Next Article in Journal
Discovery of a Small Molecule with an Inhibitory Role for RAB11
Next Article in Special Issue
Genome-Wide Identification of the Trihelix Transcription Factor Family and Functional Analysis of ZmTHX15 in Maize
Previous Article in Journal
Semisynthesis and Antitumour Evaluation of Natural Derivatives from ent-Kaurene ent-15α-Angeloyloxykaur-l6-en-3β-ol Isolated from Distichoselinum tenuifolium
Previous Article in Special Issue
Biological Insights and Recent Advances in Plant Long Non-Coding RNA
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
IJMS
Volume 25
Issue 23
10.3390/ijms252313223
ijms-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Carotenoid Cleavage Dioxygenase Gene CCD4 Enhances Tanshinone Accumulation and Drought Resistance in Salvia miltiorrhiza

by
Qian Tian
,
Wei Han
,
Shuai Zhou
,
Liu Yang
,
Donghao Wang
,
Wen Zhou
* and
Zhezhi Wang
*
Key Laboratory of the Ministry of Education for Medicinal Resources and Natural Pharmaceutical Chemistry, National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest of China, Shaanxi Normal University, Xi’an 710062, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(23), 13223; https://doi.org/10.3390/ijms252313223
Submission received: 22 October 2024 / Revised: 2 December 2024 / Accepted: 4 December 2024 / Published: 9 December 2024
(This article belongs to the Special Issue Molecular Research in Plant Adaptation to Abiotic Stress)
Browse Figures
Review Reports Versions Notes

Abstract

Danshen (Salvia miltiorrhiza Bunge) is a perennial herbaceous plant of the Salvia genus in the family Lamiaceae. Its dry root is one of the important traditional Chinese herbal medicines with a long officinal history. The yield and quality of S. miltiorrhiza are influenced by various factors, among which drought is one of the most significant types of abiotic stress. Based on the transcriptome database of S. miltiorrhiza, our research group discovered a carotenoid cleavage dioxygenase gene, SmCCD4, belonging to the carotenoid cleavage oxygenase (CCO) gene family which is highly responsive to drought stress on the basis of our preceding work. Here, we identified 26 CCO genes according to the whole-genome database of S. miltiorrhiza. The expression pattern of SmCCD4 showed that this gene is strongly overexpressed in the aboveground tissue of S. miltiorrhiza. And by constructing SmCCD4 overexpression strains, it was shown that the overexpression of SmCCD4 not only promotes the synthesis of abscisic acid and increases plant antioxidant activity but also regulates the synthesis of the secondary metabolites tanshinone and phenolic acids in S. miltiorrhiza. In summary, this study is the first in-depth and systematic identification and investigation of the CCO gene family in S. miltiorrhiza. The results provide useful information for further systematic research on the function of CCO genes and provide a theoretical basis for improving the yield and quality of S. miltiorrhiza.

1. Introduction

S. miltiorrhiza is a famous member of the Lamiaceae which has the effect of treating cardiovascular and cerebrovascular diseases such as coronary heart disease and myocardial infarction [1,2]. On 9 December 1997, Compound Danshen Dropping Pills passed the clinical drug application of the US FDA for the first time as a drug, achieving a historic breakthrough in the official entry of traditional Chinese medicine into the international pharmaceutical mainstream market, and was listed as one of the essential drugs for clinical emergency treatment by the country. Fang et al. [3] found that tanshinone Ⅱ A can inhibit the expression of the biomarker of cardiovascular disease (PYX3) by inhibiting the NF-κB and p38 MAPK signaling pathway to play the role of anti-atherosclerotic (AS). After giving a 0.78 mL/kg Danshen injection to streptozotocin-induced diabetes rats for 8 weeks, it was observed that the injection improved 24 h urinary protein excretion, serum creatinine, blood urea nitrogen, and other physiological functions in rats and alleviated the ultrastructural abnormalities of glomerular hypertrophy and fibrosis [4]. Danshen injection combined with clopidogrel can improve clinical efficacy in the treatment of coronary heart disease; regulate the levels of NO, thromboxane B2 (TXB2), and endothelin-1 (ET-1); promote endothelial function recovery; and have better therapeutic effects than using clopidogrel alone [5,6]. Due to its significant medicinal value, high conversion efficiency, and published genomic data, it is becoming a model plant system for studying the regulation of secondary metabolites. As shown in Table 1, the medicinal active ingredients of S. miltiorrhiza can be mainly divided into two categories: one includes lipophilic tanshinone compounds, including tanshinone I, tanshinone IIA, tanshinone IIB, and cryptotanshinone; the other includes water-soluble phenolic acid compounds, rosmarinic acid, salvianolic acid A, and salvianolic acid B [7]. These two types of compounds mainly accumulate in the roots of S. miltiorrhiza [8,9]. At present, with the extensive development of wild resources, the growth environment of S. miltiorrhiza has been severely damaged. Planting S. miltiorrhiza in arid areas is one of the important means to alleviate its resource shortage problem.
Carotenoid cleavage oxygenase (CCO) can specifically catalyze the cleavage of conjugated double bonds in carotenoid polyene chains, thereby forming various deprotonated carotenoids and their derivatives [10,11,12]. According to whether the substrate forms an epoxide structure, CCO can be further divided into carotenoid cleavage dioxygenase (CCD) and 9-cis-epoxycarotenoid dioxygenase (NCED) [13]. CCD can specifically cleave the double bonds of carotenoids, producing various aromatic compounds and pigments, as well as some plant hormones unicyclic lactones strigolactones (SLs) [14,15]. The cleavage of 9-cis-epoxycarotenoids to xanthoxin catalyzed by NCED is considered to be the key regulatory step of abscisic acid (ABA) biosynthesis [16,17]. CCO is a very ancient gene family. In 1997, the first CCO gene related to abscisic acid biosynthesis, VP14, was identified in the seeds of viviparous maize (Zea mays) with ABA-deficiency mutations [18]. Subsequently, nine homologous genes of VP14 were identified in Arabidopsis thaliana [19]. In recent years, Wang et al. has further divided eight soybean CCO genes into five subgroups, namely, CCD1, CCD4, CCD7, CCD8, and NCED [20]. However, Wei et al. further divided CCO genes from different species into six subgroups, namely, CCD1, CCD4, CCD7, CCD8, NCED, and CCD-like in 2016 [21].
CCD1 can cleave carotenoids in flowers and fruits to form aromatic compounds [22,23]. For example, LeCCD1 symmetrically cleaves multiple carotenoid substrates at the 9,10 (9′,10′) positions to produce a C14 dialdehyde and two C13 cyclohexones that vary depending on the substrate in Lycopersicon esculentum [24]. CCD4 regulates the homeostasis of carotenoids, and its expression can lead to the breakdown of carotenoids, thereby regulating the color of plants’ flowers, fruits, etc. [25,26]. Some members of the CCD family can also regulate plant growth and development, such as inhibiting AcCCD8 expression in kiwifruit, which can affect plant branch development and delay leaf senescence [27]. The ntccd8 mutants in tobacco (Nicotiana tabacum L.) had increased shoot branching, reduced plant height, an increased number of leaves and nodes, and reduced total plant biomass compared with wild-type plants [28]. CCD7 and CCD8 have also been shown to be essential genes for the biosynthesis of SLs [29]. Research has shown that members of the CCO family are widely involved in various stress responses of plants. For example, in Brassica oleracea, CCD1 (Bol044673) and CCD4 (Bol009345 and Bol029878) are highly responsive to drought and salt stress [30]. The expression of the saffron CCD4 gene can also be highly induced under drought conditions [31]. From this, it can be seen that CCDs not only affect plant odor and color and participate in the synthesis of hormones such as ABA and SLs but also regulate plant responses to various stress conditions. Abscisic acid (ABA) plays a very essential role in enhancing plant stress resistance. When plants are subjected to biological stress such as low temperature and drought, the content of ABA in the plant notably increases, causing changes in physiological and biochemical responses, thereby enhancing the plant’s stress resistance [32]. Research has shown that A. thaliana can highly induce the expression of related CCO under drought stress, and its overexpression leads to the accumulation of ABA [33]. In addition, the upregulation of LeNCED1 expression in Lycopersicum esculentum leaves also increases the seed dormancy rate [34]. These studies suggest that CCO genes can affect the ABA-mediated plant stress response by regulating ABA synthesis.
The carotenoid cleavage dioxygenase SmCCD4 is highly responsive to drought stress on the basis of our preceding work based on the transcriptome database of S. miltiorrhiza. Furthermore, all members of the CCO family in S. miltiorrhiza were screened and structurally analyzed. Finally, a total of 26 SmCCO genes were characterized according to the whole-genome database of S. miltiorrhiza. The expression pattern of SmCCD4 indicated that this gene is strongly overexpressed in the aboveground tissues of S. miltiorrhiza and showed obvious responses to ABA treatment and drought stress. The overexpression of SmCCD4 not only promotes the synthesis of ABA and increases plant antioxidant activity but also regulates the synthesis of the secondary metabolites tanshinone and phenolic acids in S. miltiorrhiza.

