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1. Gene flow from transgenic crops to feral populations and naturalized compatible relatives has been raised as one of the main issues for the deregulation of transgenic events. Creeping bentgrass, Agrostis stolonifera L., is a perennial, outcrossing grass that propagates by seeds and stolons. Transgenic Roundup Ready® glyphosate-resistant creeping bentgrass (GRCB), which is currently under USDA-APHIS regulated status, was planted in 2002 on 162 ha within a production control area in Oregon, USA. 2. We conducted a study to assess transgene flow from the GRCB fields. A survey within and around the production control area was performed during the year when the GRCB fields produced seed and for 3 years after the fields were taken out of production. Transgene flow was determined by testing creeping bentgrass and its relatives for expression of the glyphosate resistance transgene. 3. While GRCB seed-production practices were strictly regulated, evidence of transgene flow was found in all years. In 2006, 3 years after the transgene source fields were taken out of production and a mitigation programme was initiated, 62% of the 585 creeping bentgrass plants tested in situ were glyphosate-resistant (GR). Our results document not only the movement of the glyphosate resistance transgene from the fields, but also the establishment and persistence of high frequencies of GR plants in the area, confirming that it was unrealistic to think that containment or eradication of GRCB could be accomplished. 4. Synthesis and applications: These findings highlight the potential for transgene escape and gene flow at a landscape level. The survey provides empirical frequencies that can be used to design monitoring and management methods for genetically engineered (GE) varieties of outcrossing, wind-pollinated, perennial grasses and to evaluate the potential for coexistence of GE and non-GE grass seed crops. Such information should also be used in the decision-making process for authorization of field trials and deregulation of genetic engineering events.

Hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferase (HCT) is considered to be an essential enzyme for regulating the biosynthesis and composition of lignin. To investigate the properties and function of ZjHCT4, the ZjHCT4 gene was cloned from Zoysia japonica with a completed coding sequence of 1284-bp in length, encoding 428 amino acids. The ZjHCT4 gene promoter has several methyl jasmonate (MeJA) response elements. According to analysis of expression patterns, it was up-regulated by MeJA, GA3 (Gibberellin), and SA (Salicylic acid), and down-regulated by ABA (Abscisic acid). Ectopic ZjHCT4 expression in creeping bentgrass causes excessive plant elongation. In addition, the content of G-lingnin and H-lingnin fell in transgenic plants, whereas the level of S-lingnin increased, resulting in a considerable rise in the S/G unit ratio. Analysis of the expression levels of lignin-related genes revealed that the ectopic expression of ZjHCT4 altered the expression levels of a number of genes involved in the lignin synthesis pathway. Simultaneously, MeJA, SA, GA3, IAA, BR (Brassinosteroid), and other hormones were dramatically enhanced in transgenic plants relative to control plants, whereas ABA concentration was significantly decreased. Expression of ZjHCT4 impacted lignin composition and plant growth via altering the phenylpropionic acid metabolic pathway and hormone response, as revealed by transcriptome analysis. HCTs may influence plant lignin composition and plant development by altering hormone content. These findings contributed to a deeper comprehension of the lignin synthesis pathway and set the stage for further investigation and application of the HCTs gene.

Background MYB transcription factors play a crucial regulatory role in plant growth and stress response. The gene EpMYB , obtained from Endocarpon pusillum , a dominant lichen in the Tengger Desert, was transferred to creeping bentgrass to explore its effects on plant growth and response to abiotic stress. Results Compared to wild-type (WT), transgenic (TG) plants exhibited a faster growth rate, a significantly higher number of leaves per tiller, increased internode length, and longer maximum leaf length. However, some of the leaves were severely twisted. Additionally, the antioxidant enzyme content, lignin content and drought tolerance of the TG plants was significantly enhanced. RNA-seq analysis revealed that differentially expressed genes (DEGs) in the TG-vs-WT were primarily associated with pathways such as photosynthesis, wax biosynthesis, lipid metabolism, and flavonoid biosynthesis. By comparing the TG (Drought treatment)-vs-TG and WT (Drought treatment)-vs-WT groups, numerous DEGs related to growth, development, and stress tolerance were identified, including aldehyde decarbonylase gene( CER1 ), lignin synthesis gene ( HCT ), adenylate dimethylallyltransferase gene ( IPT ), peroxin-10 gene ( PEX10 ), among others. These results suggest that the EpMYB gene enhances the drought tolerance of transgenic creeping bentgrass by regulating photosynthesis, antioxidant enzyme activity, lignin synthesis, wax synthesis, and lipid metabolism. Conclusion These findings suggest that the EpMYB gene functions as a positive regulator of plant growth and development, while also playing a crucial role in the plant’s response to drought stress. Furthermore, this study demonstrates the feasibility of selecting specific functional genes from stress-tolerant microorganisms and applying them to plants to enhance stress resistance.

