Genome-Wide Identification and Characterization of miRNAs and Natural Antisense Transcripts Show the Complexity of Gene Regulatory Networks for Secondary Metabolism in Aristolochia contorta.

Aristolochia contorta Bunge is an academically and medicinally important plant species. It belongs to the magnoliids, with an uncertain phylogenetic position, and is one of the few plant species lacking a whole-genome duplication (WGD) event after the angiosperm-wide WGD. A. contorta has been an important traditional Chinese medicine material. Since it contains aristolochic acids (AAs), chemical compounds with nephrotoxity and carcinogenicity, the utilization of this plant has attracted widespread attention. Great efforts are being made to increase its bioactive compounds and reduce or completely remove toxic compounds. MicroRNAs (miRNAs) and natural antisense transcripts (NATs) are two classes of regulators potentially involved in metabolism regulation. Here, we report the identification and characterization of 223 miRNAs and 363 miRNA targets. The identified miRNAs include 51 known miRNAs belonging to 20 families and 172 novel miRNAs belonging to 107 families. A negative correlation between the expression of miRNAs and their targets was observed. In addition, we identified 441 A. contorta NATs and 560 NAT-sense transcript (ST) pairs, of which 12 NATs were targets of 13 miRNAs, forming 18 miRNA-NAT-ST modules. Various miRNAs and NATs potentially regulated secondary metabolism through the modes of miRNA-target gene-enzyme genes, NAT-STs, and NAT-miRNA-target gene-enzyme genes, suggesting the complexity of gene regulatory networks in A. contorta. The results lay a solid foundation for further manipulating the production of its bioactive and toxic compounds.

Characterization of two CYP80 enzymes provides insights into aporphine alkaloid skeleton formation in Aristolochia contorta.

Aporphine alkaloids are a large group of natural compounds with extensive pharmaceutical application prospects. The biosynthesis of aporphine alkaloids has been paid attentions in the past decades. Here, we determined the contents of four 1-benzylisoquinoline alkaloids and five aporphine alkaloids in root, stem, leaf, and flower of Aristolochia contorta Bunge, which belongs to magnoliids. Two CYP80 enzymes were identified and characterized from A. contorta. Both of them catalyze the unusual C-C phenol coupling reactions and directly form the aporphine alkaloid skeleton. AcCYP80G7 catalyzed the formation of hexacyclic aporphine corytuberine. AcCYP80Q8 catalyzed the formation of pentacyclic proaporphine glaziovine. Kingdom-wide phylogenetic analysis of the CYP80 family suggested that CYP80 first appeared in Nymphaeales. The functional divergence of hydroxylation and C-C (or C-O) phenol coupling preceded the divergence of magnoliids and eudicots. Probable crucial residues of AcCYP80Q8 were selected through sequence alignment and molecular docking. Site-directed mutagenesis revealed two crucial residues E284 and Y106 for the catalytic reaction. Identification and characterization of two aporphine skeleton-forming enzymes provide insights into the biosynthesis of aporphine alkaloids.

Mechanistic understanding of perianth traits hindering pollination in Aristolochia contorta Bunge.

Insects are vital pollinators for angiosperms, playing a crucial role in their reproductive success and fruit production. Aristolochia contorta is a perennial herbaceous vine that occurs in fragmented populations across East Asia. One notable feature of this plant is its trap flower, which employs a unique mechanism to attract, trap, retain, and release insects, ensuring effective pollination. The presence of this trap flower significantly influences the pollination system of A. contorta. Field surveys and pollination experiments were conducted to understand the processes and effectiveness of its pollination mechanism. It was allogamous and was pollinated by the species from Ceratopogonidae. During the insect attraction stage, 11.57% of the flowers contained insects, primarily Ceratopogonidae spp. Most Ceratopogonidae spp. concentrated in few flowers, indicating that although overall attraction might be modest, specific flowers acted as significant focal points for gathering. Trichomes effectively trapped Ceratopogonidae spp. inside flower tubes. In the retention stage, 26.16% of Ceratopogonidae spp. were loaded with pollen grains, but only 7.91% of those exited the flowers in the release stage. The sticky texture of the perianth's internal cavity posed challenges during this release, leading to adhesion and clogging of the narrow perianth tube. Consequently, a significant portion of Ceratopogonidae spp. became trapped on the perianth wall and perished. This highlights that despite the significant energy and resources invested in flower development, the perianth contributes to the low pollination effectiveness. This study revealed additive factors with negative effects on pollination, including the densely clustered distribution of its pollinators within only a few flowers, insufficient pollen loading onto pollinators, hindered release of entrapped pollinators due to the perianth adhesive surface, and a high rate of defective pollen grains in A. contorta. These factors account for the observed phenomenon of low fruit set (7.7%) and contribute to the diminished rate of sexual reproduction in A. contorta populations. This might lead the species to heavily rely on asexual reproduction, which could potentially lead to gene erosion within populations. The implications of these findings extend to the ecological and conservation aspects, emphasizing the need to understand and conserve the unique pollination system of A. contorta.

