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Home , Darlingtonia californica, Heliamphora, Ibicella, Sarracenia, Sarraceniaceae, Sarracenia alabamensis

Article Endocytosis and Digestion in Carnivorous Pitcher Plants of the Family

Marianne Koller-Peroutka 1,*, Stefanie Krammer 1, Anselm Pavlik 1, Manfred Edlinger 2, Ingeborg Lang 3 and Wolfram Adlassnig 1

1 Core Facility Cell Imaging and Ultrastructure Research, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria; [email protected] (S.K.); [email protected] (A.P.); [email protected] (W.A.) 2 HBLFA for and the Austrian Bundesgärten Schönbrunn, Schloß Schönbrunn, A-1130 Vienna, Austria; [email protected] 3 Department of Ecogenomics and Systems Biology, University of Vienna, Althanstraße 14, A-1090 Vienna, Austria; [email protected] * Correspondence: [email protected]; Tel.: +43-4277-57921

 Received: 21 August 2019; Accepted: 19 September 2019; Published: 24 September 2019 

Abstract: Highly evolved carnivorous plants secrete digestive for degradation of trapped animals and absorb whole macromolecules from their prey by means of endocytosis. (1) Background: In the pitcher- family Sarraceniaceae, the production of enzymes is dubious and no evidence for endocytosis is known so far. (2) Methods: nutans, , and nine taxa of are tested for cuticular pores, and for protease and endocytosis of the fluorescent protein analogue FITC-BSA, after 10–48 h of stimulation. (3) Results: Cuticular pores as a prerequisite for secretion and nutrient uptake are present in all tested . Permeable cells clusters in the inner epidermis of the pitchers, but are only little differentiated from impermeable epidermis cells. Proteases are found in S. psittacina, S. leucophylla, S. minor, S. oreophila, S. alabamensis, H. nutans, D. californica lacking only in S. flava and in S. purpurea ssp. purpurea, S. purpurea ssp. venosa, S. rosea, where enzyme production is possibly replaced by degradation via the extraordinary diverse inquiline fauna. S. leucophylla, S. minor, S. oreophila exhibit both protease production and endocytosis; S. psittacina, S. alabamensis, H. nutans, D. californica produce proteases only; no single species shows endocytosis without protease production. (4) Conclusions: Protease secretion seems to be a prerequisite for endocytotic nutrient uptake. Transport of FITC-BSA absorbed by endocytosis towards the vascular tissue of the trap suggests that endocytosis of nutrients is more than a side effect of enzyme secretion.

Keywords: Sarracenia; Heliamphora; Darlingtonia; endocytosis; carnivorous plants; pitcher traps; uptake; digestion; FITC-BSA; methylene-blue

1. Introduction The American family Sarraceniaceae comprises three genera of pitcher plants with at least 35 species: Darlingtonia, Heliamphora, and Sarracenia. Sarracenia includes eleven species and a multitude of , varieties, and hybrids, and is native to eastern and northern North America. Heliamphora comprises 23 species, which grow in northern South America, whereas Darlingtonia is a monotypic that appears only in the northwestern [1–3]. Within the family of Sarraceniaceae, prey capture is performed by cone shape leaves, which are superficially similar to the pitcher traps of and , but less sophisticated. This is not only true on the morphological level, but possibly also for the physiology. Since the first studies on Sarracenia,

