Maize Genomics and Genetics 2025, Vol.16, No.4, 202-218 http://cropscipublisher.com/index.php/mgg 213 indicating that they may be jointly regulated by ABA signals and DREB-like transcription factors. Research has found that under high-temperature conditions, ABA accumulation can activate genes such as ZmCAT1 by binding to ABRE through ABI transcription factors, while genes like DREB2A, lacking their own activation domains, need to collaborate with HSF to effectively promote the transcription of heat-resistant genes. Another example is the promoter of ZmHSF20 itself. Li et al. (2024) reported that there are HSF binding sites in its promoter region, suggesting that ZmHSF20 may be controlled by a higher-order HSF regulatory network and participate in negative feedback regulation. For key candidate genes, it is also necessary to clarify their upstream signaling pathways. Taking the ZmNAC transcription factor gene as an example, if its promoter carries the abolic acid response element ABRE, it is speculated that when the ABA signal rises, the expression of the NAC gene is enhanced, and thus the NAC protein further regulates the downstream HSP or antioxidant genes, achieving an ABA-mediated heat resistance pathway. Similarly, genes containing Ca2+/CaM response elements (such as the CGCG cassette) can be regulated by calcium signaling pathways. Studies have demonstrated that the CaM binding element on the ZmHSP70 promoter has a positive effect on its thermally induced expression (Song et al., 2016). In addition to the cis-type element of the promoter, heat-resistant genes often involve complex transcriptional regulatory networks. For instance, the aforementioned ZmHSF20 module: ZmHSF20 is heat-induced and, together with other HSFS, forms a regulatory network that can feedback inhibit the expression of ZmHsf4 and certain defense genes. Therefore, when analyzing the regulatory patterns of key genes, co-expression networks and genetic analysis should be combined to clarify which transcription factors directly act on the promoters of candidate genes and which downstream targets the candidate genes regulate. The interaction between candidate gene promoters and upstream transcription factors can be verified through experiments such as yeast single-hybrid (Y1H) and dual-luciferase reporting. For instance, Cao et al. (2021) utilized DAP-seq technology to discover that ZmGBF1 directly binds to the ZmCXE2 promoter and matches the G-box sequence, thereby clarifying the pattern by which ZmGBF1 regulates genes in the GA pathway. This type of research is crucial for the application of heat-resistant genes: by understanding their promoter characteristics and regulatory factors, it is possible to achieve a more ideal expression pattern of the target gene at high temperatures through promoter engineering or targeted editing. For instance, placing key HSP genes under the control of thermal shock promoters in corn (such as the ZmHSP17.9 promoter), or knocking out binding sites that negatively regulate their expression, may enhance the heat tolerance performance of plants. 6.3 Case study: Transcriptome-based comparative study of heat-tolerant maize varieties Specific cases can further illustrate the application value of transcriptomics in the study of corn heat tolerance and variety improvement. The study selected a pair of corn hybrids with significant differences in heat tolerance and compared their transcriptomes under high-temperature stress. After high-temperature treatment during the flowering period, heat-tolerant varieties (such as PF5411-1) and sensitive varieties (such as LH150) showed yield differences: the number of grains per panicle and the weight of a thousand grains in the former were less affected, while the latter significantly reduced yield. Transcriptome analysis identified a set of heat-upregulated genes shared by the two varieties, as well as their respective specific expression changes. It was found that the common response genes of the two varieties were concentrated in the basic defense pathways, such as heat shock proteins and antioxidation-related genes, which were all upregulated, indicating that these are common heat response pathways. However, in heat-tolerant varieties, some genes that were not significantly changed in sensitive varieties were specifically upregulated, such as the gene encoding Trehalose-6-phosphate synthase, which might enhance the carbohydrate protective effect of heat-tolerant varieties. It was also found that the expression levels of several transcription factors (such as HSF and DREB types) in heat-resistant varieties were higher than those in sensitive varieties. These differential genes are believed to endow heat-resistant varieties with stronger heat regulation capabilities. On the other hand, some genes that may be related to growth inhibition and premature aging were induced in sensitive varieties. For instance, the expression of the abscisic acid synthase gene was higher than that in heat-tolerant varieties, indicating that high temperatures caused sensitive varieties to overly enter the "survival mode", sacrificing yield formation. Based on these findings, researchers further utilized co-expression network analysis to identify several key genes that might determine the differences in heat tolerance among varieties. For instance, the regulatory modules formed by specifically upregulated ZmHsfA2 and
RkJQdWJsaXNoZXIy MjQ4ODYzNA==