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Liparis loeselii (L.) Rich, an endangered member of the Orchidaceae family, is found in alkaline fens. With the declining populations of L. loeselii, there is a pressing need to reintroduce this species in Central Europe. As in vitro germination is a crucial tool for obtaining plants for introduction into the environment, we looked at the morphological changes occurring during the early stages of L. loeselii development in vitro. As the early stages of orchid development, especially the protocorm stage, are thought to be responsible for SAM formation and the initiation of symbiotic association, we focused on cell wall elements whose epitopes have been found in similar processes in other species: the extensin and pectin rhamnogalacturonan I (RG-I) side chain epitopes. We addressed the following questions: Does the cell wall of L. loeselii change its composition during the early stages of development, as noted in other species? Are there noticeable similarities in the cell wall to organs of different species whose function is to contact microorganisms? Are there regularities that allow the recognition of individual structures on this basis? Immunolocalization revealed changes in the distribution of certain extensins (JIM11 and JIM20) and RG-I (LM5 and LM6) side chain epitopes. Extensins, a type of cell wall protein, were observed during the initial stages of the formation of PLB and the shoot apical meristem of protocorms and PLBs. RG-I, on the other hand, was found to play a significant role in the development of the protocorm and PLB. In pseudobulbs, which appeared on the protocorms, extensins occurred in their storage part. However, RG-I side chains (1→4)-β-galactans (LM5), and (1→5)-α-L-arabinans (LM6) were not found in pseudobulbs. We revealed that a common feature of protocorms and PLBs was an increased amount of extensins, which were detected with the JIM11 antibody, and pectins, which were detected with the LM5 antibody, that were present together, which may prove helpful in determining the identity of the induced structures and distinguishing them from pseudobulbs. Thus, our study unveiled the role of extensins and RG-I during the growth of protocorms and PLBs. We suggest that PLBs may mimic the wall remodelling that occurs in protocorms, which indicates that using cell wall components is an invitation to be colonised by a fungal partner. However, this needs to be tested in future research. The findings of this research can help interpret future studies on the propagation, acclimatisation, and introduction of L. loeselii into the natural environment. [ABSTRACT FROM AUTHOR]

Liparis loeselii (L.) Rich, an endangered member of the Orchidaceae family, is found in alkaline fens. With the declining populations of L. loeselii, there is a pressing need to reintroduce this species in Central Europe. As in vitro germination is a crucial tool for obtaining plants for introduction into the environment, we looked at the morphological changes occurring during the early stages of L. loeselii development in vitro. As the early stages of orchid development, especially the protocorm stage, are thought to be responsible for SAM formation and the initiation of symbiotic association, we focused on cell wall elements whose epitopes have been found in similar processes in other species: the extensin and pectin rhamnogalacturonan I (RG-I) side chain epitopes. We addressed the following questions: Does the cell wall of L. loeselii change its composition during the early stages of development, as noted in other species? Are there noticeable similarities in the cell wall to organs of different species whose function is to contact microorganisms? Are there regularities that allow the recognition of individual structures on this basis? Immunolocalization revealed changes in the distribution of certain extensins (JIM11 and JIM20) and RG-I (LM5 and LM6) side chain epitopes. Extensins, a type of cell wall protein, were observed during the initial stages of the formation of PLB and the shoot apical meristem of protocorms and PLBs. RG-I, on the other hand, was found to play a significant role in the development of the protocorm and PLB. In pseudobulbs, which appeared on the protocorms, extensins occurred in their storage part. However, RG-I side chains (1→4)-β-galactans (LM5), and (1→5)-α-L-arabinans (LM6) were not found in pseudobulbs. We revealed that a common feature of protocorms and PLBs was an increased amount of extensins, which were detected with the JIM11 antibody, and pectins, which were detected with the LM5 antibody, that were present together, which may prove helpful in determining the identity of the induced structures and distinguishing them from pseudobulbs. Thus, our study unveiled the role of extensins and RG-I during the growth of protocorms and PLBs. We suggest that PLBs may mimic the wall remodelling that occurs in protocorms, which indicates that using cell wall components is an invitation to be colonised by a fungal partner. However, this needs to be tested in future research. The findings of this research can help interpret future studies on the propagation, acclimatisation, and introduction of L. loeselii into the natural environment.

With the discovery in 2015 of the ability to induce somatic embryos in Cyathea delgadii, learning more about the relationship between the structure of apoplast and cell differentiation has become possible among ferns. In this study, the distribution of arabinogalactan proteins, pectins, extensins, and callose with specific epitopes recognized by monoclonal antibodies was investigated during direct somatic embryogenesis (SE) of C. delgadii. Eight antigens against the arabinogalactan proteins (JIM8, JIM13, LM2), pectins (JIM5, JIM7), extensins (JIM11, JIM12), and callose (anti-1 → 3-β-glucan) were selected. Two types of explants were analyzed, i.e. stipe fragments and internodes, which give rise to embryos of unicellular and multicellular origin, respectively. The study showed that embryogenic transition in C. delgadii is preceded by cell wall remodeling of initial explants. Dynamic changes in JIM13, JIM12, and anti-1 → 3-β-glucan localization were observed. The differences in the distribution of studied epitopes were observed between the cell walls of the epidermis and those located in the other layers of the explant. Moreover, within the somatic embryos, a stronger fluorescence of the examined antibodies was observed, mainly those reacting with arabinogalactan proteins, extensins, and callose. These results also implicated that, with the exception of the earlier appearance of callose in the stipe explants, the uni- and multicellular pathways of somatic embryo differentiation do not differ in the quality of cell wall components. The presented studies document the first time that SE in ferns can be regulated by changes in apoplast structure and they provide a basis for more detailed research. Key Message: The cell wall plays a significant role in somatic embryogenesis of Cyathea delgadii and changes in its composition may be a marker of embryogenicity for this species. [ABSTRACT FROM AUTHOR]

Root hair cells are important sensors of soil conditions. They grow towards and absorb water-soluble nutrients. This fast and oscillatory growth is mediated by continuous remodeling of the cell wall. Root hair cell walls contain polysaccharides and hydroxyproline-rich glycoproteins, including extensins (EXTs). Class-III peroxidases (PRXs) are secreted into the apoplastic space and are thought to trigger either cell wall loosening or polymerization of cell wall components, such as Tyr-mediated assembly of EXT networks (EXT-PRXs). The precise role of these EXT-PRXs is unknown. Using genetic, biochemical, and modeling approaches, we identified and characterized three root-hair-specific putative EXT-PRXs, PRX01, PRX44, and PRX73. prx01,44,73 triple mutation and PRX44 and PRX73 overexpression had opposite effects on root hair growth, peroxidase activity, and ROS production, with a clear impact on cell wall thickness. We use an EXT fluorescent reporter with contrasting levels of cell wall insolubilization in prx01,44,73 and PRX44-overexpressing background plants. In this study, we propose that PRX01, PRX44, and PRX73 control EXT-mediated cell wall properties during polar expansion of root hair cells. [ABSTRACT FROM AUTHOR]

