RESUMEN
The integration of morphological and molecular data is essential to understand the affinities of fossil taxa and spatio-temporal evolutionary processes of organisms. However, homoplastic morphological characters can mislead the placement of fossil taxa and impact downstream analyses. Here, we provide an example of how to mitigate effectively the effect of morphological homoplasy on the placement of fossil taxa and biogeographic inferences of Cissampelideae. We assembled three data types, morphological data only, morphological data with a molecular scaffold and combined morphological and molecular data. By removing high-level homoplastic morphological data or reweighting the morphological characters, we conducted 15 parsimony, 12 undated Bayesian and four dated Bayesian analyses. Our results show that the 14 selected Cissampelideae fossil taxa are placed poorly when based only on morphological data, but the addition of molecular scaffold and combination of morphological and molecular data greatly improve the resolution of fossil nodes. We raise the monotypic Stephania subg. Botryodiscia to generic status and discover that three fossils previously assigned to Stephania should be members of Diploclisia. The Bayesian tip-dated tree recovered by removing homoplastic morphological characters with a Rescaled Consistency Index <0.25 has the highest stratigraphic fit and consequently generates more reasonable biogeographic reconstruction for Cissampelideae. Cissampelideae began to diversify in Asia in the latest Cretaceous and subsequently dispersed to South America around the Cretaceous-Palaeogene boundary. Two dispersal events from Asia to Africa occurred in the Early Eocene and the Late Eocene-Late Oligocene, respectively. These findings provide guidelines and practical methods for mitigating the effects of homoplastic morphological characters on fossil placements and Bayesian tip-dating, as well as insights into the past tropical floristic exchanges among different continents.
Asunto(s)
Menispermaceae , Filogenia , Menispermaceae/anatomía & histología , Menispermaceae/genética , Teorema de Bayes , Filogeografía , Fósiles , Factores de TiempoRESUMEN
BACKGROUND AND AIMS: Modern tropical rainforests house the highest biodiversity of Earth's terrestrial biomes and are distributed in three low-latitude areas. However, the biogeographical patterns and processes underlying the distribution of biodiversity among these three areas are still poorly known. Here, we used Tiliacoreae, a tribe of pantropical lianas with a high level of regional endemism, to provide new insights into the biogeographical relationships of tropical rainforests among different continents. METHODS: Based on seven plastid and two nuclear DNA regions, we reconstructed a phylogeny for Tiliacoreae with the most comprehensive sampling ever. Within the phylogenetic framework, we then estimated divergence times and investigated the spatiotemporal evolution of the tribe. KEY RESULTS: The monophyletic Tiliacoreae contain three major clades, which correspond to Neotropical, Afrotropical and Indo-Malesian/Australasian areas, respectively. Both Albertisia and Anisocycla are not monophyletic. The most recent common ancestor of Tiliacoreae occurred in Indo-Malesia, the Afrotropics and Neotropics in the early Eocene, then rapidly diverged into three major clades between 48 and 46 Ma. Three dispersals from Indo-Malesia to Australasia were inferred, one in the middle Eocene and two in the late Oligocene-late Miocene, and two dispersals from the Afrotropics to Indo-Malesia occurred in the late Eocene-Oligocene. CONCLUSIONS: The three main clades of Anisocycla correspond to three distinct genera [i.e. Anisocycla sensu stricto and two new genera (Georgesia and Macrophragma)]. Epinetrum is a member of Albertisia. Our findings highlight that sea-level fluctuations and climate changes in the Cenozoic have played important roles in shaping the current distribution and endemism of Tiliacoreae, hence contributing to the knowledge on the historical biogeography of tropical rainforests on a global scale.
