A genetic screen for mutants defective in IAA1-LUC degradation in Arabidopsis thaliana reveals an important requirement for TOPOISOMERASE6B in auxin physiology

Abstract

Many plant growth and developmental processes are modulated by the hormone auxin. Auxin-modulated proteolysis of Aux/IAAs, a family of transcriptional repressors, represents a major mode of auxin action. Auxin facilitates the interaction of Aux/IAAs with TIR1/AFB F-box proteins, promoting their ubiquitination by the SCF^TIR1/AFB ubiquitin E3 ligase leading to subsequent degradation by the 26S proteasome. To identify new genes regulating Aux/IAA proteolysis in Arabidopsis thaliana, we took a genetic approach, identifying individuals with altered degradation of an IAA1-luciferase fusion protein (IAA1-LUC). A mutant with 2-fold slower IAA1-LUC degradation rate compared with wild-type was isolated. Positional cloning identified the mutant as an allele of TOPOISOMERASE6B, named top6b-7. TOP6B encodes a subunit of a plant and archea-specific enzyme regulating endoreduplication, DNA damage repair and transcription in plants. T-DNA insertion alleles (top6b-8 and top6b-9) were also analyzed. top6b-7 seedlings are less sensitive to exogenous auxin than wild-type siblings in primary root growth assays, and experiments with DR5:GUS. Additionally, top6b-7 seedlings have a 40% reduction in the amount of endogenous IAA. These data suggest that increased IAA1-LUC half-life in top6b-7 probably results from a combination of both lower endogenous IAA levels and reduced sensitivity to auxin.

Keywords:

• Arabidopsis thaliana
• Aux/IAA
• auxin
• DR5:GUS
• indole-3-acetic acid
• luciferase fusions
• protein degradation
• TOPOISOMERASE6B

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors gratefully acknowledge support from the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, US. Department of Energy (contracts DE-FG02–03ER15416 and DE-FG02–09ER16077) to JC. In addition, the initial development of the genetic screen was supported in part by a grant from the National Science Foundation (IBN 0212659) to JC. Additional support was from the Paul K. and Ruth R. Stumpf Endowed Professorship in Plant Biochemistry to J. Callis. We wish to acknowledge the UC-Davis Controlled Environment Facility (CEF) for assistance in the propagation of transgenic plants. We would like to thank Joey Lasiter and Gurjeet Bath for help with positional cloning and genotyping, Tania Gonzalez for assistance with auxin dose-response experiments, and Marissa Simon for advice on GUS experiments. We thank former and current members of the Callis laboratory and Dr. Anne Britt (UC-Davis) for helpful discussions.