Arsenic and nutrient absorption characteristics and antioxidant response in different leaves of two ryegrass (Lolium perenne) species under arsenic stress.

Arsenic (As), a heavy metal element, causes soil environmental concerns in many parts of the world, and ryegrass has been considered as an effective plant species for bioremediation of heavy metal pollution including As. This study was designed to investigate As content, nutrient absorption and antioxidant enzyme activity associated with As tolerance in the mature leaves, expanded leaves and emerging leaves of perennial ryegrass (Lolium perenne) and annual ryegrass (Lolium multiflorum) under 100 mg·kg-1 As treatment. The contents of As, calcium (Ca), magnesium (Mg), manganese (Mn) in the leaves of both ryegrass species were greatest in the mature leaves and least in the emerging leaves. The nitrogen (N), phosphorus (P), potassium (K) contents of both ryegrass species were greatest in the emerging leaves and least in the mature leaves. The As treatment reduced biomass more in the mature leaves and expanded leaves relative to the emerging leaves for annual ryegrass and reduced more in emerging leaves relative to the mature and expanded leaves for perennial ryegrass. Perennial ryegrass had higher As content than annual ryegrass in all three kinds of leaves. The As treatment increased hydrogen peroxide (H2O2) in expanded leaves of two ryegrass species, relative to the control. The As treatment increased the ascorbate peroxidase (APX) activity in the expanded leaves of perennial ryegrass and the mature leaves of annual ryegrass, the catalase (CAT) activity in the mature and expanded leaves of perennial ryegrass and the emerging leaves of annual ryegrass, relative to the control. The As treatment reduced peroxidase (POD) activity in all three kinds of leaves of annual ryegrass and superoxide dismutase (SOD) activity in expanded leaves of perennial ryegrass, relative to the control. The results of this study suggest that As tolerance may vary among different ages of leaf and reactive oxygen species (ROS) and antioxidant enzyme activity may be associated with As tolerance in the ryegrass.

DArT, SNP, and SSR analyses of genetic diversity in Lolium perenne L. using bulk sampling.

BACKGROUND: Lolium perenne L. is the most important forage grass species in temperate regions. It is also considered as a sustainable source of biomass for energy production. However, improvement in biomass yield has been limited by comparison with other major crops. More efficient utilisation of genetic resources and improved breeding schemes are required to advance L. perenne breeding. In an attempt to elucidate the extent of genetic diversity in L. perenne, 1384 DArT, 182 SNP and 48 SSR markers were applied to 297 accessions (Set I) contributed by three German breeding companies and the IPK Genebank. Due to the heterogeneous nature of Lolium accessions, bulk samples were used. Apart from germplasm set I, additional set II and set III was used to determine the reproducibility of marker system and judge the feasibility of bulk strategy in this study. RESULTS: By assessing different bulk sizes, 24 individuals per sample were shown to be a representative number of plants to discriminate different accessions. Among the 297 accessions, all marker types revealed a high polymorphism rate; 1.99, 2.00 and 8.19 alleles, were obtained per locus on average using DArTs, SNPs and SSRs, respectively. The Jaccard distance for DArT markers ranged from 0.00 to 0.73, the Modified Roger's distance (MRD) for SNP markers ranged from 0.03 to 0.52, and for SSR markers from 0.26 to 0.76. Gene diversity for dominant DArT and co-dominant SNP and SSR markers was found to be 0.26, 0.32 and 0.45, respectively. DArT markers showed the highest consistency and reproducibility. CONCLUSION: The resulting data were evaluated using a number of different classification methods, but none of the methods showed a clear differentiation into distinct genetic pools. With regard to hybrid breeding, this will possibly impede substantial progress towards increased biomass yields of L. perenne by utilising heterosis.

Five species, many genotypes, broad phenotypic diversity: When agronomy meets functional ecology.

