Source: PENNSYLVANIA STATE UNIVERSITY submitted to
FUNCTIONAL CHARACTERIZATION OF MAIZE NITROGEN USE EFFICIENCY QTL AND THEIR ROLE IN NITROGEN-PHOSPHORUS SIGNALING
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1028203
Grant No.
2022-67013-37038
Cumulative Award Amt.
$650,000.00
Proposal No.
2021-07502
Multistate No.
(N/A)
Project Start Date
Apr 1, 2022
Project End Date
Mar 31, 2026
Grant Year
2022
Program Code
[A1152]- Physiology of Agricultural Plants
Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
408 Old Main
UNIVERSITY PARK,PA 16802-1505
Performing Department
Plant Science
Non Technical Summary
Most agricultural soils do not provide sufficient Nitrogen (N) or Phosphorus (P) to fulfill the yield potential of our staple crop species. Although the problem can be mitigated by fertilization, current levels of application are not sustainable and are associated with unacceptable environmental costs. In the US, greater quantities of synthetic fertilizers are applied to maize than to any other crop. Nonetheless, we know very little about the genetic basis of maize nutrient use efficiency. Studies in model plant systems have revealed the importance of coordinate regulation of plant responses to N and P. Our teams at Penn State and the University of Illinois have established that plant N-P signaling components modulate nutrient use efficiency in maize. Here, we will combine analyses of natural, induced, and designed genetic variants to understand the functions of key genes integrating plant N and P signaling, and evaluate which combinations of these variants can enhancenutrient use efficiencyin field trials with maize hybrids.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2031510108040%
2031510102030%
2031510104030%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1510 - Corn;

