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Functional domains of the trihelix family transcription factor protein PETAL LOSS in arabidopsis

posted on 16.02.2017, 02:54 by Quon, Tezz Leigh
PETAL LOSS (PTL) acts to repress growth between developing sepals in Arabidopsis thaliana. The loss of ptl function results in overgrowth in the inter-sepal zone and a disruption to auxin regulation which causes a downstream loss of second whorl organ initiation. PTL encodes a member of the GT-2 clade of the trihelix transcription factor family and contains two trihelix DNA binding domains, a central α-helical domain, a C-terminal activation region and three nuclear localisation sequences. This study reports novel tests of the biological importance of each domain in protein function, and using a developed method examines protein-protein interactions in planta. By mutating key residues in each trihelix, I was able to show that each is individually required for function, and to provide evidence for the involvement of charged residues in DNA interaction. By replacing each trihelix in PTL with the corresponding domain from related proteins I was able to show that sequence targets are differentially shared, with the N-terminal trihelix domain swaps showing the strongest ability to replace PTL function. This, combined with the inability of full length related trihelix proteins to replace PTL when expressed in the PTL domain, revealed the importance of other unidentified regions of PTL for function. Through the use of a novel method I developed to visualise interactions between proteins transiently expressed in planta, I confirmed that PTL forms homodimers. By dissecting the PTL protein I revealed the importance of the central α-helical domain in this interaction. In addition, PTL interacted with other GT-2 clade proteins and the kinase AKIN10 through the central domain. Interaction of PTL with the KNOX protein STM was instead found to occur through the trihelix domains. PTL has been shown to be able to activate transcription. This study is the first demonstration of the role this plays in its biological function. The removal of the endogenous C-terminal activation region significantly reduced its complementation ability, whilst its substitution with the VP16 activation domain was able to completely compensate for the lack of the endogenous domain. The glutaredoxin ROXY1 shares a similar mutant phenotype to PTL. To test whether there is an overlap in function, I first showed that PTL and ROXY1 proteins can interact. I was unable to provide evidence that any of the cysteines tested were targets for reduction by ROXY1. However I showed that the presence of PTL is required for ROXY1 to be able to maintain second whorl organ number in experiments involving both the ectopic expression of ROXY1 and loss of function mutants. I also obtained evidence that PTL and ROXY1 act redundantly to control growth within sepals and to maintain correct organ identity in the second whorl. Thus, I have revealed the importance for each trihelix in PTL function, showed PTL is able to form dimers mostly through the α-helical domain, provided further evidence that PTL acts as an activator of transcription and showed that PTL and ROXY1 share function but PTL is unlikely to be a direct target of ROXY1.


Campus location


Principal supervisor

David Robert Smyth

Year of Award


Department, School or Centre

Biological Sciences


Doctor of Philosophy

Degree Type



Faculty of Science