Research Accomplishment Reports 2009

26S Proteasome-Dependent Control of Cytokinin Sensitivity

J.A. Smalle, J. Kurepa
Department of Plant and Soil Sciences

 

Non-Technical Summary

Cytokinins are plant hormones that play a central role in cell division, shoot and root development, distribution of nutrients between plant organs, senescence and many other essential processes. The aim of this work is to identify proteins that control the strenght of the cytokinin response. Studies on plant hormone regulation have revealed an essential role for protein degradation in the control of protein activities that mediate the hormone signal to response ratio. In many cases, the strenght of the hormone response is directly related to the degradation rate of key regulatory proteins. The purpose of this project is to identify the protein degradation-dependent control points in the cytokinin response pathway.

2009 Project Description

The main goal of this project was to identify the 26S proteasome-dependent control points of the cytokinin response pathway. Early cytokinin signaling involves the activation of Type-B Arabidopsis response regulators (ARRs), transcriptional activators that promote cytokinin responses. In addition, Type-B ARRs also promote the upregulation of Type-A ARR transcripts that encode inhibitors of cytokinin responses, thus providing feedback-inhibition control.

Immediately after the initiation of this project we were confronted with two unexpected results when we attempted to classify different proteasome mutants according to the strength of the proteolysis defect. We discovered that these mutants as expected have decreased Ub-dependent proteolysis but surprisingly have increased Ub-independent proteolysis rates. Furthermore, we discovered another unusual feature of proteasome mutants, in that they have an overall increase in cell size in shoot organs which is accompanied by a decrease in cell number, implying reduced cell division activity. Both of these unexpected proteasome mutant characteristics were important for the correct interpretation of the cytokinin response phenotypes of these mutants, and they have been investigated as an integral part of this project.

Our subsequent experiments revealed that these unusual proteasome mutant phenotypes did not invalidate the starting hypothesis of this project, proteasome mutants have decreased cytokinin sensitivity in long term growth responses because of the stabilization of a cytokinin response inhibitor. This was confirmed by the identification of ARR5, a type-A ARR, as a target for 26S proteasome-dependent proteolysis. Surprisingly, our experiments also revealed the stabilization of ARR1, an activator of cytokinin responses, in proteasome mutants. Contrary to ARR5, the ARR1 protein was destabilized by cytokinin treatments, a feature that is common to the activation of many transcriptional activators. Taken together, the data show that the combined stabilization of an activator (ARR1) as well as an inhibitor (ARR5) of cytokinin signaling provides cells with a powerful dampening mechanism to prevent sustained cytokinin responses and allow rapid readjustment to changes in cytokinin concentrations.

In addition, we have identified a family of two ubiquitin ligases, named as ARR1 controlling E3 1 and 2 (ACE1 and 2) that act as negative regulators of cytokinin signaling by catalyzing the proteasome-dependent proteolysis of ARR1.

2009 Impact

It was previously shown that feedback inhibition of the cytokinin response is accomplished by the cytokinin induction of response inhibitors. This project has revealed that feedback inhibition is also accomplished by the accelerated degradation of a response activator. This double negative control of early cytokinin action reveals an essential need of plant cells to limit the strength and the duration of the cytokinin response, suggesting that cytokinin signaling needs to be exceptionally accurate to allow proper plant development.

A second new perspective relates to the essential role played by the Ubiquitin/26S proteasome (UPP) pathway in early cytokinin signaling. Although previous studies had already documented the importance of the UPP in the control of plant hormone signaling pathways, the identities of the UPP targets and E3's in the cytokinin response pathway remained to be shown. The identification of ARR1 and ARR5 as proteasome target proteins, and the identification of ACE1 and 2 as E3's that control ARR1-dependent cytokinin action, provide abundant evidence for an essential role for Ub/26S proteasome-dependent proteolysis in early cytokinin signaling. Cytokinins control several agriculturally important traits, including seed yield, leaf senescence and nutrient allocation. Accordingly, the identification of ACE1 and ACE2 as key regulators of cytokinin sensitivity provides the plant research community with new tools for the genetic engineering of crop species with the aim of introducing beneficial traits.

Finally, this project has also resulted in a novel perspective on proteasome function in plants. We have shown that loss of 26S proteasome function leads to increased 20S proteasome activity resulting in enhanced oxidative stress tolerance. This implies that the ratio of 26S to 20S proteasome abundance is important for plant stress tolerance levels.

Secondly, we showed that mild defects in 26S proteasome activity result in a general increase in plant shoot organ size. This observation provides novel insight into the mechanisms that control final plant size.

2009 Publications

Kurepa J., S. Wang, Y. Li, and J. Smalle. 2009. Proteasome regulation, plant growth and stress tolerance. Plant Signaling & Behavior 4, 924-927.

Wang S., J. Kurepa, and J. Smalle. 2009. Arabidopsis RPN1a is a non-essential 26S proteasome subunit isoform required for optimal plant growth and stress responses. Plant & Cell Physiology 50, 1721-1725.

Kurepa J., S. Wang, Y. Li Y, D. Zaitlin, A. J. Pierce, and J. Smalle. 2009. Loss of 26S proteasome function leads to increased cell size and decreased cell number in Arabidopsis shoot organs. Plant Physiology 150, 178-189.

Kurepa J., and J. Smalle. 2008. To misfold or to lose structure Detection and degradation of oxidized proteins by the 20S proteasome. Plant Signaling & Behavior 3, 418-419.

Kurepa J., A. Toh-e, and J. Smalle. 2008. 26S proteasome regulatory particle mutants have increased oxidative stress tolerance. The Plant Journal 53, 102-114.

Kurepa J., and J. Smalle. 2008. Structure, function and regulation of plant proteasomes. Biochimie 90, 324-335.