Triticeae Genomics and Genetics, 2025, Vol.16, No.6, 269-277 http://cropscipublisher.com/index.php/tgg 270 metabolism and yield of wheat under different nitrogen conditions. The significance of this research lies in providing a new biotechnological approach to reduce nitrogen fertilizer input, increase wheat yield, mitigate environmental impact, and thereby promote sustainable agricultural development. 2 Molecular and Physiological Mechanisms of Nitrogen Uptake and Utilization 2.1 Main nitrogen uptake pathways in wheat roots (nitrate and ammonium ions) Transport is just one link. After nitrogen is absorbed, it still needs to enter the metabolic process. In wheat, the genes of the NRT and AMT families are respectively responsible for bringing nitrate and ammonium into cells, while glutamine synthase (GS) acts like a processing plant, converting the incoming ammonium into amino acids. Interestingly, GS also has several "models", and isoenzymes like GS2 play different roles in different tissues. In addition to these functional genes, some regulatory factors such as WFZP or TaSYD are also involved, mainly regulating the rhythm of root development and gene expression (Liu et al., 2020; Tiong et al., 2021; Kaur et al., 2022; Yao et al., 2025). On the surface, it seems that only the root is changing, but in fact, behind it lies a complex regulatory network in operation. 2.2 Key genes related to nitrogen metabolism Some key genes in wheat regulate nitrogen absorption and assimilation, including the nitrate transporter gene (NRT), the ammonium transporter gene (AMT), and enzymes involved in nitrogen metabolism, such as glutamine synthase (GS). The NRT gene family, such as NRT1 and NRT2, mediates the absorption and transport of nitrate, while the AMT gene promotes the absorption of ammonium. Glutamine synthase (GS) plays a core role in the process of assimilating ammonium into amino acids, and different isoenzymes, such as GS2, are crucial in the nitrogen assimilation pathway. Transcription factors and chromatin remodeling factors, such as WFZP and TaSYD, regulate the expression of these nitrogen-related genes and coordinate root development and nitrogen absorption efficiency (Liu et al., 2020; Tiong et al., 2021; Kaur et al., 2022; Yao et al., 2025). 2.3 Evaluation indicators and phenotypic traits for nitrogen use efficiency (NUE) When it comes to evaluating NUE, there is actually no universal standard. Common phenotypic data such as root length, root surface area, number of lateral roots, and even the biomass and grain yield of the aboveground part can all provide clues. If we look at it in more detail, we also need to examine the absorption efficiency of nitrogen, the assimilation rate, and the distribution of metabolic products. Some people will also rely on molecular markers, such as monitoring the expression levels of key genes like NRTand GS, as an indirect judgment method (Figure 1) (Wang et al., 2024; Du et al., 2025; Govta et al., 2025). However, no matter what method is used, the performance of high nitrogen efficiency mostly cannot do without a common point: only when the root system is strong enough and the metabolism is fast enough can stable output be maintained under low-nitrogen conditions. 3 Screening and Characteristics of Root-Specific Promoters 3.1 Common root-specific promoters (e.g., RCc3, OsRSP, AtML1): origin and function In plant genetic engineering, promoters that can express target genes only in the roots are actually not uncommon. Examples like RCc3 in rice, OsRSP or AtML1 in Arabidopsis thaliana are several that have been studied quite frequently. They are widely used not only because their expression is concentrated in the roots, but also because they are of natural origin - these promoters originally come from some genes that are mainly active in the roots and are often related to root development or nutrient transport. Interestingly, the OsAER1 promoter is not only concentrated in expression but also controlled by a bunch of cis-acting elements. This combination enhances its regulatory effect. In addition, root-specific promoters such as p8463 of corn have long been discovered through genomic mining and their "specificity" has been verified in experiments (Huang et al., 2015; Apriana et al., 2019; Li et al., 2019). Limiting the expression region of transgenic organisms through these promoters can reduce the interference to the above-ground parts, making research and application more targeted. 3.2 Methods for promoter activity identification (e.g., GUS reporter gene, fluorescent markers) To verify whether a promoter is truly "expressed only in the root", one cannot rely on guessing. A common approach is to carry the reporter gene and work together. For instance, GUS or GFP can be attached behind the candidate promoters, and then tissue staining or in vivo observation can be conducted to determine exactly where
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