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.     

Improved fructan accumulation in perennial ryegrass transformed with the onion fructosyltransferase genes 1-SST and 6G-FFT

Carbohydrate limitation has been identified as a main cause of inefficient nitrogen use in ruminant animals, which feed mainly on fresh forage, hay and silage. This inefficiency results in suboptimal meat and milk productivity. One important molecular breeding strategy is to improve the nutritional value of ryegrass (Lolium perenne) by increasing the fructan content through expression of heterologous fructan biosynthetic genes. We developed perennial ryegrass lines expressing sucrose:sucrose 1-fructosyltransferase and fructan:fructan 6G-fructosyltransferase genes from onion (Allium cepa) which exhibited up to a 3-fold increased fructan content. Further, the high fructan content was stable during the growth period, whereas the fructan content in an elite variety, marketed as a high sugar variety, dropped rapidly after reaching its maximum and subsequently remained low.     

Transforming a fructan:fructan 6G-fructosyltransferase from perennial ryegrass into a sucrose:sucrose 1-fructosyltransferase

Fructosyltransferases (FTs) synthesize fructans, fructose polymers accumulating in economically important cool-season grasses and cereals. FTs might be crucial for plant survival under stress conditions in species in which fructans represent the major form of reserve carbohydrate, such as perennial ryegrass (Lolium perenne). Two FT types can be distinguished: those using sucrose (S-type enzymes: sucrose:sucrose 1-fructosyltransferase [1-SST], sucrose:fructan 6-fructosyltransferase) and those using fructans (F-type enzymes: fructan:fructan 1-fructosyltransferase [1-FFT], fructan:fructan 6G-fructosyltransferase [6G-FFT]) as preferential donor substrate. Here, we report, to our knowledge for the first time, the transformation of an F-type enzyme (6G-FFT/1-FFT) into an S-type enzyme (1-SST) using perennial ryegrass 6G-FFT/1-FFT (Lp6G-FFT/1-FFT) and 1-SST (Lp1-SST) as model enzymes. This transformation was accomplished by mutating three amino acids (N340D, W343R, and S415N) in the vicinity of the active site of Lp6G-FFT/1-FFT. In addition, effects of each amino acid mutation alone or in combination have been studied. Our results strongly suggest that the amino acid at position 343 (tryptophan or arginine) can greatly determine the donor substrate characteristics by influencing the position of the amino acid at position 340. Moreover, the presence of arginine-343 negatively affects the formation of neofructan-type linkages. The results are compared with recent findings on donor substrate selectivity within the group of plant cell wall invertases and fructan exohydrolases. Taken together, these insights contribute to our knowledge of structure/function relationships within plant family 32 glycosyl hydrolases and open the way to the production of tailor-made fructans on a larger scale.     

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.     

Molecular breeding of transgenic perennial ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.) with altered fructan biosynthesis through the expression of fructosyltransferases

Perennial ryegrass is the most important forage grass used in temperate agriculture. Transgenic perennial ryegrass events with altered fructan biosynthesis have the potential not only to increase animal production by improving digestibility of the grasses, but also to increase pasture intake by the animal due to lower neutral detergent fibre (NDF) concentrations. Transgenic perennial ryegrass plants were shown to have increased (P?<?0.05) in fructan concentrations of leaf blades in transgenic T₀ events and have thus an increase in water-soluble carbohydrate and in vivo dry matter digestibility concentrations as well as a decrease in the NDF concentration within the plant in spring and summer. These changes in nutritive value have led to an increase in metabolisable energy of up to 1.7 MJ ME·kg DM^?1 in selected T₀ events compared to FLp418-20 during spring and summer, with no differences in autumn or winter. The field evaluation of these events and the further development of these events using molecular breeding technologies are described in this paper.     

Differential growth response and carbohydrate metabolism of global collection of perennial ryegrass accessions to submergence and recovery following de-submergence

Submergence can severely affect the growth of perennial grasses. The variations in growth and the physiological responses of perennial grass germplasm to submergence stress are not well understood. The objective of this study was to characterize the responses of diverse perennial ryegrass accessions to submergence and their recovery following de-submergence. One hundred globally collected perennial ryegrass accessions were submerged for 7d followed by 7d of recovery in two experiments (Exp 1 and Exp 2), respectively. Compared to the pattern of the controls, the overall distribution in leaf color, chlorophyll fluorescence, plant height (HT), and growth rate (GR) shifted toward a high frequency of lower values under submergence in both experiments. The accessions were generally grouped into three types: fast growth with maintenance of color (escape, T1), slow growth with maintenance of color (quiescence, T2), and slow growth with loss of color (susceptible, ST). Under submergence, T1 had higher HT and GR than the other two groups except for GR of T2 in Exp 2 and had higher water-soluble carbohydrate (WSC) and fructan concentrations, as well as fructan to WSC ratio, than ST in Exp 1. Recovery of HT and GR were generally close to that of the control level except for HT of ST in Exp 2, but the carbohydrates fully recovered in all types of plants after 7d of de-submergence. Differential responses of perennial ryegrass accessions to submergence are useful in creating more tolerant materials and in further characterizing physiological and molecular mechanisms of submergence tolerance.     