Asunto(s)
Menispermaceae , Bosque Lluvioso , Filogenia , Filogeografía , Menispermaceae/genética , Plastidios/genéticaRESUMEN
Neotropical rainforests cover about half of the world's tropical rainforests and house most of the biodiversity available on Earth. Australasia has been suggested as a potential source for Neotropical diversity. However, it remains unclear whether megathermal lineages could indeed have migrated to South America though Antarctica. The Neotropical Anomospermeae (Menispermaceae) consists of large, canopy lianas and is entirely restricted to tropical lowland rainforests. The sister relationship identified between this group and its Australasian ally represents an excellent model to test hypotheses regarding past connections between those landmasses. In this study, we used six chloroplast and two nuclear DNA markers to reconstruct phylogenetic relationships within the Neotropical Anomospermeae (Menispermaceae). The phylogeny of this group was then used as basis to reconstruct its biogeographical history. The phylogenetic framework reconstructed here strongly supports the monophyly of the Neotropical Anomospermeae and recovers the species of Anomospermum in three different clades: (i) Anomospermum sect. Anomospermum plus Orthomene; (ii) Anomospermum grandifolium and A. solimoesanum (Anomospermum sect. Elissarrhena); and (iii) Anomospermum bolivianum (Anomospermum sect. Elissarrhena). Each of these clades is recognized as a different genus and the necessary taxonomic changes are proposed. Furthermore, the Neotropical Anomospermeae seems to have split from its Australasian sister-group at c. 62â¯Ma. Ancestral area reconstructions support an Australasian origin for the Neotropical Anomospermeae, providing additional support for the hypothesis that Australasia is a source of Neotropical diversity, with megathermal lineages having dispersed via Antarctica. The Neotropical Anomospermeae differentiated in the late Eocene and subsequently diversified rapidly into seven lineages, suggesting that Neotropical lowland rainforests resembling modern rainforests physiognomically and structurally might not have developed until the late Eocene. The Neotropical Anomospermeae exemplifies the contributions of Australasian migration to Neotropical diversity.
Asunto(s)
Sitios Genéticos , Menispermaceae/clasificación , Menispermaceae/genética , Filogenia , Filogeografía , Clima Tropical , Regiones Antárticas , Australasia , Biodiversidad , Núcleo Celular/genética , Variación Genética , Funciones de Verosimilitud , América del Sur , Factores de TiempoRESUMEN
Taiwan is a continental island lying at the boundary between the Eurasian and the Philippine tectonic plates and possesses high biodiversity. Southern Taiwan, viz. Hengchun Peninsula, is notably floristically different from northern Taiwan. The floristic origin and relationships of the Hengchun Peninsula have been rarely investigated in a phylogenetic context. In this study, data from six plastid and nuclear sequences were used to reconstruct phylogenetic relationships within Burasaieae (Menispermaceae), which mainly inhabits tropical rainforests. The tree-based comparisons indicate that the position of Tinospora sensu stricto conflicts significantly between the cpDNA and ITS trees. However, alternative hypothesis tests from the ITS data did not reject the result of the cpDNA data, which suggests that tree-based comparisons might sometimes generate an artificial incongruence, especially when markers with high homoplasy are used. Based on the combined cpDNA and ITS data, we present an inter-generic phylogenetic framework for Burasaieae. Sampled species of Tinospora are placed in three different clades, including Tinospora dentata from southern Taiwan and T. sagittata from mainland China in an unresolved position alongside six lineages of Burasaieae. By integrating lines of evidence from molecular phylogeny, divergence times, and morphology, we recognize the three Tinospora clades as three different genera, including Tinospora sensu stricto, a new genus (Paratinospora) for T. dentata and T. sagittata, and Hyalosepalum resurrected. Tinospora dentata, now endemic to the Hengchun Peninsula, originated from the Late Eocene (ca. 39Ma), greatly predating the formation of Taiwan. Our study suggests that the flora of the Hengchun Peninsula contains some ancient components that might have migrated from mainland China.
