Evolution of Xylan Substitution Patterns in Gymnosperms and Angiosperms: Implications for Xylan Interaction with Cellulose

The interaction between cellulose and xylan is important for the load-bearing secondary cell wall of flowering plants. Based on the precise, evenly spaced pattern of acetyl and glucuronosyl (MeGlcA) xylan substitutions in eudicots, we recently proposed that an unsubstituted face of xylan in a 2-fold helical screw can hydrogen bond to the hydrophilic surfaces of cellulose microfibrils. In gymnosperm cell walls, any role for xylan is unclear, and glucomannan is thought to be the important cellulose-binding polysaccharide. Here, we analyzed xylan from the secondary cell walls of the four gymnosperm lineages (Conifer, Gingko, Cycad, and Gnetophyta). Conifer, Gingko, and Cycad xylan lacks acetylation but is modified by arabinose and MeGlcA. Interestingly, the arabinosyl substitutions are located two xylosyl residues from MeGlcA, which is itself placed precisely on every sixth xylosyl residue. Notably, the Gnetophyta xylan is more akin to early-branching angiosperms and eudicot xylan, lacking arabinose but possessing acetylation on alternate xylosyl residues. All these precise substitution patterns are compatible with gymnosperm xylan binding to hydrophilic surfaces of cellulose. Molecular dynamics simulations support the stable binding of 2-fold screw conifer xylan to the hydrophilic face of cellulose microfibrils. Moreover, the binding of multiple xylan chains to adjacent planes of the cellulose fibril stabilizes the interaction further. Our results show that the type of xylan substitution varies, but an even pattern of xylan substitution is maintained among vascular plants. This suggests that 2-fold screw xylan binds hydrophilic faces of cellulose in eudicots, early-branching angiosperm, and gymnosperm cell walls.The plant secondary cell wall is a complex network of various polysaccharides and phenolic compounds that act in concert to provide strength to the cell wall (Kumar et al., 2016). Cellulose, formed of strong crystalline fibrils of linear β1,4 glucan, comprises about 40% of dry plant biomass. The other secondary cell wall polysaccharides, largely xylan and glucomannan, comprise about 30% of dry plant biomass. The abundance and structure of these hemicelluloses vary with plant species and tissues, but they have in common that they are tightly associated with cellulose. It is believed that the most important biological role of hemicelluloses is their contribution to strengthening the cell wall by interaction with cellulose and, in some walls, with lignin (Scheller and Ulvskov, 2010). However, it is unclear how hemicelluloses interact with cellulose in the cell wall (Cosgrove and Jarvis, 2012). In this work, we were interested in the interaction of cellulose with xylan, one of the most abundant polysaccharides in nature.To understand polysaccharide interactions in the cell wall, we need to know not only the hemicellulose primary structure, but also the conformation of the polysaccharide chains. The glucan chains in cellulose are similar to a flat ribbon known as a 2-fold helical screw and associate through lateral hydrogen bonding into sheets. The sheets of glucan chains stack on top of each other, resulting in highly ordered crystalline cellulose. It is still unclear how many glucan chains form a microfibril and whether the microfibril has a hexagonal or rectangular cross section. However, recent studies shed some light onto these questions (Fernandes et al., 2011; Newman et al., 2013; Thomas et al., 2013; Cosgrove, 2014; Oehme et al., 2015; Thomas et al., 2015; Wang and Hong, 2016; Vandavasi et al., 2016). Whichever model is favored, hydrophobic (e.g. 100 or 200) and hydrophilic (e.g. 110 or 010) crystal faces are exposed for interaction with other molecules such as hemicelluloses (Zhao et al., 2014; Cosgrove, 2014; Li et al., 2015).The β1,4 xylan backbone is always further modified, often by acetyl (Ac), arabinosyl (Ara), and glucuronosyl (MeGlcA) side-chain substitutions. These substitutions are supposed to be necessary to maintain xylan solubility (Mikkelsen et al., 2015). Unsubstituted xylan forms crystalline fibers of chains adopting a 3-fold screw helix (Nieduszynski and Marchessault, 1971). Consequently, xylan substitutions are essential for xylan function and vascular plant viability (Mortimer et al., 2010; Xiong et al., 2013, 2015). In vitro experiments and in silico modeling suggest xylan interacts with cellulose, and it is widely accepted that this is partly through interactions on the hydrophobic faces of the cellulose fibrils (Bosmans et al., 2014; Köhnke et al., 2011; Kabel et al., 2007; Busse-Wicher et al., 2014). In contrast to the binding to the hydrophobic faces, the backbones of highly substituted hemicelluloses are thought to be unable to hydrogen bond effectively with the hydrophilic surfaces of cellulose fibrils because of steric hindrance. For example, hydrogen bonding of the xyloglucan backbone to cellulose would be blocked by steric restrictions of the side chains (Finkenstadt et al., 1995; Zhang et al., 2011). How then does the naturally occurring, highly substituted, xylan interact with cellulose? Our recent findings in the eudicot Arabidopsis (Arabidopsis thaliana) revealed that the majority of xylan bears substitutions solely on alternate xylosyl residues. Every second Xyl is acetylated (Busse-Wicher et al., 2014; Chong et al., 2014), and MeGlcA side chains reside on evenly spaced xylosyl residues, largely at 6-, 8-, 10-, or 12-residue intervals (Bromley et al., 2013). In this scenario, on a xylan backbone in the ribbon-like 2-fold helical screw conformation, all the decorations will face one side, creating an unsubstituted xylan surface. Therefore, in addition to forming stacking interactions on the hydrophobic surface, this xylan structure is compatible with hydrogen bonding to the hydrophilic surface of cellulose (Busse-Wicher et al., 2014; Busse-Wicher et al., 2016).Both xylan and glucomannan are substantial components of vascular plant secondary cell walls (Timell, 1967; Willfor et al., 2005; McKee et al., 2016). In conifers (gymnosperms, Pinales), the main hemicellulose is glucomannan, but eudicots possess relatively little glucomannan (Scheller and Ulvskov, 2010; Huang et al., 2015), and the secondary cell walls are dominated by xylan, suggesting xylan might have adopted additional functions in these flowering plants that produce hardwoods (Dammström et al., 2009). Conifers, providing softwood for the paper, pulp, and construction industries, are of major ecological and economical value. Consequently, understanding the function and architecture of the cell wall components of softwoods and hardwoods is of great importance.To investigate whether the precise arrangement of xylan decorations on evenly spaced xylosyl residues, as seen in eudicots, is a novel feature of hardwood xylan, we analyzed the pattern of xylan substitution in various gymnosperms and angiosperms. In addition to conifers, there are three further gymnosperm lineages: Cycad, Gingko and Gnetophyta (Fig. 1). There has been a debate whether Gnetophyta are the gymnosperm lineage most closely related to the angiosperms (Davis and Schaefer, 2011; Uddenberg et al., 2015). There are few studies across gymnosperm lineages to determine any divergence in the structure of xylans.Open in a separate windowFigure 1.Schematic representation of the phylogenetic relationship between gymnosperm and angiosperm species studied in this work. The distances do not correspond to phylogenetic distances.Our work shows that some gymnosperm xylans have decorations and decoration patterns that are different to those of eudicot xylans. Nevertheless, these modifications largely reside on even xylosyl residues on the backbone. Molecular dynamics simulations support the hypothesis that this highly conserved organization of substitutions allows an unsubstituted surface of xylan to bind stably to hydrophilic faces of cellulose fibrils.     

