Abstract
Annual bluegrass (Poa annua) is a turfgrass species prone to winterkill-induced damage such as from ice encasement stress. This research aimed to determine whether different levels of soil volumetric water content (SWC) influence cold acclimation and recovery from ice encasement. Annual bluegrass was exposed to 8%, 12%, and 20% SWC treatments during cold acclimation in growth chambers. After cold acclimation, plants were subjected to ice encasement by misting at –3 °C until a 2.5-cm ice layer was formed. On 0 (no ice encasement exposure), 40, and 80 days of treatment, plants were analyzed for recovery (percent green canopy cover over time), and leaf, crown, and root tissues were harvested for lipid peroxidation and total nonstructural carbohydrates (TNC) including storage carbohydrates and water-soluble carbohydrates (WSC). Low SWC during cold acclimation enhanced recovery from cold temperatures and ice encasement. Root carbohydrates were influenced by SWC regimes during cold acclimation since day 0 plant roots exposed to the 8% SWC treatment had 143.9% higher TNC and 137.6% higher WSC compared with day 0 plants exposed to 12% and 20% SWC. Root levels of carbohydrates and lipid peroxidation were least influenced by cold and ice encasement among the organs evaluated. Prolonged freezing conditions and ice encasement reduced leaf and crown tissue carbohydrates and increased lipid peroxidation compared with day 0 plants not exposed to freezing temperatures and ice encasement. After 40 days of ice encasement, plants exposed to the 8% SWC treatment recovered faster than plants cold acclimated at higher soil moisture levels. Average percent canopy cover after 36 days of recovery in the greenhouse was 71.9% higher for 8% SWC treated plants than in 12% and 20% SWC treated plants. Turfgrass managers may benefit from annual bluegrass putting green management strategies to reduce fall soil moisture. Given that soil moisture did not significantly influence carbohydrate or lipid peroxidation results, except for in roots, additional research is needed to understand the mechanism associated with these findings.
Cold temperature–induced changes associated with cold acclimation are critical for the winter survival of perennial grass species. Adequate cold acclimation or hardening requires major physiological changes, including changes in cellular water status, altered membrane fatty acid ratios, altered rates of carbohydrate metabolism, and carbohydrate allocation shifts (Thomashow 1999). Fall weather can be variable and does not always provide optimal conditions for cold acclimation processes, leaving plants susceptible to winter damage. Climate change may increase the incidence of variable conditions during cold acclimation, calling for an understanding of how critical environmental factors influence cold acclimation and winterkill stress survival. Low soil moisture in alfalfa (Medicago media) seedlings increased cold hardening and freezing survival (Paquin and Mehuys 1980). How soil moisture content influences cold acclimation and survival of severe winter stresses of perennial grass species has not been investigated.
For highly managed turfgrass areas, such as annual bluegrass (Poa annua) putting greens, irrigation levels can be tailored to attempt to provide ideal soil moisture levels. Variable fall conditions and precipitation levels may cause soil moisture to be higher or lower than desired. An understanding of how soil water content influences winterkill-related stresses is needed to make recommendations to turfgrass managers related to fall irrigation practices or winter preparatory strategies. Annual bluegrass is more susceptible to ice encasement damage compared with other cool-season putting green species, such as creeping bentgrass [Agrostis stolonifera (Beard 1964)]. However, controlled research studies evaluating whether irrigation or soil moisture content during the fall season influences cold acclimation and overwintering of turfgrasses are lacking. Additionally, knowledge of the effects of soil moisture content on physiological changes in turfgrass species during acclimation could reveal novel strategies to promote acclimation or prime plants for enhanced acclimation and winter tolerance, such as through stress preconditioning.
Cold acclimation-induced changes in freezing tolerance of perennial grasses have been associated with the level of total nonstructural carbohydrates (TNC) and specific carbohydrates in various species (Fry et al. 1993; Hoffman et al. 2010; Zhang et al. 2011). In annual bluegrass, fructans were found at high levels after nonfreezing cold acclimation conditions, and sucrose content in crown tissues was associated with freezing tolerance (Dionne et al. 2001). Perennial ryegrass (Lolium perenne) crown tissue water-soluble carbohydrates (WSC) increased during cold acclimation to a greater extent in a freezing-tolerant cultivar compared with a susceptible one (Hoffman et al. 2010). Determining whether soil moisture conditions factor into differential impacts on carbohydrate production and reserves during cold acclimation is needed.
