Carnivorous syndrome in Asian pitcher plants of the genus Nepenthes

BACKGROUND AND AIMS: Pitcher plants Nepenthes alata and N. mirabilis are carnivorous species with leaves composed of a photosynthetic part (lamina) and a pitcher trap. This characteristic permitted direct physiological and anatomical comparison between these two distinct parts of the leaves to determine those features involved in the 'carnivorous syndrome', which include low net photosynthetic assimilation rate (A(N)) and low photosynthetic nitrogen use efficiency (PNUE). METHODS: Photosynthetic rate (A(N)) and respiration rate (R(d)) were measured gasometrically, chlorophyll concentration was determined spectrophotometrically and nitrogen concentration was determined using a CHN elemental analyser in lamina and trap separately. Anatomy of N. alata was observed using light, fluorescence and transmission electron microscopy. A(N), foliar nitrogen and chlorophyll concentration were also compared with values for other carnivorous plant species (genera Sarracenia, Drosera) that combine both autotrophic and carnivorous functions into the same physical organ. KEY RESULTS: It was found that the A(N) in Nepenthes lamina was low and PNUE was only slightly higher or similar in comparison with other carnivorous plants. It was not observed that the pitcher had a higher R(d) than the lamina, but A(N) in the pitcher was significantly lower than in the lamina. Nepenthes possesses a cluster of characters that could result in reduced photosynthesis in the pitcher and be responsible for carnivorous function of the leaf: replacement of chlorophyll-containing cells with digestive glands, low chlorophyll and nitrogen concentration, compact mesophyll with a small portion of intercellular spaces, absence of palisade parenchyma and low stomatal density. CONCLUSION: Low photosynthetic capacity, nitrogen efficiency, chlorophyll and nitrogen concentration of Nepenthes pitchers was found, together with a set of features that characterized the carnivorous syndrome. Dual use of leaves for photosynthesis and nutrient gain can decrease photosynthetic efficiency in carnivorous plants in general.     

Longevity, lignin content and construction cost of the assimilatory organs of Nepenthes species

Background and Aims

This study examined level of causal relationships amongst functional traits in leaves and conjoint pitcher cups of the carnivorous Nepenthes species.

Methods

Physico-chemical properties, especially lignin content, construction costs, and longevity of the assimilatory organs (leaf and pitcher) of a guild of lowland Nepenthes species inhabiting heath and/or peat swamp forests of Brunei, northern Borneo were determined.

Key Results

Longevity of these assimilatory organs was linked significantly to construction cost, lignin content and structural trait of tissue density, but these effects are non-additive. Nitrogen and phosphorus contents (indicators of Rubisco and other photosynthetic proteins), were poor predictors of organ longevity and construction cost, suggesting that a substantial allocation of biomass of the assimilatory organs in Nepenethes is to structural material optimized for prey capture, rigidity and escape from biotic and abiotic stresses rather than to light interception. Leaf payback time – a measure of net carbon revenue – was estimated to be 48–60 d. This is in line with the onset of substantial mortality by 2–3 months of tagged leaves in many of the Nepenthes species examined. However, this is a high ratio (i.e. a longer minimum payback time) compared with what is known for terrestrial, non-carnivorous plants in general (5–30 d).

Conclusions

It is concluded that the leaf trait bivariate relationships within the Nepenthes genus, as in other carnivorous species (e.g. Sarraceniaceae), is substantially different from the global relationship documented in the Global Plant Trait Network.Key words: Botanical carnivory, carbon gain, functional traits, leaf chemistry, leaf lifespan, leaf mass per unit area, Nepenthes, pitcher, payback time     

Proteomic analysis of secreted protein induced by a component of prey in pitcher fluid of the carnivorous plant Nepenthes alata

The Nepenthes species are carnivorous plants that have evolved a specialized leaf organ, the 'pitcher', to attract, capture, and digest insects. The digested insects provide nutrients for growth, allowing these plants to grow even in poor soil. Several proteins have been identified in the pitcher fluid, including aspartic proteases (nepenthesin I and II) and pathogenesis-related (PR) proteins (β-1,3-glucanase, class IV chitinase, and thaumatin-like protein). In this study, we collected and concentrated pitcher fluid to identify minor proteins. In addition, we tried to identify the protein secreted in response to trapping the insect. To make a similar situation in which the insect falls into the pitcher, chitin which was a major component of the insect exoskeleton was added to the fluid in the pitcher. Three PR proteins, class III peroxidase (Prx), β-1,3-glucanase, and class III chitinase, were newly identified. Prx was induced after the addition of chitin to the pitcher fluid. Proteins in the pitcher fluid of the carnivorous plant Nepenthes alata probably have two roles in nutrient supply: digestion of prey and the antibacterial effect. These results suggest that the system for digesting prey has evolved from the defense system against pathogens in the carnivorous plant Nepenthes.     

