Differential Regulation of Amino Acids and Nitrogen for Drought Tolerance and Poststress Recovery in Creeping Bentgrass

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, New Brunswick, NJ 08901

Effects of amino acids and nitrogen on plant regrowth or recovery from drought stress remain largely unknown. The objectives of this study were to examine how gamma-aminobutyric acid (GABA) or proline, alone and in combination, or inorganic nitrogen [ammonium nitrate (NN)] may differentially affect turf performance during drought stress and rewatering, and to determine which specific endogenous amino acids regulated by GABA, proline, or NN priming were associated with plant tolerance to drought stress and postdrought recuperation in cool-season grass species. Creeping bentgrass (Agrostis stolonifera cv. Penncross) planted in porous ceramic fritted clay medium were exposed to well-watered conditions or drought stress by withholding irrigation for 21 days in growth chambers. Plants were treated with water (untreated control), GABA, or proline alone and in combination, or NN through foliar spray before drought stress and every 7 days during the 21-day stress period. For postdrought recovery, at 21 days of drought treatment, plants were rewatered for 14 days to return soil water content to prestress levels. Plants treated with GABA or proline alone or in combination maintained higher turf quality (TQ), dark green color index (DGCI), and stolon length by 21 days of drought stress, whereas proline-treated plants also maintained higher leaf relative water content (RWC) during drought as well as longer stolon length during rewatering. Plants treated with NN maintained higher TQ and leaf RWC during drought and had improved percent canopy cover, DGCI, and stolon length during postdrought rewatering. Accumulation of endogenous amino acids under drought stress, including proline and alanine, for proline-treated creeping bentgrass may have contributed to the enhancement of drought tolerance and postdrought regrowth. Nitrogen-enhanced accumulation of GABA, proline, and glutamic acid may have played a role in active amino acid assimilation and subsequent postdrought regrowth. Results from this study indicate that GABA or proline were mainly effective in promoting the tolerance of creeping bentgrass to drought stress while inorganic NN was effective in promoting rapid postdrought recovery and regrowth potential through the activation of amino acid metabolism. Endogenous amino acids, including GABA, proline, alanine, and glutamic acid, may be used as biomarkers to select for drought-tolerant plants and biostimulant components for improvement of drought stress tolerance and poststress recovery in cool-season turfgrass species.

Abstract

Effects of amino acids and nitrogen on plant regrowth or recovery from drought stress remain largely unknown. The objectives of this study were to examine how gamma-aminobutyric acid (GABA) or proline, alone and in combination, or inorganic nitrogen [ammonium nitrate (NN)] may differentially affect turf performance during drought stress and rewatering, and to determine which specific endogenous amino acids regulated by GABA, proline, or NN priming were associated with plant tolerance to drought stress and postdrought recuperation in cool-season grass species. Creeping bentgrass (Agrostis stolonifera cv. Penncross) planted in porous ceramic fritted clay medium were exposed to well-watered conditions or drought stress by withholding irrigation for 21 days in growth chambers. Plants were treated with water (untreated control), GABA, or proline alone and in combination, or NN through foliar spray before drought stress and every 7 days during the 21-day stress period. For postdrought recovery, at 21 days of drought treatment, plants were rewatered for 14 days to return soil water content to prestress levels. Plants treated with GABA or proline alone or in combination maintained higher turf quality (TQ), dark green color index (DGCI), and stolon length by 21 days of drought stress, whereas proline-treated plants also maintained higher leaf relative water content (RWC) during drought as well as longer stolon length during rewatering. Plants treated with NN maintained higher TQ and leaf RWC during drought and had improved percent canopy cover, DGCI, and stolon length during postdrought rewatering. Accumulation of endogenous amino acids under drought stress, including proline and alanine, for proline-treated creeping bentgrass may have contributed to the enhancement of drought tolerance and postdrought regrowth. Nitrogen-enhanced accumulation of GABA, proline, and glutamic acid may have played a role in active amino acid assimilation and subsequent postdrought regrowth. Results from this study indicate that GABA or proline were mainly effective in promoting the tolerance of creeping bentgrass to drought stress while inorganic NN was effective in promoting rapid postdrought recovery and regrowth potential through the activation of amino acid metabolism. Endogenous amino acids, including GABA, proline, alanine, and glutamic acid, may be used as biomarkers to select for drought-tolerant plants and biostimulant components for improvement of drought stress tolerance and poststress recovery in cool-season turfgrass species.

Drought inhibition of plant growth is associated with interruption of various physiological and metabolic processes, including nitrogen and amino acid metabolism (He and Dijkstra, 2014; Hopkins and Hüner, 2004; Khasanova et al., 2013; Waraich et al., 2011). Rapid recovery of plants from stress damage is critical for re-establishment of plant stands and is a highly desirable trait for perennial grasses, such as turfgrasses (Beard, 1973). Amino acids are not only important for plant tolerance to drought stress, but they may also regulate plant regrowth or recovery from drought stress on rewatering when water becomes available. Understanding drought regulation of nitrogen metabolism involving inorganic nitrogen and amino acids is of great significance for developing measures to improve drought tolerance and poststress recovery.

