Progress 08/01/07 to 07/31/10
Outputs
OUTPUTS: The goal of this project was to investigate the physiological and molecular effects of nitrogen (N) on early kernel development. Our general approach was to grow maize in the field under conditions of N stress, use an in vitro kernel culture system to manipulate amino acid supply during the developmental window when N-stress often induces kernel abortion, and then assay both cultured kernels and kernels from field grown plants for changes in amino acid accumulation and gene expression. The first objective of the project was to assess N-responsive changes in growth and metabolism among developing kernels from genotypes that show extreme differences in kernel number and grain yield responses to supplemental N. This objective was completed. Five hybrid maize genotypes (B73 crossed to each of Illinois High Protein1, Illinois Low Protein1, Mo17, MS71 and Oh7b) were chosen for comparison. Maize plants were grown in adjacent field plots that contained either sufficient N to maximize grain yield, or insufficient N and hence showed evidence of N stress. Plants were hand pollinated and two days later, cob sections with attached kernels were dissected and cultured in a laboratory system. Samples were taken from developing ears and kernels of both field-grown plants and kernel cultures at two, four and seven days after pollination, as well as physiological maturity. In the kernel culture system, two N treatments (no supplemental N, sufficient supplemental N) were provided via nutrient media. Cob and kernel growth were monitored at each sampling stage, and samples were collected for analysis of free amino acid profiles and gene expression. Free amino acid profiles were obtained via high-throughput reverse-phase HPLC. The second objective was to obtain RNA expression profiles for the same tissue samples generated in the experiment described above. The initial approach was to use oligonucleotide microarrays. However, due to the discontinued production of the microarrays and the emergence of sequence-based RNA profiling methods, it was decided to employ the RNA-Seq technology for the RNA profiling experiment. The data was generated during the summer of 2010 and the data analysis is ongoing. It is expected the publication of the results will occur in 2011. Key products include new fundamental knowledge about genetic variability in how nitrogen availability impacts maize ear and seed development. Most importantly, the genetic variability observed in field experiments was faithfully reproduced in the kernel culture system, which enables the use of the kernel culture system to investigate the genetic basis for N utilization by developing maize ears. New scientists were trained in conducting the kernel culture experiments. PARTICIPANTS: Principal investigators: Stephen Moose and Fred Below Research Associates: Jayanand Boddu and Julian Seebauer. A team of seven undergraduate students assisted part-time throughout the project. TARGET AUDIENCES: Results of this project are expected to reach and benefit a wide spectrum of audiences. Anticipated results will have considerable economic impact on crop breeding and productivity. Improving nitrogen use efficiency is becoming increasingly important to reducing the energy inputs and greenhouse gas emissions associated with agriculture. These gains must occur in the face of greater global demand for food, feed, and energy derived from maize production. Reducing the N requirements for cereal grain production, this research will address two major USDA goals: enhancing economic opportunities for agricultural producers and protecting the Nation's natural resource base and environment. PROJECT MODIFICATIONS: The RNA profiling experiments proposed in this project were delayed because the production of maize oligonucleotide arrays by the University of Arizona was discontinued, and improved methods for RNA sequencing using the Illumina technology became available. The Illumina RNA-Seq experiments were conducted in 2010.
Impacts
The primary outcome from this project is new knowledge about genetic variability in N utilization within the developing ear and improvement of experimental methods to characterize changes in N-responsive gene expression within cob and seed tissues. Key results are that genotypic differences in N-responsive growth and amino acid profiles observed among field grown plants were confirmed in the kernel culture system. Each hybrid showed limited growth in low N field and culture treatments, with growth being enhanced by N in all genotypes. The B73 x Oh7B hybrid showed the most dramatic growth increases in response to N, followed by B73 x ILP1, B73 x Mo17, B73 x MS71, and B73 x IHP1. All genotypes exhibited an increase in total free amino acids and in particular asparagine in response to N treatments, with the B73 x IHP1 hybrids showing a significantly greater accumulation of N compared to any of the other genotypes, B73 x ILP1 had the lowest rate of N accumulation. The B73 x Mo17 hybrid was selected for transcriptome profiling experiments because genome sequences of both parental lines are available (assembled for B73, in draft form for Mo17). Changes in action include application of knowledge gained which confirms that genetic variability exists for how maize ears and kernels respond to N in terms of both growth and N accumulation. Although kernel culture is not a new method, its application to experiments investigating N utilization is novel. Additional scientists were trained in approaches that integrate crop physiology and functional genomics. Nitrogen (N) is an essential and often limiting nutrient to crop yields. Improving N utilization (biomass achieved per unit of acquired plant N) is an important goal to sustainably increasing crop yields. This can be achieved in part by understanding the physiological, molecular, and genetic basis for phenotypic variation in N utilization. In cereal crops, N stress reduces grain yield primarily by decreasing grain number per plant. Genes whose expression is responsive to N in developing kernels and cob tissues are expected to mediate the enhanced grain yield in response to N supply. Discovery of genes associated with N utilization can provide valuable insights into nitrogen metabolism in plants that can in turn be used for crop improvement via molecular breeding approaches. The eventual development of maize genotypes with improved N utilization can lead to better energy balance of crop production, lower input costs, and reduce environmental impacts associated with N fertilizer use.
