Abstract
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 treatment 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 soybean as a model, the purity of the root hair was assessed by comparing the expression level of genes previously identified as soybean root hair specific between preparations 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.
Highlights
Plant root is an organ composed of multiple cell types with different functions
To utilize full potential of this attractive single cell type as a model in root systems biology, root hairs must be evenly treated preliminary to their isolation from the rest of the root system in quantities compatible with any -omic analysis, and a fortiori, transgenic root hair cells must be isolated to perform functional genomic studies at the level of a single cell type. To reach these two goals, we developed the method described below combining the use of an ultrasound aeroponic system to generate and evenly treat a large population of root hair cells and the purification of frozen root hair cells using a highly selective filtration system
We found that the number of nodules per root system is lower in the aeroponic system grown plants compared to vermiculite grown plants, the NOD hypernodulating phenotype is observed in the aeroponic system
Summary
OF CURRENT TECHNIQUES The isolation of single differentiated root cell types is limited by: (1) the accessibility to the root system; (2) the cell wall which confers the rigidity of the plant and its overall structure. The nature of the abiotic stresses allowed by the aeroponic system is diverse including: (1) changes of the chemical composition of the nutritive solution to analyze root hair response to nutrient deprivation, low and high pHs, salinity or heavy metal contaminations, etc.; (2) changes of the environmental conditions such as temperature, water potential, etc.; (3) inoculation of plants with pathogenic and symbiotic microorganisms The latter was validated by inoculating 2 weeks-old root systems of the hypernodulation soybean mutants [i.e., NOD1-3, NOD2-4, and NOD3-7; Ito et al, 2007] with a bacterial suspension of B. japonicum, the soybean nitrogenfixing symbiont. Stereomicroscopic observations revealed that these roots carry an impressive number of transgenic root hair cells (Figure 5C)
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