The carbohydrate content of plant tissues may also influence recovery from ice encasement stress, and ice encasement may influence available carbohydrates during stress recovery. McKersie et al. (1981) found that crown tissue of winter cereals such as winter wheat (Triticum aestivum) with higher tolerance to ice encasement had greater levels of total available carbohydrate and reducing sugars compared with cultivars less tolerant to ice encasement. The sensitive cultivar used in the study did not lose or use carbohydrates; thus, the loss of viability was not associated with the depletion of carbohydrate reserves. In a different study, cold acclimation increased TNC levels in crown tissues of winter wheat, which were then decreased after 7 d of ice encasement (Gao et al. 1983). Evaluating the impact that ice encasement has on carbon availability and whether carbohydrate content of plant tissues may be associated with enhanced recovery is important to understand the physiology associated with annual bluegrass susceptibility to ice encasement stress.
Lipid health and degree of saturation in plant membranes play major roles in plant overwintering. Fatty acid ratios shift to higher levels of unsaturated compared with saturated fatty acids during cold acclimation of plant tissues (Baird et al. 1998; Hoffman et al. 2010; Shang et al. 2006). Whether cold acclimation and ice encasement influence lipid health associated with lipid peroxidation has not been thoroughly evaluated for turfgrass species. Cold acclimation elicits an increase in lipid peroxidation, particularly in photosynthetic structures (Wise and Naylor 1987). Levels of hydrogen peroxide have been found to increase during cold acclimation, which can lead to oxidative stress (Prasad et al. 1994). Anoxic conditions, often found during prolonged ice encasement, can cause lipid peroxidation, particularly after plant reaeration, and less lipid peroxidation is associated with greater tolerance of anoxic conditions (Blokhina et al. 1999, 2001). In annual bluegrass, Laskowski and Merewitz (2021) found that leaf and crown tissues had similar levels of lipid peroxidation after cold acclimation and overwintering stresses. Lipid peroxidation levels increased in both tissues following 80 d of cold temperatures or ice encasement treatment. Annual bluegrass plants showing low levels of recovery had high levels of lipid peroxidation in crown and leaf tissues, indicating that lipid peroxidation damage of crown tissue may be a good indicator for overwintering recovery of annual bluegrass.
Therefore, the objective of the study was to expose annual bluegrass plants to low, optimal, and high soil moisture levels during cold acclimation to compare plant carbohydrate content and carbon allocation of plant tissues, lipid peroxidation levels, and recovery percentages after cold acclimation and cold and ice encasement stress. On the basis of limited background literature, we hypothesized that drier soil moisture conditions may increase plant recovery and could have lower associated levels of lipid peroxidation and higher levels of carbohydrates compared with plants exposed to optimal or higher levels of soil moisture.
Materials and Methods
Plant material and growth conditions.
Annual bluegrass sod plugs were collected from the Hancock Turfgrass Research Center (East Lansing, MI, USA) from a mature, perennial-type research putting green field mown at 0.3 cm. The plugs (10.2 cm diameter) were cut free of roots (1.3 cm deep) for optimal root regeneration during potting and were brought to a greenhouse on 6 Aug 2021 and 3 May 2022. Greenhouse conditions were maintained at an average day/night 23/16 °C, 14 h photoperiod, average 400 μmol·m−2·s−1 photosynthetically active radiation (PAR) supplemented with high pressure sodium lamps. Each sod piece was cut into four pieces and placed in a sandy loam soil (65.9% sand, 14.9% silt, 19.2% clay; Typic Hapludult) in a deep pot (6.0 cm diameter by 35.0 cm deep) to allow for adequate root growth. The plants were allowed to become established and spread to the entire diameter of the pot in the greenhouse for ∼60 d. There were 144 pots for sufficient plants to test three soil moisture regimes with eight replications for three sampling dates and two experiments (Expts. 1 and 2). The plants were fertilized weekly with half-strength Hoagland’s solution (Hoagland and Arnon 1950) during greenhouse establishment. Plants were trimmed to maintain a putting green height of 1.3 cm (to allow for sufficient leaf tissue for analysis) and watered as needed. Following pot establishment, all pots were moved to a growth chamber for cold acclimation treatment.
