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A major question in plant biology concerns the specification and functional differentiation of cell types. This is in the context of constraints imposed by networks of cell walls that both adhere cells and contribute to the form and function of developing organs. Here, we report the identification of a glycan epitope that is specific to phloem sieve element cell walls in several systems. A monoclonal antibody, designated LM26, binds to the cell wall of phloem sieve elements in stems of Arabidopsis (Arabidopsis thaliana), Miscanthus × giganteus, and notably sugar beet (Beta vulgaris) roots where phloem identification is an important factor for the study of phloem unloading of Suc. Using microarrays of synthetic oligosaccharides, the LM26 epitope has been identified as a 𝛽-1,6-galactosyl substitution of 𝛽-1,4-galactan requiring more than three backbone residues for optimized recognition. This branched galactan structure has previously been identified in garlic (Allium sativum) bulbs in which the LM26 epitope is widespread throughout most cell walls including those of phloem cells. Garlic bulb cell wall material has been used to confirm the association of the LM26 epitope with cell wall pectic rhamnogalacturonan-I polysaccharides. In the phloem tissues of grass stems, the LM26 epitope has a complementary pattern to that of the LM5 linear 𝛽-1,4-galactan epitope, which is detected only in companion cell walls. Mechanical probing of transverse sections of M. x giganteus stems and leaves by atomic force microscopy indicates that phloem sieve element cell walls have a lower indentation modulus (indicative of higher elasticity) than companion cell walls.

Cell walls of the brown algae contain a diverse range of polysaccharides with useful bioactivities. The precise structures of the sulfated fucan/fucoidan group of polysaccharides and their roles in generating cell wall architectures and cell properties are not known in detail. Four rat monoclonal antibodies, BAM1 to BAM4, directed to sulfated fucan preparations, have been generated and used to dissect the heterogeneity of brown algal cell wall polysaccharides. BAM1 and BAM4, respectively, bind to a non-sulfated epitope and a sulfated epitope present in the sulfated fucan preparations. BAM2 and BAM3 identified additional distinct epitopes present in the fucoidan preparations. All four epitopes, not yet fully characterised, occur widely within the major brown algal taxonomic groups and show divergent distribution patterns in tissues. The analysis of cell wall extractions and fluorescence imaging reveal differences in the occurrence of the BAM1 to BAM4 epitopes in various tissues of Fucus vesiculosus. In Ectocarpus subulatus, a species closely related to the brown algal model Ectocarpus siliculosus, the BAM4 sulfated epitope was modulated in relation to salinity levels. This new set of monoclonal antibodies will be useful for the dissection of the highly complex and yet poorly resolved sulfated polysaccharides in the brown algae in relation to their ecological and economic significance.

The leaf outer epidermal cell wall acts as a barrier against pathogen attack and desiccation, and as such is covered by a cuticle, composed of waxes and the polymer cutin. Cutin monomers are formed by the transfer of fatty acids to glycerol by glycerol-3-phosphate acyltransferases, which facilitate their transport to the surface. The extent to which cutin monomers affect leaf cell wall architecture and barrier properties is not known. We report a dual functionality of pathogen-inducible GLYCEROL-3-PHOSPHATE ACYLTRANSFERASE 6 (GPAT6) in controlling pathogen entry and cell wall properties affecting dehydration in leaves. Silencing of Nicotiana benthamiana NbGPAT6a increased leaf susceptibility to infection by the oomycetes Phytophthora infestans and Phytophthora palmivora, whereas overexpression of NbGPAT6a-GFP rendered leaves more resistant. A loss-of-function mutation in tomato SlGPAT6 similarly resulted in increased susceptibility of leaves to Phytophthora infection, concomitant with changes in haustoria morphology. Modulation of GPAT6 expression altered the outer wall diameter of leaf epidermal cells. Moreover, we observed that tomato gpat6-a mutants had an impaired cell wall–cuticle continuum and fewer stomata, but showed increased water loss. This study highlights a hitherto unknown role for GPAT6-generated cutin monomers in influencing epidermal cell properties that are integral to leaf–microbe interactions and in limiting dehydration.

