Pectic-AGP is a major form of Arabidopsis AGPs.

As a key component in cell walls of numerous organisms ranging from green algae to higher plants, AGPs play principal roles in many biological processes such as cell-cell adhesion and regulating Ca2+ signaling pathway as a Ca2+-capacitor. Consistently, AGP structures vary from species to species and from tissue to tissue. To understand the functions of AGPs, it is vital to know their structural differences relative to their location in the plant. Thus, AGPs were purified from different Arabidopsis tissues. Analyses of these AGPs demonstrated that the AGPs comprised covalently linked pectin and AGP, referred to as pectic-AGPs. Importantly, these pectic-AGPs were glycosylated with a remarkable variety of polysaccharides including homogalacturonan, rhamnogalacturonan-I, and type II arabinogalactan at different ratios and lengths. This result not only suggests that pectic-AGP is a major form of Arabidopsis AGPs, but also supports AGPs serve as crosslinkers covalently connecting pectins with structures tailored for tissue-specific functions.

Type II arabinogalactans initiated by hydroxyproline-O-galactosyltransferases play important roles in pollen-pistil interactions.

Arabinogalactan-proteins (AGPs) are hydroxyproline-rich glycoproteins containing a high sugar content and are widely distributed in the plant kingdom. AGPs have long been suggested to play important roles in sexual plant reproduction. The synthesis of their complex carbohydrates is initiated by a family of hydroxyproline galactosyltransferase (Hyp-GALT) enzymes which add the first galactose to Hyp residues in the protein backbone. Eight Hyp-GALT enzymes have been identified so far, and in the present work a mutant affecting five of these enzymes (galt2galt5galt7galt8galt9) was analyzed regarding the reproductive process. The galt25789 mutant presented a low seed set, and reciprocal crosses indicated a significant female gametophytic contribution to this mutant phenotype. Mutant ovules revealed abnormal callose accumulation inside the embryo sac and integument defects at the micropylar region culminating in defects in pollen tube reception. In addition, immunolocalization and biochemical analyses allowed the detection of a reduction in the amount of glucuronic acid in mutant ovary AGPs. Dramatically low amounts of high-molecular-weight Hyp-O-glycosides obtained following size exclusion chromatography of base-hydrolyzed mutant AGPs compared to the wild type indicated the presence of underglycosylated AGPs in the galt25789 mutant, while the monosaccharide composition of these Hyp-O-glycosides displayed no significant changes compared to the wild-type Hyp-O-glycosides. The present work demonstrates the functional importance of the carbohydrate moieties of AGPs in ovule development and pollen-pistil interactions.

The graft framework: Quantitative changes in cell wall matrix polysaccharides throughout the tomato graft union formation.

Plant cell walls provide essential functions in cell recognition, differentiation, adhesion and wound responses. Therefore, it is tempting to hypothesize that cell walls play a key role in grafting, but to date there are no quantitative studies targeting on cell wall changes during grafting. The aim of this work was to investigate the dynamics of pectic and hemicellulosic polysaccharides at the graft junctions in tomato homografts throughout the first 12 days after grafting. Cell wall fractionation, combined with ATR-FTIR spectroscopy and gas-chromatography, evidenced a marked increase in pectin content and a decrease in the degree of methyl-esterification of homogalacturonan in scion and rootstock throughout grafting. Also, recovery of tightly-bound hemicelluloses decreased at late times after grafting suggesting an increase of cross-linked hemicelluloses along grafting. In addition, immuno-dot assays revealed an increase in xyloglucan and arabinogalactan proteins in the first days after grafting, pointing to a presumed role in tissue adhesion-cohesion.

Glucuronidation of type II arabinogalactan polysaccharides function in sexual reproduction of Arabidopsis.

