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15 pages, 10118 KiB

Open AccessArticle

Overexpression of AtMYB2 Promotes Tolerance to Salt Stress and Accumulations of Tanshinones and Phenolic Acid in Salvia miltiorrhiza

by Tianyu Li, Shuangshuang Zhang, Yidan Li, Lipeng Zhang, Wenqin Song and Chengbin Chen

Int. J. Mol. Sci. 2024, 25(7), 4111; https://doi.org/10.3390/ijms25074111 - 8 Apr 2024

Cited by 1 | Viewed by 1091

Abstract

Salvia miltiorrhiza is a prized traditional Chinese medicinal plant species. Its red storage roots are primarily used for the treatment of cardiovascular and cerebrovascular diseases. In this study, a transcription factor gene AtMYB2 was cloned and introduced into Salvia miltiorrhiza for ectopic expression. [...] Read more.

Salvia miltiorrhiza is a prized traditional Chinese medicinal plant species. Its red storage roots are primarily used for the treatment of cardiovascular and cerebrovascular diseases. In this study, a transcription factor gene AtMYB2 was cloned and introduced into Salvia miltiorrhiza for ectopic expression. Overexpression of AtMYB2 enhanced salt stress resistance in S. miltiorrhiza, leading to a more resilient phenotype in transgenic plants exposed to high-salinity conditions. Physiological experiments have revealed that overexpression of AtMYB2 can decrease the accumulation of reactive oxygen species (ROS) during salt stress, boost the activity of antioxidant enzymes, and mitigate oxidative damage to cell membranes. In addition, overexpression of AtMYB2 promotes the synthesis of tanshinones and phenolic acids by upregulating the expression of biosynthetic pathway genes, resulting in increased levels of these secondary metabolites. In summary, our findings demonstrate that AtMYB2 not only enhances plant tolerance to salt stress, but also increases the accumulation of secondary metabolites in S. miltiorrhiza. Our study lays a solid foundation for uncovering the molecular mechanisms governed by AtMYB2 and holds significant implications for the molecular breeding of high-quality S. miltiorrhiza varieties. Full article

(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants)

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16 pages, 9579 KiB

Open AccessArticle

Metabolic Pathway Engineering Improves Dendrobine Production in Dendrobium catenatum

by Meili Zhao, Yanchang Zhao, Zhenyu Yang, Feng Ming, Jian Li, Demin Kong, Yu Wang, Peng Chen, Meina Wang and Zhicai Wang

Int. J. Mol. Sci. 2024, 25(1), 397; https://doi.org/10.3390/ijms25010397 - 28 Dec 2023

Cited by 3 | Viewed by 1432

Abstract

The sesquiterpene alkaloid dendrobine, widely recognized as the main active compound and a quality control standard of medicinal orchids in the Chinese Pharmacopoeia, demonstrates diverse biological functions. In this study, we engineered Dendrobium catenatum as a chassis plant for the production of dendrobine [...] Read more.

The sesquiterpene alkaloid dendrobine, widely recognized as the main active compound and a quality control standard of medicinal orchids in the Chinese Pharmacopoeia, demonstrates diverse biological functions. In this study, we engineered Dendrobium catenatum as a chassis plant for the production of dendrobine through the screening and pyramiding of key biosynthesis genes. Initially, previously predicted upstream key genes in the methyl-D-erythritol 4-phosphate (MEP) pathway for dendrobine synthesis, including 4-(Cytidine 5′-Diphospho)-2-C-Methyl-d-Erythritol Kinase (CMK), 1-Deoxy-d-Xylulose 5-Phosphate Reductoisomerase (DXR), 2-C-Methyl-d-Erythritol 4-Phosphate Cytidylyltransferase (MCT), and Strictosidine Synthase 1 (STR1), and a few downstream post-modification genes, including Cytochrome P450 94C1 (CYP94C1), Branched-Chain-Amino-Acid Aminotransferase 2 (BCAT2), and Methyltransferase-like Protein 23 (METTL23), were chosen due to their deduced roles in enhancing dendrobine production. The seven genes (SG) were then stacked and transiently expressed in the leaves of D. catenatum, resulting in a dendrobine yield that was two-fold higher compared to that of the empty vector control (EV). Further, RNA-seq analysis identified Copper Methylamine Oxidase (CMEAO) as a strong candidate with predicted functions in the post-modification processes of alkaloid biosynthesis. Overexpression of CMEAO increased dendrobine content by two-fold. Additionally, co-expression analysis of the differentially expressed genes (DEGs) by weighted gene co-expression network analysis (WGCNA) retrieved one regulatory transcription factor gene MYB61. Overexpression of MYB61 increased dendrobine levels by more than two-fold in D. catenatum. In short, this work provides an efficient strategy and prospective candidates for the genetic engineering of D. catenatum to produce dendrobine, thereby improving its medicinal value. Full article

(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants)

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Figure 1
Multigene reconstruction to enhance dendrobine production. (A) Physical map of the multigene constructs used for integration and expression of the synthetic operons. The selected target genes are depicted as light blue boxes. Promotor Prrn is shown in dark yellow and terminator TrbcL in orange. The HPTII selectable marker gene for transformation is represented as a light yellow box. An intercistronic expression element (IEE) was put between STR1 and CYP94C1 operons to ensure the downstream cistron expression under the same promotor. (B) Not I-digestion analysis of pYLTAC380H-multigene (EV, TG, FG, and SG) constructs. M: DNA ladder marker. (C) Dendrobine content in D. catenatum leaves infiltrated with Agrobacterium tumefaciens carrying multigene constructs. There are three replicates for each sample. (D) The relative expression of each gene in a specific multigene construct. EV: empty vector; TG: two genes; FG: five genes; SG: seven genes. Asterisks indicate significance based on the Student’s t-test. * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. Full article ">Figure 2
Functional verification of MCT in dendrobine synthesis. (A) MCT expression was checked by qRT-PCR with samples being collected 6 h after infiltration (n = 3). (B) Transiently infiltrated D. catenatum leaves were harvested (5 dpi) for dendrobine measurement (n = 3). (C) CRISPRi was performed to knock down MCT expression. The kinase-dead version of Cas9 (dCas9) was used to block transcription in the promotor region of MCT. Empty vector without dCas9 served as the control. MCT knock-down was verified by qRT-PCR, with samples being collected 6 h after infiltration (n = 3). (D) Leave samples were collected at 5 dpi and subjected to dendrobine measurement (n = 3). ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. Full article ">Figure 3
Generation and identification of multigene-transgenic plants. (A) Transgenic D. catenatum plantlets of SG-multigene (11-month-old) and EV control (13-month-old). (B) Analysis of the hptII transgene in SG-multigene and EV control transgenic D. catematum plantlets. DNA isolated from wild-type plants was used as the negative control “−”, while the hptII gene in the EV plasmid was used as the positive control “+”. DcActin was PCR amplified to demonstrate an equal amount of loading. nptII was amplified to avoid bacterial contamination. (C) FPPS expression was checked to demonstrate activation of the dendrobine synthesis pathway. EV transgenic D. catenatum served as the control (Ctrl). (D) SG-transgenic D. catenatum grown in pine-bark pots for 10 months. (E) Molecular characterization of the hptII transgene in SG-transgenic D. catenatum grown in pine-bark pots. (F) Molecular characterization of the hptII transgene in SG-transgenic Arabidopsis. (G) Expression analysis of individual genes in SG-transgenic Arabidopsis by qRT-PCR. Scale bar in (A) represents 1 cm. **** p ≤ 0.0001 represents significance. Full article ">Figure 4
SG-transgenic Arabidopsis tolerant to salinity stress. (A) Plant growth in response to various concentrations of NaCl (0, 100, 120, and 200 mM). (B) Plant growth in terms of fresh weight under 100 mM NaCl for four weeks. (C) Cell damage in terms of MDA release under 100 mM NaCl for four weeks. (D) Representative image showing the transgenic plants growing in earth-pot for one month. (E) Comparison of plant height for the transgenic plants growing in earth-pot for one month. Data are represented as means ± SE from three replicates. *** p ≤ 0.001 and **** p ≤ 0.0001 are of significance compared to EV controls. Scale bar represents 1 cm. The center line in (E) represents median. Full article ">Figure 5
SG-transgenic Arabidopsis was tolerant to drought stress. (A) Plant growth in response to different concentrations of PEG6000 (0, 250, 400, and 550 g/L). (B) Plant growth in terms of fresh weight under varied concentrations of PEG6000 for four weeks. (C) Cell damage in terms of MDA release under varied concentrations of PEG6000 for four weeks. * p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001 represent significance. Scale bar represents 1 cm. Full article ">Figure 6
Dendrobine synthesis-related gene screening by Venn diagram and KEGG analysis. (A) Venn distribution of DEGs for transcriptomes of different tissues (stem vs. root; leaf vs. root) from three Dendrobium species (D. houshanense; D. catenatum, and D. moniliforme). (B) KEGG pathway enrichment of 648 DEGs from (A). The x-axis represents the enrichment ratio and the y-axis represents the pathway name. (C) Venn diagram representation of the number of DEGs in samples from protocorm-like bodies (PLBs), samples under MF23 treatment, and the 648 DEGs in (A). (D) KEGG pathway enrichment of DEGs from (C). Full article ">Figure 7
Dendrobine synthesis-related hub gene screening by WGCNA. (A) Hierarchical cluster dendrogram showing six expression modules of co-expressed genes. Each leaf in the tree represents an individual gene, with the branch representing a module of highly connected genes. The designated color rows below correspond to module membership. (B) Scale-free fit index at different threshold values (β). Asterisk indicates the selected soft-thresholding power. (C) Heatmap of connectivity of eigengenes. (D) Module-trait correlations and corresponding p-values (in parenthesis). The color in the box indicates −log(P) and the color scale indicates the p-value from the Fisher exact test. Treatment means MF23 infection. Full article ">Figure 8
Functional verification of downstream genes in dendrobine synthesis. (A) CMEAO overexpression was verified by qRT-PCR. Samples were collected at 24 h post-infiltration. (B) Transiently infiltrated D. catenatum leaves (5 dpi) were harvested and subjected to dendrobine measurement. Empty vectors are used as controls. (C) qRT-PCR verification of MYB61 overexpression in transiently infiltrated one-year-old D. catenatum leaves. (D) Dendrobine content in D. catenatum leaves transiently overexpressing MYB61. Statistical significance was demonstrated as following ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Full article ">

