1, building a community map
Plant leaves are composed of cells with different shapes and specific functions, and different cell types have different gene expression patterns. The cell map of plant leaves is constructed by single cell transcriptome sequencing, which enables us to deeply understand the composition of cell types in plant leaves and obtain the unique transcription information of each cell, so as to identify the identity and function of cells.
1. Case 1
In the article [1] published in the journal Molecular Plants in June, 2020, the researchers used the cotyledons of 5-day-old Arabidopsis seedlings as materials to sequence the transcriptome of single cells, and * * * obtained 12844 cells. Through cell clustering and annotation analysis, these cells are divided into 1 1 different cell groups, including stomatal development. Such as meristem mother cells (MMCs), guard mother cells (GMCs), flat cells (PC), mesophyll cells (MPCs) and guard cells (GCs).
2. Case 2
In the article [2] published in the Journal of Plant Biotechnology in June, 20021year, the researchers sequenced the single cell transcriptome of 7-day-old peanut seedling leaves, * * * obtained 68 15 cells, and constructed the first single cell gene expression map of peanut. Through cell clustering and annotation analysis, these cells were divided into 8 main cell groups, including palisade mesophyll cells and sponge leaves. By constructing cell map, it is shown that plant leaves are composed of highly heterogeneous cells.
Second, the subdivision of cell subsets
In the study of plant organ development, rough cell grouping often can't fully describe the development track of cells, and subcellular groups have the relationship between upstream and downstream development and differentiation, so the development track of cells can be analyzed in detail, so the subdivision of cell subsets is particularly important.
1. Case 1
In the article [3] published in Plant Cell in May, 20021year, the researchers sequenced the single-cell transcriptome of mature leaves of 6-week-old Arabidopsis thaliana to reveal the different characteristics of phloem cells in leaves. Vascular bundle cell cluster 4, cluster 10 and cluster 18 are subdivided into subgroups, and the cell types of subgroups are determined according to the enrichment genes. They are C4.1.1(XP1), C4. 1.2(XP2), c4.2 (bs2), c4.3 (bs 1) and c/kloc-0. C 10.2 (PP 1), C 18. 1 (PP2) and C 18.2(XP3), the cell subgroup subdivision determines the cell group XP-PCXP-PCPP-PP of vascular bundle cells.
2. Case 2
In the article [4] published in Plant Cells in June, 20021year, the researchers sequenced the transcriptome of single cell in maize leaves and analyzed the bundle sheath differentiation of maize. It was found that bundle sheath cells were further divided into two subgroups, namely, distal bundle sheath cells (abb) and paraxial bundle sheath cells (adBS), which were dorsal-ventrally differentiated, and the analysis of cell subsets could be used for cell development.
Thirdly, identification of marker genes.
In multicellular organisms, the generation of cell types and cell-specific functions largely stems from the differential expression of different genes in cells. In the process of single cell transcriptome data analysis, the characteristic genes of a cell subgroup are identified mainly by differential analysis, and then the cell types are identified by combining marker genes. Therefore, mining new marker genes is helpful to clarify the heterogeneity of cells and is of great significance to identify cell groups with unknown cell types during plant development.
For example, in the article [1] published in the journal Molecular Plants in June 2020, the researchers used cotyledons of 5-day-old Arabidopsis seedlings as materials to sequence single-cell transcriptome, and verified the identified cell types by using several known marker genes involved in regulating the development of stomatal lineage cells. It was found that FAMA, TMM, HIC and SCRM were specifically expressed in specific cell types, while other marker genes were not specifically expressed in specific cell types, thus identifying new markers of different cell groups during the development of stomatal lineage cells, laying a foundation for further exploring the potential regulatory factors of stomatal lineage cell development and analyzing the gene expression profiles of different cell groups.
Fourthly, research on development track.
