Cytochemical and ultrastructural aspects of aquatic carnivorous plant turions

Turions, which are modified shoot apices, are vegetative, dormant overwintering organs produced by perennial aquatic plants. In this study, the turion cytochemistry and ultrastructure of Aldrovanda vesiculosa, Utricularia vulgaris and U. stygia were compared with particular emphasis placed on storage substances. These three aquatic, rootless carnivorous plant species were studied at the end of their winter dormancy. At this stage, the turions of all species had starch as their main storage material. In contrast with A. vesiculosa, Utricularia turions were rich in protein storage vacuoles, and proteins were also accumulated as crystalline inclusions in the nuclei. All examined species accumulated lipid droplets in cells of epidermal glands.     

Rootless Aquatic Plant Aldrovanda Vesiculosa: Physiological Polarity, Mineral Nutrition, and Importance of Carnivory

Various ecophysiological investigations are presented in Aldrovanda vesiculosa, a rootless aquatic carnivorous plant. A distinct polarity of N, P, and Ca tissue content per dry mass (DM) unit was found along Aldrovanda shoots. Due to effective re-utilization, relatively small proportions of N (10 – 13 %) and P (33 – 43 %) are probably lost with senescent leaf whorls, while there is complete loss of all Ca, K, and Mg. The total content of starch and free sugars was 26 – 47 % DM along adult shoots, with the maximum in the 7^th – 10^th whorls. About 30 % of the total maximum sugar content was probably lost with dead whorls. The plant was found to take up 5 – 7 times more NH₄ ⁺ to NO₃ ^– from a mineral medium. Under nearly-natural conditions in an outdoor cultivation container, catching of prey led to significantly more rapid growth than in unfed plants. DM of the fed controls was 48 % higher than in the unfed plants. The controls produced 0.69 branches per plant, while the unfed plants did not produced any. However, the N and P content per DM unit increased by 6 – 25 % in the apices and the first 6 whorls in the unfed variant, as compared to the fed controls. It may be suggested that carnivory is very important for Aldrovanda.     

Feeding on prey increases photosynthetic efficiency in the carnivorous sundew Drosera capensis

Backround and Aims

It has been suggested that the rate of net photosynthesis (A_N) of carnivorous plants increases in response to prey capture and nutrient uptake; however, data confirming the benefit from carnivory in terms of increased A_N are scarce and unclear. The principal aim of our study was to investigate the photosynthetic benefit from prey capture in the carnivorous sundew Drosera capensis.

Methods

Prey attraction experiments were performed, with measurements and visualization of enzyme activities, elemental analysis and pigment quantification together with simultaneous measurements of gas exchange and chlorophyll a fluorescence in D. capensis in response to feeding with fruit flies (Drosophila melanogaster).

Key Results

Red coloration of tentacles did not act as a signal to attract fruit flies onto the traps. Phosphatase, phophodiesterase and protease activities were induced 24 h after prey capture. These activities are consistent with the depletion of phosphorus and nitrogen from digested prey and a significant increase in their content in leaf tissue after 10 weeks. Mechanical stimulation of tentacle glands alone was not sufficient to induce proteolytic activity. Activities of β-D-glucosidases and N-acetyl-β-D-glucosaminidases in the tentacle mucilage were not detected. The uptake of phosphorus from prey was more efficient than that of nitrogen and caused the foliar N:P ratio to decrease; the contents of other elements (K, Ca, Mg) decreased slightly in fed plants. Increased foliar N and P contents resulted in a significant increase in the aboveground plant biomass, the number of leaves and chlorophyll content as well as A_N, maximum quantum yield (F_v/F_m) and effective photochemical quantum yield of photosystem II (Φ_PSII).

Conclusions

According to the stoichiometric relationships among different nutrients, the growth of unfed D. capensis plants was P-limited. This P-limitation was markedly alleviated by feeding on fruit flies and resulted in improved plant nutrient status and photosynthetic performance. This study supports the original cost/benefit model proposed by T. Givnish almost 30 years ago and underlines the importance of plant carnivory for increasing phosphorus, and thereby photosynthesis.     