13 pages, 4335 KiB

Open AccessArticle

Technology Invention and Mechanism Analysis of Rapid Rooting of Taxus × media Rehder Branches Induced by Agrobacterium rhizogenes

by Ying Wang, Xiumei Luo, Haotian Su, Ge Guan, Shuang Liu and Maozhi Ren

Int. J. Mol. Sci. 2024, 25(1), 375; https://doi.org/10.3390/ijms25010375 - 27 Dec 2023

Viewed by 1013

Abstract

Taxus, a vital source of the anticancer drug paclitaxel, grapples with a pronounced supply–demand gap. Current efforts to alleviate the paclitaxel shortage involve expanding Taxus cultivation through cutting propagation. However, traditional cutting propagation of Taxus is difficult to root and time-consuming. Obtaining [...] Read more.

Taxus, a vital source of the anticancer drug paclitaxel, grapples with a pronounced supply–demand gap. Current efforts to alleviate the paclitaxel shortage involve expanding Taxus cultivation through cutting propagation. However, traditional cutting propagation of Taxus is difficult to root and time-consuming. Obtaining the roots with high paclitaxel content will cause tree death and resource destruction, which is not conducive to the development of the Taxus industry. To address this, establishing rapid and efficient stem rooting systems emerges as a key solution for Taxus propagation, facilitating direct and continuous root utilization. In this study, Agrobacterium rhizogenes were induced in the 1–3-year-old branches of Taxus × media Rehder, which has the highest paclitaxel content. The research delves into the rooting efficiency induced by different A. rhizogenes strains, with MSU440 and C58 exhibiting superior effects. Transcriptome and metabolome analyses revealed A. rhizogenes’ impact on hormone signal transduction, amino acid metabolism, zeatin synthesis, and secondary metabolite synthesis pathways in roots. LC-MS-targeted quantitative detection showed no significant difference in paclitaxel and baccatin III content between naturally formed and induced roots. These findings underpin the theoretical framework for T. media rapid propagation, contributing to the sustainable advancement of the Taxus industry. Full article

(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants)

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The technical process of branch rooting of T. media. (a) Girdling treatment of 1–3-year-old branches. (b) Plant high-pressure propagation box fixing at the girdling site. (c) Swelling and formation of root apical meristem at the upper girdling site. (d) Formation of young roots at the upper girdling site. (e) Elongation and growth of branch adventitious roots. Full article ">Figure 2
Demonstration of branch rooting. (a) Morphology of roots after opening the high-pressure propagation box, with new shoots emerging from the branch. (b) The morphology of roots formed under the treatment of different A. rhizogenes strains, showing their growth from the girdling site. CK means the normally formed roots of T. media branches without the treatment of A. rhizogenes. (c) The rooting rate of T. media branches under the treatment of different A. rhizogenes strains that, without the treatment of A. rhizogenes, was as a control (CK). (d) The number of adventitious roots in T. media branches under treatments of different A. rhizogenes strains that, without the treatment of A. rhizogenes, was as a control (CK). (e) The growth status of the rooting branches induced by A. rhizogenes strains MSU440 and C58 after being transplanted into pots. Each experiment was repeated three times. Each replicate contains at least 35 branch rooting treatments. Asterisks indicate significant differences (*** p < 0.001). Full article ">Figure 3
Analysis of differentially expressed genes (DEGs) in TR vs. WR; gene ontology (GO); and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs in TR vs. WR. (a) Differentially expressed genes in TR vs. WR. (b) GO enrichment analysis showed that biological processes, cellular anatomical entity, and oxidoreductase activity were involved in the biological process, cellular component, and molecular function, respectively. (c) KEGG enrichment analysis showed that metabolic pathways, biosynthesis of secondary metabolites, and phenylpropanoid biosynthesis were the top three significant enrichment pathways. Full article ">Figure 4
Analysis of differentially expressed metabolites (DEMs) in TR vs. WR. (a) Differentially expressed metabolites in TR vs. WR. (b) Principal component analysis revealed clear differences between the metabolites in TR and WR. (c) Cluster analysis of DEMs. (d) KEGG enrichment analysis showed that DEMs were the main components for the biosynthesis of amino acids and their derivatives. Full article ">Figure 5
Combined analysis of transcriptome and metabolome (DEGs/DEMs) in TR vs. WR. (a) Top ten KEGG pathways correlated with DEGs/DEMs in TR vs. WR. (b) Plant hormone signal transduction pathway in the correlated DEGs/DEMs in TR vs. WR. (c) Zeatin biosynthesis pathway in the correlated DEGs/DEMs in TR vs. WR. In (b,c), the red rectangles indicate the DEGs encoded in the protein were all upregulated, the blue rectangles indicate the DEGs encoded in the protein were all downregulated and the yellow rectangle indicates the genes encoded in the protein contain both upregulated and downregulated DEGs. Full article ">Figure 6
The quantitative detection of paclitaxel and baccatin III. (a) The LC-MS/MS analysis of paclitaxel and baccatin III in 20-year-old roots, WR, and TR. The retention time (RT) of paclitaxel is 3.08 min and that of baccatin III is 2.86 min. (b) The content of paclitaxel in 20-year-old roots, WR, and TR. (c) The content of baccatin III in 20-year-old roots, WR, and TR. Asterisks indicate significant differences (*** p < 0.001). Full article ">

15 pages, 4163 KiB

Open AccessArticle

Comparison of the Metabolomics of Different Dendrobium Species by UPLC-QTOF-MS

by Tingting Zhang, Xinxin Yang, Fengzhong Wang, Pengfei Liu, Mengzhou Xie, Cong Lu, Jiameng Liu, Jing Sun and Bei Fan

Int. J. Mol. Sci. 2023, 24(24), 17148; https://doi.org/10.3390/ijms242417148 - 5 Dec 2023

Viewed by 1315

Abstract

Dendrobium Sw. (family Orchidaceae) is a renowned edible and medicinal plant in China. Although widely cultivated and used, less research has been conducted on differential Dendrobium species. In this study, stems from seven distinct Dendrobium species were subjected to UPLC-QTOF-MS/MS analysis. A total [...] Read more.

Dendrobium Sw. (family Orchidaceae) is a renowned edible and medicinal plant in China. Although widely cultivated and used, less research has been conducted on differential Dendrobium species. In this study, stems from seven distinct Dendrobium species were subjected to UPLC-QTOF-MS/MS analysis. A total of 242 metabolites were annotated, and multivariate statistical analysis was employed to explore the variance in the extracted metabolites across the various groups. The analysis demonstrated that D. nobile displays conspicuous differences from other species of Dendrobium. Specifically, D. nobile stands out from the remaining six taxa of Dendrobium based on 170 distinct metabolites, mainly terpene and flavonoid components, associated with cysteine and methionine metabolism, flavonoid biosynthesis, and galactose metabolism. It is believed that the variations between D. nobile and other Dendrobium species are mainly attributed to three metabolite synthesis pathways. By comparing the chemical composition of seven species of Dendrobium, this study identified the qualitative components of each species. D. nobile was found to differ significantly from other species, with higher levels of terpenoids, flavonoids, and other compounds that are for the cardiovascular field. By comparing the chemical composition of seven species of Dendrobium, these qualitative components have relevance for establishing quality standards for Dendrobium. Full article

(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants)

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Representative total ion current (TIC) chromatograms of different species of DD (Dendrobium denneanum Kerr.), DC (D. chrysotoxum Lindl.), DN (D. nobile Lindl.) DF (D. fimbriatum Hook.), DT (D. thyrsiflorum Rchb. f.), DO (D. officinale Kimura. et Migo.), DV (D. devonianum Paxton.) (A) and of DN alone (B) under positive ionization mode. Full article ">Figure 2
Proposed schematic of the mass fragmentation pathways of (A) (compound 195, caryoptosidic acid); (B) (compound 10, naringenin chalcone); (C) (compound 33, mangiferin); (D) (compound 63, N-Oxysophocarpine); (E) (compound 197, 6,7-dihydroxy-4-methyl coumarin); and (F) (compound 134, lusianthridin). Full article ">Figure 3
Principal component analysis of different species of DD (Dendrobium denneanum Kerr.), DC (D. chrysotoxum Lindl.), DN (D. nobile Lindl.), DF (D. fimbriatum Hook.), DT (D. thyrsiflorum Rchb. f.), DO (D. officinale Kimura. et Migo.), DV (D. devonianum Paxton.) (A); a comparison of verified DN and other species of Dendrobium (B). Full article ">Figure 4
A Venn diagram of variance components (A); a heat map of the differential compositions (B). Full article ">Figure 5
The outcomes of the metabolic pathway analysis. The main differentiating metabolites were annotated and analyzed about metabolic pathways. Each bubble on the bubble diagram represents a metabolic pathway. Technical term abbreviations are explained upon their first use. The horizontal axis and the size of the bubble indicate the size of the pathway influencing factor in the topology analysis. The greater the scale, the bigger the influencing factor. The vertical position of the bubble shows the p-value of the enrichment analysis. Full article ">Figure 6
The key differential metabolites of DN and different species of Dendrobium were mapped to metabolic pathways, and the results of metabolic pathway analysis are shown as KEGG pathway results: (A,D) galactose metabolism; (B,F) flavonoid biosynthesis; (C) cysteine and methionine metabolism; (E) flavone and flavonol biosynthesis. Full article ">

17 pages, 2190 KiB

Open AccessArticle

Evaluation of Reference Genes for Normalizing RT-qPCR and Analysis of the Expression Patterns of WRKY1 Transcription Factor and Rhynchophylline Biosynthesis-Related Genes in Uncaria rhynchophylla

by Detian Mu, Yingying Shao, Jialong He, Lina Zhu, Deyou Qiu, Iain W. Wilson, Yao Zhang, Limei Pan, Yu Zhou, Ying Lu and Qi Tang

Int. J. Mol. Sci. 2023, 24(22), 16330; https://doi.org/10.3390/ijms242216330 - 15 Nov 2023

Cited by 4 | Viewed by 1147

Abstract

Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the [...] Read more.

Uncaria rhynchophylla (Miq.) Miq. ex Havil, a traditional medicinal herb, is enriched with several pharmacologically active terpenoid indole alkaloids (TIAs). At present, no method has been reported that can comprehensively select and evaluate the appropriate reference genes for gene expression analysis, especially the transcription factors and key enzyme genes involved in the biosynthesis pathway of TIAs in U. rhynchophylla. Reverse transcription quantitative PCR (RT-qPCR) is currently the most common method for detecting gene expression levels due to its high sensitivity, specificity, reproducibility, and ease of use. However, this methodology is dependent on selecting an optimal reference gene to accurately normalize the RT-qPCR results. Ten candidate reference genes, which are homologues of genes used in other plant species and are common reference genes, were used to evaluate the expression stability under three stress-related experimental treatments (methyl jasmonate, ethylene, and low temperature) using multiple stability analysis methodologies. The results showed that, among the candidate reference genes, S-adenosylmethionine decarboxylase (SAM) exhibited a higher expression stability under the experimental conditions tested. Using SAM as a reference gene, the expression profiles of 14 genes for key TIA enzymes and a WRKY1 transcription factor were examined under three experimental stress treatments that affect the accumulation of TIAs in U. rhynchophylla. The expression pattern of WRKY1 was similar to that of tryptophan decarboxylase (TDC) under ETH treatment. This research is the first to report the stability of reference genes in U. rhynchophylla and provides an important foundation for future gene expression analyses in U. rhynchophylla. The RT-qPCR results indicate that the expression of WRKY1 is similar to that of TDC under ETH treatment. It may coordinate the expression of TDC, providing a possible method to enhance alkaloid production in the future through synthetic biology. Full article

(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants)

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The biosynthetic pathway of rhynchophylline and isorhynchophylline in U. rhynchophylla. Solid arrows represent genes identified in the pathway and dashed arrows represent presumed genes. Full article ">Figure 2
The Ct values of 10 candidate reference genes in all samples. The expression data are displayed as the Ct value of each reference gene in the sample of U. rhynchophylla. The boxplot indicates the 25th/75th percentiles and it contains the mean, maximum, and minimum values. Full article ">Figure 3
Average expression stability values (M) of the ten candidate reference genes using GeNorm. The expression stability was evaluated in samples from leaves of U. rhynchophylla subjected to low temperatures, MeJA (100 μmol), ETH (100 μmol), control (not treated), and total (all treated samples). The least stable genes are on the left with higher M values and the most stable genes are on the right with lower M values. Full article ">Figure 4
The pairwise variation (Vn/n + 1) scores of ten candidates measured by GeNorm. Different treatments are identified by different colors. The value used to determine the appropriate number of reference genes for RT-qPCR normalization is 0.15. Full article ">Figure 5
The relative expression of the WRKY1 transcription factor and key enzyme genes in response to MeJA, ETH, and low temperatures. Different colors represent different times. (a) MeJA treatment; (b) ethylene treatment; (c) low-temperature treatment. The error bars represent the mean ± SD from three biological replicates, and asterisks indicate statistically significant differences compared with the controls (0 h).* p < 0.05; ** p < 0.01. Full article ">

22 pages, 10575 KiB

Open AccessArticle

Reference Genes Screening and Gene Expression Patterns Analysis Involved in Gelsenicine Biosynthesis under Different Hormone Treatments in Gelsemium elegans

by Yao Zhang, Detian Mu, Liya Wang, Xujun Wang, Iain W. Wilson, Wenqiang Chen, Jinghan Wang, Zhaoying Liu, Deyou Qiu and Qi Tang

Int. J. Mol. Sci. 2023, 24(21), 15973; https://doi.org/10.3390/ijms242115973 - 4 Nov 2023

Cited by 3 | Viewed by 1137

Abstract

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an accurate method for quantifying gene expression levels. Choosing appropriate reference genes to normalize the data is essential for reducing errors. Gelsemium elegans is a highly poisonous but important medicinal plant used for analgesic and [...] Read more.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) is an accurate method for quantifying gene expression levels. Choosing appropriate reference genes to normalize the data is essential for reducing errors. Gelsemium elegans is a highly poisonous but important medicinal plant used for analgesic and anti-swelling purposes. Gelsenicine is one of the vital active ingredients, and its biosynthesis pathway remains to be determined. In this study, G. elegans leaf tissue with and without the application of one of four hormones (SA, MeJA, ETH, and ABA) known to affect gelsenicine synthesis, was analyzed using ten candidate reference genes. The gene stability was evaluated using GeNorm, NormFinder, BestKeeper, ∆CT, and RefFinder. The results showed that the optimal stable reference genes varied among the different treatments and that at least two reference genes were required for accurate quantification. The expression patterns of 15 genes related to the gelsenicine upstream biosynthesis pathway was determined by RT-qPCR using the relevant reference genes identified. Three genes 8-HGO, LAMT, and STR, were found to have a strong correlation with the amount of gelsenicine measured in the different samples. This research is the first study to examine the reference genes of G. elegans under different hormone treatments and will be useful for future molecular analyses of this medically important plant species. Full article

(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants)

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14 pages, 3086 KiB

Open AccessArticle

Synthesis of Crocin I and Crocin II by Multigene Stacking in Nicotiana benthamiana

by Lei Xie, Zuliang Luo, Xunli Jia, Changming Mo, Xiyang Huang, Yaran Suo, Shengrong Cui, Yimei Zang, Jingjing Liao and Xiaojun Ma

Int. J. Mol. Sci. 2023, 24(18), 14139; https://doi.org/10.3390/ijms241814139 - 15 Sep 2023

Cited by 3 | Viewed by 2108

Abstract

Crocins are a group of highly valuable water-soluble carotenoids that are reported to have many pharmacological activities, such as anticancer properties, and the potential for treating neurodegenerative diseases including Alzheimer’s disease. Crocins are mainly biosynthesized in the stigmas of food–medicine herbs Crocus sativus [...] Read more.

Crocins are a group of highly valuable water-soluble carotenoids that are reported to have many pharmacological activities, such as anticancer properties, and the potential for treating neurodegenerative diseases including Alzheimer’s disease. Crocins are mainly biosynthesized in the stigmas of food–medicine herbs Crocus sativus L. and Gardenia jasminoides fruits. The distribution is narrow in nature and deficient in resources, which are scarce and expensive. Recently, the synthesis of metabolites in the heterologous host has opened up the potential for large-scale and sustainable production of crocins, especially for the main active compounds crocin I and crocin II. In this study, GjCCD4a, GjALDH2C3, GjUGT74F8, and GjUGT94E13 from G. jasminoides fruits were expressed in Nicotiana benthamiana. The highest total content of crocins in T1 generation tobacco can reach 78,362 ng/g FW (fresh weight) and the dry weight is expected to reach 1,058,945 ng/g DW (dry weight). Surprisingly, the primary effective constituents crocin I and crocin II can account for 99% of the total crocins in transgenic plants. The strategy mentioned here provides an alternative platform for the scale-up production of crocin I and crocin II in tobacco. Full article

(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants)

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The crocin biosynthetic pathway in Crocus sativus L. (pink) and Gardenia jasminoides (blue). PSY, phytoene synthase; PDS, phytoene desaturase; Z-ISO, ζ-carotene isomerase; ZDS, ζ-carotene desaturase; CRTISO, carotenoid isomerase; LCYB, lycopene β-cyclase; BHY, β-carotene hydrolase; GjCCD4a and CsCCD2, carotenoid cleavage dioxygenase; GjALDH and CsALDH3l1, aldehyde dehydrogenase; GjUGT74F8, GjUGT94E13, CsUGT74AD1, and UGT709G1, UDP-glucosyltransferase. Full article ">Figure 2
Genetic transformation of AU-CU vector plants of T0 generation. (A). T-DNA region of the multigene vector AU-CU that was transformed into the tobacco plants. (B). Compare the leaves, roots, and stems of AU-CU transgenic plants with those of the wild-type. Full article ">Figure 3
Genetic Transformation of Nicotiana benthamiana with multigene vector synthesizing crocins and molecular identification. (A) The relative gene expression level analysis of crocins synthesis genes in transgenic tobacco lines. The Nbactin was used as an internal control and the wild-type was set to 1. The data are presented as the mean values ± SDs, n = 3 biologically independent samples. (B) PCR detection of crocins biosynthetic genes. The first lane is the DNA maker (4500 bp), the second is the wild type, and then from left to right are transgenic Nicotiana benthamiana lines N16, N18, N20, N22, and N34. (C) PCR to identify whether the T1 generation carries SgCCD4a and SgUGT74F8. The first lane of each row is the DNA maker (2000 bp), the second is the wild type, and the rest are the T1 generations. Full article ">Figure 4
Comparison of the production of crocins crocetin between Crocus sativus L. stigmas, Gardenia jasminoides fruits, and transgenic tobacco. The red curve represents HPLC-MS/MS analysis of crocins in Crocus sativus L. stigmas and Gardenia jasminoides fruits. The blue curve represents HPLC-MS/MS analysis of crocins and crocetin standards. The orange curve represents HPLC-MS/MS analysis of crocins in N18, N20, and N34 lines; t-I, c-I, t-II, c-II, t-III, c-III, t-IV, and t-V represent trans-crocin I, cis-crocin I, trans-crocin II, cis-crocin II, trans-crocin III, cis-crocin III, trans-crocin IV and trans-crocin V, respectively. Full article ">Figure 5
Probability density distribution curve of T1 generation crocin I and crocin II. (A). Probability density distribution curve of crocin I. (B). Probability density distribution curve of crocin II. The red curve represents the T1 generations of the N16 line; the green represents the T1 generations of the N18 lines; and the blue represents the T1 generations of the N20 line. The black represents the T1 generations of the N22 line and the pink represents the T1 generations of the N34 line. The curve plotting data are the mean values ± SDs, n = 3 biologically independent samples. Full article ">Figure 6
Accumulation of crocins in transgenic tobacco lines of T1 generation. The data are presented as the mean values ± SDs, n = 3 biologically independent samples. Full article ">

15 pages, 3372 KiB

Open AccessArticle

Characterization of Volatile Organic Compounds in Five Celery (Apium graveolens L.) Cultivars with Different Petiole Colors by HS-SPME-GC-MS

by Yue Sun, Mengyao Li, Xiaoyan Li, Jiageng Du, Weilong Li, Yuanxiu Lin, Yunting Zhang, Yan Wang, Wen He, Qing Chen, Yong Zhang, Xiaorong Wang, Ya Luo, Aisheng Xiong and Haoru Tang

Int. J. Mol. Sci. 2023, 24(17), 13343; https://doi.org/10.3390/ijms241713343 - 28 Aug 2023

Cited by 4 | Viewed by 1639

Abstract

Celery (Apium graveolens L.) is an important vegetable crop cultivated worldwide for its medicinal properties and distinctive flavor. Volatile organic compound (VOC) analysis is a valuable tool for the identification and classification of species. Currently, less research has been conducted on aroma [...] Read more.

Celery (Apium graveolens L.) is an important vegetable crop cultivated worldwide for its medicinal properties and distinctive flavor. Volatile organic compound (VOC) analysis is a valuable tool for the identification and classification of species. Currently, less research has been conducted on aroma compounds in different celery varieties and colors. In this study, five different colored celery were quantitatively analyzed for VOCs using HS-SPME, GC-MS determination, and stoichiometry methods. The result revealed that γ-terpinene, d-limonene, 2-hexenal,-(E)-, and β-myrcene contributed primarily to the celery aroma. The composition of compounds in celery exhibited a correlation not only with the color of the variety, with green celery displaying a higher concentration compared with other varieties, but also with the specific organ, whereby the content and distribution of volatile compounds were primarily influenced by the leaf rather than the petiole. Seven key genes influencing terpenoid synthesis were screened to detect expression levels. Most of the genes exhibited higher expression in leaves than petioles. In addition, some genes, particularly AgDXS and AgIDI, have higher expression levels in celery than other genes, thereby influencing the regulation of terpenoid synthesis through the MEP and MVA pathways, such as hydrocarbon monoterpenes. This study identified the characteristics of flavor compounds and key aroma components in different colored celery varieties and explored key genes involved in the regulation of terpenoid synthesis, laying a theoretical foundation for understanding flavor chemistry and improving its quality. Full article

(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants)

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Figure 1

Figure 1
Total ion flow diagram of volatile compounds in leaves (A) and petioles (B) of five celery varieties. Different colors of writing screened colors of celery, including green for ‘Hanyu Nencuixiqin’ and ‘Siji Lvxiangqin’, white for ‘Jingpin Saixue’, purple for ‘Ziyu Xiangqin’, and yellow for ‘Shanghai Huangxinqin’. Full article ">Figure 2
Analysis of compound species of five celery varieties. (A) A Venn diagram showed the differences and common compounds of five celery varieties. (B) The percentage stacking chart of eight kinds of compounds in five celery varieties. Full article ">Figure 3
Characteristic compound analysis of high-content compounds (Benzene,-1-methyl-3-(1-methylethyl)-abbreviated as Isopropyl M-Tolyl Sulfide). (A) The nine principle compounds in celery. Each error line represents the mean ±SD (standard deviation), t-test with significance level of 0.05 (p < 0.05). The different letters with lowcase (a–e) indicate significance differences, the same letter indicating no significance difference. (B) The correlation between tissues, cultivars, and VOCs. The horizontal axis stands for five cultivars. On the x-axis, from A to E, there were five types of celery. A for ‘Hanyu Nencuixiqin’, B for ‘Jingpin Saixue’, C for ‘Ziyu Xiangqin’, D for ‘Shanghai Huangxinqin’, and E for ‘Siji Lvxiangqin’; 1 for leaves, 2 for petioles. Full article ">Figure 4
PCA analysis of two tissues, the leaf and petiole, of five celery varieties. (A)The summary of PC1 and PC2 and significant variability between different celery varieties and different celery parts. (B) Principle analysis of the influence of compounds in different samples. Full article ">Figure 5
OPLS-DA chart of 18 compounds in five celery varieties. (A) Orthogonal partial least squares discriminant analysis of 18 compounds. The score t1 (first component) explains the largest variation of the X space, followed by t2. The numbers besides t[1] and t[2] shown the weight of eh regrression coefficient. (B) Variable importance for the projection numbers. (C) All compounds differ between two tissues in five celery varieties. The letters (A–J) shows the ten examples of celery, for leaf and petiole of ‘Hanyu Nencuixiqin’, ‘Jingpin Saixue’, ‘Ziyu Xiangqin’, ‘Shanghai Huangxinqin’, ‘Siji Lvxiangqin’, respectively. Full article ">Figure 6
Analysis of key regulatory genes in the terpenoid synthesis pathway. (A) Gene expression in distinctively colored celery. (B) Key genes and compounds in MVA and MEP pathways. (C) Gene expression in distinctive tissues. (D) RT-qPCR analysis of gene expression levels in pathway of terpenoid synthesis. Column chart in the same color with the expression of the leaf on the left side and the petiole on the right side. Each error line represents the mean ± SD (standard deviation), t-test with significance level of 0.05 (p < 0.05). The different letters with lowcase (a–e) indicate significance differences, the same letter indicating no significance difference. Full article ">

19 pages, 4059 KiB

Open AccessArticle

Biosynthesis of α-Bisabolol by Farnesyl Diphosphate Synthase and α-Bisabolol Synthase and Their Related Transcription Factors in Matricaria recutita L.

by Yuling Tai, Honggang Wang, Ping Yao, Jiameng Sun, Chunxiao Guo, Yifan Jin, Lu Yang, Youhui Chen, Feng Shi, Luyao Yu, Shuangshuang Li and Yi Yuan

Int. J. Mol. Sci. 2023, 24(2), 1730; https://doi.org/10.3390/ijms24021730 - 15 Jan 2023

Cited by 3 | Viewed by 2996

Abstract

The essential oil of German chamomile (Matricaria recutita L.) is widely used in food, cosmetics, and the pharmaceutical industry. α-Bisabolol is the main active substance in German chamomile. Farnesyl diphosphate synthase (FPS) and α-bisabolol synthase (BBS) are key enzymes related to the [...] Read more.

The essential oil of German chamomile (Matricaria recutita L.) is widely used in food, cosmetics, and the pharmaceutical industry. α-Bisabolol is the main active substance in German chamomile. Farnesyl diphosphate synthase (FPS) and α-bisabolol synthase (BBS) are key enzymes related to the α-bisabolol biosynthesis pathway. However, little is known about the α-bisabolol biosynthesis pathway in German chamomile, especially the transcription factors (TFs) related to the regulation of α-bisabolol synthesis. In this study, we identified MrFPS and MrBBS and investigated their functions by prokaryotic expression and expression in hairy root cells of German chamomile. The results suggest that MrFPS is the key enzyme in the production of sesquiterpenoids, and MrBBS catalyzes the reaction that produces α-bisabolol. Subcellular localization analysis showed that both MrFPS and MrBBS proteins were located in the cytosol. The expression levels of both MrFPS and MrBBS were highest in the extension period of ray florets. Furthermore, we cloned and analyzed the promoters of MrFPS and MrBBS. A large number of cis-acting elements related to light responsiveness, hormone response elements, and cis-regulatory elements that serve as putative binding sites for specific TFs in response to various biotic and abiotic stresses were identified. We identified and studied TFs related to MrFPS and MrBBS, including WRKY, AP2, and MYB. Our findings reveal the biosynthesis and regulation of α-bisabolol in German chamomile and provide novel insights for the production of α-bisabolol using synthetic biology methods. Full article

(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants)

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14 pages, 5021 KiB

Open AccessArticle

A Novel R2R3-MYB Transcription Factor SbMYB12 Positively Regulates Baicalin Biosynthesis in Scutellaria baicalensis Georgi

by Wentao Wang, Suying Hu, Jing Yang, Caijuan Zhang, Tong Zhang, Donghao Wang, Xiaoyan Cao and Zhezhi Wang

Int. J. Mol. Sci. 2022, 23(24), 15452; https://doi.org/10.3390/ijms232415452 - 7 Dec 2022

Cited by 13 | Viewed by 1680

Abstract

Scutellaria baicalensis Georgi is an annual herb from the Scutellaria genus that has been extensively used as a traditional medicine for over 2000 years in China. Baicalin and other flavonoids have been identified as the principal bioactive ingredients. The biosynthetic pathway of baicalin [...] Read more.

Scutellaria baicalensis Georgi is an annual herb from the Scutellaria genus that has been extensively used as a traditional medicine for over 2000 years in China. Baicalin and other flavonoids have been identified as the principal bioactive ingredients. The biosynthetic pathway of baicalin in S. baicalensis has been elucidated; however, the specific functions of R2R3-MYB TF, which regulates baicalin synthesis, has not been well characterized in S. baicalensis to date. Here, a S20 R2R3-MYB TF (SbMYB12), which encodes 263 amino acids with a length of 792 bp, was expressed in all tested tissues (mainly in leaves) and responded to exogenous hormone methyl jasmonate (MeJA) treatment. The overexpression of SbMYB12 significantly promoted the accumulation of flavonoids such as baicalin and wogonoside in S. baicalensis hairy roots. Furthermore, biochemical experiments revealed that SbMYB12 is a nuclear-localized transcription activator that binds to the SbCCL7-4, SbCHI-2, and SbF6H-1 promoters to activate their expression. These results illustrate that SbMYB12 positively regulates the generation of baicalin and wogonoside. In summary, this work revealed a novel S20 R2R3-MYB regulator and enhances our understanding of the transcriptional and regulatory mechanisms of baicalin biosynthesis, as well as sheds new light on metabolic engineering in S. baicalensis. Full article

(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants)

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Figure 1

Figure 1
Expression profiles of SbMYB12 in different tissues of S. baicalensis. The data represent the means of three biological replicates, and the error bars indicate the standard deviation (SD). Significant differences between means were identified (depicted by different letters; p < 0.05) using one-way ANOVA (followed by Tukey’s comparisons). Full article ">Figure 2
Prediction of SbMYB12 protein structure and domains. (A) Predicted protein secondary structure. (B) Predicted protein domains. (C) Predicted tertiary structure. Full article ">Figure 3
Analysis of SbMYB12 compared with related sequences. (A) SbMYB12 gene phylogenetic analysis along with 17 representatives of the R2R3-MYB S20 subgroup. (B) Analysis of sequence alignments between SbMYB12 and other R2R3-MYB S20 subgroup proteins from different plants. Red frames indicate conserved R2 and R3 domains, and blue frames indicate the WxPRL core sequence. Full article ">Figure 4
Subcellular localization and transactivation activities of the SbMYB12 protein. (A) Subcellular location of SbMYB12 in Arabidopsis mesophyll protoplasts. (B) Transcriptional activation activity analysis of BD-SbMYB12 in yeast (Scale bar: 8mm). Full article ">Figure 5
Identification of transgenic hairy roots of S. baicalensis. (A) Transgenic hairy root lines and observations with red fluorescent protein. A4: ArA4 lines (negative control); EV: ArA4 strain that harbors the pK7WG2R plasmids (positive control); MYB12-1~3: SbMYB12 overexpression hairy root lines. (B) PCR identification of transgenic hairy roots of S. baicalensis. PCR screening of SbMYB12 overexpressing lines. M: DNA marker (DL2000); P: ArA4 strain containing recombinant plasmid (positive control); N: S. baicalensis sterile plantlet (negative control); W: wild seedling of S. baicalensis. (C) Real-time quantitative PCR analysis of SbMYB12 in transgenic lines. CK1-3: ArA4 lines; OE1–3: SbMYB12 overexpression transgenic line; ** represents significant difference (p < 0.01) via Student’s t-test. Full article ">Figure 6
SbMYB12 promoted the accumulation of flavonoids (baicalin and wogonoside) in S. baicalensis transgenic hairy roots. (A) Phenotypes of the two-month-old SbMYB12-overexpression (OE) lines and the control (CK). (B) Concentrations of flavonoids in the transgenic hairy roots and the control (CK). Significant differences between the OE lines and the CK were identified (depicted by * p < 0.05) via Student’s t-test. Full article ">Figure 7
Expression analyses of enzyme genes for the flavonoid biosynthesis pathway in SbMYB12 overexpressed transgenic hairy roots. (A) Proposed biosynthetic pathway for flavonoids in S. baicalensis. (B) Flavonoid biosynthesis pathway gene expression analysis in SbMYB12 overexpressed strain. Significant differences between the OE lines and the CK were identified (depicted by * p < 0.05, ** p < 0.01) via Student’s t-test. Full article ">Figure 8
SbMYB12 binds to the SbCCL7-4, SbCHI-2, and SbF6H-1 promoters. (A) Distribution of the MYB-binding sites in the SbCCL7-4, SbCHI-2, and SbF6H-1 promoters. (B) Y1H assays indicated interactions between SbMYB12 and the promoter regions of SbCCL7-4, SbCHI-2, and SbF6H-1. MRE: AACCTAA; MBS1: CAACTG; MBS3: TAACTG. (Scale bar: 6 mm). Full article ">

15 pages, 3769 KiB

Open AccessArticle

Transcription Factor SmSPL2 Inhibits the Accumulation of Salvianolic Acid B and Influences Root Architecture

by Xiangzeng Wang, Yao Cao, Jiaxin Yang, Tong Zhang, Qianqian Yang, Yanhua Zhang, Donghao Wang and Xiaoyan Cao

Int. J. Mol. Sci. 2022, 23(21), 13549; https://doi.org/10.3390/ijms232113549 - 4 Nov 2022

Cited by 5 | Viewed by 1590

Abstract

The SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factor play vital roles in plant growth and development. Although 15 SPL family genes have been recognized in the model medical plant Salvia miltiorrhiza Bunge, most of them have not been functionally characterized to date. Here, we [...] Read more.

The SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factor play vital roles in plant growth and development. Although 15 SPL family genes have been recognized in the model medical plant Salvia miltiorrhiza Bunge, most of them have not been functionally characterized to date. Here, we performed a careful characterization of SmSPL2, which was expressed in almost all tissues of S. miltiorrhiza and had the highest transcriptional level in the calyx. Meanwhile, SmSPL2 has strong transcriptional activation activity and resides in the nucleus. We obtained overexpression lines of SmSPL2 and rSmSPL2 (miR156-resistant SmSPL2). Morphological changes in roots, including longer length, fewer adventitious roots, decreased lateral root density, and increased fresh weight, were observed in all of these transgenic lines. Two rSmSPL2-overexpressed lines were subjected to transcriptome analysis. Overexpression of rSmSPL2 changed root architectures by inhibiting biosynthesis and signal transduction of auxin, while triggering that of cytokinin. The salvianolic acid B (SalB) concentration was significantly decreased in rSmSPL2-overexpressed lines. Further analysis revealed that SmSPL2 binds directly to the promoters of Sm4CL9, SmTAT1, and SmPAL1 and inhibits their expression. In conclusion, SmSPL2 is a potential gene that efficiently manipulate both root architecture and SalB concentration in S. miltiorrhiza. Full article

(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants)

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Figure 1

Figure 1
Expression profiles of SmSPL2. (A) Expression level of SmSPL2 in different tissues. Different letters indicate significant differences between means at the p < 0.05 level by one-way ANOVA test (followed by Tukey’s comparisons). (B–J) GUS staining signals of transgenic Arabidopsis expressing SmSPL2pro::GUS. (B–D) Seedlings after germination at two (B), five (C), and nine (D) days. (E–I) Stem leaf (E), rosette leaf (F), stem (G), flower (H), and root (I) at the flowering stage. (J) Silique. Full article ">Figure 2
Subcellular location and transactivation activity analysis of SmSPL2 protein. (A) Subcellular location of SmSPL2 in onion epidermal cells. The fluorescence signal was observed via a laser scanning confocal microscope. DAPI, 4′,6-diamidino-2-phenylindole is a blue-fluorescent DNA stain used to indicate the nucleus region. (B) Transcriptional activation analysis of SmSPL2 protein. Yeast colonies with three different dilutions were cultured on SD/-Trp and SD/-Trp/-Ade/-His/X-α-gal media. X-α-gal: 5-Bromo-4-chloro-3-indolyl-α-D-galactoside medium, the color reaction substrate of α-galactosidase. Full article ">Figure 3
Phenotype of transgenic S. miltiorrhiza plantlets overexpressing SmSPL2 (SmSPL2-OE) and miR156-resistant SmSPL2 (rSmSPL2-OE). (A–C) Phenotype of 15-day-old (A), one-month-old (B), and two-month-old (C) transgenic S. miltiorrhiza plants. (D) Expression levels of SmSPL2 in the roots of different transgenic lines. (E–G) Adventitious root (AR) length (E), AR numbers (F), fresh weight of root (G) of two-month-old transgenic lines. All data are mean values of three biological replicates, with the error bar representing SD. * and ** indicate significant differences from the control at p < 0.05 and p < 0.01 level, respectively, by Student’s t-test. Full article ">Figure 4
Changes in transcriptional levels of genes for cytokinin and auxin biosynthesis and signal transduction. (A) Proposed biosynthetic and signal transduction pathway for cytokinin. DMAPP, dimethylallyl pyrophosphate; IPT, isopentenyl transferases; CYP, cytochrome P450 enzyme; LOG, LONELY GUY; AHPs, histidine phosphotransfer proteins; CRF, cytokinin response factors; B-AAR, type-B response regulators; A-AAR, type-A response regulators. (B) DEGs involved in cytokinin biosynthesis and the signal transduction pathway. For each gene, the relative expression (rSmSPL2-OE2 vs. control and rSmSPL2-OE7 vs. control) was expressed in Log2FC. Blue boxes (gene expression is down-regulated); Red boxes (gene expression is up-regulated). * indicates differentially expressed genes. (C) Concentrations of cytokinin in root extracts from control and transgenic line rSmSPL2-OE2 and rSmSPL2-OE7. *, values are significantly different from control at p < 0.05 by Student’s t-test. (D) Proposed biosynthetic and signal transduction pathways for auxin. TAA, Tryptophan aminotransferase of Arabidopsis; TAR, Tryptophan aminotransferase related; YUC, YUCCA; AIM, Indole-3-acetamide hydrolase; AAO, Aldehyde oxidase; TIR1, Transport Inhibitor Response 1; AUX/IAA, auxin/indole acetic acid; GH3, Gretchen Hagen 3; SAUR, small auxin-up RNA. (E) DEGs involved in auxin biosynthesis and signal transduction pathway. For each gene, the relative expression (rSmSPL2-OE2 vs. control and rSmSPL2-OE7 vs. control) was expressed in Log2FC. Blue boxes (gene expression is down-regulated); Red boxes (gene expression is up-regulated). * indicates differentially expressed genes. Gene ID were listed in <a href="#app1-ijms-23-13549" class="html-app">Table S2</a>. (F) Concentrations of auxin in root extracts from control and transgenic line rSmSPL2-OE2 and rSmSPL2-OE7. **, values are significantly different from control at p < 0.01 by Student’s t-test. Full article ">Figure 5
Overexpression of miR156-resistant SmSPL2 (rSmSPL2) decreased the accumulation of rosmarinic acid (RA) and salvianolic acid B (SalB). (A) Transcriptome analysis of enzyme genes for salvianolic acid biosynthesis pathway in transgenic lines (rSmSPL2-OE2 and rSmSPL2-OE7). For each gene, the relative expression (rSmSPL2-OE2 vs. control and rSmSPL2-OE7 vs. control) was expressed as Log2FC. Blue boxes (gene expression is down-regulated); Red boxes (gene expression is up-regulated). TAT, tyrosine aminotransferase; HPPR, hydroxyl phenylpyruvate reductase; PAL, phenylalanine ammonia lyase; C4H, cinnamate 4-hydroxylase; 4CL, hydroxycinnamate-CoA ligase; RAS, rosmarinic acid synthase; and CYP, cytochrome P450 enzymes. (B) Validation by qRT-PCR of six enzyme genes involved in salvianolic acid biosynthesis in transgenic lines and control. (C) Chromatograms of RA and SalB in transgenic lines and the control. (D) RA and SalB concentrations in the roots of two-month-old transgenic lines and the control. All data are mean values of three biological replicates, with the error bar representing SD. * and ** indicate significant difference from the control at p < 0.05 and p < 0.01 level, respectively, by Student’s t-test. Full article ">Figure 6
SmSPL2 binds to the promoter regions of Sm4CL9, SmTAT1, and SmPAL1 and inhibits their expression. (A) GTAC motifs in the promoter regions of Sm4CL9, SmCYP98A14, SmTAT1, and SmPAL1. Black rectangles represent the GTAC motif. (B) Yeast one-hybrid detected interactions between the SmSPL6 and the promoters of Sm4CL9, SmCYP98A14, SmTAT1, and SmPAL1. The p53HIS2/pGADT7-p53 and p53HIS2/pGADT7 were the positive and negative controls, respectively. (C) Schematic diagram of constructs used in assays of transient transcriptional activity. (D) SmSPL2 inhibited the expression of Sm4CL9, SmTAT1, and SmPAL1. Effector SmSPL2 was co-transformed with reporter p4CL9-LUC, pTAT1-LUC, or pPAL1-LUC. All data are mean values of three biological replicates, with the error bar representing SD. * and ** indicate significant differences from the control at p < 0.05 and p < 0.01 levels, respectively, by Student’s t-test. Full article ">

18 pages, 4844 KiB

Open AccessArticle

Integrating Metabolomics and Transcriptomics to Unveil Atisine Biosynthesis in Aconitum gymnandrum Maxim

by Lingli Chen, Mei Tian, Baolong Jin, Biwei Yin, Tong Chen, Juan Guo, Jinfu Tang, Guanghong Cui and Luqi Huang

Int. J. Mol. Sci. 2022, 23(21), 13463; https://doi.org/10.3390/ijms232113463 - 3 Nov 2022

Cited by 4 | Viewed by 2142

Abstract

Diterpene alkaloids (DAs) are characteristic compounds in Aconitum, which are classified into four skeletal types: C₁₈, C₁₉, C₂₀, and bisditerpenoid alkaloids. C₂₀-DAs are thought to be the precursor of the other types. Their biosynthetic [...] Read more.

