Progress 06/01/22 to 05/31/23
Outputs
Target Audience:Plant scientists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Training activities for graduate and undergraduate students occurred via one-on-one work with a mentor (PD Kelley or CoPD Walley). Professional development activities included attendance at the 2022 Maize Genetics Conference by PD Kelley, CoPD Walley, three graduate students (Craig Cowling, Maxwell McReynolds, and Linkan Dash), and three undergraduates (Melissa Draves, Rebekah Muench, and Jackson Marshall). How have the results been disseminated to communities of interest?Results have been published and are available as open-access preprints on BioRxiv. PD Kelley has performed three Science Bound Saturday outreach activities with one graduate student (Craig Cowling) and one undergraduate student (Melissa Draves) to increase public awareness and knowledge of maize roots and hormone responses. These Science Bound Saturday events reached 60 pre-college students of color who are interested in pursuing STEM degrees at a university. What do you plan to do during the next reporting period to accomplish the goals?We plan to finish up the data acquisition for Objective 2 and submit three manuscripts for publication (one on ZmARF27, another on ZmPILS6, and a third on ZmPILS2).
Impacts
What was accomplished under these goals?
Impact statement: Roots are a key organ for nutrient and water acquisition in crops. Root formation is influenced by plant hormones such as auxin, which is a common growth regulator used in agriculture. This project is focused on identifying the genetic basis for auxin-driven root development in Zea mays (maize or corn) using molecular and genetic approaches. These data will be used to inform breeding strategies for improved corn root systems and auxin applications in the field. Objective 1: To define roles for ZmARF27 in early root development, we performed a reverse genetics screen using two UniformMu transposon alleles of ZmARF27 in the W22 background. Both alleles were characterized to be loss-of-function recessive alleles with reduced expression of ZmARF27. Phenotypic analysis of arf27 mutants (designated arf27-1 and arf27-2) demonstrated that ZmARF27 is required for proper root hair formation and primary root growth. In addition, transcriptomic analysis of arf27-1 and W22 in the presence and absence of auxin indicates that this AUXIN RESPONSE FACTOR transcription factor is required for auxin-responsive gene expression. Using the transcriptomic data, an unsupervised gene regulatory network was generated for ZmARF27 using SC-ION, which contained >600 targets, which overlapped significantly with previously published DAP-seq data for ZmARF27. Several of the ZmARF27 predicted targets include classical maize genes such as RTCS1, RTH3, and RTH6, which have known roles in maize crown root architecture and root hair formation. In addition, stable transgenic tagged lines have been generated for ZmARF27 (pUBIL:ZmARF27-GSYellow) in B104 to enable ChIP-seq and microscopic experiments. Altogether these experiments define a novel role for ZmARF27 in maize root morphogenesis and provide an inroad into understanding the molecular basis of maize root formation. Objective 2: To uncover the functions of ZmPILS2 and ZmPILS6 in promoting maize root morphogenesis, we have characterized loss-of-function alleles for both genes using publicly available UniformMu and Mu_illumina lines. For ZmPILS6, we have two null alleles (designated pils6-1 and pils6-3) that are in the W22 and B73 backgrounds, respectively. These alleles were confirmed using RT-PCR and RT-qPCR. Both alleles have reduced crown root architecture compared to W22, with shorter total lengths and reduced root network area. In addition, pils6-1 and pils6-3 have reduced lateral root formation compared to W22. ZmPILS6 is localized to the endoplasmic reticulum (ER), similar to Arabidopsis PILS orthologs. ZmPILS6 can transport radiolabeled indole-3-acetic acid (IAA or auxin) when expressed in yeast. Using LC-MS, we measured endogenous levels of IAA in primary maize roots. IAA levels are highest in the stele (vasculature) and meristematic zone, compared to the elongation zone and cortex. In pils6-1 roots, radiolabeled IAA transport is altered in the meristem and elongation zone. Proteomics (global and phospho) datasets for W22 and pils6-1 with and without auxin were analyzed to determine enriched Gene Ontology (GO) terms and dysregulated proteins in the absence of ZmPILS6. For ZmPILS2, we bulked seed stocks to account for germplasm loss in 2021 due to seed storage failure. During the summer of 2022, pils6 alleles were crossed to pils2 alleles to create a novel double mutant for root phenotyping. Weighted gene correlation network analysis (WGCNA) was performed using W22 and pils6-1 proteomics data. The WGCNA analysis identified 19 modules, 1299 predicted proteins co-expressed with ZmPILS6, and 415 protein candidates that are highly correlated with ZmPILS6. In addition, stable transgenic tagged lines have been generated for ZmPILS6 (pUBIL:ZmPILS6-GSYellow) on B104 to enable protein-protein studies and microscopy. Objective 3: We have generated predictive multi-scale auxin-responsive regulatory networks that underpin maize root architecture using transcriptome data for primary roots dissected into four regions (meristematic zone, elongation zone, cortex, and stele) in response to auxin at two time points using SC-ION. This network contained 15,856 nodes and 86,461 directed edges. It contained a total of 1,372 transcription factors and 27 out of the 33 annotated maize ARFs. From this network, we reconstructed subnetworks for all 27 maize ARFs and found little overlap between the predicted targets for these transcription factors.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Draves M.A., Muench R.L., Lang M.G., Kelley D.R. (2022) Maize Seedling Growth and Hormone Response Assays Using the Rolled Towel Method. Current Protocols, first published 04 October 2022. DOI: https://doi.org/10.1002/cpz1.562
- Type:
Theses/Dissertations
Status:
Published
Year Published:
2022
Citation:
Maxwell Reid McReynolds (2022) Exploring the functions of hormone signaling using omics profiling. URI: https://dr.lib.iastate.edu/handle/20.500.12876/8zn7MIEw.