Arabinogalactan proteins (AGPs) are complex, hyperglycosylated plant cell wall proteins with little known about the biological roles of their glycan moieties in sexual reproduction. Here, we report that GLCAT14A, GLCAT14B, and GLCAT14C, three enzymes responsible for the addition of glucuronic acid residues to AGPs, function in pollen development, polytubey block, and normal embryo development in Arabidopsis. Using biochemical and immunolabeling techniques, we demonstrated that the loss of function of the GLCAT14A, GLCAT14B, and GLCAT14C genes resulted in disorganization of the reticulate structure of the exine wall, abnormal development of the intine layer, and collapse of pollen grains in glcat14a/b and glcat14a/b/c mutants. Synchronous development between locules within the same anther was also lost in some glcat14a/b/c stamens. In addition, we observed excessive attraction of pollen tubes targeting glcat14a/b/c ovules, indicating that the polytubey block mechanism was compromised. Monosaccharide composition analysis revealed significant reductions in all sugars in glcat14a/b and glcat14a/b/c mutants except for arabinose and galactose, while immunolabeling showed decreased amounts of AGP sugar epitopes recognized by glcat14a/b and glcat14a/b/c mutants compared with the wild type. This work demonstrates the important roles that AG glucuronidation plays in Arabidopsis sexual reproduction and reproductive development.

Gold Nanoparticles-Induced Modifications in Cell Wall Composition in Barley Roots.

The increased use of nanoparticles (NP) in different industries inevitably results in their release into the environment. In such conditions, plants come into direct contact with NP. Knowledge about the uptake of NP by plants and their effect on different developmental processes is still insufficient. Our studies concerned analyses of the changes in the chemical components of the cell walls of Hordeum vulgare L. roots that were grown in the presence of gold nanoparticles (AuNP). The analyses were performed using the immunohistological method and fluorescence microscopy. The obtained results indicate that AuNP with different surface charges affects the presence and distribution of selected pectic and arabinogalactan protein (AGP) epitopes in the walls of root cells.

Non-functional GoFLA19s are responsible for the male sterility caused by hybrid breakdown in cotton (Gossypium spp.).

Hybrid breakdown (HB) functions as a common reproductive barrier and reduces hybrid fitness in many species, including cotton. However, the related genes and the underlying genetic mechanisms of HB in cotton remain unknown. Here, we found that the photosensitive genetic male sterile line CCRI9106 was a hybrid progeny of Gossypium hirsutum and Gossypium barbadense and probably a product of HB. Fine mapping with F2 s (CCRI9106 × G. hirsutum/G. barbadense lines) identified a pair of male sterility genes GoFLA19s (encoding fasciclin-like arabinogalactan family protein) located on chromosomes A12 and D12. Crucial variations occurring in the fasciclin-like domain and the arabinogalactan protein domain were predicted to cause the non-functionalization of GbFLA19-D and GhFLA19-A. CRISPR/Cas9-mediated knockout assay confirmed the effects of GhFLA19s on male sterility. Sequence alignment analyses showed that variations in GbFLA19-D and GhFLA19-A likely occurred after the formation of allotetraploid cotton species. GoFLA19s are specifically expressed in anthers and contribute to tapetal development, exine assembly, intine formation, and pollen grain maturation. RNA-sequencing and quantitative reverse transcriptase-polymerase chain reaction analyses illustrated that genes related to these biological processes were significantly downregulated in the mutant. Our research on male sterility genes, GoFLA19s, improves the understanding of the molecular characteristics and evolutionary significance of HB in interspecific hybrid breeding.

BRITTLE PLANT1 is required for normal cell wall composition and mechanical strength in rice.

A series of nucleotide sugar interconversion enzymes (NSEs) generate the activated sugar donors required for biosynthesis of cell wall matrix polysaccharides and glycoproteins. UDP-glucose 4-epimerases (UGEs) are NSEs that function in the interconversion of UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal). The roles of UDP-glucose 4-epimerases in monocots remain unclear due to redundancy in the pathways. Here, we report a brittle plant (bp1) rice mutant that exhibits brittle leaves and culms at all growth stages. The mutant culms had reduced levels of rhamnogalacturonan I, homogalacturonan, and arabinogalactan proteins. Moreover, the mutant had altered contents of uronic acids, neutral noncellulosic monosaccharides, and cellulose. Map-based cloning demonstrated that OsBP1 encodes a UDP-glucose 4-epimerase (OsUGE2), a cytosolic protein. We also show that BP1 can form homo- and hetero-protein complexes with other UGE family members and with UDP-galactose transporters 2 (OsUGT2) and 3 (OsUGT3), which may facilitate the channeling of Gal to polysaccharides and proteoglycans. Our results demonstrate that BP1 participates in regulating the sugar composition and structure of rice cell walls.

Idioblasts and peltate hairs as distribution networks for water absorbed by xerophilous leaves.

