Identification of ABC transporters in the S. miltiorrhiza genome
A total of 204 homologous ABC transporters were annotated in the S. miltiorrhiza genome on the basis of the sequence alignment with all the ABC transporters in the Arabidopsis TAIR11 database. The ABC transporters of S. miltiorrhiza were verified by confirming the integrity of the conserved domains and motifs of ABC proteins. Finally, 114 genes encoding ABC transporters were identified in the S. miltiorrhiza genome (Table 1). Considering that a typical full-sized ABC protein contained at least 1,200 amino acid residues , these ABC transporters ranged in length from 186 to 1,978 amino acid residues in S. miltiorrhiza (Table 1), indicating that some predicted ABC proteins with short sequences might be pseudogene or not full-length ABC transporters. Amongst the 114 ABC transporters, 86 were intrinsic membrane proteins with TMDs. Of these intrinsic membrane proteins, 50 were putative full-sized ABC transporters, containing at least two TMD and two NBD domains, which were distributed in ABCB, ABCG and ABCC subfamilies (Table 1). Of the other 36 intrinsic membrane proteins, 31 proteins were half-sized ABC transporters, with one TMD and one NBD domain, primarily distributed in the ABCF, ABCG and ABCI subfamilies (Table 1). The remaining five proteins were non-integrated proteins harbouring two TMD domains and one NBD domain or two NBD domains and one TMD domain, most of which belonged to ABCB and ABCG subfamilies (Table 1). In addition, the remaining 28 genes were identified as non-intrinsic proteins, which encoded proteins lacking TMD. Eighteen of these non-intrinsic proteins were covered in five subfamilies (ABCB, ABCD, ABCE, ABCF and ABCG), and 10 proteins encoded a group of non-intrinsic ABC proteins (NAPs) and were divided into the ABCI subfamily (Table 1).
Fifteen motifs of candidate ABC transporters in S. miltiorrhiza were predicted and identified by the MEME Web server to characterise the diversity of ABC proteins. The results showed that the conserved motifs amongst the ABC proteins were similar. For example, the motifs of ABC signatures, Walker A and Walker B were present in these proteins (Additional file 1: Figure S1). Moreover, the motifs of the ABC proteins belonging to the same subfamily were distributed in the same position (Additional file 1: Figure S1). The integrity of the full-sized transporter could be checked by analysing the arrangement of these three motifs in the ABC transporters. The ABC proteins with high similarity had the same motif and gene structure, whereas ABC proteins containing different motifs indicated the diversity of gene functions. Moreover, the subcellular localisation of these ABC transporters has been predicted, together with the gene names (Additional file 2: Table S1), indicating that most of these transporters were located in the plasma membrane.
Phylogenetic analysis and subfamily classification
Phylogenetic analysis was used to confirm the subfamily classification of S. miltiorrhiza ABC transporters. The 114 ABC transporters were divided into eight subfamilies, including 3 ABCA, 31 ABCB, 14 ABCC, 2 ABCD, 1 ABCE, 7 ABCF, 46 ABCG and 10 ABCI transporter genes (Fig. 1). The distribution of the ABC subfamily of S. miltiorrhiza was similar to that of other plants, and the number of genes in the ABCG subfamily was significantly higher than that of other subfamily. A phylogenetic tree was constructed using the ABC transporter of S. miltiorrhiza and the ABC proteins identified in other plants to infer the function of the transporter of S. miltiorrhiza. Such functional ABC proteins identified in other plants were listed in Additional file 3: Table S2.
Subfamily analysis of ABC transporters in S.miltiorrhiza
The plant ABCA subfamily included one full-sized ABC one homolog (AOH) and several half-sized ABC two homologs (ATH). In Arabidopsis, AtABCA1, also known as AtAOH1, was the only full-sized ABCA transporter and was the largest ABC protein encoding 1,882 amino acid residues with domains arranged in a forward direction (TMD1-NBD1-TMD2-NBD2) [2, 8]. AtAOH1 was probably involved in pollen germination and seed lipid accumulation and mobilisation . ATHs were half-sized transporters with domains ranging in forward direction (TMD1-NBD1). These transporters were only found in plants and prokaryotes [26, 27]. The expression of AtATH14 and AtATH15 was down-regulated when the seedlings were grown in hydroponic culture with long-term NaCl treatment .
