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.

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.

AsHSP26.2, a creeping bentgrass chloroplast small heat shock protein positively regulates plant development.

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.

Genome Resources for Four Clarireedia Species Causing Dollar Spot on Diverse Turfgrasses.

Dollar spot (DS) is a destructive fungal disease impacting almost all warm- and cool-season turfgrasses worldwide. Multiple fungal species in the genus Clarireedia are causal agents of DS. Here, we present whole-genome assemblies of nine fungal isolates in the genus Clarireedia, including four species (C. paspali, C. hainanense, C. jacksonii, and C. monteithiana) causing DS on seashore paspalum (Paspalum vaginatum Sw.), creeping bentgrass (Agrostis stolonifera L.), and Kentucky bluegrass (Poa pratensis L.) in China. This work provides valuable baseline genomic data to support further research and management of DS pathogens on turfgrasses.

Functional annotation of creeping bentgrass protein sequences based on convolutional neural network.

BACKGROUND: Creeping bentgrass (Agrostis soionifera) is a perennial grass of Gramineae, belonging to cold season turfgrass, but has poor disease resistance. Up to now, little is known about the induced systemic resistance (ISR) mechanism, especially the relevant functional proteins, which is important to disease resistance of turfgrass. Achieving more information of proteins of infected creeping bentgrass is helpful to understand the ISR mechanism. RESULTS: With BDO treatment, creeping bentgrass seedlings were grown, and the ISR response was induced by infecting Rhizoctonia solani. High-quality protein sequences of creeping bentgrass seedlings were obtained. Some of protein sequences were functionally annotated according to the database alignment while a large part of the obtained protein sequences was left non-annotated. To treat the non-annotated sequences, a prediction model based on convolutional neural network was established with the dataset from Uniport database in three domains to acquire good performance, especially the higher false positive control rate. With established model, the non-annotated protein sequences of creeping bentgrass were analyzed to annotate proteins relevant to disease-resistance response and signal transduction. CONCLUSIONS: The prediction model based on convolutional neural network was successfully applied to select good candidates of the proteins with functions relevant to the ISR mechanism from the protein sequences which cannot be annotated by database alignment. The waste of sequence data can be avoided, and research time and labor will be saved in further research of protein of creeping bentgrass by molecular biology technology. It also provides reference for other sequence analysis of turfgrass disease-resistance research.

Expression of a Chlorophyll b Reductase Gene from Zoysia japonica Causes Changes in Leaf Color and Chlorophyll Morphology in Agrostis stolonifera.

The NYC-like (NOL) enzyme is considered as an essential enzyme for chlorophyll b degradation, which catalyzes the formation of 7-hydroxymethyl chlorophyll a from chlorophyll b. The ZjNOL gene was cloned from Zoysia japonica with a completed coding sequence of 981-bp in length, encoding 326 amino acids. ZjNOL was localized on the stroma side of the thylakoid membrane, and co-localized with ZjNYC in the chloroplasts. Multiple photoregulatory elements and hormone regulatory elements were identified in the promoter region of the ZjNOL gene, and the expression level of the ZjNOL gene was dramatically up-regulated in senescence leaves, which were regulated by a variety of plant hormones. ZjNOL's ectopic expression in creeping bentgrass produced yellow leaves, thicker cortex, and smaller vascular column cells. Additionally, transgenic plants exhibited morphological alterations in their chloroplast structure, and the number of grana and thylakoids per grana stack reduced dramatically. Transgenic plants also had a lower photosynthetic rate and Fm/Fv than the control. The transgenic plants displayed a decreased chlorophyll content and a greater rate of ion leakage. The properties and activities of ZjNOL will serve as a foundation for future research into gene functions and regulatory processes.

Expression of a Hydroxycinnamoyl-CoA Shikimate/Quinate Hydroxycinnamoyl Transferase 4 Gene from Zoysia japonica (ZjHCT4) Causes Excessive Elongation and Lignin Composition Changes in Agrostis stolonifera.

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.

Real-Time PCR Detection of Clarireedia spp., the Causal Agents of Dollar Spot in Turfgrasses.

