Different Heat Tolerance of Two Creeping Bentgrass Cultivars Related to Altered Accumulation of Organic Metabolites

Abstract

High-temperature stress is one of the main limiting factors for the cultivation and management of cool-season creeping bentgrass (Agrostis stolonifera). The objectives of the current study were to compare physiological changes in heat-tolerant PROVIDENCE and heat-sensitive PENNEAGLE and further identify differential organic metabolites associated with thermotolerance in leaves. Two cultivars were cultivated under optimal conditions (23/19 °C) and high-temperature stress (38/33 °C) for 15 days. Heat stress significantly reduced leaf relative water content, chlorophyll content, and photochemical efficiency, and also resulted in severe oxidative damage to PROVIDENCE and PENNEAGLE. Heat-tolerant PROVIDENCE exhibited 10% less water deficit, 11% lower chlorophyll loss, and significantly lower oxidative damage as well as better cell membrane stability compared with PENNEAGLE under heat stress. Metabolomic analysis further found that PROVIDENCE accumulated more sugars (fructose, tagatose, lyxose, ribose, and 6-deoxy-D-glucose), amino acids (norleucine, allothreonine, and glycine), and other metabolites (lactic acid, ribitol, arabitol, and arbutin) than PENNEAGLE. These metabolites play positive roles in energy supply, osmotic adjustment, antioxidant, and membrane stability. Heat stress significantly decreased the accumulation of tricarboxylic acid cycle-related organic acids in two cultivars, resulting in a metabolic deficit for energy production. However, both PROVIDENCE and PENNEAGLE significantly up-regulated the accumulation of stigmasterol related to the stability of cell membrane systems under heat stress. The current findings provide a better understanding of differential thermotolerance in cool-season turfgrass species. In addition, the data can also be utilized in breeding programs to improve the heat tolerance of other grass species. However, the current study only focused on physiological and metabolic responses to heat stress between two genotypes. It would be better to utilize molecular techniques in future studies to better understand and validate differential heat tolerance in creeping bentgrass species.

1. Introduction

With the acceleration of global warming, the negative impact of high temperature on cool-season crops sweeps across the globe during hot summer months [1]. High temperature leads to various harmful changes in the stability of cell membranes and cytoskeleton structure, antioxidant defense systems, photosynthesis, and the accumulation and catabolism of primary and secondary metabolites, which negatively affects seed germination, plant growth, blossoming, and yield [2,3,4]. Plants enhance self-generated thermotolerance via a complex metabolic regulatory network to ensure internal metabolic homeostasis [5,6]. The accumulation of carbohydrates is propitious to maintain cellular osmotic equilibrium and also provides an essential energy resource for growth and development under heat stress [7]. Amino acids play multiple important roles in the biosynthesis of functional proteins, osmotic adjustment (OA) as compatible osmolytes, bioprotectants for the stability of intracellular biomacromolecules and membrane systems, and reactive oxygen species (ROS) scavenging when plants are subjected to high-temperature environments [8,9]. Organic acids are involved in glycolysis, the tricarboxylic acid (TCA) cycle, and fatty acid metabolism associated with energy and matter conversion in plants [10]. It has been found that heat stress results in a deficit of intermediate metabolites in the TCA cycles of many cool-season grass species, thereby reducing energy production and conversion for the maintenance of normal life activity [10,11,12].

Creeping bentgrass (Agrostis stolonifera) is widely used on golf-course putting greens as an excellent cool-season turfgrass and is best known for its vigorous procumbent growth habit, fine texture, and strong low-mowing tolerance [13]. Heat stress accelerates leaf senescence and also reduces the turf quality of creeping bentgrass, ultimately leading to increased maintenance and management costs [14,15]. Exogenous application of morphactin, chitosan, and γ-aminobutyric acid could effectively mitigate heat-induced growth inhibition and leaf senescence in creeping bentgrass by suppressing chlorophyll (Chl) degradation, enhancing photosynthetic and antioxidant capacities, and regulating nutrient accumulation [16,17,18]. In addition, different creeping bentgrass genotypes that originate in diverse geographical positions and ecoregions often exhibit differential heat tolerance, which provides important germplasms for breeding and utilization in different worldwide zones [19]. Our prior study systematically evaluated the heat tolerance of 41 creeping bentgrass genotypes. Among them, LOFTSL-93, PROVIDENCE, and 13M were identified as the top three heat-tolerant genotypes, whereas PENNEAGLE, W66569, and W66570 were ranked as heat-sensitive genotypes [20]. Further findings showed that the better tolerance of PROVIDENCE was related to improved Chl biosynthesis and reduced Chl degradation compared with other genotypes under high-temperature conditions [20].

