Genome-wide analysis of ATP-binding cassette transporter provides insight to genes related to bioactive metabolite transportation in Salvia miltiorrhiza

- miltiorrhiza, we analysed and identified the candidate genes that likely involved in the bioactive metabolite transportation of this medicinal plant, starting with the members of the ABC transporter family..
- Results: A total of 114 genes encoding ABC transporters were identified in the genome of S.
- In particular, we found 18 highly expressed transporters in the roots of S.
- Ferruginol, the catalytic product of CYP76AH1, is an important intermediate product in the biosynthetic pathway of tanshinone [2].
- Significant progress has been made in the understanding of the biosynthetic pathways of these active ingredients in S.
- Here, we describe the first complete analysis of the ABC transporter superfamily in the S.
- On the basis of the co-expression analysis of key enzyme genes involved in the biosynthetic pathways of the active ingredients in S.
- miltiorrhiza, we predicted that three ABCG and one ABCC subfamily ABC transporter genes were involved in the transport of the bioactive metabolites tanshinone and SA, respectively.
- In addition, the ABC proteins involved in the transport of plant hormones, sec- ondary metabolites, ions, and other substances were pre- dicted in S.
- Identification of ABC transporters in the S.
- A total of 204 homologous ABC transporters were annotated in the S.
- miltiorrhiza genome on the basis of se- quence alignment with all of the ABC transporters in the Arabidopsis TAIR11 database (Araport11 genome re- lease).
- Ultimately, 114 genes encoding for ABC trans- porters were identified in the S.
- Of the other 36 intrinsic membrane proteins, 31 were half-sized ABC transporters with one TMD and one NBD domain, and they were primarily distributed in the ABCF, ABCG and ABCI subfamilies (Table 1).
- The integrity of the full- sized transporter was verified by analyzing the arrange- ment of these three motifs in the ABC transporters.
- Moreover, the motifs of the ABC proteins belonging to the same subfamily were distributed in the same position.
- Three genes (SmABCA1–3) were annotated to be ABCAs in the S.
- Compared to other plant tissues, SmABCA1 was highly expressed in the roots of S.
- miltiorrhiza (Table 1), implying that SmABCA1 might have an important function in the roots of S.
- In contrast, SmABCA2 and SmABCA3 were half-sized transporters in the S.
- Half-sized ABCB transporters are involved in the biogenesis of Fe-S clusters in the mitochondria [33].
- SmABCB30 was highly expressed in the roots of S.
- miltior- rhiza, particularly in the periderm (Table 1).
- The tissue- specific expression of SmABCB30 was similar to that of the berberine transporter CjABCB2 in Coptis chinensis [38], indi- cating that SmABCB30 might be involved in the transport of secondary metabolites in S.
- The full-sized transporter SmABCB14 was highly expressed in the flowers, while SmABCB28 and SmABCB18 were actively expressed in the roots (Table 1)..
- These three transporters in Arabidopsis are involved in the biogen- esis of Fe/S clusters [33], and their expression is up-regulated after methyl jasmonate (MeJA) treatment, which was similar to the MeJA-induced expression profile of SmABCB9.
- Most ABCC transporters in plants are located in the vacuole mem- brane, and a few have been reported to reside on the plasma membrane [43, 44].
- SmABCC2 and SmABCC1 were expressed more highly in the roots of S.
- CsABCC4a is involved in the trans- port of crocin in C.
- 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 secondary metabolites in S.
- SmABCC13 was highly expressed in the leaves and roots (Table 1) and clustered with Arabidopsis AtABCC6 and AtABCC3 (Fig.
- The ABCD subfamily is located in the peroxisome mem- brane.
- Two ABCD members, SmABCD1 and SmABCD2, were found in the S.
- AtABCE2 catalyzes the conversion of mRNA to DNA and participates in the biogenesis of the ribosome and in the initiation of translation in Arabidopsis [58]..
- Only SmABCE1 was assigned to the ABCE subfamily in the S.
- Most of the charac- terised ABCGs are located in the plasma membrane [60, 61].
- SpTUR2, one of the first identified transporter pro- teins in the ABCG subfamily, is involved in the transport of sclareol and herbicide resistance [62].
- Moreover, transporters in the ABCG subfamily have been found to be related to the transport of paraquat, and may thereby modulate the tolerance of plants to herbicides [63]..
- ABCG transporters are widely involved in the transport of various compounds in plants [64, 65].
- The ABCG proteins of Arabidopsis are involved in the transport of epidermal wax (AtABCG11) [66], plant hormones (ABA, IBA, cytokinin) [65], pathogen resistance [67] and kana- mycin resistance [68].
- Four genes (SmABCG40, SmABCG46, SmABCG4, and SmABCG44) had tissue-specific expression profiles in this subfamily, all of which were highly expressed in the roots.
- Notably, SmABCG4 was the most highly expressed gene in the periderm of S.
- Given that tanshinone is synthesized and ac- cumulates in large amounts in the roots of S.
- miltiorrhiza, particularly in the periderm tissues [24], it is possible that these four transporters might be related to the transport of tanshinone in S.
- Another full-sized transporter, SmABCG45, possessing the same gene structure and abundance as SmABCG46, was also highly expressed in the roots of S.
- Although it has the same gene structure as SmABCG46, SmABCG35 was only expressed in the flowers (Table 1 and Additional file 1:.
- Figure S1), which suggests that this gene might be involved in the transport of substances in the flowers of S..
- SmABCG32 was a full-sized transporter and highly expressed in the leaves.
- In addition, 6 half-sized ABCG trans- porters were expressed in various organs and showed higher expression levels in the flowers.
- For example, SmABCG12 showed higher expression levels in the flowers compared to other tissues.
