Anti-herbivory defences delivered by Epichloë fungal endophytes: a quantitative review of alkaloid concentration variation among hosts and plant parts.

BACKGROUND AND AIMS: In the subfamily Poöideae (Poaceae), certain grass species possess anti-herbivore alkaloids synthesized by fungal endophytes that belong to the genus Epichloë (Clavicipitaceae). The protective role of these symbiotic endophytes can vary, depending on alkaloid concentrations within specific plant-endophyte associations and plant parts. METHODS: We conducted a literature review to identify articles containing alkaloid concentration data for various plant parts in six important pasture species, Lolium arundinaceum, Lolium perenne, Lolium pratense, Lolium multiflorum|Lolium rigidum and Festuca rubra, associated with their common endophytes. We considered the alkaloids lolines (1-aminopyrrolizidines), peramine (pyrrolopyrazines), ergovaline (ergot alkaloids) and lolitrem B (indole-diterpenes). While all these alkaloids have shown bioactivity against insect herbivores, ergovaline and lolitrem B are harmful for mammals. KEY RESULTS: Loline alkaloid levels were higher in the perennial grasses L. pratense and L. arundinaceum compared to the annual species L. multiflorum and L. rigidum, and higher in reproductive tissues than in vegetative structures. This is probably due to the greater biomass accumulation in perennial species that can result in higher endophyte mycelial biomass. Peramine concentrations were higher in L. perenne than in L. arundinaceum and not affected by plant part. This can be attributed to the high within-plant mobility of peramine. Ergovaline and lolitrem B, both hydrophobic compounds, were associated with plant parts where fungal mycelium is usually present, and their concentrations were higher in plant reproductive tissues. Only loline alkaloid data were sufficient for below-ground tissue analyses and concentrations were lower than in above-ground parts. CONCLUSIONS: Our study provides a comprehensive synthesis of fungal alkaloid variation across host grasses and plant parts, essential for understanding the endophyte-conferred defence extent. The patterns can be understood by considering endophyte growth within the plant and alkaloid mobility. Our study identifies research gaps, including the limited documentation of alkaloid presence in roots and the need to investigate the influence of different environmental conditions.

Stress memory gene FaHSP17.8-CII controls thermotolerance via remodeling PSII and ROS signaling in tall fescue.

High temperature is the most limiting factor in the growth of cool-season turfgrass. To cope with high-temperature stress, grass often adopt a memory response by remembering one past recurring stress and preparing a quicker and more robust reaction to the next stress exposure. However, little is known about how stress memory genes regulate the thermomemory response in cool-season turfgrass. Here, we characterized a transcriptional memory gene, Fa-heat shock protein 17.8 Class II (FaHSP17.8-CII) in a cool-season turfgrass species, tall fescue (Festuca arundinacea Schreb.). The thermomemory of FaHSP17.8-CII continued for more than 4 d and was associated with a high H3K4me3 level in tall fescue under heat stress (HS). Furthermore, heat acclimation or priming (ACC)-induced reactive oxygen species (ROS) accumulation and photosystem II (PSII) electron transport were memorable, and this memory response was controlled by FaHSP17.8-CII. In the fahsp17.8-CII mutant generated using CRISPR/Cas9, ACC+HS did not substantially block the ROS accumulation, the degeneration of chloroplast ultra-structure, and the inhibition of PSII activity compared with HS alone. However, overexpression of FaHSP17.8-CII in tall fescue reduced ROS accumulation and chloroplast ultra-structure damage, and improved chlorophyll content and PSII activity under ACC+HS compared with that HS alone. These findings unveil a FaHSP17.8-CII-PSII-ROS module regulating transcriptional memory to enhance thermotolerance in cool-season turfgrass.

Soil chemical legacies trigger species-specific and context-dependent root responses in later arriving plants.

Soil legacies play an important role for the creation of priority effects. However, we still poorly understand to what extent the metabolome found in the soil solution of a plant community is conditioned by its species composition and whether soil chemical legacies affect subsequent species during assembly. To test these hypotheses, we collected soil solutions from forb or grass communities and evaluated how the metabolome of these soil solutions affected the growth, biomass allocation and functional traits of a forb (Dianthus deltoides) and a grass species (Festuca rubra). Results showed that the metabolomes found in the soil solutions of forb and grass communities differed in composition and chemical diversity. While soil chemical legacies did not have any effect on F. rubra, root foraging by D. deltoides decreased when plants received the soil solution from a grass or a forb community. Structural equation modelling showed that reduced soil exploration by D. deltoides arose via either a root growth-dependent pathway (forb metabolome) or a root trait-dependent pathway (grass metabolome). Reduced root foraging was not connected to a decrease in total N uptake. Our findings reveal that soil chemical legacies can create belowground priority effects by affecting root foraging in later arriving plants.

