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TURFGRASS SCIENCE

Hormone-Containing Products' Impact on Antioxidant Status of Tall Fescue and Creeping Bentgrass Subjected to Drought

Department of Crop and Soil Environmental Sciences, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0404 USA

rschmidt{at}vt.edu

	ABSTRACT

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This study was conducted to determine whether the plant endogenous antioxidant concentration is responsive to exogenous hormone-containing products (HCPs) in two turfgrass species subjected to drought. Two-week-old seedlings of tall fescue (Festuca arundinacea Schreb.) and creeping bentgrass (Agrostis palustris Huds. A.) were treated with two HCPs, seaweed extract (SWE) at 326 g ha^-1 or humic acid (HA, 25% a.i.) at 5 L ha^-1, applied alone or in combination and grown under either -0.03 or -0.5 MPa soil moisture for 5 wk. Growth and antioxidant status of leaves were determined subsequently. The HCP treatments significantly improved leaf water status (LWS) and shoot and root growth of the grasses grown under high (-0.03 MPa) and low (-0.5 MPa) soil moisture. -Tocopherol concentration increased significantly and ascorbic acid concentration remained unchanged for drought-stressed compared with nonstressed turfgrass. The HCP treatments significantly increased -tocopherol and ascorbic acid concentration of the grasses grown under high and low soil moisture. Positive correlation between antioxidants and shoot or root growth was found in the two grass species. Improvement of growth and LWS of turfgrass treated with HCPs may be related to its high antioxidant concentration.

Abbreviations: ANOVA, analysis of variance • HA, humic acid • HCP, hormone-containing product • HPLC, high performance liquid chromatography • LWP, leaf water potential • LWS, leaf water status • PSII, photosystem II • ROS, reactive oxygen species • SWE, seaweed extract • WS, water stress

	INTRODUCTION

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WATER STRESS is a major limiting factor in turfgrass management and a wide variation exists among turfgrass species in terms of drought tolerance (White et al., 1993). Tall fescue shows better drought tolerance than creeping bentgrass (Turgeon, 1991). Although the good drought tolerance of tall fescue has been attributed to its deep root system, the physiological basis of drought tolerance still remains unclear.

The two antioxidants, -tocopherol and ascorbic acid, are concentrated in the chloroplast and protect the photosynthetic apparatus photosystem II (PSII) when a plant is subjected to environmental stresses by scavenging excess reactive oxygen species (ROS) (Smirnoff, 1995). Drought stress damages plant cells via excess accumulation of ROS (Lawlor, 1995; Moran et al., 1994; Price and Hendry, 1991; Quartacci and Navarri-Izzo, 1992; Smirnoff, 1993). The antioxidant concentration of a plant is closely associated with its stress tolerance (Smirnoff, 1995). The severity of ROS-induced damage depends largely on the status of antioxidant systems and their efficiency in removing toxic ROS and protecting plant cells from lipid peroxidation and inactivation of enzymes that occur under stress (Smirnoff, 1993).

Although plant antioxidant status differs with species (Foyer, 1993; Hess, 1993), a close relationship exists between antioxidant activity and drought tolerance (Smirnoff, 1993). Price and Hendry (1989) indicated that the antioxidant -tocopherol concentration increased significantly in response to water stress (WS) in 9 out of 10 grass species examined. Others reported that drought induced a significant increase in the antioxidant status in plants (Moran et al., 1994; Mukherjee and Choudhuri, 1983; Smirnoff and Colombe, 1988).

Proper application of certain HCPs not only can improve turfgrass growth but also can enhance stress tolerance (Schott and Walter, 1991; Schmidt and Zhang, 1997). Seaweed extract and HA contain organic compounds that generate auxin- or cytokinin-like activity. When these materials are applied to plants, they enhance plant tolerance to salinity, drought, chilling, and other environmental stresses; increase antioxidant activity; and improve turfgrass quality (Crouch and Van Staden, 1993; Fagbenro and Agboola, 1993; Finnie and Van Staden, 1985; Nabati, 1991; O'Donnell, 1973; Nelson and Van Staden, 1984; Schmidt and Zhang, 1997; Zhang and Schmidt, 1999).

The objective of this study was to examine the influence of exogenous HCPs on endogenous antioxidant concentrations and growth of tall fescue and creeping bentgrass subjected to drought.

