Single-cell RNA Sequencing Reveals Spatial Distribution Of Biosynthetic Pathway Of Monoterpene Indole Alkaloid In Catharanthus Roseus Leaves

Catharanthus roseus(L.)G.Don synthesizes various monoterpene indole alkaloids(MIAs),which is not only a model plant for studying the MIA biosynthesis,but also the sole source of anti cancer drugs vinblastine and vincristine.Elucidation of the biosynthesis,regulation and transportation of MIA in C.roseus will provide a strong theoretical and technical support for the production and research and development of MIA drugs.Singlecell RNA sequencing(scRNA-seq)is an emerging technique for the study of spatiotemporal gene expression profiles at the single cell level.It has immense potential in the research of plant development and environmental stimulus.For plants,single-cell experiments require removal of the cell wall by enzymatic digestion(protoplasting)before collecting a representative(unbiased)pool of cells.The application of scRNA-seq profiling in plants has been limited by the difficulty in generating protoplasts from the complexity of plant tissues.scRNA-seq using highthroughput platforms have been published in plants,most of which focuses on model plants and important crops such as Arabidopsis,rice and maize,and there is no publication in medicinal plants up to date.In this paper,we first set up an effective protoplasting protocol suitable for scRNA-seq of C.roseus leaves by investigating the effects of leaf ages,enzyme concentration,enzymolysis time,centrifugal force and centrifugal times on separation and purification of protoplasts.Using the youngest leaves as experimental material.The results showed that the best enzyme combinations for protoplast isolation were 1.0%(w/v)cellulase R-10+0.15%(w/v)pectinase,the optimum enzymolysis time was 2h,and the best purification effect was obtained after centrifugation at 100×g and repeat twice.A total of 0.5g leave tissues were collected and digested into protoplasts,and protoplast yield and protoplast viability were measured by using hemocytometer counting and trypan blue staining,respectively.About 25 000 cells were initially mixed with 10x Genomics single-cell reaction regents to construct scRNA-seq library.The library was sequenced by Illumina platform.A gene expression matrix was generated with Cell Ranger,resulting in a pool of 10 695 cells with a total of 18 987 genes.Seurat2 software package was used for further quality control and filter of the data,and low-quality cells such as double-cell multicellular or dead cells were filtered out.A total of 8741 high-quality cells were finally obtained,with 3435 unique molecular identifiers per cell,and 1393 expressed genes per cell.Based on cluster analysis,these high-quality cells were classified into 11 clusters,and known marker genes were used to successfully identify epidermal cells(EC),mesophyll cells(MC),internal phloem associated parenchyma cells(IPAP),vascular bundle cells(VC)and idioblast cells(IC),and a series of specific marker genes of each cluster was identified.KEGG enrichment analysis further revealed that biological processes highly enriched in each cluster corresponded to cell functions.The differentially expressed genes(DEGs)in mesophyll cells were involved in the photosynthetic carbon fixed,antenna protein synthesis.DEGs in IC were involved in the synthesis of monoterpene biosynthesis,plant-pathogen interaction alkaloids and biosynthesis of secondary metabolites.In this paper,expression patterns of all genes involved in MIA pathway in different cells were analyzed to elucidate the spatial distribution of MIA pathway in leaves.IPAP cells host the first 15 steps of iridoid biosynthesis,including the whole MEP pathway followed by the first seven reactions of the secoiridoid pathway.MIA-associated cells(MIAC)also constitute a site of MIA modifications by hosting 13 enzymatic steps.These include strictosidine synthesis catalyzed by 3 enzymatic steps,indole pathway and 8 enzymatic steps of downstream pathway.Dihydroprecondylocarpineor synthesized in the MIAC,and transported to EC to form catharanthine and tabersonine.The first four steps to catalyze tabersonine to form vindoline taken place in ECs.However,the last three steps of vindoline pathway are confirmed to localize IC.Finally,the final dimeric MIAs vinblastine and vincristine occur in epidermal cells.In conclusion,the single cell transcriptional atlas of c.roseus leaves was successfully constructed at single-cell resolution.The spatiotemporal expression characteristics of MIA biosynthesis pathway genes in c.roseus leaf were described at the single cell level for the first time.This study provides a novel way for the study of the spatial distribution of biosynthesis pathways of secondary metabolites in tissue,and will promote the further application of scRNA-seq in the study of medicinal plants.