Overexpression of MtIPT gene enhanced drought tolerance and delayed leaf senescence of creeping bentgrass (Agrostis stolonifera L.).

BACKGROUND: Isopentenyltransferases (IPT) serve as crucial rate-limiting enzyme in cytokinin synthesis, playing a vital role in plant growth, development, and resistance to abiotic stress. RESULTS: Compared to the wild type, transgenic creeping bentgrass exhibited a slower growth rate, heightened drought tolerance, and improved shade tolerance attributed to delayed leaf senescence. Additionally, transgenic plants showed significant increases in antioxidant enzyme levels, chlorophyll content, and soluble sugars. Importantly, this study uncovered that overexpression of the MtIPT gene not only significantly enhanced cytokinin and auxin content but also influenced brassinosteroid level. RNA-seq analysis revealed that differentially expressed genes (DEGs) between transgenic and wild type plants were closely associated with plant hormone signal transduction, steroid biosynthesis, photosynthesis, flavonoid biosynthesis, carotenoid biosynthesis, anthocyanin biosynthesis, oxidation-reduction process, cytokinin metabolism, and wax biosynthesis. And numerous DEGs related to growth, development, and stress tolerance were identified, including cytokinin signal transduction genes (CRE1, B-ARR), antioxidase-related genes (APX2, PEX11, PER1), Photosynthesis-related genes (ATPF1A, PSBQ, PETF), flavonoid synthesis genes (F3H, C12RT1, DFR), wax synthesis gene (MAH1), senescence-associated gene (SAG20), among others. CONCLUSION: These findings suggest that the MtIPT gene acts as a negative regulator of plant growth and development, while also playing a crucial role in the plant's response to abiotic stress.

γ-Aminobutyric acid (GABA) priming alleviates acid-aluminum toxicity to roots of creeping bentgrass via enhancements in antioxidant defense and organic metabolites remodeling.

MAIN CONCLUSION: γ-Aminobutyric acid alleviates acid-aluminum toxicity to roots associated with enhanced antioxidant metabolism as well as accumulation and transportation of citric and malic acids. Aluminum (Al) toxicity has become the main limiting factor for crop growth and development in acidic soils and is further being aggravated worldwide due to continuous industrial pollution. The current study was designed to examine effects of GABA priming on alleviating acid-Al toxicity in terms of root growth, antioxidant defense, citrate and malate metabolisms, and extensive metabolites remodeling in roots under acidic conditions. Thirty-seven-day-old creeping bentgrass (Agrostis stolonifera) plants were used as test materials. Roots priming with or without 0.5 mM GABA for 3 days were cultivated in standard nutrient solution for 15 days as control or subjected to nutrient solution containing 5 mM AlCl3·6H2O for 15 days as acid-Al stress treatment. Roots were sampled for determinations of root characteristics, physiological and biochemical parameters, and metabolomics. GABA priming significantly alleviated acid-Al-induced root growth inhibition and oxidative damage, despite it promoted the accumulation of Al in roots. Analysis of metabolomics showed that GABA priming significantly increased accumulations of organic acids, amino acids, carbohydrates, and other metabolites in roots under acid-Al stress. In addition, GABA priming also significantly up-regulated key genes related to accumulation and transportation of malic and citric acids in roots under acid-Al stress. GABA-regulated metabolites participated in tricarboxylic acid cycle, GABA shunt, antioxidant defense system, and lipid metabolism, which played positive roles in reactive oxygen species scavenging, energy conversion, osmotic adjustment, and Al ion chelation in roots.

MicroRNA169 integrates multiple factors to modulate plant growth and abiotic stress responses.

MicroRNA169 (miR169) has been implicated in multi-stress regulation in annual species such as Arabidopsis, maize and rice. However, there is a lack of experimental functional and mechanistic studies of miR169 in plants, especially in perennial species, and its impact on plant growth and development remains unexplored. Creeping bentgrass (Agrostis stolonifera L.) is a C3 cool-season perennial turfgrass of significant environmental and economic importance. In this study, we generated both miR169 overexpression and knockdown transgenic creeping bentgrass lines. We found that miR169 acts as a positive regulator in abiotic stress responses but is negatively associated with plant growth and development, playing multiple critical roles in the growth and environmental adaptation of creeping bentgrass. These roles include differentiated spatial hormone accumulation patterns associated with growth and stress accommodation, elevated antioxidant activity that alleviates oxidative damage induced by stress, ion-channelling membrane components for maintaining homeostasis under saline conditions, and potential cross-talks with stress-regulating transcription factors such as AsHsfA and AsWRKYs. Our results unravel the role of miR169 in modulating plant development and stress responses in perennial grass species. This underlines the potential of manipulating miR169 to generate crop cultivars with desirable traits to meet diverse agricultural demands.