Does fructan have a functional role in physiological traits? Investigation by quantitative trait locus mapping

* The role of fructan in growth and drought-stress responses of perennial ryegrass (Lolium perenne) was investigated in an F(2) mapping family that segregates for carbohydrate metabolism. * A quantitative trait locus approach was used to compare the genetic control of traits. * Growth and drought-stress traits were extremely variable within the family. Most traits had high broad-sense heritability. Quantitative trait loci (QTL) were identified for most traits; the maximum number of QTL per trait was four. Between 11% and 75% of total phenotypic variation was explained. Few growth-trait QTL coincided with previously identified fructan QTL. A cluster of drought-trait QTL was close to two previously identified regions of the genome with tiller base fructan QTL in repulsion. * The high sugar parent contributed few alleles that increased 'reserve-driven' growth or performance during drought-stress. Correlation of growth and drought-stress traits with fructan content was low and increasing fructan content per se would not appear to improve drought resistance. Complex patterns of carbohydrate partitioning and metabolism within the cell may explain contradictory relationships between carbohydrate content and growth/stress-resistance traits.     

Altered fructan accumulation in transgenic Lolium multiflorum plants expressing a Bacillus subtilis sacB gene

Ryegrasses, like many C3 plants, accumulate fructan, which plays an important role in assimilate partitioning, as the major non-structural storage carbohydrate. The present study describes the transformation of a Bacillus subtilis sacB gene, with vacuolar targeting signal sequences and driven by constitutive promoters, into Italian ryegrass (Lolium multiflorum Lam.) by microprojectile bombardment of embryogenic suspension cells. The expression of the chimeric sacB genes in transgenic ryegrass plants and the concomitant accumulation of low levels of bacterial levan were found to substantially distort the native grass fructan synthesis pattern. High-molecular-weight native fructan was depleted, and the pattern of accumulation of oligosaccharides in the range of 5-35 degree of polymerization was altered. The levan-accumulating sacB-transgenic ryegrass plants had a lower level of total fructose, unchanged sucrose levels, and slightly reduced hexose levels compared to the isogenic controls. Growth of the levan-accumulating sacB-transgenic ryegrass plants slowed down with the onset of the reproductive phase. Flowering plants were stunted and had narrower leaves and poorly developed roots. The association between the manipulated fructan metabolism and the phenotype of the levan-accumulating sacB-transgenic ryegrass plants is discussed.     

A novel DNA-based diagnostic test for the detection of annual and intermediate ryegrass contamination in perennial ryegrass

Perennial ryegrass (Lolium perenne L.) is a preferred choice for the turf grass industry due to its ability to provide a durable turf cover. Genetic or physical contamination of annual (L. multiflorum Lam.) or intermediate (L. hybridum) ryegrass species in perennial ryegrass is one of the major problems affecting the grass seed industry. At present, seedling root fluorescence (SRF), a biochemical marker, is used for the detection of annual ryegrass contamination. Due to the unreliability of the SRF test, the seed industry is seeking an alternative, more reliable and accurate detection method. Currently, there are no DNA tests available in ryegrass for detecting contamination with annual and intermediate ryegrass types. We developed a novel quantitative polymerase chain reaction (Q-PCR)-based DNA test for the detection of annual and/or intermediate ryegrass types in perennial ryegrass. This DNA test was designed using an insertion/deletion (InDel) site in the LpVRN2_2 (Vernalization 2) gene, which is one of the several genes controlling vernalization in ryegrass. The new DNA test is more reliable, accurate and cost-effective in detecting contamination, with a high sensitivity of 0.04% in a sample size of 5,000 seeds. Use of larger sample sizes (12.5-fold higher compared to SRF test) provided additional accuracy in detecting the level of contamination. The method has produced consistent results in 68 perennial, 26 annual and 14 intermediate ryegrass lines.     