Capparis odoratissima is a tree species native to semi-arid environments of South America where low soil water availability coexists with frequent night-time fog. A previous study showed that water applied to leaf surfaces enhanced leaf hydration, photosynthesis and growth, but the mechanisms of foliar water uptake are unknown. Here, we combine detailed anatomical evaluations with water and dye uptake experiments in the laboratory, and use immunolocalization of pectin and arabinogalactan protein epitopes to characterize water uptake pathways in leaves. Abaxially, the leaves of C. odoratissima are covered with peltate hairs, while the adaxial surfaces are glabrous. Both surfaces are able to absorb condensed water, but the abaxial surface has higher rates of water uptake. Thousands of idioblasts per cm2 , a higher density than stomata, connect the adaxial leaf surface and the abaxial peltate hairs, both of which contain hygroscopic substances such as arabinogalactan proteins and pectins. The highly specialized anatomy of the leaves of C odoratissima fulfils the dual function of minimizing water loss when stomata are closed, while maintaining the ability to absorb liquid water. Cell-wall related hygroscopic compounds in the peltate hairs and idioblasts create a network of microchannels that maintain leaf hydration and promote water uptake.

Immunodetection of Pectic Epitopes, Arabinogalactan Proteins, and Extensins in Mucilage Cells from the Ovules of Pilosella officinarum Vaill. and Taraxacum officinale Agg. (Asteraceae).

The main aim of this study was to compare the cytological difference between ovular mucilage cells in two Asteraceae species-Pilosella officinarum and Taraxacum officinale-in order to determine whether pectic epitopes, arabinogalactan proteins, or extensins are present. The immunocytochemical technique was used. Both the Taracacum and Pilosella genera have been used recently as models for understanding the mechanisms of apomixis. Knowledge of the presence of signal molecules (pectic epitopes, arabinogalactan proteins, and extensins) can help better understand the developmental processes in these plants during seed growth. The results showed that in Pilosella officinarum, there was an accumulation of pectins in the mucilage, including both weakly and highly esterified pectins, which was in contrast to the mucilage of Taraxacum officinale, which had low amounts of these pectins. However, Taraxacum protoplasts of mucilage cells were rich in weakly methyl-esterified pectins. While the mucilage contained arabinogalactan proteins in both of the studied species, the types of arabinogalactan proteins were different. In both of the studied species, extensins were recorded in the transmitting tissues. Arabinogalactan proteins as well as weakly and highly esterified pectins and extensins occurred in close proximity to calcium oxalate crystals in both Taraxacum and Pilosella cells.

Cell Wall Composition as a Marker of the Reprogramming of the Cell Fate on the Example of a Daucus carota (L.) Hypocotyl in Which Somatic Embryogenesis Was Induced.

Changes in the composition of the cell walls are postulated to accompany changes in the cell's fate. We check whether there is a relationship between the presence of selected pectic, arabinogalactan proteins (AGPs), and extensins epitopes and changes in cell reprogramming in order to answer the question of whether they can be markers accompanying changes of cell fate. Selected antibodies were used for spatio-temporal immunolocalization of wall components during the induction of somatic embryogenesis. Based on the obtained results, it can be concluded that (1) the LM6 (pectic), LM2 (AGPs) epitopes are positive markers, but the LM5, LM19 (pectic), JIM8, JIM13 (AGPs) epitopes are negative markers of cells reprogramming to the meristematic/pluripotent state; (2) the LM8 (pectic), JIM8, JIM13, LM2 (AGPs) and JIM11 (extensin) epitopes are positive markers, but LM6 (pectic) epitope is negative marker of cells undergoing detachment; (3) JIM4 (AGPs) is a positive marker, but LM5 (pectic), JIM8, JIM13, LM2 (AGPs) are negative markers for pericycle cells on the xylem pole; (4) LM19, LM20 (pectic), JIM13, LM2 (AGPs) are constitutive wall components, but LM6, LM8 (pectic), JIM4, JIM8, JIM16 (AGPs), JIM11, JIM12 and JIM20 (extensins) are not constitutive wall components; (5) the extensins do not contribute to the cell reprogramming.

Structural features in tension wood and distribution of wall polymers in the G-layer of in vitro grown poplars.