Global Metabolites Reprogramming Induced by Spermine Contributing to Salt Tolerance in Creeping Bentgrass.

Soil salinization has become a serious challenge to modern agriculture worldwide. The purpose of the study was to reveal salt tolerance induced by spermine (Spm) associated with alterations in water and redox homeostasis, photosynthetic performance, and global metabolites reprogramming based on analyses of physiological responses and metabolomics in creeping bentgrass (Agrostis stolonifera). Plants pretreated with or without 0.5 mM Spm were subjected to salt stress induced by NaCl for 25 days in controlled growth chambers. Results showed that a prolonged period of salt stress caused a great deal of sodium (Na) accumulation, water loss, photoinhibition, and oxidative damage to plants. However, exogenous application of Spm significantly improved endogenous spermidine (Spd) and Spm contents, followed by significant enhancement of osmotic adjustment (OA), photosynthesis, and antioxidant capacity in leaves under salt stress. The Spm inhibited salt-induced Na accumulation but did not affect potassium (K) content. The analysis of metabolomics demonstrated that the Spm increased intermediate metabolites of γ-aminobutyric acid (GABA) shunt (GABA, glutamic acid, and alanine) and tricarboxylic acid (TCA) cycle (aconitic acid) under salt stress. In addition, the Spm also up-regulated the accumulation of multiple amino acids (glutamine, valine, isoleucine, methionine, serine, lysine, tyrosine, phenylalanine, and tryptophan), sugars (mannose, fructose, sucrose-6-phosphate, tagatose, and cellobiose), organic acid (gallic acid), and other metabolites (glycerol) in response to salt stress. These metabolites played important roles in OA, energy metabolism, signal transduction, and antioxidant defense under salt stress. More importantly, the Spm enhanced GABA shunt and the TCA cycle for energy supply in leaves. Current findings provide new evidence about the regulatory roles of the Spm in alleviating salt damage to plants associated with global metabolites reprogramming and metabolic homeostasis.

Polyamines Metabolism Interacts with γ-Aminobutyric Acid, Proline and Nitrogen Metabolisms to Affect Drought Tolerance of Creeping Bentgrass.

Due to increased global warming and climate change, drought has become a serious threat to horticultural crop cultivation and management. The purpose of this study was to investigate the effect of spermine (Spm) pretreatment on metabolic alterations of polyamine (PAs), γ-aminobutyric acid (GABA), proline (Pro), and nitrogen associated with drought tolerance in creeping bentgrass (Agrostis stolonifera). The results showed that drought tolerance of creeping bentgrass could be significantly improved by the Spm pretreatment, as demonstrated by the maintenance of less chlorophyll loss and higher photosynthesis, gas exchange, water use efficiency, and cell membrane stability. The Spm pretreatment further increased drought-induced accumulation of endogenous PAs, putrescine, spermidine, and Spm, and also enhanced PAs metabolism through improving arginine decarboxylases, ornithine decarboxylase, S-adenosylmethionine decarboxylase, and polyamine oxidase activities during drought stress. In addition, the Spm application not only significantly improved endogenous GABA content, glutamate content, activities of glutamate decarboxylase and α-ketoglutarase, but also alleviated decline in nitrite nitrogen content, nitrate reductase, glutamine synthetase, glutamate synthetase, and GABA aminotransferase activities under drought stress. The Spm-pretreated creeping bentgrass exhibited significantly lower ammonia nitrogen content and nitrite reductase activity as well as higher glutamate dehydrogenase activity than non-pretreated plants in response to drought stress. These results indicated beneficial roles of the Spm on regulating GABA and nitrogen metabolism contributing towards better maintenance of Tricarboxylic acid (TCA) cycle in creeping bentgrass. Interestingly, the Spm-enhanced Pro metabolism rather than more Pro accumulation could be the key regulatory mechanism for drought tolerance in creeping bentgrass. Current findings provide a comprehensive understanding of PAs interaction with other metabolic pathways to regulate drought tolerance in grass species.