Publications
- No publications reported this period
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Progress 08/01/08 to 07/31/09
Outputs
OUTPUTS: The primary outputs for the 2009 project period were activities associated with the conduct of experiments and analysis of results. Five hybrid maize genotypes (B73 crossed to each of Illinois High Protein1, Illinois Low Protein1, Mo17, MS71 and Oh7b) that showed different responses in ear growth when provided supplemental N were chosen for comparison. Maize plants were grown in adjacent field plots that contained either sufficient N to maximize grain yield, or insufficient N and hence showed evidence of N stress. Plants were hand pollinated and two days later, cob sections with attached kernels were dissected and cultured in a laboratory system. Samples were taken from developing ears and kernels of both field-grown plants and kernel cultures at two, four and seven days after pollination, as well as physiological maturity. In the kernel culture system, two N treatments (no supplemental N, sufficient supplemental N) were provided via nutrient media. Cob and kernel growth were monitored at each sampling stage, and samples were collected for analysis of free amino acid profiles and gene expression. Free amino acid profiles were obtained via high-throughput reverse-phase HPLC. RNAs were isolated for gene expression analysis. Key products include new fundamental knowledge about genetic variability in how nitrogen availability impacts maize ear and seed development. Most importantly, the genetic variability observed in field experiments was faithfully reproduced in the kernel culture system, which enables the use of the kernel culture system to investigate the genetic basis for N utilization by developing maize ears. New scientists were trained in conducting the kernel culture experiments. PARTICIPANTS: Principal investigators: Stephen Moose and Fred Below. Research associates: Jayanand Boddu and Julian Seebauer. A team of seven undergraduate students assisted part-time with project. TARGET AUDIENCES: Results of this project are expected to reach and benefit a wide spectrum of audiences. Anticipated results will have considerable economic impact on crop breeding and productivity. Improving nitrogen use efficiency is becoming increasingly important to reducing the energy inputs and greenhouse gas emissions associated with agriculture. These gains must occur in the face of greater global demand for food, feed, and energy derived from maize production. By reducing the N requirements for cereal grain production, this research will address two major USDA-CSREES goals: enhancing economic opportunities for agricultural producers and protecting the nation's natural resource base and environment. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The primary outcome from this project is new knowledge about genetic variability in N utilization within the developing ear and improvement of experimental methods to characterize changes in N-responsive gene expression within cob and seed tissues. Key results are that genotypic differences in N-responsive growth and amino acid profiles observed among field grown plants were confirmed in the kernel culture system. Each hybrid showed limited growth in low N field and culture treatments, with growth being enhanced by N in all genotypes. The B73 x Oh7B hybrid showed the most dramatic growth increases in response to N, followed by B73 x ILP1, B73 x Mo17, B73 x MS71, and B73 x IHP1. All genotypes exhibited an increase in total free amino acids and in particular asparagine in response to N treatments, with the B73 x IHP1 hybrids showing a significantly greater accumulation of N compared to any of the other genotypes, B73 x ILP1 had the lowest rate of N accumulation. The B73 x Mo17 hybrid was selected for a transcriptome profiling experiment because both parental lines have extensive available resources for functional genomics analysis. Changes in action include application of knowledge gained which confirms that genetic variability exists for how maize ears and kernels respond to N in terms of both growth and N accumulation. Although kernel culture is not a new method, its application to experiments investigating N utilization is novel. Additional employees are being trained in approaches that integrate crop physiology and functional genomics. Nitrogen (N) is an essential and often limiting nutrient to crop yields. Finding an efficient and inexpensive alternative for better utilization of available nitrogen in plants is a highly sought after strategy for elite crop management. This can be achieved in part by understanding the intrinsic ability of diversified crop species that differ in their "nitrogen use efficiency (NUE)". In cereal crops, N stress reduces grain yield primarily by decreasing grain number per plant. Genes whose expression is responsive to N in developing kernels and cob tissues are expected to mediate the growth enhancements in grain yield due to N. Finding these genes can provide valuable insights into nitrogen metabolism in plants that can in turn be used for crop improvement via molecular breeding approaches. The eventual development of maize genotypes with improved N utilization can lead to better energy balance of crop production, lower input costs, and reduce environmental impacts associated with N fertilizer use.
