Progress 12/15/15 to 12/14/19
Outputs
Target Audience:The target audiences are academics and industry scientists working in the fields of plant breeding, plant biology and plant genetics. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Below is a list of conferences and symposia, and other professional development experiences: Postdoc (J. Arp) attendance at PAG 2019 with presentation of science in poster form Maize Genetics Conference attendance by PI Allen, co-PI Moose, postdoc Arp, Jinga and Rhodes (graduate students in Moose lab). Professional training at the Danforth Center regarding responsible conduct of research, harassment, unconscious bias and respecting diversity in the work place and maintaining a safe work place. How have the results been disseminated to communities of interest?Posters were presented at the Danforth Center Scientific Retreat, Fall Symposium, and at international conferences such as PAG and the Maize Genetics Conference. The results of this work support a provisional patent filed on October 15, 2019. We anticipate submission of the manuscript describing the pepck single mutant by the end of this year. 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: Identify and characterize Ds and Mu transposon insertions in components of the PEPCK pathway in maize (Year 1-3) a. Establish screening populations of Ds donors in close proximity to AspAT-m, AspAT-bs and PEPCK2 as platforms for mobilization. b. Conduct reverse-genetic screens to identify additional insertion alleles into target genes. (Year2; 100% complete) Completed in previous reporting period c. Self-pollinate and create single and double mutant combinations of pepck1 x pepck2 and pepck1 x dct2 to examine genetic hierarchy of interactions. (Years 1-2; 100% complete) Populations carrying heterozygous alleles in both aspartate aminotransferase and PEPCK1 were grown in the field and self-pollinated to generate segregating double mutant populations. Segregating populations of pepck1;pepck2 double mutants containing newly identified pepck2 alleles were also grown in the field to advance populations toward homozygous double mutant stocks. d. Continue to screen databases of Mu and Ac/Ds collections for additional insertions alleles in target genes. (Year 1-2; 100% complete) Ac/Ds lines were screened by three undergraduate researchers under the supervision of postdoc Arp in summer 2019. Two new alleles in alanine aminotransferase (a gene involved in the PEPCK pathway not previously identified as a target gene in this project) were identified. e. Introgress mutant alleles into multiple genetic backgrounds including H99 (for transformation), W22 (common genetic background), ILP (Illinois low protein) and IHP (Illinois high protein) (Year 2, 50% complete) Introgression efforts into IHP and ILP are ongoing, using greenhouse and field nurseries to achieve 2 generations per year; the introgressions are currently at the BC2S1 and BC3 stage Objective 2: Employ Cas9-mediated genome editing to generate mutations in the PEPCK pathway genes for which transposon mutants are not available. (Years 1-3) a. Design CRISPR/Cas9 constructs for creating loss-of-function alleles of OMT1, NADP-MDH and three transcription factors (Years 1-2; 100% complete) Completed in previous reporting period b. Transform constructs into inbred H99 through biolistics-based transformation (Years 1-3; 100% complete) Completed in previous reporting period c. Recover transgenics and identify CRISPR-induced lesions (Years 2-3; 90% complete) For each of the OMT1 and NADP-MDH editing experiments, a number of selected callus lines were obtained that tested positive for integration of each of the selectable marker, Cas9, and gRNA components. Among the three callus lines regenerated into plants with the NADP-MDH editing construct, one showed a small DNA deletion at the expected site of Cas9 cleavage. This plant appeared to harbor a bi-allelic mutation, but unfortunately this plant did not produce any reproductive structures. For OMT1, regeneration was attempted for 20 callus lines, many of the plantlets produced atypical white leaf sectors that could indicate reductions or loss of OMT1 function. Following transfer to soil, only three regenerants survived, which were fully green and showed no evidence of OMT1 mutations when assayed by PCR. In parallel to the above experiments, we also attempted CRISPR-mediated mutagenesis of the gene encoding L-asparaginase (ASNase), which modulates asparagine cycling. Knockouts of ASNase represent an alternative test of the hypothesis presented in the original proposal that carbon fixation through the PEPCK pathway may be most important under low N conditions. This experiment succeeded in generating plants with asnase mutations that have produced seeds, and await confirmation of inherited mutations. d. Self-pollinate to create fixed lines