5-Methylcytosine (m5C) is a well-characterized DNA modification,and is also predominantly reported in abundant non-coding RNAs in both prokaryotes and eukaryotes.However,the distribution and biological functions of m5C in plant mRNAs remain largely unknown.Here,we report transcriptome-wide profiling of RNA m5C in Arabidopsis thaliana by applying m5C RNA immunoprecipitation followed by a deepsequencing approach (m5C-RIP-seq).LC-MS/MS and dot blot analyses reveal a dynamic patte of m5C mRNA modification in various tissues and at different developmental stages.m5C-RIP-seq analysis identified 6045 m5C peaks in 4465 expressed genes in young seedlings.We found that m5C is enriched in coding sequences with two peaks located immediately after start codons and before stop codons,and is associ ated with mRNAs with low translation activity.We further demonstrated that an RNA (cytosine-5)-methyltransferase,tRNA-specific methyltransferase 4B (TRM4B),exhibits m5C RNA methyltransferase activity.Mutations in TRM4B display defects in root development and decreased m5C peaks.TRM4B affects the transcript levels of the genes involved in root development,which is positively correlated with their mRNA stability and m5C levels.Our results suggest that m5C in mRNA is a new epitranscriptome marker inArabidopsis,and that regulation of this modification is an integral part of gene regulatory networks un derlying plant development.