Temporal And Spatial Expression Patterns Of Phosphate Transporter Genes In Pht1Family In Rice

Phosphorus (P) is an essential macronutrient for plant growth and development. Despite abundant content of total P in most of natural soils, P is one of the most limiting nutrients for plants due to its low concentration of the soluble form and slow rate of diffusion to the root surface. Therefore, expression of membrane phosphate (Pi) transporters (PTs), particularly the P-deficiency induced expression of high affinity PTs, is essential for efficient uptake and translocation of P by plants. Rice is a major food crop that feeds about one half of the world’s population. In the genome of cultivar of rice (Oryza Sativa ssp. Japonica cv. Nipponbare), all13Phtl genes belong to nH+/Pi symporter in Phtl family were identified. However, the expression patterns, regulation and physiological functions of these PTs in the acquisition and translocation of Pi remain unclear except OsPht1;11(OsPT11) and OsPht1;13(OsPT13) whose expression was mycorrhiza-regulated. Since these Phtl genes were named as OsPTs in previous publication, we also use the same name for each gene for simplicity and uniform in this thesis. In this study, we constructed individual putative promoter of the rice Pht1genes with β-glucuronidase (GUS) and introduced into the model cultivar of rice by Agrobacterium-mediated transformation. The temporal and spatial expression and activity of GUS and RT-PCR analyses of expression of these Phtl genes were assayed in the rice cultivar treated with Pi-starvation and Pi-sufficiency. The main results are summarized as follows:1. Analyses of subsecular localization of the PTs using a software of WOLFPSORT (http://wolfpsort.org/) showed that the all13Phtl genes are localized in the plasma membranes of plant cells. Phylogenetic analyses of the PTs within rice Phtl family together with36PTs isolated from other species suggest that the13rice Phtl genes are classified into3different functional groups. Each putative promoter region of these Phtl genes contais at least two of the three elements with different number of copies, W-box, PHO-like element and PlBS elements which were involed in regulation of P-deficiency induced gene expression. 2. RT-PCR analysis indicated that OsPT1, OsPT2, OsPT3, OsPT4, OsPT5, OsPT6, OsPT7, OsPT8and OsPT12genes were expressed in Pi-deprived roots. The transcript levels of OsPT2,OsPT3, OsPT6and OsPT7were significantly enhanced by Pi-starvation in the roots. OsPTl, OsPT2, OsPT3, OsPT5, OsPT6, OsPT7,OsPT8and OsPT12were expressed in Pi-deprived leaves. The transcript levels of OsPT1, OsPT3,OsPT6,OsPT7and OsPT12were enhanced by Pi-starvation in the leaves. In our study, transcripts of OsPT9, OsPTIO, OsPT11and OsPT13were not detected in rice grown in the nutrient solution.3. The OsPT1promoter-GUS showed a increased expression of the reporter gene in the root tips, lateral roots, root-shoot junction and leaves under Pi deficient, reporter gene expression was also observed in stamens, caryopsis and germinated seeds. OsPT2was predominantly expressed in root tips, primary roots, lateral roots, root-shoot junction, particularly the root stele but not in epidermal and cortical cells, and was strongly induced enhancive by Pi deprivation, the GUS expression driven by the OsPT2promoter was detected both in caryopsis and germinated seeds, but not in stamens. Transgenic plants containing the OsPT3promoter fused to GUS showed weak reporter gene activity in root tips, lateral roots under Pi-deprived conditions, caryopsis and germinated seeds were also weakly expression. The results showed that OsPT4::GUS fusion protein was expressed weakly in root under both Pi-deficient and sufficient conditions. No expression was detected in leaves, stamens and caryopsis. For plants containing the OsPT5promoter::GUS gene, roots tips, lateral roots, root-shoot junction, and leaves activities were weak under Pi-deficient condition, and no reporter gene activity was observed in stamens, caryopsis and germinated seeds under Pi-sufficient conditions. Our research has demonstrated that OsPT6promoter::GUS gene are significantly induced by Pi-starvation, and the expression was observed in the root tips, lateral roots, root-shoot junction and leaves under P-deficiency, there was weak expression in the mid-rib of the stamens and strong expression in germinated seeds, but not in caryopsis. The GUS activity driven by OsPT7promoters were up-regulated by Pi starvation in lateral roots, but in root tips, we did not observe the GUS activity. In leaves, the GUS activity was also induced enhancive by Pi starvation. The reporter gene expression shows a weak level in stamens, and no expression was observed in caryopsis and germinated seeds. The plants containing the OsPT8promoter::GUS/GFP constructs were detected for GUS/GFP activity either with or without supply of Pi. Strong GUS/GFP activity was detected in root tips, lateral roots and leaves under both of the conditions, but not in the root caps. In stamens, caryopsis and germinated seeds, GUS/GFP activity was also observed. We did not observe any reporter gene activity in any tissues with the OsPT9, OsPT10OsPT11and OsPT13promoters under the conditions in which plants were grown in the nutrient solution.4. According to the results of RT-PCR and transgenic rice, we concluded that the W-box and PlBS motifs have an important functional role for response to the Pi-starvation in rice. In contrast, the PHO-like element was not affected by Pi status.5. The results suggested that OsPT1, OsPT2, OsPT3, OsPT5, OsPT6and OsPT7have a role for Pi uptake from the soil solution. OsPT1, OsPT2, OsPT6, OsPT7and OsPT8were working when the P is mobilized and redistributed to young organs or to the starved root. During the seed germination, OsPT1, OsPT2, OsPT3, OsPT4, OsPT6and OsPT8transported phosphate to growing parts of the plant for their growth and development.