Isolation and characterisation of a sucrose: sucrose 1-fructosyltransferase gene from perennial ryegrass (Lolium perenne)

A sucrose: sucrose 1-fructosyltransferase (1-SST) gene and cDNA (Lp 1-SST) from perennial ryegrass (Lolium perenne) were isolated. The Lp 1-SST gene was fully sequenced and shown to contain three exons and two introns. Nucleotide sequence analysis of the 4824 bp Lp 1-SST genomic sequence revealed 1618 bp of 5' UTR and an open reading frame of 1962 bp encoding a protein of 653 amino acids. Lp 1-SST is 95% identical to the tall fescue 1-SST and contains plant fructosyltransferase functional domains. Lp 1-SST corresponds to a single copy gene in perennial ryegrass, and is expressed in young leaf bases and mature leaf sheaths. The recombinant Lp 1-SST protein from corresponding cDNA expression in Pichia pastoris showed 1-SST activity.     

Coordinated expression of functionally diverse fructosyltransferase genes is associated with fructan accumulation in response to low temperature in perennial ryegrass

* Fructan is the major nonstructural carbohydrate reserve in temperate grasses. To understand regulatory mechanisms in fructan synthesis and adaptation to cold environments, the isolation, functional characterization and genetic mapping of fructosyltransferase (FT) genes in perennial ryegrass (Lolium perenne) are described. * Six cDNAs (prft1-prft6) encoding FTs were isolated from cold-treated ryegrass plants, and three were positioned on a perennial ryegrass linkage map. Recombinant proteins were produced in Pichia pastoris and enzymatic activity was characterized. Changes in carbohydrate levels and mRNA levels of FT genes during cold treatment were also analysed. * One gene encodes sucrose-sucrose 1-fructosyltransferase (1-SST), and two gene encode fructan-fructan 6G-fructosyltransferase (6G-FFT). Protein sequences for the other genes (prfts 1, 2 and 6) were similar to sucrose-fructan 6-fructosyltransferase (6-SFT). The 1-SST and prft1 genes were colocalized with an invertase gene on the ryegrass linkage map. The mRNA levels of prft1 and prft2 increased gradually during cold treatment, while those of the 1-SST and 6G-FFT genes first increased, but then decreased before increasing again during a longer period of cold treatment. * Thus at least two different patterns of gene expression have developed during the evolution of functionally diverse FT genes, which are associated in a coordinated way with fructan synthesis in a cold environment.     

Allelic variation in the perennial ryegrass FLOWERING LOCUS T gene is associated with changes in flowering time across a range of populations

The Arabidopsis (Arabidopsis thaliana) FLOWERING LOCUS T (FT) gene and its orthologs in other plant species (e.g. rice [Oryza sativa] OsFTL2/Hd3a) have an established role in the photoperiodic induction of flowering response. The genomic and phenotypic variations associated with the perennial ryegrass (Lolium perenne) ortholog of FT, designated LpFT3, was assessed in a diverse collection of nine European germplasm populations, which together constituted an association panel of 864 plants. Sequencing and genotyping of a series of amplicons derived from the nine populations, containing the complete exon and intron sequences as well as 5' and 3' noncoding sequences of LpFT3, identified a total of seven haplotypes. Genotyping assays designed to detect the genomic variation showed that three haplotypes were present in approximately equal proportions and represented 84% of the total, with a fourth representing a further 11%. Of the three major haplotypes, two were predicted to code for identical protein products and the third contained two amino acid substitutions. Association analysis using either a mixed model with a relationship matrix to correct for population structure and relatedness or structured association with further correction using genomic control indicated significant associations between LpFT3 and variation in flowering time. These associations were corroborated in a validation population segregating for the same major alleles. The most "diagnostic" region of genomic variation was situated 5' of the coding sequence. Analysis of this region identified that the interhaplotype variation was closely associated with sequence motifs that were apparently conserved in the 5' region of orthologs of LpFT3 from other plant species. These may represent cis-regulatory elements involved in influencing the expression of this gene.     

Fructosyltransferase and invertase genes evolved by gene duplication and rearrangements: rice, perennial ryegrass, and wheat gene families.