Asunto(s)
Menispermaceae/clasificación , China , ADN de Plantas/genética , Genes de Plantas , Especiación Genética , Menispermaceae/genética , Filogenia , Filogeografía , Proteínas de Plantas/genética , Plastidios/genética , Análisis de Secuencia de ADN , TaiwánRESUMEN
BACKGROUND: Recent progress toward the elucidation of benzylisoquinoline alkaloid (BIA) metabolism has focused on a small number of model plant species. Current understanding of BIA metabolism in plants such as opium poppy, which accumulates important pharmacological agents such as codeine and morphine, has relied on a combination of genomics and metabolomics to facilitate gene discovery. Metabolomics studies provide important insight into the primary biochemical networks underpinning specialized metabolism, and serve as a key resource for metabolic engineering, gene discovery, and elucidation of governing regulatory mechanisms. Beyond model plants, few broad-scope metabolomics reports are available for the vast number of plant species known to produce an estimated 2500 structurally diverse BIAs, many of which exhibit promising medicinal properties. RESULTS: We applied a multi-platform approach incorporating four different analytical methods to examine 20 non-model, BIA-accumulating plant species. Plants representing four families in the Ranunculales were chosen based on reported BIA content, taxonomic distribution and importance in modern/traditional medicine. One-dimensional (1)H NMR-based profiling quantified 91 metabolites and revealed significant species- and tissue-specific variation in sugar, amino acid and organic acid content. Mono- and disaccharide sugars were generally lower in roots and rhizomes compared with stems, and a variety of metabolites distinguished callus tissue from intact plant organs. Direct flow infusion tandem mass spectrometry provided a broad survey of 110 lipid derivatives including phosphatidylcholines and acylcarnitines, and high-performance liquid chromatography coupled with UV detection quantified 15 phenolic compounds including flavonoids, benzoic acid derivatives and hydroxycinnamic acids. Ultra-performance liquid chromatography coupled with high-resolution Fourier transform mass spectrometry generated extensive mass lists for all species, which were mined for metabolites putatively corresponding to BIAs. Different alkaloids profiles, including both ubiquitous and potentially rare compounds, were observed. CONCLUSIONS: Extensive metabolite profiling combining multiple analytical platforms enabled a more complete picture of overall metabolism occurring in selected plant species. This study represents the first time a metabolomics approach has been applied to most of these species, despite their importance in modern and traditional medicine. Coupled with genomics data, these metabolomics resources serve as a key resource for the investigation of BIA biosynthesis in non-model plant species.
Asunto(s)
Alcaloides/metabolismo , Bencilisoquinolinas/metabolismo , Magnoliopsida/genética , Metaboloma , Proteínas de Plantas/genética , Berberidaceae/genética , Berberidaceae/metabolismo , Magnoliopsida/metabolismo , Menispermaceae/genética , Menispermaceae/metabolismo , Papaveraceae/genética , Papaveraceae/metabolismo , Proteínas de Plantas/metabolismo , Ranunculaceae/genética , Ranunculaceae/metabolismoRESUMEN
BACKGROUND: Benzylisoquinoline alkaloids (BIAs) represent a diverse class of plant specialized metabolites sharing a common biosynthetic origin beginning with tyrosine. Many BIAs have potent pharmacological activities, and plants accumulating them boast long histories of use in traditional medicine and cultural practices. The decades-long focus on a select number of plant species as model systems has allowed near or full elucidation of major BIA pathways, including those of morphine, sanguinarine and berberine. However, this focus has created a dearth of knowledge surrounding non-model species, which also are known to accumulate a wide-range of BIAs but whose biosynthesis is thus far entirely unexplored. Further, these non-model species represent a rich source of catalyst diversity valuable to plant biochemists and emerging synthetic biology efforts. RESULTS: In order to access the genetic diversity of non-model plants accumulating BIAs, we selected 20 species representing 4 families within the Ranunculales. RNA extracted from each species was processed for analysis by both 1) Roche GS-FLX Titanium and 2) Illumina GA/HiSeq platforms, generating a total of 40 deep-sequencing transcriptome libraries. De novo assembly, annotation and subsequent full-length coding sequence (CDS) predictions indicated greater success for most species using the Illumina-based platform. Assembled data for each transcriptome were deposited into an established web-based BLAST portal ( www.phytometasyn.ca) to allow public access. Homology-based mining of libraries using BIA-biosynthetic enzymes as queries yielded ~850 gene candidates potentially involved in alkaloid biosynthesis. Expression analysis of these candidates was performed using inter-library FPKM normalization methods. These expression data provide a basis for the rational selection of gene candidates, and suggest possible metabolic bottlenecks within BIA metabolism. Phylogenetic analysis was performed for each of 15 different enzyme/protein groupings, highlighting many novel genes with potential involvement in the formation of one or more alkaloid types, including morphinan, aporphine, and phthalideisoquinoline alkaloids. Transcriptome resources were used to design and execute a case study of candidate N-methyltransferases (NMTs) from Glaucium flavum, which revealed predicted and novel enzyme activities. CONCLUSIONS: This study establishes an essential resource for the isolation and discovery of 1) functional homologues and 2) entirely novel catalysts within BIA metabolism. Functional analysis of G. flavum NMTs demonstrated the utility of this resource and underscored the importance of empirical determination of proposed enzymatic function. Publically accessible, fully annotated, BLAST-accessible transcriptomes were not previously available for most species included in this report, despite the rich repertoire of bioactive alkaloids found in these plants and their importance to traditional medicine. The results presented herein provide essential sequence information and inform experimental design for the continued elucidation of BIA metabolism.
Asunto(s)
Alcaloides/metabolismo , Bencilisoquinolinas/metabolismo , Magnoliopsida/genética , Proteínas de Plantas/genética , Transcriptoma , Berberidaceae/genética , Berberidaceae/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Magnoliopsida/metabolismo , Menispermaceae/genética , Menispermaceae/metabolismo , Datos de Secuencia Molecular , Papaveraceae/genética , Papaveraceae/metabolismo , Proteínas de Plantas/metabolismo , Ranunculaceae/genética , Ranunculaceae/metabolismo , Análisis de Secuencia de ADNRESUMEN
Tinospora sagittata (Oliv.) Gagnep. is an important medicinal tetraploid plant in the Menispermaceae family. Its tuber, Radix Tinosporae, used in traditional Chinese medicine, is rich in diterpenoids and benzylisoquinoline alkaloids (BIAs). To enhance our understanding of medicinal compounds' biosynthesis and Menispermaceae's evolution, we herein report assembling a high-quality chromosome-scale genome with both PacBio HiFi and Illumina sequencing technologies. PacBio Sequel II generated 2.5 million circular consensus sequencing (CCS) reads, and a hybrid assembly strategy with Illumina sequencing resulted in 4483 contigs. The assembled genome size was 2.33 Gb, consisting of 4070 scaffolds (N50 = 42.06 Mb), of which 92.05% were assigned to 26 pseudochromosomes. T. sagittata's chromosomal-scale genome assembly, the first species in Menispermaceae, aids Menispermaceae evolution and T. sagittata's secondary metabolites biosynthesis understanding.
Asunto(s)
Genoma de Planta , Plantas Medicinales , Tinospora , Tinospora/genética , Plantas Medicinales/genética , Cromosomas de las Plantas/genética , Menispermaceae/genéticaRESUMEN
Menispermaceae species, as early-diverging eudicots, can synthesize valuable benzylisoquinoline alkaloids (BIAs) like bisbenzylisoquinoline alkaloids (bisBIAs) and sinomenines with a wide range of structural diversity. However, the evolutionary mechanisms responsible for their chemo-diversity are not well understood. Here, a chromosome-level genome assembly of Menispermum dauricum is presented and demonstrated the occurrence of two whole genome duplication (WGD) events that are shared by Ranunculales and specific to Menispermum, providing a model for understanding chromosomal evolution in early-diverging eudicots. The biosynthetic pathway for diverse BIAs in M. dauricum is reconstructed by analyzing the transcriptome and metabolome. Additionally, five catalytic enzymes - one norcoclaurine synthase (NCS) and four cytochrome P450 monooxygenases (CYP450s) - from M. dauricum are responsible for the formation of the skeleton, hydroxylated modification, and C-O/C-C phenol coupling of BIAs. Notably, a novel leaf-specific MdCYP80G10 enzyme that catalyzes C2'-C4a phenol coupling of (S)-reticuline into sinoacutine, the enantiomer of morphinan compounds, with predictable stereospecificity is discovered. Moreover, it is found that Menispermum-specific CYP80 gene expansion, as well as tissue-specific expression, has driven BIA diversity in Menispermaceae as compared to other Ranunculales species. This study sheds light on WGD occurrences in early-diverging eudicots and the evolution of diverse BIA biosynthesis.
