Previously, several long non-coding RNAs (lncRNAs) were characterized as regulators in phosphate (Pi) starvation responses. However, systematic studies of novel lncRNAs involved in the Pi starvation signaling pathways have not been reported.
Here, researchers from Tsinghua University used a genome-wide sequencing and bioinformatics approach to identify both poly(A) + and poly(A)- lncRNAs that responded to Pi starvation in Arabidopsis thaliana. They sequenced shoot and root transcriptomes of the Arabidopsis seedlings grown under Pi-sufficient and Pi-deficient conditions, and predicted 1212 novel lncRNAs, of which 78 were poly(A)- lncRNAs. By employing strand-specific RNA libraries, the researchers discovered many novel antisense lncRNAs for the first time. They further defined 309 lncRNAs that were differentially expressed between P+ and P- conditions in either shoots or roots. Through Gene Ontology enrichment of the associated protein-coding genes (co-expressed or close on the genome), they found that many lncRNAs were adjacent or co-expressed with the genes involved in several Pi starvation related processes, including cell wall organization and photosynthesis. In total, they identified 104 potential lncRNA targets of PHR1, a key regulator for transcriptional response to Pi starvation. Moreover, they identified 16 candidate lncRNAs as potential targets of miR399, another key regulator of plant Pi homeostasis.
Flowchart of identification of lncRNAs responsive
to Pi starvation in Arabidopsis
1. Plant treatment and poly(A) + and poly(A)– RNA extractions and purifications. 2. Construction of strand-specific cDNA libraries and sequencing. 3. RNA-Seq data mapping and assembly. 4. Novel lncRNAs were obtained after three filter steps, including overlap with annotation, calculation of length, and coding potential. 5. Characterization of novel lncRNAs at different levels, such as transcript length, exon number, polyadenylation, expression, epigenetic signature, and transcriptional and post-transcriptional regulation.