RESUMO
BACKGROUND AND AIMS: The cotyledons of Lupinus angustifolius contain large amounts of cell wall storage polysaccharide (CWSP) composed mainly of (1-->4)-beta-linked D-galactose residues in the form of branches attached to a rhamnogalacturonan core molecule. An exo-(1-->4)-beta-galactanase with a very high specificity towards (1-->4)-beta-linked D-galactan has been isolated from L. angustifolius cotyledons, and shown to vary (activity and specific protein) in step with CWSP mobilization. This work aimed to confirm the hypothesis that galactan is the main polymer retrieved from the wall during mobilization at the ultrastructural level, using the purified exo-galactanase as a probe. METHODS: Storage mesophyll cell walls ('ghosts') were isolated from the cotyledons of imbibed but ungerminated lupin seeds, and also from cotyledons of seedlings after the mobilization of the CWSP. The pure exo-(1-->4)-beta-galactanase was coupled to colloidal gold particles and shown to be a specific probe for (1-->4)-beta-D-galactan. They were used to localize galactan in ultrathin sections of L. angustifolius cotyledonary mesophyll tissue during CWSP mobilization. KEY RESULTS: On comparing the morphologies of isolated cell walls, the post-mobilization 'ghosts' did not have the massive wall-thickenings of pre-mobilization walls. Compositional analysis showed that the post-mobilization walls were depleted in galactose and, to a lesser extent, in arabinose. When pre-mobilization ghosts were treated with the pure exo-galactanase, they became morphologically similar to the post-mobilization ghosts. They were depleted of approximately 70% of the galactose residues that would have been mobilized in vivo, and retained all the other sugar residues originally present. Sharply defined electron-transparent wall zones or pockets are associated with CWSP mobilization, being totally free of galactan, whereas wall areas immediately adjacent to them were apparently undepleted. CONCLUSIONS: The exo-(1-->4)-beta-galactanase is the principal enzyme involved in CWSP mobilization in lupin cotyledons in vivo. The storage walls dramatically change their texture during mobilization as most of the galactan is hydrolysed during seedling development.
Assuntos
Parede Celular/metabolismo , Cotilédone/metabolismo , Galactanos/metabolismo , Glicosídeo Hidrolases/metabolismo , Lupinus/embriologia , Lupinus/enzimologia , Parede Celular/ultraestrutura , Cotilédone/ultraestrutura , Monossacarídeos/metabolismoRESUMO
The xyloglucan from cotyledons of Hymenaea courbaril was hydrolysed with endo-(1,4)-beta-D-glucanase (cellulase) and analysed by TLC and HPAEC. The limit digest was different from those obtained from xyloglucans of Tamarindus indica and Copaifera langsdorffii. On treatment with nasturtium beta-galactosidase, two main oligosaccharides were detected by TLC and HPAEC. Using a process of enzymatic sequencing involving alternate treatments with a pure xyloglucan oligosaccharide-specific alpha-xylosidase, and a pure beta-glucosidase, both from nasturtium, their structures were deduced to be XXXG and a new oligosaccharide XXXXG. These structures were confirmed by 1H NMR. The relative proportions of XXXG and XXXXG indicate that approximately half of the subunits in Hymenaea xyloglucan are based on the new oligosaccharides. In the native polymer the XXXXG subunits are likely to carry galactosyl substituents in varying proportions, since cellulase hydrolysates contained many bands which were converted to XXXXG on hydrolysis with nasturtium beta-galactosidase. Although no comparative studies on the physico-chemical properties of Hymenaea courbaril xyloglucan have yet been performed, our results indicate that this polymer is less interactive with iodine when compared with T. indica and C. langsdorffii xyloglucans, suggesting that changes in conformation may occur due to the presence of XXXXG.