bims-polyam 2019-09-01 papers

bims-polyam

Biomed News

on Polyamines

Issue of 2019‒09‒01
thirteen papers selected by
Alexander Ivanov
Engelhardt Institute of Molecular Biology

A polyamine-independent role for S -adenosylmethionine decarboxylase.
Polyamines alleviate the inhibitory effect of the DNA cross-linking agent mitomycin C on root growth.
Effects of Polyamine Binding on the Stability of DNA i-Motif Structures.
Altered polyamine profiling in the hair of patients with androgenic alopecia and alopecia areata.
Arginine biosynthesis modulates pyoverdine production and release in Pseudomonas putida as part of the adaptation mechanism to oxidative stress.
Selection and application of ssDNA aptamers against spermine based on Capture-SELEX.
Mucilaginibacter limnophilus sp. nov., isolated from a lake.
Role and dynamics of an agmatinase-like protein (AGM-1) in Neurospora crassa.
Identification of phenylamide phytoalexins and characterization of inducible phenylamide metabolism in wheat.
Ozone-induced changes in the serum metabolome: Role of the microbiome.
Latent Redox Reporter of 4-Methoxyphenol as Electrochemical Signal Proxy for Real-Time Profiling of Endogenous H2O2 in Living Cells.
Cardioprotection of post-ischemic moderate ROS against ischemia/reperfusion via STAT3-induced the inhibition of MCU opening.
Reduction of Pyrrolizidine Alkaloid Levels in Comfrey (Symphytum officinale) Hairy Roots by RNAi Silencing of Homospermidine Synthase.

Biochem J. 2019 Aug 29. pii: BCJ20190561. [Epub ahead of print]

A polyamine-independent role for S -adenosylmethionine decarboxylase.

Bin Li, Shin Kurihara, Sok Ho Kim, Jue Liang, Anthony J Michael.

  The only known function of S -adenosylmethionine decarboxylase (AdoMetDC) is to supply, with its partner aminopropyltransferase enzymes such as spermidine synthase (SpdSyn), the aminopropyl donor for polyamine biosynthesis. Polyamine spermidine is probably essential for growth of all eukaryotes, most archaea and many bacteria. Two classes of AdoMetDC exist, the prokaryotic class 1a and 1b forms, and the eukaryotic class 2 enzyme, which is derived from an ancient fusion of two prokaryotic class 1b genes. Herein, we show that "eukaryotic" class 2 AdoMetDCs are found in bacteria and are enzymatically functional. However, the bacterial AdoMetDC class 2 genes are phylogenetically limited and were likely acquired from a eukaryotic source via transdomain horizontal gene transfer, consistent with the class 2 form of AdoMetDC being a eukaryotic invention. We found that some class 2 and thousands of class 1b AdoMetDC homologues are present in bacterial genomes that also encode a gene fusion of an N-terminal membrane protein of the Major Facilitator Superfamily (MFS) class of transporters and a C-terminal SpdSyn-like domain. Although these AdoMetDCs are enzymatically functional, spermidine is absent, and an entire fusion protein or its SpdSyn-like domain only, does not biochemically complement a SpdSyn deletion strain of E. coli This suggests that the fusion protein aminopropylates a substrate other than putrescine, and has a role outside of polyamine biosynthesis. Another integral membrane protein found clustered with these genes is DUF350, which is also found in other gene clusters containing a homologue of the glutathionylspermidine synthetase family and occasionally other polyamine biosynthetic enzymes.

Keywords:  biosynthesis; enzyme activity; evolutionary biology; genomics; metabolism; polyamines

DOI:  https://doi.org/10.1042/BCJ20190561
Plant Signal Behav. 2019 Aug 26. 1-5

Polyamines alleviate the inhibitory effect of the DNA cross-linking agent mitomycin C on root growth.

Hidenori Tanaka, Kazuya Takeda, Akihiro Imai.

