Over‐expression of specific HvCslF cellulose synthase‐like genes in transgenic barley increases the levels of cell wall (1,3;1,4)‐β‐d‐glucans and alters their fine structure

Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)‐β‐d ‐glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)‐β‐d ‐glucans in the Poaceae is mediated, in part at least, by the cellulose synthase‐like CslF family of genes. Over‐expression of the barley CslF6 gene under the control of an endosperm‐specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)‐β‐d ‐glucan content in grain of transgenic barley. Analyses of (1,3;1,4)‐β‐d ‐glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)‐β‐d ‐glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)‐β‐d ‐glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)‐β‐d ‐glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)‐β‐d ‐glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)‐β‐d ‐glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)‐β‐d ‐glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses.     

Bacteria may induce the secretion of mucin‐like proteins by the diatom Phaeodactylum tricornutum

Benthic diatoms live in photoautotrophic/heterotrophic biofilm communities embedded in a matrix of secreted extracellular polymeric substances. Closely associated bacteria influence their growth, aggregation, and secretion of exopolymers. We have studied a diatom/bacteria model community, in which a marine Roseobacter strain is able to grow with secreted diatom exopolymers as a sole source of carbon. The strain influences the aggregation of Phaeodactylum tricornutum by inducing a morphotypic transition from planktonic, fusiform cells to benthic, oval cells. Analysis of the extracellular soluble proteome of P. tricornutum in the presence and absence of bacteria revealed constitutively expressed newly identified proteins with mucin‐like domains that appear to be typical for extracellular diatom proteins. In contrast to mucins, the proline‐, serine‐, threonine‐rich (PST) domains in these proteins were also found in combination with protease‐, glucosidase‐ and leucine‐rich repeat‐domains. Bioinformatic functional predictions indicate that several of these newly identified diatom‐specific proteins may be involved in algal defense, intercellular signaling, and aggregation.     

Immunolocalization of 1,3‐β‐Glucanases Secreted by Gaeumannomyces graminis var. tritici in Infected Wheat Roots

The distribution of extracellular 1,3‐β‐glucanase secreted by Gaeumannomyces graminis var. tritici (Ggt) was investigated in situ in inoculated wheat roots by immunogold labelling and transmission electron microscopy. Antiserum was prepared by subcutaneously injecting rabbits with purified 1,3‐β‐glucanase secreted by the pathogenic fungus. A specific antibody of 1,3‐β‐glucanase, anti‐GluGgt, was purified and characterized. Double immunodiffusion tests revealed that the antiserum was specific for 1,3‐β‐glucanase of Ggt, but not for 1,3‐β‐glucanase from wheat plants. Native polyacrylamide gel electrophoresis of the purified and crude enzyme extract and immunoblotting showed that the antibody was monospecific for 1,3‐β‐glucanase in fungal extracellular protein populations. After incubation of ultrathin sections of pathogen‐infected wheat roots with anti‐1,3‐β‐glucanase antibody and the secondary antibody, deposition of gold particles occurred over hyphal cells and the host tissue. Hyphal cell walls and septa as well as membranous structures showed regular labelling with gold particles, while few gold particles were detected over the cytoplasm and other organelles such as mitochondria and vacuoles. In host tissues, cell walls in contact with the hyphae usually exhibited a few gold particles, whereas host cytoplasm and cell walls distant from the hyphae were free of labelling. Furthermore, over lignitubers in the infected host cells labelling with gold particles was detected. No gold particles were found over sections of non‐inoculated wheat roots. The results indicate that 1,3‐β‐glucanase secreted by Ggt may be involved in pathogenesis of the take‐all fungus through degradation of callose in postinfectionally formed cell wall appositions, such as lignitubers.     