Plants 2019, 8, 367; doi:10.3390/plants8100367 www.mdpi.com/journal/plants Plants 2019, 8, 367 2 of 10 the production of digestive enzymes for prey degradation was questioned [4]. Gallie and Chang [5] report nucleases, proteases, and phosphatases in S. purpurea, whereas Smith [6] found no evidence for an accelerated degradation of meat in the fluid of S. purpurea compared to distilled water. On the other hand, a multitude of mutualistic pitcher inquilines has been described (summarized by [7–9]), ranging from to larvae, which contribute to prey degradation. Therefore, most authors assumed that enzyme production played at maximum a minor role in Sarracenia. Recent research, however, suggested the presence of digestive enzymes at least in part of Sarracenia [10,11]. Furthermore, prey utilization in Sarracenia seems to be less sophisticated with regards to the absorption of nutrients into living cells. Uptake is performed via selective carriers for inorganic ions and small organic molecules [12], as in presumably all other carnivorous plants [13]. Endocytosis, however, was suggested as a more advanced mechanism for nutrient uptake in carnivorous plants, which would enable the absorption of whole proteins or large fractions thereof [14]. Using fluorescent labelled proteins, the authors of this study were able to confirm this hypothesis by detecting endocytotic nutrient uptake in multiple genera of carnivorous plants, including , Dionaea, Nepenthes, Cephalotus, , and , but not in S. purpurea [15]. In eukaryotic cells, the secretion of proteins, including digestive enzymes, is usually performed via exocytosis [16]. Since the membrane surface of a non-growing cell is constant, exocytosis is usually coupled with endocytosis, resulting in the uptake of the external medium, including any dissolved compounds. In all aforementioned carnivorous plants with endocytotic nutrient uptake, the secretion of digestive enzymes has been demonstrated beyond reasonable doubt (summarized by [11,13]). Thus, the capability to absorb nutrients via endocytosis may have developed primarily as a side effect of the exocytosis of digestive enzymes. This study aims to test the hypothesis of a relationship between enzyme secretion and endocytotic uptake of whole proteins in the genus Sarracenia, supplemented by other Sarraceniaceae. The primary objective is to establish a correlation between autonomous digestion and endocytosis. Secondary objectives include screening for digestion, the fate of the absorbed proteins after endocytosis, and the characterization of pores in the impermeable [17] cuticle of the traps, which are a prerequisite of any nutrient uptake [18,19] regardless of the cellular mechanism.

2. Results The habitus of seven selected species are depicted in Figure1. Methylene blue staining (Figure2 and Supplementary Figure S1) demonstrated the presence of cuticular pores in all investigated species. Pores were restricted to the absorptive zone for uptake which is, following the terminology of Juniper et al. [20], zone 4 for Sarracenia and Heliamphora, and zone 5 for Darlingtonia (Supplementary Figure S2). These cuticular pores are completely lacking in the retention zone of the pitchers. Though no morphologically differentiated glands are developed in the absorptive zones of the pitchers in Sarraceniaceae, three types of epidermal cells can be distinguished: (1) Trichoblasts protruding into retentive hairs (opposed to atrichoblasts) never absorb methylene blue, neither to the cell wall, nor to the cytoplasm. (2) Some epidermal cells do not absorb methylene blue into the cytoplasm, but sometimes into the cell wall. (3) Other epidermal cells of the same shape but of a slightly smaller surface area (Supplementary Figure S3) accumulate methylene blue in the cytoplasm. The absorbing cells usually form isolated clusters surrounded by non-absorbing cells (Figure2 and Supplementary Figure S1). No evidence for digestion of gelatin emulsion (of analogue photographic film) was found in S. purpurea, S. rosea, and S. flava. In all other analyzed species of Sarracenia, as well as Heliamphora nutans and Darlingtonia californica, degradation of the gelatin indicated secretion of digestive enzymes upon stimulation (Figure3c–e). No evidence for absorption of FITC-BSA into the cytoplasm was found in S. purpurea (Figure4a), S. rosea, S. flava, S. psittacina, S. alabamensis, and Heliamphora nutans and Darlingtonia californica. In these species, FITC-fluorescence was visible in the cell walls of normal epidermal cells but not in the trichoblasts. In S. leucophylla, S. minor, and S. oreophila uptake of FITC-BSA Plants 2019, 8, 367 3 of 10

wasPlants found2019, 8, inx FOR isolated PEER REVIEW clusters of epidermal cells (Figure4b–d), similar to the uptake of methylene3 of 10 blue. Quantitative analysis of the FITC-fluorescence displayed highly significant differences between absorbingdifferences and between non-absorbing absorbing cellsand non-absorbing in S. oreophila, andcells comparedin S. oreophila to S., and purpurea compared(p < 0.01to S. inpurpurea all cases, (p see< 0.01 Figure in all5 ).cases, see Figure 5). Uptake patterns didifferedffered betweenbetween cellscells ofof thethe same section: FITC-BSA might be found in vesicles of varying size,size, inin thethe cytoplasm, cytoplasm, or or in in the the vacuole. vacuole. The The highest highest fluorescence fluorescence intensity intensity was was found found in the in centralthe central vacuole, vacuole, whereas whereas chloroplasts chloroplasts and the and nucleus the nu remaincleus remain dark. Atdark. an early At an stage early of stage uptake, of afteruptake, up toafter 15 hup (Figure to 15 4he), (Figure FITC-BSA 4e), FITC-BSA was exclusively was excl foundusively in the found epidermal in the cells.epidermal Later on,cells. the Later mesophyll, on, the andmesophyll, in particular and in the particular vascular the bundles, vascular are bundles, stained are as well stained (Figure as well4f). (Figure Table1 summarizes 4f). Table 1 summarizes all results. all results.

