Comparative proteomics in tall fescue to reveal underlying mechanisms for improving Photosystem II thermotolerance during heat stress memory.

BACKGROUND: The escalating impacts of global warming intensify the detrimental effects of heat stress on crop growth and yield. Among the earliest and most vulnerable sites of damage is Photosystem II (PSII). Plants exposed to recurring high temperatures develop heat stress memory, a phenomenon that enables them to retain information from previous stress events to better cope with subsequent one. Understanding the components and regulatory networks associated with heat stress memory is crucial for the development of heat-resistant crops. RESULTS: Physiological assays revealed that heat priming (HP) enabled tall fescue to possess higher Photosystem II photochemical activity when subjected to trigger stress. To investigate the underlying mechanisms of heat stress memory, we performed comparative proteomic analyses on tall fescue leaves at S0 (control), R4 (primed), and S5 (triggering), using an integrated approach of Tandem Mass Tag (TMT) labeling and Liquid Chromatography-Mass Spectrometry. A total of 3,851 proteins were detected, with quantitative information available for 3,835 proteins. Among these, we identified 1,423 differentially abundant proteins (DAPs), including 526 proteins that were classified as Heat Stress Memory Proteins (HSMPs). GO and KEGG enrichment analyses revealed that the HSMPs were primarily associated with the "autophagy" in R4 and with "PSII repair", "HSP binding", and "peptidase activity" in S5. Notably, we identified 7 chloroplast-localized HSMPs (HSP21, DJC77, EGY3, LHCA4, LQY1, PSBR and DEGP8, R4/S0 > 1.2, S5/S0 > 1.2), which were considered to be effectors linked to PSII heat stress memory, predominantly in cluster 4. Protein-protein interaction (PPI) analysis indicated that the ubiquitin-proteasome system, with key nodes at UPL3, RAD23b, and UCH3, might play a role in the selective retention of memory effectors in the R4 stage. Furthermore, we conducted RT-qPCR validation on 12 genes, and the results showed that in comparison to the S5 stage, the R4 stage exhibited reduced consistency between transcript and protein levels, providing additional evidence for post-transcriptional regulation in R4. CONCLUSIONS: These findings provide valuable insights into the establishment of heat stress memory under recurring high-temperature episodes and offer a conceptual framework for breeding thermotolerant crops with improved PSII functionality.

High-resolution mass spectrometry-based non-targeted metabolomics reveals toxicity of naphthalene on tall fescue and intrinsic molecular mechanisms.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous at relatively high concentrations by atmospheric deposition, and they are threatening to the environment. In this study, the toxicity of naphthalene on tall fescue and its potential responding mechanism was first studied by integrating approaches. Tall fescue seedlings were exposed to 0, 20, and 100 mg L-1 naphthalene in a hydroponic environment for 9 days, and toxic effects were observed by the studies of general physiological studies, chlorophyll fluorescence, and root morphology. Additionally, Ultra Performance Liquid Chromatography - Electrospray Ionization - High-Resolution Mass Spectrometry (UPLC-ESI-HRMS) was used to depict metabolic profiles of tall fescue under different exposure durations of naphthalene, and the intrinsic molecular mechanism of tall fescue resistance to abiotic stresses. Tall fescue shoots were more sensitive to the toxicity of naphthalene than roots. Low-level exposure to naphthalene inhibited the electron transport from the oxygen-evolving complex (OEC) to D1 protein in tall fescue shoots but induced the growth of roots. Naphthalene induced metabolic change of tall fescue roots in 12 h, and tall fescue roots maintained the level of sphingolipids after long-term exposure to naphthalene, which may play important roles in plant resistance to abiotic stresses.

Dig up tall fescue plastid genomes for the identification of morphotype-specific DNA variants.