Soil contamination by lead (Pb) has become one of the major ecological threats to the environment. Understanding the mechanisms of Pb transport and deposition in plants is of great importance to achieve a global Pb reduction. We exposed a collection of 360 Arabidopsis thaliana natural accessions to a Pb-polluted soil. Germination rates, growth, and leaf Pb concentrations showed extensive variation among accessions. These phenotypic data were subjected to genome wide association studies (GWAs) and we found a significant association on chromosome 1 for low leaf Pb accumulation. Genes associated with significant SNP markers were evaluated and we selected EXTENSIN18 (EXT18) and TLC (TRAM-LAG1-CLN8) as candidates for having a role in Pb homeostasis. Six Pb-tolerant accessions, three of them exhibiting low leaf Pb content, and three of them with high leaf Pb content; two Pb-sensitive accessions; two knockout T-DNA lines of GWAs candidate genes (ext18, tlc); and Col-0 were screened under control and high-Pb conditions. The relative expression of EXT18, TLC, and other genes described for being involved in Pb tolerance was also evaluated. Analysis of Darwinian fitness, root and leaf ionome, and TEM images revealed that Pb-tolerant accessions employ two opposing strategies: (1) low translocation of Pb and its accumulation into root cell walls and vacuoles, or (2) high translocation of Pb and its efflux to inactive organelles or intracellular spaces. Plants using the first strategy exhibited higher expression of EXT18 and HMA3, thicker root cell walls and Pb vacuolar sequestration, suggesting that these genes may contribute to the deposition of Pb in the roots. On the other hand, plants translocating high amounts of Pb showed upregulation of TLC and ABC transporters, indicating that these plants were able to properly efflux Pb in the aerial tissues. We conclude that EXT18 and TLC upregulation enhances Pb tolerance promoting its sequestration: EXT18 favors the thickening of the cell walls improving Pb accumulation in roots and decreasing its toxicity, while TLC facilitates the formation of dictyosome vesicles and the Pb encapsulation in leaves. These findings are relevant for the design of phytoremediation strategies and environment restoration.

Soil contamination by lead (Pb) has become one of the major ecological threats to the environment. Understanding the mechanisms of Pb transport and deposition in plants is of great importance to achieve a global Pb reduction. We exposed a collection of 360 Arabidopsis thaliana natural accessions to a Pb-polluted soil. Germination rates, growth, and leaf Pb concentrations showed extensive variation among accessions. These phenotypic data were subjected to genome wide association studies (GWAs) and we found a significant association on chromosome 1 for low leaf Pb accumulation. Genes associated with significant SNP markers were evaluated and we selected EXTENSIN18 (EXT18) and TLC (TRAM-LAG1-CLN8) as candidates for having a role in Pb homeostasis. Six Pb-tolerant accessions, three of them exhibiting low leaf Pb content, and three of them with high leaf Pb content; two Pb-sensitive accessions; two knockout T-DNA lines of GWAs candidate genes (ext18, tlc); and Col-0 were screened under control and high-Pb conditions. The relative expression of EXT18 , TLC , and other genes described for being involved in Pb tolerance was also evaluated. Analysis of Darwinian fitness, root and leaf ionome, and TEM images revealed that Pb-tolerant accessions employ two opposing strategies: (1) low translocation of Pb and its accumulation into root cell walls and vacuoles, or (2) high translocation of Pb and its efflux to inactive organelles or intracellular spaces. Plants using the first strategy exhibited higher expression of EXT18 and HMA3 , thicker root cell walls and Pb vacuolar sequestration, suggesting that these genes may contribute to the deposition of Pb in the roots. On the other hand, plants translocating high amounts of Pb showed upregulation of TLC and ABC transporters, indicating that these plants were able to properly efflux Pb in the aerial tissues. We conclude that EXT18 and TLC upregulation enhances Pb tolerance promoting its sequestration: EXT18 favors the thickening of the cell walls improving Pb accumulation in roots and decreasing its toxicity, while TLC facilitates the formation of dictyosome vesicles and the Pb encapsulation in leaves. These findings are relevant for the design of phytoremediation strategies and environment restoration. [ABSTRACT FROM AUTHOR]

Abstract Background Grafting is a technique widely used in horticulture. The processes involved in grafting are diverse, and the technique is commonly employed in studies focusing on the mechanisms that regulate cell differentiation or response of plants to abiotic stress. Information on the changes in the composition of the cell wall that occur during the grafting process is scarce. Therefore, this study was carried out for analyzing the composition of the cell wall using Arabidopsis hypocotyls as an example. During the study, the formation of a layer that covers the surface of the graft union was observed. So, this study also aimed to describe the histological and cellular changes that accompany autografting of Arabidopsis hypocotyls and to perform preliminary chemical and structural analyses of extracellular material that seals the graft union. Results During grafting, polyphenolic and lipid compounds were detected, along with extracellular deposition of carbohydrate/protein material. The spatiotemporal changes observed in the structure of the extracellular material included the formation of a fibrillar network, polymerization of the fibrillar network into a membranous layer, and the presence of bead-like structures on the surface of cells in established graft union. These bead-like structures appeared either “closed” or “open”. Only three cell wall epitopes, namely: LM19 (un/low-methyl-esterified homogalacturonan), JIM11, and JIM20 (extensins), were detected abundantly on the cut surfaces that made the adhesion plane, as well as in the structure that covered the graft union and in the bead-like structures, during the subsequent stages of regeneration. Conclusions To the best of our knowledge, this is the first report on the composition and structure of the extracellular material that gets deposited on the surface of graft union during Arabidopsis grafting. The results showed that unmethyl-esterified homogalacturonan and extensins are together involved in the adhesion of scion and stock, as well as taking part in sealing the graft union. The extracellular material is of importance not only due to the potential pectin–extensin interaction but also due to its origin. The findings presented here implicate a need for studies with biochemical approach for a detailed analysis of the composition and structure of the extracellular material.

Plant cell wall associated hydroxyproline-rich glycoproteins (HRGPs) are involved in several aspects of plant growth and development, including wood formation in trees. HRGPs such as arabinogalactan-proteins (AGPs), extensins (EXTs), and proline rich proteins (PRPs) are important for the development and architecture of plant cell walls. Analysis of publicly available gene expression data revealed that many HRGP encoding genes show tight spatio-temporal expression patterns in the developing wood of Populus that are indicative of specific functions during wood formation. Similar results were obtained for the expression of glycosyl transferases putatively involved in HRGP glycosylation. In situ immunolabelling of transverse wood sections using AGP and EXT antibodies revealed the cell type specificity of different epitopes. In mature wood AGP epitopes were located in xylem ray cell walls, whereas EXT epitopes were specifically observed between neighboring xylem vessels, and on the ray cell side of the vessel walls, likely in association with pits. Molecular mass and glycan analysis of AGPs and EXTs in phloem/cambium, developing xylem, and mature xylem revealed clear differences in glycan structures and size between the tissues. Separation of AGPs by agarose gel electrophoresis and staining with β-D-glucosyl Yariv confirmed the presence of different AGP populations in phloem/cambium and xylem. These results reveal the diverse changes in HRGP-related processes that occur during wood formation at the gene expression and HRGP glycan biosynthesis levels, and relate HRGPs and glycosylation processes to the developmental processes of wood formation.

