Antioxidative responses in roots and shoots of creeping bentgrass under high temperature: effects of nitrogen and cytokinin

It has been previously reported that either nitrogen (N) or cytokinin (CK) applications can alleviate heat stress injury on creeping bentgrass, with some studies reporting enhanced antioxidant metabolism being related to stress protection. The objective of this research was to investigate the simultaneous effects of CK and N on the antioxidant enzyme activity and isoforms of heat stressed creeping bentgrass. 'L-93' creeping bentgrass treated with three rates of CK (trans-zeatin riboside, tZR, 0, 10 and 100μM, designated by CK0, 10, and 100) and two nitrogen rates (2.5 and 7.5kgNha(-1) biweekly, low and high N) in a complete factorial arrangement was maintained in a 38/28°C (day/night) growth chamber for 28d and then harvested. Grass grown at high N (averaged across CK rates) had higher O(2)(-) production, H(2)O(2) concentration, and malondialdehyde content in roots. The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and guaiacol peroxidase (POD) in roots were enhanced 19%, 22%, and 24%, respectively, by high N relative to low N. Twenty-eight days of heat stress resulted in either the development of new isoforms or enhanced isoform intensities of SOD, APX, and POD in roots compared to plant responses prior to heat stress. However, no apparent differences were observed across treatments. Both SOD and POD showed different isoform patterns between roots and shoots, suggesting the function of these isoforms could be tissue specific. Interestingly, no CK effects on these antioxidant parameters were found in this experiment. These results demonstrate the impacts of N on antioxidant metabolism of creeping bentgrass under heat stress with some differences between roots and shoots, but no simultaneous impacts of CK and N.     

Plant growth regulator and nitrogen fertilizer effects on soil organic carbon sequestration in creeping bentgrass fairway turf

The objective of this study was to determine the effects of plant growth regulator (PGR) (no PGR, trinexapac-ethyl, and paclobutrazol) and N fertilizer (zero N, an average of 37 kg N ha^?1 month^?1, 6 and 12 kg N ha^?1 week^?1) on soil organic C (SOC) and soil N in creeping bentgrass (Agrostis stolonifera L.) fairway turf. After 4 years of field experiments soil samples were obtained from soil depths of 0–2.5, 2.5–5, 5–7.5, 7.5–10, 10–15, 15–20, and 20–30 cm. Soil bulk density, SOC, total N, NO ₃ ^? –N, and NH ₄ ⁺ –N concentrations were determined. Paclobutrazol and trinexapac-ethyl application increased SOC. The 37 kg N ha^?1 month^?1 application increased SOC at the 0–2.5 cm depth with both PGRs. When paclobutrazol was used, N fertilizer always increased SOC; however, the greatest increase was observed with the 12 kg N ha^?1 week^?1 application when compared to other rates, inversely related to the NH ₄ ⁺ –N concentration. Nitrogen application increased soil total N and NO ₃ ^? –N in the upper three depths. The application of PGRs and N fertilizer to creeping bentgrass fairway turf is an effective strategy for promoting C sequestration.     

Metabolite Responses to Exogenous Application of Nitrogen,Cytokinin, and Ethylene Inhibitors in Relation to Heat-Induced Senescence in Creeping Bentgrass

The exogenous application of ethylene inhibitors, cyotkinins, or nitrogen has previously been shown to suppress heat-induced senescence and improve heat tolerance in cool -season grasses. The objectives of this study were to examine metabolic profiles altered by exogenous treatment of creeping bentgrass with an ethylene inhibitor, cytokinin or nitrogen under heat stress and to determine metabolic pathways regulated by those compounds in association with their effectiveness for improving heat tolerance. Creeping bentgrass (Agostis stolonifera) plants (cv. Penncross) were foliar sprayed with 18 mM carbonyldiamide (N source), 25μM aminoethoxyvinylglycine (AVG, ethylene inhibitor), 25μM zeatin riboside (ZR, cytokinin), or a water control, and then exposed to 20/15°C (day/night) or 35/30°C (heat stress) in growth chambers. All three exogenous treatments suppressed leaf senescence, as manifested by increased turf quality and chlorophyll content, and reduced electrolyte leakage under heat stress. Polar metabolite profiling identified increases in the content of certain organic acids (i.e. citric and malic acid), sugar alcohols, disaccharides (sucrose), and decreased accumulations of monosaccharides (i.e. glucose and fructose) with exogenous treatment of N, AVG, or ZR at the previously mentioned concentrations when compared to the untreated control under heat stress. Nitrogen stimulated amino acid accumulation whereas AVG and ZR reduced amino acid accumulation compared to the untreated control under heat stress. These results revealed that the alleviation of heat-induced leaf senescence by N, AVG, and ZR could be due to changes in the accumulation of metabolites involved in osmoregulation, antioxidant metabolism, carbon and nitrogen metabolism, as well as stress signaling molecules.     