BACKGROUND: Tall fescue (Festuca arundinacea Schreb.) is an important cool-season perennial grass species. Hexaploid tall fescue has three distinct morphotypes used either as forage or turf purposes. Its chloroplast genome is conserved due to it being maternally inherited to the next generation progenies. To identify morphotype-specific DNA markers and the genetic variations, plastid genomes of all three tall fescue morphotypes, i.e., Continental cv. Texoma MaxQ II, Rhizomatous cv. Torpedo, and Mediterranean cv. Resolute, have been sequenced using Illumina MiSeq sequencing platform. RESULTS: The plastid genomes of Continental-, Rhizomatous-, and Mediterranean tall fescue were assembled into circular master molecules of 135,283 bp, 135,336 bp, and 135,324 bp, respectively. The tall fescue plastid genome of all morphotypes contained 77 protein-coding, 20 tRNAs, four rRNAs, two pseudo protein-coding, and three hypothetical protein-coding genes. We identified 630 SNPs and 124 InDels between Continental and Mediterranean, 62 SNPs and 20 InDels between Continental and Rhizomatous, and 635 SNPs and 123 InDels between Rhizomatous and Mediterranean tall fescue. Only four InDels in four genes (ccsA, rps18, accD, and ndhH-p) were identified, which discriminated Continental and Rhizomatous plastid genomes from the Mediterranean plastid genome. Here, we identified and reported eight InDel markers (NRITCHL18, NRITCHL35, NRITCHL43, NRITCHL65, NRITCHL72, NRITCHL101, NRITCHL104, and NRITCHL110) from the intergenic regions that can successfully discriminate tall fescue morphotypes. Divergence time estimation revealed that Mediterranean tall fescue evolved approximately 7.09 Mya, whereas the divergence between Continental- and Rhizomatous tall fescue occurred about 0.6 Mya. CONCLUSIONS: To our knowledge, this is the first report of the assembled plastid genomes of Rhizomatous and Mediterranean tall fescue. Our results will help to identify tall fescue morphotypes at the time of pre-breeding and will contribute to the development of lawn and forage types of commercial varieties.

Biochemical and in silico molecular study of caffeic acid-O-methyltransferase enzyme associated with lignin deposition in tall fescue.

Caffeic acid-O-methyltransferase (COMT), an important enzyme governing the process of lignification in plants, functions at the level of caffeic acid methylation along with 3-O-methylation of monolignol precursors. The present investigation was carried out to decipher the role of COMT in tall fescue lignification and to clone and characterize the COMT gene. The study on COMT activity variation at different growth stages of tall fescue exhibited a significant increase in activity over all the growth stages of tall fescue. A significant relative increase of 47.8% was observed from the first vegetative to reproductive stage. COMT activity exhibited a strong positive correlation with lignin content suggesting it to be an important enzyme of tall fescue lignification. Amplification and sequencing of tall fescue COMT gene resulted in an amplicon of size 1662 (Accession No.-MW442832) and an ORF of 346 amino acids. The deduced protein was hydrophobic, thermally stable and acidic with molecular formula C1679H2623N445O482S20, molecular mass 37.4 kDa and theoretical pI of 6.12. The protein possesses a conserved dimerization domain with a highly conserved SAM binding site. The COMT protein was found to be a homo-dimer with 1 catalytic SAH/SAM ligand per monomer interacting with 14 amino acid residues within 4 Å region.

Host Genetic Background Effect on Vertical Seed Transmission of Epichloë Endophyte Strains in Tall Fescue.

Tall fescue (Lolium arundinaceum (Schreb.) Darbysh.) is a cool-season perennial grass widely grown for forage and turf. Tall fescue lives in association with a fungal endophyte that helps the grass overcome abiotic and biotic stressors. The endophyte is asexual and transmits vertically from the tall fescue plant to the next generation through the seed. Producers of endophyte-infected tall fescue must have endophyte infection in at least 70% of their seed. Therefore, endophyte seed transmission is vital in breeding and seed production. Transfer of endophytes from their native host to different backgrounds of elite tall fescue cultivars can lead to a low seed transmission of the endophyte to the seed. This study screened 23 previously uncharacterized endophyte strains for transmissibility when artificially inoculated into continental and Mediterranean-type host tall fescue. We found no correlation between the rate of successful inoculation and the seed transmission rate of the endophyte in the new host. Nor did the seed transmission rate of the endophyte strains in their native host correlate with the seed transmission rate of the endophyte in the new host. Five strains exhibited seed transmission above 70% in both Mediterranean and Continental host backgrounds and will be characterized further for potential use in cultivar development.

Methane emission, nutrient digestibility, and rumen microbiota in Holstein heifers fed 14 different grass or clover silages as the sole feed.

