Hydroxyproline-rich Glycoproteins Research Articles

An intronless form of the tobacco extensin gene terminator strongly enhances transient gene expression in plant leaves.

We have found interesting features of a plant gene (extensin) 3' flanking region, including extremely efficient polyadenylation which greatly improves transient expression of transgenes when an intron is removed. Its use will greatly benefit studies of gene expression in plants, research in molecular biology, and applications for recombinant proteins. Plants are a promising platform for the production of recombinant proteins. To express high-value proteins in plants efficiently, the optimization of expression cassettes using appropriate regulatory sequences is critical. Here, we characterize the activity of the tobacco extensin (Ext) gene terminator by transient expression in Nicotiana benthamiana, tobacco, and lettuce. Ext is a member of the hydroxyproline-rich glycoprotein (HRGP) superfamily and constitutes the major protein component of cell walls. The present study demonstrates that the Ext terminator with its native intron removed increased transient gene expression up to 13.5-fold compared to previously established terminators. The enhanced transgene expression was correlated with increased mRNA accumulation and reduced levels of read-through transcripts, which could impair gene expression. Analysis of transcript 3'-ends found that the majority of polyadenylated transcripts were cleaved at a YA dinucleotide downstream from a canonical AAUAAA motif and a UG-rich region, both of which were found to be highly conserved among related extensin terminators. Deletion of either of these regions eliminated most of the activity of the terminator. Additionally, a 45 nt polypurine sequence ~ 175 nt upstream from the polyadenylation sites was found to also be necessary for the enhanced expression. We conclude that the use of Ext terminator has great potential to benefit the production of recombinant proteins in plants.

Co-inoculation of lucerne (Medicago sativa) with an AM fungus and a rhizobium reduces occurrence of spring black stem and leaf spot caused by Phoma medicaginis

Spring black stem and leaf spot of lucerne (alfalfa, Medicago sativa L.), caused by Phoma medicaginis, is an important disease in temperate regions of the world. It is now a serious disease threatening global lucerne production. This experiment was designed to test the combined effects of the arbuscular mycorrhizal (AM) fungus Funneliformis mosseae and the rhizobium Sinorhizobium medicae on growth, nutrient uptake and disease severity in lucerne. The results showed that F. mosseae increased plant phosphorus and nitrogen uptake and plant dry weight, and this beneficial effect was enhanced when in association with S. medicae. Rhizobial and AM fungal effects were mutually promoting; inoculation with AM fungus significantly increased the formation of root nodules, and inoculation with rhizobium increased the percentage of root length colonised by AM fungus (P < 0.05). After infection with P. medicaginis, typical leaf spot symptoms with the lowest disease incidence and disease index occurred on plants that were host to both F. mosseae and S. medicae. Plants with both symbiotic microorganisms had higher activities (concentrations) of phenylalanine ammonia-lyase, chitinase, β-1,3-glucanase, lignin, hydroxyproline-rich glycoprotein and jasmonic acid. Therefore, the tested AM fungus (F. mosseae) and rhizobium (S. medicae) have the potential to reduce damage and yield loss in lucerne from spring black stem and leaf spot caused by P. medicaginis.

Polysaccharide associated protein (PSAP) from the green microalga Botryococcus braunii is a unique extracellular matrix hydroxyproline-rich glycoprotein

The green colonial microalga Botryococcus braunii produces large amounts of liquid hydrocarbons that can be converted into transportation fuels. Colony cells are held together by a complex extracellular matrix (ECM) made up of a cross-linked long-chain hydrocarbon network around which liquid hydrocarbons are stored, a retaining wall for holding hydrocarbons within the cross-linked hydrocarbon network, and a polysaccharide fibrillar sheath radiating from the retaining wall and surrounding the entire colony. Analysis of “shells” shed from cell apical regions during cell division and containing the retaining wall and polysaccharide fibers shows association of a single protein where the fibers meet the retaining wall, suggesting involvement of this protein in polysaccharide fiber formation. Here we use peptide mass fingerprinting and bioinformatics to identify this protein called polysaccharide associated protein (PSAP). PSAP does not show similarity to any protein in databases, but contains several Proline-rich domains. Staining studies confirm PSAP as a glycoprotein, and mass spectrometry analysis identified ten N-linked glycosylation sites comprising seven different glycans containing mainly mannose and N-acetylglucosamine. Three of these glycans also contain fucose, with one of these glycans being unusual since it also contains arabinose. Additionally, four hydroxyproline residues have short O-linked glycans of mainly arabinose and galactose, with one also containing a 6-deoxyhexose. PSAP secretion and localization to shell material is confirmed using western blot analysis and microscopy. These studies indicate PSAP contains unique glycans and suggest its involvement in ECM polysaccharide fiber biosynthesis.

