Rice Genomics and Genetics 2025, Vol.16, No.4, 199-210 http://cropscipublisher.com/index.php/rgg 200 the level of each cell, helping us to clearly understand the transcriptional characteristics and regulatory networks of each type of cell. This technology can help us identify new grouting regulatory factors and cell-specific pathways, enabling us to better understand how gene regulation changes in time and space during grain development (Kim et al., 2017). The main objective of this study is to investigate the gene regulatory networks of different cell types in rice grain filling using scRNA-seq technology. We plan to map the transcriptional profiles of each cell type, hoping to identify the important regulatory factors, signaling pathways and gene modules that affect grouting and quality formation. These results will provide useful references for improving rice yield and quality in the future, and may also help us find new breeding ideas in terms of stress resistance and grain trait improvement. 2 Overview of Rice Grain Filling Process: Stages, Cell Types, and Regulation 2.1 Stages of grain filling and key physiological changes Grouting is not accomplished in an instant; rather, it starts from fertilization and progresses bit by bit. The process can roughly be divided into three stages: the early stage involves rapid cell division and endosperm expansion; the middle stage mainly involves the accumulation of a large amount of nutrients such as starch; and finally, it enters the stage of maturation and water reduction (Durbha et al., 2024). Of course, there are no clear boundaries between these stages, and they often overlap. The most obvious change in the early stage was the rapid proliferation of endosperm cells, which then began to convert sucrose into starch - the main storage substance in the grains. Meanwhile, the carbon in the leaves and stems is also re-mobilized to become the raw material needed for grout filling. At this time, the enzymes related to starch synthesis become active, and the metabolic direction shifts from maintaining life to reserve accumulation (Wang et al., 2021). However, these rhythms are not fixed. Changes in environmental conditions such as temperature and light can directly affect the speed and duration of grout filling, thereby influencing yield and rice quality (Shimoyanagi et al., 2021). 2.2 Major cell types involved in grain filling and their functions There is not just one type of cell in a rice grain. The endosperm, embryo, seed coat and pericarp each have their own functions (Liu et al., 2025). The endosperm is the most important storage organ, occupying the majority of the space in the grain, where starch and protein accumulate. The path for transporting sucrose from the maternal tissue to the endosperm is not simple either, as it has to pass through multiple "channels" such as the parenchyma cells of the vascular vessels, the tubercle process, and the tubercle epidermis. Among them, sugar transporters like OsSWEET11 and OsSWEET14 play a key role (Fei et al., 2021). In addition, the peel and aleurone layer are also involved in material transfer and are not merely "shells". They also play a role in signal regulation during development (Ren et al., 2021). 2.3 Hormonal and metabolic regulation during grain filling The role of hormones in the grouting process is hard to ignore. Cytokinin, auxin, abscisic acid (ABA), gibberellin and ethylene were all major participants (Figure 1) (Panda et al., 2018). They regulate not only cell division, but also the expression of genes related to starch synthesis and the "reservoir strength" of grains. For instance, cytokinin can promote the proliferation of endosperm cells and also facilitate the "filling" of grains. ABA accelerates grouting under adverse conditions and is a typical coping hormone (Yang et al., 2001). The concentration and duration of action of these hormones must be properly coordinated so that there will be no problems with grouting. There are also key links in metabolism. For instance, the process of converting sucrose into starch relies on several important enzymes, such as ADP-glucose pyrophosphorylase and starch synthase. The activities of these enzymes are influenced by both genes and the environment (Zhang et al., 2019), so the entire process is actually very subtle. It is easy to have problems, but it is very difficult to achieve coordination. 3 Principles and Advantages of scRNA-seq in Plant Research 3.1 Overview of scRNA-seq workflow and technical considerations In plant research, the scRNA-seq technology has indeed changed the way people view differences in cell expression. It can perform transcriptional analysis at the single-cell level. Cell differences that were previously "averaged out" in mixed samples can now be seen separately (Bawa et al., 2022).
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