Functional Analysis Of Phosphate Starvation Response Gene OsSQD1 On Growth,phosphate Uptake And Utilization In Rice

In soils,the major P source for plants to Uptake is orthophosphate(Pi).However,this kind of P source is in a tow level in soils,which makes the low availability of soil P become the main limitation to the growth and devetbpment of crops.Stud ies in Arabidopsis(Arabidopsis thaliana),rice and other model species revealed an array of adaptive spatiotemporally regulated morphophysiological and molecular responses.Among these adaptive responses,Pi deficiency triggers remodeling of photosynthetic membrane lipids by substituting phospholipids with anionic non-phosphorous sulfolipid(sulfbquinovosyldiacylglycerol[SQDG])and galactolipid(digalactosyl-diacylglycerol[DGDG])fecilitating scavenging and conserving Pi in Pi-deprived plants.SQDG is a kind of sulfolipid,which consists in photosystemll complex The Synthetic pathway of SQDG has been confirmed already in Arabidopsis:UDP-sulphoquinovose synthase(SQD1)catalyzes UDP-Glucose(UDP-Glu)to UDP-sulphoquinovose(UDP-SQ);and then UDP-sulphoquinovose could be catalyzed by sulfoquinovosyldiacylglycerol synthase(SQD2)to sulfequinovosyldiacylglycerol(SQDG).However,the function of SQD1 in rice was still not clear.In this thesis,we isolated the ortho logue gene of UDP-sulphoquinovose synthase gene(AtSQD1)in Arabidopsis,and designated as OsSQDI.To detect the function of OsSQD1 in rice growth and development and Pi iPtake,translocation and utilization,the transgenic rice plants carrying pOsSQD1-GUS,mutant,RNAi interference and overexpression lines were analyzed in different index,The main results are as follows:1.One orthologs gene was found in rice according to the sequence of AtSQD1 in Arabidopsis.We found that OsSQDl is located in the 5th chromosomes in rice.The identity of nucleic acid in OsSQDI is 71%,and the identity of amino acid is 64%compared to AtSQD1.Protein domain analysis showed that OsSQD1 contains a NAD-bing Rossman domaia According to the blastp analysis,the phylogenetic tree of the orthologue genes in Brachypodium(Brachypodium distachyon),tomato(Solanum lycopersicum)and wild tobacco(Nicotiana attenuate),etc.It was found that AtSQD1 and OsSQD1 were located in two different branches of the monocotyledonous and dicotyledous.however evolutionary relationships between them are very close.Promoter analysis showed that it contains 8 W-box,4 PHO-like and 1 P1 BS motif in the promoter of OsSQD1.2.By searching the rice chip database,qRT-PCR detection and tissue location analysis,we found that OsSQD1 constitutivcly expressed in various stages and various organs in rice.The relative expression of OsSQD1 in shoots is higher than that in roots.2027 bp upstream of OsSQD1 translation start codon was amplified for driving the expression of the reporter GUS gene in rice.We found that the expression of OsSQD1 was observed throughout the entire root with the maximum at the root tip,lateral root primordial and lateral root.F urther,a cross-section analysis of the root revealed the GUS expression localized to the endodermis,xylem parenchyma,cortex and phkiem.Significant GUS activity was also observed in the leaf blade and the cross section indicated its confinement largely to the mesophyll cells.GUS activity was also conspicuous in the junction of shoot and root,node and floret.In addition,the expression of OsSQD1 was induced by Pi and K-deficiency,and reduced by S-deficiency in both shoots and roots.Tobacco subcellular localization showed that OsSQD1 was located in chloroplast.3.The purified OsSQD1 in E.coli was incubated with UDPG and sodium sulfite and found that the OsSQD1 catalyzed UDPG and sulfite to UDP-SQ detecting by HPLC.In addition,the concentrations of UDPG and sulfite concentration in the mutation in QsSQD1 by T-DNA were significantly higher than wild-type plants.4.Through analysis the performance of wild-type,ossqd1,and OsSQD1-RNAi lines in vegetative growth stage,it was found that the mutation and silencing of OoSQD1 caused seriously growth and developmental defects in vegetative growth phase.The primary and adventitious root lengths of the mutant and OsSQD1-RNAi lines were much shorter than wild-type.5.Through analysis the performance of wild-type,ossqdl,OsSQD1-RNAi and OsSQD1-Ox lines in reproductive growth stage,we found that OsSQD1 boost the growth and development in reproductive growth stage,especially facilitated the development of reproductive tissues.There was a significant reduction in plant height,tiller number,seed-setting rate and grain yield per plant in flowering and maturation period in the mutants and