2. Results

2.1. Identification and Sequence Analysis

A total of 26 CCO family members were identified in the S. miltiorrhiza genome database, with 1500 bp upstream of the start codon as the promoter sequence. The length of the gene, the coding sequence and protein, the isoelectric point (PI), and the molecular weight of the SmCCO gene family members are shown in Table S1. The length of the SmCCO gene ranges from 252 bp to 6508 bp. The length of the SmCCO CDS and protein ranges from 252 bp and 83 aa to 2409 bp and 82 aa. Among them, seven genes have the same length as the CDS, indicating that these genes only have exons and no introns. The predicted MW of the proteins ranges from 9.61 kDa to 88.49 kDa, and the PI ranges from 4.55 to 9.45. As shown in Figure 1A, a neighbor-joining phylogenetic tree of 26 SmCCO protein sequences using default parameters was constructed. According to the phylogenetic analysis, 26 SmCCO proteins could be classified into six groups. The gene structure analysis of SmCCO is shown in Figure 1B. There are a total of seven genes with only one exon and no introns, while the number of introns in other genes ranges from one to three. The result of the motif analysis is shown in Figure 1C. SmCCO members in the same group have similar motif composition.

2.2. Cis-Acting Elements Analysis

The cis-acting elements in the 1500 bp upstream promoter region of all SmCCO genes were analyzed by PlantCARE, as shown in Figure 2. Most elements are associated with hormonal and abiotic/biological responses. In plants, there are two types of cis-acting elements involved in transcriptional regulation in response to drought stress: ABRE and DRE [35]. Among them, ABRE is a homeotropic element that responds to abscisic acid and is possessed by ABA-dependent genes [36]. DRE is an ABA-independent gene that responds to dehydration and homeopathy [37]. According to the analysis, 17 out of 26 genes in the SmCCO gene family contain ABRE, and 2 genes contain DRE. Among them, SmCCD4 is an ABA-dependent gene and acts on drought stress.

2.3. Characterization and Expression Profile of SmCCD4

Many studies have shown that CCD4 regulates the homeostasis of carotenoids, and its expression can lead to the breakdown of carotenoids, thereby regulating the color of plants such as flowers and fruits. And it is highly responsive to abiotic stress in plants. The phylogenetic analysis of the SmCCD4 gene and the A. thaliana CCO gene family revealed that SmCCD4 and AtCCD4 are clustered together as orthologous genes. And the SmCCD4 gene has a structural domain, RPE65 (retinal pigment epithelial membrane protein), unique to the CCO gene family, located at 100–450 bp of the gene. The coding region of the SmCCD4 gene is 1101 bp, encoding 367 amino acids, with an isoelectric point of 5.36 and a molecular weight of 40.03 kD. As shown in Figure 3, the expression levels of SmCCD4 gene in different tissues and under different treatment conditions were analyzed by RT-qPCR. Compared with the expression level of SmCCD4 in roots, the expression level in the aboveground part of S. miltiorrhiza is significantly higher than that in roots, especially in flowers. It is obvious that hormone treatment and drought stress can induce SmCCD4 to produce different levels of response. Under ABA induction, the expression level of the SmCCD4 gene was the highest at 0.5 h, which was 2.7 times higher than that at the control time of 0 h. Under the stress treatment of PEG8000 solution, the expression level of SmCCD4 increased sharply at 0.25 h, which was 2.7 times higher than that at 0 h.

2.4. Acquisition and Validation of SmCCD4 Transgenic Strain

We constructed the SmCCD4 overexpression vector by using Gateway technology and then obtained SmCCD4 transgenic overexpression plants of S. miltiorrhiza (OE) through A. tumefaciens-mediated leaf disc transformation. The 926 bp fragment of the CaMV35S promoter was detected by PCR in different strains (Figure S1). The relative expression levels of SmCCD4 in the WT and eight OE strains are shown in Figure S2. The expression levels of SmCCD4 in the OE strains was significantly higher than that in the WT, especially in OE-2, OE-5, and OE-8. However, considering the need for sufficient plant materials for the detection of secondary metabolite content and physiological indicators, we ultimately chose three strains, OE-4, O-6, and OE-7, for subsequent experiments.

2.5. SmCCD4 Regulates Tanshinone and Phenolic Acid Biosynthesis in S. miltiorrhiza

To investigate the role of SmCCD4 in plant growth, we overexpressed SmCCD4 in the S. miltiorrhiza WT strain under the control of the CaMV35S promoter. Due to the significantly higher expression level of SmCCD4 in the aboveground part of S. miltiorrhiza compared with the roots, subsequent experimental materials were selected from the aboveground part of plants. The concentrations of four types of tanshinones in the OE strains were significantly higher than those in the WT (Figure 4A). Among them, tanshinone Ⅰ increased by 3.5 times in OE-6, tanshinone IIA increased by 6.0 times in OE-4, dihydrotanshinone increased by 2.5 times in OE-6, and cryptotanshinone increased by 5.9 times in OE-6. Compared with the WT, the content of rosmarinic acid increased by 1.4 and 1.5 times in OE-4 and OE-7 respectively, while the content of salvianolic acid in the OE strains showed no significant difference (Figure 4B). The transcription levels of seven genes, SmDXS1, SmHMGR1, SmGPPS, SmGGPPS, SmCPS1, SmKSL1, and SmCYP76AH1, selected from the tanshinone biosynthesis pathway were evaluated by using RT-qPCR (Figure 5A). Except for SmGPPS1 and SmKSL1, which showed no significant difference in expression levels among different strains, the other genes were highly expressed in the OE strains, especially SmCPS1. On the contrary, the expression level of SmCYP76AH1 in the OE strains was suppressed. Seven genes, SmPAL1, SmC4H1, Sm4CL1, SmTAT1, SmHPPR1, SmRAS1, and SmCYP98A14, were selected from the phenolic acid biosynthesis pathway (Figure 5B). The expression levels of SmPAL1 in the phenylpropanoid metabolic pathway, SmHPPR in the tyrosine metabolic pathway, and SmRAS in two key downstream pathways were increased compared with the WT. The two genes Sm4CL1 and SmTAT1 were strongly suppressed in the OE strains.

