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Dep. of Plant Biology and Pathology, Rutgers Univ., New Brunswick, NJ 08901
* Corresponding author (huang{at}aesop.rutgers.edu).
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: ABA, abscisic acid • DW, dry weight • E, transpiration rate • EL, electrolyte leakage • ELISA, enzyme linked immunosorbent assay • FD, full soil profile drying • FW, fresh weight • gs, stomatal conductance • Pn, leaf net photosynthetic rate • PVC, polyvinyl chloride • RWC, relative water content • SD, surface soil drying • TDR, time-domain reflectometry • TQ, turf quality • TW, turgid weight • WS, water status
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plant responses to drought stress involve changes in various morphological, physiological, and metabolic characteristics (Nilsen and Orcutt, 1996; Shinozaki and Yamaguchi-Shinozaki, 1997). Early drought responses include growth inhibition and closure of stomata, which regulate water loss through transpiration. The conventional view is that WS is the major controlling factor regulating shoot physiological responses to drought stress. Hydraulic limitation, as manifested by the decrease in leaf WS, can directly limit growth and cause stomatal closure (Kramer, 1988). Stomatal conductance, however, is not always positively correlated with leaf WS, particularly under mild drought stress. In some species, changes in gs are observed before changes in leaf WS, particularly during early or mild phases of soil dry-down, and stomatal behavior in these species is more closely correlated with changes in the WS of the soil rather than the leaves (Zhang and Davies, 1989; Ali et al., 1999; Bahrun et al., 2002; Ismail et al., 2002). As a result, another hypothesis has been proposed on the likelihood of a nonhydraulic, chemical signal moving from roots to shoots in response to localized soil drying that induces stomatal closure and reduces water loss (Blackman and Davies, 1985; Zhang and Davies, 1989; Davies et al., 2002). This has been tested in experiments with split-root systems in which part of the root system is exposed to drying soil while the remaining roots are maintained in moist soil (Zhang and Davies, 1989; Gowing et al., 1990). The split-root technique has shown that restrictions in gs and growth occur even though roots in the well-watered soil provide sufficient moisture to maintain favorable leaf WS.
Abscisic acid is believed to be involved in hormonal control of plant response to drying soil (Wilkinson and Davies, 2002). Many studies in annual crops and woody species have shown negative correlation of gs and leaf growth with ABA accumulation (Gowing et al., 1990; Blum et al., 1991; Davies and Zhang, 1991; Tardieu et al., 1992; Bano et al., 1993; Auge et al., 1995; Jackson et al., 1995; Borel et al., 1997; Croker et al., 1998; Stoll et al., 2000; Auge and Moore, 2002), suggesting that chemical signaling is an important factor regulating physiological responses to drought, particularly mild to moderate stress. It is believed that ABA is synthesized in roots exposed to dry soil and transported to shoots, where it triggers a signal transduction cascade, eventually leading to a reduction in guard cell turgor and stomatal closure (McAinsh et al., 1997; Assmann and Shimazaki, 1999; Wilkinson and Davies, 2002). Abscisic acid is also involved in reduction of water loss by inhibiting leaf growth, which reduces transpirational area and, thus, water loss (Bacon et al., 1998; Alves and Setter, 2000).
The objectives of this study were to: (i) examine effects of SD on growth, water relations, gas exchange, and endogenous level of ABA in roots and shoots for Kentucky bluegrass, and (ii) determine whether physiological effects of localized soil drying are related to hydraulic or chemical control for the cool-season grass species. Two cultivars were examined to determine whether the effects are consistent among cultivars.
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MATERIALS AND METHODS |
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Plants were first grown in a greenhouse for 60 d at 21/15°C (day/night), 14-h photoperiod, and watered three times per week until water ran freely from bottoms of tubes. Turf was hand clipped weekly to approximately 6 cm in height. Controlled release granular fertilizer (16-4-8 N-P-K) was topdressed twice before the beginning of soil moisture treatments. Plants were then moved to a growth chamber (22/18°C day/night temperature, 14-h photoperiod, and photosynthetically active radiation of 600 µmol m–2 s–1) and allowed to acclimate for 15 d before irrigation treatments were imposed.
Treatments
The experiment consisted of three soil moisture treatments: (i) well-watered control, irrigated three times per week until water ran freely from the bottom of the tubes to maintain soil moisture at field capacity; (ii) SD, irrigation withheld from the upper 20 cm soil while lower layer (20–40 cm soil) was watered three times per week by subirrigating through the bottom of the PVC tube; and (iii) FD, irrigation withheld from the entire soil profile (0–40 cm). The treatments were terminated when the majority of the plants in the FD treatment were completely desiccated and brown (32 d).
Measurements
Turf quality was rated visually based on color, density, and uniformity of turf on a 1-to-9 scale [1 = brown, dry, dead; 9 = fully turgid, green, and dense]. A rating of 6 indicates minimum acceptable TQ. Shoot extension rate was measured every 2 to 3 d by measuring the difference in canopy height.
