Integrated mRNA and microRNA transcriptome variations in the multi-tepal mutant provide insights into the floral patterning of the orchid Cymbidium goeringii

Abstract

Background: Cymbidium goeringii is a very famous traditional orchid plant in China, which is well known for its spectacular and diverse flower morphology. In particular, the multi-tepal mutants have considerable ecological and cultural value. However, the current understanding of the molecular mechanisms of floral patterning and multi-tepal development is limited. In this study, we performed expression profiling of both microRNA (miRNA) and mRNA from wild-type and typical multi-tepal-mutant flowers of C. goeringii for the first time, to identify the genes and pathways regulating floral morphogenesis in C. goeringii.

Results: Total clean reads of 98,988,774 and 100,188,534 bp were obtained from the wild-type and mutant library, respectively, and de novo assembled into 98,446 unigenes, with an average length of 989 bp. Among them, 18,489 were identified as differentially expressed genes between the two libraries according to comparative transcript profiling. The majority of the gene ontology terms and Kyoto Encyclopedia of Genes and Genomes pathway enrichment responses were for membrane-building and ploidy-related processes, consistent with the excessive floral organs and altered cell size observed in the mutant. There were 29 MADS-box genes, as well as a large number of floral-related regulators and hormone-responsive genes, considered as candidates regulating floral patterning of C. goeringii. Small RNA sequencing revealed 132 conserved miRNA families expressed in flowers of C. goeringii, and 11 miRNAs corresponding to 455 putative target genes were considered to be responsible for multi-tepal development. Importantly, integrated analysis of mRNA and miRNA sequencing data showed two transcription factor/microRNA-based genetic pathways contributing to the multi-tepal trait: well-known floral-related miR156/SPL and miR167/ARF regulatory modes involved in reproductive organ development; and the miR319/TCP4-miR396/GRF regulatory cascade probably regulating cell proliferation of the multi-tepal development.

Conclusions: Integrated mRNA and miRNA profiling data provided comprehensive gene expression information on the wild-type and multi-tepal mutant at the transcriptional level that could facilitate our understanding of the molecular mechanisms of floral patterning of C. goeringii. These data could also be used as an important resource for investigating the genetics of floral morphogenesis and various biological mechanisms of orchid plants.

Keywords: Cymbidium goeringii; Floral patterning; Floral transcriptome; MiR396; MicroRNA; Multi-tepal mutant.

Fig. 1

Flower morphology of C. goeringii wild-type plant ‘Songmei’ and the multi-tepal mutant ‘Yuhudie’. a & b: Wild-type flower with three sepals and three petals. Two petals are similar to each other; the third is highly evolved, with an ovate to triangular shape, and is known as the labellum or lip. The male and female reproductive organs are highly fused to form a gynostemium. c: The gynostemium is replaced by newly emerged flowers in the multi-tepal mutant, and this ectopic flower continues to produce sepals and petals centripetally. d & e: Detailed compositions of two of the newly emerged flowers (indicated with red circle in c). The lips are misshapen; the gynostemia are fused to the margin of the sepals and lack an organized four-pollinia structure. Bar = 1 mm. Se, sepal; Pe, petal; Li, lip; Gy, Gynostemium

Fig. 2

COG function classification of assembled unigenes

Fig. 3

GO classification of assembled unigenes

Fig. 4

Predicted transcription factors of C. goeringii

Fig. 5

GO classification of unigenes differentially expressed between the wild-type and the mutant

Fig. 6

Comparative analysis of transcriptome sequencing based expression data with the real-time RT-PCR expression data. a: Normalized fold expression of the MADS-box genes: FPKM value of each individual gene normalized with the putative ubiquitin gene. b: real-time RT-PCR analysis of MADS-box gene expression in the wild-type and the mutant (normalized to the expression of Ubiquitin). The y-axis indicates fold change in expression among the samples. Error bars indicate the standard deviation of the mean (SD) (n = 3). Three replicates were analyzed, with similar results

Fig. 7

Transcription factors differentially expressed between the wild-type and the mutant. a: Number of up-regulated (blue) and down-regulated (red) transcripts were quantified in the mutant compared to the wild-type (based on the FPMK value with |log₂(fold change)| > 1 and Q-value < 0.05). b: percentage of up- and down-regulated genes after normalization to the total number of family members

Fig. 8

DEG-enrichment of the plant hormone signal transduction pathway by KEGG annotation. The key regulatory components in multiple hormone response pathways are presented as their names (red, up-regulated; green, down-regulated). Gene IDs and fold changes in transcript abundance are indicated in Additional file 4

Fig. 9

Comparison of complementarity profiles for miR396 with GRF-like genes. Free energies of the duplex structures were calculated using RNA hybrid software (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid)

Fig. 10

Sequence complementary to miR396 in four of the GRF-like genes. The comparison of complementary site of miR396 to GRF-like genes is indicated by the red frame

Fig. 11

Transcript levels of miR396 (a) and GRF-like genes (b) in different floral organs of C. goeringii ‘Songmei’ (WT) and the multi-tepal mutant ‘Yuhudie’ (Mu) using a real-time RT-PCR assay. The ubiquitin gene served as the internal control. Error bars indicate the standard deviation of the mean (SD) (n = 3). Three replicates were analyzed, with similar results

Fig. 12

Transcript levels of miR319 (a) and TCP-like genes (b) in different floral organs of C. goeringii ‘Songmei’ (WT) and the multi-tepal mutant ‘Yuhudie’ (Mu) using a real-time RT-PCR assay. The ubiquitin gene served as the internal control. Error bars indicate the standard deviation of the mean (SD) (n = 3). Three replicates were analyzed, with similar results

Fig. 13

Comparison of C. goeringii floral transcriptome similarity with C. sinense and C. ensifolium. a: Similarity search of C. goeringii sequences against C. ensifolium and C. sinense sequences. b. Functional classification of unigenes unique to C. goeringii

Fig. 14

MiRNA/transcription factor networks contributing to the multi-tepal trait of C. goeringii