Under the effect of disturbances, like unbalanced stem, but also during normal development, poplar trees can develop a specific secondary xylem, called "tension wood" (TW), which is easily identifiable by the presence of a gelatinous layer in the secondary cell walls (SCW) of the xylem fibers. Since TW formation was mainly performed on 2-year-old poplar models, an in vitro poplar that produces gelatinous fibers (G-fibers) while offering the same experimental advantages as herbaceous plants has been developed. Using specific cell wall staining techniques, wood structural features and lignin/cellulose distribution were both detailed in cross-sections obtained from the curved stem part of in vitro poplars. A supposed delay in the SCW lignification process in the G-fibers, along with the presence of a G-layer, could be observed in the juvenile plants. Moreover, in this G-layer, the immunolabeling of various polymers carried out in the SCW of TW has allowed detecting crystalline cellulose, arabinogalactans proteins, and rhamnogalacturonans I; however, homogalacturonans, xylans, and xyloglucans could not be found. Interestingly, extensins were detected in this typical adaptative or stress-induced structure. These observations were corroborated by a quantitation of the immunorecognized polymer distribution using gold particle labeling. In conclusion, the in vitro poplar model seems highly convenient for TW studies focusing on the implementation of wall polymers that provide the cell wall with greater plasticity in adapting to the environment.

Arabinogalactan proteins mediate intercellular crosstalk in the ovule of apple flowers.

KEY MESSAGE: AGP-rich glycoproteins mediate pollen-ovule interactions and cell patterning in the embryo sac of apple before and after fertilization. Glycoproteins are significant players in the dialog that takes place between growing pollen tubes and the stigma and style in the angiosperms. Yet, information is scarce on their possible involvement in the ovule, a sporophytic organ that hosts the female gametophyte. Apple flowers have a prolonged lapse of time between pollination and fertilization, offering a great system to study the developmental basis of glycoprotein secretion and their putative role during the last stages of the progamic phase and early seed initiation. For this purpose, the sequential pollen tube elongation within the ovary was examined in relation to changes in arabinogalactan proteins (AGPs) in the tissues of the ovule before and after fertilization. To evaluate what of these changes are developmentally regulated, unpollinated and pollinated flowers were compared. AGPs paved the pollen tube pathway in the ovules along the micropylar canal, and the nucellus entrance toward the synergids, which also developmentally accumulated AGPs at the filiform apparatus. Glycoproteins vanished from all these tissues following pollen tube passage, strongly suggesting a role in pollen-ovule interaction. In addition, AGPs marked the primary cell walls of the haploid cells of the female gametophyte, and they further built up in the cell walls of the embryo sac and developing embryo, layering the interactive walls of the three generations hosted in the ovule, the maternal sporophytic tissues, the female gametophyte, and the developing embryo.

Analysis of AGP contribution to the dynamic assembly and mechanical properties of cell wall during pollen tube growth.

Arabinogalactan proteins as cell wall structural proteins are involved in fundamental processes during plant development and growth. The aim of this study was to evaluate AGP function in the distribution of pectin, cellulose and callose along Fragaria x ananassa pollen tube and to associate the cell wall structure with local mechanical properties. We used Yariv reagent which interacts with AGPs and allows the observation of the assembly of cell walls without AGPs performing their function. Cytochemical, immunofluorescence labelling and atomic force microscope have been used to characterize the changes in cell wall structure and stiffness. It was shown that disordering of the structure of AGP present in cell walls affects the localization of cellulose, pectins and the secretion of callose. Changes in cell wall assembly are relevant to pollen tube mechanical properties. The stiffness gradient lengthwise through the axis of the pollen tube has demonstrated a significantly higher Young's modulus of the shank region than the growth zone. It has been revealed that the apex of the pollen tube cultured in the presence of Yariv reagent is stiffer (1.68 MPa) than the corresponding region of the pollen tube grown under control conditions (0.13-0.27 MPa). AGP affects the structure of the cell wall by changing the distribution of other components and the modification of their localization, and hence it plays a significant role in the mechanical properties of the cell wall.

Comparative analysis of remodelling of the plant-microbe interface in Pisum sativum and Medicago truncatula symbiotic nodules.

