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Dep. of Crop and Soil Environmental Sciences, Virginia Tech, Blacksburg, VA 24061-0404
* Corresponding author (ervin{at}vt.edu).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol (110%) and ZR (38%) contents. This is the first known report indicating that these natural products contain cytokinins and that their application resulted in increased endogenous cytokinin levels, possibly leading to improved creeping bentgrass drought resistance.
Abbreviations: ELISA, enzyme-linked immunosorbent assay • HA, humic acid • iPA, isopentenyl adenosine • PE, photochemical efficiency • SWE, seaweed extract • ZR, zeatin riboside
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Drought is a major limiting factor of turfgrass culture in many parts of the world. Drought suppresses growth and causes a loss of turf quality (White, 1996; Carrow and Duncan, 2003). Accumulation of reactive oxygen species (ROS) such as superoxide radicals 
, hydroxyl radicals (OH·), and hydrogen peroxide (H2O2) under drought causes damage to cell membranes and an accumulation of lipid peroxides (Smirnoff, 1995). Plants have developed various antioxidant defense systems to cope with ROS toxicity. Cytokinins, a class of phytohormones, also function as antioxidants and have been shown to improve drought resistance (Musgrave, 1994).
-Tocopherol functions as a lipid soluble antioxidant and membrane stabilizer and is an effective quenching agent for both singlet O2 and for alkyl peroxides (Hess, 1993). Price and Hendry (1989) concluded that -tocopherol biosynthetic capacity increases readily in response to the demands of oxidative stress associated with drought and indicated that -tocopherol may respond as a first line of defense to oxidative assault. Studies with three cool season turfgrass species showed that -tocopherol content increased significantly in response to imposed drought and positive correlations between -tocopherol and turf quality were observed (Zhang and Schmidt, 1999, 2000).
Cytokinins exhibit antisenescence properties that are related to their antioxidant activity (Musgrave, 1994). The repression of lipoxygenase by cytokinins contributes to their overall antisenescence functions. Thimann (1987) noted that cytokinins delay the senescence process probably by maintaining the integrity of the tonoplast membrane. The most common naturally occurring cytokinins are zeatin and its sugar derivatives—zeatin riboside and zeatin ribotide—dihydrozeatin, and isopentenyl adenine (Srivastava, 2002). In the isoprenoid pathway, zeatin is thought to be the final and most biologically active compound, either arising from isopentenyl adenosine, isopentenyl adenine or zeatin riboside. Reported endogenous levels of free cytokinins in plant tissues are from 1 to 100 µg g–1 FW (Srivastava, 2002).
Natural products, which contain phytohormones or exhibit hormone-like activity, have received increasing attention for use as nutrient supplements in agriculture and horticulture (Adani et al., 1998; Zhang and Schmidt, 1999). Seaweed extracts (SWE) and humic acids (HA) are in common use as major components in turfgrass and ornamental biostimulant formulations. Auxin- and cytokinin-like activities of humic acids have been reported (Cacco and Dell'Agnola, 1984; Nardi et al., 1988, 1994; Piccolo et al., 1992; Clapp et al., 1998). Further, auxins in humic acids have been identified and quantified by GC-MS and ELISA (Muscolo et al., 1998; Nardi et al., 2000). Cytokinins and auxin have been identified and quantified in SWE (Tay et al., 1985; Sanderson and Jameson, 1986; Sanderson et al., 1987). These natural products (SWE and HA) have been shown to enhance turfgrass tolerance to abiotic stress (Nabati et al., 1994; Zhang and Schmidt, 1997; Ervin et al., 2003), delaying senescence and improving turf quality (Goatley and Schmidt, 1990; Liu et al., 1998; Zhang and Schmidt, 2000).
Although it has been documented that these natural products contain cytokinins and indole acetic acid (IAA), no research has reported an association between cytokinin levels in SWE or HA with a change in cytokinin levels in the treated plant tissue itself. Further, no research is available regarding the variability of cultivar response to foliar application of these natural products. Therefore, the objectives of this study were to: (i) determine if cultivar variation exists in response to drought and natural product foliar application; (ii) test previous reports of an improvement of creeping bentgrass drought resistance due to foliar application of these materials; (iii) examine if these natural products affect creeping bentgrass leaf tissue levels of -tocopherol and cytokinins; and (iv) determine if changes in -tocopherol and cytokinins are associated with improved drought resistance.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To investigate the mode of action of the natural products, an additional trial was conducted on the same open-metal bench and during the same period as Exp. 2. The SWE and HA materials were ashed in a Thermolyne furnace (Barnstead International, Dubuque, IA) at 500°C for 8 h. The nonashed and ashed SWE and HA were mixed in water and applied to Penncross using the same rates and procedures as described previously.