A chloroplast heat shock protein modulates growth and abiotic stress response in creeping bentgrass.

Small heat shock proteins (sHSPs), a family of the ubiquitous stress proteins in plants acting as molecular chaperones to protect other proteins from stress-induced damage, have been implicated in plant growth and development as well as plant response to environmental stress, especially heat stress. In this study, a chloroplast-localized sHSP, AsHSP26.8, was overexpressed in creeping bentgrass (Agrostis stolonifera L.) to study its role in regulating plant growth and stress response. Transgenic (TG) creeping bentgrass plants displayed arrested root development, slow growth rate, twisted leaf blades and are more susceptible to heat and salt but less sensitive to drought stress compared to wild-type (WT) controls. RNA-seq analysis revealed that AsHSP26.8 modulated the expression of genes in auxin signalling and stress-related genes such as those encoding HSPs, heat shock factors and other transcription factors. Our results provide new evidence demonstrating that AsHSP26.8 negatively regulates plant growth and development and plays differential roles in plant response to a plethora of diverse abiotic stresses.

Contrasted tolerance of Agrostis capillaris metallicolous and non-metallicolous ecotypes in the context of a mining technosol amended by biochar, compost and iron sulfate.

Metal(loid) contamination of soil, resulting from the mining activities, is a major issue worldwide, due to its negative effects on the environment and health. Therefore, these contaminated soils need to be remediated. One realistic method is the assisted phytostabilization, which aims at establishing a vegetation cover on the soil that will reduce metal(loid) bioavailability and spreading through the prevention of wind erosion and water leaching. In addition, amendments are applied to improve soil conditions and ameliorate plant growth. In this goal, biochar and compost showed good results in terms of amelioration of soil fertility and reduction in lead bioavailability. However, they usually have a negative effect on arsenic. On the contrary, iron sulfate showed capacity to reduce arsenic mobility through interaction with its iron hydroxides. Finally, the choice of the appropriate plant species is crucial for the success of assisted phytostabilization. One good option is to use endemic species, adapted to the metal(loid) stress, with a fast growth and large shoot and root systems. The aims of this study were to (1) evaluate the effects of applying biochar, compost and iron sulfate, alone or combined, to a former mine soil on the soil properties and Agrostis capillaris growth, and (2) assess the difference between two Agrostis capillaris ecotypes, an endemic metallicolous ecotype and a non-metallicolous ecotype. Results of the mesocosm experiment showed that amendment application improved soil properties, i.e., reduced soil acidity, increased nutrient availability and lower metal(loid) stress, the best being the combination biochar-compost-iron sulfate. These ameliorations allowed a better plant growth. Finally, the metallicolous ecotype performed better in terms of growth than the non-metallicolous one and could thus be used in an assisted phytostabilization process on the former mine site.

Chitosan regulates metabolic balance, polyamine accumulation, and Na+ transport contributing to salt tolerance in creeping bentgrass.

