A Nuphar lutea plant active ingredient, 6,6'-dihydroxythiobinupharidine, ameliorates kidney damage and inflammation in a mouse model of chronic kidney disease.

Chronic Kidney Disease (CKD) associated complications are associated with increased inflammation through the innate immune response, which can be modulated with anti-inflammatory agents. An active ingredient derived from the Nuphar lutea aquatic plant, 6,6'-dihydroxythiobinupharidine (DTBN) has anti-inflammatory properties, mainly through the inhibition of NF-κB. We tested the effects of DTBN on mice with CKD. After preliminary safety and dosing experiments, we exposed 8 weeks old male C57BL/6J mice to adenine diet to induce CKD. Control and CKD animals were treated with IP injections of DTBN (25 µg QOD) or saline and sacrificed after 8 weeks. Serum urea and creatinine were significantly decreased in CKD-DTBN Vs CKD mice. Kidney histology showed a decrease in F4/80 positive macrophage infiltration, damaged renal area, as well as decreased kidney TGF-ß in CKD-DTBN Vs CKD mice. Kidney inflammation indices (IL-1ß, IL-6 and P-STAT3) were significantly decreased in CKD-DTBN as compared to CKD mice. DTBN treatment showed no apparent damage to tissues in control mice, besides a decrease in weight gain and mild hypoalbuminemia without proteinuria. Thus, DTBN significantly improved renal failure and inflammation indices in CKD mice. Therefore, this and similar substances may be considered as an additional treatment in CKD patients.

In Vitro and In Vivo Therapeutic Potential of 6,6'-Dihydroxythiobinupharidine (DTBN) from Nuphar lutea on Cells and K18-hACE2 Mice Infected with SARS-CoV-2.

We have previously published research on the anti-viral properties of an alkaloid mixture extracted from Nuphar lutea, the major components of the partially purified mixture found by NMR analysis. These are mostly dimeric sesquiterpene thioalkaloids called thiobinupharidines and thiobinuphlutidines against the negative strand RNA measles virus (MV). We have previously reported that this extract inhibits the MV as well as its ability to downregulate several MV proteins in persistently MV-infected cells, especially the P (phospho)-protein. Based on our observation that the Nuphar extract is effective in vitro against the MV, and the immediate need that the coronavirus disease 2019 (COVID-19) pandemic created, we tested here the ability of 6,6'-dihydroxythiobinupharidine DTBN, an active small molecule, isolated from the Nuphar lutea extract, on COVID-19. As shown here, DTBN effectively inhibits SARS-CoV-2 production in Vero E6 cells at non-cytotoxic concentrations. The short-term daily administration of DTBN to infected mice delayed the occurrence of severe clinical outcomes, lowered virus levels in the lungs and improved survival with minimal changes in lung histology. The viral load on lungs was significantly reduced in the treated mice. DTBN is a pleiotropic small molecule with multiple targets. Its anti-inflammatory properties affect a variety of pathogens including SARS-CoV-2 as shown here. Its activity appears to target both pathogen specific (as suggested by docking analysis) as well as cellular proteins, such as NF-κB, PKCs, cathepsins and topoisomerase 2, that we have previously identified in our work. Thus, this combined double action of virus inhibition and anti-inflammatory activity may enhance the overall effectivity of DTBN. The promising results from this proof-of-concept in vitro and in vivo preclinical study should encourage future studies to optimize the use of DTBN and/or its molecular derivatives against this and other related viruses.

Dearomatization of Heteroarenium Salts with ArBpin Reagents. Application to the Total Synthesis of a Nuphar Alkaloid.

Rhodium-catalyzed enantioselective addition of aryl and heteroaryl boron pinacol esters to pyridinium and quinolinium salts is developed for the synthesis of enantioenrichred dihydroheteroarenes. The methodology has enabled the synthesis of 2-heteroaryl-substituted dihydropyridines in high yield and ee, which provided efficient synthetic access to a nuphar alkaloid.

Inhibition of Cysteine Proteases by 6,6'-Dihydroxythiobinupharidine (DTBN) from Nuphar lutea.

