A novel B-galactosidase or exo-(1-4)-B-galactanase from the cotyledons of germinated Lupinus angustifolius L. seeds, and its role in post-germinative cell wall metabolism

Abstract

The cotyledons of Lupinus angustifolius contain large amounts of a polysaccharide composed of ( l-»4 )-fi-linked D- galactose residues, probably in the form of branches attached to a rhamnogalacturonan core molecule . Parker (1984) documented the ultrastructural changes in cell walls of L.angustifolius cotyledons following germination and proposed that these changes correspond to the mobilisation of cell wall polysaccharides. Crawshaw and Reid (1984) showed that the main monosaccharides mobilised from the cotyledons of L.angustifolius after germination are galactose and arabinose. The aim of this thesis was to purify the principal enzymes responsible for the mobilisation of the (1-4)-li- galactan from the cell walls of L. angustifolius cotyledons and perhaps to contribute to an understanding of the relevance of galactan in the overall development of the seedling. A novel enzyme, capable of hydrolysing ( 1-4)-fi-linked galactan specifically, was isolated from L.angustifolius cotyledons using ion exchange chromatography and affinity chromatography. On SDS-PAGE, one band with M,=60kDa (major) and another band at Mj=45kDa (minor) were obtained after purification. Antibodies raised in rabbit against the 60kDa band and affinity-purified using the major band (60kDa) still cross-reacted with the minor band. The two bands had exactly the same N-terminal sequence (performed at Unilever Laboratories-Colworth House, by Dr.Amanda Heyler). The exo- ( 1-4 )-B-galactanase did not hydrolyse li-linked galactose from any plant cell wall polysaccharides other than from lupin galactan. The related Ji-galactosidases without action on fl-galactan were partially purified.Western immunoblotting and activity measurements showed that exo-( 1-4 )-li-galactanase was synthesised de novo and that its activity and specific protein varied pari passu with cell wall mobilisation. When isolated storage mesophyll lupin cell walls were treated with pure exo-galactanase the morphological changes observed were remarkably similar to those accompanying in vivo mobilisation. In vitro, the exo-{1-4 )-ii-galactanase was capable of hydrolysing ca.60% of the galactose present in the cell walls. Exo-( 1-4 )-ii-galactanase coupled to gold particles was used as a cytochemical probe to detect galactan for the first time in ultrathin sections of L.angustifolius mesophyll cotyledonary tissue following germination. Using the gold-probe, the pattern of galactan mobilisation was observed directly in the electron microscope. The decrease in galactan content after germination (estimated using the exo-galactanase) showed a close correlation with increase in cotyledonary area and changes in plasticity and elasticity of the mesophyll storage tissue of L. angustifolius cotyledons. These results showed clearly that exo(1-4)-ft- galactanase is the main enzyme responsible for degradation of the cell walls in vivo. They also suggest that galactan is probably a dual-purpose molecule in L.angustifolius cotyledons, serving as a reserve substance and at the same time playing a role in cotyledonary expansion during seedling development.