Integrative Regulation Of The PHR1 Subfamily In Phosphate Signaling And Homeostasis In Rice

Phosphorus （P） is one of the indispensable nutrient elements for plant growth, which is also essential for agricultural system. The shortage of P in external environment will seriously decrease the crop yield. Although the overall P content in soil is high, P is one of the limiting factors for plant growth due to its rapid immobilization by soil organic and inorganic components in many natural and agricultural ecosystems, which lead an increasing application of P fertilizer in agricultural production process. That not only increased the cost of agricultural production, but also brought the problem of environmental pollution. Therefore, cultivating crops with high phosphate use efficiency is an important way to keep the sustainable development of agriculture.In plant Pi regulation network, PHR1 （Phosphate Starvation Response 1） act as a central regulator, which regulates Pi starvation responses by binding to the motif of P1BS （PHR1 binding site）. Although much work had carried on how PHR1 control the Pi-signaling and homoeostasis in different species, how the rice PHR1 subfamily genes coordinately regulate Pi-signaling network is still obscure. Therefore, this study aims to uncover the coordinate regulation mechanism of PHR1 subfamily genes in regulating Pi-signaling and homoeostasis in rice.According to the homolog alignment, the PHR1 homologous gene OsPHR3 was analysed in rice,OsPHR3 belongs to the MYB-CC domain subfamily and locates on rice chromosome 2 （2079149-2075496）. Bioinformatic analysis shows that OsPHR3 contains 1404bp, encoding a protein of 468 amino acids, with 7 exons and 6 introns. We bought the Tos-17 insertion mutant of OsPHR3 from the mutant database （http://orygenesdb.cirad.fr/）, named phr3 （NE3009₀₁02₁A）, from the mutant base.The temporal and spatial expression patterns of three Pi-signaling regulation factors OsPHRl, OsPHR2 and OsPHR3 （here briefly named PHR1-3） were analyzed by qRT-PCR and histological section. qRT-PCR analysis showed that PHR1-3 expressed in the whole growth period, and the relative expression level of OsPHRl and OsPHR2 was not significantly changed in different growth stage; while the expression level of OsPHR3 was induced significantly in the heading stage. Histological analysis indicated that the OsPHRl is mainly located in the vascular tissue, OsPHR3 mainly located in vascular tissue and the mesophyll cells, while OsPHR2 covered all the expression tissues of OsPHR1 and OsPHR3.The histological analysis results imply that PHR1-3 may be functional redundancy in rice. Hence, in order to further understand the function of PHR1-3, this study developed the related mutants of PHR1-3; including single mutant phrl, phr2, phr3, double mutant phrl/2, phr1/3, ph-2/3, and triple mutant phrl/2/3. qRT-PCR analysis showed that the Pi starvation responsive genes of OsIPS1,OsPT2 and OsmiR827, under the regulation of PHR1-3, was repressed in related mutants of PHR1-3. The growth of phr2, phrl/2, phr2/3 and phrl/2/3 were inhibited even under phosphate sufficient condition （200μM Pi）, while the Pi content of phrl, phr2, phr3 have no significant difference compared to wild type （WT）, only the Pi content of triple mutant of phrl/2/3 siginificantly decreased compared with that of WT. And then the root hair length of PHR1-3 related mutants was observed under the Pi-free condition （0μM Pi）. The results showed that the root hair elongation of phrl, phr2, phr3, phrl/2, phrl/3, phr2/3 and phrl/2/3 were inhibited compared with wild type （WT）. And the degree of triple mutant phrl/2/3 was the most serious repressed. As the microarray analysis results shown, the number of induced transcripts in phrl mutant was 78 and repressed was 158.In phr3 mutant 169 transcripts were induced and 155 transcripts were repressed. While there were 2115 transcripts induced and 2224 transcripts repressed in the triple mutant of phrl/2/3. Besides, part of transcripts （33 induced and 32 repressed） displayed similar pattern in both phrl and phr3 mutant, but most are different, indicating the regulation difference of OsPHRl and OsPHR3 in some process. These results demonstrated that PHR1, PHR2, PHR3 are functional redundancy and divergency as well.In addition, this study analyzed the affect of PHR1-3 over expression transgenic plants on Pi-signal and homeostasis. The Pi accumulate in shoot of OsPHR3 overexpression lines （OsPHR3-Ov）, under phosphate sufficient condition （200μM Pi）, higher than that of OsPHR1 but lower than OsPHR2. While there is no significant difference of Pi content compared with wild type under Pi-deficient condition （10μM Pi）, indicating that OsPHR3 is involved in regulation Pi-homeostasis in rice. Besides, over expression OsPHR3 also can promote the root hair elongation as OsPHRl and OsPHR2 under phosphate sufficient condition （200μM Pi）. The root hair elongated degree of OsPHR2 is more efficient than OsPHRl and OsPHR3, indicating that the three PHR transcription factors were function diversity. Furthermore, we found that over expression of OsPHR3 did not inhibit the normal growth of plant under both phosphate sufficient and deficient conditions. Therefore, the field experiment was conducted under three phosphate fertilizer conditions. The results indicated that the biomass and yield of OsPHR3-Ov were higher than wild type on different Pi gradients, suggesting that OsPHR3 is a candidate gene for the cultivation of phosphate use efficiency plants. Therefore,OsPHR3 was used to improve the rice variety Xiushui 134 （XS134）, a high yield variety in China, and the results also showed that over expression of OsPHR3 in XS134 was also benefit to plant growth under low phosphate condition.In order to further understand the molecular mechanism of OsPHR3-Ov tolerance to low phosphate stress, we investigated the DNA binding affinities of PHR1-3 to different P1BS motif. Basing on Y1H （Yeast-one-hybridization） analysis and EMS A （Electrophoretic mobility shift assay, EMS A） technology, we found that PHR1-3 had different DNA binding affinities to different P1BS sequences. DNA binding dynamics analysis of PHR1-3 also indicated that the affinity of OsPHR3 binding to P1BS motif is weaker than OsPHRl and OsPHR2.In summary, this study reveals the coordinated regulation of transcription factors PHR1-3 on Pi-signaling and homeostasis in rice. Also over expression of OsPHR3 can improve low phosphate tolerance in rice, which provides a new gene resource for crop genetic improvement.