Systems identification and characterization of \u03b2-glucuronosyltransferase genes involved in arabinogalactan-protein biosynthesis in plant genomes

Significant changes have occurred in plant cell wall composition during evolution and diversification of tracheophytes. As the sister lineage to seed plants, knowledge on the cell wall of ferns is key to track evolutionary changes across tracheophytes and to understand seed plant-specific evolutionary innovations. Fern cell wall composition is not fully understood, including limited knowledge of glycoproteins such as the fern arabinogalactan proteins (AGPs). Here, we characterize the AGPs from the leptosporangiate fern genera Azolla, Salvinia, and Ceratopteris. The carbohydrate moiety of seed plant AGPs consists of a galactan backbone including mainly 1,3- and 1,3,6-linked pyranosidic galactose, which is conserved across the investigated fern AGPs. Yet, unlike AGPs of angiosperms, those of ferns contained the unusual sugar 3-O-methylrhamnose. Besides terminal furanosidic arabinose, Ara (Araf), the main linkage type of Araf in the ferns was 1,2-linked Araf, whereas in seed plants 1,5-linked Araf is often dominating. Antibodies directed against carbohydrate epitopes of AGPs supported the structural differences between AGPs of ferns and seed plants. Comparison of AGP linkage types across the streptophyte lineage showed that angiosperms have rather conserved monosaccharide linkage types; by contrast bryophytes, ferns, and gymnosperms showed more variability. Phylogenetic analyses of glycosyltransferases involved in AGP biosynthesis and bioinformatic search for AGP protein backbones revealed a versatile genetic toolkit for AGP complexity in ferns. Our data reveal important differences across AGP diversity of which the functional significance is unknown. This diversity sheds light on the evolution of the hallmark feature of tracheophytes: their elaborate cell walls.

Arabinogalactan-proteins (AGPs) are a large, complex, and highly diverse class of heavily glycosylated proteins that belong to the family of cell wall hydroxyproline-rich glycoproteins. Approximately 90% of the molecules consist of arabinogalactan polysaccharides, which are composed of arabinose and galactose as major sugars and minor sugars such as glucuronic acid, fucose, and rhamnose. About half of the AGP family members contain a glycosylphosphatidylinositol (GPI) lipid anchor, which allows for an association with the outer leaflet of the plasma membrane. The mysterious AGP family has captivated the attention of plant biologists for several decades. This diverse family of glycoproteins is widely distributed in the plant kingdom, including many algae, where they play fundamental roles in growth and development processes. The journey of AGP biosynthesis begins with the assembly of amino acids into peptide chains of proteins. An N-terminal signal peptide directs AGPs toward the endoplasmic reticulum, where proline hydroxylation occurs and a GPI anchor may be added. GPI-anchored AGPs, as well as unanchored AGPs, are then transferred to the Golgi apparatus, where extensive glycosylation occurs by the action of a variety glycosyltransferase enzymes. Following glycosylation, AGPs are transported by secretory vesicles to the cell wall or to the extracellular face of the plasma membrane (in the case of GPI-anchored AGPs). GPI-anchored proteins can be released from the plasma membrane into the cell wall by phospholipases. In this review, we present an overview of the accumulated knowledge on AGP biosynthesis over the past three decades. Particular emphasis is placed on the glycosylation of AGPs as the sugar moiety is essential to their function. Recent genetics and genomics approaches have significantly contributed to a broader knowledge of AGP biosynthesis. However, many questions remain to be elucidated in the decades ahead.

The aim of this report is to provide general information on the molecular structure and synthesis of arabinogalactan proteins (AGPs) in association to their physiological significance. Assessment of genetic modifications of the activity of enzymes involved in the AGP biosynthesis is an efficient tool to study AGP functions. Thus, P4H (prolyl 4 hydroxylase) mutants, GLCAT (β-glucuronosyltransferase) mutants, and GH43 (glycoside hydrolase family 43) mutants have been described. We focused on the overview of AGPs modifications observed at the molecular, cellular, and organ levels. Inhibition of the hydroxylation process results in an increase in the intensity of cell divisions and thus, has an impact on root system length and leaf area. In turn, overexpression of P4H genes stimulates the density of root hairs. A mutation in GLCAT genes responsible for the transfer of glucuronic acid to the AGP molecule revealed that the reduction of GlcA in AGP disrupts the substantial assembly of the primary cell wall. Furthermore, silencing of genes encoding GH43, which has the ability to hydrolyze the AGP glycan by removing incorrectly synthesized β-1,3-galactans, induces changes in the abundance of other cell wall constituents, which finally leads to root growth defects. This information provides insight into AGPs as a crucial players in the structural interactions present in the plant extracellular matrix.

