Map-Based Cloning And Functional Analysis Of A Plastidic Oxidoreductase TSV In Rice(Oryza Sativa L.)

Chloroplast is not only the organelle for photosynthesis,but also a place for the synthesis andmetabolism of some important substances of plant.So the early development of chloroplast is a crucial progress for the growth of plant.The time window for the early development of the chloroplast might be just a few hours.The disturbance of this progress can not be reversed if a certain time point has been passed.In previous studies,the early development of chloroplast was mainly studied in the Arabidopsis thaliana,while the research on chloroplast development in monocots is rare.The chloroplasts development and the leaf morphological characteristics in monocot and dicot are very different.Therefore,study the early development of the chloroplasts in the monocot plant could give us more understanding of higher plants.Compared with other cereals such as wheat and barley,rice is more susceptible to cold stress,especially during its seedling stage,which is known to be one of the most sensitive stages to low temperature.Cold is a major abiotic stress that affects plant growth and plastid development.Under field conditions,sudden low-temperature phases often occur during earlyseedling development in spring,which directly affect agricultural production.Lowtemperature causes the development of the leaf,chlorophyll synthesis and the development of the chloroplasts.However,mechanisms of chloroplast development in rice seedlings under cold stress have remained poorly understood.Here we isolateda temperature sensitive virescent(tsv)mutant thatis extremely sensitive to cold stress.We demonstrated that TSY,encoding a putative enzymeof the oxidoreductase family,protected chloroplast development from cold stress,by interacting with OsTrxZ,which participated both in chlorophyll synthesis and chloroplast biogenesis.These results enhanced our understanding of there gulatory mechanism of protecting chloroplast biogenesis and chlorophyll synthesis from cold stress in higher plants.1.The rice tsv mutant was isolated from a pool of indica rice cultivar 9311 mutants created by 60 Co-irradiation.It displayed defective chloroplasts,decreased chlorophyll and zero survivorship under cold stress.The tsv mutant displayed a virescent leaf phenotype under a normal alternating light/dark cycle(C30/20,12-h-light at 30?/12-h-dark at 20?)in a growth chamber.Under cold temperature(C20,12-h-light/12-h-dark at constant 20?),tsv mutants displayed albino leaves with no chlorophyll accumulation until death.Overall,tsv mutants developed virescent leaves and displayed growth retardation,suggesting that TSV could play a role in chlorophyll synthesis and plant growth.2.We investigated the chloroplast ultrastructure of tsv mutants grown under cold stress.In tsv mutants grown under C20 condition reduced thylakoid membrane networks,vesiculation of the thylakoids,and ultimately breakdown,were found in all chloroplasts in the third leaves,while normally stacked grana and thylakoid membranes were seen in the wild type.By contrast,most chloroplasts from the third leaves developed normally with well-formed lamellar structures in tsv mutants grown under C30 condition,similar to the wild type.This suggests that in the wild type seedlings TSV may protect the development of chloroplasts from damage due to low temperature stress.3.Genetic mapping of the TSV gene was performed using the F2 and F2 3 populations from a cross between tsv and cultivar N22.We mapped the tsv gene to a 103 kb genomic region on chromosome 5 between the SSR makers B4-24 and B4-25 using 324 F2 and 1379 F2.3 mutant plants.There are 14 open reading frames(ORFs)predicted in this region by published data(http://www.gramene.org).LOC_Os05g34040 in tsv has a 2 bp deletion in the 3 rd exon at the position 2,315 bp from the ATG start codon.This 2-bp deletion creates a frame-shift causing premature termination by introducing a stop codon,removal of a translation initiation codon and deletion of a functional domain.Together,TSV rescued phenotypes of the tsv mutant plants,and LOC_Os05g34040.1 indeed corresponded to the TSV gene.There is only one copy of the TSV gene in the rice genome,which encodes a polypeptide of 719 amino acids.TSV encodes a chloroplast-localized protein and contains a chloroplast-targeting signal.Confocal microscopy showed that the green fluorescent signals of TSV-GFP fusion proteins were co-localized with the auto-fluorescent signals of chlorophylls in the chloroplasts.The activity of plastid-encoded RNA polymerase(PEP)and expression of genes participating in chlorophyll synthesis were severely reduced in tsv mutant under cold stress,but not at normal temperatures.TSV was found required for protecting PEP activity and chlorophyll synthesis under cold stress.4.TrxZ was identified in a proteomic study as acomponent of transcriptionally-active plastomes and more recently as a subunit of acomplex formed with the plastid-encoded RNA polymerase(PEP).TSV encoded a putative oxidoreductase family protein with a pyridine nucleotide-disulfide oxidoreductase conserved domain(240-327 aa)and an uncharacterized FAD-dependent dehydrogenase multi-domain(140-699 aa).We demonstrated that TSV interacted with OsTrxZ by yeast two-hybrid assays,pull-down assays and BiFC.We further investigated that TSV-OsTrxZ interaction was regulated by redox states.These results further validated that TSV enhanced the stability of OsTrxZ under low temperatures and protected chloroplast development from cold stress.