However, it is still unclear whether or not the superior heat tolerance of PROVIDENCE is associated with alterations in the accumulation and conversion of global organic metabolites. The objectives of the current study were to compare the physiological differences of heat-tolerant PROVIDENCE and heat-sensitive PENNEAGLE in response to persistent high-temperature stress and further investigate the common and differential metabolites contributing to the difference in heat tolerance between the two genotypes based on the analysis of non-targeted metabolomes. The heat-tolerant genotype could probably accumulate more organic metabolites for OA, osmoprotection, and energy metabolism than the heat-sensitive genotype under heat stress. The findings will be conducive to revealing the metabolic mechanism of heat tolerance in different creeping bentgrass genotypes and will also provide a theoretical basis for the cultivation and utilization of creeping bentgrass in different geographical and ecological regions.

2. Materials and Methods

2.1. Plant Materials and Treatments

Sods of two creeping bentgrass cultivars (PROVIDENCE and PENNEAGLE) were separated into 2 cm × 2 cm patches, and each patch was then planted in a white polyvinyl chloride pipe (12 cm in diameter and a height of 32 cm) which was filled in mixtures of half sands and half loams in growth chambers (23 °C and 14 h lights at 700 mmol·m^–2·s^–1 PAR in the daytime, and 19 °C and 10 h darkness at night). All plants were maintained at 1.5 cm mowing height and irrigated by half-strength nutrient solution (472.5 mg/L calcium nitrate tetrahydrate, 253 mg/L potassium nitrate, 40 mg/L ammonium nitrate, 68 mg/L potassium dihydrogen phosphate, 246.5 mg/L magnesium sulfate, 1.25 mL/L iron salt solution, and 2.5 mL/L micronutrient solution) twice a week until the percentage of turf coverage reached 100% [21]. All plants were then divided into four treatments and each treatment had four biological replicates (four tubes): (1) PROVIDENCE was cultivated under optimal conditions, as mentioned above, for 15 days (PR-C); (2) PENNEAGLE was cultivated under optimal conditions for 15 days (PE-C); PROVIDENCE was placed in a high-temperature growth chamber (38 °C/33 °C, day/night) for 15 days (PR-H); (3) PENNEAGLE was placed in a high-temperature growth chamber (38 °C/33 °C, day/night) for 15 days (PE-H). For heat treatments (PR-H and PE-H), plants were irrigated using tap water every day to avoid soil drought. Leaf samples were taken from four independent biological replicates of each treatment to detect physiological parameters and non-targeted metabolomics.

2.2. Measurement of Physiological Parameters

A total of 0.1 g of leaf tissue was ground in 1.5 mL of 150 mM phosphate-buffered saline (PBS) (pH 7.0) on ice and then centrifuged at 12,000× g for 20 min. The supernatant was collected for measuring malondialdehyde (MDA) content. Supernatant (0.5 mL) was added to 1 mL of reaction solution (20% trichloroacetic acid and 0.5% thiobarbituric acid). The mixture was heated at 100 °C for 5 min and cooled to room temperature. The absorbance of the reaction mixture was measured at 532 nm and 600 nm using a UV-2102PC UV spectrophotometer [22]. Leaf electrolyte leakage (EL) and relative water content (RWC) were detected based on the procedures described by Blum and Ebercon [23] and Barrs and Weatherley [24], respectively. For the extraction of chlorophyll (Chl) from fresh leaves, 0.1 g of leaves was immediately immersed in 15 mL of dimethyl sulfoxide (DMSO) solution in the dark at room temperature for 3 days until all leaves turned completely white. The absorbance of extracts was measured at 663 nm and 645 nm using the spectrophotometer mentioned above to calculate the contents of total Chl, Chl a, and Chl b and the Chl a/b ratio [25]. Leaves were adapted to the dark for 15 min, and the photochemical efficiency, including the ratio of variable to maximal fluorescence (Fv/Fm) and the performance index on absorption basis (PIabs), were recorded using a Chl fluorometer (Pocket PEA) [26].