A unique resource mutualism between the giant Bornean pitcher plant, Nepenthes rajah, and members of a small mammal community

The carnivorous pitcher plant genus Nepenthes grows in nutrient-deficient substrates and produce jug-shaped leaf organs (pitchers) that trap arthropods as a source of N and P. A number of Bornean Nepenthes demonstrate novel nutrient acquisition strategies. Notably, three giant montane species are engaged in a mutualistic association with the mountain treeshrew, Tupaia montana, in which the treeshrew defecates into the pitchers while visiting them to feed on nectar secretions on the pitchers' lids.Although the basis of this resource mutualism has been elucidated, many aspects are yet to be investigated. We sought to provide insights into the value of the mutualism to each participant. During initial observations we discovered that the summit rat, R. baluensis, also feeds on sugary exudates of N. rajah pitchers and defecates into them, and that this behavior appears to be habitual. The scope of the study was therefore expanded to assess to what degree N. rajah interacts with the small mammal community.We found that both T. montana and R. baluensis are engaged in a mutualistic interaction with N. rajah. T .montana visit pitchers more frequently than R. baluensis, but daily scat deposition rates within pitchers do not differ, suggesting that the mutualistic relationships are of a similar strength. This study is the first to demonstrate that a mutualism exists between a carnivorous plant species and multiple members of a small mammal community. Further, the newly discovered mutualism between R. baluensis and N. rajah represents only the second ever example of a multidirectional resource-based mutualism between a mammal and a carnivorous plant.     

Nutritional benefit from leaf litter utilization in the pitcher plant Nepenthes ampullaria

The pitcher plant Nepenthes ampullaria has an unusual growth pattern, which differs markedly from other species in the carnivorous genus Nepenthes. Its pitchers have a reflexed lid and sit above the soil surface in a tighly packed 'carpet'. They contain a significant amount of plant-derived materials, suggesting that this species is partially herbivorous. We tested the hypothesis that the plant benefits from leaf litter utilization by increased photosynthetic efficiency sensu stricto cost/benefit model. Stable nitrogen isotope abundance indicated that N. ampullaria derived around 41.7 ± 5.5% of lamina and 54.8 ± 7.0% of pitcher nitrogen from leaf litter. The concentrations of nitrogen and assimilation pigments, and the rate of net photosynthesis (A(N)), increased in the lamina as a result of feeding, but did not increase in the trap. However, maximal (F(v) /F(m)) and effective photochemical quantum yield of photosystem II (Φ(PSII)) were unaffected. Our data indicate that N. ampullaria benefits from leaf litter utilization and our study provides the first experimental evidence that the unique nitrogen sequestration strategy of N. ampullaria provides benefits in term of photosynthesis and growth.     

The carnivorous syndrome in Nepenthes pitcher plants: Current state of knowledge and potential future directions

Nepenthes is the largest genus of pitcher plants, with its center of diversity in SE Asia. The plants grow in substrates that are deficient in N and offset this deficiency by trapping animal prey, primarily arthropods. Recent research has provided new insights into the function of the pitchers, particularly with regard to prey tapping and retention. Species examined to date use combinations of wettable peristomes, wax layers and viscoelastic fluid to trap and retain prey. In many respects, this has redefined our understanding of the functioning of Nepenthes pitchers. In addition, recent research has shown that several Nepenthes species target specific groups of prey animals, or are even evolving away from a strictly carnivorous mode of operation. Future research into nutrient sequestration strategies and mechanisms of prey attraction would no doubt further enhance our knowledge of the ecology of this remarkable genus.Key words: carnivory, mutualism, Nepenthes, pitcher plants     

