Functional Characterization Of UDP-Glucose/Sugar Pyrophosphorylase,OsUGPase1 And OsUSPase1,in Maintaining Phosphorus Homeostasis In Rice

Phosphorus(P)is one of the major macro-nutrients indispensable for plant growth and development,and is the key component of many biological macromolecules,such as nucleic acids,phospholipids,adenosine triphosphate(ATP),and P-containing enzymes.In addition,P is also widely involved in important physiological and biochemical processes,such as material synthesis,energy transfer and signal transduction in plants.The major form of P for plants to acquire from the soil is inorganic orthophosphate(Pi).Since soluble Pi is easily converted to organic form by soil microorganisms or fixed by metal cations such as calcium,magnesium,aluminum and iron,its mobility and availability in the soil is very poor.Pi is one of the main limiting factors of plant growth in farmland and natural ecosystems.In order to adapt to the starvation of P in soils,plants have evolved a suite of physiological and biochemical responses to increase the availability of P in the soil and enhance the ability to acquire P by the plants.All these mechanisms are regulated by a series of complex and sophisticated molecular networks.Sugar,especially sucrose,plays an important role as signaling molecule in response to P starvation in plants.Plant responses to low Pi availability are preceded by increased carbohydrate contents in the shoot and phloem.Therefore,it is of great significance to reveal the regulation mechanism of the crosstalk between P and carbon metabolism pathways in crops and to breed cultivars with high P use efficiency(PUE).The synthesis of sucrose in plant mainly occurs in cytoplasm,using UDP-Glc as precursor.There are two enzymes that can catalyze the reversible reaction of using Glc-1-P as substrate to synthesize UDP-Glc:UTP+Glc-1-P(?)UDP-Glc+PPi,uridine diphosphate-glucose pyrophosphorylase(UGPase)and uridine diphosphate-sugar pyrophosphorylase(USPase).Together with another enzyme,UDP-N-acetyl glucosamine pyrophosphorylase(UAPase),they formed UDP-sugar producing pyrophosphorylase family.In rice(Oryza sativa)genome,it has been reported that two members of this family,OsUGPase1 and OsUGPase2,affect the reproductive growth of rice.However,the physiological function and regulation mechanism of UDP-sugar producing pyrophosphorylase family members in rice are still unknown.In this study,the response of UDP-sugar producing pyrophosphorylase family genes to low P stress was analyzed by using rice model variety Nipponbare(Oryza sativa L.ssp japonica)as experimental material.On this basis,two genes which were strongly in response to P deficiency stress,OsUGPase1 and OsUSPase1,were selected for detailed characterization.Since the effect of OsUGPase1 knockout on rice growth and development was very severe while the effect of OsUSPase1 knockout was much less severe,the possible molecular mechanism of OsUGPase1 regulation in sugar metabolism mediated P homeostasis in rice was identified and compared with OsUSPase1 effect in this paper.The main results obtained are summarized as follows:1.In order to identify the genes encoding uridine diphosphate-sugar producing pyrophosphorylase in rice genome,bioinformatics was used to analyze the gene that was homologous to Os UGasel and other reported members of UGPase in plant,and a total of 7 genes were found.Four of them are UGPase,one is USPase and two are UAPase.The promoter regions of OsUGasel and OsUSPase1 have three cis elements of P1BS,W-box and PHO.Therefore,it is assumed that the rice OsUGPase1 and OsUSPase1 gene can respond to the P starvation signal.2.In order to clarify the response of UDP-sugar producing pyrophosphorylase family genes to P supply levels,the expression pattern of genes in responses to main nutrition deficiency was analyzed by RT-qPCR,and the results showed that 3 members(Os UGPase1,OsUGPase2 and OsUSPase1)were strongly and specifically induced by P starvation in leaf blades.On this basis,OsUGasel and OsUSasel for further analysis.To investigate the spatio-temporal expression pattern of OsUGPase1 and OsUSPase1,RT-qPCR technique was used and as a result,OsUGPase1 and OsUSPase1 could be strongly induced by P starvation in both leaf blades and leaf sheaths.The transcript abundance of them in shoot gradually increased as extending of phosphate starvation duration,and then decreased rapidly to a basal level by re-supply of high Pi.The GUS reporter gene was fused with the promoter