2.3. Extraction, Separation, and Measurement of Leaf Metabolites

Fresh leaves were freeze-dried in a freeze dryer (LGJ-10C, Chengdu, China) until the sample weight stabilized. Freeze-dried samples were then ground into a fine powder and extracted in 80% methanol [10]. Gas chromatography–mass spectrometry (GC-MS, Pegasus 4D, LECO Corporation) was used to separate metabolites following the method of Qiu et al. (2007), using helium gas as a carrier gas at a constant flow rate of 1.0 mL/min through a DB-5MS capillary column [27]. The initial GC temperature was maintained at 80°C for 5 min, and then increased to 180°C at a rate of 10°C/min, to 240°C at 5°C/min, to 280°C at 20°C/min, and finally held at 280°C for 11 min. Data were collected in full scan mode (m/z 30–550). After raw data were collected, the TURBOMASS 4.1.1 software (PerkinElmer Inc, Waltham, MA, USA) and commercial compound libraries (NIST 2005 and Wiley 7.0) were used to identify various metabolites including amino acids, organic acids, sugars, and other primary and secondary metabolites according to relative retention times, mass to charge ratios, chemical structure, and the peak profile of each metabolite.

2.4. Statistical Analysis

The data were analyzed using two-way ANOVA together with the Tukey Test at p ≤ 0.05 (SPSS 26.0, IBM, Armonk, NY, USA). All data were presented as means ± standard error (SE) (n = 4). Metabolic pathways were created using Excel 2019 (Microsoft, Redmond, WA, USA).

3. Results

3.1. Physiological Responses of Two Genotypes to High-Temperature Stress

There were no significant differences in leaf RWC, EL, and MDA content between the two genotypes under normal conditions (Figure 1A–C). Heat stress disrupts cellular water homeostasis and cell membrane stability, resulting in a significant decline in leaf RWC and increased EL and MDA content (Figure 1A–C). Under heat stress, the RWC of PROVIDENCE was 1.48 times higher than PENNEAGLE (Figure 1A). However, PROVIDENCE exhibited significantly lower EL level and MDA content compared with PENNEAGLE under heat stress (Figure 1B,C).

High-temperature stress significantly reduced the total Chl, Chl a, and Chl b contents but did not significantly affect the Chl a/b ratios in the two genotypes (Figure 2A–D). Under high-temperature stress, PROVIDENCE exhibited 53.32% and 50.49% higher total Chl content and Chl a content than PENNEAGLE, respectively (Figure 2A,B). No significant difference in Chl b content was detected between PE-H and PR-H (Figure 2C). High temperatures also led to significant declines in Fv/Fm and PIabs in the two genotypes, but PROVIDENCE maintained 22.11% and 47.73% higher Fv/Fm and PIabs than PENNEAGLE under heat stress (Figure 2E,F).

3.2. Change in Metabolomics in the Two Genotypes in Response to Heat Stress

A total of 38 differentially accumulated metabolites (DAMs) were identified, including 8 amino acids, 13 organic acids, 8 sugars, and 9 other metabolites (Figure 3A). In PR-C vs. PE-C, 26% and 46% DAMs significantly increased and decreased, respectively (Figure 3B). In PR-H vs. PE-H, 38% and 46% DAMs significantly increased and decreased, respectively (Figure 3B). In PR-H vs. PR-C and PE-H vs. PE-C, 36%and 44% DAMs significantly decreased, respectively. In PR-H vs. PR-C and PE-H vs. PE-C, 49% and 51% DAMs significantly increased, respectively (Figure 3B). As shown in Figure 3C, high-temperature stress resulted in significant increases in the contents of sugars, amino acids, and other metabolites in the leaves of two cultivars, whereas the contents of organic acids significantly decreased in PROVIDENCE and PENNEAGLE under heat stress (Figure 3C). In response to heat stress, PR-H exhibited significantly higher contents of sugars and amino acids but a significantly lower content of organic acids than PE-H (Figure 3C). No significant difference in the contents of other metabolites was detected between the two cultivars under normal conditions or heat stress (Figure 3C). The PCA plot shows the better metabolite reproducibility of each treatment (Figure S1).