PREMISE OF THE STUDY: Current ecological theory can provide insight into the causes and impacts of plant domestication. However, just how domestication has impacted intraspecific genetic variability (ITV) is unknown. We used 50 ecotypes and 35 cultivars from five grassland species to explore how selection drives functional trait coordination and genetic differentiation. METHODS: We quantified the extent of genetic diversity among different sets of functional traits and determined how much genetic diversity has been generated within populations of natural ecotypes and selected cultivars. KEY RESULTS: In general, the cultivars were larger (e.g., greater height, faster growth rates) and had larger and thinner leaves (greater SLA). We found large (average 63%) and trait-dependent (ranging from 14% for LNC to 95.8% for growth rate) genetic variability. The relative extent of genetic variability was greater for whole-plant than for organ-level traits. This pattern was consistent within ecotypes and within cultivars. However, ecotypes presented greater ITV variability. CONCLUSIONS: The results indicated that genetic diversity is large in domesticated species with contrasting levels of heritability among functional traits and that selection for high yield has led to indirect selection of some associated leaf traits. These findings open the way to define which target traits should be the focus in selection programs, especially in the context of community-level selection.

Markers associated with heading and aftermath heading in perennial ryegrass full-sib families.

BACKGROUND: Heading and aftermath heading are important traits in perennial ryegrass because they impact forage quality. So far, genome-wide association analyses in this major forage species have only identified a small number of genetic variants associated with heading date that overall explained little of the variation. Some possible reasons include rare alleles with large phenotypic affects, allelic heterogeneity, or insufficient marker density. We established a genome-wide association panel with multiple genotypes from multiple full-sib families. This ensured alleles were present at the frequency needed to have sufficient statistical power to identify associations. We genotyped the panel via partial genome sequencing and performed genome-wide association analyses with multi-year phenotype data collected for heading date, and aftermath heading. RESULTS: Genome wide association using a mixed linear model failed to identify any variants significantly associated with heading date or aftermath heading. Our failure to identify associations for these traits is likely due to the extremely low linkage disequilibrium we observed in this population. However, using single marker analysis within each full-sib family we could identify markers and genomic regions associated with heading and aftermath heading. Using the ryegrass genome we identified putative orthologs of key heading genes, some of which were located in regions of marker-trait associations. CONCLUSION: Given the very low levels of LD, genome wide association studies in perennial ryegrass populations are going to require very high SNP densities. Single marker analysis within full-sibs enabled us to identify significant marker-trait associations. One of these markers anchored proximal to a putative ortholog of TFL1, homologues of which have been shown to play a key role in continuous heading of some members of the rose family, Rosaceae.

Targeted genotyping-by-sequencing permits cost-effective identification and discrimination of pasture grass species and cultivars.

KEY MESSAGE: A targeted amplicon-based genotyping-by-sequencing approach has permitted cost-effective and accurate discrimination between ryegrass species (perennial, Italian and inter-species hybrid), and identification of cultivars based on bulked samples. Perennial ryegrass and Italian ryegrass are the most important temperate forage species for global agriculture, and are represented in the commercial pasture seed market by numerous cultivars each composed of multiple highly heterozygous individuals. Previous studies have identified difficulties in the use of morphophysiological criteria to discriminate between these two closely related taxa. Recently, a highly multiplexed single nucleotide polymorphism (SNP)-based genotyping assay has been developed that permits accurate differentiation between both species and cultivars of ryegrasses at the genetic level. This assay has since been further developed into an amplicon-based genotyping-by-sequencing (GBS) approach implemented on a second-generation sequencing platform, allowing accelerated throughput and ca. sixfold reduction in cost. Using the GBS approach, 63 cultivars of perennial, Italian and interspecific hybrid ryegrasses, as well as intergeneric Festulolium hybrids, were genotyped. The genetic relationships between cultivars were interpreted in terms of known breeding histories and indistinct species boundaries within the Lolium genus, as well as suitability of current cultivar registration methodologies. An example of applicability to quality assurance and control (QA/QC) of seed purity is also described. Rapid, low-cost genotypic assays provide new opportunities for breeders to more fully explore genetic diversity within breeding programs, allowing the combination of novel unique genetic backgrounds. Such tools also offer the potential to more accurately define cultivar identities, allowing protection of varieties in the commercial market and supporting processes of cultivar accreditation and quality assurance.

