Map based cloning of a gene in Arabidopsis encoding novel membrane-associated metalloprotease that is required for ethylene-dependent hypocotyl gravitropism and chloroplast development

Plants use ethylene gas as a signal to regulate myriad developmental processes and stress responses. Based on the stimulation effect of ethylene on hypocotyl gravitropism of light-grown seedlings, a screening method was established to screen mutants in Arabidopsis. A mutant, edgy1-1 (e&barbelow;thylene-d&barbelow;ependent shoot g&barbelow;ravitropism deficiency and y&barbelow;ellow-green) was identified and characterized to have pleiotropic phenotype including defective ethylene stimulated hypocotyl gravitropism in light-grown seedlings and yellow-green aerial tissues. EDGY1 gene was isolated by map based cloning to encode a novel metalloprotease that belongs to M50 family and it contains a 10-bp deletion in edgy1-1 . Successful complementation with wild type EDGY1 gene and identification of two new alleles containing T-DNA insertion in EDGY1 gene confirm that the mutation in EDGY1 is responsible for the observed phenotypes. Close homologs of EDGY1 are found in Arabidopsis, rice and cyanobacteria. Microscope examination with EDGY1-GFP fusion protein and the immunoblot with EDGY1 antibodies indicate the chloroplast localization of EDGY1 protein. The chloroplast ultrastructure is defective in edgy1 to contain poorly developed thylakoid membrane, and the accumulation of LHC (l&barbelow;ight h&barbelow;arvesting c&barbelow;omplex) protein is significantly reduced. GUS reporter-aided promoter analysis indicated EDGY1 gene is transcribed ubiquitously in various organs. Immunoblot analysis revealed EDGY1 protein is associated with membrane fraction and its protein accumulation varies from organ to organ, being most prominent in leaf and stem and is regulated by light and increased in response to ethylene. Recombinant EDGY1 protein carries proteolytic activity against beta-casein in an ATP-independent but metal dependent manner. The possible target substrates of EDGY1 and its action mode are discussed in detail to explore the possible function of EDGY1 in vivo. All the data together support an important role of EDGY1, as a membrane associated metalloprotease of plastid in ethylene stimulated gravitropism and chloroplast development.