Infection of host cells by nitrogen-fixing soil bacteria, known as rhizobia, involves the progressive remodelling of the plant-microbe interface. This process was examined by using monoclonal antibodies to study the subcellular localisation of pectins and arabinogalactan proteins (AGPs) in wild-type and ineffective nodules of Pisum sativum and Medicago truncatula. The highly methylesterified homogalacturonan (HG), detected by monoclonal antibody JIM7, showed a uniform localisation in the cell wall, regardless of the cell type in nodules of P. sativum and M. truncatula. Low methylesterified HG, recognised by JIM5, was detected mainly in the walls of infection threads in nodules of both species. The galactan side chain of rhamnogalacturonan I (RG-I), recognised by LM5, was present in the nodule meristem in both species and in the infection thread walls in P. sativum, but not in M. truncatula. The membrane-anchored AGP recognised by JIM1 was observed on the plasma membrane in nodules of P. sativum and M. truncatula. In P. sativum, the AGP epitope recognised by JIM1 was present on mature symbiosome membranes of wild-type nodules, but JIM1 labelling was absent from symbiosome membranes in the mutant Sprint-2Fix- (sym31) with undifferentiated bacteroids, suggesting a possible involvement of AGP in the maturation of symbiosomes. Thus, the common and species-specific traits of cell wall remodelling during nodule differentiation were demonstrated.

Arabinogalactan proteins: Distribution during the development of male and female gametophytes.

Arabinogalactan proteins (AGPs), i.e. a subfamily of hydroxyproline-rich proteins (HRGPs), are widely distributed in the plant kingdom. For many years, AGPs have been connected with the multiple phases of plant reproduction and developmental processes. Currently, extensive knowledge is available about their various functions, i.e. involvement in pollen grain formation, initiation of pollen grain germination, pollen tube guidance in the transmission tissue of pistil and ovule nucellus, and function as a signaling molecule during cell-cell communication. Although many studies have been performed, the mechanism of action, the heterogeneous molecule structure, and the connection with other extracellular matrix components have not been sufficiently explained. The aim of this work was to gather and describe the most important information on the distribution of AGPs in gametophyte development. The present review provides a summary of the first reports about AGPs and the most recent knowledge about their functions during male and female gametophyte formation.

Cell Wall Epitopes and Endoploidy as Reporters of Embryogenic Potential in Brachypodium Distachyon Callus Culture.

Effective regeneration of callus tissue into embryos and then into whole plants is essential for plant biotechnology. The embryonic potential is often low and can further decrease with time in culture, which limits the utilisation of calli for transformation procedures and in vitro propagation. In this study, we show that the loss of embryogenic potential in callus cultures of Brachypodium distachyon is progressive over time. Flow cytometry analyses indicated endoploidy levels increased in 60- and 90-day-old calli with effective loss of the 2C DNA content peak in the latter. Analysis of indolic compounds content revealed a decrease in 60- and 90-day-old calli compared to either freshly isolated explants or 30-day-old calli. Immunohistochemical analysis revealed a decrease in arabinogalactan proteins (AGP) signal with the time of culture, but extensin (EXT) epitopes either increased (JIM12 epitopes) or decreased (JIM11 epitopes). The transcript accumulation levels of AGPs and EXTs confirmed these results, with most of AGP and EXT transcripts gradually decreasing. Some chimeric EXT transcripts significantly increased on the 30th day of culture, perhaps because of an increased embryogenic potential. Selected somatic embryogenesis-related genes and cyclins demonstrated a gradual decrease of transcript accumulation for YUCCA (YUC), AINTEGUMENTA-LIKE (AIL), BABY BOOM (BBM), and CLAVATA (CLV3) genes, as well as for most of the cyclins, starting from the 30th day of culture. Notably, WUSCHEL (WUS) transcript was detectable only on the 30th and 60th day and was not detectable in the zygotic embryos and in 90-day-old calli.

Pollen wall development in mango (Mangifera indica L., Anacardiaceae).

