BACKGROUND AND AIMS: 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. METHODS: 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). KEY RESULTS: 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. CONCLUSIONS: 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.

Department of Biophysics Faculty of Science Palacký University Šlechtitelů 27 CZ 783 71 Olomouc Czech Republic

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Adamec L. 2010. Dark respiration of leaves and traps of terrestrial carnivorous plants: are there greater energetic costs in traps? Central European Journal of Biology 5: 121–124.

Adamec L, Matušíková I, Pavlovič A. 2021. Recent ecophysiological, biochemical and evolutional insights into plant carnivory. Annals of Botany 128: 241–259. PubMed PMC

An CI, Fukusaki E, Kobayashi A. 2001. Plasma-membrane H+-ATPases are expressed in pitchers of the carnivorous plant Nepenthes alata Blanco. Planta 212: 547–555. PubMed

Baby S, Johnson AJ, Zachariah EJ, Hussain AA. 2017. Nepenthes pitchers are CO2-enriched cavities, emit CO2 to attract preys. Scientific Reports 7: 11281. PubMed PMC

Barthlott W, Szarzynski J, Vlek P, Lobin W, Korotkova N. 2009. A torch in the rain forest: thermogenesis of the Titan arum (Amorphophallus titanum). Plant Biology 11: 499–505. PubMed

Blehová A, Švubová R, Lukačová Z, Moravčíková J, Matušíková I. 2015. Transformation of sundew: pitfalls and promises. Plant, Cell, Tissue and Organ Culture 120: 681– 687.

Bohn HF, Federle W. 2004. Insect aquaplaning: Nepenthes pitcher plants capture prey with the peristome, a fully wettable water-lubricated anisotropic surface. Proceedings of the National Academy of Sciences of the United States of America 101: 14138–14143. PubMed PMC

Bräutigam K, Dietzel L, Kleine T, et al. . 2009. Dynamic plastid redox signals integrate gene expression and metabolism to induce distinct metabolic states in photosynthetic acclimation in Arabidopsis. The Plant Cell 21: 2715–2732. PubMed PMC

Capó-Bauçà S, Font-Carrascosa M, Ribas-Carbó M, Pavlovič A, Galmés J. 2020. Biochemical and mesophyll diffusional limits to photosynthesis are determined by prey and root nutrient uptake in the carnivorous pitcher plant Nepenthes × ventrata. Annals of Botany 126: 25–37. PubMed PMC

Chia TF, Aung HH, Osipov AN, Goh NK, Chia LS. 2004. Carnivorous pitcher plant uses free radicals in the digestion of prey. Redox Report 9: 255–261. PubMed

Clarke CM. 1997. Nepenthes of Borneo. Kota Kinabalu: Malaysia Natural History Publication.

Dahal K, Martyn GD, Alber NA, Vanlerberghe GC. 2017. Coordinated regulation of photosynthetic and respiratory components is necessary to maintain chloroplast energy balance in varied growth conditions. Journal of Experimental Botany 68: 657–671. PubMed PMC

Del-Saz NF, Ribas-Carbo M, McDonald AE, Lambers H, Fernie AR, Florez-Sarasa I. 2018. An in vivo perspective of the role(s) of the alternative oxidase pathway. Trends in Plant Science 23: 206–219. PubMed

Dkhar J, Bhaskar YK, Lynn A, Pareek A. 2020. Pitchers of Nepenthes khasiana express several digestive-enzyme encoding genes, harbor mostly fungi and probably evolved through changes in the expression of leaf polarity genes. BMC Plant Biology 20: 524. PubMed PMC

Escobar MA, Geisler DA, Rasmusson AG. 2006. Reorganization of the alternative pathways of the Arabidopsis respiratory chain by nitrogen supply: opposing effects of ammonium and nitrate. The Plant Journal 45: 775–788. PubMed

Givnish TJ, Burkhardt EL, Happel RE, Weintraub JD. 1984. Carnivory in the bromeliad Brocchinia reducta, with a cost/benefit model for the general restriction of carnivorous plants to sunny, moist nutrient-poor habitats. American Naturalist 124: 479–497.