Recent ecophysiological,biochemical and evolutional insights into plant carnivory

BackgroundCarnivorous plants are an ecological group of approx. 810 vascular species which capture and digest animal prey, absorb prey-derived nutrients and utilize them to enhance their growth and development. Extant carnivorous plants have evolved in at least ten independent lineages, and their adaptive traits represent an example of structural and functional convergence. Plant carnivory is a result of complex adaptations to mostly nutrient-poor, wet and sunny habitats when the benefits of carnivory exceed the costs. With a boost in interest and extensive research in recent years, many aspects of these adaptations have been clarified (at least partly), but many remain unknown.ScopeWe provide some of the most recent insights into substantial ecophysiological, biochemical and evolutional particulars of plant carnivory from the functional viewpoint. We focus on those processes and traits in carnivorous plants associated with their ecological characterization, mineral nutrition, cost–benefit relationships, functioning of digestive enzymes and regulation of the hunting cycle in traps. We elucidate mechanisms by which uptake of prey-derived nutrients leads to stimulation of photosynthesis and root nutrient uptake.ConclusionsUtilization of prey-derived mineral (mainly N and P) and organic nutrients is highly beneficial for plants and increases the photosynthetic rate in leaves as a prerequisite for faster plant growth. Whole-genome and tandem gene duplications brought gene material for diversification into carnivorous functions and enabled recruitment of defence-related genes. Possible mechanisms for the evolution of digestive enzymes are summarized, and a comprehensive picture on the biochemistry and regulation of prey decomposition and prey-derived nutrient uptake is provided.     

Field growth characteristics of two aquatic carnivorous plants,Aldrovanda vesiculosa andUtricularia australis

Basic growth characteristics of two species of free-floating submerged carnivorous plants, the very rare and stenotopicAldrovanda vesiculosa and the very common and eurytopicUtricularia australis, were investigated in a 10/11-day field growth experiment within three nylon enclosures at two artificialAldrovanda sites in the T?eboň region, S Bohemia, Czech Republic, at the peak of a growing season. Growth ofAldrovanda was best at a meso-eutrophic site (biomass doubling time,T ₂, 8.4–10.7 days, mean growth of new leaf whorls 0.96 whorls days^?1, 1.6 developed branches per shoot) and slower at an oligo-mesotrophic site (T ₂ 17.2–21.5 days, growth of whorls 1.01 whorls days^?1, 0.1–0.5 branches per shoot). Growth ofUtricularia was similar at both sites (T ₂ 19.8–33.2 days or 9.1–16.8 days, growth of whorls 3.1 or 2.7 whorls days^?1, 1.5–2.1 or 0.8–1.4 developed branches per shoot at the former or latter site, respectively). Throughout the experiment, both species at the meso-eutrophic site allocated relatively more biomass to the production and growth of branches, than to that of new whorls. The results show thatAldrovanda, although usually considered as competitively weaker, can grow faster during the growing season peak thanUtricularia due to frequent branching and the subsequent rapid growth and separation of daughter shoots. Very rapid growth of rootless aquatic carnivorous plants in nutrient-poor habitats allows the consideration of ecophysiological adaptations that enable the plants to gain limiting mineral nutrients. These adaptations include carnivory, efficient nutrient reutilization from senescent shoots, and very high affinity for mineral nutrient uptake from water. Comparison of growth rates of rare and stenotopicA. vesiculosa and very common and eurytopicU. australis shows that differences in their rarity do not seem to be based on differences of growth rate.     

