Carotene-enhanced Heat Tolerance in Creeping Bentgrass in Association with Regulation of Enzymatic Antioxidant Metabolism

in Journal of the American Society for Horticultural Science
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  • 1 Department of Plant Biology, Rutgers, The State University of New Jersey, 59 Dudley Road, New Brunswick, NJ 08901

Heat-induced leaf senescence has been associated with stress-induced oxidative damage. The major objective of this study was to determine whether exogenous application of β-carotene may improve heat tolerance in creeping bentgrass (Agrostis stolonifera cv. Penncross) by suppressing leaf senescence and activating antioxidant metabolism. Plants were subjected to heat stress at 35/30 °C (day/night) or at the optimal temperature of 22/18 °C (day/night), and were treated with either β-carotene (1 mm) or water (untreated control) by foliar spraying every 7 days for 28 days in controlled-environment growth chambers. β-Carotene application suppressed heat-induced leaf senescence, as demonstrated by an increase in turf quality (TQ) and leaf chlorophyll content as well as a reduction in electrolyte leakage (EL). β-Carotene-treated plants had a significantly lower malondialdehyde (MDA) content and significantly greater activity of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) from 14 through 28 days of heat stress, and ascorbate peroxidase (APX) activity from 21 through 28 days of heat stress. These findings suggest that β-carotene may promote heat tolerance by enhancing antioxidant activity to suppress leaf senescence.

Abstract

Heat-induced leaf senescence has been associated with stress-induced oxidative damage. The major objective of this study was to determine whether exogenous application of β-carotene may improve heat tolerance in creeping bentgrass (Agrostis stolonifera cv. Penncross) by suppressing leaf senescence and activating antioxidant metabolism. Plants were subjected to heat stress at 35/30 °C (day/night) or at the optimal temperature of 22/18 °C (day/night), and were treated with either β-carotene (1 mm) or water (untreated control) by foliar spraying every 7 days for 28 days in controlled-environment growth chambers. β-Carotene application suppressed heat-induced leaf senescence, as demonstrated by an increase in turf quality (TQ) and leaf chlorophyll content as well as a reduction in electrolyte leakage (EL). β-Carotene-treated plants had a significantly lower malondialdehyde (MDA) content and significantly greater activity of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) from 14 through 28 days of heat stress, and ascorbate peroxidase (APX) activity from 21 through 28 days of heat stress. These findings suggest that β-carotene may promote heat tolerance by enhancing antioxidant activity to suppress leaf senescence.

One of the typical symptoms of heat stress in cool-season plant species is leaf senescence, which has been related to oxidative damage resulting from the overproduction of reactive oxygen species (ROS) that attack membrane lipids, DNA, and proteins (Roy and Basu, 2009). During ROS-induced membrane lipid peroxidation, MDA accumulates as a byproduct and can be quantified as a measure of membrane stability (Davey et al., 2005; Halliwell and Gutteridge, 1989). Plants produce antioxidant enzymes, such as CAT, POD, SOD, and APX, in response to oxidative stress to reduce the excitation energy of ROS, but antioxidant metabolism is hindered by heat stress (Asada, 1999; Liu and Huang, 2000). To understand more fully how to reduce leaf senescence in cool-season plants exposed to heat stress, it is critical to determine the pathways and natural products that may ameliorate ROS-mediated damage in heat-stressed plants.

The carotenoid family encompasses various pigments used in the light-harvesting reactions of photosynthesis, and are known to serve as antioxidants that quench triplet chlorophyll (3Chl*), an excited state of chlorophyll, as well as its toxic product, singlet oxygen (1O2), which is formed when 3Chl* reacts with oxygen, while also stabilizing the phospholipid bilayer in the thylakoid membrane of chloroplasts (Boucher et al., 1977; Croce et al., 1999; Demmig-Adams and Adams, 1996; Foote et al., 1970; Havaux, 1998). Carotenoids can be subcategorized as either carotenes or xanthophylls, depending on their molecular structure and where they are located in the chloroplast (Zaripheh and Erdman, 2002). More specifically, carotenes are localized in the core complexes of photosystems I and II, whereas xanthophylls are predominantly bound to the light-harvesting complexes of chloroplasts, where they scavenge ROS to protect plants against oxidative stress (Croce et al., 1999; Qin et al., 2015; Su et al., 2017; Umena et al., 2011). The remainder of carotenoids is distributed through lipid bilayers, offering structural protection to the membrane (Dall’Osto et al., 2010; Havaux, 1998). Carotenoids have been shown to accumulate and confer stress tolerance in plants exposed to various stresses, including drought, salt, and heat stress (Havaux et al., 1996; Kim et al., 2012; Li et al., 2020; Ramel et al., 2012; Shawon et al., 2018; Zhang et al., 2021). In heat-treated potato (Solanum tuberosum) leaves, a greater endogenous content of zeaxanthin (a xanthophyll) led to decreased membrane permeability and protected the plants against heat damage (Havaux et al., 1996). The association of antioxidant effects of carotenoids with improved heat tolerance in cool-season grass species is not yet well established.

