Oxalate oxidases and differentiating surface structure in wheat: germins

Abstract

Oxalate oxidases (OXOs) have been found to be concentrated in the surface tissues of wheat embryos and grains: germin is concentrated in root and leaf sheaths that surround germinated embryos; pseudogermin (OXO-ψ) is concentrated in the epidermis and bracts that ‘encircle’ mature grains. Most strikingly, the epidermal accumulation of OXO-ψ was found to presage the transition of a delicate ‘skin’, similar to the fragile epidermis of human skin, into the tough shell (the miller's ‘beeswing’) that is typical of mature wheat grains. A narrow range of oxalate concentration (1-2 mM) in the hydrated tissues of major crop cereals (barley, maize, oat, rice, rye and wheat) contrasted with wide variations in their OXO expression, e.g. cold-tolerant and cold-sensitive varieties of maize have similar oxalate contents but the former was found to contain approx. 20-fold more germin than did the latter. Well-known OXOs in sorghum, a minor cereal, and beet, a dicotyledon, were found to have little antigenic relatedness to the germins, but the beet enzyme did share some of the unique stability properties that are peculiar to the germin-like OXOs that are found only in the major crop cereals. Their concentration in surface structures of domesticated wheat suggests a biochemical role for germin-like OXOs: programmed cell death in surface tissues might be a constitutive as well as an adaptive form of differentiation that helps to produce refractory barriers against tissue invasion by predators. Incidental to the principal investigation, and using an OXO assay (oxalate-dependent release of CO2) that did not rely on detecting H2O2, which is often fully degraded in cell extracts, it was found that OXO activity in soluble extracts of wheat was manifested only in standard solution assays if the extract was pretreated in a variety of ways, which included preincubation with pepsin or highly substituted glucuronogalactoarabinoxylans (cell-wall polysaccharides).

Cultivar-specific seedling vigor and expression of a putative oxalate oxidase germin-like protein in sugar beet (Beta vulgaris L.)

“…Germin-like proteins have been found in all plants examined and are a subset of the conserved, functionally diverse cupin superfamily of proteins that, in plants, includes at least three separate groups (Khuri et al 2001). At least 12 GLP genes reside in the Arabidopsis genome, and the role(s) of the enzymes encoded by these genes in dicots is still being debated (see Lane 2000;MembrØ et al 2000). Germin-like proteins are involved in biotic and abiotic stress responses, and are primarily regulated at the transcriptional level (Berna and Bernier 1999).…”

“…Germin is a water-soluble, pepsin-resistant, SDS-insensitive, thermostable, hexameric cell-wall glycoprotein that functions as an oxalate oxidase (Dumas et al 1993;Lane et al 1993Lane et al , 1994Woo et al 1998). Beet germin has been shown to exhibit thermostability, but does not cross react with wheat germin antibodies (Lane 2000).…”

Cotton fiber germin-like protein. I. Molecular cloning and gene expression

“…5). In germinating cereal embryos, water uptake, germin expression and vacuolation occur simultaneously (Lane 2000). Similarly, expression of GhGLP1 overlaps the period when a large central vacuole develops and fiber cells are expanding.…”

Cotton-fiber germin-like protein. II: Immunolocalization, purification, and functional analysis

“…For the true germins, detection of protein localization pattern is possible by cytochemical staining for OxO activity Zhou et al 1998); however, the resolution of this method is insufficient to determine the precise subcellular location of germin. Immuno-detection methods using alkaline phosphatase-conjugated (Lane 2000) or colloidal gold-conjugated secondary antibody (Lane et al 1992) show that germin is localized in the cell wall of germinating wheat embryos. Interestingly, tobacco plants expressing wheat germin localize the transgenic gene product in the cell wall (Membre´et al 2000) even though cell wall structure in tobacco is quite distinct from that in wheat.…”