The invertase enzyme family is responsible for carbohydrate metabolism in rice, perennial ryegrass, and wheat. Fructan molecules accumulate in cell vacuoles of perennial ryegrass and wheat and are associated with abiotic stress tolerance. High levels of amino acid similarity between the fructosyltransferases responsible for fructan accumulation indicates that they may have evolved from invertase-like ancestral genes. In this study, we have applied comparative genomics to determine the mechanisms that lead to the evolution of fructosytransferase and invertase genes in rice, perennial ryegrass, and wheat. Duplications and rearrangements have been inferred to generate variant forms of the rice invertases since divergence from a common grass progenitor. The occurrence of multiple copies of fructosyltransferase genes indicated that duplication events continued during evolution of the wheat and perennial ryegrass lineages. Further gene rearrangements were evident in perennial ryegrass genes, albeit at a reduced level compared with the rice invertases. Gene orthologs were largely static after duplication during evolution of the wheat lineage. This study details evolutionary events that contribute to fructosyltransferase and invertase gene variation in grasses.     

Isolation and characterization of cold-regulated transcriptional activator <Emphasis Type="Italic">LpCBF3</Emphasis> gene from perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

In plants, low temperatures can activate the CBF cold response pathway playing a prominent role in cold acclimation by triggering a set of cold-related gene expressions. CBF homologous gene, designated as LpCBF3, from a cold-tolerant perennial ryegrass (Lolium perenne L.) accession was identified. It carries the sequences for nuclear localization signal (NLS), AP2 DNA-binding domains and an acidic activation present in most of the plant CBF proteins. Southern analysis indicated the presence of three homologs of LpCBF3 gene in perennial ryegrass genome, and only one amino acid variation in LpCBF3 protein between cold-tolerant and -sensitive perennial ryegrass accessions. In their putative promoter regions, some differential regions were found. Northern blotting and RT-PCR analysis found that LpCBF3 reached the highest expression after 1.5 h of cold treatment (4 degrees C). The COR homologous gene, a downstream gene of CBF, can be expressed in the plant stem of cold-tolerant perennial ryegrass accessions without cold treatment. Without cold treatment, the COR gene cannot be activated in cold-sensitive perennial ryegrass accessions. Cold treatment can prompt expression levels of COR homologous genes in both perennial ryegrass accessions. In transgenic Arabidopsis, the overexpression of LpCBF3 with the 35S promoter resulted in dwarf-like plants, later flowering and greater freezing tolerance.     

Molecular genetics of fructan metabolism in perennial ryegrass

Fructans are the main storage carbohydrates of temperate grasses, sustaining regrowth immediately after defoliation, as well as contributing to the nutritive value of feed. Fructan metabolism is based on the substrate sucrose and involves fructosyltransferases (FTs) for biosynthesis and fructan exohydrolases (FEHs) for degradation. Sucrose is also utilized by invertases (INVs), which hydrolyse it into its constituent monosaccharides for use in metabolism. The isolation, molecular characterization, functional analysis, and phylogenetic relationships of genes encoding FTs, FEHs, and INVs from temperate grasses are reviewed, with an emphasis on perennial ryegrass (Lolium perenne L.). The roles these enzymes play in fructan accumulation and remobilization, and future biotechnological applications in molecular plant breeding are discussed.     

氧化石墨烯对多年生黑麦草逆境生理及光合特征的影响

为探讨不同浓度氧化石墨烯（GO）对多年生黑麦草生长、生理及光合特征的影响,该文采用盆栽试验,在土壤中添加0、10、20、30、40、50 mg·g^-1 GO进行多年生黑麦草培养,并测定植物生长指标、光合色素含量、保护酶活性、丙二醛（MDA）含量、叶片质膜透性、可溶性蛋白含量和光合参数。结果表明：（1）10、20 mg·g^-1 GO处理对多年生黑麦草生长无显著影响;30～50 mg·g^-1 GO处理对多年生黑麦草生长具有抑制作用,在50 mg·g^-1 GO浓度下多年生黑麦草株高和生物量均最小,较对照分别降低了16.8%和27.1%。（2）当GO浓度达到30 mg·g^-1时,总叶绿素和类胡萝卜素的含量显著降低,在50 mg·g^-1 GO处理时达到最低。（3）高浓度的GO处理(40、50 mg·g^-1)虽降低了多年生黑麦草的叶片净光合速率（P_n）、气孔导度（G_s）和蒸腾速率（T_r     

