N6- methyladenosine (m6A) detection in ammonium treated seedlings through RNA immunoprecipitation

Epitranscriptomics modifications constitute a gene expression checkpoint in all living organism including plants. Considering the relevance of nitrogen nutrition and metabolism for the correct plant growth and development, it can be hypothesized that epitranscriptome changes must regulate every biological process in plants including nitrogen nutrition. In the present work, the epritranscriptomics changes in maritime pine roots caused by ammonium nutrition have been monitored through RIP-seq. The main transcriptome responses to ammonium nutrition affected to transcripts involved in nitrogen and carbon metabolisms, defense response, hormone synthesis and signaling, and translation. The m6A deposition and its dynamics have been identified, which seems to be important regulators of translation when compared with the proteomic profiles of the same samples. The present study suggest that the epitranscriptome must modulate the responses to development and environmental changes, including ammonium nutrition, through buffering, filtering and focusing the final products of the gene expression. Overall design: Ammonium nutrition was studied at short times (24 hours) in roots from one month-old seedlings of maritime pine. Control seedlings were irrigated with 80 mL of water (C) and the experimental seedlings with 80 mL of 3 mM NH4Cl. Root samples were collected at 24 hours post-irrigation and immediately frozen in liquid N. This experiment was carried out three independent times. The polyA-RNA was isolated, immunoprecipited (m6A) and sequenced with Illumina technology.