Arabinogalactan proteins are proteoglycans found on the cell surface and in the cell walls of higher plants. The carbohydrate moieties of most arabinogalactan proteins are composed of β-1,3-galactan main chains and β-1,6-galactan side chains, to which other auxiliary sugars are attached. For the present study, an endo-β-1,3-galactanase, designated FvEn3GAL, was first purified and cloned from winter mushroom Flammulina velutipes. The enzyme specifically hydrolyzed β-1,3-galactan, but did not act on β-1,3-glucan, β-1,3:1,4-glucan, xyloglucan, and agarose. It released various β-1,3-galactooligosaccharides together with Gal from β-1,3-galactohexaose in the early phase of the reaction, demonstrating that it acts on β-1,3-galactan in an endo-fashion. Phylogenetic analysis revealed that FvEn3GAL is member of a novel subgroup distinct from known glycoside hydrolases such as endo-β-1,3-glucanase and endo-β-1,3:1,4-glucanase in glycoside hydrolase family 16. Point mutations replacing the putative catalytic Glu residues conserved for enzymes in this family with Asp abolished activity. These results indicate that FvEn3GAL is a highly specific glycoside hydrolase 16 endo-β-1,3-galactanase.

BackgroundThe cell wall and chloroplast are two fundamental structures determining plant mechanical strength and grain yield. Therefore, understanding mechanisms that improve plants’ ability to develop a robust cell wall and well-developed chloroplast is of utmost importance for agricultural activities.ResultsIn this study, we report the functional characterization of a novel rice mutant, brittle stem and zebra leaf (bz1), which displays altered cell wall composition and collapsed chloroplast membrane. Molecular and biochemical analysis revealed that BZ1 encodes a functional UDP-galactose/glucose epimerase (UGE) and is ubiquitously expressed with higher expression in stem and leaf tissues. Multiple techniques analyses, including immunoblots, immuno-gold, and cryogenic scanning electron microscopy, demonstrated a significantly impaired glycosylation of arabinogalactan proteins (AGPs) and disordered cellulose microfibril deposition in bz1. Lipid profiling assay showed that the amount of monogalactosyldiacylglycerols (MGDG), a major chloroplast membrane glycolipid, was significantly decreased in bz1. Taken together, these results strongly demonstrate that BZ1 participates in UDP-galactose supply for the sugar chains biosynthesis of AGPs and MGDG, which thereby, respectively, results in altered cell wall and abnormal chloroplast development. Due to inferior mechanical strength and reduced photosynthesis, bz1 plants displayed detrimental agronomic traits, whereas BZ1 overexpressing lines showed enhanced plant growth. Transcriptome analysis of stems and leaves further showed that numerous key genes involved in AGPs biosynthesis and photosynthesis metabolism were substantially suppressed in bz1.ConclusionsOur finding identifies BZ1 as a dual-targeting UGE protein for glycosylation of AGPs and MGDG and suggests a strategy for breeding robust elite crops.

Streptophyte algae are the closest relatives to land plants; their latest common ancestor performed the most drastic adaptation in plant evolution around 500 million years ago: the conquest of land. Besides other adaptations, this step required changes in cell wall composition. Current knowledge on the cell walls of streptophyte algae and especially on the presence of arabinogalactan-proteins (AGPs), important signalling molecules in all land plants, is limited. To get deeper insights into the cell walls of streptophyte algae, especially in Charophyceae, we performed sequential cell wall extractions of four Chara species. The three species Chara globularis, Chara subspinosa and Chara tomentosa revealed comparable cell wall compositions, with pectins, xylans and xyloglucans, whereas Chara aspera stood out with higher amounts of uronic acids in the pectic fractions and lack of reactivity with antibodies binding to xylan- and xyloglucan epitopes. Search for AGPs in the four Chara species and in Nitellopsis obtusa revealed the presence of galactans with pyranosidic galactose in 1,3-, 1,6- and 1,3,6-linkage, which are typical galactan motifs in land plant AGPs. A unique feature of these branched galactans was high portions of 3-O-methylgalactose. Only Nitellopsis contained substantial amounts of arabinose A bioinformatic search for prolyl-4-hydroxylases, involved in the biosynthesis of AGPs, revealed one possible functional sequence in the genome of Chara braunii, but no hydroxyproline could be detected in the four Chara species or in Nitellopsis obtusa. We conclude that AGPs that is typical for land plants are absent, at least in these members of the Charophyceae.