Transcriptional responses to water stress and recovery in a drought-tolerant fescue wild grass (Festuca ovina; Poaceae).

Water stress associated with drought-like conditions is a major factor limiting plant growth and impacts productivity of natural plant communities and agricultural crops. Molecular responses of plants to water stress have been studied most extensively in model species and crops, few of which have evolved natural drought tolerance. In the current study, we examined physiological and transcriptomic responses at multiple timepoints during increasing water stress and following initial recovery from stress in a drought-tolerant C3 species, Festuca ovina. Results demonstrated non-linear transcriptomic changes during increasing stress, but largely linear declines in physiological measurements during this same period. Transcription factors represented approximately 12.7% of all differentially expressed genes. In total, 117 F. ovina homologs of previously identified and molecularly characterized drought-responsive plant genes were identified. This information will be valuable for further investigations of the molecular mechanisms involved in drought tolerance in C3 plants.

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.

Efficient root metabolism improves drought resistance of Festuca arundinacea.

Festuca arundinacea is a model to work on the mechanisms of drought resistance in grasses. The crucial components of that resistance still remain not fully recognized. It was suggested that deep root system could be a crucial trait for drought avoidance strategy but the other components of root performance under water deficit have not paid much attention of scientists. In this study, two genotypes of F. arundinacea with a different ability to withstand soil water deficit were selected to perform comprehensive research, including analysis of root architecture, phytohormones, proteome, primary metabolome and lipidome under progressive stress conditions, followed by a rewatering period. The experiments were performed in tubes, thus enabling undisturbed development of root systems. We demonstrated that long roots are not sufficient to perfectly avoid drought damage in F. arundinacea and to withstand adverse environmental conditions without a disturbed cellular metabolism (with respect to leaf relative water potential and cellular membrane integrity). Furthermore, we proved that metabolic performance of roots is as crucial as its architecture under water deficit, to cope with drought stress via avoidance, tolerance and regeneration strategies. We believe that the presented studies could be a good reference for the other, more applied experiments, in closely related species.

SMRT and Illumina RNA sequencing reveal novel insights into the heat stress response and crosstalk with leaf senescence in tall fescue.

BACKGROUND: As a cool-season grass species, tall fescue (Festuca arundinacea) is challenged by increasing temperatures. Heat acclimation or activation of leaf senescence, are two main strategies when tall fescue is exposed to heat stress (HS). However, lacking a genome sequence, the complexity of hexaploidy nature, and the short read of second-generation sequencing hinder a comprehensive understanding of the mechanism. This study aims to characterize the molecular mechanism of heat adaptation and heat-induced senescence at transcriptional and post-transcriptional levels. RESULTS: Transcriptome of heat-treated (1 h and 72 h) and senescent leaves of tall fescue were generated by combining single-molecular real-time and Illumina sequencing. In total, 4076; 6917, and 11,918 differentially expressed genes (DEGs) were induced by short- and long-term heat stress (HS), and senescence, respectively. Venn and bioinformatics analyses of DEGs showed that short-term HS strongly activated heat shock proteins (Hsps) and heat shock factors (Hsfs), as well as specifically activated FK506-binding proteins (FKBPs), calcium signaling genes, glutathione S-transferase genes, photosynthesis-related genes, and phytohormone signaling genes. By contrast, long-term HS shared most of DEGs with senescence, including the up-regulated chlorophyll catabolic genes, phytohormone synthesis/degradation genes, stress-related genes, and NACs, and the down-regulated photosynthesis-related genes, FKBPs, and catalases. Subsequently, transient overexpression in tobacco showed that FaHsfA2a (up-regulated specifically by short-term HS) reduced cell membrane damages caused by HS, but FaNAC029 and FaNAM-B1 (up-regulated by long-term HS and senescence) increased the damages. Besides, alternative splicing was widely observed in HS and senescence responsive genes, including Hsps, Hsfs, and phytohormone signaling/synthesis genes. CONCLUSIONS: The short-term HS can stimulate gene responses and improve thermotolerance, but long-term HS is a damage and may accelerate leaf senescence. These results contribute to our understanding of the molecular mechanism underlying heat adaptation and heat-induced senescence.