Two New Aristolochic Acid Analogues from the Roots of Aristolochia contorta with Significant Cytotoxic Activity.

Twelve compounds, including two new aristolochic acid analogues with a formyloxy moiety (9-10) and 10 known aristolochic acid derivates (1-8 and 11-12), were obtained from the roots of Aristolochiacontorta. Their structures were elucidated using extensive spectroscopic methods. Their cytotoxic activity in human proximal tubular cells HK-2 was evaluated by the MTT method, which has been widely used to assess cell viability. Among these molecules, compounds 3 and 9 were found to be more cytotoxic. Furthermore, molecular modeling was used to evaluate, for the first time, the interactions of compounds 3 and 9 with the target protein organic anionic transporter 1 (OAT1) that plays a key role in mediating aristolochic acid nephropathy. Structure-activity relationships are briefly discussed.

Pollen ultrastructure in Aristolochia manshuriensis and A. contorta (Aristolochiaceae).

Pollen ultrastructure has been studied in two relict and rare species of the genus Aristolochia, A. contorta Bunge and A. manshuriensis Kom. (Aristolochiaceae). Both species have inaperturate, spheroidal, sometimes distally monocolpate or distally bicolpate pollen grains. The equatorial and polar axes of pollen grain in A. manshuriensis are 48.5 and 44.0 µm, respectively. The percentage of defective pollen grains in A. manshuriensis is 3.4%. The fossulate, perforated exine is up to 2.3 µm in thickness; the sexine and the nexine are almost equal in thickness. In A. contorta, the equatorial axis of pollen grain is 36.6 µm: the defectiveness percentage, 24.5%. The exine is verrucate, up to 0.3 µm in thickness, while the sexine is two to three times thicker than the nexine. The pollen germination experiments have shown that pollen of A. manshuriensis, in contrast to A. contorta, can germinate in 10-20% sucrose at 22°Ð¡. These data and the high percentage of pollen defectiveness in A. contorta indicate that the androecium function in this species is reduced. The reduction of the androecium function is evidenced by a small amount of pollen grains in anthers or empty anthers and a high percentage of defective pollen grains.

Aristololactam derivatives from the fruits of Aristolochia contorta Bunge.

Three new aristololactam derivatives, aristololactam W-Y (1-3), and three known compounds (4-6) were isolated from the fruits of Aristolochia contorta Bunge. Compounds 1 and 2 represent the first example of an N-CH2OCH3 aristololactam derivative from natural products. Their structures were elucidated by 1D/2D NMR and HRESIMS spectra. All of the isolated compounds were evaluated for their insecticidal activity against 4th instar larvae of Aedes aegypti. Compound 4 displayed insecticidal activity with LC50 value of 3.54 µg/mL.

Molecular Structure and Phylogenetic Analyses of Complete Chloroplast Genomes of Two Aristolochia Medicinal Species.

The family Aristolochiaceae, comprising about 600 species of eight genera, is a unique plant family containing aristolochic acids (AAs). The complete chloroplast genome sequences of Aristolochia debilis and Aristolochia contorta are reported here. The results show that the complete chloroplast genomes of A. debilis and A. contorta comprise circular 159,793 and 160,576 bp-long molecules, respectively and have typical quadripartite structures. The GC contents of both species were 38.3% each. A total of 131 genes were identified in each genome including 85 protein-coding genes, 37 tRNA genes, eight rRNA genes and one pseudogene (ycf1). The simple-sequence repeat sequences mainly comprise A/T mononucletide repeats. Phylogenetic analyses using maximum parsimony (MP) revealed that A. debilis and A. contorta had a close phylogenetic relationship with species of the family Piperaceae, as well as Laurales and Magnoliales. The data obtained in this study will be beneficial for further investigations on A. debilis and A. contorta from the aspect of evolution, and chloroplast genetic engineering.

Characterization and quantitation of aristolochic acid analogs in different parts of Aristolochiae Fructus, using UHPLC-Q/TOF-MS and UHPLC-QqQ-MS.

Aristolochiae Fructus, a Chinese herbal medicine derived from the fruit of Aristolochia contorta Bge., contains nephrotoxic aristolochic acid analogues (AAAs). According to ancient medical texts, various medicinal parts of the fruit of A. contorta were ever used. In order to reveal which part could be safely and effectively used, it is necessary to analyze the chemical profiles of different medicinal parts. Herein we compared the chemical compositions and determined aristolochic acid I (AA-I) and aristolochic acid II (AA-II) in the four parts viz. outer pericarp, inner pericarp, septum, and seed. Ultra-high performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) was applied for chemical profiling. Ultra-high performance liquid coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) was employed to quantify AA-I and AA-II in different parts. It was found that the chemical compositions of the four parts varied both qualitatively and quantitatively. A total of 10 AAAs, including 5 aristolochic acids and 5 aristolactams, together with 3 alkaloids, were unambiguously or tentatively identified by UHPLC-QTOF-MS. The quantitatively analytical results obtained by UHPLC-QqQ-MS showed that AA-I and AA-II exclusively accumulate in the seeds of A. contorta. These findings provide supporting data for the rational selection of medicinal parts.