	Materials and methods

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Hormone-Containing Product Application and Drought Stress Treatment
Creeping bentgrass (`Penncross') and tall fescue (`Rebel Jr.') seeds were sown at 0.06 and 0.25 g, respectively, in 10-cm-diameter polyvinyl chloride rings as described by Schmidt and Zhang (1997). When the seedlings were 2 wk old, soluble SWEs (Ascophyllum nodosum L.) at a rate of 326 g ha^-1 and HA (25% a.i.) at a rate of 5 L ha^-1 were applied alone or in the combination on the foliage at 35 mL solution m^-2. Both HA and SWE were supplied by Plant Wise Biostimulant Co. (Louisville, KY). The SWE contained 500 mg L^-1 of kinetin according to a bioassay using radish (Raphanus sativus L.) cotyledon reported by Acadian Seaplants Limited, Nova Scotia, Canada. The HA was extracted from leonardite, and its auxin-like activity has been identified in the bioassay by O'Donnell (1973).

Groseclose silt loam topsoil (clayey, mixed, mesic Typic Hapludult) was air-dried for 2 wk and then sieved through a 2-mm screen. This soil was mixed with appropriate water to obtain moisture levels of 11.6 and 20.1% (w/w), which were equivalent to a water potential of -0.5 and -0.03 MPa, respectively (Nabati, 1991). The soil moisture level was confirmed using Thetaprobe soil moisture sensor (type ML1; Delta-T Devices Ltd., Cambridge, UK). A 20-8.8-16.6 (N-P-K) soluble fertilizer was mixed with water to provide 34 kg N ha^-1. After thoroughly mixing the soil with water, the soil was placed in a plastic bag, sealed, and allowed to equilibrate for 72 h.

Terrarium-like boxes (30 by 40 by 75 cm) were constructed with 0.15-mm-thick clear plastic sides and tops. Four terrariums were used for each grass, with two terrariums for each soil moisture level. Fifteen kilograms of soil were placed into each terrarium and then the HCP-treated plugs were transplanted to the terrariums 1 wk after HCP treatments. The soil moisture level in which the grasses were established was approximately the same as the soil moisture in the terrariums at the time of transplanting. The HCP treatments were replicated four times, with a total of 16 plugs from each grass randomly arranged in each of the two terrariums with the same soil moisture level. The terrarium tops were sealed with clear plastic impervious to water but not gas, and the terrariums were placed randomly in a greenhouse at Virginia Tech, Blacksburg, VA, and rotated one-quarter turn three times per week. The seedlings were grown under natural light in January 1995. The average air temperature inside the terrarium was 23°C at 0800 h and 26°C at 1400 h, respectively, during the period of the experiment.

Leaf water status for all treatments was measured with a hydraulic leaf press (Campbell Scientific, Logan, UT) 7 wk after germination according to the procedure described by Nabati (1991). The last fully emerged leaf was removed from a tiller and placed in a leaf press. The leaf press was activated by a hydraulic pump that pressed the leaf against a plexiglass plate. The pressure that initially caused water to be exuded from the leaf edges was recorded. The greater the pressure required to cause water exudation, the lower the leaf water content (Nabati, 1991). The relationship between leaf water potential (LWP) and LWS has been established with ryegrass (Lolium spp.) by Yan (1993). The equation is expressed as LWP = 3.24 exp(-0.0145x), , where x is LWS measured by hydraulic press, and LWP data were measured by pressure bomb.

At the time of LWS measurement, clippings were measured and fresh leaves of the 7-wk-old seedlings were sampled from each treatment. The leaves were frozen with a small amount of liquid N, and then stored at -20°C for antioxidant analysis. Soil was washed from the roots, and root mass was measured immediately after sampling of leaves.

Antioxidant Analysis
-Tocopherol analysis was based on the methods of Tanaka et al. (1990). For ascorbic acid analysis, frozen leaf segments (1000 mg) were homogenized in 7 mL of 5% (w/v) metaphosphoric acid with a Polytron (PT3000, Kinematia, AG, Littau, Switzerland) at a speed of 4000 g for 3 min and kept cooled with ice. The homogenate was then centrifuged in a refrigerated centrifuge at 4000 g for 40 min.

The clear supernatant was passed through a Nylon Acrodisc 13 mm by 0.2 µm filter before being analyzed by high performance liquid chromatography (HPLC). The HPLC system consisted of a pump (SPD-6A), with a 20-µL injection loop, a variable wavelength ultraviolet detector (Shimadzu RF 535 fluorescence HPLC monitor), a HPLC system controller (Shimadzu SCL-6B), and a CR 501 chromatopac (Shimadzu, Japan). -Tocopherol was determined on an analytical column of Supelcosil LC-8-BD (4.6 by 150 mm) from Supelco (Bellafonte, PA) and a guard column (50 by 4.6 mm). A 15:85 ratio of methanol and 4.3 mM hexane sulfornate with 0.1 triethylamine (the pH was adjusted to 2.8 with phosphoric acid) was used as the mobile phase. Ascorbic acid was detected by measuring the absorbance intensity at 245 nm and compared with a standard curve developed from ascorbic acid obtained from Aldrich Chemical (Milwaukee, WI).

