However, in support of such a role for subfamily I receptors, histidine kinase activity has been reported for the ETR1 kinase domain expressed in yeast ( 15). The role of the histidine kinase activity in receptor signaling could not be fully addressed in previous studies because no loss-of-function ERS1 mutant had been identified. If transmission of the ethylene signal occurs through canonical two-component phosphotransfer, ETR1 and ERS1 could perform a specialized function given that only ETR1 and ERS1 possess fully conserved histidine kinase domains. The target of receptor-CTR1 signaling is thought to be the NRAMP-related EIN2 protein ( 14), null mutations in which cause global ethylene insensitivity, which is thought to act downstream of the ethylene receptors and CTR1 based on epistasis analysis ( 12). According to this model, ethylene acts as an inverse agonist by negatively regulating receptor-CTR1 signaling. This model accounts for the observations that ( i) loss-of-function mutations in either CTR1 or combinations of receptor isoform genes leads to a constitutive response phenotype ( 12, 13) and ( ii) dominant gain-of-function mutations in any of the five receptor isoforms confer global ethylene insensitivity in the plant, presumably by locking the receptor into an active signaling state. According to this model, the five receptor isoforms interact with CTR1, a RAF-related kinase, to negatively regulate response pathways. The roles of ethylene receptor isoforms in signal transduction must be considered in the context of the prevailing model for the signal transduction pathway based on genetic studies in Arabidopsis ( 2, 11). Based on these distinguishing features and overall sequence similarity, the members of the ethylene receptor family from Arabidopsis can be divided into two subfamilies: subfamily I, which includes ETR1 and ERS1, and subfamily II, which includes ETR2, EIN4, and ERS2. The residues thought to be essential for histidine kinase activity are conserved in ETR1 and ERS1 ( 7, 8), but are not completely conserved in ETR2, EIN4, and ERS2 ( 9, 10). The bacterial systems transduce signal via autophosphorylation of a histidine residue in the kinase transmitter domain, followed by transfer of phosphate to an aspartate residue in the receiver domain of a response regulator protein ( 6). In all receptor isoforms, the ethylene sensor domain is followed by a domain showing varying degrees of sequence similarity to the histidine kinase catalytic domains characteristic of bacterial two-component regulators. Additional studies with ETR1 indicated that ethylene binding is mediated through a copper cofactor ( 5). All members contain an N-terminal, membrane-associated sensor domain that shows high-affinity ethylene binding when expressed in yeast ( 3, 4). However, transformation of either the ers1-2 etr1-6 or ers1-2 etr1-7 mutant with a kinase-inactivated ETR1 genomic clone also resulted in complete restoration of normal growth and ethylene responsiveness in the double-mutant background, leading to the conclusion that canonical histidine kinase activity by receptors is not required for ethylene receptor signaling.Īlthough the five members of the ethylene receptor family from Arabidopsis share a high degree of sequence similarity, each has distinguishing characteristics. Subfamily I constructs restored normal growth, whereas subfamily II constructs failed to rescue the double mutant, providing evidence for a unique role for subfamily I in receptor signaling. Chimeric transgene constructs in which the ETR1 promoter was used to drive expression of cDNAs for each of the five receptor isoforms were transferred into the ers1-2 etr1-7 double-mutant plants. The adult ers1-2 etr1-6 and ers1-2 etr1-7 phenotypes included miniature rosette size, delayed flowering, and both male and female sterility, whereas etiolated-seedling responses were less affected. The double mutants exhibited a severe constitutive ethylene response phenotype consistent with the negative regulator model for receptor function. To examine the role of the conserved histidine kinase domain in receptor signaling, ers1 etr1 loss-of-function double mutants were generated. Of the five members of the Arabidopsis ethylene receptor family, members of subfamily I (ETR1 and ERS1) contain completely conserved histidine kinase domains, whereas members of subfamily II (ETR2, EIN4, and ERS2) lack conserved residues thought to be necessary for kinase activity. Ethylene signaling in plants is mediated by a family of receptors related to bacterial two-component histidine kinases.
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