Molecular Mechanisms Of The Increased NH₄⁺ Sensitivity Of Mutant Hsn1 In Arabidopsis

A mutant was isolated from an ethyl methane sulfonate(EMS)mutagenized Arabidopsis(Col-0)mutant library.The mutant is hypersenstitive to NH₄⁺.The phenotypic and physiological characterizations of the mutant were conducted under different concentrations of NH₄⁺.Using map-based cloning method,we identified a point mutation in a gene encoding GDP-mannose pyrophosphorylase(GMP),which is involved in the AsA synthesis and N-glycoslation in Arabidopsis.The GMP enzyme activity,AsA concentration and N-glycoslation were measured in the mutant and wild type(WT)under different concentrations of NH₄⁺.The UPR and cell death in the mutant and WT were investigated.The results are summarized as follows:1.When cultured on the 1/2 MS media with NH₄⁺,the growth of the mutant, especially the elongation of roots,is increasingly inhibited by rising concentrations of NH₄⁺.The mutant could be restored when grown on the SN media without NH₄⁺. These observations suggest that the growth of the hsn1 mutant,particularly its root elongation,is more sensitive to NH₄⁺ than WT.The NH₄⁺ concentration did not differ significantly between WT and hsn1 mutant in both aerial and root tissues on either 1/2 MS or SN media,reflecting uptaking and assimilation of the NH₄⁺ in WT and the mutant are similar.The NH₄⁺ concentration in the aerial and root tissues of WT and hsn1 seedlings were all significantly higher on 1/2 MS medium compared with their corresponding values on SN medium.2.An F₂ population was developed from a cross between the mutant and Landsberg erecta(Ler).The segregation of mutated phenotypic traits among the F₂ population indicated that the mutant possesses a recessive mutation at a single locus. The mutated gene was mapped on chromosome 2 flanked by the markers H13 and H16 based on a map-based cloning strategy using 1056 F₂ mutants.Sequencing analysis revealed a point mutation in the gene(At2g39770)encoding GDP-mannose pyrophosphorylase.Allelic mutant analysis and transgenic complementation further improve it.The in planta GMP activity levels of mutants were all significantly lower than those of WT in both the aerial and root tissues,on either 1/2 MS or SN media. Either WT and mutant seedlings or the aerial and root tissues,higher NH₄⁺ content, lower GMP activity.The experiment of recombinant WT GMP and hsn1 enzymes activities under three different pH regimes supports the NH₄⁺ effect.3.The determination of the total AsA concentration and the growth of vtc4-KO mutant indicated that decreased AsA concentration is not responsible for the enhanced NH₄⁺ sensitivity of the mutant.N-glycosylation of proteins analysis revealed that N-glycosylation of proteins is impaired in the root tissues of hsn1 and vtc1,and that the impairment is enhanced when NH₄⁺ is present in the culture medium.N-glycosylation of proteins is also slightly impaired in the roots of WT in the presence of NH₄⁺,although much less severe than in hsn1 and vtc1 roots.The results indicated that the impairment of N-glycosylation of proteins is mainly for the mutant phenotype.4.Through analyse of the UPR and cell death of WT and the mutants,we found that the UPR was trigged and cell death was found when NH₄⁺ is present in the culture medium,and it was more serious in the mutant compared with WT.5.The results demonstrate that ammonium represses protein glycosylation, activates UPR,cell death,when function of GDP-mannose pyrophosphorylase was impaired,the repression effect will be enhanced.Our findings will help us to further understanding the molecular mechanism of ammonium toxicity in plants.