Process employed for scRNA-seq of tea plant roots.

(A) Tea seedlings were used for theanine detection and scRNA-seq. Tea plant roots (∼2.0 cm in length from the root tip) were harvested and used for scRNA-seq. (B) Theanine contents of leaf, stem, cotyledon, and root. (C) Cross section of tea plant roots (from the root tip back ∼2.0 cm). Co, Cortex; Xy, Xylem; Ph, Phloem; Ca, Cambium; Pe, Pericycle; En, Endodermis; EP, Epidermis. Scale bar = 200 μm. (D) Flow chart of scRNA-seq analyses.

Annotation of cell clusters based on scRNA-seq analysis of tea plant roots.

(A) t-SNE visualization plot of 10,435 tea plant root cells showed that they were grouped into 8 cell clusters. Each dot denoted a single cell. (B) t-SNE visualization of cell-type marker genes of root cells. Color bar indicates gene expression level. (C) In situ RT-PCR of CsWAT1, CsPP2, CsAAP1 and CsLHW. The blue areas of sliced tissue represent regions where genes are expressed. The red box represents the magnified areas of root sections. Scale bar = 200 μm. (D) Violin plot showed the expression patterns for the top 5 marker genes of each cell clusters. The height of violin represents the gene expression level, and the width represents the proportion of cells expressing in the cluster. Asterisks represent homologous Arabidopsis thaliana marker genes that can be found in PlantscRNAdb. (E) Schematic of the tea plant root anatomy, cell types and their associated clusters.

Trajectory of root cell differentiation in the tea plant root.

(A) Schematic representation of root meristem organization (in median longitudinal section). t-SNE visualization and cell type annotation of Cluster 3 (bottom). (B) Simulation of the successive differentiation trajectory of the stem cell niche (SCN) over pseudo-time. Each dot denotes a unique cell. Pseudo-time analysis of all 10,435 tea plant root cells (center), and the detailed distribution of 8 cell clusters along pseudo-time trajectory (outer). Colors of the dots represent the pseudo-time score. Red and blue line mark major differentiation trajectories, black lines mark minor differentiation trajectories. (C) Heatmap illustrating the expression patterns of differential genes, along the pseudo-time trajectory, during cell differentiation of the vasculature and epidermis. Each row represents one gene. Color bars indicate the relative expression levels. Biological processes are given on the right. (D) Proposed tea plant roots cell differentiation trajectories for cell clusters shown on the t-SNE visualization plots. Dotted arrows represent assumed cell differentiation routes.

Cell heterogeneity of nitrogen (N) transport and assimilation in tea plant roots.

(A) Schematic of N transport and metabolism pathway in tea plant roots. Membrane-located transporter NRTs and AMTs uptake nitrate and ammonium into root cells, respectively, and subsequently NO3- is assimilated into NH4+ and then metabolized into various amino acids. (B) Heatmap shows cell cluster expression patterns of N transport and assimilation genes in tea plant roots. Row normalization for gene expression according to “normalized” mode. (C) In situ RT-PCR of CsAMT1.1, CsAMT3.1, CsNRT1.1 and CsNRT3.2. The blue areas of sectioned tissues represent regions where genes are expressed. The red boxes represent the magnified areas of root sections shown on the right. Scale bar = 200 μm. (D) Expression of amino acid synthesis pathway genes in different cell clusters. Color bars indicate the expression levels of amino acids synthesis pathway genes. Dot size indicates the percentage of amino acid synthesis genes expressed in a cell cluster.

Cell heterogeneity of theanine synthesis, transport, and regulation in tea plant roots.

(A) Model of the putative theanine synthesis, transport, and regulation in tea plant root cells. Black full lines represent unique metabolic processes in tea plants; grey full lines represent common metabolic pathways in plants; dotted lines indicate the multi-step process. (B) Heatmap for cell cluster expression patterns of genes encoding theanine transporters CsAAPs, key enzymes and transcription factors in the theanine metabolic pathway. Gene expression is presented in “normalized” mode. (C) t-SNE visualization of CsTSI, CsAlaDC and their overlap map. Blue color bar indicates CsTSI expression level, red color bar indicates CsAlaDC expression level in scRNA-seq. (D)Tissue localization of CsTSI and CsAlaDC using in situ PCR. Blue signal indicates gene expression in cells. The red boxes represent magnified areas of root sections shown below. Scale bar = 200 μm.

