The Charophycean green algae as model systems to study plant cell walls and other evolutionary adaptations that gave rise to land plants

The Charophycean green algae (CGA) occupy a key phylogenetic position as the evolutionary grade that includes the sister group of the land plants (embryophytes), and so provide potentially valuable experimental systems to study the development and evolution of traits that were necessary for terrestrial colonization. The nature and molecular bases of such traits are still being determined, but one critical adaptation is thought to have been the evolution of a complex cell wall. Very little is known about the identity, origins and diversity of the biosynthetic machinery producing the major suites of structural polymers (i. e., cell wall polysaccharides and associated molecules) that must have been in place for land colonization. However, it has been suggested that the success of the earliest land plants was partly based on the frequency of gene duplication, and possibly whole genome duplications, during times of radical habitat changes. Orders of the CGA span early diverging taxa retaining more ancestral characters, through complex multicellular organisms with morphological characteristics resembling those of land plants. Examination of gene diversity and evolution within the CGA could help reveal when and how the molecular pathways required for synthesis of key structural polymers in land plants arose.     

Characterization of three plant biomass-degrading microbial consortia by metagenomics- and metasecretomics-based approaches

The selection of microbes by enrichment on plant biomass has been proposed as an efficient way to develop new strategies for lignocellulose saccharification. Here, we report an in-depth analysis of soil-derived microbial consortia that were trained to degrade once-used wheat straw (WS1-M), switchgrass (SG-M) and corn stover (CS-M) under aerobic and mesophilic conditions. Molecular fingerprintings, bacterial 16S ribosomal RNA (rRNA) gene amplicon sequencing and metagenomic analyses showed that the three microbial consortia were taxonomically distinct. Based on the taxonomic affiliation of protein-encoding sequences, members of the Bacteroidetes (e.g. Chryseobacterium, Weeksella, Flavobacterium and Sphingobacterium) were preferentially selected on WS1-M, whereas SG-M and CS-M favoured members of the Proteobacteria (e.g. Caulobacter, Brevundimonas, Stenotrophomonas and Xanthomonas). The highest degradation rates of lignin (~59 %) were observed with SG-M, whereas CS-M showed a high consumption of cellulose and hemicellulose. Analyses of the carbohydrate-active enzymes in the three microbial consortia showed the dominance of glycosyl hydrolases (e.g. of families GH3, GH43, GH13, GH10, GH29, GH28, GH16, GH4 and GH92). In addition, proteins of families AA6, AA10 and AA2 were detected. Analysis of secreted protein fractions (metasecretome) for each selected microbial consortium mainly showed the presence of enzymes able to degrade arabinan, arabinoxylan, xylan, β-glucan, galactomannan and rhamnogalacturonan. Notably, these metasecretomes contain enzymes that enable us to produce oligosaccharides directly from wheat straw, sugarcane bagasse and willow. Thus, the underlying microbial consortia constitute valuable resources for the production of enzyme cocktails for the efficient saccharification of plant biomass.     

High‐throughput screening of Erwinia chrysanthemi pectin methylesterase variants using carbohydrate microarrays

Pectin methylesterases (PMEs) catalyse the removal of methyl esters from the homogalacturonan (HG) backbone domain of pectin, a ubiquitous polysaccharide in plant cell walls. The degree of methyl esterification (DE) impacts upon the functional properties of HG within cell walls and plants produce numerous PMEs that act upon HG in muro. Many microbial plant pathogens also produce PMEs, the activity of which renders HG more susceptible to cleavage by pectin lyase and polygalacturonase enzymes and hence aids cell wall degradation. We have developed a novel microarray‐based approach to investigate the activity of a series of variant enzymes based on the PME from the important pathogen Erwinia chrysanthemi. A library of 99 E. chrysanthemi PME mutants was created in which seven amino acids were altered by various different substitutions. Each mutant PME was incubated with a highly methyl esterified lime pectin substrate and, after digestion the enzyme/substrate mixtures were printed as microarrays. The loss of activity that resulted from certain mutations was detected by probing arrays with a mAb (JIM7) that preferentially binds to HG with a relatively high DE. Active PMEs therefore resulted in diminished JIM7 binding to the lime pectin substrate, whereas inactive PMEs did not. Our findings demonstrate the feasibility of our approach for rapidly testing the effects on PME activity of substituting a wide variety of amino acids at different positions.     

The Immunofluorescent Localization of Subventral Pharyngeal Gland Epitopes of Preparasitic Juveniles of Heterodera glycines Using Laser Scanning Confocal Microscopy

Laser scanning confocal microscopy (LSCM) was used to localize the reactivity of a monoclonal antibody (Sv2) that binds to the subventral pharyngeal glands of preparasitic juveniles of Heterodera glycines. The greater resolution, magnification, and image analysis of LSCM compared with conventional epifluorescent microscopy enabled Sv2 binding to be localized much more precisely to the periphery of the secretory granules. A linear increase of about 55% in fluorescent intensity was found over a 23-μm length of subventral pharyngeal gland just distal to the terminal ampullae. LSCM is a rapid and effective technique for precise immunolocalization of epitopes.     

