Genetic engineering of grass cell wall polysaccharides for biorefining

Grasses represent an abundant and widespread source of lignocellulosic biomass, which has yet to fulfil its potential as a feedstock for biorefining into renewable and sustainable biofuels and commodity chemicals. The inherent recalcitrance of lignocellulosic materials to deconstruction is the most crucial limitation for the commercial viability and economic feasibility of biomass biorefining. Over the last decade, the targeted genetic engineering of grasses has become more proficient, enabling rational approaches to modify lignocellulose with the aim of making it more amenable to bioconversion. In this review, we provide an overview of transgenic strategies and targets to tailor grass cell wall polysaccharides for biorefining applications. The bioengineering efforts and opportunities summarized here rely primarily on (A) reprogramming gene regulatory networks responsible for the biosynthesis of lignocellulose, (B) remodelling the chemical structure and substitution patterns of cell wall polysaccharides and (C) expressing lignocellulose degrading and/or modifying enzymes in planta. It is anticipated that outputs from the rational engineering of grass cell wall polysaccharides by such strategies could help in realizing an economically sustainable, grass‐derived lignocellulose processing industry.     

Ultrastructural aspects of cell wall synthesis inSpirogyra

Summary Filaments ofSpirogyra were fixed in 2% osmium tetroxide dehydrated in alcohol and embedded in Araldite. The fine structure of cells with regard to wall synthesis was studied. The cell wall was shown to have four layers. The inner one contains microfibrils and is considered to be the cell wall proper. The outer three layers are components of the slime layer. The innermost of these, the second layer of the wall, was shown to be between 1m to 3m and the third 0.3m to 1m. The fourth layer appears as no more than a dark black line measuring 10 nm across. In the cytoplasm two types of vesicles were seen. The largest of these has contents similar in appearance to the slime layer of the wall. This same material was also seen in the large vesicles attached to the Golgi bodies. It is suggested that the smaller vesicles are derived from the larger vesicles and later fuse with the cell membrane. The Golgi bodies were found to be fairly large measuring up to 5m across. Small electron opaque blobs and flecks on the outside of the plasmalemma and in between the microfibrils of the cell wall proper are considered to be mucilage droplets travelling to the slime layer. It cannot be excluded that some of the material of the large vesicles is released directly into the cytoplasm and is transferred without vesicles through the plasma membrane. The negative contrast appearance of the microfibrils seen in the cell wall is thought to be due to the spaces between them being filled with this electron opaque mucilage.Intercisternal rodlets measuring 2.5 nm across were seen in the Golgi bodies.Transverse microtubules were found to occur near the plasmalemma having the same orientation as some of the microfibrils.Lomasome-like structures sometimes with many 5 nm fibrils in their vicinity were seen.     

The cell wall of Chlamydomonas eugametos. Immunological aspects

An antiserum was raised against the major cell wall glycoprotein of Chlamydomonas eugametos which after purification reacted specifically with all individual wall components but not with intact cell walls. The antigenic sites in intact walls appear to be cryptic but become exposed on partial enzymatic degradation or in situ during daughter-cell release when the insoluble component is digested. Using the antiserum as a specific label for cell walls in various stages of disintegration, cell wall digestion during asexual and sexual reproduction was studied. It is also shown that while cell wall material is associated with isolated flagella, it is not normally associated with the flagella of intact cells.     

Laying down the bricks: logistic aspects of cell wall biosynthesis

Plant cell wall polysaccharides are synthesised at the plasma membrane and in the Golgi apparatus. Current research efforts mainly try to address how these molecules are synthesised or modified. However, it is clear that polysaccharide synthesis in the two compartments needs to be carried out in a coordinated fashion, and that carbohydrates and proteins that are delivered from the Golgi to the cell surface have to undergo a range of modifications. Consequently, there appears to be a need for a fine-tuned system that coalesces signals from the wall, synthesis of carbohydrate-based molecules and vesicle shuttling. Several recent papers have scratched the surface for an initial understanding of these linked processes. For example, the impairment of the proton pumping activity in the trans-Golgi network, which is part of the cell's trafficking system, results in growth defects, changes in Golgi stack morphology and cellulose deficiency. An increased understanding of how cell wall synthesis is coordinated with the secretory machinery may facilitate avenues for modulating cell wall contents and therefore overall plant biomass.     