3.3. Changes in the Contents of Carbohydrates and Amino Acids in the Two Genotypes in Response to Heat Stress

High-temperature stress resulted in significant increases in the contents of fructose, tagatose, galactinol, lyxose, and ribose in leaves of PENNEAGLE. However, compared with the normal conditions, the 6-deoxy-D-glucose content significantly decreased in PENNEAGLE under heat stress (Figure 4). High-temperature stress also led to significant increases in fructose, tagatose, lyxose, ribose, and 6-deoxy-D-glucose but decreased galactinol content in the leaves of PROVIDENCE (Figure 4). There were no significant changes in erythrose contents among PE-C, PR-C, PE-H, and PR-H. Compared with PENNEAGLE, PROVIDENCE exhibited 1.17, 1.28, 1.71, or 2.83 times higher fructose, tagatose, galactinol, or ribose under normal conditions, respectively. Under heat-stress conditions, PROVIDENCE showed 1.45, 1.35, 2.16, 2.34, or 1.70 times higher fructose, tagatose, lyxose, ribose, or 6-deoxy-D-glucose than PENNEAGLE, respectively (Figure 4).

The contents of serine and threonic acid significantly declined in two cultivars under heat stress (Figure 5). However, high-temperature stress resulted in significant increases in the contents of alanine, norleucine, isoleucine, L-allothreonine, and oxoproline in the leaves of PENNEAGLE. High-temperature stress also induced significant increases in the contents of alanine, norleucine, isoleucine, L-allothreonine, and glycine in the leaves of PROVIDENCE. The content of glycine significantly decreased in PENNEAGLE but significantly increased in PROVIDENCE under heat stress (Figure 5). Under high-temperature conditions, PROVIDENCE exhibited 2.09, 1.45, and 2.56 times higher contents of norleucine, L-allothreonine, and glycine than PENNEAGLE, respectively.

3.4. Changes in the Contents of Organic Acids and Other Metabolites in the Two Genotypes in Response to Heat Stress

High-temperature stress affected the accumulation of eight TCA cycle-related organic acids and four fatty acids in leaves (Figure 6A,B). Under normal conditions, PROVIDENCE exhibited significant increases in succinic acid, malic acid, 4-hydroxybutyrate, maleic acid, and glycolic acid compared with PENNEAGLE, respectively (Figure 6A,B). There were no significant differences in the contents of glutaric acid, itaconic acid, fumaric acid, and aconitic acid between PE-C and PR-C under normal conditions (Figure 6A). Under heat stress, PROVIDENCE had significantly lower contents of itaconic acid, fumaric acid, malic acid, aconitic acid, and glycolic acid than PENNEAGLE, but the contents of glutaric acid, succinic acid, 4-hydroxybutyrate, and pyruvic acid did not show significant differences between PE-H and PR-H (Figure 6A,B). Heat stress significantly increased the accumulation of malic acid, pelargonic acid, and lactic acid but reduced the accumulation of D-erythronolactone in two cultivars (Figure 6B). Compared to PENNEAGLE, PROVIDENCE showed 1.23 and 1.26 times higher lactic acid and maleic acid contents under heat stress, respectively (Figure 6B).

In response to high-temperature stress, PE-H had 8.48, 4.09, 1.25, 7.01, 3.42, and 2.39 times higher contents of phytol, stigmasterol, glycerol, threitol, ribitol, and arabitol than PE-C in leaves, respectively (Figure 7). Conversely, uridine and myo-inositol in PE-H significantly decreased by 88.62% and 9.12%, respectively, compared with PE-C (Figure 7). High-temperature stress also resulted in significant increases in phytol, stigmasterol, glycerol, threitol, ribitol, and arabitol in the leaves of PROVIDENCE but significantly decreased contents of uridine and myo-inositol (Figure 7). Under normal conditions, PROVIDENCE exhibited a significant increase in arbutin content and a decline in uridine content compared with PENNEAGLE, and no significant changes in other metabolites were detected between PE-C and PR-C. Under heat stress, PROVIDENCE showed 1.36, 2.57, and 2.14 times higher contents of arbutin, threitol, and arabitol than PENNEAGLE, respectively (Figure 7). The phytol content in PR-H significantly decreased by 35.98% compared with PE-H, whereas the contents of other metabolites (uridine, stigmasterol, myo-inositol, glycerol, and ribitol) showed no significant differences between PE-H and PR-H (Figure 7). Figure 8 shows the changes in organic metabolites in different metabolic pathways. Sugar metabolism interacted with amino acid metabolism, and then sugars from the glycolytic pathway were further metabolized into the TCA cycle (Figure 8).