Experimental treatments during acclimation.
For cold acclimation, plants were transferred from the greenhouse to a low-temperature growth chamber (LTCB-19; Biochambers, Winnipeg, MB, Canada). The cold acclimation periods lasted for ∼40 d for Expt. 1 (3 Sep to 15 Oct 2021) and 33 d for Expt. 2 (16 Jul to 17 Aug 2022). Plants were fertilized once halfway through the 10 °C cold acclimation period with half-strength Hoagland’s solution. For both experiments, growth chamber (LTCB-19) conditions were initially maintained at an average day/night 18/16 °C, 12-h photoperiod, average 400 μmol·m−2·s−1 PAR, and the chamber settings were changed to 4 °C with a 10-h photoperiod and 200 μmol·m−2·s−1 PAR for the last 2 weeks of cold acclimation.
Water content treatments.
Soil volumetric water content (SWC) was recorded and regulated for each pot to maintain the surface soil moisture at ∼8%, 12%, and 20% during cold acclimation, measured daily using time domain reflectometry using a handheld soil moisture meter (TDR 150; Spectrum Technologies Inc., Aurora, IL, USA) with rods (3.8 cm) completely inserted into the soil. Daily SWC was used to determine the amount of water added to plants to maintain similar treatment levels, with the upper 3.8 cm of soil measured. Plants were watered with deionized water as follows: 8% SWC watered every other day with 10 mL, 12% SWC watered daily with 10 mL, and 20% SWC watered daily with 20 mL to maintain proposed treatment SWC levels. Because root length was determined during sampling to be ∼30.0 cm long for day 0 plants and a 3.8-cm SWC probe was used, our treatments may be interpreted as a soil surface drying treatment for the low moisture condition. Leaf relative water content (RWC) was only determined in 2022 by standard methods from Barrs and Weatherley (1962).
Ice encasement treatments.
After the cold acclimation and water treatment periods, plants were exposed to –3 °C temperatures, 10-h photoperiod, with a 200 µmol·m−2·s−1 light level and were then separated into ice and no ice (0 d) treatments within the growth chamber (LTCB-19). Ice treatment began on 3 Dec 2021 for Expt. 1 and 18 Aug 2022 for Expt. 2. Day 0 plants were not subjected to freezing temperatures since they were cold hardened to 4 °C. The ice encasement–treated plants received mist with deionized water every 30 min until a 2.5-cm ice layer was formed. The ice layer was maintained for the duration of the study by misting plants as needed. After 0, 40, or 80 d of treatment, pots were cut in half. Half of the plant was destructively sampled to separate leaf, crown, and root tissues and were immediately frozen in liquid nitrogen and stored at –80 °C until further analysis. The other half was put at 4 °C for 2 d and then placed in the greenhouse (24 °C) for recovery analysis. Greenhouse conditions were the same as those described earlier.
Recovery analysis.
Digital images were taken daily during the recovery period to determine percent regrowth. The percent regrowth was estimated by the percentage of canopy green cover using digital analysis software (Canopeo; Patrignani and Ochsner 2015). For plants sampled on 40 d of ice encasement for Expt. 1, images were taken from 4 Jan to 3 Mar 2022, for a total of 43 d of recovery. For plants exposed to 80 d of ice encasement in Expt. 1, readings were taken from 22 Feb to 4 Apr 2022, for a total of 41 d of recovery. For Expt. 2, images of day 0 plants, not exposed to ice encasement, were taken from 17 Aug to 25 Aug 2022, for a total of 9 d of recovery. For plants exposed to 40 d of ice encasement, images were taken from 27 Sep to 1 Nov 2022, for a total of 36 d of recovery. For plants exposed to 80 d of ice encasement, images were taken from 5 Nov to 15 Dec 2022, for a total of 40 d of recovery.