UDP-glucose dehydrogenase (UGD) plays a key role in the nucleotide sugar biosynthetic pathway, as its product UDP-glucuronic acid is the common precursor for arabinose, xylose, galacturonic acid, and apiose residues found in the cell wall. In this study we characterize an Arabidopsis thaliana double mutant ugd2,3 that lacks two of the four UGD isoforms. This mutant was obtained from a cross of ugd2 and ugd3 single mutants, which do not show phenotypical differences compared with the WT. In contrast, ugd2,3 has a strong dwarfed phenotype and often develops seedlings with severe root defects suggesting that the UGD2 and UGD3 isoforms act in concert. Differences in its cell wall composition in comparison to the WT were determined using biochemical methods indicating a significant reduction in arabinose, xylose, apiose, and galacturonic acid residues. Xyloglucan is less substituted with xylose, and pectins have a reduced amount of arabinan side chains. In particular, the amount of the apiose containing side chains A and B of rhamnogalacturonan II is strongly reduced, resulting in a swollen cell wall. The alternative pathway to UDP-glucuronic acid with the key enzyme myo-inositol oxygenase is not up-regulated in ugd2,3. The pathway also does not complement the ugd2,3 mutation, likely because the supply of myo-inositol is limited. Taken together, the presented data underline the importance of UDP GlcA for plant primary cell wall formation.

Plant type II arabinogalactan (AG) polysaccharides are attached to arabinogalactan proteins (AGPs) at hydroxyproline residues, and they are very diverse and heterogeneous structures. The AG consists of a β-(1→3)-linked galactan backbone with β-(1→6)-galactan side chains that are modified mainly with arabinose, but they may also contain glucuronic acid, rhamnose or other sugars. Here, we studied the positions of fucose substitutions in AGPs, and we investigated the functions of this fucosylation. Monosaccharide analysis of Arabidopsis leaf AGP extracts revealed a significant reduction in L-Fucose content in the fut4 mutant, but not in the fut6 mutant. In addition, Fucose was reduced in the fut4 mutant in root AGP extracts and was absent in the fut4/fut6 mutant. Curiously, in all cases reduction of fucose was accompanied with a reduction in xylose levels. The fucosylated AGP structures in leaves and roots in wild type and fut mutant plants were characterised by sequential digestion with AG specific enzymes, analysis by Polysaccharide Analysis using Carbohydrate gel Electrophoresis, and Matrix Assisted Laser Desorption/Ionisation (MALDI)-Time of Flight Mass spectrometry (MS). We found that FUT4 is solely responsible for the fucosylation of AGPs in leaves. The Arabidopsis thaliana FUT4 and FUT6 genes have been previously proposed to be non-redundant AG-specific fucosyltransferases. Unexpectedly, FUT4 and FUT6 enzymes both fucosylate the same AGP structures in roots, suggesting partial redundancy to each other. Detailed structural characterisation of root AGPs with high energy MALDI-Collision Induced Dissociation MS and NMR revealed an abundant unique AG oligosaccharide structure consisting of terminal xylose attached to fucose. The loss of this structure in fut4/fut6 mutants explains the reduction of both fucose and xylose in AGP extracts. Under salt-stress growth conditions the fut4/fut6 mutant lacking AGP fucosylation exhibited a shorter root phenotype than wild type plants, implicating fucosylation of AGPs in maintaining proper cell expansion under these conditions.

The placenta of hornworts is unique among bryophytes in the restriction of transfer cells that are characterized by elaborate wall labyrinths to the gametophyte generation. During development, cells around the periphery of the sporophyte foot elongate, forming smooth-walled haustorial cells that interdigitate with gametophyte cells. Using immunogold labeling with 22 antibodies to diverse cell wall polymers, we examined compositional differences in the developmentally and morphologically distinct cell walls of gametophyte transfer cells and sporophyte haustorial cells in the placenta of Phaeoceros. As detected by Calcofluor White fluorescence, cellulose forms the cell wall scaffolding in cells on both sides of the placenta. Homogalacturonan (HG) and rhamnogalacturonan I (RG-I) pectins are abundant in both cell types, and haustrorial cells are further enriched in methyl-esterified HGs. The abundance of pectins in placental cell walls is consistent with the postulated roles of these polymers in cell wall porosity and in maintaining an acidic apoplastic pH favorable to solute transport. Xyloglucan hemicellulose, but not mannans or glucuronoxylans, are present in cell walls at the interface between the two generations with a lower density in gametophytic wall ingrowths. Arabinogalactan proteins (AGPs) are diverse along the plasmalemma of placental cells and are absent in surrounding cells in both generations. AGPs in placental cell walls may play a role in calcium binding and release associated with signal transduction as has been speculated for these glycoproteins in other plants. Callose is restricted to thin areas in cell walls of gametophyte transfer cells. In contrast to studies of transfer cells in other systems, no reaction to the JIM12 antibody against extensin was observed in Phaeoceros.

