Functional Studies Of Rice Sulfate Transporters OsSULTR1s In Sulfate Uptake And Sulfur Signaling

Rice is the second largest food crop in the world,which not only provides abundant energy for human being,but is also an important source of mineral nutritions.Sulfur（S）is one of the essential macronutrients for organisms and plays an important role in plant growth and development,crop yield and quality,and stress tolerance.Sulfate（SO42-）is the main form of inorganic sulfur absorbed by plant roots,and is reduced to sulfur-containing amino acids such as cysteine（Cys）through a series of assimilation pathway for plant utilization.Sulfate transporters（SULTR）,as the main carriers of sulfate uptake and transportation,are not only responsible for the supply and redistribution of sulfur in plants,but also participate in the response and transmission of sulfur signals,and thus are essential for maintaining sulfur homeostasis in plants.However,the functions of SULTRs and the molecular mechanisms underlying sulfate uptake and transport in rice are still not fully understood.In this study,we focused on exploring the functions of the members of rice sulfate transporter family I（OsSULTR1s）in sulfate uptake and transport,response to sulfur deficiency signals,and rhizosphere microbiome composition.The specific research contents and results are as follows:1.Heterologous expression in yeast showed that only OsSULTR1;1,OsSULTR1;2,OsSULTR1;3,OsSULTR3;6 and OsSULTR4;1 had sulfate transport activity.By quantitative RT-PCR analysis,it was found that OsSULTR1;1 and 1;2 were mainly expressed in roots,and the other OsSULTRs were mainly expressed in shoots or in both shoots and roots.The expression of OsSULTR1;1,2;1,2;2 and 4;1 were induced by sulfur deficiency in shoots and OsSULTR1;1,1;2 and 4;1 were induced in roots,while the expressions of other OsSULTRs were not altered significantly under sulfur deficiency.2.In order to further explore the function of the OsSULTR1 s subfamily,we used the promoter-driven GUS expression lines to investigate the tissue expression pattern of OsSULTR1 s.GUS staining results showed that all three members of OsSULTR1 s were expressed in shoots,roots and embryos.The expression level of OsSULTR1;3 in leaf sheaths was significantly higher than that of OsSULTR1;1 and OsSULTR1;2.The results of transverse section of the root elongation zone showed that OsSULTR1;1 was mainly expressed in epidermal cells,OsSULTR1;2 was expressed in the pericycle cells,and OsSULTR1;3 was expressed in both epidermal cells and pericycle cells.Transient expression in tobacco and rice protoplasts revealed that OsSULTR1;1,OsSULTR1;2 and OsSULTR1;3 were all localized to plasma membranes.3.By creating ossultr1;1,ossultr1;2,ossultr1;3 single knockout mutants,ossultr1;1&1.2（dko）double knockout mutant and ossultr1;1&1.2&1;3（tko）triple knockout mutant,it was found that the biomass of the mutant was significantly smaller than that of the wild type under the low sulfur treatment（4.5 μM sulfate）.The photosynthetic efficiency of all mutants except ossultr1;3 was decreased compared to the wild-type.The sulfate and total sulfur concentrations in wild type and ossultr1 s mutants were determined under three different sulfate treatment conditions.The sulfate concentrations in ossultr1;1,ossultr1;3,and dko were decreased by approcimately 50%,and total sulfur content decreased by about 30% under 4.5 μM sulfate treatment.In tko,sulfate concentrations decreased by about 70%,and total sulfur concentrations decreased by about 56%,whereas knockout of OsSULTR1;2 had no effect on sulfate and total sulfur content,indicating that the sulfate uptake by rice roots was mainly controlled by OsSULTR1;1 and OsSULTR1;3 while OsSULTR1;2 had snall contribution.The concentrations of different tissue elements in the wild-type and ossultr1 s mutants at mature stage was measured,and it was found that the knockout of OsSULTR1;1 significantly inhibited the sulfate in the flag leaf,as well as the total sulfur concentrations in the flag sheaths and nodes.Agronomic trait investigations showed that the seed setting rates of ossultr1;3 and tko were reduced by half compared with the wild type,while the other mutants were not significantly different from the wild type.The total sulfur concentrations in grains of all ossultr1 s mutants were not significantly different from that of the wild type,indicating that rice may preferentially allocate sulfur to filling grains under the condition of insufficient sulfur supply.In addition,the accumulation of molybdenum（Mo）and selenium（Se）in shoots and roots of ossultr1;1,ossultr1;3,dko,and tko under low-sulfur conditions were significantly lower than those in wild type,indicating that OsSULTR1;1 and OsSULTR1;3 may also be involved in molybdate and selenate uptake in roots.4.By complementation of yeast sul1 sul2 double mutant strains,it was found that rice