Unique occurrence of pectin-like fibrillar cell wall deposits in xylem fibres of poplar

Using light and electron microscopic techniques, we studied the unique occurrence of fibrillar cell wall deposits in mature xylem fibres from poplar. These cell wall deposits lined the lumen-facing side of the wall, mainly in fibres next to vessel elements. Different lines of evidence point to the pectin-like nature of these fibrillar cell wall deposits. First, specific staining by Alcian Blue 8GX, a dye with high affinity for pectic substances. Second, the strongly reduced staining of the cell wall deposits in microscopic sections treated with pectolytic enzyme. Third, concomitant staining of pits, which are known to consist mainly of pectic substances. Given the pectin-like nature of the fibrillar cell wall deposits as well as their preferred occurrence in fibres neighbouring water-conducting vessel elements, a function for the fibrillar cell wall deposits in lateral water diffusion and stem water storage is hypothesised. The hypothesis is supported by the increased abundance of these cell wall deposits in wood tissue of a drought-sensitive poplar species.     

Unique N-glycan moieties of the 66-kDa cell wall glycoprotein from the red microalga Porphyridium sp

We report here the structural determination of the N-linked glycans in the 66-kDa glycoprotein, part of the unique sulfated complex cell wall polysaccharide of the red microalga Porphyridium sp. Structures were elucidated by a combination of normal phase/reverse phase HPLC, positive ion MALDI-TOF MS, negative ion electrospray ionization, and MS/MS. The sugar moieties of the glycoprotein consisted of at least four fractions of N-linked glycans, each composed of the same four monosaccharides, GlcNAc, Man, 6-O-MeMan, and Xyl, with compositions Man(8-9)Xyl(1-2)Me(3)GlcNAc(2). The present study is the first report of N-glycans with the terminal Xyl attached to the 6-mannose branch of the 6-antenna and to the 3-oxygen of the penultimate (core) GlcNAc. Another novel finding was that all four glycans contain three O-methylmannose residues in positions that have never been reported before. Although it is known that some lower organisms are able to methylate terminal monosaccharides in glycans, the present study on Porphyridium sp. is the first describing an organism that is able to methylate non-terminal mannose residues. This study will thus contribute to understanding of N-glycosylation in algae and might shed light on the evolutionary development from prokaryotes to multicellular organisms. It also may contribute to our understanding of the red algae polysaccharide formation. The additional importance of this research lies in its potential for biotechnological applications, especially in evaluating the use of microalgae as cell factories for the production of therapeutic proteins.     

The fermentation of cell wall substances in grass silage and in potato pulp

Summary The amount of acid formed in grass silage was greater than could have been formed from the soluble sugars present, even when only a lactic fermentation took place. This seemed to point to fermentation of cell wall substances by lactic acid bacteria. Lactic acid fermentation in potato pulp always takes place with cell wall substances as substrates, as sugars are absent. It was found that galactose, probably occurring as galactan, and also some pectic acid were fermented in potato pulp. Some lactobacilli were isolated from potato pulp; streptobacteria which could ferment galactan but no pectic or galacturonic acid, and betabacteria which could ferment galacturonic acid but no galactan or pectic acid. A number of homofermentative lactobacilli were all found to belong to the speciesStreptobacterium casei. It was shown that a strain of this species could ferment galactan in potato pulp sterilised previously with ethylene oxide. Part of this work was carried out at the Netherlands Institute for Dairy Research, Ede, Netherlands.     

Cell wall cementing materials of grass leaves

Treatment of grass leaves with either a purified pectin lyase of Aspergillus japonicus or a purified xylanase of Trichoderma viride could lead to the isolation of some single leaf cells. However, a mixture of pectin lyase and xylanase brought about more rapid isolation of single cells than did either of the two enzymes alone, indicating a synergistic effect. Analysis of the components released from oat cell walls by the enzymes indicated that both homogalacturonans with a high degree of esterification and a kind of glucuronoarabinoxylan with ferulic acid ester may play a role in cell wall cementing in grass leaves.     

The comparative aspects of cell wall chemistry in the green algae (chlorophyta)

Summary The origin of a cell wall was an event of fundamental importance in the evolution of plants. In the green algae, cell walls apparently had independent origins in at least three lines of evolution. In this paper, the components of the cell wall were determined and compared in four filamentous green algae representing the charophycean, chlorophycean and ulvacean evolutionary lines. The walls of all four have hydroxyproline-containing proteins which separate into five or six bands upon SDS gel electrophoresis. Variation does exist, with the charophyte possessing fast moving electrophoretic bands and high hydroxyproline content, the chlorophytes having intermediate movement of bands and lower hydroxyproline content, and the ulvacean representative possessing slow moving bands and a very low, if not questionable, hydroxyproline and saccharide content. Qualitative and quantitative estimates of wall proteins and sugars have been determined and compared. A hypothetical scheme of cell wall evolution based on these data, those of previous analyses, and recent phylogenetic schemes is presented. Although sound conclusions cannot be made until more information is available, the scheme might help to emphasize the areas most in need of additional research.This work was supported by National Science Foundation Grant DEB 78-03554.     