Plant species‐specific recognition of long and short β‐1,3‐linked glucans is mediated by different receptor systems

Plants survey their environment for the presence of potentially harmful or beneficial microbes. During colonization, cell surface receptors perceive microbe‐derived or modified‐self ligands and initiate appropriate responses. The recognition of fungal chitin oligomers and the subsequent activation of plant immunity are well described. In contrast, the mechanisms underlying β‐glucan recognition and signaling activation remain largely unexplored. Here, we systematically tested immune responses towards different β‐glucan structures and show that responses vary between plant species. While leaves of the monocots Hordeum vulgare and Brachypodium distachyon can recognize longer (laminarin) and shorter (laminarihexaose) β‐1,3‐glucans with responses of varying intensity, duration and timing, leaves of the dicot Nicotiana benthamiana activate immunity in response to long β‐1,3‐glucans, whereas Arabidopsis thaliana and Capsella rubella perceive short β‐1,3‐glucans. Hydrolysis of the β‐1,6 side‐branches of laminarin demonstrated that not the glycosidic decoration but rather the degree of polymerization plays a pivotal role in the recognition of long‐chain β‐glucans. Moreover, in contrast to the recognition of short β‐1,3‐glucans in A. thaliana, perception of long β‐1,3‐glucans in N. benthamiana and rice is independent of CERK1, indicating that β‐glucan recognition may be mediated by multiple β‐glucan receptor systems.     

Heterologous expression and periplasmic secretion of an antifungal Bacillus amyloliquefaciensBLB369 endo‐β‐1,3‐1,4‐glucanase in Escherichia coli

The endo‐β‐1,3‐1,4‐glucanases are glycoside hydrolases involved in the enzymatic depolymerization of 1,3‐1,4 β‐glucans and showed an antifungal activity against some fungi. Bacillus amyloliquefaciensBLB369 has a high antagonistic activity against phytopathogenic fungi. Its glu369 full‐coding sequence of the endo‐β‐1,3‐1,4‐glucanase gene (732 bp) was sequenced, cloned and successfully expressed in Escherichia coli Top10. The encoded protein (243 amino acids) has a calculated molecular mass of 27.3 kDa. To simplify the purification procedure, the glu369 coding sequence was cloned into the vector pKJD4. The produced OmpA‐His‐Glu369 harboured OmpA signal sequence for E. coli periplasmic localization and followed by a 6His residues for its purification. The purified His‐tagged proteins revealed two bands on SDS‐PAGE analysis with molecular masses of about 30.5 (His‐Glu369) and 32.5 kDa (OmpA‐His‐Glu369). They had the ability to inhibit the growth of phytopathogenic fungus Alternaria alternata. These favourable properties make the endo‐β‐1,3‐1,4‐glucanase a good candidate for biotechnological applications.     

A model for carbohydrate metabolism in the diatom Phaeodactylum tricornutum deduced from comparative whole genome analysis

Background

Diatoms are unicellular algae responsible for approximately 20% of global carbon fixation. Their evolution by secondary endocytobiosis resulted in a complex cellular structure and metabolism compared to algae with primary plastids.

Methodology/Principal Findings

The whole genome sequence of the diatom Phaeodactylum tricornutum has recently been completed. We identified and annotated genes for enzymes involved in carbohydrate pathways based on extensive EST support and comparison to the whole genome sequence of a second diatom, Thalassiosira pseudonana. Protein localization to mitochondria was predicted based on identified similarities to mitochondrial localization motifs in other eukaryotes, whereas protein localization to plastids was based on the presence of signal peptide motifs in combination with plastid localization motifs previously shown to be required in diatoms. We identified genes potentially involved in a C4-like photosynthesis in P. tricornutum and, on the basis of sequence-based putative localization of relevant proteins, discuss possible differences in carbon concentrating mechanisms and CO₂ fixation between the two diatoms. We also identified genes encoding enzymes involved in photorespiration with one interesting exception: glycerate kinase was not found in either P. tricornutum or T. pseudonana. Various Calvin cycle enzymes were found in up to five different isoforms, distributed between plastids, mitochondria and the cytosol. Diatoms store energy either as lipids or as chrysolaminaran (a β-1,3-glucan) outside of the plastids. We identified various β-glucanases and large membrane-bound glucan synthases. Interestingly most of the glucanases appear to contain C-terminal anchor domains that may attach the enzymes to membranes.

Conclusions/Significance

Here we present a detailed synthesis of carbohydrate metabolism in diatoms based on the genome sequences of Thalassiosira pseudonana and Phaeodactylum tricornutum. This model provides novel insights into acquisition of dissolved inorganic carbon and primary metabolic pathways of carbon in two different diatoms, which is of significance for an improved understanding of global carbon cycles.     