Plant cell wall associated hydroxyproline-rich glycoproteins (HRGPs) are involved in several aspects of plant growth and development, including wood formation in trees. HRGPs such as arabinogalactan-proteins (AGPs), extensins (EXTs), and proline rich proteins (PRPs) are important for the development and architecture of plant cell walls. Analysis of publicly available gene expression data revealed that many HRGP encoding genes show tight spatio-temporal expression patterns in the developing wood of Populus that are indicative of specific functions during wood formation. Similar results were obtained for the expression of glycosyl transferases putatively involved in HRGP glycosylation. In situ immunolabelling of transverse wood sections using AGP and EXT antibodies revealed the cell type specificity of different epitopes. In mature wood AGP epitopes were located in xylem ray cell walls, whereas EXT epitopes were specifically observed between neighboring xylem vessels, and on the ray cell side of the vessel walls, likely in association with pits. Molecular mass and glycan analysis of AGPs and EXTs in phloem/cambium, developing xylem, and mature xylem revealed clear differences in glycan structures and size between the tissues. Separation of AGPs by agarose gel electrophoresis and staining with β-D-glucosyl Yariv confirmed the presence of different AGP populations in phloem/cambium and xylem. These results reveal the diverse changes in HRGP-related processes that occur during wood formation at the gene expression and HRGP glycan biosynthesis levels, and relate HRGPs and glycosylation processes to the developmental processes of wood formation. [ABSTRACT FROM AUTHOR]

Extensins (EXTs) are major protein components in plant cell walls that play crucial roles in higher plants. The function of EXTs has been reported in several plants but is limited in tomato, especially in fruit ripening. In this study, we identified 83 EXTs in tomato, and divided them into seven groups. The gene intron-exon structure and protein-motif composition of SlEXTs were similar within each group but different among groups. SlEXT genes showed different expression patterns in roots, leaves, flowers and fruits, and some SlEXT gene expressions in flowers could be regulated by treatments of auxin, gibberellic acid and jasmonic acid. In particular, SlSEXT8 had higher and increased expression during tomato fruit ripening, and its expression could be induced by ethylene, suggesting SlSEXT8 may be involved in tomato fruit softening. The result provides insights into the function of EXTs, and will facilitate to further study EXT roles in tomato fruit ripening. • Eighty-three extensins in tomato were identified by genome-wide identification. • Extensins have different expressions in tomato organs and by hormone treatments. • Some extensins may be involved in fruit softening as high expression in ripe fruits. [ABSTRACT FROM AUTHOR]

Summary: The key technical bottleneck for exploiting plant hairy root cultures as a robust bioproduction platform for therapeutic proteins has been low protein productivity, particularly low secreted protein yields. To address this, we engineered novel hydroxyproline (Hyp)‐O‐glycosylated peptides (HypGPs) into tobacco hairy roots to boost the extracellular secretion of fused proteins and to elucidate Hyp‐O‐glycosylation process of plant cell wall Hyp‐rich glycoproteins. HypGPs representing two major types of cell wall glycoproteins were examined: an extensin module consisting of 18 tandem repeats of 'Ser‐Hyp‐Hyp‐Hyp‐Hyp' motif or (SP4)18 and an arabinogalactan protein module consisting of 32 tandem repeats of 'Ser‐Hyp' motif or (SP)32. Each module was expressed in tobacco hairy roots as a fusion to the enhanced green fluorescence protein (EGFP). Hairy root cultures engineered with a HypGP module secreted up to 56‐fold greater levels of EGFP, compared with an EGFP control lacking any HypGP module, supporting the function of HypGP modules as a molecular carrier in promoting efficient transport of fused proteins into the culture media. The engineered (SP4)18 and (SP)32 modules underwent Hyp‐O‐glycosylation with arabino‐oligosaccharides and arabinogalactan polysaccharides, respectively, which were essential in facilitating secretion of the fused EGFP protein. Distinct non‐Hyp‐O‐glycosylated (SP4)18‐EGFP and (SP)32‐EGFP intermediates were consistently accumulated within the root tissues, indicating a rate‐limiting trafficking and/or glycosylation of the engineered HypGP modules. An updated model depicting the intracellular trafficking, Hyp‐O‐glycosylation and extracellular secretion of extensin‐styled (SP4)18 module and AGP‐styled (SP)32 module is proposed. [ABSTRACT FROM AUTHOR]

The perception and relay of cell-wall signals are critical for plants to regulate growth and stress responses, but the underlying mechanisms are poorly understood. We found that the cell-wall leucine-rich repeat extensins (LRX) 3/4/5 are critical for plant salt tolerance in Arabidopsis. The LRXs physically associate with the RAPID ALKALINIZATION FACTOR (RALF) peptides RALF22/23, which in turn interact with the plasma membrane-localized receptor-like protein kinase FERONIA (FER). The lrx345 triplemutant aswell as fer mutant plants display retarded growth and salt hypersensitivity, which are mimicked by overexpression of RALF22/23. Salt stress promotes S1P protease-dependent release of mature RALF22 peptides. Treatment of roots with mature RALF22/23 peptides or salt stress causes the internalization of FER. Our results suggest that the LRXs, RALFs, and FER function as a module to transduce cell-wall signals to regulate plant growth and salt stress tolerance. [ABSTRACT FROM AUTHOR]

Extensins are cell wall glycoproteins, belonging to the hydroxyproline-rich glycoprotein (HRGP) family, which are involved in many biological functions, including plant growth and defence. Several reviews have described the involvement of HRGPs in plant immunity but little focus has been given specifically to cell wall extensins. Yet, a large set of recently published data indicates that extensins play an important role in plant protection, especially in root–microbe interactions. Here, we summarise the current knowledge on this topic and discuss the importance of extensins in root defence. We first provide an overview of the distribution of extensin epitopes recognised by different monoclonal antibodies among plants and discuss the relevance of some of these epitopes as markers of the root defence response. We also highlight the implication of extensins in different types of plant interactions elicited by either pathogenic or beneficial micro-organisms. We then present and discuss the specific importance of extensins in root secretions, as these glycoproteins are not only found in the cell walls but are also released into the root mucilage. Finally, we propose a model to illustrate the impact of cell wall extensin on root secretions. [ABSTRACT FROM AUTHOR]

The oomycete pathogens produce important diseases in many plant species. To identify extensin genes expressed during the oomycete Phytophthora nicotianae-Nicotiana megalosiphon interaction, we used the SuperSAGE technology. Using this approach, we detected a N. megalosiphon extensin gene (NmEXT) triggered during the interaction. The extensin gene accumulation induced by the pathogen correlated with disease resistance in different Nicotiana species. Transient expression of NmEXT gene in susceptible Nicotiana tabacum enhanced the resistance to P. nicotianae. Our date indicated that NmEXT gene served a positive role in N. tabacum resistance against P. nicotianae. [ABSTRACT FROM AUTHOR]

Extensins (EXTs) are highly repetitive plant O -glycoproteins that require several post-translational modifications (PTMs) to become functional in plant cell walls. First, they are hydroxylated on contiguous proline residues; then they are O -glycosylated on hydroxyproline and serine. After secretion into the apoplast, O -glycosylated EXTs form a tridimensional network organized by inter- and intra-Tyr linkages. Recent studies have made significant progress in the identification of the enzymatic machinery required to process EXTs, which includes prolyl 4-hydroxylases, glycosyltransferases, papain-type cysteine endopeptidases, and peroxidases. EXTs are abundant in plant tissues and are particularly important in rapidly expanding root hairs and pollen tubes, which grow in a polar manner. Small changes in EXT PTMs affect fast-growing cells, although the molecular mechanisms underlying this regulation are unknown. In this review, we highlight recent advances in our understanding of EXT modifications throughout the secretory pathway, EXT assembly in cell walls, and possible sensing mechanisms involving the Catharanthus roseus cell surface sensor receptor-like kinases located at the interface between the apoplast and the cytoplasmic side of the plasma membrane. [ABSTRACT FROM AUTHOR]