fragment of OsUGPase1 and OsUSPase1,they were found to be expressed in primary root,lateral root,leaf blade and leaf sheath by GUS staining.In addition,subcellular localization analysis indicated that OsUGPase1 is a cytosol-localized protein.3.In order to observe the function of OsUGPase1 and OsUSPasel in maintaining rice growth and development,the OsUGasel overexpression lines and knockout mutants were obtained by introducing the constructs into rice calli via Agrobacterium tumefaciens-mediated transformation and the CRISPR/Cas9 gene editing technique.Knockout of OsUGase1 severely affected the fertility of rice which nearly led to sterility.Thus,OsUGPase1 knockdown plant was achieved by using RNA interference(RNAi)technology.In addition,OsUGPase1 overexpression lines,RNAi knockdown lines and osugpase1 knockout mutant lines all displayed a remarkably shorter plant height and decreased biomass than wild type(WT)in yield.The same techniques were used to create OsUSPasel overexpression and knockdown lines,while these transgenic plants phenotype showed no significant difference compared to wild type in paddy field.4.In order to identify the function of OsUGPase1 and OsUSPasel in maintaining carbohydrate and P content in rice,OsUGPase1 overexpression,knockdown and knockout lines were cultured hydroponically treated with high-Pi(HP,200 μM)and low-Pi(LP,10μM)conditions,then the carbohydrate and Pi accumulation were determined and analyzed.The results showed that overexpression of OsUGPase1 could increase the accumulation of sucrose in shoot and root of rice under HP condition.Inhibiting the expression of OsUGPase1 could decrease the sucrose content in shoot and root under both HP and LP conditions and promote the accumulation of starch.Notably,OsUSPasel overexpression increased the sucrose content in shoot and root under HP level,similar to that effect of Os UGPasel overexpression,however,the increased proportion was obviously lower.5.The 32P-labelled Pi uptake assay indicated that overexpression OsUGPase1 led to a significant increase in root Pi uptake rate under high Pi supply level.In comparison to WT,OsUGPase1 overexpression lines showed significant higher content of perchloric acid extracted Pi and total P in roots and shoots.Both knockout and knockdown of Os UGPase1 could reduce the absorption rate of Pi in root under LP condition.Besides,mutation of OsUGPase1 led to the increase of the content of vacuolar Pi and the descrease of the content of cytoplasmic Pi in leaf blades.Moreover,they also significantly increased the contents of Pi and total P in shoot.Besides,the overexpression of Os USPasel increased the contents of Pi and total P in shoots and roots under HP condition,while the knockdown of OsUSPase1 could result in the accumulation of Pi in leaf blades at HP level.However,the effect of knockdown OsUSPase1 on P accumulation in shoots was lower than that of knockdown Os UGPase1.6.The effect of OsUGPase1 knockout on known Pi transporters belonging to PHT1 family in rice was analyzed using RT-qPCR techinique.The results showed that when supplied with sufficient Pi,SPX1,SPX2 and SQD2 were up-regulated in both roots and shoots of osugpasel mutants.However,OsPHT1;4 and OsPHT1;8 were down-regulated in roots while OsPHT1;2 was up-regulated in shoots.In Pi-starved roots of osugpase1 mutants compared with that of WT plants,two PHT1 genes(OsPHT1;2 and OsPHT1;4)were down-regulated,while OsPHT1;8 and a subset of genes involved in Pi signaling were up-regulated.Meantime,in Pi-starved shoots of osugpase1 mutants three PHT1 genes(OsPHT1;2,OsPHT1;4 and OsPHT1;8)as well as several PSI genes were up-regulated.It is assumed that the up-regulation of PSI genes in osugpase1 mutants could be a direct consequence of decreased cyt-Pi.7.In order to clarify the ability of OsUGasel catalyzing the synthesis of UDP-Glc,the E.coli heterologous expression systems was used,and the results indicated that OsUGPase1 display UGPase enzyme activity,the same as previously reported in literature.Taken together,we characterized OsUGPase1 and OsUSPase1 are two rice uridine diphosphate-glucose pyrophosphorylase,which mediates carbohydrate synthesis and distribution.However,overexpression Os UGPasel accumulates more sucrose in shoots and roots than OsUSPase1.Both enzymes affect Pi uptake and accumulation under low phosphate regimes.Our findings provide the molecular evidence for revealing the interaction between carbon and P metabolic pathways and to breed cultivars with high PUE.