The cell-wall polysaccharides of Arabidopsis thaliana L. rosette leaves and callus

Abstract

The cell-wall polysaccharides of Arabidopsis thaliana rosette leaves and callus were characterised using an integrated approach involving chemical analysis; atomic force microscopy (AFM); fast-freeze, deep-etch, electron microscopy (EM); and solid-state 13C nuclear magnetic resonance (NMR) spectroscopy. The leaf contained cells with mostly unlignified primary cell walls, as determined histochemically. Chemical analysis of the leaf cell-wall polysaccharides showed the cell walls contained pectic polysaccharides (~ 42%), cellulose (~ 36%), xyloglucans (~ 16%), (galacto-)glucomannans (~ 6%) and small amounts of heteroxylans. This polysaccharide composition is typical of primary cell walls of dicotyledons. A solid callus culture, established from a leaf explant and cultured under conditions giving a minimal number of xylem tracheary elements, contained mostly unlignified primary cell walls, as determined histochemically. The results of chemical analysis on these cell walls were similar to those obtained for the leaf cell walls. The main exception was in the leaf cell walls, galactans were the major neutral pectic polysaccharides, whereas in the callus cell walls the major neutral pectic polysaccharides were arabinans. The leaf cell walls were fractionated by sequential chemical extraction. Analysis of the fractions showed that Fraction 1 (extracted from the cell walls using CDTA followed by sodium carbonate) was composed predominantly of pectic polysaccharides. Fraction 2 (extracted from the cell-wall residue using 4 M KOH) contained predominantly xyloglucans and heteroxylans. The residue after this sequential extraction (Fraction 3, a-cellulose) contained predominantly cellulose, although some pectic polysaccharides and (galacto-)glucomannans were also present. Solid-state 13C NMR spectroscopy was used to characterise the molecular ordering of the cellulose in the cell walls of A. thaliana rosette leaves and callus. Five different spin-relaxation time constants were used for the leaf cell walls, and it was found that proton rotating-frame relaxation with the time constant Tlp(H) provided the best discrimination between cellulose and non-cellulosic polysaccharides. This showed, in both leaf and callus cell walls, all the cellulose was in a crystalline (cellulose I) rather than a non-crystalline state. The percentage of cellulose molecules assigned to the interior of the crystallite was approximately 39%, which is consistent with a cellulose crystallite of cross-sectional diameter of 3.0 x 2.7 nm. Resolution-enhancement showed there were equal proportions of the triclinic (la) and monoclinic (1(3) crystal forms of cellulose. No evidence was found f significant interaction between the surfaces of the cellulose crystallites and xyloglucans, as expected from previous cell-wall models. Residues obtained by sequential chemical extraction of the A. thaliana leaf cell'walls were also examined using solid-state 13C NMR spectroscopy. These residues were obtained by extraction with CDTA and sodium carbonate (Residue 1A); CDTA, sodium carbonate and 1 M KOH (Residue 2A); and CDTA, sodium carbonate and 4 M KOH (Residue 3A). Using the proton rotating-frame relaxation time constant Tlp(H), it was shown that sequential extraction of the cell walls with CDTA and sodium carbonate did not affect the molecular ordering of cellulose. Sequential extraction with CDTA, sodium carbonate and 1 M KOH also did not affect the molecular ordering of cellulose, but was effective in removing disordered material. However, the spectra of Residues 1A and 2A indicated that the sequential chemical extraction caused non-cellulosic polysaccharides, possibly xyloglucans and (galacto)glucomannans, to become well-ordered through association with the cellulose crystallites. It was also shown that sequential extraction with CDTA, sodium carbonate and 4 M KOH converted cellulose I to cellulose II and amorphous cellulose. The callus cell walls and cell-wall residues after extraction with CDTA and sodium carbonate (Residue 1) or CDTA, sodium carbonate and 4 M KOH (Residue 2), were visualised using AFM and fast-freeze, deep-etch EM. The cell walls of onion were also visualised for comparison. The cell walls of both callus and onion were found to be polylamellate, with each layer composed of an interwoven mesh of microfibrils. The microfibrils in the callus cell walls were 5.8 nm in diameter, and those in the onion cell walls were 4.4 nm in diameter, when measured using both AFM and EM. The interwoven microfibril mesh observed by AFM and EM in Residue 1 appeared more open and porous compared with the unextracted cell walls. The diameter of microfibrils in Residue 1, measured using both AFM and EM was ~3.2 nm. The microfibrils in Residue 2 from the callus cell walls were swollen (~7.9 nm). Lateral striations, possibly non-cellulosic polysaccharides, were visualised on the surface of the microfibrils in onion cell walls using AFM, but not EM. Striations were not observed on the microfibrils in the callus cell walls. Using the electron microscopy freeze-fracture technique, evidence was found for a helicoidal arrangement of microfibrils in onion cell walls, but this was not seen in the callus cell walls. A model of the primary cell wall is proposed, in which each microfibril is thought to be composed of a cellulose crystallite surrounded by a hydrated matrix of non-cellulosic polysaccharides.