In response to drought stress, the accumulation of some amino acids in plants, such as gamma-aminobutyric acid (GABA) and proline, can provide protection from drought damage by scavenging toxic reactive oxygen species or maintaining cell water balance and cell membrane stability through osmotic adjustment (Bouché and Fromm, 2004; Farooq et al., 2009; Krasensky and Jonak, 2012; Salehi-Lisar and Bakhshayeshan-Agdam, 2016; Szabados and Savouré, 2010). Exogenous application of either GABA or proline has been shown to enhance drought tolerance in various plant species, including wheat (Triticum aestivum), creeping bentgrass (Agrostis stolonifera), arabidopsis (Arabidopsis thaliana), black cumin (Nigella sativa), and fennel (Foeniculum vulgare), as manifested by increased antioxidant activity, photosynthetic activities, chlorophyll content, and relative water content associated with osmoregulation (Farooq et al., 2017; Li et al., 2019; Merewitz, 2016; Moustakas et al., 2011; Rezaei-Chiyaneh et al., 2018; Zali and Ehsanzadeh, 2018). Improvement of drought tolerance through GABA application in creeping bentgrass was associated with an increase in leaf hydration status and cell membrane stability, as well as a decrease in leaf senescence, as observed by higher chlorophyll content and greater photochemical efficiency (Li et al., 2016, 2017). Increased endogenous proline content has been shown to contribute to drought tolerance in turfgrass, such as in a drought-tolerant cultivar of tall fescue (Schedonorus arundinaceus cv. Van Gogh), and was associated with increased leaf water content and endogenous content of the stress response hormone, abscisic acid (ABA), as well as the growth-promoting hormone, cytokinin, during both drought stress and postdrought rewatering (Man et al., 2011). Most studies have focused on the effects of GABA or proline application alone on drought tolerance in different plant species, such as perennial ryegrass [Lolium perenne (Krishnan et al., 2013)] or creeping bentgrass (Li et al., 2017); however, there is little known about the effectiveness of GABA or proline alone on postdrought recovery and the combinatory use of the two amino acids on both drought stress and postdrought recovery. In addition, there is an overall lack of knowledge as to how the metabolic fates of these nitrogen-enriched compounds may affect postdrought recovery in relation to assimilation of other amino acids during drought stress.

Recovery from drought stress for perennial grasses, such as turfgrasses, is dependent on rapid re-greening of the canopy, regeneration of new leaves, and expansion of lateral stems, such as stolons, that can quickly fill in drought-damaged areas on rewatering to improve canopy coverage (Steinke et al., 2013; Zhang et al., 2019). The objectives of this study were to determine effects of GABA or proline, alone and in combination, or inorganic ammonium nitrate (NN) alone on turf canopy performance during drought stress and rewatering for creeping bentgrass and to evaluate how these nitrogen-enriched compounds might be involved in regulating the endogenous accumulation of other amino acids during drought stress, contributing to postdrought recovery.

Materials and Methods

Plant materials and growing conditions.

Mature sod plugs of creeping bentgrass cv. Penncross were collected from a Rutgers University turfgrass research farm (East Brunswick, NJ) on 4 Dec. 2017, washed free of soil, and placed in individual plastic pots (10 cm diameter × 40 cm depth) filled with porous ceramic fritted clay medium (Profile Products, LLC, Buffalo Grove, IL). Plants were maintained in a greenhouse for 70 d under the following conditions: an average temperature of 22/20 °C (day/night) and average of 700 μmol·m−2·s−1 photosynthetically active radiation (PAR) using natural sunlight and supplemental lighting above the plant canopy. Plants were watered daily, trimmed to a height of 2 cm every 3 d, and fertilized weekly with a half-strength concentration of Hoagland’s nutrient solution (Hoagland and Arnon, 1950). After plant establishment, pots were placed into four controlled-environment growth chambers (Environmental Growth Chambers, Chagrin Falls, OH) controlled at an average temperature of 22/18 °C (day/night), 60% relative humidity, and 650 µmol·m−2·s−1 PAR with a 14-h photoperiod. Plants acclimated to the growth chamber conditions for 21 d before initiation of drought stress on 6 Mar. 2018.

Treatments and experimental design.

After the acclimation period, plants were either exposed to well-watered (irrigated daily until water dripped from bottom of pots) or drought stress (water completely withheld) conditions for 21 d (from 6 to 27 Mar. 2018). Gamma-aminobutyric acid (0.5 mm), proline (10 mm), a mixture of GABA and proline [GABA (0.5 mm) + proline (10 mm)], NN (30 mm in equivalent N quantity as in GABA or proline treatment), or water (untreated control) were applied to the turf canopy as a foliar spray before the initiation of drought stress (on 6 Mar. 2018) and then every 7 d during the drought stress period (on 13, 20, and 27 Mar. 2018) using a volume of 15 mL to saturate the canopy. The concentration for each treatment chosen was based on results gathered from preliminary screening experiments as well as previous research, which demonstrated the effectiveness of that concentration for improving drought tolerance (Li et al., 2017, 2019; Moustakas et al., 2011). At 21 d of drought stress (on 27 Mar. 2018), all of the aboveground tissues were trimmed off of the plant, leaving only the basal crown tissue, and all plants were rewatered to pot capacity to begin a 14-d postdrought recovery assessment (from 28 Mar. to 10 Apr. 2018). Plants were not treated with GABA, proline, or NN during the recovery period.

The experiment was arranged as a split-plot design with irrigation treatment as the main plot (well-watered or drought stress) repeated in four growth chambers, and amino acid and NN treatments as the subplots with six replicates (six pots of plants) used for each treatment. Pots were randomized among and within each growth chamber every 3 d to eliminate the potential effects of environmental variation in the chambers.

Soil and plant water status.

The volumetric soil water content (SWC) of each pot was measured daily throughout 21 d of drought stress and 14 d of recovery using a three-pronged buriable waveguide probe (Soil Moisture Equipment Corp., Goleta, CA) measuring 20 cm in length. Each probe was equipped to a time domain reflectometry system (Trase 1 System; Soil Moisture Equipment Corp.) to monitor the level of soil water deficit during the dry-down period, using the technique described in Topp et al. (1980). Probes were inserted into the root zone of both well-watered and drought-stressed plants and remained undisturbed until the end of the experiment. The SWC in pots under well-watered conditions was maintained between 27% and 30% (pot capacity) for the entire duration of the experiment. The SWC steadily declined in pots exposed to drought stress, reaching between 5% and 8% by the end of the 21-d drought stress period. On rewatering, the SWC of previously drought-stressed pots was again maintained at the pot capacity between 27% and 30%.