2.6. SmCCD4 Overexpression Increases ABA Accumulation and Improves Drought Tolerance in S. miltiorrhiza

The content of ABA was detected in SmCCD4 OE strains and WT strains of the control group. As shown in Figure 6A, the content of ABA in the OE strains significantly increased, especially in OE-6 and OE-7. In order to further investigate the drought resistance function of SmCCD4, plant materials grown for 30 days after drought treatment with 2.5% concentration of PEG8000 were collected. The contents of MDA, H2O2, CAT, and SOD in the leaves were measured (Figure 6B). Under drought stress, MDA and H2O2 contents in transgenic plants were obviously lower than those in the wild type, while SOD activity was higher than that in the wild type, and CAT activity showed no significant difference among different strains. The aforementioned results reveal that the overexpression of SmCCD4 enhances drought tolerance in S. miltiorrhiza.

3. Discussion

With the deepening of research on the pharmacological effects of S. miltiorrhiza [2,38,39,40], its commercial value is being gradually recognized, and the market demand is constantly increasing. However, wild resources and existing artificial cultivation yield and quality are unable to meet the demand [41]. Expanding the planting area of S. miltiorrhiza and increasing the content of medicinal active ingredients are very necessary. Drought stress is a common type of stress faced by plants [42], and planting S. miltiorrhiza in arid areas is one of the important means to alleviate its resource shortage problem. As an important enzyme gene for carotenoid catalysis and ABA synthesis, research has shown that the CCO gene family plays an important role in regulating plant resistance to stress [43,44]. Studying the molecular mechanism of drought resistance and the accumulation mechanism of secondary metabolites in S. miltiorrhiza is a prerequisite and a foundation for improving the yield and quality of S. miltiorrhiza.
S. miltiorrhiza is a model plant among medicinal plants [45]. Its growth is often affected by various types of environmental stress [46,47,48]. Therefore, in-depth research on the regulatory mechanism of stress resistance genes in S. miltiorrhiza is of great practical significance. The CCO gene family can regulate plant responses to various types of abiotic stress and has been extensively studied in various plants [49,50,51]; the identification and characterization of CCO based on the whole-genome sequence of S. miltiorrhiza were reported for the first time. In this study, 26 CCO members were characterized in S. miltiorrhiza. Compared with A. thaliana [19], Glycine max [52], and Malus domestica [49], S. miltiorrhiza has a larger CCO family. CCO is a type of double-cleavage diooxygenase that contains the RPE65 domain and generates secondary metabolites during carotenoid metabolism [53]. The secondary metabolites are crucial for carotenoid metabolism and aroma volatiles. Fe2+ can activate the catalytic activity of the enzyme [54,55]. The four conserved histidine residues in CCO can regulate their binding to Fe2+. In addition, several studies have confirmed that the conservation of CCO protein sequences is not high. Carotenoids are precursors of important plant hormones, such as ABA [56]. The latter plays an important role in many aspects of the plant life cycle, especially in the response of plants to various types of environmental stress [57].
Most studies on the function of the CCD4 gene focus on plant coloration [58,59,60,61]. However, there is almost no information on the regulation of medicinal active ingredients and drought resistance by CCD4 in medicinal plants. Therefore, this study first cloned the coding sequence of SmCCD4 in S. miltiorrhiza and found through RT-qPCR that SmCCD4 responds to ABA induction and PEG stress. Secondly, an overexpression vector for SmCCD4 was constructed, and a total of eight transgenic strains were obtained. By measuring the content of salvianolic acids and tanshinones in different strains, the SmCCD4 gene in S. miltiorrhiza can upregulate key genes in the biosynthesis pathways of these two secondary metabolites, thereby promoting the accumulation of the compounds. PEG was used to simulate drought stress conditions, and it was found that the ABA content in SmCCD4 OE strains was significantly increased, and compared with WT strains, the OE strains showed better drought resistance.

4. Materials and Methods

4.1. Identification of SmCCO Family and Sequence Analysis

The nucleotide and amino acids sequences of the reported A. thaliana CCO amino acid sequences AtCCD1 (AT3G63520.1), AtCCD4 (AT4G19170.1), AtCCD7 (AT2G44990.3), AtNCED2 (AT4G18350.1), AtNCED3 (AT3G14440.1), and AtNCED5 (AT1G30100.1) were employed as a query to search against the S. miltiorrhiza genome assembly of our lab by the BLASTn algorithm. PFAM (pfam-legacy.xfam.org) analyzed conserved domains, deleted sequences with incomplete RPE65 domains [62], and identified all members of the SmCCO family. We used online websites to once again determine that each member has a complete conserved domain (http://www.ebi.ac.uk/interpro/search accessed on 9 September 2024) and calculate its molecular weight and isoelectric point (https://web.expasy.org/compute_pi/ accessed on 9 September 2024). We used MEGA 6.0 to construct an evolutionary tree for the S. miltiorrhiza CCO family by using the neighbor-joining method [63]. We used online websites for motif analysis (http://meme-suite.org/tools/meme accessed on 9 September 2024), gene structure analysis (http://gsds.cbi.pku.edu.cn accessed on 9 September 2024), cis-acting component analysis (http://bioinformatics.psb.ugent.be/webtools/plantcare/ accessed on 9 September 2024), and signal peptide prediction, transmembrane domains, and hydrophilicity/hydrophobicity analysis (http://www.detaibio.com/tools/ accessed on 9 September 2024). We predicted the tertiary structure of the SmCCD4 protein by using the SWISS-MODEL online website (https://swissmodel.expasy.org/ accessed on 9 September 2024).

4.2. Plant Materials and Treatments

The cultivation of sterile S. miltiorrhiza seedlings was carried out according to the previously described procedure [64]. We treated two-month-old S. miltiorrhiza plants by spraying 100 μM ABA solution and 20% PEG8000 solution, while simultaneously spraying the corresponding solvent 10% ethanol solution and water as controls. We collected samples at 0 h, 0.25 h, 0.5 h, 1 h, 3 h, 6 h, 12 h, and 24 h and froze them in liquid nitrogen. Each sample had three biological replicates.

