Functional Analysis Of Plastid Ribosome Assembly Factor EMB15 C-Terminal Domain In Maize And Arabidopsis

Chloroplast is a photosynthetic organelle in plant cells,which uses solar radiation to generate metabolic energy equivalents.Chloroplast,as a semiautomous organelle,has its own genome and translation machinery.Ribosome is a place for protein synthesis,of which biosynthesis is tightly regulated to ensure the accuracy and efficiency of protein synthesis.Chloroplast contains the bacterial-type 70S ribosome,composed of a 50S large subunit and a 30S small subunit.Assembly of the 30S ribosomal subunits begins with the transcription of 16S rRNA,which is facilitated by assembly factors.In Escherichia coli,RimM is a key assembly factor in the assembly process of the 30S subunit.Strains with deleted RimM also exhibit slow growth,incomplete 16S rRNA processing,and reduced accumulation of 30S and 50S subunits with few polysomes.An independent RimM protein does not exist in land plants,instead,a homologous RimM is fused with a putative UAP domain in land plants that is named as EMB15 in maize.The deletion of EMB15 in maize leads to either an embryo defective or albino phenotype,depending on the genetic background.EMB15 complements the growth defect of an Escherichia coli ArimM strain.EMB15 might interact with PRPS19 whose homolog interacts with RimM in E.coli.Plastid 16S rRNA precursors accumulated in emb15,suggesting that maturation of the 30S small subunit is retarded.These results indicate that the N-terminal RimM domain of EMB15 functions as the ribosome 30S subunit assembly factor in plastid.The function of the C-terminal domain of EMB15 remians to be uncovered.To explore the complete function of EMB15,Emb15 homologous gene AtEmb15 was identified in Arabidopsis thaliana,and null mutants(Atemb15)and C-terminal domain truncation mutants(Atemb15ΔC)were created with the CRISPR/Cas9 technology.Unlike the maize emb15 mutant,Atemb15 showed etiolated and short-roots.The 16S rRNA precursors were accumulated in Atemb15ΔC and Atemb15,whereas this phenomenon was not detected in the emb15 overexpressing EMB15N.These results suggest that AtEMB 15 and its C-terminal domain both function in the assembly of plastid 30S subunit,which is different from the function of EMB15 C-terminus in maize.Evolutionary analysis showed that the N-terminal domain of EMB15 originated from prokaryotes and the C-terminal domain originated from fungi.The two proteins fuse in charophyceae,the ancestor of land plants,suggesting that the fusion might benefit the adaptation of land plants to the terrestrial environment.After a series of stress treatments,we found that anthocyanin accumulation of Atemb15 and Atemb15ΔC is insufficient under cold stress.These results suggests that AtEMB15 may participate in the response to cold stress by affecting anthocyanin accumulation.The detailed molecular mechanism are to be further investigated.