Tuning of color contrast signals to visual sensitivity maxima of tree shrews by three Bornean highland Nepenthes species

Three species of Nepenthes pitcher plants (Nepenthes rajah, Nepenthes lowii and Nepenthes macrophylla) specialize in harvesting nutrients from tree shrew excreta in their pitchers. In all three species, nectaries on the underside of the pitcher lid are the focus of the tree shrews' attention. Tree shrews are dichromats, with visual sensitivity in the blue and green wavebands. All three Nepenthes species were shown to produce visual signals, in which the underside of the pitcher lid (the area of highest nectar production) stood out in high contrast to the adjacent area on the pitcher (i.e., was brighter), in the blue and green wavebands visible to the tree shrews. N. rajah showed the tightest degree of “tuning,” notably in the green waveband. Conversely, pitchers of Nepenthes burbidgeae, a typical insectivorous species sympatric with N. rajah, did not produce a color pattern tuned to tree shrew sensitivity maxima.     

温带针阔混交林叶片性状随树冠垂直高度的变化规律

自然界中,森林植物叶片的生长随树冠高度呈现明显的垂直分布现象;然而,有关叶片性状随着树冠垂直高度增加的变化规律仍不清楚。为了更好地揭示植物叶片对光环境变化的适应策略以及对资源的利用能力,有必要深入探讨叶片性状与冠层高度的定量关系及其内在调控机制。以中国广泛分布的温带针阔混交林为对象,选取8种主要树种为研究对象(白桦、蒙古栎、水曲柳、大青杨、色木槭、千金榆、核桃楸和红松),通过测定这些物种9个冠层高度的叶片比叶面积(SLA)、叶片干物质含量(LDMC)、叶片氮含量(N)、叶片磷含量(P)、氮磷比(N∶P)和叶绿素含量(Chl)等属性,探讨了针阔混交林叶片性状的差异以及各性状之间的相关关系,进而揭示叶片性状随树冠垂直高度的变化规律。实验结果表明:1)温带针阔混交林内优势树种的部分叶片性状在不同冠层高度之间差异显著。2)随着树冠垂直高度的增加,SLA、LDMC、N、P、N∶P和Chl呈现不同的变化趋势。其中,阔叶树种SLA随着树冠垂直高度的增加而减小;所有树种的LDMC随着树冠垂直高度的增加而增加;不同树种的N、P、N∶P和Chl随着树冠垂直高度的变化规律存在差异。3)对于温带针阔混交林冠层中,SLA与N、P、N∶P均存在显著的正相关关系,高SLA伴随着高的N、P、N∶P,表明植物通过SLA与N、P等性状的协同来提高叶片的光合作用(或对光热资源的利用效率)。本研究通过定量分析探讨温带针阔混交林叶片性状随冠层高度的变化规律,一定程度地揭示了树木对光、热和水资源竞争的适应机制,以及植物叶片的资源利用和分配策略,不仅拓展了传统性状研究的范畴,其相关研究结论也有助于树木生长模型的构建和优化。     

Slippery or sticky? Functional diversity in the trapping strategy of Nepenthes carnivorous plants

The pitcher-shaped leaves of Nepenthes carnivorous plants have been considered as pitfall traps that essentially rely on slippery surfaces to capture insects. But a recent study of Nepenthes rafflesiana has shown that the viscoelasticity of the digestive fluid inside the pitchers plays a key role. Here, we investigated whether Nepenthes species exhibit diverse trapping strategies. We measured the amount of slippery wax on the pitcher walls of 23 taxa and the viscoelasticity of their digestive liquid and compared their retention efficiency on ants and flies. The amount of wax was shown to vary greatly between species. Most mountain species exhibited viscoelastic digestive fluids while water-like fluids were predominant in lowland species. Both characteristics contributed to insect trapping but wax was more efficient at trapping ants while viscoelasticity was key in trapping insects and was even more efficient than wax on flies. Trap waxiness and fluid viscoelasticity were inversely related, suggesting the possibility of an investment trade-off for the plants. Therefore Nepenthes pitcher plants do not solely employ slippery devices to trap insects but often employ a viscoelastic strategy. The entomofauna specific to the plant's habitat may exert selective pressures, favouring one trapping strategy at the expense of the other.     

