Polysaccharide Structures in the Outer Mucilage of Arabidopsis Seeds Visualized by AFM

Abstract

Evidence is presented that the polysaccharide rhamnogalacturonan I (RGI) can be biosynthesized in remarkably organized branched configurations and surprisingly long versions and can self-assemble into a plethora of structures. AFM imaging has been applied to study the outer mucilage obtained from wild-type (WT) and mutant (bxl1-3 and cesa5-1) Arabidopsis thaliana seeds. For WT mucilage, ordered, multichain structures of the polysaccharide RGI were observed, with a helical twist visible in favorable circumstances. Molecular dynamics (MD) simulations demonstrated the stability of several possible multichain complexes and the possibility of twisted fibril formation. For bxl1-3 seeds, the imaged polymers clearly showed the presence of side chains. These were surprisingly regular and well organized with an average length of ∼100 nm and a spacing of ∼50 nm. The heights of the side chains imaged were suggestive of single polysaccharide chains, while the backbone was on average 4 times this height and showed regular height variations along its length consistent with models of multichain fibrils examined in MD. Finally, in mucilage extracts from cesa5-1 seeds, a minor population of chains in excess of 30 μm long was observed.

Cell wall-localized BETA-XYLOSIDASE4 contributes to immunity of Arabidopsis against Botrytis cinerea

“…BXL1, a protein closely related to BXL4, has been shown to be a β- d -xylosidase/α- l -arabinofuranosidase that removes arabinan side-chains from RG-I in Arabidopsis seed mucilage ( Goujon et al, 2003 ; Minic et al, 2004 ; Arsovski et al, 2009 ; Williams et al, 2020 ). Mutations in BXL1 result in seed mucilage with higher levels of arabinan that, following hydration, extrudes from the seed coat much more poorly than that of WT ( Arsovski et al, 2009 ).…”

“…Only BXL1 has been studied in detail. It was shown to be a bifunctional β-D-xylosidase/α-L-arabinofuranosidase ( Goujon et al, 2003 ; Minic et al, 2004 ; Arsovski et al, 2009 ) that is important for extrusion of pectin rich mucilage upon hydration of Arabidopsis seeds ( Arsovski et al, 2009 ) as it removes the side chains present in RG-I ( Williams et al, 2020 ) and is associated with tissues undergoing secondary cell wall thickening ( Goujon et al, 2003 ). We provide evidence that another member of this protein family, BXL4, localizes to the plant cell wall, where it most likely acts on both xylans and arabinans.…”

Rhamnogalacturonan-I forms mucilage: behind its simplicity, a cutting-edge organization

“…Thanks to the great work realized by Zhang and collaborators, the tangled ‘ball of wool’ which represents the regulation of pectin synthesis is a little bit clearer now. At the mature stage, mucilage has been characterized as a plethora of unbranched RG-I polymers with an average size of 600 kDa; therefore, it is formed by ~1800 units of Rha–GalA disaccharide ( Box 2A ; Macquet et al , 2007a ; Williams et al , 2020 ; Saez-Aguayo et al , 2021 ). Elongation of this ‘giant’ polymer is initiated by the coordinated action of enzymes localized in the Golgi apparatus and in the cytosol ( Fabrissin et al , 2019 ; Saez-Aguayo et al , 2021 ).…”

“…However, the exact xylan association with the RG-I structure in mucilage still remains unclear, although MUM5 and IRX14 have been characterized as xylosyl transferases adding xylose into xylan structures in mucilage polysaccharides ( Voiniciuc et al , 2015b ; Fabrissin et al , 2019 ). During RG-I maturation, arabinan and galactan side chains are removed by MUM2 and BXL1 ( Macquet et al , 2007b ; Williams et al , 2020 ). No evidence clearly explains this strong change of structure but, considering that the presence of galactans and arabinans in the RG-I structure reduces the hydration ability of mucilage ( Dean et al , 2007 ; Arsovski et al , 2009 ; Rautengarten et al , 2011 ), it seems that the synthesis of RG-I side chains is required for compaction and organization into the dehydrated mucilage pocket, and their degradation would ensure the hydration and liberation of mucilage in the presence of water.…”

Seed mucilage evolution: diverse molecular mechanisms generate versatile ecological functions for particular environments

“…A convenient method to attest the presence of SM is pectin staining directly performed on hydrated seeds with ruthenium red or section staining with a generic and polychromatic stain such as toluidine blue (Western 2001). Further characterizations are obtained by immunolabeling with cell wall epitope antibodies directly performed on whole mount hydrated seeds or on sections (Ben-Tov et al 2018), chemical analysis facilitated by the easy extraction of mucilage (Zhao, Qiao & Wu 2017;Poulain, Botran, North & Ralet 2019) or ultrastructural analysis by scanning electron microscopy and atomic force microscopy (Kreitschitz & Gorb 2018;Williams et al 2020). In Arabidopsis, released SM is composed by an adherent layer bound to the seed and a non-adherent layer both enriched in poorly branches type I rhamnogalacturonan (RGI) pectin domains ( Figure 1; Macquet et al 2007;Poulain et al2019) while in flax the SM is composed by four contrasted layers enriched in RGI, arabinoxylans and xyloglucans/cellulose, respectively (Figure 1; Kreitschitz & Gorb 2017;Miart et al 2019)).…”