Respiration and photosynthesis of bladders and leaves of aquatic utricularia species

In aquatic species of carnivorous utricularia, about 10 - 50 % of the total biomass consists of bladders. Utricularia bladders are physiologically very active organs though their chlorophyll content may greatly be reduced. To specify energetic costs of carnivory, respiration (RD) and net photosynthetic rate (PN) were compared in bladders and leaves or shoot segments of six aquatic utricularia species with differentiated (U. ochroleuca, U. intermedia, U. floridana) or non-differentiated shoots (U. vulgaris, U. australis, U. bremii) under optimum conditions (20 degrees C, [CO (2)] 0.20 mM, 400 micromol m (-2) s (-1) PAR). RD of bladders of six utricularia species (5.1 - 8.6 mmol kg (-1)(FW) h (-1)) was 75 - 200 % greater, than that in leaves in carnivorous or photosynthetic shoots (1.7 - 6.1 mmol kg (-1)(FW) h (-1)). Within individual species, this difference was statistically significant at P < 0.002 - 0.01. However, PN in photosynthetic leaves/shoots (40 - 117 mmol kg (-1)(FW) h (-1)) exceeded that in bladders (5.2 - 14.7 mmol kg (-1)(FW) h (-1)) 7 - 10 times. RD of empty bladders of U. ochroleuca was exactly the same as that in bladders containing prey. Though utricularia bladders are essential for uptake of growth limiting mineral nutrients N and P from prey as the main benefit of carnivory, the current results support previous work showing that bladder function requires greater metabolic (maintenance) cost and very low photosynthetic efficiency (great RD : PN ratio).     

Capture of algae promotes growth and propagation in aquatic Utricularia

Background and Aims Some carnivorous plants trap not only small animals but also algae and pollen grains. However, it remains unclear if these trapped particles are useless bycatch or whether they provide nutrients for the plant. The present study examines this question in Utricularia, which forms the largest and most widely spread genus of carnivorous plants, and which captures prey by means of sophisticated suction traps.Methods Utricularia plants of three different species (U. australis, U. vulgaris and U. minor) were collected in eight different water bodies including peat bogs, lakes and artificial ponds in three regions of Austria. The prey spectrum of each population was analysed qualitatively and quantitatively, and correlated with data on growth and propagation, C/N ratio and δ¹⁵N.Key Results More than 50 % of the prey of the Utricularia populations investigated consisted of algae and pollen, and U. vulgaris in particular was found to capture large amounts of gymnosperm pollen. The capture of algae and pollen grains was strongly correlated with most growth parameters, including weight, length, budding and elongation of internodes. The C/N ratio, however, was less well correlated. Other prey, such as moss leaflets, fungal hyphae and mineral particles, were negatively correlated with most growth parameters. δ¹⁵N was positively correlated with prey capture, but in situations where algae were the main prey objects it was found that the standard formula for calculation of prey-derived N was no longer applicable.Conclusions The mass capture of immotile particles confirms the ecological importance of autonomous firing of the traps. Although the C/N ratio was little influenced by algae, they clearly provide other nutrients, possibly including phosphorus and trace elements. By contrast, mosses, fungi and mineral particles appear to be useless bycatch. Correlations with chemical parameters indicate that Utricularia benefits from nutrient-rich waters by uptake of inorganic nutrients from the water, by the production of more traps per unit of shoot length, and by the capture of more prey particles per trap, as nutrient-rich waters harbour more prey organisms.     

Micronutrient content does not reflect the status of health in the aquatic carnivorous plant <Emphasis Type="Italic">Aldrovanda vesiculosa</Emphasis>

Tissue micronutrient content (B, Fe, Mn, Zn, Cu, Mo, Co) was compared in healthy and ill shoots of a rootless aquatic carnivorous plant Aldrovanda vesiculosa growing in an outdoor culture, in plants in the field, in different accessions from around the world, and in shoots and turions. On the basis of microelemental analyses, the observed disorder of shoot apices cannot be explained as deficiency of any of the above micronutrients.     

A novel insight into the cost–benefit model for the evolution of botanical carnivory

