Pool 2 of aged seedsshowedhigher am

Pool 2 of aged seedsshowedhigher amounts of RG,XGand galactans and lower amounts ofXGAand xylans, suggesting separation of highly branched regions from less branched regions. The decrease in rhamnosyl residues along with the increase in arabinose content in Pool 1 of aged seeds corroborates the aforementioned (Fig. 4). Linkage analysis. The most prominent change in the WSP of aged beans was the increase in the amounts of t-manp and 2-manp (15
and 3%) and the decrease in the 5-araf levels (Fig. 5). Aged beanWSP contained lower amounts of 5-araf residues (16%) which resulted in a 5% decrease arabinan content (Figs. 5 and 6). The decrease in 1,5-linked arabinofuranosyl residues suggests that the arabinans of WSP in aged seeds are shorter. On the other hand, the increase of mannosyl residues is, similar to the cotyledon, a consequence of the reduction in the amount of pectins in relation to the mannose-rich
polymers derived from the extraction enzymes. A degree of branching of the XG of control and aged beans was
calculated from the amounts of 4-glcp and 4,6-glcp using the data from Fig. 5 (Fig. 6). Low branching values were found in the WIP for both control and aged samples (8 and 7%, respectively). In the WSP, no XG branching was detected in control seeds, while the degree of branching was 15% higher in aged seeds (Fig. 6). Using the same principle, the degree of branching for arabinan was also calculated. In the WIP of aged beans, arabinan degree of branching is 7% lower
than in control. In the WSP, the arabinan degree of branching did not change with bean aging. The decrease of galactosyl and glucosyl residues (2-galp and 4- glcp and in 3 and 9%, respectively and 5-araf and 3,5-araf, both in 2%) in WIP suggests a decrease in XG amounts and the presence of low-branched arabinans in the WIP (Figs. 4 and 6). On the other hand, 4-xylp + 2-xylp values in the WIP of aged beans increased 10% and could be indicative of an increase in the xylan
contents (Fig. 6). The increase of the UA and t-xylp levels (5 and 1%, respectively) in the WIP (Figs. 2 and 4) suggests that XGA and or galacturonans insolubilization also occurred. Small amounts of XGA may also became less soluble, and the increase of hull darkening and galacturonans insolubilization (HG and XGA) (Figs. 1 and 5) may occur due to phenolic compound polymerization. The start of a lignification-like mechanism is suggested as one cause of seeds hardening in various studies conducted with leguminous seeds as described in Liu (1995). Hence, in the WIP there was a
decrease of XG and a slight increase of xylan which, along with a substantial increase of arabinans contents, may explain the significative decrease in the total xylopiranosil ratios observed in Fig. 2. The insolubilization of galacturonans is corroborated by the increase of WIP amounts in aged seeds. The increase in the t-xylp residues and decrease in the 4- and
4,6-glcp residues and 2,5-araf and 3,5-araf residues in the WIP of aged seed suggest that arabinans and XG are less branched and more fragmented in this fraction (Figs. 2 and 6). The decrease of arabinans (10%) and increase of highly branched XG (14%) in the WSP of aged seeds hulls, as well as the decrease in the WSP amounts suggests the insolubilization of straight regions of xyloglucans and the high molecular mass neutral pectic material in aged seed hulls. The acidic rhamnogalacturonans also became more branched (almost 11%) in the WSP compared to control and may
be evidence of homogalacturonan insolubilization. Ageing caused a selective insolubilization of wall polysaccharides, resulting in an increase in the WIP fraction and a decrease in the WSP fraction. The WIP fraction of the aged beans was characterized by a higher content of low-branched acidic pectin (homogalacturonan) and lower content of branched neutral polysaccharides (XG and arabinans). The WSP fraction of the aged beans was composed of highly branched XG and rhamnogalacturonans (highly soluble polymers).

Fig. 5. Linkage analysis of bean hull cell wall polysaccharides and the related polysaccharides. WSP: water-soluble polysaccharides; WIP: water-insoluble polysaccharides; RG: rhamnogalacturonans; XG: xyloglucan; XGA: xylogalacturonans. Linkages were deduced from partially methylated alditol acetates; t-araf: terminal arabinofuranose; t-xylp: terminal-xylopyranose; 5-araf: an arabinosyl residue containing a C-5 linkage; 2-xylp: 2-linked xylopyranose; 3,5-araf: arabinosyl residue containing C-3 and C-5 linkages.

Fig. 6. Relative percentage of polymers related to the partially methylated monosaccharides and degree of branching of bean hull cell wall polysaccharides.WSP: water-soluble
polysaccharides; WIP: water-insoluble polysaccharides; rhamnogalacturonans (RG; 2-rha, 2,4-rha); galactans (t-galp, 3-galp, 4-galp, 6-galp); xyloglucans (XG; t-glcp, 4-glcp,
4,6-glcp, t-fuc, 2-galp, 2,4-galp, t-xylp); xylans (4-xylp + 2-xylp); arabinans (t-araf, 5-araf, 2,5-araf, 3,5-araf, Ara (OAC)5). Linkages were deduced from partially methylated
alditol acetates.