Rice Genomics and Genetics 2025, Vol.16, No.4, 199-210 http://cropscipublisher.com/index.php/rgg 204 the two transcription factors ONAC127 and ONAC129 are very high. They control the expression of sugar transporters (such as OsMST6 and OsSWEET4) and some calmodulin-related genes (Ren et al., 2021). These genes affect the efficiency of sugar transport from the mother to the endosperm. Meanwhile, mitochondrial pyruvate kinase complexes like OsPK3-OsPK1 and OsPK4 also play a role in the transport of sucrose. The expression of these genes is phased and usually occurs in the tissues responsible for sucrose transport, thereby helping grains obtain nutrients efficiently during the filling period (Hu et al., 2020). Overall, these regulatory networks act like a "coordination center", integrating the signals between the parent and the grains to make the distribution of carbohydrates more reasonable. 6.3 GRNs involved in stress response and grain quality traits When environmental conditions change rapidly, especially at high temperatures, it is very easy to interfere with grain filling. At this point, some cell type-specific regulatory networks begin to come into play. Heterodimers like ONAC127 and ONAC129 not only regulate nutrient transport but also participate in responses to stresses such as high temperatures. If they are knocked out or overexpressed, the grain filling will deteriorate and the tolerance to high temperatures will also decrease. Meanwhile, plant hormones such as ABA (abscisic acid), IAA (growth hormone), and polyamines are also involved in these regulatory processes. Interestingly, some spikelets often have poor filling and low grain weight due to relatively low hormone content (Zhang et al., 2016). Micrornas are no outsiders either. They help plants better adapt to adverse conditions by regulating hormone synthesis and signal transduction, and also influence the quality of the final grains. 7 Case Study 7.1 Identification of endosperm-specific transcription factors regulating starch genes It has been repeatedly mentioned in the research that some transcription factors are expressed only in the endosperm or at particularly high levels there, and these factors can directly regulate genes related to starch synthesis. For example, OsbZIP58 has a particularly high expression level when endosperm starch synthesis is most active, and it can directly bind to the promoters of six key genes, namely OsAGPL3, Wx, OsSSIIa, SBE1, OsBEIIb and ISA2, to control their expression (Wang et al., 2013). In addition, there are two proteins, OsNAC24 and OsNAP. The complex composed of them can regulate OsGBSSI and OsSBEI. The regulatory process is more like "fine-tuning" (Jin et al., 2023). NF-YC12 is also an example. It specifically regulates FLO6 and OsGS1;3 in the endosperm. Affecting the accumulation of starch and stored protein (Xiong et al., 2019). As for OsbZIP60, also known as OPAQUE3, it is involved not only in the synthesis of starch and protein, but also in maintaining the stability of the endoplasmic reticulum (Figure 2) (Cao et al., 2022). 7.2 Construction and analysis of starch-related GRNs from scRNA-seq data By combining laser microdissection technology with transcriptome analysis, it is now possible to map out the gene regulatory network (GRN) within the endosperm of rice, down to specific spatial positions. Studies have found that in the early stage of grouting, most of the genes related to starch synthesis are first expressed in the central starch endosperm (CSE), and then extend to the lateral and dorsal regions (Ishimaru et al., 2021). Network analysis shows that there is synergy between transcription factors and metabolic genes at different times and in different Spaces, and hormone signals and regulatory factors related to cell death have also been integrated into these networks. Recent large-scale GRN analyses have also identified hundreds of transcription factors that can regulate starch synthase. One enzyme may even be regulated by multiple factors, which also indicates that the entire system is regulated very complexly and there is redundancy (Huang et al., 2025). 7.3 Functional validation through transgenic and CRISPR approaches To verify the effects of these regulatory factors, researchers employed methods such as transgenic overexpression, knockout, and CRISPR/Cas9 (Chen and Zhang, 2024). For instance, knocking out OsbZIP58 or OsNAC24 will directly alter the starch content, the ratio of amylose to amylopectin in the grains, as well as the grain quality. These results also confirm their regulatory roles. For instance, knocking out Waxy/GBSSI with CRISPR/Cas9 reduces amylose and simultaneously triggers compensatory responses in the expression of other related genes,
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