This experiment investigated the variation in enteric methane production and associated gas exchange parameters, nutrient digestibility, rumen fermentation, and rumen microbiome when a range of silages based on different forage types (grass or clover), and different species within the 2 types, were fed as the sole feed to heifers. Three grass species (perennial ryegrass, festulolium, and tall fescue) and 2 clover species (red clover and white clover) were included. Perennial ryegrass was harvested at 2 maturity stages in the primary growth, white clover was harvested once in the primary growth, and 4 cuts of festulolium and tall fescue and 3 cuts of red clover were harvested during the growing season, giving 14 different silage batches in total. Sixteen Holstein heifers 16 to 21 mo old and 2 to 5 mo in pregnancy were fed the silages ad libitum as the sole feed in an incomplete crossover design. Each silage was fed to 4 heifers, except for the 2 perennial ryegrass silages, which were fed to 8 heifers; in total 64 observations. The CH4 production was measured for 3 d in respiration chambers. Heifers fed clover silages had higher dry matter intake (DMI) compared with heifers fed grass silages, and heifers fed tall fescue silages had the numerically the lowest DMI. Compared with grass silages, feeding clover silages led to higher crude protein digestibility but lower neutral detergent fiber (NDF) digestibility. Rumen pH was higher in heifers fed clover silages compared with those fed grass silages. Based on composition analysis, the rumen microbiota of the heifers clustered clearly according to forage type and species. More specifically, 7 of the 34 dominating rumen bacterial genus-level groups showed higher relative abundances for the clover silages, whereas 7 genus-level groups showed higher abundances for the grass silages. Methane yield was higher for heifers fed grass silages than for those fed clover silages when methane production was related to dry matter and digestible organic matter intake, whereas the opposite was seen when related to NDF digestion. The gross energy lost as methane (CH4 conversion factor, %) reduced from 7.5% to 6.7%, equivalent to an 11% reduction. The present study gives the outlines for choosing the optimal forage type and forage species with respect to nutrient digestibility and enteric methane emission in ruminants.

Evaluation of molasses on fermentation profiles of wheat straw ensiled with tall fescue in the southwest Tibet.

Two experiments were carried out to determine the optimal proportion of mixed silage made with wheat straw and tall fescue, and further to evaluate the effects of molasses on fermentation quality. In Experiment 1, wheat straw and tall fescue were mixed at proportions of 10:0 (Control), 8:2 (WT20), 6:4 (WT40) and 4:6 (WT60) on fresh weight (FW) basis. Inclusion of tall fescue significantly (p < 0.05) increased lactic acid, water-soluble carbohydrate contents and ratio of lactic to acetic acid, and significantly (p < 0.05) decreased pH and contents of dry matter, NH3 -N and volatile fatty acids. WT60 had the highest (p < 0.05) lactic acid content, and the lowest (p < 0.05) pH and butyric acid content. In Experiment 2, the mixture of wheat straw and tall fescue (4/6) were treated with 0%, 3%, 4% and 5% molasses on FW basis (defined as control, WTM3, WTM4 and WTM5 respectively). Molasses addition significantly (p < 0.05) increased lactic acid and water-soluble carbohydrate contents, and significantly (p < 0.05) decreased pH and ammonia-nitrogen content as compared with control. Acetic acid content slightly (p > 0.05) decreased during ensiling, while trace amounts of propionic and butyric acids were observed. WTM5 had the lowest pH and the highest (p < 0.05) lactic acid, water-soluble carbohydrate contents and ratio of lactic to acetic acid at end of ensiling. In conclusion, the fermentation quality was maximally improved when the addition rate of molasses was 5% in 40% wheat straw ensiled with 60% tall fescue.

Genetic parameters and selection in full-sib families of tall fescue using best linear unbiased prediction (BLUP) analysis.

BACKGROUND: Better understanding of genetic structure of economic traits is crucial for identification and selection of superior genotypes in specific breeding programs. Best linear unbiased prediction (BLUP) is the most efficient method in this regard, which is poorly used in forage plant breeding. The present study aimed to assess genetic variation, estimate genetic parameters, and predict breeding values of five essential traits in full sib families (recognized by EST-SSR markers) of tall fescue using REML/BLUP procedure. METHOD: Forty-two full-sib families of tall fescue (included of 120 individual genotypes), recognized by EST-SSR markers along with twenty-one their corresponding parental genotypes were assessed for biomass production and agro-morphological traits at three harvests (spring, summer, and autumn) in the field during 4 years (2017-2020). RESULTS: Considerable genotypic variability was observed for all traits. Low narrow-sense heritability (h2n) for dry forage yield (DFY) at three harvest indicates that non-additive gene actions may play an important role in the inheritance of this trait. Higher h2n of yield related traits and flowering time and also significant genetic correlation of these traits with forage yield, suggests that selection based on these traits may lead to indirect genetic improvement of DFY. CONCLUSION: Our results showed the adequacy of REML/BLUP procedure for identification and selection of preferable parental genotypes and progenies with higher breeding values for future breeding programs such as variety development in tall fescue. Parental genotypes 21 M, 1 M, and 20 L were identified as superior and stable genotypes and could also produce the best hybrid combinations when they were mostly used as maternal parent.

