The development of the digestive glands and enzymes in the pitchers of three Nepenthes species: N. alata, N. tobaica, and N. ventricosa (Nepenthaceae)

The Development of the Digestive Glands and Enzymes in the Pitchers of Three NepenthesSpecies: N. alata , N. tobaica , and N. ventricosa (Nepenthaceae)Author(s): Andrew H. Thornhill, Ian S. Harper, and Neil D. HallamSource: International Journal of Plant Sciences, Vol. 169, No. 5 (June 2008), pp. 615-624Published by: The University of Chicago PressStable URL: http://www.jstor.org/stable/10.1086/533599 .

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THE DEVELOPMENT OF THE DIGESTIVE GLANDS AND ENZYMES IN THE PITCHERSOF THREE NEPENTHES SPECIES: N. ALATA, N. TOBAICA, AND

N. VENTRICOSA (NEPENTHACEAE)

Andrew H. Thornhill,1,* Ian S. Harper,y and Neil D. Hallamz

*School of Botany and Zoology, Australian National University, Canberra, Australian Capital Territory 0200, Australia;yMonash Micro Imaging, School of Biomedical Sciences, Monash University, Clayton, Victoria 3800, Australia;

and zDepartment of Biological Sciences, Monash University, Clayton, Victoria 3800, Australia

The development of digestive glands in pitchers of three species of Nepenthes (N. alata Blanco, N. tobaicaDanser, and N. ventricosa Blanco) was studied by LM and SEM, and the presence of digestive enzymes wasexamined by enzyme cytochemistry. Pitchers at various developmental stages were studied, corresponding toprepitcher inflation, early pitcher inflation, postinflation (before lid opening), and fully mature open pitcher.Pitchers from all species showed the presence of digestive glands in the pitcher cavity, and each speciesexhibited a unique gland structure. Digestive glands formed from epidermal cell division, most glands havingdirect connections with underlying vascular bundles. Enzyme presence was observed in both opened andunopened pitchers for all enzymes assayed and was localized in digestive glands. These results indicate that thegenus Nepenthes digestive glands are capable of producing a wide range of digestive enzymes both during andafter pitcher development. They are thus functionally carnivorous.

Keywords: development, digestive enzymes, glands, Nepenthaceae, Nepenthes, pitcher plant, SEM.

There are 9 families, 17 genera, and 550 species of plantsthat are considered carnivorous, growing in all continents ofthe world apart from Antarctica (Cheers 1992). The numer-ous trapping structures and methods that plants have evolvedhave been well documented (Arber 1941; Lloyd 1942; Juni-per and Burras 1962; Adams and Smith 1977; Juniper et al.1989). DNA analysis has indicated that various trap formsarose independently in six angiosperm lineages (Albert et al.1992), and pitcher plant families are not considered closelyrelated to one another (DeBuhr 1975). The three plant fami-lies that develop specialized pitcher traps to catch prey are Sar-raceniaceae, Nepenthaceae, and Cephalotaceae. Within thesefamilies, there are five genera, Sarracenia, Darlingtonia, Heli-amphora, Nepenthes, and Cephalotus, which represent an ex-tensive worldwide distribution, including North and SouthAmerica, Madagascar, Southeast Asia, and Australia. The ge-nus Nepenthes is distributed from northern Australia through-out Southeast Asia to southern China, the majority of speciesgrowing in the tropical conditions of Borneo and Sumatra(Clarke 1997). There are 82 species in this genus, of which 74occur in the Malesiana region (Jebb and Cheek 1997).

The pitchers of Nepenthes initiate as an extension and infla-tion of the leaf midrib, and pitcher structure has been describedextensively (Arber 1941; Lloyd 1942; Adams and Smith 1977;Moran 1996; Moran et al. 1999). During development, thepitcher is filled with sterile fluid (Jentsch 1972; Tokes et al.1974; Heslop-Harrison 1978), while the lid is sealed shut to

the rest of the pitcher by interconnecting hairs (Owen andLennon 1999). Once the pitcher has nearly completed devel-opment the lid opens to reveal a ridged peristome, ending insharp downward-pointing teeth on the inside and an even col-lar on the outside (Lloyd 1942). The inside of the pitcher canbe divided into three zones (Lloyd 1942; Parkes 1980; Schulzeet al. 1999). Zone 1, the lid, covers the opening of the mouthand contains nectaries to attract insects (Parkes 1980). Zone 2is recognized as the upper part of the pitcher containing de-tachable wax scales that block up the hairs on the footpads ofinsects, thereby not allowing the use of microscopic spaces asfootholds particularly on vertical surfaces (Juniper and Burras1962). Zone 3 is the lower part of the pitcher that contains di-gestive glands and is the reported area of secretion and absorp-tion (Juniper et al. 1989).

