Transcription factors WRKY2 and WRKY34 control LATERAL ORGAN BOUNDARIES DOMAIN10 expression in pollen vegetative cell nuclei

The intricate regulation of gene expression determining cell fate during male gametogenesis involves a complex interplay of multiple transcriptional regulators. In Arabidopsis (Arabidopsis thaliana), the LATERAL ORGAN BOUNDARIES DOMAIN 10 (LBD10) transcription factor is prominent in early microspores and both the germ and vegetative cells of bicellular pollen, playing an important role in pollen development. However, in mature pollen, LBD10 exclusively localizes in the vegetative cell nucleus. Here, we identify cis-acting elements and trans-acting factors responsible for the specific expression of LBD10 in the vegetative cell nucleus during pollen maturation. Using a series of LBD10 promoter deletion constructs fused with GUS or GFP reporters, we pinpoint two crucial core promoter sequences. These sequences are situated within two 200 bp regions upstream of the start codon and independently govern LBD10 expression in the vegetative cell nucleus. We demonstrate that a W-box motif (AGTCAC) at -770 bp is essential for activating the expression of LBD10 in vegetative cells during pollen maturation. Our transient gene expression assays using Arabidopsis protoplasts and chromatin immunoprecipitation assays show that the transcription factors WRKY2 and WRKY34 recognize the LBD10 promoter region containing W-box motifs. Collectively, our findings suggest that WRKY2 and WRKY34 binding to the W-box motifs plays a role in the vegetative cell nucleus-specific expression of LBD10 in pollen. This interaction may contribute to male gametophyte development, shedding light on the intricate regulatory network governing this critical biological process.