Gonzàlez-Meler MA, Giles L, Thomas RB, Siedow JN. 2001. Metabolic regulation of leaf respiration and alternative oxidase activity in response to phosphate supply. Plant Cell and Environment 24: 205–215.

Gorb E, Kastner V, Peressadko A, et al. . 2004. Structure and properties of the glandular surface in the digestive zone of the pitcher in the carnivorous plant Nepenthes ventrata and its role in insect trapping and retention. The Journal of Experimental Biology 207: 2947–2963. PubMed

He J, Zain A. 2012. Photosynthesis and nitrogen metabolism of Nepenthes alata in response to inorganic NO3– and organic prey N in the greenhouse. International Scholarly Research Network Botany. Article ID 263270.

Huq S, Palmer JM. 1978. Isolation of a cyanide-resistant duroquinol oxidase from Arum maculatum mitochondria. FEBS Letters 95: 217–220. PubMed

Jones W. 2013. Olfactory carbon dioxide detection by insects and other animals. Molecules and Cells 35: 87–92. PubMed PMC

Juszczuk IM, Wagner AM, Rychter AM. 2001. Regulation of alternative oxidase activity during phosphate deficiency in bean roots (Phaseolus vulgaris). Physiologia Plantarum 113: 185–192. PubMed

Karagatzides JD, Ellison AM. 2009. Construction costs, payback times, and the leaf economics of carnivorous plants. American Journal of Botany 96: 1612–1619. PubMed

Knutson RM. 1974. Heat production and temperature regulation in eastern skunk cabbage. Science 186: 746–747. PubMed

Kruse J, Gao P, Honsel A, et al. . 2014. Strategy of nitrogen acquisition and utilization by carnivorous Dionaea muscipula. Oecologia 174: 839–851. PubMed

Laakkonen L, Jobson RW, Albert VA. 2006. A new model for the evolution of carnivory in the bladderwort plant (Utricularia): adaptive changes in cytochrome c oxidase (COX) provide respiratory power. Plant Biology 8: 758–764. PubMed

Lee L, Zhang Y, Ozar B, Sensen CW, Schriemer DC. 2017. Carnivorous nutrition in pitcher plants (Nepenthes spp.) via an unusual complement of endogenous enzymes. Journal of Proteome Research 15: 3108–3117. PubMed

Maxwell K, Johnson GN. 2000. Chlorophyll fluorescence–a practical guide. Journal of Experimental Botany 51: 659–668. PubMed

Maxwell DP, Wang Y, McIntosh L. 1999. The alternative oxidase lowers mitochondrial reactive oxygen production in plant cells. Proceedings of the National Academy of Sciences of the United States of America 96: 8271–8276. PubMed PMC

Meeuse BJD, Raskin I. 1988. Sexual reproduction in the arum lily family, with emphasis on thermogenicity. Sexual Plant Reproduction 1: 3–15.

Millenaar FF, Lambers H. 2003. The alternative oxidase: in vivo regulation and function. Plant Biology 5: 2–15.

Morales F, Pavlovič A, Abadía A, Abadía J. 2018. Photosynthesis in poor nutrient soils, in compacted soils, and under drought. In: Adams WW III, Terashima I. eds. The leaf: a platform for performing photosynthesis. Berlin: Springer, 371–399.

Nagy KA, Odell DK, Seymour RS. 1972. Temperature regulation by the inflorescence of Philodendron. Science 178: 1195–1197. PubMed

Noguchi K, Terashima I. 2006. Responses of spinach leaf mitochondria to low N availability. Plant, Cell & Environment 29: 710–719. PubMed

Owen TP Jr, Lennon KA. 1999. Structure and development of the pitchers from the carnivorous plant Nepenthes alata (Nepenthaceae). American Journal of Botany 86: 1382–1390. PubMed

Parsons HL, Yip JY, Vanlerberghe GC. 1999. Increased respiratory restriction during phosphate-limited growth in transgenic tobacco cells lacking alternative oxidase. Plant Physiology 121: 1309–1320. PubMed PMC

Pavlovič A, Masarovičová E, Hudák J. 2007. Carnivorous syndrome in Asian pitcher plants of the genus Nepenthes. Annals of Botany 100: 527–536. PubMed PMC