Background The cost–benefit model for the evolution of botanical carnivory provides a conceptual framework for interpreting a wide range of comparative and experimental studies on carnivorous plants. This model assumes that the modified leaves called traps represent a significant cost for the plant, and this cost is outweighed by the benefits from increased nutrient uptake from prey, in terms of enhancing the rate of photosynthesis per unit leaf mass or area (A_N) in the microsites inhabited by carnivorous plants.Scope This review summarizes results from the classical interpretation of the cost–benefit model for evolution of botanical carnivory and highlights the costs and benefits of active trapping mechanisms, including water pumping, electrical signalling and accumulation of jasmonates. Novel alternative sequestration strategies (utilization of leaf litter and faeces) in carnivorous plants are also discussed in the context of the cost–benefit model.Conclusions Traps of carnivorous plants have lower A_N than leaves, and the leaves have higher A_N after feeding. Prey digestion, water pumping and electrical signalling represent a significant carbon cost (as an increased rate of respiration, R_D) for carnivorous plants. On the other hand, jasmonate accumulation during the digestive period and reprogramming of gene expression from growth and photosynthesis to prey digestion optimizes enzyme production in comparison with constitutive secretion. This inducibility may have evolved as a cost-saving strategy beneficial for carnivorous plants. The similarities between plant defence mechanisms and botanical carnivory are highlighted.     

Mineral nutrient uptake from prey and glandular phosphatase activity as a dual test of carnivory in semi-desert plants with glandular leaves suspected of carnivory

Background and Aims

Ibicella lutea and Proboscidea parviflora are two American semi-desert species of glandular sticky plants that are suspected of carnivory as they can catch small insects. The same characteristics might also hold for two semi-desert plants with glandular sticky leaves from Israel, namely Cleome droserifolia and Hyoscyamus desertorum. The presence of proteases on foliar hairs, either secreted by the plant or commensals, detected using a simple test, has long been considered proof of carnivory. However, this test does not prove whether nutrients are really absorbed from insects by the plant. To determine the extent to which these four species are potentially carnivorous, hair secretion of phosphatases and uptake of N, P, K and Mg from fruit flies as model prey were studied in these species and in Roridula gorgonias and Drosophyllum lusitanicum for comparison. All species examined possess morphological and anatomical adaptations (hairs or emergences secreting sticky substances) to catch and kill small insects.

Methods

The presence of phosphatases on foliar hairs was tested using the enzyme-labelled fluorescence method. Dead fruit flies were applied to glandular sticky leaves of experimental plants and, after 10–15 d, mineral nutrient content in their spent carcasses was compared with initial values in intact flies after mineralization.

Key Results

Phosphatase activity was totally absent on Hyoscyamus foliar hairs, a certain level of activity was usually found in Ibicella, Proboscidea and Cleome, and a strong response was found in Drosophyllum. Roridula exhibited only epidermal activity. However, only Roridula and Drosophyllum took up nutrients (N, P, K and Mg) from applied fruit flies.

Conclusions

Digestion of prey and absorption of their nutrients are the major features of carnivory in plants. Accordingly, Roridula and Drosophyllum appeared to be fully carnivorous; by contrast, all other species examined are non-carnivorous as they did not meet the above criteria.Key words: Roridula gorgonias, Drosophyllum lusitanicum, Proboscidea parviflora, Ibicella lutea, Cleome droserifolia, Hyoscyamus desertorum, phosphatase, phosphomonoesters, fruit flies, N, P, K, Mg uptake from prey     

Uniformity of organellar DNA in Aldrovanda vesiculosa, an endangered aquatic carnivorous species, distributed across four continents

Organellar DNA from the widely distributed but rare and critically endangered aquatic carnivorous plant Aldrovanda vesiculosa (Droseraceae) was examined. Six chloroplast intergenic regions (3700 nt in total) were sequenced before analyzing the Southern-RFLP (Restriction Fragment Length Polymorphism) of 2 mt gene flanking regions. Only two different chloroplast haplotypes among 15 A. vesiculosa accessions from Africa, Australia, Europe, and Japan were found, generally distinguishing European and non-European plants, with two exceptions. Genetic variation observed in A. vesiculosa appears to be even lower than in other aquatic species with a similar world-wide distribution. A recent bottleneck followed by long-distance dispersal by water birds or low mutation rates could be responsible for the observed genetic uniformity. Estimation of genetic distances based on six chloroplast intergenic regions led to the conclusion that the chloroplast genome of A. vesiculosa matches more closely to that of Drosera regia than Dionaea muscipula, a sister genus sharing snapping traps. The inconsistency between genetic distance estimates based on nuclear and cytoplasmic markers may reflect a chloroplast capture. In A. vesiculosa, a four amino acids substitution (TGWS) in the amino acid sequence of ATP synthase alpha subunit (ATP1), highly conserved mitochondrial protein, was discovered, unique among all organisms based on current knowledge.     