Leaf relative water content (RWC) was analyzed weekly throughout the 21-d drought stress period and 14-d rewatering period as a measure of leaf hydration status, following the methods described by Barrs and Weatherley (1962). Fresh leaf tissue (≈0.1 g) was collected from each plant and immediately weighed on a mass balance to determine fresh weight (FW). Samples were incubated in deionized water for 12 h at 4 °C, after which leaves were blotted dry and weighed to assess turgid weight (TW). Leaf samples were then dried in an oven at 80 °C for 3 d and dry weight (DW) was measured. Leaf RWC was calculated using the formula RWC% = [(FW – DW)/(TW – DW)] × 100.

Turfgrass growth characteristics.

Each plant was visually assessed for turf quality (TQ) weekly throughout the drought stress and postdrought recovery phases of the experiment. TQ was measured using a rating scale of 1 to 9, where a score of 9 represented healthy, green turf, a score of 1 represented brown and dead or desiccated turf, and a score of 6 represented the minimum acceptable quality. TQ ratings were an overall assessment of plant health based on uniformity, density, and color of the turfgrass leaves and canopy, according to the National Turfgrass Evaluation Program standard (Morris and Shearman, 1999).

Percent canopy cover, dark green color index (DGCI), and stolon length were analyzed weekly to assess changes in canopy growth or color throughout both the drought stress and postdrought recovery periods. Digital photographs of the plant canopy were taken weekly under identical light conditions from a height of 1 m, and the methods described by Richardson et al. (2001) were used to quantify percent canopy cover. Computer software (SigmaScan Pro version 5.0; Systat Software Inc., San Jose, CA) was used to quantify red, blue, and green pixels within each photograph, and values for hue (H), saturation (S), and brightness (B) were assigned for DGCI evaluation. The DGCI values were calculated using the equation, DGCI = [(H – 60)/60 + (1 – S) + (1 – B)]/3 (Karcher and Richardson, 2003). To assess lateral spread of stolons, image analysis software (Digimizer; MedCalc Software, Ltd., Ostend, Belgium) was used to measure the length of stolons in centimeters for each photo.

Quantification of endogenous amino acid content.

Leaf tissue was collected after 21 d of irrigation treatments for both well-watered and drought-stressed plants, immediately frozen in liquid nitrogen, and stored in a freezer set to −80 °C for further endogenous amino acid analysis. To quantify endogenous amino acids, hydrophilic interaction (HILIC) ultra-high performance liquid chromatography (UHPLC) with tandem mass spectrometry was conducted using a UHPLC instrument (1290 Infinity II UHPLC; Agilent Technologies, Inc., Santa Clara, CA) in system with triple quadrupole liquid chromatography–mass spectrometry (LC/MS) (6470 Triple Quadrupole LC/MS; Agilent Technologies, Inc.) The unit was equipped with an HILIC ethylene bridged hybrid Amide column (1.7 µm, 2.1 mm × 100 mm) (ACQUITY; Waters Corp., Milford, MA). The analysis was used according to Gao et al. (2016), Guo et al. (2013), and Tsochatzis et al. (2017). The current experiment modified these methods to improve sensitivity and chromatographic performance, as described by Yuan et al. (2020). To prepare samples for LC/MS analysis, ≈0.1 g of frozen leaf tissue was ground using liquid nitrogen and extracted by vortexing and sonicating for 20 min with 10 mL of 0.1 m HCl aqueous solution and storing at 4 °C overnight. Once extracted, 0.1 mL of each sample was added to 0.9 mL of acetonitrile acidified with 100 mm HCl, and mixtures were vortexed and centrifuged for 10 min at 16,000 gn. After centrifugation, 0.5 μL of supernatant from each sample was injected into the system for LC/MS analysis, following the methods of Yuan et al. (2020).

Statistical analysis.

The effects of irrigation treatment and amino acid or NN treatments were analyzed for physiological parameters, endogenous amino acid content, and canopy growth parameters, using analysis of variance and the general linear model procedure (PROC GLM) in a statistical program (SAS version 9.2; SAS Institute Inc., Cary, NC). Differences between treatment means at a given day of treatment were separated by Fisher’s protected least significant difference test (P ≤ 0.05).

Results

Effects of GABA, proline, and nitrogen on leaf water status and turfgrass canopy characteristics.

Leaf RWC was unaffected by any of the treatments under well-watered conditions (Fig. 1A), but was significantly higher (by 16% and 37%) for proline and NN-treated plants, respectively, at 21 d drought in comparison with untreated control plants (Fig. 1B). At 7 and 14 d of rewatering, the RWC of previously drought-stressed plants increased to the nonstress level, which was not significantly different among GABA, proline, GABA + proline, and NN treatments, indicating that all plants were fully rehydrated on rewatering.

Fig. 1.
Fig. 1.

Changes in leaf relative water content (RWC) of creeping bentgrass (Agrostis stolonifera cv. Penncross) treated with water (untreated control), gamma-aminobutyric acid (GABA), proline, GABA + proline, or ammonium nitrate (NN) for 21 d under either (A) well-watered or (B) drought conditions, and 14 d of rewatering. rec = recovery/rewatering period. Vertical lines indicate least significant difference values for the comparison of treatments at P ≤ 0.05 at a given day of treatment.

Citation: Journal of the American Society for Horticultural Science 147, 4; 10.21273/JASHS05215-22

TQ ratings of plants under well-watered conditions were not significantly affected by NN, GABA, or proline alone, or the combined treatment of GABA + proline, throughout most of the experiment (Fig. 2A). Turf quality of GABA-treated plants was 6% and 24% higher than the untreated control plants at 10 and 14 d of drought stress, respectively, whereas TQ of proline-treated plants was 11%, 20%, and 41% higher at 10, 14, and 21 d of drought stress, respectively (Fig. 2B). Proline-treated plants also had TQ ratings that were 6% higher at 14 d of rewatering following drought in comparison with the untreated control plants. Plants treated with GABA + proline had TQ ratings that were 13%, 25%, and 19% higher than the untreated control plants at 10, 14, and 21 d of drought stress, respectively. The TQ of NN-treated plants was 6%, 11%, and 33% higher at 10, 14, and 21 d of drought stress, respectively, and was 16% higher at 7 d of rewatering in comparison with untreated control plants.