Genetic diversity analysis in Tunisian perennial ryegrass germplasm as estimated by RAPD, ISSR, and morpho-agronomical markers.

Tunisia is rich in diverse forage and pasture species including perennial ryegrass. In order to enhance forage production and improve agronomic performance of this local germplasm, a molecular analysis was undertaken. Random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR) and morpho-agronomical traits markers were used for genetic diversity estimation of ryegrass germplasm after screening 20 spontaneous accessions, including a local and an introduced cultivars. Same mean polymorphism information content values were obtained (0.37) for RAPD and ISSR suggesting that both marker systems were equally effective in determining polymorphisms. The average pairwise genetic distance values were 0.57 (morpho-agronomical traits), 0.68 (RAPD), and 0.51 (ISSR) markers data sets. A higher Shannon diversity index was obtained with ISSR marker (0.57) than for RAPD (0.54) and morpho-agronomical traits (0.36). The Mantel test based on genetic distances of a combination of molecular markers and morpho-agronomical data exhibited a significant correlation between RAPD and ISSR data, suggesting that the use of a combination of molecular techniques was a highly efficient method of estimating genetic variability levels among Tunisian ryegrass germplasm. In summary, results showed that combining molecular and morpho-agronomical markers is an efficient way in assessing the genetic variability among Tunisian ryegrass genotypes. In addition, the combined analysis provided an exhaustive coverage for the analyzed diversity and helped us to identify suitable accessions showed by Beja and Jendouba localities, which present large similarities with cultivated forms and can be exploited for designing breeding programmes, conservation of germplasm and management of ryegrass genetic resources.

Genetic diversity and relationships in cultivars of Lolium multiflorum Lam. using sequence-related amplified polymorphism markers.

Sequence-related amplified polymorphism (SRAP) markers were used to analyze and estimate the genetic variability, level of diversity, and relationships among 20 cultivars and strains of annual ryegrass (Lolium multiflorum Lam.). Eighteen SRAP primer combinations generated 334 amplification bands, of which 298 were polymorphic. The polymorphism information content ranged from 0.4715 (me10 + em1) to 0.5000 (me5 + em7), with an average of 0.4921. The genetic similarity coefficient ranged from 0.4304 to 0.8529, and coefficients between 0.65 and 0.90 accounted for 90.00%. The cluster analysis separated the accessions into five groups partly according to their germplasm resource origins.

Early induction of Fe-SOD gene expression is involved in tolerance to Mn toxicity in perennial ryegrass.

Manganese (Mn) toxicity limits plant growth in acid soils. Although Mn toxicity induces oxidative stress, the role of superoxide dismutase (SOD, EC.1.15.1.1) isoforms in conferring Mn tolerance remains unclear. Seedlings of ryegrass cultivars Nui (Mn-sensitive) and Kingston (Mn-tolerant) were hydroponically grown at 2.4 (optimal) or 750 µM Mn (toxic) concentration, and harvested from 2 to 48 h. Kingston showed higher shoot Mn than Nui at 2.4 µM Mn. At toxic supply, shoot Mn concentration steadily increased in both cultivars, with Kingston having the highest accumulation at 48 h. An early (2 h) increase in lipid peroxidation under Mn excess occurred, but it returned (after 6 h) to the basal level in Kingston only. Kingston exhibited higher SOD activity than Nui, and that difference increased due to toxic Mn. In general, Mn-induced gene expression of Mn- and Cu/Zn-SOD isoforms was higher in Nui than Kingston. Nevertheless, under Mn excess, we found a greater Fe-SOD up-regulation (up to 5-fold) in Kingston compared to Nui. Thus, Fe-SOD induction in Kingston might explain, at least partly, its high tolerance to Mn toxicity. This is the first evidence that Mn toxicity causes differential gene expression of SOD isoforms in ryegrass cultivars in the short-term.

Association of candidate genes with drought tolerance traits in diverse perennial ryegrass accessions.