Physiological and Metabolic Effects of 5-Aminolevulinic Acid for Mitigating Salinity Stress in Creeping Bentgrass

The objectives of this study were to determine whether foliar application of a chlorophyll precursor, 5-aminolevulinic acid (ALA), could mitigate salinity stress damages in perennial grass species by regulating photosynthetic activities, ion content, antioxidant metabolism, or metabolite accumulation. A salinity-sensitive perennial grass species, creeping bentgrass (Agrostis stolonifera), was irrigated daily with 200 mM NaCl for 28 d, which were foliar sprayed with water or ALA (0.5 mg L⁻¹) weekly during the experiment in growth chamber. Foliar application of ALA was effective in mitigating physiological damage resulting from salinity stress, as manifested by increased turf quality, shoot growth rate, leaf relative water content, chlorophyll content, net photosynthetic rate, stomatal conductance and transpiration rate. Foliar application of ALA also alleviated membrane damages, as shown by lower membrane electrolyte leakage and lipid peroxidation, which was associated with increases in the activities of antioxidant enzymes. Leaf content of Na⁺ was reduced and the ratio of K⁺/Na⁺ was increased with ALA application under salinity stress. The positive effects of ALA for salinity tolerance were also associated with the accumulation of organic acids (α-ketoglutaric acid, succinic acid, and malic acid), amino acids (alanine, 5-oxoproline, aspartic acid, and γ -aminobutyric acid), and sugars (glucose, fructose, galactose, lyxose, allose, xylose, sucrose, and maltose). ALA-mitigation of physiological damages by salinity could be due to suppression of Na⁺ accumulation and enhanced physiological and metabolic activities related to photosynthesis, respiration, osmotic regulation, and antioxidant defense.     

Overexpression of Arabidopsis ABF3 gene confers enhanced tolerance to drought and heat stress in creeping bentgrass

The Arabidopsis, abscisic acid responsive element-binding factor 3, ABF3 is known to play an important role in stress responses via regulating the expression of stress-responsive genes. In this study, we introduced pCAMBIA3301 vector harboring the ABF3 gene into creeping bentgrass (Agrostis stolonifera) through Agrobacterium-mediated transformation in order to develop a stress-tolerant variety of turfgrass. After transformation, putative transgenic plants were selected using the herbicide resistance assay. Genomic integration of the transgene was confirmed by genomic PCR and Southern blot analysis, and gene expression was validated by northern blot analysis. Under drought-stressed condition, the transgenic plants overexpressing ABF3 displayed significantly enhanced drought tolerance with higher water content and slower water loss rate than the control plants. Furthermore, the stomata of the ABF3 transgenic plants closed more than those of wild-type creeping bentgrass plants, under both non-stressed and ABA treatment conditions. In addition, the transgenic plants showed enhanced tolerance to heat stress. These results suggest that the overexpression of the ABF3 gene in creeping bentgrass might enhance survival in water-limiting and high temperature environments through increased stomatal closure and reduced water losses.     

