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 phosphorus (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 LP 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 LP 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 expression genome-wide association studies 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 2 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 LP stress.