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.

A changing climate for grassland research.

Here, we review the current genetic approaches for grass improvement and their potential for the enhanced breeding of new varieties appropriate for a sustainable agriculture in a changing global climate. These generally out-breeding, perennial, self-incompatible species present unique challenges and opportunities for genetic analysis. We emphasise their distinctiveness from model species and from the in-breeding, annual cereals. We describe the modern genetic approaches appropriate for their analysis, including association mapping. Sustainability traits discussed here include stress resistance (drought, cold and pathogeneses) and favourable agronomic characters (nutrient use efficiency, carbohydrate content, fatty acid content, winter survival, flowering time and biomass yield). Global warming will predictably affect temperature-sensitive traits such as vernalisation, and these traits are under investigation. Grass biomass utilisation for carbon-neutral energy generation may contribute to reduced atmospheric carbon emissions. Because the wider potential outcomes of climate change are unpredictable, breeders must be reactive to events and have a range of well-characterised germplasm available for new applications.

Dissecting the regulation of fructan metabolism in perennial ryegrass (Lolium perenne) with quantitative trait locus mapping.

Quantitative trait locus (QTL) mapping, which can be a useful tool for dissecting complex traits, has been used here to study the regulation of fructan metabolism in temperate forage grasses. An F2 mapping family, derived from a high water-soluble carbohydrate (WSC) x low WSC cross, was used to map fructans and the other components of WSC (sucrose, glucose and fructose) in leaves and tiller bases of perennial ryegrass (Lolium perenne) in spring and autumn. To characterize regions of the genome that control basic carbohydrate metabolism, a strategy to minimize the impact of genotype (G) x environment (E), and E-effects on the characterization of G-effects, was adopted. Most traits were highly variable within the family. There was also considerable year-to-year environmental variation. However, significant genetic effects were detected, and several traits had high broad-sense heritability. QTL were identified on chromosomes 1, 2, 5 and 6. Leaf and tiller base QTL did not coincide. Individual QTL explained between 8 and 59% of the total phenotypic variation in the traits. Fructan turnover, metabolism and their genetic control, and the effect of environment, are discussed in the context of the results.

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.

Structure of fructans from excised leaves of New Zealand flax.

The accumulation of total water-soluble carbohydrate, and specifically sucrose and fructan, by excised leaves of Phormium tenax and P. cookianum (family Phormiaceae J. G. Agardh, order Asparagales) was investigated. Total water-soluble carbohydrate content of excised leaves of P. tenax and P. cookianum increased during 48 h of continuous illumination at an average rate of 1.3 and 0.9 mg g(-1) fresh weight leaf per hour, respectively. The sucrose content of excised leaves increased throughout the experimental period. The fructan content of excised leaves of P. tenax increased slightly throughout the experimental period, whilst that of P. cookianum was variable and showed no overall change. Chemical and spectroscopic analysis of the fructans obtained from the two Phormium species showed that they were similar to each other and contained mostly 1-linked and terminal fructofuranosyl (Fruf) residues, together with smaller amounts of 6-linked Fruf, 1,6-branched Fruf, terminal and 6-linked glucopyranosyl residues. Separation of the fructans by thin-layer and high-performance anion-exchange chromatography revealed the presence of a complex mixture of fructo-oligosaccharides and higher molecular weight fructan. The branched structure of the fructans isolated from excised leaves of Phormium resembles that of fructans and fructo-oligosaccharides isolated from some related species within the order Asparagales (Agave vera cruz, Cordyline australis and Urginea maritima), but is distinct from the linear structure of fructans from others (Allium cepa and Asparagus officinalis). The structural heterogeniety of fructans within both the order Asparagales and superorder Liliiflorae may be a useful chemotaxonomic aid.

Characterisation of extracellular polysaccharides from suspension cultures of members of the poaceae.