Three genes (SMil_00000810, SMil_00004803, SMil_00004804) were annotated to be ABCAs in the S. miltiorrhiza genome (Fig. 2). SMil_00000810 was a full-sized ABCA transporter with high sequence homology to AtABCA1, namely, SmABCA1 (Table 1). SmABCA1 was also a large ABC transporter of S. miltiorrhiza, encoded by 1,978 amino acid residues. AtABCA1 might play a role in lipid accumulation and transport during seed maturation and germination in Arabidopsis, respectively . Compared with other tissues, SMil_00000810 was highly expressed in the roots of S. miltiorrhiza (Table 1), implying that SmABCA1 might have a similar function to AtABCA1 in Arabidopsis. On the contrary, SMil_00004803 and SMil_00004804 were half-sized transporters and might be ATH homologues in the S. miltiorrhiza genome.
The ABCB subfamily, the second largest subfamily, consisted of full-sized members, known as MDR or P-glycoprotein (PGP), and half-sized transporters TAP and ATM . The domains of ABCB transporters were arranged in a forward direction: TMD1-NBD1-TMD2-NBD2. A full-sized MDR encoded approximately 1,200 amino acid residues . Most MDRs from animals were plasma membrane efflux pumps, which were capable of transporting amphiphilic cations and cationic phospholipids . AtMDR1 (AtPGP1) was the first cloned and identified ABC transporter with multiple herbicide tolerances in plants . MDR played an important role in bidirectional auxin transport , stomatal regulation  and metal tolerance in Arabidopsis , most of which were located in the plasma membrane of plants . ATM was involved in the biogenesis of Fe-S clusters in the mitochondria .
Thirty-one genes were assigned to the ABCB subfamily in S. miltiorrhiza, 17 of which were full-sized transporters (Table 1; Fig. 3). SMil_00017134 (SmABCB10), SMil_00002175 (SmABCB11) and SMil_00005832 (SmABCB13), encoding full-sized transporters, had sequence homology with Arabidopsis AtABCB1  and AtABCB19  (Fig. 3) and OsABCB14  and tomato SlABCB4 , all of which were involved in auxin transport. The expression profiles of these three transporter genes had no tissue specificity in S. miltiorrhiza (Table 1). SMil_00020066 (SmABCB30) was highly expressed in the roots of S. miltiorrhiza, particularly in the periderm (Table 1). The tissue-specific expression of SmABCB30 was similar to that of the berberine transporter CjABCB2 in Coptis chinensis , indicating that SmABCB30 might be involved in the transport of secondary metabolites of S. miltiorrhiza. We also found that SMil_00018186 (SmABCB29), SmABCB30 and SMil_000 15238 (SmABCB31) showed sequence homology with AtABCB4/AtPGP4 and AtABC21 (Fig. 3), whereas the latter two transporters were responsible for the auxin transport in Arabidopsis [41, 42]. The full-sized transporters of SMil_00016160 (SmABCB14) were highly expressed in the flowers, whereas SMil_00001053 (SmABCB28) and SMil_00015518 (SmABCB18), likely to SmABCB10, were actively expressed in the roots (Table 1). SMil_00009650 was clustered nearly to AtABCB15, which was implicated in auxin transport of Arabidopsis . We hypothesised that these SmABCB genes might participate in auxin transport of S. miltiorrhiza. The half-sized transporter SMil_00021941 (SmABCB9) was the only ATM homologue in S. miltiorrhiza, which was particularly similar to the ATM of AtABCB23, AtABCB24 and AtABCB25 in Arabidopsis (Fig. 3). These three transporters in Arabidopsis were involved in the biogenesis of Fe/S clusters , and their expression was up-regulated after methyl jasmonate (MeJA) treatment, which was similar to the MeJA-induced expression profile of SmABCB9. The half-sized transporter SMil_00007974 (SmABCB4) was highly expressed in all organs (Table 1). It clustered closer to AtTAP1 (Fig. 3), which was involved in aluminium sequestration . We hypothesised that SmABCB9 and SmABCB4 might be related to the metal tolerance of S. miltiorrhiza.
