Functional studies on ARIADNE genes of Arabidopsis

Recently, we identified and classified the ARIADNE (ARI) protein family in the model plant Arabidopsis thaliana (Mladek et al., 2003; www.arabidopsis.org/info/genefamily/ARIADNE.html). Members of this family share two C3HC4 RING (really interesting genes)-finger motifs, which are separated by a central C6HC domain called the "in between RING-finger" (IBR), B-box or DRIL (double RING finger-linked) domain. Similar proteins are widespread in eukaryotes, including humans. There is increasing evidence that RING-finger proteins mediate ubiquitination of proteins targeted for proteolysis via the 26S proteasome pathway. Furthermore, RING-finger proteins play a central role in determining substrate specificity. Ubiquitin-mediated proteolysis is involved in many aspects of plant development including regulation of hormone signaling, embryogenesis, cell cycle, photomorphogenesis, circadian rhythms, flower development, leaf senescence, disease resistance and wax synthesis. At present, around 30 genes have been assigned to this pathway. Nevertheless, only four are RING-finger proteins and none belongs to the ARI class. Using sequence comparison and phylogenetic analyses we have grouped the 16 AtARI proteins of Arabidopsis into three subclasses. Interestingly, the members of two subclasses are absent in invertebrates and vertebrates, possibly indicating a plant- specific function. The objective of the project is to understand i) the functional role, ii) the biochemical activity and iii) the regulation of some members of the ARI gene family in Arabidopsis. The expression level at diverse developmental stages and different organs has already been characterized in our group (Mladek et al., 2003). Furthermore, promoter:reporter constructs were already generated for half of the members of the ARI family. We also prepared the phenotypic analysis of double and triple mutants by isolating knockouts and generating the required crosses. For three of the AtARI proteins we generated specific peptide antibodies for cell biological and biochemical analyses and produced recombinant proteins in E. coli. With these tools at hands the project will determine the role of ARI proteins in plants.

Recently, we identified and classified the ARIADNE (ARI) protein family in the model plant Arabidopsis thaliana (Mladek et al., 2003; www.arabidopsis.org/info/genefamily/ARIADNE.html). Members of this family share two C3HC4 RING (really interesting genes)-finger motifs, which are separated by a central C6HC domain called the "in between RING-finger" (IBR), B-box or DRIL (double RING finger-linked) domain. Similar proteins are widespread in eukaryotes, including humans. There is increasing evidence that RING-finger proteins mediate ubiquitination of proteins targeted for proteolysis via the 26S proteasome pathway. Furthermore, RING-finger proteins play a central role in determining substrate specificity. Ubiquitin-mediated proteolysis is involved in many aspects of plant development including regulation of hormone signaling, embryogenesis, cell cycle, photomorphogenesis, circadian rhythms, flower development, leaf senescence, disease resistance and wax synthesis. At present, around 30 genes have been assigned to this pathway. Nevertheless, only four are RING-finger proteins and none belongs to the ARI class. Using sequence comparison and phylogenetic analyses we have grouped the 16 AtARI proteins of Arabidopsis into three subclasses. Interestingly, the members of two subclasses are absent in invertebrates and vertebrates, possibly indicating a plant- specific function. The objective of the project is to understand i) the functional role, ii) the biochemical activity and iii) the regulation of some members of the ARI gene family in Arabidopsis. The expression level at diverse developmental stages and different organs has already been characterized in our group (Mladek et al., 2003). Furthermore, promoter:reporter constructs were already generated for half of the members of the ARI family. We also prepared the phenotypic analysis of double and triple mutants by isolating knockouts and generating the required crosses. For three of the AtARI proteins we generated specific peptide antibodies for cell biological and biochemical analyses and produced recombinant proteins in E. coli. With these tools at hands the project will determine the role of ARI proteins in plants.