Bioinformatic Identification and Analysis of Cell Wall Extensins in ThreeArabidopsisSpecies

Premise of research. Extensins (EXTs) are members of the cell wall hydroxyproline-rich glycoprotein superfamily and play important roles in plant growth, development, and defense. Bioinformatic analysis has identified EXTs for a number of plant species, including the model plant Arabidopsis thaliana, but none of these studies examined plants from the same genus.Methodology. Here we report on the bioinformatic identification and analysis of EXTs in Arabidopsis lyrata and Arabidopsis halleri, two of the closest relatives of A. thaliana, based on amino acid motif searches using the BIO OHIO 2.0 program. Phylogenetic trees for the various EXT subclasses were constructed using both maximum likelihood and maximum parsimony methods. Homologous proteins were revealed using the OrthoMCL program.Pivotal results. The total numbers and subclasses of EXTs were similar in all three species. In A. lyrata, 61 EXTs were identified, including 15 classical EXTs, 10 short EXTs, eight leucine-rich repeat EXTs (LRXs), 14 proli...

Systemic protection against pearl millet downy mildew disease induced by cell wall glucan elicitors from Trichoderma hamatum UOM 13

Abstract The obligate oomycete Sclerospora graminicola (Sacc.) Schroet, is the incitant of downy mildew disease, which is the main constraint in pearl millet production worldwide. Different elicitors from Trichoderma hamatum UOM 13, e.g. mycelial extract and cell wall glucans, were assessed for their resistance elicitation efficiency and the possible underlying mechanisms. Both mycelial extract and cell wall glucans of T. hamatum UOM 13 positively influenced seed quality parameters of pearl millet, significantly enhanced seed germination and seedling vigor in comparison to the untreated control. Seed priming with cell wall glucan elicitors of T. hamatum UOM 13 suppressed downy mildew on susceptible pearl millet seedlings under greenhouse conditions by induction of systemic host resistance. Of the different elicitor delivery methods tested, transplant root dip was more effective than seed treatment and foliar spray. A combination of transplant root dip + seed treatment + foliar spray was significantly more effective than the single delivery methods. The induced resistance corresponded to up regulation of genes of important defense proteins upon pathogen inoculation. Transcripts of genes of defense enzymes glucanase, phenylalanine ammonia lyase, peroxidase and polyphenoloxidase were significantly increased due to the T. hamatum UOM elicitor effect. Expression of hydroxyproline-rich glycoprotein genes, known to play an important role in cell wall cross-linking, were also up regulated in response to T. hamatum UOM cell wall glucan treatment. This study emphasizes the role of T. hamatum UOM as a potential elicitor of downy mildew resistance in pearl millet and presents novel insights into the involvement of important defense proteins mediating such as resistance trigger.

Proteomic profiling of the weed feverfew, a neglected pollen allergen source

Feverfew (Parthenium hysterophorus), an invasive weed from the Asteraceae family, has been reported as allergen source. Despite its relevance, knowledge of allergens is restricted to a partial sequence of a hydroxyproline-rich glycoprotein. We aimed to obtain the entire sequence for recombinant production and characterize feverfew pollen using proteomics and immunological assays. Par h 1, a defensin-proline fusion allergen was obtained by cDNA cloning and recombinantly produced in E. coli. Using two complementary proteomic strategies, a total of 258 proteins were identified in feverfew pollen among those 47 proteins belonging to allergenic families. Feverfew sensitized patients’ sera from India revealed IgE reactivity with a pectate lyase, PR-1 protein and thioredoxin in immonoblot. In ELISA, recombinant Par h 1 was recognized by 60 and 40% of Austrian and Indian sera, respectively. Inhibition assays demonstrated the presence of IgE cross-reactive Par h 1, pectate lyase, lipid-transfer protein, profilin and polcalcin in feverfew pollen. This study reveals significant data on the allergenic composition of feverfew pollen and makes recombinant Par h 1 available for cross-reactivity studies. Feverfew might become a global player in weed pollen allergy and inclusion of standardized extracts in routine allergy diagnosis is suggested in exposed populations.