Ectocarpus is a filamentous brown alga, which cell wall is composed mainly of alginates and fucans (80%), two non-crystalline polysaccharide classes. Alginates are linear chains of epimers of 1,4-linked uronic acids, β-D-mannuronic acid (M) and α-L-guluronic acid (G). Previous physico-chemical studies showed that G-rich alginate gels are stiffer than M-rich alginate gels when prepared in vitro with calcium. In order to assess the possible role of alginates in Ectocarpus , we first immunolocalised M-rich or G-rich alginates using specific monoclonal antibodies along the filament. As a second step, we calculated the tensile stress experienced by the cell wall along the filament, and varied it with hypertonic or hypotonic solutions. As a third step, we measured the stiffness of the cell along the filament, using cell deformation measurements and atomic force microscopy. Overlapping of the three sets of data allowed to show that alginates co-localise with the stiffest and most stressed areas of the filament, namely the dome of the apical cell and the shanks of the central round cells. In addition, no major distinction between M-rich and G-rich alginate spatial patterns could be observed. Altogether, these results support that both M-rich and G-rich alginates play similar roles in stiffening the cell wall where the tensile stress is high and exposes cells to bursting, and that these roles are independent from cell growth and differentiation.

Zygotes from Fucus species have been used extensively to study cell polarization and rhizoid outgrowth, and in this model system cell wall deposition aligns with the establishment of polarity. Monoclonal antibodies are essential tools for the in situ analysis of cell wall glycans, and here we report the characteristics of six monoclonal antibodies to alginates (BAM6–BAM11). The use of these, in conjunction with monoclonal antibodies to brown algal sulfated fucans, has enabled the study of the developmental dynamics of the Fucus zygote cell walls. Young zygotes are spherical and all alginate epitopes are deposited uniformly following cellulose deposition. At germination, sulfated fucans are secreted in the growing rhizoid wall. The redistribution of cell wall epitopes was investigated during treatments that cause reorientation of the growth axis (change in light direction) or disrupt rhizoid development (arabinogalactanprotein- reactive Yariv reagent). Alginate modeling was drastically impaired in the latter, and both treatments cause a redistribution of highly sulfated fucan epitopes. The dynamics of cell wall glycans in this system have been visualized in situ for the first time, leading to an enhanced understanding of the early developmental mechanisms of Fucus species. These sets of monoclonal antibodies significantly extend the available molecular tools for brown algal cell wall studies.

The derivation of two sensitive monoclonal antibodies directed to heteroxylan cell wall polysaccharide preparations has allowed the identification of potential inter-linkages between xylan and pectin in potato tuber cell walls and also between xylan and arabinogalactan-proteins in oat grain cell walls. Plant cell walls are complex composites of structurally distinct glycans that are poorly understood in terms of both in muro inter-linkages and developmental functions. Monoclonal antibodies (MAbs) are versatile tools that can detect cell wall glycans with high sensitivity through the specific recognition of oligosaccharide structures. The isolation of two novel MAbs, LM27 and LM28, directed to heteroxylan, subsequent to immunisation with a potato cell wall fraction enriched in rhamnogalacturonan-I (RG-I) oligosaccharides, is described. LM27 binds strongly to heteroxylan preparations from grass cell walls and LM28 binds to a glucuronosyl-containing epitope widely present in heteroxylans. Evidence is presented suggesting that in potato tuber cell walls, some glucuronoxylan may be linked to pectic macromolecules. Evidence is also presented that suggests in oat spelt xylan both the LM27 and LM28 epitopes are linked to arabinogalactan-proteins as tracked by the LM2 arabinogalactan-protein epitope. This work extends knowledge of the potential occurrence of inter-glycan links within plant cell walls and describes molecular tools for the further analysis of such links.