Development and implementation of a multiplexed single nucleotide polymorphism genotyping tool for differentiation of ryegrass species and cultivars

Perennial ryegrass (Lolium perenne L.) and Italian ryegrass (Lolium multiflorum Lam.) are important temperate forage grasses which are closely related, generating fertile interspecific hybrids. All groups are represented by multiple cultivars in the commercial pasture seeds market. Due to the close taxonomic relationship between the two species, differentiation based on morphophysiological criteria is not always readily achievable. In addition, an obligate outbreeding reproductive habit produces high levels of individual heterozygosity and intrapopulation diversity, which presents problems for discrimination between cultivars. Molecular genetic marker polymorphism provides an effective means of addressing these challenges. An iterative process of resequencing from loci distributed across the perennial ryegrass genome was used to identify single nucleotide polymorphism (SNP) markers, which were then validated and formatted in a highly multiplexed (384-plex) assay system. SNP genotyping was then performed across samples of 48–192 individuals from a total of 27 ryegrass cultivars (19 of perennial ryegrass, seven of Italian ryegrass and one hybrid cultivar). SNP markers from perennial ryegrass exhibited a high level of transfer to Italian ryegrass. Data analysis permitted quantification of intra- and inter-species diversity, as well as discrimination between cultivars within each species, including diploid and autotetraploid cultivars of perennial ryegrass. Lower levels of SNP-based diversity were detected in Italian ryegrass than in perennial ryegrass. A neighbour-joining tree based on genetic distance analysis located a hybrid cultivar to an intermediate position between the two species-specific cultivar groups. The resulting catalogue of ryegrass cultivars will provide support for the processes of cultivar accreditation and quality assurance.     

Degradation of fructans by epiphytic and inoculated lactic acid bacteria and by plant enzymes during ensilage of normal and sterile hybrid ryegrass

Degradation of grass fructans by epiphytic or inoculated lactic acid bacteria during ensilage was examined using both normal and sterile hybrid ryegrass. It was clear that even in the absence of bacteria fructan degradation occurred, but at a significantly slower rate than in normal grass which had not been inoculated with lactic acid bacteria. Fructan degradation in sterile herbage suggests that plant fructan hydrolases were partially responsible for this process in all herbages, irrespective of treatment. Inoculation of sterile herbage with a strain of Lactobacillus plantarum known to lack the ability to degrade grass fructans resulted in a slower rate of fructan breakdown than when inoculated with Lactobacillus casei subsp. paracasei , a confirmed fructan degrader. In the later stages of the fermentation of uninoculated normal herbage when water-soluble carbohydrate appeared to be limiting, lactic acid was fermented to acetic acid. However, this fermentation pathway was not observed in either of the inoculated silages. The results suggest that silage inoculant bacteria possessing fructan hydrolase activity may have potential for improving silage fermentation, particularly when late cut, low sugar grass containing a high proportion of fructans is ensiled.     

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.     

Functional analyses of caffeic acid O-Methyltransferase and Cinnamoyl-CoA-reductase genes from perennial ryegrass (Lolium perenne)

Cinnamoyl CoA-reductase (CCR) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. We identified candidate genes encoding these two enzymes in perennial ryegrass (Lolium perenne) and show that the spatio-temporal expression patterns of these genes in planta correlate well with the developmental profile of lignin deposition. Downregulation of CCR1 and caffeic acid O-methyltransferase 1 (OMT1) using an RNA interference-mediated silencing strategy caused dramatic changes in lignin level and composition in transgenic perennial ryegrass plants grown under both glasshouse and field conditions. In CCR1-deficient perennial ryegrass plants, metabolic profiling indicates the redirection of intermediates both within and beyond the core phenylpropanoid pathway. The combined results strongly support a key role for the OMT1 gene product in the biosynthesis of both syringyl- and guaiacyl-lignin subunits in perennial ryegrass. Both field-grown OMT1-deficient and CCR1-deficient perennial ryegrass plants showed enhanced digestibility without obvious detrimental effects on either plant fitness or biomass production. This highlights the potential of metabolic engineering not only to enhance the forage quality of grasses but also to produce optimal feedstock plants for biofuel production.     

Bioactivity of a pentapeptide isolated from corn gluten hydrolysate on Lolium perenne L.

Corn gluten hydrolysate (CGH) has been observed to inhibit root formation of germinating grass seeds and has the potential for use as a natural herbicide. Five dipeptides have been isolated from the aqueous solution of CGH and proved to have greater root-inhibiting activity than the crude extract of CGH. The objective of this study was to isolate and identify other biologically active compounds from CGH with herbicidal properties. A perennial ryegrass (Lolium perenne L.) Petri dish bioassay was used to test for the bioactivity. A pentapeptide, Leu-Ser-Pro-Ala-Gln, was isolated from CGH by using Sephadex G-15 gel filtration and two-step C18 reversed phase high performance liquid chromatography procedures. The compound suppressed growth of both the root and the shoot of germinating perennial ryegrass. It required 0.5 mg/mL of the pentapeptide to inhibit 50% of root length in the perennial ryegrass bioassay, and this compound is more active than any of the five dipeptides isolated previously from CGH.Abbreviations CGM corn gluten meal - CGH corn gluten hydrolysate - HPLC high performance liquid chromatography - RP reversed phase - MeOH methanol - AUFS absorbance units of full scale - Rt retention time - TFA trifluoroacetic acid - PTH phenylthiohydantoin - ANOVA analysis of variance     

An enhanced molecular marker based genetic map of perennial ryegrass (Lolium perenne) reveals comparative relationships with other Poaceae genomes.