BACKGROUND: Chitosan (CTS), a natural polysaccharide, exhibits multiple functions of stress adaptation regulation in plants. However, effects and mechanism of CTS on alleviating salt stress damage are still not fully understood. Objectives of this study were to investigate the function of CTS on improving salt tolerance associated with metabolic balance, polyamine (PAs) accumulation, and Na+ transport in creeping bentgrass (Agrostis stolonifera). RESULTS: CTS pretreatment significantly alleviated declines in relative water content, photosynthesis, photochemical efficiency, and water use efficiency in leaves under salt stress. Exogenous CTS increased endogenous PAs accumulation, antioxidant enzyme (SOD, POD, and CAT) activities, and sucrose accumulation and metabolism through the activation of sucrose synthase and pyruvate kinase activities, and inhibition of invertase activity. The CTS also improved total amino acids, glutamic acid, and γ-aminobutyric acid (GABA) accumulation. In addition, CTS-pretreated plants exhibited significantly higher Na+ content in roots and lower Na+ accumulation in leaves then untreated plants in response to salt stress. However, CTS had no significant effects on K+/Na+ ratio. Importantly, CTS enhanced salt overly sensitive (SOS) pathways and also up-regulated the expression of AsHKT1 and genes (AsNHX4, AsNHX5, and AsNHX6) encoding Na+/H+ exchangers under salt stress. CONCLUSIONS: The application of CTS increased antioxidant enzyme activities, thereby reducing oxidative damage to roots and leaves. CTS-induced increases in sucrose and GABA accumulation and metabolism played important roles in osmotic adjustment and energy metabolism during salt stress. The CTS also enhanced SOS pathway associated with Na+ excretion from cytosol into rhizosphere, increased AsHKT1 expression inhibiting Na+ transport to the photosynthetic tissues, and also up-regulated the expression of AsNHX4, AsNHX5, and AsNHX6 promoting the capacity of Na+ compartmentalization in roots and leaves under salt stress. In addition, CTS-induced PAs accumulation could be an important regulatory mechanism contributing to enhanced salt tolerance. These findings reveal new functions of CTS on regulating Na+ transport, enhancing sugars and amino acids metabolism for osmotic adjustment and energy supply, and increasing PAs accumulation when creeping bentgrass responds to salt stress.

Nitric Oxide Signal, Nitrogen Metabolism, and Water Balance Affected by γ-Aminobutyric Acid (GABA) in Relation to Enhanced Tolerance to Water Stress in Creeping Bentgrass.

γ-Aminobutyric acid (GABA) plays an important role in regulating stress tolerance in plants. Purposes of this study was to determine the effect of an exogenous supply of GABA on tolerance to water stress in creeping bentgrass (Agrostis stolonifera), and further reveal the GABA-induced key mechanisms related to water balance, nitrogen (N) metabolism and nitric oxide (NO) production in response to water stress. Plants were pretreated with or without 0.5 mM GABA solution in the roots for 3 days, and then subjected to water stress induced by -0.52 MPa polyethylene glycol 6000 for 12 days. The results showed that water stress caused leaf water deficit, chlorophyll (Chl) loss, oxidative damage (increases in superoxide anion, hydrogen peroxide, malondialdehyde, and protein carbonyl content), N insufficiency, and metabolic disturbance. However, the exogenous addition of GABA significantly increased endogenous GABA content, osmotic adjustment and antioxidant enzyme activities (superoxide dismutase, catalase, dehydroascorbate reductase, glutathione reductase and monodehydroascorbate reductase), followed by effectively alleviating water stress damage, including declines in oxidative damage, photoinhibition, and water and Chl loss. GABA supply not only provided more available N, but also affected N metabolism through activating nitrite reductase and glutamine synthetase activities under water stress. The supply of GABA did not increase glutamate content and glutamate decarboxylase activity, but enhanced glutamate dehydrogenase activity, which might indicate that GABA promoted the conversion and utilization of glutamate for maintaining Chl synthesis and tricarboxylic acid cycle when creeping bentgrass underwent water stress. In addition, GABA-induced NO production, depending on nitrate reductase and NO-associated protein pathways, could be associated with the enhancement of antioxidant defense. Current findings reveal the critical role of GABA in regulating signal transduction and metabolic homeostasis in plants under water-limited condition.

Identification and Pathogenicity of Bacteria Associated with Etiolation and Decline of Creeping Bentgrass Golf Course Putting Greens.

Bacterial etiolation and decline has developed into a widespread issue with creeping bentgrass (CBG) (Agrostis stolonifera) putting green turf. The condition is characterized by an abnormal elongation of turfgrass stems and leaves that in rare cases progresses into a rapid and widespread necrosis and decline. Recent reports have cited bacteria, Acidovorax avenae and Xanthomonas translucens, as causal agents; however, few cases exist where either bacterium were isolated in conjunction with turf exhibiting bacterial disease symptoms. From 2010 to 2014, turfgrass from 62 locations submitted to the NC State Turf Diagnostic Clinic exhibiting bacterial etiolation and/or decline symptoms were sampled for the presence of bacterial pathogens. Isolated bacteria were identified using rRNA sequencing of the 16S subunit and internal transcribed spacer region (16S-23S or ITS). Results showed diverse bacteria isolated from symptomatic turf and A. avenae and X. translucens were only isolated in 26% of samples. Frequently isolated bacterial species were examined for pathogenicity to 4-week-old 'G2' CBG seedlings and 8-week-old 'A-1' CBG turfgrass stands in the greenhouse. While results confirmed pathogenicity of A. avenae and X. translucens, Pantoea ananatis was also shown to infect CBG turf; although pathogenicity varied among isolated strains. These results illustrate that multiple bacteria are associated with bacterial disease and shed new light on culturable bacteria living in CBG turfgrass putting greens. Future research to evaluate additional microorganisms (i.e., bacteria and fungi) could provide new information on host-microbe interactions and possibly develop ideas for management tactics to reduce turfgrass pests.

