Characterization of SPX exclusive family members in plant Pi sensing and regulation

The macronutrient phosphorus is vital to plant growth but the readily absorbed form of inorganic phosphate (Pi) is often environmentally limited. To increase Pi acquisition and use efficiency, plants have developed strategies involving a group of proteins, characterized by a highly conserved N-terminal SPX domain, which play critical roles. In this project, 14 SPX domain-containing proteins were identified in potato (Solanum tuberosum) and classified into four plant SPX domain-containing protein families. Gene expression analysis of SPX-exclusive family members using quantitative real-time PCR showed StSPX2, StSPX3 and StSPX5 are transcriptionally up-regulated during Pi starvation responses, suggesting the pervasive involvement of SPX proteins in Pi regulation among different plant species. The P1BS cis-element was also found in the promoter region of potato SPX genes, suggesting these genes are likely to be regulated by a potato homologue of AtPHR1, a Myb-CC family transcription factor which binds this sequence in other plant species. Recent studies in Arabidopsis demonstrated the SPX exclusive family protein AtSPX1 can compete with DNA to physically interact with the transcription factor AtPHR1 in a Pi-dependent way. However, the interaction mechanism is poorly understood. To investigate this, Glutathione-S-transferase-SPX1 and Maltose-binding-protein-PHR1 fusion proteins were expressed and purified from E. coli. AtPHR1 was shown to be a monomeric protein in solution that dimerizes upon associating with P1BS. SPR data also showed that AtSPX1 cannot displace AtPHR1 associated with DNA, but only interact with monomeric AtPHR1 in the presence of either 5 mM Pi or 500 µM InsP6. Therefore I propose a new model that in the Pi restored condition, AtSPX1 can bind to monomeric AtPHR1 and therefore regulate PSI gene expression by tuning the AtPHR1-DNA binding equilibrium. This regulation also generates a negative feedback loop on the expression of AtSPX1 itself, providing a tight control of PSI gene expression under Pi sufficient conditions.