Functional Analysis Of A Phosphate Transporter Gene OsPht1;8in Rice

Phosphorus (P) is essential for plant growth and development due to its involvement in the processes of energy metabolism and synthesis of nucleic acids and membranes. However, the low availability of soil P is a major constraint for crop production in many agricultural systems worldwide. Higher plants thus alter their architecture and metabolism to acquire sparingly soluble P from soil. It was reported that plants are able to mobilize P by acidification of the rhizosphere by release of H+from the roots to balance excess intake of cations over anions. Pi uptake into the root symplasm involves transport from the apoplast where Pi concentration is less than2μM, across the plasma membrane (PM), and to the cytosol where Pi concentration ranges from5-17mM. Rice is one of the most important crops, feeding about one half of the world’s population. In the genome of rice, there are a total of13genes encoding proteins that belong to the Phtl high affinity PT family. Consequently, research on functional characterization of rice PM phtl family genes with regard to rice nutrients uptake and translocation is of extreme importance for understanding of the mechanism and germplasm enhancement.In this thesis, we report the tissue localization, expression pattern and the in planta function of one member of rice Phtl family. In addition, we provide electrophysiological and yeast evidence specifically demonstrating plant PT’s function in a heterologous expression system. The main results are summarized as follows:1. Sequence alignment between genomic and cDNA sequences showed that OsPT8does not have any intron. OsPT8is localized on chromosome10, and has a single copy in the genome of Nipponbare. The deduced amino acid sequence of OsPT8suggests that OsPT8is a typical intergrating membrane protein and contains consensus site for N-glycosylation. Moreover, subcelular localization analysis by OsPT8::GFP fusion proteins in onion epidermal cells confirmed that it is localized in the cell plasma membrane.2. Both Real-time RT-PCR and semi-quantitative RT-PCR indicate that the expression of OsPT8increased in roots upon Pi starvation. The expression level of OsPT8in leaves is lower than that in root, and did not show dramatic change in response to varying Pi supply. We tested the transport activity of OsPT8in two heterologous expression systems, yeast and Xenopus oocytes. OsPT8exhibited an apparent Km of21μM and was able to complement Pi uptake mutant yeast in the high affinity concentration range. In oocyte expression system the Km is27μM. The pH experiments in yeast heterologous expression systems suggested that the activity of OsPT8is dependent on electrochemical membrane potential mediates by H+-ATPase. Thus, we concluded that OsPT8might be a high affinity phosphate transporter.3. By transforming the construct of overexpression and knock-down of OsPT8into Nipponbare cultivar, we successfully obtained the transgenic lines with overexpression and knock-down of OsPT8. We found that the transgenic lines with overexpressed OsPT8displayed Pi toxicity phenotype in the shoots under normal Pi supply condition. Significant reduction in plant growth rate, necrosis in old leaves and dead old leaves were observed in the transgenic plants. The Pi concentration of root, clum, young leaf and old leaf in overexpression lines were all2-fold as high as that of wild type plants. In contrast, the Pi concentration of root, clum, young leaf and old leaf in knock-down lines were all only70%of that in wild type plants. All the data suggest that OsPT8plays very important roles in plants, although the underlying mechanism for Pi over-accumlation in OsPT8overexpressed plants still needs to be further investigated.4. We also carried out pot experiment with four levels of fertilizer P. The total P concentration in unfilled rice hull is2-3folds than that in wild type under all Pi supply, while the total P concentration in brown rice enhance20%than wild type. Under high Pi conditions, the total P concentration in panicle axis are2.2-folds and3.2-folds than wild type, respectively. We also measured the total P concentration of panicle axis, unfilled rice hull, brown rice in RNAi transgenic plants and T-DNA insertion mutant. The data acquired by analysis of RNAi plants showed that the total P in panicle axis enhance30%than WT, but the total P in unfilled rice was only70%of that in WT. All these data showed that OsPT8is involved in the Pi translocation from vegetative organs to reproductive organs in rice.5. The transgenic lines with overexpressed OsPT8also show higher sensitivity to Pi starvation with enhanced induction rate of primary root and adventitious roots than the WT. Interestingly, the overexpression lines have abundant long root hair in normal solution culture comparing with WT palnts. This phenotype is similar to that observed in the OsPHR2overexpressing plants. Considering that the expression of OsPT8was up-regulated in OsPHR2overexpressing plants, OsPT8might function downstream of OsPHR2.6. We analyzed the relative expression of the other Phtl family members in the OsPT8overexpression and knock-down plants by the Real-time PCR, the data shown that there might be partial functional redundancy between the Phtl family members with regarding to Pi uptake and translocation.Taken together, all these results indicated that OsPT8played very important roles in Pi uptake and translaction at each stage of rice development. Notably, OsPT8was also found to be involved in Pi translocation from vegetative organs to reproductive organs in rice. Our results pave the way for making rice plants with high P use efficiency.