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

Area of Science:

• Plant Molecular Biology
• Plant Physiology
• Biochemistry

Background:

• Phosphorus is crucial for plant growth but often scarce in soils.
• The transcription factor PHR1 (phosphate starvation response 1) controls phosphate starvation-induced (PSI) genes by binding to P1BS DNA sequences.
• SPX proteins regulate AtPHR1/OsPHR2 activity via protein-protein interactions in plants like Arabidopsis and rice.

Purpose of the Study:

• To investigate the kinetic and affinity of the interaction between AtPHR1 and P1BS DNA sites.
• To elucidate the role of AtSPX1 in regulating AtPHR1 DNA-binding activity.
• To understand the phosphate-dependent mechanism controlling PSI gene expression.

Main Methods:

• Surface plasmon resonance (SPR) was used to measure binding kinetics and affinity.
• MBPAtdPHR1 (a fusion protein) was used to study interactions with single and tandem P1BS sequences.
• The effect of AtSPX1, phosphate (Pi), and inositol hexakisphosphate (InsP6) on AtPHR1-DNA binding was assessed.

Main Results:

• A tandem P1BS sequence exhibited approximately 50-fold higher affinity for MBPAtdPHR1 than a single site, primarily due to a slower dissociation rate, indicating protein co-operativity.
• AtSPX1 did not alter the dissociation rate of MBPAtdPHR1 from P1BS when added with Pi or InsP6.
• Pre-incubation of AtSPX1 with MBPAtdPHR1 in the presence of Pi or InsP6 significantly reduced the DNA-binding signal, suggesting AtSPX1 inhibits binding in a phosphate-dependent manner.

Conclusions:

• AtSPX1 binds to monomeric AtPHR1 in solution under phosphate-restored conditions, modulating the AtPHR1-DNA binding equilibrium.
• This phosphate-dependent regulation by AtSPX1 provides a mechanism for tight control of PSI gene expression.
• A negative feedback loop is established where Pi-dependent regulation of AtPHR1-DNA binding also impacts AtSPX1 expression.