Effects of biological and physical properties of microalgae on disruption induced by a low-frequency ultrasound

Ultrasonication has drawn an increasing attention as one of cell disruption methods for extracting cellular compounds or controlling algal blooms. However, the effects of biological and physical properties of microalgae on cell disruption were not well understood. In this work, cell disruption of six microalgae, namely, Chlamydomonas reinhardtii, Chlorella pyrenoidosa, Microcystis aeruginosa (three strains: PCC 7806, FACHB 469, and FACHB 1343), and Synechococcus elongatus, was compared mutually based on their characteristics induced by a low-frequency ultrasound (35 kHz, 0.043 W mL−1). Results showed that the most sensitive strain was C. reinhardtii which has a hydroxyproline-rich-glycoproteins cell wall and a larger cell size (normally 10 μm in diameter). More than 80% of the cells of C. reinhardtii were ruptured after sonication for 5 min. In comparison, C. pyrenoidosa, a cellulose-rich-wall algal species with a medium size of 4–6 μm, and M. aeruginosa FACHB 1343, a peptidoglycan-wall species with a smaller average size of 2.3 μm, were highly resistant to ultrasound. Only 7.5 and 7.7% of cell disruption were achieved for C. pyrenoidosa and M. aeruginosa FACHB 1343, respectively, when they were sonicated for 60 min. Declumping effect was dominant in these strains. This suggested that cellulose-rich-wall algal species might be much more resistant than hydroxyproline-rich glycoproteins, and peptidoglycan-wall species to sonication. It also revealed that the larger cell size was more susceptible to sonication the cell would be. This research provides useful insights into choosing the low-cost microalgae for extraction or controlling specific microalgal blooms in water systems using ultrasound.

Changes in quality and defense resistance of kiwifruit in response to nitric oxide treatment during storage at room temperature

The effects of nitric oxide (NO) treatment on quality and disease resistance of kiwifruit (Actinidia deliciosa) cv. ‘Bruno’ were investigated after the fruits were immersed in 0.2mM sodium nitroprusside or water (as control) for 10min and then stored at room temperature. NO treatment reduced diseases incidence; delayed the increase in soluble solid content and the loss of vitamin C (Vc); increased the activities phenylalanine ammonialyase (PAL), peroxidase (POD), and β-1,3-glucanase, with accompanying up-regulation of PAL, POD and chitinase (CHT) expression; and elevated the level of total phenolics, flavonoids, hydroxyproline-rich glycoprotein, and lignin during storage. Thus, NO treatment was beneficial to the maintenance of fruit quality and improvement of disease resistance of kiwifruit during storage.

Insights into the Evolution of Hydroxyproline-Rich Glycoproteins from 1000 Plant Transcriptomes.

The carbohydrate-rich cell walls of land plants and algae have been the focus of much interest given the value of cell wall-based products to our current and future economies. Hydroxyproline-rich glycoproteins (HRGPs), a major group of wall glycoproteins, play important roles in plant growth and development, yet little is known about how they have evolved in parallel with the polysaccharide components of walls. We investigate the origins and evolution of the HRGP superfamily, which is commonly divided into three major multigene families: the arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins. Using motif and amino acid bias, a newly developed bioinformatics pipeline, we identified HRGPs in sequences from the 1000 Plants transcriptome project (www.onekp.com). Our analyses provide new insights into the evolution of HRGPs across major evolutionary milestones, including the transition to land and the early radiation of angiosperms. Significantly, data mining reveals the origin of glycosylphosphatidylinositol (GPI)-anchored AGPs in green algae and a 3- to 4-fold increase in GPI-AGPs in liverworts and mosses. The first detection of cross-linking (CL)-EXTs is observed in bryophytes, which suggests that CL-EXTs arose though the juxtaposition of preexisting SPn EXT glycomotifs with refined Y-based motifs. We also detected the loss of CL-EXT in a few lineages, including the grass family (Poaceae), that have a cell wall composition distinct from other monocots and eudicots. A key challenge in HRGP research is tracking individual HRGPs throughout evolution. Using the 1000 Plants output, we were able to find putative orthologs of Arabidopsis pollen-specific GPI-AGPs in basal eudicots.