Transporters for ammonium, amino acids and peptides are expressed in pitchers of the carnivorous plant Nepenthes

Insect capture and digestion contribute substantially to the nitrogen budget of carnivorous plants. In Nepenthes, insect-derived nitrogenous compounds are imported from the pitcher fluid and transported throughout the plant via the vascular tissue to support growth. Import and distribution of nutrients may require transmembrane nitrogen transporters. Representatives of three classes of genes encoding transporters for the nitrogenous compounds ammonium, amino acids and peptides were identified in Nepenthes pitchers. The expression at the cellular level of an ammonium transporter gene, three amino acid transporter genes, and one peptide transporter gene were investigated in the insect trapping organs of Nepenthes. Expression of the ammonium transporter gene NaAMT1 was detected in the head cells of digestive glands in the lower part of the pitcher where NaAMT1 may function in ammonium uptake from the pitcher fluid. One amino acid transporter gene, NaAAP1, was expressed in bundle sheath cells surrounding the vascular tissue. To understand the locations where transmembrane transport could be required within the pitcher, symplasmic and apoplasmic continuity was probed using fluorescent dyes. Symplasmic connections were not found between cortical cells and vascular bundles. Therefore, the amino acid transporter encoded by NaAAP1 may be involved in transport of amino acids into the vascular tissue. In contrast, expression of the peptide transporter gene NaNTR1 was detected in phloem cells of the vascular tissue within pitchers. NaNTR1 may function in the export of nitrogen from the pitcher by loading peptides into the phloem.     

Distribution of naphthoquinones, plumbagin, droserone, and 5-O-methyl droserone in chitin-induced and uninduced Nepenthes khasiana: molecular events in prey capture

Prey capture and digestion in Nepenthes spp. through their leaf-evolved biological traps involve a sequence of exciting events. Sugar-rich nectar, aroma chemicals, narcotic alkaloid secretions, slippery wax crystals, and other biochemicals take part in attracting, capturing, and digesting preys in Nepenthes pitchers. Here we report the distribution of three potent naphthoquinones in Nepenthes khasiana and their roles in prey capture. Plumbagin was first detected in N. khasiana, and its content (root: 1.33 ± 0.02%, dry wt.) was the highest found in any natural source. Chitin induction enhanced plumbagin levels in N. khasiana (root: 2.17 ± 0.02%, dry wt.). Potted N. khasiana plants with limited growth of roots and aerial parts, showed higher levels of plumbagin accumulation (root: 1.92 ± 0.02%; root, chitin induction: 3.30 ± 0.21%, dry wt.) compared with field plants. Plumbagin, a known toxin, insect ecdysis inhibitor, and antimicrobial, was also found embedded in the waxy layers at the top prey capture region of N. khasiana pitchers. Chitin induction, mimicking prey capture, produced droserone and 5-O-methyl droserone in N. khasiana pitcher fluid. Both these naphthoquinone derivatives provide antimicrobial protection to the pitcher fluid from visiting preys. A two-way barrier was found between plumbagin and its two derivatives. Plumbagin was never detected in the pitcher fluid whereas both its derivatives were only found in the pitcher fluid on chitin induction or prey capture. The three naphthoquinones, plumbagin, droserone, and 5-O-methyl droserone, act as molecular triggers in prey capture and digestion in the carnivorous plant, N. khasiana.     

Degradation of a peptide in pitcher fluid of the carnivorous plant Nepenthes alata Blanco

Carnivorous plants acquire substantial amounts of nitrogen from insects. The tropical carnivorous plant Nepenthes produces trapping organs called pitchers at the tips of tendrils elongated from leaf ends. Acidic fluid is secreted at the bottoms of the pitchers. The pitcher fluid includes several hydrolytic enzymes, and some, such as aspartic proteinase, are thought to be involved in nitrogen acquisition from insect proteins. To understand the nitrogen-acquisition process, it is essential to identify the protein-degradation products in the pitcher fluid. To gain insight into protein degradation in pitcher fluid, we used the oxidized B-chain of bovine insulin as a model substrate, and its degradation by the pitcher fluid of N. alata was investigated using liquid chromatography-mass spectrometry (LC-MS). LC-MS analysis of the degradation products revealed that the oxidized B-chain of bovine insulin was initially cleaved at aromatic amino acids such as phenylalanine and tyrosine. These cleavage sites are similar to those of aspartic proteinases from other plants and animals. The presence of a series of peptide fragments as degradation products suggests that exopeptidase(s) is also present in the pitcher fluid. Amino acid analysis and peptide fragment analysis of the degradation products demonstrated that three amino acids plus small peptides were released from the oxidized B-chain of bovine insulin, suggesting that insect proteins are readily degraded to small peptides and amino acids in the pitcher fluid of N. alata.     