Two Festuca Species-F. arundinacea and F. glaucescens-Differ in the Molecular Response to Drought, While Their Physiological Response Is Similar.

Impact of photosynthetic and antioxidant capacities on drought tolerance of two closely related forage grasses, Festuca arundinacea and Festuca glaucescens, was deciphered. Within each species, two genotypes distinct in drought tolerance were subjected to a short-term drought, followed by a subsequent re-watering. The studies were focused on: (i) analysis of plant physiological performance, including: water uptake, abscisic acid (ABA) content, membrane integrity, gas exchange, and relative water content in leaf tissue; (ii) analysis of plant photosynthetic capacity (chlorophyll fluorescence; gene expression, protein accumulation, and activity of selected enzymes of the Calvin cycle); and (iii) analysis of plant antioxidant capacity (reactive oxygen species (ROS) generation; gene expression, protein accumulation and activity of selected enzymes). Though, F. arundinacea and F. glaucescens revealed different strategies in water uptake, and partially also in ABA signaling, their physiological reactions to drought and further re-watering, were similar. On the other hand, performance of the Calvin cycle and antioxidant system differed between the analyzed species under drought and re-watering periods. A stable efficiency of the Calvin cycle in F. arundinacea was crucial to maintain a balanced network of ROS/redox signaling, and consequently drought tolerance. The antioxidant capacity influenced mostly tolerance to stress in F. glaucescens.

Adjustment of Photosynthetic and Antioxidant Activities to Water Deficit Is Crucial in the Drought Tolerance of Lolium multiflorum/Festuca arundinacea Introgression Forms.

Lolium multiflorum/Festuca arundinacea introgression forms have been proved several times to be good models to identify key components of grass metabolism involved in the mechanisms of tolerance to water deficit. Here, for the first time, a relationship between photosynthetic and antioxidant capacities with respect to drought tolerance of these forms was analyzed in detail. Two closely related L. multiflorum/F. arundinacea introgression forms distinct in their ability to re-grow after cessation of prolonged water deficit in the field were selected and subjected to short-term drought in pots to dissect precisely mechanisms of drought tolerance in this group of plants. The studies revealed that the form with higher drought tolerance was characterized by earlier and higher accumulation of abscisic acid, more stable cellular membranes, and more balanced reactive oxygen species metabolism associated with a higher capacity of the antioxidant system under drought conditions. On the other hand, both introgression forms revealed the same levels of stomatal conductance, CO2 assimilation, and consequently, intrinsic water use efficiency under drought and recovery conditions. However, simultaneous higher adjustment of the Calvin cycle to water deficit and reduced CO2 availability, with respect to the accumulation and activity of plastid fructose-1,6-bisphosphate aldolase, were clearly visible in the form with higher drought tolerance.

Lipidomic reprogramming associated with drought stress priming-enhanced heat tolerance in tall fescue (Festuca arundinacea).

Stress priming by exposing plants to a mild or moderate drought could enhance plant tolerance to subsequent heat stress. Lipids play vital roles in stress adaptation, but how lipidomic profiles change, affecting the cross-stress tolerance, is largely unknown. The objectives of this study were to perform lipidomics, to analyse the content, composition, and saturation levels of lipids in leaves of tall fescue (Festuca arundinacea) following drought priming and subsequent heat stress, and to identify major lipids and molecular species associated with priming-enhanced heat tolerance. Plants were initially exposed to drought for 8 days by withholding irrigation and subsequently subjected to 25 days of heat stress (38/33°C day/night) in growth chambers. Drought-primed plants maintained significantly higher leaf relative water content, chlorophyll content, photochemical efficiency, and lower electrolyte leakage than nonprimed plants under heat stress. Drought priming enhanced the accumulation of phospholipids and glycolipids involved in membrane stabilization and stress signalling (phosphatidic acid, phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, and digalactosyl diacylglycerol) during subsequent exposure to heat stress. The reprogramming of lipid metabolism for membrane stabilization and signalling in response to drought priming and subsequent exposure to heat stress could contribute to drought priming-enhanced heat tolerance in cool-season grass species.