The mango (Mangifera indica) is a woody perennial crop currently cultivated worldwide in regions with tropical and subtropical climates. Despite its importance, an essential process such as pollen development, and, specifically, cell wall composition that influences crosstalk between somatic cells and the male germline, is still poorly understood in this species and in the Anacardiaceae as a whole. A detailed understanding of this process is particularly important to know the effect of low temperatures during flowering on pollen development that can be a limiting factor for fertilization and fruit set. To fill this gap, we performed a thorough study on the cell wall composition during pollen development in mango. The results obtained reveal a clear differentiation of the cell wall composition of the male germline by pectins, AGPs and extensins from the early developmental stages during microsporogenesis and microgametogenesis reflecting a restricted communication between the male germline and the surrounding somatic cells that is very sensitive to low temperatures. The combination of the results obtained provides an integrated study on cell wall composition of the male germline in mango that reveals the crucial role of the sporophyte and the gametophyte and the vulnerability of the process to low temperatures.

Targeting Endothelial Ligands: ICAM-1/alicaforsen, MAdCAM-1.

Specific blockade of the endothelial ligands intercellular adhesion molecule-1 [ICAM-1] and mucosal addressin cell adhesion molecule [MAdCAM] involved in leukocyte recruitment to the site of inflammation as therapeutic targets in inflammatory bowel disease [IBD] has been recognized from their overexpression in the inflamed mucosa and successful intervention based on these ligands in preclinical animal models. Interventions to target ICAM-1 in human IBD are confined to the ICAM-1 anti-sense oligonucleotide alicaforsen. While results with parenteral formulations of alicaforsen in Crohn's disease have largely been negative, efficacy signals derived from studies with an enema formulation in ulcerative colitis and pouchitis are promising and have led to a Food and Drug Administration Fast-Track designation for the latter. A large phase III programme in pouchitis is underway. Phase II studies with the anti-MAdCAM-1 antibody [SHP647] delivered positive results in ulcerative colitis and anti-inflammatory signals in Crohn's disease. Furthermore, it was shown that SHP647 does not affect the number and composition of cells in cerebrospinal fluid, suggesting that the compound is not affecting immune surveillance in the central nervous system. In addition, both alicaforsen and SHP647 are promising compounds based on the clear safety profile observed so far.

Immunolocalization of AGPs and Pectins in Quercus suber Gametophytic Structures.

The arabinogalactan proteins (AGPs) are highly glycosylated proteins, ubiquitous in plants that have been linked to numerous aspects of sexual reproduction in several plant species, including the monoecious tree species Quercus suber. AGPs are found in cell membranes and cell walls of all types of tissues, including reproductive cells and organs. Pectins are cell wall components that also have been shown to change in composition and quantity during the maturations of the male and female gametophyte in cork oak. These findings were only possible to reveal, due to the histological study of AGP and pectins epitopes by immunolabeling. The immunofluorescence microscopy technique uses antibodies linked to fluorophores and relies on the specificity of the antibody binding to its antigen, labeling the epitope with a fluorescent dye.In the method presented here, we explore the immunolocalization technique performed in male and female flowers of Quercus suber, using London Resin (LR-White) as the embedding medium, after vacuum fixation with formaldehyde/glutaraldehyde. An extensive description of all the aspects of this technique is provided, from the plant material developmental stages selection to the critical analysis of results performed, continuously supported by troubleshooting recommendations.

Non-cellulosic polysaccharide distribution during G-layer formation in poplar tension wood fibers: abundance of rhamnogalacturonan I and arabinogalactan proteins but no evidence of xyloglucan.

MAIN CONCLUSION: RG-I and AGP, but not XG, are associated to the building of the peculiar mechanical properties of tension wood. Hardwood trees produce tension wood (TW) with specific mechanical properties to cope with environmental cues. Poplar TW fibers have an additional cell wall layer, the G-layer responsible for TW mechanical properties. We investigated, in two poplar hybrid species, the molecules potentially involved in the building of TW mechanical properties. First, we evaluated the distribution of the different classes of non-cellulosic polysaccharides during xylem fiber differentiation, using immunolocalization. In parallel, G-layers were isolated and their polysaccharide composition determined. These complementary approaches provided information on the occurrence of non-cellulosic polysaccharides during G-fiber differentiation. We found no evidence of the presence of xyloglucan (XG) in poplar G-layers, whereas arabinogalactan proteins (AGP) and rhamnogalacturonan type I pectins (RG-I) were abundant, with an apparent progressive loss of RG-I side chains during G-layer maturation. Similarly, the intensity of immunolabeling signals specific for glucomannans and glucuronoxylans varies during G-layer maturation. RG-I and AGP are best candidate matrix components to be responsible for TW mechanical properties.