ABCC, also known as MRP, was encoded by at least 1,500 amino acid residues and comprised only the full-sized ABC transporter in Arabidopsis . Compared with MDR, MRP harboured the ABCC-specific additional hydrophobic N-terminal transmembrane domain (TMD0) . The domains of the ABCCs were arranged in a forward direction (TMD0-TMD1-NBD1-TMD2-NBD2) . Most MRPs in plants were located in the vacuole membrane, and few have been reported to reside on the plasma membrane [10, 45, 46]. MRPs have been identified in Arabidopsis and maize [44, 47, 48]. MRP involved heavy metal tolerance [49, 50], stomatal regulation, glutathione S-conjugate transport  and phytate storage in plants . In addition, MRP was responsible for the transport of secondary metabolites in several plants. For example, ZmMRP3 transported anthocyanins in corn , and CsABCC4 of saffron mediated crocin accumulation in the vacuoles .
The transporter genes of the ABCC subfamily were expressed in all organs and tissues of S. miltiorrhiza (Table 1). SMil_00016360 (SmABCC2) and SMil_00016361 (SmABCC1) were preferentially expressed in the roots of S. miltiorrhiza (Table 1), and they were homologous to AtABCC11, AtABCC12, AtABCC1 and AtABCC2 in A.thaliana (Fig. 4). Given that AtABCC1 and AtABCC2 were involved in the transport of folic acid, glutathione S-conjugates and chlorophyll , and the function of AtABCC11 and AtABCC12 was unknown, SmABCC1 was predicted to be related with the transport of primary metabolites in S. miltiorrhiza. SMil_00015789 (SmABCC5) was constitutively expressed in all organs (Table 1) and clustered with CsABCC4a and AtABCC4 (Fig. 4). Considering that CsABCC4a acted as a crocin transporter located on the stigma vacuole membrane of Crocus sativus (saffron) , whereas AtABCC4 participated in the transport of folic acid and glutathione S-conjugate to regulate stomatal movement , we presumed that SmABCC5 might be similar to its homologous protein in S. miltiorrhiza.
SMil_00002827 (SmABCC4) was highly homologous to ZmMRP3 in maize  and VvABCC1 in grape , and the latter two transporters were related to anthocyanin transport (Fig. 4). Compared with other organs, the expression of SmABCC4 in the leaves was higher under MeJA induction (Table 1), and this ABC transporter might be involved in the transport of anthocyanin in S. miltiorrhiza leaves. SMil_00028509 (SmABCC8) was located on another branch near SMil_00002827 and was highly expressed in the leaves (Table 1), implying that SmABCC8 might also participate in the transportation of substances in the leaves (Fig. 4). SMil_00004040 (SmABCC11) was highly expressed in the flowers and roots, and its homologue AtABCC5 in Arabidopsis was related to the storage of phytate and loading of InsP6 in the seeds , indicating that SmABCC11 might contribute to the accumulation of phytic acid in S. miltiorrhiza. SMil_00020247 (SmABCC13) was highly expressed in the leaves and roots (Table 1) and clustered with AtABCC3 and AtABCC6 (Fig. 4). AtABCC3 and AtABCC6 were related to heavy metal tolerance [56, 57], inferring that SmABCC13 might be involved in the heavy metal tolerance of S. miltiorrhiza.
The ABCD subfamily, also known as PMP, was located in the peroxisome membrane. In plants, this subfamily contained full-sized and half-sized transporters. The full-sized transporter AtABCD1 was related to the import of long-chain fatty acyl-CoA into peroxisomes in Arabidopsis  and transport of 12-oxophytodienoic acid  and jasmonic acids . Two ABCD members, SMil_00013326 (SmABCD1) and SMil_00009714 (SmABCD2), were found in the S. miltiorrhiza genome (Table 1; Fig. 5). SmABCD1 was constitutively expressed in all organs and was homologous to AtABCD1 (Table 1; Fig. 5). We hypothesized that SmABCD1 had a similar function to AtABCD1 in S. miltiorrhiza.
ABCE and ABCF subfamilies
ABCE, also known as RNase L inhibitor (RLI), was a soluble protein with only two conserved NBDs and without any detectable TMD, which was conserved in eukaryotes and archaea. In Arabidopsis, AtABCE1 and AtABCE2 (AtRLI2) were involved in RNA interference (RNAi) regulation other than transport [61, 62]. AtRLI2 catalysed the conversion of mRNA and participated in the biogenesis of the ribosome and initiation of translation in Arabidopsis . ABCF, also known as a GCN homologue, similar to ABCE, was a soluble protein containing only two fused NBDs. Yeast GCN20, as the translational regulator of GCN4, elongated the ribosome during the activation of eIF2a kinase GCN2 .
