As the continent's main source of food and feed, the yield of maize is vital for agriculture and animal husbandry. Increasing the yield per unit area of maize, especially the number of grains per panicle, is the key to ensuring the balance between supply and demand of maize in mainland China. The spatiotemporal-specific expression of key genes determines the development of female ears of maize, which in turn affects the yield of maize per ear. However, conventional plant transcriptome techniques often ignore the heterogeneity of cells and fail to obtain spatiotemporal expression information of key genes. In addition, we still know little about which types of ginseng are associated with the morphogenesis of maize ear stalks. Therefore, the current use of forward genetics to mine the relevant genes regulating the development of female ears and apply them to the improvement of corn ear traits is relatively low. On May 14, 2024, Nature Plants published an article online in collaboration between Huazhong Agricultural University and Shenzhen BGI Research Institute, which constructed the first spatial transcriptome map of maize female ears, and finally obtained the transcriptome and spatial location information of 12 cell types, and identified and verified 4 new cell types. Using the advantages of spatial omics, two MADS-box genes that were specifically expressed in the apex of floret meristem and determined their differentiation certainty were identified. By integrating single-cell transcriptome and spatial transcriptome data, the researchers explored a new method for high-throughput gene mining, and identified a number of new genes that potentially control the formation of maize ears by constructing cell-specific gene co-expression networks. Firstly, the researchers optimized the experimental conditions of Stereo-seq, developed a spatial transcriptome sequencing method suitable for maize female ears, and selected 6 mm female ears in the critical growth and development period as the experimental subjects, and performed spatial transcriptome and single-cell transcriptome sequencing on them, respectively. After analyzing the spatial transcriptome data, the researchers came up with transcriptomes and spatial locations for 12 cell types. Referring to the description of the anatomy of maize ears in existing studies, cell population identity can be quickly defined. Ultimately, the researchers found that most of the cell types were located within the known female spike development organs, but four entirely new cell types were also identified, including three cells distributed in the inflorescence meristem and one cell distributed in the center of the floret meristem. In order to further verify the reliability of spatial transcription maps, the researchers randomly selected 55 marker genes identified and published in spatial transcriptome data with spatially specific characteristics, and verified their expression patterns by in situ hybridization experiments. The experimental results showed that 74% of the genes were expressed in situ hybridization and were consistent with the spatial transcriptome results, indicating the feasibility and reliability of spatial transcriptome sequencing (Fig. 1).
Fig.1 Spatial transcriptome map of 6 mm female ear of maize was constructed
The spatial transcriptome map of the 6 mm female spike constructed based on Stereo-seq technology could clearly distinguish different types of meristems. Therefore, according to their different spatial positions, the researchers extracted the inflorescence meristem and floret meristem transcriptome of the 6 mm female spike and performed recluster analysis. The results showed that these meristems could be further distinguished into three different cell types, which were distributed at the tip of the inflorescence meristem, the periphery of the floret meristem, and the tip of the floret meristem. A series of differentially expressed genes between the three cell types were detected, including two MADS-box genes, ZmMADS8 and its homologous gene ZmMADS14, which were specifically expressed in DMT, and their expression patterns were verified by mRNA in situ hybridization. Previous studies have shown that MADS-box family genes play an important role in plant organ development. Therefore, in order to verify the function of ZmMADS8 and ZmMADS14, the researchers generated dual knockout mutants of ZmMADS8 and ZmMADS14 by CRISPR/Cas9 gene editing technology. It was observed that in the single mutants of ZmMADS8 and ZmMADS14, the floret meristem could develop normally and produce normal epiflorets. In the CR-Zmmads8/14 double mutant, the floret meristem does not differentiate from the lower floral organ, but reverses to an uncertain long branch, which cannot produce a normal floral organ (Fig. 2). These results indicate that ZmMADS8 and ZmMADS14 are the key factors in determining the fate of floret meristems. These results suggest that the spatial transcriptome data obtained based on Stereo-seq technology can distinguish highly similar meristem types, which allows us to further study the subtle changes produced by meristems during differentiation and helps to identify key developmental regulators.
Figure 2. Stereo-seq data were used to identify ZmMADS8 and ZmMADS14 to determine the meristem certainty of florets
Next, the researchers used the STRIDE analysis workflow to integrate the single-cell transcriptome and spatial transcriptome data, annotated six cell types in the single-cell transcriptome data through spatial mapping, and constructed a gene regulatory network with spatial expression specificity based on the transcriptome information of different cells. Among them, the researchers identified a co-expression network with OCL5 (OUTER CELL LAYER 5) AND OCL3 (OUTER CELL LAYER 3) GENES AS HUB NODES IN THE EPIDERMAL CELLS OF MAIZE FEMALE EAR MERISTEM, AND THIS CO-EXPRESSION NETWORK ALSO INCLUDED THREE OTHER TYPES OF GENES, NAMELY 3-ketoacyl-CoA synthase gene and epidermal wax synthesis gene (glossy). genes)。 The conclusion that OCL gene plays an important role in the formation of the stratum corneum of the grain epidermis has been reported in Arabidopsis thaliana and maize, and the spatial transcriptome map of maize female ear shows that OCL5 gene is clearly expressed in epidermal cells of female ear meristem. Therefore, the researchers speculated that there was a gene regulatory network related to stratum corneum synthesis at the core of the OCL gene in this site, which was highly consistent with the biological functions performed by meristem epidermal cells. In addition, the researchers identified a co-expression network specifically expressed in the inflorescence meristem, which contains multiple genes associated with trehalose-6-phosphate synthesis. According to past studies, trehalose-6-phosphate is very important for the growth and development of plants. Within this co-expression network, transcription factors of another NAC family NACTF25 identified as hub genes and are specifically expressed in inflorescence meristems. Therefore, the researchers speculated that NACTF25 may affect inflorescence meristem development by regulating the expression of genes associated with trehalose-6-phosphate synthesis (Figure 3).
Figure 3. Integrate single-cell transcriptome and spatial transcriptome data to construct cell type-specific co-expression networks
In summary, this study constructed a spatial transcriptome map of maize females by combining the single-cell and spatial transcriptome data of maize females during the critical developmental period. The use of this atlas enables the identification of highly similar meristem types, which allows us to further study the subtle changes produced by meristems during differentiation and helps to identify key developmental regulators. The expression patterns (expression levels and sites) of all genes in the map, as well as the marker genes identified in different cell types, can be queried through https://db.cngb.org/stomics/mdesta/ access.
Prof. Ning Yang and Prof. Lei Liu from the State Key Laboratory of Crop Genetic Improvement and Hubei Hongshan Laboratory of Huazhong Agricultural University and Prof. Huan Liu from Shenzhen Huada Research Institute are the co-corresponding authors of the paper. Dr. Yuebin Wang, Dr. Yun Luo, Dr. Yunfu Li, Jiali Yan, and Dr. Xing Guo from Huazhong Agricultural University are the co-first authors of the paper. Prof. Yan Jianbing and Prof. Zhang Zuxin from Huazhong Agricultural University, Prof. David Jackson from Cold Spring Harbor Laboratory in the United States, and Prof. Xu Xun from Shenzhen Huada Research Institute participated in the work. Zhuo Lin, Wei Wenjie, Ding Qian, and Bai Minji from Huazhong Agricultural University, and Wei Xiaofeng, Shao Wenwen, Chen Lihua, Li Li, Yang Tao, and Chen Jing from Shenzhen Huada Research Institute provided technical support for the research. This work has been supported by the Major Project of Agricultural Biological Breeding, the National Natural Science Foundation of China Outstanding Young Scientist Fund, and the National Key Research and Development Program.
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