Soil Biochemical Responses to Nitrogen Addition in a Bamboo Forest

Many vital ecosystem processes take place in the soils and are greatly affected by the increasing active nitrogen (N) deposition observed globally. Nitrogen deposition generally affects ecosystem processes through the changes in soil biochemical properties such as soil nutrient availability, microbial properties and enzyme activities. In order to evaluate the soil biochemical responses to elevated atmospheric N deposition in bamboo forest ecosystems, a two-year field N addition experiment in a hybrid bamboo (Bambusa pervariabilis × Dendrocalamopsis daii) plantation was conducted. Four levels of N treatment were applied: (1) control (CK, without N added), (2) low-nitrogen (LN, 50 kg N ha⁻¹ year⁻¹), (3) medium-nitrogen (MN, 150 kg N ha⁻¹ year⁻¹), and (4) high-nitrogen (HN, 300 kg N ha⁻¹ year⁻¹). Results indicated that N addition significantly increased the concentrations of NH₄ ⁺, NO₃ ⁻, microbial biomass carbon, microbial biomass N, the rates of nitrification and denitrification; significantly decreased soil pH and the concentration of available phosphorus, and had no effect on the total organic carbon and total N concentration in the 0–20 cm soil depth. Nitrogen addition significantly stimulated activities of hydrolytic enzyme that acquiring N (urease) and phosphorus (acid phosphatase) and depressed the oxidative enzymes (phenol oxidase, peroxidase and catalase) activities. Results suggest that (1) this bamboo forest ecosystem is moving towards being limited by P or co-limited by P under elevated N deposition, (2) the expected progressive increases in N deposition may have a potential important effect on forest litter decomposition due to the interaction of inorganic N and oxidative enzyme activities, in such bamboo forests under high levels of ambient N deposition.     

Accumulation of Trehalose and Sucrose in Cyanobacteria Exposed to Matric Water Stress

The drought-resistant cyanobacteria Phormidium autumnale, strain LPP₄, and a Chroococcidiopsis sp. accumulated trehalose, sucrose, and both trehalose and sucrose, respectively, in response to matric water stress. Accumulated sugar concentrations reached values of up to 6.2 μg of trehalose per μg of chlorophyll in P. autumnale, 6.9 μg of sucrose per μg of chlorophyll in LPP₄, and 4.1 μg of sucrose and 3.2 μg of trehalose per μg of chlorophyll in the Chroococcidiopsis sp. The same sugars were accumulated by these cyanobacteria in similar concentrations under osmotic water stress. Cyanobacteria that did not show drought resistance (Plectonema boryanum and Synechococcus strain PCC 7942) did not accumulate significant amounts of sugars when matric water stress was applied.     

A PCR-based linkage map of Agrostis stolonifera and identification of QTL markers for dollar spot resistance

Creeping bentgrass (Agrostis stolonifera L.) is the most widely utilized cool-season turf species for intensively managed sports playing surfaces, including bowling greens and golf course putting greens, tees, and fairways. One of the biggest disease problems affecting creeping bentgrass is dollar spot disease caused by Sclerotinia homoeocarpa F.T. Bennett. Relative to traditional food crops, little attention has been paid to applying molecular technology to traditional creeping bentgrass breeding programs. The objective of this study was to develop a PCR-based linkage map of creeping bentgrass and identify quantitative trait loci (QTLs) associated with dollar spot resistance. Mapping populations segregating for dollar spot resistance were created, phenotyped for disease resistance, and genotyped for simple sequence repeat, conserved intron scanning primer, intron length polymorphism, and amplified fragment length polymorphism markers. As expected, 14 linkage groups (LGs) were detected for each parental map, covering a total of 1,424 and 1,374 cM for the 7418-3 and the L93-10 parental maps, respectively. A total of eight QTL regions (23 markers) for dollar spot resistance were observed for three isolates (Crenshaw, PRG, and UMass1) in our creeping bentgrass mapping populations. LGs 1, 4, and 5 contained at least two overlapping QTL regions to different isolates, indicating that these regions may play a significant role in dollar spot resistance. Identification of QTLs associated with disease resistance will help to facilitate marker-assisted selection in traditional creeping bentgrass breeding programs.     

Adjusting Nitrogen Application in Accordance with Soil Water Availability Enhances Yield and Water Use by Regulating Physiological Traits of Maize under Drip Fertigation

