Maize Genomics and Genetics 2025, Vol.16, No.4, 219-228 http://cropscipublisher.com/index.php/mgg 223 5 Phenotypic and Physiological Effects of Overexpression 5.1 Changes in phosphate uptake efficiency and accumulation Not all genes are so "efficient", but when phosphate transporters like ZmPT7 or ZmVPEs are overexpressed, the absorption and accumulation of phosphorus by plants do indeed increase significantly. For instance, the transgenic strain of ZmPT7 can absorb phosphorus more effectively than the wild type and has a significantly higher total phosphorus content in its body. This extent of enhancement is actually directly related to the intensity of transgenic expression. As for ZmVPEs, its effect is a bit different - mainly more effective in transferring phosphorus from old leaves to new ones. This internal adjustment, especially when there is a phosphorus deficiency, will enable the plants to maintain a better phosphorus balance and utilization efficiency. 5.2 Alterations in root architecture and growth dynamics The changes in the fundamental structure are sometimes more intuitive than the absorption of nutrients themselves. Regulatory factors like ZmPHR1 and ZmPHR2, after overexpression, significantly increase the number of lateral roots in corn and make the total root weight larger. This is equivalent to enhancing the ability to "find phosphorus" from the source. Interestingly, there are also some genes like ZmARF1 and ZmPP2AA1 that not only regulate roots but also incidentally control the expression of phosphorus transporters. As a result, more root branches and a larger root system surface area emerged. Overall, these changes in the roots are often accompanied by alterations in the above-ground parts, such as more vigorous buds and longer leaves. 5.3 Impact on yield, biomass, and stress tolerance Ultimately, whether overexpression treatment can lead to an increase in output is the most concerning issue for everyone. The answer is: In a low-phosphorus environment, it is indeed possible. Like ZmPT7 and ZmPTF1, after overexpression, the dry weight of the buds increased, the grains became plumper, and the quantity also increased (Li et al., 2011). Some genes, such as ZmNF-YC1 and ZmAPRG, not only affect yield but also enhance photosynthesis under low phosphorus conditions. More importantly, these transgenic lines usually perform well under adverse conditions as well - not only phosphorus deficiency, but also under drought, saline-alkali and even osmotic stress, they are more resilient than the control (Li et al., 2022). Stable nutritional status and strong root systems are the core elements that support this tolerance. 6 Molecular and Biochemical Characterization of Overexpression Lines 6.1 Transcriptomic profiling to assess gene expression changes Not all transcriptome analyses are so complex that they are hard to understand. Methods like RNA sequencing and qRT-PCR have long been used to study the expression changes of phosphate transporters after overexpression. Once the regulatory factors ZmPHR1 and ZmPHR2 are overexpressed, the expression of many genes will be mobilized in a low-phosphorus environment, such as those involved in phosphorus signaling, hormone pathways, metal ion transport, and photosynthesis (Hu et al., 2024). However, this is not the only example. The overexpression of ZmNF-YC1 and ZmAPRG can also affect the expression of a batch of lipid metabolism and photosynthetic genes, and ultimately enhance the tolerance of plants to low phosphorus. In conclusion, this type of analysis can help us clearly understand the direct or indirect impact of transporters at the gene expression level. 6.2 Protein localization studies to confirm transporter targeting Although some proteins have been expressed, it is also necessary to confirm exactly where they function. At this point, the GFP fusion protein comes in handy. Like ZmPT7, it is ultimately located on the plasma membrane in corn cells, a fact that can be replicated in different systems, which also indicates that it is indeed involved in the absorption and transport of phosphorus. Another transport protein called ZmVPE has a different location and has been confirmed to be on the vacuole membrane, which precisely matches its function of "mobilizing" phosphorus from the vacuole (Guo et al., 2024b). So, in essence, this kind of localization experiment is to confirm whether these proteins have "gone to where they should". 6.3 Enzyme activity and metabolic flux analyses for phosphate-related pathways From a biochemical perspective, the changes brought about by overexpression of transporters are also considerable. The Pi absorption capacity of the ZmPT7 overexpression strain is stronger, and it can also transport
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