Somatic embryogenesis is a reliable system for in vitro plant regeneration, with biotechnological applications in trees, but the regulating mechanisms are largely unknown. Changes in cell wall mechanics controlled by methylesterification of pectins, mediated by pectin methylesterases (PMEs) and pectin methyl esterase inhibitors (PMEIs) underlie many developmental processes. Arabinogalactan proteins (AGPs) are highly glycosylated proteins located at the surface of plasma membranes, in cell walls, and in extracellular secretions, with key roles in a range of different processes. In this study, we have investigated changes in two cell wall components, pectins and AGPs, during somatic embryogenesis in Quercus suber, a forest tree of high economic and ecologic value. At early embryogenesis stages, cells of proembryogenic masses showed high levels of esterified pectins and expression of QsPME and QsPMEI genes encoding a PME and a putative PMEI, respectively. At advanced stages, differentiating cells of heart, torpedo and cotyledonary embryos exhibited walls rich in de-esterified pectins, while QsPME gene expression and PME activity progressively increased. AGPs were detected in cell walls of proembryogenic masses and somatic embryos. QsLys-rich-AGP18, QsLys-rich-AGP17, and QsAGP16L1 gene expression increased with embryogenesis progression, as did the level of total AGPs, detected by dot blot with β-glucosyl Yariv reagent. Immuno dot blot, immunofluorescence assays and confocal analysis using monoclonal antibodies to high- (JIM7, LM20) and low- (JIM5, LM19) methylesterified pectins, and to certain AGP epitopes (LM6, LM2) showed changes in the amount and distribution pattern of esterified/de-esterified pectins and AGP epitopes, that were associated with proliferation and differentiation and correlated with expression of the PME and AGP genes analyzed. Pharmacological treatments with catechin, an inhibitor of PME activity, and Yariv reagent, which blocks AGPs, impaired the progression of embryogenesis, with pectin de-esterification and an increase in AGP levels being necessary for embryo development. Findings indicate a role for pectins and AGPs during somatic embryogenesis of cork oak, promoting the cell wall remodeling during the process. They also provide new insights into the regulating mechanisms of somatic embryogenesis in woody species, for which information is still scarce, opening up new possibilities to improve in vitro embryo production in tree breeding.

Arabinogalactan proteins (AGPs) are highly glycosylated cell wall proteins essential for plant growth and reproduction. AGPs are extensively decorated with arabinogalactan polysaccharides, composed primarily of arabinose and galactose, along with minor sugars such as glucuronic acid, fucose, and rhamnose. Their glycosylation patterns and glycosylphosphatidylinositol anchor enable interactions with receptors, modulating signal transduction pathways critical for reproduction. AGPs also associate with cell wall components like pectin and hemicellulose, impacting cellulose deposition and cell wall integrity. Recent research highlights AGPs' role as calcium (Ca2+) capacitors, regulating Ca2+ storage and release during crucial reproductive stages. Despite significant progress, their precise molecular mechanisms remain elusive. In this review, we explore the multifaceted roles of AGPs in plant reproduction, shedding light on the recent progress in their involvement in signalling pathways, cell wall interactions, and Ca2+ homeostasis, while highlighting the ongoing research needed to fully understand their mechanisms in reproductive success.

A variety of Southern African resurrection plants were surveyed using high-throughput cell wall profiling tools. Species evaluated were the dicotyledons, Myrothamnus flabellifolia and Craterostigma plantagineum; the monocotyledons, Xerophyta viscosa, Xerophyta schlecterii, Xerophyta humilis and the resurrection grass Eragrostis nindensis, as well as a pteridophyte, the resurrection fern, Mohria caffrorum. Comparisons were made between hydrated and desiccated leaf and frond material, with respect to cell wall composition and polymer abundance, using monosaccharide composition analysis, FT-IR spectroscopy and comprehensive microarray polymer profiling in combination with multivariate data analysis. The data obtained suggest that three main functional strategies appear to have evolved to prepare plant cell walls for desiccation. Arabinan-rich pectin and arabinogalactan proteins are found in the resurrection fern M. caffrorum and the basal angiosperm M. flabellifolia where they appear to act as 'pectic plasticizers'. Dicotyledons with pectin-rich walls, such as C. plantagineum, seem to use inducible mechanisms which consist of up-regulating wall proteins and osmoprotectants. The hemicellulose-rich walls of the grass-like Xerophyta spp. and the resurrection grass E. nindensis were found to contain highly arabinosylated xylans and arabinogalactan proteins. These data support a general mechanism of 'plasticising' the cell walls of resurrection plants to desiccation and implicate arabinose-rich polymers (pectin-arabinans, arabinogalactan proteins and arabinoxylans) as the major contributors in ensuring flexibility is maintained and rehydration is facilitated in these plants.