The specificity of inhibition by 6,6'-dihydroxythiobinupharidine (DTBN) on cysteine proteases was demonstrated in this work. There were differences in the extent of inhibition, reflecting active site structural-steric and biochemical differences. Cathepsin S (IC50 = 3.2 µM) was most sensitive to inhibition by DTBN compared to Cathepsin B, L and papain (IC50 = 1359.4, 13.2 and 70.4 µM respectively). DTBN is inactive for the inhibition of Mpro of SARS-CoV-2. Docking simulations suggested a mechanism of interaction that was further supported by the biochemical results. In the docking results, it was shown that the cysteine sulphur of Cathepsin S, L and B was in close proximity to the DTBN thiaspirane ring, potentially forming the necessary conditions for a nucleophilic attack to form a disulfide bond. Covalent docking and molecular dynamic simulations were performed to validate disulfide bond formation and to determine the stability of Cathepsins-DTBN complexes, respectively. The lack of reactivity of DTBN against SARS-CoV-2 Mpro was attributed to a mismatch of the binding conformation of DTBN to the catalytic binding site of Mpro. Thus, gradations in reactivity among the tested Cathepsins may be conducive for a mechanism-based search for derivatives of nupharidine against COVID-19. This could be an alternative strategy to the large-scale screening of electrophilic inhibitors.

6,6'-Dihydroxythiobinupharidine (DTBN) Purified from Nuphar lutea Leaves Is an Inhibitor of Protein Kinase C Catalytic Activity.

Water lily (Nuphar) bioactive extracts have been widely used in traditional medicine owing to their multiple applications against human ailments. Phyto-active Nuphar extracts and their purified and synthetic derivatives have attracted the attention of ethnobotanists and biochemists. Here, we report that 6,6'-dihydroxythiobinupharidine (DTBN), purified from extracts of Nuphar lutea (L.) Sm. leaves, is an effective inhibitor of the kinase activity of members of the protein kinase C (PKC) family using in vitro and in silico approaches. We demonstrate that members of the conventional subfamily of PKCs, PKCα and PKCγ, were more sensitive to DTBN inhibition as compared to novel or atypical PKCs. Molecular docking analysis demonstrated the interaction of DTBN, with the kinase domain of PKCs depicting the best affinity towards conventional PKCs, in accordance with our in vitro kinase activity data. The current study reveals novel targets for DTBN activity, functioning as an inhibitor for PKCs kinase activity. Thus, this and other data indicate that DTBN modulates key cellular signal transduction pathways relevant to disease biology, including cancer.

Nuphar lutea Extracts Exhibit Anti-Viral Activity against the Measles Virus.

Different parts of Nuphar lutea L. (yellow water lily) have been used to treat several inflammatory and pathogen-related diseases. It has shown that Nuphar lutea extracts (NUP) are active against various pathogens including bacteria, fungi, and leishmanial parasites. In an effort to detect novel therapeutic agents against negative-stranded RNA (- RNA) viruses, we have tested the effect of a partially-purified alkaloid mixture of Nuphar lutea leaves on the measles virus (MV). The MV vaccine's Edmonston strain was used to acutely or persistently infect cells. The levels of several MV proteins were detected by a Western blot and immunocytochemistry. Viral RNAs were quantitated by qRT-PCR. Virus infectivity was monitored by infecting African green monkey kidney VERO cells' monolayers. We showed that NUP protected cells from acute infection. Decreases in the MV P-, N-, and V-proteins were observed in persistently infected cells and the amount of infective virus released was reduced as compared to untreated cells. By examining viral RNAs, we suggest that NUP acts at the post-transcriptional level. We conclude, as a proof of concept, that NUP has anti-viral therapeutic activity against the MV. Future studies will determine the mechanism of action and the effect of NUP on other related viruses.

6-Hydroxythiobinupharidine Inhibits Migration of LM8 Osteosarcoma Cells by Decreasing Expression of LIM Domain Kinase 1.

BACKGROUND/AIM: Osteosarcoma is the most malignant type of bone tumor. Patients with osteosarcoma metastases have a poorer prognosis than those without metastases. Thus, the prognosis of osteosarcoma patients with metastases must be improved. MATERIALS AND METHODS: The present study investigated the inhibitory effects of 6-hydroxythiobinupharidine isolated from Nuphar pumilum on migration of LM8 murine osteosarcoma cells by a migration assay and also examined the expression of proteins related to actin dynamics by western blot. The present study also developed an automatic cell counting system using machine learning to count migrated cells by Fiji and Trainable Weka Segmentation. RESULTS: 6-Hydroxythiobinupharidine inhibited migration of LM8 osteosarcoma cells in a dose-dependent manner, and decreased protein expression of Lin11, Isl-1, and Mec-3 domain kinase 1 (LIMK1) and the levels of phosphorylated Cofilin. CONCLUSION: 6-Hydroxythiobinupharidine suppressed migration of LM8 osteosarcoma cells by decreasing expression of LIMK1. 6-Hydroxythiobinupharidine could be potentially used as an anti-metastatic compound.