Figure 1. 1. HabitusHabitus of oftested tested Sarraceniaceae. Sarraceniaceae. (a) Sarracenia (a) purpurea ssp. purpureassp. purpurea (b) Sarracenia(b) (c) minorSarracenia(c) Sarracenialeucophylla leucophylla(d) Sarracenia(d) flavaSarracenia (e) Sarracenia flava (e oreophila) Sarracenia (f) Heliamphora oreophila (f) nutansHeliamphora (g) Sarracenia nutans (psittacinag) Sarracenia. psittacina.

Plants 2019, 8, 367 4 of 10 Plants 2019, 8, x FOR PEER REVIEW 4 of 10

Figure 2. Methylene blue staining of cuticular pores in the absorptive zone (a) Figure 2. Methylene blue staining of cuticular pores in the absorptive zone (a) Sarracenia alabamensis (b) (c) Sarracenia minor (d) (e) Darlingtonia californica (f) Sarracenia (b) Sarracenia leucophylla (c) Sarracenia minor (d) Sarracenia oreophila (e) Darlingtonia californica (f) psittacina. Arrows indicate trichoblasts. Scale bar: 100 µm. . Arrows indicate trichoblasts. Scale bar: 100 µm. Table 1. Results of the physiological analysis for enzyme formation, endocytosis, and methylene Tableblue 1. Results uptake. of the physiological analysis for enzyme formation, endocytosis, and methylene blue uptake.

SpeciesSpecies Cuticular Pores Pores Digestive Digestive EnzymesEnzymes Endocytosis Endocytosis S. purpurea ssp. ssp.purpurea purpurea ++ -- - - S. purpureapurpureassp. ssp.venosa venosa ++ --- - S.S. rosea rosea ++ --- - S.S. psittacinapsittacina + + + + - - S. flavaflavavar. var.flava flava ++ --- - S.S. leucophyllaleucophylla ++ ++ ++ S. minor var. minor + + + S. minorS. oreophila var. minor ++ ++ ++ S.S. alabamensis oreophila ++ ++ -+ HeliamphoraS. alabamensis nutans ++ ++ -- DarlingtoniaHeliamphora californica nutans ++ ++ -- Darlingtonia californica + + -

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FigureFigure 3. Enzyme3. Enzyme test test for proteases;for proteases; (a) preparing (a) preparingDarlingtonia Darlingtonia californica californica, Sarracenia, Sarracenia psittacina psittacina, Sarracenia, minorSarracenia, and Sarracenia minor, and alabamensis Sarracenia alabamensisfor the enzyme for the test enzyme by attaching test by attaching little pieces little of pieces film wetted of film withwetted BSA ontowith the BSA absorptive onto the zones absorptive of the zones pitchers; of the (b) pitchers; incubation (b) in incubation wet chambers in wet for chambers 18–48 h; for (c) developing18–48 h; (c) of thedeveloping film pieces of in the a filmfilm developer;pieces in a (filmd) positive developer; enzyme (d) positive test for enzymeS. minor test;(e for) positive S. minor enzyme; (e) positive test for Darlingtoniaenzyme test californica for Darlingtonia. californica.