Physiological effects induced by aluminium and fluoride stress in tall fescue (Festuca arundinacea Schreb).

Aluminium (Al) and fluoride (F) are phytotoxic elements that can inhibit plant growth and development. Al3+ and F- can react with each other to form complexes in the soil which will induce alteration of toxicity of single element. However, the mechanisms of plant response to aluminium fluoride induced toxicity are not very clear. In the present study, tall fescue (Festuca arundinacea Schreb) cultivar 'Houndog 5' was treated by 0, 0.4, 4, 20 mg·L-1 Al2(SO4)3 and 0, 0.5, 5 mg·L-1 NaF, respectively. After 25 days of treatment, leaf samples were collected for physiological evaluation. The results showed that several forms of Al-OH and Al-F complexes such as Al(OH)2+, AlOH2+, Al(OH)3, Al(OH)4-, Al2(OH)24+, Al3(OH)45+, AlF2+, AlF2+, AlF3 and AlF4- were formed in Al3+ and F- combined solution. The nutrient uptake including Al, P and K were improved by Al3+ and F-. Under Al3+ stress, the MDA (malondialdehyde) content and EL (electrolyte leakage) dramatically increased after high concentration of F- treatment, while relative low concentration of F induced decrease of MDA content and EL. On the contrary, chlorophyll content decreased significantly after high concentration of F treatment. The photosynthesis efficiency parameters, including φP0 (Fv/Fm), δR0 and PIABS, decreased remarkably after high concentration of Al and F treatment. However, L-band incresed after high concentration of Al3+ and F- treatment. The results of correlation analysis showed that MDA content and EL negatively correlated with other indexes, and Al-F complex significantly correlated with MDA, Pro and EL but negatively correlated with Chl and φP0. These results suggested that low concentration of F could alleviate the damage induced by Al stress in tall fescue, but high concentration of Al and F combined solution had negative effects on the growth and development of tall fescue.

Near-Infrared Hyperspectral Imaging Pipelines for Pasture Seed Quality Evaluation: An Overview.

Near-infrared (800-2500 nm; NIR) spectroscopy coupled to hyperspectral imaging (NIR-HSI) has greatly enhanced its capability and thus widened its application and use across various industries. This non-destructive technique that is sensitive to both physical and chemical attributes of virtually any material can be used for both qualitative and quantitative analyses. This review describes the advancement of NIR to NIR-HSI in agricultural applications with a focus on seed quality features for agronomically important seeds. NIR-HSI seed phenotyping, describing sample sizes used for building high-accuracy calibration and prediction models for full or selected wavelengths of the NIR region, is explored. The molecular interpretation of absorbance bands in the NIR region is difficult; hence, this review offers important NIR absorbance band assignments that have been reported in literature. Opportunities for NIR-HSI seed phenotyping in forage grass seed are described and a step-by-step data-acquisition and analysis pipeline for the determination of seed quality in perennial ryegrass seeds is also presented.

Paclobutrazol Ameliorates Low-Light-Induced Damage by Improving Photosynthesis, Antioxidant Defense System, and Regulating Hormone Levels in Tall Fescue.

