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Type: Thesis
Title: Identification and Functional Characterization of Long Noncoding RNAs Involved in Endosperm Development of Arabidopsis thaliana
Author: Do, Quang Trung
Issue Date: 2018
School/Discipline: School of Biological Sciences
Abstract: Elucidating the molecular events underlying endosperm and embryo development in angiosperms are important both in terms of understanding plant development and developing new methods to enhance crop productivity. Seeds arguably undergo one of the most complex developmental programs of any plant organ, and are therefore subject to many gene regulatory mechanisms. In recent years, it has become clear that various classes of noncoding ribonucleic acid (ncRNA) and covalent histone modifications have important roles in gene regulation. Of these ncRNAs, small RNAs (20 to 25 nucleotides) are beginning to be understood; however, less is known about the role and complexity of long noncoding RNAs (lncRNAs). Here, we detail the methodology for purifying specific cell types, RNA sequencing, bioinformatic annotation of lncRNAs and investigation of biological function, using the reference plant Arabidopsis thaliana. We also detail methodology for highly reproducible bisulfite treatment of RNA, efficient locus-specific PCR amplification, detection of 5-methycytosine that includes sequencing on the Illumina MiSeq platform and bioinformatic calling of converted and non-converted cytosines. Next, we investigated the contribution of transposable elements (TEs) to long intergenic noncoding RNAs (lincRNAs) during plant development and abiotic stress tolerance. Using deep Illumina sequencing, we identified 47, 611 and 398 TE-associated lincRNAs (TE-lincRNAs) from Arabidopsis, rice and maize, respectively. We demonstrated that some of these TE-lincRNAs were tissue specifically transcribed and others were expressed after salt, abscisic acid (ABA) or cold treatments. After identification and characterization of about 50 TE-lincRNA mutants, the mutant TE-linc11195 was identified as having less sensitivity to ABA. The TE-linc11195 mutant had longer roots and higher shoot mass when compared with wildtype in the presence of ABA. Our data suggest that TE-lincRNAs might be a promising reservoir to adapt to changing environmental conditions. We also explored the potential roles of lncRNAs in regulating epigenetic modifications deposited by the Polycomb Repressive Complex 2 (PRC2) complex. We immunoprecipitated PRC2-associated lncRNAs and sequenced the bound RNAs by Illumina sequencing. We validated the expression of these PRC2-associated lncRNAs by strand-specific reverse transcription polymerase chain reaction (RT-PCR), and computationally predicted their functions in seed development by association with H3K27me3-targeted (PRC2) genes. Interestingly, the data also showed that G-tract motifs (G2L1-4) are significantly enriched among PRC2-binding transcripts. This dataset provides an initial insight into PRC2-associated RNAs and may contribute towards understanding PRC2 function. Further, we identified 615 lncRNAs from Arabidopsis thaliana one day after pollination (DAP) of siliques using high-throughput Illumina sequencing. Next, we showed that some of these lncRNAs could be transcribed in an organ-specific manner or more broadly transcribed in root, flower and silique organs. Among the broadly transcribed lncRNAs, some were differentially abundant, while others were similarly abundant across all three tissue types. We also investigated the function of 42 lncRNAs by using either artificial microRNAs or RNAi to knockdown the targets. Of these, the knockdown plants of lnc1246 were observed to have smaller cells and organs in all tested tissues: roots, cotyledons and seeds. We also demonstrated with open reading frame analysis that LNCRNA_1246 was unlikely to encode for a functional protein. Functional analysis using a recessive lnc1246 mutant allele and reciprocal crosses demonstrated that LNCRNA_1246 acted maternally to reduce seed size. This could be a result of smaller cells within the outer integument layer and a smaller integument. Together, our results demonstrate that lncRNAs are broadly transcribed and at least one plays an important role in seed size. Overall, this thesis focuses on the genome-wide identification and characterization of lncRNAs from A. thaliana 1DAP silique and the possible functions of lncRNAs in plant development by interacting with their partners, such as TEs and FIS2–PRC2 complexes. It also illustrates the potential effects of lncRNAs on diverse biological processes during plant evolution.
Advisor: Searle, Iain
David, Rakesh
Dissertation Note: Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2018
Keywords: Arabidopsis thaliana
seed size
transposable element
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
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