3. Discussion3. Discussion InIn addition addition to to thethe trapping of of animals, animals, carnivor carnivorousous plants, plants, by definition, by definition, digest digestand utilise and their utilise theirprey prey [1,20]. [1,20 The]. The absorption absorption of ofsmall small molecules molecules into into the the epidermis epidermis by bymeans means of ofmethylene methylene blue blue stainingstaining was was confirmed confirmed for allfor investigatedall investigated species, species, as was as towas be expectedto be expected from earlier from resultsearlier [12results,21,22 ]. Protease[12,21,22]. production Protease andproduction digestion and within digestion 48 hwithin after 48 stimulation h after stimulation seems to seems be present to be present in the majorityin the ofmajority Sarraceniaceae, of Sarraceniaceae, including Darlingtoniaincluding Darlingtoniaand Heliamphora and Heliamphora[23], but lacking[23], but in lacking the closely in the related closely [24 ] speciesrelatedS. [24] rosea speciesand S. S. purpurearosea and. S. purpurea rosea is. a S. sister rosea tois a both sisterS. to purpurea both S. purpureassp. purpurea ssp. purpureaand S. purpureaand S. purpurea ssp. venosa [2,24], which show similar physiological features in this study, and are enclosed ssp. venosa [2,24], which show similar physiological features in this study, and are enclosed in the in the Sarracenia purpurea complex [24]. Previous data on protease production in S. purpurea are Sarracenia purpurea complex [24]. Previous data on protease production in S. purpurea are contradictory; contradictory; one study which found evidence for protein digestion [5] suggests that protease one study which found evidence for protein digestion [5] suggests that protease activity, if any, peaks activity, if any, peaks after >4 days. A recent study by Young et al. [25] indicates that protease activity after >4 days. A recent study by Young et al. [25] indicates that protease activity in the pitcher fluid of in the pitcher fluid of S. purpurea remains at baseline levels if antibiotics are added. S. purpureaUptakeremains of whole at baseline proteins levels was restricted if antibiotics to only are added.three Sarracenia species (Table 2). All species withUptake endocytosis of whole also proteins secreted was prot restrictedeases, but to not only vice three versa.Sarracenia Secretionspecies of proteases (Table 2by). Allexocytosis species can with endocytosisbe expected also to secretedbe accompanied proteases, by but the not recyclin vice versa.g of membranes Secretion ofby proteases endocytosis. by exocytosisHowever, canthis be expectedmembrane to be recycling accompanied alone seems by the not recycling to result of in membranes the uptake byof detectable endocytosis. amounts However, of FITC-BSA. this membrane The recyclingabsorption alone of visible seems notquantities to result seems in the to require uptake sp ofecific detectable cellular amounts mechanisms of FITC-BSA., which are The present absorption only ofin visible a few quantities species of seemsSarracenia to require, and are specific accompanied cellular by mechanisms, mechanisms whichfor theare transport present of only FITC-BSA in a few speciestowards of Sarraceniathe mesophyll., and are accompanied by mechanisms for the transport of FITC-BSA towards the mesophyll. Table 2. Number of species/subspecies showing or lacking digestion or endocytosis respectively. Table 2. Number of species/subspecies showing or lacking digestion or endocytosis respectively. Digestion Lacking Digestion Present Total Endocytosis lacking Digestion Lacking 4 Digestion Present 4 8Total Endocytosis present 0 3 3 Endocytosis lacking 4 4 8 EndocytosisTotal present 04 3 7 11 3 Total 4 7 11

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Plants 2019, 8, x FOR PEER REVIEW 6 of 10 On the cellular level uptake into the cytoplasm is restricted to specialized cells. Though these permeableOn the cells cellular are virtually level uptake indistinguishable into the cytoplasm from the is surroundingrestricted to specialized impermeable cells. cells, Though they seemthese to bepermeable as specialized cells as are the virtually morphologically indistinguishable differentiated from the specialized surrounding glands impermeable of other carnivorous cells, they seem plants. Withinto be theas specialized cells, the uptake as the dimorphologicallyffers from the differ patternentiated found specialized in glands and of other Nepenthaceae carnivorous [15 ]: inplants. these families,Within the a cells, multitude the uptake of small differs vesicles from the is foundpattern which found fusein Droseraceae to large endosomes, and Nepenthaceae partially displacing[15]: in these the centralfamilies, vacuole. a multitude In Sarracenia of small vesicles, fluorescent is found vesicles which seem fuse to to large be inconspicuous, endosomes, partially whereas FITC-BSAdisplacing accumulates the central invacuole. the cytoplasm In Sarracenia and, particularly fluorescent vesicles in the central seem to vacuole. be inconspicuous, Thus, the degradation whereas ofFITC-BSA the absorbed accumulates protein seems in the to follow cytoplasm a diff erentand pathway.particularly However, in the transportcentral vacuole. towards Thus, the vascular the systemdegradation is found of as the in Droseraabsorbedor proteinGenlisea seems[15]. to follow a different pathway. However, transport towards the vascular system is found as in Drosera or Genlisea [15].

FigureFigure 4. 4.Uptake Uptake of of the the fluorescent fluorescent protein protein FITC-BSA. FITC-BSA. The The yellow yellow color color shows shows FITC-fluorescence FITC-fluorescence in in the detectionthe detection wavelength wavelength 500–550 500–550 nm. (a )nm.Sarracenia (a) Sarracenia purpurea purpureaepidermal epidermal cells show cells no show uptake; no uptake; (b) Sarracenia (b) minorSarracenia;(c,d,f ) minorSarracenia; (c,d oreophila,f) Sarracenia;(e) Sarracenia oreophila; leucophylla(e) Sarracenia(the blueleucophylla color shows(the blue autofluorescence color shows of cuticleautofluorescence and , of cuticle detection and chlorophyll, wavelength de 626–672tection wavelength nm). Scale 626–672 bar: 50 µ nm).m. Scale bar: 50 µm.