Drought is a major environmental stress limiting growth of perennial grasses in temperate regions. Plant drought tolerance is a complex trait that is controlled by multiple genes. Candidate gene association mapping provides a powerful tool for dissection of complex traits. Candidate gene association mapping of drought tolerance traits was conducted in 192 diverse perennial ryegrass (Lolium perenne L.) accessions from 43 countries. The panel showed significant variations in leaf wilting, leaf water content, canopy and air temperature difference, and chlorophyll fluorescence under well-watered and drought conditions across six environments. Analysis of 109 simple sequence repeat markers revealed five population structures in the mapping panel. A total of 2520 expression-based sequence readings were obtained for a set of candidate genes involved in antioxidant metabolism, dehydration, water movement across membranes, and signal transduction, from which 346 single nucleotide polymorphisms were identified. Significant associations were identified between a putative LpLEA3 encoding late embryogenesis abundant group 3 protein and a putative LpFeSOD encoding iron superoxide dismutase and leaf water content, as well as between a putative LpCyt Cu-ZnSOD encoding cytosolic copper-zinc superoxide dismutase and chlorophyll fluorescence under drought conditions. Four of these identified significantly associated single nucleotide polymorphisms from these three genes were also translated to amino acid substitutions in different genotypes. These results indicate that allelic variation in these genes may affect whole-plant response to drought stress in perennial ryegrass.

Association study between the gibberellic acid insensitive gene and leaf length in a Lolium perenne L. synthetic variety.

BACKGROUND: Association studies are of great interest to identify genes explaining trait variation since they deal with more than just a few alleles like classical QTL analyses. They are usually performed using collections representing a wide range of variability but which could present a genetic substructure. The aim of this paper is to demonstrate that association studies can be performed using synthetic varieties obtained after several panmictic generations. This demonstration is based on an example of association between the gibberellic acid insensitive gene (GAI) polymorphism and leaf length polymorphism in 'Herbie', a synthetic variety of perennial ryegrass. METHODS: Leaf growth parameters, consisted of leaf length, maximum leaf elongation rate (LERmax) and leaf elongation duration (LED), were evaluated in spring and autumn on 216 plants of Herbie with three replicates. For each plant, a sequence of 370 bp in GAI was analysed for polymorphism. RESULTS: Genetic effect was highly significant for all traits. Broad sense heritabilities were higher for leaf length and LERmax with about 0.7 in each period and 0.5 considering both periods than for LED with about 0.4 in each period and 0.3 considering both periods. GAI was highly polymorphic with an average of 12 bp between two consecutive SNPs and 39 haplotypes in which 9 were more frequent. Linkage disequilibrium declined rapidly with distance with r 2 values lower than 0.2 beyond 150 bp. Sequence polymorphism of GAI explained 8-14% of leaf growth parameter variation. A single SNP explained 4% of the phenotypic variance of leaf length in both periods which represents a difference of 33 mm on an average of 300 mm. CONCLUSIONS: Synthetic varieties in which linkage disequilibrium declines rapidly with distance are suitable for association studies using the "candidate gene" approach. GAI polymorphism was found to be associated with leaf length polymorphism which was more correlated to LERmax than to LED in Herbie. It is a good candidate to explain leaf length variation in other plant material.

Conflicts in maintaining biodiversity at multiple scales.

Biodiversity consists of multiple scales, including functional diversity in ecological traits, species diversity and genetic diversity within species, and is declining across the globe, largely in response to human activities. While species extinctions are the most obvious aspect of this, there has also been a more insidious loss of genetic diversity within species. While a vast literature concerns each of these scales of biodiversity, less is known about how different scales affect one another. In particular, genetic and species diversity may influence each other in numerous ways, both positively and negatively. However, we know little about the mechanism behind these patterns. In this issue of Molecular Ecology, Nestmann et al. (2011) experimentally explore the effect of species and functional diversity and composition of grassland plant communities on the genetic structure of one of the component species. Increasing species richness led to greater changes in the genetic composition of the focal populations over 4 years, primarily because of genetic drift in smaller population sizes. However, there were also genetic changes in response to particular plant functional groups, indicating selective differences driven by plant community composition. These results suggest that different levels of biodiversity can trade-off in communities, which may prove a challenge for conservation biologists seeking to preserve all aspects of biodiversity.

