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

Biochem J. 2017 Oct 23;474(21):3675-3687. doi: 10.1042/BCJ20170522.

Abstract

Phosphorus is an essential macronutrient for plant growth and is deficient in ∼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 (PHR1-binding sequences). In Arabidopsis and rice, activity of AtPHR1/OsPHR2 is regulated in part by their downstream target SPX (Syg1, Pho81, Xpr1) proteins through protein-protein interaction. Here, we provide kinetic and affinity data for interaction between AtPHR1 and P1BS sites. Using surface plasmon resonance, a tandem P1BS sequence showed ∼50-fold higher affinity for MBPAtdPHR1 (a fusion protein comprising the DNA-binding domain and coiled-coil 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 2× P1BS-binding site, suggesting an important role for protein co-operativity. 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.

Keywords: Arabidopsis thaliana; eukaryotic gene expression; myb transcription factor; phosphate; surface plasmon resonance.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / chemistry*
  • Arabidopsis / genetics
  • Arabidopsis / metabolism
  • Arabidopsis Proteins / chemistry*
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism
  • DNA, Plant / chemistry*
  • DNA, Plant / genetics
  • DNA, Plant / metabolism
  • Gene Expression Regulation, Plant / physiology
  • Nuclear Proteins / chemistry*
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism
  • Phytic Acid / chemistry
  • Phytic Acid / metabolism
  • Protein Binding
  • Protein Domains
  • Transcription Factors / chemistry*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • Arabidopsis Proteins
  • DNA, Plant
  • Nuclear Proteins
  • PHR1 protein, Arabidopsis
  • SPX1 protein, Arabidopsis
  • Transcription Factors
  • Phytic Acid