  Polyamines (putrescine, spermidine and spermine) are ubiquitously present in various types of cells of living organisms. They are involved in a variety of cellular processes, including cell proliferation and cell differentiation, and are required for abiotic stress tolerances in plants. However, it is still not understood whether polyamines are involved in the plant growth inhibition caused by DNA-damaging agents. In this study, we examined the effects of polyamines on the inhibition of plant root growth and gene expression in Arabidopsis thaliana treated with mitomycin C (MMC), a genotoxic agent that induces DNA interstrand crosslinks. We found that polyamines alleviated the inhibitory effect caused by MMC on root growth. In addition, we also found that polyamines alleviated the increased expression of AtBRCA1 and AtRAD51 genes induced by MMC treatment. Our study provides the first evidence that polyamines contribute to tolerance against plant-growth inhibition caused by a DNA-damaging chemical.

Keywords:  Arabidopsis thaliana; DNA damage; Polyamine; mitomycin C; root growth

DOI:  https://doi.org/10.1080/15592324.2019.1659687
ACS Omega. 2019 May 31. 4(5): 8967-8973

Effects of Polyamine Binding on the Stability of DNA i-Motif Structures.

Michael M Molnar, Shelby C Liddell, Randy M Wadkins.

B-form DNA can adopt alternative structures under conditions such as superhelical duress. Alternative DNA structures are favored when there is asymmetric distribution of guanosine and cytosine on complimentary DNA strands. A guanosine-rich strand can form a four-stranded structure known as a quadruplex (G4). The complimentary cytosine-rich strand can utilize intercalating cytosine-cytosine base pairing to form a four-stranded structure known as the i-motif (iM). Both secondary structures are energetically uphill from double-strand DNA (dsDNA), meaning that additional factors are needed for their formation. Most iMs require slightly acidic conditions for structure stabilization. However, crowding agents such as polyethylene glycols and dextrans can shift the pK a of the iM to near-physiological pH ≈ 7. Nucleic acids have long been known to be bound and stabilized by polyamines such as putrescine, spermidine, and spermine. Polyamines have very high concentrations in cells (0.1-30 mM), and their binding to DNA is driven by electrostatic interactions. Polyamines typically bind in the minor groove of DNA. However, because of the unusual structure of iMs, it was unknown whether polyamines might also bind and stabilize iMs. The study described here was undertaken to analyze polyamine-iM interactions. The thermal stability and pH dependence of iM structures were determined in the presence of polyamines. In contrast to dsDNA, our results suggest that polyamines have considerably weaker interactions with iMs, as demonstrated by the minimal change in iM pH dependence and thermal stability. Our results suggest that polyamines are unlikely to provide a significant source of iM stabilization in vivo.

DOI: https://doi.org/10.1021/acsomega.9b00784
J Dermatol. 2019 Aug 28.

Altered polyamine profiling in the hair of patients with androgenic alopecia and alopecia areata.

Yu Ra Lee, Bark Lynn Lew, Woo Young Sim, Jeongae Lee, Jongki Hong, Bong Chul Chung.

  Hair follicles are among the most highly proliferative tissues. Polyamines are associated with proliferation, and several polyamines including spermidine and spermine play anti-inflammatory roles. Androgenic alopecia results from increased dihydrotestosterone metabolism, and alopecia areata is an autoimmune disease. This study aimed to investigate differences in polyamine profiles in hair samples between patients with androgenic alopecia and alopecia areata. Polyamine concentrations were determined through high-performance liquid chromatography-mass spectrometry. Hair samples were derivatized with isobutyl chloroformate. Differences in polyamine levels were observed between androgenic alopecia and alopecia areata compared with normal controls. In particular, polyamine levels were higher in alopecia areata patients than in normal controls. Certain polyamines displayed different concentrations between the androgenic alopecia and alopecia areata groups, suggesting that some polyamines, particularly N-acetyl putrescine (P = 0.007) and N-acetyl cadaverine (P = 0.0021), are significantly different in androgenic alopecia. Furthermore, spermidine (P = 0.021) was significantly different in alopecia areata. Our findings suggest that non-invasive quantification of hair polyamines may help distinguish between androgenic alopecia and alopecia areata. Our study provides novel insights into physiological alterations in patients with androgenic alopecia and those with alopecia areata and reveals some differences in polyamine levels in hair loss diseases with two different modes of action.

