Progress 04/01/16 to 03/31/18
Outputs
Target Audience:The research accomplished within this project will contribute to the body of knowledge about plant nitrogen response and was disseminated to other scientists working in plant sciences at Plant and Animal Genome (PAG) and the Maize Genetics Conference. I plan to attend the Project Director's Meeting in Washington D.C. in August 2018. Changes/Problems:In May 2017, I graduated from University of Illinois at Urbana Champaign and deposited thesis: Discovery of Novel Regulators and Genes in Nitrogen Utilization Pathways in Maize. Upon graduation, I accepted a position at the Donald Danforth Plant Science Center as a postdoctoral associate advised by Dr. Thomas Brutnell and Dr. Douglas Allen, started May 2017. After the termination of Dr. Thomas Brutnell by the Danforth Center for sexual harassment, I am currently advised by Dr. Douglas Allen and Dr. Todd Mockler. Activity on this USDA research was completed on 5/15/2017, and remaining funds should be returned to the USDA by the University of Illinois at Urbana-Champaign. What opportunities for training and professional development has the project provided?Graduation from the University of Illinois at Urbana Champaign and deposit of thesis: Discovery of Novel Regulators and Genes in Nitrogen Utilization Pathways in Maize, May 2017. Accepted position at Donald Danforth Plant Science Center as a postdoctoral associate advised by Dr. Thomas Brutnell and Dr. Douglas Allen, started May 2017. Attendance at Plant and Animal Genome Conference, January 2017, 2018. Attendance and poster presentation at Maize Genetics Conference March 2017, 2018. Poster Award at both UIUC Pioneer Plant Sciences Symposium, September 2016 and UIUC Science and Spirits, October 2016. List of Teachers Ranked as Excellent by their Students: Spring 2016, Fall 2016 (Outstanding). A student I mentored, Kat Holan, presented at Maize Genetics Conference in spring 2016 and begangraduate studies at Iowa State University in the Fall of 2016. How have the results been disseminated to communities of interest?The results of each objective have been disseminated as posters at international conferences and a summary of all results will be presented at the Project Directors Meeting in August 2018. Objective 1:Characterization of a Tissue-Specific Knockout of zap1 (Maize Genetics Conference, St. Louis, MO. March 2017). Objective 2:Dynamic Changes in Nitrogen Utilization Genes from a Century of Selection for Grain Protein (Maize Genetics Conference, Jacksonville, FL. March 2016). Objective 3:Asparagine Cycling Alleles Strongly Impact Grain Protein Concentration (Danforth Center Fall Symposium: Genetics and Genomics of Crop Improvement, St. Louis, MO. September 2016). What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1: From the nitrogen responsive genes and genes which were co-expressed with important nitrogen metabolism genes, eight candidate genes were selected for characterization of mutations resulting from Uniform Mu transposon insertion. The mutant plants were grown in the N-responsive nursery in 2016, and sampled for RNA during a developmental time where the native gene would be expected to be abundant, therefore giving the greatest possible transcriptome effect. Then, qPCR was performed to confirm knockout or knockdown of each mutant gene. Unfortunately, despite selecting transposon insertions in exon and promoter regions, most mutants did not decrease expression of the gene of interest. However, the zap1-mum1 mutant gave a tissue-specific knockdown of zap1 gene expression only in the leaf. The zap1 gene is closely related to APETALA1 from Arabidopsis, which has been well characterized for its role in floral organogenesis. Recent evidence has shown maize zap1 may also have a functional role in the leaf. The zap1-mum1 mutant line retained full gene expression in ear tissues, so no floral morphology defects were detected in these plants. To understand genes regulated by zap1 in vegetative tissues, RNAseq profiles were compared between the zap1-mum1 and wild-type, from leaves harvested at anthesis from plants grown with either deficient or sufficient amounts of N. 876 unique genes were differentially expressed in zap1-mum1 for any N treatment. Among the differentially expressed genes are a large number of transcription factors. The zap1-mum1 mutant showed upregulation of EREB, WRKY, and Myb transcription factors, while MYBR and MADS genes were less expressed in the mutant. In the leaf, zap1 may control expression of key transcription factors to control developmental processes. Key Outcomes: Unfortunately, most of the transposon insertion lines failed to decrease expression of the candidate genes. The zap1 gene showed decreased expression in leaves, and loss of zap1 expression resulted in differential expression of a suite of nitrogen-related