Biofunctional Effects of Thiohemiaminal-Type Dimeric Sesquiterpene Alkaloids from Nuphar Plants.

Dimeric sesquiterpene thioalkaloids from the rhizomes of Nuphar pumilum exhibited immunosuppressive effects using a sheep erythrocyte plaque forming cell (PFC) assay, as well as an anti-metastasis effect, and rapid apoptosis-inducing effects in tumor cell lines. In particular, dimeric sesquiterpene thioalkaloids with a hydroxy group (6-hydroxythiobinupharidine, 6,6'-dihydroxythiobinupharidine, 6-hydroxythionuphlutine B) showed substantial effects, whereas dimeric sesquiterpene thioalkaloids lacking the hydroxy group (thiobinupharidine, thionuphlutine B, 6'-hydroxythionuphlutine B, neothiobinupharidine, thionuphlutine B ß-sulfoxide, neothiobinupharidine ß-sulfoxide) and monomeric sesquiterpene alkaloids (nupharidine, 7-epideoxynupharidine, nupharolutine) showed weak activity. In this review, we summarize our studies of the biofunctional effects of these alkaloids.

Nuphar alkaloids induce very rapid apoptosis through a novel caspase-dependent but BAX/BAK-independent pathway.

Nuphar alkaloids, originally isolated from water lilies, induce apoptosis in mammalian cells in less than 1 h, making them possibly the fastest known inducers. However, the mechanism by which this rapid apoptosis occurs remains unknown. We have investigated canonical aspects of apoptosis to determine how the nuphar alkaloid, (+)-6-hydroxythiobinupharidine (6HTBN), induces apoptosis. 6HTBN induced rapid apoptosis in various leukemia, lymphoma, and carcinoma cell lines, suggesting that its mechanism is cell-type independent. It also circumvented resistance of patient-derived chronic lymphocytic leukemia cells generated by co-culture on survival-promoting stroma. Intriguingly, 6HTBN failed to induce apoptosis in platelets. The mechanism of apoptosis involves activation of caspase 9 and caspase 3, but not caspase 8 as previously reported. The release of cytochrome c from mitochondria occurred even in the absence of BAX/BAK and in cells that retained mitochondrial membrane potential. These results suggest a novel mechanism of apoptosis that has previously not been reported. The molecular target of the nuphar alkaloids remains to be determined.

Cytotoxicity of sesquiterpene alkaloids from Nuphar plants toward sensitive and drug-resistant cell lines.

Multi-drug resistance (MDR) is a critical problem in cancer chemotherapy. MDR causes the overexpression of ATP-binding cassette (ABC) transporters and mutations in tumor suppressor genes and oncogenes. To tackle this issue, in this study, we focused on Nuphar plants, which have been traditionally used as food. Sesquiterpene alkaloids (1-3) were isolated from N. japonicum and dimeric sesquiterpene thioalkaloids (4-10) were isolated from N. pumilum. P-glycoprotein-overexpressing CEM/ADR5000 cells were cross-resistant to 6,6'-dihydroxythiobinupharidine (10). Using in silico molecular docking, we calculated the binding energies and simulated the interactions of these compounds with the corresponding amino acid residues at the binding site of P-gp. In addition, we investigated the cytotoxicity of these compounds towards cell lines overexpressing other ABC transporters (BCRP, ABCB5), cell lines with a knocked out tumor suppressor gene TP53 or cell lines overexpressing a deletion-activated EGFR oncogene. These cell lines were sensitive or only minimally cross-resistant to these compounds compared with their corresponding wild-type cell lines.

Variable importance for sustaining macrophyte presence via random forests: data imputation and model settings.

