Relationship between epigenetic marks and the behavior of 45S rDNA sites in chromosomes and interphase nuclei of Lolium-Festuca complex.

The grasses of the Lolium-Festuca complex show a prominent role in world agricultural scenario. Several studies have demonstrated that the plasticity of 45S rDNA sites has been recently associated with the possible fragility of the loci. Often, these fragile sites were observed as extended sites and gaps in metaphases. This organization can be evaluated in relation to their transcriptional activity/accessibility through epigenetic changes. Thus, this study aimed to investigate the relationship of the 5-methylcytosine and histone H3 lysine-9 dimethylation in different conformations of 45S rDNA sites in interphase nuclei and in metaphase chromosomes of L. perenne, L. multiflorum and F. arundinacea. The FISH technique using 45S rDNA probes was performed sequentially after the immunolocalization. The sites showed predominantly the following characteristics in the interphase nuclei: intra- and perinucleolar position, decondensed or partially condensed and hypomethylated and hyper/hypomethylated status. Extranucleolar sites were mainly hypermethylated for both epigenetic marks. The 45S rDNA sites with gaps identified in metaphases were always hypomethylated, which justifies it decondensed and transcriptional state. The frequency of sites with hypermethylated gaps was very low. The structural differences observed in these sites are directly related to the assessed epigenetic marks, justifying the different conformations throughout the cell cycle.

Functional co-expression of a fungal ferulic acid esterase and a ß-1,4 endoxylanase in Festuca arundinacea (tall fescue) modifies post-harvest cell wall deconstruction.

MAIN CONCLUSION: Improved post-harvest cell wall deconstruction of tall fescue leaves has been demonstrated by in-planta co-expression of a constitutively expressed ferulic acid esterase together with a senescence-induced ß-1,4 endoxylanase. Tall fescue plants (Festuca arundinacea) constitutively expressing vacuole- or apoplast-targeted ferulic acid esterase from Aspergillus niger were retransformed with a senescence-induced and apoplast-targeted ß-1,4 endo-xylanase from Trichoderma reesei. Enzyme activities in co-expressing plants stabilized after repeated vegetative propagation, with xylanase activity in senescent leaves increasing and ferulic acid esterase activity decreasing after tillering. Plants co-expressing both enzymes in the apoplast, with the lowest levels of ferulate monomers and dimers and the lowest levels of cell wall arabinoxylans, released ten times more cell wall hydroxycinnamic acids and five times more arabinoxylan from the cell wall on autodigestion compared to expression of ferulic acid esterase or xylanase alone. These plants also showed a 31 % increase in cellulase-mediated release of reducing sugars, a 5 % point increase in in vitro dry matter digestibility and a 23 % increase in acetyl bromide-soluble lignin. However, plant growth was adversely affected by expressing FAE in the apoplast, giving plants with narrower shorted leaves, and a 71 % decrease in biomass.

An RNA sequencing transcriptome analysis of the high-temperature stressed tall fescue reveals novel insights into plant thermotolerance.

BACKGROUND: Tall fescue (Festuca arundinacea Schreb.) is major cool-season forage and turf grass species worldwide, but high-temperature is a major environmental stress that dramatically threaten forage production and turf management of tall fescue. However, very little is known about the whole-genome molecular mechanisms contributing to thermotolerance. The objectives of this study were to analyzed genome-wide gene expression profiles in the leaves of two tall fescue genotypes, heat tolerant 'PI578718' and heat sensitive 'PI234881' using high-throughput RNA sequencing. RESULTS: A total of 262 million high-quality paired-end reads were generated and assembled into 31,803 unigenes with an average length of 1,840 bp. Of these, 12,974 unigenes showed different expression patterns in response to heat stress and were categorized into 49 Gene Ontology functional subcategories. In addition, the variance of enrichment degree in each functional subcategory between PI578718 and PI234881 increased with increasing treatment time. Cell division and cell cycle genes showed a massive increase in transcript abundance in heat-stressed plants and more activated genes were detected in PI 578718 by Kyoto Encyclopedia of Genes and Genomes pathways analysis. Low molecular weight heat shock protein (LMW-HSP, HSP20) showed activated in two stressed genotypes and high molecular weight HSP (HMW-HSP, HSP90) just in PI578718. Assimilation such as photosynthesis, carbon fixation, CH4, N, S metabolism decreased along with increased dissimilation such as oxidative phosphorylation. CONCLUSIONS: The assembled transcriptome of tall fescue could serve as a global description of expressed genes and provide more molecular resources for future functional characterization analysis of genomics in cool-season turfgrass in response to high-temperature. Increased cell division, LMW/HMW-HSP, dissimilation and antioxidant transcript amounts in tall fescue were correlated with successful resistance to high temperature stress.