Pipeline to Identify Hydroxyproline-Rich Glycoproteins.

Intrinsically disordered proteins (IDPs) are functional proteins that lack a well-defined three-dimensional structure. The study of IDPs is a rapidly growing area as the crucial biological functions of more of these proteins are uncovered. In plants, IDPs are implicated in plant stress responses, signaling, and regulatory processes. A superfamily of cell wall proteins, the hydroxyproline-rich glycoproteins (HRGPs), have characteristic features of IDPs. Their protein backbones are rich in the disordering amino acid proline, they contain repeated sequence motifs and extensive posttranslational modifications (glycosylation), and they have been implicated in many biological functions. HRGPs are evolutionarily ancient, having been isolated from the protein-rich walls of chlorophyte algae to the cellulose-rich walls of embryophytes. Examination of HRGPs in a range of plant species should provide valuable insights into how they have evolved. Commonly divided into the arabinogalactan proteins, extensins, and proline-rich proteins, in reality, a continuum of structures exists within this diverse and heterogenous superfamily. An inability to accurately classify HRGPs leads to inconsistent gene ontologies limiting the identification of HRGP classes in existing and emerging omics data sets. We present a novel and robust motif and amino acid bias (MAAB) bioinformatics pipeline to classify HRGPs into 23 descriptive subclasses. Validation of MAAB was achieved using available genomic resources and then applied to the 1000 Plants transcriptome project (www.onekp.com) data set. Significant improvement in the detection of HRGPs using multiple-k-mer transcriptome assembly methodology was observed. The MAAB pipeline is readily adaptable and can be modified to optimize the recovery of IDPs from other organisms.

Bioinformatic Identification and Analysis of Hydroxyproline-Rich Glycoproteins in Populus trichocarpa.

BackgroundHydroxyproline-rich glycoproteins (HRGPs) constitute a plant cell wall protein superfamily that functions in diverse aspects of growth and development. This superfamily contains three members: the highly glycosylated arabinogalactan-proteins (AGPs), the moderately glycosylated extensins (EXTs), and the lightly glycosylated proline-rich proteins (PRPs). Chimeric and hybrid HRGPs, however, also exist. A bioinformatics approach is employed here to identify and classify AGPs, EXTs, PRPs, chimeric HRGPs, and hybrid HRGPs from the proteins predicted by the completed genome sequence of poplar (Populus trichocarpa). This bioinformatics approach is based on searching for biased amino acid compositions and for particular protein motifs associated with known HRGPs with a newly revised and improved BIO OHIO 2.0 program. Proteins detected by the program are subsequently analyzed to identify the following: 1) repeating amino acid sequences, 2) signal peptide sequences, 3) glycosylphosphatidylinositol lipid anchor addition sequences, and 4) similar HRGPs using the Basic Local Alignment Search Tool (BLAST).ResultsThe program was used to identify and classify 271 HRGPs from poplar including 162 AGPs, 60 EXTs, and 49 PRPs, which are each divided into various classes. This is in contrast to a previous analysis of the Arabidopsis proteome which identified 162 HRGPs consisting of 85 AGPs, 59 EXTs, and 18 PRPs. Poplar was observed to have fewer classical EXTs, to have more fasciclin-like AGPs, plastocyanin AGPs and AG peptides, and to contain a novel class of PRPs referred to as the proline-rich peptides.ConclusionsThe newly revised and improved BIO OHIO 2.0 bioinformatics program was used to identify and classify the inventory of HRGPs in poplar in order to facilitate and guide basic and applied research on plant cell walls. The newly identified poplar HRGPs can now be examined to determine their respective structural and functional roles, including their possible applications in the areas plant biofuel and natural products for medicinal or industrial uses. Additionally, other plants whose genomes are sequenced can now be examined in a similar way using this bioinformatics program which will provide insight to the evolution of the HRGP family in the plant kingdom.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0912-3) contains supplementary material, which is available to authorized users.