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
McReynolds M.R., Dash L., Montes C., Draves M.A., Lang M.G., Walley J.W., Kelley D.R. (2022) Temporal and spatial auxin responsive networks in maize primary roots. Quantitative Plant Biology, Volume 3, 2022, e1. DOI: https://doi.org/10.1017/qpb.2022.17
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Interdisciplinary Plant Science Consortium (2023) Inclusive collaboration across plant physiology and genomics: Now is the time! Plant Direct, first published 17 May 2023 https://doi.org/10.1002/pld3.493
- Type:
Book Chapters
Status:
Awaiting Publication
Year Published:
2023
Citation:
Clark N.M, Hurgobin B., Kelley D.R., Lewsey M.G., Walley J.W. (2022) Inference of multi-omics networks in plant systems. Accepted at Plant Gene Regulatory Networks, 2nd Edition, Methods in Molecular Biology, Springer Nature. Zenodo DOI: https://doi.org/10.5281/zenodo.6962317.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2023
Citation:
Cowling C.L., Triebe A.R., Dash L., Kelley D.R. Let us count the ways: roles of auxin in maize growth and development. Journal of Experimental Botany, invited review article.
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Progress 06/01/21 to 05/31/22
Outputs
Target Audience:This project has served two female undergraduate researchers, one male Hispanic undergraduate researcher, 30 underserved middle school students (Black and Hispanic) from central Iowa via Science Bound Saturday outreach activities, and two male graduate students (one who is a US Veteran). Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?This project has provided training for two graduate students (one a US Veteran) and three undergraduates (two females and one Hispanic male) in molecular biology, genetics, transcriptomics, proteomics, microscopy, and systems biology. Professional development, including one-on-one work with a mentor (PI Kelley and Co-PI Walley), attendance at the Maize Genetics conferences in summer 2021 and spring 2022, giving seminars, weekly study groups among the students, and individual study. PI Kelley and Co-PI Walley gave invited talks at local and national conferences and seminar series. How have the results been disseminated to communities of interest?McReynolds M.R. *, Dash, L. *, Montes, C., Draves, M.A. *, Lang, M.G. *, Walley, J.W., Kelley, D.R. (2021) Temporal and spatial auxin responsive networks in maize primary roots. BioRxiv doi: https://doi.org/10.1101/2022.02.01.478706. PI Kelley has given several talks in which the results of this work have been disseminated to communities of interest: 1. 2022 Invited speaker, Pre-Maize Development Meeting, Maize Genetics Meeting, St. Louis MO. Regulation of maize root growth by auxin pathways. 2. 2021 Invited Early Career Speaker, Corn Breeding Research Meeting, virtual. Roles of auxin pathways driving maize growth. 3. 2022 Invited speaker, Phenomics Phridays. Discovering genetic drivers of auxin dependent phenotypes in maize. 4. 2022 Invited speaker, ISU Genetics Club. Discovering auxin regulated phenotypes from proteotypes. 5. 2021 Invited speaker, Local Hormone Meeting, Duke University. Roles of auxin pathways driving maize root growth. 6. 2021 Invited speaker, University of Massachusetts Amherst Plant Bio seminar. Linking proteotype to phenotype during root development. 7. 2021 Invited speaker, Crop Bioengineering Center Annual Meeting. Base editing in maize using a Cas9-nickase to assess the influence of posttranslational protein modifications in vivo. 8. 2021 Speaker, ISU CALS Building Community Digital/Precision Agriculture Research. Roles of auxin in plant growth. 9. 2021 Speaker, ISU Bioinformatics and Computational Biology graduate program seminar. Proteotypes to phenotypes. What do you plan to do during the next reporting period to accomplish the goals?We plan to submit two manuscripts for publication, finalize the publication of two manuscripts, and present our work at the Maize Genetics Conference in 2023. We plan to continue our collaborations with the Strader lab at Duke University to complete our PILS studies and finalize our PILS co-expression networks.