Diterpene alkaloids (DAs) are characteristic compounds in Aconitum, which are classified into four skeletal types: C₁₈, C₁₉, C₂₀, and bisditerpenoid alkaloids. C₂₀-DAs are thought to be the precursor of the other types. Their biosynthetic pathway, however, is largely unclear. Herein, we combine metabolomics and transcriptomics to unveil the methyl jasmonate (MJ) inducible biosynthesis of DAs in the sterile seedling of A. gymnandrum, the only species in the Subgenus Gymnaconitum (Stapf) Rapaics. Target metabolomics based on root and aerial portions identified 51 C₁₉-DAs and 15 C₂₀-DAs, with 40 inducible compounds. The highest content of C₂₀-DA atisine was selected for further network analysis. PacBio Isoform sequencing integrated with RNA sequencing not only provided the full-length transcriptome but also their response to induction, revealing 1994 genes that exhibited up-regulated expression. Further, 38 genes involved in terpenoid biosynthesis were identified, including 7 diterpene synthases. In addition to the expected function of the four diterpene synthases, AgCPS5 was identified to be a new ent-8,13-CPP synthase in Aconitum and could also combine with AgKSL1 to form the C₂₀-DAs precursor ent-atiserene. Combined with multiple network analyses, six CYP450 and seven 2-ODD genes predicted to be involved in the biosynthesis of atisine were also identified. This study not only sheds light on diterpene synthase evolution in Aconitum but also provides a rich dataset of full-length transcriptomes, systemic metabolomes, and gene expression profiles, setting the groundwork for further investigation of the C₂₀-DAs biosynthesis pathway. Full article

(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants)

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Figure 1

Figure 1
The production of Das is a major metabolic response of A. gymnandrum sterile seedlings to induction. MJ and MJL refer to the sample of root and aerial portions. (a) Principal component analysis of A. gymnandrum of different plant portions shows compounds with peak accumulation occurring at the indicated time, demonstrating that MJ has a greater impact on the root than aerial portions. (b) Principal component analysis of 77 metabolites in the root; the five compounds most representative of the first principal component are atisine, compound 45, compound 56, compound 63, and compound 66. (c) Cluster analysis of 40 metabolites in the root from A. gymnandrum at different induction times. The value of each compound was taken from relative content Log2 standardization. (d) Plots to demonstrate the increasing accumulation of the atisine in the root (error bars represent the standard error). Untreated and treated refer to the control group and experimental group, respectively. Full article ">Figure 2
Venn diagram of up-regulated DEGs by different induction groups in the A. gymnandrum root. Full article ">Figure 3
The expression profile for the genes involved in terpenoid biosynthesis in the root of A. gymnandrum. The transcriptome expression of each identified gene is Log2 normalization of fpkm values. Abbreviations: AACT, aceto-acetyl-CoA thiolase; CMK, 4-(cytidine 50-diphospho)-2-C-methyl-D-erythritol kinase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; DXR, 1-deoxy-D-xylulose-5-phosphate reductoisomerase; FPS, farnesyl pyrophosphate synthase; HDR, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate reductase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; GGPPS, geranylgeranyl pyrophosphate synthase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; IDI, isopentenyl diphosphate isomerase; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; MDD, mevalonate diphosphate decarboxylase; MDS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; MVK, mevalonate kinase; PMK, phosphomevalonate kinase; TPS, terpene synthases (including monoterpene synthases and sesquiterpene synthases); CPS, copalyl diphosphate synthase; KSL, kaurene synthase. Full article ">Figure 4
GC-MS analysis of AgCPSs and AgKSL1 reaction products obtained from in vitro assays. (a) 1~5: The product of AgCPS1/2/4/5 and ZmCPS2 (specific to ent-CPP) enzymatic reaction with GGPP, it is proved that the products of AgCPS1/2/4 and AgCPS 5 were CPP and 8,13-CPP; 6~8: The product of AgCPS1/2/4/5 and ZmCPS2 combined with AtKS (specific to ent-kaurene), it is proved that the products of AgCPS1/2/4 were the ent-configuration; 9~12: the product of AgCPS1/2/4/5 and ZmCPS2 combined with AgKSL1/IrKSL4 (specific to ent-atiserene), it is proved that the products of AgKSL1 was ent-atiserene. The enzymes ZmCPS2, PcTPS1, IrKSL4, and AtKS were used as controls. (b) Extracted ion chromatograms (EIC) of the product in different combinations above. Peak1: ent-CPP; Peak2: ent-8,13-CPP; Peak3: ent-kaurene; Peak4: ent-atiserene. Full article ">Figure 5
Identification of co-expression network modules in A. gymnandrum. (a) Gene dendrogram was obtained by clustering the dissimilarity based on consensus topological overlap with the corresponding module colors indicated by the color row. (b) Modular eigenvector clustering heatmap. The heatmap shows the relatedness of the 6 co-expression modules identified in WGCNA, with red indicating highly related and blue indicating not related. (c) Module-atisine correlation heatmap. The right transverse panel with red-blue indicates a color scale for module–trait correlation, from –0.5 to 0.5. Full article ">Figure 6
Co-expression analysis in module green of A. gymnandrum. Transcript 45862, transcript 49735, transcript 49882, transcript 19239, transcript 65043, transcript 42527, transcript 29295, transcript 33670, and transcript 49129 were filtered as hub genes by degree. The circle depicts genes co-expressed with 10 hub genes (r > 0.8; 77 genes in total). The size and color (red to green) of the circle were arranged according to the degree, and the lines indicate the p-value between the genes (purple is the largest and yellow is the smallest). Full article ">Figure 7
The predicted biosynthetic pathway of atisine and the involved enzyme genes in A. gymnandrum, red colors represent functional qualification, blue colors were screened from transcripts, and orange colors were filtered in the module green. Abbreviations: KO: ent-kaurene oxidase, SDC: serine decarboxylase, TA: transaminase. Full article ">

18 pages, 2698 KiB

Open AccessArticle

Plant Metabolic Engineering by Multigene Stacking: Synthesis of Diverse Mogrosides

by Jingjing Liao, Tingyao Liu, Lei Xie, Changming Mo, Xiyang Huang, Shengrong Cui, Xunli Jia, Fusheng Lan, Zuliang Luo and Xiaojun Ma

Int. J. Mol. Sci. 2022, 23(18), 10422; https://doi.org/10.3390/ijms231810422 - 9 Sep 2022

Cited by 6 | Viewed by 2940

Abstract

Mogrosides are a group of health-promoting natural products that extracted from Siraitia grosvenorii fruit (Luo-han-guo or monk fruit), which exhibited a promising practical application in natural sweeteners and pharmaceutical development. However, the production of mogrosides is inadequate to meet the need worldwide, and [...] Read more.

Mogrosides are a group of health-promoting natural products that extracted from Siraitia grosvenorii fruit (Luo-han-guo or monk fruit), which exhibited a promising practical application in natural sweeteners and pharmaceutical development. However, the production of mogrosides is inadequate to meet the need worldwide, and uneconomical synthetic chemistry methods are not generally recommended for structural complexity. To address this issue, an in-fusion based gene stacking strategy (IGS) for multigene stacking has been developed to assemble 6 mogrosides synthase genes in pCAMBIA1300. Metabolic engineering of Nicotiana benthamiana and Arabidopsis thaliana to produce mogrosides from 2,3-oxidosqualene was carried out. Moreover, a validated HPLC-MS/MS method was used for the quantitative analysis of mogrosides in transgenic plants. Herein, engineered Arabidopsis thaliana produced siamenoside I ranging from 29.65 to 1036.96 ng/g FW, and the content of mogroside III at 202.75 ng/g FW, respectively. The production of mogroside III was from 148.30 to 252.73 ng/g FW, and mogroside II-E with concentration between 339.27 and 5663.55 ng/g FW in the engineered tobacco, respectively. This study provides information potentially applicable to develop a powerful and green toolkit for the production of mogrosides. Full article

(This article belongs to the Special Issue Biosynthesis and Regulatory Mechanism of Secondary Metabolites in Medicinal Plants)

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