Evolutionary potential of a widespread clonal grass under changing climate.

Adaptive responses are probably the most effective long-term responses of populations to climate change, but they require sufficient evolutionary potential upon which selection can act. This requires high genetic variance for the traits under selection and low antagonizing genetic covariances between the different traits. Evolutionary potential estimates are still scarce for long-lived, clonal plants, although these species are predicted to dominate the landscape with climate change. We studied the evolutionary potential of a perennial grass, Festuca rubra, in western Norway, in two controlled environments corresponding to extreme environments in natural populations: cold-dry and warm-wet, the latter being consistent with the climatic predictions for the country. We estimated genetic variances, covariances, selection gradients and response to selection for a wide range of growth, resource acquisition and physiological traits, and compared their estimates between the environments. We showed that the evolutionary potential of F. rubra is high in both environments, and genetic covariances define one main direction along which selection can act with relatively few constraints to selection. The observed response to selection at present is not sufficient to produce genotypes adapted to the predicted climate change under a simple, space for time substitution model. However, the current populations contain genotypes which are pre-adapted to the new climate, especially for growth and resource acquisition traits. Overall, these results suggest that the present populations of the long-lived clonal plant may have sufficient evolutionary potential to withstand long-term climate changes through adaptive responses.

Abscisic acid mediation of drought priming-enhanced heat tolerance in tall fescue (Festuca arundinacea) and Arabidopsis.

Abscisic acid (ABA) may play roles in mediating cross stress tolerance in plants. The objectives of this study were to investigate the priming effects of drought and ABA on heat tolerance and to determine how ABA may be involved in enhanced heat tolerance by drought. Focusing on the transcriptional level, two independent experiments were conducted, using a perennial grass species, tall fescue (Festuca arundinacea) and Arabidopsis. In experiment 1, tall fescue plants were exposed to mild drought by withholding irrigation for 8 days (drought priming) and foliar sprayed with ABA or an ABA-synthesis inhibitor (fluridone). After that they were subsequently subjected to heat stress (38/33°C day/night) for 25 days in growth chambers. In experiment 2, Arabidopsis Columbia ecotype (wild-type) and ABA-deficient mutant (aba3-1, CS157) were pre-treated with drought priming and then exposed to heat stress (45/40°C) for 3 days. The physiological analysis demonstrated that both drought priming and foliar application of ABA-enhanced heat tolerance in tall fescue, while drought priming had no significant effects on heat tolerance in ABA-deficient Arabidopsis plants. Application of fluridone to tall fescue and ABA-deficient mutants of Arabidopsis exhibited diminished or attenuated positive effects of drought priming on heat tolerance. ABA mediation of acquired heat tolerance by drought priming was associated with the upregulation of CDPK3, MPK3, DREB2A, AREB3, MYB2, MYC4, HsfA2, HSP18, and HSP70. Our study revealed the roles of ABA in drought priming-enhanced heat tolerance, which may involve transcriptional regulation for stress signaling, ABA responses and heat protection.

Physiological consequences of gamma ray irradiation in tall fescue with elimination potential of Epichloë fungal endophyte.

