A Feedback Regulation Of The Central Regulator For Phosphate Signaling And Homeostasis

Systemic phosphate (Pi) starvation signaling is regulated by the conserved central regulators, transcription factor AtPHRl and its homolog OsPHR2in rice. The SPX-domain protein, SPXI, genetically functions as a negative regulator of PHR2was also reported. The mechanism of inactivating PHR2by the SPX-domain protein(s), however, is still unknown.In rice, six SPX proteins defined as the proteins exclusively contain SPX-domain were identified. Among them, two SPX proteins, SPX1and SPX2are localized exclusively in the nucleus. In this study, we find that SPX1and SPX2are positively responsive to Pi-starvation at transcriptional levels, and their Pi-starvation-induced expression dominantly under control of PHR2. The results suggest a negative feedback regulation of the central Pi-signaling pathway and Pi-homeostasis under control of PHR2and SPX proteins.Mutant analyses reveal that mutation of SPX1or SPX2leads to increased Pi concentration, and Pi accumulation is dramatically enhanced in spxl/spx2double mutant under Pi-sufficient condition. Moreover, these mutants show all increased Pi uptake ability from roots and root-to shoot translocation compared with the wild type plants. It is noted that the Pi-starvation induced (PSI) marker genes downstream of PHR2are much induced in double mutant spx1/spx2mutant, IPS1, miR399and PT2are up-regulated in spxl single mutant, while only PT2is highly up-regulated in spx2single mutant. Time course analysis showed the supersensitive to Pi-starvation of spx1, spx2and spx1/spx2mutants. Furthermore, overexpression of SPXI and SPX2prolonged elapsed time of PHR2-mediated Pi-starvation signaling. These data indicate that SPXI and SPX2additively repress PHR2activity.The gene and protein tissue-expression patterns were indicated by in situ hybridization and histochemical GUS assay. SPX1and SPX2are expressed in mesophyll cell and vascular cylinder in leaves, and all of the cells with exception of epidermis in lateral roots as a similar pattern, while in primary roots, distinct patterns are observed between them. The mainly expression of SPX2in exodermis and sclerenchyma layer is found compared to the constitutive expression of SPX1. In addition, the exclusively nuclear location of SPX1and SPX2requires their SPX domains, suggesting that SPX domain is essential for SPX1and SPX2protein function. Coimmunoprecipitation (co-IP) showed that SPX1and SPX2interacts with PHR2under both Pi-sufficient and Pi-deficient conditions, and SPX domain plays an important role in this interaction. Besides, the C-terminus of PHR2required for P1BS binding is responsible for its interaction with SPX1and SPX2. The interaction between PHR2and SPX1or SPX2and their interaction domain are confirmed by bimolecular fluorescence complementation (BiFC) assay and the interaction fluorescence signals are shown in the nucleus.Using transient expression in tobacco leaves, overexpression of SPX1and SPX2inhibits PHR2from transcribing LUC (firefly luciferase) reporter gene driven by4×P1BS promoter, and the N-terminal functional domain containing entire SPX domain (SPX1-N195and SPX2-N191) are definded. Furthermore, chromatin immunoprecipitation analysis (ChIP) indicate that the interaction of SPX1, SPX2and their SPX domain with PHR2inhibits the binding ability of PHR2to its czs-element P1BS and there is different negative effect of SPX1and SPX2on PHR2binding target genes’promoter in vivo.The feedback regulation of SPX1, SPX2and their SPX domain on PHR2was supported by genetic interaction analysis using the plants with overexpressied PHR2-FLAG alone and simultaneously overexpressed PHR2-FLAG and SPX1, SPX2or SPX domain(SPX1-N195and SPX2-N191), and the double mutants, phr2/spxl, phr2/spx2, spxl/pt2and spx2/pt2. The results showed that the up-regulated PS1gene expression and shoot excessive Pi accumulation caused by overexpression of PHR2-FLAG can be rescued by overexpressed SPX1, SPX2or their SPX domain. By contrast, the mimic Pi-starvation signaling and Pi accumulation endowed by mutation of SPX1and SPX2are recovered in phr2mutant, and PHR2-mediated inducting of PT2is responsible for the most of increased Pi concentration in spxl and spx2mutants.In summary, the present study reveals that a feedback regulation controlled by PHR2and SPX-domain proteins underlies the central Pi-signaling pathway and Pi-homeostasis in plants, and the N-terminal functional domain containing entire SPX domain plays the negative function on PHR2same as the full length SPXland SPX2proteins. This finding provides the new knowledge on a moderate response to Pi deprivation in cells coordinated by positive and negative factor, which is required for adaptive response of plants to environmental Pi stress.