4.3. Expression Pattern Analysis

Total RNA isolation kits (Vazyme, Nanjing, China) were used to extract total RNA from the abovementioned plants. The integrity of RNA was detected by agarose gel electrophoresis, and the concentration of RNA was detected by a spectrophotometer. ATGScript Reverse Transcriptase was used to reverse-transcribe total RNA into cDNA (Vazyme, Nanjing, China). The expression pattern of SmCCD4 was examined by using RT-qPCR and calculated by using the comparative CT method (2−ΔΔCT) [65]. By using SmACT (DQ243702) [66] as an internal reference, the gene expression levels of different samples were detected. The expression analysis of SmCCD4 in all samples was performed by using three biological replicates and three technical replicates. Asterisks indicate significance differences from 0 h by using the t-test. Data are shown as means ± SDs, n = 3.

4.4. SmCCD4 Gene Cloning and Vector Construction

The full-length CDS of SmCCD4 obtained from the S. miltiorrhiza genome database is 1101 bp, and the primers were designed by using Vector NT1Suits (Table S2). The cDNA of S. miltiorrhiza flower was used as the template for PCR amplification. The PCR product was subjected to agarose gel electrophoresis, and the target band of about 1100 bp was cut and recovered by using the agarose gel DNA recovery kit (Vazyme, Nanjing, China). After recycling, the product was measured for concentration by using a spectrophotometer and sequenced for comparison. We constructed overexpression vectors by using Gateway technology [67]. Firstly, a BP reaction was performed to connect the CDS of SmCCD4 fragments with attB sites to pENTR207 vectors with attP sites, resulting in the production of the pENTR207-SmCCD4 recombinant plasmid. Next, an LR reaction was performed to recombine the pENTR207-SmCCD4 from the previous step with the target pEarlyGate202 vector containing the attR site, resulting in the overexpression vector pEarleyGate202-SmCCD4 recombinant plasmid.

4.5. Genetic Transformation and Transformant Selection

We transformed the constructed recombinant plasmid pEarleyGate202-SmCCD4 into Agrobacterium tumefaciens EHA105 [64]. We cut the leaves of two-month-old sterile seedlings of S. miltiorrhiza into 1 cm × 1 cm squares, with the leaves facing downwards, and placed them on solid MS medium supplemented with 1 µg/mL 6-Benzylaminopurine and 0.1 µg/mL naphthylacetic acid for two days at 25 °C with a 16 h photoperiod at 108 µmol/m2/s. The pre-cultured explants were immersed in the diluted bacterial solution (OD 0.2–0.3) for transfection and shook at 25 °C and 100 rpm for cultivation. After 15 min, the explants were taken out to absorb the remaining surface solution and placed on solid 6N medium (25 °C in the dark). Two days later, the explants were put on solid 6N medium (supplemented with 0.1 µg/mL thidiazuron and 50 µg/mL cefotaxime) for callus induction (25 °C under a 16 h photoperiod). We changed the medium every 10 days until callus tissue formed and buds differentiated.
Finally, the buds were transferred from the callus to 1/2 MS solid medium supplemented with 0.1 µg/mL thidiazuron and 50 µg/mL cefotaxime to induce rooting and form complete transgenic plants of S. miltiorrhiza. We extracted genomic DNA from different S. miltiorrhiza lines and detected positive transgenic lines by amplification using CaMV35S promoter specific primers [68]. Total RNA was also isolated to evaluate gene expression levels through RT-qPCR.

4.6. Determination of Tanshinone and Phenolic Acid Contents

The roots of 2-month-old OE-SmCCD4 and WT S. miltiorrhiza plants were harvested and dried at 30 °C until constant weigh. The secondary metabolites were extracted as previously described. An Agilent 1260 HPLC system coupled to an Agilent 6460 QQQ LC/MS system (Agilent, Clara, CA, USA) was used to determine phenolic acid and tanshinone contents under previously described detection conditions [69]. Chromatography was performed on a Welch Ultimate XB-C18 column with a 0.4 mL/min flow rate and a 5 µL injection volume. The mobile phase included Solvent A (acetonitrile) and Solvent B (0.1% formic acid in deionized water), and tanshinones and phenolic acids followed the following gradient distributions: Tanshinones: 0–5 min, A 75–90%, B 25–10%; 5–6 min, A 90–25%, B 10–75%; 6–10 min, A 25%, B 75%. Phenolic acids: 0–6 min, A 20–60%, B 80–40%; 6–7 min, A 60–20%, B 40–80%; 7–10 min, A 20%, B 80%. Analytes were detected in positive ionization mode, and the drying gas flow was 1 L/min.

4.7. Physiological Assays

Two-month-old WT, EV, and OE S. miltiorrhiza seedlings were analyzed for drought resistance. Superoxide dismutase (SOD), malondialdehyde (MDA), catalase (CAT), and hydrogen peroxide (H2O2) concentrations were measured by using the Plant Micro Assay Kit (Solarbio, Beijing, China), with three biological and three technical replicates.

4.8. Statistics

All experiments were repeated by using three biological replicates and three technical replicates unless otherwise specified. The data were calculated as the means ± standard error of the mean, and significance analysis was performed by using the t-test calculated by GraphPad Prism 7.0.4.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms252313223/s1.

