Journal of Plant Research

, Volume 131, Issue 3, pp 555–562 | Cite as

Identification, characterization and expression analysis of genes involved in steroidal saponin biosynthesis in Dracaena cambodiana

Regular Paper

Abstract

Dracaena cambodiana is a traditional medicinal plant used for producing dragon’s blood. The plants and dragon’s blood of D. cambodiana contain a rich variety of steroidal saponins. However, little is known about steroidal saponin biosynthesis and its regulation in D. cambodiana. Here, 122 genes encoding enzymes involved in steroidal saponin biosynthesis were identified based on transcriptome data, with 29 of them containing complete open reading frames (ORF). Transcript expression analysis revealed that several genes related to steroidal saponin biosynthesis showed distinct tissue-specific expression patterns; the expression levels of genes encoding the key enzymes involved in the biosynthesis and early modification of steroidal saponins were significantly down-regulated in the stems in response to the inducer of dragon’s blood, exhibiting positive correlations with the content of steroidal saponins. These results provide insights on the steroidal saponins biosynthetic pathway and mechanisms underlying induced formation of dragon’s blood in D. cambodiana.

Keywords

Dracaena cambodiana Dragon’s blood Steroidal saponin Gene expression 

Introduction

As important secondary metabolites, steroidal saponins have broad pharmacological functions, and ubiquitously exist in plants (Pérez-Labrada et al. 2011; Wang et al. 2015). Steroidal saponins have been commonly used as important starting materials for the synthesis of various steroidal drugs to treat many diseases (Pérez-Labrada et al. 2011; Zhang et al. 2013). The steroidal saponins are widely distributed in monocots, such as some species of Liliaceae, Dioscoreaceae, Agave and Palmae (Osbourn 2003; Sparg et al. 2004; Wang et al. 2015). Steroidal saponins in plants are biosynthesized from isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) through the cytosolic mevalonate (MVA) pathway and plastidial 2-C-methl-d-erythritol-4-phospate (MEP) pathway (Singh et al. 2017). Then, the first key step in steroidal saponin biosynthesis is the cyclization of 2,3-oxidosqualene catalyzed by oxidosqualene cyclase (OSC). After cyclization, the downstream processes of steroidal saponin biosynthesis include a set of modifications catalyzed by cytochrome P450-dependent monoxygenases (CYPs) and glycosyltransferases (GTs) (Augustin et al. 2011; Ciura et al. 2017; Schaller 2004; Singh et al. 2017; Upadhyay et al. 2014).

Dracaena plants are members of Agave subfamily within the monocot family, which are the resources of dragon’s blood (Gupta et al. 2008). Dragon’s blood is a red resin produced mainly in the stem xylem of dragon trees when encountered external stresses and stimulations (Gupta et al. 2008; Wang et al. 2011). Dragon’s blood has been widely used for promoting blood circulation, relieving pain, antimicrobial, and other diseases (Gupta et al. 2008; Wang et al. 2011). Many chemical studies have revealed that Dracaena plants contain a number of steroidal saponins, which have broad biological activities, such as anticancer, antimicrobial, anti-inflammatory, antiviral and antifungal activities (He et al. 2004; Kougan et al. 2010; Moharram and El-Shenawy 2007; Shen et al. 2014; Tapondjou et al. 2008; Xu et al. 2010). The steroidal saponin content in the fresh stems of Dracaena plants can reach to 60% of the total chemical compound content. Interestingly, after formation of dragon’s blood resin, main chemical components are polyphenol compounds (mainly flavonoids), with steroidal saponin representing only a small proportion (Gao et al. 2014; Zheng et al. 2004).