MicroRNA319 (miR319) has been demonstrated to regulate plant development and responses to stress such as drought and salt. However, its role in thermotolerance, particularly in cool season grasses, remains unclear. Here we report that miR319 plays a negative role in heat tolerance of creeping bentgrass (Agrostis stolonifera). A basic helix–loop–helix (bHLH) transcription factor, AsbHLH094 was identified as the target gene of miR319, and its expression was significantly downregulated in the miR319-overexpressing (OE319) transgenic creeping bentgrass lines. Functional characterisation revealed that overexpression of AsbHLH094 enhanced heat tolerance of the transgenic tobacco plants. Furthermore, protein–protein interaction assays confirmed that AsbHLH094 physically interacts with AsIAA1, an Aux/IAA protein involved in auxin signalling. Transcriptomic analysis showed that auxin biosynthesis genes such as TARs, YUCCAs, along with auxin-response genes including Auxin/IAAs and ARFs were downregulated in the OE319 transgenic creeping bentgrass plants, leading to reduced auxin accumulation, while elevated auxin levels and induced changes in auxin biosynthesis- and response-related genes were observed in the AsbHLH094 overexpression tobacco. Endogenous indole-3-acetic acid (IAA) levels in creeping bentgrass were significantly increased under high-temperature conditions, and exogenous application of IAA at appropriate concentrations improved heat tolerance in creeping bentgrass. Together, our findings reveal a previously uncharacterized miR319-AsbHLH094 regulatory module that modulates auxin biosynthesis and signalling, thereby contributing to heat stress responses in creeping bentgrass

Recently, there has been renewed interest in reducing excess inputs into turf, with a special emphasis on reducing water use. One potential mechanism to achieve this goal is the use of plant growth–promoting rhizobacteria (PGPR) applications. Plant growth–promoting rhizobacteria research in turfgrass is limited, but the few studies that have been conducted show that PGPR can reduce biotic and abiotic stress in turfgrass. Two creeping bentgrass ( Agrostis stolonifera ) cultivars (Penncross and 007) were treated with either PGPR Blend 20, PGPR DH44, water, or nitrogen fertilizer before being subjected to a dry-down period during which half of each treatment was either irrigated or drought stressed. Results indicate that PGPR DH44 may help maintain greater quality in drought-stressed ‘007’ creeping bentgrass compared with nitrogen-treated plots; however, quality did not differ between DH44- and water-treated plots. When drought stressed, PGPR did not help creeping bentgrass maintain clipping yield compared with nitrogen-fertilized or water-treated plots, nor did PGPR affect root biomass. More research is needed before recommendations can be made regarding PGPR applications to turfgrass.

Background Small heat shock proteins (sHSPs) are critical for plant response to biotic and abiotic stresses, especially heat stress. They have also been implicated in various aspects of plant development. However, the acting mechanisms of the sHSPs in plants, especially in perennial grass species, remain largely elusive. Results In this study, AsHSP26.8a , a novel chloroplast-localized sHSP gene from creeping bentgrass ( Agrostis stolonifera L.) was cloned and its role in plant response to environmental stress was studied. AsHSP26.8a encodes a protein of 26.8 kDa. Its expression was strongly induced in both leaf and root tissues by heat stress. Transgenic Arabidopsis plants overexpressing AsHSP26.8a displayed reduced tolerance to heat stress. Furthermore, overexpression of AsHSP26.8a resulted in hypersensitivity to hormone ABA and salinity stress. Global gene expression analysis revealed AsHSP26.8a-modulated expression of heat-shock transcription factor gene, and the involvement of AsHSP26.8a in ABA-dependent and -independent as well as other stress signaling pathways. Conclusions Our results suggest that AsHSP26.8a may negatively regulate plant response to various abiotic stresses through modulating ABA and other stress signaling pathways.

Plant defense against heat stress involves adjustments in amino acid metabolism. The objective of this study was to identify major amino acids and associated metabolic pathways differentially regulated by γ-aminobutyric acid (GABA) and proline that may contribute to augmentation of heat tolerance in cool-season grass species. Creeping bentgrass (Agrostis stolonifera L. cv. ‘Penncross’) was exposed to non-stress (22/18 °C, day/night) or heat stress (35/30 °C, day/night) conditions for 35 d in controlled-environment growth chambers. Non-stressed and heat-stressed plants were foliar-sprayed with water (untreated control), GABA, proline, or ammonium nitrate (N) as a nitrogen source control. Under heat stress, foliar application of GABA, proline, or N significantly increased turf quality and leaf chlorophyll content compared to untreated control plants. N application had nutritional effects, resulting in increases in the content of all amino acids under heat stress. Application of GABA under heat stress significantly increased endogenous content of glutamic acid, GABA, and threonine. Plants treated with proline under heat stress had significantly higher endogenous levels of proline, GABA, glutamic acid, aspartic acid, lysine, isoleucine, leucine, valine, serine, alanine, threonine, and tryptophan compared to untreated controls. The improved heat tolerance in creeping bentgrass pathway by GABA was mainly associated with regulation of amino acid metabolism in the GABA shunt and oxaloacetate pathways. Proline-enhanced heat tolerance involved the regulation of five metabolic pathways (GABA shunt, oxaloacetate, 3-phosphoglycerate, secondary metabolism, and pyruvate). GABA and proline, as well as their responsive amino acids could be used as biomarkers to improve heat tolerance in cool-season grass species.