Field Of Science
1040 - Molecular biology; 1080 - Genetics; 1020 - Physiology;
Goals / Objectives
Most agricultural soils do not provide sufficient Nitrogen (N) or Phosphorus (P) to fulfill the yield potential of staple crop species. Although the problem can be mitigated by fertilization, current levels of application are not sustainable and are associated with unacceptable environmental costs. In the US, greater quantities of synthetic fertilizers are applied to maize than to any other crop. We will combine analyses of natural, induced, and designed genetic variants to understand the functions of key genes integratingmaize N and P signaling, and evaluate which combinations of these variants can enhance N use efficiencyin field trials with maize hybrids.Specifically, we will:Characterize the role of the maize NRT1.1-SPX4 module in NP signallingCharacterize the effects of allelic variation at the maize Pho1;2a locus on N use efficiencyEvaluate the cumulative effect of Nrt1.1b and Pho1;2a variants on maize N use efficiency
Project Methods
AIM 1. Characterize the role of the NRT1.1-SPX4 module in NP signaling1.1 Functional characterization of nrt1.1 mutantsWe will characterize nrt1.1b and nrt1.1abc mutants for their impact on nitrogen utilization efficiency (NUtE) as hybrids with both IHP1 and NILs spanning the Nrt1.1b QTL interval. We will measure P and N accumulation in leaves, stalk, and grain. Unless otherwise stated, field evaluation will be carried out in low N plots at U. Illinois. Each genotype will be planted in at least three replicate paired split-plots, where one receives no supplemental N, and the other fertilizer applied at a rate of 150 kg N/hectare. Fertilizer is typically applied at the V3 growth stage. In addition to in-season measures of plant growth such as flowering time and plant height, we will collect relative leaf greenness via image capture from unmanned aerial vehicles, end-point estimates of biomass, N, and P accumulation in stalks, leaves, cobs and grain will be obtained from plants harvested at physiological maturity.1.2 Description of the maize Spx gene family and characterization of spx4 mutantsWe have obtained/will generate spx4 mutants for functional analysis and eventual combination with nrt1.1 material. Five publicly available Mutator transposon insertional alleles of the maize Spx4 gene are available; two of these insertions are exonic and predicted to result in loss of function; three insertions are upstream of the translational start and may condition more subtle regulatory effects on NP signalling. We will also perform CRISPR mutagenesis of Spx4 using the H99 multiplex editing systems used to generate the nrt1.1 mutations. We will characterize the impact of spx4 alleles on Spx4 transcript accumulation, seedling growth under different NP availability and the induction of N and P responsive transcripts. We will use a sand-fertigation system to follow plant growth up to 25 days after emergence under a range of N and P regimes. We will evaluate chlorophyll accumulation, leaf surface area, final root and shoot biomass, and total N and P accumulation. We will generate an antibody against SPX4 and assay protein levels in both wild-type and spx4 over a range of NP availability. We will assay transcriptional responses using a small number of marker transcripts selected from published whole transcriptome data and previously optimized in real time PCR assays.1.3 Interaction of Nrt1.1 and Spx4 in global transcriptional responses to NPHaving characterized spx4 loss-of-function mutants in the H99 background, we will select a suitable mutant allele to generate nrt1.1b; spx4 double mutants and the quadruple mutant nrt1.1abc; spx4. We will grow these higher order mutants and controls to 25 days after emergence as described above, under nutrient replete conditions, low N, low P and combined low NP stress. We will again use antibodies to follow NRT1.1B and SPX4 protein levels; our prediction is that SPX4 is stabilized under low P in nrt1.1 mutants, suppressing the P starvation response. To evaluate global nutrient stress responses, we will extract RNA for sequencing from root and shoot tissue. Library construction and sequencing will be carried out at the UI core facility.1.4. Field evaluation of NUE and PUE in spx4 and nrt1.1 mutantsBased on Aim 1.2, we will select spx4 mutant alleles for field evaluation of NUE and PUE alongside the nrt1.1b and nrt1.1abc mutants and the appropriate wild-type controls. We will evaluate mutants per se and as a test cross to the IHP1 inbred. We will evaluate responses to low P stress, using long term low P plots available at Penn State, historically treated with acidified alumina to bring available P down to <10 ppm.AIM 2. Characterize phenotypic effects of allelic variation at Pho1;2a on NUtE2.1 Field evaluation of qNUtE4 NILs and pho1;2a mutantsWe have obtained BC4 NIL stocks containing reciprocal introgression of the qNUtE4/Pho1;2a region between B73 and Mo17. We have generated/obtained two pho1;2a loss-of-function alleles in the W22 background: an 8bp transposon footprint allele and an exonic Mutator transposon insertion from a public resource. As for Spx4, we will initially work with available mutants, combining with other material in W22 or using existing introgressions of pho1;2a into B73, while initiating CRISPR/Cas editing of Pho1;2a in the H99 background for later combination with nrt1.1 candidates. The qNUtE4 NILs, B73, Mo17, W22 and the pho1;2a mutants will be evaluated for NUtE per se following protocols established in our characterization of nrt1.1. Each of these genotypes will also be crossed to the IHP1 tester used in the initial QTL mapping experiment.2.2 Fine mapping of functional variation at qNUtE4We will continue to fine map qNUtE4. Pho1;2a remains our principal candidate and we have identified small insertion/deletion polymorphisms between the B73 and Mo17 Pho1;2a sequences that we have used to design PCR markers to follow this locus. Additional genotyping across the interval will be carried out using KASP markers, leveraging the assembly of complete genomic sequence for both parents and the ready availability of usable single nucleotide polymorphism (SNP) variants. In addition to fine mapping, we will directly target Pho1;2a variation using CRISPR/Cas gene editing. We have built a CRISPR/Cas construct containing a pair of guides located in the second and third exons of Pho1;2a, respectively, positioned to allow simple PCR for screening small deletion events. We will cross our CRISPR/Cas transformants to B73 and to a NIL carrying the qNUtE4 Mo17 allele in the B73 background. As a stretch aim in this project, we will look to isolate editing events in both B73 and Mo17 alleles of Pho1;2a for functional comparison. Such material would provide a definitive link between variation at Pho1;2a and NUtE.2.3 Exploration of an interaction between qNUtE4 and Pho1;2bThe rice Pho1;2 gene is represented in maize by the pair of paralogous genes Pho1;2a and Pho1;2b. We have generated a Ds transposon insertion in Pho1;2b. We have introduced the pho1;2b allele into the B73 background through six generations of back-crossing. We will cross the pho1;2b allele to the B73 NIL carrying the qNUtE4 Mo17 allele. We will generate sibling families fixed for all combinations of B73 and Mo17 at qNUtE4 and the wild-type and mutant allele at pho1;2b. We will evaluate NUtE in all stocks, evaluating how the effect at qNUtE4 is modified by loss-of-function at pho1;2b. We will also cross fixed stocks to generate qNUtE4 heterozygotes in wild-type and pho1;2b backgrounds. These latter stocks will allow us to test for a heterotic effect at qNUtE4 - consistent with the original testcross mapping design - and explore the requirement of any such effect on pho1;2b.AIM 3. Evaluate the cumulative effect of Nrt1.1b and Pho1;2a variants on NUtE3.1 Field NUE of Nrt1.1b/Pho1;2a NILsWe will build genetic stocks based on a previously identified NIL (B087) that contains Mo17 alleles at both qNUtE1 and qNUtE4 loci in a B73 background (BC4). We have crossed this line to B73 and will perform a first round of self pollination prior to the start of the funding period. We will use KASP markers based on SNPs identified from comparison of the B73 and Mo17 genome assemblies to follow the two QTL. With each QTL having three possible genotypes (B73/B73, B73/Mo17, Mo17/Mo17), we will generate a total of 6 stocks to evaluate all combinations of the two loci, replicated over two pedigrees. With this scheme, we will be in a position to accurately estimate additive QTL effects, dominance and possible epistasis by the Summer of 2023, with a repeated evaluation in 2024. Material will be evaluated in UI as described above.

