Cotton Genomics and Genetics 2025, Vol.16, No.3, 107-116 http://cropscipublisher.com/index.php/cgg 111 5.3 Network rewiring under environmental and hormonal cues The environment is changing, and the regulatory network will certainly not remain unchanged. Take water stress as an example. The JAZ1a module is manifested under this pressure, and it can mobilize a group of genes related to stress response (You et al., 2016). In addition to the environment, hormone signals will also "manipulate" the network structure. Some transcription factors related to hormone response will be integrated into the regulatory module, causing the entire network structure to adjust. Other studies have combined chromatin openness and gene expression and found that these environmental and hormonal factors can actually directly affect which fiber-related genes will be turned on and which will be temporarily closed (Chen et al., 2023). From these analyses, it can be seen that the regulatory system of cotton fiber is not a rigid model, but a dynamic system composed of multiple functional modules, which will change continuously according to internal and external conditions. The modules are interconnected, and the relationship between the central gene, signal pathway, and environmental factors is also quite complex, but it is these changes that jointly determine the final characteristics of cotton fiber. 6 Case Study: Gossypium hirsutumFiber Development Network 6.1 Integration of RNA-seq and ChIP-seq data to identify regulatory circuits RNA-seq alone can actually reveal a lot of things. For example, in different developmental stages of upland cotton, researchers have discovered thousands of genes with significantly different expressions through transcriptome analysis, some of which are transcription factors, and some are key players related to metabolism or signal transduction (Zhang et al., 2022). These genes may not play a role alone, but they are indeed inseparable from fiber growth. However, if you want to figure out how it is regulated, these expression data alone are not enough. At this time, co-expression network analysis tools such as WGCNA come in handy. It can screen out modules and central genes related to fiber traits, at least it can help us straighten out the threads and provide a general direction for the subsequent construction of regulatory pathways (Jiao et al., 2023). As for ChIP-seq, some studies use it a lot, while others have not been developed too much. Although the current literature does not systematically explain how RNA-seq and ChIP-seq can be used together, this does not hinder the research from moving forward. As long as the high-throughput sequencing results and network analysis are integrated, we can indeed see some clear maps, especially in understanding the regulatory mechanisms specific to cotton fibers, where signs have begun to emerge. 6.2 Dissection of the GhMYB25-like-centered regulatory cascade in fiber initiation GhMYB25-like genes are a very important core factor in the development of upland cotton (G. hirsutum) fibers. Some co-expression network studies have found that transcription factors such as MYB and bHLH are critical "pivot points" in the initiation and elongation stages (Figure 2) (Yang et al., 2021). These transcription factors regulate genes related to cell wall synthesis, cytoskeleton arrangement, and hormone conduction, thereby affecting the differentiation and growth process of fiber cells at the beginning of development. 6.3 Validation of network predictions through gene knockout and overexpression Scientists have verified the predicted regulatory networks through gene knockout and overexpression experiments. For example, some genes that are considered to be "hubs" encode actin, Rho GTPase activating proteins or specific transcription factors, which have been verified experimentally, indicating that they are indeed involved in fiber development (Zou et al., 2019). For example, after overexpressing the candidate gene GhTPR, it was found that the roots of Arabidopsis thaliana became longer, which supports the regulatory role of GhTPR in cotton fiber elongation (Xiao et al., 2023). These experimental methods provide very direct evidence that the genes and modules predicted in the network are indeed related to the biological processes of cotton fibers. 7 Challenges and Knowledge Gaps 7.1 Limited spatial resolution of transcriptomic and epigenomic profiling in fiber cells Technology is changing, and research methods are becoming more and more detailed, especially in the field of single cells and spatial transcriptomes. It seems that there has been a lot of progress, but it is still a bit early to really understand the spatial expression pattern of cotton fiber cells clearly. The problem is not just whether the
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