A WHY1-NADK3-PEX5 module confers drought tolerance by maintaining peroxisomal ROS homeostasis in Brassica napus.

Drought severely limits rapeseed productivity, but the role of NAD kinase in this process remains unclear. Here, we identify BnaA07.NADK3 as a drought- and ABA-inducible peroxisomal NADK in Brassica napus. Overexpression of BnaA07.NADK3 enhanced drought tolerance, whereas CRISPR/Cas9-edited lines showed reduced drought tolerance. Mechanistically, overexpression of BnaA07.NADK3 elevates NADK activity and NADPH accumulation, thereby promoting the scavenging of drought-induced reactive oxygen species (ROS). Transcriptome profiling of BnaA07.NADK3-overexpressing plants revealed significant enrichment of genes involved in ROS detoxification and root development. Both in vivo and in vitro assays demonstrate that BnaA07.NADK3 physically interacts with the peroxisomal import receptor BnaA03.PEX5 (Peroxin 5), suggesting that BnaA07.NADK3 is associated with the peroxisomal protein import machinery required for maintaining ROS homeostasis under stress. In addition, BnaC08.WHY1 directly binds a conserved TTTTTGAC element in the BnaA07.NADK3 promoter and activates its transcription. In summary, our findings uncover a previously unrecognized WHY1-NADK3-PEX5 regulatory module that integrates plastid retrograde signaling, peroxisomal redox metabolism, and drought adaptation in Brassica napus, providing promising targets for molecular breeding of drought-tolerant rapeseed cultivars.