Ⅰ.Functional Study, Subcellular Localization And Degradation Pathway Research On MxIRT1, An Iron Regulated Transporter From Malus Xiaojinensis Cheng Et Jiang. Ⅱ.Study On Substrate Identification Of OsNAT7.

Iron is an essential element with numerous cellular functions,and plays a critical role in important biochemical processes.Although aboundant in the soil,iron is often available in limited amounts because the neutral or basic pH of soil.Thus,low iron availability affects plant normal growth and development.Plants have enolved two effective iron acquisition systems known as strategyâ… and strategyâ…¡to cope with iron deficiency.The two also called reduction strategy and chelation strategy.However,little is known about iron homeostasis in fruit trees,especially in cell level.And functional study of rice up-regulated genes under iron-deficient conditions has not been fully understood.In this work,research on substrate range,subcellular localization and degradation pathway of MxIRT1(Iron Regulated Transporter) was studied.In addition, substrate analysis of OsNAT7(Nucleobase-Ascorbate Transporter),one up-regulated gene responded to iron deficiency from rice,was performed.â… .Functional study,subcellular localization and degradation pathway research on MxIRT1Fruit trees belong to Strategyâ… Plants.Plasma membrane proteins play essential role in iron absorb especially under iron starvation conditions.Malus xiaojinensis Cheng et Jiang is an iron efficient genotype in the genus Malus.A cDNA clone,MxIRT1,was previously isolated from a library constructed from roots of M.xiaojinensis under iron-deficient conditions.Transcript level analyses indicated that the expression of MxIRT1 was strongly enhanced in roots under iron limitation.In this section,research following previous statement of MxIRT1 was outline as below:a.MxIRT1 is an iron and cadmium transporterMxIRT1 is highly homologous to other iron-regulated transporters belonging to the ZIP(ZRT/IRT like Protein) family.To identify the substrates of MxIRT1 transporting,expression vectors was transformed into different yeast mutants.MxIRT1 could complement iron uptake activity of Saccharoomyces cerevisiae mutant strain DEY1453(fet3fet4).Also,it presented strong effect on Cd transport for transformed DY1457 presents strong sensitivity of Cd.Changes of metal content,measured by ICP-MS,in transformed yeast cells support the complementation results.It implies MxIRT1 is an iron and cadmium transporter.MxIRT1,nevertheless,expression in yeast could not complete the growth defect of zinc,manganese copper transport yeast mutants and enhance metal uptake and accumulation.And DY1457,transformed with MxIRT1 dose not present sensitivity of Co and Ni.It indicates MxIRT1 could not transport zinc,manganese, copper,cobalt and nickel.b.His-rich region in MxIRT1 plays different role in iron and cadmium transportDue to MxIRT1 is an iron and cadmium transporter,it is essential to do cation selectivity research to guarantee that only certain ions across the membrane.It is presume that His-rich repeat in variable region may be involved in metal binding.To investigate the functional of the motif,the region was removed through PCR-based method.Functional expression experiment suggested His rich region of MxIRT1 was significant in Fe transport but no effect on Cd absorb.The result different from point mutation in Connolly' lab,which illustrate the secondary structure may vital for its function.c.Protein secretory is essential for the functional MxIRT1Considering that iron-regulated transporter is a membrane-spanning protein,subcellular localization was visualized by MxIRT::GFP fusion protein.The expression of MxIRT1::GFP protein in transformed yeast cells are localized in plasma membrane and vesicles.The result indicated that MxIRT1 was targeted into the plasma membrane through a protein trafficking processes via membrane vesicles under iron limitation conditions(BPDS or Ferrozine added).Meanwhile,signal peptides removed MxIRT1 could not have the ability of Fe and Cd transport illustrated that MxIRT1 secretion not only is a signal peptides dependent process,but also an essential way for it function,especially under iron-deficient conditions.d.Iron content in culture medium have effect on subcellular localization and protein abundanceMxIRT1::GFP fusion proteins present dynamic localization in response to changing metal levels.Under iron-deficient conditions,green fluorescence localized in cytosol dot-like structure.Under YPD culture medium,however,green fluorescence restrict in special region of plasma membrane.And with increasing of iron concentration and extending the culture time,level of green fluorescence declined.It suggests that the degradation process of MxIRT1.To further investigate the degradation pathway,expression vector with an induced promoter was constructed. And green fluorescence localized in vacuole of yeast cells with induced expression of MxIRTI::GFP.The fluorescence enhanced when high iron was added.It implies that turnover of MxIRT1 is a vacuole dependent process and iron could accelerate this process.Western blot analysis show that MxIRT1::GFP decreases under iron-replete conditions.But little change found in iron limiting conditions.In endocytosis defect mutant,end4,green fluorescence could also observed in vacuole.It indicates degradation pathway is not dependent endocytosis.Newly synthesized transporters with the secretory pathway could account for the regulated degradation.In vacuolar protease mutant,pep4,green fluorescence accumulates in vacuole.It implies the degradation is a PEP4 dependent process.e.MxIRT1 localized in plasma membrane and endomembrane system in plant cellsTo comprehend the subcellular localization of MxIRT1 in plant cells,transient and stable expression was carry out in protoplast and BY-2 suspension cells respectively.And MxIRT1 localized in plasma membrane and endomembrane in the two systems.In iron-deficient or iron-sufficient medium,the localization presents same pattern.It suggested that plant have different regulation mechanism from yeast cells.Plant response to iron status may lie on whole plant or some specific organ.f.No obvious differences of metal content were found in sense and anti-sense transgenic rice seedsTo evaluated iron content in rice seeds,rice transformation was made.Identification of transgenic rice was indicated sense and anti-sense MxIRT1 was integrated into rice genome respectively.But little difference was found in metal content of wild type,sense and anti-sense transgenic rice seeds of T₁ generation.Possibly, it may due to iron transportation and homeostasis in plant is a tightly controled process. â…¡.Study on substrate identification of OsNAT7OsNAT7,belonging to NAT(Nucleobase-Ascorbate Transporter) gene family,is an up-regulated gene under iron deficient conditions in rice roots at transcriptional level.It was found in cDNA microarray analysis for transcriptome change in rice roots under iron deficient and sufficient conditions.The ratio of -Fe/EDTA is 9.605.But little is known about NAT function in iron metabolism.In this section,researches of OsNAT7 are outline below:To know the transcriptional change of OsNAT7,Real-time PCR was performed.The result indicated that it was highly expressed under iron deficient conditions at day 5.But the transcription level decline in day 3 after iron starvation.And no significant change was found in day7.It suggests the transcription change follow the iron conditions.Transient and stable expression of OsNAT7::GFP was mainly localized in plasma membrane of onion epidermal cells and BY-2 cells.Part of fluoscence was observed in ednomembrane.It suggested that it plays a role in substance transport.Although many attempts were made to elucidate the functional activity of OsNAT7 proteins using heterologous expression in different yeast cells,no clear results were obtained.The substrates were tested including adenine,hypoxanthine,guanine,cytosine,uracil,uric acid,adenosine,guanosine,xanthine and so on.It implies heterologous expression in yeast dose not suitable for substrate identification of NAT family. Further studies are required to shed light on the heterologous expression in A.nidulans.