Cell grouping is essentially to represent that the development of plants is a discontinuous process, but the development of organisms or organs should be a continuous process and complicated. By analyzing the differentiation path of plant leaf cells with time, we can understand its dynamic development process. Quasi-time series analysis refers to sorting individual cells along the trajectory according to the similarity of expression patterns between cells, so as to infer the differentiation trajectory of cells or the evolution process of cell subtypes during development.
For example, in the article [2] published in the Journal of Plant Biotechnology in June, 20021,the researchers sequenced the single cell transcriptome of peanut leaves, which not only revealed the heterogeneity of peanut leaves, but also reconstructed the development track of peanut leaves, and found four small branches near the main stem. Different cell subsets show the temporal heterogeneity of the main pathways of cell differentiation. In addition, quasi-time series analysis revealed 10 key marker genes, which can be used to divide all single cells into nine states of leaf cell development and differentiation.
Analysis of verb (Verb's abbreviation) Gene Regulatory Network
The difference of cell transcription state leads to the heterogeneity of cells, and the gene regulatory network dominated by transcription factors maintains and stabilizes the specificity of transcription state. By analyzing the regulatory network of transcription factors in different cell types, we can understand the regulatory mechanism of plant leaf development.
The article [2] published in Plant Biotechnology Journal in June, 20021analyzed the expression level of transcription factors in different cell groups, and found that compared with other groups, the transcription factors identified by 9 1 had a downward trend in sponge tissue, which means that the expression of transcription inhibition transcription factors may determine the differentiation state of peanut plant leaf sponge cells. The homologous interaction network of transcription factors in Arabidopsis thaliana was analyzed. The results show that the ethylene (ETH) and jasmonic acid (JA) pathways have key characteristics, forming a protein interaction network with ERF4/6, WRKY40, MYC2 and several JAZ proteins as the core.
Sixth, the study of stress response mechanism
Abiotic stress is an important environmental factor in plant growth and development. By sequencing the transcriptome of a single cell, exploring the composition changes of cell types under different treatment conditions and analyzing the stress response mechanism of organisms will help us understand the mechanism of plant cells and developmental biology at the single cell level.
In the article [5] published in 5438+2020 10 June, the response mechanism of rice under two abiotic stresses of high salt and low nitrogen was studied. Firstly, the expression of cell type-specific differential genes under different environmental stresses was studied, and it was found that the differentially expressed genes under low nitrogen treatment also showed differential expression under high salt treatment, which means that plants may respond to different abiotic stresses and adopt the strategy of * * * restructuring the regulatory network. Then, the changes of cell size under different environmental stresses were studied. It was found that plants growing in high salinity showed a decrease in the overall size of primordia and epidermal cells, which may be related to the decrease of cell wall swelling pressure caused by the increase of osmotic pressure. These results reveal the changes of cell type specificity under abiotic stress. Finally, the changes of cell population composition under different environmental stresses were studied by developmental trajectory analysis. It was found that the delay of mesophyll cell development under the condition of high salt and low nitrogen was not only beneficial to reduce the energy consumption of photosynthesis, but also to improve the resistance of rice seedlings to harsh environment.
7. Review of research ideas on single cell in plant leaves.
At present, the study of plant leaf single cells has been carried out in Arabidopsis, peanut and rice, with the focus on mesophyll cells and stomatal related cells in general, in order to reveal the development regulation mechanism of mesophyll cells and stomatal lineage cells. The research idea is to construct a single cell map of species leaves, analyze cell development and its regulation mechanism through cell clustering, cell subgroup subdivision, marker gene identification, development trajectory and gene regulation network analysis, and explore the response mechanism of plant leaves under abiotic stress environment. It is believed that with the breakthrough of some technical problems in experiments and analysis, in the future research, it will become a hot topic to conduct more in-depth analysis of other cell types, more research on non-model species, more excavation and verification of marker genes, and multidimensional analysis of plant cell development mechanism through single cell analysis. The field of plant single cells has shown a strong momentum of vigorous development since its rise, which will surely set off a great revolution in plant transcriptome research in the future!