Structural and functional characteristics of S-like ribonucleases from carnivorous plants

Although the S-like ribonucleases (RNases) share sequence homology with the S-RNases involved in the self-incompatibility mechanism in plants, they are not associated with this mechanism. They usually function in stress responses in non-carnivorous plants and in carnivory in carnivorous plants. In this study, we clarified the structures of the S-like RNases of Aldrovanda vesiculosa, Nepenthes bicalcarata and Sarracenia leucophylla, and compared them with those of other plants. At ten positions, amino acid residues are conserved or almost conserved only for carnivorous plants (six in total). In contrast, two positions are specific to non-carnivorous plants. A phylogenetic analysis revealed that the S-like RNases of the carnivorous plants form a group beyond the phylogenetic relationships of the plants. We also prepared and characterized recombinant S-like RNases of Dionaea muscipula, Cephalotus follicularis, A. vesiculosa, N. bicalcarata and S. leucophylla, and RNS1 of Arabidopsis thaliana. The recombinant carnivorous plant enzymes showed optimum activities at about pH 4.0. Generally, poly(C) was digested less efficiently than poly(A), poly(I) and poly(U). The kinetic parameters of the recombinant D. muscipula enzyme (DM-I) and A. thaliana enzyme RNS1 were similar. The k _cat/K _m of recombinant RNS1 was the highest among the enzymes, followed closely by that of recombinant DM-I. On the other hand, the k _cat/K _m of the recombinant S. leucophylla enzyme was the lowest, and was ~1/30 of that for recombinant RNS1. The magnitudes of the k _cat/K _m values or k _cat values for carnivorous plant S-like RNases seem to correlate negatively with the dependency on symbionts for prey digestion.     

Functional characteristics of traps of aquatic carnivorous Utricularia species

Firing and resetting of traps in aquatic Utricularia species are associated with water flow and trap volume changes. In this study, trap thickness was used as a measure of water flow and was monitored automatically using an electronic position sensor. The basic characteristics of mechanically stimulated firing and resetting were measured in isolated traps from 13 aquatic Utricularia species and in two trap size categories in Utricularia reflexa. This allowed to study the relationship between these trap functions and trap thickness and length (criteria of trap size). Additionally, the characteristics of spontaneous firings (without any mechanical stimulation) were compared for U. reflexa traps fed or denied prey during a 1-day period. On the absolute scale, the 13 Utricularia species differed considerably in their firing and resetting rates. Significant interspecific differences were also found in the magnitude of firing (in total 3.7–4.2 times) and resetting rates (10–24 times) per unit trap thickness or length. Overall, traps of Utricularia australis, Utricularia stellaris and Utricularia inflata showed the greatest firing and resetting rates. The relative magnitude of firing per unit trap thickness or length showed a highly significant negative correlation with both trap thickness and length and the same also held for the relative resetting rates. Smaller and narrower traps are thus relatively more effective at trap firing and resetting than larger traps. Neither firing nor resetting characteristics were significantly different between unfed and prey-fed traps of U. reflexa and this was also true for the occurrence of spontaneous firings. A strict linear resetting rate, without any lag-period, was found during the first 3 min after trap firing in U. reflexa. This suggests that water is pumped out of the trap continuously and probably recirculates. Given the concepts of spontaneous firing and water recirculation, an ecological model based on the literature data has been devised to quantify the daily N and P gain from the ambient water by Utricularia traps devoid of animal prey. The model shows that the total N and P content estimated in the trap fluid is too high to be accumulated from only the ambient water. This implies that the prey-free traps do not take up N or P from the trap fluid but rather exude a quantity of these nutrients to the fluid to support the microbial community. Therefore, the trap microorganisms behave more as parasites than commensals and represent an additional ecological cost for trap maintenance.     

