Function and regulatory mechanism of HvSPX4 in maintaining phosphate homeostasis in barley

Low phosphorus (LP) tolerance varies among plant species and genotypes within a species, and the molecular mechanisms underlying P signaling in barley (Hordeum vulgare L.) remain unclear. Here, we report the function of HvSPX4, a member of the SPX (SYG1/Pho81/XPR1) subfamily, in maintaining P homeostasis and regulating low P responses in barley. HvSPX4 exhibited lower expression in the LP-tolerant genotype Zaoaibai than the LP-sensitive Salooni2. Knocking out HvSPX4 resulted in growth inhibition due to over-accumulation of P under normal P conditions. Overexpression of HvSPX4 resulted in reduced biomass under both normal and low P conditions and less P uptake, especially in shoots under normal P conditions. HvSPX4 directly interacted with transcription factors Phosphate Starvation Response 1/2/4 (HvPHR1/2/4) to inactivate PSI genes such as Phosphate Transporter 1;6 (HvPHT1;6). An eGWAS analysis of HvSPX4 expression revealed that a 50-bp insertion in the promoter region of HvSPX4 enhances its expression, probably due to the presence of two CAAT boxes in this insertion. Moreover, the absence of the insertion was associated with improved plant growth in a natural population of barley under LP conditions. These findings suggest that fine-tuning the expression of HvSPX4 could be a promising strategy to improve plant adaptation to low P stress.