Plant Physiology and Biochemistry 104 (2016) 11e16 Contents lists available at ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy Research article A carnivorous sundew plant prefers protein over chitin as a source of nitrogen from its traps * Andrej Pavlovic a, b, , Miroslav Krausko b, Lubomír Adamec c a Department of Biophysics, Centre of the Region Hana for Biotechnological and Agricultural Research, Faculty of Science, Palacký University, Slechtitelu27, CZ-783 71, Olomouc, Czech Republic b Department of Plant Physiology, Faculty of Natural Sciences, Comenius University in Bratislava, Ilkovicova 6, Mlynska dolina, SK-842 15, Bratislava, Slovakia c Institute of Botany of the Czech Academy of Sciences, Section of Plant Ecology, Dukelska 135, CZ-379 82, Trebon, Czech Republic article info abstract Article history: Carnivorous plants have evolved in nutrient-poor wetland habitats. They capture arthropod prey, which Received 19 January 2016 is an additional source of plant growth limiting nutrients. One of them is nitrogen, which occurs in the Received in revised form form of chitin and proteins in prey carcasses. In this study, the nutritional value of chitin and protein and 4 March 2016 their digestion traits in the carnivorous sundew Drosera capensis L. were estimated using stable nitrogen Accepted 4 March 2016 isotope abundance. Plants fed on chitin derived 49% of the leaf nitrogen from chitin, while those fed on Available online 5 March 2016 the protein bovine serum albumin (BSA) derived 70% of its leaf nitrogen from this. Moreover, leaf ni- trogen content doubled in protein-fed in comparison to chitin-fed plants indicating that the proteins Keywords: fi Carnivorous plant were digested more effectively in comparison to chitin and resulted in signi cantly higher chlorophyll Drosera contents. The surplus chlorophyll and absorbed nitrogen from the protein digestion were incorporated Chitin into photosynthetic proteins e the light harvesting antennae of photosystem II. The incorporation of Nitrogen uptake insect nitrogen into the plant photosynthetic apparatus may explain the increased rate of photosynthesis Chlorophyll and plant growth after feeding. This general response in many genera of carnivorous plants has been Photosynthesis reported in many previous studies. Plant chitinase © 2016 Elsevier Masson SAS. All rights reserved. 1. Introduction nutrient supply; particularly in foliar/shoot nitrogen and phos- phorus contents (Farnsworth and Ellison, 2008; Pavlovic et al., Carnivorous plants have independently evolved several times by 2009, 2014; He and Zain, 2012; Kruse et al., 2014; Gao et al., the process of convergent evolution (Ellison and Gotelli, 2009). 2015). The greatest enhancement in photosynthetic gains from They usually grow in sunny, wet and nutrient-poor habitats where prey capture occurs under conditions of soil nutrient shortage, the nutritional benefit gained from captured prey exceeds the costs together with sufficient humidity and light. Such conditions have of modifying leaves into traps (Givnish et al. 1984; Pavlovic et al. favoured the evolution of botanical carnivory. It has been suggested 2009). The costs of carnivory represents extra energy costs with that the mechanism accounting for the increased AN in response to prey attraction (production of lures), capture (production of traps) nitrogen uptake from prey is an increased concentration in and digestion (production of digestive enzymes) being required. photosynthetic proteins (mainly Rubisco) (Givnish et al., 1984); There is also a decreased rate of photosynthesis (AN) and in some however, this has never been tested. This suggestion seems species an increased rate of respiration (RD) as a result of leaf reasonable because Rubisco is present at very high levels in the adaptation for carnivory (Givnish et al., 1984; Ellison, 2006; photosynthesizing cells of C3 plants and may contribute up to 50% Pavlovic and Saganova, 2015). On the other hand, a potential of soluble leaf protein and 20e30% of total leaf N (Evans, 1989; benefit from carnivory is an increase in AN through improved Feller et al., 2008). The second major fraction of nitrogen directly related to photosynthesis consists of the pigmenteprotein com- plexes in thylakoid membranes (Evans, 1989). * Corresponding author. Department of Biophysics, Centre of the Region Hana for Carnivorous plants obtain a substantial amount of nutrients Biotechnological and Agricultural Research, Faculty of Science, Palacký University, fi from prey capture and have evolved ve basic trapping mecha- Slechtitelu 27, CZ-783 71, Olomouc, Czech Republic. E-mail address: [email protected] (A. Pavlovic). nisms (Juniper et al., 1989; Krol et al., 2012; Pavlovic and Saganova, http://dx.doi.org/10.1016/j.plaphy.2016.03.008 0981-9428/© 2016 Elsevier Masson SAS. All rights reserved. 