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

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.

Seasonal Variations of Rosmarinic Acid and Its Glucoside and Expression of Genes Related to Their Biosynthesis in Two Medicinal and Aromatic Species of Salvia subg. Perovskia.

Salvia abrotanoides Kar. and Salvia yangii B.T. Drew are medicinal and aromatic plants belonging to the subgenus Perovskia and used as herbal medicines in Asia. Derivatives of caffeic acid, mainly rosmarinic acid (RA), are the major phenolic compounds identified in these plants. Understanding the factors and molecular mechanisms regulating the accumulation of pharmacologically and ecologically relevant phenolic metabolites is essential for future biotechnological and medical applications. Up to date, no studies of phenylpropanoid biosynthetic pathway at the transcriptional level has been performed in the Perovskia subgenus. Using a combined qRT-PCR transcriptional activity analysis with LC-MS based metabolic profiling of roots and leaves at the beginning, in the middle and at the end of vegetation season, we have identified the following gene candidates with properties correlating to phenolic acid biosynthesis in S. abrotanoides and S. yangii: PAL, C4H, 4CL, TAT, HPPR, RAS1, RAS2 and Cyp98A14. A comparison of phenolic acid profiles with gene transcript levels revealed the transcriptional regulation of RA biosynthesis in the roots but not the leaves of the studied species. Additionally, RAS1 and Cyp98A14 were identified as rate-limiting steps regulating phenylpropanoid biosynthesis on a transcription level. In the future, this will facilitate the gene-based metabolic enhancement of phenolic compounds production in these promising medicinal herbs.

Phytochemical Profile and Antioxidant Activities of Coleus amboinicus Lour. Cultivated in Indonesia and Poland.

Coleus amboinicus Lour., Lamiaceae, is a perennial herb that is native to Indonesia and also cultivated in Africa, Asia and Australia. The major phytochemicals responsible for its bioactivity are rosmarinic acid (RA) and its analogues, flavonoids and abietane diterpenoids. The possibility of cultivation in a colder climate would extend the use of this herb and provide new opportunities to herb growers and livestock farmers. Our study to compare feed value and phytochemical composition of C. amboinicus plants cultivated in its original region, Indonesia, and in Poland. The crude protein content was significantly higher in plants cultivated in Indonesia compared to those cultivated in Poland-21% and 13% of dry matter, respectively. The higher ADF contents were detected in C. amboinicus cultivated in Indonesia, 38-41%, in comparison to 34% in plants cultivated in Poland. The phytochemical composition was also significantly influenced by the cultivation location. Polish samples were higher in polyphenols (RA and its analogues), and also had 1.5-2-fold higher antioxidant potential, as measured by DPPH scavenging, phosphomolybdenum reduction and Fenton reaction driven lipid peroxidation. The Indonesian samples contained more diterpenoid compounds, such as dihydroxyroyleanone, and the sum of terpenoids was ca. 10 times higher than in samples from Poland (15.59-23.64 vs. 1.87 µg/g of extracts). In conclusion, C. amboinicus is suitable for cultivation in non-optimal climatic conditions but some nutritional properties and bioactivity are significantly affected.

Metabolomics and DNA-Based Authentication of Two Traditional Asian Medicinal and Aromatic Species of Salvia subg. Perovskia.

Subgenus Perovskia of the extended genus of Salvia comprises several Central Asian medicinal and aromatic species, of which S. yangii and S. abrotanoides are the most widespread. These plants are cultivated in Europe as robust ornamentals, and several cultivars are available. However, their medicinal potential remains underutilized because of limited information about their phytochemical and genetic diversity. Thus, we combined an ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS) based metabolomics with DNA barcoding approach based on trnH-psbA and ITS2 barcodes to clarify the relationships between these two taxa. Metabolomic analysis demonstrated that aerial parts are more similar than roots and none of the major compounds stand out as distinct. Sugiol in S. yangii leaves and carnosic acid quinone in S. abrotanoides were mostly responsible for their chemical differentiation, whereas in roots the distinction was supported by the presence of five norditerpenoids in S. yangii and two flavonoids and one norditerpenoid in S. abrotanoides. To verify the metabolomics-based differentiation, we performed DNA authentication that revealed S. yangii and S. abrotanoides to be very closely related but separate species. We demonstrated that DNA barcoding coupled with parallel LC-MS profiling constitutes a powerful tool in identification of taxonomically close Salvia species.