Photosynthesis and dark respiration of leaves of terrestrial carnivorous plants

Using CO₂ gasometry, net photosynthetic (P _N) and dark respiration rates (R _D) were measured in leaves or traps of 12 terrestrial carnivorous plant species usually grown in the shade. Generally, mean maximum P _N (60 nmol CO₂ g⁻¹(DM) s⁻¹ or 2.7 μmol m⁻² s⁻¹) was low in comparison with that of vascular non-carnivorous plants but was slightly higher than that reported elsewhere for carnivorous plants. After light saturation, the facultatively heliophytic plants behaved as shade-adapted plants. Mean R _D in leaves and traps of all species reached about 50% of maximum P _N and represents the high photosynthetic (metabolic) cost of carnivory.     

Strategy of nitrogen acquisition and utilization by carnivorous Dionaea muscipula

Plant carnivory represents an exceptional means to acquire N. Snap traps of Dionaea muscipula serve two functions, and provide both N and photosynthate. Using ¹³C/¹⁵N-labelled insect powder, we performed feeding experiments with Dionaea plants that differed in physiological state and N status (spring vs. autumn plants). We measured the effects of ¹⁵N uptake on light-saturated photosynthesis (A _max), dark respiration (R _D) and growth. Depending on N status, insect capture briefly altered the dynamics of R _D/A _max, reflecting high energy demand during insect digestion and nutrient uptake, followed by enhanced photosynthesis and growth. Organic N acquired from insect prey was immediately redistributed, in order to support swift renewal of traps and thereby enhance probability of prey capture. Respiratory costs associated with permanent maintenance of the photosynthetic machinery were thereby minimized. Dionaea’s strategy of N utilization is commensurate with the random capture of large prey, occasionally transferring a high load of organic nutrients to the plant. Our results suggest that physiological adaptations to unpredictable resource availability are essential for Dionaea’s success with regards to a carnivorous life style.     

Metabolomic analysis reveals reliance on secondary plant metabolites to facilitate carnivory in the Cape sundew,Drosera capensis

Background and AimsSecondary metabolites are integral to multiple key plant processes (growth regulation, pollinator attraction and interactions with conspecifics, competitors and symbionts) yet their role in plant adaptation remains an underexplored area of research. Carnivorous plants use secondary metabolites to acquire nutrients from prey, but the extent of the role of secondary metabolites in plant carnivory is not known. We aimed to determine the extent of the role of secondary metabolites in facilitating carnivory of the Cape sundew, Drosera capensis.MethodsWe conducted metabolomic analysis of 72 plants in a time-series experiment before and after simulated prey capture. We used ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) and the retention time index to identify compounds in the leaf trap tissue that changed up to 72 h following simulated prey capture. We identified associated metabolic pathways, and cross-compared these compounds with metabolites previously known to be involved in carnivorous plants across taxa.Key ResultsFor the first time in a carnivorous plant, we have profiled the whole-leaf metabolome response to prey capture. Reliance on secondary plant metabolites was higher than previously thought – 2383 out of 3257 compounds in fed leaves had statistically significant concentration changes in comparison with unfed controls. Of these, ~34 compounds are also associated with carnivory in other species; 11 are unique to Nepenthales. At least 20 compounds had 10-fold changes in concentration, 12 of which had 30-fold changes and are typically associated with defence or attraction in non-carnivorous plants.ConclusionsSecondary plant metabolites are utilized in plant carnivory to an extent greater than previously thought – we found a whole-metabolome response to prey capture. Plant carnivory, at the metabolic level, likely evolved from at least two distinct functions: attraction and defence. Findings of this study support the hypothesis that secondary metabolites play an important role in plant diversification and adaptation to new environments.     

Trap diversity and evolution in the family Droseraceae

We review trapping mechanisms in the carnivorous flowering plant family Droseraceae (order Caryophyllales). Its members are generally known to attract, capture, retain and digest prey animals (mainly arthropods) with active snap-traps (Aldrovanda, Dionaea) or with active sticky flypaper traps (Drosera) and to absorb the resulting nutrients. Recent investigations revealed how the snap-traps of Aldrovanda vesiculosa (waterwheel plant) and Dionaea muscipula (Venus’ flytrap) work mechanically and how these apparently similar devices differ as to their functional morphology and shutting mechanics. The Sundews (Drosera spp.) are generally known to possess leaves covered with glue-tentacles that both can bend toward and around stuck prey. Recently, it was shown that there exists in this genus a higher diversity of different tentacle types and trap configurations than previously known which presumably reflect adaptations to different prey spectra. Based on these recent findings, we finally comment on possible ways for intrafamiliar trap evolution.     