Alien introgression in the grasses Lolium perenne (perennial ryegrass) and Festuca pratensis (meadow fescue): the development of seven monosomic substitution lines and their molecular and cytological characterization.

BACKGROUND AND AIMS: To address the issues associated with food security, environmental change and bioenergy in the context of crop plants, the production, identification and evaluation of novel plant phenotypes is fundamental. One of the major routes to this end will be wide hybridization and introgression breeding. The transfer of chromosomes and chromosome segments between related species (chromosome engineering or alien introgression) also provides an important resource for determining the genetic control of target traits. However, the realization of the full potential of chromosome engineering has previously been hampered by the inability to identify and characterize interspecific introgressions accurately. METHODS: Seven monosomic substitution lines have been generated comprising Festuca pratensis as the donor species and Lolium perenne as the recipient. Each of the seven lines has a different L. perenne chromosome replaced by the homoeologous F. pratensis chromosome (13 L. perenne + 1 F. pratensis chromosome). Molecular markers and genomic in situ hybridization (GISH) were used to assign the F. pratensis chromosomes introgressed in each of the monosomic substitutions to a specific linkage group. Cytological observations were also carried out on metaphase I of meiosis in each of the substitution lines. RESULTS: A significant level of synteny was found at the macro-level between L. perenne and F. pratensis. The observations at metaphase I revealed the presence of a low level of interspecific chromosomal translocations between these species. DISCUSSION: The isolation of the seven monosomic substitution lines provides a resource for dissecting the genetic control of important traits and for gene isolation. Parallels between the L. perenne/F. pratensis system and the Pooideae cereals such as wheat, barley, rye, oats and the model grass Brachypodium distachyon present opportunities for a comparison across the species in terms of genotype and phenotype.

How meristem plasticity in response to soil nutrients and light affects plant growth in four Festuca grass species.

Investigation of responses of meristems to environmental conditions is important for understanding the mechanisms and consequences of plant phenotypic plasticity. Here, we examined how meristem plasticity to light and soil nutrients affected leaf growth and relative growth rate (RGR) in fast- and slow-growing Festuca grass species. Activity in shoot apical meristems was measured by leaf appearance rate, and that in leaf meristems by the duration and rate of cell production, which was further divided into single cell cycle time and the number of dividing cells. Light and soil nutrients affected activity in shoot apical meristems similarly. The high nutrient supply increased the number of dividing cells, which was responsible for enhancement of cell production rate; shaded conditions extended the duration of cell production. As a result, leaf length increased under high nutrient and shaded conditions. The RGR was correlated positively with the total meristem size of the shoot under a low nutrient supply, implying inhibition of RGR by cell production under nutrient-limited conditions. Fast-growing species were more plastic for cell production rate and specific leaf area (SLA) but less plastic for RGR than slow-growing species. This study demonstrates that meristem plasticity plays key roles in characterizing environmental responses of plant species.

Cytogenetics of Festulolium (Festuca x Lolium hybrids).

Grasses are the most important and widely cultivated crops. Among them, ryegrasses (Lolium spp.) and fescues (Festuca spp.) provide high quality fodder for livestock, are used for turf and amenity purposes, and play a fundamental role in environment protection. Species from the two genera display complementary agronomic characteristics and are often grown in mixtures. Breeding efforts to combine desired features in single entities culminated with the production of Festuca x Lolium hybrids. The so called Festuloliums enjoy a considerable commercial success with numerous cultivars registered all over the world. They are also very intriguing from a strictly cytogenetic point of view as the parental chromosomes recombine freely in hybrids. Until a decade ago this phenomenon was only known in general quantitative terms. The introduction of molecular cytogenetic tools such as FISH and GISH permitted detailed studies of intergeneric chromosome recombination and karyotyping of Festulolium cultivars. These tools were also invaluable in revealing the origin of polyploid fescues, and facilitated the development of chromosome substitution and introgression lines and physical mapping of traits of interest. Further progress in this area will require the development of a larger set of cytogenetic markers and high-resolution cytogenetic maps. It is expected that the Lolium-Festuca complex will continue providing opportunities for breeding superior grass cultivars and the complex will remain an attractive platform for fundamental research of the early steps of hybrid speciation and interaction of parental genomes, as well as the processes of chromosome pairing, elimination and recombination.

Simultaneous overexpression of both CuZn superoxide dismutase and ascorbate peroxidase in transgenic tall fescue plants confers increased tolerance to a wide range of abiotic stresses.