4. Discussion

It has been widely reported that prolonged heat stress negatively affects water homeostasis and cell membrane stability in the leaves of creeping bentgrass, leading to reduced leaf RWC and increased membrane lipid peroxidation levels [10,11,17]. These findings are consistent with our results, which show that sustained high-temperature stress resulted in water loss and significant increases in MDA content and EL in the leaves of both PENNEAGLE and PROVIDENCE, indicating heat-induced physiological drought and cell membrane system damage. However, the heat-tolerant PROVIDENCE exhibited higher RWC as well as lower EL and MDA levels than the heat-sensitive PENNEAGLE after heat stress. In addition, significantly higher Chl content, Fv/Fm, and PIabs were also detected in PROVIDENCE compared with PENNEAGLE in response to heat stress. As a main photosynthetic pigment in chloroplasts, Chl is responsible for light energy absorption and conversion. Chl a is more sensitive to ROS and degrades faster than Chl b under stress conditions [28,29]. Fv/Fm reflects the primary photochemical efficiency of PSII, and PIabs provides a comprehensive indication of the maximum photochemical efficiency of PSII, serving as a sensitive indicator of plant health under abiotic stress [29,30]. Therefore, Chl content, Fv/Fm, and PIabs have been widely used to screen and identify heat-tolerant creeping bentgrass genotypes [19,20]. Roossi and Huang reported that when creeping bentgrass plants were subjected to heat stress, Chl synthesis was significantly inhibited, but Chl degradation was accelerated, resulting in increased leaf senescence [16]. The mitigation of Chl loss by the exogenous application of morphactin could effectively delay heat-induced leaf senescence [16]. In the current study, heat-induced declines in Chl content, Fv/Fm, and PIabs were smaller in PROVIDENCE compared with PENNEAGLE, demonstrating the better photosynthetic capacity of PSII and lower leaf senescence of PROVIDENCE against prolonged heat stress.

Heat stress disrupts normal metabolic processes, leading to metabolic disturbance due to damaged cell membrane systems, increased leaf wilting, and accelerated leaf senescence [2]. The alteration of the accumulation of primary metabolites, including many sugars, amino acids, and organic acids, is an important adaptive response to heat stress [10,31]. These primary metabolites play essential roles in energy metabolism, redox equilibrium, OA, osmoprotection, and key enzyme biosynthesis [32]. The analysis of metabolomics found that heat stress significantly increased the accumulation of sugars in PROVIDENCE and PENNEAGLE. Moreover, PROVIDENCE accumulated more fructose, tagatose, lyxose, ribose, and 6-deoxy-D-glucose than PENNEAGLE under heat stress. Carbohydrates are essential for maintaining osmotic and redox balances and also act as signaling molecules to regulate plant growth [33,34,35]. Fructose plays multiple roles as an energy source for growth and a regulator of osmotic potential for water balance, helping to alleviate heat stress [36]. Fructose and lyxose were found to accumulate significantly in heat-stressed chickpea (Cicer arietinum) plants, contributing to OA and energy supply under heat stress [37]. Additionally, tagatose was shown to significantly contribute to heat-stress recovery in tall fescue (Festuca arundinaceae) and improve the salt tolerance of creeping bentgrass [38,39]. Ribose serves as a precursor for energy metabolism and a defensive metabolite against heat stress [40]. Heat-tolerant PROVIDENCE accumulated more of these sugars, which can enhance OA and energy metabolism under heat stress. Previous findings from metabolomic analysis also demonstrated that enhanced sugar metabolism is an important adaptive response to heat stress in different plants [41,42,43].

