The tea tree is an important woody leaf gardening crop. The tea made from its fresh leaves is deeply loved by people because of its unique flavor and natural compounds. The accumulation of flavor related secondary metabolites in tea is the material basis for the formation of the unique flavor of tea, such as catechin, especially ester catechin, is one of the sources of the bitter taste of tea, and theanine is the source of the freshness of tea. With the analysis of tea genome, remarkable achievements have been made in the research on the synthesis and regulation mechanism of major secondary metabolism in tea, but there are few reports on the biosynthesis and distribution deep into the cell level.
Single cell sequencing, as a high-resolution analysis technology that can analyze transcriptome at the single cell level, makes it possible to analyze the metabolic characteristics of different cell types. Recently, Professor songchuankui’s team from the State Key Laboratory of tea biology and resource utilization of Anhui Agricultural University published the title”single cell trans” in the well-known botanical journal plant biotechnology Journal (SCI impact factor 13.263 in 2021)criptome atlas reveals developmental trajectories and a novel meTabolic path of catechin esters in tea leaves. This study established a rapid isolation system of protoplasts from tea leaves, studied 16977 single-cell transcriptome, constructed the first single-cell map of woody plant leaves, mapped the development trajectory of tea leaves, and found a new type of ester catechin glycosyltransferase, obtaining evidence of ester catechin glycosidation.
Because the cell wall of tea leaves is thick, secondary metabolism is rich, woody plant tissues are difficult to digest and release protoplasts, and tea growth cycle is long, there are few specific marker genes of tea cell type, which increases the difficulty of tea cell type identification. In order to overcome these difficulties, Professor Song Chuankui’s team used different methods to detect the structure of tea leaves, observe tea cells, and establish a rapid separation method of tea leaf protoplasts according to the characteristics of tea leaves. On this basis, 16977 single cell transcriptome were sequenced, and the single cells of tea leaves were divided into 16 cell clusters. Then, through the same enzymatic hydrolysis method, the author separated six sub components of leaves, such as epidermis, mesophyll, vascular tissue, and verified the relative expression level of genes specifically highly expressed in each cell cluster in the six components. Combined with the homologous annotation of cell marker genes in model plants, the author included the main cell types in tea leaves.
The team compared the transcriptome differences between young leaves and old leaves, mapped the differentiation and development trajectory of young leaf cells, and marked the highly expressed genes in each state and branch. Based on single-cell transcriptome data and cell type identification, we can reduce the background noise caused by unrelated cells, so as to improve the prediction accuracy of regulatory networks. Therefore, through the coexpression network analysis of the specific expression matrix in the mesophyll, we found a glycosyltransferase gene ugt72b23, which is highly expressed in the mesophyll and is significantly related to catechin metabolism. Its ester catechin glycosyltransferase activity was verified by in vivo and in vitro activities. Ester catechin glycosides were detected in plant leaves, and the space-time specific catechin ester glucoside synthesis pathway mediated by ugt72b23 at the single cell level was mapped.
This study used a variety of methods to verify the cell marker genes, which provided important reference information for the mapping of woody plant leaf cells. Its rapid protoplast separation technology also provided a new idea for the rapid separation of other lignified tissues. A novel ester catechin glycosyltransferase was found by screening the expression matrix of specific cell types, which provided evidence for the glycosidation of tea ester catechins.
Our university is the first signatory, with postdoctoral Wang Qiang and Master Wu Yi of the State Key Laboratory of tea plant biology and resource utilization as the first authors, and Professor Song Chuankui and Wilfried Schwab of Munich University of technology as the corresponding authors. Dr. Cui Jilai of Xinyang Normal University and Dr. Tian Kai of Nanyang Normal University participated in the study. The project was supported by the National Natural Science Foundation of China for outstanding young scholars, the international cooperation project, the German Science Foundation and other projects.