Composition and desiccation-induced alterations of the cell wall in the resurrection plant Craterostigma wilmsii

Resurrection plants have the unique capacity to revive from an air-dried state. In order to tolerate desiccation they have to overcome a number of stresses, mechanical stress being one. In leaves of the Craterostigma species, an extensive shrinkage occurs during drying as well as a considerable cell wall folding. Our previous microscopically analysis using immunocytochemistry on the resurrection plant Craterostigma wilmsii , has shown an increase in labelling of xyloglucan and unesterified pectins in the cell wall during drying. In this study, we have undertaken a biochemical approach to separate, quantify and characterize major cell wall polysaccharides in fully hydrated and dry leaves of C. wilmsii . Our results show that the overall cell wall composition of C. wilmsii leaves was similar to that of other dicotyledonous plants with respect to the pectin content. However, the structure of the hemicellulosic polysaccharide xyloglucan was characterized to be XXGG-type. The data also demonstrate marked changes in the hemicellulosic wall fraction from dry plants compared to hydrated ones. The most conspicuous change was a decrease in glucose content in the hemicellulosic fraction of dry plants. In addition, xyloglucan from the cell wall of dry leaves was relatively more substituted with galactose than in hydrated walls. Together these findings show that dehydration induces significant alteration of polysaccharide content and structure in the cell wall of C. wilmsii , which in turn might be involved in the modulation of the mechanical properties of the wall during dehydration.     

Immunocytochemical characterization of early-developing flax fiber cell walls

Summary The deposition and formation of a thick secondary wall is a major event in the differentiation of flax (Linum usitatissimum) fibers. This wall is cellulose-rich; but it also contains significant amounts of other matrix polymers which are noncellulosic such as pectins. We have used immunocytochemical techniques with antibodies specific for various epitopes associated with either pectins or arabinogalactan proteins (AGPs) to investigate the distribution of these polymers within the walls of differentiating young fibers of 1- and 2-week-old plants. Our results show that different epitopes exhibit distinct distribution patterns within fiber walls. Unesterified pectins recognized by polygalacturonic acid-rhamnogalacturonan I (PGA/RG-I) antibodies and rhamnogalacturonan II recognized by anti-RG-II-borate complex antibodies are localized all over the secondary wall of fibers. PGA/RG-I epitopes, but not RG-II epitopes, are also present in the middle lamellae and cell junctions. In marked contrast, -(14) galactans recognized by the LM5 monoclonal antibody and AGP epitopes recognized by anti--(16) galactan and LM2 antibodies are primarily located in the half of the secondary wall nearest the plasma membrane. LM2 epitopes, present in 1-week-old fibers, are undetectable later in development, suggesting a regulation of the expression of certain AGP epitopes. In addition, localization of cellulose with the cellobiohydrolase I-gold probe reveals distinct subdomains within the secondary walls of young fibers. These findings indicate that, in addition to cellulose, early-developing flax fibers synthesize and secrete different pectin and AGP molecules.     

Extensin,an underestimated key component of cell wall defence?

BackgroundExtensins are plant cell wall hydroxyproline-rich glycoproteins known to be involved in cell wall reinforcement in higher plants, and in defence against pathogen attacks. The ability of extensins to form intra- and intermolecular cross-links is directly related to their role in cell wall reinforcement. Formation of such cross-links requires appropriate glycosylation and structural conformation of the glycoprotein.ScopeAlthough the role of cell wall components in plant defence has drawn increasing interest over recent years, relatively little focus has been dedicated to extensins. Nevertheless, new insights were recently provided regarding the structure and the role of extensins and their glycosylation in plant–microbe interactions, stimulating an interesting debate from fellow cell wall community experts. We have previously revealed a distinct distribution of extensin epitopes in Arabidopsis thaliana wild-type roots and in mutants impaired in extensin arabinosylation, in response to elicitation with flagellin 22. That study was recently debated in a Commentary by Tan and Mort (Tan L, Mort A. 2020. Extensins at the front line of plant defence. A commentary on: ‘Extensin arabinosylation is involved in root response to elicitors and limits oomycete colonization’. Annals of Botany 125: vii–viii) and several points regarding our results were discussed. As a response, we herein clarify the points raised by Tan and Mort, and update the possible epitope structure recognized by the anti-extensin monoclonal antibodies. We also provide additional data showing differential distribution of LM1 extensin epitopes in roots between a mutant defective in PEROXIDASES 33 and 34 and the wild type, similarly to previous observations from the rra2 mutant defective in extensin arabinosylation. We propose these two peroxidases as potential candidates to specifically catalyse the cross-linking of extensins within the cell wall.ConclusionsExtensins play a major role within the cell wall to ensure root protection. The cross-linking of extensins, which requires correct glycosylation and specific peroxidases, is most likely to result in modulation of cell wall architecture that allows enhanced protection of root cells against invading pathogens. Study of the relationship between extensin glycosylation and their cross-linking is a very promising approach to further understand how the cell wall influences root immunity.     

Characterization and localization of laccase forms in stem and cell cultures of sycamore

A laccase-type polyphenoloxidase (EC 1.10.3.2.), abundantly secreted by suspension-cultured sycamore (Acer pseudoplatanus) cells was purified to homogeneity. This laccase form is a glycoprotein (molecular weight 110000) with high mannose and complex glycans. The polypeptide moiety has a molecular weight of 66 000, indicating that the glycoprotein is 40% carbohydrate. Laccase is abundantly present in both the cell wall and the culture medium of suspension-cultured sycamore cells, but it is not detected in the cytoplasm, indicating that this large protein is efficiently secreted by the cells. Polyclonal rabbit antiserum was raised against the deglycosylated protein and was used to probe extracts of sycamore stem tissues. A second laccase form (molecular weight 56 000), antigenically related to laccase from cell cultures, is abundant in the epidermis of sycamore stems. In addition, this 56 kDa laccase form co-localizes with lignin precursors on tissue prints from sycamore stems. A polypeptide (molecular weight 50 000-56 000), antigenically related to sycamore laccase, was also immunodetected in most plant organs previously described in the literature as polyphenoloxidase-rich.     

The role of high-mannose and complex asparagine-linked glycans in the secretion and stability of glycoproteins

Suspension-cultured cells of sycamore (Acer pseudoplatanus L.) secrete a number of acid hydrolases and other proteins that have both highmannose and complex asparagine-linked glycans. We used affinity chromatography with concanavalin A and an antiserum specific for complex glycans in conjunction with in vivo-labeling studies to show that all of the secreted proteins carry glycans. The presence of complex glycans on secretory proteins indicates that they are passing through the Golgi complex on the way to the extracellular compartment. The sodium ionophore, monensin, did not block the transport of proteins to the extracellular medium, even though monensin efficiently inhibited the Golgi-mediated processing of complex glycans. The inhibition of N-glycosylation by tunicamycin reduced by 76% to 84% the accumulation of newly synthesized (i.e. radioactively labeled) protein that was secreted by the sycamore cells, while cytoplasmic protein biosynthesis was not affected by this antibiotic. However, in the presence of glycoprotein-processing inhibitors, such as castanospermine and deoxymannojirimycin, the formation of complex glycans was prevented but glycoprotein secretion was unchanged. These results support the conclusion that N-linked glycan processing is not necessary for sorting, but glycosylation is required for accumulation of secreted proteins in the extracellular compartment.