Plant cell wall proteins play major roles during plant development and in response to environmental cues. A bioinformatic search for functional domains has allowed identifying the PAC domain (Proline-rich, Arabinogalactan proteins, conserved Cysteines) in several proteins (PDPs) identified in cell wall proteomes. This domain is assumed to interact with pectic polysaccharides and O-glycans and to contribute to non-covalent molecular scaffolds facilitating the remodeling of polysaccharidic networks during rapid cell expansion. In this work, the characteristics of the PAC domain are described in detail, including six conserved Cys residues, their spacing, and the predicted secondary structures. Modeling has been performed based on the crystal structure of a Plantago lanceolata PAC domain. The presence of β-sheets is assumed to ensure the correct folding of the PAC domain as a β-barrel with loop regions. We show that PDPs are present in early divergent organisms from the green lineage and in all land plants. PAC domains are associated with other types of domains: Histidine-rich, extensin, Proline-rich, or yet uncharacterized. The earliest divergent organisms having PDPs are Bryophytes. Like the complexity of the cell walls, the number and complexity of PDPs steadily increase during the evolution of the green lineage. The association of PAC domains with other domains suggests a neo-functionalization and different types of interactions with cell wall polymers

Small molecule fluorescent probes that bind selectively to plant cell wall polysaccharides have been instrumental in elucidating the localization and function of these glycans. Arabinogalactan proteins (AGPs) are cell wall proteoglycans implicated in essential functions such as cell signaling, plant growth, and programmed cell death. There is currently no small molecule probe capable of fluorescently labeling AGPs. The Yariv reagents are the only small molecules that bind AGPs, and have been used to study AGP function and isolate AGPs via precipitation of an AGP-Yariv complex. However, the Yariv reagents are not fluorescent, rendering them ineffective for localization studies using fluorescence microscopy. A fluorescent version of a Yariv reagent that is capable of both binding as well as imaging AGPs would provide a powerful tool for studying AGPs in planta. Herein, we describe the synthesis of an azido analog of the Yariv reagent that can be further functionalized with a fluorophore to provide a glycoconjugate that binds AGPs and is fluorescent. We show that the modified reagent binds gum arabic in in vitro binding assays when used in conjunction with the βGlcYariv reagent. Fluorescent imaging of AGPs in fixed maize leaf tissue enables localization of AGPs to cell walls in the leaf. Significantly, imaging can also be carried out using fresh tissue. This represents the first small molecule probe that can be used to visualize AGPs using fluorescence microscopy.

Arabinogalactan proteins (AGPs), present in cell walls, plasma membranes and extracellular secretions, are massively glycosylated hydroxyproline-rich proteins that play a key role in several plant developmental processes. After stress treatment, microspores cultured in vitro can reprogramme and change their gametophytic developmental pathways towards embryogenesis, thereby producing embryos which can further give rise to haploid and double haploid plants, important biotechnological tools in plant breeding. Microspore embryogenesis constitutes a convenient system for studying the mechanisms underlying cell reprogramming and embryo formation. In this work, the dynamics of both AGP presence and distribution were studied during pollen development and microspore embryogenesis in Brassica napus, by employing a multidisciplinary approach using monoclonal antibodies for AGPs (LM2, LM6, JIM13, JIM14, MAC207) and analysing the expression pattern of the BnAGP Sta 39-4 gene. Results showed the developmental regulation and defined localization of the studied AGP epitopes during the two microspore developmental pathways, revealing different distribution patterns for AGPs with different antigenic reactivity. AGPs recognized by JIM13, JIM14 and MAC207 antibodies were related to pollen maturation, whereas AGPs labelled by LM2 and LM6 were associated with embryo development. Interestingly, the AGPs labelled by JIM13 and JIM14 were induced with the change of microspore fate. Increases in the expression of the Sta 39-4 gene, JIM13 and JIM14 epitopes found specifically in 2-4 cell stage embryo cell walls, suggested that AGPs are early molecular markers of microspore embryogenesis. Later, LM2 and LM6 antigens increased progressively with embryo development and localized on cell walls and cytoplasmic spots, suggesting an active production and secretion of AGPs during in vitro embryo formation. These results give new insights into the involvement of AGPs as potential regulating/signalling molecules in microspore reprogramming and embryogenesis.