Function Analysis Of Rice Transcription Factors OsPHR1 And OsPHR2 Involved In Signalling Of Phosphorus Starvation

It has been known that AtPHR1, a Myb-like transcription factor, plays a key role on phosphorus (P) signalling system in Arabidopsis Whether the homologous genes in rice are important regulators in the P-signalling pathways, however, is still largely unknown. According to sequence similarity analysis, we have isolated two putative homologues of AtPHR1 from rice (Oryza sativa L), designated as OsPHR1 and OsPHR2, respectively. Detailed bioinformatics and function characterizations of these two homologous genes are investigated. The results are summarized as follows:1. OsPHR1 and OsPHR2 are two novel members of MYB-CC gene family with conserved domains shared by AtPHR1 and CrPSR1. Phylogenetic analysis within this family reveals that OsPHR1 and OsPHR2 are belong to the same subgroup of AtPHR1 and CrPSR1, and more closely related to AtPHR1. OsPHR1 shows higher sequence similarity to AtPHR1 than OsPHR2 does. Sequence comparison analysis indicates that OsPHR1 and OsPHR2 locate on chromosome 3 (120024-124948bp) and chromosome.7 (24959-30561bp) with a single copy, respectively. Both of the genes have a similar gene structure except one more exon and intron in OsPHR2 genomic sequence.2. RT-PCR analysis indicates that both OsPHR1 and OsPHR2 show a similar constitutive expression pattern in all tissues, with highest expression in leaves and roots. Both of the genes are not responsive to phosphate deprivation.3. Subsecular localization of GFP fused OsPHR1 and OsPHR2 proteins demonstrate that they are located in nucleus. In addition, the amino acid sequences at N terminus of these two proteins have strong transcriptional activities in yeast. These results suggest that OsPHR1 and OsPHR2 are functional Myb-like transcription factors4. Transgenic lines with overexpressed OsPHR2 displays a Pi toxicity phenotype with over accumulated Pi in shoot under Pi sufficient conditions. Significant reduction in plant growth rate, tiller number and seed setting rate are observed in the transgenic plants. These toxicity phenotypes can be rescued under low Pi supplied conditions with a comparable Pi concentration in shoot of transgenic plants to that of the wild-type. These results indicate that 0sPHR2 is involved in the regulation of phosphorus homeostasis under Pi sufficient conditions in rice.5. The overexpression of OsPHR2 triggers the expresstion of a subset of phosphate starvation responsive genes under Pi sufficient conditions and also increases their expression levels under Pi deprived conditions. Reversely, the down expression of OsPHR2 by RNA interference reduces the expressions of the Pi-starvation-inducible genes. These results indicate that OsPHR2 plays an important role in phosphorus starvation signalling pathway in rice. The transgenic lines with overexpressed OsPHR2 also show higher sensitivity to Pi starvation with enhanced induction rate of primary root and adventitious roots than the wild-type. More interestingly, the overexpression lines have abundant long root hair in normal solution culture compared to the wild type.6. Some high affinity phosphate transporters under Pi supplied conditions were up-regulated in both shoot and root of OsPHR2 overexpressed lines. The ectopic expression of these transporters may be attributable to the Pi over accumulation in shoot.7. OsPHR2 also controls the expression of miR399 gene family in rice like AtPHR1 dose in Arabidopsis. But the reciprocal expression of OsmiR399 and 0sPH02 was not observed in rice, suggesting a different regulatory mechanism in controlling the Pi homeostasis between rice and Arabidopsis.8. Unlike 0sPHR2, the overexpression or RNAi of OsPHR1 did not show obvious phenotypes in transgenic plants, while it is also involved in the P signalling pathway, suggesting the function of OsPHR1 and OsPHR2 may have partial redundant in Pi signalling pathway.