Legume Genomics and Genetics 2025, Vol.16, No.2, 63-71 http://cropscipublisher.com/index.php/lgg 68 quickly after rewatering. And such phenotypic changes are often associated with the high expression of WRKY. Interestingly, some non-Wrky stress genes are also up-regulated, but their changes do not seem as stable or significant as those of WRKY (Borhani et al., 2019). 6.3 Implications for chickpea improvement throughWRKYgene manipulation Not every gene is suitable for breeding targets, but WRKY is clearly an exception. With the support of expression data and the results of functional verification, such genes naturally become the key focus of molecular breeding. Regulating the expression of WRKY through genetic modification, gene editing or traditional molecular marker breeding methods has been proposed as a feasible strategy to enhance the drought resistance of chickpeas. Especially if the expression profile information of WRKY can be integrated into the breeding process, it may not be far off to select and breed new germplasms adapted to arid regions (Figure 2) (Sen et al., 2017). Of course, all of this still needs to be based on a deeper understanding of the regulatory mechanism of the WRKYgene. 7 Functional Validation through Transgenic and Omics Approaches 7.1 Overexpression and silencing studies in model plants To verify the function of the WRKY gene, it is not necessary to start with the chickpeas themselves. Often, researchers will first conduct overexpression or silencing experiments using model plants such as tobacco. For instance, after CaWRKY50 was overexpressed in tobacco, the plants showed signs of early flowering and accelerated senescence. This indicates that it not only participates in the response to adverse conditions but is also related to the growth and development of plants. Similarly, once CaWRKY70 is overexpressed in chickpeas, it supposes the salicylic acid pathway and multiple defense responses, resulting in the plants being more susceptible to Fusarium infection. But things are not black and white. For instance, when researchers silenced the downstream gene CaHDZ12, the plants became more vulnerable to drought and salt. Although such experiments ostensibly only regulate the expression of a few genes, they actually reveal the key position of WRKY in the adversity regulatory network (Chakraborty et al., 2020). Figure 2 Functional characterization of CaHDZ12 transformed chickpea plants under salt and drought stresses. (A & B) Phenotype of control, CaHDZ12-OE and CaHDZ12-silenced chickpea plants treated with 200 mM NaCl (salinity) or kept for 10 days without water (drought). (C) DAB staining represents the extent of H2O2 production in CaHDZ12-OE and silenced plants under above mentioned conditions. (D) NBT staining represents the extent of formation in CaHDZ12-OE and silenced plants under above-mentioned conditions. (E) Analysis of antioxidant enzyme activities (CAT, SOD, APX) in CaHDZ12-OE and silenced plants before and after stress treatments. Data are means ± SEM from three biological replicates. Significant difference of transformed plants compared with WT plants is done by one way ANOVA using Duncan’s multiple range test (DMRT); significance level indicates P< 0.05 (Adopted from Sen et al., 2017) 7.2 Transcriptomics and proteomics-based validation Not all gene functions can be verified by transgenic methods, and omics technology offers another approach in this regard. Transcriptome data, especially the expression profiles under abiotic and biological stresses, reveal which WRKY genes are involved in the stress response of plants. Among them, some WRKY are frequent visitors who exhibit differential expression under multiple stressful conditions. After further analysis through the
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