A GmWRKY20–GmbZIP9 transcriptional module synergistically activates GmANKTM21 to confer drought tolerance in soybean

Soybean yields are increasingly curtailed by drought; however, the native transcription factor pairs that translate water-deficit signals into a robust antioxidant response have not been fully characterized. Here, we show that CRISPR/Cas9-mediated knockout of GmWRKY20 reduces antioxidant-enzyme activities and decreases drought tolerance, confirming its essential role in soybean drought tolerance. The bZIP factor GmbZIP9, isolated as a GmWRKY20 interactor, is nuclear-localized and rapidly induced upon dehydration. Overexpression of GmbZIP9 alone elevated reactive oxygen species (ROS) scavenging capacity and enhanced drought tolerance. Critically, co-expression of GmWRKY20 and GmbZIP9 in both yeast and soybean hairy-root assays demonstrated a powerful synergistic effect, conferring superior osmotic-stress tolerance and survival under drought compared with either gene alone. We further identified GmANKTM21, encoding a plasma-membrane ankyrin-repeat protein, as the direct downstream target of this complex. The GmWRKY20-GmbZIP9 heterodimer binds directly to the GmANKTM21 promoter and synergistically drives its transcription, leading to enhanced antioxidant defense. Our findings unveil a key transcriptional regulatory module and provide a rational strategy for pyramiding GmWRKY20 and GmbZIP9 to breed drought-resilient soybean cultivars.