Data sets plagued with missing data and performance-affecting model parameters represent recurrent issues within the field of data mining. Via random forests, the influence of data reduction, outlier and correlated variable removal and missing data imputation technique on the performance of habitat suitability models for three macrophytes (Lemna minor, Spirodela polyrhiza and Nuphar lutea) was assessed. Higher performances (Cohen's kappa values around 0.2-0.3) were obtained for a high degree of data reduction, without outlier or correlated variable removal and with imputation of the median value. Moreover, the influence of model parameter settings on the performance of random forest trained on this data set was investigated along a range of individual trees (ntree), while the number of variables to be considered (mtry), was fixed at two. Altering the number of individual trees did not have a uniform effect on model performance, but clearly changed the required computation time. Combining both criteria provided an ntree value of 100, with the overall effect of ntree on performance being relatively limited. Temperature, pH and conductivity remained as variables and showed to affect the likelihood of L. minor, S. polyrhiza and N. lutea being present. Generally, high likelihood values were obtained when temperature is high (>20 °C), conductivity is intermediately low (50-200 mS m-1) or pH is intermediate (6.9-8), thereby also highlighting that a multivariate management approach for supporting macrophyte presence remains recommended. Yet, as our conclusions are only based on a single freshwater data set, they should be further tested for other data sets.

Total Synthesis of Unsymmetrically Oxidized Nuphar Thioalkaloids via Copper-Catalyzed Thiolane Assembly.

An asymmetric total synthesis of (+)-6-hydroxythiobinupharidine (1b) and (-)-6-hydroxythionuphlutine (2b), a set of hemiaminal containing dimeric sesquiterpenes isolated from yellow water lilies of the Nuphar genus, is described. The central bis-spirocyclic tetrahydrothiophene ring was forged through the Stevens rearrangement of a sulfonium ylide, generated in situ from the coupling of a copper-carbene with a spirocyclic thietane. This strategy diverges both from the proposed biosynthesis1 and previous syntheses of this family of alkaloids,2,3 all of which employ dimerization of symmetric monomers to form the aforementioned thiaspirane. The coupling of unsymmetrical monomers allowed access to the unsymmetrically oxidized product 2b for the first time.

Permanently open stomata of aquatic angiosperms display modified cellulose crystallinity patterns.

Most floating aquatic plants have stomata on their upper leaf surfaces, and usually their stomata are permanently open. We previously identified 3 distinct crystallinity patterns in stomatal cell walls, with angiosperm kidney-shaped stomata having the highest crystallinity in the polar end walls as well as the adjacent polar regions of the guard cells. A numerical bio-mechanical model suggested that the high crystallinity areas are localized to regions where the highest stress is imposed. Here, stomatal cell wall crystallinity was examined in 4 floating plants from 2 different taxa: basal angiosperms from the ANITA grade and monocots. It appears that the non-functional stomata of floating plants display reduced crystallinity in the polar regions as compared with high crystallinity of the ventral (inner) walls. Thus their guard cells are both less flexible and less stress resistant. Our findings suggest that the pattern of cellulose crystallinity in stomata of floating plants from different families was altered as a consequence of similar evolutionary pressures.

Stereoselective Synthesis and Biological Evaluation of C1-Epimeric and Desmethyl Monomeric Nuphar Analogues.

A class of monomeric nuphar analogues that are either epimeric at C1 and C1' or lack the naturally occurring methyl group at those positions were synthesized and evaluated for biological activity. The syntheses feature enantioselective vinylogous Mukaiyama-Mannich (vM-Mannich) reactions catalyzed by chiral phosphoric acids that proceed with excellent diastereoselectivity. Biological assays reveal that both the desmethyl and C1-epimeric monomeric nuphar analogous are able to induce rapid apoptosis.

Genetic diversity and population structure of Nuphar submersa (Nymphaeaceae), a critically endangered aquatic plant endemic to Japan, and implications for its conservation.

