Gene expression profiling before and after internode culture for adventitious shoot formation in ipecac

Abstract

Background: In ipecac (Carapichea ipecacuanha (Brot.) L. Andersson), adventitious shoots can be induced simply by placing internodal segments on phytohormone-free culture medium. The shoots form locally on the epidermis of the apical region of the segments, but not the basal region. Levels of endogenous auxin and cytokinin transiently increase in the segments after 1 week of culture.

Results: Here, we conducted RNA-seq analysis to compare gene expression patterns in apical and basal regions of segments before culture and after 1 week of culture for adventitious shoot formation. The results revealed 8987 differentially expressed genes in a de novo assembly of 76,684 genes. Among them, 276 genes were upregulated in the apical region after 1 week of culture relative to before culture and the basal region after 1 week of culture. These genes include 18 phytohormone-response genes and shoot-formation-related genes. Validation of the gene expression by quantitative real-time PCR assay confirmed that the expression patterns were similar to those of the RNA-seq data.

Conclusions: The transcriptome data show that expression of cytokinin biosynthesis genes is induced along with the acquisition of cellular pluripotency and the initiation of cell division by wounding in the apical region of internodal segments, that trigger adventitious shoot formation without callusing.

Keywords: Adventitious shoot; Carapichea ipecacuanha; De novo transcriptome assembly; Internodal segment; RNA-seq; Transcriptional change.

Fig. 1

Sample preparation for RNA-seq analysis. Internodal segments (5 mm in length) were cut and divided into apical and basal regions before or after 1 week of culture on hormone-free B5 medium. 0a, apical region without culture; 0b, basal region without culture; 1a, apical region after 1 week of culture; 1b, basal region after 1 week of culture. n = 12 in 0a and 0b and 8 in 1a and 1b. Bar, 5 mm

Fig. 2

Multidimensional scaling (MDS) plot of gene expression in 0a, 0b, 1a, and 1b samples. 0a, apical region without culture, 0b, basal region without culture; 1a, apical region after 1 week of culture; 1b, basal region after 1 week of culture

Fig. 3

MA plots of differentially expressed genes (DEGs) between (a) 0a and 0b, (b) 1a and 1b, (c) 1a and 0a, and (d) 1a and 0a. 0a, apical region without culture; 0b, basal region without culture; 1a, apical region after 1 week of culture; 1b, basal region after 1 week of culture. Colored numbers indicate numbers of (red) upregulated and (blue) downregulated genes (log FC > |1|, FDR < 0.01)

Fig. 4

Venn diagrams of numbers of DEGs. a Upregulated in 1a relative to 0a and 1b (1a-up). b Downregulated in 1a relative to 0a and 1b (1a-down). c Upregulated in 1b relative to 0b and 1a (1b-up). d Downregulated in 1b relative to 0b and 1a (1b-down). 0b, basal region without culture; 1a, apical region after 1 week of culture; 1b, basal region after 1 week of culture (log FC > |1|, FDR < 0.01)

Fig. 5

GO enrichment analysis of DEGs in apical region: GO terms enriched in a 1a-up and b 1a-down. 1a, apical region after 1 week of culture

Fig. 6

GO enrichment analysis of DEGs in basal region: GO terms enriched in a 1b-up and b 1b-down. 1b, basal region after 1 week of culture

Fig. 7

Relative expression of phytohormone-response genes and shoot-formation-related genes. Data are means ± SE (n = 4). Eight segments were used in each experiment. EF-1 was used as an internal standard. 0a, apical region without culture; 0b, basal region without culture; 1a, apical region after 1 week of culture; 1b, basal region after 1 week of culture. Different letters indicate significant differences among segments (Tukey’s HSD, P < 0.05)

Fig. 8

Hypothetical model of adventitious shoot formation in ipecac. In the early stage (after 1 week of culture), phytohormone biosynthesis genes, phytohormone-response genes, and shoot-formation-related genes are expressed in the apical region of internodal segments. The appropriate expression triggers the meristem formation of adventitious shoots in the late stage (after 4 or 5 weeks of culture)