Dollar spot is one of the most economically important diseases of turfgrasses. Recent taxonomic revisions have placed the dollar spot fungal pathogens in the new genus Clarireedia, with five species described. The main goal of this study was to develop a quantitative real-time PCR (qPCR) molecular detection assay based on the internal transcribed spacer (ITS) of the ribosomal RNA genes to quantify the abundance of Clarireedia spp. from environmental (field) samples. The qPCR assay was able to detect isolates of the four tested Clarireedia spp. but did not cross react with nontarget fungi, including closely related taxa, other turfgrass pathogens, or other fungal species commonly isolated from turfgrass. The assay is capable of detecting as little as 38.0 fg (3.8 × 10-14 g) of Clarireedia genomic DNA in 3 h. The qPCR assay detected Clarireedia spp. in both symptomatic and asymptomatic creeping bentgrass (Agrostis stolonifera) foliar tissue. Clarireedia spp. were rarely detected in the thatch or soil, indicating that these pathogens are not widely distributed in these areas of the environment. The fact that the pathogen was detected in asymptomatic tissue suggests that creeping bentgrass may be able to tolerate a certain quantity of the pathogens in leaves before disease symptoms appear; however, further research is needed to validate this hypothesis.

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.

Recent developments in metabolomics-based research in understanding transgenic grass metabolism.

BACKGROUND: Transgenic herbicide-resistant (HR) turfgrass together with its associated, broad spectrum herbicides promise cheap, selective and efficient weed control by excluding infested weeds resulting in turf lawn with high uniformity and aesthetic value. The concept of this "weeding program" initiated from modern biotechnology has been widely implemented in several principal crops including maize, soybean, canola and cotton as early as the 1990s. Transgenic HR turfgrass classified as a genetically modified organism (GMO) has undoubtedly caused public concern with respect to its biosafety and legalities similar to well-established HR crops. Nevertheless, applying metabolomics-based approaches which focuses on the identification of the global metabolic state of a biological system in response to either internal or external stimuli can also provide a comprehensive characterization of transgenic grass metabolism and its involvement in biosecurity and public perception. AIM OF REVIEW: This review summaries the recent applications of metabolomics applied to HR crops to predict the molecular and physiological phenotypes of HR turfgrass species, glyphosate-resistant Kentucky bluegrass (Poa pratensis L.) and glufosinate-resistant creeping bentgrass (Agrotis stonifera L.). Additionally, this review also presents background knowledge with respect to the application of metabolomics, transformation of HR crops and its biosafety concerns, turfgrass botanical knowledge and its economic and aesthetic value. KEY SCIENTIFIC CONCEPTS OF REVIEW: The purpose of this review is to demonstrate the molecular and physiological phenotypes of HR turfgrass based on several lines of evidence primarily derived from metabolomics data applied to HR crops to identify alterations on HR turfgrass metabolism as a result of genetic modification that confers resistant traits.

Regulation of Heat Shock Factor Pathways by γ-aminobutyric Acid (GABA) Associated with Thermotolerance of Creeping Bentgrass.

Activation and enhancement of heat shock factor (HSF) pathways are important adaptive responses to heat stress in plants. The γ-aminobutyric acid (GABA) plays an important role in regulating heat tolerance, but it is unclear whether GABA-induced thermotolerance is associated with activation of HSF pathways in plants. In this study, the changes of endogenous GABA level affecting physiological responses and genes involved in HSF pathways were investigated in creeping bentgrass during heat stress. The increase in endogenous GABA content induced by exogenous application of GABA effectively alleviated heat damage, as reflected by higher leaf relative water content, cell membrane stability, photosynthesis, and lower oxidative damage. Contrarily, the inhibition of GABA accumulation by the application of GABA biosynthesis inhibitor further aggravated heat damage. Transcriptional analyses showed that exogenous GABA could significantly upregulate transcript levels of genes encoding heat shock factor HSFs (HSFA-6a, HSFA-2c, and HSFB-2b), heat shock proteins (HSP17.8, HSP26.7, HSP70, and HSP90.1-b1), and ascorbate peroxidase 3 (APX3), whereas the inhibition of GABA biosynthesis depressed these genes expression under heat stress. Our results indicate GABA regulates thermotolerance associated with activation and enhancement of HSF pathways in creeping bentgrass.

Transgenic creeping bentgrass plants expressing a Picea wilsonii dehydrin gene (PicW) demonstrate improved freezing tolerance.