In our study, the objectives were to examine how exogenous application of β-carotene may improve heat tolerance in creeping bentgrass (Agrostis stolonifera), a widely used turfgrass species, by reducing chlorophyll loss and supporting membrane stability, and to examine whether these effects may be attributed to alterations in antioxidant metabolism. Understanding how β-carotene may affect heat tolerance in cool-season grass species is of great significance for improving heat tolerance in cool-season turfgrasses by using β-carotene as a biomarker for germplasm selection and incorporating it in management practices as a biostimulant component for improving abiotic stress tolerance.

Materials and Methods

Plant materials and growing conditions.

Creeping bentgrass (cv. Penncross) sod plugs (diameter, 10 cm) were harvested from mature field plots at the Rutgers Horticultural Research Farm II in North Brunswick, NJ, and planted directly in 16 pots (diameter, 15 cm; height, 15 cm) containing fine sand, with each pot having one plug. Plants grew for 28 d in a greenhouse at average temperatures of 24/16 °C (day/night) under natural and sodium vapor lighting at a light intensity of 700 µmol⋅m–2⋅s–1 photosynthetically active radiation (PAR). Plants were irrigated every 2 d, trimmed to a height of 3.5 cm twice per week, and fertilized with three-quarter-strength Hoagland’s nutrient solution (Hoagland and Arnon, 1950) weekly. After the 28-d acclimation period, plants were transferred immediately into environmental growth chambers (Environmental Growth Chambers, Chagrin Falls, OH) controlled at 22/17 °C (day/night), 60% relative humidity, and 700 µmol⋅m–2⋅s–1 PAR for a 14-d acclimation period.

Treatments and experimental design.

Individual plants were foliar-treated at 7-d intervals with 40 mL β-carotene (400 µm) or water only (untreated control), and were subjected to optimal growth temperature (22/17 °C day/night) or heat stress (35/30 °C day/night) for an experimental period of 28 d. The β-carotene used was a crystalline powder sourced from Sigma-Aldrich (217538; Sigma-Aldrich, St. Louis, MO) and was solubilized in dimethyl sulfoxide (DMSO) at 24 °C to create a 1-m stock concentration that could be diluted subsequently to 400 µm with water. The concentration of β-carotene used in our study was determined by a prior screening to be optimal for promoting turfgrass health and performance under heat stress. Our study was designed as a split-plot experiment, with temperature (nonstress or heat stress) as the main plot and chemical treatment (β-carotene or water) as the subplot. For the untreated control or β-carotene treatment, there were four total growth chambers used, two set to optimal control temperature (22/17 °C day/night) and the other two to heat stress (35/30 °C day/night) conditions. There were four replicate plants for each temperature treatment, and plants were randomized among growth chambers every 3 d to avoid potential confounding effects of variable environmental conditions in different growth chambers.

Measurement of physiological parameters.

To evaluate leaf senescence and heat tolerance as affected by β-carotene application, TQ, leaf chlorophyll content, and EL were examined. TQ was evaluated weekly on a scale of 1 through 9, based on turf greenness, uniformity, texture, and canopy density, where 1 was assigned to completely dead turf and 9 was assigned to completely healthy, uniform, dense, and green turf (Beard, 1972). The lowest value at which turf was rated to be of minimally adequate quality was a 6.

Leaf chlorophyll content was measured at 7-d intervals according to the procedure of Hiscox and Israelstam (1979), with some alterations. About 0.1 g of leaves was harvested from each plant and placed into centrifuge tubes containing 10 mL DMSO. Samples remained in total darkness for 72 h to allow chlorophyll to be extracted, and absorbance of the chlorophyll solution was read at 663- and 645-nm wavelengths on a spectrophotometer. After leaves were desiccated at 80 °C for 72 h in a convection oven, dry weights were measured. To calculate chlorophyll content, absorbance values and weights were substituted into equations given by Arnon (1949).

Leaf EL was measured every 7 d by sectioning ≈0.2 g of leaves into 1-cm pieces, submerging the samples in 35 mL deionized water, and shaking the samples on a horizontal shaker. After 8 h of shaking, initial conductance (Ci) was read using a conductance meter (model 32; YSI, Yellow Springs, OH), and samples were heat-killed in an autoclave set to a cycle of 30 min at 121 °C. Killed samples were shaken for an additional 8 h, and maximum conductance (Cmax) was measured. Using the equation given in Blum and Ebercon (1981), EL was calculated by dividing Ci by Cmax and multiplying by 100 to express EL as a percentage.

Quantification of antioxidant enzyme activity.

Leaf tissue (250 mg) was excised from plants every 7 d and frozen immediately in liquid nitrogen, ground manually into a powder, and stored at –80 °C in a low-temperature freezer. To prepare the ground tissue for analysis in a series of antioxidant enzyme reaction and lipid peroxidation assays, the tissue was extracted with a 1-mL mixture of 0.2 mm ethylenedinitriletetraacetic acid (EDTA), 50 mm phosphate buffer, and 1% polyvinylpolypyrrolidone and ground over an ice bath for 5 min. An additional 2 mL of extract solution was added to the sample solution before centrifuging at 15,000 gn for 20 min at 4 °C. The supernatant was stored for future use in the antioxidant enzyme activity reactions and MDA content quantification assay.