OsSQDl-RNAi lines.The overexpression of OsSQDI resulted in the increase of grain yield per plants,grain weight and width of seeds.In addition,the mutation and silent of OaSQD1 reduced the pollen fertility,and defected the development of pollen and anther.The relative expression of OsC6 and OsABCG26 were decreased in flowers of mutants and OsSQD1-RNAi lines.6.Results of different phosphate(Pi)treatment hydroponic showed that OsSQDI promoted Pi UPtake and accumulation in the vegetative growth stage,and this promotion is more pronounced under-P condition OsSQD1 promoted plant growth and development under both Pi-sufficient(+P)and Pi-deficient(-P)conditions.Mutation and silencing of OsSQD1 inhibited the growth of seminal,lateral,and adventitious roots at different Pi concentrations,and the inhibition was more significant under-P conditio a The overexpression of OsSQDI increased the shoot and root biomass under different Ri treatments.Mutation and silencing of OsSQDI reduced the total P and Pi concentration in roots and shoots in Pi deficiency.The overexpression of OsSQDI did not affect total P concentration,but significantly increased Pi concentration.7.Results of different Pi treatment pot experiment showed that OsSQDI facilitated the translocation of Pi from root to shoot and its accumulation in reproductive tissues.Mutation and silencing of OsSQDI reduced total P concentrations in leaf sheaths,panicle axis and seeds under both+ P and-P conditions,and significantly decreased the partition ratio of total P in reproductive and vegetative organs;overexpression of OsSQD1 reduced the total P concentration in old leaf blades under-P condition,while increased it in culms,panicles,and seeds under both+P and-P conditions.The partition ratios of total P in reproductive and vegetative organs also increased significantly.8.Analysis of the relative expression of PSI(Pi-starvation induced)genes in wild-type,ossqdl,OsSQDl-RNAi and OsSQDI-Ox lines by qRT-PCR showed that OsSQDI mainly affected the relative expression of PSI genes-OsIPS1,OsPAPIO,OsSQD2 in varying degrees under-P conditions.The mutation and silencing of OsSQDI induced the expression of OsIPS1 in roots under-P condition,reduced CsSQD2 under both+P and-P condition and OsPAPIO under-P conditioa The overexpression of OsSQDI induced OsSQD2 expression level under both+P and-P condition,and there was no significant effect on the relative expression of OsIPSl and OsPAPIO.In addition,OsSQDI also more significant affected on the relative expression of Phtl family phosphate transporter genes under-P condition compared with+P condition Under+P condition,the expression of OsPT2 and 4 in OsSQDI mutants and RNAi lines was significantly decreased,and the relative expression of OsPT1 and 2 in OsSQD1 overexpression lines were significantly up-regulated.Under+P conditions,The relative expression of OsPT2,4,6,and 8 in OsSQD1 mutants and RNAi lines was significantly down-regulated,and the expression of OsPT1,2,4,and 6 in OsSQD1 overexpression lines was significantly induced.9.Determination of sulfete(SO42-),sulfite(SO32-)and total sulfur(S)in wild-type,mutant,and OsSQD1-RNAi lines revealed that although the mutation and silencing of OsSQD1 inhibited the accumulation of phosphorus,it significantly increased the S accumulation.The relative expression level of SO42-transporter genes were detected.Compared with the wild type,the OsSULTR1.1 in roots of the mutant and RNAi lines were up-regulated,and in shoots the OsSULTR2.1 was down-regulated under both+P and-P conditions.The relative expression of osSULTR2.2 was down-regulated in shoots under-P conditions.The expression level of OsSULTR3.4 did not change significantly.These results suggest that the mutation and silencing of OsSQD1 may lead to the accumulation of sulfur,and this accumulation does not response to the supply of different Pi treatments.In conclusion,OsSQD1 catalyzed the conversion of UDPG and sulfite to UDP-sulfoquinovose,and involved in the regulation of sulfite metabolism.OsSQD1 facilitates the uptake,translocation and accumulation of Pi under different Pi conditions in vegetative growth stage,and may promote the conversion of organic phosphorus into inorganic phosphorus under-P condition.It promotes the Pi translocation and redistribution under different Pi conditions during the reproductive growth stage.OsSQD1 also plays various roles in rice growth and development,especially the development of anther and pollen.In addition,it was also found that the mutation and silencing of OsSQD1 increased the accumulation of sulfur in rice,and thfe accumulation did not response to the Pi deficiency.