Structural And Functional Analysis Of The Endosomal-type NHX Antiporter In Mulberry(MaNHX6)

Worldwide,soil salinity is a major limiting factor in the productivity of agricultural crops.High soil salinity leads to osmotic stresses by inhibiting the plants normal water uptake,and high Na⁺ concentrations in the cytosol of plants are toxic to important physiological and biochemical processes.Furthermore,due to competition between Na⁺ and K⁺ for uptake mechanisms,high Na⁺concentrations decrease K⁺,an essential macronutrient and the most abundant inorganic cation in plant cells,availability.Thus,maintaining the cytosolic Na⁺ concentration at a normal level is critical to plants under salt stress.Plant cell have two strategies to maintain a high concentration of K⁺ and a low concentration of Na⁺ in the cytosol,Na⁺ efflux and vacuolar compartmentalization of Na⁺,the plants Na⁺,K⁺/H⁺ antiporter play very important role in the process.In plants,Na⁺,K⁺/H⁺ antiporter(NHX)-type exchangers play important roles in salt tolerance,as well as the ion and p H homeostases of intracellular compartments,because they are the critical regulators of vesicular trafficking and cell volume.NHX-like antiporters usually have a highly conserved N-terminus,which includes membrane-spanning pores and is responsible for cation binding and exchange,and a not conserved hydrophilic C-terminus,which regulates antiporter activity.The plant NHXs can be classified into three distinct subfamilies,the plasma membrane-localized NHXs and the intracellular-localized NHXs,which contains the vacuolar-type NHXs,and endosomal-type NHXs.Na⁺ efflux is dependent on a plasma membrane Na⁺/H⁺exchanger(NHX)and the vacuolar NHXs catalyze the compartmentalization of Na⁺ into vacuoles.The endosomal-type NHXs play important roles in protein trafficking,and K⁺ and vesicle p H homeostases,depending on their cellular localization to the Golgi bodies,trans-Golgi network(TGN),and prevacuolar compartment(PVC).The regarding the structure governing the ion-transport mechanism and the role of the C-terminal tail in plant plasma membrane and vacuolar-type NHXs are clear,but the endosomal-type NHXs remain unclear.Mulberry is an economically important perennial woody plan.Mulberry leaves are the exclusive source of food for the silkworm,Bombyx mori,and its fruits are very popular and nutritious.In addition,mulberry has better adaptation to many different environments stresses,including saline environments.Thus mulberry can be used as a potential ecologically plant in saline-alkali soil,to date,little is known regarding the molecular mechanisms.As the important salt stress-responsive genes,the functions of NHX in mulberry remain unclear.Therefore,it is significant the studying of mulberry NHXs for exploring the molecular mechanisms of mulberry under salt stress,and also help to improve our understanding of the roles played by the C-terminal tail in the endosomal-type NHX and the ion transport mechanism of endosomal-type NHX.In this study,we identified the mulberry NHX gene family by bioinformatics method and gene cloning.The tissue-specific pattern of mulberry NHXs in the two mulberry species by transcriptome data and quantitative real-time PCR.The Ma NHX genes' expression under high salinity,drought and signal molecules treatment were analyzed by quantitative real-time PCR.The function of the mulberry NHX gene family was investigated by utilizing heterologous expression in yeast.MnNHX6 was selected as the focus for further study.MnNHX6 was transformed into Arabidopsis and the tolerance of the overexpression transgenic plants to salt stress was analyzed.The ion transport mechanism study of the MnNHX6,was investigated by homology modeling and mutagenesis in yeast.In addition,mutagenesis and nuclear magnetic resonance(NMR)structural analysis to investigate the function and structure of MnNHX6's C-terminal tail.Yeast two-hybrid(Y2H)were used to identify the potential regulatory protein interactions with C-terminal of MnNHX6.The main results are as follows:1.Molecular characterization and expression analysis of the mulberry Na⁺/H⁺ exchanger gene familyIn this study,we identified and cloned seven NHX genes from mulberry using bioinformatics methods.Multiple sequence alignment analysis and the topological analysis predicted that Ma NHXs have a highly conserved N-terminus,which contain 12 transmembrane(TM)domains,and a hydrophilic C-terminus.The genomic organization,multiple sequence alignment analysis and the topological analysis of Ma NHX1-7 showed that the proteins form three distinct subgroups: Ma NHX1-5 belong to the vacuolar-type NHXs and Ma NHX6 and 7 belong to endosomal-type and plasma membrane