In the grass species Brachypodium distachyon,the production of mixed‐linkage (1,3;1,4)‐β‐glucan (MLG) occurs in the Golgi apparatus

Mixed‐linkage (1,3;1,4)‐β‐glucan (MLG) is a glucose polymer with beneficial effects on human health and high potential for the agricultural industry. MLG is present predominantly in the cell wall of grasses and is synthesized by cellulose synthase‐like F or H families of proteins, with CSLF6 being the best‐characterized MLG synthase. Although the function of this enzyme in MLG production has been established, the site of MLG synthesis in the cell is debated. It has been proposed that MLG is synthesized at the plasma membrane, as occurs for cellulose and callose; in contrast, it has also been proposed that MLG is synthesized in the Golgi apparatus, as occurs for other matrix polysaccharides of the cell wall. Testing these conflicting possibilities is fundamentally important in the general understanding of the biosynthesis of the plant cell wall. Using immuno‐localization analyses with MLG‐specific antibody in Brachypodium and in barley, we found MLG present in the Golgi, in post‐Golgi structures and in the cell wall. Accordingly, analyses of a functional fluorescent protein fusion of CSLF6 stably expressed in Brachypodium demonstrated that the enzyme is localized in the Golgi. We also established that overproduction of MLG causes developmental and growth defects in Brachypodium as also occur in barley. Our results indicated that MLG production occurs in the Golgi similarly to other cell wall matrix polysaccharides, and supports the broadly applicable model in grasses that tight mechanisms control optimal MLG accumulation in the cell wall during development and growth. This work addresses the fundamental question of where mixed linkage (1,3;1,4)‐β‐glucan (MLG) is synthesized in plant cells. By analyzing the subcellular localization of MLG and MLG synthase in an endogenous system, we demonstrated that MLG synthesis occurs at the Golgi in Brachypodium and barley. A growth inhibition due to overproduced MLG in Brachypodium supports the general applicability of the model that a tight control of the cell wall polysaccharides accumulation is needed to maintain growth homeostasis during development.     

KRE genes are required for β‐1,6‐glucan synthesis,maintenance of capsule architecture and cell wall protein anchoring in Cryptococcus neoformans

The polysaccharide β‐1,6‐glucan is a major component of the cell wall of Cryptococcus neoformans, but its function has not been investigated in this fungal pathogen. We have identified and characterized seven genes, belonging to the KRE family, which are putatively involved in β‐1,6‐glucan synthesis. The H99 deletion mutants kre5Δ and kre6Δskn1Δ contained less cell wall β‐1,6‐glucan, grew slowly with an aberrant morphology, were highly sensitive to environmental and chemical stress and were avirulent in a mouse inhalation model of infection. These two mutants displayed alterations in cell wall chitosan and the exopolysaccharide capsule, a primary cryptococcal virulence determinant. The cell wall content of the GPI‐anchored phospholipase B1 (Plb1) enzyme, which is required for cryptococcal cell wall integrity and virulence, was reduced in kre5Δ and kre6Δskn1Δ. Our results indicate that KRE5, KRE6 and SKN1 are involved in β‐1,6‐glucan synthesis, maintenance of cell wall integrity and retention of mannoproteins and known cryptococcal virulence factors in the cell wall of C. neoformans. This study sets the stage for future investigations into the function of this abundant cell wall polymer.     

Identification of a Vitis vinifera endo‐β‐1,3‐glucanase with antimicrobial activity against Plasmopara viticola

Inducible plant defences against pathogens are stimulated by infections and comprise several classes of pathogenesis‐related (PR) proteins. Endo‐β‐1,3‐glucanases (EGases) belong to the PR‐2 class and their expression is induced by many pathogenic fungi and oomycetes, suggesting that EGases play a role in the hydrolysis of pathogen cell walls. However, reports of a direct effect of EGases on cell walls of plant pathogens are scarce. Here, we characterized three EGases from Vitis vinifera whose expression is induced during infection by Plasmopara viticola, the causal agent of downy mildew. Recombinant proteins were expressed in Escherichia coli. The enzymatic characteristics of these three enzymes were measured in vitro and in planta. A functional assay performed in vitro on germinated P. viticola spores revealed a strong anti‐P. viticola activity for EGase3, which strikingly was that with the lowest in vitro catalytic efficiency. To our knowledge, this work shows, for the first time, the direct effect against downy mildew of EGases of the PR‐2 family from Vitis.     