Heat stress affects the distribution of JIM8-labelled arabinogalactan proteins in pistils of Solanum lycopersicum cv Micro-Tom

Arabinogalactan-proteins are a family of highly glycosylated hydroxyproline-rich glycoproteins widely distributed in the plant kingdom and mainly located at the cell surface. Because of their great heterogeneity, abundance and ubiquitous localization, arabinogalactan-proteins are thought to play important and different roles in plant growth and development. Many evidences also indicate a role of arabinogalactan-proteins during reproduction as well as in response to stress conditions. In the present work, we investigated the distribution of arabinogalactan-proteins recognised by JIM8 antibody in pistils of Solanum lycopersicum cv Micro-Tom heat-stressed for 3 h at 42 °C at different developmental stages (5 and 10 days before anthesis). Our results indicate that high temperature strongly affects the distribution and content of arabinogalactan-proteins in stigma and ovule, particularly in samples heat-stressed 5 days before anthesis. In stigmas, cells exhibited an altered pattern of JIM8-labelled AGPs, dispersed and less abundant. In ovules, the embryo sac-surrounding cells showed a clear reduction in the content of JIM8-labelled arabinogalactan proteins. These evidences suggest that heat stress affects both content and distribution of AGPs. Considering the role of AGPs in plant reproduction, from the acquisition of stigmatic receptivity to pollen guidance into the ovule, we can suppose that high temperature affects all these processes through the alteration of AGPs.

Association mapping in Brassica napus (L.) accessions identifies a major QTL for blackleg disease resistance on chromosome A01

Blackleg disease caused by the fungus Leptosphaeria maculans is one of the most devastating diseases of Brassica napus. Association mapping was used to evaluate the response of 139 B. napus accessions originated from 16 countries with 4 growth habit types to inoculation with L. maculans isolates from pathogenicity group 4. All accessions were inoculated at the seedling stage and 37,346 single nucleotide polymorphism markers based upon genotyping-by-sequencing were used for analysis. One major QTL associated with the blackleg disease was identified on chromosome A01 at 9.66 Mbp and explains about 14.7 % of phenotypic variation (p value <2.2E−05). Orthologs of Arabidopsis thaliana hydroxyproline-rich glycoprotein family protein, acyl-CoA oxidase 1 and cysteine-/histidine-rich C1 domain family protein that are involved in plant defense mechanism were identified in this QTL region. At a lower significance (p value <0.00139), thirty-five additional markers were identified which are located on 13 other chromosomes. Stepwise regression identified that these markers belong to ten QTL regions and together explain 51.04 % of phenotypic variations. Additional twenty orthologs of A. thaliana disease resistance genes, transcription factors, genes in phytohormone pathway and signaling in plant defense pathway have been identified to be associated with the blackleg disease. Based on the haplotype at the most significant QTL, a total of 22 genotypes were evaluated in the greenhouse. As expected with the haplotype, we had 100 % success in predicting the phenotype. These markers could further be used for identification of phenotype that can be used in the breeding program.

Nanospherical arabinogalactan proteins are a key component of the high-strength adhesive secreted by English ivy