Keywords:  alopecia areata; androgenic alopecia; autoimmune disease; hair follicle; polyamine

DOI:  https://doi.org/10.1111/1346-8138.15063
J Bacteriol. 2019 Aug 26. pii: JB.00454-19. [Epub ahead of print]

Arginine biosynthesis modulates pyoverdine production and release in Pseudomonas putida as part of the adaptation mechanism to oxidative stress.

Laura Barrientos-Moreno, María Antonia Molina-Henares, Marta Pastor-García, María Isabel Ramos-González, Manuel Espinosa-Urgel.

Iron is essential for most lifeforms. Under iron-limiting conditions, many bacteria produce and release siderophores, molecules with high affinity for iron, which are then transported into the cell in their iron-bound form, allowing incorporation of the metal into a wide range of cellular processes. However, free iron can also be a source of reactive oxygen species that cause DNA, protein and lipid damage. Not surprisingly, iron capture is finely regulated, and linked to oxidative stress responses. Here we provide evidence indicating that in the plant-beneficial bacterium Pseudomonas putida KT2440, the amino acid L-arginine is a metabolic connector between iron capture and oxidative stress. Mutants defective in arginine biosynthesis show reduced production and release of the siderophore pyoverdine, and altered expression of certain pyoverdine-related genes, resulting in higher sensitivity to iron limitation. Although this amino acid is not part of the siderophore side chain, addition of exogenous L-arginine restores pyoverdine release in the mutants, and increased pyoverdine production is observed in the presence of polyamines (agmatine and spermidine), of which arginine is a precursor. Spermidine also has a protective role against hydrogen peroxide in P. putida, whereas defects in arginine and pyoverdine synthesis result in increased production of reactive oxygen species.IMPORTANCE The results from this study show a previously unidentified connection between arginine metabolism, siderophore turnover, and oxidative stress in Pseudomonas putida Although the precise molecular mechanisms involved are yet to be characterized in full detail, our data are consistent with a model in which arginine biosynthesis, and the derived pathway leading to polyamine production, function as a homeostasis mechanism that helps maintaining the balance between iron uptake and oxidative stress response systems.

DOI: https://doi.org/10.1128/JB.00454-19
Anal Chim Acta. 2019 Nov 12. pii: S0003-2670(19)30839-6. [Epub ahead of print]1081 168-175

Selection and application of ssDNA aptamers against spermine based on Capture-SELEX.

Huili Tian, Nuo Duan, Shijia Wu, Zhouping Wang.

  Spermine contamination ranks as one of the food safety issues, it will cause some adverse reactions if the intake of spermine is excessive in human body. So it is of great significance to establish fast and efficient analysis method to detect spermine in foods. In this study, the spermine aptamers with high affinity and specificity were obtained by the capture systematic evolution of ligands by exponential enrichment (Capture-SELEX) technique. Forty-one aptamer sequences were obtained by cloning and sequencing, and were divided into eight families based on homology and secondary structure analysis. The affinity and specificity of candidate aptamers was analyzed by isothermal titration calorimetry (ITC) and fluorescence assay. The aptamers named APJ-6 was picked out as the optimal aptamer that recognizes spermine specifically with the Kd value of 9.648 ± 0.896 nM. In order to verify the practicability of the selected aptamers, the sensitive aptamer-based fluorescene assay was designed. Under optimized conditions, this aptasensor exhibited a low detection limit of 0.052 nM, as well as a linear within the range of 0.1-20 nM. Besides, it has been further applied for the determination of spermine in pork samples and the recoveries ranged from 86.45% to 98.15%, showing its great potential for sensitive analysis in food safety control.