SOD activity was analyzed by measuring the inhibition in reduction of Nitro blue tetrazolium chloride (NBT) by SOD according to the procedure outlined in Giannopolitis and Ries (1977), with the modifications outlined in Du et al. (2009). An enzyme extract (100 µL) from each plant was added to a mixture of 0.195 m methionine, 50 mm phosphate buffer (pH 7.8), 60 µM riboflavin, 3 µm EDTA, and 1.125 mm NBT and exposed to fluorescent lights to begin the reduction of NBT. After 20 min of light exposure, the reaction was stopped by placing the plants in total darkness for 10 min, and SOD activity was quantified by reading the colorimetric reaction at a wavelength of 560 nm on a spectrophotometer. A single unit of SOD activity was defined as the amount of SOD required to inhibit the photochemical reduction of NBT by 50%.

The methods of Chance and Maehly (1955), modified by Du et al. (2009), were used to analyze CAT and POD activity. An enzyme extract (100 µL) from each sample was added to a mixture consisting of 50 mm phosphate buffer (pH 7) and 45 mm hydrogen peroxide (H2O2), and the CAT-mediated decay of H2O2 was quantified by measuring absorbance at a wavelength of 240 nm every 10 s using a spectrophotometer. One unit of CAT enzyme activity was defined as a decline in absorbance by 0.01 U/min. To measure the activity of POD, an enzyme extract (50 µL) was combined with a solution comprised of 100 mm acetic acid and sodium acetate (CH3COOH-CH3COONa) buffer (pH 5), 50% guaiacol, and 0.75% H2O2, and the oxidation of guaiacol by H2O2 in the presence of POD was measured by reading the absorbance of samples at 460 nm at 10-s intervals for 1 min on a spectrophotometer.

APX activity was assessed using the procedure provided by Nakano and Asada (1981), with modifications. To prevent decline in activity resulting from prolonged storage, fresh leaf tissue was extracted immediately as outlined earlier, and this extract was used to quantify APX activity. The extract (100 µL) was combined with a mixture of 100 mm CH3COOH-CH3COONa buffer (pH 5.8), 0.17 mm ascorbic acid, 0.05 µm EDTA, and 0.08 mm H2O2, and the APX-mediated oxidation of ascorbic acid was measured by reading the absorbance of samples at 290 nm on a spectrophotometer at 10-s intervals for 1 min (Du et al., 2013). One unit of APX activity was expressed as a 0.01 U/min change in absorbance.

Protein was extracted from each sample by combining the enzyme extract solution with 20% trichloroacetic acid (TCA) and incubating the solution at 4 °C for 1 h until the protein precipitated on the bottom of each test vial. Samples were centrifuged for 15 min at 11,500 gn, and after decanting the supernatant, the protein pellet was air-dried and resuspended into 1 m sodium hydroxide. The Bradford protein assay was used to determine protein content, by generating a standard curve by combining a standard, bovine serum albumin, with varying volumes of Coomassie Brilliant Blue G-250 Dye (Bio-Rad Laboratories, Hercules, CA) set up in a serial dilution. The absorbance of protein extract from each sample for each dilution factor was measured at 595 nm using a spectrophotometer, and these values were substituted into the standard curve to quantify protein content, which was used to calculate antioxidant enzyme activities.

MDA content was analyzed as a measure of lipid peroxidation, through the thiobarbituric acid (TBA) reaction assay of Heath and Packer (1968) and the methods of Dhindsa et al. (1981), with some alterations. An enzyme extract (1 mL) was added to a 2-mL volume consisting of 0.5% TBA and 20% TCA, and the contents were heated at 95 °C for 30 min to initiate the reaction. To stop the reaction, the solution was cooled immediately in an ice-water bath, and air bubbles were removed by agitating the reaction vials. Samples were centrifuged at 10,000 gn for 10 min, and absorbance of the supernatant was read at 532 and 600 nm using a spectrophotometer so that the values could be substituted into the equation as detailed by Heath and Packer (1968) and Kwon et al. (1965).

Statistical analysis.

Significant effects of temperature and chemical treatments, and treatment interactions were analyzed through two-way analysis of variance using the general linear model procedure in SAS (version 9.2; SAS Institute, Cary, NC). Fisher’s protected least significant difference test was used at a probability level of P = 0.05 to separate difference in means between treatments.

Results

Physiological effects of β-carotene on creeping bentgrass.

Under nonstress conditions, TQ, chlorophyll content, or EL remained unchanged during the 28-d experimental period. There were no significant differences in TQ, chlorophyll content, or EL between untreated control and β-carotene-treated plants under nonstress conditions (Figs. 1A, 2A, and 3A).

Fig. 1.
Fig. 1.

Turf quality (TQ) evaluations ranging from 1 to 9, as indicated for β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control (A) or heat stress (B) temperature conditions. TQ of 1 signifies the poorest quality turf that is brown and dead, whereas a 9 is assigned to the highest quality turf that is uniformly green and healthy. A 6 is the rating at which turf is considered minimally acceptable. Bars define the significant differences between the untreated control and the heat stress treatments, n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 0.3309 for plants under control conditions and 0.4478 for those under heat stress.

Citation: Journal of the American Society for Horticultural Science 147, 3; 10.21273/JASHS05201-22

Fig. 2.
Fig. 2.

Leaf chlorophyll content for β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control (A) or heat stress (B) temperature conditions. Bars define the significant differences between the untreated control and the heat stress treatments, where n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 2.3978 for plants under control conditions and 2.5414 for those under heat stress. DW = dry weight.

