Hans-Joachim G. Jung

Supplementation with dietary fiber improves fecal incontinence.

BackgroundHuman studies have shown that dietary fiber affects stool composition and consistency. Because fecal incontinence has been shown to be exacerbated by liquid stools or diarrhea, management strategies that make stool consistency less loose or liquid may be useful. ObjectiveTo compare the effects of a fiber supplement containing psyllium, gum arabic, or a placebo in community-living adults who were incontinent of loose or liquid stools. Mechanisms underlying these effects (e.g., fermentation of the fibers and water-holding capacity of stools) were examined. MethodsThirty-nine persons with fecal incontinence of loose or liquid stools prospectively recorded diet intake and stool characteristics and collected their stools for 8 days prior to and at the end of a 31-day fiber supplementation period. During the fiber supplementation period, they ingested psyllium, gum arabic, or a placebo by random assignment. ResultsIn the baseline period, the groups were comparable on all variables measured. In the fiber supplementation period, (a) the proportion of incontinent stools of the groups ingesting the fiber supplements was less than half that of the group ingesting the placebo, (b) the placebo group had the greatest percentage of stools that were loose/unformed or liquid, and (c) the psyllium group had the highest water-holding capacity of water-insoluble solids and total water-holding capacity. The supplements of dietary fiber appeared to be completely fermented by the subjects as indicated by non-significant differences in total fiber, short chain fatty acids and pH in stools among the groups in the baseline or fiber supplementation periods. ConclusionsSupplementation with dietary fiber from psyllium or gum arabic was associated with a decrease in the percentage of incontinent stools and an improvement of stool consistency. Improvements in fecal incontinence or stool consistency did not appear to be related to unfermented dietary fiber.

Genetic modification of herbaceous plants for feed and fuel.

Referee: Dr. E. Charles Brummer, Forage Breeding and Genetics, 1204 Agromonomy, Iowa State University, Ames, IA 50011 Much of the research on the genetic modification of herbaceous plant cell walls has been conducted to improve the utilization of forages by ruminant livestock. The rumen of these animals is basically an anaerobic fermentation vat in which the micro flora break down the complex polysaccharides of plant cell walls into simpler compounds that can be further digested and absorbed by the mammalian digestive system. Research on improving the forage digestibility of switchgrass, Panicum virgatum L., and other herbaceous species has demonstrated that genetic improvements can be made in forage quality that can have significant economic value. To meet future energy needs, herbaceous biomass will need to be converted into a liquid fuel, probably ethanol, via conversion technologies still under development. If feedstock quality can be genetically improved, the economics and efficiency of the conversio...

Using RNA-Seq for gene identification, polymorphism detection and transcript profiling in two alfalfa genotypes with divergent cell wall composition in stems

BackgroundAlfalfa, [Medicago sativa (L.) sativa], a widely-grown perennial forage has potential for development as a cellulosic ethanol feedstock. However, the genomics of alfalfa, a non-model species, is still in its infancy. The recent advent of RNA-Seq, a massively parallel sequencing method for transcriptome analysis, provides an opportunity to expand the identification of alfalfa genes and polymorphisms, and conduct in-depth transcript profiling.ResultsCell walls in stems of alfalfa genotype 708 have higher cellulose and lower lignin concentrations compared to cell walls in stems of genotype 773. Using the Illumina GA-II platform, a total of 198,861,304 expression sequence tags (ESTs, 76 bp in length) were generated from cDNA libraries derived from elongating stem (ES) and post-elongation stem (PES) internodes of 708 and 773. In addition, 341,984 ESTs were generated from ES and PES internodes of genotype 773 using the GS FLX Titanium platform. The first alfalfa (Medicago sativa) gene index (MSGI 1.0) was assembled using the Sanger ESTs available from GenBank, the GS FLX Titanium EST sequences, and the de novo assembled Illumina sequences. MSGI 1.0 contains 124,025 unique sequences including 22,729 tentative consensus sequences (TCs), 22,315 singletons and 78,981 pseudo-singletons. We identified a total of 1,294 simple sequence repeats (SSR) among the sequences in MSGI 1.0. In addition, a total of 10,826 single nucleotide polymorphisms (SNPs) were predicted between the two genotypes. Out of 55 SNPs randomly selected for experimental validation, 47 (85%) were polymorphic between the two genotypes. We also identified numerous allelic variations within each genotype. Digital gene expression analysis identified numerous candidate genes that may play a role in stem development as well as candidate genes that may contribute to the differences in cell wall composition in stems of the two genotypes.ConclusionsOur results demonstrate that RNA-Seq can be successfully used for gene identification, polymorphism detection and transcript profiling in alfalfa, a non-model, allogamous, autotetraploid species. The alfalfa gene index assembled in this study, and the SNPs, SSRs and candidate genes identified can be used to improve alfalfa as a forage crop and cellulosic feedstock.

