Nepenthes Research Articles

Determining the legality of newly described CITES-listed species in the horticulture trade of tropical pitcher plants (Nepenthes).

Due diligence is a fundamental component of ensuring a sustainable and legal wildlife trade that is also supportive of the livelihoods and businesses that depend on the trade. This is particularly true with species listed on the Convention on International Trade in Endangered Species (CITES) that are considered threatened or may become threatened by trade. Undertaking due diligence exercises requires access to information on which to base such decisions; however, the extent to which information is available is unclear. We used the trade in tropical pitcher plants (Nepenthes) for horticultural purposes as a case study to determine the extent to which information is available. A systematic survey of online trade was conducted for species described from 1996 to 2016. For the species found in trade, these were cross-referenced with the CITES trade database, and inquiries were made to the relevant CITES Management Authorities and National Focal Points Access and Benefit Sharing (ABS). Of 83 newly described species, 61% were offered for sale online in 2018. Despite all Nepenthes species being listed on CITES, only 23% (n=19) of the species being sold online were reported in trade on the CITES Trade Database, and only 3 were from the countries of origin. Thirty-two of these species had no international trade recorded according to the database. Management authorities of CITES for the countries of origin confirmed trade had been permitted for 5 of 32 species. Lack of CITES records may be explained by trade under "Nepenthes spp." or as exempt parts and derivatives. However, permits to collect and commercialize are likely to be required as part of the Nagoya Protocol on ABS from the Convention on Biological Diversity. The ABS National Focal Points were contacted to determine whether collection or commercialization permits had been issued for the remaining species. Only 2 of 7 focal points replied, and both stated no permits had been issued. Lack of traceability information or response related to the issuance of collection and commercialization permits is concerning and hinders the due diligence of businesses and consumers wanting to ensure their trade is legal, sustainable, and ethical.

Inhibition of SARS-CoV-2 Nsp9 ssDNA-Binding Activity and Cytotoxic Effects on H838, H1975, and A549 Human Non-Small Cell Lung Cancer Cells: Exploring the Potential of Nepenthes miranda Leaf Extract for Pulmonary Disease Treatment.

Carnivorous pitcher plants from the genus Nepenthes are renowned for their ethnobotanical uses. This research explores the therapeutic potential of Nepenthes miranda leaf extract against nonstructural protein 9 (Nsp9) of SARS-CoV-2 and in treating human non-small cell lung carcinoma (NSCLC) cell lines. Nsp9, essential for SARS-CoV-2 RNA replication, was expressed and purified, and its interaction with ssDNA was assessed. Initial tests with myricetin and oridonin, known for targeting ssDNA-binding proteins and Nsp9, respectively, did not inhibit the ssDNA-binding activity of Nsp9. Subsequent screenings of various N. miranda extracts identified those using acetone, methanol, and ethanol as particularly effective in disrupting Nsp9's ssDNA-binding activity, as evidenced by electrophoretic mobility shift assays. Molecular docking studies highlighted stigmast-5-en-3-ol and lupenone, major components in the leaf extract of N. miranda, as potential inhibitors. The cytotoxic properties of N. miranda leaf extract were examined across NSCLC lines H1975, A549, and H838, focusing on cell survival, apoptosis, and migration. Results showed a dose-dependent cytotoxic effect in the following order: H1975 > A549 > H838 cells, indicating specificity. Enhanced anticancer effects were observed when the extract was combined with afatinib, suggesting synergistic interactions. Flow cytometry indicated that N. miranda leaf extract could induce G2 cell cycle arrest in H1975 cells, potentially inhibiting cancer cell proliferation. Gas chromatography-mass spectrometry (GC-MS) enabled the tentative identification of the 19 most abundant compounds in the leaf extract of N. miranda. These outcomes underscore the dual utility of N. miranda leaf extract in potentially managing SARS-CoV-2 infection through Nsp9 inhibition and offering anticancer benefits against lung carcinoma. These results significantly broaden the potential medical applications of N. miranda leaf extract, suggesting its use not only in traditional remedies but also as a prospective treatment for pulmonary diseases. Overall, our findings position the leaf extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and antiviral therapies, warranting further investigation into its molecular mechanisms and potential clinical applications.

