Study On The Relationship Between Cotton Mitochondrial Atp1 Gene RNA Editing And Fiber Growth And Development

Cotton(Gossypium hirsutum)is one of the most economically important crops.In recent years,with the genome sequencing of G.arboreum,G.raimondii,and G.hirsutum,great advances have been made in revealing the molecular mechanisms of cotton fiber initiation and elongation.Cotton fiber metabolic pathways are complex,regulated processes,and most studies have focused on gene regulation at the transcriptional level.However,the mechanism of posttranscriptional processing of key genes and its effect on fiber cell elongation remain largely unknown.RNA editing is a posttranscriptional modification process that results in mature mRNA differing from the DNA coding sequence via insertion,deletion and conversion of nucleotides in the RNA sequence.RNA editing is found in a wide variety of organisms,including viruses,fungus,plants,and animals.The nucleotide modifications by RNA editing span a range of changes,including C-to-U and U-to-C conversion in plant organelle genes and C-to-U and A-to-I changes in the nucleus of animals.In higher plants,RNA editing occurs most frequently in coding regions of chloroplasts and mitochondrial genomes.However,the molecular functions and physiological roles of RNA editing are still poorly understood.In this study,we identified 692 RNA editing sites in G.hirsutum mitochondrial genes using high-throughput sequencing and found Ghatpl was significantly different for editing efficiency at five sites between fiber cells and ovules.The editing efficiency of C1292 and C1415 sites in Ghatp1 positively correlated with the ATP/ADP ratio during fiber elongation periods,and application of ATP in vitro significantly promoted cotton fiber cell elongation.Genetic and molecular analysis indicated that the two vital editing sites edited Ghatp1 were required for cell growth and ATP accumulation in yeast.Yeast two-hybrid,bimolecular fluorescence complementation and pull-down assays showed that these two crucial editing sites were essential for the interaction between GhAtp1 and GhAtp?,which is necessary for ATP production.Overexpression of C1292 and C1415 site-edited Ghatp1 in Arabidopsis caused the number of trichomes and root length to significantly increase,accompanied by elevation of the ATP/ADP ratio and ATP synthase activity.Our results suggest that RNA editing at C1292 and C1415 sites are essential for GhAtp1 to interact with GhAtp?,thus playing an important role for ATP accumulation in fiber cell elongation stages.The main finding can be concluded as follows:1.Mitochondrial RNA-sequencing(RNA-seq)was conducted with the samples from fiber and ovules at 10 days post-anthesis.Comparative analysis revealed that 692 RNA editing sites were identified in mitochondrial genes.The distribution of editing sites showed that 422 editing sites were in CDS regions.We also analyzed the preference for specific amino acid changes and found that C-to-U editing tended to change proline to leucine.Editing sites were mainly located at the second or first codon positions in protein-coding genes.2.We conducted a comparative transcriptome analysis of mitochondrial protein coding genes and investigated differences of RNA editing in mitochondrial transcripts between G.hirsutum var.Xuzhou142(WT)fiber and its near isogenic line,fuzzless-lintless mutant(fl)ovule.No significant difference was observed in the distribution of RNA editing sites in the non-coding regions between fiber and ovule cells.However,the editing efficiency of Ghatp1,which encodes a subunit of ATP synthase in complex V,had a prominent difference between fiber and ovule.To verify this observation,we sequenced mitochondria-encoded genes in fiber and ovule samples using traditional clone-sequencing methods.Notably,we found that Ghatp1 exhibited the most significant difference among all investigatedgenes,with five editing sites identified in fiber and none in ovule.3.The editing efficiency of the five sites during fiber elongation was evaluated using direct sequencing and clone sequencing and found that the editing efficiency of C1292 and C1415 sites significantly increased,and reached the peak value around 10 dpa.Further analysis showed that both ATP synthase activity and the ATP/ADP ratio had positive correlations with WT fiber growth,and reached peak values around 10 dpa,2?3 times higher than those in fl ovules.Meanwhile,application of an ATP in vitro significantly increased fiber cell elongation and ATP biosynthesis inhibitor inhibited fiber cell elongation.4.A series of yeast expression vectors harbouring nine edited versions Ghatp1 were individually constructed and transformed into Scatp1? mutant cells.Our results showed C1292 and C1415 sites edited Ghatpl restored the viability of ScatplA mutant cells.Mutant yeast cells expressing C1292 and C1415 sites edited Ghatpl contained almost identical ATP accumulation and produced large and elliptical cells as the wild type.5.We performed a yeast two-hybrid assay to determine which editing sites in Ghatp1 are crucial for this interaction.We found that C1292 and C1415 sites edited GhAtp1 proteins interacted with GhAtp?,which was further verified by the BiFC and Pull-down assays.6.We constructed a Scatpl and Scatp? double mutant strain(Scatp1?Scatp??),and subsequently transformed variants of edited Ghatpl with or without Ghatp? to better understand the interaction between GhAtp1 and GhAtp? in vivo.When nine versions of edited Ghatpl was transformed into the ScatplAScatp?? mutants without Ghatp?,the phenotypes of complemented cells were similar to those of double mutant yeast,which had a slower growth rate,lower ATP accumulation,and virgulate cells.When Ghatp? and the nine edited variants of Ghatpl were co-transformed into Scatp1?Scatp?? mutant cells,only the cells expressing C1292 and C1415 sites edited GhAtpl showed phenotypic improvement and that the levels of growth and ATP production were recovered to wild-type.7.To explore the function of the five editing sites at Ghatp1 in Arabidopsis,the edited Ghatp1 variants were individually sub-cloned into the PQG110 expression vector.In total nine A.thaliana transgenic lines were obtained.In contrast to the wild-type plants,the transgenic Atp1-4,Atp1-5,Atp1-4,5,or Atp1-E lines had a considerable increase in leaf trichomes and longer roots.