Bioscience Methods 2025, Vol.16, No.4, 204-217 http://bioscipublisher.com/index.php/bm 208 3.2 Nitrogen-regulated transcription factors Plants use a series of transcriptional regulatory factors to sense and respond to external nitrogen levels, thereby coordinating the expression of many functional genes. NIN-like proteins (NLP) are important nitrogen signal transcription factors discovered in recent years. In model plants, AtNLP7 has been shown to regulate the nitrate-responsive gene network. Functional studies of the rice NLP family have shown that OsNLP1 and OsNLP3 are involved in regulating the expression of related genes under both nitrogen-sufficient and nitrogen-deficient conditions. OsNLP1 responds most quickly to nitrogen starvation. When nitrogen is deficient, its active form rapidly translocates into the nucleus, inducing the expression of a series of nitrate transport and assimilation genes, thereby improving the plant's ability to absorb and utilize nitrogen. Rice overexpressing OsNLP1 significantly increased grain yield under low-nitrogen field conditions, and maintained higher nitrogen accumulation and utilization efficiency under high nitrogen conditions (Alfatih et al., 2020; Zhang et al., 2022). OsNLP3 mainly plays a role when nitrate is sufficient, promoting the expression of genes related to nitrogen absorption and assimilation, optimizing plant nitrogen metabolism, and enabling rice to obtain higher yield and NUE under normal nitrogen application conditions. OsNLP4 has been reported to be a key factor in improving rice NUE: field experiments have shown that knocking out OsNLP4 will lead to a significant decrease in rice yield and NUE under different nitrogen application levels, while overexpressing OsNLP4 can increase yield by about 30% and NUE by about 47% under moderate nitrogen application. In addition to the NLP family, DOF and MYB transcription factors are also involved in efficient nitrogen regulation. Some members of the DOF (DNA binding and one finger protein) family can affect the coordination of carbon-nitrogen metabolism. For example, exogenous introduction of maize ZmDof1 can enhance photosynthesis and assimilation during the grain filling period of rice, significantly improving growth and nitrogen accumulation under low nitrogen conditions. Rice's own DOF factor OsDof substances (such as OsRDD1) have also been shown to play a role in nitrogen absorption and assimilation. Their overexpression can promote the root system's absorption of ammonium ions and other nutrients and accumulate more assimilates. In terms of MYB transcription factors, OsMYB305 is a transcriptional activator that senses nitrogen in roots. OsMYB305 is upregulated when nitrogen is deficient, and overexpression of this gene significantly increases the number of tillers, aboveground biomass, and nitrogen content of rice plants under low nitrogen conditions. Mechanistically, OsMYB305 activates the expression of high-affinity nitrate transport system-related genes (such as OsNRT2.1/2.2, OsNAR2.1) and nitrogen assimilation enzyme genes, increases the root system's uptake rate of nitrate nitrogen, and inhibits root lignin synthesis-related genes to allow more carbohydrates to be used for nitrogen assimilation (Wang et al., 2020). The activities of key enzymes such as nitrate reductase (NiR) and GOGAT in the roots of its overexpression strains were significantly enhanced, promoting nitrogen assimilation and plant growth. A series of transcription factors such as NLP, DOF, and MYB regulate the nitrogen response network through different pathways: some directly bind to nitrate response elements to activate downstream genes (such as NLP); some indirectly improve nitrogen utilization by affecting carbon metabolism and hormone pathways (such as DOF and MYB). The discovery of these factors provides new molecular targets for improving the intrinsic nitrogen efficiency of crops. 3.3 Application of molecular breeding and gene editing technologies to improve nitrogen efficiency Based on the understanding of nitrogen efficiency-related genes, molecular breeding has opened up a new path for breeding nitrogen-efficient rice. On the one hand, quantitative trait loci (QTL) positioning and association analysis have identified many genes that affect NUE. For example, a genome-wide association study found that there is a "hotspot" on chromosome 1 that is significantly enriched in NUE-related genes, among which an excellent haplotype of OsNPF6.1 can improve nitrogen absorption and yield under low nitrogen conditions. According to statistics, more than 1,000 genes related to NUE have been reported, including a large number of transporters and transcription factors. By integrating multi-omics data, the range of candidate genes can be further narrowed and key control genes can be locked. These genetic resources provide rich materials for the breeding of nitrogen-efficient varieties. On the other hand, gene editing technology (such as CRISPR/Cas9) has shown great potential in improving crop NUE. Site-specific editing of existing NUE alleles in rice can quickly create ideal
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