Identification and Functional Prediction of Poplar Root circRNAs Involved in Treatment With Different Forms of Nitrogen

Abstract

Circular RNAs (circRNAs) are a class of noncoding RNA molecules with ring structures formed by covalent bonds and are commonly present in organisms, playing an important regulatory role in plant growth and development. However, the mechanism of circRNAs in poplar root responses to different forms of nitrogen (N) is still unclear. In this study, high-throughput sequencing was used to identify and predict the function of circRNAs in the roots of poplar exposed to three N forms [1 mM NO₃ ^- (T1), 0.5 mM NH₄NO₃ (T2, control) and 1 mM NH₄ ⁺ (T3)]. A total of 2,193 circRNAs were identified, and 37, 24 and 45 differentially expressed circRNAs (DECs) were screened in the T1-T2, T3-T2 and T1-T3 comparisons, respectively. In addition, 30 DECs could act as miRNA sponges, and several of them could bind miRNA family members that play key roles in response to different N forms, indicating their important functions in response to N and plant growth and development. Furthermore, we generated a competing endogenous RNA (ceRNA) regulatory network in poplar roots treated with three N forms. DECs could participate in responses to N in poplar roots through the ceRNA regulatory network, which mainly included N metabolism, amino acid metabolism and synthesis, response to NO₃ ^- or NH₄ ⁺ and remobilization of N. Together, these results provide new insights into the potential role of circRNAs in poplar root responses to different N forms.

Keywords: Populus × canescens; ceRNA regulatory network; circular RNAs; different nitrogen forms; roots.

Figure 1

Genomic features of identified circRNAs in the roots of P. × canescens treated with NO₃⁻(T1), NH₄⁺NO₃⁻(T2), and NH₄⁺(T3) conditions for 21 days. (A) The expression level of circRNAs along the 19 chromosomes. (B) Chromosomal distribution of the three types (exonic, intronic, and intergenic circRNAs). (C) The proportion of the three types of identified circRNAs. (D) Percentage of hosting protein-coding genes that generated different circRNA candidates. (E) Length distribution of circRNAs. (F) The number of exons per circRNA.

Figure 2

Validation of circRNAs in the roots of P. X canescens. (A,C) A detailed depiction of two circRNA circularization and Sanger sequence validation using divergent primers. (B,D) PCR amplification results for four predicted circRNAs in cDNA and genomic DNA samples. Convergent and divergent primer sets worked on both cDNA and genomic DNA. M: marker.

Figure 3

Statistical analysis of identified DECs in Tl, T2, and T3 of P. × canescens. (A) The number of upregulated and downregulated DECs in each comparison; (B) Venn diagram analysis of DECs; (C) heatmap of DECs; (D) Quantitative RT-qPCR validation of DECs.

Figure 4

The DEC–miRNA–DEmRNA networks in the roots of P. × canescens treated with Tl, T2 and T3. Diamond, triangle, and circular nodes represent circRNAs, miRNAs, and mRNAs, respectively.

Figure 5

DECs and their possibly regulated mRNAs in the ceRNA network were verified by RT–qPCR in P. × canescens root treated with T1, T2 and T3. (A): RT–qPCR results of T1 vs. T2. (B): RT–qPCR results of T3 vs. T2. (C): RT–qPCR results of T1 vs. T3.

Figure 6

A schematic model of the ceRNA regulatory network involved in the P. × canescens root response to different N forms.