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.

Chitosan (CTS) Alleviates Heat-Induced Leaf Senescence in Creeping Bentgrass by Regulating Chlorophyll Metabolism, Antioxidant Defense, and the Heat Shock Pathway.

Chitosan (CTS) is a deacetylated derivative of chitin that is involved in adaptive response to abiotic stresses. However, the regulatory role of CTS in heat tolerance is still not fully understood in plants, especially in grass species. The aim of this study was to investigate whether the CTS could reduce heat-induced senescence and damage to creeping bentgrass associated with alterations in antioxidant defense, chlorophyll (Chl) metabolism, and the heat shock pathway. Plants were pretreated exogenously with or without CTS (0.1 g L-1) before being exposed to normal (23/18 °C) or high-temperature (38/33 °C) conditions for 15 days. Heat stress induced detrimental effects, including declines in leaf relative water content and photochemical efficiency, but significantly increased reactive oxygen species (ROS) accumulation, membrane lipid peroxidation, and Chl loss in leaves. The exogenous application of CTS significantly alleviated heat-induced damage in creeping bentgrass leaves by ameliorating water balance, ROS scavenging, the maintenance of Chl metabolism, and photosynthesis. Compared to untreated plants under heat stress, CTS-treated creeping bentgrass exhibited a significantly higher transcription level of genes involved in Chl biosynthesis (AsPBGD and AsCHLH), as well as a lower expression level of Chl degradation-related gene (AsPPH) and senescence-associated genes (AsSAG12, AsSAG39, Asl20, and Ash36), thus reducing leaf senescence and enhancing photosynthetic performance under heat stress. In addition, the foliar application of CTS significantly improved antioxidant enzyme activities (SOD, CAT, POD, and APX), thereby effectively reducing heat-induced oxidative damage. Furthermore, heat tolerance regulated by the CTS in creeping bentgrass was also associated with the heat shock pathway, since AsHSFA-6a and AsHSP82 were significantly up-regulated by the CTS during heat stress. The potential mechanisms of CTS-regulated thermotolerance associated with other metabolic pathways still need to be further studied in grass species.

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.

New Phytotoxic Cassane-like Diterpenoids from Eragrostis plana.

Eragrostis plana (Nees) is an allelopathic plant with invasive potential in South American pastures. To isolate and identify phytotoxic compounds from leaves and roots of E. plana, a bioassay-directed isolation of the bioactive constituents was performed. This is the first report on a new diterpene carbon skeleton, neocassane, and of three new neocassane diterpenes, neocassa-1,12(13),15-triene-3,14-dione, 1; 19-norneocassa-1,12(13),15-triene-3,14-dione, 2; and 14-hydroxyneocassa-1,12(17),15-triene-3-one, 3, identified from the roots. Compounds 1, 2, and 3 inhibited the growth of duckweed by 50% at concentrations of 109 ± 28, 200 ± 37, and 59 ± 15 µM, respectively. Compound 2 was fungicidal to Colletotrichum fragariae, Colletotrichum acutatum, and Colletotrichum gloeosporioides. The compounds identified here could explain the allelopathy of E. plana. The description of the newly discovered compounds, besides contributing to the chemical characterization of the species, may be the first step in the study of the potential of these compounds as bioherbicides.

2, 3-Butanediol activated disease-resistance of creeping bentgrass by inducing phytohormone and antioxidant responses.

Brown patch, caused by Rhizoctonia solani, is a serious disease in Agrostis stolonifera. 2, 3-butanediol (2, 3-BD) is the major component of volatile organic compounds and was found to initiate induced systemic resistance (ISR). To investigate the induced resistance mechanism of 2, 3-BD, we examined the effects of resistance by area affected, along with changes in the content of phytohormones (Zeatin (ZT), Abscisic Acid (ABA) and Indole-3-Acetic Acid (IAA)), the activities of three phenylpropanoid metabolic enzymes (Phenylalaninammo-Nialyase (PAL), Chalcone Isomerase (CHI) and 4-Coumarate:Coenzyme A Ligase (4CL)) and the level of secondary metabolites (total phenols, flavonoid and lignin). The result showed that 2, 3-BD treatment at 250 µmoL/L had the best induction effect with the area affected decreased from 95% of the control to 55%. Compared to the controls, treatment with 250 µmoL/L 2, 3-BD induced higher levels of PAL, CHI and 4CL activity and increased total phenols, flavonoid and lignin levels. While 2, 3-BD treatment decreased the content of ZT and ABA but increased the content of IAA compared to controls. This study provides a basis for elucidating the mechanism of 2, 3-BD as a new plant disease control agent.