Only SMil_00000396 (SmABCE1) was assigned to the ABCE subfamily of the S. miltiorrhiza genome, and it was constitutively expressed in all organs (Table 1; Fig. 5). Based on the functions of their homologues AtABCE1 and AtABCE2 in Arabidopsis, SmABCE1 might play roles in the regulation of gene silencing. The ABCF subfamily of S. miltiorrhiza contained seven members, four of which (SMil_00025510, SMil_00013582, SMil_00023741 and SMil_00004895) were highly expressed in all organs (Table 1; Fig. 5). Amongst the members, SMil_00013582 (SmABCF6) was significantly expressed in high abundance in the leaves and was down-regulated after treatment with MeJA (Table 1). Considering that the homologues of SmABCF6 in yeast and humans were involved in the regulation of gene expression , SmABCF6 might negatively regulate the expression of leaf tissue-specific genes under MeJA-induced conditions.
The ABCG subfamily was the largest subfamily in plants, which was represented by full-sized PDR and half-sized WBC transporters. The NBD-TMD domains of this subfamily were arranged in opposite directions. Most of the characterised ABCGs were located in the plasma membrane [64, 65]. To date, SpTUR2 was an early identified transporter protein of the ABCG subfamily, which involved in the transport of sclareol and resistance of herbicides . In addition, the transporters of the ABCG subfamily were related to the transport of paraquat, thereby changing the tolerance of plants to herbicides . ABCG transporters were widely involved in the transport of various compounds in plants [68, 69]. Lr34 was involved in the resistance of wheat to various fungal pathogens , and CrTPT2 was responsible for the transport of vinblastine in Catharanthus roseus . CsPDR8 and CsPDR12 were related to the hormone response of cucumber . StPDR2 conferred resistance to the biotic and abiotic stresses in tomato , and PhPDR2 was identified as a petuniasterone transporter in leaves and trichomes of Petunia hybrida . Four PDR genes have been identified in tobacco, amongst which, NpABC1 and NtPDR1 were involved in the secretion of antifungal terpenoids [75, 76]. NbABCG1/2 was involved in the export of antimicrobial diterpenes and capsidiol for defence against Phytophthora infestans , and NtPDR3 was induced to express iron deficiency in the culture medium .
WBC was involved in the transport of epidermal wax (AtABCG11) , plant hormones (ABA, IBA, cytokinin) , pathogen resistance (AtPDR8)  and kanamycin resistance (AtWBC19) in Arabidopsis . WBC was also responsible for the synthesis of pollen walls (AtABCG1 and AtABCG16) , lignin biosynthesis  and exine formation on the pollen surface (AtABCG26) . GhWBC1 was considered as an important factor for cotton fibre development and cotton elongation .
ABCG was also the largest subfamily of ABC transporters in S. miltiorrhiza, including 20 PDRs and 26 WBCs (Table 1; Fig. 6). Four genes (SMil_00010949, SMil_00020022, SMil_00027268 and SMil_00004712) had tissue-specific expression profiles in this subfamily, all of which were highly expressed in the roots of S. miltiorrhiza (Table 1). Notably, SMil_00027268 (SmABCG4) was the most highly expressed gene in the periderm of S. miltiorrhiza roots (Table 1). Given that tanshinone was synthesized and accumulated in large amounts in the roots of S. miltiorrhiza, particularly in the periderm tissues , these four transporters might be related to the transport of tanshinone in S. miltiorrhiza. Phylogenetic analysis showed that SmABCG4 and SMil_00010949 (SmABCG40) were clustered relatively near to the ginsenoside transporter PgPDR3  and the antifungal terpenoid transporter NpABC1 and NtPDR1 [75, 76] (Fig. 6). SMil_00020022 (SmABCG46) and SMil_00004712 (SmABCG44) were closely related to AtABCG39/AtPDR11  and AtABCG34  in Arabidopsis, which participated in the stress response of Arabidopsis. MeJA induced the expression of SmABCG46 and SmABCG44 at different levels, which was identical to the MeJA induction of AtABCG34 in Arabidopsis (Table 1). Another full-sized transporter, SMil_00016963 (SmABCG45), possessing the same gene structure and abundance as SmABCG46, was highly expressed in the roots of S. miltiorrhiza (Additional file 1: Figure S1; Table 1). These five genes of the SmABCG subfamily might be involved in terpenoid transport in S. miltiorrhiza, which might mediate the stress responses of this medicinal plant. SMil_00023314 (SmABCG35) was only expressed in the flowers, although it has the same gene structure as SmABCG46, suggesting that this gene might be involved in the transport of substances in the flowers of S. miltiorrhiza (Table 1; Additional file 1: Figure S1).