Although the overall structure of Nepenthes pitchers iswell known, there is little information on the formation ofdigestive glands (Owen and Lennon 1999; Nishida and Ta-kahashi 2001; Gaume et al. 2002) and their associated en-zymes during pitcher development. Species of Nepenthes arecapable of secreting enzymes to digest prey (Nakayama andAmagase 1968; Amagase et al. 1969, 1972; Amagase 1972;Jentsch 1972; Tokes et al. 1974; Owen and Lennon 1999;An et al. 2001, 2002), but there is scant literature on thesynthesis of these enzymes in unopened pitchers. To addressthe issue of gland formation and enzyme activity during pitcherformation in Nepenthes, we studied gland structure devel-opment by LM and SEM and in conjunction conducted cy-tochemical assays for the localization of proteases, esterases,and acid phosphatases in both the glands and surroundingtissues.

1 Author for correspondence; e-mail: [email protected].

Manuscript received September 2005; revised manuscript received October

2007.

615

Int. J. Plant Sci. 169(5):615–624. 2008.

� 2008 by The University of Chicago. All rights reserved.

1058-5893/2008/16905-0003$15.00 DOI: 10.1086/533599



Material and Methods

Plants of three Nepenthes species (N. alata Blanco, N. to-baica Danser, and N. ventricosa Blanco) were grown on-sitein sphagnum moss without fertilizer and under natural lightin a humidity-controlled (65%–80%) glasshouse (14�–39�C).Pitchers were sampled at four different stages. Nepenthes

pitchers develop at varied rates, depending on species andgrowing conditions (Clarke 1997), and therefore pitchers wereselected for study based on the following morphological crite-ria using visual selection (fig. 1): preinflation of the leaf tendril(stage 1), early inflation of the pitcher (stage 2), postinflationof the pitcher with closed lid (stage 3), and open pitcher(stage 4).

Fig. 1 The four Nepenthes pitcher stages studied. a, Preinflation of pitcher. b, Early pitcher inflation. c, Closed pitcher, postinflation. d, Fully

mature, open pitcher. Bar ¼ 1 cm.

Fig. 2 Stage 1 pitcher anatomy. a, Transverse section, young Nepenthes alata pitcher shows no apparent digestive glands. b, Viewed at low

magnification, developing pitchers are covered by a dense mass of trichomes. c, Magnified view of immature N. tobaica pitcher shows divided cellsof pitcher epidermal surface. Bar ¼ 50 mm in a; 250 mm in b; 25 mm in c.

616 INTERNATIONAL JOURNAL OF PLANT SCIENCES



Light Microscopy

Tissue used for LM was dissected in a solution of 5% glu-taraldehyde fixative made from 25% glutaraldehyde mixedwith 0.03 M PIPES buffer (pH 6.8–7.0) in a 1 : 4 glutaralde-hyde : buffer ratio; 0.1 g of caffeine was added for every 10mL of glutaraldehyde immediately before use to stop pheno-lics from leaching into the cytoplasm during fixation (Muel-ler and Greenwood 1978). Fixed tissue was rinsed in bufferand dehydrated through a graded ethanol series (10%–100%)at 10% intervals, followed by infiltration over 4 d with LRWhite medium grade. During infiltration, the resin : alcoholratio was increased daily by 33%, with three changes of resinper day, and samples were immersed in 100% resin for the fi-nal 2 d to improve infiltration. Polymerization was conductedunder a UV light in an oxygen-free atmosphere for 24 h. Resinblocks were sectioned at 2-mm thickness with a Leica Ultrami-crotome using a glass knife, and sections were stained usingtoluidine blue (pH 4.5). Whole pieces of stage 4 tissue sampleswere fixed and dehydrated through a 10% ethanol series and

then cleared using the method of Shobe and Lersten (1967).The transparent tissue was then stained with chlorozal blackE to quantify digestive gland–vascular bundle connectivity. Alltissue was examined with an Olympus Provis AX70 micro-scope equipped with an Olympus DP50 digital camera andAnalySIS (Soft Imaging System, Marburg, Germany) software.