The Impact of Molecular Methods on the Systematics of Angiosperms

A comparison is made between the systematics of selected orders and families based on morphology and other “classical” characters on the one hand, and the results of molecular methods on the other hand. It can be shown that taxa defined by a broad array of characters from morphology, anatomy, embryology and phytochemistry usually are confirmed by molecular results. On the other hand a family like the Saxifragaceae s.l. delimited solely on the basis of floral morphology has been shown to be grossly polyphyletic. Some quite surprising results of the molecular analyses usually agree with some embryological or phytochemical characters, and sometimes even with characters of vegetative morphology and anatomy. As a special case “unequal ancient splits” are discussed, where one clade contains few genera and species usually retaining many primitive characters, and the other shows great diversity and contains the more advanced members.     

Molecular Data from the Chloroplast rpoC1 Gene Suggest a Deep and Distinct Dichotomy of Contemporary Spermatophytes into Two Monophyla: Gymnosperms (Including Gnetales) and Angiosperms

Partial sequences of the rpoC1 gene from two species of angiosperms and three species of gymnosperms (8330 base pairs) were determined and compared. The data obtained support the hypothesis that angiosperms and gymnosperms are monophyletic and none of the recent groups of the latter is sister to angiosperms. Received: 20 November 1998 / Accepted: 26 April 1999     

Biochemical and Molecular Basis of Pesticide Degradation by Microorganisms

Abstract

Fungal arachidonic acid (ARA)-rich oil is an important microbial oil that affects diverse physiological processes that impact normal health and chronic disease. In this article, the historic developments and technological achievements in fungal ARA-rich oil production in the past several years are reviewed. The biochemistry of ARA, ARA-rich oil synthesis and the accumulation mechanism are first introduced. Subsequently, the fermentation and downstream technologies are summarized. Furthermore, progress in the industrial production of ARA-rich oil is discussed. Finally, guidelines for future studies of fungal ARA-rich oil production are proposed in light of the current progress, challenges and trends in the field.     

Radial Oxygen Loss from Roots: The Theoretical Basis for the Manipulation of Flux Data Obtained by the Cylindrical Platinum Electrode Technique

A résumé is given of the cylindrical platinum electrode technique for measuring the rate of oxygen release from the submerged roots of intact plants. Methods are then described for manipulating the oxygen flux data to quantify the following root characteristics: total effective internal diffusional resistance, non-metabolic (pore-space) resistance, internal apical oxygen concentration, effective diffusion coefficient of internal transport and fractional porosity, and the respiratory contribution to internal transport. The diffusional resistance of the root wall is discussed and the method formerly suggested for converting low temperature flux data to the appropriate room temperature values (Armstrong 1971) is revised. Finally, suggestions are made for overcoming the difficulties encountered in using flux data for comparative work if the roots differ in their apical radii.     

Biochemical and Molecular Genetic Basis of Hydrogenases

Hydrogenases catalyse the reversible reduction of protons to molecular hydrogen. Applied research is focused on structure and catalytic function under the aspect of hydrogen formation. In this review we summarize the current knowledge about properties and physiological roles of hydrogenases in pro- and eukaryotes and compile molecular genetical data about structural features of prokaryotic hydrogenases. Finally, prospects are given for the possible application of hydrogenases or ‘hydrogenase-like catalysts’ in energy production.     

Small Circles of Helical DNA Obtained on the Basis of Transcriptional Pause Sites Sequence

Abstract

The 21-base pair synthetic DNA duplexes with basic ‘pause-motif’ site (‘CATGC’) were ligated head-to-tail to produce linear and circular multimers. This also was done from other closely related sequences. Electrophoretic mobilities of the linear multimers in Polyacrylamide gels were determined under the standard and modified conditions. We revealed that small linear multimers (～ 90 bp) were characterized by comparable value of gel retardation relative to the well known curved DNA, while longer multimers (130–170 bp) had only slightly expressed mobility anomaly. Nevertheless these multimers containing nontruncated ‘pause-motif were capable of cyclization, in particular, formation of unusually small circles while truncated ones were not. We conclude that basic ‘pause-motif site increases the closure ability while the multimers based on truncated ‘pause motif fail to curve into the small circles. We tend to explain this situation as a result of intrinsic bending as well as the influence of the thermal fluctuations of DNA, the latter most probably can be associated with ‘pause motif. We have estimated the equilibrial and maximal bend angles per 10.5 bp to be 12°～16° and 32° accordingly under experimental conditions of our study.     

