Plants are often subjected to extreme environmental conditions and must adapt rapidly. The phytohormone abscisic acid (ABA) accumulates under abiotic stress conditions, signaling transcriptional changes that trigger physiological responses. Epigenetic modifications are also required to facilitate transcription, particularly at genes exhibiting temporal, tissue-specific and environmentally induced expression. In maize (Zea mays), MEDIATOR OF PARAMUTATION 1 (MOP1) is required for progression of an RNA-dependent epigenetic pathway that regulates transcriptional silencing of loci across the genome. As critical regulators of gene expression, MOP1 and ABA pathways predictably regulate specific genes in a coordinated manner. In one project, the amino acid sequence of DNG103 and the gene promoter region were analyzed for conserved domains and cis-responsive elements, respectively. DNG103 is similar to the Arabidopsis ROS1, contains the conserved domains of a DNA glycosylase with DNA demethylase activity, and contains ABA-responsive elements (ABREs) in its promoter region. Transcript levels of Dng103, and the ABA-responsive gene Viviparous 1 (Vp1), were monitored in maturing embryos from two genotypes placed in culture under different conditions. Expression of both genes decreased after culture in hormone-free medium and was induced by ABA in Mop1 wildtype. Dng103 and Mop1 showed decreased expression in mop1-1 and dng103 mutants, respectively. Dng103 is not responsive to ABA in the mop1-1 mutant and Vp1 has reduced sensitivity. Therefore, DNG103, MOP1 and ABA might have common regulatory targets. Protoplast isolation and transfection protocols were standardized alongside the production of reporter constructs containing the Dng103 and Vp1 promoters. This technology will allow for the promoter characterization of genes of interest through quantifiable luciferase expression, using different conditions and genotypes. To identify genome-wide ABA-induced, MOP1-dependent and independent transcriptional responses, mop1-1 and Mop1 homozygous seedlings were subjected to exogenous ABA and RNA-sequencing. A total of 3,242 differentially expressed genes (DEGs) were identified in four pairwise comparisons. Overall, the loss of MOP1 exaggerated some ABA-induced changes in gene expression. The highest number of DEGs were identified in ABA-induced mop1-1 mutants, including many transcription factors. A gene regulatory network was used to predict relationships between DEGs; suggesting multifaceted regulatory scenarios including direct and indirect transcriptional responses to genetic disruption (mop1-1) and/or stimulus-induction of a hierarchical, cascading network of responsive genes. Additionally, a modest increase in CHH methylation at putative MOP1-RdDM loci in response to ABA was observed in some genotypes, suggesting that MOP1 might be necessary to achieve environmentally induced transcriptional responses in maize. To understand the multistep ABA response of identified ABA-induced genes, an ABA-time course was carried out in another project using four different time points. The previously predicted ABA-responsive transcription factors Hb41 and Bzip4 showed an early induction to ABA and the late embryogenesis abundant Rab17 gene was induced during the transition from early to late response. Together, these results indicate that MOP1 and ABA act at multiple levels within complex, connected transcriptional networks to mediate tissue-specific growth and responses to some abiotic stresses. LIST OF SUPPLEMENTARY FILES 1. Significant differentially expressed genes in analysis groups I-VIII. 2. GO term enrichment in groups I, II, V and VI. 3. Arabidopsis and maize homologous transcription factors and target genes in ABA transcription factor hierarchical network and their corresponding gene expression. 4. Transcription factors and target genes in ABA transcription factor hierarchical network separated by transcriptional levels. 5. Group model parameters per gene. 6. Genome-wide siRNA changes in mop1-1 mutant. 7. TGS2 target genes. 8. Sequence Capture (SeqCap) DNA methylation ratios in all sequence contexts. 9. Promoter DNA methylation for Mop1 wildtype ABA-responsive DEGs. 10. MOP1-ABA targets with a loss of siRNA and DNA methylation at ABRE sites. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / 2019 / November 6, 2019. / Abscisic acid (ABA), Epigenetics, Mediator of paramutation (Mop1), RNA-dependent DNA-Methylation (RdDM), siRNAs, Zea mays / Includes bibliographical references. / Karen M. McGinnis, Professor Directing Dissertation; Hong Li, University Representative; Henry W. Bass, Committee Member; Brian P. Chadwick, Committee Member; Jonathan H. Dennis, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_753070 |
| Contributors | Vendramin Alegre, Stefania (author), McGinnis, Karen M. (professor directing dissertation), Li, Hong (university representative), Bass, Hank W. (committee member), Chadwick, Brian P. (committee member), Dennis, Jonathan Hancock (committee member), Florida State University (degree granting institution), College of Arts and Sciences (degree granting college), Department of Biological Science (degree granting departmentdgg) |
| Publisher | Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text, doctoral thesis |
| Format | 1 online resource (167 pages), computer, application/pdf |
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