A role for arabinogalactan-proteins in plant cell expansion: evidence from studies on the interaction of β-glucosyl Yariv reagent with seedlings of Arabidopsis thaliana

Seedlings of Arabidopsis thaliana were germinated and grown in medium containing β-glucosyl Yariv reagent (βGlcY), a synthetic phenyl glycoside that interacts specifically with arabinogalactan-proteins (AGPs), a class of plant cell surface proteoglycans. The effect of βGlcY on the seedlings was to reduce the overall growth of both the root and the shoot. βGlcY only accumulated in the root tissues and the reduced growth of the shoot appeared to be an indirect effect of impaired root growth. Reduced root growth was a consequence of a reduction in cell elongation during the postproliferation phase of elongation at the root apex and this was associated with extensive radial expansion of root epidermal cells. βGlcY penetrated roots as far as the endodermis and it is suggested that the interaction of βGlcY with AGPs in the load-bearing cell layers inhibited root elongation. When βGlcY was added to carrot suspension-cultured cells that had been induced to elongate rather than proliferate, cell elongation was inhibited. The AGP-unreactive α-galactosyl Yariv reagent (αGalY) had no biological activity in either of these systems.     

Side chains of pectic polysaccharides are regulated in relation to cell proliferation and cell differentiation

The occurrence and function of the side chains occurring in the rhamnogalacturonan I domain of pectic poly- saccharides have been investigated during carrot cell development using monoclonal antibodies to defined epitopes of (1-->4)-beta-D-galactan and (1-->5)-alpha-L-arabinan. Immunolocalization studies of carrot root apices indicated that cell walls in the central region of the meristem contained higher levels of (1-->5)-alpha-arabinan than the cell walls of surrounding cells. In contrast (1-->4)-beta-galactan was absent from the cell walls of the central meristematic cells but appeared abundantly at a certain point during root cap cell differentiation and also appeared in cell walls of differentiating stele and cortical cells. This developmental pattern of epitope occurrence was also reflected in a suspension-cultured carrot cell line that can be induced to switch from proliferation to elongation by altered culture conditions. (1-->4)-beta-galactan occurred at a low level in cell walls of proliferating cells but accumulated rapidly in cell walls following induction, before any visible cell elongation, while (1-->5)-alpha-arabinan was present in cell walls of proliferating cells but was absent from cell walls of elongated cells. Immunochemical assays of the cultured cells confirmed the early appearance of (1-->4)-beta-galactan during the switch from cell proliferation to cell elongation. Anion-exchange chromatography confirmed that (1-->4)-beta-galactan was attached to acidic pectic domains and also indicated that it was separate from a distinct homogalacturonan-rich component. These results indicate that the neutral components of pectic polysaccharides may have important roles in plant cell development.     

Vacuolar reticulum in oomycete hyphal tips: An additional component of the Ca2+Regulatory system?

Cultures of Achlya sp., Phytophthora cinnamomi, Saprolegnia diclina, S. ferax, and S. parasitica, treated with 6-carboxyfluorescein diacetate solution, accumulate 6-carboxyfluorescein in a reticulate system of fine tubules. The network shows longitudinal polarity within the hyphae, tubules being finest toward the hyphal tips. In more mature subapical regions the network is connected with large vacuoles that also accumulate 6-carboxyfluorescein. A morphologically similar system has also been identified in freeze-substituted hyphae of S. ferax. The network is considered to be vacuolar, but differs from the tubular vacuole system of true fungi in that tubules are less motile, more frequently branched, and do not alternate with clusters of spherical vacuoles. The appearance of the network resembles patterns of calcium-sensitive dye staining and it is suggested that the vacuolar reticulum in the tip region of oomycete hyphae may act as a Ca2+ sink. The tubular reticulum in oomycetes is very fragile and can be shown with 6-carboxyfluorescein in only those hyphal tips with a motility and organelle distribution characteristic of growing hyphae with normal morphology. Diverse abnormal hyphae show a range of other fluorochrome localizations. These include large irregular compartments filled with fluorochrome, and fluorescent cytoplasm with organelles and vacuoles standing out in negative contrast. These localizations in abnormal hyphae are correlated with other structural changes indicative of damage. Special care is required in experiments with oomycetes to avoid such artefacts of localization. Copyright 1997 Academic Press. Copyright 1997 Academic Press     

Synthetic methyl hexagalacturonate hapten inhibitors of anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7

A range of synthetic methyl hexagalacturonates were used as potential hapten inhibitors in competitive-inhibition enzyme-linked immunosorbent assays (ELISAs) with anti-homogalacturonan monoclonal antibodies LM7, JIM5 and JIM7. The selective inhibition of these antibodies by different haptens provides insight into the structures of the partially methyl-esterified pectin epitopes of these widely used monoclonal antibodies.     

Molecular mechanism of ligand recognition by membrane transport protein,Mhp1

The hydantoin transporter Mhp1 is a sodium‐coupled secondary active transport protein of the nucleobase‐cation‐symport family and a member of the widespread 5‐helix inverted repeat superfamily of transporters. The structure of Mhp1 was previously solved in three different conformations providing insight into the molecular basis of the alternating access mechanism. Here, we elucidate detailed events of substrate binding, through a combination of crystallography, molecular dynamics, site‐directed mutagenesis, biochemical/biophysical assays, and the design and synthesis of novel ligands. We show precisely where 5‐substituted hydantoin substrates bind in an extended configuration at the interface of the bundle and hash domains. They are recognised through hydrogen bonds to the hydantoin moiety and the complementarity of the 5‐substituent for a hydrophobic pocket in the protein. Furthermore, we describe a novel structure of an intermediate state of the protein with the external thin gate locked open by an inhibitor, 5‐(2‐naphthylmethyl)‐L‐hydantoin, which becomes a substrate when leucine 363 is changed to an alanine. We deduce the molecular events that underlie acquisition and transport of a ligand by Mhp1.