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Molecular Genetic Analysis of a Novel Feedback Inhibition Mechanism in the Cytokinin Response Pathway
Department of Plant and Soil Sciences
Cytokinins are a class of hormones that play essential roles in plant development. By controlling the development and activities of shoot and root meristems, they influence agriculturally important traits such as nutrient allocation and distribution, and flower, fruit and seed yield.
Although great progress has been made in identifying the components of the cytokinin response pathway, our knowledge of how plants control the intensity and the duration of cytokinin responses has remained limited. This type of information is however essential for the correct understanding of meristem development and function.
The project will address the important question in cytokinin hormone regulation of how plants respond to the cytokinin signal while maintaining sensitivity to fluctuations in cytokinin concentrations. This type of flexibility is essential for maintaining accuracy in developmental processes, and yet how plants do this has remained elusive.
2011 Project Description
We have generated novel analytical tools for studying type-B ARR proteolysis mechanisms. To monitor type-B ARR protein abundances we have generated antisera to the type-B ARRs 1, 10 and 12. These three family members have been shown to mediate most of the cytokinin response. The antisera approach was chosen because the use of tagged versions of the ARR1 protein resulted in decreased cytokinin sensitivity compared to ectopic expression of the unmodified protein. Furthermore, the posttranslational control of tagged ARR1 versions was different from the unmodified form precluding their use for uncovering ARR1 proteolysis controls.
Cytokinin and proteaosme-dependent posttranslational control of the endogenous ARR1 and ARR10 proteins have been investigated in wild type and proteasome mutant plants and also in a set of mutants that are defective at key steps in the cytokinin response pathway.
Transgenic lines have been isolated that express different versions of the ARR1 and ARR10 proteins, including the wild type, phosphomimic and phosphoblock versions. The phosphomimic version contains an aminoacid substitution at the conserved residue in the receiver domain of type-B ARRs which is believed to be the phospho-acceptor site in the cytokinin signal transduction mechanism. Accordingly, the phosphomimic version of a type-B ARR protein resembles the phosphorylated version and is predicted to be constitutively active. In contrast, a phosphoblock version contains an aminoacid substitution at the same conserved position, but which does not resemble the phosphorylated form and therefore is expected to keep the protein inactive by making it insensitive to the cytokinin signal. Double mutants have been isolated that have both members of the candidate type-B- ARR targeting ubiquitin ligase gene pair knocked out.
In addition, we have characterized other cytokinin-regulated genes that encode ubiquitin ligases, with the long-term aim of further increasing our understanding of the proteolysis controls of this hormone response pathway.
We found an essential difference in the proteolysis controls of ARR1 and ARR10. While both proteins are unstable and targeted for 26S proteasome-dependent proteolysis, we find that cytokinin only controls ARR1 stability but not ARR10. We also find confirmation of this when using various cytokinin signaling mutants. This observation suggests that cytokinin signaling is more complex than previously perceived.
The above observations have been confirmed when using transgenic plants that ectopically express the wild type and mutant versions of ARR1 and ARR10. We find that phosphomimic mutations affect the stability of both proteins. Surprisingly however, we find that phosphomimic ARR1 stability remains affected by cytokinin treatments. This suggests that cytokinin signaling controls the ARR1 protein in more than one way.
We find that the phosphomimic ARR1 and ARR10 lines indeed display constitutive cytokinin response phenotypes. However, contrary to ARR10, phosphomimic ARR1 lines are also cytokinin hypersensitive resulting in a response that is stronger than any response that can be induced by treating the wild type with high concentrations of the hormone.
The double knockout mutant of the ubiquitin ligase gene pair did not have an enhanced phenotype compared to the single mutants. We concluded from this that other ubiquitin ligases are also involved in type-B ARR proteolysis and that these E3's provide partial complementation for the loss of function of both genes. We are currently investigating other candidate ligases for their involvement in type-B ARR turnover.