Volumetric soil water content was measured with the time-domain reflectometry (TDR) method (Topp et al., 1980) by three pronged buriable waveguide TDR probes (20 cm long) installed vertically in the 0- to 20- and 20- to 40-cm soil layers (Soil Moisture Equipment, Santa Barbara, CA). The cable from TDR probes in the 20- to 40-cm soil layer was taped up against the inner wall of the PVC tube and projected out above the surface of the soil in the upper PVC section. Soil water content in the both upper and lower layers of control and the lower layer of SD tubes was maintained close to field capacity (approximately 30 ± 2.9%, mean of eight replications ± standard error) throughout the experimental period. Moisture content in the 0- to 20-cm soil layer of SD and FD treatments decreased to approximately 5 to 7% (v/v) after 32 d of treatment, corresponding to the permanent wilting point of the soil.
Leaf RWC was determined weekly with 10 to 15 first and second fully expanded leaves per pot. Leaves were clipped and weighed (fresh weight, FW), then placed into small Petri dishes filled with water. They were soaked in water for approximately 18 h at 4°C and then weighed immediately after excess moisture was removed with paper towels (turgid weight, TW). The leaves were then dried in an oven at 75°C for 72 h to determine DW. Leaf RWC was calculated as (FW – DW)/(TW – DW) x 100.
Cell membrane stability was estimated by calculating percentage of electrolyte leakage (EL) from cells. Approximately 10 first and second fully expanded leaves per pot were carefully excised and cut into 2-cm pieces. They were rinsed three times with distilled water and placed into vials containing 20 mL distilled water. After shaking for 6 h, initial conductivity (Ci) of the bathing solution containing fresh leaves was measured with a conductivity meter (YSI, Inc., Yellow Springs, OH). Leaves were then killed in an autoclave, and placed on a shaker for 24 h before final conductivity (Cf) of the bathing solution was measured. The EL was calculated as Ci/Cf x 100.
Single-leaf photosynthetic rate, gs, and transpiration rate (E) were measured with a portable gas exchange system (LI-6400, LI-COR, Inc., Lincoln, NE). Three to four fully expanded leaves per pot were placed within a 6-cm2 leaf chamber which was suspended over the top of the pots with a camera tripod. While still attached, individual leaves were enclosed in the leaf chamber at a PAR of 800 µmol m–2 s–1 and temperature of 23°C and allowed to equilibrate for approximately 1 min before data were recorded.
On the same day as gas exchange measurements, approximately 0.3 g of shoots (FW) were harvested for leaf ABA analysis. Leaves were excised and wrapped in aluminum foil, and then immediately submerged into liquid nitrogen. Samples were stored at –80°C until further quantification. Abscisic acid extraction and analysis were performed with the indirect enzyme linked immunosorbent assay (ELISA) method as described by Alves and Setter (2000) and modified by Wang et al. (2003). Leaves were extracted in 80% [v/v] methanol with 1% glacial acetic acid [v/v] and 10 mg L–1 butylated hydroxytoluene (sample to extraction = 1:10). Supernatants were vacuum dried and resuspended in 20% methanol. For purification, aliquots were applied onto C18 chromatography columns (Supelco, Bellefonte, PA). These ABA-containing fractions were eluted with 50% methanol (with 1% glacial acetic acid, v/v), vacuum dried, and redissolved with Tris-buffered saline solution (pH 7.5). Abscisic acid was quantified with anti-ABA monoclonal antibodies (Agdia, Inc., Elkhart, IN) by indirect ELISA.
At the end of the experimental period, roots were harvested separately from each soil layer. Roots were placed in test tubes containing extraction solution for ABA immediately after brushing clean of soil. Approximately 0.3 g root FW was used for ABA analysis. The remaining root portions were washed free of soil and dried in an oven at 75°C for DW determination.
Experimental Design and Statistical Analysis
The experiment consisted of two factors (two cultivars and three soil moisture treatments) with eight replications for each treatment in a completely randomized block design. Treatment effects were determined by ANOVA according to the general linear model procedure of the Statistical Analysis System V.8.2 (SAS Institute, Cary, NC). Differences among means for treatments for each cultivar were determined by the LSD test at the 0.05 probability level.
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RESULTS |
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After 7 d of FD, significant increases in ABA content of shoots were detected, and these continued to amplify throughout the remainder of the drought stress (Fig. 6) . Under SD, significant increases in ABA shoot content were observed at 4, 8, 29, and 32 d for Award and 4, 8, and 15 d for Nuglade.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The highest proportion of root biomass typically occurred in the upper 20 cm of soil. Under SD, however, the proportion of roots in the 20- to 40-cm soil layer increased compared with corresponding layers under well-watered and FD conditions. Deep rooting is considered to be an important mechanism for drought avoidance in many species, including turfgrasses, which allows water uptake from the deeper soil profile when surface soil is dry and avoids or delays tissue dehydration (Qian et al., 1997; Huang, 1999; Volaire and Lelievre, 2001). Keeley and Koski (2002) and Bonos and Murphy (1999) reported drought-tolerant Kentucky bluegrass cultivars had greater percentages of root systems in deeper soil layers compared with susceptible cultivars. Enhanced rooting may be due to increased carbon reallocation from shoots to roots at deeper soil depths. Huang and Fu (2000) investigated carbon metabolic responses to SD for Kentucky bluegrass and tall fescue and found that both species exhibited decreased respiration rates in shoots and roots in the upper drying layer, but enhanced carbon allocation to roots in the lower, wet soil layer.