Nuphar submersa (Nymphaeaceae) is a critically endangered freshwater macrophyte indigenous to central Japan, with only four small extant populations represented across its entire range. We investigated the genotypic and genetic diversity as well as the genetic structure of all extant individuals of N. submersa based on analysis of 15 microsatellite loci. Among 278 individual ramets, 52 multilocus genotypes were detected: 30 genotypes in Nikko City (NIK), 18 in Nasukarasuyama City (NAS), 3 in Mooka City (MOK), and 1 in Sakura City (SAK). The average number of alleles per locus ranged from 1.20 to 1.93, whereas the observed and expected heterozygosities ranged from 0.11 to 0.33 and from 0.10 to 0.24, respectively. With the exception of SAK, all populations contained multiple clones, but our results indicated low levels of within-population genetic diversity. The populations NIK and NAS comprised few large or middle-sized genets and many small genets. The populations NIK and NAS were suggested to comprise large old, old fragmented, and/or young small genets resulting from seedling establishment. All four populations were differentiated, and gene flow between the populations was restricted (average level of gene flow (Nm) = 0.122, G' ST  = 0.639). Of the total genetic diversity, 67.20 and 9.13% were attributable to inter- and intra-population diversity, respectively. STRUCTURE analysis revealed two or three well-differentiated groups of populations. Cluster I comprised one population (NIK) and cluster II comprised the remaining populations at K = 2. The populations NIK, NAS, and the remaining populations were assigned to clusters I, II, and III, respectively, at K = 3. For conservation practices, we recommend that each cluster be regarded as a different management unit. We further suggest that artificial gene flow among MOK and SAK populations is an appropriate option, whereas NIK should not be reinforced with genotypes from the remaining populations.

Toward the Synthesis of Nuphar Sesquiterpene Thioalkaloids: Stereodivergent Rhodium-Catalyzed Synthesis of the Thiolane Subunit.

A stereodivergent approach to the central thiolane subunit of Nuphar sesquiterpene thioalkaloids has been developed. This approach features a rhodium-catalyzed Stevens-type rearrangement in conjunction with an enzyme resolution reaction. Further elaboration into a polycyclic ring system via alcohol oxidation and ring-closing metathesis is also described.

Action mechanism of 6, 6'-dihydroxythiobinupharidine from Nuphar japonicum, which showed anti-MRSA and anti-VRE activities.

BACKGROUND: Multidrug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant enterococci (VRE), cause serious infections at clinical sites, for which the development of new drugs is necessary. We screened candidates for new antibiotics and investigated its action mechanism. METHODS: An antimicrobial compound was isolated from an extract of Nuphar japonicum. Its chemical structure was determined by NMR, MS, and optical rotation. We measured its minimum inhibitory concentration (MIC) using the microdilution method. The effects of the compound on DNA gyrase and DNA topoisomerase IV were investigated with DNA supercoiling, decatenation, and cleavage assay. RESULTS: We isolated and identified 6,6'-dihydroxythiobinupharidine as the antimicrobial compound. The MIC of this compound was 1-4 µg/mL against various MRSA and VRE strains. We also demonstrated that this compound inhibited DNA topoisomerase IV (IC50 was 10-15 µM), but not DNA gyrase in S. aureus, both of which are known to be the targets of quinolone antibiotics and necessary for DNA replication. However, this compound only exhibited slight cross-resistance to norfloxacin-resistant S. aureus, which indicated that DTBN might inhibit other targets besides topoisomerase IV. These results suggest that 6,6'-dihydroxythiobinupharidine may be a potent candidate or seed for novel antibacterial agents. CONCLUSIONS: DTBN from N. japonicum showed anti-MRSA and anti-VRE activities. DTBN might be involved in the inhibition of DNA topoisomerase IV. GENERAL SIGNIFICANCE: DTBN might be useful as a seed compound. The information on the inhibition mechanism of DTBN will be useful for the modification of DTBN towards developing novel anti-MRSA and anti-VRE drug.

Anti-inflammatory effect of a Nuphar lutea partially purified leaf extract in murine models of septic shock.

ETHNOPHARMACOLOGICAL RELEVANCE: Various plant organs of Nuphar lutea (L.) SM. (Nymphaeaceae) are used in traditional medicine for the treatment of arthritis, fever, aches, pains and inflammation. The main purpose of this study was to determine the anti-inflammatory effect of Nuphar lutea leaf extract (NUP) in two septic shock models: (1) Survival of mice challenged with a lethal dose of LPS, determination of pro-inflammatory and anti-inflammatory cytokines in serum, as well as in peritoneal macrophages in cell culture. (2) The effect of NUP in a murine model of fecal-induced peritonitis. MATERIALS AND METHODS: NUP pre-treatment partially protected mice in two models of acute septic shock. We concluded that NUP is anti-inflammatory by inhibiting the NF-κB pathway, modulating cytokine production and ERK phosphorylation. RESULTS: A significant average survival rate (60%) of LPS lethally-challenged mice was achieved by pre-treatment with NUP. In addition, NUP pre-treatment reduced nuclear NF-κB translocation in peritoneal macrophages. The production of pro-inflammatory cytokines, TNF-α, IL-6 and IL-12, in the sera of LPS-treated mice or in the supernatants of peritoneal macrophages stimulated with LPS for 2-6 h was also decreased by NUP. Pre-treatment with NUP caused a significant increase in the anti-inflammatory cytokine IL-10. The NUP pre-treatment reduced and delayed mortality in mice with fecal-induced peritonitis. Our studies also revealed that NUP pre-treatment induced a dose-dependent phosphorylation of ERK in peritoneal macrophages. Since most of the reports about the anti-inflammatory effect of Nuphar lutea refer to rhizome and root powder and extracts, it is important to clarify the effectiveness of leaf extract as a source for such activity. CONCLUSION: NUP pre-treatment partially protected mice in two models of acute septic shock. We concluded that NUP is anti-inflammatory by inhibiting the NF-κB pathway, modulating cytokine production and ERK phosphorylation.

