AtPME3, a ubiquitous cell wall pectin methylesterase of Arabidopsis thaliana, alters the metabolism of cruciferin seed storage proteins during post-germinative growth of seedlings.

AtPME3 (At3g14310) is a ubiquitous cell wall pectin methylesterase. Atpme3-1 loss-of-function mutants exhibited distinct phenotypes from the wild type (WT), and were characterized by earlier germination and reduction of root hair production. These phenotypical traits were correlated with the accumulation of a 21.5-kDa protein in the different organs of 4-day-old Atpme3-1 seedlings grown in the dark, as well as in 6-week-old mutant plants. Microarray analysis showed significant down-regulation of the genes encoding several pectin-degrading enzymes and enzymes involved in lipid and protein metabolism in the hypocotyl of 4-day-old dark grown mutant seedlings. Accordingly, there was a decrease in proteolytic activity of the mutant as compared with the WT. Among the genes specifying seed storage proteins, two encoding CRUCIFERINS were up-regulated. Additional analysis by RT-qPCR showed an overexpression of four CRUCIFERIN genes in the mutant Atpme3-1, in which precursors of the α- and ß-subunits of CRUCIFERIN accumulated. Together, these results provide evidence for a link between AtPME3, present in the cell wall, and CRUCIFERIN metabolism that occurs in vacuoles.

Characterization of mucilage polysaccharides, arabinogalactanproteins and cell-wall hemicellulosic polysaccharides isolated from flax seed meal: A wealth of structural moieties.

The present study aimed at analyzing the structural features of seed mucilage and cell-wall polysaccharides which accounted for 41% of the mass of flax meal (FM). A combination of high molar-mass mucilage-like polysaccharides (rhamnogalacturonan and arabinoxylan) was released from FM in water, together with arabinogalactan proteins and glucans. About half of FM homogalacturonans was extracted using a calcium chelator and boiling water. Hemicellulosic xyloglucans and xylans were further extracted with 1M KOH, in ∼13% FM-sugars yield. Structural characterization of the xyloglucan using specific enzyme hydrolysis, ion exchange chromatography (HPAEC) and matrix assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectroscopy showed the presence of XXXG type xyloglucan, but also that of XXGG-structure, possibly characteristic of flax seeds. Hydrolysis of xylans with endo-(1→4)-ß-D-xylanase, and analysis of the neutral and acidic oligosaccharides by MALDI-TOF-MS showed that xylan consisted of ß-(1→4)-linked-D-xylopyranose backbone with some zones (DP 5-7) substituted with 4-O-MeGlcA\GlcA\Glc residues.

Polysaccharide hydrolases are released with mucilages after water hydration of flax seeds.

Water imbibition of flax seed induces secretion of mucilages whose physico-chemical properties vary according to genotype and environment. The viscosity and composition of mucilage have ecological implications and also affect the utility of the crop. Several types of enzymes are secreted along with the mucilage. Our objective was to study these enzymes in the context of the composition and physical properties of the mucilage. The kinetics of production by flax seeds (variety Eden) of i) mucilages, ii) glycosidases and iii) endo-hydrolases were followed over 48 h under sterile conditions. The impact of enzymatic activities on mucilage was investigated by SEC-MALLS, viscosimetry and sugar composition. The mucilages consisted mainly of rhamnogalacturonan-I (RG-I, 52-62%) and arabinoxylan (AX, 27-36%). RG-I related enzyme activities (rhamnogalacturonase and ß,d-galactosidase) were quantified, together with AX related activity of α,l-arabinofuranosidase, ß,d-xylosidase and ß-xylanase. Maximal xylanase activity was reached after 4 h seed-hydration, when the minimal viscosity of the polysaccharides was observed, and the AX/RG-I ratio was the lowest. At that time, poly and oligosaccharides mainly contained pectic sugars. From 24 to 48 h water-hydration, when mucilages more tightly associated with cell walls were released, the glycosidase activities per g mucilage became maximal; the percentage, average molar-mass and viscosity of the polysaccharides decreased. Glucose, xylose and arabinose were the main sugars in the oligomer fraction. Our data confirmed the presence of ß-d xylosidase and α-l-arabinofuranosidase activities and provided evidence for significant pectinase activities in flax mucilages. They also indicate an impact of enzymatic activities on the physicochemical properties of mucilages.

Characteristics of cadmium tolerance in 'Hermes' flax seedlings: contribution of cell walls.

Most flax (Linum usitatissimum) varieties are described as tolerant to high concentrations of Cd. The aim of the present paper was to better characterize this tolerance, by studying the responses of flax plantlets, cv Hermes, to 18d growth on 0.5mM Cd. In Cd-treated seedlings, the majority of Cd was compartmentalized in the roots. Analysis of other elements showed that only Fe concentration was reduced, while Mn increased. Growth parameters of Cd treated flax were only moderately altered, with similar mass tolerance-indices for roots and shoots. Tissue anatomy was unaffected by treatment. The effect on lipid peroxidation, protein carbonylation and antioxidative activities appeared low but slightly higher in roots. The most important impacts of Cd were, in all organs, cell expansion, cell-wall thickening, pectin cross-linking and increase of cell-wall enzymatic activities (pectin methylesterase and peroxidase). Thus, the role of the cell wall in Cd tolerance might be important at two levels: (i) in the reinforcement of the tissue cohesion and (ii) in the sequestration of Cd.

Temporal regulation of cell-wall pectin methylesterase and peroxidase isoforms in cadmium-treated flax hypocotyl.

BACKGROUND AND AIMS: In hypocotyls of flax (Linum usitatissimum) cadmium-induced reorientation of growth (i.e. an increase in expansion and a decrease in elongation) coincides with marked changes in the methylesterification and cross-linking of homogalacturonans within various cell-wall (CW) domains. The aim of the present study was to examine the involvement of pectin methylesterase (PME) and peroxidase (PER) in this cadmium-induced CW remodelling. METHODS: CW proteins were extracted from hypocotyls of 10- and 18-d-old flax that had been treated or not treated with 0.5 mm Cd(NO(3))(2). PME and PER expression within these extracts was detected by LC/MS, by isoelectric focusing and enzyme activity assays. Transcript expression by RT-PCR of known flax PME and PER genes was also measured in corresponding samples. KEY RESULTS: In cadmium-treated seedlings, PME activity increased as compared with controls, particularly at day 10. The increased activity of PME was accompanied by increased abundance of both a basic protein isoform (B2) and a particular transcript (Lupme5). In contrast, induction of PER activity by cadmium was highest at day 18. Among the four reported PER genes, Flxper1 and 3 increased in abundance in the presence of cadmium at day 18. CONCLUSIONS: The temporal regulation of Lupme and Flxper genes and of their respective enzyme activities fits the previously reported cadmium-induced structural changes of homogalacturonans within the CWs. After PME-catalysed de-esterification of homogalacturonans, their cross-linking would depend on the activity of PERs interacting with calcium-dimerized blocks and reinforce the cell cohesion during the cadmium-induced swelling.