OsSULTR1;1,OsSULTR1;2 and OsSULTR1;3 all possessed sulfate transport activity.Site-directed mutation of conserved amino acid sites OsSULTR1;1E381,OsSULTR1;2E371 and OsSULTR1;2E375 to glutamine severely inhibited the sulfate transport activity,but did not affect the sensing and transmission of sulfur signals,indicating that rice OsSULTR1;1,OsSULTR1;2 and OsSULTR1;3 also have both sulfate transport and signal sensing and transmission activities.We further used the yeast two-hybrid assay to detect whether the OsSULTR1 s protein interacted with rice O-acetylserine（thiol）lyases（OASTLs）.By testing the ineteration of OsSULTR1 s with 10 OsOASTLs,we found that the STAS domain of OsSULTR1;1（OsSULTR1;1-LS）was able to interact with OASTL2-114 A and OASTL8-201 A.The STAS domain of OsSULTR1;3（OsSULTR1;3-LS）interacted with OASTL2-114 A,OASTL4-Y130,OASTL7-F133 and OASTL8-201 A.However,the STAS domain of OsSULTR1;2 did not interact with any OsOASTL.These results suggest that the involvement of OsSULTR1;1 and OsSULTR1;3 in sulfur signal sensing and transmission may require the interaction with OsOASTL.5.Through the rice root splitting experiment combined with transcriptome analysis,we investigated whether OsSULTR1 s was involved in the response to sulfur deficiency and the response to sulfate resupply after sulfur deficiency.A series of differentially expressed genes（DEGs）involved in the response to sulfur deficiency were identified in both roots and shoots of rice seedlings under sulfur deficiency for 15 days.The number of DEGs in the root was 4.2 times higher than that of the shoot,indicating that the response to sulfur deficiency in the root was much stronger than that in the shoot.Most of these DEGs were enriched in pathways such as amino acid metabolism.After 12 h of sulfate resupply,only the expressions of a few DEGs were recovered in the shoots and roots after sulfate resupply for 12 h,and most of the DEGs remained unchanged.By comparing root samples under different sulfur treatments,the local response,systemic response and both local and systemic response genes to sulfate resupply were identified.The systemic response genes were mainly related to calcium transport and signalling.Genes involved in both local and systemic sulfur signaling include OsSULTR1;1,APR,MYB,EIF,etc.In addition,genes responding to heterogeneous and homogeneous sulfate resupply in shoots were identified,including APR and OASTL genes.Through q RT-PCR analysis of the expression levels of genes related to sulfur deficiency and sulfate resupply in ossultr1 s mutants,it was found that the knockout of OsSULTR1 s affected the expressions of sulfur deficiency responsive genes and sulfate resupply-related genes,indicating that OsSULTR1 s may be involved in the local and systemic response to sulfur deficiency and sulfate resupply.6.Through 16 s r DNA sequencing of rhizosphere microorganisms in the wild-type and five ossultr1 s mutant grown under flooded and unflooded conditions,it was found that the core microbiome in rhizosphere were Chloroflexi,Proteobacteria and Acidobacteria.Taking the non-rhizosphere soils as controls,it was found that the difference in microbial community composition between the non-rhizosphere soil and the rhizosphere soil was greater than the differences of rhizosphere soils between the wild type and various mutants.The abundance of Proteobacteria and Actinobacteria in rhizosphere soil was significantly higher than that in non-rhizosphere soil under flooded or drained conditions.There were also differences in the rhizosphere microbiota between different mutants and wild type.Under unflooded conditions,the genus Megasphaera was the most abundance rhizosphere microorganisms differentially among ossultr1;3,dko,tko and the wild-type.The Treponema₂ was the most abundant genus of ossultr1;1,ossultr1;2,ossultr1;3 and tko compared with the wild type.In addition,the relative abundance of sulfate-reducing bacteria（SRB）in the rhizosphere of tko under unflooded condition was higher than that in wild type,indicating that knockout of OsSULTR1 s reduced plant sulfate uptake and increased sulfate content in rhizosphere pore water,thereby causing the increased abundance of SRB in rhizosphere soils of tko.In summary,in this study,we investigated the functions of the rice sulfate transporter OsSULTR1 s,and found that OsSULTR1;1 and OsSULTR1;3 is mainly responsible for the rice root sulfate uptake.OsSULTR1 s were found to have both sulfate transport activity and sulfur signaling in the yeast system.A series of genes involved in local and systemic sulfur response were identified through transcriptome analysis,and knockdown of OsSULTR1 s affected the expression of some genes involved in sulfur response.By analyzing the rhizosphere microbiome of wild-type and ossultr1 s mutants,the effect of knocking out OsSULTR1 s on rice rhizosphere microbiome was initially explored.