Hetero‐trans‐β‐glucanase,an enzyme unique to Equisetum plants,functionalizes cellulose

Cell walls are metabolically active components of plant cells. They contain diverse enzymes, including transglycanases (endotransglycosylases), enzymes that ‘cut and paste’ certain structural polysaccharide molecules and thus potentially remodel the wall during growth and development. Known transglycanase activities modify several cell‐wall polysaccharides (xyloglucan, mannans, mixed‐linkage β‐glucan and xylans); however, no transglycanases were known to act on cellulose, the principal polysaccharide of biomass. We now report the discovery and characterization of hetero‐trans‐β‐glucanase (HTG), a transglycanase that targets cellulose, in horsetails (Equisetum spp., an early‐diverging genus of monilophytes). HTG is also remarkable in predominantly catalysing hetero‐transglycosylation: its preferred donor substrates (cellulose or mixed‐linkage β‐glucan) differ qualitatively from its acceptor substrate (xyloglucan). HTG thus generates stable cellulose–xyloglucan and mixed‐linkage β‐glucan–xyloglucan covalent bonds, and may therefore strengthen ageing Equisetum tissues by inter‐linking different structural polysaccharides of the cell wall. 3D modelling suggests that only three key amino acid substitutions (Trp → Pro, Gly → Ser and Arg → Leu) are responsible for the evolution of HTG's unique specificity from the better‐known xyloglucan‐acting homo‐transglycanases (xyloglucan endotransglucosylase/hydrolases; XTH). Among land plants, HTG appears to be confined to Equisetum, but its target polysaccharides are widespread, potentially offering opportunities for enhancing crop mechanical properties, such as wind resistance. In addition, by linking cellulose to xyloglucan fragments previously tagged with compounds such as dyes or indicators, HTG may be useful biotechnologically for manufacturing stably functionalized celluloses, thereby potentially offering a commercially valuable ‘green’ technology for industrially manipulating biomass.     

The cytoplasmic localization of the catalytic site of CSLF6 supports a channeling model for the biosynthesis of mixed‐linkage glucan

Mixed‐linkage glucan (MLG) is a significant cell wall carbohydrate in grasses and an important carbon source for human consumption and biofuel production. MLG biosynthesis depends on the biochemical activity of membrane spanning glucan synthases encoded by the CSLH and CSLF cellulose synthase‐like gene families. CSLF proteins are the best characterized to date but relatively little information is known about their topology with respect to the biosynthetic membranes. In this study, we report on the topology of CSLF6 protein derived from the model grass species Brachypodium distachyon (BdCSLF6) when it is expressed in heterologous systems. Using live cell imaging and immuno‐electron microscopy analyses of tobacco epidermal cells expressing BdCSLF6, we demonstrate that a functional yellow fluorescent protein (YFP) fusion of BdCSLF6 is localized to the Golgi apparatus and that the Golgi localization of BdCSLF6 is sufficient for MLG biosynthesis. By implementing protease protection assays of BdCSLF6 expressed in the yeast Pichia pastoris, we also demonstrate that the catalytic domain, the N‐terminus and the C‐ terminus of the protein are exposed in the cytosol. Furthermore, we found that BdCSLF6 is capable of producing MLG not only in tobacco cells but also in Pichia, which generally does not produce MLG. Together, these results support the conclusion that BdCSLF6 can produce both of the linkages present in the (1,3;1,4)‐β‐d ‐glucan chain of MLG and that the product is channelled at the Golgi into the secretory pathway for deposition into the cell wall.     