Proper cell wall assembly is crucial during pollen tube growth. Leucine‐rich repeat extensins (LRXs) are extracellular glycoproteins which belong to the hydroxyproline‐rich glycoprotein (HRGP) family. They contain a conserved N‐terminal leucine‐rich repeat (LRR) domain and a highly variable C‐terminal extensin domain. Here, we characterized four LRX proteins (LRX8 through LRX11) from pollen of Arabidopsis thaliana. To investigate the role of LRX8‐LRX11 in pollen germination and pollen tube growth, multiple T‐DNA lrx mutants were obtained. The lrx mutants display abnormal pollen tubes with an irregular deposition of callose and pectin. They also show serious alterations in pollen germination and segregation ratio. Our results suggest that LRXs are involved in ensuring proper cell wall assembly during pollen tube growth. [ABSTRACT FROM AUTHOR]

Summary: Plant lodging resistance is an important integrative agronomic trait of grain yield and quality in crops. Although extensin proteins are tightly associated with plant cell growth and cell wall construction, little has yet been reported about their impacts on plant lodging resistance. In this study, we isolated a novel extensin‐like (OsEXTL) gene in rice, and selected transgenic rice plants that expressed OsEXTL under driven with two distinct promoters. Despite different OsEXTL expression levels, two‐promoter‐driven OsEXTL‐transgenic plants, compared to a rice cultivar and an empty vector, exhibited significantly reduced cell elongation in stem internodes, leading to relatively shorter plant heights by 7%–10%. Meanwhile, the OsEXTL‐transgenic plants showed remarkably thickened secondary cell walls with higher cellulose levels in the mature plants, resulting in significantly increased detectable mechanical strength (extension and pushing forces) in the mature transgenic plants. Due to reduced plant height and increased plant mechanical strength, the OsEXTL‐transgenic plants were detected with largely enhanced lodging resistances in 3 years field experiments, compared to those of the rice cultivar ZH11. In addition, despite relatively short plant heights, the OsEXTL‐transgenic plants maintain normal grain yields and biomass production, owing to their increased cellulose levels and thickened cell walls. Hence, this study demonstrates a largely improved lodging resistance in the OsEXTL‐transgenic rice plants, and provides insights into novel extensin functions in plant cell growth and development, cell wall network construction and wall structural remodelling. [ABSTRACT FROM AUTHOR]

Premise of research. Extensins (EXTs) are members of the cell wall hydroxyproline-rich glycoprotein super family 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 proline-rich EXT-like receptor kinases (PERKs), five formin homology EXTs (FHXs), four hybrid hydroxyproline-rich glycoproteins (HRGPs), and five other chimeric EXTs. In A. halleri, 65 EXTs were identified, including 13 classical EXTs, 14 short EXTs, eight LRXs, 14 PERKs, six FHXs, four hybrid HRGPs, and six other chimeric EXTs. For comparison, 69 EXTs were identified in A. thaliana, including 19 classical EXTs, 13 short EXTs, 11 LRXs, 12 PERKs, six FHXs, four hybrid HRGPs, and four other chimeric EXTs. Phylogenetic trees and cluster analysis revealed a number of potential orthologous and paralogous proteins among the three species. Conclusions. The identified EXTs and their homologous proteins among A. lyrata, A. halleri, and A. thaliana provide insight into the evolution and functions of EXTs in related species within the same genus. This project offers an excellent example of studying a model system consisting of three closely related species, in which diverse genetic resources and biological data make it possible to address fundamental evolutionary questions that cannot be addressed in a single species. [ABSTRACT FROM AUTHOR]

* Background and Aims Within the ovary, the obturator bridges the pathway of the pollen tube from the style to the ovule. Despite its widespread presence among flowering plants, its function has only been studied in a handful of species, and the molecules involved in pollen tube--obturator cross-talk have not been explored hitherto. This work evaluates the involvement of glucans and glycoproteins on pollen tube growth in the obturator of apple flowers (Malus x domestica). * Methods Pollen tube kinetics were sequentially examined in the pistil and related to changes occurring on the obturator using histochemistry and inmunocytochemistry. To discriminate between changes in the obturator induced by pollen tubes from those developmentally regulated, both pollinated and unpollinated pistils were examined. * Key Results Pollen tube growth rates were slow in the stigma, faster in the style and slow again in the ovary. The arrival of pollen tubes at the obturator was concomitant with the secretion of proteins, saccharides and glycoprotein epitopes belonging to extensins and arabinogalactan proteins (AGPs). While some of these secretions -- extensions and AGPs labelled by JIM13 -- were developmentally regulated, others -- AGPs labelled by JIM8 -- were elicited by the presence of pollen tubes. Following pollen tube passage, all these glycoproteins were depleted. * Conclusions The results show a timely secretion of glycoproteins on the obturator surface concomitant with pollen tube arrival at this structure. The fact that their secretion is depleted following pollen tube passage strongly suggests their role in regulating pollen tube access to the ovule. Remarkably, both the regulation of the secretion of the different glycoproteins, as well as their association with the performance of pollen tubes exhibit similarities with those observed in the stigma, in line with their common developmental origin. [ABSTRACT FROM AUTHOR]

EXTENSINS (EXTs) are a 65-member subfamily of hydroxyproline-rich glycoproteins (HRGP) 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 (3 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 (4 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.

Background: Hydroxyproline-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). Results: The 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. Conclusions: The 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. [ABSTRACT FROM AUTHOR]

The biological conversion of cellulosic biomass to biofuel is hindered by cell wall recalcitrance, which can limit the ability of cellulases to access and break down cellulose. The purpose of this study was to investigate whether hydroxyproline-rich cell wall proteins (extensins) are present in poplar stem biomass, and whether these proteins may contribute to recalcitrance. Three classical extensin genes were identified in Populus trichocarpa through bioinformatic analysis of poplar genome sequences, with the following proposed names: PtEXTENSIN1 (Potri.001G019700); PtEXTENSIN2 (Potri.001G020100); PtEXTENSIN3 (Potri.018G050100). Tissue print immunoblots localized the extensin proteins in poplar stems to regions near the vascular cambium. Different thermochemical pretreatments reduced but did not eliminate hydroxyproline (Hyp, a proxy for extensins) from the biomass. Protease treatment of liquid hot waterpretreated poplar biomass reduced Hyp content by a further 16% and increased subsequent glucose yield by 20%. These data suggest that extensins may contribute to recalcitrance in pretreated poplar biomass, and that incorporating protease treatment into pretreatment protocols could result in a small but significant increase in the yield of fermentable glucose. [ABSTRACT FROM AUTHOR]

Extensins (EXTs) are a family of plant cell wall hydroxyproline-rich glycoproteins (HRGPs) that are implicated to play important roles in plant growth, development, and defense. Structurally, EXTs are characterized by the repeated occurrence of serine (Ser) followed by three to five prolines (Pro) residues, which are hydroxylated as hydroxyproline (Hyp) and glycosylated. Some EXTs have Tyrosine (Tyr)-X-Tyr (where X can be any amino acid) motifs that are responsible for intramolecular or intermolecular cross-linkings. EXTs can be divided into several classes: classical EXTs, short EXTs, leucine-rich repeat extensins (LRXs), proline-rich extensin-like receptor kinases (PERKs), formin-homolog EXTs (FH EXTs), chimeric EXTs, and long chimeric EXTs. To guide future research on the EXTs and understand evolutionary history of EXTs in the plant kingdom, a bioinformatics study was conducted to identify and classify EXTs from 16 fully sequenced plant genomes, including Ostreococcus lucimarinus, Chlamydomonas reinhardtii, Volvox carteri, Klebsormidium flaccidum, Physcomitrella patens, Selaginella moellendorffii, Pinus taeda, Picea abies, Brachypodium distachyon, Zea mays, Oryza sativa, Glycine max, Medicago truncatula, Brassica rapa, Solanum lycopersicum, and Solanum tuberosum, to supplement data previously obtained from Arabidopsis thaliana and Populus trichocarpa. A total of 758 EXTs were newly identified, including 87 classical EXTs, 97 short EXTs, 61 LRXs, 75 PERKs, 54 FH EXTs, 38 long chimeric EXTs, and 346 other chimeric EXTs. Several notable findings were made: (1) classical EXTs were likely derived after the terrestrialization of plants; (2) LRXs, PERKs, and FHs were derived earlier than classical EXTs; (3) monocots have few classical EXTs; (4) Eudicots have the greatest number of classical EXTs and Tyr-X-Tyr cross-linking motifs are predominantly in classical EXTs; (5) green algae have no classical EXTs but have a number of long chimeric EXTs that are absent in embryophytes. Furthermore, phylogenetic analysis was conducted of LRXs, PERKs and FH EXTs, which shed light on the evolution of three EXT classes. [ABSTRACT FROM AUTHOR]