Author Contributions

Conceptualization, Q.T. and W.H.; methodology, S.Z. and L.Y.; validation, W.Z., D.W. and Z.W.; formal analysis, L.Y. and W.H.; investigation, S.Z. and L.Y.; writing—original draft preparation, Q.T. and W.H.; writing—review and editing, Z.W. and W.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by National Natural Science Foundation of China (No. 32170378).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Cheng, T.O. Danshen: A versatile Chinese herbal drug for the treatment of coronary heart disease. Int. J. Cardiol. 2006, 113, 437–438. [Google Scholar] [CrossRef] [PubMed]
  2. Wang, Z.; Peters, R.J. Tanshinones: Leading the way into Lamiaceae labdane-related diterpenoid biosynthesis. Curr. Opin. Plant Biol. 2022, 66, 102189. [Google Scholar] [CrossRef] [PubMed]
  3. Fang, J.; Chen, Q.; He, B.; Cai, J.; Yao, Y.; Cai, Y.; Xu, S.; Rengasamy, K.R.R.; Gowrishankar, S.; Pandian, S.K.; et al. Tanshinone IIA attenuates TNF-α induced PTX3 expression and monocyte adhesion to endothelial cells through the p38/NF-κB pathway. Food Chem. Toxicol. 2018, 121, 622–630. [Google Scholar] [CrossRef] [PubMed]
  4. Yin, D.; Yin, J.; Yang, Y.; Chen, S.; Gao, X. Renoprotection of Danshen Injection on streptozotocin-induced diabetic rats, associated with tubular function and structure. J. Ethnopharmacol. 2014, 151, 667–674. [Google Scholar] [CrossRef]
  5. Zhang, Z.; Wang, Y.; Tan, W.; Wang, S.; Liu, J.; Liu, X.; Wang, X.; Gao, X. A Review of Danshen Combined with Clopidogrel in the Treatment of Coronary Heart Disease. Evid.-Based Complement. Altern. Med. 2019, 2019, 2721413. [Google Scholar] [CrossRef]
  6. Lin, S.; He, X.; Zhai, G.; Wang, C.; Xue, H.; Lin, S. Prospective study of the effect of sulfotanshinone sodium combined with tirofiban on vascular endothelial function and indicators of plaque stability in elderly patients with acute coronary syndrome. J. Clin. Pharm. Ther. 2021, 46, 319–327. [Google Scholar] [CrossRef]
  7. Li, S.; Wang, L.; Yan, X.; Wang, Q.; Tao, Y.; Li, J.; Peng, Y.; Liu, P.; Liu, C. Salvianolic Acid B Attenuates Rat Hepatic Fibrosis via Downregulating Angiotensin II Signaling. Evid.-Based Complement. Altern. Med. 2012, 2012, 160726. [Google Scholar] [CrossRef]
  8. Li, Y.-G.; Song, L.; Liu, M.; Zhi Bi, H.; Wang, Z.-T. Advancement in analysis of Salviae miltiorrhizae Radix et Rhizoma (Danshen). J. Chromatogr. A 2009, 1216, 1941–1953. [Google Scholar] [CrossRef]
  9. Wang, X.; Morris-Natschke, S.L.; Lee, K.-H. New developments in the chemistry and biology of the bioactive constituents of Tanshen. Med. Res. Rev. 2007, 27, 133–148. [Google Scholar] [CrossRef]
  10. Giuliano, G.; Al-Babili, S.; von Lintig, J. Carotenoid oxygenases: Cleave it or leave it. Trends Plant Sci. 2003, 8, 145–149. [Google Scholar] [CrossRef]
  11. Yu, J.; Wang, Y.; Bai, H.; Zhang, X.; Wang, R. Genome-wide identification and expressional analysis of carotenoid cleavage oxygenase (CCO) gene family in Betula platyphylla under abiotic stress. BMC Genom. 2024, 25, 872. [Google Scholar] [CrossRef] [PubMed]
  12. Sui, X.; Farquhar, E.R.; Hill, H.E.; von Lintig, J.; Shi, W.; Kiser, P.D. Preparation and characterization of metal-substituted carotenoid cleavage oxygenases. J. Biol. Inorg. Chem. 2018, 23, 887–901. [Google Scholar] [CrossRef] [PubMed]
  13. Auldridge, M.E.; Block, A.; Vogel, J.T.; Dabney-Smith, C.; Mila, I.; Bouzayen, M.; Magallanes-Lundback, M.; DellaPenna, D.; McCarty, D.R.; Klee, H.J. Characterization of three members of the Arabidopsis carotenoid cleavage dioxygenase family demonstrates the divergent roles of this multifunctional enzyme family. Plant J. 2006, 45, 982–993. [Google Scholar] [CrossRef] [PubMed]
  14. Fukamatsu, Y.; Tamura, T.; Hihara, S.; Oda, K. Mutations in the CCD4 Carotenoid Cleavage Dioxygenase Gene of Yellow-Flesh Peaches. Biosci. Biotechnol. Biochem. 2013, 77, 2514–2516. [Google Scholar] [CrossRef] [PubMed]
  15. Aly, R.; Dubey, N.K.; Yahyaa, M.; Abu-Nassar, J.; Ibdah, M. Gene silencing of CCD7 and CCD8 in Phelipanche aegyptiaca by tobacco rattle virus system retarded the parasite development on the host. Plant Signal. Behav. 2014, 9, e29376. [Google Scholar] [CrossRef]
  16. Lefebvre, V.; North, H.; Frey, A.; Sotta, B.; Seo, M.; Okamoto, M.; Nambara, E.; Marion-Poll, A. Functional analysis of Arabidopsis NCED6 and NCED9 genes indicates that ABA synthesized in the endosperm is involved in the induction of seed dormancy. Plant J. 2006, 45, 309–319. [Google Scholar] [CrossRef]
  17. Jia, K.-P.; Baz, L.; Al-Babili, S. From carotenoids to strigolactones. J. Exp. Bot. 2018, 69, 2189–2204. [Google Scholar] [CrossRef]
  18. Schwartz, S.H.; Tan, B.C.; Gage, D.A.; Zeevaart, J.A.; McCarty, D.R. Specific oxidative cleavage of carotenoids by VP14 of maize. Science 1997, 276, 1872–1874. [Google Scholar] [CrossRef]
  19. Tan, B.C.; Joseph, L.M.; Deng, W.T.; Liu, L.J.; Li, Q.B.; Cline, K.; McCarty, D.R. Molecular characterization of the Arabidopsis 9-cis epoxycarotenoid dioxygenase gene family. Plant J. 2003, 35, 44–56. [Google Scholar] [CrossRef]
  20. Wang, R.-K.; Wang, C.-E.; Fei, Y.-Y.; Gai, J.-Y.; Zhao, T.-J. Genome-wide identification and transcription analysis of soybean carotenoid oxygenase genes during abiotic stress treatments. Mol. Biol. Rep. 2013, 40, 4737–4745. [Google Scholar] [CrossRef]
  21. Wei, Y.; Wan, H.; Wu, Z.; Wang, R.; Ruan, M.; Ye, Q.; Li, Z.; Zhou, G.; Yao, Z.; Yang, Y. A Comprehensive Analysis of Carotenoid Cleavage Dioxygenases Genes in Solanum Lycopersicum. Plant Mol. Biol. Report. 2016, 34, 512–523. [Google Scholar] [CrossRef]
  22. Ilg, A.; Beyer, P.; Al-Babili, S. Characterization of the rice carotenoid cleavage dioxygenase 1 reveals a novel route for geranial biosynthesis. FEBS J. 2009, 276, 736–747. [Google Scholar] [CrossRef] [PubMed]