Citation: Journal of the American Society for Horticultural Science 147, 3; 10.21273/JASHS05201-22

Fig. 3.
Fig. 3.

Leaf electrolyte leakage (EL) for β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control (A) or heat stress (B) temperature conditions. Bars define the significant differences between the untreated control and the heat stress treatments, where n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 9.375 for plants under control conditions and 4.4322 for those under heat stress.

Citation: Journal of the American Society for Horticultural Science 147, 3; 10.21273/JASHS05201-22

TQ and chlorophyll content declined through the duration of heat stress whereas EL increased during the 28-d experimental period in both untreated control and β-carotene-treated plants that were exposed to heat stress (Figs. 1B, 2B, and 3B). Plants treated with β-carotene maintained significantly greater TQ than untreated control plants during a prolonged period (21 and 28 d) of heat stress, which was 10.53% and 26.32% greater at 21 and 28 d, respectively (Fig. 1B). Similarly, chlorophyll content was also increased to a significantly greater level in β-carotene-treated plants compared with untreated control plants at later phases of heat stress, with increases of 11.87% and 15.36% at 21 and 28 d, respectively (Fig. 2B). β-Carotene-treated plants maintained significantly lower EL at 21 and 28 d of heat stress (by 25.6% and 16.09%, respectively) compared with untreated controls (Fig. 3B).

Effects of β-carotene on lipid peroxidation and antioxidant enzyme activity.

Under nonstress conditions, MDA content in leaves did not show changes throughout the duration of the experiment in untreated control or β-carotene-treated plants (Fig. 4A). There were also no significant differences in MDA content between untreated control and β-carotene treatments (Fig. 4A).

Fig. 4.
Fig. 4.

Leaf malondialdehyde (MDA) content measuring lipid peroxidation in β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control (A) or heat stress (B) temperature conditions. Bars define the significant differences between the untreated control and the heat stress treatments, where n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 2.5508 for plants under control conditions and 7.7238 for those under heat stress. FW = fresh weight.

Citation: Journal of the American Society for Horticultural Science 147, 3; 10.21273/JASHS05201-22

Leaf MDA content increased through the duration of heat stress in plants treated with β-carotene and untreated controls, but the increase was to a lesser extent in β-carotene-treated plants than in untreated control plants (Fig. 4B). In addition, compared with that in untreated control plants, leaf MDA content was significantly less in plants treated with β-carotene after 14 d of heat stress. β-Carotene application reduced MDA content by 15.47%, 16.68%, and 19.29% at 14, 21, and 28 d of heat stress, respectively (Fig. 4B).

The activity of antioxidant enzymes (SOD, POD, CAT, and APX) was unchanged during the 28-d experimental period in untreated control plants or the β-carotene treatment under nonstress conditions (Figs. 5A, 6A, 7A, and 8A). None of the enzymes showed significant differences in their activity between untreated control and β-carotene treatment under nonstress conditions (Figs. 5A, 6A, 7A, and 8A). In plants exposed to heat stress, the activity of all four antioxidant enzymes decreased in untreated control and β-carotene-treated plants, but the decreases were less pronounced in those treated with β-carotene (Figs. 5B, 6B, 7B, and 8B).

Fig. 5.
Fig. 5.

Leaf antioxidant enzyme activity of superoxide dismutase (SOD) in β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control conditions (A) or heat stress (B). Bars define the significant differences between the untreated control and the heat stress treatments, where n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 10.091 for plants under control conditions and 10.202 for those under heat stress. FW = fresh weight.

Citation: Journal of the American Society for Horticultural Science 147, 3; 10.21273/JASHS05201-22

Fig. 6.
Fig. 6.

Leaf antioxidant enzyme activity of peroxidase (POD) in β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control conditions (A) or heat stress (B). Bars define the significant differences between the untreated control and the heat stress treatments, where n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 5.5329 for plants under control conditions and 5.6978 for those under heat stress. FW = fresh weight.

Citation: Journal of the American Society for Horticultural Science 147, 3; 10.21273/JASHS05201-22

Fig. 7.
Fig. 7.

Leaf antioxidant enzyme activity of catalase (CAT) in β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control conditions (A) or heat stress (B). Bars define the significant differences between the untreated control and the heat stress treatments, where n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 0.4809 for plants under control conditions and 0.78 for those under heat stress. FW = fresh weight.

Citation: Journal of the American Society for Horticultural Science 147, 3; 10.21273/JASHS05201-22

Fig. 8.
Fig. 8.

Leaf antioxidant enzyme activity of ascorbate peroxidase (APX) in β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control conditions (A) or heat stress (B). Bars define the significant differences between the untreated control and the heat stress treatments, where n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 0.3712 for plants under control conditions and 0.8716 for those under heat stress. FW = fresh weight.

Citation: Journal of the American Society for Horticultural Science 147, 3; 10.21273/JASHS05201-22

Under heat stress, β-carotene application resulted in significant increases in SOD activity, which was 10.15%, 19.86%, and 23.77% greater than the untreated control at 14, 21, and 28 d, respectively (Fig. 5B). The activity of POD was 10.83%, 19.00%, and 43.23% greater in β-carotene-treated plants than the untreated control at 14, 21, and 28 d of heat stress, respectively (Fig. 6B). The activities of CAT were increased by 46.70%, 171.55%, and 227.38% at 14, 21, and 28 d of heat stress, respectively, as a result of β-carotene application in comparison with that of the untreated control (Fig. 7B). APX activity was significantly greater in plants treated with β-carotene at 21 and 28 d of heat stress (by 54.84% and 224.07%, respectively) (Fig. 8B).

