Functional Characterization And Mechanism Dissection Of OsPPR16 Related To Early Chloroplast Development In Rice

Plastid-encoded genes are coordinately transcribed by the nucleus-encoded plastid RNA polymerase?NEP?and the plastid-encoded RNA polymerase?PEP?.The resulting primary transcripts are frequently subject to RNA editing by C-to-U conversions at specific sites.However,the biological function and its regulation mechanism of many RNA editing events are still largely unknown.The DYW subgroup of pentatricopeptide repeat?PPR?protein encoded by nuclear genes is the major RNA editing factor.In this study,the CRISPR/Cas9 technique was used to to knock out 30 DYW type PPR proteins,respectively,which are all predicted to localize to the chloroplast.The genotype and phenotype of transgenic plants were analyzed systematically.Later on,the function of Os PPR16 was chosen for further study to investigate its role in chloroplast RNA editing.Furthermore,molecular biological and genetic analysis strategies were applied to investigate the effects of Os PPR16 on chloroplast development.The main results are as follows:1.Using CRISPR/Cas9 gene editing technique,28 out of 30 PPR genes were confirmed to be knocked out,respectively.A striking developmental phenotype was observed in the first 2 leaves of tillers of osppr16 and osppr67 homozygous and biallelic mutants,which were pale at the early developmental stage,but turned to green at later stages.2.Knock-out of Os PPR16 leads to a pale phenotype during early leaf development.The first 4 leaves of osppr16 T₁ seedlings grown in growth chamber were pale and then turned to green before the five-leaf stage.A very similar phenotype of T₁ plants was seen as T₀ plants when they were transferred to the paddy field,i.e.the first 2 leaves of tellers were pale and then gradually turned green at the tillering stage.By contrast,after the tillering stage,leaf color of osppr16 mutant plants grown in the field was not different from the wild type?WT?.Correlatedly,the chlorophyll accumulation in osppr16 mutant leaves corresponded well with the visual observation of the greening process.3.The main agronomic traits of osppr16 mutant and WT plants were analyzed,which showed that inactivation of Os PPR16 would reduce the rice biomass and yield.4.Os PPR16 plays an important role in the biogenesis of chloroplasts during early leaf development.The chloroplasts in the pale leaves of osppr16 mutant lacked organized thylakoid membranes at the three-leaf stage.By contrast,the chloroplasts in re-green leaves contained well-organized thylakoids and stacked grana were present at the five-leaf stage.These results indicate that the Os PPR16 gene product plays an important role in the biogenesis of chloroplasts during early leaf development.5.Expression of an Os PPR16-YFP?GFP?fusion protein in rice protoplasts indicating that Os PPR16 is localized in chloroplast nucleoids.6.Os PPR16 is required for the editing of RNA editing site rpo B-545 in rice plastids.RNA editing efficiency of rpo B-545 in osppr16 mutant plants was 0%,whereas that of was 84%in the WT.Moreover,all other 23 editing sites in osppr16 chloroplasts were edited normally and with similar efficiency as in the WT.In addition,the expression level of Os PPR16 in RNAi plants correlated with editing efficiency of rpo B-545.Genetic complementation studies confirmed that Os PPR16 is required for the editing of rpo B-545,and Os PPR16 disruption is the direct cause of the pale leaves of osppr16 during early leaf development.7.Inactivation of Os PPR16 reduces the accumulation of Rpo B protein during early leaf development.At the three-leaf stage,Rpo B accumulation was much reduced in the pale leaves of osppr16 mutant compared to the WT.At the five-leaf stage,the accumulation of Rpo B was partly restored in osppr16 re-green leaves and only slightly lower than that in WT plants at the same stage.In addition,accumulation of Rpo B in the WT was considerably higher at the three-leaf stage than at the five-leaf stage.These results suggest that Rpo B accumulation at an early developmental stage is severely affected in osppr16 and more PEP is required for early chloroplast development than at later developmental stages.8.RNA editing of rpo B-545 resulting in an amino acid change from hydrophilic serine to hydrophobic leucine at position 182 of the PEP?-subunit.The crystal structure of the Thermus aquaticus RNA polymerase-promoter open complex indicates that the hydrophilic serine at position 182 may reduce the stability of Rpo B protein when the editing of rpo B-545 is impaired.9.Os PPR16 affects the trn E expression through transcriptional ability of PEP,which in turn affects the synthesis of chlorophyll during early leaf development.In the pale leaves of osppr16 mutant plants,the expression of PEP-dependent genes was strongly reduced at the three-leaf stage.Interestingly,at the five-leaf stage,the expression of most PEP-dependent genes in re-green leaves of osppr16 mutant plants had nearly fully recovered and was similar to that in the WT.These results indicate that the transcriptional ability of PEP in osppr16 mutants is decreased during early leaf development,but recovers at later developmental stages.At the three-leaf stage,accumulation of a PEP-dependent and chlorophyll synthesis gene trn E in pale leaves of osppr16 mutant plants was very low.By contrast,at the five-leaf stage,trn E accumulation in the re-green leaves of osppr16 mutants had recovered and nearly reached WT levels.These results suggest that the chang of trn E accumulation is responsible for the virescence phenotype of osppr16 mutants.In conclusion,our results clearly indicated that the inactivation of Os PPR16 would result in impaired RNA editing of rpo B-545,consequently leads to impaired accumulation of Rpo B,reduced expression of PEP-dependent genes,and a pale phenotype during early plant development.Thus,by editing the rpo B m RNA,Os PPR16 is required for faithful plastid transcription,which in turn is required for chlorophyll synthesis and chloroplast development during early leaf development.Our results not only reveal the physiological role of rpo B-545 editing site,but also provide new insights into the interconnection of the finely tuned regulatory mechanisms that operate at the transcriptional and post-transcriptional levels of plastid gene expression during early leaf development.