Function Research About Zinc Or Iron Transporter Protein In Arabidopsis Thaliana And Leymus Chinensis

Zinc?Zn? and iron?Fe? are essential trace elements to maintain plant growth and development.When Zn or Fe is deficient or excessive,it will affect plant growth and even cause plant death.The zinc and iron homeostasis is critical to normal growth of plants.At present,some gene families have been found in plants to participate in Zn transport,Fe transport or Zn/Fe transport,which take part in maintaining Zn homeostasis and Fe homeostasis.In this thesis,we worked on the physiological and molecular functions of the Arabidopsis thaliana Zn transporter At ZNE1?ZINC NUTRITION ESSENTIAL 1?which previously discovered by our team.At ZNE1homologous protein is widely present in important crops and its structure is conservative.Therefore,the research of At ZNE1 provides a theoretical basis for molecular breeding to increase the zinc content of crops.Leymus chinensis,as a high-quality forage grass,is a constructive species in the grasslands of Inner Mongolia.The research on the regulation mechanism of its zinc and iron mineral nutrition is still unclear.Through transcriptome analysis of Leymus chinensis,mining the gene encoding the zinc or iron transport protein in Leymus chinensis.The study of zinc or iron transport in Leymus chinensis will provide a theoretical basis for bio-fortification of Leymus chinensis on mineral nutrition.The results are as follows:?1?At ZNE1 participates in the zinc and iron dependent growth of Arabidopsis,but does not participate in the accumulation of zinc and iron in Arabidopsis.Under the conditions of high zinc concentration of 75?M or low iron concentration of 0?M,the growth of Arabidopsis seedling At ZNE1 knockout mutant atzne1 was inhibited compared with wild-type Col-0,and the leaves turned yellow and died.The genetically complementary material atzne1-1/At ZNE1-GFP restores wild-type phenotypes in high-zinc or low-iron media.The leaves of adult Arabidopsis mutant atzne1 also turned yellow and even died under hydroponic conditions with high zinc or low iron.The determination of wild-type Col-0 and mutant atzne1showed no significant difference in the zinc and iron content of rosette leaves and roots under high-zinc or low-iron hydroponic conditions.The Col-0 and atzne1 were cultured in total nutrient hydroponic culture solution until the seeds were mature,and the zinc and iron content of the seeds was measured,and there was no significant difference between the wild-type Col-0 and the mutant atzne1.?2?At ZNE1 has Zn²⁺ transport function.At ZNE1 is not a member of the Arabidopsis ZIP family and has two RNA splicing forms At ZNE1.1 and At ZNE1.2.The At ZNE1.2 transcript contains one more exon at its 5?end and the encoded protein contains 24 more amino acids at its N-terminus than At ZNE1.1.Since only At ZNE1.1was successively amplified from c DNA transcribed from total RNA,we focused on this RNA splicing form in the current study.Complementary yeast zinc or iron transport-deficient mutant found that At ZNE1 could restore the growth phenotype of the hyper-zinc-sensitive yeast mutant?zrc1/?cot1?vacuolar zinc transporter deletion mutant?in excess-Zn²⁺medium,but could not restore the growth phenotypes of the low-zinc-sensitive yeast mutant?zrt1/?zrt2?cell membrane zinc transporter deletion mutant?in Zn²⁺-deficient medium and low-iron sensitive yeast mutant?fet3/?fet4?cell membrane iron transporter deletion mutant?in Fe²⁺-deficient medium.It shows that At ZNE1 can mediate the transport of Zn²⁺from low concentration to high concentration,but can not mediate the transport of Zn²⁺or Fe²⁺from high concentration to low concentration.?3?The non-transmembrane domains facing the cytoplasmic side Cyto?and non-cytoplasmic side Non-Cyto?and Non-Cyto?of At ZNE1 can bind to Zn²⁺.The histidine in Non-Cyto?is the Zn²⁺binding site and is also necessary for At ZNE1 to transport Zn²⁺.All non-transmembrane domains cannot bind to Fe²⁺.It is predicted that At ZNE1 contains 14 transmembrane domains,and the non-transmembrane domain of At ZNE1 is artificially synthesized.Through isothermal titration,it was found that facing the cytoplasmic side Cyto?and non-cytoplasmic side Non-Cyto?and Non-Cyto?can bind to Zn²⁺.The histidine in Non-Cyto? is mutated to alanine,which causes loss of Zn²⁺binding function.At the same time,the mutation of At ZNE1 corresponding to histidine in Non-Cyto?to alanine?At ZNE1?H131A?complements the high zinc sensitive yeast mutant?zrc1/?cot1 cannot restore its growth phenotype in excess-Zn²⁺medium,indicating that the histidine in Non-Cyto?is necessary for Zn²⁺transport.All non-transmembrane domains reacted with Fe²⁺without heat change,indicating that they could not combine with Fe²⁺.?4?At ZNE1 co-localizes with Golgi marker protein and vesicle transport marker protein and the deletion of At ZNE1 does not affect the accumulation of Golgi Zn²⁺in Arabidopsis cells.The transient expression of At ZNE1-GFP in Arabidopsis mesophyll cell protoplasts was localized to the Golgi apparatus,and