Paclobutrazol (PBZ) is a plant-growth regulator (PGR) in the triazole family that enhances plant tolerance to environmental stresses. Low-light (LL) intensity is a critical factor adversely affecting the growth of tall fescue (Festuca arundinacea Schreb.). Therefore, in this study, tall fescue seedlings were treated with PBZ under control and LL conditions to investigate the effects of PBZ on enhancing LL stress resistance by regulating the growth, photosynthesis, oxidative defense, and hormone levels. Our results reveal that LL stress reduced the total biomass, chlorophyll (Chl) content, photosynthetic capacity, and photochemical efficiency of photosystem II (PSII) but increased the membrane lipid peroxidation level and reactive oxygen species (ROS) generation. However, the application of PBZ increased the photosynthetic pigment contents, net photosynthetic rate (Pn), maximum quantum yield of PSII photochemistry (Fv/Fm), ribulose-1,5-bisphosphate carboxylase (RuBisCO) activity, and starch content. In addition, PBZ treatment activated the antioxidant enzyme activities, antioxidants contents, ascorbate acid-glutathione (AsA-GSH) cycle, and related gene expression, lessening the ROS burst (H2O2 and O2∙-). However, the gibberellic acid (GA) anabolism was remarkably decreased by PBZ treatment under LL stress, downregulating the transcript levels of kaurene oxidase (KO), kaurenoic acid oxidase (KAO), and GA 20-oxidases (GA20ox). At the same time, PBZ treatment up-regulated 9-cis-epoxycarotenoid dioxygenase (NCED) gene expression, significantly increasing the endogenous abscisic acid (ABA) concentration under LL stress. Thus, our study revealed that PBZ improves the antioxidation and photosynthetic capacity, meanwhile increasing the ABA concentration and decreasing GA concentration, which ultimately enhances the LL stress tolerance in tall fescue.

Evaluation of rhizoremediation and methane emission in diesel-contaminated soil cultivated with tall fescue (Festuca arundinacea).

Rhizoremediation, CH4 emission, and bacterial community dynamics were evaluated in diesel-contaminated soil cultivated with tall fescue via a pot experiment. At the beginning of the experiment, total petroleum hydrocarbons (TPHs) removal efficiency was 30.2% in tall fescue-cultivated soil, which was significantly higher than that of unplanted soil (19.4%). However, when compost was added as a soil amendment, TPHs removal efficiency increased to 39.2% in tall fescue-cultivated soil. Interestingly, potential CH4 emissions were more affected by the initial diesel concentration than by compost addition or tall fescue planting. Specifically, the potential CH4 emission was approximately 3.8 times higher in the treatment with the highest initial diesel concentration (T-WC38) than that of the treatment with the lowest initial diesel concentration (T-WC5). Functional gene analysis revealed that TPHs removal had a linear correlation with the alkB/16S gene ratio, whereas potential CH4 emission had a linear correlation with pmoA gene copy numbers. Initial diesel concentrations in soil also affected bacterial community structures and the genera Rhizobium, Halothiobacillus, and Geobacter were found to be positively linked to diesel-contaminated soil rhizoremediation. Therefore, this study provides useful insights into the development of strategies to enhance rhizoremediation efficiency and CH4 emission mitigation in diesel-contaminated soils.

Effects of harvest date and grass species on silage cell wall components and lactation performance of dairy cows.

This study evaluated the effect of harvest date and forage species on the concentration of hydroxycinnamic acids in silage and its relationship to dairy cow performance. Tall fescue and timothy were harvested at a regular date on June 27 and July 8, respectively, or at a late date on July 8 and 25, respectively, in the first regrowth. Forage was treated with a salt-based additive and ensiled in hard-pressed round bales. Forty-seven lactating dairy cows were used in a block design. Cows received 1 of 4 treatments: (1) tall fescue harvested at regular date (RTF), (2) timothy harvested at regular date (RTI), (3) tall fescue harvested at late date (LTF), and (4) timothy harvested at late date (LTI). Diets were formulated to have the same forage-to-concentrate ratio (46:54 on a dry matter basis). Harvesting at late date increased fiber components, but only for timothy, where LTI contained greater neutral detergent fiber, acid detergent fiber, and acid detergent lignin concentrations than the other silages. Concentrations of hydroxycinnamic acids were affected by forage species, where concentrations of esterified ferulic acid and p-coumaric acid were greater for tall fescue silages than for timothy silages. Cows fed the RTI diet showed the greatest intakes of dry matter, organic matter, and crude protein. Feeding diets containing timothy silages increased milk yield and energy-corrected milk yield compared with tall fescue diets when averaged over harvest dates. Cows fed the RTI diet had greater milk protein yield than cows fed the RTF and LTF diets, and milk lactose yield was greater for cows fed diets containing timothy silage compared with tall fescue silage when averaged over harvest dates. Cows fed the LTF diet showed greater urinary N excretion compared with the LTI diet, but RTI showed the lowest urinary N and urea N excretions when calculated as percent of N intake. Cows fed diets containing timothy silage excreted more uric acid than cows fed tall fescue diets. Allantoin excretion was greater for cows eating the RTI and LTI diets compared with cows eating the RTF diet. Cows fed the RTI diet had a greater estimated microbial N flow and a greater excretion of hippuric acid than the RTF and LTF groups. In conclusion, besides the effect of harvest date on increasing the fiber components of timothy, concentrations of hydroxycinnamic acids were mainly affected by forage species; consequently, milk production was only affected by forage species. This indicates that hydroxycinnamic acids, such as ferulic acid, which cross-links to glucuronoarabinoxylans, was a major factor regulating milk production of cows fed tall fescue- and timothy silage-based diets, where lower hydroxycinnamic acid concentrations were responsible for greater milk yield.