Determination of Pseudomonas aeruginosa by Biochemical Test Methods

A rapid and simple test method for the detection of acylamidase activity of Pseudomonas aeruginosa was devised. One loopful of a nutrient agar overnight culture of a test organism was inoculated into 1 ml of a test medium consisting of 0.2% KH₂PO₄, 0.01% MgSO₄·7H₂O, 0.5% NaCl and 0.1% acetamide (final pH 6.8). After aerobic incubation at 37C for 6 hr, one drop of Nessler's reagent was dropped into the test medium. A reddish-brown sediment appeared immediately if results were positive. Of 40 test strains of P. aeruginosa 39 gave strongly positive results. A strain showed a weakly positive result after 6 hr incubation, but the reaction became stronger after 18 hr culture. Other species of Pseudomonas, and various species of bacteria such as genera Vibrio and Aeromonas, and family Enterobacteriaceae were negative in this test. From these experimental results, the acylamidase test was considered to be highly specific for strains of P. aeruginosa, and therefore useful as a reliable method for the identification of this species.     

Determination of Pseudomonas aeruginosa by Biochemical Test Methods

A new test method was devised for microbial gluconate oxidation, using an ammonium molybdate reagent. One loopful (about 2 mg wet wt.) of the test organism, grown on a nutrient agar plate for 18 hr, is transferred into 1 ml of the test liquid medium consisting of (NH₄)₂SO₄ 0.5 mg, potassium gluconate 10 mg, NaCl 5 mg, KH₂PO₄ 2 mg, MgSO₄·7H₂O 0.1 mg, and 1 ml of distilled water, incubated at 37 C for 6 hr without shaking, and then mixed with 3 ml of 1% aqueous solution of ammonium molybdate and 0.2 ml of glacial acetic acid. The mixture is heated in boiling water for 5 min, followed by abrupt cooling with running water. A deep blue colour appears in a positive result. A total of 39 strains of Pseudomonas aeruginosa showed positive results by this method, whereas Aeromonas, Vibrio, Proteus group, Klebsiella, Citrobacter and Enterobacter A group were all negative. Though some strains of Enterobacter B group showed a weak blue colour, it could be easily differentiated from the deep blue colour of Pseudomonas. Longer incubation of test microbes in the test medium, and longer heating of the reaction mixture gave unsatisfactory results.     

Differences in Velocity of Auxin Movement Obtained by Intercept and Peak Arrival Methods

Velocity of IAA movement was determined by noting the time of arrival of an ether-soluble auxin wave, at a fixed distance, after presentation of an auxin pulse to bean (Phaseolus vulgaris L. cv. Pinto) hypocotyl segments. The effects of IAA adsorption on wave symmetry were reduced by monitoring the arrival of ether-soluble auxin molecules. Velocity of auxin movement, as estimated by wave arrival, was slower than velocity estimated by the intercept method in controls and in both iodoacetate (IOAA) and 2,3,5-triiodobenzoic acid (TIBA) treated tissue. Velocity of wave migration was reduced by both treatments but intercept velocity was reduced only by TIBA treatment. Velocity of IAA migration was faster in segments (independent of method of measurement) than from segments into agar by a factor of 4 to 8. The rate limiting step of auxin migration in the traditional agar-plant sandwich is the partitioning of IAA between the tissue and agar. It was suggested that arrival curves for pulsed auxin migration are analogous to elution profiles of chromatographic columns and that at least two populations of mobile molecules with different velocities exist. It was also suggested that the two velocities represent migration of auxin on two different pathways: the faster velocity representing auxin movement of water films which coast highly crosslinked polymers in the segment and the slower component representing a population which moves primarily within the matrices of crosslinked polymers. Velocity of both populations may be a function of tissue hydration and charge interactions of mobile molecules and matrix polymers.     

