Map-based Cloning And Function Analysis Of OsBT1-3 Controlling White Leaf At Seedling Stage In Rice

Nucleotides are among the most essential cellular components for plant growth,development,and metabolism and are required for the synthesis of DNA and RNA.Moreover,nucleotides function as enzyme co-factors in the accumulation of proteins,sugars,and lipids and act as signalling molecules in the cell.In plants,the de novo synthesis of adenine nucleotides occurs exclusively in plastids,and reports have not indicated that nucleotides are transported at high rates throughout the entire plant.All types of cells must be able to synthesize nucleotides de novo.Newly synthesized adenine nucleotides,which used for metabolism in the cytosol and other organelles,are exported into the cytosol from plastids.The carriers,which export adenine nucleotides to the cytosol,are called plastidial net adenine nucleotide transporters(pANTs).An Arabidopsis pANTs(AtBT1)was shown to reside in plastids and mediate the unidirectional transport of' adenylated nucleotides(Kirchberger et al.,2008).However,limited research has been perf'ormed on pANTs in monocotyledon.In this study,a rice(Oryza sativa)mutant exhibiting early chloroplast development(seedling leaf albino(sla))was isolated from a filial generation via hybridization breeding.The leaves of the sla mutant appeared albino at the first and second leaves,whereas the third leaf appeared a normal green.The contents of chlorophyll a,chlorophyll b,carotenoid and total chlorophyll in sla mutant seedlings at the first and second leaves were drastically lower than those in pOsBT1-3-T1(1?3)and 93-11.The levels of these pigments in the sla mutant increased and became similar to pOsBT1-3-T1(1?3)and 93-11 in the third leaf.These data show that the albino phenotype of sla mutant seedlings is caused by a reduction in total chlorophyll content.Using transmission electron microscopy to compare the ultrastructure of chloroplasts in the sla mutant,pOsBT1-3-T1 and 93-11 of the first and second leaves,we found chloroplasts were abundant in mesophyll cells of pOsBTl-3-T1 and 93-11 at first and second leaves.However,the chloroplasts were quite sparse in mesophyll cells of sla mutant at the first and second leaves.Only few mesophylls contain few immature proplastids.These data indicate the differentiation of proplastid is blocked seriously.Genetic analysis revealed that the phenotype of sla mutant is recessive and controlled by a single gene.Molecular analysis of an F2 population from the cross RPY jing × sla mutant placed the sla mutant gene between the markers RM6298 and RM7424 on chromosome 6.Five simple sequence repeat markers were developed between RM6298 and RM7424.The sla mutant gene locus was further narrowed down to an 80-kb region,which includes 13 putative open reading frames.We sequenced all ORFs and found a 4-bp deletion in LOC_Os06g40050(OsBT1-3),causing a premature stop codon.The protein sequence analysis of OsBT1-3 indicated it belongs to mitochondrial carrier family(MCF).The subcellular location analysis of OsBT1-3 indicated it is located in chloroplast.Western blotting using a peptide-specific antibody further indicated location of OsBT1-3 in the chloroplast envelope.Recombinant Trx-His6-S-OsBT1-3 protein was expressed in E.coli cells,and biochemical function of OsBT1-3 was analyzed by isotopic tracer method.The results indicate that OsBT1-3 is a unidirectional transporter of adenine nucleotide.We have also studied the temporal and spatial expression mode of OsBT1-3,the result showed that OsBT1-3 gene highly expressed in the first and second leaves during developing stage,and much lower expression in mature stage and after the second leaf.This expression character of OsBT1-3 coincided with the albino phenotype of sla mutant.According to these results,we proposed that OsBT1-3 is a plastidial adenine nucleotides uniporter used to export newly synthesized nucleotides into cytosol.At the same time,we have also analyzed the expression pattern of OsBT1-2,which is the homology of OsBT1-3.The results show that OsBT1-2 highly expressed in leaf sheath and after the second leaf,whereas,the expression of OsBT1-2 is quite low in roots and the first and second leaf.The results of subcellular location also reveal that both OsBT1-2 and OsBT1-3 positioning in the chloroplast.It seemed that OsBT1-2 can period complementary Osbtl-3 function.In conclusion,the adenine nucleotides transportion of rice regulated by both Osbt1-2 and Osbtl-3,different from the pattern of dicot such as Arabidopsis,which transport adenine nucleotides by single transporter.