and cross pollinate nadp-mdh mutants to dct2 (Years 2-3; 0% complete). Because we did not successfully recover nadp-mdh mutants, this objective was not completed. Objective 3: Characterize single mutants at biochemical, physiological, and transcriptional levels. (Years 1-3). a. Mutant alleles will be grown in the field and greenhouse under low and high N and effects on biomass, seed weight, seed protein content and seed number will be characterized. (Years 1-3; 100% complete) Field-based phenotyping was performed for each of Years 1, 2, 3, and 4 in the nitrogen response field in Urbana. Whole plant sampling was performed at maturity to determine the biomass and nitrogen content of vegetative and reproductive components of the plants. Yield component traits were measured including kernel number, kernel weight, kernel volume, kernel row number, ear length, ear width, and cob weight. Seeds were measured for protein using non-destructive, whole seed near infrared reflectance methods. b. RNAseq library construction will be performed on mutant alleles on seedling and mature plant tissues under low and high N. (Year 2; 100% complete) Completed in previous reporting period c. Stable isotope assimilation assays and metabolic flux analysis will be performed on plants grown at high and low N. (Years 2-3; 100% complete) An experiment using 13CO2­ labeling was performed to compare pepck1::Ds to wild-type plants grown at high and low N. Plants were sampled at ten time points (0, 10, 15, 20, 30, 60, 90, 180, 300, and 600 seconds) to determine the rate of label incorporation into central carbon metabolites. Metabolites including amino acids and sugar phosphates were quantified using a HILIC-based method on a QTRAP 6500 mass spectrometer. Total pool sizes for each metabolite were measured separately from the unlabeled control samples. Additionally, biomass traits (plant height, stem circumference) were measured from the same plants used for stable isotope labeling. Isotopomer quantification was performed using Analyst software and in-house R scripts. Label incorporation into malate and aspartate occurred rapidly, and appreciable amounts of 13C labeled malate and aspartate were measured in the first time point (10s). Label incorporation into Calvin Cycle metabolites was slower, with a brief lag before label was observed in the second time point. This lag is reflective of labeled carbon coming into the Calvin Cycle through the C4 shuttle. Pool sizes for metabolites were compared between the mutant and wild-type and between nitrogen treatments. Overall, pool sizes for all metabolites were smaller at low N in both genotypes. Many metabolites also had smaller pools in the mutant compared to wild type. The exception is aspartate, which accumulated to very high levels in pepck1::Ds for both N treatments. This is consistent with the mutant having decreased PEPCK function, as aspartate is one reaction upstream of PEPCK activity.
Publications
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Progress 12/15/17 to 12/14/18
Outputs
Target Audience:The target audiences are academics and industry scientists working in the fields of plant breeding, plant biology and plant genetics. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Below is a list of conferences and symposia, and other professional development experiences: Postdoc (J. Arp) and PI's attendance (Allen) at PAG 2019 with presentation of science in poster form by Dr. Arp. Maize Genetics Conference attendance by co-PI Moose, postdoc Arp, Jinga and Rhodes (graduate students in Moose lab). Intended participation by all at upcoming Maize Genetics Conference spring 2019 with possible presentation of science. Professional training at the Danforth Center regarding responsible conduct of research, harassment, unconscious bias and respecting diversity in the work place and maintaining a safe work place. Scientific training by Li-Cor on measurement of photosynthetic parameters with new instruments and techniques. How have the results been disseminated to communities of interest?Posters were presented at the Danforth Center Scientific Retreat, Fall Symposium, and at international conferences such as PAG and the Maize Genetics Conference. Currently we are composing a manuscript that will be based on analysis of the pepck single mutant. What do you plan to do during the next reporting period to accomplish the goals?During the coming year we intend to: Complete analysis of field phenotypic data Continue to make crosses using Ds insertion lines to generate double mutant and introgression lines Perform 13CO2 labeling experiments and analysis Confirm genome edits from CRISPR constructs in transformed maize plants Obtain nadp-mdh, omt, and asparaginase mutants and perform initial phenotyping Report results in talks and publication(s)
Impacts
What was accomplished under these goals?