Cytotoxicity and Multi-Enzyme Inhibition of Nepenthes miranda Stem Extract on H838 Human Non-Small Cell Lung Cancer Cells and RPA32, Elastase, Tyrosinase, and Hyaluronidase Proteins

The carnivorous pitcher plants of the genus Nepenthes have long been known for their ethnobotanical applications. In this study, we prepared various extracts from the pitcher, stem, and leaf of Nepenthes miranda using 100% ethanol and assessed their inhibitory effects on key enzymes related to skin aging, including elastase, tyrosinase, and hyaluronidase. The cytotoxicity of the stem extract of N. miranda on H838 human lung carcinoma cells were also characterized by effects on cell survival, migration, proliferation, apoptosis induction, and DNA damage. The cytotoxic efficacy of the extract was enhanced when combined with the chemotherapeutic agent 5-fluorouracil (5-FU), indicating a synergistic effect. Flow cytometry analysis suggested that the stem extract might suppress H838 cell proliferation by inducing G2 cell cycle arrest, thereby inhibiting carcinoma cell proliferation. Gas chromatography–mass spectrometry (GC–MS) enabled the tentative identification of the 15 most abundant compounds in the stem extract of N. miranda. Notably, the extract showed a potent inhibition of the human RPA32 protein (huRPA32), critical for DNA replication, suggesting a novel mechanism for its anticancer action. Molecular docking studies further substantiated the interaction between the extract and huRPA32, highlighting bioactive compounds, especially the two most abundant constituents, stigmast-5-en-3-ol and plumbagin, as potential inhibitors of huRPA32’s DNA-binding activity, offering promising avenues for cancer therapy. Overall, our findings position the stem extract of N. miranda as a promising source of natural compounds for anticancer therapeutics and anti-skin-aging treatments, warranting further investigation into its molecular mechanisms and potential clinical applications.

Do Reactive Oxygen and Nitrogen Species Have a Similar Effect on Digestive Processes in Carnivorous Nepenthes Plants and Humans?

Carnivorous plants attract animals, trap and kill them, and absorb nutrients from the digested bodies. This unusual (for autotrophs) type of nutrient acquisition evolved through the conversion of photosynthetically active leaves into specialised organs commonly called traps. The genus Nepenthes (pitcher plants) consists of approximately 169 species belonging to the group of carnivorous plants. Pitcher plants are characterised by specialised passive traps filled with a digestive fluid. The digestion that occurs inside the traps of carnivorous plants depends on the activities of many enzymes. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) also participate in the digestive process, but their action is poorly recognised. ROS and RNS, named together as RONS, exhibit concentration-dependent bimodal functions (toxic or signalling). They act as antimicrobial agents, participate in protein modification, and are components of signal transduction cascades. In the human stomach, ROS are considered as the cause of different diseases. RNS have multifaceted functions in the gastrointestinal tract, with both positive and negative impacts on digestion. This review describes the documented and potential impacts of RONS on the digestion in pitcher plant traps, which may be considered as an external stomach.

Mechanics reveals the role of peristome geometry in prey capture in carnivorous pitcher plants (Nepenthes)

Carnivorous pitcher plants (Nepenthes) are a striking example of a natural pitfall trap. The trap's slippery rim, or peristome, plays a critical role in insect capture via an aquaplaning mechanism that is well documented. While the peristome has received significant research attention, the conspicuous variation in peristome geometry across the genus remains unexplored. We examined the mechanics of prey capture using Nepenthes pitcher plants with divergent peristome geometries. Inspired by living material, we developed a mathematical model that links the peristomes' three-dimensional geometries to the physics of prey capture under the laws of Newtonian mechanics. Linking form and function enables us to test hypotheses related to the function of features such as shape and ornamentation, orientation in a gravitational field, and the presence of "teeth," while analysis of the energetic costs and gains of a given geometry provides a means of inferring potential evolutionary pathways. In a separate modeling approach, we show how prey size may correlate with peristome dimensions for optimal capture. Our modeling framework provides a physical platform to understand how divergence in peristome morphology may have evolved in the genus Nepenthes in response to shifts in prey diversity, availability, and size.