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.

Unravelling the bioherbicide potential of Eucalyptus globulus Labill: Biochemistry and effects of its aqueous extract.

In the worldwide search for new strategies in sustainable weed management, the use of plant species able to produce and release phytotoxic compounds into the environment could be an effective alternative to synthetic herbicides. Eucalyptus globulus Labill. is known to be a source of biologically active compounds responsible for its phytotoxic and allelopathic properties. Our previous results demonstrated the bioherbicide potential of eucalyptus leaves incorporated into the soil as a green manure, probably through the release of phytotoxins into the soil solution. Thus, the aims of this study were to understand the phytotoxicity of the eucalyptus leaves aqueous extract applied in pre- and post-emergence, and to identify and quantify its potentially phytotoxic water-soluble compounds. The effects were tested on the germination and early growth of the model target species Lactuca sativa and Agrostis stolonifera, and on physiological parameters of L. sativa adult plants after watering or spraying application. Dose-response curves and ED50 and ED80 values for eucalyptus aqueous extracts revealed pre-emergence inhibitory effects on both target species, effects being comparable to the herbicide metolachlor. While spraying treatment reduced the aerial and root biomass and increased the dry weight/fresh weight ratio of lettuce adult plants, watering application reduced protein contents and chlorophyll concentrations with respect to control, reflecting different modes of action depending on the site of phytotoxin entry. Via HPLC analyses, a total of 8 phenolic compounds (chlorogenic, two ρ-coumaric derivatives, ellagic, hyperoside, rutin, quercitrin, and kaempferol 3-O-glucoside) and other 5 low weight organic acids (citric, malic, shikimic, succinic and fumaric acids) were obtained from aqueous extract, the latter being identified for the first time in E. globulus. Despite some phytotoxic effects were found on lettuce adult plants, the use of eucalyptus aqueous extract would be discarded in post-emergence, whereas it was promising as a pre-emergence bioherbicide.

Isolation of a phytotoxic isocoumarin from Diaporthe eres-infected Hedera helix (English ivy) and synthesis of its phytotoxic analogs.

BACKGROUND: The fungus Diaporthe eres was isolated from a fungal pathogen-infected leaf of Hedera helix (English ivy) exhibiting necrosis. It is hypothesized that the causative fungus produces phytotoxins as evidenced by necrotic lesions on the leaves. RESULTS: The fungus was isolated and grown in Czapek Dox broth culture medium and potato dextrose broth culture medium and identified as Diaporthe eres. The ethyl acetate extracts of the culture broths were phytotoxic to lettuce (Lactuca sativa) and bentgrass (Agrostis stolonifera). 3,4-Dihydro-8-hydroxy-3,5-dimethylisocoumarin (1) and tyrosol (2) were isolated and identified as the phytotoxic constituents. Six analogs of 3,4-dihydro-isocoumarin were synthesized and shown to be phytotoxic. The synthesized 3,4-dihydro-8-hydroxy-3,7-dimethylisocoumarin and 3,4-dihydro-8-hydroxy-3,3,7-trimethylisocoumarin were two- to three-fold more phytotoxic than the naturally occurring 1 in a Lemna paucicostata growth bioassay. CONCLUSION: Synthesis and herbicidal activities of the several new analogs of 1 are reported for the first time. These promising molecules should be used as templates for synthesis and testing of more analogs. © 2017 Society of Chemical Industry.

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.

Transcriptome analysis of creeping bentgrass exposed to drought stress and polyamine treatment.