Objective 1: Identify and characterize Ds and Mu transposon insertions in components of the PEPCK pathway in maize (Year 1-3; 90% complete) a. Establish screening populations of Ds donors in close proximity to AspAT-m, AspAT-bs and PEPCK2 as platforms for mobilization. b. Conduct reverse-genetic screens to identify additional insertion alleles into target genes. (Year2; 100% complete) To date 10,500 seedlings have been screened for insertion alleles in AspAT-bs, and 5,500 seedlings have been screened for PEPCK2. From the screens, two alleles in PEPCK2 and one in AspAT-bs were identified and confirmed to be heritable. The resulting Ds insertion alleles were self-pollinated to bulk seed stock and outcrossed with pepck1 alleles and each other to begin building the double mutants. We were unable to target AspAT-m through a Ds approach. c. Self-pollinate and create single and double mutant combinations of pepck1 x pepck2 and pepck1 x dct2 to examine genetic hierarchy of interactions. (Years 1-2; 100% complete) Double mutants combining pepck1 x pepck2: double heterozygous plants were grown and self-pollinated in the field during Summer 2018. Homozygous double mutants will be identified from the resulting ears during the winter of 2018-2019. Segregating double mutant populations for pepck1::Ds x dct2::Ac were generated in 2018. Resulting pepck1; dct2 double mutants phenotyped from the segregating population in the greenhouse. The population segregated into three phenotypic groups that included a set of wild-type and pepck1 mutants that grow normally, dct2::Ac homozygous mutants that show light green coloration and stunted growth, and pepck1::Ds;dct2::Ac double mutants that were also light green and increasingly stunted. The homozygous pepck1::Ds;dct2::Ac plants were unable to grow to maturity and died after approximately two weeks, once the plants had exhausted germinative seed reserves. d. Continue to screen databases of Mu and Ac/Ds collections for additional insertions alleles in target genes. (Year 1-2; 100% complete) Mutator lines containing insertions in AspAT-m were self-pollinated to generate bulk stocks of homozygous mutant plants, and the lines were crossed to the other mutant lines to generate double mutant populations. e. Introgress mutant alleles into multiple genetic backgrounds including H99 (for transformation), W22 (common genetic background), ILP (Illinois low protein) and IHP (Illinois high protein) (Year 2, 50% complete) Introgression efforts into IHP and ILP are ongoing, using greenhouse and field nurseries to achieve 2 generations per year; the introgressions are currently at the BC1S1 and BC2 stage Objective 2: Employ Cas9-mediated genome editing to generate mutations in the PEPCK pathway genes for which transposon mutants are not available. (Years 1-3; 50% complete) a. Design CRISPR/Cas9 constructs for creating loss-of-function alleles of OMT1, NADP-MDH and three transcription factors (Years 1-2; 100% complete) Single guide RNAs (sgRNAs) for both OMT1 and NADP-MDH were cloned into a vector downstream of a RNA polymerase III promoter; however, these sgRNAs did not produce confirmed genome edits. The Moose lab adapted methods described for genome editing in wheat (Liang et al., 2018, Nature Protocols), to conduct in vitro assays for the efficiency of target cleavage by candidate sgRNAs. Assays were conducted for the optimal sgRNA targeting either OMT1 or NADP-MDH. Both sgRNAs showed good cleavage efficiencies, which suggests they should be effective in planta. We have also modified our cloning strategy using newly available vectors from the Dan Voytas lab. In our earlier experiments, we followed the approach of expressing Cas9 and sgRNAs from purified fragment(s) delivered by biolistics. We observed that integration of intact Cas9 fragments and co-integration of Cas9 and sgRNA fragments occurred at lower than expected frequencies, perhaps due to the large size (>8-kbp). The new system enables rapid cloning of multiple sgRNAs into a smaller vector that produces both Cas9 and sgRNAs from the same transcript, greatly increasing frequency of co-delivery of both required factors. We have now produced vectors in the new system for both OMT1 and NADP-MDH. b. Transform constructs into inbred H99 through biolistics-based transformation (Years 1-3; 100% complete) We have conducted three rounds of transformation experiments to generate genome edits. The first round, in Year 1, did not yield edited plants. In a large experiment this summer, 20 events tested positive by PCR for sgRNAs and Cas9 and are in the plant regeneration phase to generate stable transgenic plants. In a third experiment, we delivered