Improvement of endogenous polyamine (PA) levels by exogenous spermidine (Spd) application in leaves may promote drought tolerance of plants. In this study, leaves of creeping bentgrass (cv. ‘Penn-A4’) were pretreated with 0.2 mmol Spd every another day for three cycles before water was limited for 15 days in a growth chamber. The application of exogenous Spd elevated the accumulation of endogenous PA, including putrescine, Spd and spermine in leaves of creeping bentgrass under drought stress. Exogenous Spd effectively alleviated the damage effects from drought stress, as demonstrated by lower O₂·⁻generation rate, H₂O₂and malondialdehyde content, higher relative water content, chlorophyll content and antioxidant enzyme activities (superoxide dismutase, peroxidase, catalase, and ascorbate peroxidase) as compared to untreated plants. Additionally, the pretreatment with Spd resulted in a lower accumulation of indole-3-acetic acid (IAA) and gibberellin A₃(GA₃) after 15 days of drought stress, but an unchanged accumulation of abscisic acid compared to the untreated ones. These results suggest that ameliorating drought stress through exogenously applied Spd may be associated with increased antioxidant protection capability and after a longer stress treatment with changed IAA and GA₃accumulation level on account of improved endogenous PA content. In addition, drought tolerance mechanisms related to PA regulation of phytohormones are very complex and deserve further investigation.

Heat-induced leaf senescence may be related to protein hydrolysis into amino acids due to increased protease enzyme activities. Protease inhibitors may delay heat-induced leaf senescence and improve stay-green qualities in cool-season, perennial plants that are sensitive to high temperatures. The objectives of this study included examining whether exogenous application of protease inhibitors in the cysteine, serine, or aspartic classes may suppress heat-induced leaf senescence in the heat-sensitive grass species, creeping bentgrass ( Agrostis stolonifera ) and determining whether their effects on heat tolerance are associated with alterations in protein degradation and amino acid metabolism. Creeping bentgrass plants (cv. Penncross) were exposed to heat stress (35/30 °C, day/night) or non-stress control (22/18 °C, day/night) temperatures for 35 d in environment-controlled growth chambers and were foliar-sprayed weekly with 1 µM leupeptin, 10 µM aprotinin, or 10 µM pepstatin A. Turf quality was significantly higher from 14 to 35 d of heat stress in leupeptin- or pepstatin A-treated plants and from 14 to 28 d of heat stress in aprotinin-treated plants. Photochemical efficiency was significantly higher in plants treated with any of the protease inhibitors from 21 to 35 d of heat stress. Chlorophyll content was significantly higher in leupeptin- and aprotinin-treated from 7 to 35 d of heat stress and in pepstatin A-treated plants at 7 d and from 21 to 35 d of heat stress. Under heat stress, endogenous protein content was significantly higher in all protease inhibitor-treated plants while general protease activity and the content of a majority of proteinogenic amino acids were significantly lower, suggesting that the rate of protein hydrolysis was lower. These findings indicate that exogenous application of protease inhibitors can suppress heat-induced leaf senescence and enhance turf performance by suppressing proteolysis.

Key message The creeping bentgrass small heat shock protein AsHSP26.2 positively regulates plant growth and is a novel candidate for use in crop genetic engineering for enhanced biomass production and grain yield. Small heat shock proteins (sHSPs), a family of proteins with high level of diversity, significantly influence plant stress tolerance and plant development. We have cloned a creeping bentgrass chloroplast-localized sHSP gene, AsHSP26.2 responsive to IAA, GA and 6-BA stimulation. Transgenic creeping bentgrass overexpressing AsHSP26.2 exhibited significantly enhanced plant growth with increased stolon number and length as well as enlarged leaf blade width and leaf sheath diameters, but inhibited leaf trichomes initiation and development in the abaxial epidermis. These phenotypes are completely opposite to those displayed in the transgenic plants overexpressing AsHSP26.8 , another chloroplast sHSP26 isoform that contains additional seven amino acids (AEGQGDG) between the consensus regions III and IV (Sun et al., Plant Cell Environ 44:1769–1787, 2021). Furthermore, AsHSP26.2 overexpression altered phytohormone biosynthesis and signaling transduction, resulting in elevated auxin and gibberellins (GA) accumulation. The results obtained provide novel insights implicating the sHSPs in plant growth and development regulation, and strongly suggest AsHSP26.2 to be a novel candidate for use in crop genetic engineering for enhanced plant biomass production and grain yield.