Creeping bentgrass is an important cool-season turfgrass species sensitive to drought. Treatment with polyamines (PAs) has been shown to improve drought tolerance; however, the mechanism is not yet fully understood. Therefore, this study aimed to evaluate transcriptome changes of creeping bentgrass in response to drought and exogenous spermidine (Spd) application using RNA sequencing (RNA-Seq). The high-quality sequences were assembled and 18,682 out of 49,190 (38%) were detected as coding sequences. A total of 22% and 19% of genes were found to be either up- or down-regulated due to drought while 20% and 34% genes were either up- or down- regulated in response to Spd application under drought conditions, respectively. Gene ontology (GO) and enrichment analysis were used to interpret the biological processes of transcripts and relative transcript abundance. Enriched or differentially expressed transcripts due to drought stress and/or Spd application were primarily associated with energy metabolism, transport, antioxidants, photosynthesis, signaling, stress defense, and cellular response to water deprivation. This research is the first to provide transcriptome data for creeping bentgrass under an abiotic stress using RNA-Seq analysis. Differentially expressed transcripts identified here could be further investigated for use as molecular markers or for functional analysis in responses to drought and Spd.

New Pesticidal Diterpenoids from Vellozia gigantea (Velloziaceae), an Endemic Neotropical Plant Living in the Endangered Brazilian Biome Rupestrian Grasslands.

Vellozia gigantea is a rare, ancient, and endemic neotropical plant present in the Brazilian Rupestrian grasslands. The dichloromethane extract of V. gigantea adventitious roots was phytotoxic against Lactuca sativa, Agrostis stolonifera, and Lemna paucicostata, and showed larvicidal activity against Aedes aegypti. Phytotoxicity bioassay-directed fractionation of the extract revealed one new isopimaradiene, 8(9),15-isopimaradien-1,3,7,11-tetraone, and three new cleistanthane diterpenoids, 7-oxo-8,11,13-cleistanthatrien-3-ol, 3,20-epoxy-7-oxo-8,11,13-cleistanthatrien-3-ol, and 20-nor-3,7-dioxo-1,8,11,13-cleistanthatetraen-10-ol. These new structures are proposed based on interpretation of ¹H, 13C, COSY, NOESY, HSQC, and HMBC NMR data. 8(9),15-isopimaradien-1,3,7,11-tetraone was especially phytotoxic with an IC50 value (30 µM) comparable to those of commercial herbicides clomazone, EPTC, and naptalam. In addition, 7-oxo-8,11,13-cleistanthatrien-3-ol provided 100% mortality at a concentration of 125 ppm against one-day-old Ae. aegypti larvae. Our results show that ancient and unique plants, like the endangered narrowly endemic neotropical species V. gigantea present in the Rupestrian grasslands, should also be protected because they can be sources of new bioactive compounds.

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.

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.

Poly(3-hydroxybutyrate)/metribuzin formulations: characterization, controlled release properties, herbicidal activity, and effect on soil microorganisms.

Slow-release formulations of the herbicide metribuzin (MET) embedded in the polymer matrix of degradable poly-3-hydroxybutyrate [P(3HB)] in the form of microparticles, films, microgranules, and pellets were developed and tested. The kinetics of polymer degradation, MET release, and accumulation in soil were studied in laboratory soil microecosystems with higher plants. The study shows that MET release can be controlled by using different techniques of constructing formulations and by varying MET loading. MET accumulation in soil occurs gradually, as the polymer is degraded. The average P(3HB) degradation rates were determined by the geometry of the formulation, reaching 0.17, 0.12, 0.04, and 0.05 mg/day after 60 days for microparticles, films, microgranules, and pellets, respectively. The herbicidal activities of P(3HB)/MET formulations and commercial formulation Sencor Ultra were tested on the Agrostis stolonifera and Setaria macrocheata plants. The parameters used to evaluate the herbicidal activity were plant density and the weight of fresh green biomass measured at days 10, 20, and 30 after sowing. All P(3HB)/MET formulations had pronounced herbicidal activity, which varied depending on MET loading and the stage of the experiment. In the early phases of the experiment, the herbicidal effect of P(3HB)/MET formulations with the lowest MET loading (10 %) was comparable with that of the commercial formulation. The herbicidal effect of P(3HB)/MET formulations with higher MET loadings (25 and 50 %) at later stages of the experiment were stronger than the effect of Sencor Ultra.