Mono‐ and digalactosyldiacylglycerol composition of the marennine‐producing diatom,Haslea ostrearia: Comparison to a selection of pennate and centric diatoms

Diatoms are one of the largest groups of primary producers in the oceans, yet despite their environmental importance little is known about their plastidial lipid biochemistry. It has been previously reported that Skeletonema species contain primarily C₁₆/C₁₆ and C₂₀/C₁₆ forms of mono‐ and digalactosyldiacylglycerol (MGDG and DGDG, respectively). Likewise, it was also reported that Phaeodactylum tricornutum contains primarily C₁₆/C₁₆ and C₂₀/C₂₀ forms of MGDG and DGDG. We seek to relate their studies to other diatoms, both in the centrics and pennates, with particular focus on the marennine‐producing pennate diatom, Haslea ostrearia. To this end, the composition and positional distribution of fatty acids of MGDG and DGDG were examined using positive‐ion electrospray ionization/mass spectrometry (ESI/MS). Two centric diatoms, Skeletonema marinoi and Thalassiosira weissflogii, and the pennate diatom, P. tricornutum, contained primarily C₂₀/C₁₆ (sn‐1/sn‐2) and C₁₈/C₁₆ forms of MGDG and DGDG. The other pennate diatoms, H. ostrearia and Navicula perminuta, contained primarily C₁₈/C₁₆ or C₁₈/C₁₈ forms of MGDG and DGDG, indicating a previously unrecognized fatty acid diversity in diatom MGDG and DGDG.     

Regulation of phosphoribulokinase and glyceraldehyde 3‐phosphate dehydrogenase in a freshwater diatom,Asterionella formosa1

The regulation of phosphoribulokinase (PRK) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was investigated in a freshwater pennate diatom, Asterionella formosa Hassall, and compared to the well‐studied chlorophyte Chlamydomonas reinhardtii P. A. Dang. As has been reported for a marine centric diatom, in A. formosa, PRK was not regulated by reduction with dithiothreitol (DTT) apart from a weak induction in the presence of NADPH and DTT. However, NADPH‐GAPDH was strongly activated when reduced, in contrast to a previous report on a diatom. Surprisingly, it was inhibited by NADPH, unlike in C. reinhardtii, while NADH‐GAPDH was not affected. NADH‐GAPDH was also strongly activated by DTT in contrast to most other photosynthetic cells. In A. formosa, unlike C. reinhardtii, 1,3‐bisphosphoglycerate, the substrate of GAPDH, activated this enzyme, even in the absence of DTT, when using both NADH and NADPH as cofactors. Some of these kinetic behaviors are consistent with regulation by protein–protein interactions involving CP12, a small protein that links PRK and GAPDH in cyanobacteria, green algae, and higher plants. This conclusion was supported by immunodetection of CP12 in crude extracts of A. formosa, using antibodies raised against CP12 from C. reinhardtii. This is the first report of the existence of CP12 in a diatom, but CP12 may be a common feature of diatoms since a bioinformatic search suggested that it was also present in the Thalassiosira pseudonana Hasle et Heimdal genome v3.0. Despite the presence of CP12, this work provides further support for the differential regulation of Calvin cycle enzymes in diatoms compared to green algae.     

The intracellular distribution of inorganic carbon fixing enzymes does not support the presence of a C4 pathway in the diatom <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis>

Diatoms are unicellular algae and important primary producers. The process of carbon fixation in diatoms is very efficient even though the availability of dissolved CO₂ in sea water is very low. The operation of a carbon concentrating mechanism (CCM) also makes the more abundant bicarbonate accessible for photosynthetic carbon fixation. Diatoms possess carbonic anhydrases as well as metabolic enzymes potentially involved in C4 pathways; however, the question as to whether a C4 pathway plays a general role in diatoms is not yet solved. While genome analyses indicate that the diatom Phaeodactylum tricornutum possesses all the enzymes required to operate a C4 pathway, silencing of the pyruvate orthophosphate dikinase (PPDK) in a genetically transformed cell line does not lead to reduced photosynthetic carbon fixation. In this study, we have determined the intracellular location of all enzymes potentially involved in C4-like carbon fixing pathways in P. tricornutum by expression of the respective proteins fused to green fluorescent protein (GFP), followed by fluorescence microscopy. Furthermore, we compared the results to known pathways and locations of enzymes in higher plants performing C3 or C4 photosynthesis. This approach revealed that the intracellular distribution of the investigated enzymes is quite different from the one observed in higher plants. In particular, the apparent lack of a plastidic decarboxylase in P. tricornutum indicates that this diatom does not perform a C4-like CCM.