Research EnzymaticBlackwell Publishing Ltd. activities in traps of four aquatic species of the carnivorous genus Utricularia Dagmara Sirová1, Lubomír Adamec2 and Jaroslav Vrba1,3 1Faculty of Biological Sciences, University of South Bohemia, BraniSovská 31, CZ−37005 Ceské Budejovice, Czech Republic; 2Institute of Botany AS CR, Section of Plant Ecology, Dukelská 135, CZ−37982 Trebo˜, Czech Republic; 3Hydrobiological Institute AS CR, Na Sádkách 7, CZ−37005 Ceské Budejovice, Czech Republic Summary Author for correspondence: • Here, enzymatic activity of five hydrolases was measured fluorometrically in the Lubomír Adamec fluid collected from traps of four aquatic Utricularia species and in the water in Tel: +420 384 721156 which the plants were cultured. Fax: +420 384 721156 • In empty traps, the highest activity was always exhibited by phosphatases (6.1– Email: [email protected] 29.8 µmol l−1 h−1) and β-glucosidases (1.35–2.95 µmol l−1 h−1), while the activities Received: 31 March 2003 of α-glucosidases, β-hexosaminidases and aminopeptidases were usually lower by Accepted: 14 May 2003 one or two orders of magnitude. Two days after addition of prey (Chydorus sp.), all doi: 10.1046/j.1469-8137.2003.00834.x enzymatic activities in the traps noticeably decreased in Utricularia foliosa and U. australis but markedly increased in Utricularia vulgaris. • Phosphatase activity in the empty traps was 2–18 times higher than that in the culture water at the same pH of 4.7, but activities of the other trap enzymes were usually higher in the water. Correlative analyses did not show any clear relationship between these activities. • Trap comensals (Euglena) could be partly responsible for production of some trap enzymes. The traps can produce phosphatases independently of catching prey. Tak- ing into account the enzymatic activities in traps, phosphorus uptake from prey might be more important than that of nitrogen for the plants. Key words: aquatic carnivorous plants, trap fluid pH, extracellular enzymatic activity, phosphatase, aminopeptidase, glucosidase, β-hexosaminidase, chitinase. © New Phytologist (2003) 159: 669–675 prey brushes against the trigger hairs, it is sucked in because Introduction of the underpressure maintained inside the utricle. After The genus Utricularia (Lentibulariaceae), with over 210 firing, the trap restores the underpressure by removing species, is the most widespread of the carnivorous plants water from the lumen until the original compressed shape ( Juniper et al., 1989; Taylor, 1989). Many species from is reached. After this process, which lasts about 30 min, is this rootless genus are aquatic and form floating shoots in completed, the trap is ready to fire again (Sydenham & quiet, pollution-free ponds and humic waters where they Findlay, 1975). are important components of the freshwater flora. Like Little is known about the mechanisms of digestion in other carnivorous plants, Utricularia supplements normal Utricularia. There have been no recent or detailed studies on photolithotrophic nutrition by trapping and utilizing prey, in situ enzymatic activity in the actual trap fluid. Standard typically aquatic crustaceans, mites, rotifers and protozoa biochemical techniques have provided evidence for the pres- ( Jobson & Morris, 2001; Richards, 2001). The trap is a ence of proteases in the traps (von Luetzelberg, 1910; Adowa, hollow utricle, mostly two cells thick, filled with water. Traps 1924; Hada, 1930). Protease (Parkes, 1980; Vintéjoux, 1973, are usually 1– 4 mm long. They have a doorway obstructed by 1974), acid phosphatase and esterase (Heslop-Harrison, 1975; a trapdoor which opens inwards upon irritation. The trapdoor Parkes, 1980) were localized cytochemically in the quadrifid is surrounded by trigger hairs and other appendages. After the digestive glands. Utricles also support a diverse community © New Phytologist (2003) 159: 669–675 www.newphytologist.com 669 670 Research of microorganisms, including many species of living bacteria, with filtered (mesh size 44 µm) culture water. The cuttings algae, rotifers and protozoa (Cohn, 1875; Hegner, 1926; were collected from younger parts of the plants to ensure full Schumacher, 1960; Botta, 1976; Jobson & Morris, 2001; trap functionality. A portion of the material was used Richards, 2001). Species of Euglena (Euglenophyta) appar- immediately to collect trap fluid for estimation of enzymatic ently even reproduced in this environment (Hegner, 1926; activity in empty traps. Fine zooplankton, commonly found Botta, 1976). It is therefore reasonable to assume that a con- in habitats of all aquatic Utricularia species (Chydorus sp., size siderable proportion of enzymatic activity in the trap fluid is 0.5–0.6 mm, fresh weight c. 50–100 µg), was added to the derived from this community. This supports the hypothesis remaining material and was carefully removed after 8 h. By that Utricularia plants benefit more