NHXs,respectively.Compared to other species NHXs,the endosomal-type and plasma membrane NHXs of mulberry have the only one,and not the plasma membrane NHXs like At NHX8,which is Li+transporter.The phylogenetic analysis showed that,except for Ma NHX5 are clustered firstly with At NHX4,all of the Ma NHX genes are clustered firstly with those of the P.euphratica NHX-type exchangers.The results suggest that mulberry is more closely related to poplar than to other species.Expression analysis of mulberry NHX genes of M.atropurpurea Roxb.cv.Yuesang and Morus alba cv.Cesha were detected in this study.The results showed that Ma NHX1-7 expressed ubiquitously but differentially expressed in various tissues.The expression patterns of Ma NHX1,2and 3 may be different in the two mulberry species,because of the different growing environments.M.atropurpurea Roxb.cv.Yuesang is a native to southern China's Guangdong Province.However,Morus alba cv.Cesha grows in northwest China's Xinjiang Province,which is more drought prone and has higher annual temperature differences compare with Guangdong Province.It suggests that the Ma NHX1,2 and 3 may play different roles in the abiotic stress response and normal physiological functions.The expression patterns of mulberry NHX genes responses to high salinity,drought and signaling molecules were detected in this study.The results showed that the expression levels of mulberry NHX genes were not only affected by high salinity,drought and ABA treatment,like other species NHXs,but also by responses to SA,Me JA and H₂O₂ in some tissues.Our results showed that SA,JA and ROS(Reactive oxygen species),not just by ABA,signaling pathways might be involved in the regulation of Ma NHX genes.It suggested that at least four different Ma NHX signaling pathways exist in mulberry responding to ionic stress or osmotic stress in different tissues.The other signaling pathways were first discovered,except for ABA-dependent signaling pathway of osmotic stress responding and ABA-independent signaling pathway of ionic stress responding.2.Functional analysis of the endosomal-type NHX antiporter in mulberry(MnNHX6)Drop tests were used to compare the halotolerance levels of the MnNHX family members(MnNHX1-6)in the salt sensitive yeast.The results showed that MnNHX2 and 3 play a major role in the function of the vacuolar-type NHXs.Compared to the vacuolar-type NHXs,the endosomal-type MnNHX6 greatly enhanced the tolerance to salt-stress,and to hygromycin B in particular.MnNHX6's molecular mechanism of saline tolerant was investigated,overexpression of MnNHX6 in Arabidopsis can reduce the salt damage of seedling growth and development of roots.Further analysis indicated overexpression of MnNHX6 improves proline and chlorophyll content of transgenic plant but reduces MDA content under salt stress.Above analysis indicates that the overexpression of MnNHX6 significantly enhanced salt-stress tolerance in Arabidopsis.Subcellular localization of MnNHX6 in yeast was analyzed by FM4-64 staining and subcellular co-localization with Sc NHX1.The results showed that the plant endosomal-type NHXs was located in membranes of the PVC,which was the same localization as yeast Sc NHX1.In contrast,the vacuolar-type NHXs was predominantly localized in the vacuole and additional punctate bodies in the yeast.In addition,compared to the vacuolar-type NHXs,the endosomal-type MnNHX6 greatly enhanced the tolerance to hygromycin B in the salt sensitive yeast,it suggested that MnNHX6 may regulates the vacuole p H homeostases and protein sorting,like Sc NHX1.Above analysis indicates that the plant endosomal-type NHX serves a similar function with yeast Sc NHX1.To investigate MnNHX6's ion transport mechanism,MnNHX6's three-dimensional structure was established by homology modeling and transmembrane region analysis.Structure analysis showed that MnNHX6 contains unusual 13 transmembrane helices,unlike the 12 transmembrane helices of other NHXs,but the structural core formed by TM5-TM12 assembly is conserved along with the TM4-11 assembly of other NHXs.In addition,the MnNHX6 model suggested a charge-compensated pattern similar(but not identical)to that of NHE1,with Asp176(TM5),Glu332(TM10),Arg367(TM11),and Arg402(TM12)corresponding to Asp238(TM4),Glu391(TM9),Arg425(TM10),and Arg458(TM11)in NHE1,respectively.A mutagenesis analysis showed that these four conserved charge residues are essential for the function of MnNHX6.The subcellular localization experiments showed that Arg402 is important for protein stability of MnNHX6.Further analysis indicated the MnNHX6 might share similar catalytic mechanisms with their human counterparts in NHE1.MnNHX6 model shares a typical “funnel” fold as