mRNA and miRNA Expression Analysis Reveal the Regulation for Flower Spot Patterning in Phalaenopsis 'Panda'

Abstract

Phalaenopsis cultivar 'Panda' is a beautiful and valuable ornamental for its big flower and unique big spots on the petals and sepals. Although anthocyanins are known as the main pigments responsible for flower colors in Phalaenopsis, and the anthocyanins biosynthetic pathway in Phalaenopsis is generally well known, the detailed knowledge of anthocynins regulation within the spot and non-spot parts in 'Panda' flower is limited. In this study, transcriptome and small RNA libraries analysis from spot and non-spot sepal tissues of 'Panda' were performed, and we found PeMYB7, PeMYB11, and miR156g, miR858 is associated with the purple spot patterning in its sepals. Transcriptome analyses showed a total 674 differentially expressed genes (DEGs), with 424 downregulated and 250 upregulated (Non-spot-VS-Spot), and 10 candidate DEGs involved in anthocyanin biosynthetic pathway. The qPCR analysis confirmed that seven candidate structure genes (PeANS, PeF3'H, PeC4H, PeF3H, PeF3H1, Pe4CL2, and PeCHI) have significantly higher expressing levels in spot tissues than non-spot tissues. A total 1552 differentially expressed miRNAs (DEMs) were detected with 676 downregulated and 876 upregulated. However, microRNA data showed no DEMs targeting on anthocyanin biosynthesis structure gene, while a total 40 DEMs target transcription factor (TF) genes, which expressed significantly different level in spot via non-spot sepal, including 2 key MYB regulator genes. These results indicated that the lack of anthocyanidins in non-spot sepal may not directly be caused by microRNA suppressing anthocyanidin synthesis genes rather than the MYB genes. Our findings will help in understanding the role of miRNA molecular mechanisms in the spot formation pattern of Phalaenopsis, and would be useful to provide a reference to similar research in other species.

Keywords: Phalaenopsis; anthocyanin biosynthesis; microRNA; molecular mechanism; transcriptome.

Figure 1

Anatomical structures and anthocyanin content of Phalaenopsis ‘Panda’ sepal. (A) Sepal (bar = 1 cm); (B) Upper epidermal cell in spot area (bar = 10 μm); (C) Upper epidermal cell in non-spot area (bar = 10 μm); (D) UV-visible spectrophotometer scanning of non-spot area; (E) UV-visible spectrophotometer scanning of spot area. Red number of 531 nm represents the absorption wavelength of anthocyanin.

Figure 2

Volcano plot of differentially expressed genes (DEGs). X axis: log2 transformed fold change; Y axis: −log10 transformed significance; Red points: upregulated DEGs; Blue points: downregulated DEGs. Gray points: non-DEGs.

Figure 3

The most enrichment pathway of DEGs (TOP20). X axis: enrichment factor; Y axis: pathway name; The color: the q-value (high: white, low: blue), the lower q-value indicates the more significant enrichment; Point size: DEG number (The bigger dots refer to larger amount); Rich Factor: the value of enrichment factor, which is the quotient of foreground value (the number of DEGs) and background value (total Gene amount), the larger the value, the more significant enrichment. Red arrows represent the pathways related directly to the anthocyanin biosynthesis.

Figure 4

Heatmap of differentially expressed structure genes (DEGs) related to anthocyanin biosynthesis in Phalaenopsis. Bold arrow means up/downregulations of genes. (PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumarate--CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, Flavonoid 3’-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, Anthocyanidin synthase; DHK, dihydrokaempferol; DHQ, dihydroquercetin; DHM, dihydromyricetin; LC, leucocyanidin; Cy, cyaniding).

Figure 5

Volcano plot of DEMs. X axis represents log2 transformed fold change. Y axis represents −log10 transformed significance. Red points represent upregulated DEMs. Blue points represent downregulated DEMs. Gray points represent non-DEMs.

Figure 6

The expression level of structure gene and regulate gene Associated with Anthocyanin Biosynthesis, * represent there are significant differences between spot and non-spot area; ** represent there are extremely significant differences; red: spot area, blue: non-spot area.

Figure 7

The expression level of mtr-miR156g-3p and miR858. ** represent an extremly significant difference (p < 0.01); * represent an significant difference (p < 0.05).

Figure 8

Identification of PeMYB7, PeMYB11 and their target sites by miR156g, miR858. (A) Phylogenetic tree inferred from the MYB sequences of Phalaenopsis equestris and Oryza sativa; (B) Identification of target sites of miR156 and miR858 in PeMYB7 and PeMYB11.

Figure 9

A proposed model summarizing of spot formation pattern in Phalaenopsis ‘Panda’ sepal. The purple segment illustrates the accumulation of anthocyanidin in spot area of ‘Panda’. MIR156g, MIR858 target on key regulate genes PeMYB7 and PeMYB11, suppressed the transcript level of Pe4CL2, PeF3H, PeF3′H, PeANS and resulted in reduced anthocyanidin production in the non-spot area, while relatively lower expression levels of MIR156g and MIR858 and high levels of transcription of these genes in spot area cause accumulation of anthocyanidin. The black dashed arrows represent low levels of transcription, while the black solid-line arrows represent high levels of transcription. The yellow solid-line arrows represent promoting transcription, while yellow dashed arrow represent lack of promotion function. The blue bold solid-line T-arrow represents interfering transcription, while blue bold dashed arrow represents lack of interference function. The red bold arrow represents upregulation, and the green bold arrow represents downregulation. DHK, dihydrokaempferol; DHQ, dihydroquercetin; DHM, dihydromyricetin; LC, leucocyanidin; LD, leucodelphinidin; Cy, cyanidin; De, delphinidin.

Figure 10

The flower tissue of Phalaenopsis ‘Panda’. Bars = 1 cm. (A) Full bud; (B) Sepal; I. spot area; II. non-spot area. (C) Full bloom stage.