Despite pharmacological importance of steroidal saponins, the transcriptome and genome data of Dracaena plants are limited, which hinder the investigation of the molecular mechanisms underlying steroidal saponin biosynthesis and regulation. Injection of the inducer (patent No. CN103283515A) in stems could stimulate the defense responses of Dracaena cambodiana Pierre ex Gagnep., resulting in induced formation of dragon’s blood (Jiang et al. 2015). Recently, we established de novo transcriptome assembly for D. cambodiana using the stems before and after injection of the inducer, and found that genes involved in flavonoid accumulation were significantly up-regulated in the process of the formation of dragon’s blood (Zhu et al. 2016). In this study, 122 genes involved in steroidal saponin biosynthesis in D. cambodiana were identified at the transcriptome level. Steroidal saponin content and expression profiles of steroidal saponin-related genes after injection of the inducer were analyzed to elucidate the correlation between them. This study provides provide insights on the steroidal saponins biosynthetic pathway and mechanisms underlying induced formation of dragon’s blood formation in D. cambodiana.

Materials and methods

Plant materials, RNA extraction and cDNA synthesis

Dracaena cambodiana plants were grown in plantation at the Institute of Tropical Bioscience and Biotechnology (Haikou, China). The stems of D. cambodiana plants were injected with 10% inducer (patent No. CN103283515A) and were collected at 0, 3 and 6 days after treatment. Different tissues (roots, stems, leaves and flowers) were washed with distilled H2O, and then immediately frozen in liquid nitrogen. Total RNAs were isolated from all tissues using Plant Total RNA Isolation Kit (FOREGENE, China). cDNAs were synthesized using a PrimeScript RT reagent kit (Takara, China) according to the manufacturer’s protocol. Three technical replicates and three biological replicates for each sample were performed.

Determination of total steroidal saponin

The content of total steroidal saponin was determined using colorimetry with diosgenin as a reference substance. The stem cortex from three biological replications of D. cambodiana were crushed to powder and extracted three times with 95% EtOH under reflux. The combined extracts were dissolved in 5 mL of methanol, and filtered through 0.45 μm microporous membrane filter. Absorbance was measured at 209 nm using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., USA). Results were expressed as micrograms of steroidal saponin equivalent per gram of fresh weight using a calibration curve of diosgenin. The calibration curve (y = 0.0156x + 0.0118, where y is absorbance and x is sample concentration) ranged from 10 to 100 μg mL−1 (R 2 = 0.9968). Experiments were conducted in triplicate, and displayed as the mean ± SD. Statistical analysis was conducted by using one-way analysis of variance, and the mean values were considered statistically different when P < 0.05 or significantly different when P < 0.01.

Function annotation of unigenes involved in steroidal saponin biosynthesis

Based on transcriptome data in our previous study (Zhu et al. 2016), the assignments of polypeptides encoded by unigenes generated from combined assembly were mapped into terpenoid backbone biosynthesis and steroid biosynthesis according to the KEGG database. All the members of gene identified were analyzed and validated using BLAST search using NCBI database. The theoretical molecular weight (MW) and isoelectric point (pI) of deduced proteins were obtained using online ProtParam software ExPASy. The functional domains were predicted by the NCBI Conserved Domain Search.

Expression analysis

The gene expression level and the transcripts abundances were calculated using reads fragment per kilobase of exon model per million mapped reads (FPKM) method. Genes with FPKM value ≥ 2 were screened for further expression analysis. For tissue expression, the transcripts levels of genes coding for enzymes involved in steroidal saponin biosynthesis in four tissues (roots, stems, leaves and flowers) were analyzed by quantitative real-time quantitative-PCR (qPCR). For genes represented by multiple sequences, the major isoform with highest FPKM value was selected for analysis. Differentially expressed genes (DEGs) under the inducer treatment were identified according to a threshold of fold change ≥ 2 and a false discovery rate (FDR) ≤ 0.001. DEGs were validated by qPCR. qPCR was performed in a Mx3005P Real-Time PCR System (Agilent, America) using a SYBR Premix kit (Takara, China) by specific primers. Specific primer pairs were designed using the IDT PrimerQuest Tool (http://sg.idtdna.com/Primerquest), verified by determining the melting curves at the end of each run and by sequencing the PCR product. For genes represented by multiple sequences, specific primers were designed in the regions with sequence variation. The reaction volume for qPCR was 20 μL: 10 μL 2 × SYBR mix, 1 μL of each the forward and reverse primers (10 μM), 1 μL of the cDNA template (20 ng mL cDNA−1 for each gene), and 7 μL of ddH2O. The qPCR conditions were performed as follows: 95 °C for 30 s, 40 cycles at 94 °C for 10 s, 60 °C for 30 s, and 72 °C for 20 s. The qPCR data are presented as the average relative quantities ± SE of three different replicates. The D. cambodiana actin gene was employed as an internal control. The relative expression levels were calculated using \({2^{ - \Delta \Delta {{\text{C}}_t}}}\) method. All primers pairs used in this study were listed in Table S1.

