Triticeae Genomics and Genetics, 2025, Vol.16, No.3, 138-147 http://cropscipublisher.com/index.php/tgg 142 4.3 Roles of non-coding RNAs in spatiotemporal regulation As for non-coding RNA, it may not have received much attention in the past, but now it can be seen that its "behind-the-scenes operations" should not be underestimated either. Especially small molecules like miRNA have been confirmed to be involved in the detailed regulation of the grain filling process. They usually do not directly express functional proteins but rather "target" core genes in key pathways, such as nutrient absorption, hormone response, and stress response, which have all been identified (Gupta et al., 2021). Of course, the specific role of non-coding RNA in wheat grout filling is still under continuous exploration. However, one point has basically become a consensus: they are playing an increasingly crucial role in regulating the spatiotemporal rhythm of gene expression, especially in ensuring the final development and quality stability of grains, and their status is rapidly rising. 5 Case Studies: Functional Validation of Key Genes and Pathways 5.1 Spatiotemporal expression profiles of starch synthesis-related genes As soon as the grouting period begins, the synthesis of starch is never idle. However, it is not the result of a few genes working alone, but rather a complete regulatory network. TaTPP-7A is one of the ones that has been proven to have a very obvious effect. This trehalose 6-phosphatase gene is specifically expressed during the grain development period. Once overexpressed, it not only activates the genes related to starch synthesis but also directly increases the thousand-grain weight (TKW) and yield. How was it done? The core pathway is T6P-SnRK1, and together with the signal "dialogue" between sugar and ABA, they coordinate the decomposition, flow and utilization of sucrose (Figure 2) (Liu et al., 2023). These regulations are not "cast the net evenly". Spatial transcriptome data further reveal how transcription factors such as TaNAC019 and TaNF-Y regulate starch synthesis genes in specific cell types, thereby affecting endosperm structure and grain size (Li et al., 2025). 5.2 Protein accumulation-related genes and the molecular basis of quality formation Not all proteins accumulate in large quantities simultaneously in the grains, nor does it all depend on the timing. The location of the tissue and the stage of development jointly determine which types of proteins are expressed in which area. Both proteomics and transcriptomics have shown that storage proteins, including gliadin and gliadin, each have their own distinct expression regions. For instance, low-molecular-weight glutenin is particularly active in the strong gluten genotype, especially in the later stage of filling, which directly contributes to bread quality (Araya-Flores et al., 2020). At the spatial distribution level, the accumulation sites of disulfide isomerase and glutamine under the endosperm and alar layers precisely illustrate how macropolymers are formed step by step - this is a key step directly related to the final quality (Zhang et al., 2021). In addition, cell type specific proteomics studies have also shown that the regulatory mechanisms of nitrogen metabolism and protein synthesis in different regions within endosperm are not the same, and these minor differences will eventually affect the nutritional level of grains (Zhang et al., 2023). 5.3 Case studies of stress-responsive genes during the grain filling stage Not only is the regulation under normal conditions worthy of attention, but the genetic response in adverse circumstances is also a greater test of the system's resilience. Once stress such as high temperature and shading occurs, the gene expression pattern of wheat will immediately "switch". In some heat-tolerant varieties, transcription factors such as TaAP2/ERF are up-regulated, and the ethylene signaling pathway is also activated to maintain the stable development of seeds (Magar et al., 2024). For instance, "emergency components" such as heat shock proteins and catalase can also be induced to help regulate osmotic pressure, enhance resilience and grouting efficiency (Rangan et al., 2020; Sihag et al., 2023; Hou et al., 2024). Of course, not all coercion can be withstood. For instance, shading can inhibit the key pathways of starch synthesis and photosynthesis, resulting in small grains and low starch content. However, such research is not all about bad news. Some candidate genes (such as TaLFNR1-7A and TaFd-7A) have been discovered, and they can still maintain a relatively high photosynthetic efficiency under low light. The functional verification of such genes also provides more precise targets for breeding, especially in enhancing the stability and resilience of crops in adverse conditions.
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