Keywords:  Aptamer; Capture-SELEX; Isothermal titration calorimetry; Pork; Spermine

DOI:  https://doi.org/10.1016/j.aca.2019.07.031
J Microbiol. 2019 Aug 28.

Mucilaginibacter limnophilus sp. nov., isolated from a lake.

Shih-Yi Sheu, Yi-Ru Xie, Wen-Ming Chen.

  A polyphasic taxonomy approach was used to characterize strain YBJ-36T, isolated from a freshwater lake in Taiwan. Phylogenetic analyses, based on 16S rRNA gene sequences and coding sequences of an up-to-date bacterial core gene set (92 protein clusters), indicated that strain YBJ-36T formed a phylogenetic lineage in the genus Mucilaginibacter. 16S rRNA gene sequence similarity indicated that strain YBJ-36T is closely related to species within the genus Mucilaginibacter (93.8-97.8% sequence similarity) and is most similar to Mucilaginibacter fluminis TTM-2T (97.8%), followed by Mucilaginibacter roseus TTM-1T (97.2%). Microbiological analyses demonstrated that strain YBJ-36T is Gram-negative, aerobic, non-motile, rod-shaped, surrounded by a thick capsule, and forms pink-colored colonies. Strain YBJ-36T grew between 20-40°C (optimal range, 35-37°C), pH 5.5-7.0 (optimal pH of 6) and 0-2% NaCl (optimal concentration, 0.5%). The predominant fatty acids of strain YBJ-36T are iso-C15:0 and summed feature 3 (C16:1ω7c and/or C16:1ω6c), the major polar lipid is phosphatidylethanolamine, the major polyamine is homospermidine, and the major isoprenoid quinone is MK-7. The draft genome is approximately 4.63 Mb in size with a G+C content of 42.8 mol%. Strain YBJ-36T exhibited less than 35% DNA-DNA relatedness with Mucilaginibacter fluminis TTM-2T and Mucilaginibacter roseus TTM-1T. Based on phenotypic and genotypic properties and phylogenetic inference, strain YBJ-36T should be classified in a novel species of the genus Mucilaginibacter, for which the name Mucilaginibacter limnophilus sp. nov. is proposed. The type strain is YBJ-36T (= BCRC 81056T = KCTC 52811T = LMG 30058T).

Keywords:  Bacteroidetes; Mucilaginibacter limnophilus sp. nov.; Sphingobacteriaceae; Sphingobacteriales; Sphingobacteriia; polyphasic taxonomy

DOI:  https://doi.org/10.1007/s12275-019-9146-z
Fungal Genet Biol. 2019 Aug 26. pii: S1087-1845(19)30037-4. [Epub ahead of print] 103264

Role and dynamics of an agmatinase-like protein (AGM-1) in Neurospora crassa.

Luis L Pérez-Mozqueda, Rafael Vazquez-Duhalt, Ernestina Castro-Longoria.

  Agmatinase is known as a metalloenzyme which hydrolyzes agmatine to produce urea and putrescine, being crucial in the alternative pathway to produce polyamines. In this study, an agmatinase-like protein (AGM-1) (NCU 01348) in the filamentous fungus Neurospora crassa is reported. Purified AGM-1 from N. crassa displays enzymatic activity hydrolyzing agmatine; therefore, it can be considered as an agmatinase-like protein. However, its role in the alternative pathway to produce polyamines apparently is not its main function since only a slight reduction of polyamines concentration was detected in the Δagm-1het strain. Moreover, the null mutant Δagm-1 (homokaryon strain) was unable to grow and the deficiency of agm-1 in the heterokaryon strain provoked a decrease in elongation rate, conidia and biomass production, despite of having de constitutive pathway via the ornithine decarboxylase (ODC). Additionally, mature hyphae of the Δagm-1het strain presented unusual apical branching and a disorganized Spitzenkörper (Spk). Trying to reveal the role of AGM-1in N. crassa, the protein was tagged with GFP and interestingly the dynamics and intracellular localization of AGM-1 closely resembles the F-actin population. This finding was further examined in order to elucidate if AGM-1is in a close association with F-actin. Since polyamines, among them agmatine, have been reported to act as stabilizers of actin filaments, we evaluated in vitro G-actin polymerization in the presence of agmatine and the effect of purified AGM-1 from N. crassa on these polymerized actin filaments. It was found that polymerization of actin filaments increases in the presence of agmatine and the addition of purified AGM-1 from N. crassa depolymerizes these actin filaments. Also, it was determined that an intact substrate binding site of the enzyme is necessary for the localization pattern of the native AGM-1. These results suggest that in N. crassa AGM-1 has a close association with the F-actin population via its substrate agmatine, playing an essential role during cell development.