Cas9-sgRNA ribonucleoprotein complexes assembled in vitro to a large population of immature embryos, and then rapidly induced these embryos to regenerate. Encouraging results have already been obtained--a few plants regenerated following delivery of the OMT1 sgRNAs are producing atypical white or yellow sectors that might be an expected phenotype for loss of OMT1 function. c. Recover transgenics and identify CRISPR-induced lesions (Years 2-3; 75% complete) Efforts to characterize gene edited maize plants are ongoing using restriction enzyme, Sanger, and high-throughput sequencing approaches. d. Self-pollinate to create fixed lines and cross pollinate nadp-mdh mutants to dct2 (Years 2-3; 0% complete). We expect nadp-mdh mutants in 2019. Objective 3: Characterize single mutants at biochemical, physiological, and transcriptional levels. (Years 1-3). a. Mutant alleles will be grown in the field and greenhouse under low and high N and effects on biomass, seed weight, seed protein content and seed number will be characterized. (Years 1-3; 75% complete) Field-based phenotyping was performed for each of Years 1, 2 and 3 in the nitrogen response field in Urbana. Whole plant sampling was performed at maturity to determine the biomass and nitrogen content of vegetative and reproductive components of the plants. Yield component traits were measured including kernel number, kernel weight, kernel volume, kernel row number, ear length, ear width, and cob weight. Seeds were measured for protein using non-destructive, whole seed near infrared reflectance methods. Data analysis for the combined multi-year dataset is ongoing. To characterize a stronger nitrogen deficit under conditions where the PEPCK pathway may be more physiologically important, a hydroponics N treatment was also performed in a low CO2 (~200 parts per million) growth chamber. The pepck1::Ds mutant seedlings and wild-type, W22 control plants were grown under deficient (1.5mM) nitrogen in the growth chamber for 80 days. Differences were observed in nitrogen stress response between the wild-type and mutant plants. b. RNAseq library construction will be performed on mutant alleles on seedling and mature plant tissues under low and high N. (Year 2; 100% complete) Samples were taken in June 2017 from plants at the V8 growth stage from high and low nitrogen plots. Leaf segments were taken from four points along the developmental gradient of the 13th leaf. RNA was extracted, and library preparation was performed. RNA sequencing was done by the W.M. Keck Center / Roy J. Carver Biotechnology Center at the University of Illinois Urbana-Champaign. Reads were aligned to the W22 genome and differential gene expression was called using EdgeR. c. Stable isotope assimilation assays and metabolic flux analysis will be performed on plants grown at high and low N. (Years 2-3; 50% complete) Pilot experiments have been performed to optimize a labeling procedure for maize plants 35 days after sowing. A new 13CO2­ labeling chamber was constructed to deliver 13CO2 to a consistent, small area of the leaf. An experiment to compare pepck1::Ds to wild-type plants grown at high and low N, for eight time points will be conducted in early 2019 using plants that are growing now.
Publications
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Progress 12/15/16 to 12/14/17
Outputs
Target Audience:The target audiences are academics and industry scientists working in the fields of plant breeding, plant biology and plant genetics. Changes/Problems:We have not yet been able to utilize gRNA's to successfullytarget the MDH and OMT genes in the H99 background and will thus seek to collaborate with DuPont Pioneer to utilize their Baby Boom/Wuschel transformation technology to generate edits in the W22 germplasm. If successful, this would greatly accelerate the progress of transgenics. What opportunities for training and professional development has the project provided?Postdoctoral Associate Arp attended the 2018 PAG conference to present her results at a poster session and had the opportunity to network with experts in the field. She also received a travel award from the Danforth Center to attend the Maize Genetics Conference to present her results in March 2018. In the summer of 2018 she will mentor an undergraduate student as part of the Danforth outreach program. How have the results been disseminated to communities of interest?Presentation have been made at the Plant and Animal Genome conference by Arp. What do you plan to do during the next reporting period to accomplish the goals?This is summarized in "what was accomplished under these goals"
Impacts
What was accomplished under these goals?