Drought Stress Treatment
One week after treatment, half of the containers were subjected to dry down and the remaining containers were irrigated to approximately maintain field capacity (–0.01 MPa; 200 mL per container) by hand three times a week. The containers within each factor were rearranged randomly three times a week. The turfgrass was mowed twice per week at 2 cm with electric clippers. Mowing ceased 2 wk before the end of each trial to provide sufficient leaf tissue for cytokinin and -tocopherol sampling.
Soil moisture content was measured with a ThetaProbe soil moisture sensor (Type mL1, Delta-T Devices Ltd, Cambridge, UK) once a week. Moisture retention at –0.01 MPa and –1.5 MPa of the soil was estimated before treatment initiation with a standard pressure plate method (Klute, 1983). Three samples per matric potential were used.
Photochemical Efficiency, Quality Rating, and Growth Measurement
Photochemical efficiency (PE; Fv/Fm) of the turf canopy was measured with a chlorophyll fluorometer once a week according to the methods of Zhang et al. (2003a). Weekly turfgrass visual quality was rated based on a scale of 1 to 9, with 1 indicating the poorest quality–complete wilting, 9 indicating the best quality, and 6 indicating minimum commercial acceptability.
Four weeks after initiation of dry down, when average soil moisture readings at the 0- to 8-cm depth had reached the approximate permanent wilting point of –1.5 MPa (or 5% volumetric soil moisture), both experimental runs were terminated and clipping weights were determined. The soil was then washed from the roots and the roots were dried in a forced-air oven at 60°C for 24 h before being weighed. The grasses were not mowed two weeks before sampling. For Exp. 2 only, leaves were sampled and frozen with a small amount of liquid N, and then stored at –80°C for subsequent -tocopherol and cytokinin analyses.
-Tocopherol Extraction and Assay
-Tocopherol analysis was based on the methods of Tanaka et al. (1990) and Zhang and Schmidt (1997). An HPLC system was used for quantification. The HPLC system consisted of a pump (SPD-6A), a variable wavelength ultraviolet detector (Shimadzu RF 535 fluorescence monitor), a 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). -Tocopherol was detected by measuring the fluorescence intensity at 325 nm while exciting at 292 nm. An -tocopherol standard from Sigma Chemical (St. Louis, MO) was used to develop a standard curve for the calculation of sample concentration. A recovery rate of 67% was obtained.
Cytokinin Extraction and Assay
Twelve 0.1-g samples from each of the two natural products (HA and SWE) were randomly collected and six samples of HA were mixed at the appropriate treatment ratio with six samples of SWE so that six samples each were obtained for HA, SWE, and HA + SWE. Of the six samples, three were ashed in the muffle furnace as described above. Subsequently, three ashed and three non-ashed samples from each treatment were used in an enzyme-linked immunosorbent assay (ELISA) to estimate cytokinin concentrations. Additionally, frozen leaf samples (0.5 g fresh wt) collected from each container at the end of Exp. 2, and the ashing experiment, were used for cytokinin ELISA as described below. Extraction and purification procedures for the cytokinins—zeatin riboside (ZR) and isopentenyl adenosine (iPA)—were conducted on representative samples of the natural products themselves following the methods of Turnbull et al. (1997), with minor modifications. A recovery rate greater than 90% was obtained on the basis of the internal standards.
Zeatin riboside and iPA were analyzed by ELISA as described by Trione et al. (1985) with clarifications provided by Banowetz (2002, personal communication), the senior author of Trione et al. (1985). Wells of a 96-unit plate were coated with 100 µL/well of ZR conjugated to bovine serum albumin (BSA) (1:2000 dilution), incubated overnight at 5°C, emptied, and washed three times with phosphate buffered saline (PBS)–Tween (PBS containing 0.05% Tween 20). The reaction was "blocked" with 200 µL of 1% BSA in PBS (37°C, 30 min) to prevent nonspecific protein adsorption. After the plate was washed twice with PBS–Tween, 50 µL of the cytokinin extracts or standards and 50 µL of the antibody tZR3 (1:200 dilution) were added to the wells and the plates were incubated at 37°C for 60 min, emptied and washed three times with PBS–Tween. The A and B rows in the plate were used to develop a standard curve. A series of ZR concentrations (0, 2.5, 5, 10, 25, 50 ng mL–1) were made from the stock solution. Appropriate dilutions were prepared for the natural product samples. Each standard or sample was repeated three times and the averages were used for data analysis.
