AtSPX1 affects the AtPHR1 -DNA binding equilibrium by binding monomeric AtPHR1 in solution

Phosphorus is an essential macronutrient for plant growth, and is deficient in about 50% of agricultural soils. The transcription factor Phosphate Starvation Response 1 (PHR1) plays a central role in regulating the expression of a subset of Phosphate Starvation Induced (PSI) genes through binding to a cis acting DNA element termed P1BS. In Arabidopsis and rice, activity of AtPHR1/OsPHR2 is regulated in part by their downstream target SPX proteins through protein-protein interaction. Here we provide kinetic and affinity data for interaction between AtPHR1 and P1BS sites. Using SPR, a tandem P1BS sequence showed ~50-fold higher affinity for MBPAtdPHR1 (a fusion protein comprising the DNA binding domain and coiled-coiled domain of AtPHR1 fused to maltose binding protein) than a single site. The affinity difference was largely reflected in a much slower dissociation rate from the 2x P1BS binding site, suggesting an important role for protein cooperativity. Injection of AtSPX1 in the presence of phosphate or inositol hexakisphosphate (InsP6) failed to alter the MBPAtdPHR1-P1BS dissociation rate, while pre-mixing of these two proteins in the presence of either 5 mM Pi or 500 µM InsP6 resulted in a much lower DNA binding signal from MBPAtdPHR1. These data suggest that in the Pi restored condition, AtSPX1 can bind to monomeric AtPHR1 in solution and therefore regulate PSI gene expression by tuning the AtPHR1-DNA binding equilibrium. This Pi-dependent regulation of AtPHR1-DNA binding equilibrium also generates a negative feedback loop on the expression of AtSPX1 itself, providing a tight control of PSI gene expression.