Total nonstructural carbohydrate analysis.
Previously frozen crown, leaf, and root tissues were analyzed for total nonstructural carbohydrates (Chatterton et al. 1989; Westhafer et al. 1982). Tissues were dried initially at 100 °C for at least 1 h to prevent metabolism in plant material and then maintained in the oven at 70 °C for 72 h. Approximately 50 mg of tissue was placed into 2-mL tubes and was ground using a tissue homogenizer (1600 MiniG; SPEX SamplePrep LLC, Metuchen, NJ, USA). For extraction, 1 mL of 92% ethanol was added to each tube and centrifuged (5430R; Eppendorf North America, Enfield, CT, USA) for 10 min at 14,000 gn at room temperature. The supernatant was transferred to a clean 15-mL tube, and the pellet was resuspended twice more in 1 mL of 92% ethanol resulting in a total of 3 mL extraction solution, which was diluted to 10 mL with deionized water. The remaining pellet of plant tissue was placed in the oven at 70 °C to dry overnight for storage carbohydrate analysis.
Reducing sugar content was determined as in Ting (1956) and Smith (1969). A 0.2-mL aliquot of ethanol extraction solution was added to 1.25 mL of alkaline ferricyanide solution and 0.8 mL of deionized water in a large test tube. This mixture was heated to 100 °C for 10 min and then immediately cooled in an ice bath. Then, 2.5 mL of 2 N sulfuric acid was added to the cooled solution and the test tube was shaken vigorously. Arsenomolybdate solution (1 mL) was added to this solution, and it was then diluted to 25 mL with deionized water.
For sucrose hydrolysis, 2 mL of the extraction solution was added to 2 mL of 4% sulfuric acid (w/v). This solution was mixed and boiled at 100 °C for 15 min. Solutions were then allowed to cool at room temperature and 1 mL of 1 N NaOH was added to neutralize the solution. The preparation for the quantification of sucrose hydrolysis was performed as described for reducing sugars.
To determine the total storage carbohydrates, the dried tissues from the ethanol extraction were resuspended in 0.5 mL deionized water and heated at 100 °C for 10 min then allowed to cool to room temperature. Acetate buffer (0.4 mL, 200 mM, pH 5.1) and 0.1 mL enzyme solution including amyloglucosidase and alpha-amylase (Fu and Dernoeden 2008) was added to the tube. Tubes were vortexed and incubated at 55 °C for 16 h (Thermomixer; Eppendorf North America, Inc.). The next day, samples were centrifuged at 14,000 gn for 20 min until a solid pellet was formed. The supernatant was poured into a 15-mL centrifuge tube and diluted to 10 mL with deionized water.
To determine starch content, 0.9 mL of the water extraction solution and 0.1 mL 1N sulfuric acid were added to a test tube. This tube was then mixed and boiled at 100 °C for 15 min and then was left to cool to room temperature. Sodium hydroxide (0.1 mL 1N NaOH) was added to neutralize the solution. For quantifying all sugar extractions, absorbances were measured at 515 nm using a spectrophotometer (Genesys 10S ultraviolet-VIS; Thermo Fisher Scientific Inc., Waltham, MA, USA). Sugar extractions were quantified based on a glucose standard curve from methods of Ting (1956).
Lipid peroxidation.
Lipid peroxidation was determined based on malondialdehyde (MDA) content using modified methods from Dhindsa et al. (1981) and Zhang and Kirkham (1994). Previously separated and frozen crown, leaf, and root turfgrass samples were weighed to ∼200 mg and ground using an automated tissue homogenizer (1600 MiniG). Samples remained frozen using liquid nitrogen throughout the entire process and MDA was extracted using a 50 mM phosphate buffer with a pH of 7.0 along with 1% polyvinylpolypyrrolidone; 0.8 mL of this solution was used with 0.4 mL 20% trichloroacetic acid (w/v) and 0.5% thiobarbituric acid (w/v), then heated at 95 °C for 30 min. The absorbance at 532 and 600 nm was measured and MDA content was calculated.
Experimental design and statistical methods.