from the byproducts of this time, most of the traps had fired and contained prey. this community than from carnivory itself (Richards, 2001). Trap fluid from the fed traps of U. foliosa, U. australis and The aim of our study was to measure in situ activity of five U. vulgaris was collected 2 d later (and also after 4 d in U. common hydrolases in the trap fluid collected from four vulgaris), during which the plastic jars were placed in the same aquatic Utricularia species and in the water in which the light and temperature conditions as those under which the plants were cultured, in order to estimate pH optima and plants were cultivated. No zooplankton was added to traps of changes in activity of these enzymes at time intervals follow- U. aurea. ing prey capture. In this way, we determined the direct avail- Trap fluid from the largest traps (usually > 2 mm) was col- ability of the enzymes in the traps for prey digestion. lected by glass pipettes with fine, 0.4-mm wide tips attached to a plastic syringe. The trap door was carefully pushed inwards by the pipette tip to avoid damage to plant cells and Materials and Methods consequent contamination. The fluid was sucked into the tip by capillary action and forced into plastic 0.5 ml Eppendorf Plant material filtration vials placed on ice. The fluid (volume c. 0.5 ml) collected Adult plants of Utricularia vulgaris L. and Utricularia australis from 90 to 300 traps of each Utricularia species was pooled R.Br. (collected in the Czech Republic) were cultivated into a vial equipped with a filter (0.2 µm) and centrifuged at outdoors in plastic containers (area 2 m2, 750 l for the former 600–1200 g for 10–15 min. This procedure filtered out thou- species; area 0.8 m2, 220 l for the latter species; Adamec, sands of algal cells, mainly Euglena sp., which could burst 1997a,b). Utricularia foliosa L. (collected in northern Florida, upon freezing and contaminate the samples. Several vials with USA) was grown in a glasshouse in a similar container to that filtrate were prepared in this way and frozen at −20°C. The used for Utricularia australis. In these cultures, water depth abundance of Euglena cells in parallel fluid samples was esti- −1 was kept at 25–35 cm and tap water (NO3-N 0.7–1.1 mg l ; mated using a Petroff–Hausser counting chamber (depth + −1 −1 × NH4 -N < 10 µg l ; PO4-P < 10 µg l ) was added to 0.04 mm) and a light microscope (magnification 200). This compensate for water loss. Utricularia aurea Lour. (collected was repeated 10 times for each Utricularia species. in Malaysia) was grown indoors in 3-l aquaria (Adamec, The pH of the trap fluid was estimated roughly using a pH 1999). Litter of robust sedge species was used as a cultivation paper (Lachema, Brno, Czech Rep.). A piece of the pH paper, substrate in all of the above cultures, and approximately 3 × 3 mm, was placed on clean white plastic pad and soaked simulated natural conditions. From the concentrations of in the fluid pooled from 10 traps (volume c. 20 µl). The col- + + −1 – – −1 NH4 (NH4 -N 5–10 µg l ), NO3 (NO3 -N 28–40 µg l ), our change of the pH paper was compared with the standard 2– −1 HPO4 (PO4-P 12–15 µg l ) and humic substances colour scale. This was repeated five times for each of the Utri- (humic acids + tannins 4–10 mg l−1), the water in these cularia species. cultures was considered oligotrophic and humic (Adamec, 1997b). The pH of the cultivation media ranged from 7.3 to Enzymatic assay 7.9, dissolved oxygen concentration from 0.22 to 0.3 m, and free CO2 concentration from 0.03 to 0.1 m. Cultiva- A common fluorometric method (Hoppe, 1983, 1993) was tion methods used were analogous to those described for the adopted for the microplate assay (Marx et al., 2001) and aquatic carnivorous plant Aldrovanda vesiculosa (Adamec, modified to determine enzymatic activity in both trap fluid 1997a,b, 1999). Fine zooplankton (Chydorus sp., Bosmina sp. and ambient water samples. We used five fluorogenic and Cyclops sp.) was added repeatedly to the cultures to promote substrates: -leucine 7-amino-4-methylcoumarin hydrochloride, plant growth. Feeding was interrupted approximately 2 wk 4-methylumbelliferyl (MUF) phosphate, MUF N-acetyl- before trap sampling. β--glucosaminide, MUF α--glucoside, and MUF β-- glucoside (Glycosynth, Warrington, UK), to estimate the activity of aminopeptidases, phosphatases, β-hexosaminidases, Collection of trap fluid, pH measurements α-glucosidases, and β-glucosidases, respectively. We used Trap-bearing leaves of the four Utricularia species studied special, black 96-well microplates for fluorescence detection were cut from adult plants and placed into plastic jars (0.2 l) (Nunc, Roskilde, Denmark) in two different microplate www.newphytologist.com © New Phytologist (2003) 159: 669–675 Research 671 set-ups as follows. For activity in traps, all wells were filled Determination of pH optimum with 300 µl of 2 m acetate buffer (pH 4.7).