Knowledge of the interactive effects of water and nitrogen (N) on physio-chemical traits of maize (Zea mays L.) helps to optimize water and N management and improve productivity. A split-plot experiment was conducted with three soil water conditions (severe drought, moderate drought, and fully water supply referring to 45%–55%, 65%–75%, and 85%–95% field capacity, respectively) and four N application rates (N₀, N₁₅₀, N₂₄₀, and N₃₃₀ referring to 0, 150, 240, 330 kg N ha^–1 respectively) under drip fertigation in 2014 and 2015 in the Huang-Huai-Hai Plain of China. The results indicated that drought stress inhibited physiological activity of plants (leaf relative water content, root bleeding sap, and net photosynthetic rate), resulting in low dry matter accumulation after silking, yield, and N uptake, whereas increased WUE and NUE. N application rates over than 150 kg ha^–1 aggravated the inhibition of physiological activity under severe drought condition, while it was offset under moderate drought condition. High N application rates (N₃₃₀) still revealed negative effects under moderate drought condition, as it did not consistently enhance plant physiological activity and significantly reduced N uptake as compared to the N₂₄₀ treatment. With fully water supply, increasing N application rates synergistically enhanced physiological activity, promoted dry matter accumulation after silking, and increased yield, WUE, and N uptake. Although the N₂₄₀ treatment reduced yield by 5.4% in average, it saved 27.3% N under full water supply condition as compared with N₃₃₀ treatment. The results indicated that N regulated growth of maize in aspects of physiological traits, dry matter accumulation, and yield as well as water and N use was depended on soil water status. The appropriate N application rates for maize production was 150 kg ha^–1 under moderate drought or 240 kg ha^–1 under fully water supply under drip fertigation, and high N supply (>150 kg ha^–1) should be avoided under severe drought condition.     

Response to inoculation and N fertilization for increased yield and biological nitrogen fixation of common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) is able to fix 20–60 kg N ha^–1 under tropical environments in Brazil, but these amounts are inadequate to meet the N requirement for economically attractive seed yields. When the plant is supplemented with N fertilizer, N₂ fixation by Rhizobium can be suppressed even at low rates of N. Using the ¹⁵N enriched method, two field experiments were conducted to compare the effect of foliar and soil applications of N-urea on N₂ fixation traits and seed yield. All treatments received a similar fertilization including 10 kg N ha^–1 at sowing. Increasing rates of N (10, 30 and 50 kg N ha^–1) were applied for both methods. Foliar application significantly enhanced nodulation, N₂ fixation (acetylene reduction activity) and yield at low N level (10 kg N ha^–1). Foliar nitrogen was less suppressive to nodulation, even at higher N levels, than soil N treatments. In the site where established Rhizobium was in low numbers, inoculation contributed substantially to increased N₂ fixation traits and yield. Both foliar and soil methods inhibited nodulation at high N rates and did not significantly increase bean yield, when comparing low (10 kg N ha^–1) and high (50 kg N ha^–1) rates applied after emergence. In both experiments, up to 30 kg N ha^–1 of biologically fixed N₂ were obtained when low rates of N were applied onto the leaves.     

Yield and yield components of hybrid corn (Zea mays L.) as affected by mycorrhizal symbiosis and zinc sulfate under drought stress

With respect to the significance of improving hybrid corn performance under stress, this experiment was conducted at the Islamic Azad University, Arak Branch, Iran. A complete randomized block design with three levels of irrigations (at 100%, 75% and 50% crop water requirement), two levels of arbuscular mycorrhizal (AM) fungi (Glumus intraradisis) (including control), and three levels of zinc (Zn) sulfate (0, 25 and 45 kg ha⁻¹), was performed. Results of the 2-year experiments indicated that irrigation treatment significantly affected corn yield and its components at P = 1%. AM fungi and increasing Zn levels also resulted in similar effects on corn growth and production. Although AM fungi did not significantly affect corn growth at the non-stressed irrigation treatment, at moderate drought stress AM fungi significantly enhanced corn quality and yield relative to the control treatment. The combined effects of AM fungi and Zn sulfate at 45 kg ha⁻¹ application significantly affected corn growth and production. In addition, the tripartite treatments significantly enhanced corn yield at P = 1%. Effects of Zn and AM fungi on plant growth under drought stress is affected by the stress level.     

Comparative Genome Analysis between Agrostis stolonifera and Members of the Pooideae Subfamily,including Brachypodium distachyon