A molecular-marker linkage map has been constructed for perennial ryegrass (Lolium perenne L.) using a one-way pseudo-testcross population based on the mating of a multiple heterozygous individual with a doubled haploid genotype. RFLP, AFLP, isoenzyme, and EST data from four collaborating laboratories within the International Lolium Genome Initiative were combined to produce an integrated genetic map containing 240 loci covering 811 cM on seven linkage groups. The map contained 124 codominant markers, of which 109 were heterologous anchor RFLP probes from wheat, barley, oat, and rice, allowing comparative relationships between perennial ryegrass and other Poaceae species to be inferred. The genetic maps of perennial ryegrass and the Triticeae cereals are highly conserved in terms of synteny and colinearity. This observation was supported by the general agreement of the syntenic relationships between perennial ryegrass, oat, and rice and those between the Triticeae and these species. A lower level of synteny and colinearity was observed between perennial ryegrass and oat compared with the Triticeae, despite the closer taxonomic affinity between these species. It is proposed that the linkage groups of perennial ryegrass be numbered in accordance with these syntenic relationships, to correspond to the homoeologous groups of the Triticeae cereals.     

Genetic Differentiation in Response to Selection for Water-Soluble Carbohydrate Content in Perennial Ryegrass (<Emphasis Type="Italic">Lolium perenne</Emphasis> L.)

Plant carbohydrates are of increasing interest as renewable feedstocks to replace petrochemicals in the generation of fuels and production of high-value chemicals. Greater understanding of the genetic control of diversity in fructan synthesis and accumulation would facilitate more directed channelling of feedstock to process in a ryegrass biorefinery. Divergent populations produced by phenotypic selection for water-soluble carbohydrate content have been used to investigate relationships between traits, and to identify patterns of genetic differentiation which indicate genomic regions under high and low selection pressure. Selection for high water-soluble carbohydrate content was associated with increased synthesis of large fructan polymers and increased accumulation of above-ground plant biomass, particularly during spring. Three rounds of selection and two rounds of recombination resulted in widespread genetic differentiation across the whole genome, causing reduced allelic richness and increasing homozygosity at some loci. A smaller number of loci were shown to be subject to high selection pressure. Breeding material subjected to many years of selection for water-soluble carbohydrate also showed allelic differences which may reflect the consequences of high selection pressure at some of these same loci. However, some of the loci unaffected in the divergent selection experiment showed similar effects. This might arise from differences in linkage disequilibrium in these two sets of plant materials, but more likely from the different genetic background of the germplasm. This illustrates the complex nature of the water-soluble carbohydrate trait in perennial ryegrass.     

Fructans,but not the sucrosyl-galactosides,raffinose and loliose,are affected by drought stress in perennial ryegrass

The aim of this study was to evaluate the putative role of the sucrosyl-galactosides, loliose [alpha-D-Gal (1,3) alpha-D-Glc (1,2) beta-D-Fru] and raffinose [alpha-D-Gal (1,6) alpha-D-Glc (1,2) beta-D-Fru], in drought tolerance of perennial ryegrass and to compare it with that of fructans. To that end, the loliose biosynthetic pathway was first established and shown to operate by a UDP-Gal: sucrose (Suc) 3-galactosyltransferase, tentatively termed loliose synthase. Drought stress increased neither the concentrations of loliose and raffinose nor the activities of loliose synthase and raffinose synthase (EC 2.4.1.82). Moreover, the concentrations of the raffinose precursors, myoinositol and galactinol, as well as the gene expressions of myoinositol 1-phosphate synthase (EC 5.5.1.4) and galactinol synthase (EC 2.4.1.123) were either decreased or unaffected by drought stress. Taken together, these data are not in favor of an obvious role of sucrosyl-galactosides in drought tolerance of perennial ryegrass at the vegetative stage. By contrast, drought stress caused fructans to accumulate in leaf tissues, mainly in leaf sheaths and elongating leaf bases. This increase was mainly due to the accumulation of long-chain fructans (degree of polymerization > 8) and was not accompanied by a Suc increase. Interestingly, Suc but not fructan concentrations greatly increased in drought-stressed roots. Putative roles of fructans and sucrosyl-galactosides are discussed in relation to the acquisition of stress tolerance.