Modifying crops to increase cell wall digestibility

Improving digestibility of roughage cell walls will improve ruminant animal performance and reduce loss of nutrients to the environment. The main digestibility impediment for dicotyledonous plants is highly lignified secondary cell walls, notably in stem secondary xylem, which become almost non-digestible. Digestibility of grasses is slowed severely by lignification of most tissues, but these cell walls remain largely digestible. Cell wall lignification creates an access barrier to potentially digestible wall material by rumen bacteria if cells have not been physically ruptured. Traditional breeding has focused on increasing total dry matter digestibility rather than cell wall digestibility, which has resulted in minimal reductions in cell wall lignification. Brown midrib mutants in some annual grasses exhibit small reductions in lignin concentration and improved cell wall digestibility. Similarly, transgenic approaches down-regulating genes in monolignol synthesis have produced plants with reduced lignin content and improved cell wall digestibility. While major reductions in lignin concentration have been associated with poor plant fitness, smaller reductions in lignin provided measurable improvements in digestibility without significantly impacting agronomic fitness. Additional targets for genetic modification to enhance digestibility and improve roughages for use as biofuel feedstocks are discussed; including manipulating cell wall polysaccharide composition, novel lignin structures, reduced lignin/polysaccharide cross-linking, smaller lignin polymers, enhanced development of non-lignified tissues, and targeting specific cell types. Greater tissue specificity of transgene expression will be needed to maximize benefits while avoiding negative impacts on plant fitness.cauliflower mosiac virus (CaMV) 35S promoter.

Analysis of Forage Fiber and Cell Walls in Ruminant Nutrition

Analysis of the fiber or cell wall present in forages is of major concern in ruminant nutrition because diets often contain large amounts of forage, and the fiber fraction affects both feed intake and animal performance. Traditional extractive, gravimetric methods such as crude fiber and neutral detergent fiber recover variable amounts of the plant cell wall, but they remain popular because of their ease of use and the large feed data bases available for these methods. More intensive chemical methods utilizing chromatography and spectrometric analysis provide greater detail on cell wall composition and structure, but they have been used little in ruminant nutrition. Lignin analysis has remained problematic because no definitive reference method exists. Recently attention has focused on the measurement of lignin composition and cell wall phenolic acids; however, these methods have yet to be widely adopted in ruminant nutrition. The detergent fiber methods have been semi-automated to increase sample handling capacity. Near-infrared spectroscopy is routinely used for prediction of fiber concentration in forages and has greatly increased the ease of obtaining fiber analysis of forage samples. Widespread adoption in ruminant nutrition of the more sophisticated methods of cell wall analysis is unlikely to occur until these methods can be demonstrated to improve diet formulation and prediction of animal performance.

Optimizing on-farm pretreatment of perennial grasses for fuel ethanol production.

Switchgrass (Panicum virgatum L.) and reed canarygrass (Phalaris arundinacea L.) were pretreated under ambient temperature and pressure with sulfuric acid and calcium hydroxide in separate experiments. Chemical loadings from 0 to 100g (kg DM)(-1) and durations of anaerobic storage from 0 to 180days were investigated by way of a central composite design at two moisture contents (40% or 60% w.b.). Pretreated and untreated samples were fermented to ethanol by Saccharomyces cerevisiae D5A in the presence of a commercially available cellulase (Celluclast 1.5L) and beta-glucosidase (Novozyme 188). Xylose levels were also measured following fermentation because xylose is not metabolized by S. cerevisiae. After sulfuric acid pretreatment and anaerobic storage, conversion of cell wall glucose to ethanol for reed canarygrass ranged from 22% to 83% whereas switchgrass conversions ranged from 16% to 46%. Pretreatment duration had a positive effect on conversion but was mitigated with increased chemical loadings. Conversions after calcium hydroxide pretreatment and anaerobic storage ranged from 21% to 55% and 18% to 54% for reed canarygrass and switchgrass, respectively. The efficacy of lime pretreatment was found to be highly dependent on moisture content. Moreover, pretreatment duration was only found to be significant for reed canarygrass. Although significant levels of acetate and lactate were observed in the biomass after storage, S. cerevisiae was not found to be inhibited at a 10% solids loading.

Maize stem tissues: ferulate deposition in developing internode cell walls

It has been hypothesized that ferulates are only deposited in the primary cell wall of grasses. To test this hypothesis, the fourth elongating, above-ground internode of maize (Zea mays l.) was sampled from three maize hybrids throughout development. Cell wall composition was determined by the Uppsala Dietary Fibre method. Ester- and ether-linked ferulates were determined by HPLC analysis of ferulic acid released from the internodes by low and high temperature alkaline treatments. Internode length increased from 9 to 152 mm over 96 days of growth, with elongation being complete in the first 12 days. More than half of the cell wall material in the maize internodes accumulated after elongation had ended. Deposition of cell wall material appeared to reach its maximum extent 40 days after sampling began, well before physiological maturity of the maize plants. Galactose and arabinose began to accumulate early in cell wall development which was presumed to be associated with primary wall growth during internode elongation. The major secondary wall constituents (analyzed as glucose, xylose, and Klason lignin) did not begin to accumulate rapidly until shortly before internode elongation ended. Ferulate ester deposition began before ferulate ethers were observed in the cell wall, but both forms of ferulate continued to accumulate in secondary cell walls, long after internode elongation had ceased. These data clearly show that contrary to the hypothesis, ferulate deposition was not restricted to the primary wall and that active lignin/polysaccharide cross-linking mediated by ferulates occurs in the secondary wall.

