Functional Analyses Of AtMGT6 In Mg-transport In Arabidopsis

Magnesium(Mg²⁺)is abundant in plant cells and plays a critical role in many physiological processes.It is the central atom of chlorophyll and participates in many enzymatic reactions.So far,the transport mechanism of magnesium is well known in the bacteria.But it is less understood about the transportation of magnesium in plant from the molecular levels.A 10-member genes family AtMGTs?also known as AtMRS2?and single gene family AtMHX were identified in Arabidopsis,which belongs to a eukaryote subset of the CorA super-family,functioning as magnesium transporters by functional complementation in bacterial Salmonella and yeast mutant strains.Some family members of AtMGTs functional complement bacterial mutant or yeast mutant lacking Mg²⁺ transport capability,While there's less report about the physiological function of them in plant.By screening T-DNA mutants or RNAi transgenic lines of AtMGTs and AtMHX gene families,we found the AtMGT6/AtMRS2-4 RNAi transgenic plants are hypersensitive to magnesium deficiency and displayed growth retardation,suggesting a correlation between AtMGT6/AtMRS2-4 and Mg²⁺ uptake or transport in plant.Here we report the data on the AtMGT6 that displayed in Mg²⁺ uptake in Arabidopsis:By RT-PCR,we cloned AtMGT6 cDNA sequence which contains 1311 base pairs of nucleotides in the open-reading-frame?ORF?,encoding a putative protein of 437 amino acid residues.Functional complementation in bacterial Salmonella mutant strain MM281 indicated that AtMGT6 could complement MM281 strain deficient in Mg²⁺ uptake.The ⁶³Ni²⁺ tracer analysis in the bacterial model showed that AtMGT6 functions as a low-affinity Mg²⁺ transporter.Although AtMGT6 might be able to transport other divalent cations such as Zn²⁺?Cd²⁺?Cu²⁺?Fe²⁺?Mn²⁺?Co²⁺,the concentrations required for activity went beyond physiological range for those cations that are considered as trace elements for plant nutrition and exist well below the micromolar levels.These results indicate that AtMGT6 is capable of transporting Mg²⁺ under physiological conditions.No AtMGT6 T-DNA insertion line is available,so we constructed two types of RNAi transgenic plants?constitutive MGT6-RNAi?C?and inducible MGt6-RNAi?I??for AtMGT6.Both transgenic plants are hypersensitive to magnesium deficiency and displayed growth retardation compared to the wild-type,particularly,the roots of transgenic plants were significantly shorter than those of wild type plants.Furthermore,when sufficient Mg²⁺ were replenished,the Mg-deficiency phenotype of MGT6-RNAi can be restored.The Mg²⁺ content in MGTd-RNAi transgenic and wild-type plants were measured by inductively coupled plasma-mass spectrometry?ICP-MS?.Under 50?M Mg²⁺ circumstance,Mg²⁺ content in MGT6-RNAi transgenic plants were about 55%lower than those in wild-type plants.Under magnesium-replete condition?3mM?,however,there showed no obvious divergence in Mg²⁺ content between MGT6-RNAi and wild-type plants.Furthermore,the restoration of low-magnesium phenotype of MGT6-RNAi plants was accompanied by the restoration of Mg accumulation in planta.In a word,our data demonstrated that the Mg-deficiency phenotype of MGT6-RNAi transgenic plants is consistent with the alteration of Mg accumulation in planta.AtMGT6 mRNA levels increased within several hours and peaked after 12 hr with a 7.8-fold induction,these results clearly demonstrated AtMGT6 expression is upregulated by Mg deficiency in the root.Strong GUS staining was observed in the root when plants were grown under Mg deficiency.A cross section in the region with the strongest staining revealed that cortex and endoderm layers are stained.35S-driven AtMGT6-GFP fusion proteins were transiently expressed in Arabidopsis mesophyll protoplasts.The GFP signals were detected under confocal microscopy,and localized to the plasma membrane of Arabidopsis cells,indicating AtMGT6 is plasma membrane-localised.The inductive coupling plasma mass spectrum?ICP-MS?and the ⁶³Ni²⁺ isotopic labeling research indicated that AtMGT6 mediated Mg²⁺uptake of plant.The data conformed to a Michaelis-Menten function and,when expressed as a double reciprocal Lineweaver-Burk plot,enabled us to calculate apparent Km and Vmax of 326?M and 55.25 mg g-1 hr-1 by the inductive coupling plasma mass spectrum?ICP-MS?respectively,and an apparent Km and Vmax of 467?M and 74.07 mg g-1 hr-1 by ⁶³Ni²⁺isotopic labeling respectively.Those indicated that AtMGT6 could mediated low-affinity Mg²⁺ uptake of plant.Together,our results demonstrated that AtMGT6 plays an important role in the Mg uptake required for plant growth and development under Mg limitation?...