Microscopic examination of suspension- cultured cells of Phleum pratense L., Panicum miliaceum L., Phalarisaquatica L. and Oryza sativa L. showed that they were comprised of numerous root primordia. Polysaccharides secreted by these suspension cultures contained glycosyl linkages consistent with the presence of high proportions of root mucilage-like polysaccharides. In contrast, suspension-cultured cells of Hordeum vulgare L. contained mostly undifferentiated cells more typical of plant cells in suspension culture. The polysaccharides secreted by H. vulgare cultures contained mostly linkages consistent with the presence of glucuronoarabinoxylan. The soluble polymers secreted by cell-suspension cultures of Phleum pratense contained 70% carbohydrate, 14% protein and 6% inorganic material. The extracellular polysaccharides were separated into four fractions by anion-exchange chromatography using a gradient of imidazole-HCl at pH 7.0. From glycosyl-linkage analyses, five polysaccharides were identified: an arabinosylated xyloglucan (comprising 20% of the total polysaccharide), a glucomannan (6%), a type-II arabinogalactan (an arabinogalactan-protein; 7%), an acidic xylan (3%), and a root-slime-like polysaccharide, which contained features of type-II arabinogalactans and glucuronomannans (65%).

Model fitting and projection of the AIDS epidemic.

Previously it was possible to fit detailed models to incidence data (for example, of AIDS) only by trial and error and good judgment; the large number of parameters obstructed optimization of, for example, the (approximate) likelihood. Here, we analyze a model for the spread of AIDS in a homosexual population and identify a minimal set of primary components that dictate the dynamics of the Model: the initial growth rate theta, the basic reproductive ratio R0, and the heterogeneity coefficient S. It is then shown that it is sufficient to maximize the likelihood over these three primary components; further maximization over the remaining secondary parameters does not produce a significant improvement in the fit or affect the projection of the epidemic. This method also allows construction of confidence limits for the projected incidence curve, allowing us to quantify the uncertainties associated with such model fitting procedures. The method is tested on simulation data to analyze how the accuracy of estimates and projections changes as we gain more data.

Epidemics in heterogeneous populations: II. Nonexponential incubation periods and variable infectiousness.

Two stochastic models for the spread of an infection through a heterogeneous population are considered. First, we consider a model where the incubation period has an increasing hazard rate but constant infectiousness; in the second model, the incubation period is the sum of p exponentially distributed stages, each with its own mean and level of infectiousness. By using multitype birth-death and branching processes as approximations to each epidemic model, it is shown that the epidemics initially have underlying exponential growth. Furthermore, the growth rate theta is an increasing function of the Frobenius root of the matrix of reproductive ratios R0. The results have applications in long-term sensitivity analyses, model fitting, and the determination of optimal vaccination strategies.

Epidemics in heterogeneous populations: aspects of optimal vaccination policies.

Three models for epidemics among m groups with applications to human disease, especially socially and geographically heterogeneous populations, are considered. It is shown that the initial growth of each is an increasing function of the Frobenius root of R0, the matrix of reproductive ratios. A new way of looking at optimal vaccination is presented by linking policies to the growth rate of a new epidemic. Aspects of how to minimize the initial growth rate are analysed. In particular, we see that, when R0 has positive eigenvalues, we can find an explicit solution for the final optimal policy, and that there exists an extension of this policy which always gives the least possible growth rate at all stages of vaccination.

Colorimetric microtiter plate assay of glucose and fructose by enzyme-linked formazan production: applicability to the measurement of fructosyl transferase activity in higher plants.

A rapid, enzyme-linked colorimetric assay, for the sequential determination of nanomole quantities of glucose and fructose in the same sample, has been developed for the measurement of fructosyl transferase activity in plant extracts. The assay extends the conventional dehydrogenase-linked assay for these sugars by utilizing the intermediary electron carrier, phenazine methosulfate, to couple NADP reduction to the production of a formazan dye from the tetrazolium salt, thiazolyl blue, in a form suitable for measurement using a microtiter plate reader. When the microtiter plate assay was used to measure the activities of yeast invertase and sucrose:sucrose fructosyl transferase from Lolium temulentum, results obtained were very similar to results obtained using the conventional procedure. The rapidity, small scale, and ease of execution of the method offers considerable advantages over the conventional hexose assay and is particularly suitable for screening of large numbers of small samples, exploiting both the speed of the microtiter plate reader and the facility of for microcomputer processing of data. The potential of this method for use with other enzyme systems and other metabolites is discussed.