Triticeae Genomics and Genetics, 2025, Vol.16, No.4, 175-183 http://cropscipublisher.com/index.php/tgg 178 conditions and stress conditions. For example, whole transcriptome analysis has found that when barley encounters abiotic stress, the expression levels of the MYB, NAC and GATA transcription factor families change. Researchers usually use real-time fluorescence quantitative PCR (qRT-PCR) to confirm these results (Zhang et al., 2004; Tombuloglu et al., 2013). There is also a method called DAP-seq (DNA affinity purification sequencing), which can more accurately find out which transcription factors bind to specific DNA sequences. For example, scientists have used it to find GATA-type transcription factors related to drought response in barley (Wang et al., 2023). 4.2 Co-expression network and GO/KEGG functional analysis Some methods, such as WGCNA (weighted gene co-expression network analysis), can group genes with similar expression trends. This grouping method can help find regulatory modules and possible transcription factors related to root traits or stress resistance (Chen et al., 2023; Li et al., 2023). Next, researchers often use GO (gene ontology) and KEGG (genomic pathway) enrichment analysis to assign functional and pathway labels to these gene groups. This allows us to see whether these genes are involved in processes such as hormone signaling, stress response, or growth and development. These integrated methods can draw regulatory network diagrams. For example, some studies have found that the two transcription factors NAC and WRKY are core regulatory factors for barley to adapt to environmental changes (Xu et al., 2022). 4.3 Functional validation via gene editing and overexpression systems In order to determine the role of a transcription factor, researchers often use gene editing technologies such as CRISPR/Cas9, or overexpress it to see what changes will occur in the plant. In barley, after overexpression of a transcription factor (such as Ant1), the accumulation of anthocyanins in the grain increased significantly, indicating that this gene does have an effect (Zhou et al., 2021). This method can help us confirm whether this gene is a key factor affecting root structure or stress resistance. In addition to CRISPR, some other methods are also commonly used, such as virus-induced gene silencing (VIGS) or transgenic experiments. They can also help us understand the specific role of transcription factors in plant development or stress response (Shang et al., 2020). 5 Case Studies of Functional Transcription Factors in Barley 5.1 HvNAC005: a key transcription factor regulating root development and stress response NAC-type transcription factors in barley, such as some members of the HvC3H family, are closely related to root development and plant coping with adversity. Genome analysis found that the expression levels of NAC and CCCH-type zinc finger transcription factors such as HvC3H1, HvC3H2 and HvC3H13 increased significantly under various stress conditions or after treatment with plant hormones. This shows that they play an important role in the adaptation and recovery of the root system to stress. The expression of these transcription factors varies at different growth stages and under stress environments. This feature makes them an important target for improving barley root performance and resistance in breeding (Ai et al., 2022). 5.2 HvWRKY23: mediating immune responses against root pathogen invasion WRKY-type transcription factors are very important for barley to resist pathogens, especially for root diseases. Genomic studies have found more than a hundred WRKY genes in barley, and duplication and diversification among these genes have been found. Many WRKY genes are involved in immune response and stress resistance mechanisms. Their diversity may have been selected during the domestication of barley. Overall, the WRKY family is considered to be a key factor in helping barley roots cope with biotic and environmental stresses (Figure 2) (Pandey et al., 2018; Kan et al., 2021). 5.3 HvMYB1: regulating root hair development under phosphorus deficiency MYB is another important class of transcription factors, especially the R2R3-MYB subgroup, which has been shown to regulate root hair development and help plants cope with nutrient deficiency. In barley, through transcriptome analysis, researchers have discovered many MYB transcription factors that show responsiveness when faced with stress, such as boron toxicity or other abiotic stresses. The MYB family has a direct effect on root hair formation and nutrient uptake, which also indicates that they play an important role in plant adaptation to low-P soils (Rubio-Somoza et al., 2006; Tombuloglu et al., 2013).
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