Functional Analysis Of The Golgi Localized Mn²⁺ Transporter GMT1 In Arabidopsis

Manganese?Mn?is a necessary micronutrient for plant growth and development.Mn serves as the component or cofactor for metalloenzymes,for example,manganese superoxide dismutase?MnSOD?in mitochondria which is involved in antioxidant activity.In chloroplasts,Mn is the core atom of oxygen-evolving complex in photosystem ??PS??and catalyzes the water-splitting reaction.Besides,Mn also acts as activator of many enzymes to participate in the metabolism in cells,such as the DNA synthesis,sugar metabolism or protein modification.The distribution of Mn in plant cells relies on a variety of Mn2+ transporters existing on the plasma membrane and various organelles.The previous research of our lab demonstrated an inner envelope Mn2+transporter CMT1 functioning in Mn2+ uptake across chloroplast.CMTI belongs to an evolutionarily conserved family of UPF0016?Uncharacterized protein family 0016?,which possesses cation transport activity.There are five members in UPF0016 family in Arabidopsis,so we speculated other members might also function as Mn2+transporter.In this thesis,we characterized another member of this family,At5g36290,and designated it as GMT1?Golgi Manganese Transporter 1?based on its subcellular localization and functions.The molecular mechanism of GMT1 was analyzed by various methods,including genetics,molecular biology,histochemical staining,ionomics and yeast complementary assay.The main results are as follows:?1?GMT1 affected plant growth and development under Mn-deficient conditionIn this thesis,we acquired two homozygous T-DNA insertion lines?CS320336 and SALK₀97998?of GMT1,and named them as gmtl-1 and gmtl-2 respectively.RT-PCR analysis showed that there was no GMT1 transcript detected in the gmt1-1 mutant,whereas gmtl-2 still retains partial GMT1 mRNA albeit at a reduced level as compared to the wild type.So gmtl-1 and gmt1-2 were knockout and knockdown mutant respectively.In the normal MS medium,there was no obvious difference between mutants and the wild type.Under Mn2+-deficient condition,both gmt1-1 and gmtl-2 mutants showed the curled and wrinkled leaves,and the smaller shoot compared with the wild type.As the increasing Mn2+ in the Mn-limited medium,the phenotype of mutants were gradually recovered.To confirm that the phenotype was caused by loss-function-of GMT1,the 35S::GMT1-GFP construct was introduced into Agrobacterium GV3101 and transformed into gmt1-1 mutant for complementation test.The phenotype of over-expressed plants?gmt135S:AtGMT1?was successfully recovered to the wild type level under Mn2+-deficient condition,proving that the phenotype of mutants under Mn2+-deficient condition was indeed caused by the mutation of GMT1.?2?GMT1 is located in cis-GolgiTo investigate the subcellular localization of GMT],the fusion expression vector GMT1-GFP was constructed and transiently introduced into Arabidopsis protoplasts.We found that the green fluorescence signal of GMT1 showed punctate distribution,and speculated GMT]might be localized on endomembrane compartments.To clarify the precise localization of GMT1,we co-expressed GMT1-GFP with marker proteins localized to different endosystems in Arabidopsis protoplasts.The results showed that GMT1 was co-localized with MAN1-mCherry,a cis-Golgi marker,but not with TGN marker SYP61-mCherry and PVC marker VSR2-RFP.Therefore,these results indicated that GMT1 is located in the cis-Golgi.?3?GMT1 functions in Mn2+ transport in yeastThe full-length version and truncated version without signal peptide??N25GMT1?of GMT1 were introduced into the ?smf1 yeast strain which is defective in Mn uptake.The results showed that the truncated version of GMT1 could complement the ?smf1 growth defects under Mn-limited conditions.The liquid growth curve is consistent with the plate experiment.Then the ion content of yeast cells containing the empty vector?pYES2?and truncated version of GMT1??N25GMT1?were measured by inductively coupled plasma-mass spectrometry?ICP-MS?.Under Mn-deficient or Mn-sufficient conditions,the Mn concentration of the yeast cells containing?N25GMT1 was higher than those of the yeast cells containing pYES2.These results indicated that GMT1 functions as Mn2+ transporter in vitro.In summary,we identified a novel Mn2+ transporter in Arabidopsis,which is located in the cis-Golgi.Under Mn2+-deficient condition,the mutants showed the curled and wrinkled leaves,and the smaller shoot.This phenotype could be complemented by exogenous Mn2+,and the GMT1 exhibited Mn2+transport activity in vitro.The thesis provides useful theoretical guidances and experimental evidences for molecular breeding to improve Mn2+ utilization efficiency of crops.