Abstract
BackgroundNitrogen (N) is a key macronutrient essential for plant growth, and its availability has a strong influence on crop development. The application of synthetic N fertilizers on crops has increased substantially in recent decades; however, the applied N is not fully utilized due to the low N use efficiency of crops. To overcome this limitation, it is important to understand the genome-wide responses and functions of key genes and potential regulatory factors in N metabolism.ResultsHere, we characterized changes in the rice (Oryza sativa) transcriptome, including genes, newly identified putative long non-coding RNAs (lncRNAs), and microRNAs (miRNAs) and their target mRNAs in response to N starvation using four different transcriptome approaches. Analysis of rice genes involved in N metabolism and/or transport using strand-specific RNA-Seq identified 2588 novel putative lncRNA encoding loci. Analysis of previously published RNA-Seq datasets revealed a group of N starvation-responsive lncRNAs showing differential expression under other abiotic stress conditions. Poly A-primed sequencing (2P-Seq) revealed alternatively polyadenylated isoforms of N starvation-responsive lncRNAs and provided precise 3′ end information on the transcript models of these lncRNAs. Analysis of small RNA-Seq data identified N starvation-responsive miRNAs and down-regulation of miR169 family members, causing de-repression of NF-YA, as confirmed by strand-specific RNA-Seq and qRT-PCR. Moreover, we profiled the N starvation-responsive down-regulation of root-specific miRNA, osa-miR444a.4-3p, and Degradome sequencing confirmed MADS25 as a novel target gene.ConclusionsIn this study, we used a combination of multiple RNA-Seq analyses to extensively profile the expression of genes, newly identified lncRNAs, and microRNAs in N-starved rice roots and shoots. Data generated in this study provide an in-depth understanding of the regulatory pathways modulated by N starvation-responsive miRNAs. The results of comprehensive, large-scale data analysis provide valuable information on multiple aspects of the rice transcriptome, which may be useful in understanding the responses of rice plants to changes in the N supply status of soil.
Highlights
Nitrogen (N) is a key macronutrient essential for plant growth, and its availability has a strong influence on crop development
All samples were analyzed by strand-specific RNA-Seq and small RNA-Seq. Poly A-primed sequencing (2P-Seq) and Degradome sequencing were applied to selected samples based on the analysis of the strand-specific RNA-Seq and small RNA-Seq datasets
These reads were used for expression profiling of International Rice Genome Sequencing Project (IRGSP)-annotated genes (RAP-DB), and identifying previously unannotated putative long non-coding RNAs (lncRNAs) based on the reference annotationbased transcript (RABT) assembly using Cufflinks [42]
Summary
Nitrogen (N) is a key macronutrient essential for plant growth, and its availability has a strong influence on crop development. The application of synthetic N fertilizers on crops has increased substantially in recent decades; the applied N is not fully utilized due to the low N use efficiency of crops. To overcome this limitation, it is important to understand the genome-wide responses and functions of key genes and potential regulatory factors in N metabolism. Nitrogen (N) is a key macronutrient for plants and has a strong influence on crop development and productivity. Improving crop NUE while maintaining crop productivity has several economic and environmental benefits
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