Although the activities of all antioxidant enzymes were increased by β-carotene in plants exposed to heat stress, the degree of increase varied among the four enzymes (SOD, POD, CAT, and APX). Under heat stress, CAT exhibited the most remarkable increases in its activity in response to β-carotene, followed by APX, whereas SOD and POD activity increased to a lesser extent compared with CAT and APX.

Discussion

Multiple studies have reported the ameliorative effects that accumulation of β-carotene has on plants exposed to various abiotic stresses, such as drought stress in Chinese cabbage (Brassica rapa) and carrot (Daucus carota), salt stress in sweetpotato (Ipomoea batatas) and tobacco (Nicotiana tabacum), high light stress in arabidopsis (Arabidopsis thaliana), and heat stress in potato (S. tuberosum) (Havaux et al., 1996; Kim et al., 2012; Li et al., 2020; Ramel et al., 2012; Shawon et al., 2018; Zhang et al., 2021). Our study demonstrates the positive effects of exogenous application of β-carotene on enhancing heat tolerance in a cool-season turfgrass species, as shown by an improvement in TQ and chlorophyll content as well as a reduction of EL in plants treated with β-carotene. β-Carotene-enhanced heat tolerance in creeping bentgrass could be related to the roles of β-carotene in regulating antioxidant metabolism, as discussed next.

β-Carotene, having antioxidant properties, can scavenge ROS directly, including 1O2 and peroxyl radical, to protect plants against oxidative stress (Croce et al., 1999; Hirayama et al., 1994; Moon and Shibamoto, 2009). A number of studies also found that β-carotene also activates antioxidant enzymes, including SOD, CAT, POD, and APX, to detoxify ROS induced by abiotic stresses, although the responses of specific antioxidant enzymes to β-carotene may vary depending on plant species, type, and duration of stress (Asada, 1999; Smirnoff, 1993). For example, in β-carotene-accumulating celery (Apium graveolens) mutants exposed to salinity stress, it was found that accumulation of β-carotene enhanced the activity of SOD and POD (Yin et al., 2020). In β-carotene-accumulating sweetpotato (I. batatas) mutants, the activity of SOD, POD, and CAT increased significantly under salt stress (Li et al., 2017). In our study, β-carotene promoted the activity of SOD, POD, CAT, and APX under heat stress, and the elevation of antioxidant enzyme activity correlated to the reduction in MDA content and EL mediated by β-carotene under heat stress, suggesting that β-carotene may have mitigated lipid peroxidation and augmented enzymatic antioxidant activities during oxidative stress caused by heat stress.

ROS scavenging requires the coordination of multiple antioxidant enzymes. SOD serves as the initial defense for scavenging superoxide into H2O2. Hydrogen peroxide molecules are reduced into water by APX and other enzymes in the ascorbate–glutathione (AsA-GSH) cycle and by the CAT enzyme in the water–water cycle. In the water–water cycle, CAT decomposes H2O2 into molecular oxygen without using a substrate, whereas APX uses ascorbate as a substrate to detoxify H2O2 into monodehydroascorbate and water in the AsA-GSH cycle (Asada, 1999; Chelikani et al., 2004). In our study, CAT and APX were most responsive to β-carotene application at 28 d of heat stress (by 2.27- and 2.24-fold, respectively) in comparison with SOD and POD (0.24- and 0.43-fold, respectively). These findings indicate that β-carotene prevented oxidative stress in creeping bentgrass exposed to heat stress by activating ROS scavenging activity in the AsA-GSH and water–water cycles during oxidative stress caused by heat stress.

In summary, application of β-carotene enhanced heat tolerance effectively in creeping bentgrass by stimulating the metabolism of enzymatic antioxidants, especially those in the AsA-GSH and water–water cycles, offering the plants protection when the activity of antioxidant enzymes was greatly hindered by heat stress. Our findings indicate that β-carotene could be used as a biomarker and biological agent for improving heat tolerance in cool-season turfgrass species in locations with supraoptimal temperatures where plants suffer from oxidative stress caused by prolonged heat stress.

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  • Foote, C.S., Chang, Y.C. & Denny, R.W. 1970 Chemistry of singlet oxygen: X. Carotenoid quenching parallels biological protection J. Amer. Chem. Soc. 92 5216 5218

  • Giannopolitis, C.N. & Ries, S.K. 1977 Superoxide dismutases: I. Occurrence in higher plants Plant Physiol. 59 309 314 https://doi.org/10.1104/pp.59.2.309

  • Halliwell, B. & Gutteridge, J.M.C. 1989 Free radicals in biology and medicine 2nd ed. Oxford University Press Oxford, UK https://doi.org/10.1016/0748-5514(85)90140-0

    • Search Google Scholar
    • Export Citation
  • Havaux, M 1998 Carotenoids as membrane stabilizers in chloroplasts Trends Plant Sci. 3 147 151 https://doi.org/10.1016/S1360-1385(98)01200-X