Using a Genome-Based PCR Primer Prediction Pipeline to Develop Molecular Diagnostics for the Turfgrass Pathogen Acidovorax avenae.

Acidovorax avenae is the causal agent of bacterial etiolation and decline (BED) of creeping bentgrass, a poorly understood and often misdiagnosed disease that can result in considerable aesthetic and functional damage to golf course putting greens. Current diagnostics of BED are based on laborious culture-based methods. In this work, we employed a novel alignment-free primer prediction pipeline to design diagnostic primers for turfgrass-pathogenic A. avenae using 15 draft genomes of closely related target and nontarget Acidovorax spp. as input. Twenty candidate primer sets specific to turfgrass-pathogenic A. avenae were designed. The specificity and sensitivity of these primer sets were validated via a traditional polymerase chain reaction (PCR) and a real-time PCR assay. Primer sets 0017 and 0019 coupled with an internal oligo probe showed optimal sensitivity and specificity when evaluated with the target pathogen, closely related bacterial species, and microorganisms that inhabit the same host and soil environment. Finally, the accuracy of the newly developed real-time PCR assay was evaluated to detect BED pathogens from BED-symptomatic and asymptomatic turfgrass samples. The diagnostic results produced by the real-time PCR assay were consistent with results of a cultural-based method. This assay will allow quicker and more effective detection of the BED pathogen, thus potentially reducing misdiagnoses and unnecessary usage of fungicides.

Alteration of Transcripts of Stress-Protective Genes and Transcriptional Factors by γ-Aminobutyric Acid (GABA) Associated with Improved Heat and Drought Tolerance in Creeping Bentgrass (Agrostis stolonifera).

Gamma-aminobutyric acid (GABA) may play a positive role in regulating plant tolerance to drought or heat stress. The objectives of this study were to investigate the physiological effects of GABA on tolerance of creeping bentgrass (Agrostis stolonifera) to heat and drought stress and to determine whether enhanced heat and drought tolerance due to GABA treatment was associated with the up-regulation of selected genes and transcriptional factors involved in stress protection. Creeping bentgrass (cultivar "Penncross") plants were treated with 0.5 mM GABA or water (untreated control) as a foliar spray and were subsequently exposed to heat stress (35/30 °C, day/night), drought stress by withholding irrigation, or non-stress conditions in controlled-environment growth chambers. Exogenous application of GABA significantly improved plant tolerance to heat and drought stress, as reflected by increased leaf water content, cell membrane stability, and chlorophyll content. The analysis of gene transcript level revealed that exogenous GABA up-regulated the expression of ABF3, POD, APX, HSP90, DHN3, and MT1 during heat stress and the expression of CDPK26, MAPK1, ABF3, WRKY75, MYB13, HSP70, MT1, 14-3-3, and genes (SOD, CAT, POD, APX, MDHAR, DHAR, and GR) encoding antioxidant enzymes during drought stress. The up-regulation of the aforementioned stress-protective genes and transcriptional factors could contribute to improved heat and drought tolerance in creeping bentgrass.

Metabolic pathways regulated by abscisic acid, salicylic acid and γ-aminobutyric acid in association with improved drought tolerance in creeping bentgrass (Agrostis stolonifera).

