Establishment Of Gene Regulatory Network And Function Analysis Of Regulators In Somatic Embryogenesis Of Arabidopsis

Plant cell totipotency refers to the ability of cells to retain the full genetic potential characteristics of developing into a complete organism, and thus enables the cell to regenerate an entire plant under favorable culture conditions. It has been observed that there are many genetic and physiological factors that influence the plant cell totipotency. Somatic embryos(SEs) have been identified as structures that arise from somatic cells but resemble zygotic embryos. Somatic embryogenesis is recognized as a model to understand the molecular biological events occurring during plant embryo development and the mechanisms of plant cell totipotency. The origin of SEs, which has been described from a single-cell or groups of cells in direct or indirect way, remains confused. Here, we focused on studying the cell origin of the SEs and the mechanisms of somatic embryogenesis in Arabidopsis. We identified essential regulators that regulated plant cell totipotency during SE induction. Through analyzing the regulation relationship of these factors, we established a gene network of plant cell totipotency. Furthermore, we analyzed the functions of the regulators in somatic embryogenesis. The main results are as follows:(1) SEs initiate directly from single somatic cellsWe established an in vitro system of somatic embryogenesis to analyze the regulatory mechanisms of cell totipotency during this process. Overexpression of LEAFY COTYLEDON 2 (LEC2) resulted in the induction of SEs, and SEs were directly induced from cells localized in the tips of the cotyledons of 7-day-old transgenic seedlings without formation of embryonic callus. Through microscopy analysis, we observed that SEs initiated from single cells of the cotyledon epidermis, which were defined as totipotent stem cells.(2) The genetic characteristics of totipotent stem cellsEmbryo specific genes including LECI and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE 1 (SERK1) are involved in zygotic embryogenesis. These genes were specifically expressed in the totipotent stem cells but not in the cotyledon cells. Then,the expression signals of DR5::GUS, pYUC4::GUS and TCS::GFP were also specially detected in the totipotent stem cells, suggesting that auxin and cytokinin play essential roles in SE induction. We analyzed the dynamic changes of the microfilament cytoskeleton and endoplasmic reticulum in the totipotent stem cells. We observed the depolymerized actin filaments in totipotent stem cells, with punctiform and granular distribution. While the actin filaments were clearly polymerized in other cotyledon cells. The endoplasmic reticulum had a short and tubular appearance in the totipotent stem cells. In contrast, the endoplasmic reticulum sheets and tubules were both present in other cotyledon cells, implying the low differentiation of the totipotent stem cells.(3) Establishment of the gene regulatory network on plant cell totipotencyWe preformed RNA-Sequence analysis in three types of cells including the SERK1-marked totipotent stem cells, cells surrounding the totipotent stem cells, and other cotyledon cells without SE induction. We identified a total of 2078 genes with much higher expression levels in the totipotent stem cells than in the surrounding cells and other cotyledon cells. Through bioinformatics analysis, we analyzed the regulation relationship of these factors and established a gene regulatory network, which consists of 56 genes including 24 transcription factors. We defined three regions of the gene network based on the expression levels and interrelationship among these genes. Eight genes localized in the first region (the central region with red color), and of them, LEC2 and ABSCISIC ACID INSENSITIVE 3(ABI3) that have been reported to be regulators on plant cell totipotency. Most of the genes in this region regulated the transcription of other genes in the network. There were 33 genes,which included the totipotency regulators MYB DOMAIN PROTEIN 118 (MYB118) and PICKLE (PKL) in the second region (orange color), and they were closely associated with the genes in the first region. Moreover, there were 15 genes in the third region (green color)which were closely associated with those in the second region.(4) Function analysis of regulators in somatic embryogenesisWe analyzed the expression patterns and functions of the essential genes shown in the regulatory network, and found that TOTIPOTENCY 1 (TP1), TP2 and CUP-SHAPED COTYLEDON 1 (CUC1) genes were not only specifically expressed in the totipotent stem cells but also in the zygotic embryos, implying that these regulators of cell totipotency are also required for zygote embryogenesis. Overexpression of TP1, TP2 or HYDROXYSTEROID DEHYDROGENASE 1 (HSD1) resulted in the induction of SEs, suggesting that they play essential roles in the regulation of plant cell totipotency. We further identified unknown genes named UNKNOWN TOTIPOTENCY 1 (UTP1) and UTP2, which were specifically expressed in the totipotent stem cells and also required for zygotic embryogenesis. In addition,we identified the genes that had high homology with cell totipotency regulators in animals,and they were named TOTIPOTENCY IN ANIMAL 1 (TPA1), TPA2, TPA3 and TPA4. These genes were also required for zygotic embryogenesis, suggesting that the functions of the cell totipotency regulators are conserved in plant and animal cells.