress reduced MDA contents at 20/35°C and 35/35°C, but application at 0 d had an earlier effect on the inhibition of MDA accumulation. Also, the effectiveness of ZR increased with increasing concentrations. Plants treated with 10, 1, or 0.1 µmol ZR at 0 or 14 d of heat stress had significantly lower MDA contents than untreated plants at both 20/35°C and 35/35°C. Applying 0.01 µmol ZR at 0 or 14 d of heat stress did not significantly affect MDA content at 20/35°C but significantly reduced MDA content at 56 d of 35/35°C.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Leaf electrolyte leakage (EL) increased, whereas chlorophyll content decreased under high soil temperature alone or in combination with high air temperature, but the combination was more detrimental. Increases in EL and loss of chlorophyll usually are attributed to membrane damage and leaf senescence (Simon, 1974; Liu and Huang, 2000a). These results suggested that high soil temperature accelerated leaf senescence in creeping bentgrass, particularly when combined with high air temperature. This is consistent with our observation of turf quality decline induced by high soil and/or air temperatures (Liu et al., 2002).

Applying ZR at 1 and 10 µM reduced EL and chlorophyll loss under both high soil and air/soil temperatures. Cytokinins were reported to retard senescence by inhibiting losses of chlorophyll and carotenoid in Xanthium pensylvanicum Wallr. and Avena sativa L. (Tetley and Thimann, 1974; Musgrave et al., 1987). These results demonstrated that the alleviating effects of ZR on shoot and root growth of creeping bentgrass reported by Liu et al. (2002) could be due to the protective effects of the cytokinin on cell membranes and leaf senescence. The application of ZR before plants were exposed to heat stress had an earlier and more effective influence on leaf senescence than application after heat stress occurred.

Malonydiadehyde is a product of peroxidation of unsaturated fatty acids in phospholipids, and lipid peroxidation is responsible for cell membrane damage (Halliwell and Gutteridge, 1989). High soil and air/soil temperatures increased MDA production, which is consistent with the increase in cell membrane permeability at high soil and high air/soil temperatures. Applying ZR inhibited the accumulation of MDA under high temperatures, suggesting that ZR could inhibit the lipid peroxidation of cell membranes in creeping bentgrass resulting from high temperature injury. Benzyladenine and zeatin played the same roles as antioxidants in P. vulgaris, i.e., scavenging active oxygen and protecting membranes from ozone damage (Pauls and Thompson, 1982). Cytokinins react with superoxide in organic solvents to produce the corresponding amides (Leshem et al., 1979). The {alpha}-carbon atom of the amine bond donates a hydrogen atom and provides a partner for the unpaired electron in free radicals. Cytokinins also could retard leaf senescence partly by scavenging active oxygen through lowering endogenous lipoxygenase, as was found in P. sativum and Vigna unguiculata L., or by inhibiting lipid peroxidation (Grossman and Leshem, 1978; Swamy and Suguna, 1992). Lipoxygenase catalyzed the hydroperoxidation of unsaturated fatty acids (Axelrod et al., 1981).

Superoxide dismutase and CAT are two key enzymes in the active-oxygen scavenging system. They quench super oxygen free radicals and H2O2, respectively, and thus suppress the production of active oxygen species (Elstner, 1982; Asada, 1992; Bowler et al., 1992; Scandalious, 1993; 1994). In our study, the declines in SOD and CAT activities under high soil or air/soil temperatures were mitigated by exogenous ZR, especially at the higher concentrations (1 and 10 µM). Therefore, alleviation of heat injury in creeping bentgrass by ZR could be related to the maintenance of the scavenging ability of antioxidants at high temperatures. As a result, production of super oxygen free radicals and H2O2 would be inhibited. Applying exogenous ZR at high concentrations could inhibit lipid peroxidation either by direct quenching of free radicals or by increasing SOD and CAT activities.

In summary, results in this study suggested that ZR could alleviate heat stress injury in creeping bentgrass by maintaining active antioxidants and reducing lipid peroxidation. The treatment with 10 µmol ZR was most effective in protecting cell membranes from heat injury and slowing leaf senescence.

Received for publication December 12, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 




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