| Phosphorus(P)is an essential macronutrient required for plant growth and development.In addition to its role as an important structural component of biological macromolecules such as nucleic acids,ATP,and phospholipids in plants,phosphorus(P)has effects on photosynthesis,respiration,material metabolism,and energy transfer.The major form of P uptake by plants is inorganic orthophosphate(Phosphate,Pi)in soils.Pi,the only form of P that can be absorbed and utilized by plants directly,is often limited in soils as Pi forms precipitate with metal cations or microorganisms.Several numbers of families of phosphate transporters(PTs)are essential for the uptake of Pi from the soil and its internal transport and utilization within plants.To date,most members belong to the high-affinity PHT1 family have been isolated and identified in rice,and the research on functional characterization of rice PHT2 family phosphate transporter in rice has not been further studied.In this thesis,we focused on the PHT2 family phosphate transporter gene OsPHT2;1 in rice using Nipponbare,a model cultivated variety of japonica rice.Using the qRT-PCR,yeast heterologous expression system,transgenic rice plants of carrying pOsPHT2;1-GUS,OsPHT2;1-Ox and mutant transgenic plants,we analyzed the expression characteristics and functions of OsPHT2;1.The main results were summarized as follows:1.The bioinformatics analysis showed that OsPHT2;1 gene is located on chromosome 2 in the rice genome,and OsPHT2;1 contains 3 exons and 2 introns.The full-length of CDS(coding sequence)is 1719 bp,which encodes a total of 572 amino acids.Subcellular localization analysis in rice protoplast cells indicated that OsPHT2;1 is a chloroplast-envelope-located protein.2.The spatio-temporal expression pattern of OsPHT2;1 was analyzed by the rice chip database,qRT-PCR and transgenic rice plants of carrying pOsPHT2;1-GUS.The results showed that OsPHT2;1 had the highest expression abundance in rice leaves while its expression was barely detectable in roots,and it was induced by Pi starvation and light in two parallel pathways in leaves.At the flowering stage,the expression of OsPHT2;1 was higher in the new leaves than in the old leaves,and OsPHT2;1 was also expressed in the node,peduncle,rachis and spikelet.OsPHT2;1 was detected in the node and rice husk at the filling stage.After GUS staining,we found that OsPHT2;1 was expressed in the shoot-root junctions,leaf sheaths and auricles,and GUS staining was also detected in the rudimentary glumes,lemma and palea in developing rice seeds.3.The results of yeast heterologous expression system showed that OsPHT2;1 was able to complement a yeast mutant strain defective in Pi uptake only under high Pi conditions(10 mM Pi),indicating that OsPHT2;1 has Pi transport activity and shows low-affinity in heterologous system.4.Wild type(WT),OsPHT2;1-Ox and ospht2;1 mutant plants were cultured hydroponically under Pi-sufficient(200 μM Pi)and Pi-deficient(10 μM Pi)conditions to analyze the growth performance and Pi accumulation.The results showed that both OsPHT2;1-Ox and ospht2;1 mutant plants caused a phenotype of decreased plant height and biomass in the vegetative growth stage,especially under Pi-sufficient conditions.The Pi concentrations in the leaves of OsPHT2;1-Ox plants were higher than that of wild type under both Pi-sufficient and Pi-deficient conditions,and there was no difference in the roots.Under Pi-sufficient conditions,no differences were detected in leaves or roots of Pi concentrations between ospht2;1 and WT plants.However,under Pi-deficient conditions,the Pi concentrations in the leaves of ospht2;1 mutants were reduced compared with WT,and the mutation of OsPHT2;1 did not change the Pi concentrations in roots.The results of the 32P-labelled Pi uptake assay showed a significantly lower accumulation of 32P in the shoots of ospht2;1 mutants.However,no significant 32P activity was observed in the roots.Besides,the ratio of 32P distribution in the shoots and roots of ospht2;1 mutants was reduced compared with the WT.These results indicated that OsPHT2;1 affects the accumulation of Pi in leaves and the distribution of Pi in rice.5.WT,OsPHT2;1-Ox and ospht2;1 mutant plants were cultured hydroponically under Pi-sufficient and Pi-deficient conditions to determined the photo synthetic rate and sucrose and starch concentrations.The results showed that the photosynthetic rates of OsPHT2;1-Ox plants were higher