Results

Candidate genes involved in steroidal saponin biosynthesis

Steroidal saponins are synthesized from terpenoid backbone biosynthesis, followed by squalene and cycloartenol biosynthesis, and then undergo various modifications by specific CYPs and GTs for formation of various steroidal saponins (Fig. 1). Acetyl-CoA is a common precursor in the biosynthesis of steroidal saponins and flavonoids. Based on the annotation results, a total of 122 unigenes encoding 31 enzymes possibly involved in steroidal saponin biosynthesis were discovered in the transcriptome data (Table S2). In most of the cases, more than one unigene was assigned to the same enzyme. These candidate genes almost cover the entire biosynthetic pathway.

Fig. 1

Putative pathways for steroidal saponin biosynthesis. Candidate genes found in this study are shown in bold. For each gene abbreviation, its full name can be referenced in Table S3. Up and down arrows indicate increased and decreased expression of representative gene transcripts after injecting the inducer

Molecular characterization of genes with full length sequences

Among 122 genes involved in steroidal saponin biosynthesis, 29 transcripts coding for 24 kinds of enzymes have complete open reading frames. All of those transcripts are expressed at high abundance in the flesh stems of D. cambodiana, suggesting they may be the major isoforms in steroidal saponin biosynthesis. The deduced proteins of these genes vary greatly, ranging from 215 to 760 amino acids, with molecular weight from 24.17 to 252.28 kDa, and isoelectric points from 5.07 to 9.05 (Table 1 and Supplemental Data Set S1). As shown in Fig. 1, the cyclization of 2,3-oxidosqualene, catalyzed by cycloartenol synthase (CAS), is the rate-limited step for leads to the formation of steroidal saponins. It is noteworthy that a full-length CAS gene (DcCAS1) was found in the transcriptome sequences. The deduced DcCAS1 protein showed close identities to CAS from other plants and exhibited the typical structure of plant CAS (Fig. S1). DcCAS1 with other reported OSC sequences contained four QxxxxxW motifs, stabilizing the OSCs structure (Poralla et al. 1994) and one highly conserved DCTAE motif, involving in substrate binding and protonation (Abe and Prestwich 1995).

Table 1

Information of full-length genes involved in steroidal saponin biosynthesis

Gene

Unigene ID

FPKM

Unigene length

ORF

Predicted protein

Size (aa)

MW (kDa)

