Exogenous auxin alleviates cadmium toxicity in hulless barley by modulating the transcriptional network of the HbPIN gene family.

Hulless barley (Hordeum vulgare L. var. nudum) is a climate-resilient crop, but cadmium (Cd) contamination severely threatens its development on the Qinghai-Xizang Plateau. We identified 12 conserved HbPIN gene family members. Physiological tests revealed that 50 μM Cd stress severely suppressed seedling growth and drastically repressed the expression of HbPIN2, HbPIN4, HbPIN9, and HbPIN12. Interestingly, applying exogenous indole-3-acetic acid (IAA) alone also reduced root length, directly supporting the "auxin optimum principle". Crucially, Cd + IAA co-application successfully rescued growth inhibition and fully restored the transcript profiles of HbPIN4, HbPIN9, and HbPIN12. Spatially decoupled LC-MS/MS metabolomics unraveled that this rescue counteracts an auxin transport stalemate under Cd stress, where active auxins paradoxically accumulate in shoots due to transport blockades while roots suffer intensive catabolism. Transcriptome profiling revealed that this bottleneck is initiated by the synchronized transcriptional shutdown of upstream regulatory master switches, including MADS-box, ERF, and WRKY families. Furthermore, downstream qRT-PCR assays demonstrated that the restored HbPIN profiles systematically modulate transmembrane heavy metal routing gatekeepers; upon exogenous IAA administration, the transcript abundance of the xylem-loading facilitator HbHMA2 and the shootward allocation driver HbCAL1 is remarkably recovered under Cd pressure, thereby contributing to the systemic reconfiguration of dynamic ion homeostasis. In summary, our study explains the key regulatory roles of the HbPIN family, providing vital candidate genes and a strong theoretical basis for breeding heavy-metal-tolerant cereal cultivars.