Creeping bentgrass (Agrostis stolonifera, allotetraploid 2n = 4x = 28) is one of the major cool-season turfgrasses. It is widely used on golf courses due to its tolerance to low mowing and aggressive growth habit. In this study, we investigated genome relationships of creeping bentgrass relative to the Triticeae (a consensus map of Triticum aestivum, T. tauschii, Hordeum vulgare, and H. spontaneum), oat, rice, and ryegrass maps using a common set of 229 EST-RFLP markers. The genome comparisons based on the RFLP markers revealed large-scale chromosomal rearrangements on different numbers of linkage groups (LGs) of creeping bentgrass relative to the Triticeae (3 LGs), oat (4 LGs), and rice (8 LGs). However, we detected no chromosomal rearrangement between creeping bentgrass and ryegrass, suggesting that these recently domesticated species might be closely related, despite their memberships to different Pooideae tribes. In addition, the genome of creeping bentgrass was compared with the complete genome sequence of Brachypodium distachyon in Pooideae subfamily using both sequences of the above-mentioned mapped EST-RFLP markers and sequences of 8,470 publicly available A. stolonifera ESTs (AgEST). We discovered large-scale chromosomal rearrangements on six LGs of creeping bentgrass relative to B. distachyon. Also, a total of 24 syntenic blocks based on 678 orthologus loci were identified between these two grass species. The EST orthologs can be utilized in further comparative mapping of Pooideae species. These results will be useful for genetic improvement of Agrostis species and will provide a better understanding of evolution within Pooideae species.     

Transgenic creeping bentgrass overexpressing Osa‐miR393a exhibits altered plant development and improved multiple stress tolerance

MicroRNA393 (miR393) has been implicated in plant growth, development and multiple stress responses in annual species such as Arabidopsis and rice. However, the role of miR393 in perennial grasses remains unexplored. Creeping bentgrass (Agrostis stolonifera L.) is an environmentally and economically important C3 cool‐season perennial turfgrass. Understanding how miR393 functions in this representative turf species would allow the development of novel strategies in genetically engineering grass species for improved abiotic stress tolerance. We have generated and characterized transgenic creeping bentgrass plants overexpressing rice pri‐miR393a (Osa‐miR393a). We found that Osa‐miR393a transgenics had fewer, but longer tillers, enhanced drought stress tolerance associated with reduced stomata density and denser cuticles, improved salt stress tolerance associated with increased uptake of potassium and enhanced heat stress tolerance associated with induced expression of small heat‐shock protein in comparison with wild‐type controls. We also identified two targets of miR393, AsAFB2 and AsTIR1, whose expression is repressed in transgenics. Taken together, our results revealed the distinctive roles of miR393/target module in plant development and stress responses between creeping bentgrass and other annual species, suggesting that miR393 would be a promising candidate for generating superior crop cultivars with enhanced multiple stress tolerance, thus contributing to agricultural productivity.     

Effects of SAG12-ipt expression on cytokinin production,growth and senescence of creeping bentgrass (<Emphasis Type="Italic">Agrostis stolonifera</Emphasis> L.) under heat stress

The study was conducted to determine the effects of expression of a transgene encoding adenine isopentenyl transferase (ipt), which controls cytokinin synthesis, on growth and leaf senescence of creeping bentgrass (Agrostis stolonifera L.), subjected to heat stress. Creeping bentgrass (cv. Penncross) was transformed with ipt ligated to a senescence-activated promoter (SAG12). Eight SAG12-ipt transgenic lines exhibiting desirable turf quality and a transgenic control line (transformed with the empty vector) were evaluated for morphological and physiological changes under normal growth temperature (20°C) and after 14 days of heat stress (35°C) in growth chambers. Six of the SAG12-ipt lines developed more tillers than the control line during establishment under normal growth temperature of 20°C. Following 14 days of heat stress, four of the SAG12-ipt lines had increased 65–83% of roots and for all six SAG12-ipt lines root elongation continued, whereas root production ceased and total root length decreased for the control line. Root isopentenyl adenine (iPA) content increased 2.5–3.5 times in five of the SAG12-ipt lines, whereas in the control line iPA decreased 20% after 14 days at 35°C. Total zeatin riboside (ZR) content was maintained at the original level or increased in five of the SAG12-ipt lines, whereas in the control line ZR decreased under heat stress. Our results suggest expression of SAG12-ipt in creeping bentgrass stimulated tiller formation and root production, and delayed leaf senescence under heat stress, suggesting a role for cytokinins in regulating cool-season grass tolerance to heat stress.     