Fig. 2.
Fig. 2.

Changes in visual turf quality (TQ) of creeping bentgrass (Agrostis stolonifera cv. Penncross) treated with water (untreated control), gamma-aminobutyric acid (GABA), proline, GABA + proline, or ammonium nitrate (NN) for 21 d under either (A) well-watered or (B) drought conditions, and 14 d of rewatering. TQ was measured on a rating scale of 1 to 9; 9 = healthy and green turf, 1 = brown and dead turf, and 6 = the minimum acceptable quality rating. rec = recovery/rewatering period. Vertical lines indicate least significant difference values for the comparison of treatments at P ≤ 0.05 at a given day of treatment.

Citation: Journal of the American Society for Horticultural Science 147, 4; 10.21273/JASHS05215-22

The application of GABA or proline alone, GABA + proline, or NN had no significant effects on percent canopy cover under either well-watered (Fig. 3A) or drought (Fig. 3B) conditions. The NN treatment resulted in significantly higher (by 49%) percent canopy cover at 7 d of rewatering from drought in comparison with untreated control plants.

Fig. 3.
Fig. 3.

Changes in percent canopy cover of creeping bentgrass (Agrostis stolonifera cv. Penncross) treated with water (untreated control), gamma-aminobutyric acid (GABA), proline, GABA + proline, or ammonium nitrate (NN) for 21 d under either (A) well-watered or (B) drought conditions, and 14 d of rewatering. rec = recovery/rewatering period. Vertical lines indicate least significant difference values for the comparison of treatments at P ≤ 0.05 at a given day of treatment.

Citation: Journal of the American Society for Horticultural Science 147, 4; 10.21273/JASHS05215-22

DGCI for well-watered plants was not significantly affected by the application of GABA or proline alone, GABA + proline, or NN during most of the experimental period (Fig. 4A). The DGCI at 21 d of drought stress was significantly higher (by 6% and 5%) for plants treated with either proline or the combination of GABA + proline, respectively, compared with untreated control plants (Fig. 4B). During the rewatering phase, DGCI for NN-treated plants that were previously drought-stressed was significantly higher (by 14%) at 7 d of rewatering compared with untreated control plants. All treatments produced significantly higher DGCI at 14 d of rewatering in comparison with the untreated control plants.

Fig. 4.
Fig. 4.

Changes in dark green color index (DGCI) of creeping bentgrass (Agrostis stolonifera cv. Penncross) treated with water (untreated control), gamma-aminobutyric acid (GABA), proline, GABA + proline, or ammonium nitrate (NN) for 21 d under either (A) well-watered or (B) drought conditions, and 14 d of rewatering. rec = recovery/rewatering period. Vertical lines indicate least significant difference values for the comparison of treatments at P ≤ 0.05 at a given day of treatment.

Citation: Journal of the American Society for Horticultural Science 147, 4; 10.21273/JASHS05215-22

Stolon length in well-watered plants was not significantly affected by GABA or proline alone or in combination, but was significantly higher (by 6%) in the NN treatment compared with untreated control treatment at 21 d (Fig. 5A). Stolons in drought-stressed plants were significantly longer due to treatment with GABA (by 15%, 15%, and 11%), proline (by 10%, 12%, and 10%), or GABA + proline (by 17%, 15%, and 11%) at 7, 14, and 21 d of drought stress, respectively, and due to treatment with NN (by 20%) at 7 d of drought stress in comparison with the untreated control plants (Fig. 5B). During the rewatering of drought-stressed plants, only plants treated with either proline or NN had greater stolon length compared with untreated control plants, and stolon length was 8% and 6% higher for proline-treated plants and 12% and 22% higher for NN-treated plants at 7 and 14 d of rewatering, respectively. Photos taken at 21 d of drought stress (Fig. 6A) depict treatment differences at the end of drought stress, whereas those captured at 14 d of rewatering (Fig. 6B) exhibit differences seen at the end of postdrought recovery.

Fig. 5.
Fig. 5.

Changes in stolon length of creeping bentgrass (Agrostis stolonifera cv. Penncross) treated with water (untreated control), gamma-aminobutyric acid (GABA), proline, GABA + proline, or ammonium nitrate (NN) for 21 d under either (A) well-watered or (B) drought conditions, and 14 d of rewatering. rec = recovery/rewatering period. Vertical lines indicate least significant difference values for the comparison of treatments at P ≤ 0.05 at a given day of treatment.

Citation: Journal of the American Society for Horticultural Science 147, 4; 10.21273/JASHS05215-22

Fig. 6.
Fig. 6.

Creeping bentgrass (Agrostis stolonifera cv. Penncross) plants at (A, top photos) 21 d of drought stress and (B, bottom photos) 14 d of rewatering treated with water (untreated control), gamma-aminobutyric acid (GABA), proline, GABA + proline, or ammonium nitrate.

Citation: Journal of the American Society for Horticultural Science 147, 4; 10.21273/JASHS05215-22

Differential responses of endogenous amino acids to exogenous application of GABA, proline, or nitrogen under well-watered and drought stress conditions.

To identify specific amino acids regulated by GABA, proline, or NN that may be associated with improved turfgrass performance during drought stress and postdrought recovery, comparative analysis for endogenous amino acid content affected by GABA, proline, or NN treatment in comparison with the untreated control treatment under either well-watered or drought stress conditions are shown in Fig. 7. Endogenous amino acids exhibited differential responses to exogenous application of GABA, proline, or NN under well-watered and drought stress conditions, as indicated in the following.

Fig. 7.
Fig. 7.

Heat map of changes in endogenous content of 21 amino acids in creeping bentgrass (Agrostis stolonifera cv. Penncross) at 21 d in response to exogenous gamma-aminobutyric acid (GABA), proline, or ammonium nitrate (NN) application under either well-watered or drought stress conditions. Fold-change shows effects of each amino acid or NN treatment under each irrigation treatment. Orange indicates an upregulation, and green indicates a downregulation. Endogenous content of cysteine was not detected for untreated control under well-watered conditions, and thus could not be used in calculations. n.d = not detected.

