Source: UNIVERSITY OF MISSOURI submitted to NRP
SINGLE CELL CHARACTERIZATION OF THE TRANSCRIPTIONAL PROGRAMS CONTROLLING PLANT ROOT ORGAN INITIATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1031776
Grant No.
2022-67013-41793
Cumulative Award Amt.
$542,458.13
Proposal No.
2023-11863
Multistate No.
(N/A)
Project Start Date
Oct 1, 2023
Project End Date
Mar 31, 2026
Grant Year
2024
Program Code
[A1152]- Physiology of Agricultural Plants
Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211
Performing Department
(N/A)
Non Technical Summary
The goal of this proposal is to determine the molecular programs controlling the initiation and early development of lateral roots and nodules in three legume species (Medicago, soybean, and common bean). We will state-of-the-art technology on selected plant cells to precisely capture the activity of the genes of the meristematic and differentiated cells composing the lateral roots and nodules. We will compare the sets of differentially expressed genes between cells and across species to identify patterns of evolutionary conservation and divergence. We will validate the roles of differentially expressed transcription factor genes by evaluating lateral organ initiation in knockdown, knockout, and overexpressing mutants. Together, we expect to determine key regulatory genes that dictate the initiation and development of lateral roots and nodules in legumes. These will be ideal targets for manipulation by biotechnology or breeding to develop better-adapted and more efficient root systems. Ultimately, this new generation of crops will be resilient to adverse environmental conditions such as drought stress and will promote sustainable agricultural practices through optimized water and nutrient uptakes without reducing crop yields.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011419104070%
2061820103030%
Knowledge Area
206 - Basic Plant Biology; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1419 - Leguminous vegetables, general/other; 1820 - Soybean;

Field Of Science
1040 - Molecular biology; 1030 - Cellular biology;
Goals / Objectives
We propose to reveal these programs and the extent of their conservation between legume species (i.e., G. max, P. vulgaris, M. truncatula) by applying single-cell resolution RNA-seq technology on the cells composing the emerging nodules and lateral roots. These cells will be identified and isolated from transgenic roots expressing auxin and CK-inducible reporters. The major goals of this project are to: 1) reveal the dynamic transcriptional programs occurring in the meristems of the nodule and lateral root using sNucRNA-seq technology and quantitative imaging of cell-level hormone dynamics; 2) determine the levels of conservation and divergence of these programs to determine conserved and distinct developmental programs among species and between lateral organs; and 3) characterize the role of key developmental regulators of these programs in organ identity and development through reverse genetic approaches. We expect to reveal the core programs involved in meristem initiation and the shared and unique transcriptomic programscontrolling the early development of lateral roots and determinate and indeterminate nodules at single-cell resolution. The knowledge gained in this project will reveal molecular components and programs that determine the development of root organs. This fundamental knowledge will lead to the development of new strategies to control RSA to optimize its adaptation to soil quality (i.e., most soils are characterized by the heterogeneous distribution of resources) and environmental stresses (e.g., drought stress). This project will also identify overlapping and distinct developmental programs used by legume plants to develop root nodules.
Project Methods
This project includes three very distinctive and complementary collections of methods:- Microscopic method to record and quantify the activity of auxin and cytokinin reported during the early stage of nodule development in soybean, common bean, and medicago plants. These observations are critical to bridge phenotypic and molecular datasets.- Single-cell-omics. Upon applying nuclei sorting to increase the representation of the cells composing the emerging lateral root and nodule of the legume plants, we apply single-cell RNA-seq technology to establish high-resolution transcriptomes.- Computational methods. The molecular datasets are currently processed using different computational packages such as Seurat.

Progress 10/01/23 to 09/30/24

Outputs
Target Audience: Root biologists and experts in biological nitrogen fixation will benefit the most from this work. Upon completion, our study across three different legume species would be of interest to evolutionary biologists. Changes/Problems:The NCGR underspent on its budget due in part to staffing changes on other projects that have impacted the availability of the NCGR personnel assigned to this one. The NCGR has been assessing feasibility of making a new hire to cover the shortages on these other projects in order to focus previously assigned staff in the analysis-focused phases of the project that have begun. What opportunities for training and professional development has the project provided?Two MS graduate students Aniket Singh and Gurdeep Singh were trained in composite plant generation, multiphoton imaging, image analysis, gene cloning, experimental design, data documentation and interpretation of results. A post-doctoral associate Dr. Bhanu Petla was trained in image analysis, composite plant generation, undergraduate student research advising and grant proposal development. Workshops for undergraduate/graduate-level students on single-cell analysis have incorporated methods and data associated with the project. See further details under the next item. How have the results been disseminated to communities of interest?Results were disseminated in the form of posters and oral presentations at conferences. Relevant datasets added to the Legume InformationSystem added from the Libault lab during the past year include soybean root and nodule scRNA-seq datasets: https://shinycell.legumeinfo.org/glyma.expr.Cervantes-Perez_Zogli_2024/ Some new features have also been added to the tool to help better integrate it with other gene-oriented reports and views available at the site through the linkout mechanisms used by other tools on the site. Our previous deployment of the shinyCell application on Medicago root data for the legume community is now featured among the visualization resources for the Plant Cell Atlas community: https://www.plantcellatlas.org/tools-and-repositories.html?locale=es In addition, this deployment of the tool is being featured in ongoing single-cell analysis workshops hosted by NCGR under the auspices of the NM-INBRE project: https://inbre.ncgr.org/single-cell-workshop/shinycell.html. What do you plan to do during the next reporting period to accomplish the goals?We will continue assessing the results of the integration of each species data independently and further explore strategies for differential expression analysis in the single-cell context. In addition, we have begun investigating BENGAL (https://github.com/Papatheodorou-Group/BENGAL) a nextflow-based workflow for cross-species single cell integration, which includes assessments of various strategies both with respect to preservation in the integration space of biologically oriented properties (e.g. consistent cell ontology terms represented in clusters for cells from different species) as well as batch-mixing effectiveness (ie how much cross-species representation occurs within clusters). An attractive aspect of the framework in the context of plant data is the ability to assess different strategies for handling cross-species gene relationships other than one-to-one orthology. Microscopically, we will complete three different analyses: 1- The analysis of ACRO ratios in lateral root primordia 2-The imaging of root nodules 3- The analysis of ACRO ratios in root nodules Ultimately, we will generate and evaluate composite plans expressing promoter:GUS constructs for validation

