GmbZIP4a/b Positively Regulate Nodule Number by Affecting Cytokinin Biosynthesis in Glycine max

Legumes have the capability to form nodules that facilitate symbiotic nitrogen fixation (SNF) with rhizobia. Given the substantial energy consumption during the process of SNF, legumes need to optimize nodule number in response to everchanging environmental scenarios. The TGACG BINDING FACTOR1/4 (TGA1/4) are key players in the basal immune response of plants. In this study, both β-glucuronidase staining and quantitative reverse transcription PCR (qRT-PCR) demonstrated that both <i>GmbZIP4a</i> and <i>GmbZIP4b</i> are inducible upon rhizobial inoculation. To investigate their roles further, we constructed <i>gmbzip4a</i>/<i>b</i> double mutants using CRISPR/Cas9 system. Nodulation assessments revealed that these double mutants displayed a reduction in the number of infection threads, which subsequently resulted in a decreased nodule number. However, the processes associated with nodule development including nodule fresh weight, structural characteristics, and nitrogenase activity, remained unaffected in the double mutants. Subsequent transcriptome analyses revealed that zeatin biosynthesis was downregulated in <i>gmbzip4a</i>/<i>b</i> mutants post rhizobial inoculation. Supporting these findings, genes associated with cytokinin (CTK) signaling pathway were upregulated in Williams 82 (Wm82), but this upregulation was not observed in the double mutants after rhizobial treatment. These results suggest that GmbZIP4a/b positively influences nodule formation by promoting the activation of the CTK signaling pathway during the early stages of nodule formation.