Transcriptome-wide Analysis of Protein-coding Genes and Non-coding RNAs in Response to Nitrogen Supply Status in Rice

Abstract

Nitrogen is a key nutrient in plant growth and development, and usage of nitrogen fertilizer is directly involved in crop yields. To increase crop yield, the enormous amount of chemical nitrogen fertilizer has been used. However, limitation on nitrogen-use-efficiency of crop plant make amounts of fertilizer to be remained in soil, and leaching of those remaining nitrogen sources to river and ocean occur environmental problems, such as eutrophication. Non-coding RNAs, including miRNAs and long non-coding RNAs, are a class of RNAs that are not be translated into protein. In recent studies, it is known that some of those miRNAs and long non-coding RNAs function as key regulator and regulate expression level of its target gene through various molecular mechanisms. Moreover, most of those non-coding RNAs also respond to various stimuli and stresses, and they could control the changes of plant physiology in response to those stimuli and stresses. However, roles of non-coding RNAs on nitrogen supply status in rice have not been deeply studied yet. To comprehensively investigate and understand transcriptome-wide responses of rice to nitrogen starvation, multimodal RNA-Seq methods were applied: strand-specific RNA-Seq, small RNA-Seq, and 2P-Seq. Using strand-specific RNA-Seq, expression profiling and analysis of annotated genes including nitrogen metabolism-related genes were performed. Also, nitrogen-responsive miRNAs were identified in nitrogen-starved rice roots, and the relationship between representative nitrogen supply status-responsive miRNA, miR169, and its target genes, NF-YAs, were analyzed by expression pattern analysis and 5 RLM-RACE. Using strand-specific RNA-Seq dataset, identification of putative lncRNA-deriving loci and analysis of their expression patterns were performed. Moreover, identifying 3 end of transcripts was tried by analyzing 2P-Seq data and those sites detected by 2P-Seq were confirmed by 3 RACE against putative lncRNA candidate. Dataset analyzed in this study will be used as a fundamental dataset for further studies about roles of genes and non-coding RNAs in response of rice to change of nitrogen supply status.