transcription factors. These results were disseminated as a poster at the Maize Genetics Conference in St. Louis in March 2017. Objective 2: Begun in 1896, the Illinois Long Term Selection Experiment (ILTSE) is the longest running genetic experiment in higher plants, with more than 110 cycles of divergent recurrent selection producing known extremes for grain nitrogen (N) concentration. The ILTSE is a unique resource for maize functional genomics because of its genetic variation for N uptake, utilization, and growth response to N, each of which are influenced by many genes. Based on RNA sequencing of inbred lines derived from Illinois High Protein (IHP1) and Illinois Low Protein (ILP1), 7% of genes on average were differentially expressed in the leaf, earshoot or seeds. Additional candidate selected regions were identified by finding changes in the gene expression networks generated for IHP1 and ILP1. Similarly, only 5% of SNP loci were found to be fixed for different alleles between IHP and ILP populations after 65 cycles of selection. Meanwhile, surprisingly high levels of allelic diversity remained within these populations, even after 100 cycles of selection. Integration of expression differences between IHP1 and ILP1, N-responsive gene expression in one or both of these genotypes, and strong divergence of SNP allele frequencies by cycle 65 of IHP and ILP identified a small set of 10 genes that may contribute to N utilization. These and other candidate genes found to be important in the ILTSE population could be utilized to improve nitrogen use efficiency in breeding maize and other crops. Key Outcomes: Using three different approaches: differential expression between IHP1 and ILP1; nitrogen responsive gene expression; and divergent allele frequency between IHP and ILP, I identified a small set of candidate genes involved in selection for grain protein. Objective 3: The Illinois Long Term Selection Experiment (ILTSE) for grain protein concentration began in 1896 and over the course of 118 cycles has generated phenotypic extremes for grain protein and nitrogen use efficiency traits. Illinois High Protein (IHP) plants also distinctly hyperaccumulate the amino acid asparagine that functions in storage and transport of N in maize. In maize, asparagine concentration is modulated by two genes: asparagine synthetase and asparaginase. Previous work identified lesions in the promoters of both genes in IHP that appear to cause changes in expression and have been driven to fixation in the ILTSE populations. To test the role of these asparagine cycling alleles, near isogenic lines (NILs) were generated. Forward markers were used for selection, and background markers were determined on completed NILs using genotyping by sequencing. The NILs differed from the selection lines for grain protein as measured by near infrared reflectance and an fl2-RFP marker phenotype, which uses the red color to quantify expression of the maize storage protein, zein. For the IHP background, grain protein decreased by 0.6% protein and 1.1% protein with the addition of ILP alleles for Asparagine synthetase3 (As3) and Asparaginase (Asnase), respectively. Conversely, in the ILP background, grain protein increased by 0.4%, 0.9%, and 1.1% protein with the addition of asparagine synthetase3-IHP, asparaginase-IHP, or both. Free asparagine concentrations followed the same pattern of decrease in the IHP background and increase in the ILP background. In addition, the NILs were crossed to diverse inbred lines with varied allele combinations for As3 and Asnase. The hybrid lines were grown in the field in 2016 and phenotyped for grain protein and related characteristics. Among the hybrids, grain protein was most strongly affected by the parent from the asparagine cycling NIL rather than the alleles provided by the diverse parent. The effect of ILTSE alleles for asparagine cycling variants, and asparaginase in particular, was able to significantly alter grain protein concentration and related yield traits. However, removing the hyperaccumulation of asparagine was not able to account for all of the high protein phenotype in IHP--seeds still had over 21% grain protein--rather than a catastrophic decrease, so additional genes must play a role in determining the high protein phenotype in IHP. Key Outcomes: Near isogenic lines were developed and grown in the field in Urbana in summer 2016. Genotyping-by-sequencing was performed to identify the size of the introgressed regions, which ranged from less than 1Mb to 48Mb around asparagine synthetase and asparaginase. IHP-derived lines with introgressions of ILP alleles decreased in grain protein compared to IHP.ILP-derived lines where a single IHP allele was introgressed increased in grain protein. The introgression line with IHP alleles for both asparagine cycling alleles showed the largest increasein grain protein.
