Mg²⁺Transportation Features And Functions Of OsHKT2;4

Land salinization is the main factor leading to rice drop-production in China.The high affinity K+ transporter HKT plays an important role in rice salt tolerance.HKT proteins are highly conserved with the K+ transporters of bacteria(KtrB,TrkH)and fungi(Trk),which together constitute a large family of Ktr/TRK/HKT.The view that HKT family members are sodium-selective uniporters or sodium-potassium symporters is widely held.By analyzing the topological structure of HKT protein,HKT contains eight transmembrane regions and four selective filtration pores,Wherein the first filter pore is a domain that determines its ion transport properties.According to the different amino acid sequence of HKT,the HKT is divided into two subfamilies.Subfamily 1 is a kind of Na+ selective transporter and subfamily 2 is a kind of Na+/K+ synergetic transporter.Rice contains nine genes encoding HKT protein,including subfamily 1 and subfamily 2.Among them,OsHKT2;4 is a member of the subfamily 2,found to transport K+,Na+,as well as Mg2+ and Ca2+ and other divalent cations by heterologous expression in yeast and Xenopuslaevis oocytes.However,physiological functions relating with these cations in planta remain poorly understood and the permeability of Mg2+ and Ca2+ in heterologous Xenopuslaevis oocyte expression system was argued.Magnesium (Mg2+) is the most abundant divalent cation in plant cells that is required for important functions involved in photosynthesis and various metabolic pathways,which is crucial to the growth and reproduction of plants.The absorption and transport of Mg2+ by plants is mediated by the corresponding transporter.The AtMGTs gene family in model plant Arobidopsis thaliana encoding 10 magnesium transport proteins had been identified.Among them,MGT6 localized in the plasma membrane is a high affinity Mg2+ transporter,which is the key component to absorb Mg2+ from the soil,so that the plant adapts to the low Mg2+ environment.In order to further explore the Mg2+ transport properties of OsHKT2;4 and its possible physiological functions in plants,in this paper,OsHKT2;4 was heterologous expressed Mg2+absorption defects Salmonella mutant MM281,Xenopuslaevis oocytes,and Arabidopsis thaliana,by analytical chemistry,bipolar voltage clamps and plant physiology methods,obtaining the following main findings:1.The heterologous single-cell expression system demonstrates that OsHKT2;4 has the characteristics of low affinity Mg2+ transporter.Through the functional complement experiment Salmonella mutant MM281,we found that,OsHKT2;4 can replenish Mg2+ absorption defects function of MM281,but in 10 ?M low Mg2+ conditions,no such function.Electrophysiological experiments of Xenopuslaevis oocytes have shown that it can exist Mg2+ current in the oocyte mM-Mg2+ conditions.These results indicated that OsHKT2;4 can transport Mg2+ under high magnesium conditions and has the characteristics of low affinity Mg2+ transporter.2.Transgenic plants with OsHKT2;4 in Arabidopsis mutant atmgt6 showes a stress growth phenotype under high Mg2+.Overexpressing the promoter and coding sequence of OsHKT2;4 to Arabidopsis mutant atmgt6,there were no significant phenotypic differences in overexpressions and mutant under low Mg2+ conditions and the shoot of overexpressions showed growth stress under high Mg2+.The results indicated that the shoot of OsHKT2;4 overexpression was sensitive to high Mg2+.3.OsHKT2;4 mediates the distribution of Mg2+ in plants.The contents of Mg2+ in root and shoot under different Mg2+ concentrations were determined by analytical chemistry.We found that the Mg2+ content in the mutant root was higher than that in OsHKT2;4 overexpression,and the Mg2+ content in the shoot of the overexpression was higher than that in the mutant.The Mg2+content of OsHKT2;4 overexpression under high Mg2+ was higher than that in the control by analyzing the distribution ratio of Mg2+ in the shoots and roots,indicating that OsHKT2;4 mediates distribution of Mg2+ in plants under the high Mg2+ condition.4.OsHKT2;4 regulates Mg2+/Ca2+ homeostasis of plant shoot.Adding Ca2+ could alleviate the stress growth phenotype of shoot under high Mg2+ of OsHKT2;4 overexpression indicating that Ca2+ could alleviate the high Mg2+ toxicity and maintain the normal Mg2+/Ca2+ homeostasis.This suggested that OsHKT2;4 overexpression changes the Mg2+/Ca2+ homeostasis.Together,our results demonstrate that OsHKT2;4 has the characteristics of low affinity Mg2+ transporter responsible for Mg2+ transport to aerials and regulates Mg2+/Ca2+ homeostasis in plants under high Mg2+ conditions.This also verifies the previous studies that OsHKT2;4 is a nonselective cation channel.These results provide powerful theoretical basis and reference value for the molecular mechanism of OsHKT2 in Mg2+ uptake and the function of OsHKT2 in rice.