Objective 1: Identify and characterize Ds and Mu transposon insertions in components of the PEPCK pathway in maize (Year 1-3; 50% complete) The primary goal of this objective is to establish the populations needed to alter the flux of both carbon and nitrogen intermediates that are utilized during photosynthesis. a. Establish screening populations of Ds donors in close proximity to AspAT-m, AspAT-bs and PEPCK2 as platforms for mobilization. (Year2; 100% complete) Approximately 30 to 40 crosses were made to tag AspAT-bs and PEPCK2, and around 10,000 seed were harvested to use in the Ds screen. The Ds populations for AspAT-bs and PEPCK2 are being screened this winter in the greenhouse. Currently, 2,500 seedlings have been screened for insertion alleles in AspAT-bs, and 1,000 seedlings have been screened for PEPCK2. Resulting Ds insertion alleles will be self-pollinated to bulk the seed stock and outcrossed to pepck1 alleles to begin building the double mutants. b. Conduct reverse-genetic screens to identify additional insertion alleles into target genes (Year 2; 50% complete, see above). c. Self-pollinate and create single and double mutant combinations of pepck1 x pepck2 and pepck1 x dct2 to examine genetic hierarchy of interactions. (Years 1-2; 50% complete) pepck1 x pepck2plants were self-pollinated and segregating progeny will be screened this winter to identify double mutants. We will also screen for additional Ds insertions in pepck2 as it likely that the Mu allele of pepck2 is a weak allele.pepck1anddct2plants were crossed in the field in 2017, and the resulting double heterozygous plants will be self-pollinated this winter in both the winter nursery in Mexico and in the greenhouse. Resultingpepck1;dct2double mutants are not expected to be viable, so they will be phenotyped from the segregating population in summer 2018. d. Continue to screen databases of Mu and Ac/Ds collections for additional insertions alleles in target genes. (Year1-2; 100% complete) Four newMutatorinsertion lines with AspAT-m alleles were identified and seeds will be self-pollinated and outcrossed to W22 in the greenhouse. These alleles will also be crossed to the pepck1 and dct2 lines in the winter greenhouse to begin generation of double mutant lines. e. Introgress mutant alleles into multiple genetic backgrounds including H99 (for transformation), W22 (common genetic background), ILP (Illinois low protein) and IHP (Illinois high protein) (Year2, 25% complete) Plants have been crossed to IHP, ILP, and W22. Given recent advancements in transformation (Lowe et al., 2016, Plant Cell 28:1998-2015), we anticipate that it will be possible to transform multiple maize inbred lines soon. The DDPSC has recently signed an agreement with Pioneer and will initiate experiments soon to transform W22. A second generation of backcrossing will be performed in the greenhouse this winter. Multiple plantings of IHP, ILP, and W22 will be done in order to match the flowering time to the mutants. Objective 2: Employ Cas9-mediated genome editing to generate mutations in the PEPCK pathway genes for which transposon mutants are not available. (Years 1-2; 30% complete) a. Design CRISPR/Cas9 constructs for creating loss-of-function alleles of OMT1, NADP-MDH and three transcription factors (Years 1; 50% complete) Guide RNAs to target OMT1 have been designed, and transformations will occur this winter into lines carrying Cas9. Seed from transformed plants is expected by the end of summer 2018. b. Transform constructs into inbred H99 through biolistics-based transformation (Years1-2; 50% complete) Cas9-transformed H99 inbreds are being testing using ELISA to determine expression of Cas9 protein. c. Recover transgenics and identify CRISPR-induced lesions (Years 2-3; 25% complete) We are currently screening plants to identify edits in MDH and have designednew gRNAs to target NADP-MDH and maintain guide activity. d. Self-pollinate to create fixed lines and cross pollinate nadp-mdh mutants to dct2 to test hypothesis that carbon flux will increase through the PEPCK1 pathway.(Years 2-3; 0% complete). Plants with edited NADP-MDH are expected to be grown this winter (see part c). Objective 3: Characterize single mutants at biochemical, physiological, and transcriptional levels. (Years 1-3; 50% complete). a. Mutant alleles will be grown in the field and greenhouse under low and high N and effects on biomass, seed weight, seed protein content and seed number will be characterized. (Years 1-3; 66% complete) Field-based phenotyping was performed for a second year in the nitrogen response field in Urbana.Greenhouse N treatment is planned for this winter in order to characterize a strong N deprivation response in the pepck1-1 and 1-2 mutant lines and will be performed in coordination with13CO2labeling experiments (see part c). b. RNAseq library construction will be performed on mutant alleles on seedling and mature plant tissues under low and high N. (Year2; 75% complete) Samples were taken in June 2017 from plants at the V8 growth stage from high and low nitrogen plots. Leaf segments were taken from four points along the developmental gradient of the 13thleaf. RNA was extracted and library preparation was performed. Due to problems with the Lexogen library preparation, samples were of low quality and not sequenced. Additional troubleshooting and optimization of the protocol is underway, and libraries should be ready for sequencing in late February.
Publications
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Progress 12/15/15 to 12/14/16
Outputs
Target Audience:Work over the past year has focussed on generating the genetic materials and biological samples that will enable a systems approach to understanding carbon-nitrogen relations in maize leaves. The target audience are both academics and industry scientists working in the fields of plant breeding, plant biology and plant genetics. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?1. RNA will be extracted from mutant and wild-type samples for RNAseq analysis. 2. CRISPR/Cas9 genome editing events will be characterized. 3. Double mutants will be generated. 4. 13C and 15N labeling experiments will be initiated.
