Analysis Of The Dual Function Of Osbt3,a Novel Plastid Atp/adp Transporter,in Rice(Oryza Sativa L.)

Chloroplasts are key organelles for higher plants,whose main function is photosynthesis that converts the energy from sun irradiation into chemical energy(ATP)for all living organisms to consume.Thus,chloroplast development,maintenance and function are of fundamental importance to all life forms on the earth.Differentiation from proplastids into chloroplasts is an elaborate process which needs the coordinated expression of chloroplast-and nuclear-encoded genes because most chloroplast proteins(more than 90%)are encoded by the nuclear genome and imported into the chloroplast,where they are assembled together with the chloroplast-encoded proteins to form functional photosynthetic and metabolic complexes.Thus any related gene mutation may lead to poor chloroplast development resulting in varying types of leaf color variation.A major function of chloroplast and photosynthetic reactions is to generate ATP,which is a universal energy supply for all organisms.However,due to the size and charge,adenylates cannot cross the biomembranes freely and require a series of intracellular nucleotide transporters to carry adenylates across many internal cell membranes in order to supply energy for the vast numbers of metabolic reactions in different cellular compartments.So far,three main types of nucleotide transporters have been identified at the biochemical and molecular level in higher plants.The first type is ADP/ATP carrier(AAC),which belongs to the mitochondrial carrier family(MCF)and mediates the export of ATP from mitochondrial into cytosol.The second type of adenylates transporter is the plastidial nucleotide translocator(NTT).NTT catalyzes ATP/ADP antiport,it mediates an uptake of ATP into heterotrophic non-green plastids and autotrophic plastids which lose the photosynthetic activity at night.A third type of nucleotide transporter in plastids was named after the maize(Zea mays)mutant brittlel(BT1).The mutant seeds collapse during ripening and appear brittle-like.Hence the gene is nominated as Brittlel.Apart from their transport function,we know nothing about their contribution to plant development and metabolic.In this study,we identified and characterized a novel MCF-type ATP/ADP transporter OsBrittle3(OsBT3)which was essential for chloroplast development at early leaf stage.A single base deletion in OsBT3 led to the white striped leaf(wsl)phenotype in seedlings,accompanied by abnormal chloroplast development,low photosynthesis efficiency and reduced chlorophyll content.OsBT3 located in the chloroplasts and acted as an ATP/ADP carrier.We found that intracellular ATP might function as a novel signal which was required for the seedling development of rice.Moreover,OsBT3 was demonstrated to interact with PORA,an essential enzyme in chlorophyll biosynthesis and chloroplast differentiation.Our study provided a novel insight into the chaperone-like activity of OsBT3 in chloroplast development in rice.The results are as followed:1)The differences of phenotype between wsl2 and the wild-type.wsl2 exhibits white striped leaves before five-leaf-stage especially at the third leaf so as the chlorophyll contents.There is no PLB in proplastids of wsl2 etiolated seedlings which has an effect in chloroplast development.The mutant expresses reduced photosynthetic rate and electron transport rate which resulted in the decline in plant height and 1000-garin weight.Multiple retrograde signals from chloroplast to nucleus are affected in wsl2 which lead to a repression in genes expression level involved with chlorophyll synthesis and plastid development Activity of PORA falls down causing the accumulation of ROS in wsl2.2)For genetic analysis of the wsl2 mutant,the phenotype of albino leaf in the wsl2 mutant is controlled by a single recessive nuclear locus.We identify the OsBT3 by map-based cloning.The wsl2 allele contains a single base deletion at 326bp of the first exon leading to an advanced stop.OsBT3 is a putative adenine transporter belonging to MCF which consists of three tandem repeats of?100 residues showing 6 putative transmembrane helices.There are three homologous of BTs in rice with totally deferent expression pattern We constructed and transformed RNAi vector 'pUbi-dsRNAiBT3,into japonica variety Nipponbare.All of the positive transgenic lines displayed visible white striped leaves with lower chlorophyll at the early stage as the mutant.Genes involved with plastid development also display a lower expression level.3)OsBT3 is a plastid ATP/ADP transporter.OsBT3 display a subcellular localization in chloroplast.The experiment,using Escherichia coli for heterologous expression,shows that OsBT3 is an ATP/ADP transporter.We test much more ATP in chloroplast while less in the cytosol in wsl2 mutant.In addition,adding ATP in vitro can regulate the expression of photosynthetic genes.4)OsBT3 has an activity for holdase of PORA.We find the interaction relationship between OsBT3 and PORA,an important rate-limiting enzyme in chlorophyll synthesis.We also detect the activity of OsBT3 for protecting PORA from aggregation caused by heat shock and oxidative stress.At the same time,we find that OsBT3 may function in the accumulation of PORA protein during night.5)The holdase activity of OsBT3 is depend on the ATP/ADP binding sites.All three different site-directed mutated proteins have lost the ability to transport ATP.And also they no longer have the activities to protect the stability of PORA as the mutant.These experiments indicate the possibility of something new functions of adenine transporters.This finding provide us a more comprehensive understanding of the function of MCF proteins in rice.