pI

ACC1

comp114069_c0_seq1

6.62

7782

6795

2264

252.28

6.15

HMGS1

comp43397_c0_seq1

148.01

1934

1413

470

51.96

6.39

HMGR1

comp103062_c0_seq2

73.84

1959

1578

577

61.46

8.18

MVK1

comp86095_c0_seq1

10.17

1503

1182

393

42.12

5.85

PMK1

comp104525_c0_seq1

12.62

1906

1535

512

55.41

5.77

IPPI1

comp102580_c0_seq2

21.63

3244

738

245

28.14

5.07

DXS1

comp43442_c0_seq1

19.73

2551

2166

722

79.13

6.52

DXS2

comp104432_c0_seq3

31.73

2361

2139

712

76.51

8.22

DXR1

comp95406_c0_seq2

7.29

1787

1416

471

50.89

6.21

CMK1

comp69046_c0_seq1

6.29

1648

1314

437

48.13

8.72

MDS1

comp93862_c0_seq1

18.39

1008

696

231

24.99

9.05

HDS1

comp68642_c0_seq1

28.52

3089

2247

748

82.81

5.60

HDR1

comp43842_c0_seq1

69.55

2075

1392

463

51.82

5.67

GPPS1

comp103858_c0_seq2

17.94

1497

1101

366

38.80

6.17

GPPS2

comp106371_c0_seq14

18.54

2995

1023

340

36.52

5.54

SQE1

comp43900_c0_seq1

180.01

2116

1569

522

56.41

8.72

CAS1

comp98551_c0_seq2

192.43

2825

2283

760

86.20

6.33

SMT1

comp99563_c0_seq1

8.06

1428

1077

358

39.88

6.23

MSMO1

comp99887_c0_seq1

131.56

1481

894

297

34.23

6.59

CYP51A1

comp95661_c0_seq1

2810.70

1869

1350

489

55.51

8.79

D14SR1

comp96161_c0_seq2

28.78

1278

1110

369

41.77

9.22

CDI1

comp104445_c2_seq1

64.58

1759

648

215

24.17

8.55

CDI2

comp140495_c0_seq1

3.30

1062

657

218

24.72

9.07

DHCR1

comp101533_c0_seq1

278.70

1918

1308

435

49.39

8.67

D24SR1

comp93987_c0_seq1

473.81

2217

1692

563

65.72

8.74

D24SR2

comp98495_c0_seq1

29.74

1995

1683

560

65.01

8.84

SGT1

comp99674_c0_seq2

16.39

2410

1773

590

65.28

6.23

BGL1

comp43518_c0_seq1

25.68

1759

1470

489

55.93

5.12

BGL2

comp86314_c0_seq5

5.45

2393

1884

627

68.01

7.20

FPKM values show expression levels of genes in the fresh stems before inducer treatment

Expression of genes related to steroidal saponin biosynthesis in different tissues

To investigate steroidal saponin metabolism in D. cambodiana, we performed tissue-specific expression analysis of 30 selected candidate genes. Those genes were highly expressed in stems of D. cambodiana and may be the major isoforms involved in steroidal saponin biosynthesis. The results showed that that all of the 30 genes were differentially expressed in roots, stems, flowers, and leaves (Fig. 2). We compared expression patterns of these genes in each tissue and found the following predominant expression in various tissues: 22 genes (HMGS1, HMGR1, MVK1, PMK2, MVD1, DXS3, DXR1, MCT1, MEK1, HDS1, HDR1, IPPI1, GPPS1, SQS1, SQE1, CAS1, SMO1, CYC1, CHDI1, DHCR1, D24SR1 and BGL1) in leaves, five genes (MDS1 GPPS2, SMT1,CYP51A1 and SGT1) in stems, AACT1 in roots and FPPS1 in flowers. Most genes mentioned above showed highest expression in the leaves, followed by stems, indicating that leaves and stems may be the main place for synthesizing the precursors of steroidal saponins.

Fig. 2

Expression profiles of genes in different tissues. The qPCR analyses were performed using total RNA extracted from roots (R), stems (S), flowers (F) and leaves (L) of D. cambodiana under normal culture conditions. qPCR results represent the mean (± SD) of three biological replicates

Steroidal saponin content and expression profiles of related genes under the inducer treatment

It has been showed that the total steroidal saponin content is significantly reduced in dragon’s blood resin comparison with that in the flesh stems (Gao et al. 2014). Therefore, we measured the contents of steroidal saponin in stems before and after injecting the inducer. Total steroidal saponin content, which was 363.81 μg g−1 at 0 day sample, significantly decreased to 128.44 μg g−1 (P < 0.01) at 6 days after injecting the inducer (Fig. 3).

