Session 4: Hormone Signalling
Chair: Caren Chang, University of Maryland, USA
email:cc203@umail.umd.edu
Caren Chang (University of Maryland) presented evidence that the histidine protein kinase domains of the ETR1 and ERS ethylene receptors can physically associate with the amino-terminal domain of the CTR1 protein kinase. These interactions were detected using the yeast two-hybrid assay and an in vitro protein-binding assay. The interacting portion of CTR1 is the presumed regulatory domain, suggesting that the regulation of CTR1 activity involves direct interaction with the ethylene receptors.
Next, G. Eric Schaller (University of New Hampshire) presented data demonstrating that the ETR1 ethylene receptor contains histidine protein kinase activity. This activity had been postulated based on sequence motifs shared with bacterial two-component regulators, but it was unknown whether the ETR1 receptor necessarily contained this activity. Auto-phosphorylation was observed in vitro using a purified glutathione-S-transferase fusion to the ETR1 histidine kinase domain. Mutations that eliminated either the presumed phosphorylation site (His353), or catalytic residues within the kinase domain, were found to abolish auto-phosphorylation.
Tatsuo Kakimoto (Osaka University) presented results from two types of screens for cytokinin signalling mutants. One screen was for EMS-induced mutations that confer hypersensitivity to cytokinins in callus. This screen yielded recessive mutations in two complementation groups, ckh1 and ckh2. The CKH1 and CKH2 genes may represent negative regulators of cytokinin signalling. In a different strategy, activation-tagged calli were screened for typical cytokinin responses in the absence of cytokinin. Two loci (CKI1 and CKI2) were tagged and cloned: CKI1 is a member of the two-component family of histidine protein kinases, and CKI2, which has not been completely cloned, also appears to be a histidine protein kinase. An interesting possibility is that these genes encode cytokinin receptors.
Dario Bonetta, from Peter McCourt's lab (University of Toronto) described the vegetative defects of a mutant, era1, which was isolated based on having an enhanced seed dormancy response to exogenous abscisic acid. ERA1 is postulated to be a negative regulator of abscisic acid responses. Prior cloning of ERA1 indicated that the ERA1 gene product is a farnesyl transferase. The era1 mutant displays reduced branching, increased leaf size and twice as many floral buds per inflorescence. These defects indicate potential new roles for abscisic acid (and/or protein farnesylation) in vegetative development.
Mark Estelle (Indiana University) discussed similarities between genes from various systems that, taken together, suggest that ubiquitin-mediated events play an important role in auxin response. AXR1 and TIR1 are known to act synergistically in auxin signalling in Arabidopsis. AXR1 has similarity to the ubiquitin-activating enzyme E1, and is similar to yeast ENR2, which is required for specific ubiquitination events in cell cycle regulation. TIR1 is similar to both human SKP2, which is required for the G1/S transition, and yeast GRR1, which is required for cyclin degradation. TIR1 also contains an F-box, which in fungi and mammals binds to a protein (SKP1) that is involved in target recognition in ubiquitin conjugation.
Ingela Fridborg from Peter Engstrom's lab (Uppsala University, Sweden) presented the cloning of SHI, a new putative zinc-finger protein gene. SHI was isolated from an Ac/Ds transposon tagging system designed to recover gain-of-function mutants. The shi mutant phenotype is caused by over-expression of the SHI gene, initiated from a CaMV 35S promoter in the Ds element. The mutant phenotype, which resembles that of gibberellin biosynthesis mutants, is not rescued by exogenous gibberellin. SHI is a potential negative regulator of gibberellin induced stem cell elongation.