Plant species diversity and composition of experimental grasslands affect genetic differentiation of Lolium perenne populations.

Contrasting hypotheses exist about the relationship between plant species diversity and genetic diversity. However, experimental data of species diversity effects on genetic differentiation among populations are lacking. To address this, Lolium perenne was sown with an equal number of seeds in 78 experimental grasslands (Jena Experiment) varying in species richness (1, 2, 4, 8 to 16) and functional group richness and composition (1-4; grasses, legumes, small herbs, tall herbs). Population sizes were determined 4years after sowing, and single-nucleotide polymorphism (SNP) DNA markers based on bulk samples of up to 100 individuals per population were applied. Genetic distances between the field populations and the initially sown seed population increased with sown species richness. The degree of genetic differentiation from the original seed population was largely explained by actual population sizes, which suggests that genetic drift was the main driver of differentiation. Weak relationships among relative allele frequencies and species diversity or actual population sizes, and a positive correlation between actual population sizes and expected heterozygosity also supported the role of genetic drift. Functional composition had additional effects on genetic differentiation of L. perenne populations, indicating a selection because of genotype-specific interactions with other species. Our study supports that genetic diversity is likely to be lower in plant communities with a higher number of interspecific competitors. Negative effects of species richness on population sizes may increase the probability of genetic drift, and selection because of genotype-specific interactions depending on species and genotypic community composition may modulate this relationship.

Evolutionary history of tall fescue morphotypes inferred from molecular phylogenetics of the Lolium-Festuca species complex.

BACKGROUND: The agriculturally important pasture grass tall fescue (Festuca arundinacea Schreb. syn. Lolium arundinaceum (Schreb.) Darbysh.) is an outbreeding allohexaploid, that may be more accurately described as a species complex consisting of three major (Continental, Mediterranean and rhizomatous) morphotypes. Observation of hybrid infertility in some crossing combinations between morphotypes suggests the possibility of independent origins from different diploid progenitors. This study aims to clarify the evolutionary relationships between each tall fescue morphotype through phylogenetic analysis using two low-copy nuclear genes (encoding plastid acetyl-CoA carboxylase [Acc1] and centroradialis [CEN]), the nuclear ribosomal DNA internal transcribed spacer (rDNA ITS) and the chloroplast DNA (cpDNA) genome-located matK gene. Other taxa within the closely related Lolium-Festuca species complex were also included in the study, to increase understanding of evolutionary processes in a taxonomic group characterised by multiple inter-specific hybridisation events. RESULTS: Putative homoeologous sequences from both nuclear genes were obtained from each polyploid species and compared to counterparts from 15 diploid taxa. Phylogenetic reconstruction confirmed F. pratensis and F. arundinacea var. glaucescens as probable progenitors to Continental tall fescue, and these species are also likely to be ancestral to the rhizomatous morphotype. However, these two morphotypes are sufficiently distinct to be located in separate clades based on the ITS-derived data set. All four of the generated data sets suggest independent evolution of the Mediterranean and Continental morphotypes, with minimal affinity between cognate sequence haplotypes. No obvious candidate progenitor species for Mediterranean tall fescues were identified, and only two putative sub-genome-specific haplotypes were identified for this morphotype. CONCLUSIONS: This study describes the first phylogenetic analysis of the Festuca genus to include representatives of each tall fescue morphotype, and to use low copy nuclear gene-derived sequences to identify putative progenitors of the polyploid species. The demonstration of distinct tall fescue lineages has implications for both taxonomy and molecular breeding strategies, and may facilitate the generation of morphotype and/or sub-genome-specific molecular markers.

Effects of elevated CO(2) on growth, photosynthesis, elemental composition, antioxidant level, and phytochelatin concentration in Lolium mutiforum and Lolium perenne under Cd stress.