Publications
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Progress 04/01/16 to 03/31/17
Outputs
Target Audience:The research accomplished within this project will contribute to the body of knowledge about plant nitrogen response and was disseminated to other scientists working in plant sciences at Plant and Animal Genome (PAG), the Danforth Center Fall Symposium, the University of Illinois Corn Breeders' School, and the Maize Genetics Conference. The course I taught in Spring and Fall 2016, Horticulture 105 - Vegetable Gardening, is an introductory course for non-majors and predominantly attracts upperclassmen from outside the biological sciences. Additionally, I mentored undergraduates in the Moose Lab. In particular, I worked closely with Kat Holan to complete a transcriptome project which she presented at the Maize Genetics Conference. During this year, she has graduated and is a graduate student at Iowa State University. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Attendance at Plant and Animal Genome Conference, January 2017. Attendance and poster presentation at additional conferences: Danforth Center Fall Symposium, Illinois Corn Breeders' School, and Maize Genetics Conference. Poster Award at both UIUC Pioneer Plant Sciences Symposium, September 2016 and UIUC Science and Spirits, October 2016. List of Teachers Ranked as Excellent by their Students: Spring 2016 and Fall 2016. A student I mentored, Kat Holan, presented at Maize Genetics Conference in spring 2016 and begangraduate studies at Iowa State University in the fall of 2016. Continued mentoring to improve my teaching skills, and I was observed teaching both a lecture and laboratory section of Hort105 in fall 2016. How have the results been disseminated to communities of interest?The results of each objective have been disseminated as posters at international conferences. Objective 1: Characterization of a Tissue-Specific Knockout of zap1 (Maize Genetics Conference, St. Louis, MissouriMarch 2017). Objective 2: Dynamic Changes in Nitrogen Utilization Genes from a Century of Selection for Grain Protein (Maize Genetics Conference, Jacksonville, Florida March 2016). Objective 3: Asparagine Cycling Alleles Strongly Impact Grain Protein Concentration (Danforth Center Fall Symposium: Genetics and Genomics of Crop Improvement, St. Louis, Missouri September 2016). What do you plan to do during the next reporting period to accomplish the goals?Dr. Moose and I will work closely to finalize the manuscripts for the results of each objective. The results will be disseminated in peer-reviewed journals. Additionally, I expect to graduate during the next year; my dissertation is written and revisions are in progress.
Impacts
What was accomplished under these goals?
Objective 1: Grew eight mutant lines in the N responsive nursery in Urbana, Illinois during summer 2016. Phenotypic data was taken throughout the growing season and at harvest. RNA was sampled from individual plants and mutant gene expression was determined using qPCR. Seven of the eight mutants were found to retain full gene expression. One mutant, zap1, was knocked out in the leaf tissue only. RNA was sampled from the zap1 mutant, and the effects of the loss of zap1 gene expression on the transcriptome was assessed using RNAseq. In total, the expression of 875 genes was affected in the mutant, and the differentially expressed genes were enriched for regulatory genes, including additional MADS-box genes and other transcription factors. Objective 2: Found 6,701 genes were differentially expressed between IHP and ILP genotypes at any point in leaf or ear development. On average, 2,100 genes were differentially expressed at any point in development. 2,018 genes were responsive to field nitrogen fertilization in IHP. In any one leaf or ear sample, about 500 genes showed an N response. 3,716 genes were gene expression network hubs. About 1/3 of those genes were shared between the IHP gene expression network and the ILP gene expression network. Additionally, a comparison of the genomes of IHP and ILP plants from cycle 65 of selection found 3,060 SNPs where the two populations were each fixed for a different allele. Those SNPs were found in 1,716 genes. In total, ten genes were identified by all four methods. Although four were unannotated, the remaining six genes are good candidates for regulation of phenotypic differences between IHP and ILP. Objective 3: Near isogenic lines were developed and grown in the field in Urbana in summer 2016. Genotyping-by-sequencing was performed to identify the size of the introgressed regions, which ranged from less than 1Mb to 48Mb around asparagine synthetase and asparaginase. IHP-derived lines with introgressions of ILP alleles decreased in grain protein compared to IHP. ILP-derived lines where a single IHP allele was introgressed increased in grain protein. The introgression line with IHP alleles for both asparagine cycling alleles showed the largest increase in grain protein.
Publications
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