Maize Genomics and Genetics 2025, Vol.16, No.4, 219-228 http://cropscipublisher.com/index.php/mgg 222 of plants is enhanced. On the contrary, after the mutation, phosphorus accumulates in the old leaves but cannot be supplied by the new leaves, which indicates that it plays a significant role in the dynamic balance of phosphorus. 3.3 Involvement in phosphate signaling pathways and stress adaptation Light absorption and transportation are not enough. Plants also need to "sense" whether there is enough phosphorus and then decide what adjustments to make. The transporter is also involved in this link. Transcription factors like NIGT1.2 and ZmPHR1/2 regulate the expression of PHT1-like proteins and other related genes, and can also affect the way nitrogen is absorbed, and even change the appearance of roots. The regulatory means are not limited to one type. There are protein phosphorylation (such as ZmPT7), microRNA intervention (miR399 is one of them), and non-coding long Rnas (such as PILNCR2) that enhance the ability to resist phosphorus deficiency by stabilizing mRNA. In conclusion, this set of mechanisms enables corn to respond to the external environment, maintain the stability of phosphorus in its body, and at the same time enhance its adaptability to adverse conditions. 4 Overexpression Strategies for Functional Validation in Maize 4.1 Genetic transformation techniques in maize To verify the function of phosphate transporters in corn through overexpression, the issue of transgenic introduction must first be addressed (Chen et al., 2024). Not all methods are applicable to all materials. Currently, there are two commonly used ones: the Agrobacterium method and the gene gun method. Agrobacterium has been chosen by many because it can produce stable, single-copy transgenic insertions and is effective for many maize genotypes, especially when using stem tips or immature embryos as explants (Wang et al., 2018). Of course, Agrobacterium can not be used in all situations. Sometimes its efficiency is low, and then a gene gun is the only option. Although the gene gun method is more "hardcore" and the number of inserted copies may be large and fragmented, it can be applied to a wider range of corn varieties, which is very important. Both of these two methods have been well used in the construction of overexpression materials (Canas et al., 2020), each with its advantages and disadvantages. Which one to choose depends on the experimental objective and the type of material. 4.2 Selection of promoter systems for targeted or constitutive overexpression Whether the transgenic expression is good or not, the selection of the promoter is very crucial. Many people would directly use the maize ubiquitin promoter or the rice actin promoter. These promoters are of the "universal type" - basically highly expressed in any tissue. Doing so can help researchers quickly observe the influence of genes on the entire plant (Liu et al., 2015; 2020). However, sometimes it is not desirable to have genes "flying all over" the entire plant. For instance, if one wants to observe the performance of a certain organ or under specific stress conditions, more precise control is required. At this point, stress promoters like Arabidopsis rd29B or those specifically expressed in floral organs would be more appropriate (Nuccio et al., 2015). So, whether it is a broad expression or a targeted approach depends entirely on whether you are studying "global functions" or "fixed-point analysis". 4.3 Advantages and limitations of overexpression compared to knockout and knockdown approaches Overexpression sounds all-powerful. Indeed, it can directly cause genes to "amplify their actions", especially for those genes with multiple homologous copies that are difficult to study through knockout. Sometimes, the function of a certain gene can even be directly determined through an obvious phenotype. For example, the strains produced by the FOX-hunting system have achieved this (Abe and Ichikawa, 2016). For genes whose function loss can be fatal, overexpression is often the only option. But then again, it also has "side effects" - for instance, some overly expressed traits do not necessarily reflect the true physiological state; instead, they mask the detailed regulation. In contrast, knockout or knockdown (such as T-DNA insertion or RNAi) is more suitable for "subtraction" verification. However, these methods also have blind spots, especially when it comes to redundant or essential genes, obvious phenotypes are often not observed. Overall, it is difficult to exhaust all functional information by relying on a single approach. Combining overexpression and knockout strategies is the truly effective path for functional genomics.
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