the transient expression in tobacco epidermal cells was found to be co-localized with the vesicle transport-related marker proteins At VTI13-RFP,At CLC2-RFP and At ST1-RFP.The wild-type Col-0 and mutant atzne1 mesophyll cell protoplasts were extracted separately and treated with 300?M Zn²⁺treatment solution,and dyeing with membrane-permeable zinc ion dye Zinpyr-1 revealed that the content of Zn²⁺in the Golgi body was not significantly different from each other.?5?At ZNE1 has homologous genes in 50 plants,and the ZNE1 homologous proteins of Triticum aestivum,Setaria viridis and Brachypodium distachyon have Zn²⁺transport function.Phylogenetic analysis using the amino acid sequence of At ZNE1 found that the homologous genes of At ZNE1 are widely existed in plants.The ZNE1 homologous genes of T.aestivum,S.viridis and B.distachyon were transformed into yeast zinc or iron transport-deficient mutants and found that all of them could restore the growth phenotype of the high zinc sensitive yeast mutant?zrc1/?cot1 in excess-Zn²⁺medium,but could not restore the growth phenotype of low zinc sensitive yeast mutant?zrt1/?zrt2 in Zn²⁺-deficient medium and the growth phenotype of low iron sensitive yeast mutant?fet3/?fet4 in Fe²⁺-deficient medium.The ZNE1homologous genes of T.aestivum,S.viridis and B.distachyon can mediate the transfer of Zn²⁺ from low concentration to high concentration,but cannot mediate the transfer of Zn²⁺ or Fe²⁺from high concentration to low concentration,which has the same function as At ZNE1,indicating ZNE1 is functionally conserved during evolution.?6?Mined three Zn transporters and two Fe transporters in Leymus chinensis.Bioinformatics analysis of Leymus chinensis transcriptome data was performed to screen zinc or iron transporter encoding genes for functional identification.It was found that Lc234720 is predicted to be an At HMA4 homologous gene and located in the vacuole membrane.It could restore the growth phenotype of the hyper-zinc-sensitive yeast mutant?zrc1/?cot1 in excess-Zn²⁺medium and mediated the transfer of Zn²⁺from low concentration to high concentration.Lc228182is predicted to be a homologous gene of At HMA2 and located in the Golgi apparatus.It could restore the growth phenotype of the hyper-zinc-sensitive yeast mutant?zrc1/?cot1 in excess-Zn²⁺medium and mediated the transfer of Zn²⁺from low concentration to high concentration.Lc240210 is predicted to be a homologous gene of At ZNE1,which is located in the endoplasmic reticulum.It could restore the growth phenotype of the hyper-zinc-sensitive yeast mutant?zrc1/?cot1 in excess-Zn²⁺medium and mediated the transfer of Zn²⁺from low concentration to high concentration.The non-transmembrane domains of Lc240210 which facing the cytoplasmic side Cyto I,II,V,VIII and non-cytoplasmic side Non-Cyto I,II,III,V can bind to Zn²⁺.Lc235481 is predicted to be a homologous gene of At ZIP10 and located in the vacuole membrane.It could restore the growth phenotype of the low-iron sensitive yeast mutant?fet3/?fet4 in Fe²⁺-deficient medium and mediated Fe²⁺transport from high concentration to low concentration.Lc206852 is predicted to be a homologous gene of At ZIP1,which is located in the endoplasmic reticulum,and can restore the growth phenotype of the low-iron sensitive yeast mutant?fet3/?fet4 in Fe²⁺-deficient medium and mediated Fe²⁺ transport from high concentration to low concentration.The non-transmembrane domains of Lc206852 which facing the cytoplasmic side Cyto I and II can bind to Fe²⁺ but all non-transmembrane domains cannot bind to Fe³⁺.?7?The expression of transcripts of genes encoding Leymus chinensis Zn or Fe transporters is induced by changes in the concentration of zinc and iron in the environment.The transcription levels of Lc ZNE1,Lc HMA4,and Lc ZIP1 in adult Leymus chinensis root were up-regulated in high-zinc of 150?M Zn²⁺ or low-iron of 0?M Fe²⁺ hydroponics.The transcription levels of Lc HMA4 and Lc ZIP1 in adult Leymus chinensis root and the Lc ZIP10 transcription levels in adult Leymus chinensis leaf and root were all up-regulated in the high-iron of 500?M Fe²⁺ hydroponic culture solution.On the contrary,the transcription level of Lc HMA2 in adult Leymus chinensis root was down-regulated in the low-zinc of 0?M Zn²⁺ hydroponic culture solution.The research found that the new Zn transporter At ZNE1 is involved in mediating the Golgi Zn²⁺ transport in Arabidopsis cells,and may also be involved in regulating vesicle transport to maintain intracellular Zn²⁺ homeostasis.The absence of At ZNE1weakened the adaptability of Arabidopsis to high-zinc or low-iron environments,but did not affect the content of Zn and Fe in Arabidopsis leaves,roots and seeds,and the accumulation of Zn by the Golgi apparatus.The discovery of At ZNE1 is an important supplement to the mechanism of plants regulating Zn homeostasis.Three Zn transporters and two Fe transporters have been found and verified in Leymus chinensis.The discovery of these transporters provides a theoretical basis for the future biofortification of Leymus chinensis.