a structural basis for the alternating-access mechanism.In our MnNHX6 model,the similar two discontinuous funnels made of TM3,5,6,and 10 at the cytoplasmic side and TM3,9,and 12 at the luminal side contain Pro107(TM3),Pro108(TM3),Glu200(TM6),Asp205(TM6),and Ser292(TM9),which correspond to Pro167(TM2),Pro168(TM2),Glu262(TM5),Asp267(TM5),and Ser351(TM8)in NHE1,respectively.The mutagenesis analysis showed that these conserved MnNHX6 residues are essential for the function.In addition,the highly conserved Ser292 of MnNHX6 may participate in cation binding,mutagenesis analysis showed the negative charge at this conserved position enhanced Na⁺/H⁺ transport activity in the vacuolar-type NHX but abolished the function of the endosomal-type NHX.It suggested that the ion-transport mechanism of plant endosomal-type NHXs might be different to the vacuolar-type NHXs.Based on above analysis,we investigated the ion-transport mechanism of plant endosomal-type NHXs.MnNHX6's three-dimensional structure,established by homology modeling and transmembrane region analysis,was supported by mutagenesis analysis.MnNHX6 contains unusual 13 transmembrane helices,but the structural core formed by TM assembly is conserved,and MnNHX6 contains a conserved cation binding mechanism and a similar charge-compensated pattern as NHE1.3.Structural and functional analysis of the C-terminal tail of mulberry endosomal-type NHX antiporter(MnNHX6)The sensitivity to low p H levels increased in nhx1 mutant yeast as a result of vacuolar hyperacidification and defective trafficking to vacuoles.Overexpression of MnNHX6 in yeast conferred tolerance to the growth sensitivity to low p H.The p H measurement of yeast vacuoles was analyzed by BCECF-AM,the results showed that overexpression of MnNHX6 in yeast reduced vacuolar hyperacidification.Ste3,a vacuolar protein marker of endosome to vacuole trafficking was chosen to ascertain whether MnNHX6 suppressed the endosome to vacuole trafficking defects in the nhx1 mutant,the results showed that overexpression of MnNHX6 were complement the endosome to vacuole trafficking defects in yeast.But MnNHX6 with the completely truncated cytoplasmic C-terminus lost these function,it indicated that MnNHX6 cytoplasmic C terminus may have an important role to play in this process.To investigate the function of MnNHX6's C terminus,the functional and localization analyses of C-terminal truncations in MnNHX6 showed that the C-terminal conserved region was responsible for the function and stability of the protein.To determine whether this conserved region was important for MnNHX6 function,24 of the most highly conserved amino acids in this region were mutated to alanine.The functional and localization analyses of these mutations showed that the all of the strongly hydrophobic amino acids in the C-terminal conserved region were essential for the correct functioning of MnNHX6.The distribution of the functionally essential amino acids in the C-terminal conserved region showed distinctly regional changes,particularly at the end of the C-terminal conserved region,the results suggested that these sites could form a structurally important domain in the molecule.The NMR structural analysis provided direct evidence that the functional segments formed a stable 310 ?-helix by the residues Asp521,Arg522,and Asn523.In addition,the results showed that protein stability of MnNHX will be damaged,if the Asp521 are mutated to hydrophobic amino acids.To ascertain the minimum MnNHX6 sequence motif required for the interaction with MnSNX1,yeast two-hybrid assays between MnSNX1 and MnNHX6 C-terminal domain were performed.The results showed that the C-terminal conserved region of MnNHX6 is necessary for the interaction with MnSNX1,but not the only region.In addition,the results showed that the interaction with MnNHX6 did not affect the membrane localization of MnSNX1.The results of the yeast three-hybrid showed that MnNHX6 did not interact with the SNX1-SNX2 heterodimer,which means that the SNX2 and endosomal-type NHX may competitively bind with SNX1.Based on above analysis,we disapprove the previous view that NHX C-terminal is the random coil in the cytoplasm,not stable protein structures,which regulates antiporter activity only through the interaction with regulatory protein.In this study,our results showed that the C-terminal conserved region of the C-terminal tail in the endosomal-type NHX appears to be a functional domain that is responsible for the function and stability of the protein itself and is necessary for protein interactions,hydrophobicity and the formation of the 310 ?-helix of this functional domain play an important role to the function of endosomal-type NHXs.