Integrated Analysis of microRNAs and Transcription Factor Targets in Floral Transition of Pleioblastus pygmaeus

Abstract

Bamboo plants have erratic flowering habits with a long vegetative growth and an uncertain flowering cycle. The process of floral transition has always been one of the hot and intriguing topics in bamboo developmental biology. As master modulators of gene expression at the post-transcriptional level, miRNAs play a crucial role in regulating reproductive growth, especially in floral transition of flowering plants. Pleioblastus pygmaeus is a kind of excellent ground cover ornamental bamboo species. In this study, we performed miRNA expression profiling of the shoot buds and flower buds from the bamboo species, to investigate flowering-related miRNAs in bamboo plants. A total of 179 mature miRNAs were identified from P. pygmaeus, including 120 known miRNAs and 59 novel miRNAs, of which 96 (61 known miRNAs and 35 novel miRNAs) were differentially expressed in the shoots at different growth stages. Based on target gene (TG) prediction, a total of 2099 transcription factors (TFs) were annotated to be TGs of the 96 differentially expressed miRNAs (DEMs), corresponding to 839 recordings of DEM-TF pairs. In addition, we identified 23 known DEMs involved in flowering and six known miRNAs related to floral organ development based on previous reports. Among these, there were 11 significantly differentially expressed miRNAs, with 124 TF targets corresponding to 132 DEM-TF pairs in P. pygmaeus. In particular, we focused on the identification of miR156a-SPL (SQUAMOSA Promoter-Binding protein-Like) modules in the age pathway, which are well-known to regulate the vegetative-to-reproductive phase transition in flowering plants. A total of 36 TF targets of miR156a were identified, among which there were 11 SPLs. The Dual-Luciferase transient expression assay indicated miR156a mediated the repression of the PpSPL targets in P. pygmaeus. The integrated analysis of miRNAs and TGs at genome scale in this study provides insight into the essential roles of individual miRNAs in modulating flowering transition through regulating TF targets in bamboo plants.

Keywords: Pleioblastus pygmaeus; age pathway; floral transition; miRNAs; transcription factors.

Figure 1

Identification and classification of sRNAs in P. pygmaeus. (A) The distribution of small RNA with 18–30 nt length in each sample. (B) The classification of annotated sRNAs in P. pygmaeus. F1_1–3, the shoot buds of flowering P. pygmaeus with three biological repeats; F2_1–3, the flower buds of flowering P. pygmaeus with three biological repeats; N1_1–3, the shoot buds of non-flowering P. pygmaeus with three biological repeats; N2_1–3, the leaf buds of non-flowering P. pygmaeus with three biological repeats.

Figure 2

The classification of miRNA families in P. pygmaeus.

Figure 3

Cluster heatmap of 61 differentially expressed known miRNAs. F1, the shoot buds of flowering P. pygmaeus; F2, the flower buds of flowering P. pygmaeus; N1, the shoot buds of non-flowering P. pygmaeus; N2, the leaf buds of non-flowering P. pygmaeus.

Figure 4

Expression pattern of miRNA156a and miRNA172b in Pleioblastus pygmaeus. F1, the shoot buds of flowering P. pygmaeus; F2, the flower buds of flowering P. pygmaeus; N1, the shoot buds of non-flowering P. pygmaeus; N2, the leaf buds of non-flowering P. pygmaeus. * means p < 0.05, ** means p < 0.01.

Figure 5

Link graph of miRNA156a and TF targets and phylogenetic analysis of 11 miRNA156a-targeting PpSPLs. (A) Link graph of miRNA156a and TF targets. Different colors represent different family genes. Red icon represents miRNA156a and green icons represent 11 putative PpSPLs. The circle size means the mRNA abundance of genes, the line distance means the regulatory role of miRNA156a in down-regulating TF targets. (B) Phylogenetic analysis of the 11 putative miRNA156a-targeting PpSPLs.

Figure 6

The correlation of miR156a with four putative PpSPL targets using the Dual-Luciferase transient expression assay. (A–D) Fluorescence scanning of tobacco leaves co-transforming miR156a with PpSPL12 (Cluster-10432.20508), PpSPL13 (Cluster-10432.12439), PpSPL14 (Cluster-10432.2297), and PpSPL16 (Cluster-10432.22213), respectively. Note: the same leaf injected with pGreenII-0800-PpSPL+ 35s-miR156-NOS are shown in the top left part, pGreenII-0800-PpSPL+ 35s-NOS in the top right part, pGreenII-0800+35s-miR156-NOS in the bottom left part, and pGreenII-0800+35s-NOS in the bottom right part. Bar = 1 cm.