12 A. Pavlovic et al. / Plant Physiology and Biochemistry 104 (2016) 11e16 2015). The most important nutrients, which restrict the carnivorous approximately 15 mg of chitin from shrimp shells (95% acetylated, plant growth in nutrient-poor soils are nitrogen (N), phosphorus Sigma Aldrich, St. Louis, USA) every week for 16-week-long feeding (P) and potassium (K) (Ellison, 2006). Insect prey is a rich source of period (in total 240 mg of chitin per plant for 16 weeks). We ana- À N and P, the contents of which exceed that in leaf tissue by 5e10 lysed the nitrogen content in chitin and we found 64.8 mg N g 1 times, and these elements are markedly absorbed (Adamec, 2002; DW. Thus each fed plant obtained a total of 15.6 mg of nitrogen Pavlovic et al., 2014). This accounts for the relatively high contri- during the whole feeding period. Another seven plants were fed on bution of insect-derived N to total leaf N content in carnivorous protein with approximately 6 mg of BSA (Sigma Aldrich, St. Louis, plants which successfully capture insect prey (10e90%, Schulze USA) every week for the same feeding period (in total 96 mg BSA et al., 1991, 1997; Chapin and Pastor, 1995, 2001; Moran et al., per plant for 16 weeks). The analysed nitrogen content in BSA was À 2001; Millet et al., 2003), and is allocated mainly in the new fo- 144.2 mg N g 1 DW, thus each plant obtained in total 13.8 mg of liage (Schulze et al., 1997; Gao et al., 2015). Adamec (2002) and nitrogen during the whole feeding period. Both substances were Pavlovic et al. (2014) analysed prey carcasses after their digestion applied on two or three fully developed traps covered with sticky and found that a high amount of N (40e60%) in insect carcasses was tentacles during each feeding. Thus both groups of plants obtained unavailable for absorption by sundew traps. On the other hand, P approximately the same amount of N in the form of chitin or BSA and K were absorbed much more effectively. They hypothesized (ca. 14e16 mg) to investigate the nutritional value of both sub- that the less effective N uptake was due to the large proportion of N stances commonly found in insect prey. Seven plants served as in insect chitin exoskeletons (as poly-N-acetylglucosamine) which unfed control. After 16 weeks of feeding, the plants were allowed to is not available for absorption. Indeed, the exoskeleton of the grow for another 6 weeks without any additional feeding before digested prey does not seem to be significantly affected by the they were harvested for analysis. This period was necessary for the digestive processes (Juniper et al., 1989). However, this seems to be production of new leaves used in elemental and isotopic analyses in contrast to the recent molecular findings that several classes of which were not contaminated with remaining BSA or chitin applied chitinases in the digestive fluid in different species of carnivorous on trap surface. plants have been identified, and are even up-regulated by the presence of prey and/or chitin (Matusíkova et al., 2005; Eilenberg 2.2. Elemental analysis and isotopic composition of leaves et al., 2006; Rottloff et al., 2011; Hatano and Hamada, 2012; Renner and Specht, 2012; Paszota et al., 2014). Plant aboveground biomass was harvested from five plants In our previous work, we have shown that feeding the sundew before the feeding experiment and from seven plants per feeding plant Drosera capensis on fruit flies significantly increased its leaf group after the experiment, dried at 70 C for a week, weighed for nitrogen and phosphorus content as well as the photosynthetic rate DW determination, and ground to a fine powder. To avoid (Pavlovic et al., 2014). In this work, we focused on the importance of contamination with chitin or BSA, newly formed unfed leaves two nitrogen-rich compounds (chitin and protein) in the nutrition developed during the 6-week-long period after feeding were used of the carnivorous sundew plant D. capensis. The sundew plants for elemental analyses. All the fed leaves, which usually senesced were fed either on chitin or protein (bovine serum albumin, BSA) to within 22 (16 þ 6)-week-long period after feeding were not har- estimate the contribution of both N sources to the total N budget in vested for analysis to avoid contamination from chitin and BSA, plants. We measured the biomass, elemental composition, stable N including old leaves in control plants and plants before experi- isotopes and chlorophyll content to reveal the uptake and nutri- ments. Samples (1 mg) were packed into tin capsules and the ni- tional value of both insect nitrogen-rich compounds. In addition, trogen content was determined in a Vario MICRO Cube (Elementar, we did Western blot analyses for important photosynthetic pro- Hanau, Germany) Elemental Analyzer. A connected continuous- teins to find the role of N nutrition in the photosynthesis of flow IRMS Delta plus XL (Thermo Finnigan, Bremen, Germany) carnivorous plants. was used to analyse the stable isotope ratios of nitrogen to assess the plant's nitrogen origin. The d15N values (‰) were measured 15 2.