  23. Sun, Z.; Hans, J.; Walter, M.H.; Matusova, R.; Beekwilder, J.; Verstappen, F.W.A.; Ming, Z.; van Echtelt, E.; Strack, D.; Bisseling, T.; et al. Cloning and characterisation of a maize carotenoid cleavage dioxygenase (ZmCCD1) and its involvement in the biosynthesis of apocarotenoids with various roles in mutualistic and parasitic interactions. Planta 2008, 228, 789–801. [Google Scholar] [CrossRef] [PubMed]
  24. Simkin, A.J.; Schwartz, S.H.; Auldridge, M.; Taylor, M.G.; Klee, H.J. The tomato carotenoid cleavage dioxygenase 1 genes contribute to the formation of the flavor volatiles β-ionone, pseudoionone, and geranylacetone. Plant J. 2004, 40, 882–892. [Google Scholar] [CrossRef]
  25. Ohmiya, A.; Kishimoto, S.; Aida, R.; Yoshioka, S.; Sumitomo, K. Carotenoid cleavage dioxygenase (CmCCD4a) contributes to white color formation in chrysanthemum petals. Plant Physiol. 2006, 142, 1193–1201. [Google Scholar] [CrossRef]
  26. Yoshioka, S.; Aida, R.; Yamamizo, C.; Shibata, M.; Ohmiya, A. The carotenoid cleavage dioxygenase 4 (CmCCD4a) gene family encodes a key regulator of petal color mutation in chrysanthemum. Euphytica 2012, 184, 377–387. [Google Scholar] [CrossRef]
  27. Ledger, S.E.; Janssen, B.J.; Karunairetnam, S.; Wang, T.; Snowden, K.C. Modified CAROTENOID CLEAVAGE DIOXYGENASE8 expression correlates with altered branching in kiwifruit (Actinidia chinensis). New Phytol. 2010, 188, 803–813. [Google Scholar] [CrossRef]
  28. Gao, J.; Zhang, T.; Xu, B.; Jia, L.; Xiao, B.; Liu, H.; Liu, L.; Yan, H.; Xia, Q. CRISPR/Cas9-Mediated Mutagenesis of Carotenoid Cleavage Dioxygenase 8 (CCD8) in Tobacco Affects Shoot and Root Architecture. Int. J. Mol. Sci. 2018, 19, 1062. [Google Scholar] [CrossRef]
  29. Schwartz, S.H.; Qin, X.Q.; Loewen, M.C. The biochemical characterization of two carotenoid cleavage enzymes from Arabidopsis indicates that a carotenoid-derived compound inhibits lateral branching. J. Biol. Chem. 2004, 279, 46940–46945. [Google Scholar] [CrossRef]
  30. Kim, Y.; Hwang, I.; Jung, H.-J.; Park, J.-I.; Kang, J.-G.; Nou, I.-S. Genome-Wide Classification and Abiotic Stress-Responsive Expression Profiling of Carotenoid Oxygenase Genes in Brassica rapa and Brassica oleracea. J. Plant Growth Regul. 2016, 35, 202–214. [Google Scholar] [CrossRef]
  31. Rubio-Moraga, A.; Luis Rambla, J.; Fernandez-de-Carmen, A.; Trapero-Mozos, A.; Ahrazem, O.; Orzaez, D.; Granell, A.; Gomez-Gomez, L. New target carotenoids for CCD4 enzymes are revealed with the characterization of a novel stress-induced carotenoid cleavage dioxygenase gene from Crocus sativus. Plant Mol. Biol. 2014, 86, 555–569. [Google Scholar] [CrossRef] [PubMed]
  32. Seo, M.; Koshiba, T. Complex regulation of ABA biosynthesis in plants. Trends Plant Sci. 2002, 7, 41–48. [Google Scholar] [CrossRef] [PubMed]
  33. Huang, F.-C.; Molnar, P.; Schwab, W. Cloning and functional characterization of carotenoid cleavage dioxygenase 4 genes. J. Exp. Bot. 2009, 60, 3011–3022. [Google Scholar] [CrossRef] [PubMed]
  34. Thompson, A.J.; Jackson, A.C.; Symonds, R.C.; Mulholland, B.J.; Dadswell, A.R.; Blake, P.S.; Burbidge, A.; Taylor, I.B. Ectopic expression of a tomato 9-cis-epoxycarotenoid dioxygenase gene causes over-production of abscisic acid. Plant J. 2000, 23, 363–374. [Google Scholar] [CrossRef]
  35. Narusaka, Y.; Nakashima, K.; Shinwari, Z.K.; Sakuma, Y.; Furihata, T.; Abe, H.; Narusaka, M.; Shinozaki, K.; Yamaguchi-Shinozaki, K. Interaction between two cis-acting elements, ABRE and DRE, in ABA-dependent expression of Arabidopsis rd29A gene in response to dehydration and high-salinity stresses. Plant J. 2003, 34, 137–148. [Google Scholar] [CrossRef]
  36. Leung, J.; Giraudat, J. Cloning genes of Arabidopsis thaliana by chromosome walking. Methods Mol. Biol. 1998, 82, 277–303. [Google Scholar]
  37. Yamaguchi-Shinozaki, K.; Shinozaki, K. A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress. Plant Cell 1994, 6, 251–264. [Google Scholar] [CrossRef]
  38. Liu, B.; Du, Y.; Cong, L.; Jia, X.; Yang, G. Danshen (Salvia miltiorrhiza) Compounds Improve the Biochemical Indices of the Patients with Coronary Heart Disease. Evid.-Based Complement. Altern. Med. 2016, 2016, 9781715. [Google Scholar] [CrossRef]
  39. Ma, X.; Zhang, L.; Gao, F.; Jia, W.; Li, C. Salvia miltiorrhiza and Tanshinone IIA reduce endothelial inflammation and atherosclerotic plaque formation through inhibiting COX-2. Biomed. Pharmacother. 2023, 167, 115501. [Google Scholar] [CrossRef]
  40. Jia, Y.; Yao, D.; Bi, H.; Duan, J.; Liang, W.; Jing, Z.; Liu, M. Salvia miltiorrhiza Bunge (Danshen) based nano-delivery systems for anticancer therapeutics. Phytomedicine 2024, 128, 155521. [Google Scholar] [CrossRef]
  41. Wang, Y.; Yang, B.; Wang, H.; Yang, C.; Zhang, J.; Zhu, M.; Yang, R. Variation in microbial community structure in the rhizosphere soil of Salvia miltiorrhiza Bunge under three cropping modes. Acta Ecol. Sin. 2019, 39, 4832–4843. [Google Scholar]
  42. Ahluwalia, O.; Singh, P.C.; Bhatia, R. A review on drought stress in plants: Implications, mitigation and the role of plant growth promoting rhizobacteria. Resour. Environ. Sustain. 2021, 5, 100032. [Google Scholar] [CrossRef]
  43. Auldridge, M.E.; McCarty, D.R.; Klee, H.J. Plant carotenoid cleavage oxygenases and their apocarotenoid products. Curr. Opin. Plant Biol. 2006, 9, 315–321. [Google Scholar] [CrossRef] [PubMed]
  44. Rubio, A.; Rambla, J.L.; Santaella, M.; Gomez, M.D.; Orzaez, D.; Granell, A.; Gomez-Gomez, L. Cytosolic and plastoglobule-targeted carotenoid dioxygenases from Crocus sativus are both involved in β-ionone release. J. Biol. Chem. 2008, 283, 24816–24825. [Google Scholar] [CrossRef]
  45. Song, J.-Y.; Luo, H.-M.; Li, C.-F.; Sun, C.; Xu, J.; Chen, S.-L. Salvia miltiorrhiza as medicinal model plant. Yao Xue Xue Bao = Acta Pharm. Sin. 2013, 48, 1099–1106. [Google Scholar]
  46. Liu, H.; Wang, X.; Wang, D.; Zou, Z.; Liang, Z. Effect of drought stress on growth and accumulation of active constituents in Salvia miltiorrhiza Bunge. Ind. Crops Prod. 2011, 33, 84–88. [Google Scholar] [CrossRef]