FaHSP17.8-CII orchestrates lead tolerance and accumulation in shoots via enhancing antioxidant enzymatic response and PSII activity in tall fescue.

Tall fescue (Festuca arundinacea Schreb.) shows huge potential for lead (Pb) phytoremediation, while little is known on the molecular mechanisms involved in Pb tolerance and accumulation. Here, genetic engineering strategy was firstly used to investigate Pb tolerance and accumulation in tall fescue. The transgenic tall fescue overexpressing a class II (CII) sHSP gene FaHSP17.8-CII was generated. After exposure to 1000 mg/L Pb(NO3)2, two FaHSP17.8-CII overexpressing lines, OE#3 and OE#7, showed higher tolerance to Pb as illustrated by the reduced levels of electrolyte leakage (EL) and malondialdehyde (MDA) as compared to the wild-type (WT) plants under Pb stress. Moreover, the FaHSP17.8-CII overexpression lines, OE#3 and OE#7, exhibited 36.3% and 46.6% higher shoot Pb accumulation relative to the WT grasses. When the grasses were exposed to Pb stress, the two OE lines had higher CAT, POD and SOD activities as compared to WT. Additionally, overexpression of FaHSP17.8-CII improved the synthesis of chlorophyll and transcript abundance of FapsbC, FapsbD and FapsbE, and alleviated the photoinhibition of PSII in tall fescue under Pb stress. This study provides an initial genetic engineering strategy to improve Pb phytoremediation efficiency in tall fescue by FaHSP17.8-CII overexpression.

Gamma radiation negatively impacted seed germination, seedling growth and antioxidant enzymes activities in tall fescue infected with Epichloë endophyte.

Plants and their accompanying microorganisms growing in contaminated sites with long-lived gamma-emitting radionuclides may be affected by radiation stress. The present study aimed to investigate the effects of gamma radiation on symbiotic relationship between Epichloë endophyte and Festuca arundinacea plant along with the radio-sensitivity of a pair of clones of tall fescue with (E+) and without (E-) symbiotic Epichloë endophyte exposed to different doses of gamma radiation including 25, 50, 75, 100, 150, 200, 300, and 400 Gray (Gy) from a Cobalt-60 source. Both irradiated and non-irradiated seeds of each status were grown under controlled conditions. Seed germination indices, seedling growth and certain physiological criteria associated with plant responses to oxidative stress were examined. The results revealed that low doses (up to 75 Gy) of gamma radiation stimulated seed germination indices and seedling growth. However, high doses (100-400 Gy) significantly reduced the final germination percentage, germination rate index, coefficient of velocity of germination, and the seed reserve depletion percentage, and enhanced the mean germination time. Further, high doses of radiation reduced root and shoot lengths, root and shoot fresh weights, and activities of antioxidant enzymes (especially catalase and superoxide dismutase), and increased the content of hydrogen peroxide (H2O2) and malondialdehyde (MDA) of the seedlings. The results showed that the endophyte was present in seeds after gamma ray irradiation. However, the presence of endophyte in seedlings started to be reduced significantly (18.45% reduction rather than the control) at 50 Gy of gamma radiation. High doses (100 Gy and above) dramatically declined the presence of endophyte down to zero in seedlings compared to the control. In this study, the E- clone had higher seed germination and seedling growth as well as lower H2O2 and MDA contents under radiation stress as compared with the E+ clone. Additionally, shoot tolerance index (STI) indicated more radiation tolerance in the E- clone. According to the results of the present study, it is concluded that biological impacts of gamma radiation stress and the harmful effects on endophyte viability may cause more radio-sensitivity and changes in the growth and physio-biochemical aspects of the host plant.