  • Havaux, M., Tardy, F., Ravenel, J., Chanu, D. & Parot, P. 1996 Thylakoid membrane stability to heat stress studied by flash spectroscopic measurements of the electrochromic shift in intact potato leaves: Influence of the xanthophyll content Plant Cell Environ. 19 1359 1368 https://doi.org/10.1111/j.1365-3040.1996.tb00014.x

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Heath, R.L. & Packer, L. 1968 Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation Arch. Biochem. Biophys. 125 189 198 https://doi.org/10.1016/0003-9861(68)90654-1

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Hirayama, O., Nakamura, K., Hamada, S. & Kobayasi, Y. 1994 Singlet oxygen quenching ability of naturally occurring carotenoids Lipids 29 149 150 https://doi.org/10.1007/BF02537155

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Hiscox, J.D. & Israelstam, G.F. 1979 A method for the extraction of chlorophyll from leaf tissue without maceration Can. J. Bot. 57 1332 1334 https://doi.org/101139/b79-163

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Hoagland, D.R. & Arnon, D.I. 1950 The water-culture method for growing plants without soil Calif. Agric. Exp. Stn. Circ. 347

  • Kim, S.H., Ahn, Y.O., Ahn, M.J., Lee, H.S. & Kwak, S.S. 2012 Down-regulation of β-carotene hydroxylase increases β-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweetpotato Phytochemistry 74 69 78 https://doi.org/10.1016/j.phytochem.2011.11.003

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Kwon, T.W., Menzel, D.B. & Olcott, H.S. 1965 Reactivity of malonaldehyde with food constituents J. Food Sci. 30 808 813 https://doi.org/10.111/j.1365-2621.1965.tb01845.x

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Li, C., Ji, J., Wang, G., Li, Z., Wang, Y. & Fan, Y. 2020 Over-expression of LcPDS, LcZDS, and LcCRTISO, genes from wolfberry for carotenoid biosynthesis, enhanced carotenoid accumulation, and salt tolerance in tobacco Front. Plant Sci. 11 119 https://doi.org/10.3389/fpls.2020.00119

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Li, R., Kang, C., Song, X., Yu, L., Liu, D., He, S., Zhai, H. & Liu, Q. 2017 A ζ-carotene desaturase gene, IbZDS, increases β-carotene and lutein contents and enhances salt tolerance in transgenic sweetpotato Plant Sci. 262 39 51 https://doi.org/10.1016/j.plantsci.2017.05.014

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Liu, X. & Huang, B. 2000 Heat stress injury in relation to membrane lipid peroxidation in creeping bentgrass Crop Sci. 40 503 510 https://doi.org/2135/cropsci2000.402503x

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Moon, J.K. & Shibamoto, T. 2009 Antioxidant assays for plant and food components J. Agr. Food Chem. 57 1655 1666 https://doi.org/10.1021/jf803537k

  • Nakano, Y. & Asada, K. 1981 Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts Plant Cell Physiol. 22 867 880 https://doi.org/10.1093/oxfordjournals.pcp.a076232

    • Search Google Scholar
    • Export Citation
  • Qin, X., Suga, M., Kuang, T. & Shen, J.R. 2015 Structural basis for energy transfer pathways in the plant PSI-LHCI supercomplex Science 348 989 995 https://doi.org/10.1126/science.aab0214

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Ramel, F., Birtic, S., Cuiné, S., Triantaphylidès, C., Ravanat, J.L. & Havaux, M. 2012 Chemical quenching of singlet oxygen by carotenoids in plants Plant Physiol. 158 1267 1278 https://doi.org/10.1104/pp.111.182394

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Roy, B. & Basu, A.K. 2009 Abiotic stress tolerance in crop plants: Breeding and biotechnology New India Publishing New Delhi, India

  • Shawon, R.A., Kang, B.S., Kim, H.C., Lee, S.G., Kim, S.K., Lee, H.J., Bae, J.H. & Ku, Y.G. 2018 Changes in free amino acid, carotenoid, and proline content in Chinese cabbage (Brassica rapa subsp. pekinensis) in response to drought stress Korean J. Plant Res. 31 622 633 https://doi.org/10.7732/kjpr.2018.31.6.622

    • Search Google Scholar
    • Export Citation
  • Smirnoff, N 1993 Tansley Review No. 52. The role of active oxygen in the response of plants to water deficit and desiccation New Phytol. 125 27 58

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Su, X., Ma, J., Wei, X., Cao, P., Zhu, D., Chang, W., Liu, Z., Zhang, X. & Li, M. 2017 Structure and assembly mechanism of plant C2S2M2-type PSII-LHCII supercomplex Science 357 815 820 https://doi.org/10.1126/science.aan0327

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Umena, Y., Kawakami, K., Shen, J.R. & Kamiya, N. 2011 Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å Nature 473 55 60 https://doi.org/10.1038/nature09913

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Yin, L., Liu, J.X., Tao, J.P., Xing, G.M., Tan, G.F., Li, S., Duan, A.Q., Ding, X., Xu, Z.S. & Xiong, A.S. 2020 The gene encoding lycopene epsilon cyclase of celery enhanced lutein and β-carotene contents and confers increased salt tolerance in Arabidopsis Plant Physiol. Biochem. 157 339 347 https://doi.org/10.1016/j.plaphy.2020.10.036

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Zaripheh, S. & Erdman, J.W. Jr 2002 Factors that influence the bioavailability of xanthophylls J. Nutr. 132 531S 534S https://doi.org/10.1093/jn/132.3.531S

  • Zhang, R.R., Wang, Y.H., Li, T., Tan, G.F., Tao, J.P., Su, X.J., Xu, Z.S., Tian, Y.S. & Xiong, A.S. 2021 Effects of simulated drought stress on carotenoid contents and expression of related genes in carrot taproots Protoplasma 258 379 390 https://doi.org/10.1007/s00709-020-01570-5

    • Crossref
    • Search Google Scholar
    • Export Citation

Contributor Notes

We thank Rutgers New Jersey Agricultural Experiment Station for funding.