Fig. 3

Comparison of steroidal saponin content before and after inducer treatment. Steroidal saponins are shown on the y-axis on the right. The data represents the mean (± SD) of three biological replicates. Significant differences were assessed by ANOVA (two stars correspond to P < 0.01)

To elucidate the correlation between steroidal saponin content and expression of related genes, we tested the transcript levels of all genes involved in steroidal saponin biosynthesis. Transcripts expression analysis revealed 27 genes in the stem of D. cambodiana were differentially expressed in response to injection of the inducer, among which 18 genes (AACT1, HMGS1, HMGR1, PMK1, MVD1, SQS1-4, SQE1-3, CAS1-2, MSMO1, CYC1, CYP51A1 and D14SR1) were down-regulated and 9 genes (ACC1-3, MVK1, DXS3,GPPS1 and BGL2-4) were up-regulated (Fig. 4 and Table S3). The decreased content of steroidal saponins in dragon’s blood resin may be due to differentially expression of these DEGs related to steroidal saponin biosynthesis.

Fig. 4

Expression levels of steroidal saponin biosynthesis genes after inducer treatment. Green and red colors are used to represent low-to-high expression levels, and color scales correspond to the mean centered log2-transformed FPKM values

qPCR validation of differential gene expression

To confirm that the genes obtained in this study from transcriptome analysis were differentially expressed, 15 DEGs associated with steroidal saponin biosynthesis were chosen for qPCR assay. As shown in Fig. 5, all the selected genes were differentially expressed under the inducer treatment, showing similar pattern as reflected by FPKM values. Therefore, our results provide accurate and reliable data for further studies on the steroidal saponins biosynthetic pathway and mechanisms underlying induced formation of dragon’s blood in D. cambodiana.

Fig. 5

qPCR validations of 15 putative genes involved in steroidal saponin biosynthesis. The histograms show the qPCR results of 10 genes involved in steroidal saponin biosynthesis in stems of D. cambodiana after injecting the inducer in 0, 3, 6 days respectively; the line charts show the FPKM values of these unigenes. qPCR results represent the mean (± SD) of three biological replicates

Discussion

To date, at least 100 steroidal saponins have been isolated from Dracaena species (Gao et al. 2014; Shen et al. 2014), but the mechanisms of steroidal saponins biosynthesis and regulation in Dracaena species are unclear. In the present study, we investigated the steroidal saponins biosynthetic pathway in D. cambodiana based on our transcriptome data.

In plant, steroidal saponins are synthesized mainly via the MVA pathway (Sawai and Saito 2011). The cyclisation of 2,3-oxidosqualene catalysed by OSCs is a very important rate-limiting step because it is a branch point of the synthesis of steroidal saponin, triterpenoid saponin, and other terpenes (Augustin et al. 2011; Phillips et al. 2006). Cyclization of 2,3-oxidosqualene into cycloartenol by CAS, is the initial step in steroidal saponin biosynthesis (Benveniste 2004). In our analysis, we detected the existence of high- abundance CAS transcripts, no other OSC genes coding for β-amyrin synthase (AS), lupeol synthase, dammarenediol synthase, and lanosterol synthase was detected by RNA-sequencing. This result is consistent with the fact that steroidal saponin is the most abundant chemical compound in the stems of Dracaena plants (Gao et al. 2014).