Effect of phosphorus supplementation on growth, nutrient uptake, physiological responses, and cadmium absorption by tall fescue (Festuca arundinacea Schreb.) exposed to cadmium.

Cadmium is a common heavy metal pollutant. In some plants, its absorption is inhibited by exogenous phosphorus. Here, the effect of P supplementation on the growth of tall fescue exposed to Cd was evaluated in a hydroponic culture experiment. Plants were exposed to five concentrations of P (0, 0.25, 0.5, 0.75, and 1.0 mmol L-1) and three concentrations of Cd (50, 100, and 150 mg L-1), and plant growth, Cd content, absorption, physiological characteristics, and nutrient accumulation were investigated. P supplementation significantly reduced the Cd content, Cd translocation factor (TF), Cd removal efficiency, plant P absorption, chlorophyll content, glutathione levels, glutathione reductase levels, and superoxide dismutase (SOD) activity in tall fescue under Cd stress (P < 0.05). Moreover, it increased the vertical growth rate and biomass of tall fescue. At a constant P concentration, the biomass and vertical growth rate significantly decreased with an increasing Cd concentration, and the shoot Cd content, SOD activity, and TF significantly increased (P < 0.05). High P supplementation (0.75 and 1.0 mmol L-1) ameliorated the damage caused by 150 mg L-1 Cd stress, and the biomass, vertical shoot and vertical root growth rates were increased by 72.06-82.06%, 250.00-316.67%, 300.00-312.00%, respectively. In the plants subjected to 50 mg L-1 Cd stress, 0.5 mmol L-1 P supplementation enhanced biomass, vertical shoot and vertical root growth rates by 29.99%, 20.41%, and 21.43%, respectively, and reduced the Cd content in shoots (45.85%) and roots (9.71%). Except for the total potassium content and catalase activity, different concentrations of Cd negatively affected all parameters tested. Such negative effects were limited by P supplementation. Optimizing the nutrient composition and concentrations could minimize the potential negative impacts of Cd on plant growth.

Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses.

Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely used for precise gene editing in plants. However, simultaneous gene editing of multiple homoeoalleles remains challenging, especially in self-incompatible polyploid plants. Here, we simultaneously introduced targeted mutations in all three homoeoalleles of two genes in the self-incompatible allohexaploid tall fescue, using both CRISPR/Cas9 and LbCas12a (LbCpf1) systems. Loss-of-function mutants of FaPDS exhibited albino leaves, while knockout of FaHSP17.9 resulted in impaired heat resistance in T0 generation of tall fescue. Moreover, these mutations were inheritable. Our findings demonstrate the feasibility of generating loss-of-function mutants in T0 generation polyploid perennial grasses using CRISPR/Cas systems.

Multi-species pastures for grazing dairy cows in small-scale dairy systems in the highlands of Mexico.

Grazing in small-scale dairy systems reduces costs and enhances sustainability. However, binary pastures (BP) have low persistency, which may be increased by including more species given their flexibility to withstand different agroecological and management situations. The objective was to assess a multi-species (MS) pasture of perennial ryegrass (RG), tall fescue (TF), bromegrass (BG), red clover (RC) and white clover (WC) in comparison to a BP of perennial ryegrass (RG) - WC grazed by six Holstein dairy cows during the dry season in an on-farm double cross-over experiment, with three 14-day each experimental periods for animal variables, and a split-plot design for pasture variables at 3 cow/ha. There were differences (P < 0.05) between pastures for sward height (MS 5.0 vs. BP 4.0 ± 0.10 cm, both MS and BP) and net herbage accumulation (MS 902 vs BP 228 ± 172.4 kg DM/ha, both MS and BP) and differences (P < 0.05) for chemical composition among periods. There were differences (P < 0.01) among periods for estimated DM intake, but no differences (P > 0.05) for milk yield (mean 16.8 kg/cow/day), milk fat or protein content (mean 31.8 and 28.8 g/kg). Multi-species pastures are a viable alternative for SSDS in the highlands of central Mexico, being more resistant to harsh environment and to weed and kikuyu grass invasion.