UDP-sugar pyrophosphorylase is essential for pollen development in Arabidopsis

Arabidopsis UDP-sugar pyrophosphorylase (AtUSP) is a broad substrate enzyme that synthesizes nucleotide sugars. The products of the AtUSP reaction can act as precursors for the synthesis of glycolipids, glycoproteins, and cell wall components including pectin and hemicellulose. AtUSP has no close homologs in Arabidopsis and its biological function has not been clearly defined. We identified two T-DNA insertional mutant lines for AtUSP, usp-1 and usp-2. No homozygous individuals were identified and progeny from plants heterozygous for usp-1 or usp-2 showed a 1:1 segregation ratio under selection. Despite decreased levels of both AtUSP transcript and USP activity (UDP-GlcA→GlcA-1-P), heterozygous plants were indistinguishable from wild type at all stages of development. Reciprocal test crosses indicated the source of the segregation distortion was lack of transmission through the male gametophyte. Analysis of pollen tetrads from usp-1 in the quartet background revealed a 2:2 ratio of normal:collapsed pollen grains. The collapsed pollen grains were not viable as determined by Alexander’s viability and DAPI staining, and pollen germination tests. The pollen phenotype of usp-1 was complemented by transformation of usp-1 with the AtUSP cDNA sequence. Surface and ultrastructural analyses of pollen from wild-type and usp mutants demonstrated that the mutation had no apparent effect on the outer wall (exine) but prevented the synthesis of the pectocellulosic inner wall (intine). Evidence presented here shows that AtUSP has a critical role in pollen development.

Impact of lignin composition on cell-wall degradability in an Arabidopsis mutant†

An Arabidopsis mutant that does not deposit syringyl-type lignin was used to test the hypothesis that lignin composition impacts cell-wall degradability. Two lines of the ferulate-5-hydroxylase-deficient fah1 mutant and the wild-type control line were grown in the greenhouse. In Experiment 1, the plants were harvested at the mature seed stage. For Experiment 2, plants were harvested 5, 6, 7 and 8 weeks after sowing. In both experiments stems were collected and analysed for cell-wall concentration and composition, and in vitro degradability of cell-wall polysaccharide components by rumen micro-organisms. The absence of syringyl-type lignin was confirmed for the mutant lines by nitrobenzene oxidation and pyrolysis-GC-MS. Lignin concentration was the same for all three Arabidopsis lines, at all stages of maturity. The Arabidopsis stems were similar to forage legumes in that the potentially degradable cell-wall fraction was very quickly degraded. Cell-wall polysaccharide degradability did not differ among the Arabidopsis lines in the first experiment after 24-h fermentations, but the cell-wall polysaccharides of the fah1-2 mutant line were less degradable after 96-h than either the wild-type or the fah1-5 mutant. In contrast, in Experiment 2 no differences among lines were found for cell-wall polysaccharide degradability after either 24- or 96-h fermentations; however, signficantly higher levels of ester-bound ferulic acid were found in the walls of the fah1 mutant lines. As expected, increasing stem maturity was correlated with reduced degradation of cell-wall polysaccharides. These experiments indicate that either lignin composition, as measured by syringyl-to-guaiacyl ratio, does not alter cell-wall degradability in Arabidopsis, or that the fah1 mutation has other effects on the cell walls of these mutants such that the impact of the change in syringyl-to-guaiacyl ratio is masked.

Impact of accessibility and chemical composition on cell wall polysaccharide degradability of maize and lucerne stems.

Although lignification of forages is generally accepted as limiting cell wall degradability, prediction of degradation from cell wall composition is often difficult when forages are of similar maturity. It has been proposed that rumen microbe accessibility to potentially degradable cell walls is limited by the presence of non-disrupted cells in forage particles with lignified middle lamella/primary walls acting as barriers to microbial access. We tested this accessibility hypothesis by evaluating the impact of reducing particle size of maize and lucerne stems to the level of individual cells by ball-milling, in order to eliminate accessibility as a limiting factor. While cell wall concentration and composition were not influenced by ball-milling compared with grinding to pass a 1 mm screen in a cyclone-type mill, degradability of total cell wall polysaccharides was dramatically increased. However, only those polysaccharides (cellulose and xylan) which are most abundant in cell types with lignified middle lamella/primary and secondary walls increased in degradability owing to particle size reduction. Degradability of pectins, which are abundant in non-lignified tissues in lucerne, did not respond to ball-milling. Contrary to our expectations, ball-milled forages showed fewer correlations for cell wall composition with degradability than observed for the larger-particle-size grinding treatment. Many components of the cell wall were correlated with polysaccharide degradation for the cyclone-ground samples; however, the results were inconsistent as to which cell wall components were correlated with degradation among and within forages. This observation does not clarify the role of cell wall chemical structure as a limiting agent to wall degradation in the absence of accessibility barriers, but this study does provide support for the hypothesis that lignified middle lamella/primary walls act as barriers to microbial access for degradation.