Agrostis stolonifera L. 'Penn A-4' is a common creeping bentgrass species that is widely used in urban landscaping and golf courses. To prolong the green stage of this grass, a dehydrin gene PicW isolated from Wilson's spruce (Picea wilsonii) was transformed into plants of 'Penn A-4' cultivar via a straightforward stolon node infection system. A putative transgenic plant was obtained and its tolerance to low-temperature stress was evaluated. When the transgenic line was subjected to a freezing (- 5 °C) treatment, it showed better viability and more robust physiology than wild type, as evidenced by higher soluble sugar and proline contents, and lower relative electrical conductivity and malondialdehyde content. The transgenic line also showed tolerance to a chilling treatment (5 °C), although its performance was not significantly different from that of wild-type plants. Overall, the research here clearly revealed the explicit role of PicW in increasing freezing tolerance of grass at the whole-plant level, and demonstrated that the straightforward stolon node transformation method could be well used to genetically modify turfgrass. The obtained transgenic line might be as genetic resource for breeding program and practiced to grow in cold temperate zones.

Transcript Profiling and Gene Identification Involved in the Ethylene Signal Transduction Pathways of Creeping Bentgrass (Agrostis stolonifera) during ISR Response Induced by Butanediol.

Creeping bentgrass (Agrostis stolonifera) is the preferred green lawn grass, with excellent turf characteristics but poor disease resistance. At present, the mechanisms of disease resistance in creeping bentgrass are poorly understood, especially the ethylene signal transduction pathway under the induced systemic resistance (ISR) response. In this study, butanediol (BDO), as a new type of disease-resistance compound, was applied to creeping bentgrass seedlings to induce the ISR response. Then, we measured ethylene production and related enzyme activities. Additionally, transcript profiling and gene identification were performed in association to ethylene signal transduction pathways. The changes of ethylene production and related enzyme 1-aminocyclopropane-1-carboxylic acid oxidase (ACO) and 1-aminocyclopropane-1-carboxylic acid synthases (ACS) activities showed significant difference at 24 h after Rhizoctonia solani inoculation among five treatments of various BDO concentrations. After 100 µmol L-1 BDO treatment, ethylene production and related enzyme activities reached their peak levels. Additionally, 208,672 unigenes of creeping bentgrass were obtained by de novo assembly. In total, 15,903 annotated unigenes were grouped into 33 canonical pathways in the KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. Among those, 1803 unigenes were classified as 'signal transduction'. There were 6766 differentially expressed genes (DEGs) among B24 (inoculated-rhizobacteria in MS medium with 100 µmol L-1 BDO for 24 h), NB24, B72 and NB24 (no rhizobacteria in MS medium with 100 µmol L-1 BDO for 24 h) libraries, and 4,639 DEGs between B24 and B72 (inoculated-rhizobacteria in MS medium with 100 µmol L-1 BDO for 72 h) libraries, with 4489 DEGs in all three libraries. As suggested by the RT-PCR assay, the expression levels of ethylene-responsive and defense-related genes were variable among treated samples during the BDO-induced ISR responses. The expression levels of EIN, ERF, NPR1, PR3 and PR4 genes increased and reached their peaks in the first 24 h after R. solani infection in the BDO-induced ISR reaction compared with NB24 treatments. This results is consistent with the changes of important ethylene biosynthetic enzymes and ethylene concentrations during the BDO-induced ISR responses. We further found the intermediate substances for the signaling pathway, and the relationships between the expression levels of BDO-induced ISR disease-resistance genes and those of the response genes for ethylene signal pathway. Our findings present a genetic basis for systemic resistance of creeping bentgrass through transcriptomic analysis and our study provides a theoretical and practical basis for the improvement of turfgrass disease resistance and quality.

Ectopic expression of a cyanobacterial flavodoxin in creeping bentgrass impacts plant development and confers broad abiotic stress tolerance.

Flavodoxin (Fld) plays a pivotal role in photosynthetic microorganisms as an alternative electron carrier flavoprotein under adverse environmental conditions. Cyanobacterial Fld has been demonstrated to be able to substitute ferredoxin of higher plants in most electron transfer processes under stressful conditions. We have explored the potential of Fld for use in improving plant stress response in creeping bentgrass (Agrostis stolonifera L.). Overexpression of Fld altered plant growth and development. Most significantly, transgenic plants exhibited drastically enhanced performance under oxidative, drought and heat stress as well as nitrogen (N) starvation, which was associated with higher water retention and cell membrane integrity than wild-type controls, modified expression of heat-shock protein genes, production of more reduced thioredoxin, elevated N accumulation and total chlorophyll content as well as up-regulated expression of nitrite reductase and N transporter genes. Further analysis revealed that the expression of other stress-related genes was also impacted in Fld-expressing transgenics. Our data establish a key role of Fld in modulating plant growth and development and plant response to multiple sources of adverse environmental conditions in crop species. This demonstrates the feasibility of manipulating Fld in crop species for genetic engineering of plant stress tolerance.

