Function Of OsSPX4 And OsSPX6 On Pi-signaling And Pi-homeostasis In Rice

PHR2, belonging to MYB family transcription factor, is the central regulator of Pi-signaling pathway and responsible to for induction of a series of PSI genes under Pi deficient condition. It has been reported that SPX family proteins act as repressors of PHR2 and negatively regulate Pi starvation response. In this study, we focus on the roles of SPX4 and SPX6 in coordinately mediating Pi starvation signaling and Pi homeostasis in rice.The transcriptional level of SPX4 is not responsive to Pi status. However, the expression level of SPX6 is induced by Pi starvation and then rescued by Pi resupply. Further investigation show that there exsits three P1BS motif, which is proved to be the cis-element of PHR2, indicating that SPX6 is under the direct control of PHR2. SPX4 is ubiquitously expressed in the leaf and root, while SPX6 is mainly detected in the mesophyll cell and phlone in leaf and root tip. SPX4 and SPX6 localized both in the cytoplasm and nucleus, and the C terminal 173-199 amino acids sequence of SPX4 is essential for SPX4 nuclear localization. SPX4 is degraded rapidly under Pi starvation condition and the degradation is mainly dependent on phosphate concentration. The cell-free degradation experiment showed that C terminal of SPX4 is responsible for SPX4 degradation. Different to SPX4, SPX6 protein degraded in leaves and accumulated in roots under Pi deficient condition.Over-expression of SPX4 inhibited plant growth and altered phosphate homeostasis between leaves and roots. SPX6 over-expression lines also showed plant growth inhibition and Pi concentration decrease in leaves. Furthermore, the overexpression of SPX4 and SPX6 both affected the Pi-signaling pathway.We obtained spx4 and spx6 mutant by TALEN technology, which causes 175 site and 177-179 sites amino acids deletion of the SPX4 protein and 81-84 sites amino acids deletion of the SPX6 protein locating between sub-domain 2 and sub-domain 3 in the SPX domain respectively. The spx4 mutant showed necrosis in the tips of old leaves and plant growth inhibition. The Pi concentration in the spx4 leaves is about 2.5 times of wild-type plants, while in root it is about 1.75 times. The necrosis symptom disappears when grown in low Pi condition. The spx6 mutant also showed retarded growth, necrosis in the old leaf tips and Pi accumulation in the leaf and root. The SPX4 and SPX6 also affect the elongation of root hair which mimic the phenotype of PHR2 overexpresion. We obtained spx4/spx6 double mutant by hybridization and it showed more severe symptom than spx4 and spx6, indicating that spx4/spx6 double mutant showed additive effect and suggesting that SPX4 and SPX6 coordinately regulate rice Pi signaling.Pull-down and Y2H assays showed that SPX4 and SPX6 interact directly with PHR2, but not mutated SPX4 and SPX6. BiFC and Co-IP results further confirmed this interaction in vivo. In addition, the BiFC assay also suggested that SPX4 and SPX6 could affect subcellular localization of PHR2. Transient co-expression of SPX4.SPX6 and PHR2 in the tobacco leaves confirmed the suggestion. Furthermore, the EMSA assay displayed that SPX4 and SPX6 interacted with C terminal of PHR2 (containing MYB domain and CC domain) and the interaction PHR2 lost the ability of binding to the P1BS motif upon binding to SPX4 or SPX6. Genetic evidence that SPX6-Ov/PHR2-Ov and SPX6-Ov/PHR2-Ov can rescue PHR2-Ov phenotype further confirms the genetic interaction between SPX4, SPX6 and PHR2. At last we propose the regulation model of SPX4 and SPX6 in rice Pi starvation responses.In summary, this study investigated the function of two novel SPX domain containing proteins SPX4 and SPX6 in Pi starvation signaling and Pi homeostasis. SPX4 is degraded under-Pi condition and SPX6 protein is degraded in the leaves. SPX4 and SPX6 affect PHR2 subcellular localization through interacting with PHR2. Therefore, in the+P condition the SPX4 and SPX6 protein can prevent PHR2 from binding to P1BS motif; while in-P condition SPX4 and SPX6 are degraded, PHR2 was released and translocated into the nucleus to induced Pi starvation response. The combination of biochemical and genetic evidence revealed that SPX4 and SPX6 coordinately regulate rice phosphate deficient response, providing new knowledge for how plant adapted to phosphate starvation environment.