Asunto(s)
Bencilisoquinolinas , Sistema Enzimático del Citocromo P-450 , Menispermaceae , Bencilisoquinolinas/metabolismo , Bencilisoquinolinas/química , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Menispermaceae/genética , Menispermaceae/metabolismo , Menispermaceae/química , Alcaloides/metabolismo , Filogenia , Evolución Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
PREMISE OF THE STUDY: This work surveys endocarp morphology of Menispermaceae in the context of a well-supported molecular phylogeny. The study is important since menispermaceous endocarps appear often in the fossil record and indicate the presence of a wet forest ecosystem. ⢠METHODS: Three chloroplast regions were used to derive phylogenies for 53 genera and 60 species. Endocarps of 47 genera and 92 species were dissected and morphological characters scored. Photographs of key features are presented. We superimposed our morphological matrix onto the phylogeny to explore character evolution. A detailed key to fruits is presented, allowing identification of extant and fossil specimens to the level of clade or genus. ⢠KEY RESULTS: Menispermaceae consists of two major subfamilies: Tinosporoideae and Menispermoideae. Within Tinosporoideae, tribe Coscineae is basal. Within Menispermoideae, tribe Menispermeae is basal. Tinosporoideae consists mainly of taxa with apical style scars, bilateral curvature, subhemispherical condyles, and foliaceous cotyledons with divaricate or imbricate orientation. Menispermoideae consists almost entirely of taxa with basal or subbasal style scars, dorsoventral curvature, bilaterally and/or dorsoventrally compressed condyles, and subterete or fleshy cotyledons oriented dorsoventrally or laterally. ⢠CONCLUSIONS: Several fruit characters differentiate major clades, and further synapomorphies are diagnostic of various subclades. Fruit characters that can be inferred as ancestral in the family are basal or subbasal stylar scars, endocarps with dorsoventral curvature, endocarp walls woody or bony, presence of a condyle, locule without ribs, sublateral vascular traces, presence of endosperm, and foliaceous or subterete cotyledons.
Asunto(s)
Frutas/genética , Menispermaceae/genética , Menispermaceae/fisiología , Filogenia , Flores , Fósiles , Frutas/anatomía & histología , Especificidad de la EspecieRESUMEN
⢠Modern tropical rainforests have the highest biodiversity of terrestrial biomes and are restricted to three low-latitude areas. However, the actual timeframe during which tropical rainforests began to appear on a global scale has been intensely disputed. Here, we used the moonseed family (Menispermaceae), an important physiognomic and structural component of tropical rainforests on a worldwide basis, to obtain new insights into the diversification of this biome. ⢠We integrated phylogenetic, biogeographic and molecular dating methods to analyse temporal and spatial patterns of global diversification in Menispermaceae. ⢠Importantly, a burst of moonseed diversification occurred in a narrow window of time, which coincides with the Cretaceous-Paleogene (K-Pg) boundary. Our data also suggest multiple independent migrations from a putative ancestral area of Indo-Malay into other tropical regions. ⢠Our data for Menispermaceae suggest that modern tropical rainforests may have appeared almost synchronously throughout the three major tropical land areas close to, or immediately following, the K-Pg mass extinction.