To mitigate the oxidative damage inflicted by biotic or abiotic stresses, plants have evolved complex anti-oxidative defense mechanisms that involve induction of antioxidant and anti-oxidative enzymes, such as superoxide dismutase (SOD) and ascorbate peroxidase (APX). To determine whether overexpression of the genes encoding copper-zinc SOD (CuZnSOD) and APX in plants is capable of decreasing reactive oxygen species (ROS) produced in response to abiotic stresses, we generated transgenic tall fescue plants expressing the CuZnSOD and APX genes in chloroplasts under the control of the oxidative stress-inducible promoter, sweet potato peroxidase anionic 2 (SWPA2). Transgenic plants were generated by Agrobacterium-mediated genetic transformation, and genotypes were confirmed by DNA blot analysis. Transgenic plants were exposed to several ROS-generating abiotic stresses, such as methyl viologen (MV), H(2)O(2), and the heavy metals copper, cadmium, and arsenic, and their tolerance was evaluated. High levels of CuZnSOD and APX gene transcripts in the transgenic plants under these treatments suggested that the transgenes were functionally expressed. Compared to transgenic plants, higher amounts of ROS were generated in the leaves of control plants exposed to abiotic stresses, resulting in increased thiobarbituric acid reactive substances (TBARS), ion leakage, and chlorophyll degradation. These parameters were significantly lower in transgenic plants. Enzyme activity assays and native polyacrylamide gel electrophoresis (PAGE) showed that total SOD and APX were highly active in transgenic plants under the abiotic stresses examined. We conclude that one of the mechanisms of increased anti-oxidative defense in transgenic tall fescue plants is overexpression of the CuZnSOD and APX genes, which are utilized in scavenging ROS and thus provide improved tolerance to abiotic stresses.

Reducing cell wall feruloylation by expression of a fungal ferulic acid esterase in Festuca arundinacea modifies plant growth, leaf morphology and the turnover of cell wall arabinoxylans.

A feature of cell wall arabinoxylan in grasses is the presence of ferulic acid which upon oxidative coupling by the action of peroxidases forms diferuloyl bridges between formerly separated arabinoxylans. Ferulate cross-linking is suspected of playing various roles in different plant processes. Here we investigate the role of cell wall feruloyaltion in two major processes, that of leaf growth and the turnover of cell wall arabinoxylans on leaf senescence in tall fescue using plants in which the level of cell wall ferulates has been reduced by targeted expression of the Aspergillus niger ferulic acid esterase A (FAEA) to the apoplast or Golgi. Analysis of FAE expressing plants showed that all the lines had shorter and narrower leaves compared to control, which may be a consequence of the overall growth rate being lower and occurring earlier in FAE expressing leaves than in controls. Furthermore, the final length of epidermal cells was shorter than controls, indicating that their expansion was curtailed earlier than in control leaves. This may be due to the observations that the deposition of both ether and ester linked monomeric hydroxycinnamic acids and ferulate dimerization stopped earlier in FAE expressing leaves but at a lower level than controls, and hydroxycinnamic acid deposition started to slow down when peroxidase levels increased. It would appear therefore that one of the possible mechanisms for controlling overall leaf morphology such as leaf length and width in grasses, where leaf morphology is highly variable between species, may be the timing of hydroxycinnamic acid deposition in the expanding cell walls as they emerge from cell division into the elongation zone, controlled partially by the onset of peroxidase activity in this region.

Genotyping of somatic hybrids between Festuca arundinacea Schreb. and Triticum aestivum L.

In order to genotype hybrid genomes of distant asymmetric somatic hybrids, we synthesized hybrid calli and plants via PEG-mediated protoplast fusion between recipient tall fescue (Festuca. arundinacea Schreb.) and donor wheat (Triticum aestivum L.). Seventeen and 25 putative hybrid clones were produced from the fusion combinations I and II, each with the donor wheat protoplast treated by UV light for 30 s and 1 min, respectively. Isozyme and RAPD profiles confirmed that ten hybrid clones were obtained from combination I and 19 from combination II. Out of the 29 hybrids, 12 regenerated hybrid plants with tall fescue phenotype. Composition and methylation-variation of the nuclear and cytoplasmic genomes of some hybrids, either with or without regenerative ability, were compared by genomic in situ hybridization, restriction fragment length polymorphism, and DNA methylation-sensitive amplification polymorphism. Our results indicated that these selected hybrids all contained introgressed nuclear and cytoplasmic DNA as well as obvious methylation variations compared to both parents. However, there were no differences either in nuclear/cytoplasmic DNA or methylation degree between the regenerable and non-regenerable hybrid clones. We conclude that both regeneration complementation and genetic material balance are crucial for hybrid plant regeneration.