Compared to the fresh stems, the content of steroidal saponins dramatically reduced after formation of dragon’s blood resin, while the content of flavonoids were markedly increased (Gao et al. 2014; Zheng et al. 2004), which indicating that steroidal saponin biosynthesis may be suppressed and flavonoid biosynthesis may be accelerated during dragon’s blood formation. The previous evidence showed that the inducer of dragon’s blood could increase the accumulation of flavonoids in the stems of D. cambodiana via up-regulating flavonoid biosynthesis-related genes, while phenylpropanoid pathway had no significantly change (Zhu et al. 2016). In this study, we found that the content of steroidal saponins in stems of D. cambodiana significantly decreased after injecting the inducer. The reaction of acetyl-CoA catalyzed by acetyl-CoA C-acetyltransferase (AACT) is the first step in MVA pathway for steroidal saponin biosynthesis; acetyl-CoA is also catalyzed by acetyl-CoA carboxylase (ACC), forming malonyl-CoA, an important precursor for biosynthesis of flavonoids. The expression levels of three ACC genes and AACT1 in stems after injecting the inducer were up-regulated and down-regulated respectively, exhibiting positive correlations with the contents of flavonoids and steroidal saponins. It seemed that such branch point genes (ACC and AACT) may impact respective branch flux by redirecting the precursor pool toward flavonoid and steroidal saponin biosynthesis during formation of dragon’s blood.

It has been shown that the HMGR, SQS, SQE and CAS enzymes represent the key regulatory enzymes in saponin biosynthesis (Hwang et al. 2015; Ryder 1991). Transcripts expression analysis revealed that transcripts encoding the key steroidal saponin biosynthetic enzymes (HMGR, SQS, SQE and CAS) were significantly down-regulated in the stem of D. cambodiana after injecting the inducer. In addition, some genes (MSMO1, CYC1, CYP51A1 and D14SR1) corresponding to early modifications of cycloartenol were also inhibited under the inducer treatment. The decreased expression of these transcripts in response to the inducer of dragon’s blood showed that steroidal saponin biosynthesis was suppressed, which further supported the fact that the steroidal saponin content is significantly reduced in dragon’s blood resin comparison with that in the flesh stems (Gao et al. 2014). Interestingly, three members of beta-glucosidase gene family (BGL2-3) were up-regulated in the stem of D. cambodiana after injecting the inducer. Except for modification of steroidal saponin, beta-glucosidase is also involved in the hydrolysis of other beta-glycosides (Henrissat and Davies 1997; Morant et al. 2008). Those three up-regulated beta-glucosidase genes may be related to the modification of other secondary metabolites. In this study, we also found that the expression levels of genes encoding AACT, HMGS, HMGR, PMK and MVD in MVA pathway were obviously suppressed, while no transcript in MEP pathway was evidently down-regulated after injecting the inducer, which further proved that steroidal saponins are biosynthesized from IPP mainly generated from MVA pathway.

In conclusion, we performed the first transcriptome-wide analysis of genes involved in steroidal saponin biosynthesis in D. Cambodian based on the transcriptome sequences. A total of 122 unigenes encoding 31 enzymes possibly involved in steroidal saponin biosynthesis were identified. The results of expression analysis revealed that several genes involved in steroidal saponin biosynthesis showed distinct tissue-specific expression patterns. The low content of steroidal saponins in the stems after injecting the inducer was associated with decreased transcriptional levels of genes encoding the key enzymes involved in biosynthesis and early modification of steroidal saponins. The results provide insights on the steroidal saponins biosynthetic pathway and mechanisms underlying induced formation of dragon’s blood in D. cambodiana.

Notes

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Nos. 81773845; 31400297), Central Public-interest Scientific Institution Basal Research Fund for Chinese Academy of Tropical Agricultural Sciences (Nos. 17CXTD-15, 1630052016013) and Innovation Research Group Program of Hainan Province Natural Science Foundation (No. 2017CXTD020).