Keywords:  Actin; Agmatinase; Agmatine; Cell development; N. crassa; Polyamines

DOI:  https://doi.org/10.1016/j.fgb.2019.103264
Phytochemistry. 2019 Aug 23. pii: S0031-9422(19)30414-5. [Epub ahead of print]167 112098

Identification of phenylamide phytoalexins and characterization of inducible phenylamide metabolism in wheat.

Naoki Ube, Daiyu Harada, Yuhka Katsuyama, Kumiko Osaki-Oka, Takuji Tonooka, Kotomi Ueno, Shin Taketa, Atsushi Ishihara.

  Changes in specialized metabolites were analyzed in wheat leaves inoculated with Bipolaris sorokiniana, the causal agent of spot blotch of Poaceae species. HPLC analysis detected the accumulation of six compounds in B. sorokiniana-infected leaves. Of these, we purified two compounds by silica gel and ODS column chromatography and preparative HPLC, and identified them as cinnamic acid amides, N-cinnamoyl-9-hydroxy-8-oxotryptamine and N-cinnamoyl-8-oxotryptamine, by spectroscopic analyses. The remaining four compounds were predicted to be p-coumaric acid amides of hydroxyputrescine, hydroxyagmatine, hydroxydehydroagmatine, and agmatine by mass spectrometry. The accumulation of two cinnamic acid amides was also induced by Fusarium graminearum infection, and by treatment with CuCl2, jasmonic acid, and isopentenyladenine. Antifungal activity of these amides was shown by inhibition of conidial germination and germ tube elongation of F. graminearum and Alternaria brassicicola, indicating that they act as phytoalexins. The accumulation of these amides also detected in barley leaves treated with CuCl2. We examined the accumulation of 25 phenylamides in B. sorokiniana-infected wheat leaves using LC-MS/MS. Hydroxycinnamic acid amides of tryptamine, serotonin, putrescine, and agmatine, were induced after infection with B. sorokiniana. Thus, the induced accumulation of two groups of phenylamides, cinnamic acid amides with indole amines, and p-coumaric acid amides with putrescine and agmatine related amines, represents a major metabolic response of wheat to pathogen infection.

Keywords:  Bipolaris sorokiniana; Fusarium graminearum; Phenylamide; Phytoalexin; Poaceae; Triticum aestivum

DOI:  https://doi.org/10.1016/j.phytochem.2019.112098
PLoS One. 2019 ;14(8): e0221633

Ozone-induced changes in the serum metabolome: Role of the microbiome.

Youngji Cho, Ross S Osgood, Lauren N Bell, Edward D Karoly, Stephanie A Shore.