To each well, 100 µL of a 1:1000 dilution of alkaline phosphatase-labeled goat anti-mouse IgG (Sigma Chemical Co., St. Louis, MO) was added and the plates were incubated at 37°C for 60 min. After three washes with PBS–Tween, 100 µL of substrate solution (3 mg mL–1 of p-nitrophenyl phosphate in 10% diethanolamine buffer, pH 9.8, 0.5 mM MgCl2) were added to each well and the plates were incubated at 37°C for 50 min. The color reactions in each well were determined by measuring absorbance at 405 nm with an enzyme immunoassay microplate reader (Opsys MR, Thermo Labsystems, Chantilly, VA). Zeatin riboside concentration was calculated on the basis of the standard curve after logarithmic conversion of the data. The same procedure was applied for iPA assay. An iPA3 antibody (1:200 dilution) and iPA-BSA conjugate (1:10 000 dilution) was used.
Experimental Design and Statistical Analysis
In greenhouse Exp. 1 and 2, a split-split plot design was used with four replications. Soil moisture levels (well-watered or dry-down) were considered as main plots, cultivars as the split plots, and natural product treatment as the split-split plots. For the ashed and non-ashed natural product experiment, a randomized block design was used with four replications. The data were subjected to ANOVA and separation of means was performed with a protected LSD test at a 5% probability level (SAS, 1996).
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RESULTS |
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Statistical Analyses and Data Presentation
The three factors—moisture, cultivar, and natural product—had a significant main effect on most data parameters in both experiments. Cultivar x natural product and three-way interactions were nonsignificant, while significant two-way interactions for most data parameters included moisture x cultivar and moisture x natural product. Thus, our focus for data presentation will include both of these two-way interactions.
Moisture x Cultivar Interactions
Turf quality among the cultivars at the end of Exp. 1 did not differ under either drought or well-watered conditions (Table 2). However, when averaged over cultivars, drought did significantly reduce quality. In Exp. 2, quality did not begin to decline until Day 14 of the dry-down cycle, with Penncross having worse quality than the other two cultivars (Fig. 1). However, by the end of the dry-down (Day 28), all cultivars had declined in quality equivalently.
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As expected, photochemical efficiency (PE) declined because of drought, with the PE of L-93 declining the most in Exp. 1 (Table 2), while no clear differences in PE were apparent during the Exp. 2 dry-down cycle (Fig. 3) . Loss of visual quality beginning at about Day 14 corresponded with declining PE (Fig. 1 and 3).
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-tocopherol increased significantly across cultivars when exposed to drought rather than well-watered conditions (Table 3). However, there were no -tocopherol differences between cultivars at either moisture level. Zeatin riboside leaf tissue content at Day 28 across cultivars was not affected by moisture level, but there were differences between cultivars (Table 3). Moisture-stressed Penn G-2 leaf tissue had greater ZR relative to L-93 and Penncross, while L-93 had greater ZR than the others under nonstressed conditions. Drought resulted in less iPA content across all cultivars (Table 3). Penn G-2 had greater leaf-tissue iPA content than the other two cultivars under both moisture treatments.
Moisture x Natural Product Interactions
Under well-watered conditions, the control had reduced quality at the end of Exp. 1 compared with the three natural product treatments (Table 4). Similar results were found under well-watered conditions in Exp. 2, where the control had reduced visual quality compared to the natural product treatments at Day 21 and 28 (data not shown). Drought resulted in the greatest quality loss for the control, with the HA + SWE combination providing the greatest quality at the end of Exp. 1. During the Exp. 2 dry-down cycle, quality began to decline between Day 7 and 14 (Fig. 2). At Days 14 and 21, all three natural product treatments had greater quality than the control. By 28 d of dry-down, the HA + SWE had again resulted in the least amount of quality decline (Fig. 2).
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-tocopherol content relative to well-watered conditions when averaged over natural product treatment (Table 5). There were no significant -tocopherol differences under drought or well-watered conditions between natural product treatments. However, when averaged over moisture levels, HA + SWE increased -tocopherol content by 110%. There was no moisture x natural product interaction for cytokinins (ZR or iPA) (Table 5). However, natural product treatment did have a main effect on ZR levels, with SWE and HA + SWE increasing ZR by 48 and 38%, respectively, when averaged over moisture levels and cultivars.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Further, the data indicate that application of the natural products in nonashed form increased leaf ZR content; however, the products, when ashed before foliar application, failed to affect leaf ZR content in Penncross creeping bentgrass subjected to drought (Table 1). This supports the hypothesis that the beneficial influences of HA and SWE on turfgrass abiotic stress tolerance are associated with their hormonal components, not the mineral fraction in these materials.