Hydroxyproline O-arabinosyltransferases ( HPATs) are members of a small, deeply conserved family of plant-specific glycosyltransferases that add arabinose sugars to diverse proteins including cell wall-associated extensins and small signaling peptides. Recent genetic studies in flowering plants suggest that different HPAT homologs have been co-opted to function in diverse species-specific developmental contexts. However, nothing is known about the roles of HPATs in basal plants. We show that complete loss of HPAT function in Arabidopsis thaliana and the moss Physcomitrella patens results in a shared defect in gametophytic tip cell growth. Arabidopsis hpat1/ 2/ 3 triple knockout mutants suffer from a strong male sterility defect as a consequence of pollen tubes that fail to fully elongate following pollination. Knocking out the two HPAT genes of Physcomitrella results in larger multicellular filamentous networks due to increased elongation of protonemal tip cells. Physcomitrella hpat mutants lack cell-wall associated hydroxyproline arabinosides and can be rescued with exogenous cellulose, while global expression profiling shows that cell wall-associated genes are severely misexpressed, implicating a defect in cell wall formation during tip growth. Our findings point to a major role for HPATs in influencing cell elongation during tip growth in plants. [ABSTRACT FROM AUTHOR]

Extensins (EXTs) are hydroxyproline-rich glycoproteins (HRGPs) that are structural components of the plant primary cell wall. They are basic proteins and are highly glycosylated with carbohydrate accounting for >50% of their dry weight. Carbohydrate occurs as monogalactosyl serine and arabinosyl hydroxyproline, with arabinosides ranging in size from 1 to 4 or 5 residues. Proposed functions of EXT arabinosylation include stabilizing the polyproline II helix structure and facilitating EXT cross-linking. Here, the involvement of arabinosylation in EXT cross-linking was investigated by assaying the initial cross-linking rate and degree of cross-linking of partially or fully de-arabinosylated EXTs using an in vitro cross-linking assay followed by gel permeation chromatography. Our results indicate that EXT arabinosylation is required for EXT cross-linking in vitro and the fourth arabinosyl residue in the tetraarabinoside chain, which is uniquely α-linked, may determine the initial cross-linking rate. Our results also confirm the conserved structure of the oligoarabinosides across species, indicating an evolutionary significance for EXT arabinosylation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Leucine-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. Results: The 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. Conclusions: LRX3, 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. [ABSTRACT FROM AUTHOR]

Proline is a proteogenic amino acid and accumulates both under stress and non-stress conditions as a beneficial solute in plants. Recent discoveries point out that proline plays an important role in plant growth and differentiation across life cycle. It is a key determinant of many cell wall proteins that plays important roles in plant development. The role of extensins (EXTs), arabinogalactan proteins (AGPs) and hydroxyproline- and proline-rich proteins (H/PRPs) as important components of cell wall proteins that play pivotal roles in cell wall signal transduction cascades, plant development and stress tolerance is discussed in this review. Molecular insights are also provided here into the plausible roles of proline transporters modulating key events in plant development. In addition, the roles of proline during seed developmental transitions including storage protein synthesis are discussed. [ABSTRACT FROM AUTHOR]

The regulation of plant cell growth and early defense response involves the insolubilization of hydroxyproline-rich glycoproteins (HRGPs), such as extensin, in the primary cell wall. In tomato ( Lycopersicon esculentum ), insolubilization occurs by the formation of tyrosyl-crosslinks catalyzed specifically by the pI 4.6 extensin peroxidase (EP). To date, neither the gene encoding EP nor the protein itself has been identified. Here, we have identified tomato EP candidates using both proteomic and bioinformatic approaches. Bioinformatic screening of the tomato genome yielded eight EP candidates, which contained a putative signal sequence and a predicted pI near 4.6. Biochemical fractionation of tomato culture media followed by proteomic detection further refined our list of EP candidates to three, with the lead candidate designated ( CG5 ). To test for EP crosslinking activity, we cloned into a bacterial expression vector the CG5 open-reading frame from tomato cDNA. The CG5 was expressed in Escherichia coli , fractionated from inclusion bodies, and folded in vitro . The peroxidase activity of CG5 was assayed and quantified by ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)) assay. Subsequent extensin crosslinking assays showed that CG5 can covalently crosslink authentic tomato P1 extensin and P3-type extensin analogs in vitro supporting our hypothesis that CG5 encodes a tomato EP. [ABSTRACT FROM AUTHOR]

Compared to other plant cell wall-degrading enzymes, proteases are less well understood. In this study, the extracellular metalloprotease Prt1 from Pectobacterium carotovorum (formerly Erwinia carotovora) was expressed in Escherichia coli and characterized with respect to N-terminal processing, thermal stability, substrate targets, and cleavage patterns. Prt1 is an autoprocessing protease with an N-terminal signal pre-peptide and a pro-peptide which has to be removed in order to activate the protease. The sequential cleavage of the N-terminus was confirmed by mass spectrometry (MS) fingerprinting and N-terminus analysis. The optimal reaction conditions for the activity of Prt1 on azocasein were at pH 6.0, 50 °C. At these reaction conditions, K was 1.81 mg/mL and k was 1.82 × 10 U M. The enzyme was relatively stable at 50 °C with a half-life of 20 min. Ethylenediaminetetraacetic acid (EDTA) treatment abolished activity; Zn addition caused regain of the activity, but Znaddition decreased the thermal stability of the Prt1 enzyme presumably as a result of increased proteolytic autolysis. In addition to casein, the enzyme catalyzed degradation of collagen, potato lectin, and plant extensin. Analysis of the cleavage pattern of different substrates after treatment with Prt1 indicated that the protease had a substrate cleavage preference for proline in substrate residue position P1 followed by a hydrophobic residue in residue position P1′ at the cleavage point. The activity of Prt1 against plant cell wall structural proteins suggests that this enzyme might become an important new addition to the toolbox of cell-wall-degrading enzymes for biomass processing. [ABSTRACT FROM AUTHOR]

Cell wall components such as hydroxyproline-rich glycoproteins (HRGPs, extensins) have been proposed to be involved in aluminum (Al) resistance mechanisms in plants. We have characterized the distribution of extensin in pea ( Pisum sativum L.) root nodules apoplast under short (for 2 and 24 h) Al stress. Monoclonal antibodie LM1 have been used to locate extensin protein epitope by immunofluorescence and immunogold labeling. The nodules were shown to respond to Al stress by thickening of plant and infection thread (IT) walls and disturbances in threads growth and bacteria endocytosis. Immunoblot results indicated the presence of a 17-kDa band specific for LM1. Irrespective of the time of Al stress, extensin content increased in root nodules. Further observation utilizing fluorescence and transmission electron microscope showed that LM1 epitope was localized in walls and intercellular spaces of nodule cortex tissues and in the infection threads matrix. Al stress in nodules appears to be associated with higher extensin accumulation in matrix of enlarged thick-walled ITs. In addition to ITs, thickened walls and intercellular spaces of nodule cortex were also associated with intense extensin accumulation. These data suggest that Al-induced extensin accumulation in plant cell walls and ITs matrix may have influence on the process of IT growth and tissue and cell colonization by Rhizobium bacteria. [ABSTRACT FROM AUTHOR]

Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. These data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production. [ABSTRACT FROM AUTHOR]

Background and Aims Cell wall changes in ripening grapes (Vitis vinifera) have been shown to involve re-modelling of pectin, xyloglucan and cellulose networks. Newer experimental techniques, such as molecular probes specific for cell wall epitopes, have yet to be extensively used in grape studies. Limited general information is available on the cell wall properties that contribute to texture differences between wine and table grapes. This study evaluates whether profiling tools can detect cell wall changes in ripening grapes from commercial vineyards. Methods Standard sugar analysis and infra-red spectroscopy were used to examine the ripening stages (green, véraison and ripe) in grapes collected from Cabernet Sauvignon and Crimson Seedless vineyards. Comprehensive microarray polymer profiling (CoMPP) analysis was performed on cyclohexanediaminetetraacetic acid (CDTA) and NaOH extracts of alcohol-insoluble residue sourced from each stage using sets of cell wall probes (mAbs and CBMs), and the datasets were analysed using multivariate software. Key Results The datasets obtained confirmed previous studies on cell wall changes known to occur during grape ripening. Probes for homogalacturonan (e.g. LM19) were enriched in the CDTA fractions of Crimson Seedless relative to Cabernet Sauvignon grapes. Probes for pectic-β-(1,4)-galactan (mAb LM5), extensin (mAb LM1) and arabinogalactan proteins (AGPs, mAb LM2) were strongly correlated with ripening. From green stage to véraison, a progressive reduction in pectic-β-(1,4)-galactan epitopes, present in both pectin-rich (CDTA) and hemicellulose-rich (NaOH) polymers, was observed. Ripening changes in AGP and extensin epitope abundance also were found during and after véraison. Conclusions Combinations of cell wall probes are able to define distinct ripening phases in grapes. Pectic-β-(1,4)-galactan epitopes decreased in abundance from green stage to véraison berries. From véraison there was an increase in abundance of significant extensin and AGP epitopes, which correlates with cell expansion events. This study provides new ripening biomarkers and changes that can be placed in the context of grape berry development. [ABSTRACT FROM PUBLISHER]

Extensin, the structural motif of plant extracellular matrix proteins, possesses a unique highly glycosylated, hydrophilic, and repeating Ser1Hyp4 pentapeptide unit, and has been proposed to include post-translational hydroxylation at proline residue and subsequent oligo- L-arabinosylations at all of the resultant hydroxyprolines as well as galactosylation at serine residue. Reported herein is the stereoselective synthesis of one of the highly glycosylated motifs, Ser(Galp1)-Hyp(Araf4)-Hyp(Araf4)-Hyp(Araf3)-Hyp(Araf1). The synthesis has been completed by the application of 2-(naphthyl)methylether-mediated intramolecular aglycon delivery to the stereoselective construction of the Ser(Galp1) and Hyp(Arafn) fragments as the key step, as well as Fmoc solid-phase peptide synthesis for the backbone pentapeptide. [ABSTRACT FROM AUTHOR]

Summary: Hydroxyproline-rich glycoproteins (HRGPs) are plant cell wall proteins related to plant growth and development, and extensins (EXTs) are a subfamily of HRGPs. In this study, the function of HRGPs, especially EXTs, was investigated in the pollen tube and style cell growth of Nicotiana tabacum L. By using the techniques of protein blot and immunohistochemistry, the JIM20-recognized epitopes of EXTs were abundantly expressed in vivo for pollen tubes and transmitting tissue. A hydroxyproline synthesis inhibitor, 3,4-dehydro-l-proline (3,4-DHP), was used to investigate the functions of HRGPs. The addition of 3,4-DHP decreased the speed of pollen tube growth and shortened the length of style. Moreover, the hydroxyproline assay and JIM20 immunolocalization confirmed that 3,4-DHP treatment reduced the level of hydroxyproline and EXTs in the treated styles, respectively. These results indicate that HRGPs, most likely EXTs, may play important roles in the pollen tube and style cell growth. [ABSTRACT FROM AUTHOR]

Proline hydroxylation is a major posttranslational modification of hydroxyproline-rich glycoproteins (HRGPs) that is catalyzed by prolyl 4-hydroxylases (P4Hs). HRGPs such as arabinogalactan proteins (AGPs) and extensios play significant roles on cell wall structure and function and their implication in cell division and expansion has been reported. We used tobacco rattle virus (TRV)-based virus induced gene silencing to investigate the role of three tomato P4Hs, out of ten present in the tomato genome, in growth and development. Eight-days old tomato seedlings were infected with the appropriate TRV vectors and plants were allowed to grow under standard conditions for 6 weeks. Lower P4H mRNA levels were associated with lower hydroxyproline content in root and shoot tissues indicating successful gene silencing. P4H-silenced plants had longer roots and shoots and larger leaves. The increased leaf area can be attributed to increased cell division as indicated by the higher leaf epidermal cell number in SlP4H1- and SlP4H9-silenced plants. In contrast, SlP4H7-silenced plants had larger leaves due to enhanced cell expansion. Western blot analysis revealed that silencing of SlP4H7 and SlP4H9 was associated with reduced levels of JIM8-bound AGP and JIM11-bound extensin epitopes, while silencing of SlP4H1 reduced only the levels of AGP proteins. Collectively these results show that P4Hs have significant and distinct roles in cell division and expansion of tomato leaves. [ABSTRACT FROM AUTHOR]

Plant cell walls are composite structures mainly composed of polysaccharides, also containing a large set of proteins involved in diverse functions such as growth, environmental sensing, signaling, and defense. Research on cell wall proteins (CWPs) is a challenging field since present knowledge of their role into the structure and function of cell walls is very incomplete. Among CWPs, hydroxyproline (Hyp)-rich O-glycoproteins (HRGPs) were classified into three categories: (i) moderately glycosylated extensins (EXTs) able to form covalent scaffolds; (ii) hyperglycosylated arabinogalactan proteins (AGPs); and (iii) Hyp/proline (Pro)-Rich proteins (H/PRPs) that may be non-, weakly- or highly-glycosylated. In this review, we provide a description of the main features of their post-translational modifications (PTMs), biosynthesis, structure and function. We propose a new model integrating HRGPs and their partners in cell walls. Altogether, they could form a continuous glyco-network with non-cellulosic polysaccharides via covalent bonds or non-covalent interactions, thus strongly contributing to cell wall architecture. [ABSTRACT FROM AUTHOR]

