Transcriptional Changes in Damask Rose Suspension Cell Culture Revealed by RNA Sequencing

Abstract

Damask roses (Rosa x damascena) are widely used in cosmetics and pharmaceutics. Here, we established an in vitro suspension cell culture for calli derived from damask rose petals. We analyzed rose suspension cell transcriptomes obtained at two different time points by RNA sequencing to reveal transcriptional changes during rose suspension cell culture. Of the 580 coding RNAs (1.3%) highly expressed in the suspension rose cells, 68 encoded cell wall-associated proteins. However, most RNAs encoded by the chloroplasts and mitochondria are not expressed. Many highly expressed coding RNAs are involved in translation, catalyzing peptide synthesis in ribosomes. Moreover, the amide metabolic process producing naturally occurring alkaloids was the most abundant metabolic process during the propagation of rose suspension cells. During rose cell propagation, coding RNAs involved in the stress response were upregulated at an early stage, while coding RNAs associated with detoxification and transmembrane transport were upregulated at the late stage. We used transcriptome analyses to reveal important biological processes and molecular mechanisms during rose suspension cell culture. Most non-coding (nc) RNAs were not expressed in the rose suspension cells, but a few ncRNAs with unknown functions were highly expressed. The expression of ncRNAs and their target coding RNAs was highly correlated. Taken together, we revealed significant biological processes and molecular mechanisms occurring during rose suspension cell culture using transcriptome analyses.

Keywords: RNA sequencing; callus; rose; suspension cells; transcriptome.

Figure 1

Suspension cell culture of rose callus generated from petals of damask rose. (A) Buds of rose were sterilized on a clean bench. (B) Transfer of rose petals to plates. (C) Induction of callus from rose petals on the medium. (D) Propagation of rose callus on the medium. (E) Transfer of subculture of rose suspension cell culture to the bioreactor. (F) Suspension cell culture of rose cells in dark condition.

Figure 2

The number of coding RNAs according to expression level (FPKM value). The expression of 45,548 coding RNAs in the suspension cells was calculated based on FPKM values.

Figure 3

Top 20 highly enriched GO terms in 580 coding RNAs. The top 20 highly enriched GO terms in 580 coding RNAs were visualized by the network plots according to biological process (A), cellular component (B), and molecular function (C). The network plots display the relationship between enriched GO terms. Two GO terms (nodes) are connected if they share 20% (default) or more genes. Darker nodes are more significantly enriched gene sets. Bigger nodes represent larger gene sets. Thicker edges represent more overlapped genes. The network was generated using the ShinyGO 0.76.3 website.

Figure 4

Predicted subcellular localization of 580 proteins. Subcellular localization of 580 rose proteins was predicted using TargetP ver. 2.0 based on the presence of N-terminal presequences. Secreted proteins with signal peptide (SP), a mitochondrial protein with mitochondrial transit peptide (mTP), chloroplast proteins with chloroplast transit peptide (cTP), and others indicate proteins targeted to other subcellular localizations.

Figure 5

The number of ncRNAs according to expression level. The expression of 4969 ncRNAs in the suspension cells was calculated based on FPKM values.

Figure 6

Information on target coding RNAs for the 20 ncRNAs in which expressions were downregulated. (A) The number of target coding RNAs for the 20 downregulated ncRNAs. (B) Chromosomal location of coding RNAs indicated by red dots. The enriched regions on chromosome 2 are indicated by violet lines.

Figure 7

Quantitative real-time RT-PCR results of four selected RNAs. The relative expression of four selected RNAs was assessed via quantitative real-time RT-PCR and normalized using RhEF1. Three biological replicates were utilized in this analysis. Statistical significance was determined using a t-test. Distinct letters (e.g., “a” vs. “b”) indicate significant differences between the corresponding groups.