Supplementary material

10265_2017_1004_MOESM1_ESM.pdf (2 mb)
Supplementary material 1 (PDF 2021 KB)

References

  1. Abe I, Prestwich GD (1995) Identification of the active site of vertebrate oxidosqualene cyclase. Lipids 30:231–234CrossRefPubMedGoogle Scholar
  2. Augustin JM, Kuzina V, Andersen SB, Bak S (2011) Molecular activities, biosynthesis and evolution of triterpenoid saponins. Phytochemistry 72:435–457CrossRefPubMedGoogle Scholar
  3. Benveniste P (2004) Biosynthesis and accumulation of sterols. Annu Rev Plant Biol 55:429–457CrossRefPubMedGoogle Scholar
  4. Ciura J, Szeliga M, Grzesik M, Tyrka M (2017) Next-generation sequencing of representational difference analysis products for identification of genes involved in diosgenin biosynthesis in fenugreek (Trigonella foenum-graecum). Planta 245:977–991CrossRefPubMedPubMedCentralGoogle Scholar
  5. Gao Y, Pu DB, Li RT, Li HZ (2014) Changes of steroidal saponins in the formation of Sanguis Draconis. Yunnan J Tradit Chin Med Mater Med 35:75–78Google Scholar
  6. Gupta D, Bleakley B, Gupta RK (2008) Dragon’s blood: botany, chemistry and therapeutic uses. J Ethnopharmacol 115:361–380CrossRefPubMedGoogle Scholar
  7. He L, Wang ZH, Tu PF, Hou H (2004) Advances in study on chemical constituents and pharmacological activities in plants of Dracaena Vand. ex L. Chin Tradit Herb Drugs 35:221–228Google Scholar
  8. Henrissat B, Davies G (1997) Structural and sequence-based classification of glycoside hydrolases. Curr Opin Struct Biol 7:637–644CrossRefPubMedGoogle Scholar
  9. Hwang HS, Lee H, Choi YE (2015) Transcriptomic analysis of Siberian ginseng (Eleutherococcus senticosus) to discover genes involved in saponin biosynthesis. BMC Genom 16:180CrossRefGoogle Scholar
  10. Jiang HM, Dai HF, Wang H, Wang J, Luo YP, Mei WL (2015) Antibacterial components from artificially induced Dragon’s Blood of Dracaena cambodiana. China J Chin Materia Med 40:4002–4006Google Scholar
  11. Kougan GB, Miyamoto T, Tanaka C, Paululat T, Mirjolet JF, Duchamp O, Sondengam BL, Lacaille-Dubois MA (2010) Steroidal saponins from two species of Dracaena. J Nat Prod 73:1266–1270CrossRefPubMedGoogle Scholar
  12. Moharram FA, El-Shenawy SM (2007) Antinociceptive and anti-inflammatory steroidal saponins from Dracaena ombet. Planta Med 73:1101–1106CrossRefPubMedGoogle Scholar
  13. Morant AV, Jørgensen K, Jørgensen C, Paquette SM, Sánchezpérez R, Møller BL, Bak S (2008) Beta-glucosidases as detonators of plant chemical defense. Phytochemistry 69:1795–1813CrossRefPubMedGoogle Scholar
  14. Osbourn AE (2003) Saponins in cereals. Phytochemistry 62:1–4CrossRefPubMedGoogle Scholar
  15. Pérez-Labrada K, Brouard I, Morera C, Estévez F, Bermejo J, Rivera DG (2011) ‘Click’ synthesis of triazole-based spirostan saponin analogs. Tetrahedron 67:7713–7727CrossRefGoogle Scholar
  16. Phillips DR, Rasbery JM, Bartel B, Matsuda SP (2006) Biosynthetic diversity in plant triterpene cyclization. Curr Opin Plant Biol 9:305–314CrossRefPubMedGoogle Scholar
  17. Poralla K, Hewelt A, Prestwich GD, Abe I, Reipen I, Sprenger G (1994) A specific amino acid repeat in squalene and oxidosqualene cyclases. Trends Biochem Sci 19:157–158CrossRefPubMedGoogle Scholar