Citation: Journal of the American Society for Horticultural Science 147, 4; 10.21273/JASHS05215-22

Under well-watered conditions, several endogenous amino acids were upregulated in response to exogenous GABA treatment, including valine, proline, alanine, threonine, aspartic acid, asparagine, and GABA, whereas other amino acids were mostly unchanged in content. Under drought stress, only the endogenous levels of GABA showed a positive response to exogenous GABA application.

The endogenous content of proline and GABA increased in response to exogenous application of proline under well-watered conditions. Under drought stress, proline-treated plants exhibited positive regulation of most endogenous amino acids, including proline, leucine, isoleucine, valine, tyrosine, alanine, threonine, asparagine, arginine, histidine, lysine, and GABA.

Plants treated with NN under well-watered conditions showed positive regulation of several endogenous amino acids, including proline, asparagine, and GABA. Under drought stress, most amino acids were positively affected by NN treatment, including proline, alanine, glycine, glutamic acid, threonine, aspartic acid, glutamine, serine, asparagine, arginine, lysine, and GABA.

Amino acids with significant changes in endogenous content affected by exogenous application of GABA, proline, or nitrogen.

Among all endogenous amino acids, GABA, proline, alanine, and glutamic acid were significantly affected by exogenous application of GABA, proline, or NN under well-watered or drought stress conditions (Fig. 8).

Fig. 8.
Fig. 8.

Changes to endogenous amino acid content in creeping bentgrass (Agrostis stolonifera cv. Penncross) at 21 d of treatment due to water (untreated control), gamma-aminobutyric acid (GABA), proline, or ammonium nitrate (NN) under either (A) well-watered or (B) drought conditions. Asterisk atop bars indicates significant differences based on least significant difference test at P ≤ 0.05.

Citation: Journal of the American Society for Horticultural Science 147, 4; 10.21273/JASHS05215-22

Endogenous content of GABA was significantly increased by 42%, 59%, and 38% by exogenous application of GABA, proline, or N, respectively, in comparison with the untreated control under well-watered conditions. The endogenous content of GABA was also significantly increased by 14% due to NN treatment under drought stress in comparison with untreated control treatment.

The endogenous content of proline did not show significant differences in GABA-, proline- or NN-treated plants compared with the untreated control under well-watered conditions. Under drought stress, endogenous proline content exhibited significant increases in response to exogenously applied proline or NN treatment, by 30% and 23%, respectively, compared with untreated control treatment.

Endogenous alanine was not responsive to exogenously applied GABA, proline, or NN under well-watered conditions, but its content was significantly increased (by 28%) under drought stress due to exogenous proline treatment as compared with the untreated control.

Exogenous application of NN significantly increased the endogenous content of glutamic acid under drought stress conditions (by 18%) in comparison with the untreated control. Under well-watered conditions, glutamic acid content did not show significant responses to NN application.

Discussion

Amino acids serve as building blocks for proteins and accumulate in plants as solutes for stress defense, mediating adaptation to drought stress (Chaves et al., 2003; Krasensky and Jonak, 2012). In the current study, exogenous application of GABA or proline alone and the combination of GABA and proline had positive effects on TQ, percent canopy cover, green leaf index, and stolon length for lateral spread during drought stress or rewatering, although the extent to which each parameter was affected varied depending on treatment and stress condition. The combined GABA and proline treatment did not have additive effects relative to GABA or proline application alone on turf growth under drought stress, but plants treated with the combination of GABA + proline did experience additive effects relative to that of GABA alone in terms of TQ, percent canopy cover, and lateral spread of stolons during rewatering, indicating that the combination of GABA and proline has potential for stimulating rapid recovery of plants from drought damage.

Exogenous application of GABA significantly improved turf growth under drought stress but did not enhance re-greening or regrowth of plants once the stress was relieved during rewatering, suggesting that GABA is mostly effective in protecting plants from drought stress. Endogenous content of GABA also increased in response to exogenous treatments of GABA, proline, or NN under well-watered or drought stress conditions in this study by strengthening stress defense mechanisms. The protective roles of GABA for drought stress have been attributed to its effects on a myriad of important biological roles, such as stress-signaling, activation of antioxidant metabolism, maintenance of cell membrane stability, and osmotic regulation (Bouché and Fromm, 2004; Farooq et al., 2009; Krasensky and Jonak, 2012; Li et al., 2016, 2017, 2019; Merewitz, 2016; Rezaei-Chiyaneh et al., 2018; Salehi-Lisar and Bakhshayeshan-Agdam, 2016; Szabados and Savouré, 2010; Zali and Ehsanzadeh, 2018). Our previous study found that GABA improved heat stress tolerance in creeping bentgrass through the regulation of amino acids involved in several pathways, mainly in the oxaloacetate and GABA shunt pathways (Rossi et al., 2021). In response to oxidative stress, GABA is metabolized via the GABA shunt pathway, where the formation of succinate allows the bypass of succinyl-CoA ligase and α-ketoglutarate dehydrogenase, two enzymes involved in the tricarboxylic acid (TCA) cycle that are sensitive to oxidative stress (Bouché and Fromm, 2004). In the current study, the increase in endogenous GABA content may have occurred through activation of the GABA shunt pathway, contributing to drought tolerance and stress protection.