Application of silicon sources increases silicon accumulation in perennial ryegrass turf on two soil types

This study investigated the ability of perennial ryegrass to accumulate silicon and the factors that may influence plant silicon accumulation. Plants were grown in the greenhouse in two soil types, peat:sand mix and Hagerstown-silt-loam, amended with two commercially available sources of silicon, calcium silicate slag and wollastonite at 0, 0.5, 1, 2, 5 and 10 t/ha. Shoot tissue of nine-week-old perennial ryegrass plants was analyzed for silicon content (%) and found to reach a dry matter concentration of up to 4% in this study. Silicon accumulation in perennial ryegrass was influenced by the soil type and source, and was higher in plants grown in low-silicon peat:sand mix compared to Hagerstown-silt-loam. Silicon content (%) in the plants consistently increased with increasing rates of silicon in all four soil and source combinations. Acetic acid (HAc) extractable silicon and Ca increased in both soil types when amended with either of the silicon sources. Effects of silicon sources on soil pH varied with soil type. This study indicates that soil type, source of silicon, and rate of silicon application are important factors influencing the uptake of silicon by perennial ryegrass which is a widely used turfgrass species in golf courses, sports fields, and residential lawns in the United States.     

Advanced data-mining strategies for the analysis of direct-infusion ion trap mass spectrometry data from the association of perennial ryegrass with its endophytic fungus, Neotyphodium lolii

Direct-infusion mass spectrometry (MS) was applied to study the metabolic effects of the symbiosis between the endophytic fungus Neotyphodium lolii and its host perennial ryegrass (Lolium perenne) in three different tissues (immature leaf, blade, and sheath). Unbiased direct-infusion MS using a linear ion trap mass spectrometer allowed metabolic effects to be determined free of any preconceptions and in a high-throughput fashion. Not only the full MS(1) mass spectra (range 150-1,000 mass-to-charge ratio) were obtained but also MS(2) and MS(3) product ion spectra were collected on the most intense MS(1) ions as described previously (Koulman et al., 2007b). We developed a novel computational methodology to take advantage of the MS(2) product ion spectra collected. Several heterogeneous MS(1) bins (different MS(2) spectra from the same nominal MS(1)) were identified with this method. Exploratory data analysis approaches were also developed to investigate how the metabolome differs in perennial ryegrass infected with N. lolii in comparison to uninfected perennial ryegrass. As well as some known fungal metabolites like peramine and mannitol, several novel metabolites involved in the symbiosis, including putative cyclic oligopeptides, were identified. Correlation network analysis revealed a group of structurally related oligosaccharides, which differed significantly in concentration in perennial ryegrass sheaths due to endophyte infection. This study demonstrates the potential of the combination of unbiased metabolite profiling using ion trap MS and advanced data-mining strategies for discovering unexpected perturbations of the metabolome, and generating new scientific questions for more detailed investigations in the future.     

Overexpression of ubiquitin‐like LpHUB1 gene confers drought tolerance in perennial ryegrass

HUB1, also known as Ubl5, is a member of the subfamily of ubiquitin‐like post‐translational modifiers. HUB1 exerts its role by conjugating with protein targets. The function of this protein has not been studied in plants. A HUB1 gene, LpHUB1, was identified from serial analysis of gene expression data and cloned from perennial ryegrass. The expression of this gene was reported previously to be elevated in pastures during the summer and by drought stress in climate‐controlled growth chambers. Here, pasture‐type and turf‐type transgenic perennial ryegrass plants overexpressing LpHUB1 showed improved drought tolerance, as evidenced by improved turf quality, maintenance of turgor and increased growth. Additional analyses revealed that the transgenic plants generally displayed higher relative water content, leaf water potential, and chlorophyll content and increased photosynthetic rate when subjected to drought stress. These results suggest HUB1 may play an important role in the tolerance of perennial ryegrass to abiotic stresses.     