The cost of carnivory for Darlingtonia californica (Sarraceniaceae): evidence from relationships among leaf traits

Scaling relationships among photosynthetic rate, foliar nutrient concentration, and leaf mass per unit area (LMA) have been observed for a broad range of plants. Leaf traits of the carnivorous pitcher plant Darlingtonia californica, endemic to southern Oregon and northern California, USA, differ substantially from the predictions of these general scaling relationships; net photosynthetic rates of Darlingtonia are much lower than predicted by general scaling relationships given observed foliar nitrogen (N) and phosphorus (P) concentrations and LMA. At five sites in the center of its range, leaf traits of Darlingtonia were strongly correlated with elevation and differed with soil calcium availability and bedrock type. The mean foliar N : P of 25.2 ± 15.4 of Darlingtonia suggested that these plants were P-limited, although N concentration in the substrate also was extremely low and prey capture was uncommon. Foliar N : P stoichiometry and the observed deviation of Darlingtonia leaf traits from predictions of general scaling relationships permit an initial assessment of the "cost of carnivory" in this species. Carnivory in plants is thought to have evolved in response to N limitation, but for Darlingtonia, carnivory is an evolutionary last resort when both N and P are severely limiting and photosynthesis is greatly reduced.     

Contribution of pitcher fragrance and fluid viscosity to high prey diversity in a Nepenthes carnivorous plant from borneo

Mechanisms that improve prey richness in carnivorous plants may involve three crucial phases of trapping:attraction, capture and retention.Nepenthes rafflesiana var. typica is an insectivorous pitcher plant that is widespread in northern Borneo.It exhibits ontogenetic pitcher dimorphism with the upper pitchers trapping more flying prey than the lower pitchers.While this difference in prey composition has been ascribed to differences in attraction,the contribution of capture and retention has been overlooked.This study focused on distinguishing between the prey trapping mechanisms, and assessing their relative contribution to prey diversity.Arthropod richness and diversity of both visitors and prey in the two types of pitchers were analysed to quantify the relative contribution of attraction to prey trapping.Rate of insect visits to the different pitcher parts and the presence or absence of a sweet fragrance was recorded to clarify the origin and mechanism of attraction.The mechanism of retention was studied by insect bioassays and measurements of fluid viscosity. Nepenthes rafflesiana was found to trap a broader prey spectrum than that previously described for any Nepenthes species,with the upper pitchers attracting and trapping a greater quantity and diversity of prey items than the lower pitchers.Capture efficiency was low compared with attraction or retention efficiency.Fragrance of the peristome,or nectar rim,accounted mainly for the observed non-specific, better prey attraction by the upper pitchers, while the retentive properties of the viscous fluid in these upper pitchers arguably explains the species richness of their flying prey.The pitchers of N. rafflesiana are therefore more than simple pitfall traps and the digestive fluid plays an important yet unsuspected role in the ecological success of the species.     

In situ enzyme activity in the dissolved and particulate fraction of the fluid from four pitcher plant species of the genus Nepenthes