Mechanical aspects of chest wall distortion

During passive inflation of the respiratory system, the rib cage (RC) expands because the pressure applied to it [approximately equal to abdominal pressure (Pab)] increases. Similar Pab-tidal volume (VT) relationships between passive and spontaneous inspirations would occur only if 1) Pab acts on RC equally in the two situations (no distortion) or 2) the extradiaphragmatic inspiratory muscles expand RC, compensating for distortion. In anesthetized adult rats and in sleeping human infants the passive relationships between VT and Pab or abdomen motion (AB) were constructed by occluding the airways during expiration. For a given Pab (or AB) in active breathing VT averaged 55% (rats) and 49% (infants) of the passive volume change. With phrenic stimulation in rats VT was only slightly less than during spontaneous breathing, indicating that, in the latter case, the respiratory system was essentially driven only by the diaphragm. In both species occasional breaths with large RC expansion occurred, and VT was then equal to or larger than the passive volume at iso-Pab. We conclude that 1) RC distortion decreases VT to approximately half of the passive value and 2) being on the relaxation curve reflects "compensated" distortion and not absence of it.     

Unique and conserved aspects of gut development in zebrafish

Although the development of the digestive system of humans and vertebrate model organisms has been well characterized, relatively little is known about how the zebrafish digestive system forms. We define developmental milestones during organogenesis of the zebrafish digestive tract, liver, and pancreas and identify important differences in the way the digestive endoderm of zebrafish and amniotes is organized. Such differences account for the finding that the zebrafish digestive system is assembled from individual organ anlagen, whereas the digestive anlagen of amniotes arise from a primitive gut tube. Despite differences of organ morphogenesis, conserved molecular programs regulate pharynx, esophagus, liver, and pancreas development in teleosts and mammals. Specifically, we show that zebrafish faust/gata-5 is a functional ortholog of gata-4, a gene that is essential for the formation of the mammalian and avian foregut. Further, extraembryonic gata activity is required for this function in zebrafish as has been shown in other vertebrates. We also show that a loss-of-function mutation that perturbs sonic hedgehog causes defects in the development of the esophagus that parallel those associated with targeted disruption of this gene in mammals. Perturbation of sonic hedgehog also affects zebrafish liver and pancreas development, and these effects occur in a reciprocal fashion, as has been described during mammalian liver and ventral pancreas development. Together, these data define aspects of digestive system development necessary for the characterization of zebrafish mutants. Given the similarities of teleost and mammalian digestive physiology and anatomy, these findings have implications for developmental and evolutionary studies as well as research of human diseases, such as diabetes, liver cirrhosis, and cancer.     

Unique aspects of the regulation of the aspartate transcarbamylase of Serratia marcescens.

Aspartate trancarbamylase (ATC ase; EC 2.1.3.2) from Serratia marcescens HY has been purified 134-fold. Its properties are unique. Unlike the ATCase from Escherichia coli and Salmonella typhimurium, the S. marcescens HY enzyme activity is not feedback inhibited by any purine or pyrimidine nucleotide effectors; instead, the enzyme is activated by both cytidine 5'-triphosphate and adenosine 5'-triphosphate. Like the ATCase from E. coli and S. typhimurium, adenosine 5'-triphosphate alters the [S]0.5 of the enzyme and, in contrast, cytidine 5'-triphosphate does not alter the [S]0.5 but, instead, alters the Vmax. As has been shown for both E. coli and S . typhimurium, effector sensitivity may be selectively dissociated form catalytic activity by treatment with heat, parachloromercuribenzoate, or neohydrin. This dissociated enzyme possesses threefold higher specific activity than the native enzyme. The sedimentation coefficient of the native enzyme is approximately 11.4S, whereas the dissociated enzyme has a value of 6.0S. Whereas it has been possible to reconstitute the E. coli and the S. marcescens ATCase enzymes from their own homologous subunits, it has not been possible to make hybrid enzymes of catalytic and regulatory heterologous subunits from each other. It was not possible to detect repression of ATCase formation after growth of prototrophic strains of S. marcescens HY supplemented with 200 mug of uracil per ml, but eightfold derepression was observed after uracil withdrawal in pyrimidine auxotrophs.