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.

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 x 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 b-1,6-galactosyl substitution of b-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 b-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.

Main conclusionThe 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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-015-2375-4) contains supplementary material, which is available to authorised users.

HighlightNew monoclonal antibodies to brown algal cell wall polymers allow tracing of in situ dynamics of alginates and sulfated fucans in Fucus zygotes and in the context of growth disruption

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.

Summary The cell wall is the primary interface between plant cells and their immediate environment and must balance multiple functionalities, including the regulation of growth, the entry of beneficial microbes, and protection against pathogens. Here, we demonstrate how API, a SCAR2 protein component of the SCAR/WAVE complex, controls the root cell wall architecture important for pathogenic oomycete and symbiotic bacterial interactions in legumes. A mutation in API results in root resistance to the pathogen Phytophthora palmivora and colonization defects by symbiotic rhizobia. Although api mutant plants do not exhibit significant overall growth and development defects, their root cells display delayed actin and endomembrane trafficking dynamics and selectively secrete less of the cell wall polysaccharide xyloglucan. Changes associated with a loss of API establish a cell wall architecture with altered biochemical properties that hinder P. palmivora infection progress. Thus, developmental stage-dependent modifications of the cell wall, driven by SCAR/WAVE, are important in balancing cell wall developmental functions and microbial invasion.

Summary 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.

5Pectin is a major component of the primary plant cell wall and is important for cell expan-6 sion. However, the relationship between its chemistry and mechanical properties is not fully 7 understood, especially in vivo. In this study, a protocol for viscoelastic micro-indentation 8 using atomic force microscopy (AFM) was developed and applied to pectin in vitro and in 9 vivo. After determining that linear viscoelasticity was a suitable theoretical framework for in 10 vitro pectin analyses were conducted with both a standard linear solid and fractional Zener 11 model. These indicated a strong coupling between elastic and viscous properties over a range 12 of degrees of methyl-esterification (DM). Both elasticity and viscosity were found to vary 13 non-linearly with DM which had interesting consequences for pectin gels of mixed DM. In 14 Arabidopsis cell walls, the standard linear solid model was found to be appropriate. In this 15 in vivo composite material a weaker elastic-viscous coupling was exhibited, correlated with 16 DM. The viscoelastic testing in vivo of rapidly elongating cell walls, rich in high DM pectin, 17 displayed a longer viscous time-scale. The implications of the testing method and results are 18 discussed in the context of mechanobiology, mechano-chemistry, and cell growth.19 1 Introduction 20 Networks of hydrophilic polymer chains, known as hydrogels, are a critically important class of 21 material in biology [1]. Hydrogels made from pectin, and insights into their mechanical proper-22ties, are useful in applied [2,3] and fundamental contexts [4,5]. In the plant species Arabidopsis 23 thaliana, pectin is the single largest constituent of the primary cell wall [6], the structural layer 24 encasing growing cells which is acknowledged to be a critical arbiter of growth [7]. The umbrella 25 term 'pectin' refers to a variety of pectic polysaccharides which cohabit the cell wall including ho-26 mogalacturonan (HG), rhamnogalacturonan I, rhamnogalacturonan II and xylogalacturonan [8]. 27 In Arabidopsis, HG is the largest component by a significant margin [9]. Key features of HG are its 28 initial state, which is highly methyl-esterified, and its de-esterification when acted upon by pectin 29 methylesterase (PME) enzymes [10]. HG de-esterification can occur in a blockwise contiguous 30 mode or a non-contiguous random mode. The degree of 'blockiness' likely depends on the prop-31 erties of the PMEs themselves [11], of which there are sixty-six (66) in Arabidopsis [12]. Random 32 de-esterification can occur through non-enzymatic means with changes in pH [13]. When a methyl 33 group is removed (i.e. de-esterified) a negative charge is left in its place and two negatively charged 34 sugars can bond using Ca 2+ ions [11]. Indeed, calcium cross-linking is the primary method of gela-35 tion for low degree of methyl-esterification (DM) gels and longer 'blocks' of de-esterified units form 36 stronger bonds -provided there is sufficient free calcium nearby. In contrast, high DM HG relies 37 on hydrogen bond...