Impacts
What was accomplished under these goals?
Impact Statement: Roots are the primary organ for water and nutrient uptake in crops. Our project is focused on understanding how hormones influence corn (maize) root formation, which is instrumental in informing agricultural strategies for improved crop water stress tolerance and nutrient usage. Our genetic and molecular studies fit with the USDA NIFA strategic goal to "advance our Nation's ability to achieve global food security and fight hunger" (NIFA Strategic Plan FY2014-2018), and this project is aligned with the Physiology of Agricultural Plants plant growth and developmental processes program area priority. During this project period, two Iowa State University graduate students (one male US Veteran and one Indian male) and four undergraduate research assistants (three female and one Hispanic male) designed and conducted molecular and genetic research on hormone-regulated plant growth. In addition, the graduate and undergraduate students developed new research methods and procedures for integrating large-scale data across scales (molecular to organismal). These results were presented via posters at the 2022 Maize Genetics Meeting in St. Louis, MO, 2022 Research in the Capitol, 2022 ISU Annual Symposium on Undergraduate Research & Creative Expression (SoURCE), and the 2022 STUPKA Symposium. Finally, two manuscripts were prepared and submitted during this project period with the students as first authors; one manuscript is accepted, and the other is currently under review. Broader outcomes of this work for the real world include (1) improved research and critical thinking skills among ISU students who are the future of our US agricultural workforce, (2) diversification of our agricultural workforce (one of the undergraduate female students is now a full-time employee at Bayer and the Hispanic male undergraduate researcher is in the Interdepartmental Genetics & Genomics Ph.D. program at ISU) and (3) new fundamental knowledge of root growth and development. Objectives: In the past year, we have made significant strides toward accomplishing the objectives of this project. (1) Define roles for ZmARF27 in early root development Using genetic, transcriptomic, and proteomic approaches, we have determined that ZmARF27 is required for primary root length, lateral root formation, and proper auxin responses in vivo. We have quantified shoot length, primary root length, and lateral root density both in the presence and absence of exogenous 10 uM indole-3-acetic acid (auxin) treatment and determined that arf27-1 and arf27-2 (two recessive loss of function alleles we have characterized from the UniformMu transposon collection) roots are shorter with reduced lateral root density. In addition, the arf27-1 mutant was observed to produce twin embryos at a 5-10% frequency. The auxin response DR5:RFP marker has been crossed into the arf27-1 mutant to visualize auxin response in these mutant roots next year. We have performed ionomics analysis of arf27-1 and arf27-2 leaves and seeds and identified reduced manganese levels in these mutants compared to wild-type controls. We have generated ten independent T1 ProUBQ:ARF27-GSYellow transgenic lines that have been backcrossed to B104 once and verified for transgene expression using RT-PCR. Gene expression analysis of loss of function arf27 mutants compared to wild-type W22 both in the presence and absence of 1 hour 10 uM indole-3-acetic acid (auxin) treatment has been conducted using 3' RNA-seq (transcriptomics), proteomics, and phosphoproteomics. From these expression data, we have generated an ARF27 gene regulatory network (GRN) using a GENIE3-based unsupervised GRN software method known as SC-ION (spatiotemporal clustering and interference of omics networks) from Clark et al., Nature Communications 2021. The ARF27 GRN consists of 4,330 nodes with 364,808 edges. ARF27 is predicted to target 847 nodes directly, including 128 genes that are auxin responsive based on our transcriptomic data. This network was overlaid with the DAP-SEQ data for ARF27 from the Galli et al., 2018 Nature Communications paper to determine putative direct targets of ARF27. From this analysis, we identified a total of 431 shared first neighbor targets, which represents ~50% of our predicted targets as being shared between these two independent analyses. (2) Uncover functions of ZmPILS2 and ZmPILS6 in promoting maize root morphogenesis For this aim, two graduate students have developed an indole-3-acetic acid (IAA) transport assay for maize roots. They have carried out IAA transport assays in dissected maize roots four times using dissected B73 roots (meristematic zone, elongation zone, cortex, and stele) to determine the optimal assay conditions and quantify auxin transport in ZmPILS loss of function mutants: pils2-1,pils2-2, and pils6-1. From these assays, we can conclude that both ZmPILS2 and ZmPILS6 are required for proper auxin transport in vivo. We have carried out IAA transport assays in a heterologous system (Saccharomyces cerevisiaestrain JBY575) and determined that ZmPILS6 can move IAA across the plasma membrane. We have generated vectors containing PILS6-GSYellow and determined that ZmPIL6 is localized to the endoplasmic reticulum (ER) in Nicotiana benthimiana epidermal cells