Identification, molecular characterization and phylogenetic analysis of a novel nucleorhabdovirus infecting Paris polyphylla var. yunnanensis.

A novel betanucleorhabdovirus infecting Paris polyphylla var. yunnanensis, tentatively named Paris yunnanensis rhabdovirus 1 (PyRV1), was recently identified in Yunnan Province, China. The infected plants showed vein clearing and leaf crinkle at early stage of infection, followed by leaf yellowing and necrosis. Enveloped bacilliform particles were observed using electron microscopy. The virus was mechanically transmissible to Nicotiana bethamiana and N. glutinosa. The complete genome of PyRV1 consists of 13,509 nucleotides, the organization of which was typical of rhabdoviruses, containing six open reading frames encoding proteins N-P-P3-M-G-L on the anti-sense strand, separated by conserved intergenic regions and flanked by complementary 3'-leader and 5'-trailer sequences. The genome of PyRV1 shared highest nucleotide sequence identity (55.1%) with Sonchus yellow net virus (SYNV), and the N, P, P3, M, G, and L proteins showed 56.9%, 37.2%, 38.4%, 41.8%, 56.7%, and 49.4% amino acid sequence identities with respective proteins of SYNV, suggesting RyRV1 belongs to a new species of the genus Betanucleorhabdovirus.

Comparative transcriptome analysis reveals key genes for polyphyllin difference in five Paris species.

Paris species accumulate a large amount of steroidal saponins, which have numerous pharmacological activities and have become an essential component in many patented drugs. However, only two among all Paris species. Paris are identified as official sources due to high level of bioactive compounds. To clarify the composition of steroidal saponins and the molecular basis behind the differences between species, we investigated transcriptome and metabolic profiles of leaves and rhizomes in Paris polyphylla var. chinensis (PPC), Paris polyphylla var. yunnanensis (PPY), Paris polyphylla var. stenophylla (PPS), Paris fargesii (PF), and Paris mairei (PM). Phytochemical results displayed that the accumulation of steroidal saponins was tissue- and species-specific. PF and PPS contained more steroidal saponins in leaves than rhizomes, while PPY accumulated more steroidal saponins in rhizomes than leaves. PPC and PM contained similar amounts of steroidal saponins in leaves and rhizomes. Transcriptome analysis illustrated that most differentially expressed genes related to the biosynthesis of steroidal saponins were abundantly expressed in rhizomes than leaves. Meanwhile, more biosynthetic genes had significant correlations with steroidal saponins in rhizomes than in leaves. The result of CCA indicated that ACAT, DXS, DWF1, and CYP90 constrained 97.35% of the variance in bioactive compounds in leaves, whereas CYP72, UGT73, ACAT, and GPPS constrained 98.61% of the variance in phytochemicals in rhizomes. This study provided critical information for enhancing the production of steroidal saponins by biotechnological approaches and methodologies.

Effective prediction of biosynthetic pathway genes involved in bioactive polyphyllins in Paris polyphylla.

The genes in polyphyllins pathway mixed with other steroid biosynthetic genes form an extremely complex biosynthetic network in Paris polyphylla with a giant genome. The lack of genomic data and tissue specificity causes the study of the biosynthetic pathway notably difficult. Here, we report an effective method for the prediction of key genes of polyphyllin biosynthesis. Full-length transcriptome from eight different organs via hybrid sequencing of next generation sequencingand third generation sequencing platforms annotated two 2,3-oxidosqualene cyclases (OSCs), 216 cytochrome P450s (CYPs), and 199 UDP glycosyltransferases (UGTs). Combining metabolic differences, gene-weighted co-expression network analysis, and phylogenetic trees, the candidate ranges of OSC, CYP, and UGT genes were further narrowed down to 2, 15, and 24, respectively. Beside the three previously characterized CYPs, we identified the OSC involved in the synthesis of cycloartenol and the UGT (PpUGT73CR1) at the C-3 position of diosgenin and pennogenin in P. polyphylla. This study provides an idea for the investigation of gene cluster deficiency biosynthesis pathways in medicinal plants.

Plastome phylogenomics, biogeography, and clade diversification of Paris (Melanthiaceae).

