A tissue-specific profile of miRNAs and their targets related to paeoniaflorin and monoterpenoids biosynthesis in Paeonia lactiflora Pall. by transcriptome, small RNAs and degradome sequencing

Abstract

Paeonia lactiflora Pall. (Paeonia) has aroused many concerns due to its extensive medicinal value, in which monoterpene glucoside paeoniflorin and its derivatives are the active chemical components. However, little is known in the molecular mechanism of monoterpenoids biosynthesis, and the regulation network between small RNAs and mRNAs in monoterpenoids biosynthesis has not been investigated yet. Herein, we attempted to reveal the tissue-specific regulation network of miRNAs and their targets related to paeoniaflorin and monoterpenoids biosynthesis in Paeonia by combining mRNA and miRNA expression data with degradome analysis. In all, 289 miRNAs and 30177 unigenes were identified, of which nine miRNAs from seven miRNA families including miR396, miR393, miR835, miR1144, miR3638, miR5794 and miR9555 were verified as monoterpenoids biosynthesis-related miRNAs by degradome sequencing. Moreover, the co-expression network analysis showed that four monoterpenoids-regulating TFs, namely AP2, MYBC1, SPL12 and TCP2, were putatively regulated by five miRNAs including miR172, miR828, miR858, miR156 and miR319, respectively. The present study will improve our knowledge of the molecular mechanisms of the paeoniaflorin and monoterpenoids biosynthesis mediated by miRNA to a new level, and provide a valuable resource for further study on Paeonia.

Fig 1. Quantitative analysis of paeoniflorin in different tissues of Paeonia.

(A Sampling process and four different tissues of Paeonia, B The contents of paeoniflorin in four different tissues, a represents significant differences at p<0.05 by comparing root, b represents significant differences at p<0.05 by comparing leaf).

Fig 2. Tissue-specifically expressed genes in Paeonia.

(A The spatial expressing patterns of genes in four different tissues, B Number of differentially expressed genes between different tissues).

Fig 3. Hierarchical cluster analysis of DEGs involving in the monoterpenoids biosynthesis pathways in different tissues of Paeonia.

(A The DEGs in terpenoid backbone and monoterpenoids biosynthesis pathway, B The DEGs in limonene and pinene degradation pathway, C The DEGs in transport pathway).

Fig 4. Genetic regulation network between transcriptional factors (TFs: Round nodes) and monoterpenoids biosynthesis genes (TPs: Round rectangle nodes).

(Edges denote the association between nodes and the width of every edge denotes normalized co-expressing weight. Red edges represent high association between screened TFs and TPs. green round rectangles represent terpenoid backbone biosynthesis genes, yellow round rectangles represent monoterpenoids synthase genes and blue round rectangles represent the genes associated with post-modification stage of monoterpenoids biosynthesis).

Fig 5. Length distributions of unique miRNAs.

Fig 6. Quantity of distinct members presenting in conserved miRNA families in Paeonia.

Fig 7. Tissue-specifically expressed miRNAs in Paeonia.

(A The spatial expressing patterns of the miRNAs in four different tissues of Paeonia, B Differential expressing levels of miRNAs in four different tissues of Paeonia).

Fig 8. Annotation and enrichment analysis of targets for miRNAs.

(A GO enrichment analysis of predicted targeted genes, B KEGG enrichment analysis of predicted targeted genes).

Fig 9. MiRNAs regulatory network.

(A Network of relationship between miRNAs and targeted genes related to monoterpenoids biosynthesis, B Network of relationship between DEMs and targeted genes, Yellow squares represent target genes and purple circles represent miRNAs).

Fig 10. A combined views of the expressing level between miRNAs and their targets with differential expression in Paeonia.

(A and B showed the one-to-one mapping correlation by DEMs in Paeonia, C and D showed the one-to-one mapping correlation by DEGs in monoterpenoids biosynthesis pathway of Paeonia).

Fig 11. Expression levels of 12 miRNAs.

(The bar charts and linear diagrams indicate the RT-qPCR and TPM data of the miRNAs, separately. The R² value denotes the correlation between the RT-qPCR and TPM data).

Fig 12. Expressing levels of 11 targeted genes.

(The bar charts and linear diagrams indicate the RT-qPCR and FPKM data of the genes, separately. The R² value denotes the correlation between the RT-qPCR and FPKM data).

Fig 13. Paeoniflorin regulation mechanism of Paeonia.