than that of WT under both Pi-sufficient conditions.And the photosynthetic rates of ospht2;1 mutants were reduced compared with WT under both Pi-sufficient and Pi-deficient conditions,indicating that OsPHT2;1 plays a role in maintaining photosynthesis in rice.In addition,sucrose concentrations in the leaves of ospht2;1 mutants were higher than those in WT plants under Pi-sufficient conditions,but did not change under Pi-deficient conditions.In contrast,starch concentrations in the leaves of ospht2;1 mutants were reduced under both Pi sufficient and Pi deficient conditions.In the roots of ospht2;1,starch concentrations were higher than that those of WT under Pi-sufficient conditions,but were similar to that in WT under Pi-deficient condition.These results indicate that OsPHT2;1 plays a role in the regulation of carbohydrate concentrations.6.Field experiments were performed with WT and ospht2;1 mutant plants in Nanjing,Jiangsu Province and Ledong Li Autonomous County,Hainan Province.Although plant heights and panicle lengths of ospht2;1 mutants were reduced compared with the WT,the effective tiller number,NO.of branch per panicle,seed setting rates,1000-grain weight and yield per plant were comparable between ospht2;1 mutants and the WT in Nanjing,Jiangsu Province.However,plant heights and panicle lengths of ospht2;1 mutants were reduced compared with the wild type,whereas the seed setting rates and grain yields were dramatically decreased in ospht2;1 mutants in Ledong Li Autonomous County,Hainan Province.In addition,there were no significant differences in effective tiller number,NO.of branch per panicle and 1000-grain weight between ospht2;1 mutants and the WT.The results indicated that OsPHT2;1 modulates rice yields in different latitudes.7.WT and ospht2;1 mutant plants were used for the UV-B irradiation treatment and nitroblue tetrazolium(NBT)staining experiments.The results showed that the majority of WT plants were slightly withered,whereas the ospht2;1 plants were seriously withered after UV-B irradiation treatment.Moreover,nitroblue tetrazolium(NBT)staining results showed that the antioxidant activity of ospht2;1 mutant plants were decreased after UV-B irradiation.Taken together,these data indicated that OsPHT2;1 affects the UV-B tolerance of rice.8.Metabolites were detected and analyzed in the rice leaves of WT and ospht2;1 mutants.Approximately 112 metabolites showed differences between ospht2;1 mutants and WT among the detected metabolites,and most of the different metabolites were involved in flavonoid biosynthesis and flavone and flavonol biosynthesis,this indicated that the mutation of OsPHT2;1 significantly influenced flavonoids homeostasis in rice.9.WT and ospht2;1 mutant plants were used for the determination of flavonoid concentrations,and several key genes in the flavonoid synthesis pathway and phenylalanine synthesis pathway were detected by quantitative PCR.The results showed that the mutation of OsPHT2;1 led to a decrease in flavonoid concentration in rice leaves.Several genes related to the biosynthesis of flavonoids were analyzed in the ospht2;1 mutants,transcripts of CHI and CHS did not change in the ospht2;1 mutants,whereas C4H,4CL3,4CL4,F3H2 and FNS1 were upregulated.In the phenylalanine biosynthesis pathway,the expression of CM1;1 was comparable in ospht2;1 and WT plants,whereas CM2;2 and CAT2/4/7/11 were induced in ospht2;1 mutants.Interestingly,the expression of ADT1/MTR1 was reduced,it indicated that the reduced accumulation of phenylalanine in ospht2;1 mutants might be associated with the suppression of ADT1/MTR1.These results showed that OsPHT2;1 mediates flavonoid accumulation through its effect on phenylalanine homeostasis.In summary,by functionally characterized a phosphate transporter gene OsPHT2;1 belonging to PHT2 family in rice,we found that OsPHT2;1 is a chloroplast-envelope-located phosphate transporter.OsPHT2;1 affected the growth and development of rice during vegetative growth and participating,and involved in Pi accumulation in leaves and the distribution of Pi in rice.OsPHT2;1 also plays an important role in maintaining photosynthesis and regulating carbohydrate accumulation in rice.In addition,OsPHT2;1 mediates UV tolerance via partially regulating flavonoid homeostasis in rice,and modulates rice yields in different latitudes at least partially through the regulation of UV tolerance. |
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