Fine mapping and characterization of genes involved in maize nitrogen utilization efficiency
Maize is one of the most productive crop species on earth. A core component of this productivity comes from maize’s responsiveness to nitrogen fertilizers. Despite their importance in crop production, these fertilizers can place an economic burden on growers and can also have negative environmental and health impacts when applied in excess. In order to mitigate the negative effects of nitrogen fertilizers while also developing maize lines productive enough to feed a growing global population, improvements in nitrogen utilization efficiency (NUtE) is an important component of current and future crop research. Importantly, this research must be conducted in conditions that closely mimic commercial-scale production to ensure that the findings are applicable to modern agricultural settings. A key resource that has facilitated understanding of the genetic components associated with nitrogen utilization efficiency has been the Intermated B73 X Mo17 Recombinant Inbred Lines (IBMRIL) X Illinois High Protein (IHP) mapping population. Previous QTL mapping experiments using this population have identified 9 robust genomic regions associated with NUtE and associated traits. In order to identify candidate genes in additional QTL from the IBMRIL X IHP mapping study, several genetic techniques were utilized. Fine mapping was used to reduce the size of a NUtE QTL on chromosome 1 from 15 Mb to 2.5 Mb. This QTL’s effect on NUtE was replicated using an IBMNIL population containing introgressions of the chromosome 1 QTL genomic region of interest. This region contained 22 annotated genes which included two members of the NRT1.1 nitrate transporter gene family (NRT1.1B and NRT1.1C). The NRT1.1 gene family contains four members: NRT1.1A, NRT1.1B, NRT1.1C and NRT1.1D, of which NRT1.1A and NRT1.1B seen to function as the primary functional homologues based on cross species homology and gene expression data. Gene orthologs of NRT1.1A and NRT1.1B have been shown to play critical roles in nitrogen use traits in rice. A publicly available transposon mediated mutant of NRT1.1A was obtained in order to study its effect on NUtE traits in the field. Consistent reductions in stover nitrogen content and biomass were observed in the NRT1.1A mutant, especially under low nitrogen conditions. In addition, rooting area and rooting depth were reduced within the mutant which is consistent with this gene’s role in nitrogen uptake from the soil and regulation of root architecture. Another strategy to identify candidate genes within the NUtE QTL regions is to utilize a population genetics approach that looks at differences in allele frequencies of genetic markers between heterotic groups. Genomic regions with highly divergent allele frequencies could potentially indicate genes under selection to perform effectively within hybrid lines and may play an important role in modern germplasm. Using a measure of this heterotic stratification, which we deem Delta P, we identified candidate genes within the chromosome 4 and chromosome 9 QTL that were in close proximity to SNPs with regionally high Delta P measures. The gene on chromosome 4 was the phosphate transporter (Pho1;2a) and a null mutant of this gene was obtained and grown in the field to study how NUtE associated traits were affected. Interestingly, stover biomass and stover nitrogen measures were consistently higher in the Pho1;2a mutant when compared to wildtype which could be due to alterations in the nitrate/phosphate response pathway. The chromosome 9 QTL contained a gene known to negatively regulate autophagy and nitrogen use traits in plants (HVA22). Near isogenic lines (NILs) containing HVA22 showed changes in grain protein measures, which is consistent with previous data in maize. In an attempt to use HVA22 to further alter grain protein, a transgene was designed that overexpressed the B73 HVA22 gene using the Gamma Zein promoter and transformed into H99. Transgenic and non-transgenic lines were compared in the field under low and high nitrogen rates as both inbred and hybrids and a consistent significant increase in grain protein concentration was observed. In an effort to develop a targeted mutagenesis protocol to allow for the characterization of additional genes involved in NUtE, a genome-editing pipeline was designed that was compatible with our biolistic transformation system. The first gene-targeted using this pipeline was the maize NADP-MDH gene involved in the C4 photosynthetic pathway. This gene functions at the intersection of carbon and nitrogen balance within the plant and modification of this gene could help inform future efforts to modify this C X N balance. A single homozygous NADP-MDH edited line was developed but died as a T0 due to photosynthetic deficits which prevented further study. Another genome editing experiment was performed that targeted the NRT1.1A, NRT1.1B and NRT1.1C genes using a csy4 mediated multiplex genome editing vector that previously hadn’t been shown to function in maize. Six independent edited lines were generated that contained an array of sequence mutations. One of these lines contained a homozygous edit of the NRT1.1B gene and another line contained homozygous edits of NRT1.1A, NRT1.1B and NRT1.1C. Both mutant lines were shown to reduce transcript abundance of edited genes under certain conditions. These lines were carried forward for further characterization in both greenhouse and field trials. In order to assess the nitrate uptake capacity of these edited lines, their sensitivity to chlorate, which is a herbicide taken up through nitrate transporters, was studied. Both the single and triple NRT1.1 mutants showed decreased sensitivity to chlorate as would be expected given their decreased capacity to take up this herbicide. Nitrate treatment assays were also performed on seedlings of these mutants which showed step wise reductions in plant height as additional NRT1.1 transporters were mutated. Field studies that grew these mutant lines under low and high nitrogen conditions showed similar trends as the greenhouse experiments in that stepwise reduction in nitrogen content and biomass were seen in mutant stover and grain. However, despite these obvious indications of reduced nitrogen uptake capacity in NRT1.1B, this mutant showed phenotypes suggesting altered nitrogen response such as increased tillering and reductions in grain protein concentration. The described research validates the hypothesis that the genetic components of hybrid maize NUtE can be identified if properly designed experiments are conducted. By utilizing the extensive genetic resources available to maize researchers, as well as adapting biotechnology tools such as genome editing, we can continue to narrow the knowledge gap regarding the molecular underpinnings of this important trait. Unfortunately, many of the lines generated in this study impaired the plant's ability to efficiently utilize nitrogen, but hopefully these discoveries enable genetically informed improvements in this trait in the future.
Maize, Nitrogen Utilization Efficiency, CRISPR/Cas9, Nitrate Transporter
University of Illinois - Urbana-Champaign
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University of Illinois at Urbana-Champaign