Transcriptional Reprogramming of Legume Genomes: Perspective and Challenges Associated With Single-Cell and Single Cell-Type Approaches During Nodule Development

Transcriptomic approaches revealed thousands of genes differentially or specifically expressed during nodulation, a biological process resulting from the symbiosis between leguminous plant roots and rhizobia, atmospheric nitrogen-fixing symbiotic bacteria. Ultimately, nodulation will lead to the development of a new root organ, the nodule. Through functional genomic studies, plant transcriptomes have been used by scientists to reveal plant genes potentially controlling nodulation. However, it is important to acknowledge that the physiology, transcriptomic programs, and biochemical properties of the plant cells involved in nodulation are continuously regulated. They also differ between the different cell-types composing the nodules. To generate a more accurate picture of the transcriptome, epigenome, proteome, and metabolome of the cells infected by rhizobia and cells composing the nodule, there is a need to implement plant single-cell and single cell-types strategies and methods. Accessing such information would allow a better understanding of the infection of plant cells by rhizobia and will help understanding the complex interactions existing between rhizobia and the plant cells. In this mini-review, we are reporting the current knowledge on legume nodulation gained by plant scientists at the level of single cell-types, and provide perspectives on single cell/single cell-type approaches when applied to legume nodulation

Transcriptional Reprogramming of Legume Genomes: Perspective and Challenges Associated With Single-Cell and Single Cell-Type Approaches During Nodule Development

Transcriptomic approaches revealed thousands of genes differentially or specifically expressed during nodulation, a biological process resulting from the symbiosis between leguminous plant roots and rhizobia, atmospheric nitrogen-fixing symbiotic bacteria. Ultimately, nodulation will lead to the development of a new root organ, the nodule. Through functional genomic studies, plant transcriptomes have been used by scientists to reveal plant genes potentially controlling nodulation. However, it is important to acknowledge that the physiology, transcriptomic programs, and biochemical properties of the plant cells involved in nodulation are continuously regulated. They also differ between the different cell-types composing the nodules. To generate a more accurate picture of the transcriptome, epigenome, proteome, and metabolome of the cells infected by rhizobia and cells composing the nodule, there is a need to implement plant single-cell and single cell-types strategies and methods. Accessing such information would allow a better understanding of the infection of plant cells by rhizobia and will help understanding the complex interactions existing between rhizobia and the plant cells. In this mini-review, we are reporting the current knowledge on legume nodulation gained by plant scientists at the level of single cell-types, and provide perspectives on single cell/single cell-type approaches when applied to legume nodulation

The Carbon-Nitrogen Balance of the Nodule and Its Regulation under Elevated Carbon Dioxide Concentration

Legumes have developed a unique way to interact with bacteria: in addition to preventing infection from pathogenic bacteria like any other plant, legumes also developed a mutualistic symbiotic relationship with one gender of soil bacteria: rhizobium. This interaction leads to the development of a new root organ, the nodule, where the differentiated bacteria fix for the plant the atmospheric dinitrogen (atmN2). In exchange, the symbiont will benefit from a permanent source of carbon compounds, products of the photosynthesis. The substantial amounts of fixed carbon dioxide dedicated to the symbiont imposed to the plant a tight regulation of the nodulation process to balance carbon and nitrogen incomes and outcomes. Climate change including the increase of the concentration of the atmospheric carbon dioxide is going to modify the rates of plant photosynthesis, the balance between nitrogen and carbon, and, as a consequence, the regulatory mechanisms of the nodulation process. This review focuses on the regulatory mechanisms controlling carbon/nitrogen balances in the context of legume nodulation and discusses how the change in atmospheric carbon dioxide concentration could affect nodulation efficiency

Plant Hormones Differentially Control the Sub-Cellular Localization of Plasma Membrane Microdomains during the Early Stage of Soybean Nodulation