Progress 04/01/23 to 03/31/24

Outputs
Target Audience:Results have been communicated through teaching, seminars, scientific conferenceand group activities targeted atthe student and academic community Changes/Problems:The central goal of our work remains generation of loss-of-function mutants of the maize Spx4 gene to investigate its role in N-P signalling. We have continued to balance available transposon-based mutant collections and gene editing approaches. The availability of seed from public collections, and the need to move material from different collections into comparable backgrounds has delayed progress, but we are now in a strong position. Notably, we have three independent exonic Mutator insertions intoSpx4in, or partially introgressed, into the W22, B73 and Oh43background. In a slight modification of our original plan, we will focus on the W22materials for a transcriptomic experiment in Fall 2024, coupled with field evaluation 2025. As described in the project proposal, we will profile plants grown a combinations of high/low N and P availability. Our hypothesis is that the N requirement for the activation of the P starvation response we have previously described will be partially removed in spx4 mutants. Molecular characterization of mutiple independent spx4 alleles in this context will be an important contribution of this project. In parallel, we will continue to generate higher order mutants, combiningspx4alleles withnrt1.1mutants to further test the role of these two key components in maize N-P signaling. What opportunities for training and professional development has the project provided?PSU project members Melanie Perryman (technical support) and Sergio Perez-Limon (PhD student PSU Agricultural and Environmental Plant Science program) have gained experience in maize genetics, genotypic analysis and evaluation of nutrient use efficiency in field grown plants. Several undergraduates have also participated in the project both in the laboratory and the field.Lincoln Koehler (MSc. student) presented the project as a poster at the Maize Genetics Conference 2024, Raleigh, NC, and is now actively applying to PhD programs.Nandan Rai (postdoc) has worked with gene editing approaches and will represent the project at the annual meeting in Honolulu, HI. How have the results been disseminated to communities of interest?The Penn State and Illinois teams presented a joint poster at theMaize Genetics Conference 2024, Raleigh, NC. What do you plan to do during the next reporting period to accomplish the goals?We now have available collection of nrt1.1, pho1;2a and spx4 mutant alleles in, or partially introgressed (4 or more generations), into the W22 genetic background. Self-pollination during the Winter 2023/34 will produce stocks anticipated to segregate for these mutants/mutant combinations. During Summer 2024, we will identify homozygous mutant and sibling wild-type individuals in the field to generate working stocks for a greenhouse transcriptome experiment to be run during the Fall 2024. Introgression of nrt1.1b alleles into W22 and B73 will be completed during 2024. We will combinethese with pho1;2a and spx4 mutants to have higher order combinations available for characterization before the end of 2025. We will look to confirm our observations of increased NUtE in nrt1.1a/pho1;2a double mutants by further characterization and by generation of additional genetic stocks. Notably, we will cross the nrt1.1a mutant allele to two additional pho1;2a mutant alleles. We will evalauate per seB73 NILs carrying Mo17 introgression at Pho1;2a for NUtE during Summer 2024. We will also generate hybrids of these NILs with the high protein tester used in ouroriginal B73 x Mo17 mapping experiment for futher evaluation. ?

