The spatial regulation of Auxin Response Factors

Jingyi, Han (2021) The spatial regulation of Auxin Response Factors. PhD thesis, University of Nottingham.

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Auxin regulates plant growth and development through the transcription factors (TFs) of the AUXIN RESPONSE FACTOR (ARF) gene family. Class A ARFs, ARF5, 6, 7, 8 and 19 are transcriptional activators, and control many developmental processes. However, we only have limited understanding on how these ARFs can mediate such diverse developmental responses. In this study we investigated expression patterns of ARFs in the root and shoot apical meristems and showed that they have specific domains of expression. Through a yeast one-hybrid and protoplast assay, we identified a network of transcriptional repressors which regulated these ARFs. To validate this network, we over-expressed candidate TFs in ARF reporter lines, generated new reporter lines to check the co-expression of TFs and ARFs in specific tissues and carefully quantified auxin specific phenotypes in the mutants of candidate TFs. Collectively, these results support a mechanism in which the spatial and temporal expression of ARFs is modulated mainly by tissue specific repression.

In order to understand ARF promoter specificity in auxin responses in the most efficient manner, we saw the opportunity to improve the mechanism for creating reporter constructs. In order to improve live imaging of gene expression in its geometric context, we developed a new series of lines (DEAL) showing cellular anatomy which can efficiently combine with auxin sensors and other reporters by Greengate cloning.

Analysing the 5 ARF reporter lines, we found ARF7 has an interesting expression pattern. A broad expression of ARF7 was observed in root tips only in the reporter containing an in-frame fusion of GFP from 3kb promoter to the second exon. Whilst this region upstream of the transcriptional start site had no effect on expression in the shoot. I demonstrated that the first intron plays an important role in transcriptional regulation in the root meristem. A swap experiment in which the first intron was moved to the 5’-UTR showed the position of intron is not essential for the correct expression. Therefore, we propose that it is the sequence within this intron that is required and that key transcription factors bind to this region. Bioinformatic analysis into potential binding sites within this promoter suggests that NACs and MYBs bind in this intron to regulate ARF7 expression in the root apical meristem. Collectively these data support a role in which root- and shoot-specific binding motifs coordinate the elaborate expression patterns of ARFs.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Bishopp, Anthony
Voss, Ute
Keywords: Auxin, Plant growth, Transcription factors, TFs, Auxin Response Factors, ARF, Plant genetics
Subjects: Q Science > QK Botany > QK710 Plant physiology
Faculties/Schools: UK Campuses > Faculty of Science > School of Biosciences
Item ID: 66997
Depositing User: Han, Jingyi
Date Deposited: 18 Jan 2022 15:47
Last Modified: 08 Dec 2023 04:30

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