Transcriptional Landscape Of Rice Roots At The Single-cell Resolution

Plant roots have the function of absorbing water and minerals from the soil,which participating in the interaction between plants and biological or abiotic signals,and also playing a role in fixing plants.At present,our current knowledge of cellular and molecular mechanism driving root development is mainly based on studies of the dicot model Arabidopsis thaliana.However,little is known about the development process and biological function underlying cell type specification in the world major crop rice.Throughout the life cycle of the plant,the root tips are constantly differentiating and developing,making them very important and highly heterogeneous organs.Traditional high-throughput sequencing can not detect the difference of transcriptome between different cell types.The rapid development of single-cell sequencing technology allows us to explore the transcriptional regulation and the dynamic development trajectory of single cells.Here,we report the transcriptomes of single cells derived from root tips of two agronomically important rice cultivars: Nipponbare(Japonica,geng)and 93-11(Indica,xian)by using 10 x Genomic Single cell RNA-Seq technology,and draw a dynamic development trajectory of single cells derived from rice root tip,then followed by in situ hybridization identification of selected marker genes.1.20,000 and 16,000 high-activity protoplasts were obtained from the 5 mm primary root tips of Nip and 93-11 respectively cultured for 3 days,which were mixed with 10 x Genomics platform reagents to form a microdroplet structure for library construction and sequencing.The sequencing data was filtered to screen out 10,968 Nipponbare root cells,and 2,592 genes were detected per cell;12,56493-11 root cells and 2,636 genes per cell were detected with high-quality single-cell data for downstream analysis.2.Under unsupervised clustering,the rice root tip were divided into 8 cell types.Using in situ hybridization to identify these cell types: epidermis,endodermis,cortex,stele,root hair,root cap,xylem.We also found a new cell type: epidermis near root hair which located between the root tip epidermal cells and root hair cells.It might be a type of transition cells.In this classification,the epidermis,cortex and stele are the main cell types containing 2,328 to 4,833 cells,while the clusters of root hairs,primary xylem,root cap and endodermis have only a few cells(17-435),which is similar with the ratio of different type of cells in the root tip.These genes are conserved between these two subspecies,indicating the conservation during evolution.3.In-depth analysis of each cell type reveals that auxin biosynthesis and response genes were overrepresented in the clusters of stele and epidermis,SA response genes and BR signal genes were enriched in the clusters of cortex.GO enrichment analysis was performed on the differentially expressed genes of each cell type,and found that genes related to water and liquid transport were enriched in cortex cells,genes related to cell wall were enriched in the epidermis cells,and genes related to the response to external stimuli are enriched in the epidermis(Near the root hair).This study also tested the functional enrichment of differentially expressed genes in each cell type of Nip and 93-11,and found that most genes are related to stimuli response,indicating that Nip and 93-11 have different response mechanisms to external stimuli.4.Through the pseudo-time analysis of the single cell data,the development trajectory of root epidermal cells was plotted.Epidermal differentiation takes epidermal cells as the starting point and differentiates in two different directions: One branch eventually develops into root cap and mature epidermal cells;the other branch undergoes a transition type of epidermal cells and finally differentiates into root hair cells.5.Using published Arabidopsis root tip single cell RNA-Seq data combined the single-cell data set of rice and used orthologous genes as anchors to compare genes one by one,and use Seurat software to identify the homology of the root tip cell types of these two subspecies.Although the cell types and hormone regulatory networks were similar between these two rice subspecies,but the evolutionary analysis found that the roots of Arabidopsis and rice differed greatly,only the root hairs and xylem cells were highly conserved,but the evolution of the same tissue and organ among different species is not conserved.Therefore,it is very important to conduct single-cell research on tissues and organs of different species.Taken together,our study provides insight into the transcriptomic landscape of major cell types of the rice root tip at single-cell resolution and opens new avenues to study cell type specification,function and evolution in plants root tip.