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Figure 5. Fluorescent intensity in the cytoplasm of epidermal cell of Sarracenia purpurea and Sarracenia oreophilaoreophila (n(n == 20).

4. Materials and Methods 4.1. Plant Material 4.1. Plant Material The following Sarraceniaceae were studied (see Figure1): Sarracenia purpurea ssp. purpurea, The following Sarraceniaceae were studied (see Figure 1): Sarracenia purpurea ssp. purpurea, Sarracenia purpurea ssp. venosa, (= Sarracenia purpurea ssp. venosa var. burkii), Sarracenia Sarracenia purpurea ssp. venosa, Sarracenia rosea (= Sarracenia purpurea ssp. venosa var. burkii), Sarracenia psittacina, Sarracenia flava var. flava, Sarracenia leucophylla, Sarracenia minor var. minor, Sarracenia psittacina, var. flava, Sarracenia leucophylla, Sarracenia minor var. minor, Sarracenia oreophila var. alba, Sarracenia alabamensis (= ssp. alabamensis), Heliamphora nutans, oreophila var. alba, Sarracenia alabamensis (= Sarracenia rubra ssp. alabamensis), Heliamphora nutans, Darlingtonia californica. Darlingtonia californica. All test plants were nursed at the botanical collection of the HBLFA for horticulture and the All test plants were nursed at the botanical collection of the HBLFA for horticulture and the Österreichischen Bundesgärten, Vienna, Austria. During the experiments, plants were located at the Österreichischen Bundesgärten, Vienna, Austria. During the experiments, plants were located at the greenhouse of the Biocenter Althanstraße, University of Vienna, Austria. greenhouse of the Biocenter Althanstraße, University of Vienna, Austria. 4.2. Methylene Blue-Staining for Detection of Cuticular Pores 4.2. Methylene Blue-Staining for Detection of Cuticular Pores Cuticular pores were detected by using a 0.1–1% aqueous solution of methylene blue as described by AdlassnigCuticular et pores al. [15 ].were detected by using a 0.1–1% aqueous solution of methylene blue as described by Adlassnig et al. [15]. 4.3. Test for Proteases 4.3. Test for Proteases The presence of digestive enzymes (i.e., proteases) was tested with conventional photographic film,The as described presence byof Meyers-Ricedigestive enzymes [26], slightly (i.e., protease modified.s) wasSarracenia tested withpitchers conventional were isolated photographic from the plant,film, as cut described in halves by with Meyers-Rice a razor blade, [26], slightly functional modified. zone was Sarracenia selected, pitchers wetted were with isolated a 1% solution from the of theplant, protein cut in BSA halves (Bovine with a Serum razor blade, Albumin—cold functional ethanolzone was fraction selected, pH wetted 5.2, 96%,with Lota 1% # solution SLBWW2269, of the ≥ asprotein well asBSA Bovine (Bovine Serum Serum Albumin—lyophilized Albumin—cold ethanol powder, fraction 96%,pH 5.2, Lot ≥96%, #SLBJ8588V, Lot # SLBWW2269, both SIGMA as well Life ≥ Science)as Bovine in Serum tap water, Albumin—lyophilized in order to stimulate proteasepowder, secretion.≥96%, Lot Growth #SLBJ8588V, of trap both inhabiting SIGMA bacteria, Life Science) which mightin tap water, falsify in the order outcome to stimulate by producing protease additional secretion. proteases,Growth of wastrap inhibitedinhabiting by bacteria, adding which tetracycline might (Tetracycline.HCl,falsify the outcome research by producing grade, Serva additional Lot#35866, proteases, concentration was 0.1 inhibited mg per l).by Pieces adding of photographic tetracycline film(Tetracycline.HCl, ILFORD XPI 400 research were assembled grade, withServa the Lot#35866, gelatin emulsion concentration side onto 0.1 the tissuesmg per of thel). testPieces plants of andphotographic mostly fixed film with ILFORD staples XPI (see 400 Figure were3). assembled Subsequently, with these the gelatin prepared emulsion trap tissues side wereonto the incubated tissues inof athe humid test plants chamber and for mostly 10–48 h,fixed the filmwith wasstaples developed (see Figure by using 3). Subsequently, a conventional these developer prepared solution trap (Kodaktissues TMaxwere RSincubated developer). in a Secretion humid chamber of protease for led 10–48 to digestion h, the offilm the was gelatin developed emulsion by resulting using ina transparentconventional areas, developer whereas solution undigested (Kodak intact TMax gelatin RS de emulsionveloper). remains Secretion opaque. of protease led to digestion of the gelatin emulsion resulting in transparent areas, whereas undigested intact gelatin emulsion remains opaque.