Impacts
What was accomplished under these goals?
Objective 1. Identify and characterize Ds and Mu transposon insertions in components of the PEPCK pathway in maize (Year 1-3; 20% complete) The primary goal of this objective is to establish the populations needed to alter the flux of both carbon and nitrogen intermediates that are utilized during photosynthesis. This understanding will help to both breed and engineer more carbon and nitrogen use efficient forms of photosynthesis that will reduce required water and nitrogen inputs. We identified several specific aims and progress on each is detailed below. a. Establish screening populations of Ds donors in close proximity to AspAT-m, AspAT-bs and PEPCK2 as platforms for mobilization. (Year1; 0%) We identified Ds transposons that are within the target range for mutagenesis of AspAT-m and AspAT-bs and had planted in the summer nursery for propagation. However, due to poor field and weather conditions, we were unable to propagate these lines. We will now plant in the greenhouse for propagation and build mutagenesis populations in the summer of 2017. We did not identify Ds insertions near PEPCK2, but have recovered a Mu insertion allele in PEPCK2 as well as Mu insertions in AspAT-m and AspAT-bs. b. Conduct reverse-genetic screens to identify additional insertion alleles into target genes (Year 2; see above). c. Self-pollinate and create single and double mutant combinations of pepck1 x pepck2 and pepck1 x dct2 to examine genetic hierarchy of interactions. (Years 1-2; 25% complete) Crosses were performed between pepck1 and pepck2 single mutants in the summer nursery. Crosses between pepck1 and dct2 will be performed in the greenhouse and self-pollinations of pepck1 x pepck2 will also be performed in greenhouse. d. Continue to screen databases of Mu and Ac/Ds collections for additional insertions alleles in target genes. (Year1-2; 50% complete) This work is ongoing and will likely accelerate with the addition of newly mapped Ds lines from the Brutnell lab and Mutator insertion lines from the McCarty lab. e. Introgress mutant alleles into multiple genetic backgrounds including H99 (for transformation), W22 (common genetic background), ILP (Illinois low protein) and IHP (Illinois high protein) (Year2, 0% complete) This work will be conducted in the winter nursery as flowering time differences prevented crosses between diverse genotypes in the summer nursery. We are planning to cross H99, W22, IHP and ILP to pepck1-1, 1-2, dct2, aspat1, aspat2. Objective 2. Employ Cas9-mediated genome editing to generate mutations in the PEPCK pathway genes for which transposon mutants are not available. (Years 1-2; 30% complete) a. Design CRISPR/Cas9 constructs for creating loss-of-function alleles of OMT1, NADP-MDH and three transcription factors (Years 1; 50% complete) We have targeted MDH and OMT for disruption with two guide RNAs each and transcription factor targets will be defined following analysis of RNAseq data. b. Transform constructs into inbred H99 through biolistics-based transformation (Years1-2; 50% complete) We have generated Cas9-expressing H99 inbreds that will serve as recipients for targeted disruptions of MDH and OMT. We have also initiated bombardment experiments in which a construct containing Cas9 and guides are introduced into embryos and will be carried through the transformation process. We are also trying to regenerate events that were not under selection and screening for chimeras using PCR to test for edits. c. Recover transgenics and identify CRISPR-induced lesions (Years 2-3; 0% complete) Not yet initiated. d. Self-pollinate to create fixed lines and cross pollinate nadp-mdh mutants to dct2 to test hypothesis that carbon flux will increase through the PEPCK1 pathway.(Years 2-3; 0% complete). Not yet initiated. Objective 3. Characterize single mutants at biochemical, physiological, and transcriptional levels. (Years 1-3). a. mutant alleles will be grown in the field and greenhouse under low and high N and effects on biomass, seed weight, seed protein content and seed number will be characterized. (Years 1-3; 30% complete). Tissue was harvested from field grown single mutant alleles of pepck1-1, 1-2, aspat1, aspat2, pepck2 and the near isogenic W22 control. RNA will be extracted and used for RNAseq analysis in year 2 of the project. Phenotypic characterizations including biomass, seed weight, seed number and seed protein content are currently underway. b. RNAseq library construction will be performed on mutant alleles on seedling and mature plant tissues under low and high N. (Year2; 0% complete) Not yet initiated. c. Stable isotope assimilation assays and metabolic flux analysis will be performed on plants grown at high and low N. (Years 2-3; 0% complete) Not yet initiated.
Publications
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