Legume Genomics and Genetics 2025, Vol.16, No.2, 63-71 http://cropscipublisher.com/index.php/lgg 67 5 Regulatory Networks and Downstream Targets of WRKY Transcription Factors in Chickpea 5.1 Interaction with signaling molecules (ABA, JA, SA) The WRKY gene is indeed very active in the regulation of adverse signals, but their "selectivity" is also worth noting. Take CaWRKY70 as an example. It binds to the promoters of salicylic acid (SA) signal-related genes, such as NPR1, PR2, PR10, etc., thereby influencing the immune response and systemic acquired resistance (SAR) of plants. However, it's not the case that the more the better. Experiments have found that once CaWRKY70 is overexpressed, it will instead inhibit the endogenous SA level and the expression of its response genes, resulting in a decline in the plant's resistance to Fusarium. Another case is WRKY40, whose promoter is activated during jasmonic acid (JA) treatment and pathogen infection, but remains indifferent under SA induction (Li et al., 2024). This indicates that WRKY's hormonal response has a distinct "preference" and does not consume all signaling pathways. 5.2 Crosstalk with other transcription factor families It is not entirely accurate to say that WRKY is the core regulator. Often, they are more like a "node" in the network, working together with other families of transcription factors. For instance, they will interact with families such as ERF, MYB, NAC, and bZIP. CaWRKY70 and CaWRKY54 have been found to jointly participate in regulating defense and stress pathways, sometimes cooperating and sometimes checking and balancing each other. More interestingly, CaWRKY70 can also suppress the CaMPK9-CaWRKY40 signal path, demonstrating the role of an "intervener" (Phukan et al., 2016; Rai et al., 2024). This complex relationship where you are in me and I am in you might precisely be the key for plants to strike a balance between growth and defense. 5.3 Regulation of stress-responsive gene cascades WRKY's participation in defense responses relies not on single-point output but on a complete set of "cascading mechanisms". They can recognize W-box elements, thereby regulating a whole set of downstream stress-related genes. In chickpeas, WRKY members such as Ca-08086 have been found to co-express with some key gene modules, which are involved in typical defense responses such as callose deposition and chitin induction. More interestingly, these networks exhibit distinct activation states across different chickpea genotypes (disease resistance vs. susceptibility) (Chakraborty et al., 2019; Konda et al., 2019). This not only indicates the significance of WRKY, but also reflects their "differentiated" strategies in regulatory directions, with some activating and others inhibiting. 6 Case Study: WRKYGene Involvement in Drought Stress Tolerance in Chickpea 6.1 Selection and characterization of drought-responsive WRKYgenes Not all stress response genes are significantly expressed under drought conditions, but WRKY transcription factors are often the most active among them. Through the joint analysis of whole-genome and transcriptome data, researchers found that WRKY was upregulated in different genotypes, but to varying degrees. Especially when water is severely scarce, the expression of WRKY in drought-tolerant chickpeas is often more intense, while that in drought-tolerant ones is relatively lower (Kumar et al., 2019; Waqas et al., 2019). This difference is not accidental. Multiple RNA sequencing and qPCR studies have shown that those WRKY genes with high expression levels are likely to be associated with enhanced drought resistance. Although other transcription factors are also involved in regulation, such as bZIP or MYB, WRKY is often among the first to be upregulated in the early stages of drought stress. 6.2 Experimental setup and phenotypic observations under water deficit To verify the functions of these genes, the experimental design must be close to real drought scenarios. The common practice is to select two strains, drought-tolerant and drought-tolerant, and create a water-deficient environment by treating with polyethylene glycol or cutting off water supply. The monitoring of gene expression is carried out at multiple time points, mainly using semi-quantitative or real-time PCR techniques. In terms of appearance, drought-resistant varieties are more "resilient" during stress - they are less likely to wilt and recover
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