BACKGROUND: Paris (Melanthiaceae) is an economically important but taxonomically difficult genus, which is unique in angiosperms because some species have extremely large nuclear genomes. Phylogenetic relationships within Paris have long been controversial. Based on complete plastomes and nuclear ribosomal DNA (nrDNA) sequences, this study aims to reconstruct a robust phylogenetic tree and explore historical biogeography and clade diversification in the genus. RESULTS: All 29 species currently recognized in Paris were sampled. Whole plastomes and nrDNA sequences were generated by the genome skimming approach. Phylogenetic relationships were reconstructed using the maximum likelihood and Bayesian inference methods. Based on the phylogenetic framework and molecular dating, biogeographic scenarios and historical diversification of Paris were explored. Significant conflicts between plastid and nuclear datasets were identified, and the plastome tree is highly congruent with past interpretations of the morphology. Ancestral area reconstruction indicated that Paris may have originated in northeastern Asia and northern China, and has experienced multiple dispersal and vicariance events during its diversification. The rate of clade diversification has sharply accelerated since the Miocene/Pliocene boundary. CONCLUSIONS: Our results provide important insights for clarifying some of the long-standing taxonomic debates in Paris. Cytonuclear discordance may have been caused by ancient and recent hybridizations in the genus. The climatic and geological changes since the late Miocene, such as the intensification of Asian monsoon and the rapid uplift of Qinghai-Tibet Plateau, as well as the climatic fluctuations during the Pleistocene, played essential roles in driving range expansion and radiative diversification in Paris. Our findings challenge the theoretical prediction that large genome sizes may limit speciation.

Chloroplast phylogenomic analysis provides insights into the evolution of the largest eukaryotic genome holder, Paris japonica (Melanthiaceae).

BACKGROUND: Robust phylogenies for species with giant genomes and closely related taxa can build evolutionary frameworks for investigating the origin and evolution of these genomic gigantisms. Paris japonica (Melanthiaceae) has the largest genome that has been confirmed in eukaryotes to date; however, its phylogenetic position remains unresolved. As a result, the evolutionary history of the genomic gigantisms in P. japonica remains poorly understood. RESULTS: We used next-generation sequencing to generate complete plastomes of P. japonica, P. verticillata, Trillium govanianum, Ypsilandra thibetica and Y. yunnanensis. Together with published plastomes, the infra-familial relationships in Melanthiaceae and infra-generic phylogeny in Paris were investigated, and their divergence times were calculated. The results indicated that the expansion of the ancestral genome of extant Paris and Trillium occurred approximately from 59.16 Mya to 38.21 Mya. The sister relationship between P. japonica and the section Euthyra was recovered, and they diverged around the transition of the Oligocene/Miocene (20 Mya), when the Japan Islands were separated from the continent of Asia. CONCLUSIONS: The genome size expansion in the most recent common ancestor for Paris and Trillium was most possibly a gradual process that lasted for approximately 20 million years. The divergence of P. japonica (section Kinugasa) and other taxa with thick rhizome may have been triggered by the isolation of the Japan Islands from the continent of Asia. This long-term separation, since the Oligocene/Miocene boundary, would have played an important role in the formation and evolution of the genomic gigantism in P. japonica. Moreover, our results support the taxonomic treatment of Paris as a genus rather than dividing it into three genera, but do not support the recognition of T. govanianum as the separate genus Trillidium.

Transcriptome analyses of Paris polyphylla var. chinensis, Ypsilandra thibetica, and Polygonatum kingianum characterize their steroidal saponin biosynthesis pathway.

Steroidal saponins, one of the most diverse groups of plant-derived natural products, elicit biological and pharmacological activities; however, the genes involved in their biosynthesis and the corresponding biosynthetic pathway in monocotyledon plants remain unclear. This study aimed to identify genes involved in the biosynthesis of steroidal saponins by performing a comparative analysis among transcriptomes of Paris polyphylla var. chinensis (PPC), Ypsilandra thibetica (YT), and Polygonatum kingianum (PK). De novo transcriptome assemblies generated 57,537, 140,420, and 151,773 unigenes from PPC, YT, and PK, respectively, of which 56.54, 47.81, and 44.30% were successfully annotated, respectively. Among the transcriptomes for PPC, YT, and PK, we identified 194, 169, and 131; 17, 14, and 26; and, 80, 122, and 113 unigenes corresponding to terpenoid backbone biosynthesis; sesquiterpenoid and triterpenoid biosynthesis; and, steroid biosynthesis pathways, respectively. These genes are putatively involved in the biosynthesis of cholesterol that is the primary precursor of steroidal saponins. Phylogenetic analyses indicated that lanosterol synthase may be exclusive to dicotyledon plant species, and the cytochrome P450 unigenes were closely related to clusters CYP90B1 and CYP734A1, which are UDP-glycosyltransferases unigenes homologous with the UGT73 family. Thus, unigenes of ß-glucosidase may be candidate genes for catalysis of later period modifications of the steroidal saponin skeleton. Our data provide evidence to support the hypothesis that monocotyledons biosynthesize steroidal saponins from cholesterol via the cycloartenol pathway.