Perennial plants and their associated microorganisms grow in the areas that may be contaminated with long-lived gamma-emitting radionuclides. This will induce gamma stress response in plants and their accompanying microorganisms. The present work investigated the growth and physiological responses of Epichloe endophyte infected tall fescue to gamma radiation, as well as whether the endophyte could persist and infect the host plant once exposed to gamma radiation. Seeds of Iranian native genotype of 75B+ of tall fescue were exposed to different doses, including 5.0, 10.0, 15.0, 20.0, 30.0 and 40.0 krad of gamma ray from a 60Co source. Irradiated and unirradiated seeds were sown in pots and grown under controlled conditions in the greenhouse. The growth and physiological parameters associated with plant tolerance to oxidative stress of host plants, as well as endophytic infection frequency (% of plants infected) and intensity (mean number of endophytic hyphae per the field of view), were examined in 3 months-old seedlings. The results indicated that all gamma radiation doses (except 5.0 kr) significantly reduced the height and survival percentage of the host plant. Days to the emergence of seedling increased gradually as gamma doses rose. A dose-rate dependent induction was seen for photosynthetic pigments and proline content. Malondialdehyde (MDA) content grew with elevation of irradiation doses. Depending on the dose and time, the activities of antioxidant enzymes in the host plant responded differently to gamma radiation. Gamma radiation altered the enzyme activities with sever decline in SOD and CAT activities. However, it had barely any effect on in APX and POD activities. The results also revealed that the persistence and intensity of endophyte were affected after gamma-ray irradiation. The initial percentage of tall fescue seeds infected with the endophyte was 91% in un-irradiated seeds. Presence of the viable endophyte started to decline significantly (23%) at 5.0 kr of gamma radiation. A dramatic reduction in the presence and intensity of endophyte occurred at 10.0 to 40.0 kr intensities. Gamma radiation × trait (GT)-biplot analysis indicated positive correlations between the endophyte symbiosis and antioxidant enzyme activities. Also, negative correlations were observed between the endophyte and MDA content in the host plant. Our results suggest that radiation stress (doses over 5.0 kr) caused reduction in the growth and antioxidant enzyme activities of the host plant that accompanied by a dramatic reduction in the persistence and intensity of endophyte fungi. Our findings have provided the basic information for future studies on the effect of gamma irradiation on the interaction between endophytic fungi and its host plant.

Strigolactones Promote Leaf Elongation in Tall Fescue through Upregulation of Cell Cycle Genes and Downregulation of Auxin Transport Genes in Tall Fescue under Different Temperature Regimes.

Strigolactones (SLs) have recently been shown to play roles in modulating plant architecture and improving plant tolerance to multiple stresses, but the underlying mechanisms for SLs regulating leaf elongation and the influence by air temperature are still unknown. This study aimed to investigate the effects of SLs on leaf elongation in tall fescue (Festuca arundinacea, cv. 'Kentucky-31') under different temperature regimes, and to determine the interactions of SLs and auxin in the regulation of leaf growth. Tall fescue plants were treated with GR24 (synthetic analog of SLs), naphthaleneacetic acid (NAA, synthetic analog), or N-1-naphthylphthalamic acid (NPA, auxin transport inhibitor) (individually and combined) under normal temperature (22/18 °C) and high-temperature conditions (35/30 °C) in controlled-environment growth chambers. Exogenous application of GR24 stimulated leaf elongation and mitigated the heat inhibition of leaf growth in tall fescue. GR24-induced leaf elongation was associated with an increase in cell numbers, upregulated expression of cell-cycle-related genes, and downregulated expression of auxin transport-related genes in elongating leaves. The results suggest that SLs enhance leaf elongation by stimulating cell division and interference with auxin transport in tall fescue.

Contrasting survival and physiological responses of sub-Arctic plant types to extreme winter warming and nitrogen.

MAIN CONCLUSION: Evergreen plants are more vulnerable than grasses and birch to snow and temperature variability in the sub-Arctic. Most Arctic climate impact studies focus on single factors, such as summer warming, while ecosystems are exposed to changes in all seasons. Through a combination of field and laboratory manipulations, we compared physiological and growth responses of dominant sub-Arctic plant types to midwinter warming events (6 °C for 7 days) in combination with freezing, simulated snow thaw and nitrogen additions. We aimed to identify if different plant types showed consistent physiological, cellular, growth and mortality responses to these abiotic stressors. Evergreen dwarf shrubs and tree seedlings showed higher mortality (40-100%) following extreme winter warming events than Betula pubescens tree seedlings and grasses (0-27%). All species had growth reductions following exposure to - 20 °C, but not all species suffered from - 10 °C irrespective of other treatments. Winter warming followed by - 20 °C resulted in the greatest mortality and was strongest among evergreen plants. Snow removal reduced the biomass for most species and this was exacerbated by subsequent freezing. Nitrogen increased the growth of B. pubescens and grasses, but not the evergreens, and interaction effects with the warming, freezing and snow treatments were minor and few. Physiological activity during the winter warming and freezing treatments was inconsistent with growth and mortality rates across the plants types. However, changes in the membrane fatty acids were associated with reduced mortality of grasses. Sub-Arctic plant communities may become dominated by grasses and deciduous plants if winter snowpack diminishes and plants are exposed to greater temperature variability in the near future.

Remodeling of chloroplast proteome under salinity affects salt tolerance of Festuca arundinacea.