Tree shrew lavatories: a novel nitrogen sequestration strategy in a tropical pitcher plant

Nepenthes pitcher plants are typically carnivorous, producing pitchers with varying combinations of epicuticular wax crystals, viscoelastic fluids and slippery peristomes to trap arthropod prey, especially ants. However, ant densities are low in tropical montane habitats, thereby limiting the potential benefits of the carnivorous syndrome. Nepenthes lowii, a montane species from Borneo, produces two types of pitchers that differ greatly in form and function. Pitchers produced by immature plants conform to the ‘typical’ Nepenthes pattern, catching arthropod prey. However, pitchers produced by mature N. lowii plants lack the features associated with carnivory and are instead visited by tree shrews, which defaecate into them after feeding on exudates that accumulate on the pitcher lid. We tested the hypothesis that tree shrew faeces represent a significant nitrogen (N) source for N. lowii, finding that it accounts for between 57 and 100 per cent of foliar N in mature N. lowii plants. Thus, N. lowii employs a diversified N sequestration strategy, gaining access to a N source that is not available to sympatric congeners. The interaction between N. lowii and tree shrews appears to be a mutualism based on the exchange of food sources that are scarce in their montane habitat.     

Enzyme production in the traps of aquatic <Emphasis Type="Italic">Utricularia</Emphasis> species

We studied the influence of habitat and increased mineral phosphorus and nitrogen loading on the extracellular activity of five selected hydrolases and pH in the trap fluid of the aquatic carnivorous plants, Utricularia vulgaris, U. australis, and U. foliosa (Lentibulariaceae). Enzyme activities in the trap fluid were determined using fluorometry. Phosphatase exhibited the highest activities in the traps of the European species as well as field-grown tropical U. foliosa. Trap enzyme production appeared to be uninfluenced by elevated dissolved mineral N or P concentrations both in the trap and ambient environment and thus, it seems to be constitutive. Enzyme activity in the trap fluid was determined by species and environmental conditions and varied significantly among sites within a single species. Trap fluid pH was between 4.2–5.1 in U. vulgaris and U. australis but between 5.7–7.3 in U. foliosa and seems to be regulated by the traps.     

S-like ribonuclease gene expression in carnivorous plants

Functions of S-like ribonucleases (RNases) differ considerably from those of S-RNases that function in self-incompatibility. Expression of S-like RNases is usually induced by low nutrition, vermin damage or senescence. However, interestingly, an Australian carnivorous plant Drosera adelae (a sundew), which traps prey with a sticky digestive liquid, abundantly secretes an S-like RNase DA-I in the digestive liquid even in ordinary states. Here, using D. adelae, Dionaea muscipula (Venus flytrap) and Cephalotus follicularis (Australian pitcher plant), we show that carnivorous plants use S-like RNases for carnivory: the gene da-I encoding DA-I and its ortholog cf-I of C. follicularis are highly expressed and constitutively active in each trap/digestion organ, while the ortholog dm-I of D. muscipula becomes highly active after trapping insects. The da-I promoter is unmethylated only in its trap/digestion organ, glandular tentacles (which comprise a small percentage of the weight of the whole plant), but methylated in other organs, which explains the glandular tentacles-specific expression of the gene and indicates a very rare gene regulation system. In contrast, the promoters of dm-I, which shows induced expression, and cf-I, which has constitutive expression, were not methylated in any organs examined. Thus, it seems that the regulatory mechanisms of the da-I, dm-I and cf-I genes differ from each other and do not correlate with the phylogenetic relationship. The current study suggests that under environmental pressure in specific habitats carnivorous plants have managed to evolve their S-like RNase genes to function in carnivory.