Abscisic acid (ABA), salicylic acid (SA) and γ-aminobutyric acid (GABA) are known to play roles in regulating plant stress responses. This study was conducted to determine metabolites and associated pathways regulated by ABA, SA and GABA that could contribute to drought tolerance in creeping bentgrass (Agrostis stolonifera). Plants were foliar sprayed with ABA (5 µM), GABA (0.5 mM) and SA (10 µM) or water (untreated control) prior to 25 days drought stress in controlled growth chambers. Application of ABA, GABA or SA had similar positive effects on alleviating drought damages, as manifested by the maintenance of lower electrolyte leakage and greater relative water content in leaves of treated plants relative to the untreated control. Metabolic profiling showed that ABA, GABA and SA induced differential metabolic changes under drought stress. ABA mainly promoted the accumulation of organic acids associated with tricarboxylic acid cycle (aconitic acid, succinic acid, lactic acid and malic acid). SA strongly stimulated the accumulation of amino acids (proline, serine, threonine and alanine) and carbohydrates (glucose, mannose, fructose and cellobiose). GABA enhanced the accumulation of amino acids (GABA, glycine, valine, proline, 5-oxoproline, serine, threonine, aspartic acid and glutamic acid) and organic acids (malic acid, lactic acid, gluconic acid, malonic acid and ribonic acid). The enhanced drought tolerance could be mainly due to the enhanced respiration metabolism by ABA, amino acids and carbohydrates involved in osmotic adjustment (OA) and energy metabolism by SA, and amino acid metabolism related to OA and stress-defense secondary metabolism by GABA.

Constitutive Expression of Rice MicroRNA528 Alters Plant Development and Enhances Tolerance to Salinity Stress and Nitrogen Starvation in Creeping Bentgrass.

MicroRNA528 (miR528) is a conserved monocot-specific small RNA that has the potential of mediating multiple stress responses. So far, however, experimental functional studies of miR528 are lacking. Here, we report that overexpression of a rice (Oryza sativa) miR528 (Osa-miR528) in transgenic creeping bentgrass (Agrostis stolonifera) alters plant development and improves plant salt stress and nitrogen (N) deficiency tolerance. Morphologically, miR528-overexpressing transgenic plants display shortened internodes, increased tiller number, and upright growth. Improved salt stress resistance is associated with increased water retention, cell membrane integrity, chlorophyll content, capacity for maintaining potassium homeostasis, CATALASE activity, and reduced ASCORBIC ACID OXIDASE (AAO) activity; while enhanced tolerance to N deficiency is associated with increased biomass, total N accumulation and chlorophyll synthesis, nitrite reductase activity, and reduced AAO activity. In addition, AsAAO and COPPER ION BINDING PROTEIN1 are identified as two putative targets of miR528 in creeping bentgrass. Both of them respond to salinity and N starvation and are significantly down-regulated in miR528-overexpressing transgenics. Our data establish a key role that miR528 plays in modulating plant growth and development and in the plant response to salinity and N deficiency and indicate the potential of manipulating miR528 in improving plant abiotic stress resistance.

AsHSP17, a creeping bentgrass small heat shock protein modulates plant photosynthesis and ABA-dependent and independent signalling to attenuate plant response to abiotic stress.

Heat shock proteins (HSPs) are molecular chaperones that accumulate in response to heat and other abiotic stressors. Small HSPs (sHSPs) belong to the most ubiquitous HSP subgroup with molecular weights ranging from 12 to 42 kDa. We have cloned a new sHSP gene, AsHSP17 from creeping bentgrass (Agrostis stolonifera) and studied its role in plant response to environmental stress. AsHSP17 encodes a protein of 17 kDa. Its expression was strongly induced by heat in both leaf and root tissues, and by salt and abscisic acid (ABA) in roots. Transgenic Arabidopsis plants constitutively expressing AsHSP17 exhibited enhanced sensitivity to heat and salt stress accompanied by reduced leaf chlorophyll content and decreased photosynthesis under both normal and stressed conditions compared to wild type. Overexpression of AsHSP17 also led to hypersensitivity to exogenous ABA and salinity during germination and post-germinative growth. Gene expression analysis indicated that AsHSP17 modulates expression of photosynthesis-related genes and regulates ABA biosynthesis, metabolism and ABA signalling as well as ABA-independent stress signalling. Our results suggest that AsHSP17 may function as a protein chaperone to negatively regulate plant responses to adverse environmental stresses through modulating photosynthesis and ABA-dependent and independent signalling pathways.

Enhancing cytokinin synthesis by overexpressing ipt alleviated drought inhibition of root growth through activating ROS-scavenging systems in Agrostis stolonifera.