Fabrication of biomimetic anisotropic crescent-shaped microstructured surfaces by laser shock imprinting

Abstract The crescent-shaped microstructure bionic to the slip zone of the slippery zone of the carnivorous plant genus Nepenthes was fabricated on the surface of copper foil by laser shock imprinting (LSI). The microstructure of crescent-shaped grooves was initially fabricated on the surface of the micro-mold by etching, and then the microstructure was replicated on the surface of copper foil through plastic deformation under laser shock loading. Increasing the laser shock energy or the number of shocks can increase the degree of replication of the crescent-shaped microstructure, the height of the crescent-shaped microstructure, and the contact angle of water droplets on the surface. The wettability of the surface of the crescent microstructure is anisotropic and increases with an increase in offset distance. The anisotropy of the crescent-shaped microstructure causes the solid–liquid contact line in the direction of the bottom of the arc to become a long and approximately straight line. According to the rule that controlling LSI processing parameters can fabricate surfaces with different heights and wettability, a gradient wetting surface consisting of crescent-shaped microstructures was designed to achieve the directional spreading of droplets. By altering the distribution of crescent-shaped microstructures, a type-I flow channel with the ability to limit the spreading range of water droplets was fabricated.

Comprehensive approaches for assessing extinction risk of endangered tropical pitcher plant Nepenthes talangensis.

It has been 23 years since the conservation status of highland tropical pitcher plant Nepenthes talangensis was assessed in 2000. A number of existing threats (anthropogenic and environmental) may be increasing the risk of extinction for the species. A better understanding of the ecology and conservation needs of the species is required to manage the wild populations. Specifically, better information related to population distributions, ecological requirements, priority conservation areas, the impact of future climate on suitable habitat, and current population structure is needed to properly assess extinction risks. A better understanding of the requirements of the species in its natural habitat would benefit for successfully securing the species at Botanic Gardens. We have identified 14 new occurrence records of N. talangensis in Mount Talang. Study on the ecological requirement using Random Forest (RF) and Artificial Neural Network (ANN) suggested that elevation, canopy cover, soil pH, and slope are four important variables. The population of N. talangensis was dominated by juvenile and mature (sterile) individuals, we found only a few mature males (7 individuals) and females (4 individuals) in the sampled areas. Our modelling of current conditions predicted that there were 1,076 ha of suitable habitat to very highly suitable habitat in Mount Talang, which is 14.7% of the total area. Those predicted habitats ranged in elevation from 1,740-2,558 m. Suitable habitat in 2100 was predicted to decrease in extent and be at higher elevation in the less extreme climate change scenario (SSP 1-2.6) and extreme climate change scenario (SSP 5-8.5). We projected larger habitat loss in the SSP 5-8.5 compared to the SSP 1-2.6 climate change scenario.. We proposed the category CR B1ab(iii,v), C2a(ii) as the new conservation status of N. talangensis. The status is a higher category of threat compared to the current status of the species (EN C2b, ver 2.3). Nepenthes talangensis seedlings and cuttings established in a Botanic Garden have relatively high survival rate at about 83.4%. Sixty percent of the seeds germinated in growth media successfully grew to become seedlings.