Pavlovič A., Slováková L, Šantrůček J. 2011. Nutritional benefit from leaf litter utilization in the pitcher plant Nepenthes ampullaria. Plant Cell and Environment 34: 1865–1873. PubMed

Pavlovič A, Saganová M. 2015. A novel insight into the cost–benefit model for the evolution of botanical carnivory. Annals of Botany 15: 1075–1092. PubMed PMC

Pavlovič A, Singerová L, Demko V, Hudák J. 2009. Feeding enhances photosynthetic efficiency in the carnivorous pitcher plant Nepenthes talangensis. Annals of Botany 104: 307–314. PubMed PMC

Ribas-Carbo M, Berry JA, Yakir D, et al. . 1995. Electron partitioning between the cytochrome and alternative pathways in plant mitochondria. Plant Physiology 109: 829–837. PubMed PMC

Saganová M, Bokor B, Stolárik T, Pavlovič A. 2018. Regulation of enzyme activities in carnivorous pitcher plants of the genus Nepenthes. Planta 248: 451–464. PubMed

Seymour RS. 2010. Scaling of heat production by thermogenic flowers: limits to floral size and maximum rate of respiration. Plant, Cell and Environment 33: 1474–1485. PubMed

Schägger H. 2006. Tricine–SDS-PAGE. Nature Protocols 1: 16–22. PubMed

Schöner CR, Schöner MG, Kerth G, Grafe TU. 2013. Supply determines demand: influence of partner quality and quantity on the interactions between bats and pitcher plants. Oecologia 173: 191–202. PubMed

Schulze W, Frommer WB, Ward JM. 1999. Transporters for ammonium, amino acids and peptides are expressed in pitchers of the carnivorous plant Nepenthes. The Plant Journal 17: 637–646. PubMed

Shchennikova AV, Beletsky AV, Filyushin MA, et al. . 2021. Nepenthes × ventrata transcriptome profiling reveals a similarity between the evolutionary origins of carnivorous traps and floral organs. Frontiers in Plant Science 12: 643137. PubMed PMC

Sieger SM, Kristensen BK, Robson CA, et al. . 2005. The role of alternative oxidase in modulating carbon use efficiency and growth during macronutrient stress in tobacco cells. Journal of Experimental Botany 56: 1499–1515. PubMed

Umbach AL, Ng VS, Siedow JN. 2006. Regulation of plant alternative oxidase activity: a tale of two cysteines. Biochimica Et Biophysica Acta 1757: 135–142. PubMed

Vanlerberghe GC. 2013. Alternative oxidase: a mitochondrial respiratory pathway to maintain metabolic and signaling homeostasis during abiotic and biotic stress in plants. International Journal of Molecular Sciences 14: 6805–6847. PubMed PMC

Vanlerberghe GC, Dahal K, Alber NA, Chadee A. 2020. Photosynthesis, respiration and growth: a carbon and energy balancing act for alternative oxidase. Mitochondrion 52: 197–211. PubMed

Vanlerberghe GC, McLntosh L. 1996. Signals regulating the expression of the nuclear gene encoding alternative oxidase of plant mitochondria. Plant Physiology 111: 589–595. PubMed PMC

Wagner AM, Krab K, Wagner MJ, Moore AL. 2008. Regulation of thermogenesis in flowering Araceae: the role of the alternative oxidase. Biochimica et Biophysica Acta 1777: 993–1000. PubMed

Wasserman S, Salomon A, Frye MA. 2013. Drosophila tracks carbon dioxide in flight. Current Biology 23: 301–306. PubMed PMC

Yoshida K, Terashima I, Noguchi K. 2006. Distinct roles of the cytochrome pathway and alternative oxidase in leaf photosynthesis. Plant & Cell Physiology 47: 22–31. PubMed

Zarkovic J, Anderson SL, Rhoads DM. 2005. A reporter gene system used to study developmental expression of alternative oxidase and isolate mitochondrial retrograde regulation mutants in Arabidopsis. Plant Molecular Biology 57: 871–888. PubMed

Alternative or cytochrome? Respiratory pathways in traps of aquatic carnivorous bladderwort Utricularia reflexa