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Tall fescue (Lolium arundinaceum) is one of the primary forage and turf grasses in temperate regions of the world. A number of favourable characteristics of tall fescue are enhanced by its seed-transmissible fungal symbiont (endophyte) Epichloë coenophiala. Our approach was to assemble the tall fescue transcriptome, then identify differentially expressed genes (DEGs) for endophyte-symbiotic (E+) vs endophyte-free (E−) clones in leaf blades, pseudostems, crowns and roots. RNA-seq reads were used to construct a tall fescue reference transcriptome and compare gene expression profiles. Over all tissues examined, 478 DEGs were identified between the E+ and E− clones for at least one tissue (more than two-fold; P < 0.0001, 238 E+ > E− and 240 E− > E+), although no genes were differentially expressed in all four tissues. Gene ontology (GO) terms, GO:0010200 (response to chitin), GO:0002679 (respiratory burst during defence response) and GO:0035556 (intracellular signal transduction) were significantly overrepresented among 25 E− > E+ DEGs in leaf blade, and a number of other DEGs were associated with defence and abiotic response. In particular, endophyte effects on various WRKY transcription factors may have implications for symbiotic stability, endophyte distribution in the plant, or defence against pathogens.

Tall fescue (Festuca arundinacea Schreb.) is a popular pasture and turf grass particularly known for drought resistance, allowing for its persistence in locations that are unfavorable for other cool‐season grasses. Also, its seed‐borne fungal symbiont (endophyte) Epichloë coenophiala, which resides in the crown and pseudostem, can be a contributing factor in its drought tolerance. Because it contains the apical meristems, crown survival under drought stress is critical to plant survival as well as the endophyte. In this study, we subjected tall fescue plants with their endophyte to water‐deficit stress or, as controls with normal watering, then compared plant transcriptome responses in four vegetative tissues: leaf blades, pseudostem, crown, and roots. A transcript was designated a differentially expressed gene (DEG) if it exhibited at least a twofold expression difference between stress and control samples with an adjusted p value of .001. Pathway analysis of the DEGs across all tissue types included photosynthesis, carbohydrate metabolism, phytohormone biosynthesis and signaling, cellular organization, and a transcriptional regulation. While no specific pathway was observed to be differentially expressed in the crown, genes encoding auxin response factors, nuclear pore anchors, structural maintenance of chromosomes, and class XI myosin proteins were more highly differentially expressed in crown than in the other vegetative tissues, suggesting that regulation in expression of these genes in the crown may aid in survival of the meristems in the crown. Core Ideas Gene expression was evaluated in four tissues of stressed and unstressed tall fescue clones. Differentially expressed genes (DEGs) were identified using RNA‐seq. Gene ontology pathways were identified by DEGs in different tissues. Crown‐specific, stress‐responsive DEGs were presented. Nuclear pore anchor and class IX myosin genes involved in development were downregulated in crown under stress.

Incorporation of red clover (Trifolium pratense L.) into grass pastures can reduce the need for nitrogen fertilizer applications and increase the nutritional value of the forage. However, red clover cultivars available for Kentucky producers are highly susceptible to herbicides, such as 2,4-D (2,4-dichlorophenoxy acetic acid), used for pasture broadleaf weed control. To overcome this problem, ‘UK2014’ red clover was selected for increased tolerance to 2,4-D. We employed a transcriptome analysis approach to compare the gene expression response following 2,4-D treatment of ‘UK2014’ to that of ‘Kenland’, a 2,4-D sensitive red clover and one of the parents of ‘UK2014’. The objectives were to first determine if the increased 2,4-D tolerance in ‘UK2014’ is reflected in a change of transcription response and/or a quicker recovery of a transcriptional response following 2,4-D treatment, and second, to identify genes, whether constitutively expressed or induced by 2,4-D, which could be the basis for the increased 2,4-D tolerance. Leaf tissue from the two red clovers grown in the field was collected at 4, 24, and 72 h after 2,4-D (1.12 kg 2,4-amine a.e. ha−1) treatment from both untreated and treated plants. Global gene expression was determined with reads from Illumina Hiseq 2500 mapped against the red clover draft genome, Tpv2.1 (GenBank Accession GCA_900079335.1). Genes that displayed differential expression (DEGs) following 2,4-D treatment were selected for further analysis. The number of DEGs was higher for ‘Kenland’ than for ‘UK2014’, suggesting that a lower transcriptional response corresponds with the higher 2,4-D tolerance in the ‘UK2014’ line. Similarly, gene ontology enrichment analysis revealed that expression of photosynthesis-related genes was less affected by 2,4-D in the ‘UK2014’ line than ‘Kenland’. Although we were not able to identify any specific genes that are the basis for the increased 2,4-D tolerance of ‘UK2014’, we concluded that the increased 2,4-D tolerance of ‘UK2014’ correlates with a decreased transcription response to 2,4-D. Additionally, expression of several cytochrome P450 genes that had different isoforms between ‘UK2014’ and ‘Kenland’ increased significantly in both following 2,4-D treatment, one or more of these P450s could be mediators of 2,4-D metabolism and tolerance in red clover.

