The gaseous plant hormone ethylene influences many aspects of plant development and mediates responses to biotic and abiotic stresses. A framework of the ethylene signaling pathway has been assembled using a combination of genetic and biochemical analysis in Arabidopsis (Arabidopsis thaliana) although this pathway is not completely understood. Furthermore, ethylene influences developmental processes and responses to environmental challenges that are not part of the Arabidopsis life cycle. Mutation at the Green-ripe (Gr) and reversion to ethylene sensitivity 1 (rte1) loci, which encode homologous proteins of unknown biochemical function, influence ethylene responses in tomato (Solanum lycopersicum) and Arabidopsis thaliana, respectively. In Arabidopsis, RTE1 is required for function of the ETR1 ethylene receptor and acts predominantly through this receptor isoform via direct protein-protein interaction. In tomato, mutation at the Gr locus causes ectopic expression of GR leading to reduced ethylene responsiveness in a subset of tissues, which can be recapitulated by over-expression of GR driven by the CaMV35S promoter. The tomato genome contains two additional GR homologs designated GREEN RIPE LIKE 1 (SlGRL1) and GREEN RIPE LIKE 2 (SlGRL2), whose function, and role in ethylene signaling remain unknown.In this study, the potential role of SlGRL1 and SlGRL2 in ethylene signaling was investigated together with their relationship to SlGR and RTE1 of Arabidopsis. SlGR, SlGRL1 and SlGRL2 are differentially expressed during development and in response to ethylene treatment and each protein is predominantly localized in the Golgi. A combination of over-expression in tomato and complementation of the rte1-3 mutant allele indicates that SlGR and SlGRL1 influence distinct ethylene responses suggesting the existence of separate ethylene-signaling modules in tomato that are influenced either individually by SlGR or SlGRL1 or together by both proteins. In contrast, over-expression of SlGRL2 in tomato did not reveal any altered ethylene-related phenotypes suggesting that this gene may not be involved in ethylene signaling. Interestingly, over-expression of AtRTE1 in tomato leads to reduced ethylene responsiveness in a subset of tissues, which more closely resemble the SlGRL1 lines than SlGR lines. Phylogenetic and sequence analysis indicated an expansion of the GR/RTE1 family within the Solanaceae family of the eudicot lineage. Typically eudicot species contain a single gene closely related to AtRTE1 and SlGRL1. In contrast, members of the Solanaceae family contain a second more divergent gene, defined by SlGR that appears to be restricted to this plant family. Furthermore, putative GR orthologs are relatively divergent when compared to putative GRL1orthologs of the Solanaceae family leading to the hypothesis that they may exhibit altered functional properties. This hypothesis was confirmed through over-expression of putative GR orthologs in tomato and through complementation of the rte1-3 mutant allele. Utilizing a comparative approach, a series of amino acid residues were identified that correlate with the ability of the Solanaceae GR and GRL1 proteins to complement the rte1 mutant phenotype and these were tested through the expression of synthetic constructs which carry altered amino acids leading to the identification of a set of 10 amino acids that are important for the ability of Solanaceae GR and GRL1 orthologs to complement the rte1 mutant phenotype. Together, these data provide considerable new insight into the role of the GR/RTE1 family in controlling ethylene responses in plants.
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