Asunto(s)
Biodiversidad , Menispermaceae/fisiología , Lluvia , Árboles/fisiología , Clima Tropical , Teorema de Bayes , Funciones de Verosimilitud , Menispermaceae/genética , Filogenia , Factores de TiempoRESUMEN
The present study involves isolation of Streptomyces spp. from rhizosphere of Coscinium fenestratum Gaertn, an endangered medicinal plant from Western Ghats of Karnataka, India. Four potential isolates were identified by 16S rRNA sequencing as Streptomyces sp. RHPR3, Streptomyces puniceus RHPR9, Streptomyces sp. RHPR14 and Streptomyces mediolani RHPR25. An enrichment culture method was used for the isolation of Streptomyces spp. for biosurfactant activity. Among four potential Streptomyces spp., S. puniceus RHPR9 showed highest Emulsification index (EI) (78±0.2%) and Emulsification assay (EA) (223±0.2 EU mL-1). Thin layer chromatography, Fourier transform infrared spectroscopy (FTIR) and mass spectrometric analysis revealed that as glycolipid. Further confirmed by presence of fatty acids like hexanoic acid methyl ester, decanoic acid by Gas chromatography mass spectroscopy (GC-MS) analysis. S. puniceus RHPR9 showed a significant IAA production (41µg mL-1), solubilized P (749.1 µg mL-1), growth promotion of chilli (Capsicum annuum L.) was evaluated using paper towel method and greenhouse conditions. S. puniceus RHPR9 showed a significant increase in seed vigor index (2047) and increase in plant biomass (65%) when compared to uninoculated control. To our knowledge, this is the first report on epiphytic S. puniceus RHPR9 isolated from an endangered medicinal plant C. fenestratum Gaertn, for biosurfactant production and plant growth promotion activities.
Asunto(s)
Menispermaceae , Streptomyces , India , Menispermaceae/genética , ARN Ribosómico 16S/genética , Rizosfera , Streptomyces/genéticaRESUMEN
In Ayurveda and Thai traditional medicines, material from Coscinium fenestratum is commonly prescribed as active ingredients with diverse therapeutic purposes. However, C. fenestratum is also a seriously endangered medicinal liana. Thus, its crude material is very rare and is being substituted with substances from Arcangelisia flava or Fibraurea tinctoria (Menispermaceae), which have high morphological similarity. In this current study, nuclear 18S ribosomal RNA (rRNA) gene and nuclear ribosomal DNA internal transcribed spacer (ITS) gene sequences with the polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) technique were exploited to identify these three species. The nuclear 18S rRNA gene sequences of C. fenestratum, A. flava, and F. tinctoria consisted of 1809, 1805, and 1809 base pairs (bps), respectively, while their ITS gene regions were 694, 622, and 631 bps in length, respectively. The 18S rRNA gene of C. fenestratum digested with SmaI restriction enzyme displayed the electrophoresis profile of 729 and 790 bps; for A. flava and F. tinctoria, the digested products showed fragments of 1519 bps. Although the ITS gene regions of A. flava and F. tinctoria had unrecognized sequences with SalI, the SalI-digested ITS of C. fenestratum exhibited fragments of approximately 599 bp. Thus, the 18S rRNA gene and ITS gene sequences with PCR-RFLPs were proven to be powerful molecular markers for identifying C. fenestratum and distinguishing it from the other two Menispermaceae plants.