Stomatal conductance was inhibited by 35 to 45% when part of the root system was exposed to dry soil, even though TQ, EL, RWC, and Pn were not affected by SD. These reductions are similar to those found in other split-root experiments: 25 to 35% in sorghum (sorghum bicolor L.) (Auge et al., 1995); 50% in rice (Oryza sativa L.) (Bano et al., 1993); and 50% in maize (Zea mays L.) (Zhang and Davies, 1989). Similarly, Es were reduced by 30 to 40% for both cultivars without reduction in Pn under SD conditions, suggesting that surface-dried plants were able to maintain carbon assimilation with less water. It was evident that the extent of stomatal closure was not sufficient to have an effect on carbon fixation rates in Kentucky bluegrass exposed to SD soil. Other studies also demonstrated that gs could be substantially reduced while photosynthesis rates remained unaffected under drought stress (Nguyen et al., 1997). The reduction in gs in both cultivars occurred in leaves that did not experience water deficit under SD conditions, indicating that stomatal closure was not caused by the lack of water or hydraulic limitation, but could be chemically controlled.
Shoot extension was also significantly inhibited by SD relative to that of controls. These differences manifested themselves after approximately 2 wk of withholding irrigation from the upper soil layer. Shoot extension rate of surface-dried plants was approximately 40 to 50% lower than well-watered plants, but based on visual observation, leaves of those plants still maintained ample turgidity. Similarly, split-root experiments with other species also found that reductions in shoot growth occurred without any obvious decrease in leaf turgor (Michelena and Boyer, 1982; Passioura, 1988; Bacon et al., 1998; Ismail et al., 2002). While it has generally been assumed that leaf turgor plays a predominant role in controlling leaf expansion during drought conditions, our study and previous split-root studies indicate other factors than water may be involved in the growth inhibition. Taken together, our results suggested that a regulatory mechanism other than hydraulic limitation controlling shoot physiological responses in Kentucky bluegrass adaptation to SD. Since leaf WS could not account for the conductance and growth inhibition responses, evidence pointed toward a potential chemical constraint as a function of soil moisture availability, as discussed in the introduction.
Studies on chemical signaling have found that ABA is the primary hormonal regulator associated with changes in leaf physiology under localized soil drying. The role of ABA in regulating stomatal behavior and shoot growth is well recognized, even though the actual receptors that trigger the signal transduction events have not yet been identified (Assmann and Shimazaki, 1999; Wilkinson and Davies, 2002) and the mechanistic basis for how ABA might directly inhibit leaf expansion is not known. Bacon et al. (1998) hypothesized that an increase of ABA in the vicinity of elongation zones may decrease leaf extension rates by reducing cell wall extensibility and/or turgor of these cells. More recent work suggests there may be other interacting factors that may depend on the degree of soil drying, and so the exclusive role of ABA here is less certain (Feng, 1996; Sharp, 2001; Roberts et al., 2002; Sharp and LeNoble, 2002).
In the present study, ABA content of roots in dry soil increased substantially under SD even though roots in the lower soil profile did not change relative to that in well-watered plants. Increased ABA accumulation was detected in shoots of plants under SD, although the magnitude of change was not consistent across all days of treatment and was to a lesser extent than that in roots. This has also been shown in other species (Stoll et al., 2000). In leaves, the biosynthesis of ABA generally increases only when leaf water relations are significantly disturbed, such as when leaf turgor approaches zero (Hartung et al., 2002). This could account for the large increases in ABA content in leaves of fully dried plants. However, under conditions of localized soil drying, import of ABA from roots in dry soil would likely be responsible for increased accumulation of ABA in leaves because there was no leaf water deficit. The increases in ABA content in shoots, even though small in magnitude in some cases, may be sufficient to have effects on shoot physiological functions. Significant changes in gs can occur with only slight changes in leaf ABA content (Loveys, 1984; Stoll et al., 2000). On the basis of evidence from this and other studies, it is reasonable to assume that the increases in ABA content found in root and shoots of Kentucky bluegrass cultivars could be associated with the decline in gs and shoot growth during SD.
In summary, Kentucky bluegrass plants were able to adapt to localized soil drying by maintaining TQ, photosynthesis, leaf WS, and root growth with water in the deeper soil profile. The inhibition of gs and shoot growth, independent of WS, could be because of hormonal control, which ultimately helped maintain WS by reducing water loss under localized drought conditions. Enhanced accumulation of ABA in roots and shoots seem to play a role in this response, although a direct association for ABA as the primary chemical signal responsible for changes in shoot physiology was not determined in this study, requiring further investigation. The capability of exploiting such chemical signaling in turfgrasses would benefit the development of water-saving grasses and efficient irrigation programs. This would be an important strategy for conserving water and modifying growth characteristics without sacrificing TQ and persistence.
Received for publication August 3, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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