  47. Zhong, M.; Yu, H.; Jiang, Y.; Liao, J.; Li, G.; Chai, S.; Yang, R.; Jiang, H.; Wang, L.; Deng, X.; et al. Physiological and molecular mechanisms of carbon quantum dots alleviating Cu2+ toxicity in Salvia miltiorrhiza bunge. Environ. Pollut. 2024, 358, 124521. [Google Scholar] [CrossRef]
  48. Deng, H.; Li, Q.; Cao, R.; Ren, Y.; Wang, G.; Guo, H.; Bu, S.; Liu, J.; Ma, P. Overexpression of SmMYC2 enhances salt resistance in Arabidopsis thaliana and Salvia miltiorrhiza hairy roots. J. Plant Physiol. 2023, 280, 153862. [Google Scholar] [CrossRef]
  49. Chen, H.; Zuo, X.; Shao, H.; Fan, S.; Ma, J.; Zhang, D.; Zhao, C.; Yan, X.; Liu, X.; Han, M. Genome-wide analysis of carotenoid cleavage oxygenase genes and their responses to various phytohormones and abiotic stresses in apple (Malus domestica). Plant Physiol. Biochem. 2018, 123, 81–93. [Google Scholar] [CrossRef]
  50. Haider, M.Z.; Sami, A.; Shafiq, M.; Anwar, W.; Ali, S.; Ali, Q.; Muhammad, S.; Manzoor, I.; Shahid, M.A.; Ali, D.; et al. Genome-wide identification and in-silico expression analysis of carotenoid cleavage oxygenases gene family in Oryza sativa (rice) in response to abiotic stress. Front. Plant Sci. 2023, 14, 1269995. [Google Scholar] [CrossRef]
  51. Yao, Y.; Jia, L.; Cheng, Y.; Ruan, M.; Ye, Q.; Wang, R.; Yao, Z.; Zhou, G.; Liu, J.; Yu, J.; et al. Evolutionary Origin of the Carotenoid Cleavage Oxygenase Family in Plants and Expression of Pepper Genes in Response to Abiotic Stresses. Front. Plant Sci. 2022, 12, 792832. [Google Scholar] [CrossRef] [PubMed]
  52. Li, Q.; Zhang, J.; Deng, Y.; Yu, W.; Gao, H.; Jin, J.; Wang, N.; Wang, F.; Li, H. Cloning and expression analysis of GmNCED1 from Glycine max. Chin. J. Oil Crop Sci. 2014, 36, 455–460. [Google Scholar]
  53. Iuchi, S. Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis (vol 27, pg 325, 2001). Plant J. 2002, 30, 611. [Google Scholar]
  54. Bouvier, F.; Dogbo, O.; Camara, B. Biosynthesis of the food and cosmetic plant pigment bixin (annatto). Science 2003, 300, 2089–2091. [Google Scholar] [CrossRef]
  55. Bouvier, F.; Suire, C.; Mutterer, J.; Camara, B. Oxidative remodeling of chromoplast carotenoids: Identification of the carotenoid dioxygenase CsCCD and CsZCD genes involved in crocus secondary metabolite biogenesis. Plant Cell 2003, 15, 47–62. [Google Scholar] [CrossRef]
  56. Chernys, J.T.; Zeevaart, J.A.D. Characterization of the 9-cis-epoxycarotenoid dioxygenase gene family and the regulation of abscisic acid biosynthesis in avocado. Plant Physiol. 2000, 124, 343–353. [Google Scholar] [CrossRef]
  57. Raghavendra, A.S.; Gonugunta, V.K.; Christmann, A.; Grill, E. ABA perception and signalling. Trends Plant Sci. 2010, 15, 395–401. [Google Scholar] [CrossRef]
  58. Adami, M.; De Franceschi, P.; Brandi, F.; Liverani, A.; Giovannini, D.; Rosati, C.; Dondini, L.; Tartarini, S. Identifying a Carotenoid Cleavage Dioxygenase (ccd4) Gene Controlling Yellow/White Fruit Flesh Color of Peach. Plant Mol. Biol. Report. 2013, 31, 1166–1175. [Google Scholar] [CrossRef]
  59. Jonathan, D.-C.; Rosa, U.-C.; Yair, C.-C.; Manuel, C.-U.V.; Diana, S.-U.; Margarita, A.-E.; Renata, R.-M. Carotenoid cleavage dioxygenases 4 from woodiness plant and their relationships with herbaceous plants. Ind. Crops Prod. 2023, 205, 117529. [Google Scholar] [CrossRef]
  60. Ma, J.; Li, J.; Zhao, J.; Zhou, H.; Ren, F.; Wang, L.; Gu, C.; Liao, L.; Han, Y. Inactivation of a Gene Encoding Carotenoid Cleavage Dioxygenase (CCD4) Leads to Carotenoid-Based Yellow Coloration of Fruit Flesh and Leaf Midvein in Peach. Plant Mol. Biol. Report. 2014, 32, 246–257. [Google Scholar] [CrossRef]
  61. Ureshino, K.; Takara, H.; Miyajima, I. Comparison of Expression CCD4 Gene Levels in Petals of Evergreen Azalea Species. Hortic. J. 2019, 88, 535–540. [Google Scholar] [CrossRef]
  62. Sui, X.; Zhang, J.; Golczak, M.; Palczewski, K.; Kiser, P.D. Key Residues for Catalytic Function and Metal Coordination in a Carotenoid Cleavage Dioxygenase. J. Biol. Chem. 2016, 291, 19401–19412. [Google Scholar] [CrossRef] [PubMed]
  63. Hall, B.G. Building Phylogenetic Trees from Molecular Data with MEGA. Mol. Biol. Evol. 2013, 30, 1229–1235. [Google Scholar] [CrossRef] [PubMed]
  64. Yan, Y.; Wang, Z. Genetic transformation of the medicinal plant Salvia miltiorrhiza by Agrobacterium tumefaciens-mediated method. Plant Cell Tissue Organ Cult. 2007, 88, 175–184. [Google Scholar] [CrossRef]
  65. Schmittgen, T.D.; Livak, K.J. Analyzing real-time PCR data by the comparative CT method. Nat. Protoc. 2008, 3, 1101–1108. [Google Scholar] [CrossRef]
  66. Yang, Y.; Hou, S.; Cui, G.; Chen, S.; Wei, J.; Huang, L. Characterization of reference genes for quantitative real-time PCR analysis in various tissues of Salvia miltiorrhiza. Mol. Biol. Rep. 2010, 37, 507–513. [Google Scholar] [CrossRef]
  67. Earley, K.W.; Haag, J.R.; Pontes, O.; Opper, K.; Juehne, T.; Song, K.M.; Pikaard, C.S. Gateway-compatible vectors for plant functional genomics and proteomics. Plant J. 2006, 45, 616–629. [Google Scholar] [CrossRef]
  68. Li, L.; Wang, D.; Zhou, L.; Yu, X.; Yan, X.; Zhang, Q.; Li, B.; Liu, Y.; Zhou, W.; Cao, X.; et al. JA-Responsive Transcription Factor SmMYB97 Promotes Phenolic Acid and Tanshinone Accumulation in Salvia miltiorrhiza. J. Agric. Food Chem. 2020, 68, 14850–14862. [Google Scholar] [CrossRef]
  69. Wang, B.; Niu, J.; Li, B.; Huang, Y.; Han, L.; Liu, Y.; Zhou, W.; Hu, S.; Li, L.; Wang, D.; et al. Molecular Characterization and Overexpression of SmJMT Increases the Production of Phenolic Acids in Salvia miltiorrhiza. Int. J. Mol. Sci. 2018, 19, 3788. [Google Scholar] [CrossRef]
Ijms 25 13223 g001
Figure 1. Phylogenetic trees, gene structures, and conserved motifs of SmCCO genes. (A) SmCCO proteins were clustered into 6 subgroups, designated as S1 to S6. (B) Yellow boxes and grey lines represent exons and introns. (C) Boxes represent motif distributions.
Figure 1. Phylogenetic trees, gene structures, and conserved motifs of SmCCO genes. (A) SmCCO proteins were clustered into 6 subgroups, designated as S1 to S6. (B) Yellow boxes and grey lines represent exons and introns. (C) Boxes represent motif distributions.