Core Ideas RNA‐seq was performed on four tall fescue clone pairs. Three Epichloë coenophiala strains were evaluated. Gene expression was compared for stressed and unstressed plants. Differentially expressed unigenes were identified. Few positive endophyte effects on stress tolerance were observed. Two tall fescue [Lolium arundinaceum (Schreb.) Darbysh. = Schedonorus arundinaceus (Schreb.) Dumort. = Festuca arundinacea var. arundinacea Schreb.] plant genotypes with an Epichloë coenophiala (Morgan‐Jones & W. Gams) C.W. Bacon & Schardl common toxic endophyte (CTE), one with a nontoxic strain (NTE19) and one with another Epichloë species (FaTG‐4) were evaluated and compared with their respective endophyte‐free clones for responses to water‐deficit stress in the greenhouse. One of the plant genotypes (P27) showed a positive effect of its CTE strain on tiller production after stress and resumed watering. In transcriptome analysis of the pseudostems (leaf sheath whorls), differentially expressed genes (DEGs) were defined as having at least twofold expression difference and false discovery rate (FDR) < 0.05 in comparisons of water treatment (stressed or watered), endophyte presence or absence, or both. Stress affected 38% of the plant transcripts including those for the expected stress‐response pathways. The DEGs affected by endophyte in stressed plants were unique to individual plant genotypes. In unstressed plants, endophyte presence tended to reduce expression of genes putatively for defense against fungi, but in unstressed P27 endophyte presence there was enhanced expression of dehydrin and heat shock protein genes. Our results indicated subtle and variable effects of endophytes on tall fescue gene expression; where the endophyte confers protection, its effects on plant gene expression may help prime the plant for stress resistance.

A small gene family of phosphatidyl ethanolamine-binding proteins (PEBP) has been shown to function as key regulators in flowering; in Arabidopsis thaliana the FT protein promotes flowering whilst the closely related TFL1 protein represses flowering. Control of flowering time in soybean [Glycine max (L.) Merrill] is important for geographic adaptation and maximizing yield. Soybean breeders have identified a series of loci, the E-genes, that control photoperiod-mediated flowering time, yet how these loci control flowering is poorly understood. The objectives of this study were to evaluate the expression of GmFT-like genes in the E1 near-isogenic line (NIL) background. Of the 20 closely related PEBP proteins in the soybean genome, ten are similar to the Arabidopsis FT protein. Expression analysis of these ten GmFT-like genes confirmed that only two are detectable in the conditions tested. Further analysis of these two genes in the E1 NILs grown under short-day (SD) and long-day (LD) conditions showed a diurnal expression and tissue specificity expression commensurate with soybean flowering time under SD and LD conditions, suggesting that these were good candidates for flowering induction in soybean. Arabidopsis ft mutant lines flowered early when transformed with the two soybean genes, suggesting that the soybean genes can complement the Arabidopsis FT function. Flowering time in E1 NILs is consistent with the differential expression of the two GmFT-like genes under SD and LD conditions, suggesting that the E1 locus, at least in part, impacts time to flowering through the regulation of soybean FT expression.

Tall fescue (Lolium arundinaceum) is a highly adaptable forage, pasture and turf grass that is grown on over 14 M ha in the eastern half of the United States and in other temperate regions of the world. A significant factor in adaptability, productivity and stand persistence is in part due to the presence of an intercellular, seed-transmissible, endophytic fungus, Epichloë coenophiala. Epichloë endophytes have been shown to produce a number of alkaloid compounds only in planta, some that are beneficial in repelling insects, while others are toxic to animals. The goal of this work was to monitor the level of the ergot and loline (classified as pyrrolizidine) alkaloid accumulation in individual plants to determine the plant genotype contribution to alkaloid concentrations. The experimental design consisted of sixteen tall fescue KY31 clones in a space-planted, replicated trial over three years. Our results demonstrated that while changes in the alkaloid concentrations for each plant/endophyte genotype were observed over the three years, the overall alkaloid levels remained relatively constant when compared to other plant/endophyte genotypes combinations in the field. Additionally, overall levels of the ergot and loline alkaloid accumulation did not vary in the same way over the three years. Since the E. coenophiala endophyte genotype was the same across all clones, our results indicate that it is the plant genotype that is responsible for determining alkaloid levels in each plant, and suggest that the signal(s) from the plant to the endophyte may not be the same for ergot and loline alkaloid production.