using confocal microscopy. Both graduate students independently determined that PILS6-GSYellow co-localizes with ER-mCherry using transient expression assays in N. benthimiana. In addition, we have created a new PILS2 clone that will be used for these same assays. Towards characterizing the roles of ZmPILS2 and ZmPILS6 in root morphogenesis, one of the grad students has optimized a staining protocol for characterizing lateral root primordia. To date, he has stained lateral root primordia in pils2-1 and pils2-2 mutants and observed fewer LR primordia compared to wild-type W22 roots. We have generated stable transgenic lines UBILpro:PILS6-GSYT1 lines that were confirmed by RT-PCR. The auxin response DR5:RFP marker has been crossed into the arf27-1andpils6-1 mutant. (3) Determine predictive multi-scale auxin responsive regulatory networks that underpin maize root architecture. To characterize auxin regulated networks underlying maize root development, we have characterized auxin responsive transcription across two time points (30 and 120 minutes) and four regions of the primary root: the meristematic zone, elongation zone, cortex, and stele. Hundreds of auxin-regulated genes involved in diverse biological processes were quantified in these different root regions. In general, most auxin regulated genes are region unique and are predominantly observed in differentiated tissues compared to the root meristem. Auxin gene regulatory networks (GRNs) were reconstructed with these data to identify key transcription factors that may underlie auxin responses in maize roots. Additionally, Auxin Response Factor (ARF) subnetworks were generated to identify target genes that exhibit tissue or temporal specificity in response to auxin. These networks describe novel molecular connections underlying maize root development and provide a foundation for functional genomic studies in a key crop.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2022
Citation:
Cowling, C.L.* and Kelley, D.R. (2021) An unknown protein influences maize yield via sugar and auxin. New Phytologist, invited commentary. https://doi.org/10.1111/nph.18027
- Type:
Journal Articles
Status:
Accepted
Year Published:
2022
Citation:
McReynolds M.R. *, Dash, L. *, Montes, C., Draves, M.A. *, Lang, M.G. *, Walley, J.W., Kelley, D.R. (2021) Temporal and spatial auxin responsive networks in maize primary roots. BioRxiv doi: https://doi.org/10.1101/2022.02.01.478706. Accepted to Quantitative Plant Biology.
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Progress 06/01/20 to 05/31/21
Outputs
Target Audience:
Nothing Reported
Changes/Problems:On January 7, 2021 we lost our key collaborator, Dr. Nick Lauter. Dr. Lauter died unexpectedly and was a key personnel on our project for our proosed yield studies. I have established a new collaboration with Dr. Shawn Kaeppler at the University of Wisconsin-Madison to continue our efforts to perform these yield studies; this may push these results out an additional year. Additionally, during the COVID19 pandemic my laboratory was closed according to university guidelines for 3 months in 2020. Only essential research was conducted for a 6 month period during 2020, which hampered our productivity. I had two rotation graduate students during the 2020-2021 school year and plan to have one of these students join this project in Fall 2021. What opportunities for training and professional development has the project provided?This award has supported training of one PhD graduate student in phenotyping and integrated genomics (transcriptomics, proteomics and phosphoproteomics). It has supported the training of one undergraduate in maize genetics and phenotyping. How have the results been disseminated to communities of interest?I have presented these results at scienfitic meetings via Zoom including the ISU GDCB departmental seminar (Fall 2020), in the Early Career session of the Crop Breeding Research annual meeting (February 2021), the University of Massachussetts Amherst Plant Biology seminar (April 2021) and the ISU Crop Bioengineering Center seminar (May 2021). What do you plan to do during the next reporting period to accomplish the goals?We will evaluate the transgenic pUBQ::ARF27-GSYellow, pUBQ::PILS2-GSYellow, and pUBQ::PILS6-GSYellow lines for expression and stable genomic integration; these lines will also be backcrossed to B104. We will compare our ARF27 dependent expression data to exisiting datasets, such as DAP-seq data (Galli et. al., 2019) to determine potential direct targets of this transcription factor. We will finalize our gene expression analyses to facilite network reconconstructions. We will use RT-qPCR to determine the associated gene expression levels in the all of the UFMu alleles we have phenotyped to date. We will use CRISPR-Cas9 based gene editing to generate a ARF27 knock-out or null to expand our genetic materials for gene function studies. We will repeat our auxin transport assays on the pils mutants and perform transport assays with heterologous expression of PILS2 and PILS6 in collaboration with Lucia Strader. We will prepare two manuscripts for publication, one focused on ARF27 and the other focused on PILS2 and PILS6/PIN14. We will continue backcrossing the root mutant alleles into ExPVP lines during our summer 2021 nursery.