The experimental design was a split-plot design within one growth chamber with soil moisture treatment as the whole plot and ice encasement or no ice encasement (0 d) split within each whole plot with eight replications per treatment. The sampling time was completely randomized within the whole plot. The experiment was repeated using the same growth chamber. A similar experimental design was used previously (Laskowski et al. 2019). Normality was assessed using visual analysis of residual plots and Levene’s test for homogeneity of error. The MDA and TNC results did not conform to the assumption of normality and were transformed using the square root of x; data presented are the untransformed means, whereas P values are from the transformed analysis. Data resulting from all measured parameters were subjected to analysis of variance (ANOVA) using statistical software (RStudio ver. 4.1.0; Posit, PBC, Boston, MA, USA) with a linear mixed model to determine the main and interacting effects of the experimental factors. Mean separations were performed using Fisher’s protected least significant difference test at the P ≤ 0.05 level. The duration of ice encasement and SWC treatment were held as fixed effects. Interactions associated with years were not significant; therefore, data were pooled together across years for all measured parameters.
Results
SWC and leaf RWC.
Soil water content was about achieved to the desired experimental levels of 8%, 12%, and 20% since the average soil moisture level for low, medium, and high levels were 8.27%, 12.37%, and 24.75%, respectively (Fig. 1). The RWC of leaf tissues taken from plants exposed to 12% and 20% SWC treatment remained at an average of 79.7% and 81.2%, respectively, throughout the duration of the study (Fig. 2). The initial RWC of leaf tissues from the plants exposed to the 8% SWC treatment averaged 81.1% for the first 2 weeks and then decreased to an average of 72.4% for the following 3 weeks. The RWC for the 8% SWC treated plants was significantly lower than those for the 12% and 20% SWC treatments on 14, 21, and 28 d of acclimation (Table 1, Fig. 2).
Soil water content of annual bluegrass plants watered to achieve a treatment level of 8%, 12%, or 20% soil water content during growth chamber simulated cold acclimation. Least significant difference values are indicated on dates when significance was detected, and least significant difference values are represented by vertical bars (P ≤ 0.05) for treatment comparisons on a given day of treatment. Means from 2021 and 2022 are pooled together.
Citation: Journal of the American Society for Horticultural Science 148, 3; 10.21273/JASHS05288-22
Relative water content from 2022 of leaf tissues during cold acclimation for annual bluegrass plants exposed to 8%, 12%, or 20% soil water content treatments. Least significant difference values are indicated on dates when significance was detected and least significant difference values are represented by vertical bars (P ≤ 0.05) for treatment comparisons on a given day of treatment.
Citation: Journal of the American Society for Horticultural Science 148, 3; 10.21273/JASHS05288-22
Analysis of variance for the main treatment factors of water treatment (WT), time (T), and their interactions for relative water content (RWC), soil volumetric water content (SWC), and recovery of annual bluegrass following different SWC treatments during cold acclimation and following ice encasement treatments in growth chambers and recovery in a greenhouse. Results from the years 2021 and 2022 were pooled together. The factor T represents duration of cold acclimation for RWC and SWC values, whereas T for recovery represents the duration of the recovery period. Recovery periods were not the same for 0, 40, and 80 d.
Lipid peroxidation.
Soil water content treatment level did not influence MDA content, but the duration of cold and ice encasement did (Table 2). In crown tissues, MDA content increased from levels observed for day 0 plants after 80 d of cold and ice encasement by 32.1%. Leaf tissue MDA content increased in response to cold and ice encasement exposure of 40 or 80 d compared with day 0 plants (Fig. 3). However, prolonged ice encasement duration did not result in any differences in leaf MDA content (40 d not different from 80 d). Root tissues exhibited fewer changes associated with MDA content due to cold and ice encasement treatment and had less MDA content than crown and leaf tissue on all dates measured (Table 3). For instance, day 0 plant roots had 14.01 nmol·g−1 dry weight (DW) compared with 38.44 and 46.63 nmol·g−1 DW of MDA for crown and leaf day 0 plants, respectively.
