A gene encoding a germin-like protein, identified by a cDNA-AFLP approach, is specifically expressed during germination of Phaseolus vulgaris

Abstract

In order to identify markers of germination in Phaseolus vulgaris L., a cDNA-amplified fragment length polymorphism (AFLP) approach was conducted on mRNAs from embryo axes and from cotyledons. Among changes observed throughout the germination process, a cDNA fragment not detected 9 h after imbibition (HAI) but present specifically in axes 24 HAI was further studied. The complete cDNA was recovered by rapid amplification of cDNA ends, then cloned and sequenced. It includes an open reading frame predicting a 206-amino-acid polypeptide of 21.8 kDa. Analysis of the nucleotide sequence and deduced amino acid sequence revealed a high homology with germin-like proteins (GLPs), and particularly with an auxin-binding protein from peach, ABP19, that belongs to the GLP family. Thus, we propose that this cDNA encodes the first GLP described in P. vulgaris, designated PvGLP1. Northern blot analysis carried out on mRNAs from seed axes showed a dramatic increase in PvGLP1 expression a few hours before radicle emergence (17 HAI). Among mature vegetative tissues, PvGLP1 expression was very weak in pods and not detected in leaves, stems or roots. Immunoblot analysis using antibodies raised against AtGER3 from Arabidopsis thaliana showed that the protein could be detected only in axes from the dry seed stage onwards, at a steady-state level. Then, PvGLP1 expression seems to be associated with the early stages of embryo axis growth. The high homology indicated with ABP19 led us to study the effect of different concentrations of indole-3-acetic acid (IAA) on PvGLP1 expression during germination. Whereas no effect was noticed at low concentrations (1, 5, 10 microM), a marked decrease in PvGLP1 mRNA level was observed in axes of seeds imbibed with 100 microM IAA. Thus, PvGLP1 gene expression is not stimulated by auxin and, moreover, it might be inhibited by high concentrations of IAA.