Insights into The Diversity of Nepenthes L. (Nepenthaceae) Across Peninsular Malaysia, Including The First Sighting of an Undescribed Taxon with Flared Peristomes and Quadruple-Row Ventral Wings

The discoveries of six taxa between 2019 and 2022 have brought the total number of Nepenthesspecies occurring in Peninsular Malaysia to 18. This article serves as a comprehensive overview, incorporating the latest information and taxonomic insightsconcerningallspecies native to the peninsula. The information is derived from published taxonomic records and cumulative field observations conducted since 2013.Notably, it further reports heretoforeundocumented discoveries, making it a significant contribution to the current knowledgeof tropical pitcher plants. Theseinclude a sighting of a population of Nepenthes with unusual traits in the upper montane forest of Banjaran Titiwangsa, which may represent the 19thtaxonin Peninsular Malaysia. The plantsexhibit conspicuously expandedperistomes and an unusual wing morphology, which altogether provide compelling evidence supporting the recognition of these individuals as a distinct and previously unknown species. Therefore, a provisional taxonomic name—Nepenthessp. Titiwangsa (pesonawangsa_223) A. Amin—is assigned.The species is inferred to belongto the N. macfarlaneigroup based on its toothed peristome and the presence of fine hairs below the lid. The partial description, speciesnote, photographs,and a dichotomous keyof the proposednew speciesare provided.In addition, a velvety N. sanguineapopulation has been observed in northern Banjaran Titiwangsa and treated as a new natural variation (var. velutina) in this article.

DIVERSITY OF PITCHER PLANTS (NEPENTHES SPP.) IN RIAU ARCHIPELAGO PROVINCE, INDONESIA

Nepenthes spp. is a unique carnivorous plant with the modified leaf lamina as a pitcher to trap insects. Nepenthes usually produce lower traps near the base of the plant, and upper pitchers form a loop in the tendril, allowing it to wrap around the nearby plant. Pitcher plants, being insectivorous plants, can grow in nitrogen-deficient soils. In the Riau Archipelago Province, the intense land conversion to establish ecotourism in national parks disturbed the pitcher plant habitat, even though the pitcher plants have great potential as ornamental plants. Therefore, for in situ conservation, the existing endemic flora may benefit tourist attractions to support local ecotourism efforts. Based on the above discussion, the presented study aimed to characterize the pitcher plants’ diversity and analyze the relationship among its species using morphological markers in the islands of Bintan, Karimun, and Lingga, Indonesia. Based on the collection and identified pitcher plants, the study discovered that the genus Nepenthes comprises 25 accessions belonging to six different species, i.e., N. gracilis, N. × trichocarpa, N. reinwardtiana, N. ampullaria, N. rafflesiana, and N. × hookeriana. The cluster analysis grouped two main clusters that have a similarity coefficient of 31%–97% based on morphological characteristics among Bintan, Karimun, and Lingga accessions. However, the cluster constructions were more on the traits’ similarity than locality based. For principal component analysis (PCA), 19 morphological traits can benefit as diagnostic features to distinguish the pitcher groups. The study revealed that pitcher plants have diverse phenotypic plasticity in the Riau Archipelago, which is experiencing land conversion for tourism activities. The pitcher plants diversity has the genetic potential for ornamental plants development and is a biological wealth that requires conservation for research, tourism activities, and future generations.

The Inhibitory Effects and Cytotoxic Activities of the Stem Extract of Nepenthes miranda against Single-Stranded DNA-Binding Protein and Oral Carcinoma Cells.