Although the positive effects of proline on drought tolerance have been widely reported in various plant species, as discussed at the beginning of the article, limited information is available for the roles of proline in poststress recovery. In this study, proline showed positive effects on enhancing plant tolerance to drought stress, as manifested by increased leaf RWC and other canopy parameters, as well as postdrought recovery on rewatering in creeping bentgrass. Proline-mediated stimulation of postdrought recovery was demonstrated in this study by the enhancement of canopy density, green color, and lateral spread of stolons, suggesting that proline promoted turf regrowth and re-greening on rewatering. The mechanisms by which proline promotes drought tolerance are well-documented, such as its regulation of osmotic adjustment and antioxidant defense (Liang et al., 2013; Szabados and Savouré, 2010); however, the underlying mechanisms regarding how proline stimulates plant regrowth and re-greening for poststress recovery are largely unknown and deserve further investigation. In this study, among all amino acids, only endogenous alanine and proline exhibited significant increases in content due to exogenous application of proline in drought-stressed plants, indicating that proline could have roles in protecting plant tissues against drought damage to enable leaves to regrow when water becomes available. In our previous study, endogenous content of alanine was also significantly increased due to exogenous proline treatment under heat stress and may have influenced energy production under heat stress conditions and contributed to heat tolerance by activating pyruvate and acetyl-CoA, members of the TCA cycle (Hildebrandt et al., 2015; Rossi et al., 2021). Alanine accumulation has been connected to drought tolerance in creeping bentgrass treated with either ABA or salicylic acid and is known to play a role in stress defense pathways (Li et al., 2017; Merewitz et al., 2012). How alanine and proline accumulation occurring concomitantly during drought may contribute to regrowth of plants for postdrought recovery deserves further research.

Application of NN had the most pronounced effects on postdrought recovery, while it also improved leaf RWC and turfgrass growth under drought stress, relative to the proline or GABA treatment. The significant increases in the content of endogenous GABA, proline, and glutamic acid due to exogenous NN treatment suggests that plants actively assimilate ammonium into amino acids during drought stress. The process of ammonium assimilation into amino acids initially involves two key components, glutamine and glutamate, which can then be assimilated into other amino acids through aminotransferase reactions (Bowsher et al., 2008). Glutamate is a precursor in the biosynthesis of GABA or proline, contributing its α-amino group and carbon skeleton to their production (Bowsher et al., 2008). Higher content of these amino acids may indicate an actively working biochemical process, where an upregulation of GABA, along with its precursors, glutamine and glutamic acid (a protonated form of glutamate), might provide evidence for active amino acid assimilation since the decarboxylation of glutamate into GABA is irreversible (Bowsher et al., 2008). The current findings suggest that there is still active synthesis of both GABA and glutamic acid occurring under drought for the NN-treated plants. An enhancement in the upregulation of endogenous glutamine and glutamic acid under NN treatment may have occurred because inorganic ammonium is more readily used by the plant for amino acid assimilation. The increased content of amino acids under NN treatment in association with enhanced physiological parameters under drought stress suggests maintaining active amino acid production during drought stress could contribute to more rapid recovery from drought damage.

In summary, differential effects of GABA, proline, and NN on drought tolerance and postdrought recovery in creeping bentgrass were found in this study. GABA mainly played roles in enhancing plant tolerance to drought stress, whereas inorganic nitrogen was more effective for promoting postdrought recovery relative to GABA and proline. Proline had positive effects on both drought tolerance and postdrought recovery. The combination of GABA and proline had no additive effects on drought tolerance, but stimulated turfgrass regrowth during rewatering. The maintenance of active assimilation of endogenous GABA, proline, alanine, and glutamic acid regulated by exogenous application of GABA, proline, or NN during drought could contribute to stress protection or postdrought recovery. These four amino acids may be used as biomarkers to select for drought-tolerant plants and biostimulant components to improve plant tolerance to drought stress and poststress recovery in cool-season turfgrass species.

Literature Cited

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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
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    • Search Google Scholar
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
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    • Search Google Scholar
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
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    • Search Google Scholar
    • Export Citation
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  • Moustakas, M., Sperdouli, I., Kouna, T., Antonopoulou, C.I. & Therios, I. 2011 Exogenous proline induces soluble sugar accumulation and alleviates drought stress effects on photosystem II functioning of Arabidopsis thaliana leaves Plant Growth Regulat. 65 2 315 325 https://doi.org/10.1007/s10725-011-9604-z

    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
    • Export Citation
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    • Search Google Scholar
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    • Search Google Scholar
    • Export Citation
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Contributor Notes

We thank the New Jersey Agricultural Experiment Station for funding support.

B.H. is the corresponding author. E-mail: huang@sebs.rutgers.edu.

  • View in gallery

    Changes in leaf relative water content (RWC) of creeping bentgrass (Agrostis stolonifera cv. Penncross) treated with water (untreated control), gamma-aminobutyric acid (GABA), proline, GABA + proline, or ammonium nitrate (NN) for 21 d under either (A) well-watered or (B) drought conditions, and 14 d of rewatering. rec = recovery/rewatering period. Vertical lines indicate least significant difference values for the comparison of treatments at P ≤ 0.05 at a given day of treatment.

  • View in gallery

    Changes in visual turf quality (TQ) of creeping bentgrass (Agrostis stolonifera cv. Penncross) treated with water (untreated control), gamma-aminobutyric acid (GABA), proline, GABA + proline, or ammonium nitrate (NN) for 21 d under either (A) well-watered or (B) drought conditions, and 14 d of rewatering. TQ was measured on a rating scale of 1 to 9; 9 = healthy and green turf, 1 = brown and dead turf, and 6 = the minimum acceptable quality rating. rec = recovery/rewatering period. Vertical lines indicate least significant difference values for the comparison of treatments at P ≤ 0.05 at a given day of treatment.

  • View in gallery

    Changes in percent canopy cover of creeping bentgrass (Agrostis stolonifera cv. Penncross) treated with water (untreated control), gamma-aminobutyric acid (GABA), proline, GABA + proline, or ammonium nitrate (NN) for 21 d under either (A) well-watered or (B) drought conditions, and 14 d of rewatering. rec = recovery/rewatering period. Vertical lines indicate least significant difference values for the comparison of treatments at P ≤ 0.05 at a given day of treatment.

