Triticeae Genomics and Genetics, 2025, Vol.16, No.6, 269-277 http://cropscipublisher.com/index.php/tgg 271 they light up in the plant. GUS can display tissue localization, while fluorescent proteins can track expression dynamics in real time. This type of method has been applied to many crops - there are cases in rice, corn, tomato, tobacco, etc. For instance, SlREO and TIP2 have demonstrated stable expression activity in roots (Cui et al., 2020; James et al., 2022; Jenny et al., 2024). Of course, if one wants to further confirm the intensity and specificity, it is still necessary to rely on quantitative methods such as qRT-PCR to make up for it and ensure that the data is more convincing. Figure 1 TaSYD-B directly interacts with WFZP-A (Adopted from Du et al., 2025) Image caption: (A) Schematic diagram showing TaSYD-B and different truncated protein fragments. QLQ, SbcC, PLN03142, SnAC and Rne represent distinct domains. (B) Firefly luciferase (LUC) complementation imaging (LCI) assay showing that TaSYD-B-F1 interacts with WFZP-A in N. benthamiana leaves. (C) Yeast two-hybrid (Y2H) showing the interaction between WFZP-A-P3 and TaSYD-B-F1. The transformed yeast cells were grown on synthetic dextrose (SD) medium lacking Trp and Leu (SD-L/W), and selected on SD lacking Trp, Leu, Ade and His (SD-L/W/H/A). AD, pGADT7 vector; BD, PGBKT7 vector. (D) Co-IP assay showing that the TaSYD-B-F1 associates with WFZP-A in N. benthamianaleaves (Adopted from Du et al., 2025) 3.3 Expression specificity in different tissues/developmental stages The "specificity" of root-specific promoters is not absolute and is not equally stable at all developmental stages. Some promoters, such as OsAER1, even if mainly expressed in roots, occasionally show a low level of activity in reproductive sites like anthers or seeds. And the MtPT1 and MtPT2 promoters from alfalfa do not perform exactly the same in different root cell types and are also regulated by nutrient supply. To enhance the specificity of expression, some studies have simply designed synthetic promoters, such as SRSP. By splicing multiple root-specific cis elements, it achieves almost only "exerting force" in the root, with little response elsewhere (Apriana et al., 2019; Cui et al., 2020; Masura et al., 2024). These differences and details must all be taken into account in advance when improving the nitrogen absorption capacity of wheat.
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