Differential regulation and interactions of wheat WRKY transcription factor homoeologs in modulating iron deficiency response.

Iron (Fe) is an essential micronutrient required for plant growth and development. Plants have evolved complex regulatory networks to maintain Fe homeostasis, including regulation of gene expression involved in Fe uptake and assimilation. Among these regulatory mechanisms, the function of WRKY transcription factors (TFs) in mediating nutrient deficiencies remain unexplored. Hence, we aimed to characterize potential roles of the wheat WRKY TFs network under Fe deficiency (-Fe). Quantitative RT-PCR evaluated expression pattern of WRKY genes under -Fe, and cycloheximide treatment for different times. Yeast-two-hybrid and split-luciferase complementation assays were used to detect WRKY-WRKY protein interactions. Yeast-one-hybrid and effector-reporter assays were used to identify affinity of WRKY TFs for W-box in the promoter region of -Fe response genes. Our results suggest that, among 40 candidate TaWRKYs, 18 were significantly upregulated in -Fe conditions. Expression of TaWRKY18-A1 and TaWRKY40 homoeologs was not affected by cycloheximide, while TaWRKY70 homoeologs remained insensitive. TaWRKY70 can interact with its homoeologs and other WRKY TFs. TaWRKY40-B4, TaWRKY18-A1 and TaWRKY70-D1 show differential binding affinities towards the W-box in the zinc-induced facilitator-like proteins TaZIFL1B and TaZIFL1D promoters. There was variation in TaWRKY gene expression under -Fe, interaction strength among TaWRKY homoeologs, and binding specificity with TaZIFL1. These findings provide a scientific basis for understanding Fe homoeostasis gene networks and the role of homoeologous WRKY TFs in -Fe responses in hexaploid wheat.