Accumulation and Subcellular Localization of α-Galactosidase-Hemagglutinin in Developing Soybean Cotyledons

Abstract

We have investigated the accumulation and intracellular localization of soybean (Glycine max IL.] Merr. cv Forrest) a-galactosidase-hemagglutinin during seed development. Cotyledon tissue was embedded in Lowicryl K4M and immunocytochemical localization was accomplished through treating thin sections with a-galactosidase antisera followed by indirect labeling with protein A coupled to colloidal gold. Gold particles were localized on the Golgi apparatus and protein bodies. We interpret this to indicate that a-galactosidase-hemagutinin is transferred to and transported through the Golgi apparatus and finally deposited within the protein body by a Golgi apparatus-mediated process.a-Galactosidase (EC 3.2.1.22) is widely distributed among plant species and is generally considered to participate in the degradation of reserve raffinose-type oligosaccharides and cell wall galactomannans (for review, see 25). The enzyme, depending on its source, displays a preference for one of these substrates (for review, see 25). The a-galactosidase from legume seed cotyledons exhibits a substrate preference for the raffinose-type oligosaccharide and shows only minimal activity toward galactomannans (5, 10). Thus, the presumed function of the cotyledon a-galactosidase is to degrade the raffinose-type oligosaccharides during germination and early seedling growth (18). a-Galactosidase from mature soybean seeds is a tetrameric protein (10) with a mol wt of 160,000 D at pH 4.0 (4). At pH 7.0, the protein dissociates into subunits with mol wt of 38,000 and 40,000 D (4). Although both the monomeric and tetrameric forms are enzymically active, they display different kinetic properties (4). At pH 4.0, the enzyme displays a transitory type of hemagglutinin activity and is referred to as a-galactosidasehemagglutinin (4). Whether the hemagglutinin activity is physiologically relevent is unknown. The a-galactosidase from soybean is virtually identical in biochemical and physical properties to a-galactosidases from the cotyledons of other legume species which may or may not display hemagglutinin activity (8,9). Soybean a-galactosidase-hemagglutinin is discrete from the wellcharacterized seed lectin, SBA2 (4). Because of its lectin-like properties and because it is immunologically related to legume lectins which bind galactose (8, 9), the a-galactosidases of mung bean and soybean seeds have been of particular interest to lectin researchers.'Supported by National Science Foundation grant PCM 8205148. The present study employs high-resolution electron microscope immunocytochemical observations to examine the developmental accumulation and intracellular localization of a-galactosidase in soybean cotyledons. Our findings indicate that soybean a-galactosidase is localized in protein bodies within storage parenchyma cells. We also demonstrate that a-galactosidase is localized in the Golgi apparatus of developing seed storage parenchyma cells supporting the proposal that the Golgi apparatus mediates the deposition of the legume protein body matrix ...

Enzymatic breakdown of raffinose oligosaccharides in pea seeds

“…25 and 26) in the pea amino acid sequence, similar to the tomato acidic -galactosidase enzyme, which is derived from a single gene only (Feurtado et al 2001). Hence, pea -galactosidases seem to be targeted to vacuolar compartments, as already demonstrated for several other plant species by subcellular fractionation (Bassel et al 2001), immunolocalization (Herman and Shannon 1985) and transient expression of an -galactosidase chloramphenicol acetyltransferase fusion protein (Bassel et al 2001).…”

“…In contrast, acidic -galactosidases are thought to be routed to PSVs via clathrin-coated vesicles during seed maturation (Harley and Beevers 1989). The acidic enzymes were located in extracted PSVs of dry pea seeds, as already demonstrated for several other plant species (Herman and Shannon 1985;Bassel et al 2001). This suggests that the accumulation of RFOs during seed development may be facilitated, in part, by a cellular compartmentation of RFOs (located in the cytosol and in PSVs) and acidic -galactosidases (located in PSVs only).…”

Analysis of the Raffinose Family Oligosaccharide Pathway in Pea Seeds with Contrasting Carbohydrate Composition

“…Therefore, transport of RFOs into protein storage vacuoles may be important (Tanner et al, 1968), but is certainly not able to eliminate the inhibitory interactions in the cytoplasm outlined above. However, hydrolytic ␣-galactosidases colocalize with RFOs in protein storage vacuoles (Dey, 1984;Herman and Shannon, 1985). As a consequence, it has been speculated that RFO accumulation is regulated by synthesis and simultaneous hydrolysis (Keller and Pharr, 1996;Kuo et al, 1997).…”

A novel alkaline α-galactosidase gene is involved in rice leaf senescence

“…The precise location of the Osh69 protein within chloroplast requires further investigation. In contrast, acid a-galactosidases are found predominantly associated with vacuoles (Bhalla and Dalling, 1984;Herman and Shannon, 1985). The presence of a b-galactosidase in the chloroplast stroma of mesophyll cells of the wheat primary leaf has been reported (Bhalla and Dalling,1984), and the enzyme activity increased as the leaf aged.…”