Comparative transcriptome combined with metabolome analyses revealed key factors involved in nitric oxide (NO)-regulated cadmium stress adaptation in tall fescue.

BACKGROUND: It has been reported that nitric oxide (NO) could ameliorate cadmium (Cd) toxicity in tall fescue; however, the underlying mechanisms of NO mediated Cd detoxification are largely unknown. In this study, we investigated the possible molecular mechanisms of Cd detoxification process by comparative transcriptomic and metabolomic approaches. RESULTS: The application of Sodium nitroprusside (SNP) as NO donor decreased the Cd content of tall fescue by 11% under Cd stress (T1 treatment), but the Cd content was increased by 24% when treated with Carboxy-PTIO (c-PTIO) together with Nitro-L-arginine methyl ester (L-NAME) (T2 treatment). RNA-seq analysis revealed that 904 (414 up- and 490 down-regulated) and 118 (74 up- and 44 down-regulated) DEGs were identified in the T1 vs Cd (only Cd treatment) and T2 vs Cd comparisons, respectively. Moreover, metabolite profile analysis showed that 99 (65 up- and 34-down- regulated) and 131 (45 up- and 86 down-regulated) metabolites were altered in the T1 vs Cd and T2 vs Cd comparisons, respectively. The integrated analyses of transcriptomic and metabolic data showed that 81 DEGs and 15 differentially expressed metabolites were involved in 20 NO-induced pathways. The dominant pathways were antioxidant activities such as glutathione metabolism, arginine and proline metabolism, secondary metabolites such as flavone and flavonol biosynthesis and phenylpropanoid biosynthesis, ABC transporters, and nitrogen metabolism. CONCLUSIONS: In general, the results revealed that there are three major mechanisms involved in NO-mediated Cd detoxification in tall fescue, including (a) antioxidant capacity enhancement; (b) accumulation of secondary metabolites related to cadmium chelation and sequestration; and (c) regulation of cadmium ion transportation, such as ABC transporter activation. In conclusion, this study provides new insights into the NO-mediated cadmium stress response.

Genome-wide small RNA profiling reveals tiller development in tall fescue (Festuca arundinacea Schreb).

BACKGROUND: Tall fescue (Festuca arundinacea Schreb.) is a major cool-season forage and turfgrass species. The low tiller density and size dramatically limits its turf performance and forage yield. MicroRNAs (miRNA)-genes modules play critical roles in tiller development in plants. In this study, a genome-wide small RNA profiling was carried out in two tall fescue genotypes contrasting for tillering production ('Ch-3', high tiller production rate and 'Ch-5', low tiller production rate) and two types of tissue samples at different tillering development stage (Pre-tillering, grass before tillering; Tillering, grass after tillering). 'Ch-3', 'Ch-5', Pre-tillering, and Tillering samples were analyzed using high-throughput RNA sequencing. RESULTS: A total of 222 million high-quality clean reads were generated and 208 miRNAs were discovered, including 148 known miRNAs belonging to 70 families and 60 novel ones. Furthermore, 18 miRNAs were involved in tall fescue tiller development process. Among them, 14 miRNAs displayed increased abundance in both Ch-3 and Tillering plants compared with that in Ch-5 and Pre-tillering plants and were positive with tillering, while another four miRNAs were negative with tiller development. Out of the three miRNAs osa-miR156a, zma-miR528a-3p and osa-miR444b.2, the rest of 15 miRNAs were newfound and associated with tiller development in plants. Based on our previous full-length transcriptome analysis in tall fescue, 28,927 potential target genes were discovered for all identified miRNAs. Most of the 212 target genes of the 18 miRNAs were dominantly enriched into "ubiquitin-mediated proteolysis", "phagosome", "fatty acid biosynthesis", "oxidative phosphorylation", and "biosynthesis of unsaturated fatty acids" KEGG pathways. In addition, bdi-miR167e-3p targets two kinase proteins EIF2AK4 and IRAK4, and osa-miR397a targets auxin response factor 5, which may be the significant miRNA-genes controllers in tillering development. CONCLUSIONS: This is the first genome-wide miRNA profiles analysis to identify regulators involved in tiller development in cool-season turfgrass. Tillering related 18 miRNAs and their 212 target genes provide novel information for the understanding of the molecular mechanisms of miRNA-genes mediated tiller development in cool-season turfgrass.