Regulated expression of a wheat germin gene in tobacco: oxalate oxidase activity and apoplastic localization of the heterologous protein

Abstract

Wheat (Triticum aestivum) germin is a homopentameric glycoprotein whose synthesis is allied with seed germination. Germin pentamers show an unusual resistance to dissociation and possess an oxalate oxidase (OxO) activity. In order to increase our knowledge of germin gene expression, the function(s) of germin during development and possible uses in plant genetic engineering, an in vivo expression system is required. To this end, a gene for germin, named gf-2.8, was studied by expressing either promoter-GUS fusions or the intact gene in transgenic tobacco (Nicotiana tabacum) plants. Heterologous gene transcription was monitored in vitro and in vivo by GUS or OxO activity and was found to occur in developing seeds and in seedlings. This transcription was stimulated by auxins, as would be expected because of the presence of putative auxin-responsive elements in the promoter of the gf-2.8 gene. Auxin stimulation also extended to young leaves since OxO activity could be detected in treated but not in untreated leaves. The biochemical characteristics of wheat germin were also conserved in a transgenic host: the OxO activity was present under the form of a doublet co-migrating with germin G and G' isoforms. Also, germin distributed between a soluble and an apoplastic fractions despite the fact that wheat cell wall substantially differs from tobacco cell wall. Therefore, tobacco constitutes a suitable host for in vivo studies of this monocotyledon gene.