  • View in gallery

    Changes in dark green color index (DGCI) of creeping bentgrass (Agrostis stolonifera cv. Penncross) treated with water (untreated control), gamma-aminobutyric acid (GABA), proline, GABA + proline, or ammonium nitrate (NN) for 21 d under either (A) well-watered or (B) drought conditions, and 14 d of rewatering. rec = recovery/rewatering period. Vertical lines indicate least significant difference values for the comparison of treatments at P ≤ 0.05 at a given day of treatment.

  • View in gallery

    Changes in stolon length of creeping bentgrass (Agrostis stolonifera cv. Penncross) treated with water (untreated control), gamma-aminobutyric acid (GABA), proline, GABA + proline, or ammonium nitrate (NN) for 21 d under either (A) well-watered or (B) drought conditions, and 14 d of rewatering. rec = recovery/rewatering period. Vertical lines indicate least significant difference values for the comparison of treatments at P ≤ 0.05 at a given day of treatment.

  • View in gallery

    Creeping bentgrass (Agrostis stolonifera cv. Penncross) plants at (A, top photos) 21 d of drought stress and (B, bottom photos) 14 d of rewatering treated with water (untreated control), gamma-aminobutyric acid (GABA), proline, GABA + proline, or ammonium nitrate.

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    Heat map of changes in endogenous content of 21 amino acids in creeping bentgrass (Agrostis stolonifera cv. Penncross) at 21 d in response to exogenous gamma-aminobutyric acid (GABA), proline, or ammonium nitrate (NN) application under either well-watered or drought stress conditions. Fold-change shows effects of each amino acid or NN treatment under each irrigation treatment. Orange indicates an upregulation, and green indicates a downregulation. Endogenous content of cysteine was not detected for untreated control under well-watered conditions, and thus could not be used in calculations. n.d = not detected.

  • View in gallery

    Changes to endogenous amino acid content in creeping bentgrass (Agrostis stolonifera cv. Penncross) at 21 d of treatment due to water (untreated control), gamma-aminobutyric acid (GABA), proline, or ammonium nitrate (NN) under either (A) well-watered or (B) drought conditions. Asterisk atop bars indicates significant differences based on least significant difference test at P ≤ 0.05.

  • Barrs, H.D. & Weatherley, P.E. 1962 A re-examination of the relative turgidity technique for estimating water deficits in leaves Aust. J. Biol. Sci. 15 3 413 428 https://doi.org/10.1071/BI9620413

    • Search Google Scholar
    • Export Citation
  • Beard, J.B 1973 Turfgrass: Science and culture Prentice Hall Englewood Cliffs, NJ

  • Bouché, N. & Fromm, H. 2004 GABA in plants: Just a metabolite? Trends Plant Sci. 9 3 110 115 https://doi.org/10.1016/j.tplants.2004.01.006

  • Bowsher, C., Steer, M. & Tobin, A. 2008 Plant biochemistry Garland Science London, UK https://doi.org/10.4324/9780203833483

  • Chaves, M.M., Maroco, J.P. & Pereira, J.S. 2003 Understanding plant responses to drought: From genes to the whole plant Funct. Plant Biol. 30 3 239 264 https://doi.org/10.1071/FP0207

    • Search Google Scholar
    • Export Citation
  • Farooq, M., Nawaz, A., Chaudhry, M.A.M., Indrasti, R. & Rehman, A. 2017 Improving resistance against terminal drought in bread wheat by exogenous application of proline and gamma-aminobutyric acid J. Agron. Crop Sci. 203 6 464 472 https://doi.org/10.1111/jac.12222

    • Search Google Scholar
    • Export Citation
  • Farooq, M., Wahid, A., Kobayashi, N., Fujita, D. & Basra, S. 2009 Plant drought stress: Effects, mechanisms and management Agron. Sustain. Dev. 29 1 185 212 https://doi.org/10.1051/agro:2008021

    • Search Google Scholar
    • Export Citation
  • Gao, J., Helmus, R., Cerli, C., Jansen, B., Wang, X. & Kalbitz, K. 2016 Robust analysis of underivatized free amino acids in soil by hydrophilic interaction liquid chromatography coupled with electrospray tandem mass spectrometry J. Chromatography 1449 78 88 https://doi.org/10.1016/j.chroma.2016.04.071

    • Search Google Scholar
    • Export Citation
  • Guo, S., Duan, J.A., Qian, D., Tang, Y., Qian, Y., Wu, D., Su, S. & Shang, E. 2013 Rapid determination of amino acids in fruits of Ziziphus jujuba by hydrophilic interaction ultra-high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry J. Agr. Food Chem. 61 11 2709 2719 https://doi.org/10.1021/jf305497r

    • Search Google Scholar
    • Export Citation
  • He, M. & Dijkstra, F.A. 2014 Drought effect on plant nitrogen and phosphorus: A meta-analysis New Phytol. 204 4 924 931 https://doi.org/10.1111/nph.12952

    • Search Google Scholar
    • Export Citation
  • Hildebrandt, T.M., Nesi, A.N., Araújo, W.L. & Braun, H.P. 2015 Amino acid catabolism in plants Mol. Plant 8 11 1563 1579 https://doi.org/10.1016/j.molp.2015.09.005

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

  • Hopkins, W.G. & Hüner, N.P.A. 2004 Introduction to plant physiology 3rd ed. Wiley Hoboken, NJ

  • Karcher, D.E. & Richardson, M.D. 2003 Quantifying turfgrass color using digital image analysis Crop Sci. 43 3 943 951 https://doi.org/10.2135/cropsci2003.9430

    • Search Google Scholar
    • Export Citation
  • Khasanova, A., James, J.J. & Drenovsky, R.E. 2013 Impacts of drought on plant water relations and nitrogen nutrition in dryland perennial grasses Plant Soil 372 1 541 552 https://doi.org/10.1007/s11104-013-1747-4

    • Search Google Scholar
    • Export Citation
  • Krasensky, J. & Jonak, C. 2012 Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks J. Expt. Bot. 63 4 1593 1608 https://doi.org/10.1093/jxb/err460

