- Title
- The molecular deconstruction of the microRNA160 auxin response factor 10/16/17 expression module in Arabidopsis thaliana
- Creator
- Zimmerman, Kim
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2019
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Small RNAs (sRNAs) form a class of regulatory molecule that is central to the control of gene expression at both the transcriptional and posttranscriptional level. In plants, sRNAs have evolved to form a number of different sRNA species, including the microRNA (miRNAs), transacting small-interfering RNA (tasiRNA), natural antisense transcript siRNA (natsiRNA)and repeat-associated siRNA (rasiRNA) species. The production of each sRNA species is mediated by a DICER-LIKE (DCL) endonuclease, acting with or without the assistance of a DOUBLE-STRANDED RNA BINDING (DRB) protein via the processing of structurally distinct, double-stranded RNA (dsRNA) precursor molecules. In addition to sRNAs, plants also rely on hormonal signalling to control gene expression. Plant hormones fall into a diverse range of classes including the auxins, abscisic acid, brassinosteroid, cytokinins, ethylene, gibberellic acids, jasmonic acid, salicylic acid, and strigolactones. Of these, auxin is a crucial phytohormone that regulates diverse aspects of growth and development. Auxin can elicit molecular responses via multiple pathways. One example of this action is whereby auxin frees AUXIN RESPONSE FACTOR (ARF) transcription factors from their posttranslational inhibition by AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) proteins. Auxin-directed release allows the ARF to function as a classic transcription factor to either promote or repress AUXIN RESPONSE GENE (ARG) expression. Considering that both sRNAs and auxin are demonstrated molecular regulators, it is not a surprise that, on occasion, both regulate the same developmental or adaptive response in plants. Furthermore, sRNA-directed regulation of the protein machinery central to the auxin pathway is well documented, including the miR160/ARF10/ARF16/ARF17 and miR167/ARF6/ARF8 expression modules. To further assess sRNA-directed regulation of the auxin response pathway, a single expression module was selected for detailed molecular analysis. Initial molecular examination of the miR160/ARF10/ARF16/ARF17 expression module in wild-type Arabidopsis thaliana roots, and in the knockout mutant lines, drb1 and drb2, defective in DRB1 and DRB2 activity, respectively, indicated that both DRB proteins are central to the regulation of this expression module. Both DRB1 and DRB2 influence the production of the miR160 sRNA, and following miR160 production, DRB1 and DRB2 aid in the regulation of the abundance of the miR160 target transcripts, ARF10, ARF16, and ARF17. Following the demonstration of the requirement for DRB1 and DRB2 for homeostatic maintenance of the miR160/ARF10/ARF16/ARF17 expression module in Arabidopsis root tissues, the phenotypic and molecular consequence of synthetic auxin, (2,4-dichlorophenoxyacetic acid (2,4-D)) treatment of wild-type Arabidopsis plants and the drb1, drb2, and drb12 mutants were assessed. This analysis revealed that exogenous auxin treatment had a significant impact on root architectural development in all four plant lines, with the promotion of lateral and adventitious root development. Further, at the molecular level, this experiment provided evidence that both DRB1 and DRB2 are required for mediating miR160 production, and subsequently for regulating ARF10, ARF16, and ARF17 transcript abundance as part of the molecular response of Arabidopsis to exogenous auxin treatment. To further characterise the regulatory requirement of DRB1 and DRB2 for maintenance of the miR160/ARF10/ARF16/ARF17 expression module, miR160 resistant versions of ARF10 and ARF16, under the control of their native promoters, were introduced into wild-type Arabidopsis plants, and the drb1 and drb2 mutant backgrounds. In addition, miR160 overexpression lines were also generated in these plants, via the introduction of the MIR160B overexpression transgene. Taken together, the phenotypic and molecular analyses stemming from this experiment revealed that in the absence of DRB1 activity, DRB2 can readily direct miR160 production in root tissues and, further, that miR160-directed regulation of ARF10 and ARF16 expression appeared to be predominantly mediated via a translational repression mechanism of RNA silencing in drb1 roots. Specifically, miR160-directed translational repression of ARF10 appeared to largely control the promotion of lateral and adventitious root growth and development. In summary, the experimental component of this research thesis demonstrated that both DRB1 and DRB2 are required for miR160 production, and to subsequently control miR160-directed expression regulation of ARF10, ARF16, and ARF17 target gene expression. Moreover, in addition to the well documented mRNA cleavage mechanism of ARF10, ARF16, and ARF17 expression regulation directed by the miR160 sRNA, the findings presented here provide strong evidence that miR160-directed translational repression forms an additional layer of posttranscriptional regulatory complexity to control miR160 target gene abundance in Arabidopsis root tissues. This thesis has also identified a possible role for ARF16, in addition to that previously documented for ARF17, in promoting adventitious root growth and development in Arabidopsis.
- Subject
- microRNA; auxin; Arabidopsis; root; genetic control; hormonal control
- Identifier
- http://hdl.handle.net/1959.13/1408770
- Identifier
- uon:35883
- Rights
- Copyright 2019 Kim Zimmerman
- Language
- eng
- Full Text
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