Background The characteristics of pollen tube growth are not constant, but display distinct patterns of growth within the different tissues of the pistil. In the stigma, the growth rate is slow and autotrophic, whereas in the style, it is rapid and heterotrophic. Very little is known about the interactions between these distinct maternal tissues and the traversing pollen tube and the role of this interaction on the observed metabolism. In this work we characterise pollen tube growth in the apple flower and look for differences in glycoprotein epitope localization between two different maternal tissues, the stigma and the style. Results While immunocytochemically-detected arabinogalactan proteins were present at high levels in the stigma, they were not detected in the transmitting tissue of the style, where extensins were abundant. Whereas extensins remained at high levels in unpollinated pistils, they were no longer present in the style following pollen tube passage. Similarily, while abundant in unpollinated styles, insoluble polysaccharides such as β-glucans, were depleted in pollinated pistils. Conclusions The switch from autotropic to heterotrophic pollen tube growth correlates spatially with a change of glycoprotein epitopes between the stigma and the style. The depletion of extensins and polysaccharides following pollen tube passage in the style suggest a possible contribution to the acceleration of heterotrophic pollen tube growth, which would imply an active contribution of female tissues on prezygotic male-female crosstalk. [ABSTRACT FROM AUTHOR]

The genetic set up and the enzymes that define the O-glycosylation sites and transfer the activated sugars to the cell wall protein backbone have remained unknown for a long time. We are now beginning to see the emerging components of the molecular machinery that assembles these complex O-glycoproteins on the plant cell wall. Genes conferring the posttranslational modifications, i.e. proline hydroxylation and subsequent O-glycosylation, of the plant cell wall protein extensin subgroup have being recently identified. In this review we summarize the enzymes that define the O-glycosylation sites on the O-glycoproteins, i.e. the prolyl 4–hydroxylases (P4Hs), the glycosyltransferases that transfer arabinose units (named arabinosyltransferases, AraTs) and the one responsible for the transfer of a single galactose (galactosyltransferase, GalT) on the protein extensin backbones, and finally the EXT peroxidase-mediated crosslinking at the cell wall. We discuss the effect of posttranslational modification on the structure and function of extensins in the plant cell walls.

The pectic composition of cell wall is altered during the processes of cell differentiation, plant growth, and development. These alterations may be time-dependent, and fluctuate in distinct regions of the same cell or tissue layer, due to the biotic stress caused by the activity of the gall inducer. Among the roles of the pectins in cell wall, elasticity, rigidity, porosity, and control of cell death may be crucial during gall development. Galls on Baccharis reticularia present species-specific patterns of development leading to related morphotypes where pectins were widely detected by Ruthenium red, and the pectic epitopes were labeled with specific monoclonal antibodies (LM1, LM2, LM5, LM6, JIM5, and JIM7) in distinct sites of the non-galled and the galled tissues. In the studied system B. reticularia, the epitopes for extensins were not labeled in the non-galled tissues, as well as in those of the rolling and kidney-shaped galls. The high methyl-esterified homogalacturonans (HGA) were labeled all over the tissues either of non-galled leaves or of the three gall morphotypes, while the intense labeling for arabinogalactans was obtained just in the rolling galls. The pectic composition of non-galled leaves denotes their maturity. The kidney-shaped gall was the most similar to the non-galled leaves. The pectic dynamics in the gall tissues was particularly altered in relation to low methyl-esterified HGA, which confers elasticity and expansion, as well as porosity and adhesion to cell walls, and are related to the homogenization and hypertrophy of gall cortex, and to translocation of solutes to the larval chamber. Herein, the importance of the pectic dynamics of cell walls to the new functional design established during gall development is discussed for the first time. The repetitive developmental patterns in galls are elegant models for studies on cell differentiation. [ABSTRACT FROM AUTHOR]

Plants encode a poorly understood superfamily of developmentally expressed cell wall hydroxyproline-rich glycoproteins ( HRGPs). One, EXTENSIN3 ( EXT3) of the 168 putative HRGPs, is critical in the first steps of new wall assembly, demonstrated by broken and misplaced walls in its lethal homozygous mutant. Here we report the findings of phenotypic (not genotypic) revertants of the ext3 mutant and in-depth analysis including microarray and q RT- PCR (polymerase chain reaction). The aim was to identify EXT3 substitute(s), thus gaining a deeper understanding of new wall assembly. The data show differential expression in the ext3 mutant that included 61% ( P ≤ 0.05) of the HRGP genes, and ability to self-rescue by reprogramming expression. Independent revertants had reproducible expression networks, largely heritable over the four generations tested, with some genes displaying transgenerational drift towards wild-type expression levels. Genes for nine candidate regulatory proteins as well as eight candidate HRGP building materials and/or facilitators of new wall assembly or maintenance, in the (near) absence of EXT3 expression, were identified. Seven of the HRGP fit the current model of EXT function. In conclusion, the data on phenotype comparisons and on differential expression of the genes-of-focus provide strong evidence that different combinations of HRGPs regulated by alternative gene expression networks, can make functioning cell walls, resulting in (apparently) normal plant growth and development. More broadly, this has implications for interpreting the cause of any mutant phenotype, assigning gene function, and genetically modifying plants for utilitarian purposes. [ABSTRACT FROM AUTHOR]

Pre-zygotic interspecific incompatibility ( II) involves an active inhibition mechanism between the pollen of one species and the pistil of another. As a barrier to fertilization, II effectively prevents hybridization and maintains species identity. Transgenic ablation of the mature transmitting tract ( TT) in Nicotiana tabacum resulted in the loss of inhibition of pollen tube growth in Nicotiana obtusifolia (synonym Nicotiana trigonophylla) and Nicotiana repanda. The role of the TT in the II interaction between N. tabacum and N. obtusifolia was characterized by evaluating N. obtusifolia pollen tube growth in normal and TT-ablated N. tabacum styles at various post-pollination times and developmental stages. The II activity of the TT slowed and then arrested N. obtusifolia pollen tube growth, and was developmentally synchronized. We hypothesize that proteins produced by the mature TT and secreted into the extracellular matrix inhibit interspecific pollen tubes. When extracts from the mature TT of N. tabacum were injected into the TT-ablated style prior to pollination, the growth of incompatible pollen tubes of N. obtusifolia and N. repanda was inhibited. The class III pistil-specific extensin-like protein ( PELPIII) was consistently associated with specific inhibition of pollen tubes, and its requirement for II was confirmed through use of plants with antisense suppression of PELPIII. Inhibition of N. obtusifolia and N. repanda pollen tube growth required accumulation of PELPIII in the TT of N. tabacum, supporting PELPIII function in pre-zygotic II. [ABSTRACT FROM AUTHOR]

The Arabidopsis thaliana (L.) Heynh. atExt1 extensin gene is expressed in a cell and tissue-specific manner, in response to developmental cues, and is inducible by a wide range of biotic and abiotic stresses. Over-expression of this gene has been shown to alter stem morphology and to limit the invasiveness of virulent bacterial pathogens, indicating that this cell wall protein gene plays an important role in plant development and defense. A detailed sequence analysis of 3.2 kb of the atExt1 gene promoter region has identified a large number of putative 5′ cis-acting elements. Based on the location of clusters of putative promoter control elements, seven atExt1 5′ promoter truncations were constructed, fused upstream of the β-glucuronidase ( GUS) reporter gene, and transformed into A. thaliana. Transgenic plants carrying the various promoter constructs were challenged by wounding and pathogen attack and analysed for GUS expression - this analysis revealed a complex pattern of regulation, involving positive and negative control regions. Northern analysis using wounded tissues from transgenic Arabidopsis plants carrying the 3.2 kb-promoter:: GUS construct confirmed the transcriptional activation of the transgene. [ABSTRACT FROM AUTHOR]

