Expression Patterns And Functional Characterization Of A Magnesium Transporter Gene OsMHX1 In Rice

In the cytosol of higher plant cells,magnesium(Mg)is the most abundant divalent cation and plays a critical role in activating and mediating a number of physiological metabolic processes,such as being a component of the chlorophyll molecule,regulating the key enzymes involved in the fixation of carbon in chloroplasts,maintaining the stability of the genomic structure,being a cofactor for many enzymatic processes associated with RNA polymerases,phosphorylation,dephosphorylation,protein kinases,photosynthesis and the hydrolysis of various compounds.Furthermore,Mg also plays an important role in alleviating aluminum(Al)toxicity.However,the availability of Mg in some highly weathering acidic soils is low,affecting productivity and quality of plants.In this study,we cloned the gene of OsMHX1 by RT-PCR from the genome of rice.Bioinformatics analysis using public gene-chip data show that this gene is constitutively expressed and the highest expression in leaves and anther of rice.We fused the putative OsMHX1 promoter with GUS(?-glucuronidase)reporter for analyzing the tissue-specific expression of OsMHX1 in rice.GUS staining was evident in leaf blade,culm and sheath,root-shoot junction,root tip,root hair zone and lateral root.The GUS staining signals indicated that OsMHX1 expression was enhanced by Mg starvation.We examined the functional complementation of OsMHX1 in a Mg uptake-deficient yeast strain CM66.We observed that the strains expressing empty vector could not grow on the solid medium containing less than 4mM Mg2+,however the strains expressing OsMHX1 could grow on the medium containing 0.1 mM Mg2+.The growth curve of the strains in liquid medium was also identical with the complementation results observed on the agar plates.The results confirmed that OsMHX1 was capable of mediating Mg2+-uptake in yeast.To address the physiological function of OsMHX1 in rice plants,we generated the transgenic lines by over-expression of OsMHX1 and requested T-DNA insertional line for knockout of OsMHX1 in the genetic background of cv.Dongjin and OsMHX1-RNAi lines in the genetic background of cv.Nipponbare.We examined the growth of wild type(WT),two over-expression lines(OE1 and OE2),three OsMHX1-RNAi lines(R1,R2 and R3)and its knowckout(KO)line under different Mg supply conditions for 21 days.The results indicated that knockout of OsMHX1 decreased Mg concentration in the aerial parts and showed necrotic lesions.The Mg deficient symptoms of yellowing was much more severe in the older leaves than the new leaves under low Mg supply condition.The symptoms were relatively mild in both WT and OsMHX1-OE lines.There were no Mg deficient symptoms in all the WT,over-expression,OsMHX1-RNAi lines and KO line at high Mg2+-supply levels.The results demonstrated that OsMHX1 played the important role in Mg nutrition in rice.To further detect the function of OsMHX1 in the resistance to Al stress in rice,the WT and the transgenic lines were grown in IRRI nutrient solutions with four different combination of Mg and Al levels for 21 days.The treatments were 0 mM Mg2++ 0.05 mM Al3+,0.05 mM Mg2++ 0.05 mM Al3+,1 mM Mg2++ 0.05 mM Al3+ and 1 mM Mg2+-Al3+.In comparison to both WT and over-expression lines,OsMHX1 knockout line showed necrotic lesions in the leaves,inhibited both root elongation and shoot growth by Al presence in the low Mg solutions.The KO line contained significant lower Mg and higher Al than WT and over-expression lines.Over-expression of OsMHX1 did not have the significant effect on the growth and the concentrations of Mg and Al at Al stress conditions irrespective Mg supply levels.The results indicated that OsMHX1 is involved in Mg-mediated resistance to Al toxicity in rice.In summary,this study to our knowledge was the first time to validate the function of OsMHX1 in Mg nutrition of rice.The results suggested that OsMHX1 was capable of mediating Mg2+ uptake and distribution,and was benefical for enhancing the resistance to Al stress in rice.