The carnivorous pitcher plants of the genus Nepenthes exhibit many ethnobotanical uses, including treatments of stomachache and fever. In this study, we prepared different extracts from the pitcher, stem, and leaf extracts of Nepenthes miranda obtained using 100% methanol and analyzed their inhibitory effects on recombinant single-stranded DNA-binding protein (SSB) from Klebsiella pneumoniae (KpSSB). SSB is essential for DNA replication and cell survival and thus an attractive target for potential antipathogen chemotherapy. Different extracts prepared from Sinningia bullata, a tuberous member of the flowering plant family Gesneriaceae, were also used to investigate anti-KpSSB properties. Among these extracts, the stem extract of N. miranda exhibited the highest anti-KpSSB activity with an IC50 value of 15.0 ± 1.8 μg/mL. The cytotoxic effects of the stem extract of N. miranda on the survival and apoptosis of the cancer cell lines Ca9-22 gingival carcinoma, CAL27 oral adenosquamous carcinoma, PC-9 pulmonary adenocarcinoma, B16F10 melanoma, and 4T1 mammary carcinoma cells were also demonstrated and compared. Based on collective data, the cytotoxic activities of the stem extract at a concentration of 20 μg/mL followed the order Ca9-22 > CAL27 > PC9 > 4T1 > B16F10 cells. The stem extract of N. miranda at a concentration of 40 μg/mL completely inhibited Ca9-22 cell migration and proliferation. In addition, incubation with this extract at a concentration of 20 μg/mL boosted the distribution of the G2 phase from 7.9% to 29.2% in the Ca9-22 cells; in other words, the stem extract might suppress Ca9-22 cell proliferation by inducing G2 cell cycle arrest. Through gas chromatography-mass spectrometry, the 16 most abundant compounds in the stem extract of N. miranda were tentatively identified. The 10 most abundant compounds in the stem extract of N. miranda were used for docking analysis, and their docking scores were compared. The binding capacity of these compounds was in the order sitosterol > hexadecanoic acid > oleic acid > plumbagin > 2-ethyl-3-methylnaphtho[2,3-b]thiophene-4,9-dione > methyl α-d-galactopyranoside > 3-methoxycatechol > catechol > pyrogallol > hydroxyhydroquinone; thus, sitosterol might exhibit the greatest inhibitory capacity against KpSSB among the selected compounds. Overall, these results may indicate the pharmacological potential of N. miranda for further therapeutic applications.

Leaf Anatomical Characteristics of Nepenthes Species in Western Sarawak, Borneo

The dimorphism and phenotypic plasticity of genus Nepenthes is taxonomically challenging. They depicted a variety of pitcher morphological features that often lead to difficulty in the species delineation. However, there is an alternative that could aid to distinguish the Nepenthes species which is by observing the anatomical characteristics of their leaves. Despite few researches had reported the comparative study on leaf anatomy of certain Nepenthes species, yet, there are very scarce data showing other distinct anatomical characteristics that could be used to differentiate the Nepenthes species especially in the western of Sarawak. Hence, this research was performed to identify anatomical characteristics that could be useful taxonomic tools for Nepenthes species identification. There are nine species with one variety and three natural hybrids were included in the study namely, Nepenthes albomarginata Lobb, N. ampullaria Jack, N. gracilis Korth, N. hirsuta Hook. f., N. hispida Beck, N. mirabilis (Lour.) Druce, N. mirabilis var. echinostoma (Hook. f.) J. H. Adam & Wilcock, N. nazreeana sp. nov. ined., N. rafflesiana Jack, N. reinwardtiana Miq., N. x trichocarpa Miq, N. x hookeriana Lindl, and N. x kuchingensis Sh. Kurata. The anatomical data of leaves, i.e stomatal complex type, stomatal size and density, type of anticlinal walls, type of glandular structures and others were analysed, tabulated and discussed.

The taxonomic relevance of Naphthoquinones in tropical pitcher plants (Nepenthes L., Nepenthaceae)

The distribution of the naphthoquinones ramentaceone and plumbagin was studied among 50 taxa of the genus Nepenthes. Naphthoquinone patterns support classifications based on homology of plastid and/or nuclear genes to some extent, with plumbagin predominant in sections Nepenthes, Urceolatae, Tentaculatae, and Regiae, ramentaceone predominant in sections Insignes and Villosae, and both isomers present without clear predominance in sections Pyrophytae and Montanae. Only 9 of 96 studied species contained both isomers in the same plant. Naphthoquinone data from artificial hybrids of known parentage allowed conclusions on the biosynthesis of these compounds and the heredity of the respective enzymatic steps.

Capture of mammal excreta by Nepenthes is an effective heterotrophic nutrition strategy.