B.H. is the corresponding author. Email: huang@sebs.rutgers.edu.

  • View in gallery

    Turf quality (TQ) evaluations ranging from 1 to 9, as indicated for β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control (A) or heat stress (B) temperature conditions. TQ of 1 signifies the poorest quality turf that is brown and dead, whereas a 9 is assigned to the highest quality turf that is uniformly green and healthy. A 6 is the rating at which turf is considered minimally acceptable. Bars define the significant differences between the untreated control and the heat stress treatments, n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 0.3309 for plants under control conditions and 0.4478 for those under heat stress.

  • View in gallery

    Leaf chlorophyll content for β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control (A) or heat stress (B) temperature conditions. Bars define the significant differences between the untreated control and the heat stress treatments, where n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 2.3978 for plants under control conditions and 2.5414 for those under heat stress. DW = dry weight.

  • View in gallery

    Leaf electrolyte leakage (EL) for β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control (A) or heat stress (B) temperature conditions. Bars define the significant differences between the untreated control and the heat stress treatments, where n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 9.375 for plants under control conditions and 4.4322 for those under heat stress.

  • View in gallery

    Leaf malondialdehyde (MDA) content measuring lipid peroxidation in β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control (A) or heat stress (B) temperature conditions. Bars define the significant differences between the untreated control and the heat stress treatments, where n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 2.5508 for plants under control conditions and 7.7238 for those under heat stress. FW = fresh weight.

  • View in gallery

    Leaf antioxidant enzyme activity of superoxide dismutase (SOD) in β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control conditions (A) or heat stress (B). Bars define the significant differences between the untreated control and the heat stress treatments, where n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 10.091 for plants under control conditions and 10.202 for those under heat stress. FW = fresh weight.

  • View in gallery

    Leaf antioxidant enzyme activity of peroxidase (POD) in β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control conditions (A) or heat stress (B). Bars define the significant differences between the untreated control and the heat stress treatments, where n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 5.5329 for plants under control conditions and 5.6978 for those under heat stress. FW = fresh weight.

  • View in gallery

    Leaf antioxidant enzyme activity of catalase (CAT) in β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control conditions (A) or heat stress (B). Bars define the significant differences between the untreated control and the heat stress treatments, where n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 0.4809 for plants under control conditions and 0.78 for those under heat stress. FW = fresh weight.

  • View in gallery

    Leaf antioxidant enzyme activity of ascorbate peroxidase (APX) in β-carotene-treated or untreated control creeping bentgrass subjected to nonstress control conditions (A) or heat stress (B). Bars define the significant differences between the untreated control and the heat stress treatments, where n defines the number of measurements corresponding to each value (n = 4), and means were separated using Fisher’s protected least significant difference (lsd) procedure (P = 0.05). For comparisons among sampling dates for each given treatment, the lsd value is 0.3712 for plants under control conditions and 0.8716 for those under heat stress. FW = fresh weight.

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  • Foote, C.S., Chang, Y.C. & Denny, R.W. 1970 Chemistry of singlet oxygen: X. Carotenoid quenching parallels biological protection J. Amer. Chem. Soc. 92 5216 5218

  • Giannopolitis, C.N. & Ries, S.K. 1977 Superoxide dismutases: I. Occurrence in higher plants Plant Physiol. 59 309 314 https://doi.org/10.1104/pp.59.2.309

  • Halliwell, B. & Gutteridge, J.M.C. 1989 Free radicals in biology and medicine 2nd ed. Oxford University Press Oxford, UK https://doi.org/10.1016/0748-5514(85)90140-0

    • Search Google Scholar
    • Export Citation
  • Havaux, M 1998 Carotenoids as membrane stabilizers in chloroplasts Trends Plant Sci. 3 147 151 https://doi.org/10.1016/S1360-1385(98)01200-X

  • Havaux, M., Tardy, F., Ravenel, J., Chanu, D. & Parot, P. 1996 Thylakoid membrane stability to heat stress studied by flash spectroscopic measurements of the electrochromic shift in intact potato leaves: Influence of the xanthophyll content Plant Cell Environ. 19 1359 1368 https://doi.org/10.1111/j.1365-3040.1996.tb00014.x

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Heath, R.L. & Packer, L. 1968 Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation Arch. Biochem. Biophys. 125 189 198 https://doi.org/10.1016/0003-9861(68)90654-1

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Hirayama, O., Nakamura, K., Hamada, S. & Kobayasi, Y. 1994 Singlet oxygen quenching ability of naturally occurring carotenoids Lipids 29 149 150 https://doi.org/10.1007/BF02537155