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4.4. FITC-BSA Staining to Test for Endocytosis and Uptake FITC-BSA (Albumin, Fluorescein isothiocyanat Conjugate from Bovine, SIGMA Product Number A9771) contains the fluorescent dye FITC (molecular weight 389.4), which is conjugated with the protein BSA (Bovine Serum Albumin, molecular weight of about 66,000). A 1% FITC-BSA solution—diluted with tap water or, if available, with the native filtrated pitcher fluid, was applied. The experimental design was similar to Adlassnig et al. [15]: A droplet FITC-BSA solution was placed on those pitcher tissues where cuticular discontinuities had been found. The samples were incubated at 28–30 ◦C in humid chambers under dark conditions to prevent bleaching. After 18–48 h, the samples were thoroughly rinsed, and both surface and longitudinal hand sections were made and analyzed with a confocal scanning microscope (CLSM, Leica TCS SP5 DM-6000 CS). As negative control, a 1% BSA-solution was applied and subsequently treated and analyzed like the fluorescent test series. Fluorescence of FITC-BSA was excited with the 496 nm laser-line from the argon-laser and detected at 500–550 nm. Fluorescence intensity as well as cell area were quantified by using the Leica LAS AF Lite–Version: 3.3.10134, and compared between different treatments and stainings by using the statistical analysis software STATA 14.2. Since all samples deviated significantly from normal distribution (Skewness–Curtosis test), the non-parametric Kruskal–Wallis test with the post-hoc Dunn test were used to test for differences. p < 0.01 was regarded as highly significant. Cohen’s κ was used to assess the correlation between the presence or lack of digestion and endocytosis. The non-parametric Mann–Whitney Test was used to compare the area of permeable and impermeable cells.

5. Conclusions Most members of Sarraceniaceae produce protease, but only a few species of Sarracenia are capable of endocytotic nutrient uptake. Endocytotic nutrient uptake seems to have developed from membrane recycling in the frame of the exocytosis of enzymes. In Sarraceniaceae, physiologically specialized epidermal cells fulfil the same functions as morphologically differentiated glands in other carnivorous plants.

Supplementary Materials: The following are available online at http://www.mdpi.com/2223-7747/8/10/367/s1. Author Contributions: Conceptualization was realized by W.A. and M.K.-P.; statistics—formal analysis: W.A.; horticulture and plant material: M.E.; investigation and experiments: S.K., A.P., M.K.-P; supervision experiments: W.A., M.K.-P., I.L.; original draft preparation: W.A., M.K.-P., I.L.; review and editing: M.K.-P., W.A., I.L. All authors discussed the results and commonly contributed to this work. Funding: This research was supported by the Hochschuljubiläumsstiftung grant number H-237754/2014 to M.K.-P., and W.A. The “Back to Research Grant” of the faculty of life sciences of the University of Vienna to M.K.-P. is also greatly acknowledged. Open Access Funding was provided by the University of Vienna. Acknowledgments: The authors thanks Johanna Auer, Barbara Fux, Dylan Simon, Christian Kopeinig, Isabelle Meyer, Rebecca Steinacher, and Lisa Vrbecky for participating in this study. Special thanks are also due to Robert Wessely and his team from the HBLFA for horticulture and the Austrian Bundesgärten Schönbrunn for providing test plants. Thanks are also due to Brigitte Schmidt, Core Facility Cell Imaging and Ultrastructure Research, as well as to Thomas Joch und Andreas Schröffel from Department of Ecogenomics and Systems Biology, Biocenter Althanstraße 14, University of Vienna, Austria for taking care of the test plants during the diverse testing series for Sarraceniaceae. This work is dedicated to the 90th birthday of Walter G. Url, a pioneer in high resolution light microscopy of living plant cells. Conflicts of Interest: The authors declare no conflict of interest.

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