Screening of Rice Genes from the cDNA Catalog Using the Data Obtained by Protein Sequencing

The partial amino acid sequences of 121 rice proteins separated by two-dimensional gel electrophoresis (2D-PAGE), were determined for a protein sequence data file. In the Rice Genome Research Program (RGP), more than 20,000 cDNA clones randomly selected from rice cDNA libraries have been sequenced to construct a cDNA catalog. Complimentary DNAs encoding about 30% of proteins in the protein sequence data file could be identified in the catalog by computer search. It was deduced that 20,000–40,000 genes are present in the rice genome. Only half of about 20,000 cDNAs sequenced in the RGP, corresponding to 1/4–1/2 of genes present in the entire rice genome, should have unique sequences after considering gene redundancy. This is consistent with the fact that the cDNAs encoding about 30% of the sequenced proteins could be identified in the catalog. If the size of the cDNA catalog is enlarged further, cDNAs encoding all proteins separated by 2D-PAGE could be easily identified from the catalog by using the protein sequence data.     

A Critique of the Practice of Comparing Control Data Obtained At A Single Time Point to Experimental Data Obtained At Multiple Time Points

The normal circadian rhythm in DNA synthetic activity (DNA-SA) in the tip of the mouse tongue is presented. When this rhythm, obtained from mice which were not treated (NT) or handled, was compared to the rhythms obtained from mice treated with saline (SAL) or 25 mg/kg isoproterenol (IPR), no alteration in the rhythm was observed after either treatment. the conclusion from this chronobiological, experimental design was that IPR had no effect on DNA-SA in the tip of the tongue. However, when three single time points (08.00, 11.00 or 14.00) are selected from the SAL-treated, control rhythm and compared to the multiple time point data from the IPR-treated mice, three very different, statistically supported conclusions were reached. The common practice of obtaining data at only one time point in control animals and comparing these data to data obtained from drug-treated animals at multiple time points is an example of poor experimental design which results in erroneous conclusions and unnecessary confusion in the literature on in vivo research.     

Biochemical Basis of Obligate Autotrophy in Blue-Green Algae and Thiobacilli

Differential rates of incorporation of sugars, organic acids, and amino acids during autotrophic growth of several blue-green algae and thiobacilli have been determined. In obligate autotrophs (both blue-green algae and thiobacilli), exogenously furnished organic compounds make a very small contribution to cellular carbon; acetate, the most readily incorporated compound of those studied, contributes about 10% of newly synthesized cellular carbon. In Thiobacillus intermedius, a facultative chemoautotroph, acetate contributes over 40% of newly synthesized cellular carbon, and succinate and glutamate almost 90%. In the obligate autotrophs, carbon from pyruvate, acetate, and glutamate is incorporated into restricted groups of cellular amino acids, and the patterns of incorporation in all five organisms are essentially identical. These patterns suggest that the tricarboxylic acid cycle is blocked at the level of alpha-ketoglutarate oxidation. Enzymatic analyses confirmed the absence of alpha-ketoglutarate dehydrogenase in the obligate autotrophs, and also revealed that they lacked reduced nicotinamide adenine dinucleotide oxidase, and had extremely low levels of malic and succinic dehydrogenase. These enzymatic deficiencies were not manifested by the two facultative chemoautotrophs examined. On the basis of the data obtained, an interpretation of obligate autotrophy in both physiological and evolutionary terms has been developed.     

Hybridism and the rate of evolution in Angiosperms

Ohne Zusammenfassung

---

Hybridism and the rate of evolution in Angiosperms