Physiological And Molecular Mechanisms Of Vacuolar Phosphate Transporters OsSPX-MFS1～3 Regulating Phosphorus Homeostasis In Rice

Phosphorus is an essential nutrient for plant development and reproduction.Most of the phosphorus in the soil is fixed and can not be directly used by plants.Phosphorus deficiency has become an important factor limiting crop yields.Elucidating the molecular mechanism mediating plant phosphorus homeostasis is the basis of molecular design breeding for high phosphorus use efficiency in crops.Vacuoles store most of the phosphorus in plant cells and play an important role in balancing intracellular phosphorus homeostasis.Previous studies in the lab have shown that the three members of the OsSPX-MFS protein family,OsSPX-MFS1,OsSPX-MFS2,and OsSPX-MFS3 are rice vacuolar phosphate（Pi）transporters,but their direction of transporting Pi through the vacuolar membrane,the relative contribution of those transporters and their regulatory mechanisms are still unclear.The corresponding genetic materials of OsSPX-MFS1～3 were generated and characterized.Through various physiological and molecular experiments,the functions of OsSPX-MFS1～3 were clarified.The yeast two-hybrid system was used to screen the interaction proteins of OsSPX-MFS3,the vacuolar Pi transporter has the greatest contribution among the three OsSPX-MFSs.The corresponding genetic materials of the interaction gene OsSYP21/22 were generated and characterized.The detailed results are as follows:1.OsSPX-MFS1/2/3 all have the function of vacuolar phosphate influx transporters.Among them,OsSPX-MFS3 has the greatest contribution.Seven mutants of the OsSPX-MFS1～3 gene were obtained through CRISPR/Cas9 genome editing system,including single mutants osspx-mfs1,2,3,double mutants osspx-mfs1/2,1/3,2/3,and triple mutant osspx-mfs1/2/3.The measurement results of Pi concentration in roots,mature leaves and root vacuoles showed that OsSPX-MFS1/2/3 have the function of vacuolar Pi influx transporters,and the contribution of OsSPX-MFS3 is the largest and that of OsSPX-MFS2 is the smallest.The results of hydroponic physiological experiments showed that the loss function of OsSPX-MFS1/2/3 will change the distribution of Pi in plant cells,causing more Pi to accumulate in the cytoplasm,and then be transported to the apical tissues of the plant through xylem transport.The leaf tips and spikelets of osspx-mfs1/3 and osspx-mfs1/2/3 had necrotic symptoms caused by Pi over-accumulation.The mutants were sensitive to high Pi supply and environmental Pi fluctuations.Under low Pi supply conditions,the growth retardation and Pi over-accumulation caused necrotic symptoms of osspx-mfs1/3 and osspx-mfs1/2/3 were alleviated.In the low Pi field,the yield of osspx-mfs1 and osspx-mfs2 was significantly higher than that of the wild type,which improved the phosphorus-use efficiency.2.Molecular regulation of the vacuolar Pi transporters OsSPX-MFSsThe protein subcellular localization assay using rice protoplasts showed that under Pi sufficient,OsSPX-MFS1/2/3 were mainly localized on the vacuolar membrane,while under Pi deficient conditions,they were mainly localized in the endoplasmic reticulum（ER）.Thus,it is likely that the vacuolar phosphate influx activities of OsSPX-MFS1/2/3 are weakened under Pi deficient condition through the shift of their subcellular localization from vacuolar membrane to ER.In order to investigate the molecular mechanism of post-translational regulation of OsSPX-MFS protein,the potential interacting proteins of OsSPX-MFS3 were screened by using a split-ubiquitin membrane-based yeast two-hybrid system.Seventeen potential OsSPX-MFS3 interacting proteins were obtained,including a class of syntaxin proteins/SYP subfamily proteins,OsSYP22 and OsSYP131.Through yeast two-hybrid verification,it is found that OsSYP21/22 can interact with OsSPX-MFS1/2/3.In addition,these proteins are co-localization in membrane structures in the cytoplasm of tobacco epidermal cells.OsSYP22 is a member of the SYP2 subfamily proteins.It contains three domains:N-terminal SNARE（Syn N）,target membrane-associated SNARE（t-SNARE）,and transmembrane domain（TM）.Yeast two-hybrid showed that the three OsSPX-MFSs interact with OsSYP22 at its Syn N domain.In order to understand the function of OsSYP21/22,single and double mutants of OsSYP21/22 mediated by CRISPR/Cas9,overexpression,endogenous expression and dominant negative mutants materials of OsSYP21/22 were generated.The CRISPR/Cas9 genome editing could not produce homozygous frameshift deletion mutants of ossyp22,indicating that homozygous deletion of ossyp22 is lethal.Subcellular localization using rice protoplasts showed that OsSYP21/22 was localized in the inner membrane structure and pre-vacuolar compartments（PVC）,and OsSPX-MFS3 was mainly localized on the vacuolar membrane.When in the protoplasts of ossyp22#1 mutant,one allele of OsSYP22 are frame-shifted and the other lack of 9 amino acids,the proportion of vacuole membrane-localized OsSPX-MFS3 was significantly reduced.However,no significant changes in the localization of OsSPX-MFS3 were observed after deletion of OsSYP21.The bimolecular fluorescence complementation（Bi FC）experiment found that under the Pi sufficient condition,the OsSYP22 protein lacking the TM region(OsSYP22^△TM)can interact with OsSPX-MFS3,while under the Pi deficient condition,OsSYP22^△TM no longer interacted with OsSPX-MFS3,but it can still interact with OsSPX-MFS3^△SPX（the OsSPX-MFS3 protein lack of the SPX domain）.These results indicated that the SPX domain of OsSPX-MFS3 perceives the cellular Pi concentration and regulates the interaction between OsSPX-MFS3 and OsSYP22.In conclusion,OsSYP22 assists the localization of OsSPX-MFS3 to the vacuolar membrane through protein interaction.