Ijms 25 13223 g001
Ijms 25 13223 g002
Figure 2. The number of cis-acting elements in the SmCCO promoter. The graph was generated based on the presence of cis-acting elements in response to specific elicitors/conditions/processes (x-axis) in the SmCCO gene family members (y-axis).
Figure 2. The number of cis-acting elements in the SmCCO promoter. The graph was generated based on the presence of cis-acting elements in response to specific elicitors/conditions/processes (x-axis) in the SmCCO gene family members (y-axis).
Ijms 25 13223 g002
Ijms 25 13223 g003
Figure 3. RT-qPCR analysis of SmCCD4 gene expression level. (A) Expression level of SmCCD4 in different organs (X-axis: root, stem, leaf, and flower) of S. miltiorrhiza. (B,C) Expression level of SmCCD4 under ABA induction and PEG drought stress. Data were normalized to SmACT gene. All data represent means ± SDs of three independent experiments. Statistical significance was assessed with Student’s t-test (*** p < 0.001; ** p < 0.01; * p < 0.05). The X-axis represents the treatment time of ABA and PEG.
Figure 3. RT-qPCR analysis of SmCCD4 gene expression level. (A) Expression level of SmCCD4 in different organs (X-axis: root, stem, leaf, and flower) of S. miltiorrhiza. (B,C) Expression level of SmCCD4 under ABA induction and PEG drought stress. Data were normalized to SmACT gene. All data represent means ± SDs of three independent experiments. Statistical significance was assessed with Student’s t-test (*** p < 0.001; ** p < 0.01; * p < 0.05). The X-axis represents the treatment time of ABA and PEG.
Ijms 25 13223 g003
Ijms 25 13223 g004
Figure 4. Detection of tanshinone and phenolic acid contents in WT and overexpression strains. (A–D) Concentration of tanshinone compounds: tanshinone I (A), tanshinone IIA (B), ihydrotanshinone (C), and cryptotanshinone (D). (E,F) Concentration of phenolic acid compounds: salvianolic acid B (E) and rosmarinic acid (F). All data represent means ± SDs of three independent experiments. Statistical significance was assessed with Student’s t-test (*** p < 0.001; ** p < 0.01; * p < 0.05).
Figure 4. Detection of tanshinone and phenolic acid contents in WT and overexpression strains. (A–D) Concentration of tanshinone compounds: tanshinone I (A), tanshinone IIA (B), ihydrotanshinone (C), and cryptotanshinone (D). (E,F) Concentration of phenolic acid compounds: salvianolic acid B (E) and rosmarinic acid (F). All data represent means ± SDs of three independent experiments. Statistical significance was assessed with Student’s t-test (*** p < 0.001; ** p < 0.01; * p < 0.05).
Ijms 25 13223 g004
Ijms 25 13223 g005
Figure 5. RT-qPCR analysis of key enzyme genes involved in tanshinone (A) and phenolic acid (B) biosynthetic pathway in overexpression and WT strains. Data were normalized to SmACT gene. All data represent means ± SDs of three independent experiments. Statistical significance was assessed with Student’s t-test (*** p < 0.001; ** p < 0.01; * p < 0.05).
Figure 5. RT-qPCR analysis of key enzyme genes involved in tanshinone (A) and phenolic acid (B) biosynthetic pathway in overexpression and WT strains. Data were normalized to SmACT gene. All data represent means ± SDs of three independent experiments. Statistical significance was assessed with Student’s t-test (*** p < 0.001; ** p < 0.01; * p < 0.05).
Ijms 25 13223 g005
Ijms 25 13223 g006
Figure 6. Detection of contents of abscisic acid (A) and MDA (B), H2O2 accumulation (C), and CAT (D) and SOD activity (E) in WT and overexpression (OE) strains. All data represent means ± SDs of three independent experiments. Statistical significance was assessed with Student’s t-test (** p < 0.01; * p < 0.05).
Figure 6. Detection of contents of abscisic acid (A) and MDA (B), H2O2 accumulation (C), and CAT (D) and SOD activity (E) in WT and overexpression (OE) strains. All data represent means ± SDs of three independent experiments. Statistical significance was assessed with Student’s t-test (** p < 0.01; * p < 0.05).
Ijms 25 13223 g006
Table 1. The main medicinal active ingredients in S. miltiorrhiza.
Table 1. The main medicinal active ingredients in S. miltiorrhiza.
Liposoluble Components
(Tanshinones)		Water-Soluble Components
(Phenolic Acids)
NameMolecular StructureNameMolecular
Structure
Tanshinone IIjms 25 13223 i001Rosmarinic acidIjms 25 13223 i002
Tanshinone IIAIjms 25 13223 i003Salvianolic acid AIjms 25 13223 i004
Tanshinone IIBIjms 25 13223 i005Salvianolic acid BIjms 25 13223 i006
CryptotanshinoneIjms 25 13223 i007Salvianolic acid CIjms 25 13223 i008
Isotanshinone IIjms 25 13223 i009Protocatechuic acidIjms 25 13223 i010
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Tian, Q.; Han, W.; Zhou, S.; Yang, L.; Wang, D.; Zhou, W.; Wang, Z. Carotenoid Cleavage Dioxygenase Gene CCD4 Enhances Tanshinone Accumulation and Drought Resistance in Salvia miltiorrhiza. Int. J. Mol. Sci. 2024, 25, 13223. https://doi.org/10.3390/ijms252313223

AMA Style

Tian Q, Han W, Zhou S, Yang L, Wang D, Zhou W, Wang Z. Carotenoid Cleavage Dioxygenase Gene CCD4 Enhances Tanshinone Accumulation and Drought Resistance in Salvia miltiorrhiza. International Journal of Molecular Sciences. 2024; 25(23):13223. https://doi.org/10.3390/ijms252313223

Chicago/Turabian Style

Tian, Qian, Wei Han, Shuai Zhou, Liu Yang, Donghao Wang, Wen Zhou, and Zhezhi Wang. 2024. "Carotenoid Cleavage Dioxygenase Gene CCD4 Enhances Tanshinone Accumulation and Drought Resistance in Salvia miltiorrhiza" International Journal of Molecular Sciences 25, no. 23: 13223. https://doi.org/10.3390/ijms252313223

APA Style

Tian, Q., Han, W., Zhou, S., Yang, L., Wang, D., Zhou, W., & Wang, Z. (2024). Carotenoid Cleavage Dioxygenase Gene CCD4 Enhances Tanshinone Accumulation and Drought Resistance in Salvia miltiorrhiza. International Journal of Molecular Sciences, 25(23), 13223. https://doi.org/10.3390/ijms252313223

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop