Transcriptome Sequencing in Response to Salicylic Acid in Salvia miltiorrhiza

Abstract

Salvia miltiorrhiza is a traditional Chinese herbal medicine, whose quality and yield are often affected by diseases and environmental stresses during its growing season. Salicylic acid (SA) plays a significant role in plants responding to biotic and abiotic stresses, but the involved regulatory factors and their signaling mechanisms are largely unknown. In order to identify the genes involved in SA signaling, the RNA sequencing (RNA-seq) strategy was employed to evaluate the transcriptional profiles in S. miltiorrhiza cell cultures. A total of 50,778 unigenes were assembled, in which 5,316 unigenes were differentially expressed among 0-, 2-, and 8-h SA induction. The up-regulated genes were mainly involved in stimulus response and multi-organism process. A core set of candidate novel genes coding SA signaling component proteins was identified. Many transcription factors (e.g., WRKY, bHLH and GRAS) and genes involved in hormone signal transduction were differentially expressed in response to SA induction. Detailed analysis revealed that genes associated with defense signaling, such as antioxidant system genes, cytochrome P450s and ATP-binding cassette transporters, were significantly overexpressed, which can be used as genetic tools to investigate disease resistance. Our transcriptome analysis will help understand SA signaling and its mechanism of defense systems in S. miltiorrhiza.

Fig 1. mRNA profiling of SA induced S. miltiorrhiza cell cultures by RNA-seq.

(a) The common and unique expression profiles among sample groups. Numbers represent expressed unigenes in control (0 h) and SA (2 h and 8 h) treated cell cultures. (b) Number of DEGs found among different sample groups, according to a FDR< 0.01 and FC ≥ 2 or ≤ -2. T1, T2 belong to control group, T3, T4 and T5 belong to treatment group of SA induction for 2 h, T6, T7 and T8 belong to treatment group of SA induction for 8 h. (c) Length distribution of the 50 778 assembled unigenes (digital details see S3 Table).

Fig 2. The most enriched GO terms (level 2) in unigenes of S. miltiorrhiza cell cultures.

All 17 867 unigenes predominantly belonged to ‘Catalytic activity’ and ‘Binding’ under Molecular function, ‘Cell part’ and ‘Cell’ under Cellular component, and ‘Metabolic process’ and ‘Cellular process’ under Biological process. The number of unigenes belonging to each category are provided.

Fig 3. Functional analysis of DEGs in S. miltiorrhiza cell cultures after SA induction.

(a) Hierarchically clustered heat map for the expression profile of DEGs (reflected as log₂ FC when compared to control), which consist of 1584 up-regulated (left), 1492 down-regulated (middle) and 2240 inconsistently regulated DEGs (right) after 8h SA induction. Blue represent repression, whereas red represent induction. (b) Analysis of biological process category of DEGs including up-regulated (red) and down-regulated (green) in S. miltiorrhiza cells after 8h SA induction. Enrichment was measured by comparing the number of DEGs from each category with the total number of genes for that GO term and using Fisher’s exact test. Significance indicated p-values below 0.01 or between 0.01 and 0.05, respectively.

Fig 4. KEGG classifications of the DEGs in S. miltiorrhiza cell cultures under SA induction.

A total of 532 DEGs were assigned to 104 KEGG pathways. The DEGs predominantly belonged to ‘Plant hormone signal transduction’ and ‘Plant-pathogen interaction’. The number of DEGs belonging to each category are provided.

Fig 5. Effect of SA on antioxidative enzymes and GSH in S. miltiorrhiza cell cultures.

(a) Effect of SA on isozymograms of SOD (left) and POD (right). 1 represents control and 2 represents SA treatment for 2 h. a, b, c, d represents four bands of SOD and POD, respectively. (b) Effect of SA on enzyme activities of SOD and POD. (c) Effect of SA on the content of GSH. Significance was indicated by double or single asterisks with p-values below 0.01 or between 0.01 and 0.05, respectively.