Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583
Performing Department
Agronomy and Horticulture
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 factors 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
5%
Research Effort Categories
Basic
45%
Applied
5%
Developmental
50%
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 projects 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; and3) 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 programs controlling 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 legumes plants to develop root nodules.
Project Methods
Single-cell and nuclear approaches to define cell-type transcriptomes from a multicellular plant organ. Single-cell transcriptomes can be established by using isolated cells or nuclei. In plants, different labs described the feasibility of scRNA-seq with Arabidopsis root and cotyledon protoplasts. As an alternative to the use of protoplasts, we isolated plant nuclei to apply sNucRNA-seq technology on Arabidopsis roots then on the roots of various plant species including Medicago and soybean. Supporting the feasibility and the biological significance of plant sNucRNA-seq technology, comparative analyses revealed that the Arabidopsis root cellular and nuclear transcriptomes are highly correlated and are both highly correlated to a whole root transcriptome (Spearman's Rank Correlation Coefficients = 0.879-0.892). Our comparison of protoplast- and nucleus-based RNA-seq technologies also revealed differences:1) The effective isolation of plant protoplasts requires the development of particular cell wall-degrading enzymatic cocktails tailored for the differential biochemical composition of the cell wall between cells, cell types, and plant species, and the relative position of the cells in the organ. The enrichment for cell-types from the outer layers of the root and the absence of state II endodermal protoplasts characterized by the deposition of cutin and suberin in their cell wall suggest the limited release of plant protoplasts.2) The relatively large size of plant cells associated with the size discrimination of droplet systems might lead to the absence or relative depletion of large plant cells.3) The bursting of plant protoplasts decreases library construction efficiency while isolated nuclei are prone to transcript leakage. To minimize this leakage, we optimized our sNucRNA-seq protocol to maximize nuclear membrane integrity.4) The smaller pool of polyadenylated transcript per nucleus vs. per protoplast limits the number of expressed genes identified per biological entity. However, when considering clusters, the percentage of expressed genes per cluster is similar upon applying sNucRNA-seq or scRNA-seq technologies, and sNucRNA-seq data alone are sufficient to decode the tissue heterogeneity to a similar level as the scRNA-seq data. These may better represent dynamic transcriptional activity than the cellular transcriptome, which represents an integration of gene activity over time; a consequence of the rapid export of the transcripts from the nucleus, and their relative stability.According to our results, the use of single nuclei is a sensitive and valid approach for accessing the transcriptome of a heterogeneous population of biological entities, overcomes challenges associated with the use of protoplasts (see above), will improve detection of rapid and transient transcriptional responses to rhizobia inoculation, and will facilitate the comparison of transcriptomic datasets. In this USDA-AFRI project, we will apply our plant nuclei isolation protocol and fluorescence-activated nuclei sorting (FANS) to isolate at least 10,000 purified GFP/tdTomato-labelled nuclei from root sections where lateral root and nodule formations are initiated. We will perform these experiments on three different plant species: Medicago, soybean, and common bean, to reveal the programs activated or repressed during the early stage of development of lateral roots and determinate and indeterminate nodules. For each biological replicate and plant species, we will precisely estimate nuclei purity and number by using an epifluorescent microscope and an automated counter.Identification of cell-type marker genes in non-model systems: development of Visium Spatial Gene Expression (VSGE) technology for plants. The annotation of Arabidopsis root cell/nucleus clusters is facilitated by numerous -omic resources. For instance, previous transcriptomic studies, including the recent releases of Arabidopsis root single-cell transcriptomes, revealed cell-type-specific marker genes. However, because similar resources are more limited in legume species, we propose to apply VSGE to characterize the transcriptome of tissue cross-sections within their morphological context. This technology uses spatially-barcoded oligonucleotides that are printed on a glass microscopic slide. Upon their release from the cells, the polyadenylated transcripts are attached to the printed oligonucleotides. Reverse transcription is performed, creating a library of barcoded cDNAs. The unique location of each barcoded oligonucleotide on the capture area allows the mapping of the sequenced cDNA in the context of the morphology of the tissue. To maximize the release of mRNA from plant cross-sections, we optimized the conditions of permeabilization for legume root cross-sections and recently performed a first VSGE experiment to provide spatial transcript information from legume root sections (data under analysis).Monitoring auxin and CK response during the development of plant root lateral organs. Although the auxin-CK ratio is likely to be an important determinant of legume nodule development, the mechanism has yet to be defined. Recent studies are unraveling molecular mechanisms by which auxin and CK interact at the levels of biosynthesis, inactivation/degradation, transport, and signaling in plants. One of the first steps in elucidating mechanisms by which auxin and CK interact to dictate proper nodule development is to map and quantify the cellular outputs of these hormones. While many methods exist to measure auxin and CK distributions in root systems, fluorescence imaging allows for non-invasive observation of hormone outputs (a combination of both hormone levels and a cell's response). While both perception and transcriptional output sensors are available for auxin, only transcriptional output sensors have been developed for CK. We reasoned that using transcriptional output sensors for both hormones would allow the best possible comparison of relative cellular outputs of these hormones in specific cell types during nodule development. Therefore, we decided to use the DR5 promoter that specifically responds to auxin and the TCSn promoter which is the most sensitive and specific to CK. The use of fluorescent proteins that localize to the nucleus as output markers would enable accurate quantification, as outputs from nuclear-localized signals are unlikely to be affected by cell size and vacuolization. For example, quantification of auxin perception in specific root cell files using the R2D2 sensor in Arabidopsis root tips revealed that CK maintains the position of the transition zone by controlling both polar auxin transport and local auxin degradation. To enable simultaneous quantitative imaging of auxin and CK outputs, we generated a single construct that carried transcriptional output sensors for both hormones. The construct contained transcriptional fusions of nuclear-localized green fluorescent protein (GFP) to the auxin-responsive DR5 promoter and nuclear-localized tdTomato to the CK-responsive TCSn promoter (DR5:GFP-NLS and TCSn:tdTomato-NLS).