Phytohormones regulate the mutualistic symbiotic interaction between legumes and rhizobia, nitrogen-fixing soil bacteria, notably by controlling the formation of the infection thread in the root hair (RH). At the cellular level, the formation of the infection thread is promoted by the translocation of plasma membrane microdomains at the tip of the RH. We hypothesize that phytohormones regulate the translocation of plasma membrane microdomains to regulate infection thread formation. Accordingly, we treated with hormone and hormone inhibitors transgenic soybean roots expressing fusions between the Green Fluorescent Protein (GFP) and GmFWL1 or GmFLOT2/4, two microdomain-associated proteins translocated at the tip of the soybean RH in response to rhizobia. Auxin and cytokinin treatments are suffcient to trigger or inhibit the translocation of GmFWL1 and GmFLOT2/4 to the RH tip independently of the presence of rhizobia, respectively. Unexpectedly, the application of salicylic acid, a phytohormone regulating the plant defense system, also promotes the translocation of GmFWL1 and GmFLOT2/4 to the RH tip regardless of the presence of rhizobia. These results suggest that phytohormones are playing a central role in controlling the early stages of rhizobia infection by regulating the translocation of plasma membrane microdomains. They also support the concept of crosstalk of phytohormones to control nodulation

Unleashing the potential of the root hair cell as a single plant cell type model in root systems biology

Plant root is an organ composed of multiple cell types with different functions.This multicellular complexity limits our understanding of root biology because -omics studies performed at the level of the entire root reflect the average responses of all cells composing the organ. To overcome this difficulty and allow a more comprehensive understanding of root cell biology, an approach is needed that would focus on one single cell type in the plant root. Because of its biological functions (i.e., uptake of water and various nutrients; primary site of infection by nitrogen-fixing bacteria in legumes), the root hair cell is an attractive single cell model to study root cell response to various stresses and treatments. To fully study their biology, we have recently optimized procedures in obtaining root hair cell samples. We culture the plants using an ultrasound aeroponic system maximizing root hair cell density on the entire root systems and allowing the homogeneous teatment of the root system. We then isolate the root hair cells in liquid nitrogen. Isolated root hair yields could be up to 800 to 1000 mg of plant cells from 60 root systems. Using soy bean as a model, the purity of the root hair was assessed by comparing the expression level of genes previously identified as soy bean root hair specific between prepartions of isolated root hair cells and stripped roots, roots devoid in root hairs. Enlarging our tests to include other plant species, our results support the isolation of large quantities of highly purified root hair cells which is compatible with a systems biology approach

Decipher the Molecular Response of Plant Single Cell Types to Environmental Stresses

The analysis of the molecular response of entire plants or organs to environmental stresses suffers from the cellular complexity of the samples used. Specifically, this cellular complexity masks cell-specific responses to environmental stresses and logically leads to the dilution of the molecular changes occurring in each cell type composing the tissue/organ/plant in response to the stress.Therefore, to generate a more accurate picture of these responses, scientists are focusing on plant single cell type approaches. Several cell types are now considered as models such as the pollen, the trichomes, the cotton fiber, various root cell types including the root hair cell, and the guard cell of stomata. Among them, several have been used to characterize plant response to abiotic and biotic stresses. In this review, we are describing the various -omic studies performed on these different plant single cell type models to better understand plant cell response to biotic and abiotic stresses

Cell-Type-Specific Profiling of the \u3ci\u3eArabidopsis thaliana\u3c/i\u3e Membrane Protein-Encoding Genes

Membrane proteins work in large complexes to perceive and transduce external signals and to trigger a cellular response leading to the adaptation of the cells to their environment. Biochemical assays have been extensively used to reveal the interaction between membrane proteins. However, such analyses do not reveal the unique and complex composition of the membrane proteins of the different plant cell types. Here, we conducted a comprehensive analysis of the expression of Arabidopsis membrane proteins in the different cell types composing the root. Specifically, we analyzed the expression of genes encoding membrane proteins interacting in large complexes. We found that the transcriptional profiles of membrane protein-encoding genes differ between Arabidopsis root cell types. This result suggests that different cell types are characterized by specific sets of plasma membrane proteins, which are likely a reflection of their unique biological functions and interactions. To further explore the complexity of the Arabidopsis root cell membrane proteomes, we conducted a co-expression analysis of genes encoding interacting membrane proteins. This study confirmed previously reported interactions between membrane proteins, suggesting that the co-expression of genes at the single cell-type level can be used to support protein network predictions

