Gibberellin (GA), a classical phytohormone, plays significant roles in plant growth and development. It shares the important intermediate diphosphate precursor, GGPP, with the main lipophilic bioactive components, diterpenoid tanshinones in Salvia miltiorrhiza Bunge, one of the most important Traditional Chinese Medicine materials and an emerging model medicinal plant. Analysis of GA metabolism and regulation may help to demonstrate the biological functions of GAs and the crosstalk between GA metabolism and tanshinone biosynthesis in S. miltiorrhiza. However, genes involved in the conversion of ent-kaurene to GAs have not been systematically studied. Through genome-wide prediction and molecular cloning, twenty two candidate gibberellin metabolism pathway genes were systematically identified for the first time. It includes a SmKO, two SmKAOs, six SmGA20oxs, two SmGA3oxs and eleven SmGA2oxs, of which twenty genes are novel. The deduced proteins showed sequence conservation and divergence. Gibberellin metabolism pathway genes exhibited tissue-specific expression patterns and responded differentially to exogenous GA3 treatment, indicating differential regulation of gibberellin metabolism in different tissue types in S. miltiorrhiza. SmKAO1, SmKAO2, SmGA2ox2, and SmGA2ox4–SmGA2ox7 were significantly up-regulated; SmGA20ox2, SmGA3ox1, SmGA2ox1, SmGA2ox8, SmGA2ox10 and SmGA2ox11 were significantly down-regulated; while the responses of many other genes varied among different tissue-types and time-points of GA3 treatment, suggesting the complexity of feedback regulation. Tanshinone biosynthesis-related genes, such as SmCPS1 and SmKSL1, were up-regulated in response to GA3 treatment. Among the 22 identified genes, nine responded to yeast extract and Ag+-treatment in S. miltiorrhiza hairy roots. Moreover, tissue-specifically expressed splice variants were identified for SmKO, SmGA20ox3, SmGA2ox3 and SmGA2ox11, of which SmKOv1, SmGA20ox3v and SmGA2ox11v1 were GA3-responsive, suggesting the importance of alternative splicing in regulating GA metabolism. The results show tissue-specifically expressed, feedback-regulated, stress-responsive and alternatively spliced novel genes and reveal multiple layer regulation of GA metabolism and crosstalk between gibberellin metabolism and tanshinone biosynthesis in S. miltiorrhiza.

To systematically determine their phylogenetic relationships and develop molecular markers for species discrimination of Salvia bowleyana, S. splendens, and S. officinalis, we sequenced their chloroplast genomes using the Illumina Hiseq 2500 platform. The chloroplast genomes length of S. bowleyana, S. splendens, and S. officinalis were 151,387 bp, 150,604 bp, and 151,163 bp, respectively. The six genes ndhB, rpl2, rpl23, rps7, rps12, and ycf2 were present in the IR regions. The chloroplast genomes of S. bowleyana, S. splendens, and S. officinalis contain 29 tandem repeats; 35, 29, 24 simple-sequence repeats, and 47, 49, 40 interspersed repeats, respectively. The three specific intergenic sequences (IGS) of rps16-trnQ-UUG, trnL-UAA-trnF-GAA, and trnM-CAU-atpE were found to discriminate the 23 Salvia species. A total of 91 intergenic spacer sequences were identified through genetic distance analysis. The two specific IGS regions (trnG-GCC-trnM-CAU and ycf3-trnS-GGA) have the highest K2p value identified in the three studied Salvia species. Furthermore, the phylogenetic tree showed that the 23 Salvia species formed a monophyletic group. Two pairs of genus-specific DNA barcode primers were found. The results will provide a solid foundation to understand the phylogenetic classification of the three Salvia species. Moreover, the specific intergenic regions can provide the probability to discriminate the Salvia species between the phenotype and the distinction of gene fragments.

Polyprenyl diphosphate synthase (PPS) plays important roles in the biosynthesis of functionally important plastoquinone (PQ) and ubiquinone (UQ). However, only few plant PPS genes have been functionally characterized. Through genome-wide analysis, two PPS genes, termed SmPPS1 and SmPPS2, were identified from Salvia miltiorrhiza, an economically significant Traditional Chinese Medicine material and an emerging model medicinal plant. SmPPS1 and SmPPS2 belonged to different phylogenetic subgroups of plant trans-long-chain prenyltransferases and exhibited differential tissue expression and light-induced expression patterns. Computational prediction and transient expression assays showed that SmPPS1 was localized in the chloroplasts, whereas SmPPS2 was mainly localized in the mitochondria. SmPPS2, but not SmPPS1, could functionally complement the coq1 mutation in yeast cells and catalyzed the production of UQ-9 and UQ-10. Consistently, both UQ-9 and UQ-10 were detected in S. miltiorrhiza plants. Overexpression of SmPPS2 caused significant UQ accumulation in S. miltiorrhiza transgenics, whereas down-regulation resulted in decreased UQ content. Differently, SmPPS1 overexpression significantly elevated PQ-9 content in S. miltiorrhiza. Transgenic lines showing a down-regulation of SmPPS1 expression exhibited decreased PQ-9 level, abnormal chloroplast and trichome development, and varied leaf bleaching phenotypes. These results suggest that SmPPS1 is involved in PQ-9 biosynthesis, whereas SmPPS2 is involved in UQ-9 and UQ-10 biosynthesis.