Ozone is an asthma trigger. In mice, the gut microbiome contributes to ozone-induced airway hyperresponsiveness, a defining feature of asthma, but the mechanistic basis for the role of the gut microbiome has not been established. Gut bacteria can affect the function of distal organs by generating metabolites that enter the blood and circulate systemically. We hypothesized that global metabolomic profiling of serum collected from ozone exposed mice could be used to identify metabolites contributing to the role of the microbiome in ozone-induced airway hyperresponsiveness. Mice were treated for two weeks with a cocktail of antibiotics (ampicillin, neomycin, metronidazole, and vancomycin) in the drinking water or with control water and then exposed to air or ozone (2 ppm for 3 hours). Twenty four hours later, blood was harvested and serum analyzed via liquid-chromatography or gas-chromatography coupled to mass spectrometry. Antibiotic treatment significantly affected 228 of the 562 biochemicals identified, including reductions in the known bacterially-derived metabolites, equol, indole propionate, 3-indoxyl sulfate, and 3-(4-hydroxyphenyl)propionate, confirming the efficacy of the antibiotic treatment. Ozone exposure caused significant changes in 334 metabolites. Importantly, ozone-induced changes in many of these metabolites were different in control and antibiotic-treated mice. For example, most medium and long chain fatty acids declined by 20-50% with ozone exposure in antibiotic-treated but not control mice. Most taurine-conjugated bile acids increased with ozone exposure in antibiotic-treated but not control mice. Ozone also caused marked (9-fold and 5-fold) increases in the polyamines, spermine and spermidine, respectively, in control but not antibiotic-treated mice. Each of these metabolites has the capacity to alter airway responsiveness and may account for the role of the microbiome in pulmonary responses to ozone.

DOI: https://doi.org/10.1371/journal.pone.0221633
ACS Sens. 2019 Aug 26.

Latent Redox Reporter of 4-Methoxyphenol as Electrochemical Signal Proxy for Real-Time Profiling of Endogenous H2O2 in Living Cells.

Kesavan Manibalan, Shuang Han, Yajing Zheng, Haifang Li, Jin-Ming Lin.

Hydrogen peroxide (H2O2) plays a persuasive role in the human defense mechanism cell physiology. Developing an efficient assay platform; a high sensitive tracking and quantification of H2O2 in a physiological system is of great importance are crucial to understanding the neoplastic changes and/or redox homeostasis of cells. In this study, a novel strategy, new-of-a-kind turn-on latent electrochemical redox probe coupled electrocatalytic signal amplification strategy is proposed., A custom made readily available turn-on latent electrochemical probe 4-methoxyphenylboronic acid pinacol ester (4-MPBP) was have designed for selective detection of endogenous H2O2 endogenous in live cells. The designed electrochemical probe composed of a latent electrochemical reporter unit (4-Methoxy phenol, 4-MP) linked with bearing a target recognition unit (boronic acid pinacol ester) for H2O2 sensing. The selective analyte-triggered signal chemical transformation release free electrochemical reporter 4-MP. mechanism towards The amount of H2O2 were was evaluated electrochemically at glassy carbon electrode (GCE) in with a broad dynamic detection range of 0.5 µM to 1mM. An amplified signal response of released 4-MP from 4-MPBP to build a high sensitive assay tool was have been proficiently achieved by via replacing the GCE transducer electrode with Polydopamine@carbonnanotube-Molebtinumdisulfie hybrid modified GCE (PDA@CNT-MoS2/GCE) with as it delivered an exceptional dynamic detection range of 0.01 µM to 100 µM. The innovative blend of electrochemical molecular probe strategy, with electrocatalytic signal amplification technique has delivered outstanding assay performance at trace level sensing of H2O2. Next, we set up a platform for real-time in-vivo monitoring of endogenously produced H2O2 in Caco-2 cells, and MCF-7 cells through spermine-polyamine analogue, and phorbol 12-myristate 13-acetate (PMA) induction in SSAT/PAO gene and protein kinase C (PKC), respectively. As expected, the 4-MPBP latent probe coupled electrocatalytic signal amplification strategy delivered outstanding performance towards An efficient in-situ H2O2 release, and tracking, over time. and quantification prowess was observed electrochemically through 4-MPBP latent probe coupled electrocatalytic signal amplification approach.

DOI: https://doi.org/10.1021/acssensors.9b01049
Basic Res Cardiol. 2019 Aug 28. 114(5): 39

Cardioprotection of post-ischemic moderate ROS against ischemia/reperfusion via STAT3-induced the inhibition of MCU opening.

Lan Wu, Ji-Liang Tan, Zhong-Yan Chen, Gang Huang.

