Mechanism Analysis Of The Arabidopsis Pentatricopeptide Repeat Protein AtECB2 In Chloroplast RNA Editing

In higher plants, RNA editing mainly defines the process that alters a cytosine (C) to uracil (U) in specific positions of RNA. PPR proteins and MORF proteins were reported to be involved in the process of RNA editing. As a PPR-type protein, AtECB2 has been shown to affect the editing efficiency of 6 chloroplast transcripts (accD、ndhF、ndhG、petL、rpoA and rpoC1). However, the functional mechanism of AtECB2 in this process is still unclear. Here, we generated the transgenic plant expressing AtECB2 fused with 4xMYC tag. Then we demonstrated that AtECB2 was associated with the affected transcripts including accD、ndhF、ndhG and petL, suggesting AtECB2 is directly involved in the RNA editing of these transcripts. Meanwhile, we have identified the MORF2 protein and the porphobilinogen deaminase HEMC by co-immunoprecipitation of AtECB2, followed by mass spectrometry. This indicates that MORF2 and HEMC proteins may be involved in AtECB2-related RNA editing process. Our yeast two-hybrid experiments further demonstrated that the E domain of AtECB2 interacted with HEMC, and HEMC interacted with MORF8. It was reported that MORF2 could interact with MORF8. HEMC knockout mutant hemc-1 showed an albino and seedling lethal phenotype, and RNA editing in chloroplast was largely affected compared with WT. These results indicate that HEMC may assist AtECB2 couples with MORF proteins to perform RNA editing in vivo. Western blot assay showed that AtECB2 and MORF8 were mainly located in thylakoid membrance. BN-PAGE results showed that AtECB2 exsit predominantly in protein complexes which between 440kDa and 669kDa, while MORF8 was predominantly distributed in a broad range more than 440kDa. Thus, AtECB2 and MORF8 may exsit in the same complex. Our investigations revealed the functional mechanism of AtECB2 in RNA editing and gave new insight into the editosome.