Over 130 y have passed since Charles Darwin first discovered that the adventitious roots of English ivy (Hedera helix) exude a yellowish mucilage that promotes the capacity of this plant to climb vertical surfaces. Unfortunately, little progress has been made in elucidating the adhesion mechanisms underlying this high-strength adhesive. In the previous studies, spherical nanoparticles were observed in the viscous exudate. Here we show that these nanoparticles are predominantly composed of arabinogalactan proteins (AGPs), a superfamily of hydroxyproline-rich glycoproteins present in the extracellular spaces of plant cells. The spheroidal shape of the AGP-rich ivy nanoparticles results in a low viscosity of the ivy adhesive, and thus a favorable wetting behavior on the surface of substrates. Meanwhile, calcium-driven electrostatic interactions among carboxyl groups of the AGPs and the pectic acids give rise to the cross-linking of the exuded adhesive substances, favor subsequent curing (hardening) via formation of an adhesive film, and eventually promote the generation of mechanical interlocking between the adventitious roots of English ivy and the surface of substrates. Inspired by these molecular events, a reconstructed ivy-mimetic adhesive composite was developed by integrating purified AGP-rich ivy nanoparticles with pectic polysaccharides and calcium ions. Information gained from the subsequent tensile tests, in turn, substantiated the proposed adhesion mechanisms underlying the ivy-derived adhesive. Given that AGPs and pectic polysaccharides are also observed in bioadhesives exuded by other climbing plants, the adhesion mechanisms revealed by English ivy may forward the progress toward understanding the general principles underlying diverse botanic adhesives.

Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis.

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions.

Biochemical Defenses Induced by Mycorrhizae Fungi Glomus Mosseae in Controlling Strawberry Fusarium Wilt.

The effect of VAM on reducing wilt caused by Fusarium oxysporum Schlecht. f.sp. fragariae Winks et Williams (FO) infection in strawberry and the possible mechanisms involved were investigated. Two key substance involved in disease defenses, lignin and hydroxyproline-rich glycoprotein were induced and formed in the cell wall of strawberry root, and the peak content of lignin and hydroxyproline-rich glycoprotein occurred on the 25th day (149.52mg/g) and on the 15th day (10.08 mg/g). The activity of protective enzymes SOD, POD and CAT inoculation with VAM significantly increased when compared with the control under both CK (natural growth) and inoculated with FO. The conductivity of VAM plus FO treatment was higher than the CK treatment, but significantly was lower than the FO treatment.

EXTENSIN18 is required for full male fertility as well as normal vegetative growth in Arabidopsis.

EXTENSINS (EXTs) are a 65-member subfamily of hydroxyproline-rich glycoproteins (HRGPs) of which 20 putatively form crosslinking networks in the cell wall. These 20 classical EXTs are involved at the start of new wall assembly as evidenced by a requirement for EXT3 during cytokinesis, and the ability of some EXTs to polymerize in vitro into dendritic patterns. EXT3 was previously shown to form pulcherosine (three Tyrosines) cross-links. Little direct data exists on the other 19 classical EXTs. Here, we describe the phenotypes of ext18 mutants and rescued progeny as well as associated expression profiles of all 20 classical EXT genes. We found that EXT18 is required for full male fertility, as well as for normal vegetative growth. EXT18 has potential to form crosslinking networks via di-iso-di-tyrosine (four Tyrosines) covalent bonds, and not via pulcherosine due to deficit of lone Tyrosines. This together with ext18 defective pollen grains and pollen tubes, and reduced plant size, suggests that EXT18-type EXTs are important contributors to wall integrity, in pollen and other rapidly extending walls. The data also show that a knockout of EXT18 had a pleiotropic affect on the expression of several EXTs, as did the reintroduction of the native EXT18 gene, thus supporting the thesis that transcription of groups of EXTs are co-regulated and work in different combinations to make distinctive inputs into wall assembly of different cell types. These insights contribute to basic knowledge of cell wall self-assembly in different cell types, and potentially enable biotechnological advances in biomass increase and plant fertility control.

Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth.