The objective of this study was to investigate combined effects of Cd and elevated CO(2) on growth, physiological and physiochemical characteristics, elemental compositions in Lolium mutiforum and Lolium perenne grown in soils amended with three Cd concentrations (0, 25, 100 mg kg(-1)) under two CO(2) levels (375, 810 microLL(-1)). Elevated CO(2) increased net assimilation rate and internal CO(2) concentration, and consequently increased total plant biomass by 51 to 31%. At same spiked Cd level, malondialdehyde content in leaves was lower under elevated than under ambient CO(2), whereas superoxide dismutase activity was higher. Elevated CO(2) decreased Cd, S, and phytochelatin concentrations in roots and shoots to a various degree, depending on plant species and element, but the PC-Cd ratio was not affected. It was concluded that elevated CO(2) ameliorated Cd toxicity in both Lolium species under Cd stress, and that the increase of plant biomass and the alleviation of Cd toxicity with elevated CO(2) for the Lolium species may be more dependent on increased photosynthesis and enhanced antioxidant capacity. Results of the study may provide insights into the interaction between soil Cd contamination and atmospheric CO(2) concentration with regard to plant ability to grow and remove the Cd from soils.

Extremely high cytoplasmic diversity in natural and breeding populations of Lolium (Poaceae).

Ten chloroplast microsatellite markers were used to characterise chloroplast genetic diversity at allelic and haplotypic level in 104 accessions of Lolium perenne, other Lolium species, Festuca species and x Festulolium cultivars. Furthermore, genetic relationships between the accessions and biogeographic distribution of haplotypes were investigated using a range of Nei's population genetic diversity measures and analysis of molecular variance (AMOVA). An extremely high number (511) of haplotypes was detected in 1575 individuals. Nei's gene diversity values among L. perenne accessions ranged between 0 and 0.333. Much of the L. perenne European ecotype diversity (61%), as calculated using AMOVA, could be attributed to within-population variance and this is likely caused by, and maintained by, high levels of natural and anthropogenic seed dispersal. Plastid gene pools and maternal lineages for L. perenne could be clearly identified. Evidence was found, using AMOVA, to show a likely migration route of L. perenne from Southern regions of Europe northwards.

Optimal choice of dairy forages in eastern Australia.

Although several forage species such as perennial ryegrass are predominant, there is a wide range of forage species that could be grown in subtropical and temperate regions in Australia as dairy pastures. These species have differing seasonal patterns of growth, nutrient quality, and water-use efficiency, as demonstrated in a large experiment evaluating over 30 species at the University of Sydney (Camden, New South Wales, Australia). Some species can be grazed, whereas others require mechanical harvesting, which incurs a further cost. Previous comparisons of species that relied on yield of dry matter per unit of some input (typically land or water) did not simultaneously take into account the season in which forage is produced, or other factors related to the costs of production and delivery to the cows. To effectively compare the profitability of individual species, or combinations of species, requires the use of a whole-farm, multiperiod model. Linear programming was used to find the most profitable mix of forage species for an irrigated dairy farm in a warm temperate irrigation region of New South Wales, Australia. It was concluded that for a typical farmer facing the prevailing milk and purchased feed prices with average milk production per cow, the most profitable mix of species would include a large proportion of perennial ryegrass (Lolium perenne) and prairie grass (Bromus willdenowii). The result was robust to changes in seasonal milk pricing and a move from year-round to a more seasonal calving pattern.

Effects of perennial ryegrass cultivars on milk yield and nitrogen utilization in grazing dairy cows.

