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.
© 2017 The Author(s).
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Arabidopsis / chemistry*
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Arabidopsis / genetics
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Arabidopsis / metabolism
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Arabidopsis Proteins / chemistry*
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Arabidopsis Proteins / genetics
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Arabidopsis Proteins / metabolism
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DNA, Plant / chemistry*
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DNA, Plant / genetics
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DNA, Plant / metabolism
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Gene Expression Regulation, Plant / physiology
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Nuclear Proteins / chemistry*
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Nuclear Proteins / genetics
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Nuclear Proteins / metabolism
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Phytic Acid / chemistry
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Phytic Acid / metabolism
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Protein Binding
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Protein Domains
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Transcription Factors / chemistry*
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Transcription Factors / genetics
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Transcription Factors / metabolism
Substances
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Arabidopsis Proteins
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DNA, Plant
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Nuclear Proteins
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PHR1 protein, Arabidopsis
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SPX1 protein, Arabidopsis
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Transcription Factors
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Phytic Acid