A two-factor analysis of variance (ANOVA) and a least significant difference (LSD) test were performed on each data set. When the moisture x HCP interaction was not significant, the data were pooled across moisture treatments for HCP treatment comparisons.

	Results

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Responses of Leaf Water Status to Hormone-Containing Products
Higher LWS values, determined with the hydraulic press, are an indication of lower leaf water content. The leaf water content of tall fescue and creeping bentgrass was improved with application of the HCPs, as was indicated by the lower LWS values, regardless of soil moisture (Table 1) .

Antioxidant Status and Turfgrass Growth
Positive correlations between the antioxidants and growth traits were found in the two grasses grown under both high and low soil moisture levels (Fig. 1–4) . In Fig. 1 and 2, it can be shown that turfgrass with a high level of -tocopherol produced better shoot and root growth under high or low soil moisture environment compared with the turfgrass containing low levels of this antioxidant. A high concentration of -tocopherol was required of the grasses grown in low soil moisture to produce root mass similar to the grasses grown in high soil moisture. Positive correlations between ascorbic acid and shoot or root growth were also found in tall fescue and creeping bentgrass grown under low and high soil moisture levels (Fig. 3 and 4).

View larger version (24K): [in this window] [in a new window]   Fig. 1 Relationship between -tocopherol concentration and shoot weight in tall fescue and creeping bentgrass grown under high (-0.03 MPa, solid line) and low (-0.5 MPa, dotted line) soil moisture levels

View larger version (23K): [in this window] [in a new window]   Fig. 2 Relationship between shoot -tocopherol concentration and root weight in tall fescue and creeping bentgrass grown under high (-0.03 MPa, solid line) and low (-0.5 MPa, dotted line) soil moisture levels

View larger version (23K): [in this window] [in a new window]   Fig. 3 Relationship between ascorbic acid concentration and shoot weight in tall fescue and creeping bentgrass grown under high (-0.03 MPa, solid line) and low (-0.5 MPa, dotted line) soil moisture levels

View larger version (23K): [in this window] [in a new window]   Fig. 4 Relationship between shoot ascorbic acid concentration and root weight in tall fescue and creeping bentgrass grown under high (-0.03 MPa, solid line) and low (-0.5 MPa, dotted line) soil moisture levels

View larger version (25K): [in this window] [in a new window]   Fig. 5 Relationship between ascorbic acid and -tocopherol concentrations in tall fescue and creeping bentgrass grown under high (-0.03 MPa, solid line) and low (-0.5 MPa, dotted line) soil moisture levels

	Discussion

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Water stress resulted in significant increases in antioxidant -tocopherol concentration in the two turfgrass species. -Tocopherol, associated closely with the photosynthetic apparatus, functions as a cell membrane stabilizer and an efficient antioxidant that prevents cells from drought-induced oxidative damages. This result is consistent with previous research conducted by Moran et al. (1994), Price and Hendry (1989), Tanaka et al. (1990), and Zhang and Schmidt (1999). Tanaka et al. (1990) noted that water deficit induced a significant increase of -tocopherol in spinach (Spinacia oleracea L.) leaves. The increase of this antioxidant has been reported to be triggered by excess production of ROS in the photosynthetic apparatus under WS (Smirnoff, 1993). Water stress did not induce an increase of ascorbic acid concentration. Foyer (1993) noted that ascorbic acid not only quenches ROS but also regenerates -tocopherol. The actual concentration of ascorbic acid results from the balance between synthesis and breakdown. Smirnoff (1993) indicated that reduction of ascorbic acid levels under WS may be caused by depletion of excess ROS or utilization for cycling -tocopherol.

Exogenous HCP enhancement of -tocopherol and ascorbic acid concentrations in both turfgrass species grown under high or low soil moisture levels is supported by the studies of Kim (1988), Yokoyama and Keithly (1991), and Zhang and Schmidt (1999). Seaweed extracts contain substantial amounts of hormones that may improve growth when applied to plants. The growth improvement by application of SE may result from the activity of cytokinin (Crouch and Van Staden, 1993). Piccolo et al. (1992) showed that HA stimulates stem elongation and exhibits auxin-like activity. The significant increase of antioxidant concentration of the turfgrasses associated with low soil moisture can be attributed to the increase of hormonal activities by the exogenous application of HCPs.

The results of this study are consistent with the study of Kentucky bluegrass (Zhang and Schmidt, 1999). The high level of -tocopherol in grasses may contribute to the better drought tolerance (Price and Hendry, 1989). It appeared that, when under WS, turfgrasses with higher antioxidant levels produced better growth. Seaweed extract and HA may enhance antioxidants -tocopherol and ascorbic acid activity and thus promote growth and enhance drought tolerance.

Received for publication April 21, 1999.

	REFERENCES

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