While isotopic enrichment of nitrogen (15N) and carbon (13C) is often used to determine whether carnivorous plant species capture and assimilate nutrients from supplemental sources such as invertebrate prey or mammal excreta (heterotrophic nutrition), little is known about how successful the different strategies deployed by carnivorous plants are at obtaining supplemental nutrition. The collection of mammalian faeces by Nepenthes (tropical pitcher plants) is the result of a highly specialized biological mutualism that results in heterotrophic nitrogen gain; however, it remains unknown how effective this strategy is in comparison to Nepenthes species not known to collect mammalian faeces. We examined how isotopic enrichment varied in the diverse genus Nepenthes, among species producing pitchers for invertebrate capture and species exhibiting mutualisms for the collection of mammal excreta. Enrichment factors were calculated from δ15N and δ13C values from eight Nepenthes species and naturally occurring hybrids along with co-occurring reference (non-carnivorous) plants from three mountain massifs in Borneo: Mount Kinabalu, Mount Tambuyukon and Mount Trus Madi. All Nepenthes examined, except N. edwardsiana, were significantly enriched in 15N compared to co-occurring non-carnivorous plants, and 15N enrichment was more than two-fold higher in species with adaptations for the collection of mammal excreta compared with other Nepenthes. The collection of mammal faeces clearly represents a highly effective strategy for heterotrophic nitrogen gain in Nepenthes. Species with adaptations for capturing mammal excreta occur exclusively at high elevation (i.e. are typically summit-occurring) where previous studies suggest invertebrate prey are less abundant and less frequently captured. As such, we propose this strategy may maximize nutritional return by specializing towards ensuring the collection and retention of few but higher-value N sources in environments where invertebrate prey may be scarce.

A semi-detritivorous pitcher plant, Nepenthes ampullaria diverges in its regulation of pitcher fluid properties

ABSTRACT Tropical pitcher plants (Nepenthes) are carnivorous plants that trap and digest prey using highly modified fluid-filled leaves known as pitchers. Prey are digested by plant-secreted enzymes and pitcher symbionts. Pitchers exert control over abiotic properties of the digestive fluid such as pH levels that can influence its symbionts. Here we examine natural variation in pH and dissolved mineral concentrations in three sympatric Nepenthes species, assessing correlations between fluid properties and pitcher traits. We use addition experiments to investigate differences in protein digestion/absorption rates between species. Fluid pH and dissolved mineral levels both showed distinct patterns corresponding to pitcher developmental stages in N. gracilis and N. rafflesiana, whereas N. ampullaria differs from its congeners in exhibiting far less variation in fluid pH, as well as less clear evidence of protein depletion. This study further elucidates the properties of pitchers as habitats, revealing ways in which the host plant regulates that habitat.

The feasibility of tropical pitcher plant (Nepenthes sp.) exploration booklet as a learning media

Learning media is designed to arouse curiosity and increase students' knowledge. If only listening to verbal information from the teacher, students are more likely to ignore what is being taught. Therefore, the use of learning media has proven to be effective. Currently, learning materials are sourced from textbooks. This study aims to determine the feasibility, practicality, and effectiveness of a booklet developed from the inventory of pitcher plants (Nepenthes sp.) as a learning medium for high school/MA students. The research stages include the needs analysis stage, the booklet planning stage, the booklet creation stage, the validation stage, the trial stage, and the multiplication of booklet prints. The research instruments are in the form of validation sheets and questionnaires. Material experts and design experts assess validation. The practical tests of students and teachers include evaluating the booklet's physical appearance and overall content. The validation assessment was 88.6% and 90.4% "very valid" by design and material experts, respectively. The product's practicality based on the responses of students and teachers obtained a result of 96.7%, and the product's effectiveness got a result of 94% with the category of "excellent." This data shows Indonesia's biodiversity is a force that can be developed to meet the opportunities and challenges in education.Abstrak Media pembelajaran dirancang untuk membangkitkan rasa ingin tahu serta menambah ilmu pengetahuan siswa. Jika hanya mendengarkan informasi verbal dari guru, siswa lebih cenderung mengabaikan apa yang diajarkan. Oleh karena itu, penggunaan media pembelajaran terbukti efektif. Saat ini, materi pembelajaran bersumber pada buku teks. Penelitian ini bertujuan untuk mengetahui kelayakan, kepraktisan dan keefektifan buklet yang dikembangkan dari inventarisasi tumbuhan kantong semar (Nepenthes sp.) sebagai media pembelajaran bagi siswa SMA/MA. Tahapan penelitian yang dilakukan meliputi tahap analisis kebutuhan, tahap perencanaan buklet, tahap pembuatan buklet, tahap validasi, tahap uji coba, dan perbanyakan cetakan buklet. Instrumen penelitian berupa lembar validasi dan kuesioner. Validasi dinilai oleh ahli material, ahli desain. Uji praktis oleh respon siswa dan wawancara guru dalam menilai penampilan fisik dan keseluruhan isi buklet. Penilaian validasi yang diperoleh sebesar 88,6%, 90,4% “sangat valid” masing-masing oleh ahli desain dan ahli material. Kepraktisan produk berdasarkan respon siswa dan guru memperoleh hasil 96,7%, dan keefektifan produk memperoleh hasil 94% dengan kategori “sangat baik”. Data ini menunjukkan keanekaragaman hayati di Indonesia adalah kekuatan yang dapat dikembangkan untuk menghadapi peluang dan tantangan dalam pendidikan.

