Transcriptional profile of Taxus chinensis cells in response to methyl jasmonate

Abstract

Background: Methyl jasmonate (MeJA) has been successfully used as an effective elicitor to enhance production of taxol and other taxanes in cultured Taxus cells. However the mechanism of MeJA-mediated taxane biosynthesis remains unclear. Genomic information for species in the genus Taxus is currently unavailable. Therefore, information about the transcriptome of Taxus cells and specifically, description of changes in gene expression in response to MeJA, is needed for the better exploration of the biological mechanisms of MeJA-mediated taxane biosynthesis.

Results: In this research, the transcriptome profiles of T. chinensis cells at 16 hours (T16) after MeJA treatment and of mock-treated cells (T0) were analyzed by "RNA-seq" to investigate the transcriptional alterations of Taxus cell in response to MeJA elicitation. More than 58 million reads (200 bp in length) of cDNA from both samples were generated, and 46,581 unigenes were found. There were 13,469 genes found to be expressed differentially between the two timepoints, including all of the known jasmonate (JA) biosynthesis/JA signaling pathway genes and taxol-related genes. The qRT-PCR results showed that the expression profiles of 12 randomly selected DEGs and 10 taxol biosynthesis genes were found to be consistent with the RNA-Seq data. MeJA appeared to stimulate a large number of genes involved in several relevant functional categories, such as plant hormone biosynthesis and phenylpropanoid biosynthesis. Additionally, many genes encoding transcription factors were shown to respond to MeJA elicitation.

Conclusions: The results of a transcriptome analysis suggest that exogenous application of MeJA could induce JA biosynthesis/JA signaling pathway/defence responses, activate a series of transcription factors, as well as increase expression of genes in the terpenoid biosynthesis pathway responsible for taxol synthesis. This comprehensive description of gene expression information could greatly facilitate our understanding of the molecular mechanisms of MeJA-mediated taxane biosynthesis in Taxus cells.

Figure 1

Assembled unigenes length distribution. Distribution of all assembled unigenes lengths.

Figure 2

Go annotation of all unigenes. Annotated sequences were classified into ‘Biological Process’, ‘Molecular Function’ and ‘Cellular Component’ groups and 54 subgroups.

Figure 3

COG function classification of all-unigenes. Out of 25,812 NR hits, 5,812 sequences have a COG classification among the 25 categories. A, RNA processing and modification; B, Chromatin structure and dynamics; C, Energy production and conversion; D, Cell cycle control, cell division, chromosome partitioning; E, Amino acid transport and metabolism; F, Nucleotide transport and metabolism; G, Carbohydrate transport and metabolism; H, Coenzyme transport and metabolism; I, Lipid transport and metabolism; J, Translation, ribosomal structure and biogenesis; K, Transcription; L, Replication, recombination and repair; M, Cell wall/membrane/envelope biogenesis; N, Cell motility; O, Posttranslational modification, protein turnover, chaperones; P, Inorganic ion transport and metabolism; Q, Secondary metabolites biosynthesis, transport and catabolism; R, General function prediction only; S, Function unknown; T, Signal transduction mechanisms; U, Intracellular trafficking, secretion, and vesicular transport; V, Defense mechanisms; W, Extracellular structures; Y, Nuclear structure; Z, Cytoskeleton.

Figure 4

Expression level T0 vs T16. The expression level of T0 vs Tm16. The red points represent up-regulated unigenes, the green points represents down-regulated unigenes, and the blue points represent the non-DEGs.

Figure 5

Relative expression of putative genes involved in JA biosynthesis and JA signaling pathway as determined by qRT-PCR. qRT-PCR analysis of putative genes involved in JA biosynthesis and JA signaling pathway, *P < 0.05, **P < 0.01. PLD is short for phospholipase D, DAD1 for phospholipase A1, LOXs for lipoxygenases, AOS for allene oxide synthase, AOC for allene oxide cyclase, OPR3 for OPDA reductase, JMT for jasmonic acid carboxyl methyltransferasesignal, COI1 for coronatine-insensitive protein 1, JAZ for jasmonate ZIM-domain protein, and MYC2 is a bHLH transcription factor.

Figure 6

Relative expression of 10 selected taxol biosynthesis genes as determined by qRT-PCR. qRT-PCR analysis of 10 selected taxol biosynthesis genes, *P < 0.05, **P < 0.01. TS is short for taxadiene synthase, T5αH for taxane 5-alpha hydroxylase, TAT for 5-O-aceyltranferase, T10βH for taxane 10-beta hydroxylase, T13αH for taxane 13-alpha hydroxylase, TBT for 2-benzoyltransferase, DBAT for 10-O-aceyltransferase, BAPT for phenylpropanoyltransferase, DBTNBT for 3'-N-debenzoyltaxol N-benzoyltransferase, PAM for phenylalanine aminomutase.