Impacts
What was accomplished under these goals? The goal of this project is to characterize the molecular programs controlling the initiation and early development of plant root organs and the extent of conservation of these programs between organs (i.e., lateral root and nodules), and between legume species (Medicago, soybean, and common bean). To precisely capture the transcriptomes of the lateral root and nodule meristematic cells and the transcriptomes of the cells composing the emerging nodules and lateral roots, we propose to apply single-cell transcriptomic technology. The cells selected for analysis will be carefully selectedusing nuclear-localized green fluorescent protein to the auxin-responsive DR5 promoter and nuclear-localized Tdtomato, to the cytokinin-responsive TCSn promoter. To quantifyrelative auxin-cytokinin output (ACRO) ratios in primary root tips, transgenic roots expression of the auxin-cytokinin transcriptional output marker construct (DR5:GFP-NLS; TCSn:TdTomato-NLS) were imaged using multiphoton microscopy to collect at least six high-quality images for each species (Medicago truncatula and Common bean Phaseolus vulgaris). An image analysis pipeline was developed using ICY evaluating software reproducibility, and accuracy of nuclei detection (false positive and false negative rates). Parameters for analysis including root orientation, number of slices, projection method, region of interest, type of image for detection (Greyscale vs color), and detection sensitivity were optimized. Two-dimensional images of median optical sections of the primary roots with all cell types of interest in the primary root tip were analyzed to measure fluorescence output from each marker and ACRO ratios. Cell types and root zones (columella, quiescent center QC and meristem, and vasculature) were identified based on cell orientation and proximity of nuclei. Relative ratios were calculated for groups of nuclei at every 10 um interval. The highest ACRO ratios were observed in the QC/meristem region on the vascular end as observed in soybeans. Evaluation of raw intensity outputs indicated that cytokinin output showed a gently increasing trend from the vasculature/Qc transition point toward the root tip. In contrast, auxin output showed a sharp increase at the vasculature/Qc transition point and gently decreased towards the root tip. The high ACRO ratio in QC/meristem was primarily due to higher auxin output rather than a reduced cytokinin output. To quantifyACRO ratios in lateral roots, at least six volumes of multiphoton images have been collected for each of the following stages of lateral root development in common bean: 1a, 1b, 2, 3, 4, 5, and 6. For Medicago truncatula stages 4, 5, 6, and post emergence. ACRO ratio quantification pipelines have been developed and image analysis is in progress. Nodule imaging is in progress in both species. To validate organ and meristem-specific expression of marker genes FWL1 (nodule-specific), TMO7 (lateral root specific) and LRP1 (expressed in both) were selected. Respective promoter sequences were cloned in front of GUS and expressed in soybean composite plants. DR5:GUS and TCSn:GUS were used as positive controls. Imaging and analysis are in progress. To enable the establishment of the genetic programs governing root and nodule emergence in legumes, single-cell resolution RNA-seq data for all three species (soybean, common bean, and Medicago) have been separately processed against species-specific references augmented with the reporter gene vector construct and outputs run through the integration workflow developed from standard best-practices tools for sc/snRNA-seq, including ambient RNA noise reduction on Cellranger outputs with Cellbender; QC with scanpy/Seurat; batch effect correction/integration using Seurat. Marker genes and condition-specific differentially expressed genes for all three species have been independently generated, initially with Seurat for cluster-based assessments and with Monocle3 for continuous trajectory-localized expression assessments, and combining the top genes from the two approaches into a candidate list and enabling comparisons between condition-specific and cluster-specific genes for each species. Utilization of tradeSeq for trajectory-oriented condition-dependent expression analysis is ongoing, but initial differential topology analysis on the integration UMAPs with respect to mock/inoculated conditions has revealed some areas with enrichment for cells from replicates representing one or the other of these conditions. In addition, known marker genes supplied by the Subramanian lab and their orthologous/homoeologous counterparts have also been analyzed separately with respect to both cluster- and condition-specific expression.

Publications

  • Type: Other Journal Articles Status: Published Year Published: 2024 Citation: Grones, C., T. Eekhout, D. Shi, M. Neumann, L.S. Berg, Y. Ke, R. Shahan, K.L. Cox, Jr., F. Gomez-Cano, H. Nelissen, J.U. Lohmann, S. Giacomello, O.C. Martin, B. Cole, J.W. Wang, K. Kaufmann, M.T. Raissig, G. Palfalvi, T. Greb, M. Libault, and B. De Rybel, Best practices for the execution, analysis, and data storage of plant single-cell/nucleus transcriptomics. Plant Cell, 2024. 36(4): p. 812-828.
  • Type: Other Journal Articles Status: Published Year Published: 2023 Citation: Amini, S., J.J. Doyle, and M. Libault, The evolving definition of plant cell type. Front Plant Sci, 2023. 14: p. 1271070.