Comparative transcriptomic analysis reveals genes regulating the germination of morphophysiologically dormant Paris polyphylla seeds during a warm stratification.

We previously analyzed the expression of genes associated with Paris polyphylla var. yunnanensis seed maturation and dormancy release; however, we were unable to clarify the relationship between gene expression levels and these processes. To reveal the molecular mechanisms underlying P. polyphylla var. yunnanensis seed dormancy release during a warm stratification, the transcriptomes of dormant and germinating P. polyphylla var. yunnanensis seeds were separately analyzed by RNA sequencing and were also compared with the transcriptomes of stem-leaf and root tissues harvested during the seed maturation stage. The RNA sequencing of five tissues generated 234,331 unigenes, of which 10,137 (4.33%) were differentially expressed among the analyzed tissues. The 6,619 unigenes whose expression varied among mature dormant, sprouted, and germinated seeds included 95 metabolic and 62 signaling genes related to abscisic acid, gibberellin, auxin, brassinosteroid, cytokinin, ethylene, jasmonic acid and salicylic acid. Additionally, 243 differentially expressed genes were annotated as known seed dormancy/germination-related genes. Among these genes, 109 were regulated by hormones or involved in hormone signal transduction. Finally, 310 transcription factor unigenes, including 71 homologs of known seed dormancy/ germination-related genes, were observed to be differentially expressed during a warm stratification. These results confirm that multiple hormones and transcription factors influence P. polyphylla var. yunnanensis seed dormancy release and germination during a warm stratification. This study identified candidate genes (e.g., ABI5) that should be cloned and functionally characterized regarding their effects on the release of P. polyphylla var. yunnanensis seed morphophysiological dormancy.

Cloning and functional analysis of two sterol-C24-methyltransferase 1 (SMT1) genes from Paris polyphylla.

The biosynthetic pathways of phytosterols and steroidal saponins are located in two adjacent branches which share cycloartenol as substrate. The rate-limiting enzyme S-adenosyl-L-methionine-sterol-C24-methyltransferase 1 (SMT1) facilitates the metabolic flux toward phytosterols. It catalyzes the methylation of the cycloartenol in the side chain of the C24-alkyl group, to generate 24(28)-methylene cycloartenol. In this study, we obtained two full-length sequences of SMT1 genes from Pari polyphylla, designated PpSMT1-1 and PpSMT1-2. The full-length cDNA of PpSMT1-1 was 1369 bp long with an open reading frame (ORF) of 1038 bp, while the PpSMT1-2 had a length of 1222 bp, with a 1005 bp ORF. Bioinformatics analysis confirmed that the two cloned SMTs belong to the SMT1 family. The predicted function was further validated by performing in vitro enzymatic reactions, and the results showed that PpSMT1-1 encodes a cycloartenol-C24-methyltransferase, which catalyzes the conversion of cycloartenol to 24-methylene cycloartenol, whereas PpSMT1-2 lacked this catalytic activity. The tissue expression patterns of the two SMTs revealed differential expression in different organs of Paris polyphylla plants of different developmental stage and age. These results lay the foundation for detailed genetic studies of the biosynthetic pathways of steroid compounds, which constitute the main class of active substances found in P. polyphylla.

Chloroplast Genomic Resource of Paris for Species Discrimination.

Paris is famous in China for its medicinal value and has been included in the Chinese Pharmacopoeia. Inaccurate identification of these species could confound their effective exploration, conservation, and domestication. Due to the plasticity of the morphological characteristics, correct identification among Paris species remains problematic. In this regard, we report the complete chloroplast genome of P. thibetica and P. rugosa to develop highly variable molecular markers. Comparing three chloroplast genomes, we sought out the most variable regions to develop the best cpDNA barcodes for Paris. The size of Paris chloroplast genome ranged from 162,708 to 163,200 bp. A total of 134 genes comprising 81 protein coding genes, 45 tRNA genes and 8 rRNA genes were observed in all three chloroplast genomes. Eight rapidly evolving regions were detected, as well as the difference of simple sequence repeats (SSR) and repeat sequence. Two regions of the coding gene ycf1, ycf1a and ycf1b, evolved the quickest and were proposed as core barcodes for Paris. The complete chloroplast genome sequences provide more integrated and adequate information for better understanding the phylogenetic pattern and improving efficient discrimination during species identification.