Drought stress limits root growth and inhibits cytokinin (CK) production. Increases in CK production through overexpression of isopentenyltransferase (ipt) alleviate drought damages to promote root growth. The objective of this study was to investigate whether CK-regulated root growth was involved in the alteration of reactive oxygen species (ROS) production and ROS scavenging capacity under drought stress. Wild-type (WT) creeping bentgrass (Agrostis stolonifera L. 'Penncross') and a transgenic line (S41) overexpressing ipt ligated to a senescence-activated promoter (SAG12) were exposed to drought stress for 21 d in growth chambers. SAG12-ipt transgenic S41 developed a more extensive root system under drought stress compared to the WT. Root physiological analysis (electrolyte leakage and lipid peroxidation) showed that S41 roots exhibited less cellular damage compared to the WT under drought stress. Roots of SAG12-ipt transgenic S41 had significantly higher endogenous CK content than the WT roots under drought stress. ROS (hydrogen peroxide and superoxide) content was significantly lower and content of total and free ascorbate was significantly higher in S41 roots compared to the WT roots under drought stress. Enzymatic assays and transcript abundance analysis showed that superoxide dismutase, catalase, peroxidase, and dehydroascorbate reductase were significantly higher in S41 roots compared to the WT roots under drought stress. S41 roots also maintained significantly higher alternative respiration rates compared to the WT under drought stress. The improved root growth of transgenic creeping bentgrass may be facilitated by CK-enhanced ROS scavenging through antioxidant accumulation and activation of antioxidant enzymes, as well as higher alternative respiration rates when soil water is limited.

Responses of Poa annua and three bentgrass species (Agrostis spp.) to adult and larval feeding of annual bluegrass weevil, Listronotus maculicollis (Coleoptera: Curculionidae).

The annual bluegrass weevil (ABW), Listronotus maculicollis Kirby, is an economically important pest of short-cut turfgrass in Eastern North America. Wide spread insecticide resistance warrants the development of alternative management strategies for this pest. ABW damage typically occurs in areas with a high percentage of annual bluegrass, Poa annua L., the preferred ABW host. Damage to bentgrasses, Agrostis spp., is much rarer and usually less severe. To aid the implementation of host plant resistance as an alternative ABW management strategy we investigated the tolerance of three bentgrass species to ABW feeding. Responses of P. annua, creeping bentgrass, Agrostis stolonifera L., colonial bentgrass, Agrostis capillaris L., and velvet bentgrass, Agrostis canina L., to adult and larval feeding were compared in greenhouse experiments. Grass responses were measured as visual damage, dry weight of the grass stems and leaves, color, density and overall grass quality. To determine possible mechanisms of grass tolerance constitutive fiber and silicon content were also determined. The three bentgrass species tolerated 2-3 times higher numbers of ABW adults and larvae than P. annua before displaying any significant quality decrease. Creeping bentgrass had the lowest damage ratings. ABW infestation caused higher plant yield reduction in P. annua (up to 42%) than in bentgrasses. Observed differences among the grass species in fiber and silicon content in the plant tissue are unlikely to play a role in the resistance of bentgrasses to ABW. Our findings clearly show that A. stolonifera is the best grass species for the implementation of host plant resistance in ABW management.

Proteins associated with heat-induced leaf senescence in creeping bentgrass as affected by foliar application of nitrogen, cytokinins, and an ethylene inhibitor.

Heat stress causes premature leaf senescence in cool-season grass species. The objective of this study was to identify proteins regulated by nitrogen, cytokinins, and ethylene inhibitor in relation to heat-induced leaf senescence in creeping bentgrass (Agrostis stolonifera). Plants (cv. Penncross) were foliar sprayed with 18 mM carbonyldiamide (N source), 25 µM aminoethoxyvinylglycine (AVG, ethylene inhibitor), 25 µM 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 treatments suppressed heat-induced leaf senescence, as shown by higher turf quality and chlorophyll content, and lower electrolyte leakage in treated plants compared to the untreated control. A total of 49 proteins were responsive to N, AVG, or ZR under heat stress. The abundance of proteins in photosynthesis increased, with ribulose-1,5-bisphosphate carboxylase/oxygenase affected by all three treatments, chlorophyll a/b-binding protein by AVG and N or Rubisco activase by AVG. Proteins for amino acid metabolism were upregulated, including alanine aminotransferase by three treatments and ferredoxin-dependent glutamate synthase by AVG and N. Upregulated proteins also included catalase by AVG and N and heat shock protein by ZR. Exogenous applications of AVG, ZR, or N downregulated proteins in respiration (enolase, glyceraldehyde 3-phosphate dehydrogenase, and succinate dehygrogenase) under heat stress. Alleviation of heat-induced senescence by N, AVG, or ZR was associated with enhanced protein abundance in photosynthesis and amino acid metabolism and stress defense systems (heat shock protection and antioxidants), as well as suppression of those imparting respiration metabolism.