Asunto(s)
ADN Espaciador Ribosómico , Contaminación de Medicamentos , Medicina Tradicional de Asia Oriental , Menispermaceae/genética , ARN Ribosómico 18S , Secuencia de Bases , Electroforesis , Componentes Aéreos de las Plantas/genética , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADNRESUMEN
Plant halogenated natural products are rare and harbor various interesting bioactivities, yet the biochemical basis for the involved halogenation chemistry is unknown. While a handful of Fe(II)- and 2-oxoglutarate-dependent halogenases (2ODHs) have been found to catalyze regioselective halogenation of unactivated C-H bonds in bacteria, they remain uncharacterized in the plant kingdom. Here, we report the discovery of dechloroacutumine halogenase (DAH) from Menispermaceae plants known to produce the tetracyclic chloroalkaloid (-)-acutumine. DAH is a 2ODH of plant origin and catalyzes the terminal chlorination step in the biosynthesis of (-)-acutumine. Phylogenetic analyses reveal that DAH evolved independently in Menispermaceae plants and in bacteria, illustrating an exemplary case of parallel evolution in specialized metabolism across domains of life. We show that at the presence of azide anion, DAH also exhibits promiscuous azidation activity against dechloroacutumine. This study opens avenues for expanding plant chemodiversity through halogenation and azidation biochemistry.
Asunto(s)
Alcaloides/biosíntesis , Compuestos Ferrosos/metabolismo , Hidrolasas/metabolismo , Ácidos Cetoglutáricos/metabolismo , Menispermaceae/metabolismo , Compuestos de Espiro/metabolismo , Alcaloides/química , Alcaloides/genética , Bacterias/metabolismo , Biocatálisis , Genes de Plantas/genética , Halogenación , Menispermaceae/embriología , Menispermaceae/genética , Mutagénesis , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes , Metabolismo Secundario/genética , Alineación de Secuencia , Compuestos de Espiro/química , TranscriptomaRESUMEN
The relative significance of weak non-covalent interactions in biological context has been much debated. Here, we have addressed the contribution of Coulombic interactions to protein stability and assembly experimentally. The sweet protein monellin, a non-covalently linked heterodimeric protein, was chosen for this study because of its ability to spontaneously reconstitute from separated fragments. The reconstitution of monellin mutants containing large surface charge perturbations was compared to the thermostability of structurally equivalent single-chain monellin containing the same sets of mutations under varying salt concentrations. The affinity between monellin fragments is found to correlate with the thermostability of single chain monellin, indicating the involvement of the same underlying Coulombic interactions. This confirms that there are no principal differences in the interactions involved in folding and binding. Based on comparison with a previous mutational study involving hydrophobic core residues, the relative contribution of Coulombic interactions to stability and affinity is modest. However, the Coulombic perturbations only affect the association rates of reconstitution in contrast to perturbations involving hydrophobic residues, which affect primarily the dissociation rates. These results indicate that Coulombic interactions are likely to be of main importance for the association of protein assembly, relevant for functions of proteins.
Asunto(s)
Proteínas de Plantas/química , Estabilidad de Medicamentos , Menispermaceae/química , Menispermaceae/genética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Electricidad Estática , TermodinámicaRESUMEN
Processes shaping the African Guineo-Congolian rain forest, especially in the West African part, are not well understood. Recent molecular studies, based mainly on forest tree species, confirmed the previously proposed division of the western African Guineo-Congolian rain forest into Upper Guinea (UG) and Lower Guinea (LG) separated by the Dahomey Gap (DG). Here we studied nine populations in the area of the DG and the borders of LG and UG of the widespread liana species, Chasmanthera dependens (Menispermaceae) by amplified fragment length polymorphism (AFLP), a chloroplast DNA sequence marker, and modelled the distribution based on current as well as paleoclimatic data (Holocene Climate Optimum, ca. 6 kyr BP and Last Glacial Maximum, ca. 22 kyr BP). Current population genetic structure and geographical pattern of cpDNA was related to present as well as historical modelled distributions. Results from this study show that past historical factors played an important role in shaping the distribution of C. dependens across West Africa. The Cameroon Volcanic Line seems to represent a barrier for gene flow in the present as well as in the past. Distribution modelling proposed refugia in the Dahomey Gap, supported also by higher genetic diversity. This is in contrast with the phylogeographic patterns observed in several rainforest tree species and could be explained by either diverging or more relaxed ecological requirements of this liana species.