Characterization and quantitation of aristolochic acid analogs in different parts of Aristolochiae Fructus, using UHPLC-Q/TOF-MS and UHPLC-QqQ-MS / 中国天然药物

Aristolochiae Fructus, a Chinese herbal medicine derived from the fruit of Aristolochia contorta Bge., contains nephrotoxic aristolochic acid analogues (AAAs). According to ancient medical texts, various medicinal parts of the fruit of A. contorta were ever used. In order to reveal which part could be safely and effectively used, it is necessary to analyze the chemical profiles of different medicinal parts. Herein we compared the chemical compositions and determined aristolochic acid I (AA-I) and aristolochic acid II (AA-II) in the four parts viz. outer pericarp, inner pericarp, septum, and seed. Ultra-high performance liquid chromatography equipped with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) was applied for chemical profiling. Ultra-high performance liquid coupled with triple quadrupole mass spectrometry (UHPLC-QqQ-MS) was employed to quantify AA-I and AA-II in different parts. It was found that the chemical compositions of the four parts varied both qualitatively and quantitatively. A total of 10 AAAs, including 5 aristolochic acids and 5 aristolactams, together with 3 alkaloids, were unambiguously or tentatively identified by UHPLC-QTOF-MS. The quantitatively analytical results obtained by UHPLC-QqQ-MS showed that AA-I and AA-II exclusively accumulate in the seeds of A. contorta. These findings provide supporting data for the rational selection of medicinal parts.

Tissue Culture and Plant Regeneration of Aristolochia contorta Bge / 广州中医药大学学报

[Objective] To explore the culture conditions of inducing callus from tissue of Aristolochia contorta Bge (ACB). [Methods] The leaves of ACB were used as the explants. Basic medium (including 1/2 MS medium, MS medium and modified MS medium) containing corresponding phyto-hormones was applied for the induction of ACB callus. The influerices of different culture conditions such as three kinds of basic medium, different pH values and addition of different kinds of phyto-hormones at different concentrations into the basic medium respectively or simultaneously, on the callus induction of ACB were observed. [Results] (1) After the three kinds of basic medium were added with the phytohormones of 0.1mg/L kinetin (KT) and 0.2mg/L ?-naphthalene acetic acid (NAA) , a large amount of ACB calluses were induced in the MS medium and modified MS medium, while ACB calluses did not occur in the 1/2 MS medium. (2) When the pH value of the culture medium composed of modified MS medium and 0.1mg/L KT and 0.2mg/L NAA was at 5.8, 7.0 and 8.0, a large amount of ACB calluses were induced in the medium with pH value being 7.0 and 8.0 while a few calluses occurred in the medium with pH value being 5.8. (3) ACB calluses were induced in modified MS medium with NAA added , but calluses did not occur in modified MS medium with KT added. When the modified MS medium was added with different kinds of phyto-hormones at different concentrations, ACB calluses were loose in the medium with high-concentration NAA and this did not benefit to the differentiation of buds for too fast cell division, and the callus induction rate was low in the medium with low-concentration NAA. The optimized culture medium was modified MS medium with 1mg/L KT and 0.5mg/L NAA added. [Conclusion] The optimum culture conditions of inducing callus from ACB tissues are: MS medium or modified MS medium with 1mg/L KT and 0.5 mg/L NAA added being the culture medium, and pH value being 7.0-8.0.

Cloning, Sequencing and Homologous Analysis of Tyrosine Decarboxylase Gene from Aristolochia contorta Bge / 广州中医药大学学报

[Objective] To obtain the tyrosine decarboxylase gene (tyrDC) from Aristolochia contorta Bge.and to assay its cDNA sequence and homologous analysis, thus to remove its nephrotoxieity. [Methods] The primers designed by referring to the conservative amino acid sequences of known plant tyrDC were used to amplify a fragment of cDNA from Aristolochia contorta Bge.by reverse transcription-polymerase chain reaction (RT-PCR). The amplified cDNA sequence was cloned and sequenced to design a pair of specific primers and to amplify a full-length tyrDC cDNA from Aristolochia contorta Bge.by rapid amplification of cDNA ends (RACE). [Results] The length of cloned tyrDC cDNA is 1678 base pairs (bp), which comprises an open reading frame ( ORF) of 1551 bp encoding 516 amino acids and a downstream untranslated region (3'UTR) of 127 bp. The results of sequence comparison indicated that the amino acid sequence deduced from the nucleotide sequence of tyrDC from Aristolochia contorta Bge.shares 76% homology with issued tyrosine decarboxylase of Papaver somniferum L. and 79% homology with tyrosine /DOPA decarboxylase from Thalictrum flavum subsp. glaucum. [Conclusion] The full-length tyrDC cDNA has been amplified from Aristolochia contorta Bge. and the homologous retrieve of tyrosine decarboxylase reveals an extensive sequence similarity among tyrosine decarboxylases of different plants. This will provide evidence for the romoval of nephrotoxicity of Aristolochia contorta Bge. .