Analysis of variance for main treatment factors, water treatment (WT) and duration of cold temperature and ice encasement (ID), and their interactions for parameters measured including crown (Cr), leaf (L), and root (R) malondialdehyde (MDA) content, water soluble carbohydrates (WSC), storage carbohydrates (SC), and total nonstructural carbohydrates (TNC) of annual bluegrass following different SWC treatments during cold acclimation and following cold and ice encasement treatments in growth chambers in 2021 and 2022.
Lipid peroxidation as expressed by malondialdehyde content of annual bluegrass leaf, crown, and root tissues after 0, 40, or 80 d of ice encasement at (–3 °C) in growth chamber conditions following soil moisture treatments during cold acclimation. Means from 2021 and 2022 are pooled together. Capital letters indicate statistical differences within a given plant organ (leaf, crown, or root). All statistical letters are derived from least significant difference tests (P ≤ 0.05). The same letters indicate values that are not significantly different (P ≤ 0.05).
Citation: Journal of the American Society for Horticultural Science 148, 3; 10.21273/JASHS05288-22
Main effects for tissue type for malondialdehyde content (MDA), total nonstructural carbohydrates (TNC), water-soluble carbohydrates (WSC), and storage carbohydrates (SC) of annual bluegrass for leaves, crowns, and roots. Means from 2021 and 2022 are pooled together.
Total nonstructural carbohydrates.
The TNC contents (sum of SC and WSC) of the crown or leaf tissue were not influenced by SWC treatments (Table 2), but a significant interaction of SWC treatment and duration of ice encasement was found for the TNC of root tissues (Fig. 4). The day 0 plant roots exposed to 8% SWC had higher levels of TNC and WSC compared with the day 0 plants exposed to 12% and 20% SWC. For day 0 plants, SC was not significantly different among plants treated with different levels of SWC. On other sampling days, fewer differences associated with SWC were detected for TNC, SC, and WSC for 40- or 80-d plants.
Total nonstructural carbohydrates (TNC) as a sum of water-soluble carbohydrate (WSC) and storage carbohydrate (SC) fractions in root tissues of annual bluegrass exposed to soil water content levels of 8%, 12%, or 20% during cold acclimation. Means from 2021 and 2022 are pooled together. Different capital black letters indicate statistically different means based on least significant difference values for TNC across sampling days (P ≤ 0.05). Different white lowercase letters indicate statistical differences in SC and different gray lowercase letters indicate statistical differences for WSC.
Citation: Journal of the American Society for Horticultural Science 148, 3; 10.21273/JASHS05288-22
Cold and ice encasement for 40 and 80 d at –3 °C significantly decreased the amount of TNC and the respective fractions of SC and WSC in crown and leaf tissues compared with day 0 plants (Fig. 5). Plants exposed to 80 d of cold and ice encasement had similar levels of crown and root TNC, WSC, and SC compared with plants exposed to 40 d of ice encasement. Leaf tissue TNC and SC were lower for day 80 plants compared with day 40 plants. Root tissue TNC was less influenced by cold and ice encasement duration since root TNC and SC did not significantly change over sampling days, but WSC was lower after 40 or 80 d of ice encasement compared with day 0 plant roots.
Total nonstructural carbohydrates (TNC) as a sum of water-soluble carbohydrate (WSC) and storage carbohydrate (SC) fractions in leaf, crown, and root tissues of annual bluegrass as it relates to 0, 40, or 80 d of ice encasement. Means from 2021 and 2022 are pooled together. Different capital black letters indicate statistically different TNC means, white letters are for SC, and gray letters are for WSC (P ≤ 0.05) within a plant organ and statistical letters are derived from least significant difference tests (P ≤ 0.05).
Citation: Journal of the American Society for Horticultural Science 148, 3; 10.21273/JASHS05288-22
Percent recovery.