Neprosin belongs to a new family of glutamic peptidase based on in silico evidence

Neprosin was first discovered in the insectivorous tropical pitcher plants of Nepenthes species as a novel protease with prolyl endopeptidase (PEP) activity. Neprosin has two uncharacterized domains of neprosin activation peptide and neprosin. A previous study has shown neprosin activity in hydrolyzing proline-rich gliadin, a gluten component that triggers celiac disease. In this study, we performed in silico structure-function analysis to investigate the catalytic mechanism of neprosin. Neprosin sequences lack the catalytic triad and motifs of PEP family S9. Protein structures of neprosins from Nepenthes × ventrata (NvNpr) and N. rafflesiana (NrNpr1) were generated by ab initio methods and comparatively assessed to obtain high-quality models. Structural alignment of models to experimental structures in the Protein Data Bank (PDB) found a high structural similarity to glutamic peptidases. Further investigations reveal other resemblances to the glutamic peptidases with low optimum pH that activates the enzyme via autoproteolysis for maturation. Two highly conserved glutamic acid residues, which are stable according to the molecular dynamics simulation, can be found at the active site of the substrate cleft. Protein docking demonstrated that mature neprosins bind well with potent antigen αI-gliadin at the putative active site. Taken together, neprosins represent a new glutamic peptidase family, with a putative catalytic dyad of two glutamic acids. This study illustrates a hypothetical enzymatic mechanism of the neprosin family and demonstrates the useful application of an accurate ab initio protein structure prediction in the structure-function study of a novel protein family.

Alternative oxidase (AOX) in the carnivorous pitcher plants of the genus Nepenthes: what is it good for?

The carnivorous pitcher plants of the genus Nepenthes have evolved modified leaves that act as pitcher traps. The traps are specialized for prey attraction, capture, digestion and nutrient uptake but not for photosynthetic assimilation. In this study, we used antibodies against different photosynthetic (D1, Lhcb2, Lhcb4, RbcL) and respiratory-related (AOX, COXII) proteins for semi-quantification of these proteins in the assimilation part of the leaves and the pitcher traps of different Nepenthes species and hybrids. Different functional zones of the trap and the traps from different ontogenetic stages were investigated. The pitcher traps of the distantly related species Sarracenia purpurea ssp. venosa were used as an outgroup. In addition, chlorophyll fluorescence and infrared gas analysis were used for measurements of the net rate of photosynthesis (AN) and respiration in the dark (RD). The pitcher traps contained the same or lower abundance of photosynthesis-related proteins in accordance with their low AN in comparison to the assimilation part of the leaves. Surprisingly, all traps contained a high amount of alternative oxidase (AOX) and low amount of cytochrome c oxidase subunit II (COX II) than in the assimilation part of the leaves. Thermal imaging did not confirm the role of AOX in pitcher thermogenesis. The pitcher traps contain a high amount of AOX enzyme. The possible role of AOX in specialized pitcher tissue is discussed based on knowledge of the role and function of AOX in non-carnivorous plants. The roles of AOX in prey attraction, balance between light and dark reactions of photosynthesis, homeostasis of reactive oxygen species, digestive physiology and nutrient assimilation are discussed.