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Hiscox, J.D. & Israelstam, G.F. 1979 A method for the extraction of chlorophyll from leaf tissue without maceration Can. J. Bot. 57 1332 1334 https://doi.org/101139/b79-163

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Hoagland, D.R. & Arnon, D.I. 1950 The water-culture method for growing plants without soil Calif. Agric. Exp. Stn. Circ. 347

  • Kim, S.H., Ahn, Y.O., Ahn, M.J., Lee, H.S. & Kwak, S.S. 2012 Down-regulation of β-carotene hydroxylase increases β-carotene and total carotenoids enhancing salt stress tolerance in transgenic cultured cells of sweetpotato Phytochemistry 74 69 78 https://doi.org/10.1016/j.phytochem.2011.11.003

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Kwon, T.W., Menzel, D.B. & Olcott, H.S. 1965 Reactivity of malonaldehyde with food constituents J. Food Sci. 30 808 813 https://doi.org/10.111/j.1365-2621.1965.tb01845.x

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Li, C., Ji, J., Wang, G., Li, Z., Wang, Y. & Fan, Y. 2020 Over-expression of LcPDS, LcZDS, and LcCRTISO, genes from wolfberry for carotenoid biosynthesis, enhanced carotenoid accumulation, and salt tolerance in tobacco Front. Plant Sci. 11 119 https://doi.org/10.3389/fpls.2020.00119

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Li, R., Kang, C., Song, X., Yu, L., Liu, D., He, S., Zhai, H. & Liu, Q. 2017 A ζ-carotene desaturase gene, IbZDS, increases β-carotene and lutein contents and enhances salt tolerance in transgenic sweetpotato Plant Sci. 262 39 51 https://doi.org/10.1016/j.plantsci.2017.05.014

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Liu, X. & Huang, B. 2000 Heat stress injury in relation to membrane lipid peroxidation in creeping bentgrass Crop Sci. 40 503 510 https://doi.org/2135/cropsci2000.402503x

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Moon, J.K. & Shibamoto, T. 2009 Antioxidant assays for plant and food components J. Agr. Food Chem. 57 1655 1666 https://doi.org/10.1021/jf803537k

  • Nakano, Y. & Asada, K. 1981 Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts Plant Cell Physiol. 22 867 880 https://doi.org/10.1093/oxfordjournals.pcp.a076232

    • Search Google Scholar
    • Export Citation
  • Qin, X., Suga, M., Kuang, T. & Shen, J.R. 2015 Structural basis for energy transfer pathways in the plant PSI-LHCI supercomplex Science 348 989 995 https://doi.org/10.1126/science.aab0214

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Ramel, F., Birtic, S., Cuiné, S., Triantaphylidès, C., Ravanat, J.L. & Havaux, M. 2012 Chemical quenching of singlet oxygen by carotenoids in plants Plant Physiol. 158 1267 1278 https://doi.org/10.1104/pp.111.182394

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Roy, B. & Basu, A.K. 2009 Abiotic stress tolerance in crop plants: Breeding and biotechnology New India Publishing New Delhi, India

  • Shawon, R.A., Kang, B.S., Kim, H.C., Lee, S.G., Kim, S.K., Lee, H.J., Bae, J.H. & Ku, Y.G. 2018 Changes in free amino acid, carotenoid, and proline content in Chinese cabbage (Brassica rapa subsp. pekinensis) in response to drought stress Korean J. Plant Res. 31 622 633 https://doi.org/10.7732/kjpr.2018.31.6.622

    • Search Google Scholar
    • Export Citation
  • Smirnoff, N 1993 Tansley Review No. 52. The role of active oxygen in the response of plants to water deficit and desiccation New Phytol. 125 27 58

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Su, X., Ma, J., Wei, X., Cao, P., Zhu, D., Chang, W., Liu, Z., Zhang, X. & Li, M. 2017 Structure and assembly mechanism of plant C2S2M2-type PSII-LHCII supercomplex Science 357 815 820 https://doi.org/10.1126/science.aan0327

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Umena, Y., Kawakami, K., Shen, J.R. & Kamiya, N. 2011 Crystal structure of oxygen-evolving photosystem II at a resolution of 1.9 Å Nature 473 55 60 https://doi.org/10.1038/nature09913

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Yin, L., Liu, J.X., Tao, J.P., Xing, G.M., Tan, G.F., Li, S., Duan, A.Q., Ding, X., Xu, Z.S. & Xiong, A.S. 2020 The gene encoding lycopene epsilon cyclase of celery enhanced lutein and β-carotene contents and confers increased salt tolerance in Arabidopsis Plant Physiol. Biochem. 157 339 347 https://doi.org/10.1016/j.plaphy.2020.10.036

    • Crossref
    • Search Google Scholar
    • Export Citation
  • Zaripheh, S. & Erdman, J.W. Jr 2002 Factors that influence the bioavailability of xanthophylls J. Nutr. 132 531S 534S https://doi.org/10.1093/jn/132.3.531S

  • Zhang, R.R., Wang, Y.H., Li, T., Tan, G.F., Tao, J.P., Su, X.J., Xu, Z.S., Tian, Y.S. & Xiong, A.S. 2021 Effects of simulated drought stress on carotenoid contents and expression of related genes in carrot taproots Protoplasma 258 379 390 https://doi.org/10.1007/s00709-020-01570-5

    • Crossref
    • Search Google Scholar
    • Export Citation
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