SUMMARYHow the diverse polysaccharides present in plant cell walls are assembled and interlinked into functional composites is not known in detail. Here, using two novel monoclonal antibodies and a carbohydrate-binding module directed against the mannan group of hemicellulose cell wall polysaccharides, we show that molecular recognition of mannan polysaccharides present in intact cell walls is severely restricted. In secondary cell walls, mannan esterification can prevent probe recognition of epitopes/ligands, and detection of mannans in primary cell walls can be effectively blocked by the presence of pectic homogalacturonan. Masking by pectic homogalacturonan is shown to be a widespread phenomenon in parenchyma systems, and masked mannan was found to be a feature of cell wall regions at pit fields. Direct fluorescence imaging using a mannan-specific carbohydrate-binding module and sequential enzyme treatments with an endo-b-mannanase confirmed the presence of cryptic epitopes and that the masking of primary cell wall mannan by pectin is a potential mechanism for controlling cell wall micro-environments.

Cotton fiber cellulose is highly crystalline and oriented; when native cellulose (cellulose I) is treated with certain alkali concentrations, intermolecular hydrogen bonds are broken and Na-cellulose I is formed. At higher alkali concentrations Na-cellulose II forms, wherein intermolecular and intramolecular hydrogen bonds are broken, ultimately resulting in cellulose II polymers. Crystallinity changes in cotton fibers were observed and assigned using attenuated total reflectance Fourier transform infrared (ATR FT-IR) spectroscopy and X-ray diffraction (XRD) subsequent to sodium hydroxide treatment and compared with an in situ protein-binding methodology using cellulose-directed carbohydrate-binding modules (CBMs). Crystallinity changes observed using CBM probes for crystalline cellulose (CBM2a, CBM3a) and amorphous cellulose (CBM4-1, CBM17) displayed close agreement with changes in crystallinity observed with ATR-FTIR techniques, but it is notable that crystallinity changes observed with CBMs are observed at lower NaOH concentrations (2.0 mol dm(-3)), indicating these probes may be more sensitive in detecting crystallinity changes than those calculated using FTIR indices. It was observed that the concentration of NaOH at which crystallinity changes occur as analyzed using the CBM labeling techniques are also lower than those observed using X-ray diffraction techniques. Analysis of crystallinity changes in cellulose using CBMs offers a new and advantageous method of qualitative and quantitative assessment of changes to the structure of cellulose that occur with sodium hydroxide treatment.

The roles of non-cellulosic polysaccharides in cotton fiber development are poorly understood. Combining glycan microarrays and in situ analyses with monoclonal antibodies, polysaccharide linkage analyses and transcript profiling, the occurrence of heteromannan and heteroxylan polysaccharides and related genes in developing and mature cotton (Gossypium spp.) fibers has been determined. Comparative analyses on cotton fibers at selected days post-anthesis indicate different temporal and spatial regulation of heteromannan and heteroxylan during fiber development. The LM21 heteromannan epitope was more abundant during the fiber elongation phase and localized mainly in the primary cell wall. In contrast, the AX1 heteroxylan epitope occurred at the transition phase and during secondary cell wall deposition, and localized in both the primary and the secondary cell walls of the cotton fiber. These developmental dynamics were supported by transcript profiling of biosynthetic genes. Whereas our data suggest a role for heteromannan in fiber elongation, heteroxylan is likely to be involved in the regulation of cellulose deposition of secondary cell walls. In addition, the relative abundance of these epitopes during fiber development varied between cotton lines with contrasting fiber characteristics from four species (G. hirsutum, G. barbadense, G. arboreum and G. herbaceum), suggesting that these non-cellulosic polysaccharides may be involved in determining final fiber quality and suitability for industrial processing.

Cotton fibres develop over four stages: initiation, elongation, secondary-wall thickening, and maturation. They develop a significant crystalline structure during the secondary wall thickening stage of development. Cotton fibres were harvested from 17 days to 60 days after flowering (dpa). Transmission Electron Microscopy (TEM), Interferometry, Attenuated Total Reflectance Fourier-transform Infrared (ATR-FTIR) spectroscopy, immunofluorescence labelling, and fluorescence spectroscopy were used to characterise the cotton fibres in different stages. It was found that, secondary wall thickening and micronaire remain fairly constant from 17 to 24 dpa, after that time significant change occurs until maturity. Maturity ratio increases as the fibres develop. Birefringence increases rapidly from 17 dpa to 26 dpa, then levels off up to 60 dpa. It is evident by comparing the Lateral Order Index (LOI) and 2 results from the binding of a crystalline-cellulose binding probe (CBM3a) that there is a significant increase in the degree of cellulose crystallinity from 17 dpa to 26 dpa. Hydrogen Bond Intensity (HBI) increased to 24 dpa and decreased from 24 to 40 dpa indicating significant changes in inter-molecular hydrogen bonds. From 40 to 60 dpa an increase of HBI was observed. It is concluded that during the maturation stage of cotton fibre development, water loss from lumen allows the cellulose chains to come closer together and to form intermolecular hydrogen-bonds. TEM, Interferometry, ATR-FTIR spectroscopy, and immunofluorescence labelling combined with fluorescence spectroscopy, were demonstrated to be useful techniques in quantifying physical changes in cotton fibres during development, offering advantages over traditional analytical techniques.

No abstract