Comparative transcriptomics of two Salvia subg. Perovskia species contribute towards molecular background of abietane-type diterpenoid biosynthesis

Abstract

Tanshinones, are a group of diterpenoid red pigments present in Danshen - an important herbal drug of Traditional Chinese Medicine which is a dried root of Salvia miltiorrhiza Bunge. Some of the tanshinones are sought after as pharmacologically active natural products. To date, the biosynthetic pathway of tanshinones has been only partially elucidated. These compounds are also present in some of the other Salvia species, i.a. from subgenus Perovskia, such as S. abrotanoides (Kar.) Sytsma and S. yangii B.T. Drew. Despite of the close genetic relationship between these species, significant qualitative differences in their diterpenoid profile have been discovered. In this work, we have used the Liquid Chromatography-Mass Spectrometry analysis to follow the content of diterpenoids during the vegetation season, which confirmed our previous observations of a diverse diterpenoid profile. As metabolic differences are reflected in different transcript profile of a species or tissues, we used metabolomics-guided transcriptomic approach to select candidate genes, which expression possibly led to observed chemical differences. Using an RNA-sequencing technology we have sequenced and de novo assembled transcriptomes of leaves and roots of S. abrotanoides and S. yangii. As a result, 134,443 transcripts were annotated by UniProt and 56,693 of them were assigned as Viridiplantae. In order to seek for differences, the differential expression analysis was performed, which revealed that 463, 362, 922 and 835 genes indicated changes in expression in four comparisons. GO enrichment analysis and KEGG functional analysis of selected DEGs were performed. The homology and expression of two gene families, associated with downstream steps of tanshinone and carnosic acid biosynthesis were studied, namely: cytochromes P-450 and 2-oxoglutarate-dependend dioxygenases. Additionally, BLAST analysis revealed existence of 39 different transcripts related to abietane diterpenoid biosynthesis in transcriptomes of S. abrotanoides and S. yangii. We have used quantitative real-time RT-PCR analysis of selected candidate genes, to follow their expression levels over the vegetative season. A hypothesis of an existence of a multifunctional CYP76AH89 in transcriptomes of S. abrotanoides and S. yangii is discussed and potential roles of other CYP450 homologs are speculated. By using the comparative transcriptomic approach, we have generated a dataset of candidate genes which provides a valuable resource for further elucidation of tanshinone biosynthesis. In a long run, our investigation may lead to optimization of diterpenoid profile in S. abrotanoides and S. yangii, which may become an alternative source of tanshinones for further research on their bioactivity and pharmacological therapy.

Figure 1

Chemical structures of the most abundant or unique abietane diterpenoids detected in S. abrotanoides and S. yangii.

Figure 2

Overall expression profiles of biological replicates extracted from roots and leaves of S. abrotanoides and S. yangii. The phenomap clusters created according correlation of global samples expression.

Figure 3

Selection of differentially expressed genes involved in four comparisons: S. abrotanoides roots versus S. yangii roots (upper left), S. abrotanoides leaves versus S. yangii leaves (upper right), S. yangii roots versus leaves (lower left); and S. abrotanoides roots versus leaves (lower right). (a) MA plots of DEGs. Red and green dots depict DEGs significantly changing expression between plant organs and both species. (b) Heatmaps of significant DEGs with fluctuated expression. Expression values are Z-score scaled.

Figure 4

Visualization of differentially expressed genes. (a) Venn diagram of DEGs involved in four experimental comparisons. Numbers inside the blocks indicate DEGs assigned to particular plant organs in Salvia species. (b) Circular visualization of selected Gene Ontology terms and related DEGs in four experimental comparisons: S. abrotanoides roots versus S. yangii roots (upper left), S. abrotanoides leaves versus S. yangii leaves (upper right), S. yangii roots versus leaves (lower left); and S. abrotanoides roots versus leaves (lower right).

Figure 5

Circular visualization of trans-interactions occurring between differentially expressed protein-coding genes (DEGs) related to diterpenoid metabolism and other candidate genes revealed by transcriptome mining of roots and leaves of S. abrotanoides and S. yangii. Heatmaps show the expression profiles of DEGs in roots (green–red) and leaves (green–yellow) of both species as well as DEGs in S. yangii (blue–yellow) and S. abrotanoides (blue–red). DEGs were arranged according to increasing log2(fold change). Details of the interactions are provided in the text. Gene abbreviations are explained in the text and in the Supplementary Tables S3, S5 and S6.

Figure 6

A proposed biosynthetic pathway of abietane-type diterpenoids in S. abrotanoides and S. yangii. (a) Pathways and genes involved in the biosynthesis of abietane diterpenoids. (b) Gene expression heatmap (Z-score of the FPKM value) of the identified candidate genes in roots and leaves of S. abrotanoides and S. yangii, clustered according to their expression profile. Gene abbreviations are explained in the text and in the Table 2.

Figure 7

Alignment of regions around the four distinguishing active sites residues of CYP76AH89, CYP76AH90 and CYP76AH91 together with sequences of their homologs. Different colors refer to different amino acids, red arrows point to amino acids of 301, 306, 395 and 479, respectively.

Figure 8

Expression levels of genes related to the downstream biosynthesis pathway of abietane diterpenoids in leaves and roots of S. abrotanoides and S. yangii in three growth stages during the vegetative season (SOS – start of season, MOS – middle of season, EOS – end of season). Bars represent transcript levels normalized to ACT. NE, normalized expression. The letters above the error bars refer to statistically significant differences as follows: a – significant differences in comparison to SOS, b – significant differences in comparison to MOS, c – significant differences in comparison to S. abrotanoides, d – significant differences in comparison to leaves. The level of significance is indicated by the used fonts: p < 0.01 lower case font, p < 0.001 italic font, p < 0.0001 underlined font.