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Title:Discovery of novel regulators and genes in nitrogen utilization pathways in maize
Author(s):Arp, Jennifer Julia
Director of Research:Moose, Stephen P
Doctoral Committee Chair(s):Moose, Stephen P
Doctoral Committee Member(s):Hudson, Matthew; Huber, Steven; Vodkin, Lila
Department / Program:Crop Sciences
Discipline:Crop Sciences
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Nitrogen use efficiency (NUE)
functional genomics
gene expression
asparaginase
asparagine synthetase
artificial selection
Abstract:Nitrogen (N) is a key limiting plant nutrient and its availability is expected to have significant impacts on the expression of genes that function in nitrogen metabolism and growth responses to N. Although a key target for improving maize yield response to nitrogen, relatively little is known about the gene regulatory systems that modulate N remobilization. Advancements in sequencing and computational analysis have led to systems-level understanding of gene expression in the model plant Arabidopsis. These improvements can be applied in maize using transcriptional profiling and established populations as genetic tools for understanding the regulation of N remobilization. Elucidating the N-responsive transcriptome is the first step toward understanding the complex interaction network which regulates maize response to N. This analysis discovered candidate genes which were tested using functional genomic techniques including mutants and near-isogenic lines. RNAseq was performed to profile changes in gene expression in both leaf and developing ear tissues of plants grown in the field with different levels of soil N supply during the period of active N remobilization. RNA profiles were obtained from three genotypes that differ in N utilization: Illinois High Protein (IHP1), Illinois Low Protein (ILP1) and B73. IHP1 and ILP1 are inbred strains developed from the Illinois long term selection experiment (ILTSE) for seed nitrogen concentration. The developmental dynamics of nitrogen response were described for the reference genotype, B73, and were coordinated with physiological and environmental dynamics. Classes of genes that showed coordinated transcriptional responses to N across different tissues and developmental stages within a genotype may indicate key control points in N cycling between source and sink tissues. Expression profiling was used to identify gene expression “hubs”—genes that interact with many other N-responsive genes in a gene network. Putative mutant alleles from reverse genetics resources were obtained for candidate genes identified in chapter 2, but few were confirmed to decrease gene expression. One mutant, zap1-mum1, was knocked out in a tissue specific manner, with expression loss only in the leaves. The zap1-mum1 mutant was grown in the N-responsive field and profiled for transcriptomic and phenotypic response. In total, 875 genes were differentially expressed in the zap1-mum1 mutant compared to wild-type. In addition, the nitrogen responsive component of the transcriptome was impacted. Phenotypically, no obvious visual defects were observed in vegetative or floral structures. However, for both field N rates, the zap1-mum1 mutant had increased ear weights and kernel number, showing the indirect effect of perturbation of the vegetative transcriptome on sink tissues. Relative to B73, IHP1 is more efficient at taking up and translocating nitrogen and ILP1 exhibits a reduced ability to uptake and remobilize nitrogen; these different nitrogen use strategies were apparent in the transcriptome profiles of these genotypes. In total, over one-quarter of all maize genes exhibited variation in DNA sequence or RNA expression between IHP1 and ILP1, revealing the powerful evolutionary change resulting from the combined impacts of long-term reproductive isolation and recurrent divergent selection. Moreover, grain protein is a highly quantitative trait, and many genes were likely affected specifically by selection. Genes linked to component traits and known aspects of grain protein like nitrogen and carbon metabolism would be strong candidates for selection rather than drift, and many such genes were identified for their differences in the IHP and ILP transcriptomes. Only 376 genes were found to exhibit strong divergence in both allele frequencies and expression level among IHP and ILP, these genes are most likely to influence grain protein. In addition to allelic and differential expression between IHP and ILP, candidate genes were identified through gene expression networks and nitrogen response in IHP. Comparing all four methods of identifying candidate genes showed ten genes that were significant in all analyses. This small set and other candidate alleles could be exploited to improve maize breeding. Prior studies in our lab have shown that changes in the regulation of both the synthesis and degradation of asparagine are associated with the different N remobilization phenotypes of IHP1 and ILP1. The effect of asparagine cycling alleles was confirmed using a series of near isogenic lines where variant alleles for these genes were introgressed into the IHP1 background, and vice versa. Grain protein concentration decreased by 0.9% on average in IHP1 when ILP1 alleles for asparagine synthetase 3 or asparaginase were introgressed. Protein concentration in the ILP1 background increased commensurately, by 0.8% on average in the near isogenic lines. This change in grain protein was significant, but was still insufficient to account for all of the grain protein variation between IHP and ILP. Further characterization of the differences between IHP and ILP is necessary.
Issue Date:2017-04-21
Type:Thesis
URI:http://hdl.handle.net/2142/97436
Rights Information:Copyright 2017 Jennifer Arp
Date Available in IDEALS:2017-08-10
Date Deposited:2017-05


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