Cell-Type-Specific Profiling of the \u3ci\u3eArabidopsis thaliana\u3c/i\u3e Membrane Protein-Encoding Genes

Membrane proteins work in large complexes to perceive and transduce external signals and to trigger a cellular response leading to the adaptation of the cells to their environment. Biochemical assays have been extensively used to reveal the interaction between membrane proteins. However, such analyses do not reveal the unique and complex composition of the membrane proteins of the different plant cell types. Here, we conducted a comprehensive analysis of the expression of Arabidopsis membrane proteins in the different cell types composing the root. Specifically, we analyzed the expression of genes encoding membrane proteins interacting in large complexes. We found that the transcriptional profiles of membrane protein-encoding genes differ between Arabidopsis root cell types. This result suggests that different cell types are characterized by specific sets of plasma membrane proteins, which are likely a reflection of their unique biological functions and interactions. To further explore the complexity of the Arabidopsis root cell membrane proteomes, we conducted a co-expression analysis of genes encoding interacting membrane proteins. This study confirmed previously reported interactions between membrane proteins, suggesting that the co-expression of genes at the single cell-type level can be used to support protein network predictions

The evolving definition of plant cell type

In 1665 Robert Hooke, looking at cork through his microscope, discovered that plants are composed of elementary structures he named “cells”. Variation in the expression of a single genome in a complex eukaryotic organism guides the initiation, maturation, physiology, and biochemistry of cells with different shapes and sizes, playing different structural and functional roles in space and time. How many kinds of cells—”cell types”— an organism possesses of course depends on the organism’s cellular complexity, but the plasticity within a cell type fuels the emergence of the concept of cell state (Figure 1). The transition between cell states is driven by the developmental processes of multicellular organisms (e.g., cell determination and differentiation) and their response to environmental stresses (Wang et al., 2018). In the last decade, single cell/nucleus (sc/sn) omics, especially scRNA-seq, and spatial transcriptomics have enabled high-resolution mapping of molecular profiles of each cell, as well as mirroring cell trajectories through different states. Furthermore, unsupervised clustering of cells based on their transcriptomic (and/or epigenomic) signatures has proven effective in discovering previously unidentified cell types and cell states and providing new insight into cellular heterogeneity within a cell type (Schaum et al., 2018; Liu et al., 2021; Schumacher et al., 2021; Elmentaite et al., 2022). Classifying cells is necessary to conceptualize the biological complexity of some organs like the human brain or the plant root. But how should this be done? What criteria should be used, or, if several criteria are used, how should they be prioritized

Genetics and functional genomics of legume nodulation

http://www.soyroothair.org/publications.phpGram-negative soil bacteria (rhizobia) within the Rhizobiaceae phylogenetic family (a-proteobacteria) have the unique ability to infect and establish a nitrogen-fixing symbiosis on the roots of leguminous plants. This symbiosis is of agronomic importance, reducing the need for nitrogen fertilizer for agriculturally important plants (e.g. soybean and alfalfa). The establishment of the symbiosis involves a complex interplay between host and symbiont, resulting in the formation of a novel organ, the nodule, which the bacteria colonize as intracellular symbionts. This review focuses on the most recent discoveries relating to how this symbiosis is established. Two general developments have contributed to the recent explosion of research progress in this area: first, the adoption of two genetic model legumes, Medicago truncatula and Lotus japonicus, and second, the application of modern methods in functional genomics (e.g. transcriptomic, proteomic and metabolomic analyses).Ongoing related research in the Stacey laboratory is funded by grants from the US Department of Energy (DE-FG02-02ER15309), the National Science Foundation (NSF) (DBI-0421620), and the National Research Initiative (NRI) of the US Department of Agriculture (USDA) Cooperative State Research, Education and Extension Service (2005-35319-16192 and 2004-35604-14708)