The effects of 4 diploid perennial ryegrass cultivars that differed in water-soluble carbohydrate (WSC) concentrations on milk yield and nitrogen (N) utilization in dairy cows were evaluated in a 2-yr grazing experiment. Twelve lactating dairy cows were assigned to 1 cultivar for a 2-wk period in a 4 x 4 Latin square design with 3 replicates. Each year, the experiment lasted 8 wk. Swards were in a vegetative stage throughout the experiment. Herbage constituents were determined, and DM intake was estimated with the n-alkane technique. Nitrogen utilization was calculated as N excreted in milk divided by N intake, assuming a zero N retention. Two cultivars had consistently higher WSC concentrations and slightly lower neutral detergent fiber concentrations than the other 2 cultivars. The ranking of the cultivars in chemical composition traits in both years was rather consistent. Cows grazing the cultivar with the lowest concentration of WSC had the lowest herbage DM intake, N intake, milk yield, and milk N yield in 2002, but with a similar difference in WSC concentration, no differences among cultivars were found in 2003. In both years, milk urea N concentration was slightly higher for cows grazing the cultivar with the lowest WSC concentration, although it was significant only in 2003. Nitrogen utilization (N milk:N intake, g/g) varied between 0.241 and 0.246 in 2002 and between 0.190 and 0.209 in 2003, and in both years there was no effect of cultivar. At relatively high N concentrations in grass and only small differences among cultivars in neutral detergent fiber concentrations, cultivars with an elevated WSC concentration did not increase N utilization in grazing dairy cows.

Polyploidy and cellular mechanisms changing leaf size: comparison of diploid and autotetraploid populations in two species of Lolium.

BACKGROUND AND AIMS: Growth and development of plant organs, including leaves, depend on cell division and expansion. Leaf size is increased by greater cell ploidy, but the mechanism of this effect is poorly understood. Therefore, in this study, the role of cell division and expansion in the increase of leaf size caused by polyploidy was examined by comparing various cell parameters of the mesophyll layer of developing leaves of diploid and autotetraploid cultivars of two grass species, Lolium perenne and L. multiflorum. METHODS: Three cultivars of each ploidy level of both species were grown under pot conditions in a controlled growth chamber, and leaf elongation rate and the cell length profile at the leaf base were measured on six plants in each cultivar. Cell parameters related to division and elongation activities were calculated by a kinematic method. KEY RESULTS: Tetraploid cultivars had faster leaf elongation rates than did diploid cultivars in both species, resulting in longer leaves, mainly due to their longer mature cells. Epidermal and mesophyll cells differed 20-fold in length, but were both greater in the tetraploid cultivars of both species. The increase in cell length of the tetraploid cultivars was caused by a faster cell elongation rate, not by a longer period of cell elongation. There were no significant differences between cell division parameters, such as cell production rate and cell cycle time, in the diploid and tetraploid cultivars. CONCLUSION: The results demonstrated clearly that polyploidy increases leaf size mainly by increasing the cell elongation rate, but not the duration of the period of elongation, and thus increases final cell size.

Cloning and characterization of a putative fructosyltransferase and two putative invertase genes from the temperate grass Lolium temulentum L.

The invertases of Lolium temulentum have been characterized at the enzyme level. However, studies on the expression of the genes coding for these enzymes have been lacking. To elucidate the role of acid 'invertase-like' genes in sucrose metabolism and carbon partitioning in Gramineae further, three 'invertase-like' homologous clones were isolated from L. temulentum cDNA expression libraries based on leaf tissue, using maize soluble invertase probes. The effect of developmental stage and alterations in carbohydrate status on the expression and tissue distribution of these genes was investigated. The three highly homologous genes (Inv 1:2, Inv 1:4, and FT 2:2) show different patterns of expression and different tissue distribution. Inv 1:2 was predominantly expressed in root tissue. Expression increased during the dark in root and tiller base tissue. Minimal variations in gene expression were observed in leaf tissue following changes in carbohydrate status. Inv 1:4 was predominantly expressed in tiller bases, leaf sheath, and leaf base, with increased expression in tissue samples in the dark period. FT 2:2 was also predominantly expressed in tiller bases, leaf sheath, and leaf base. Higher expression was observed in leaf tissue following increases in carbohydrate content, in a manner that paralleled the regulation and spatial occurrence of fructan in the leaf tissue. Whilst invertases and fructosyltransferases are difficult to distinguish at the level of the whole sequence, analysis of 5' sequence and specific amino acids allows discrimination which correlates with patterns of expression within the tissue. Based on expression patterns and sequence characteristics, it is proposed that Inv 1:2 and Inv 1:4 code for soluble acid invertases, whilst FT 2:2 codes for a fructosyltransferase.