Expression Regulation And Function Of Two Rice Phosphate Transporters OsPht1;2 And OsPht1;6 In Phosphate Uptake And Translocation

In most soils, low solubility and mobilization of inorganic phosphate (Pi) in the solution often limit plant growth and crop yield. Therefore, studies on plant res ponse to phosphorus deficiency are ecologically and agriculturally important. The Ph t1 Pi transporters are assumed to be the predominant system responsible for Pi acqu isition by plant roots. Rice is one of the most important crops, feeding about one h alf of the world’s population. In the genome of rice, there are a total of 13 genes encoding proteins that belong to the Phtl high affinity PT family. However, their ro les in the acquisition and translocation of P remain unclear except for OsPhtl;11(Os PT11) and OsPht1;13(OsPT13) which were shown to be specifically activated during mycorrhizal symbiosis, Since these Pht1 genes were named as OsPTs in previous publication, we also use the same name in the present study for simplicity and unif orm. Using RT-PCR technique we observed that OsPT2 and OsPT6 are two of the most abundant transcripts among all the 13 Phtl genes in Pi starved rice roots. In t his thesis, we report for these two rice PTs their tissue localized expression pattern and in plant roles. In addition, we provide electrophysiological evidence specifically demonstrating plant PT’s electrical activity in a heterologous expression system. The main results are summarized as follows:1. Both OsPT2 and OsPT6 have a single copy in the genome of Nipponbare, the cultivar used for sequencing of the rice genome. Genomic DNA sequence anal ysis shows that both OsPT2 and OsPT6 do not have intron, OsPT2 and OsPT6 are localized on chromosome 3 and chromosome 8, respectively. Phylogenetic tree reve als that OsPT2 and OsPT6 belong to different subfamily, suggesting that the two ge nes have different functions in the plants. The deduced amino acid sequences of bot h OsPT2 and OsPT6 suggest that both are typical intergrating membrane proteins an d contain consensus sites for phosphorylation by both protein kinase C and casein k inase Ⅱ, and the consensus sites for N-glycosylation. Subcelular localization of GFP fused OsPT2 and OsPT6 proteins in onion epidermal cells demonstrates that they a re both localized in the cell plasma membrane.2. Each putative promoter region of these Phtl genes contains at least two of the three elements with different number of copies, W-box, PHO-like element and PIBS elements which were involved in regulation of P-deficiency induced gene expr ession. RT-PCR analysis indicates that the transcript levels of OsPT2 and OsPT6 w ere significantly enhanced by Pi-starvation in the roots. In the leaves, Pi deprivation enhanced OsPT2 expression moderately and OsPT6 very strongly.3. To determine the promoter regions of the two genes for their expression o f tissue specificity and Pi responsiveness, the sequences immediately upstream of the translation start for OsPT2 and OsPT6 were amplified. The 5’fragment of 1670 b p for OsPT2 and 2380 bp for OsPT6 was used in expression analysis. The P-glucur onidase (GUS) gene was used as the reporter to examine the spatial distribution of expression and quantitative analysis of promoter strength. The promoter-reporter g ene fusions were introduced into Nipponbare and progeny of the transgenic lines we re assayed for the spatial distribution of expression under low-and high-Pi supply. In the Pi-deprived rice, the reporter gene driven by the OsPT2 promoter was expres sed throughout the stele in primary roots, lateral roots, root-shoot junction, but not i n epidermal and cortical cells, Provision of high Pi in the culture solution suppresse d the expression of OsPT2 in roots. OsPT6 promoter-GUS expression was observed in the younger primary, lateral roots and root-shoot junction under P-deficiency. Fu rthermore, expression of OsPT6 was found not only in epidermal cells and througho ut the young primary and lateral roots, but also throughout the cortical and stele cel Is under Pi starved condition. As the age of the primary roots increased, the degree of OsPT6 promoter-GUS expression decreased, while expression was particularly ab undant at the site of lateral root emergence. No expression of OsPT6 promoter-GUS was observed in the root cap either with or without supply of Pi, indicating that t his transporter might play a dual function in the uPTake and translocation of Pi in nce.We also checked the GUS expression driven by either the OsPT2 or OsPT6 p romoters in Pi-starved rice leaves. A high Pi supply in the culture solution decrease d the expression of OsPT2 gene, and almost completely suppressed the expression o f OsPT6 in the leaves. We also found both OsPT2 and OsPT6 promoters also direc ted expression of GUS in some mature organs. OsPT2 promoter-GUS expression wa s observed in caryopsis and germinated seeds. OsPT6 promoter-GUS expression was observed in the stamens and germinated seeds strongly.4. We tested heterologously expression of the both OsPT2 and OsPT6 proteins in Xenopus oocytes. Expression of OsPT2 and OsPT6 resulted in a more than sev en- and five-fold increase in the Pi content of the oocyte, in comparison with the water-injected oocyte control, demonstrating that both proteins could mediate the upt ake of Pi from the external solution. Electrophysiological measurements showed that the plasma membrane potential became less negative when NaH2PO4 was supplied in the external solution of oocytes injected with OsPT2 mRNA. This extent of the electrical depolarization was dependent on the external NaH2PO4 concentration, and i ncreased from 1 to 10 mM. In contrast, either OsPT6 mRNA- or water-injected ooc ytes did not show any response to the supply of NaH2PO4 at these concentrations. The results indicated that OsPT2 is a low Pi affinity co-transporter of H+:Pi with n et positive electrical charges, mediating Pi-uptake in the mM range, while OsPT6 m ight be a high Pi affinity transporter with neutral electrical charge for the uptake.5. By transforming the construct of ubiquitin promoter fused with OsPT6 into Nipponbare cultivar, we successfully obtained the To seeds with overexpression of OsPT6 under both Pi-deprivation and sufficient conditions. We found that the ransge nic lines with overexpressed OsPT6 displayed a Pi toxicity phenotype in the shoots under normal Pi supply condition. Signiticant reduction in plant growth rate, necrosi s in old leaves and dead old leaves were observed in the transgenic plants. The pre liminary data suggest that overexpression of OsPT6 could enhance the transport of p hosphorus from roots to the shoots in rice.6. We constructed the putative promoters of OsPT6 gene containing different copies of the three motifs which might be combined with Pi-starvation enhanced tra nscription factors with GUS reporter gene and transformed them into Nipponbare cul tivar. Analysis of the transgenetic rice lines demonstrated that two motifs of P1BS and W-box were important binding elements for response to the Pi-starvation signali ng in rice. We also observed the positive relationship between the copy numbers of W-box motif in the promoter and the activities of its driven GUS reporter. At leas t one copy of W-box motif was essential for the Pi-deficient enhanced expression o f OsPT6 in rice.