Expression in grasses of multiple transgenes for degradation of munitions compounds on live-fire training ranges.

The deposition of toxic munitions compounds, such as hexahydro-1, 3, 5-trinitro-1, 3, 5-triazine (RDX), on soils around targets in live-fire training ranges is an important source of groundwater contamination. Plants take up RDX but do not significantly degrade it. Reported here is the transformation of two perennial grass species, switchgrass (Panicum virgatum) and creeping bentgrass (Agrostis stolonifera), with the genes for degradation of RDX. These species possess a number of agronomic traits making them well equipped for the uptake and removal of RDX from root zone leachates. Transformation vectors were constructed with xplA and xplB, which confer the ability to degrade RDX, and nfsI, which encodes a nitroreductase for the detoxification of the co-contaminating explosive 2, 4, 6-trinitrotoluene (TNT). The vectors were transformed into the grass species using Agrobacterium tumefaciens infection. All transformed grass lines showing high transgene expression levels removed significantly more RDX from hydroponic solutions and retained significantly less RDX in their leaf tissues than wild-type plants. Soil columns planted with the best-performing switchgrass line were able to prevent leaching of RDX through a 0.5-m root zone. These plants represent a promising plant biotechnology to sustainably remove RDX from training range soil, thus preventing contamination of groundwater.

Transcriptional Responses of Creeping Bentgrass to 2,3-Butanediol, a Bacterial Volatile Compound (BVC) Analogue.

Bacterial volatile compounds (BVCs) have been reported to enhance plant growth and elicit plant defenses against fungal infection and insect damage. The objective of this study was to determine transcriptomic changes in response to synthetic BVC that could be associated with plant resistance to Rhizoctonia solani in creeping bentgrass. The 2,3-butanediol (BD) (250 µM) was sprayed on creeping bentgrass leaves grown in jam jars. The result showed that synthetic BD induced plant defense against R. solani for creeping bentgrass. Transcriptomic analysis demonstrated that more genes were repressed by BD while less showed up-regulation. BD suppressed the expression of some regular stress-related genes in creeping bentgrass, such as pheromone activity, calcium channel activity, photosystem II oxygen evolving complex, and hydrolase activity, while up-regulated defense related transcription factors (TFs), such as basic helix-loop-helix (bHLH) TFs, cysteine2-cysteine2-contans-like (C2C2-CO) and no apical meristem TFs (NAC). Other genes related to disease resistance, such as jasmonic acid (JA) signaling, leucine rich repeats (LRR)-transmembrane protein kinase, pathogen-related (PR) gene 5 receptor kinase and nucleotide binding site-leucine rich repeats (NBS-LRR) domain containing plant resistance gene (R-gene) were also significantly up-regulated. These results suggest that BD may induce changes to the plant transcriptome in induced systemic resistance (ISR) pathways.

Copper stress-induced changes in leaf soluble proteome of Cu-sensitive and tolerant Agrostis capillaris L. populations.

Changes in leaf soluble proteome were explored in 3-month-old plants of metallicolous (M) and nonmetallicolous (NM) Agrostis capillaris L. populations exposed to increasing Cu concentrations (1-50 µM) to investigate molecular mechanisms underlying plant responses to Cu excess and tolerance of M plants. Plants were cultivated on perlite (CuSO4 spiked-nutrient solution). Soluble proteins, extracted by the trichloroacetic acid/acetone procedure, were separated with 2-DE (linear 4-7 pH gradient). Analysis of CCB-stained gels (PDQuest) reproducibly detected 214 spots, and 64 proteins differentially expressed were identified using LC-MS/MS. In both populations, Cu excess impacted both light-dependent (OEE, cytochrome b6-f complex, and chlorophyll a-b binding protein), and -independent (RuBisCO) photosynthesis reactions, more intensively in NM leaves (ferredoxin-NADP reductase and metalloprotease FTSH2). In both populations, upregulation of isocitrate dehydrogenase and cysteine/methionine synthases respectively suggested increased isocitrate oxidation and enhanced need for S-containing amino-acids, likely for chelation and detoxification. In NM leaves, an increasing need for energetic compounds was indicated by the stimulation of ATPases, glycolysis, pentose phosphate pathway, and Calvin cycle enzymes; impacts on protein metabolism and oxidative stress increase were respectively suggested by the rise of chaperones and redox enzymes. Overexpression of a HSP70 may be pivotal for M Cu tolerance by protecting protein metabolism. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD001930 (http//proteomecentral.proteomexchange.org/dataset/PXD001930).

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.