Poster: Hormones

Abs # 604: Functional domain analysis of the SLN1 protein of barley through transient expression

Presenter:	Ho, Tuan-hua D, ho@biology.wustl.edu
Authors	Ho, Tuan-hua D (A)   Zentella, Rodolfo  (A)
Affiliations:	(A): Washington University

The recent cloning of the SLN1 gene in barley revealed that this gene encodes a protein in the DELLA family. The DELLA proteins are an essential component in the gibberelling (GA) signaling cascade, acting as repressors of GA action. However, their mode of action and regulation are still unknown. Here we show that transient overexpression of SLN1 protein in wild type and sln1 mutant alerurone cells is sufficient to inhibit the GA mediated expression of alpha-amylase in a dose-dependent fashion. Amino- and carboxyl-terminal deletions revealed that the last two thirds of the protein are necessary for gene repression. Overexpression of the truncated protein encoded by the mutant allele sln1a has very mild repression activity in mutant aleurone cells, but it has a dominant negative effect when overexpressed in wild type cells. It was found that the initial 68 amino acids of this protein are responsible for the dominant negative effect. Site directed mutagenesis of conserved amino acid regions revealed that the VHIID and SH2-like motifs and the predicted bipartite nuclear localization sequence (NLS) are important for the repressive activity of SLN1. Other motifs with lesser negative effects on the repression of alpha-amylase were also identified. Protein fusions of GFP with SLN1 demonstrated that this signaling molecule is localized to the nucleus. Deletions and site-directed mutations indicated that the NLS in SLN1 is not the predicted bipartite sequence, but a conserved basic motif closer to the N-terminus. Transient transformation of mutants to elucidate the functional domains of proteins is very rapid, provided a good reporter gene or phenotype exists, can accelerate the discovery of important features of a regulatory protein, like SLN1.