Selection of reference genes for quantitative real-time PCR normalization in creeping bentgrass involved in four abiotic stresses.

KEY MESSAGE: This study identified stable reference genes for normalization of gene expression data in qRT-PCR analysis of leaf and root tissues in creeping bentgrass under four abiotic stresses. Examination of gene expression using quantitative real-time PCR (qRT-PCR) in plant responses to abiotic stresses can provide valuable information for stress-tolerance improvement. Selecting stable reference genes for qRT-PCR analysis is critically important. The objective of this study was to determine the stability of expression for eight candidate reference genes (ACT, EF1a, TUB, UPL7, GAPDH, PP2A, PEPKR1, and CACS) in two tissues (roots and leaves) of a perennial grass species under four abiotic stresses (salt, drought, cold, and heat) using four programs (GeNorm, NormFinder, BestKeeper, and RefFinder). The results showed that (1) the combinations of CACS and UPL7 or PP2A and ACT were stably expressed in salt-treated roots or leaves; (2) the combinations of GAPDH and CACS or PP2A and PEPKR1 were stable in roots and leaves under drought stress; (3) CACS and PP2A exhibited stable expression in cold-treated roots and the combination of EF1a and UPL7 was also stable in cold-treated leaves; and (4) CACS and PP2A were the two most stable reference genes in heat-stressed roots and UPL7 combined with GAPDH and PP2A was stably expressed in heat-stressed leaves. The qRT-PCR analysis of a target gene, AsSAP expression patterns in response to salinity and drought stress, confirmed the reliability of those selected and stable reference genes. Identification of stable reference genes in creeping bentgrass will improve assay accuracy for selecting stress-tolerance genes and identifying molecular mechanisms conferring stress tolerance in this species.

Characterization of a small constitutive promoter from Arabidopsis translationally controlled tumor protein (AtTCTP) gene for plant transformation.

KEY MESSAGE: A plant-derived 0.3 kb constitutive promoter was obtained from AtTCTP expression analysis, and successfully applied to the expression of a selectable marker gene for production of transgenic creeping bentgrass plants. The isolation and use of an efficient promoter is essential to develop a vector system for efficient genetic transformation of plants, and constitutive promoters are particularly useful for the expression of selectable marker genes. In this study, we characterized a small size of the constitutive promoter from the expression analysis of Arabidopsis thaliana translationally controlled tumor protein (AtTCTP) gene. Histochemical and fluorometric GUS analyses revealed that a 303 bp upstream region from the start codon of the AtTCTP gene showed strong GUS expression throughout all plant tissues, which is approximately 55 % GUS activity compared with the cauliflower mosaic virus 35S promoter (35Spro). To examine the possible application of this promoter for the development of genetically engineered crops, we introduced pCAMBIA3301 vector harboring the 0.3 kb promoter of AtTCTP (0.3kbpro) that was fused to the herbicide resistance BAR gene (0.3kb pro ::BAR) into creeping bentgrass. Our transformation results demonstrate that transgenic creeping bentgrass plants with herbicide resistance were successfully produced using 0.3kb pro ::BAR as a selectable marker. Northern blot analysis revealed that the transgenic plants with 0.3kb pro ::BAR showed reduced but comparable expression levels of BAR to those with 35S pro ::BAR. Moreover, the transcription activity of the 0.3 kb promoter could be increased by the fusion of an enhancer sequence. These results indicate that the 0.3 kb AtTCTP promoter can be used as a plant-derived constitutive promoter for the expression of selectable marker genes, which facilitates its use as an alternative to the 35S promoter for developing genetically engineered crops.