Analysis of miRNA-mediated regulation of flowering induction in Lilium × formolongi

Abstract

Background: MicroRNAs play pivotal roles in plant vegetative phase change and flowering induction via integrating into multiple flowering pathways. Lilium × formolongi is an important ornamental lily cultivar that can flower within one year after sowing. However, it remains unresolved how miRNA-mediated regulation networks contribute to the L. × formolongi characteristics of a short vegetative growth period and rapid flowering.

Results: In this study, the small RNA libraries and one degradome library were constructed for L. × formolongi during vegetative growth and flowering initiation, and 366 conserved miRNAs and 32 novel miRNAs were identified. Additionally, 84 miRNAs were significantly differentially expressed during development. A total of 396 targets of 185 miRNAs were identified and validated through degradome sequencing. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that functions of the targets were top enriched in the cold and cadmium ion responses, pentose phosphate pathway and carbon fixation in photosynthetic organisms. Furthermore, among 23 differentially expressed miRNA-target pairs, the miR156s-LfSPL2, miR172a-LfAP2 and miR164a-LfNAC pairs as well as miR159a-LfSPL2 were found to be relevant to flowering based on the correlation analysis of expression profiles in the miRNA libraries, degradome and transcriptome. A coexpression regulatory network focused on differentially expressed pairs was also constructed by WGCNA, and 14 miRNAs were considered putative key miRNAs during vegetative development and flowering induction. miR156a/ d/ e showed particularly strong relationships with other miRNAs in the coexpression network.

Conclusions: This study provides cues for the further exploration of the regulatory mechanisms of short vegetative development and flowering in L. × formolongi.

Keywords: Development; Flowering; Lilium × formolongi; miRNA.

Fig. 1

Length distribution of small RNAs sequences, identification of known miRNA families from L. × formolongi. a Length distribution of total reads and unique reads in five libraries. b Distribution of known miRNA family members. c Counts of each known miRNA family

Fig. 2

Heat map showing the expression patterns of differentially expressed miRNAs. The miRNAs were clustered by hierarchical clustering at p ≤ 0.05 according to their expression patterns during development in L. × formolongi

Fig. 3

Target plots of miRNA targets identified by degradome sequencing in lily. a-d represent miR5179, miR160a, miR164a and miR172a, respectively. Arrows represent the nucleotide position of cleavage in the target genes

Fig. 4

Gene functional classification of identified target genes. a Gene ontology enrichment and classification of target genes. b KEGG pathway analysis

Fig. 5

mRNAs simultaneously targeted by miRNAs belonging to multiple families. a Diagram of LfSPL2 and LfGAMYB. Conserved domains are included, and the boxes represent the miRNA target sites. b Alignment of the miRNA targeting sites of miR156s-LfSPL2, miR159a-LfSPL2 and miR159a/miR319f-LfGAMYB

Fig. 6

Heat map showing the expression patterns of differentially expressed miRNA-target. These miRNAs targeting differentially expressed mRNAs were clustered by hierarchical clustering at p ≤ 0.05 according to their expression patterns. The black-framed portion represents the expression level of miRNA and mRNA that showed negative correlation relationship at the expression level

Fig. 7

The expression patterns of miRNAs determined by RT-qPCR during development in L. × formolongi. a-j The expression patterns of miRNAs and their targets. k-l The tissue-specific expression profiles of miR159a and miR160a during lily development processes. The normalized miRNA and target levels at the VJ_I stage were arbitrarily set to 1. Each bar represents the mean ± SE of triplicate assays. * or ** indicates a statistically significant difference relative to the value at VJ_ I for each miRNA at p < 0.05 or 0.01, respectively

Fig. 8

The coexpression subnetwork of putative crucial flowering-related miRNAs and their targets. The coexpression subnetwork of putative crucial flowering-related miRNAs and their targets in lily. In the nodes, circles and diamonds represent miRNAs and target transcripts, respectively. Different colours of nodes represent different modules identified by WGCNA

Fig. 9

The putative miRNA-mediated molecular regulation network of flowering induction and development of L. × formolongi. The locations of the genes are determined by their expression patterns. Genes in green background displayed expression peak at the VJ stage, genes in the yellow and blue background displayed up-regulated expression at the FI stage and FD stage respectively. The numbers of differentially expressed genes are also presented. Arrows in dotted lines represents the potential regulatory relationship between two genes