Impacts
What was accomplished under these goals?
Objective 1. Define roles for ZmARF27 in early root development. Wehave quantified primary root and lateral root phenotypes in two UFMu alleles of arf27which are both promoter insertions (mu1078826 = arf27-1; mu1068742 = arf27-2). ARF27 is required for primary root growth and proper formation of root hairs. We have generated a pUBQ::GFP-ARF27 construct for transient expression in maize root protoplasts and established a working protocol for isolating protoplasts from five-day-old maize primary roots. GFP-AFR27 exhibits auxin-dependent nuclear-cytoplasmic localization when expressed transiently in maize root protoplasts. Specifically, in the absence of auxin GFP-ARF27 is primarily in the cytoplasm. Following auxin treatment GFP-ARF27 is primarily nuclear localized. These findings are in line with nuclear-cytoplasmic behaviors observed in the Arabidopsis ARF orthologs, ARF7 and ARF19 (Powers et. al., 2019).We have generated a pUBQ::ARF27-GSYellow construct (pLD17) and sent it to the ISU Plant Transformation facility to generate stably transformed maize lines (B104). Objective 2.Uncover functions of ZmPILS2 and ZmPILS6 in promoting maize root morphogenesis. We have performed auxin transport assays on pils2 and pils6 (now called pin14 by current MaizeGDB annotations) in collaboration with Lucia Strader (Duke University). From these assays we determined that ZmPILS2 and ZmPILS6 are required for normal auxin distributions in primary maize roots. In the absence of ZmPILS2, auxin transport is increased in the elongation zone of five-day-old primary roots. While in the absence of ZmPILS6 auxin transport is increased in the meristematic and elongation zones of five-day-old primary roots. In addition, we have quantified primary root and lateral root phenotypes in two UFMu alleles of pils2 and pils6.We have generated constructs for stable transformation into maize (B104),pUBQ::PILS2-GSYellow (pLD18) aand pUBQ::PILS6-GSYellow construct (pLD19). These will be verified in transient expression in N. benthimianaleaves before we send them to the ISU Plant Transformation facility for Agrobacterium mediated transformation. Objective 3.Determine predictive multi-scale auxin responsive regulatory networks that underpin maize root architecture. We have generated integrated transcriptomic, proteomic and phosphoproteomic datasets from W22, arf27, pils2 and pils6 mutants. These datasets will provide the foundation for our proposed co-expression and gene regulatory network reconstructions. Using four biological replicates of fifive-day-old roots treated 10 uM indole-3-acetic acid (IAA) or mock solvent control for one hour (60 minutes) we obtained the following data. Transcriptomics (FDR<0.05): 142 differentially expressed (DE) genes in W22 in response to auxin (124 up, 18 down), 472 DE genes in arf27-1 compared to wild-type (167 up and 305 down), 388 DE genes in pils2-1 compared to wild-type (142 up, 246 down) and 544 DE genes in pils6 compared to wild-type (242 up, 302 down). Proteomics (~13,900 proteins identified per run, FDR<0.10):1092differentially expressed (DE) proteinsin W22 in response to auxin,2730DE proteinsin arf27-1 compared to wild-type, 60 DE proteins in pils2-1 compared to wild-typeand 5977 DE proteins in pils6 compared to wild-type.Phosphoproteomics (~26,500 phosphopeptides identified per run, FDR<0.10): 315differentially expressed (DE) phosphopeptidesin W22 in response to auxin,155DE phosphopeptides in arf27-1 compared to wild-type, 16DE phosphopeptides in pils2-1 compared to wild-typeand 12,614DE phosphopeptides in pils6 compared to wild-type. In addition, we have crossed the arf27 and pils mu alleles into B104, PH207 and PHZ51 in collaboration with Nick Lauter (USDA ARS) in order to perform yield studies in these genetic backgrounds. We have also obtained other inbred lines (LH244, PHP02 and PH24E) from Shawn Kaeppler and initated a collaboration to cross our root mutants into these backgrounds for future yield trials.
Publications
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