Functional Characterization Of Ferroxidase OsLPR5 And P₅ ATPase OsPDR2 In Rice Growth Development And Phosphorus Utilization

Phosphorus(P),one of the essential macronutrients,is required for several biochemical and physiological processes and is a component of key macromolecules including nucleic acids,ATP and membrane phospholipids.P is absorbed from rhizosphere as inorganic phosphate(Pi),which is often not easily available to plants due to its slow diffusion rates in soils and/or fixation as immobile organic Pi.Limited Pi availability adversely affects growth and development of plant.In this thesis,we isolated five LPR orthologs and onePDR2 orthologs in rice,and designated as OsLPRl-5 and OsPDR2,respectively.Using the transgenic rice plants carrying pOsSIZl-GUS,transgenic rice plants over-expressing OsLPR5,RNAi OsLPR5 and RNAi OsPDR2,we analyzed the function of OsLPR5 and OsPDR2 in growth and development,and its involvement in phosphorus utilization.The main results are as follows:1.Five LPR orthologs,OsLPR1-OsLPR5 were found in rice according to the sequence of AtLPR1 in Arabidopsis.Through a TBLASTN search,we collected 53 LPR orthologs in 29 kinds of plants.Phylogenic tree analysis revealed that a clear distinction of LPR candidates between monocot and dicot plants.OsLPR2 was placed in clade a;OsLPRl and OsLPR3-OsLPR5 were placed in clade b.OsLPR3 and OsLPR5 were in a distinct single sub-branch.OsLPR1 and OsLPR3-OsLPR5 had significant Cu-oxidase ?,?,?.OsLPR1 and OsLPR3-OsLPR5 had significant Cu-oxidase?,?,and insignificant Cu-oxidase domain and insignificant Cu-oxidase domain I.Alignment of OsLPR3,OsLPR5 and AtLPRl showed that they had conservative ferroxidase amino acid residues site.3D structure model of OsLPR5 was similar to AtLPR1.Additionally,OsLPR5 is localized to endoplasmic reticulum and nuclear.2.Using the protein sequence of AtPDR2 as queries,OsPDR2 was identified in rice genome through a TBLASTN search.We cloned the full sequence of ORF of OsPDR2 buy RACE.OsPDR2 had 20 exons and 21 introns.Additionally,OsPDR2 is localized to endoplasmic reticulum and nuclear.3.Using qRT-PCR,expression of OsLPRl was detected in all the tissues.Its level was significantly higher in root compared with other tissues.Expression of OsLPR2 was significantly higher in leaf blades and low in root.Expression of OsLPR3 and OsLPR5 was largely detected in root zones and low or barely detectable expression in leaf blades and leaf sheaths.Expression of OsLPR4 was also relatively higher in basal stem,leaf sheath and root.Using GUS expression analysis,expression levels of OsLPR5 were expressed in the nodes and collars.Relative expression levels of OsLPR1 and OsLPR2 were significantly induced under-K conditions.Relative expression levels of OsLPR3 were significantly induced under-P and-K conditions,reduced under-N condition.Relative expression levels of OsLPR4 were elevated under-P and-K conditions.Relative expression levels of OsLPR5 increased under-P and-Fe conditions,decreased under-N condition.Expression of OsLPR3 and OsLPR5 showed strong induction after 6h of Pi deprivation,respectively.Expression of OsLPR4 was significant increased after 7d Pi deprivation treatment.4.The expression of OsPDR2 was investigated by quantitative reverse transcription(RT)-PCR in all the tissues,its level was higher in leaf blade and leaf sheath than other tissues.Expression of OsPDR2 was found to be significantly induced in shoots but decreased in the roots after 21d Pi deprivation treatment.5.Function of OsLPR5 was evaluated by determining ferroxidase activity of yeast and tobacco transformed with OsLPR5,and empty vector as control.Ferroxidase activity of protein extracts of yeast and tobacco transformed with OsLPR5 was significant higher than control.Ferroxidase activity in extracts of wild-type(WT),OsLPR5 over-expression plants and genetically modified negative lines had been compared.Ferroxidaseactivity in shoots extracts of OsLPR5 over-expression plants was much higher than wild-type in Pi sufficient,deficient condition,respectively.Therefore OsLPR5 coded for a ferroxidase.6.Over-expression of OsLPR5 caused seriously growth and developmental defects.There were significant differences in biomass,plant height,tiller number,panicle length,panicle branch number and seed-setting rate between OsLPR5 over-expression plants and WT.Length of primary roots,lateral roots of OsLPR5 over-expression plants were lower than WT.The results indicated that OsLPR5 plays an important role in rice growth and development.7.Over-expression of OsLPR5 influenced Pi absorption and translocation and distribution as evidenced by hydroponic and pot experiments.Pi concentrations in theshoots of OsLPR5 over-expression plants were significant increased.To precisely determine the contribution of OsLPR5 to Pi acquisition and translocation,the uptake and distribution assay using the 32P radioisotope was performedwith OsLPR5 over-expression plants and WT.32P uptake rates andthe relativeamount of 32P translocation from roots to shoots of OsLPR5 over-expression plants were much higher than WT.In pot experiment,over-expression OsLPR5 caused P accumulated in culms and sheath not in other tissues.8.Knock down of OsPDR2 caused seriously growth and developmental defects.There were significant differences in biomass,plant height,pollen fertility,tiller numbers,panicle length,panicle branch number and seed-setting rate between OsPDR2-RNAi plants and WT.Length of primary roots,lateral roots of OsPDR2-RNAi plants were much lower than WT.Moreover,cell length and diameter of maturation zone and elongation zone of OsPDR2-RNAi plants was decreased compared with WT.The results indicated that OsPDR2 plays an important role in rice growth and development.9.Knock down of OsPDR2 influenced Pi homeostasis and distribution of rice as evidenced by hydroponic experiments.Pi concentrations in the sheath and old leaf blades of OsPDR2-RNAi plants were significant increased.Ratio of Pi to total P in leaf sheath and old leaf blades of OsPDR2-RNAi plants were much higher than WT.In pot experiment,knock down of OsPDR2 caused P accumulated in culms and sheath compared with WT.The results indicated that OsPDR2 plays an important role in Pi homeostasis and distribution of rice.10.In root spit experiment,relativeamount of 32P translocation from roots in +P treatment to shoots and roots in-P treatment of OsPDR2-RNAi plants were much higher than WT,respectively.Relative expression of OsLPR5 in roots of-P treatment of OsPDR2-RNAi plants was significant induced compared with WT,which suggested that OsPDR2 could negative-regulated expression of OsLPR5 in response to local Pi signal.In conclusion,OsLPR5 and OsPDR2 played important roles in rice growth and development.OsLPR5 was involved in Piuptake,translocation and distribution,respectively;OsPDR2 plays an important role in Pi homeostasis of rice,and negative-regulation of OsLPR5 in response to local Pi signal.