Acclimation of photosynthetic apparatus to variable environmental conditions is an important component of tolerance to dehydration stresses, including salinity. The present study deals with the research on alterations in chloroplast proteome of the forage grasses. Based on chlorophyll fluorescence parameters, two genotypes of a model grass species-Festuca arundinacea with distinct levels of salinity tolerance: low salt tolerant (LST) and high salt tolerant (HST), were selected. Next, two-dimensional electrophoresis and mass spectrometry were applied under both control and salt stress conditions to identify proteins accumulated differentially between these two genotypes. The physiological analysis revealed that under NaCl treatment the studied plants differed in photosystem II activity, water content, and ion accumulation. The differentially accumulated proteins included ATPase B, ATP synthase, ribulose-1,5-bisphosphate carboxylase large and small subunits, cytochrome b6-f complex iron-sulfur subunit, oxygen-evolving enhancer proteins (OEE), OEE1 and OEE2, plastidic fructose-bisphosphate aldolase (pFBA), and lipocalin. A higher level of lipocalin, potentially involved in prevention of lipid peroxidation under stress, was also observed in the HST genotype. Our physiological and proteomic results performed for the first time on the species of forage grasses clearly showed that chloroplast metabolism adjustment could be a crucial factor in developing salinity tolerance.

Unpacking multi-trophic herbivore-grass-endophyte interactions: feedbacks across different scales in vegetation responses to Soay sheep herbivory.

Grazing can induce changes in both plant productivity and nutritional quality, which may subsequently influence herbivore carrying capacity. While research on Soay sheep (Ovis aries L.) dynamics on Hirta Island in the St. Kilda archipelago has elucidated the complexity of population drivers, including parasites, the role of herbivore-generated feedbacks as an intrinsic regulating factor remains unclear. The sheep lack large predators and every 3-9 years undergo population crashes (overcompensatory mortality). We investigated the effects of grazing on (1) sward productivity and (2) quality (toxicity) of the primary forage species, red fescue (Festuca rubra L.), which is highly infected by an alkaloid-synthesizing fungal endophyte. Grazing had a negative impact on both forage quantity and quality. At higher sheep densities, impacts on sward growth were magnified, resulting in a nonlinear relationship with plant productivity. Simultaneously, endophyte hyphal load (and by inference, toxicity) peaked close to the time of a crash. A greenhouse experiment showed that alkaloid concentration in F. rubra increased in response to artificial defoliation. We conclude that at high sheep densities, grazing-mediated reductions in productivity, together with sustained alkaloid production, are likely to influence sheep dynamics. Future research should consider the interactive effects of forage toxicity, quantity, and nutritional content.

Effect of consuming endophyte-infected fescue seed on transcript abundance in the mammary gland of lactating and dry cows, as assessed by RNA sequencing.

Ergot alkaloids in endophyte-infected grasses inhibit prolactin secretion and reduce milk production in lactating cows. However, we previously showed that prepartum consumption of infected seed throughout the dry period did not inhibit subsequent milk production and prior exposure to bromocriptine (ergot peptide) actually increased production in the next lactation. To identify changes in the transcriptome and molecular pathways mediating the mammary gland's response to ergot alkaloids in the diet, RNA sequencing (RNA-seq) was performed on mammary tissues obtained from 22 multiparous Holstein cows exposed to 1 of 3 treatments. Starting at 90 ± 4 d prepartum, cows were fed endophyte-free fescue seed (control; CON), endophyte-free fescue seed plus 3×/wk subcutaneous injections of bromocriptine (BROMO; 0.1 mg/kg of BW), or endophyte-infected fescue seed (INF) as 10% of the diet. Cows were dried off 60 ± 2 d prepartum. Mammary biopsies from 4 (BROMO, INF) or 5 (CON) cows/treatment at each of the 3 phases were obtained: 7 d before dry off during the initial lactation (L1), mid-dry period (D), and 10 d postpartum (L2). Although tissue from the same cow was preferentially used at 3 phases (L1, D, L2), tissue from additional cows were used to as necessary to provide RNA of sufficient quality. Individual samples were used to generate individual RNA-seq libraries. Normalized reads of the RNA-seq data were organized into technical and biological replicates before processing with the RSEM software package. Each lactation phase was processed separately and genes that differed between any of 3 treatments were identified. A large proportion of genes differentially expressed in at least 1 treatment (n = 866) were found to be similarly expressed in BROMO and INF treatments, but differentially expressed from CON (n = 575, total for 3 phases). Of genes differentially expressed compared with CON, 104 genes were common to the L1 and L2 phases. Consistent with the production findings, networks most affected by treatments in L1 and L2 included lipid metabolism, small molecule biochemistry, and molecular transport, whereas networks related more to developmental and cellular functions and maintenance were evident during D phase. Similar patterns of expression in BROMO and INF during late and early lactation suggest involvement of similar cell signaling pathways or mechanisms of action for BROMO and INF and the importance of prolactin messaging pathways.

