RESEARCH SUMMARY

Plant responses to phosphate (Pi) starvation represent an emblematic system for studies on regulation of gene activity. In plants, these responses involve both biochemical and developmental changes that improve Pi acquisition and recycling, and protect against the stress of Pi starvation. The induction of Pi starvation responses requires a sophisticated regulatory system that integrates information on external and internal plant Pi concentration, and on other nutrient content. Our aim is to contribute to the dissection of this regulatory system, which operates mainly at the transcriptional level but also involves post-transcriptional control.

Regarding transcriptional control, we have further demonstrated the key role of the transcription factor phosphate starvation response regulator1 (PHR1) and its binding site (P1BS) as a master trans-cis integrator module in the control of Pi stress responses. We also initiated large-scale phylogenomic footprinting to identify all cis-motifs involved in Pi starvation responsiveness. To identify regulatory mechanisms that act on PHR1, we carried out a screening for phr1 suppressors, leading to the isolation of three mutants that partially suppress phr1 mutant phenotypes. Cloning of one of the corresponding mutant genes revealed it corresponds to ALIX, a gene conserved from yeast to mammals. ALIX regulates ubiquitination and subsequent endocytosis of plasma membrane receptors, essential for signal propagation from these receptors. Our hypothesis is that ALIX regulates Pi status signalling by direct interaction with a yet unknown Pi sensor.

Concerning post-transcriptional control, we started a program to study the biochemical and physiological functions of the ubiquitin (Ub) regulatory pathway in the control of plant responses to Pi starvation. We identified a small subfamily of E3 Ub ligases (PCE1-4) that control Pi starvation responses, as indicated by characterization of Arabidopsis pce null mutants. PCE proteins are nuclear-localized and its abundance is reduced by Pi addition, indicating a potential role as repressors of Pi signalling. Using a yeast two-hybrid approach, we identified a number of potential targets of PCE1 activity, including known transcription factors involved in control of plant growth and development. The biological relevance of these interactions is currently being addressed. As a complementary approach, we are analyzing variation in the abundance of nuclear proteins that depend on Pi supply and Ub-proteasome activity. We have focused on the nuclear proteome, since nuclear proteins probably have regulatory activities. Using 2-D fluorescence difference gel electrophoresis (DiGE) techniques (available at the CNB Proteomics Facility), we have identified four proteins with a potential regulatory role in Pi signalling whose characterization is ongoing .