Leaf tissue ZR content was increased by foliar application of SWE and HA + SWE when averaged over cultivar and moisture treatments. Total leaf free cytokinin (ZR and iPA) content ranged from 22 to 41 ng g–1 DW. The cytokinin levels found in this study are in general agreement with the ELISA results of Reiber and Nueman (1999) in Phaseolus vulgaris L. (4–18 ng ZR g–1 FW), Schwartzenberg et al., (1988) in spruce (Picea spp.) needles (55–89 ng ZR g–1 DW), and Liu et al., (2002) in creeping bentgrass (5–60 ng cytokinins g–1 DW). Goatley and Schmidt (1990) indicated that the antisenescence properties of SWE may be associated with its cytokinin activity. Liu et al. (2002) found that root-injected ZR at 1 or 10 µM increased root and leaf cytokinin content of heat-stressed Penncross creeping bentgrass from 14 to 56 d after application. These increases were associated with protection of the photosynthetic apparatus and alleviation of heat damage to both shoots and roots. Similarly, exogenous application of SWE and HA + SWE, providing the equivalent of 0.26 µM ZR and 1.0 µM ZR, respectively, increased turf visual quality, root mass, PE, -tocopherol, and leaf ZR under well-watered and drought conditions (Tables 4 and 5). Visual quality and PE declines under drought were postponed by up to 7 d because of natural product treatments (Fig. 2 and 4). These results are in agreement with Zhang and Schmidt (2000) who found that HA and SWE alone or in combination resulted in greater leaf -tocopherol content and root mass in Penncross creeping bentgrass subjected to drought. Price and Hendry (1989) found that drought-tolerant species have up to three-fold higher levels of -tocopherol relative to drought-susceptible species. Fryer (1992) indicated that there is a significant correlation between the concentration of -tocopherol in chloroplasts and drought tolerance. Moran et al. (1994) found that drought caused a two- to three-fold increase of lipid and protein oxidation. Increased leaf antioxidant activity may improve drought tolerance by quenching reactive oxygen species and protecting membrane integrity (Smirnoff, 1995).
Drought reduced shoot and root mass, PE and visual quality of all three cultivars in this study (Tables 2 and 3; Fig. 1 and 3). Differences were found in root mass and cytokinin levels, but not in -tocopherol content between the cultivars. Greater root mass and ZR content was found for L-93 than the other cultivars under well-watered conditions, but not under drought. Penn G-2 contained a greater amount of ZR than Penncross under well-watered and drought conditions. However, increased ZR was not clearly associated with greater quality during drought for Penn G-2. While there are reports of L-93 having greater heat tolerance than Penncross (Xu and Huang, 2001; Huang et al., 2001), scant data are available regarding drought resistance rankings of these cultivars. In an unirrigated fairway–tee variety trial in Virginia, Penncross maintained greater quality and fall ground cover relative to Penn G-2 when averaged over the 5-yr trial period (NTEP, 1998). Clearly, further research is needed to identify cultivar differences in bentgrass drought resistance and determine morphological or physiological characteristics that may be associated with such differences.
In summary, these results indicate that small physiological differences exist between creeping bentgrass cultivars in response to drought and natural product foliar application, but these differences were not significant as concerns whole-plant drought resistance. This confirms previous reports of increased creeping bentgrass drought resistance because of foliar application of SWE and HA. As in previous research, increased drought resistance was related to higher antioxidant (-tocopherol) levels, maintenance of photochemical efficiency, and root mass. However, this is the first report indicating that these natural products contain significant amounts of cytokinins that appear to be associated with increased leaf cytokinin levels and greater drought resistance. Further research is needed to identify and quantify any IAA that may be present in these materials and determine any role this hormonal component may have in rooting and overall drought resistance effects. Finally, field research is required to quantify deficit irrigation levels that may be applied to SWE+HA-treated creeping bentgrass and conserve water while maintaining adequate turf quality.
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ACKNOWLEDGMENTS |
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Received for publication August 31, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tocopherol). Plant Cell Environ. 15:381–392.-tocopherol. p. 111–134. In R.G. Alscher and J.L. Hess (ed.) Antioxidants in higher plants. CRC Press, Inc., Boca Raton, FL.-tocopherol status in water-stressed Poa pratensis L. Int. Turfgrass Res. J. 8:1364–1373.Related articles in Crop Science:
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