  18. Ryder NS (1991) Squalene epoxidase as a target for the allylamines. Biochem Soc Trans 19:774–777CrossRefPubMedGoogle Scholar
  19. Sawai S, Saito K (2011) Triterpenoid biosynthesis and engineering in plants. Front Plant Sci 2:25CrossRefPubMedPubMedCentralGoogle Scholar
  20. Schaller H (2004) New aspects of sterol biosynthesis in growth and development of higher plants. Plant Physiol Biochem 42:465–476CrossRefPubMedGoogle Scholar
  21. Shen HY, Zuo WJ, Wang H, Zhao YX, Guo ZK, Luo Y, Xiao NL, Dai HF, Mei WL (2014) Steroidal saponins from dragon’s blood of Dracaena cambodiana. Fitoterapia 17:94–101CrossRefGoogle Scholar
  22. Singh P, Singh G, Bhandawat A, Singh G, Parma R, Seth R, Sharma RK (2017) Spatial transcriptome analysis provides insights of key gene(s) involved in steroidal saponin biosynthesis in medicinally important herbtrillium govanianum. Sci Rep 7:45295CrossRefPubMedPubMedCentralGoogle Scholar
  23. Sparg SG, Light ME, Staden JV (2004) Biological activities and distribution of plant saponins. J Ethnopharmacol 94:219–243CrossRefPubMedGoogle Scholar
  24. Tapondjou LA, Ponou KB, Teponno RB, Mbiantcha M, Djoukeng JD, Nguelefack TB, Watcho P, Cadenas AG, Park HJ (2008) In vivo anti-inflammatory effect of a new steroidal saponin, mannioside A, and its derivatives isolated from Dracaena mannii. Arch Pharm Res 31:653–658CrossRefPubMedGoogle Scholar
  25. Upadhyay S, Phukan UJ, Mishra S, Shukla RK (2014) De novo leaf and root transcriptome analysis identified novel genes involved in steroidal sapogenin biosynthesis in asparagus racemosus. BMC Genom 15:746CrossRefGoogle Scholar
  26. Wang XH, Zhang CH, Yang LL, Gomes-Laranjo J (2011) Production of dragon’s blood in Dracaena cochinchinensis plants by inoculation of Fusarium proliferatum. Plant Sci 180:292–299CrossRefPubMedGoogle Scholar
  27. Wang X, Chen DJ, Wang YQ, Xie J (2015) De novo transcriptome assembly and the putative biosynthetic pathway of steroidal sapogenins of Dioscorea Composita. PLoS One 10:e0124560CrossRefPubMedPubMedCentralGoogle Scholar
  28. Xu M, Zhang YJ, Li XC, Jacob MR, Yang CR (2010) Steroidal saponins from fresh stems of Dracaena angustifolia. J Nat Prod 73:1524–1528CrossRefPubMedGoogle Scholar
  29. Zhang X, Ito Y, Liang J, Su Q, Zhang Y, Liu J, Sun W (2013) Preparative isolation and purification of five steroid saponins from Dioscorea zingiberensis, C.H.Wright by counter-current chromatography coupled with evaporative light scattering detector. J Pharm Biomed Anal 84:117–123CrossRefPubMedPubMedCentralGoogle Scholar
  30. Zheng QA, Zhang YJ, Li HZ, Yang CR (2004) Steroidal saponins from fresh stem of Dracaena cochinchinensis. Steroids 69:111–119CrossRefPubMedGoogle Scholar
  31. Zhu JH, Cao TJ, Dai HF, Li HL, Guo D, Mei WL, Peng SQ (2016) De Novo transcriptome characterization of Dracaena cambodiana and analysis of genes involved in flavonoid accumulation during formation of dragon’s blood. Sci Rep 6:38315CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer Japan KK, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Key Laboratory of Tropical Crop Biotechnology, Ministry of Agriculture, Institute of Tropical Bioscience and BiotechnologyChinese Academy of Tropical Agricultural SciencesHaikouChina

Personalised recommendations