The genus Nepenthes, a carnivorous plant, has a pitcher to trap insects and digest them in the contained fluid to gain nutrient. A distinctive character of the pitcher fluid is the digestive enzyme activity that may be derived from plants and dwelling microbes. However, little is known about in situ digestive enzymes in the fluid. Here we examined the pitcher fluid from four species of Nepenthes. High bacterial density was observed within the fluids, ranging from 7×10(6) to 2.2×10(8) cells ml(-1). We measured the activity of three common enzymes in the fluid: acid phosphatases, β-D-glucosidases, and β-D-glucosaminidases. All the tested enzymes detected in the liquid of all the pitcher species showed activity that considerably exceeded that observed in aquatic environments such as freshwater, seawater, and sediment. Our results indicate that high enzyme activity within a pitcher could assist in the rapid decomposition of prey to maximize efficient nutrient use. In addition, we filtered the fluid to distinguish between dissolved enzyme activity and particle-bound activity. As a result, filtration treatment significantly decreased the activity in all enzymes, while pH value and Nepenthes species did not affect the enzyme activity. It suggested that enzymes bound to bacteria and other organic particles also would significantly contribute to the total enzyme activity of the fluid. Since organic particles are themselves usually colonized by attached and highly active bacteria, it is possible that microbe-derived enzymes also play an important role in nutrient recycling within the fluid and affect the metabolism of the Nepenthes pitcher plant.     

A novel resource-service mutualism between bats and pitcher plants

Mutualistic relationships between vertebrates and plants apart from the pollen and seed-dispersal syndromes are rare. At first view, carnivorous pitcher plants of the genus Nepenthes seem to be highly unlikely candidates for mutualistic interactions with animals, as they form dimorphic terrestrial and aerial pitchers that trap arthropods and small vertebrates. Surprisingly, however, the aerial pitchers of Nepenthes rafflesiana variety elongata are poor insect traps, with low amounts of insect-attractive volatile compounds and low amounts of digestive fluid. Here, we show that N. rafflesiana elongata gains an estimated 33.8 per cent of the total foliar nitrogen from the faeces of Hardwicke's woolly bats (Kerivoula hardwickii hardwickii) that exclusively roost in its aerial pitchers. This is the first case in which the faeces-trapping syndrome has been documented in a pitcher plant that attracts bats and only the second case of a mutualistic association between a carnivorous plant and a mammal to date.     

Spatial variation in leaf nutrient traits of dominant desert riparian plant species in an arid inland river basin of China

Understanding how patterns of leaf nutrient traits respond to groundwater depth is crucial for modeling the nutrient cycling of desert riparian ecosystems and forecasting the responses of ecosystems to global changes. In this study, we measured leaf nutrients along a transect across a groundwater depth gradient in the downstream Heihe River to explore the response of leaf nutrient traits to groundwater depth and soil properties. We found that leaf nutrient traits of dominant species showed different responses to groundwater depth gradient. Leaf C, leaf N, leaf P, and leaf K decreased significantly with groundwater depth, whereas patterns of leaf C/N and leaf N/P followed quadratic relationships with groundwater depth. Meanwhile, leaf C/P did not vary significantly along the groundwater depth gradient. Variations in leaf nutrient traits were associated with soil properties (e.g., soil bulk density, soil pH). Groundwater depth and soil pH jointly regulated the variation of leaf nutrient traits; however, groundwater depth explained the variation of leaf nutrient traits better than did soil pH. At the local scale in the typical desert riparian ecosystem, the dominant species was characterized by low leaf C, leaf N, and leaf P, but high leaf N/P and leaf C/P, indicating that desert riparian plants might be more limited by P than N in the growing season. Our observations will help to reveal specific adaptation patterns in relation to the groundwater depth gradient for dominant desert riparian species, provide insights into adaptive trends of leaf nutrient traits, and add information relevant to understanding the adaptive strategies of desert riparian forest vegetation to moisture gradients.     

Proteome analysis of pitcher fluid of the carnivorous plant Nepenthes alata

The genus Nepenthes comprises carnivorous plants that digest insects in pitcher fluid to supplement their nitrogen uptake. In a recent study, two acid proteinases (nepenthesins I and II) were purified from the pitcher fluid. However, no other enzymes involved in prey digestion have been identified, although several enzyme activities have been reported. To identify all the proteins involved, we performed a proteomic analysis of Nepenthes pitcher fluid. The secreted proteins in pitcher fluid were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and several protein bands were detected by silver staining. The proteins were identified by in-gel tryptic digestion, de novo peptide sequencing, and homology searches against public databases. The proteins included homologues of beta-D-xylosidase, beta-1,3-glucanase, chitinase, and thaumatin-like protein, most of which are designated "pathogenesis-related proteins". These proteins presumably inhibit bacterial growth in the pitcher fluid to ensure sufficient nutrients for Nepenthes growth.