Publications
- Boddu, J., Taylor, E., Ayodeji, A. and Moose, S.P. 2009. Transcriptome analyses of developing ear-shoot in selected genotypes of maize. Abstracts of 51st annual Maize Genetics Conference, view link at http://maizegdb.org/cgi-bin/displayrefrecord.cgiid=1232475.
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Progress 08/01/07 to 07/31/08
Outputs
OUTPUTS: Maize kernel-growth-in-culture experiments can be divided into five sections, namely: 1. Growing selected genotypes in nitrogen (N) responsive field at N-sufficient and N-deficient conditions; 2. Culture growth in N-sufficient and N-deficient conditions; 3. Microarrays; 4. qRT-PCR validation using RNA from selected genotypes; and 5. Metabolite profiling. Five hybrid genotypes called B73 X IHP1, B73 X ILP1, B73 X Mo17, B73 X MS71, and B73 X Oh7B that are expected to show differential nitrogen response were selected. The first and second sections of the experiment were completed. Depending on kernel growth stages and sampling intervals the culture growth section can be sub-divided into growth initiation, establishment, and physiological maturity stages. Sampling was done at 2 and 5 days in culture (DIC) and at physiological maturity. Kernel weights at each stage showed a distinct genotype-dependent grain filling. The maize hybrid B73 X Mo17 kernels grown in low nitrogen field and culture showed little or no growth in culture while significant growth was observed in high nitrogen field and culture conditions. Hence, this hybrid was selected for transcriptome profiling experiment. Existing knowledge on B73 X Mo17 hybrid molecular genetics and functional genomics is expected to be useful during the analysis of the microarray experiment. PARTICIPANTS: Principal investigators: Stephen Moose, Fred Below Research associates: Jayanand Boddu, Julian Seabauer TARGET AUDIENCES: Results of this project are expected to reach and benefit a wide spectrum of audiences. Anticipated results will have considerable economic impact on crop breeding, production and distribution segments of the society. Especially during this new age of burgeoning need for alternative energy, where utilization of biomass from certain crops is an imminent necessity, understanding the nuances in tightly linked nitrogen-carbon balance in plants gains immense importance. Hence, farmers, food and biomass processing industries and the consumers will be direct or indirect recipients of the benefits from the proposed work. Taken together, reducing the N requirements for cereal grain production, this research will address two major USDA-CSREES goals: enhancing economic opportunities for agricultural producers and protecting the Nation's natural resource base and environment. PROJECT MODIFICATIONS: Project proposal was aimed at conducting microarrays using the genotype B73 X Mo17. In addition to this objective, to elucidate genotype dependent nitrogen response, we selected four other genotypes namely B73 X IHP1, B73 X ILP1, B73 X MS71 and B73 X Oh7B for post-microarray validation of differentially accumulating genes.
Impacts
Nitrogen (N) is an essential and often limiting nutrient to crop yields. Even though N constitutes 78% of the atmosphere, the living organisms cannot make direct use of it. Generation of usable form of nitrogen to living organisms, especially plants, is an expensive but a necessary process. Crops require input of nitrogen fertilizer for increase in yield. Finding an efficient and inexpensive alternative for better utilization of available nitrogen in plants is a highly sought after strategy for elite crop management. This can be achieved in part by understanding the intrinsic ability of diversified crop species that differ in their "nitrogen use efficiency (NUE)". In cereal crops, N stress reduces grain yield primarily by decreasing grain number per plant besides affecting several other traits. This project proposes to integrate physiological and functional genomics approaches to explore how N promotes continued kernel growth in maize. There is well documented information on nitrogen responsive agronomy and molecular genetics of maize in terms of NUE that can be integrated into several quantitative trait loci (QTL). However, understanding is limited on specific genes that respond to nitrogen. Finding these genes can provide valuable insights into nitrogen metabolism in plants that can in turn be used for biotechnological applications. RNA expression profiling using B73 X Mo17 hybrid will provide a basal data set that can be used for qRT-PCR studies on the selected genotypes. Existing knowledge on maize whole genome sequence and its synteny with other grass species will be useful for bioinformatics studies to putatively attribute the identified genes to the QTL. This approach can simplify the effort to physically map the interesting genes. Recent studies showed that the metabolic profiling of amino acids in given samples is also useful to understand key events of nitrogen response in plants. Our recently acquired 1200 series rapid resolution HPLC system from Agilent Technologies is expected to be useful for the metabolic profiling studies.
Publications
- No publications reported this period
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