Impacts
What was accomplished under these goals? 1. Characterize the role of the maize NRT1.1-SPX4 module in NP signalling We have continued to advance stocks carrying mutations in the Spx4 target for functional characterization. Over the Winter cycle 2023/24, we moved the transposon insertional alleles (Bonn-Mu resource; mixed genetic background) spx4-5 and spx4-6 for a fifth generation to the W22 background, as well as generating segregating stocks. We have also now been able to obtain stocks for a previously unavailable allele, designated spx4-4, from the Uniform-Mu resource (W22 background). We confirmed the insertion at spx4-4 and backcrossed to W22 Summer 2023 (the original stock is reported to contain a high number of non-linked Mutator insertions). We further backcrossed and self-pollinated spx4-4 stocks during Winter 2023/24.In parallel, we have continued evaluation of material transformed with a CRISPR-CAS construct to induce gene edits in the Spx4 gene. One CRISPR knock-out allele of Spx4 has now been confirmed, and we are screening material from a furthersix independent transformation events for edits. 2. Characterize the effects of allelic variation at the maize Pho1;2a locus on N use efficiency We have continued to advance stocks carrying mutations in the Pho1;2a target for functional characterization - our previously published Activator allele, we have identified and confirmed two additional Mutator transposon insertional alleles. We have continued to advance introgression of all alleles into W22 and B73 backgrounds. We have used our PCR based assay to further isolate the Mo17 allele of Pho1;2a in a B73 background (BC5 near-isogenic line) while generating corresponding sibling families carrying the B73 allele. We have added further data of the impact of pho1;2a Activator mutant on plant height and total plant N accumulation. To date, at the level of replication we have available, we have not detected an impact of the pho1;2a mutation on N utilization efficiency (NUtEplant biomass/plant N) under low N, although we have seen a mild (non-significant) increase in NUtE under high N. However, we are beginning to document interesting synergistic effects with nrt1.1a (see below). 3. Evaluate the cumulative effect of Nrt1.1b and Pho1;2a variants on maize N use efficiency We have generated further data showing that the nrt1.1a mutation reduces plant height, while the pho1;2a mutation increases height. Interestingly, in the double mutant, nrt1.1a completely suppresses the pho1;2a effect. Conversely, pho1;2a suppresses a negative effect on NUtE observed in nrt1.1a mutants. Indeed, our preliminary data suggest that NUtE is higher in nrt1.1a/pho1;2a double mutants than in either single mutant or wildtype plants.?

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Lincoln Koehler, Eddie Ross, Leah Erdmann, Maruti Nandan Rai, Catherine Li, Stephen Moose, Melanie Perryman, Ruairidh Sawers. 2024. "Functional Genomics of Coordinated Nitrogen and Phosphorous Signaling in Maize". Maize Genetics Conference 2024, Raleigh, NC
  • Type: Journal Articles Status: Published Year Published: 2024 Citation: McLaughlin, Chloee M., Meng Li, Melanie Perryman, Adrien Heymans, Hannah Schneider, Jesse R. Lasky, and Ruairidh J. H. Sawers. 2024. Evidence That Variation in Root Anatomy Contributes to Local Adaptation in Mexican Native Maize. Evolutionary Applications 17 (3): e13673.