Metabolic pathways regulated by γ-aminobutyric acid (GABA) contributing to heat tolerance in creeping bentgrass (Agrostis stolonifera).

γ-Aminobutyric acid is a non-protein amino acid involved in various metabolic processes. The objectives of this study were to examine whether increased GABA could improve heat tolerance in cool-season creeping bentgrass through physiological analysis, and to determine major metabolic pathways regulated by GABA through metabolic profiling. Plants were pretreated with 0.5 mM GABA or water before exposed to non-stressed condition (21/19 °C) or heat stress (35/30 °C) in controlled growth chambers for 35 d. The growth and physiological analysis demonstrated that exogenous GABA application significantly improved heat tolerance of creeping bentgrass. Metabolic profiling found that exogenous application of GABA led to increases in accumulations of amino acids (glutamic acid, aspartic acid, alanine, threonine, serine, and valine), organic acids (aconitic acid, malic acid, succinic acid, oxalic acid, and threonic acid), sugars (sucrose, fructose, glucose, galactose, and maltose), and sugar alcohols (mannitol and myo-inositol). These findings suggest that GABA-induced heat tolerance in creeping bentgrass could involve the enhancement of photosynthesis and ascorbate-glutathione cycle, the maintenance of osmotic adjustment, and the increase in GABA shunt. The increased GABA shunt could be the supply of intermediates to feed the tricarboxylic acid cycle of respiration metabolism during a long-term heat stress, thereby maintaining metabolic homeostasis.

Constructing Slow-Release Formulations of Metribuzin Based on Degradable Poly(3-hydroxybutyrate).

Experimental formulations of herbicide metribuzin embedded in matrices of degradable natural polymer poly(3-hydroxybutyrate) (P3HB) and its composites with poly(ethylene glycol) (PEG), poly-ε-caprolactone (PCL), and wood powder have been prepared in the form of pressed pellets containing 75% polymeric basis (pure P3HB or its composite with a second component at a ratio of 7:3) and 25% metribuzin. Incubation of formulations in soil laboratory systems led to the degradation of the matrix and herbicide release. The most active release of metribuzin (about 60% of the embedded herbicide over 35 days) was detected for the P3HB/PEG carrier compared to the P3HB, P3HB/wood, and P3HB/PCL forms (30-40%). Thus, the study shows that herbicide release can be controlled by the matrix formulation. Metribuzin formulations exerted a significant herbicidal effect on the plant Agrostis stolonifera, used as a weed plant model. Application of these long-term formulations will make it possible to reduce environmental release of chemicals, which will restrict the rate of their accumulation in trophic chains of ecosystems and abate their adverse effects on the biosphere.

Synonymous mutation gene design to overexpress ACCase in creeping bentgrass to obtain resistance to ACCase-inhibiting herbicides.

Overexpression of a native gene can cause expression of both introduced and native genes to be silenced by posttranscriptional gene silencing (PTGS) mechanisms. PTGS mechanisms rely on sequence identity between the transgene and native genes; therefore, designing genes with mutations that do not cause amino acid changes, known as synonymous mutations, may avoid PTGS. For proof of concept, the sequence of acetyl-coA carboxylase (ACCase) from creeping bentgrass (Agrostis stolonifera L.) was altered with synonymous mutations. A native bentgrass ACCase was cloned and used as a template for the modified gene. Wild-type (WT) and modified genes were further modified with a non-synonymous mutation, coding for an isoleucine to leucine substitution at position 1781, known to confer resistance to ACCase-inhibiting herbicides. Five-hundred calli of creeping bentgrass 'Penn A-4' were inoculated with Agrobacterium containing either the WT or modified genes, with or without the herbicide-resistance mutation. Six herbicide-resistant-transgenic events containing the modified gene with the 1781 mutation were obtained. Transcription of the modified ACCase was confirmed in transgenic plants, showing that gene-silencing mechanisms were avoided. Transgenic plants were confirmed to be resistant to the ACCase-inhibiting herbicide, sethoxydim, providing evidence that the modified gene was functional. The result is a novel herbicide-resistance trait and shows that overexpression of a native enzyme with a gene designed with synonymous mutations is possible.

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).

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.