The recovery of plants was estimated by measuring the percentage of green cover over time. Recovery was significantly influenced by cold and ice encasement duration (Table 1, Fig. 6) since 40 and 80 d of ice encasement reduced the rate of recovery and maximum percent cover achieved compared with day 0 plants. For instance, after 40 d of ice encasement and a 36-d recovery period, plants had an average of 47.35% green cover, whereas after 80 d of ice encasement and a 40-d recovery period plants had an average of ∼31.15% green cover. All day 0 annual bluegrass plants fully recovered to an average of 93.82% green cover after a recovery period of 9 d in the greenhouse. Of the 0-d plants, those that were maintained at 8% SWC started at a lower percentage of green canopy cover and had significantly less green cover than the 12% and 20% SWC treated plants for 6 d out of the 9-d recovery period. Plants treated with 8% SWC had a faster rate of recovery after 40 d under ice encasement. After a 36-d greenhouse recovery period, 8% SWC-treated plants had an average green cover of 65.67%, whereas 12% and 20% soil moisture-treated plants averaged 40.56% and 35.81% green cover, respectively. No differences in recovery rate were detected among plants differing in SWC treatments following 80 d of ice encasement.
Recovery analysis as measured by green canopy cover (%) of annual bluegrass plants exposed to 8%, 12%, and 20% soil water content treatments after (A) 0 d, (B) 40 d, and (C) 80 d of ice encasement in low temperature (−3 °C) growth chamber conditions following a regrowth period in a greenhouse. Least significant difference values are indicated on dates when significance was detected, and least significant difference values are represented by vertical bars (P ≤ 0.05) for treatment comparisons on a given day of treatment. Means from 2021 and 2022 are pooled together. Note that scales are different on each x-axis.
Citation: Journal of the American Society for Horticultural Science 148, 3; 10.21273/JASHS05288-22
Discussion
Annual bluegrass plants sampled at 0 d experienced only cold acclimation conditions (4 °C) followed by de-acclimation and recovery in the greenhouse. During the cold acclimation or hardening process, high molecular weight sugars such as starches are typically hydrolyzed into smaller sugars to promote freezing tolerance (Tronsmo et al. 1993). Water-soluble carbohydrates such as sucrose and nonstructural storage carbohydrates such as fructans have been found to accumulate following cold acclimation, but neither correlated with the freezing tolerance of annual bluegrass ecotypes (Dionne et al. 2001). The latter contrasts with findings in various other grass species (Livingston 1996; Suzuki and Nass 1988). Hoffman et al. (2014) reported that higher levels of high molecular weight fructans may contribute to enhanced freezing tolerance of some annual bluegrass ecotypes for crown tissues. Our day 0 plant results expand the understanding of carbohydrate allocation in annual bluegrass following cold acclimation by including a comparison of leaf, root, and crown tissues. Storage carbohydrate quantities determined in our study can be ranked as leaf > crown > root for day 0 plants (Table 2). Low levels of photosynthesis at or near freezing conditions could still be occurring in leaf tissues (Levitt 1980), causing leaf carbohydrates to be higher than in crowns or roots. Additional cold acclimation regimes, pre-acclimation measurements, and cold acclimation durations would be needed to explain and further elucidate organ-level carbohydrate partitioning in annual bluegrass.
Ice encasement and cold duration reduced crown TNC by 205.7% and WSC by 115.1% and leaf TNC by 130.1% and WSC by 94.1% compared with day 0 plants. The experimental design does not allow for explicit linkage of carbohydrate loss specifically to ice encasement stress, but rather the combination of the duration of low temperature and ice encasement as a single treatment. Ice encasement stress has been associated with a reduction in carbohydrates (Gao et al. 1983). McKersie et al. (1981) found that crown tissue of winter wheat cultivars with higher tolerance to ice encasement had greater levels of total available carbohydrates and reduced sugars compared with cultivars less tolerant of ice encasement. The sensitive cultivar used in that study did not lose or use carbohydrates and thus the loss of viability was not associated with the depletion of carbohydrate reserves. It is not clear from our study if the loss of TNC over time was associated with stress or maintenance respiration during cold dormancy. Future research to identify individual sugars associated with organ-level changes in annual bluegrass along with tissue viability indicators may be warranted.