Cell walls of the brown algae contain a diverse range of polysaccharides with useful bioactivities. The precise structures of the sulfated fucan/fucoidan group of polysaccharides and their roles in generating cell wall architectures and cell properties are not known in detail. Four rat monoclonal antibodies, BAM1 to BAM4, directed to sulfated fucan preparations, have been generated and used to dissect the heterogeneity of brown algal cell wall polysaccharides. BAM1 and BAM4, respectively, bind to a non-sulfated epitope and a sulfated epitope present in the sulfated fucan preparations. BAM2 and BAM3 identified additional distinct epitopes present in the fucoidan preparations. All four epitopes, not yet fully characterised, occur widely within the major brown algal taxonomic groups and show divergent distribution patterns in tissues. The analysis of cell wall extractions and fluorescence imaging reveal differences in the occurrence of the BAM1 to BAM4 epitopes in various tissues of Fucus vesiculosus. In Ectocarpus subulatus, a species closely related to the brown algal model Ectocarpus siliculosus, the BAM4 sulfated epitope was modulated in relation to salinity levels. This new set of monoclonal antibodies will be useful for the dissection of the highly complex and yet poorly resolved sulfated polysaccharides in the brown algae in relation to their ecological and economic significance.

The leaf epidermal wall is covered by a cuticle, composed of cutin and waxes, which protects against dehydration and constitutes a barrier against pathogen attack. Cutin monomers are formed by the transfer of 16- or 18-carbon fatty acids to glycerol by glycerol-3-phosphate acyltransferase (GPAT) enzymes, which facilitates their transport to the plant surface. Here we address the dual functionality of pathogen-inducible Glycerol phosphate acyltransferase 6 (GPAT6) in controlling pathogen entry and dehydration in leaves. Silencing of Nicotiana benthamiana NbGPAT6a increased leaf susceptibility to the oomycetes Phytophthora infestans and P. palmivora, whereas stable overexpression of NbGPAT6a-GFP rendered leaves more resistant to infection. A loss-of-function mutation of the orthologous gene in tomato (Solanum lycopersicum), SlGPAT6, similarly resulted in increased susceptibility of leaves to Phytophthora infection concomitant with altered intracellular infection structure morphology. Conversely, Botrytis cinerea disease symptoms were reduced. Modulation of GPAT6 expression predominantly altered the outer cell wall of leaf epidermal cells. The impaired cell wall-cuticle continuum of tomato gpat6-a mutants resulted in increased water loss and these plants had fewer stomata. Our work highlights a hitherto unknown role for GPAT6-generated cutin monomers in controlling epidermal cell properties that are integral to leaf-microbe interactions and limit dehydration.

Pectin is a major component of the primary plant cell wall and is important for cell expansion. However, the relationship between its chemistry and mechanical properties is not fully understood, especially in vivo. In this study, a protocol for viscoelastic micro-indentation using atomic force microscopy (AFM) was developed and applied to pectin in vitro and in vivo. After determining that linear viscoelasticity was a suitable theoretical framework for in vitro pectin analyses were conducted with both a standard linear solid and fractional Zener model. These indicated a strong coupling between elastic and viscous properties over a range of degrees of methyl-esterification (DM). Both elasticity and viscosity were found to vary non-linearly with DM which had interesting consequences for pectin gels of mixed DM. In Arabidopsis cell walls, the standard linear solid model was found to be appropriate. In this in vivo composite material a weaker elastic-viscous coupling was exhibited, correlated with DM. The viscoelastic testing in vivo of rapidly elongating cell walls, rich in high DM pectin, displayed a longer viscous time-scale. The implications of the testing method and results are discussed in the context of mechanobiology, mechano-chemistry, and cell growth.

Population-based cancer registries generate estimates of incidence and survival that are essential for cancer surveillance, research, and control strategies. Although data on cancer stage allow meaningful assessments of changes in cancer incidence and outcomes, stage is not recorded by most population-based cancer registries. The main method of staging adult cancers is the TNM classification. The criteria for staging paediatric cancers, however, vary by diagnosis, have evolved over time, and sometimes vary by cooperative trial group. Consistency in the collection of staging data has therefore been challenging for population-based cancer registries. We assembled key experts and stakeholders (oncologists, cancer registrars, epidemiologists) and used a modified Delphi approach to establish principles for paediatric cancer stage collection. In this Review, we make recommendations on which staging systems should be adopted by population-based cancer registries for the major childhood cancers, including adaptations for low-income countries. Wide adoption of these guidelines in registries will ease international comparative incidence and outcome studies.