Functional characterization of seed coat-specific members of the barley germin gene family

The present project aimed to isolate testa-, pericarp- and epicarp-specific gene promoters for the developing caryopsis of barley (Hordeum vulgare L.) . These might be applied in transgenic plants to express antifungal agents or modify metabolic pathways. A testa-specific 379-nucleotide fragment was cloned by differential amplification and used to screen a bacterial artific ial chromosome (BAC) library of 6.3 haploid genome equivalents. Fifty-three clones containing genes encoding: for proteins of the germin family were f ound. Characterization of the clones identified a minimum of six seed coat- and eight leaf-specific germin genes. Four seed coat- and one leaf-specifi c genes were sequenced. The deduced primary structure of the proteins revea led a remarkable conservation of the manganese(II) binding His and Glu resi dues and beta-barrel secondary structure of oxalate oxidase - also in barle y, wheat, rice and Arabidopsis germins, for which an enzymatic activity has not yet been identified. The oxalate oxidase and germins of barley and oth er species an synthesized with a conserved pre-sequence of 23 or 24 amino a cids for targeting into the cell wall. beta-Glucuronidase expression with t he barley germin F gene promoter occurs specifically in the testa and epica rp of the developing barley caryopsis, while expression with the B gene pro moter is restricted to the testa. Oxalate oxidase activity is prominent in the epicarp and the root tips of the developing embryo. A family tree based on primary structure homologies of germins distinguishes three groups: oxa late oxidases, leaf-specific germins and seed coat-specific germins. (C) 20 00 Editions scientifiques et medicales Elsevier SAS.