SMil_00004104 (SmABCG32) was a full-sized transporter and highly expressed in the leaves. Its homologous protein CrTPT2 was responsible for the transport of catharanthine , suggesting that SmABCG32 might be involved in the transport of secondary metabolites in the leaves of S. miltiorrhiza. In addition, six half-sized WBCs that were expressed in various organs, including SMil_00027466 (SmABCG12), showed high expression levels in the flowers, and its homologue AtABCG25 participated in the export of abscisic acid , indicating that SmABCG12 might be involved in the transport of plant hormones in the flowers of S. miltiorrhiza. SMil_00015148 (SmABCG19) was highly expressed in the flowers and was homologous to AtABCG11  and GhWBC1 , suggesting that SmABCG19 likely played roles in the transport substances that were related to the growth and development of S. miltiorrhiza. SMil_00000399 (SmABCG27) and SMil_00005271 (SmABCG28) showed the same expression patterns, both of which were half-sized proteins and expressed in all organs other than the leaves (Table 1). Their homologue AtABCG14 mediated the root-to-shoot translocation of trans-Zeatin in Arabidopsis . Thus, SmABCG27 and SmABCG28 likely involved in the hormone transport of S. miltiorrhiza. SMil_00010332 (SmABCG15) was highly expressed in the leaves and induced by MeJA (Table 1), indicating that SmABCG15 might participate in the MeJA signal transduction pathway.
No ABCH subfamily was found in plants; thus, an additional subfamily (ABCI) containing prokaryotic-type ABC proteins was used to instead of ABCH. ABCI was designated as non-intrinsic ABC protein (NAP) with only one NBD. The Arabidopsis genome contained 15 ABCIs, whereas the rice genome contained 10 members of this subfamily [2, 8]. The ABCI subfamily of S. miltiorrhiza consisted of 10 genes (Fig. 7), all of which contained only one soluble NBD. These ABCI transporters were expressed in all organs of S. miltiorrhiza (Table 1). SMil_00009816 (SmABCI4) might be involved in the biosynthesis of Fe/S clusters in the leaves because its expression profile was similar to the homologous gene AtABCI6 . SMil_00003192 (SmABCI5) had a high degree of homology with ABCI13, which involved in the formation of plastid lipids . SMil_00001364 (SmABCI2) showed high similarity to AtABCI1, which was related to the maturation of cytochrome c . The results showed that SmABCI2 and SmABCI5 might have similar functions to their homologous transporters in Arabidopsis.