    • Search Google Scholar
    • Export Citation
  • Krishnan, S., Laskowski, K., Shukla, V. & Merewitz, E.B. 2013 Mitigation of drought stress damage by exogenous application of a non-protein amino acid γ–aminobutyric acid on perennial ryegrass J. Amer. Soc. Hort. Sci. 138 5 358 366 https://doi.org/10.21273/JASHS.138.5.358

    • Search Google Scholar
    • Export Citation
  • Liang, X., Zhang, L., Natarajan, S.K. & Becker, D.F. 2013 Proline mechanisms of stress survival Antioxid. Redox Signal. 19 9 998 1011 https://doi.org/10.1089/ars.2012.5074

    • Search Google Scholar
    • Export Citation
  • Li, Z., Yu, J., Peng, Y. & Huang, B. 2017 Metabolic pathways regulated by abscisic acid, salicylic acid and γ-aminobutyric acid in association with improved drought tolerance in creeping bentgrass (Agrostis stolonifera) Physiol. Plant. 159 1 42 58 https://doi.org/10.1111/ppl.12483

    • Search Google Scholar
    • Export Citation
  • Li, Z., Huang, T., Tang, M., Cheng, B., Peng, Y. & Zhang, X. 2019 iTRAQ-based proteomics reveals key role of γ-aminobutyric acid (GABA) in regulating drought tolerance in perennial creeping bentgrass (Agrostis stolonifera) Plant Physiol. Biochem. 145 216 226 https://doi.org/10.1016/j.plaphy.2019.10.018

    • Search Google Scholar
    • Export Citation
  • Li, Z., Peng, Y. & Huang, B. 2016 Physiological effects of γ-aminobutyric acid application on improving heat and drought tolerance in creeping bentgrass J. Amer. Soc. Hort. Sci. 141 1 76 84 https://doi.org/10.21273/JASHS.141.1.76

    • Search Google Scholar
    • Export Citation
  • Man, D., Bao, Y.X., Han, L.B. & Zhang, X. 2011 Drought tolerance associated with proline and hormone metabolism in two tall fescue cultivars HortScience 46 7 1027 1032 https://doi.org/10.21273/HORTSCI.46.7.1027

    • Search Google Scholar
    • Export Citation
  • Merewitz, E 2016 Chemical priming-induced drought stress tolerance in plants 77 103 Hossain, M., Wani, S., Bhattacharjee, S., Burritt, D. & Tran, L.S. Drought stress tolerance in plants Vol 1 Springer Cham, Switzerland https://doi.org/10.1007/978-3-319-28899-4_4

    • Search Google Scholar
    • Export Citation
  • Merewitz, E.B., Du, H., Yu, W., Liu, Y., Gianfagna, T. & Huang, B. 2012 Elevated cytokinin content in ipt transgenic creeping bentgrass promotes drought tolerance through regulating metabolite accumulation J. Expt. Bot. 63 3 1315 1328 https://doi.org/10.1093/jxb/err372

    • Search Google Scholar
    • Export Citation
  • Morris, K.N. & Shearman, R.C. 1999 NTEP turfgrass evaluation guidelines 11 May 2022. https://www.ntep.org/pdf/ratings.pdf

  • Moustakas, M., Sperdouli, I., Kouna, T., Antonopoulou, C.I. & Therios, I. 2011 Exogenous proline induces soluble sugar accumulation and alleviates drought stress effects on photosystem II functioning of Arabidopsis thaliana leaves Plant Growth Regulat. 65 2 315 325 https://doi.org/10.1007/s10725-011-9604-z

    • Search Google Scholar
    • Export Citation
  • Richardson, M., Karcher, D. & Purcell, L. 2001 Quantifying turfgrass cover using digital image analysis Crop Sci. 41 6 1884 1888 https://doi.org/10.2135/cropsci2001.1884

    • Search Google Scholar
    • Export Citation
  • Rezaei-Chiyaneh, E., Seyyedi, S.M., Ebrahimian, E., Moghaddam, S.S. & Damalas, C.A. 2018 Exogenous application of gamma-aminobutyric acid (GABA) alleviates the effect of water deficit stress in black cumin (Nigella sativa L.) Ind. Crops Prod. 112 741 748 https://doi.org/10.1016/j.indcrop.2017.12.067

    • Search Google Scholar
    • Export Citation
  • Rossi, S., Chapman, C., Yuan, B. & Huang, B. 2021 Improved heat tolerance in creeping bentgrass by γ-aminobutyric acid, proline, and inorganic nitrogen associated with differential regulation of amino acid metabolism Plant Growth Regulat. 93 2 231 242 https://doi.org/10.1007/s10725-020-00681-6

    • Search Google Scholar
    • Export Citation
  • Salehi-Lisar, S.Y. & Bakhshayeshan-Agdam, H. 2016 Drought stress in plants: Causes, consequences, and tolerance 1 16 Hossain, M., Wani, S., Bhattacharjee, S., Burritt, D. & Tran, L.S. Drought stress tolerance in plants Vol 1 Springer Cham, Switzerland https://doi.org/10.1007/978-3-319-28899-4_1

    • Search Google Scholar
    • Export Citation
  • Steinke, K., Chalmers, D.R., White, R.H., Fontanier, C.H., Thomas, J.C. & Wherley, B.G. 2013 Lateral spread of three warmseason turfgrass species as affected by prior summer water stress at two root zone depths HortScience 48 790 795 https://doi.org/10.21273/HORTSCI.48.6.790

    • Search Google Scholar
    • Export Citation
  • Szabados, L. & Savouré, A. 2010 Proline: A multifunctional amino acid Trends Plant Sci. 15 2 89 97 https://doi.org/10.1016/j.tplants.2009.11.009

  • Topp, G.C., Davis, J.L. & Annan, A.P. 1980 Electromagnetic determination of soil water content: Measurements in coaxial transmission lines Water Resour. Res. 16 574 582 https://doi.org/10.1029/WR016i003p00574

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