In response to heat stress, the contents of amino acids significantly increased but the contents of organic acids significantly decreased in the leaves of both PROVIDENCE and PENNEAGLE. Amino acids are critical osmolytes and intracellular signaling molecules, thereby helping plants to cope with abiotic stress [44]. Organic acids are involved in numerous metabolic processes within plant cells, such as photosynthesis and respiration for energy metabolism [44]. The regulation of organic acid metabolism plays a crucial role in plant adaptation to various stress conditions. Metabolomic analysis found that greater accumulations of norleucine, allothreonine, and glycine were detected in PROVIDENCE compared with PENNEAGLE under high-temperature stress conditions. The recent study of Huang et al. found that the upregulation of amino acid metabolism contributed to the adaptation mechanism of heat tolerance in tea (Camellia sinensis) plants [41]. However, the contents of many intermediate metabolites in the TCA cycle, such as succinate, malate, and aconitate, significantly decreased in both PROVIDENCE and PENNEAGLE after heat stress, indicating that heat stress causes a metabolic deficit for energy production in creeping bentgrass plants. Similar findings have also demonstrated that the contents of citrate and succinate significantly decrease in leaves due to heat damage in creeping bentgrass and annual bluegrass (Poa annua) [11]. A previous study also demonstrated that heat stress resulted in defective energy metabolism by decreasing the intermediates of the TCA cycle, such as succinate, aconitate, and citrate, in creeping bentgrass [10]. In addition, more of these intermediate metabolites were consumed in heat-tolerant PROVIDENCE, which could provide energy for the biosynthesis of organic metabolites to support stress defense. On the contrary, heat stress significantly promoted the accumulation of malic acid, pelargonic acid, and lactic acid in PROVIDENCE and PENNEAGLE. Under lead stress, the content of pelargonate in Hydrangea strigosa increased significantly in favor of the maintenance of cellular compartmentalization and normal cell membrane function [45]. Lactic acid enhances stress resistance in tobacco (Nicotiana tabacum) by activating antioxidant enzyme activities and upregulating endogenous hormone levels and flavonoid biosynthesis-related genes [46]. Persistent high-temperature stress significantly reduces lactic acid content in annual bluegrass with lower heat tolerance, but not in creeping bentgrass with better heat tolerance [11]. However, the positive function of pelargonate and lactic acid on the heat tolerance of creeping bentgrass deserves further investigation in the future.

Heat stress also induced significant changes in other metabolites, such as stigmasterol, glycerol, arbutin, threitol, and arabitol, in PROVIDENCE and PENNEAGLE. Recently, the stigmasterol was identified as a “stress sterol” because various biotic and abiotic stresses induce its accumulation [47]. Stigmasterol is a vital component in plant cell membrane systems involved in the mediation of membrane fluidity and permeability, and its accumulation also enhances defense capability to resist ROS damage to plasma membranes and organellar membranes [48,49]. Overexpression of AtCYP710A1 encoding a C22-sterol desaturase for stigmasterol biosynthesis in Arabidopsis thaliana conferred tolerance to low and high temperatures by restraining EL and Chl loss [50]. Exogenous application of diethyl aminoethyl hexanoate improved the salt tolerance of white clover (Trifolium repens) related to significant increases in sterol contents, including stigmasterol and campesterol [51]. In addition, enhanced accumulations of stigmasterol, ribitol, arabitol, and arbutin in the roots of creeping bentgrass contributed to γ-aminobutyric acid-induced heat tolerance [31]. It has been found that secondary metabolite arbutin is a bioactive substance in plants that plays a positive role in mitigating ultraviolet radiation-B-induced oxidative damage [52]. Both PROVIDENCE and PENNEAGLE significantly accumulated stigmasterol in response to heat stress, which could be an adaptive alleviation of heat-stress damage. The better heat tolerance of PROVIDENCE could be associated with larger accumulations of sugar alcohols (ribitol and arabitol) and arbutin than PENNEAGLE, since these sugar alcohols and secondary metabolites exhibit potential functions of OA and as antioxidants in plants.

5. Conclusions

The better heat tolerance of PROVIDENCE was associated with better water homeostasis, cell membrane stability, and photochemical efficiency, as well as less Chl loss and oxidative damage compared with PENNEAGLE. In response to heat stress, PROVIDENCE accumulated more organic metabolites than PENNEAGLE. These metabolites play positive roles in energy supply, OA, ROS homeostasis, and cell membrane function. However, heat stress significantly decreased the accumulation of TCA cycle-related organic acids in the two cultivars, leading to a metabolic deficit for energy production. Current findings indicate the differential heat tolerance of the two creeping bentgrass cultivars is associated with alterations in the organic metabolite profiles, which helps to explain thermotolerance in cool-season turfgrass species. In addition, the data could also be utilized in breeding programs to improve the heat tolerance of other grass species. However, the current study only focused on physiological and metabolic responses to heat stress between two genotypes. It would be better to utilize molecular techniques in future studies to better understand and validate differential heat tolerance in creeping bentgrass species.