The genetic set up and the enzymes that define the O-glycosylation sites and transfer the activated sugars to cell wall glycoprotein Extensins (EXTs) have remained unknown for a long time. We are now beginning to see the emerging components of the molecular machinery that assembles these complex O-glycoproteins on the plant cell wall. Genes conferring the posttranslational modifications, i.e., proline hydroxylation and subsequent O-glycosylation, of the EXTs have been recently identified. In this review we summarize the enzymes that define the O-glycosylation sites on the O-glycoproteins, i.e., the prolyl 4-hydroxylases (P4Hs), the glycosyltransferases that transfer arabinose units (named arabinosyltransferases, AraTs), and the one responsible for transferring a single galactose (galactosyltransferase, GalT) on the protein EXT backbones. We discuss the effects of posttranslational modifications on the structure and function of extensins in plant cell walls. [ABSTRACT FROM AUTHOR]

A monoclonal antibody, JIM 20, derived against an extensin type of hydroxyproline-rich glycoprotein (HRGP) from pea, showed high affinity for HRGP in pearl millet [ Pennisetum glaucum (L.) R. Br.]. Electrophoretic separation of Tris-SDS extracted proteins from suspension cells of pearl millet revealed a range of PM-HRGP polypeptides having a glycan epitope, which reacted with JIM 20. A high molecular mass band, probably an HRGP aggregate or polymer, and a few low molecular mass polypeptides were recognized by JIM 20 during Western blot analysis. Treatment of pearl millet suspension cells with hydrogen peroxide in the presence of an endogenous peroxidase resulted in insolubilization of HRGP polypeptides with molecular weights between 45 and 33 kDa. To investigate the gene coding for an extensin type of HRGP, a fosmid-based genomic library of pearl millet having a fourfold genome coverage was constructed. A partial sequence of 378 bp of an HRGP gene was obtained by PCR amplification of pearl millet DNA with a primer pair designed from the conserved regions of monocotyledon extensin type of HRGPs. Screening the genomic library using the homologous probe developed from the 378-bp PCR product resulted in the isolation of five fosmid clones. Restriction mapping of these fosmids resulted in an 11.8-kb region around an HRGP gene in pearl millet. The newly characterized gene, PM- HRGP, had all the characteristic features of a monocotyledon extensin type of HRGP. An intron at the 3′ untranslated region of the gene was identified by cDNA cloning. Differential expression of the PM- HRGP gene was observed during compatible and incompatible interactions of pearl millet with the downy mildew pathogen Sclerospora graminicola (Sacc) Schroet. Induced expression of the gene was observed only in case of an incompatible interaction. [ABSTRACT FROM AUTHOR]

We recently showed that yellow lupine is highly sensitive to soil water deficits since this stressor disrupts nodule structure and functioning, and at the same time triggers flower separation through abscission zone (AZ) activation in the upper part of the plant. Both processes require specific transformations including cell wall remodeling. However, knowledge about the involvement of particular cell wall elements in nodulation and abscission in agronomically important, nitrogen-fixing crops, especially under stressful conditions, is still scarce. Here, we used immuno-fluorescence techniques to visualize dynamic changes in cell wall compounds taking place in the root nodules and flower AZ of Lupinus luteus following drought. The reaction of nodules and the flower AZ to drought includes the upregulation of extensins, galactans, arabinans, xylogalacturonan, and xyloglucans. Additionally, modifications in the localization of high- and low-methylated homogalacturonans and arabinogalactan proteins were detected in nodules. Collectively, we determined for the first time the drought-associated modification of cell wall components responsible for their remodeling in root nodules and the flower AZ of L. luteus. The involvement of these particular molecules and their possible interaction in response to stress is also deeply discussed herein. [ABSTRACT FROM AUTHOR]

A light sphagnum peat mix inoculated with Trichoderma hamatum 382 consistently provided a significant (P = 0.05) degree of protection against bacterial spot of tomato and its pathogen Xanthomonas euvesicatoria 110c compared with the control peat mix, even though this biocontrol agent did not colonize aboveground plant parts. To gain insight into the mechanism by which T. hamatum 382 induced resistance in tomato, high-density oligonucleotide microarrays were used to determine its effect on the expression pattern of 15,925 genes in leaves just before they were inoculated with the pathogen. T. hamatum 382 consistently modulated the expression of genes in tomato leaves. We identified 45 genes to be differentially expressed across the replicated treatments, and 41 of these genes could be assigned to at least one of seven functional categories. T. hamatum 382-induced genes have functions associated with biotic or abiotic stress, as well as RNA, DNA, and protein metabolism. Four extensin and extensin-like proteins were induced. However, besides pathogenesis-related protein 5, the main markers of systemic acquired resistance were not significantly induced. This work showed that T. hamatum 382 actively induces systemic changes in plant physiology and disease resistance through systemic modulation of the expression of stress and metabolism genes. [ABSTRACT FROM AUTHOR]

Green algae of the order Volvocales provide an unrivalled opportunity for exploring the transition from unicellularity to multicellularity. They range from unicells, like Chlamydomonas, through homocytic colonial forms with increasing cooperation of individual cells, like Gonium or Pandorina, to heterocytic multicellular forms with different cell types and a complete division of labour, like Volvox. A fundamental requirement for the evolution of multicellularity is the development of a complex, multifunctional extracellular matrix (ECM). The ECM has many functions, which can change under developmental control or as a result of environmental factors. Here molecular data from 15 novel proteins are presented. These proteins have been identified in Chlamydomonas reinhardtii, Gonium pectorale, Pandorina morum and Volvox carteri, and all belong to a single protein family, the pherophorins. Pherophorin-V1 is shown to be a glycoprotein localized to the ‘cellular zone’ of the V. carteri ECM. Pherophorin-V1 and - V2 mRNAs are strongly induced not only by the sex inducer, which triggers sexual development at extremely low concentrations, but also by mechanical wounding. Like the extensins of higher plants, which are also developmentally controlled or sometimes inducible by wounding, the pherophorins contain a (hydroxy-)proline-rich (HR) rod-like domain and are abundant within the extracellular compartment. In contrast to most extensins, pherophorins have additional globular A and B domains on both ends of the HR domains. Therefore pherophorins most closely resemble a particular class of higher plant extensin, the solanaceous lectins (e.g. potato lectin), suggesting multivalent carbohydrate-binding functions are present within the A and B domains and are responsible for cross-linking. Our results suggest that pherophorins are used as the building blocks for the extracellular scaffold throughout the Volvocales, with the characteristic mesh sizes in different ECM structures being a result of the highly diverse extensions of the HR domains. Pherophorins have therefore been a versatile element during the evolution of ECM architecture in these green algae. [ABSTRACT FROM AUTHOR]

Summary: Plant–pathogen interactions involve highly complex series of reactions in disease development. Plants are endowed with both, resistance and defence genes. The activation of defence genes after contact with avirulence gene products of pathogens depends on signals transduced by leucine-rich repeats (LRRs) contained in resistance genes. Additionally, LRRs play roles for various actions following ligand recognition. Polygalacturonase inhibiting proteins (PGIPs), the only plant LRR protein with known ligands, are pectinase inhibitors, bound by ionic interactions to the extracellular matrix (ECM) of plant cells. They have a high affinity for fungal endopolygalacturonases (endoPGs). PGIP genes are organised in families encoding proteins with similar physical characteristics but different specificities. They are induced by infection and stress related signals. The molecular basis of PG–PGIP interaction serves as a model to understand the evolution of plant LRR proteins in recognising non-self-molecules. Extensins form a different class of structural proteins with repetitive sequences. They are also regulated by wounding and pathogen infection. Linkage of extensins with LRR motifs is highly significant in defending host tissues against pathogen invasion. Overexpression of PGIPs or expression of several PGIPs in a plant tissue, and perhaps manipulation of extensin expression could be possible strategies for disease management. [Copyright &y& Elsevier]