Red clover (Trifolium pratense L.) is an important forage crop and serves as a major contributor of nitrogen input in pasture settings because of its ability to fix atmospheric nitrogen. During the legume-rhizobial symbiosis, the host plant undergoes a large number of gene expression changes, leading to development of root nodules that house the rhizobium bacteria as they are converted into nitrogen-fixing bacteroids. Many of the genes involved in symbiosis are conserved across legume species, while others are species-specific with little or no homology across species and likely regulate the specific plant genotype/symbiont strain interactions. Red clover has not been widely used for studying symbiotic nitrogen fixation, primarily due to its outcrossing nature, making genetic analysis rather complicated. With the addition of recent annotated genomic resources and use of RNA-seq tools, we annotated and characterized a number of genes that are expressed only in nodule forming roots. These genes include those encoding nodule-specific cysteine rich peptides (NCRs) and nodule-specific Polycystin-1, Lipoxygenase, Alpha toxic (PLAT) domain proteins (NPDs). Our results show that red clover encodes one of the highest number of NCRs and ATS3-like/NPDs, which are postulated to increase nitrogen fixation efficiency, in the Inverted-Repeat Lacking Clade (IRLC) of legumes. Knowledge of the variation and expression of these genes in red clover will provide more insights into the function of these genes in regulating legume-rhizobial symbiosis and aid in breeding of red clover genotypes with increased nitrogen fixation efficiency.

Control of soybean flowering time is important for geographic adaptation and maximizing yield. Plant breeders have identified a series of genes (E genes) that condition time to flowering; however, the molecular basis in the control of flowering by these E genes, in conjunction with canonical flowering-time genes, has not been studied. Time to flowering in near-isogenic lines (NILs) at the E1 locus was tested using a reciprocal transfer experiment under short day (SD) and long day (LD) conditions. Beginning 8 days after planting, three plant samples were harvested every 3 h for a 48-h period. RNA was isolated from these plants, and RNA samples were pooled for each line and each time period for cDNA synthesis. RT-PCR analysis was performed using primers synthesized for a number of putative flowering-time genes based on homology of soybean EST and genomic sequences to Arabidopsis genes. The results of the reciprocal transfer experiment suggest that the pre-inductive photoperiod-sensitive phase of the E1 NILs responsible for inducing flowering is perceived as early as 5-7-day post-planting. No gene expression differences were found between the E1 and e1 NILs, suggesting that the E1 gene does not directly affect the flowering-time genes during the time period tested; however, differences were observed in gene expression between SD and LD treatments for the putative soybean TOC1, CO, and FT genes. The gene expression results in this study were similar to those of flowering-time genes found in other SD species, suggesting that the selected genes correspond to the soybean flowering-time orthologs.

Background The endophytic fungus, Neotyphodium coenophialum , can enhance drought tolerance of its host grass, tall fescue. To investigate endophyte effects on plant responses to acute water deficit stress, we did comprehensive profiling of plant metabolite levels in both shoot and root tissues of genetically identical clone pairs of tall fescue with endophyte (E+) and without endophyte (E-) in response to direct water deficit stress. The E- clones were generated by treating E+ plants with fungicide and selectively propagating single tillers. In time course studies on the E+ and E- clones, water was withheld from 0 to 5 days, during which levels of free sugars, sugar alcohols, and amino acids were determined, as were levels of some major fungal metabolites. Results After 2–3 days of withholding water, survival and tillering of re-watered plants was significantly greater for E+ than E- clones. Within two to three days of withholding water, significant endophyte effects on metabolites manifested as higher levels of free glucose, fructose, trehalose, sugar alcohols, proline and glutamic acid in shoots and roots. The fungal metabolites, mannitol and loline alkaloids, also significantly increased with water deficit. Conclusions Our results suggest that symbiotic N. coenophialum aids in survival and recovery of tall fescue plants from water deficit, and acts in part by inducing rapid accumulation of these compatible solutes soon after imposition of stress.

Core Ideas De novo transcriptome assembly of red clover (Trifolium pratense L.) Functional annotation of the assembled transcriptome Global identification of red clover transcripts encoding putative transcription factors Identification of tissue‐enriched gene coexpression clusters Red clover (Trifolium pratense L.) is a cool‐season forage legume grown throughout the northeastern United States and is the most widely planted forage legume after alfalfa (Medicago sativa L.). Red clover provides high‐value feed to the livestock because of high protein content and easy digestibility. To date, genomic resources for red clover are scarce. In the current study, a de novo transcriptome assembly of red clover was constructed representing different tissue types. The draft assembly consists of 37,565 contigs with N50 and average contig length of 1707 and 1262 bp, respectively. A comparative study with three other legume species displayed a high degree of sequence conservation between red clover and other legumes. The assembled transcriptome was annotated to allow identification of desirable genes. In particular, a genome‐wide identification of red clover transcripts encoding putative transcription factors was performed. A comparative gene expression analysis between different tissue types was performed using the assembled transcriptome as the reference, which revealed dynamic gene expression patterns across different tissue types and also identified spatially dynamic gene coexpression clusters. Genes representing tissue‐enriched clusters were subjected to gene ontology (GO) enrichment analysis to identify over‐represented functional groups. Identification of these tissue‐enriched gene coexpression clusters can help in future research focusing on developmental studies across tissues or in biotechnological improvement of red clover.