As meristematic regions essential to spring regeneration, crown and root tissue locations at or below the soil level facilitate the protection and insulation of crowns by foliage, thatch, and soil particles. Although obvious, these are particularly important factors of grass plant survival of winterkill stresses. The lower levels of lipid peroxidation (oxidative stress), which indicate greater membrane health, in root tissues compared with leaf and crown tissues of cold-acclimated-only (day 0) plants were likely associated with the protected location of the roots. The lack of damage to root tissue membranes observed here and the enhanced levels of carbohydrates observed during dry soil conditions compared with greater levels of soil moisture may warrant a need for more investigations of root acclimation, survival, and overwintering in annual bluegrass, particularly in field conditions. In leaf tissue of plants exposed to 40 and 80 d of ice encasement at –3 °C, MDA content was higher compared with in 0-d plants not exposed to ice encasement and prolonged time at –3 °C. Our results are consistent with the increase in MDA content found in crown and leaf tissues of annual bluegrass in response to low-temperature duration with no ice encasement and in ice-encased plants (Laskowski and Merewitz 2021). Cold acclimation and extended durations of plant tissues in cold temperatures can cause reactive oxygen species generation and lipid peroxidation, particularly given that cold temperatures influence membrane fluidity, stability, and dynamic regulation of fatty acid metabolism (Gill and Tuteja 2010). Crown tissue maintained low levels of MDA until 80 d in ice encasement, and root tissues maintained low levels throughout the study. Leaf tissue had significantly higher MDA than day 0 plants at 40 d of cold and ice encasement. Thus, crown and root tissues appear to be more resilient to lipid peroxidation than leaf tissues; therefore, they may be more protected by the soil compared with leaf tissue.
During greenhouse recovery, cold-acclimated-only plants (day 0) had a rapid resumption of leaf growth and canopy coverage, as expected. The 8%, 12%, and 20% SWC treatment levels during cold acclimation influenced annual bluegrass recovery following 40 d of cold temperature (–3 °C) and ice encasement. After 40 d under ice, the initial percent green canopy was high and then decreased to almost 0% after 2 d in the greenhouse. This is commonly seen in our ice encasement experiments and is likely due to the cold temperature preservation of chlorophyll and other leaf structures followed by posthypoxic reaeration and light exposure conditions causing tissue browning. Plants that cold-acclimated in the 8% SWC treatment recovered quicker and to a greater level of green coverage than the plants cold acclimated at higher soil moisture contents. This supports the hypothesis that maintaining low soil moisture before cold temperatures or ice encasement may result in quicker recovery after cold temperatures or ice encasement. Our analysis of TNC and MDA content in different organs shed some light on why this hypothesis was supported. Lipid peroxidation levels were not significantly influenced by soil moisture levels in any of the plant organs. Levels of TNC were not influenced by soil moisture during cold acclimation for crown or leaf tissues but were for root tissue. Root tissue levels of TNC, SC, and WSC were higher for day 0 plants in low soil moisture conditions compared with TNC levels of medium and high soil moisture-treated plants. Root carbohydrate content before overwintering may play an important role in annual bluegrass winter stress resilience.
Exposing a plant to a mild stress before a more severe or prolonged stress can precondition the plant and enhance survival. Drought preconditioning led to enhanced freezing tolerance in perennial ryegrass (Hoffman et al. 2012). In this study, leaf RWC was indicative of a mild drought stress imposition on the 8% SWC-treated annual bluegrass plants before ice encasement and low-temperature exposure. Stress response mechanisms often overlap for various abiotic stresses, and thus the pathways or mechanisms activated by mild drought stress could have increased overwintering potential. It is also possible that plants with drier soil had more available oxygen in open soil pore spaces. Oxygen limitation plays a major role in ice encasement damage. Further research into soil moisture conditions in field conditions and drought preconditioning mechanisms that may influence cold acclimation or winterkill survival of annual bluegrass is needed.
Regarding applied turfgrass management strategies, our findings suggest that preventative measures such as reducing water inputs during the fall cold acclimation period or improving drainage before fall acclimation period may reduce annual bluegrass stand loss due to ice encasement. Other management strategies such as cultivation techniques (e.g., core or solid tine aerification or removal of snow from putting greens during the fall) may assist with promoting drier soil conditions. These preventative measures should be used in conjunction with measuring and recording soil moisture levels, which could assist with forecasting potential damage associated with a given winter.
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