  Enhanced reactive oxygen species (ROS) at the beginning of reperfusion activated signal transducer and activator of transcription 3 (STAT3) in intermittent hypobaric hypoxia (IHH)-afforded cardioprotection against ischemia/reperfusion (I/R). However, its mechanism remains largely unknown. This study aimed to investigate the role and the downstream of STAT3 in exogenous enhanced post-ischemic ROS-induced cardioprotection using the model of moderate hydrogen peroxide postconditioning (H2O2PoC) mimicking endogenous ROS in IHH. Moderate H2O2PoC not only improved the post-ischemic myocardial contractile recovery and reduced the infarct size in isolated rat I/R hearts, but also alleviated mitochondrial calcium overload and ameliorated Ca2+ transients, cell contraction, and mitochondrial membrane potential in rat I/R cardiomyocytes. However, the cardioprotective effects of moderate H2O2PoC were abrogated by Janus kinase 2 (JAK2)/STAT3 inhibitor AG490 in rat hearts as well as adenovirus-delivered short hairpin RNA specific for STAT3 and the opener of mitochondrial calcium uniporter (MCU) spermine in rat cardiomyocytes. Notably, the moderate H2O2PoC-afforded cardioprotection abrogated by spermine could be rescued by STAT3 over-expression with adenovirus in rat I/R cardiomyocytes. Besides, moderate H2O2PoC enhanced mitochondrial STAT3 expression during I/R. A co-localization/interaction of STAT3 or phospho-STAT3ser727 and MCU was observed in rat cardiomyocytes with moderate H2O2PoC at 5 and 30 min of reperfusion but not in rat I/R cardiomyocytes. Further, STAT3 interacted with the N-terminal domain (NTD) of MCU in rat cardiomyocytes with moderate H2O2PoC. These findings indicated that post-ischemic moderate ROS activate STAT3 against cardiac I/R by inhibiting MCU opening via its interaction with the NTD of MCU to alleviate mitochondrial calcium overload.

Keywords:  Cardiac contraction; Hydrogen peroxide postconditioning; Mitochondrial Ca2+ concentration; Mitochondrial calcium uniporter; Post-ischemic moderate ROS; Signal transducer and activator of transcription 3

DOI:  https://doi.org/10.1007/s00395-019-0747-9
Planta Med. 2019 Aug 26.

Reduction of Pyrrolizidine Alkaloid Levels in Comfrey (Symphytum officinale) Hairy Roots by RNAi Silencing of Homospermidine Synthase.

Lars H Kruse, Thomas Stegemann, Julia Jensen-Kroll, Annika Engelhardt, Anne-Maria Wesseling, Annemarie Lippert, Jutta Ludwig-Müller, Dietrich Ober.

Comfrey is a medicinal plant, extracts of which are traditionally used for the treatment of painful inflammatory muscle and joint problems, because the plant contains allantoin and rosmarinic acid. However, its medicinal use is limited because of its toxic pyrrolizidine alkaloid (PA) content. PAs encompass more than 400 different compounds that have been identified from various plant lineages. To date, only the first pathway-specific enzyme, homospermidine synthase (HSS), has been characterized. HSS catalyzes the formation of homospermidine, which is exclusively incorporated into PAs. HSS has been recruited several times independently in various plant lineages during evolution by duplication of the gene encoding deoxyhypusine synthase (DHS), an enzyme of primary metabolism. Here, we describe the establishment of RNAi knockdown hairy root mutants of HSS in Symphytum officinale. A knockdown of HSS by 60 - 80% resulted in a significant reduction of homospermidine by ~ 86% and of the major PA components 7-acetylintermedine N-oxide and 3-acetylmyoscorpine N-oxide by approximately 60%. The correlation of reduced transcript levels of HSS with reduced levels of homospermidine and PAs provides in planta support for HSS being the central enzyme in PA biosynthesis. Furthermore, the generation of PA-depleted hairy roots might be a cost-efficient way for reducing toxic by-products that limit the medicinal applicability of S. officinale extracts.

DOI: https://doi.org/10.1055/a-0998-5125