Progress 04/01/22 to 03/31/23

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?PSU project members Melanie Perryman (technical support) and Sergio Perez-Limon (PhD student PSU Agricultural and Environmental Plant Science program) have gained experience in maize genetics, genotypic analysis and evaluation of nutrient use efficiency in field grown plants. Several undergraduates have also participated in the project both in the laboratory and the field. How have the results been disseminated to communities of interest?PSU and ISU project members have presented an internal institutional events. What do you plan to do during the next reporting period to accomplish the goals?Characterize the role of the maize NRT1.1-SPX4 module in NP signalling We will continue introgression of spx4 transposon alleles into B73 and W22 backgrounds. In parallel, we will generate segregating stocks from the most advanced W22 introgressions to perform a basic molecular characterization of the alleles with respect to production of spx4 transcripts. We will perform an initial observation of segregating stocks in the field Summer 2024 to look for obvious differences in development, growth or phenology. We will screen CRISPR-CAS material to identify spx4 gene edits and propagate material for further characterization. Characterize the effects of allelic variation at the maize Pho1;2a locus on N use efficiency We will confirm the molecular consequences of new pho1;2a alleles. We will generate test cross stocks using Pho1;2a mutant and NILs prior to field evaluation of N use efficiency in the field in Summer 2024. Evaluate the cumulative effect of Nrt1.1b and Pho1;2a variants on maize N use efficiency We will perform a second year of field evaluation of existing double mutant stocks while continuing to generate additional material for evaluation.

Impacts
What was accomplished under these goals? Characterize the role of the maize NRT1.1-SPX4 module in NP signalling We have advanced the generation of stocks carrying mutations in the Spx4 target for functional characterization. We have identified and confirmed three transposon insertional alleles from public collections (spx4-3 from the Uniform-Mu resource; spx4-5 and spx4-6 from the Bonn-Mu resource). Given the mixed genetic backgrounds of these alleles, we have used PCR-based genotyping to introduce alleles into the common inbreds W22 and B73. The spx4-3 allele was initially generated in W22; we have now also moved this through 4 generations to B73. The alleles spx4-5 and spx4-6 were identified in a mixed background; we have now moved these through 4 and 2 generations to W22 and B73, respectively. In parallel, we have generated a CRISPR-CAS construct to induce gene edits in the Spx4 gene. This construct has been transformed to the H99 background and primary transformants identified. Ten events from the first Spx4 editing experiment are now in regeneration and a second experiment is in progress. Characterize the effects of allelic variation at the maize Pho1;2a locus on N use efficiency We have advanced stocks carrying mutations in the Pho1;2a target for functional characterization. In addition to our previously published Activator allele, we have identified and confirmed two additional Mutator transposon insertional alleles. We now have advanced introgression of all alleles into W22 and B73 backgrounds. In addition, we have developed a PCR based assay to readily distinguish wild-type B73 and Mo17 alleles of Pho1;2a. Using this assay, we have confirmed the presence of Pho1;2a-Mo17 in the predominantly B73 background of an existing BC4 near-isogenic stock. We have performed an additional backcross to B73 and self-pollination to generate sibling families at BC5 homozygous for either the B73 or Mo17 allele at Pho1;2a.With a further field season, we now have robust documentation of the impact of pho1;2a Activator mutant on plant height and total plant N accumulation. Evaluate the cumulative effect of Nrt1.1b and Pho1;2a variants on maize N use efficiency We have confirmed nrt1.1a/pho1;2a double mutants. Preliminary evaluation suggests the nrt1.1a mutation to suppress the effect of pho1;2a on plant height. Material is planted for generation of nrt1.1b/pho1;2b double mutants.

Publications