[Structural and functional aspects of heterophylly in Nuphar lutea (L.) Smith: ultrastructure and photosynthesis].

Ultrastructure and functional characteristics of photosynthetic apparatus of the floating and submersed leaves of heterophyllous aquatic plant Nuphar lutea (L.) Sibth. et Sm. were examined. Differences in the mesophyll cell chloroplast ultrastructure, pigment content and chlorophyll fluorescence parameters of floating and submersed leaves as well as of submersed leaves at the different water depth were detected. The efficiency of PS II was established to reduce significantly under the chlorophyll fluorescence induction with light intensity higher than 60 micromol m(-2)s(-1). The obtained results may be considered as plant adaptation to reduced light intensity and changed light spectrum in the water.

Evolution of plant phage-type RNA polymerases: the genome of the basal angiosperm Nuphar advena encodes two mitochondrial and one plastid phage-type RNA polymerases.

BACKGROUND: In mono- and eudicotyledonous plants, a small nuclear gene family (RpoT, RNA polymerase of the T3/T7 type) encodes mitochondrial as well as chloroplast RNA polymerases homologous to the T-odd bacteriophage enzymes. RpoT genes from angiosperms are well characterized, whereas data from deeper branching plant species are limited to the moss Physcomitrella and the spikemoss Selaginella. To further elucidate the molecular evolution of the RpoT polymerases in the plant kingdom and to get more insight into the potential importance of having more than one phage-type RNA polymerase (RNAP) available, we searched for the respective genes in the basal angiosperm Nuphar advena. RESULTS: By screening a set of BAC library filters, three RpoT genes were identified. Both genomic gene sequences and full-length cDNAs were determined. The NaRpoT mRNAs specify putative polypeptides of 996, 990 and 985 amino acids, respectively. All three genes comprise 19 exons and 18 introns, conserved in their positions with those known from RpoT genes of other land plants. The encoded proteins show a high degree of conservation at the amino acid sequence level, including all functional crucial regions and residues known from the phage T7 RNAP. The N-terminal transit peptides of two of the encoded polymerases, NaRpoTm1 and NaRpoTm2, conferred targeting of green fluorescent protein (GFP) exclusively to mitochondria, whereas the third polymerase, NaRpoTp, was targeted to chloroplasts. Remarkably, translation of NaRpoTp mRNA has to be initiated at a CUG codon to generate a functional plastid transit peptide. Thus, besides AGAMOUS in Arabidopsis and the Nicotiana RpoTp gene, N. advena RpoTp provides another example for a plant mRNA that is exclusively translated from a non-AUG codon. In contrast to the RpoT of the lycophyte Selaginella and those of the moss Physcomitrella, which are according to phylogenetic analyses in sister positions to all other phage-type polymerases of angiosperms, the Nuphar RpoTs clustered with the well separated clades of mitochondrial (NaRpoTm1 and NaRpoTm2) and plastid (NaRpoTp) polymerases. CONCLUSIONS: Nuphar advena encodes two mitochondrial and one plastid phage-type RNAP. Identification of a plastid-localized phage-type RNAP in this basal angiosperm, orthologous to all other RpoTp enzymes of flowering plants, suggests that the duplication event giving rise to a nuclear gene-encoded plastid RNA polymerase, not present in lycopods, took place after the split of lycopods from all other tracheophytes. A dual-targeted mitochondrial and plastididal RNA polymerase (RpoTmp), as present in eudicots but not monocots, was not detected in Nuphar suggesting that its occurrence is an evolutionary novelty of eudicotyledonous plants like Arabidopsis.