A gene co-expression network predicted key regulators of CsAlaDC.

(A) Using bulk RNA-seq data, a gene co-expression network was generated for CsAlaDC (|correlation coefficient| ≥ 0.85 and p-Value ≤ 0.05). Red and blue dots represent differential genes positively and negatively correlated with CsAlaDC, respectively. Bulk RNA-seq data is from different types of N treatment of tea plant roots (Yang et al., 2019). (B) Heatmap showing expression of co-expressed genes with CsAlaDC in eight tea plant tissues. The black arrows denote root specific co-expressed (RSCE) genes. (C) Heatmap showing the expression pattern for RSCE genes under different types of N treatment (0 N, CK, EA, NH4+ and NO3-). Correlation between RSCE genes and CsAlaDC is shown on the heatmap at left. (D) Heatmap showing the expression level of RSCE genes in each cell cluster. (B-D) Row normalization of gene expression is according to the “normalized” mode.

Theanine synthesis regulated by the transcription factor CsLBD37.

(A) Schematic representation of the tea plant tissues used for gene detection. (B) Relative expression of CsLBD37 in different tea plants tissues. (C) The t-SNE visualization graph shows expression levels of CsLBD37 in various cell clusters. (D) Cell specificity of CsLBD37 expression in tea plant roots; blue signal represents location of gene expression. Scale bar = 200 μm. (E) Subcellular localization of CsLBD37; green fluorescence signal represents protein localization. (F) The binding of CsLBD37 to the CsAlaDC promoter, tested by yeast one-hybrid assay (Y1H). (G) EMSA assay showing the association of CsLBD37 with the CsAlaDC promoter. The red arrow points to the binding position. (H) A schematic of the effector and reportor constructs for transcriptional activity of the CsAlaDC promoter (top). LCI assays in tobacco leaves show that the CsAlaDC promoter can interact with CsLBD37. The color bar indicates the range of luminescence intensity (mid). The LUC/REN values represent relative activity of CsLBD37 on CsAlaDC expression. Significant difference was evaluated by two-tailed Student’s t-test analysis (bottom). (I-L) CsLBD37 gene silencing and overexpression in tea plant roots. The expression level of CsLBD37 and CsAlaDC in gene silenced (I) and overexpression plants (K). Ethylamine and theanine contents in gene silenced (J) and overexpression plants. (L). sODN, sense oligo nucleotides; asODN, antisense oligonucleotides, silenced CsLBD37 in tea plant root by asODN methods; EV, empty vector; CsLBD37-OE/Cs, CsLBD37 overexpression in transgenic hairy root system. Significant difference was determined by Welch’s t test.

Proposed model for theanine biosynthesis, transport and regulation at single cell resolution.

(A) Multicellular compartmentation of theanine biosynthesis. The high expression of CsTSI in pericycle cells suggests theanine is mainly synthesized in pericycle cells. CsAlaDC is highly expressed in cambium and protoxylem cells, suggesting that ethylamine (EA) is mainly synthesized in these cells. Thus, EA may move from cambium and protoxylem cells into pericycle cells, where it is a substrate for theanine biosynthesis. (B) In tea plant roots, CsAAP1 likely mediates in theanine retrieval from apoplast for its transport into cortical/endodermal cells. CsCAT2 imports theanine into vacuoles, especially in pericycle cells, which are the main cells for theanine biosynthesis. Theanine is exported, by an unidentified theanine exporter, from pericycle cells into the apoplastic pathway for entry into the xylem. (C) CsLBD37 co-regulates theanine biosynthesis and lateral root development. Increasing levels of nitrogen (N) promote theanine biosynthesis and accumulation. High accumulation of theanine and N induces CsLBD37 expression. CsLBD37 inhibits CsAlaDC expression to reduce ethylamine synthesis, which finetunes theanine biosynthesis in feedback loop. Besides, CsLBD37 is expressed in pericycle cells and also inhibits lateral root development. It is reported that apoplastic theanine can negatively regulate lateral root development, under high nitrogen levels (Chen et al., 2022). Therefore, CsLBD37 may also be involved in theanine-regulated lateral root development.