Characterization and Functional Analysis of FaHsfC1b from Festuca arundinacea Conferring Heat Tolerance in Arabidopsis.

Heat transcription factors (Hsfs) belong to a large gene family classified into A, B, and C groups, with classes A and B Hsfs being well-characterized and known for their roles in plant tolerance to abiotic stresses. The functions and roles of Class C Hsfs are not well-documented. The objectives of this study were to characterize a class C Hsf gene (FaHsfC1b) cloned from tall fescue (Festuca arundinacea), a perennial grass species, and to determine the physiological functions of FaHsfC1b in regulating heat tolerance by overexpressing FaHsfC1b in Arabidopsis thaliana. Full length cDNA of FaHsfC1b was cloned and the sequence alignment showed that it had high similarity to OsHsfC1b with typical DNA binding domain, hydrophobic oligomerization domain, and a nucleus localization signal. Transient expression with FaHsfC1b-eGFP in protoplasts of Arabidopsis leaves indicated its nucleus localization. qRT-PCR analysis showed that FaHsfC1b responded to heat, osmotic, salt, and cold stress in leaves and roots during 48-h treatment. Physiological analysis showed that FaHsfC1b overexpression enhanced plant survival rate, chlorophyll content, and photochemical efficiency, while it resulted in decreases in electrolyte leakage, H2O2 and O2- content under heat stress. qRT-PCR showed that endogenous HsfC1 was induced in transgenic plants and the expression levels of heat protection protein genes, including several HSPs, AtGalSyn1, AtRof1, and AtHSA32, as well as ABA-synthesizing gene (NCED3) were significantly upregulated in transgenic plants overexpressing FaHsfC1b under heat stress. Our results first demonstrate that HsfC1b plays positive roles in plant tolerance to heat stress in association with the induction and upregulation of heat-protective genes. HsfC1b may be used as a candidate gene for genetic modification of cool-season plant species for improving heat tolerance.

Transcriptome profilings of two tall fescue (Festuca arundinacea) cultivars in response to lead (Pb) stress.

BACKGROUND: Lead (Pb) is one of the most toxic heavy metal environmental pollutants. Tall fescue is an important cold season turf grass which can tolerate and accumulate substantial amount of Pb. To estimate genes related to Pb response and the molecular mechanism associated with Pb tolerance and accumulation, we analyzed the transcriptome of tall fescue in response to Pb treatment. RESULTS: RNA-sequencing was performed in two tall fescue cultivars, Pb tolerant Silverado and Pb sensitive AST7001. A total of 810,146 assembled unique transcripts representing 25,415 unigenes were obtained from the tall fescue leaves. Among the panel, 3,696 differentially expressed genes (DEGs) were detected between the Pb treated (1000 mg/L) and untreated samples. Gene ontology (GO) and pathway enrichment analysis demonstrated that the DEGs were mainly implicated in energy metabolism, metabolism of terpenoids and polyketides, and carbohydrate metabolism related pathways. The expression patterns of 16 randomly selected genes were in consistent with that from the Solexa analysis using quantitative reverse-transcription PCR. In addition, compared to the common transcriptional response to Pb stress in both cultivars, the regulation of numerous genes including those involved in zeatin biosynthesis, limonene and pinene degradation, phagosome was exclusive to one cultivar. CONCLUSIONS: The tall fescue assembled transcriptome provided substantial molecular resources for further genomics analysis of turfgrass in response to heavy metal stress. The significant expression difference of specific unigenes may account for Pb tolerance or accumulation in two different tall fescue cultivars. This study provided new insights for the investigation of the molecular basis of Pb tolerance and accumulation in tall fescue as well as other related turf grass species.