Gene expression profiling analysis
The gene expression profiles of the 114 putative ABC transporters were detected on the basis of the transcriptome data generated from different organs (leaf, stem, flower, root) and tissues (periderm, phloem, xylem) of S. miltiorrhiza (Table 1). The relative expression levels of these genes were analysed by the FPKM values verified by transcriptome sequencing in previous studies . According to the gene expression pattern, 13 genes (SmABCB4, SmABCB7, SmABCC1, SmABCC5, SmABCD1, SmABCE1, SmABCF3-SmABCF6 and SmABCG46) were highly expressed in all organs. By contrast, 11 genes showed low expression levels in all organs, including SmABCA3, SmABCB1, SmABCB3, SmABCC3, SmABCC6, SmABCC14, SmABCF2, SmABCG13, SmABCG33 and SmABCI2. Furthermore, a total of 46 genes were rarely expressed in all organs, including SmABCB6, SmABCB8, SmABCB12, SmABCB15-SmABCB17, SmABCB20-SmABCB23, SmABCB25-SmABCB27, SmABCB29, SmABCB31, SmABCC7, SmABCC9, SmABCC12, SmABCD2, SmABCF1, SmABCG2, SmABCG3, SmABCG5-SmABCG7, SmABCG9, SmABCG10, SmABCG14, SmABCG16-SmABCG18, SmABCG20-SmABCG23, SmABCG25, SmABCG26, SmABCG29-SmABCG31, SmABCG34, SmABCG36, SmABCG38, SmABCG39, SmABCG41 and SmABCI7. The expression of some genes showed tissue- or organ-dependent specificity. For example, we found that 14 genes were highly expressed in the roots and root tissues, including SmABCA1, SmABCB2, SmABCB5, SmABCB9, SmABCB30, SmABCC2, SmABCC4, SmABCC11, SmABCC13, SmABCG1, SmABCG4, SmABCG11, SmABCG40 and SmABCI5. Although nine genes were expressed in the flowers, stems and leaves, they were not expressed in the roots and root tissues, such as SmABCB14, SmABCB24, SmABCC10, SmABCG12, SmABCG15, SmABCG19, SmABCG32, SmABCG45 and SmABCI6. The 13 genes were preferably expressed in the flowers, stems, leaves and roots but not in the three tissues of the root, including SmABCA2, SmABCB11, SmABCB19, SmABCC8, SmABCF7, SmABCG24, SmABCG27, SmABCG28, SmABCG37, SmABCG43, SmABCI1, SmABCI3 and SmABCI8. Moreover, six genes were highly expressed in the root rather than in other tissues, including SmABCB10, SmABCB13, SmABCB18, SmABCB28, SmABCG8 and SmABCG45. The different expression profiles of these ABC genes suggested that they might perform different gene functions in S. miltiorrhiza.
Verification of gene expression of candidate transporters in the transport of tanshinone and SA
The tissue-specific expression of some transporter genes might be related to their function in specific tissues or organs. By contrast, some genes showed indistinguishable expression profiles in all tissues, suggesting that they might play a role in the transport of basic substances in cells, such as transporter genes that were involved in the transport of primary metabolites. Considering that tanshinone and SA were primarily synthesised and accumulated in the roots of S. miltiorrhiza [22, 24], we hypothesised that the highly abundant transporter genes expressed in the roots of S. miltiorrhiza might be related to the transportation of tanshinone and SA. Based on the characteristics of gene expression profiles (Table 1), we screened out 18 candidate genes that were highly expressed in the roots for qRT-PCR verification (Fig. 8), including 1 ABCA (SmABCA1), 5 ABCBs (SmABCB10, SmABCB13, SmABCB18, SmABCB28 and SmABCB30), 4 ABCCs (SmABCC1, SmABCC2, SmABCC11 and SmABCC13) and 8 ABCGs (SmABCG8, SmABCG27, SmABCG28, SmABCG40, SmABCG44, SmABCG45 and SmABCG46). Amongst these candidate ABC genes, we found that the expression patterns of SMil_00020022 (SmABCG46), SMil_00010949 (SmABCG40) and SMil_00027268 (SmABCG4) were nearly identical to that of CYP76AH1 and SmCPS1, which were key enzyme genes involved in the biosynthetic pathway of tanshinone (Fig. 9). Moreover, SMil_00016361 (SmABCC1) was co-expressed with CYP98A14 and SmRAS, which encoded the key enzymes in the biosynthetic pathway of SA in S. miltiorrhiza (Fig. 9). The subcellular localisation of these four candidate transporter genes related with tanshinone and SA transport in S. miltiorrhiza was predicted to be located in the plasma membrane (Additional file 2: Table S1). Therefore, these four candidate ABC transporters co-expressed with key enzyme genes in the biosynthesis of tanshinone and SA likely participated in the intracellular transport of these two active compounds in S. miltiorrhiza.
In addition, the 3D model of these four candidate ABC transporters was created by Swiss-model using 6vxi.1.A (ABCG2) and 6pza.1.A (ABCC8) as templates, which had a broad substrate specificity, in the Swiss database. The amino acid sequence homology between SMil_00027268, SMil_00020022, SMil_00010949 and SMil_00016361 with the template protein was 25.68%, 24.79%, 25.98% and 30.46%, respectively (Additional file 4: Figure S2).