Influence of Starvation on Potential Ammonia-Oxidizing Activity and amoA mRNA Levels of Nitrosospira briensis

The effect of short-term ammonia starvation on Nitrosospira briensis was investigated. The ammonia-oxidizing activity was determined in a concentrated cell suspension with a NO_x biosensor. The apparent half-saturation constant [K_m(app)] value of the NH₃ oxidation of N. briensis was 3 μM NH₃ for cultures grown both in continuous and batch cultures as determined by a NO_x biosensor. Cells grown on the wall of the vessel had a lower K_m(app) value of 1.8 μM NH₃. Nonstarving cultures of N. briensis showed potential ammonia-oxidizing activities of between 200 to 250 μM N h⁻¹, and this activity decreased only slowly during starvation up to 10 days. Within 10 min after the addition of fresh NH₄⁺, 100% activity was regained. Parallel with activity measurements, amoA mRNA and 16S rRNA were investigated. No changes were observed in the 16S rRNA, but a relative decrease of amoA mRNA was observed during the starvation period. During resuscitation, an increase in amoA mRNA expression was detected simultaneously. The patterns of the soluble protein fraction of a 2-week-starved culture of N. briensis showed only small differences in comparison to a nonstarved control. From these results we conclude that N. briensis cells remain in a state allowing fast recovery of ammonia-oxidizing activity after addition of NH₄⁺ to a starved culture. Maintaining cells in this kind of active state could be the survival strategy of ammonia-oxidizing bacteria in nature under fluctuating NH₄⁺ availability.     

Protein accumulation in leaves and roots associated with improved drought tolerance in creeping bentgrass expressing an ipt gene for cytokinin synthesis

Cytokinins (CKs) may be involved in the regulation of plant adaptation to drought stress. The objectives of the study were to identify proteomic changes in leaves and roots in relation to improved drought tolerance in transgenic creeping bentgrass (Agrostis stolonifera) containing a senescence-activated promoter (SAG12) and the isopentyl transferase (ipt) transgene that increases endogenous CK content. Leaves of SAG12-ipt bentgrass exhibited less severe senescence under water stress, as demonstrated by maintaining lower electrolyte leakage and lipid peroxidation, and higher photochemical efficiency (F(v)/F(m)), compared with the null transformant (NT) plants. SAG12-ipt plants had higher root/shoot ratios and lower lipid peroxidation in leaves under water stress than the NT plants. The suppression of drought-induced leaf senescence and root dieback in the transgenic plants was associated with the maintenance of greater antioxidant enzyme activities (superoxide dismutase, peroxidase, and catalase). The SAG12-ipt and NT plants exhibited differential protein expression patterns under well-watered and drought conditions in both leaves and roots. Under equivalent leaf water deficit (47% relative water content), SAG12-ipt plants maintained higher abundance of proteins involved in (i) energy production within both photosynthesis and respiration [ribulose 1,5-bisphosphate carboxylase (RuBisCO) and glyceraldehyde phosphate dehydrogenase (GAPDH)]; (ii) amino acid synthesis (methionine and glutamine); (iii) protein synthesis and destination [chloroplastic elongation factor (EF-Tu) and protein disulphide isomerases (PDIs)]; and (iv) antioxidant defence system (catalase and peroxidase) than the NT plants. These results suggest that increased endogenous CKs under drought stress may directly or indirectly regulate protein abundance and enzymatic activities involved in the above-mentioned metabolic processes, thereby enhancing plant drought tolerance.     

Clonal response to cold tolerance in creeping bentgrass and role of proline-associated pentose phosphate pathway

Single seed origin creeping bentgrass (‘Penncross’) clonal lines were screened to find genetic heterogeneity, which reflected diversity of phenolic production linked to cold stress within a cross-pollinated cultivar. In this study, total soluble phenolic and antioxidant activity varied among 20 creeping bentgrass clonal lines, confirming wide heterogeneity in this cross-pollinated species. Correlations between phenolic content and proline-associated pentose phosphate pathway were also found among the clonal lines. The active metabolic role of proline in cellular metabolic adjustment to cold stress and its support for likely energy synthesis via mitochondrial oxidative phosphorylation was inferred in creeping bentgrass clonal lines based on the activity of proline dehydrogenase. Results of photochemical efficiency of these clonal lines after cold temperature treatment (4 °C) also indicated a close association between stress tolerance and proline-associated pentose phosphate pathway regulation for phenolic biosynthesis and antioxidant response. This study provides a sound metabolic based rationale to screen bentgrass clonal lines for enhanced cold stress tolerance.