Predicting habitat suitability of critically endangered Nepenthes sumatrana

Nepenthes sumatrana is a tropical pitcher plant endemic to Sumatra. It has been categorised as critically endangered based on the IUCN Red List. It often occupies in lowland tropical forest of Sumatra. Its habitat has been threatened by land use conversion in the recent decade. Due to the high threats in the wild, the conservation efforts are necessarily required. It is not possible to assess all locations that are suitable for this species. In terms of addressing that issue, Species Distribution Modelling is considered as an alternative way to identify the regions that have the similar environmental conditions to where the species is known to occur. The occurrence records were derived from Global Biodiversity Information Facility and reliable scientific papers. Topography, soil, climate and land cover were used as predictors of the model. MaxEnt algorithm was used to predict the habitat suitability. MaxEnt produces a predictive model with the AUC value of 0.833 indicating that the model has a good performance. The suitable habitats are predicted in several different locations in Sumatra. The information about predicted suitability habitat would be useful to prioritize the protected areas of this species. Furthermore, our findings would be valuable for the discovery of unknown subpopulation.

Comparative leaf anatomy of ten Nepenthes L. species (Nepenthaceae) from Peninsular Malaysia

The genus Nepenthes (Nepenthaceae) in Peninsular Malaysia includes fourteen species that can be found from the sea-level to over 1000 m in the mountains. Our observation indicated that Nepenthes can be found in five major habitat types: specifically in tropical lowland evergreen rain forest, heath forest, peat swamp forest, montane forest, and limestone forest. Their leaves have several anatomical characters that remain underexplored. There were specific differences between species that could be potential identification characters. The following anatomical characteristics were explored for their diagnostic value and comprised of ten distinct anatomical characteristics in Nepenthes, viz., density and stomatal index (SI), hypodermis cell – cell layers, occurrence of fiber groups mixed with the hypodermis cells, adaxial cuticle thickness, vascular bundle arrangement, midrib outline shape, venation plasticity, druses appearance and appendage/trichome type.

Protease activity is maintained in Nepenthes ampullaria digestive fluids depleted of endogenous proteins with compositional changes.

Nepenthes ampullaria is a unique carnivorous tropical pitcher plant with the detritivorous capability of sequestering nutrients from leaf litter apart from being insectivorous. The changes in the protein composition and protease activity of its pitcher fluids during the early opening of pitchers (D0 and D3C) were investigated via a proteomics approach and a controlled protein depletion experiment (D3L). A total of 193 proteins were identified. Common proteins such as pathogenesis-related protein, proteases (Nep [EC:3.4.23.12], SCP [EC:3.4.16.-]), peroxidase [EC:1.11.1.7], GDSL esterase/lipase [EC:3.1.1.-], and purple acid phosphatase [EC:3.1.3.2] were found in high abundance in the D0 pitchers and were replenished in D3L samples. This reflects their importance for biological processes upon pitcher opening. Meanwhile, prey-inducible chitinases [EC:3.2.1.14] were found in D0 but not in D3C and D3L samples, which suggests their degradation in the absence of prey. Protease activity assays demonstrated the replenishment of proteases in D3L with similar levels of proteolytic activities to that of D3C samples. This supports a feedback mechanism and signaling in the molecular regulation of endogenous protein secretion, turnover, and activity in Nepenthes pitcher fluids. Furthermore, we also discovered several new enzymes (XTH [EC:2.4.1.207], PAE [EC:3.1.1.98]) with possible functions in cell wall degradation that could contribute to the detritivory habit of N. ampullaria.