Suppression of gray leaf spot (blast) of perennial ryegrass turf by Pseudomonas aeruginosa from spent mushroom substrate

Bacteria isolated from spent mushroom substrate (SMS) were evaluated for the suppression of Pyricularia grisea, the causal agent of gray leaf spot of perennial ryegrass (Lolium perenne) turf. Thirty-two of 849 bacterial isolates (3.8%) from SMS significantly inhibited the mycelial growth of P. grisea in vitro. Six bacterial isolates that afforded maximum inhibition of P. grisea were also refractory to Rhizoctonia solani, Rhizoctonia cerealis, Sclerotinia homoeocarpa, and Fusarium culmorum. Each of the six isolates was identified as Pseudomonas aeruginosa by fatty acid profile analysis. The biocontrol activity of the bacterial isolates was not compromised by a prolonged exposure to UV radiation in vitro. In controlled-environment chamber experiments, all 32 bacterial isolates were tested for suppression of gray leaf spot on Pennfine perennial ryegrass when applied as seed treatment or foliar sprays. Foliar applications of the bacteria (10⁸ cfu/ml 0.1% carboxymethylcellulose), but not the seed treatment, significantly reduced disease severity and incidence. The three most efficient isolates from foliar application treatments, which were among the six bacterial isolates identified as P. aeruginosa, were further evaluated for suppression of gray leaf spot as a function of timing of application. The three isolates of P. aeruginosa suppressed gray leaf spot in perennial ryegrass in Cone-tainers when applied at 1, 3, and 7 days prior to inoculation with P. grisea both in controlled-environment chamber experiments, and in potted ryegrass plants maintained in the field. All application intervals, regardless of the bacterial isolate, provided significant reduction of gray leaf spot severity. Suppression of gray leaf spot by isolates of P. aeruginosa under controlled-environment chamber conditions was not different from that observed in potted ryegrass plants maintained in the field. In field experiments, an isolate of P. aeruginosa provided significant suppression of gray leaf spot when applied at 1, 7, and 14 days prior to inoculation with P. grisea. The bacterium proved effective against gray leaf spot of perennial ryegrass maintained as fairway and rough heights. These results indicate that P. aeruginosa may be a potential biocontrol agent for gray leaf spot of perennial ryegrass turf.     

Expressed sequence tag-derived microsatellite markers of perennial ryegrass (Lolium perenne L.)

An expressed sequence tag (EST) library of the key grassland species perennial ryegrass (Lolium perenne L.) has been exploited as a resource for microsatellite marker development. Out of 955 simple sequence repeat (SSR) containing ESTs, 744 were used for primer design. Primer amplification was tested in eight genotypes of L. perenne and L. multiflorum representing (grand-) parents of four mapping populations and resulted in 464 successfully amplified EST-SSRs. Three hundred and six primer pairs successfully amplified products in the mapping population VrnA derived from two of the eight genotypes included in the original screening and revealed SSR polymorphisms for 143 ESTs. Here, we report on 464 EST-derived SSR primer sequences of perennial ryegrass established in laboratory assays, providing a dedicated tool for marker assisted breeding and comparative mapping within and among forage and turf grasses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Stable transformation and long-term expression of the gusA reporter gene in callus lines of perennial ryegrass (Lolium perenne L.)

Stable transformation of perennial ryegrass (Lolium perenne L.) was achieved by biolistic bombardment of a non embryogenic cell suspension culture, using the hpt and gusA gene. The transformation yielded on the average 5 callus lines per bombardment (1.4×10⁶ cells). Stable integration of the genes into the plant genome was demonstrated by Southern analysis of DNA, isolated from hygromycin-resistant callus lines. The gusA reporter gene, which was regulated by the constitutive promoter of the rice gene GOS2, was expressed in both transient and stable transformation assays, indicating that this promoter is suitable for expression of a transferred gene in perennial ryegrass. Long-term GUS expression was observed in ca. 40% of the callus lines, whereas the other callus lines showed instability after 6 months and 1 year of culture.