- SmABCG12’s homologue, AtABCG25, partici- pates in the export of abscisic acid [61], indicating that SmABCG12 might be involved in the transport of plant hor- mones in the flowers of S.
- SmABCG27 and SmABCG28 showed the same expression patterns and were more highly expressed in the flowers compared to the rest of the plant..
- SmABCG15 was highly expressed in the leaves and also induced by MeJA (Table 1), indicating that SmABCG15 might participate in the MeJA signal transduction pathway..
- SmABCI4 might be involved in the biosynthesis of Fe/S clusters in the leaves because its expression profile was similar to its homologous gene AtABCI6 [78].
- homologous to AtABCI13, and the latter is involved in the formation of plastid lipids [78].
- Moreover, 6 genes were highly expressed in the root rather than in other tissues, including SmABCB10, SmABCB13, SmABCB18, SmABCB28, SmABCG8 and SmABCG45.
- Verification of the gene expression of candidate transporters in the transport of tanshinone and salvianolic acid.
- they might play a role in the transport of basic sub- stances and primary metabolites in all cells.
- Considering that tanshinone and SA were primarily synthesised and accumulated in the roots of S.
- miltiorrhiza we hypothesised that the highly abundant transporter genes expressed in the roots of S.
- Based on gene expression profiles and transcriptome analysis (Table 1), we identified out 18 candidate genes which were highly expressed in the roots of S.
- Under the induc- tion of ABA treatment for 3 h, a total of 11 genes were strongly up-regulated in the roots of S.
- miltiorrhiza, and another 5 genes were significantly up-regulated in the roots induced by MeJA (Fig.
- For the four candidate genes, the high of SmABCG40 and SmABCG4 was induced by 12 h of the ABA treatment in the leaves (Fig.
- 7b), while in the roots, the expression of SmABCG46 and SmABCC1 was significantly induced by 3 h of ABA treatment (Fig.
- Under MeJA treatment, the gene expression levels of SmABCG46 and SmABCC1 increased significantly at different time points in the root (Fig.
- In contrast, the expression of SmABCG4 and SmABCG44 was detected to be induced by MeJA treat- ment in the leaves (Fig.
- In plants, the first ABC protein was initially identified as a transporter involved in the final detoxification process [81].
- In this study, a total of 114 ABC proteins were identi- fied in the genome of S.
- total number of genes encoding for ABC proteins was nearly identical in the two species, despite of the large differences in genome size (615 Mb versus 125 Mb) and gene content (30,478 versus 25,498 genes) [12, 21].
- 6 Co-expression profile analysis of candidate transporter genes and key enzyme genes involved in the biosynthetic pathway of tanshinone and salvianolic acid.
- The heat map showed the relative gene expression profiles of 18 candidate genes detected by qRT-PCR in the organs (root, stem, leaf and flower) and the tissues of the root, including the periderm, phloem and xylem.
- Similar to Arabidopsis [6] and grape [13], only one full-sized ABC transporter (SmABCA1) had the longest gene sequence in the S.
- 2a), indicating that these two genes may be involved in the stress.
- a The relative expression of these SmABCs in the root of 1-year old S.
- b The relative expression of these SmABCs in the leaves of 1-year old S.
- Table 2 Putative cis-elements identified in the promoter sequences of SmABC genes.
- Lr34 was involved in the resistance of wheat to various fungal pathogens [89], while CsPDR8 and CsPDR12 were related to the hormone response of cucumber [90].
- NbABCG1/2 was involved in the export of antimicrobial diterpenes and capsidiol for defence against Phytophthora infestans [94], and NtPDR3 in N.
- tabacum was induced to express iron deficiency in the culture medium [95].
- The function of AtABCG genes identi- fied in Arabidopsis are sufficient to demonstrate the diversity of gene functions in the ABCG subfamily [96].
- Some members of the ABCG subfamily may participate in the transport of these ac- tive compounds in this medicinal plant..
- The co-expression of the transporter genes with the key enzymatic genes in the secondary metabolic pathway (Fig..
- 7) provided evidence that these transporters might be in- volved in the transport of secondary metabolites in S.
- For example, CsABCC4a and CsABCC2, highly expressed in the stigmas of C.
- We anticipate that a functional study in the near future will elucidate the molecular and physio- logical functions of the lead candidate ABC transporter in- volved in tanshinone and SA transport in this important medicinal plant..
- The transporters identified in the ABCG and ABCC subfamilies might be involved in the transport of secondary metabolites of S.
- In addition, the transporters might be involved in the transport of anthocyanins, auxin and metal resistance have been identi- fied in several ABC subfamilies of S.
- Our study outlined the ABC proteins in the S.
- Identification of ABC transporter genes in the S..
- BLAST was used to align all the proteins in the S.
- miltior- rhiza genome with the ABC proteins in the Arabidopsis TAIR11 database (E value is less than 1e-5) and identify ABC homologues in S.
- miltiorrhiza (line 99–3) plants were grown in the me- dicinal plant garden of the Institute of Medicinal Plant Development.
- The candidate genes that were highly expressed in the roots of S.
- miltiorrhiza and the ex- pression patterns of these genes in the seedlings treated with ABA or MeJA were analysed by qRT-PCR.
- The motif in the SmABC proteins was identified by using Multiple Em for Motif Elicitation (MEME).
- The cis-elements identified in the promoter sequences of all the SmABC genes.
- Whole-transcriptome survey of the putative ATP-binding cassette (ABC) transporter family genes in the latex- producing laticifers of Hevea brasiliensis.
- Tomato ATP-binding cassette transporter SlABCB4 is involved in auxin transport in the developing fruit.
- The Arabidopsis concentration-dependent influx/efflux transporter ABCB4 regulates cellular auxin levels in the root epidermis

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