BackgroundLeucine-rich repeat extensins (LRXs) are extracellular proteins consisting of an N-terminal leucine-rich repeat (LRR) domain and a C-terminal extensin domain containing the typical features of this class of structural hydroxyproline-rich glycoproteins (HRGPs). The LRR domain is likely to bind an interaction partner, whereas the extensin domain has an anchoring function to insolubilize the protein in the cell wall. Based on the analysis of the root hair-expressed LRX1 and LRX2 of Arabidopsis thaliana, LRX proteins are important for cell wall development. The importance of LRX proteins in non-root hair cells and on the structural changes induced by mutations in LRX genes remains elusive.ResultsThe LRX gene family of Arabidopsis consists of eleven members, of which LRX3, LRX4, and LRX5 are expressed in aerial organs, such as leaves and stem. The importance of these LRX genes for plant development and particularly cell wall formation was investigated. Synergistic effects of mutations with gradually more severe growth retardation phenotypes in double and triple mutants suggest a similar function of the three genes. Analysis of cell wall composition revealed a number of changes to cell wall polysaccharides in the mutants.ConclusionsLRX3, LRX4, and LRX5, and most likely LRX proteins in general, are important for cell wall development. Due to the complexity of changes in cell wall structures in the lrx mutants, the exact function of LRX proteins remains to be determined. The increasingly strong growth-defect phenotypes in double and triple mutants suggests that the LRX proteins have similar functions and that they are important for proper plant development.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0548-8) contains supplementary material, which is available to authorized users.

In Silico Analysis of the Metabolic Potential and Niche Specialization of Candidate Phylum "Latescibacteria" (WS3).

The “Latescibacteria” (formerly WS3), member of the Fibrobacteres–Chlorobi–Bacteroidetes (FCB) superphylum, represents a ubiquitous candidate phylum found in terrestrial, aquatic, and marine ecosystems. Recently, single-cell amplified genomes (SAGs) representing the “Latescibacteria” were obtained from the anoxic monimolimnion layers of Sakinaw Lake (British Columbia, Canada), and anoxic sediments of a coastal lagoon (Etoliko lagoon, Western Greece). Here, we present a detailed in-silico analysis of the four SAGs to gain some insights on their metabolic potential and apparent ecological roles. Metabolic reconstruction suggests an anaerobic fermentative mode of metabolism, as well as the capability to degrade multiple polysaccharides and glycoproteins that represent integral components of green (Charophyta and Chlorophyta) and brown (Phaeophycaea) algae cell walls (pectin, alginate, ulvan, fucan, hydroxyproline-rich glycoproteins), storage molecules (starch and trehalose), and extracellular polymeric substances (EPSs). The analyzed SAGs also encode dedicated transporters for the uptake of produced sugars and amino acids/oligopeptides, as well as an extensive machinery for the catabolism of all transported sugars, including the production of a bacterial microcompartment (BMC) to sequester propionaldehyde, a toxic intermediate produced during fucose and rhamnose metabolism. Finally, genes for the formation of gas vesicles, flagella, type IV pili, and oxidative stress response were found, features that could aid in cellular association with algal detritus. Collectively, these results indicate that the analyzed “Latescibacteria” mediate the turnover of multiple complex organic polymers of algal origin that reach deeper anoxic/microoxic habitats in lakes and lagoons. The implications of such process on our understanding of niche specialization in microbial communities mediating organic carbon turnover in stratified water bodies are discussed.

Plant regeneration in leaf culture of Centaurium erythraea Rafn. Part 2: the role of arabinogalactan proteins

Arabinogalactan proteins (AGPs) are cell wall hydroxyproline-rich glycoproteins with diverse functions in plant growth and development. In this work the role of AGPs was comparatively studied in four developmental pathways that can be induced from centaury leaf explants—indirect somatic embryogenesis (ISE), indirect and direct shoot development (ISD and DSD) and direct root development (DRD). The addition of β-d-glucosyl Yariv (βGlcY) reagent, a synthetic phenylglycoside that specifically crosslinks AGPs, to the growth medium reduced the number of somatic embryos and adventitious buds formed per explant. The morphogenetic paths can be arranged in order of increasing sensitivity to βGlcY as: DRD (insensitive) < ISD < DSD ≪ ISE. The content of AGPs increased during all processes, but the accumulation of AGPs was the most rapid during ISE and when ISE and ISD occurred simultaneously. We have identified four centaury AGP transcripts, CeAGP1–CeAGP4, of which CeAGP1, 2 and 4 code for fasciclin-like AGPs. CeAGP3 is an AG peptide with conserved DUF1070 domain. Expression pattern of these genes indicated specific involvement of CeAGP1 in ISE and unspecific involvement of CeAGP3 in morphogenesis.