Transcriptome analysis reveals the regulation of brassinosteroids on petal growth in Gerbera hybrida

Abstract

Gerbera hybrida is a cut-flower crop of global importance, and an understanding of the mechanisms underlying petal development is vital for the continued commercial development of this plant species. Brassinosteroids (BRs), a class of phytohormones, are known to play a major role in cell expansion, but their effect on petal growth in G. hybrida is largely unexplored. In this study, we found that the brassinolide (BL), the most active BR, promotes petal growth by lengthening cells in the middle and basal regions of petals, and that this effect on petal growth was greater than that of gibberellin (GA). The RNA-seq (high-throughput cDNA sequencing) technique was employed to investigate the regulatory mechanisms by which BRs control petal growth. A global transcriptome analysis of the response to BRs in petals was conducted and target genes regulated by BR were identified. These differentially expressed genes (DEGs) include various transcription factors (TFs) that were activated during the early stage (0.5 h) of BL treatment, as well as cell wall proteins whose expression was regulated at a late stage (10 h). BR-responsive DEGs are involved in multiple plant hormone signal pathways, hormone biosynthesis and biotic and abiotic stress responses, showing that the regulation of petal growth by BRs is a complex network of processes. Thus, our study provides new insights at the transcriptional level into the molecular mechanisms of BR regulation of petal growth in G. hybrida.

Keywords: Brassinolide; Gerbera hybrida; Petal growth; RNA-seq; Regulation.

Figure 1. The effects of BRs and GAs on petal growth in G. hybrida.

G. hybrida grew in a greenhouse under the conditions described in ‘Materials and Methods’. Plants with inflorescences at stage 2 were sprayed with deionized water (with 0.1% ethanol) (Control), 10 µM BL, 10 µM GA₃, 10µM BRZ, a mixture of 10 µM BL and 10 µM GA₃ (BL + GA₃), or a mixture of 10 µM BL and 10 µM BRZ (BL + BRZ) and were subjected to morphological characterization (A), then the petal length (B), width (C) and length/width (D) were measured and calculated after nine days of treatment. Five to six inflorescences were measured for each treatment (Scale bar represents 1 cm. Value = means ± SE, n > 10, letters above the bars indicate significant differences between the respective values (p < 0.05)). (E) Time-course dynamics of petal length under different treatments. Detached petals were used in this experiment, at least 10 petals for each treatment were cultured for nine days.

Figure 2. The effects of BRs and GAs on cell elongation of petals in G. hybrida.

Petals of inflorescences at stage 3 were detached and placed on double filter papers soaked with deionized water (with 0.1% ethanol) (Control), 10 µM BL, 10 µM GA₃, 10µM BRZ, BL + GA₃ or BL + BRZ for nine days (A). The petals were then used for morphological characterization of adaxial epidermal cell in three different regions (top, middle and basal) using a confocal microscope (B) and measurement of elongation rate of each petal region (C), (n = 10) or cell (D), (n > 100). Three biological replicates were performed for each measurement (Scale bar represents 1 cm (A) or 50 µm (B). Value = means ± SE, letters above the bars indicate significant differences between the respective values (p < 0.05)).

Figure 3. The elongation rates of petals and global analysis of transcript profiles in different treatments.

(A) Detached petals at stage 3 were placed on double filter papers soaked with 10 µM BL for 0.5, 0.75, 1, 2, 4, 10, 12 and 24 h, and then transferred to papers soaked with deionized water. Petals were cultured for a total of 72 h and then the elongation rates were measured. Control: petals kept on double filter papers soaked with deionized water for 72 h. BL: petals kept on double filter papers soaked with10 µM BL for 72 h. (The experiment was repeated at least three times. Value = means ± SE, letters above the bars indicate significant differences between the respective values (p < 0.05)) (B) Global transcript profiles of different treatments. T01: petals without treatment (Mock), T02: H₂O treatment for 0.5 h, T03: BL treatment for 0.5 h, T04: H₂O treatment for 10 h, T05: BL treatment for 10 h. Each box plot shows the distribution of the relative transcription level (log₁₀ (RPKM)) of genes with at least one read mapped to the transcriptome of G. hybrida in one sample. (C) Venn diagram showing the distribution of DEGs at 0.5 h or 10 h of BL treatment. (D) The number of DEGs in 0.5U 10U, 0.5U 10D, 0.5D 10U and 0.5D 10D. 0.5U 10U refers to DEGs up-regulated at both 0.5 h and 10 h, 0.5U 10D refers to DEGs up-regulated at 0.5 h and down-regulated at 10 h, 0.5D 10U refers to DEGs down-regulated at 0.5 h and up-regulated at 10 h, 0.5D 10D refers to DEGs down-regulated at both 0.5 h and 10 h.

Figure 4. Heat-maps of DEGs involved in “plant hormone signal transduction” and “biotic and abiotic stress”.

(A) DEGs involved inplant hormone signal transduction”. (B) DEGs involved in “biotic and abiotic stress”. The bar represents the scale of the expression levels for each gene (log₁₀RPKM) in different samples as indicated by green/red rectangles. Green indicates down-regulation of genes, red indicates up-regulation and no change is indicated in black.

Figure 5. DEGs expression profiles and the heatmaps of some selected DEGs in their profiles.

(A) Class I indicated a down-regulated trend during 0–0.5 h of BL treatment and a gradual trend in H₂O treatment. Class II indicated an up-regulated trend during 0–0.5 h of BL treatment and a gradual trend in H₂O treatment. (B) Heatmap of DEGs selected from Class II and their annotations. (C) Class III indicated a down-regulated trend during 0.5 h–10 h of BL treatment and a gradual trend in H₂O treatment. Class IV indicated an up-regulated trend during 0.5 h to10 h of BL treatment and a gradual trend in H₂O treatment. (D) Heatmap of DEGs selected from Class III and their annotations. (E) Heatmap of DEGs selected from Class IV and their annotations.

Figure 6. Comparison of unigene expression levels revealed by qRT-PCR and RNA-seq (RPKM).

(A) DEGs involved in “Plant hormone signal transduction”. (B) DEGs involved in “hormone biosynthesis”. (C) Four DEGs involved in “plant-pathogen interaction”. (D) DEGs selected from the four classes (nine in Class II, six in Class III and four in Class IV) (E) Nine DEGs selected randomly. ACTIN (AJ763915) of G. hybrida was used as the normalization control. Three biological repeats were included for each condition.

Figure 7. Coefficient analysis of fold change data between qRT-PCR and RNA-seq.

Fourteen random unigenes were selected for this analysis. The data of “Mock” was seted as one unit, scatterplots were generated by the ratios of the other four samples from qRT-PCR (x-axis) and RNA-seq (y-axis).