Transcriptomics analyses reveal the key genes involved in stamen petaloid formation in Alcea rosea L

Abstract

Alcea rosea L. is a traditional flower with a long cultivation history. It is extensively cultivated in China and is widely planted in green belt parks or used as cut flowers and potted ornamental because of its rich colors and flower shapes. Double-petal A. rosea flowers have a higher aesthetic value compared to single-petal flowers, a phenomenon determined by stamen petaloid. However, the underlying molecular mechanism of this phenomenon is still very unclear. In this study, an RNA-based comparative transcriptomic analysis was performed between the normal petal and stamen petaloid petal of A. rosea. A total of 3,212 differential expressed genes (DEGs), including 2,620 up-regulated DEGs and 592 down-regulated DEGs, were identified from 206,188 unigenes. Numerous DEGs associated with stamen petaloid were identified through GO and KEGG enrichment analysis. Notably, there were 63 DEGs involved in the plant hormone synthesis and signal transduction, including auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinosteroid, jasmonic acid, and salicylic acid signaling pathway and 56 key transcription factors (TFs), such as MADS-box, bHLH, GRAS, and HSF. The identification of these DEGs provides an important clue for studying the regulation pathway and mechanism of stamen petaloid formation in A. rosea and provides valuable information for molecular plant breeding.

Keywords: Alcea rosea L.; Flower morphology; Plant hormone; Stamen petaloid; Transcriptomics.

Fig. 1

Torch-type flower and petal of A. rosea. (a) Torch-type flower of A. rosea. (b) Stamen petaloid petals (PP). (c) Normal petals (NP)

Fig. 2

Annotations of unigenes. (a) Statistics of e-value distribution in the NR database. (b) Statistics of similarity distribution in the NR database. (c) GO functional annotation. (d) KEGG classification

Fig. 3

Gene expression comparisons. (a) Volcano map of DEGs. The horizontal ordinate denotes the multiple change value of unigenes’ expression difference in PP vs. NP; The vertical ordinate denotes the statistical test value of unigenes’ expression difference in PP vs. NP, that is, the p-value. The dots represent the unigenes: the red dots indicate up-regulation, the green dots indicate down-regulation, and the gray dots indicate no difference. (b) Clustering heat map of DEGs.The top is the tree diagram of unigenes clustering. The shorter the distance between the two unigene branches, the closer the expression level. On the left is the tree diagram of sample clustering, and on the right is the name of the sample

Fig. 4

Functional enrichment analysis of DEGs. (a) GO enrichment analysis of DEGs. (b) KEGG Pathway enrichment of DEGs. The X-axis is the enrichment rate, and the formula for calculating the Enrich factor is GeneRatio/BgRatio. The Y-axis represents the GO/KEGG term, which belongs to a classification described as the Class legend information on the right. Each dot represents a GO/KEGG term; the larger the dot, the more differentially expressed the genes are

Fig. 5

A. rosea stamen petaolid associated DEGs heat map clustering (a) Heat map of unigenes involved in plant hormone biosynthesis and signal transduction pathway. (b) Heat map of transcription factors associated with stamen petaloid. The bar represents the scale of the expression levels for each gene (log₁₀RPKM) in PP vs. NP. The red bars represent up-regulated genes, the green bars represent down-regulated genes, and the black bars represent genes that do not differ significantly

Fig. 6

Analysis of plant hormone biosynthesis and signal transduction pathways. (a) Auxin signaling pathway. (b) Cytokinin signaling pathway. (c) Gibberellin signaling pathway. (d) Abscisic acid signaling pathway. (e) Ethylene signaling pathway. (f) Brassinosteroid signaling pathway. (g) Jasmonic acid signaling pathway. (h) Salicylic acid signaling pathway. The bar represents the scale of the expression levels for each gene (log₁₀RPKM) in PP vs. NP. The red bars represent up-regulated genes, while the green bars represent down-regulated genes

Fig. 7

Validation of A. rosea stamen petaloid identified DEGs. (a) Relative expression levels of the twelve identified DEGs by qRT-PCR (bar chart, left Y-axes) and by FPKMs (lines, right Y-axes). 18s rRNA gene was used as reference for relative expression measurement in both qRT-PCR and RNAseq (FPKMs). Error bars indicate the standard deviation of three independent replicates (in qRT-PCR). (b) Fold-change value correlation analysis. RNA-seq fold change refers to the ratios of FPKM values of PP to NP, while qRT-PCR fold change is the relative quantity of PP normalized to the expression level of NP