Scanning Electron Microscopy

SEM tissue was fixed using the same procedures as for LMfollowed by critical-point drying using a Balzers CPD 030.Dried samples were mounted on aluminum stubs with Elec-trodag glue and sputter-coated with gold using a BalzersSCD 005. Specimens were viewed at 10–15 kV on a HitachiS570 SEM.

Enzyme Analysis

Fresh tissue fragments (ca. 5 mm2) were placed in reactionmixtures for specific staining of esterase (Pearse 1972) and

Fig. 3 Stage 2 pitcher anatomy. a, Transverse section, Nepenthes tobaica pitcher showing young digestive glands that form from division

of epidermal cells. b, Transverse section, N. ventricosa pitcher show younger rounded digestive glands. c, Longitudinal section, young pitcher

showing first appearance of glands. Note gradient of gland maturity. d, Glands toward top of pitcher are less mature than those at base and were

noticeable in hollow depressions of epidermis. e, Glands at base of pitchers show maturity and delineation from surrounding epidermal cells.Bar ¼ 50 mm in a and b; 500 mm in c; 10 mm in d and e.

617THORNHILL ET AL.—GLANDS AND ENZYMES OF NEPENTHES



acid phosphatase (Pearse 1968). Stained tissue fragments wererinsed in distilled water, fixed in 3% phosphate-buffered glu-taraldehyde overnight at room temperature, dehydrated in a10% ethanol series over 24 h, cleared in xylene, and perma-nently mounted in MOWIOL (6 g glycerol; 2.4 g MOWIOL4-88; 6 mL distilled H2O; 12 mL phosphate-buffered saline).Control tissue was boiled for 5 min to inactivate tissue en-zymes. Paired enzyme and control tests were examined underthe LM with the same exposure setting.

Protease presence was investigated using a revised versionof the substrate film technique of Fratello (1968). The colorfilm is made from nitrocellulose backing on which there arethree layers of gelatin, containing dyes of each primary colorsuperimposed (Pearse 1980). Unexposed color reversal film(Kodak Ektachrome Film) was developed and then moistenedin distilled H2O for 1 h. Fresh tissue samples were pressedfirmly into contact with the film and incubated at 37�C for

24 h. Tissue fragments were then removed and the film rinsedin distilled water to remove any digestion products. Proteaseactivity is revealed by the resulting color after progressivedigestion of the colored layers. Lack of protease activity is in-dicated by the film remaining black or brown, whereas diges-tion progressively changes the film to red or white (completedigestion of all pigment layers). Results were recorded bylight microphotography using similar settings for all images.

Results

Digestion Zone Development Morphology

Stage 1. Developing digestive glands were absent fromthe epidermal layer in all species (fig. 2a). The epidermal layerformed as a single row of vertically elongated, rectangular-shaped cells. A high percentage of cell division appeared in

Fig. 4 Stage 3 pitcher anatomy. a, Developed gland of Nepenthes ventricosa lying level with epidermis. Head gland cells appear as small

square cells, subtended by two layers of highly compressed gland cells. b, Mature gland of N. tobaica consisting of four cell layers. Elongated

gland head cells are darkly stained, while deeper gland cells are flattened and do not stain as intensely. Gland is in contact with a nearby vascular

bundle by vessels. c, Surface view shows mature glands of N. ventricosa. d, At higher magnification, gland delineation from the epidermis was notnoticeable. Bar ¼ 50 mm in a, b, and d; 250 mm in c.




the mesophyll layers, and many vascular bundles were alsoobserved. A dense surround of darkly stained trichome cellsformed around the outer epidermis. SEM showed an epider-mal surface layer devoid of developing glands. An overviewof longitudinally sectioned pitchers exhibited a high densityof trichomes covering the outer epidermis (fig. 2b). At highermagnification, the surface appeared smooth but slightly cob-bled on account of the patterning of square epidermal cells(fig. 2c).

Stage 2. Cross-sectioned pitchers of this stage revealedthe first indication of distinct glands (fig. 3a). Immature glandsformed in epidermal depressions from the periclinal divisionof epidermal cells. In cross section, glands of Nepenthes ven-

tricosa were seen as a double layer, four to five cells in length,protruding above the epidermal layer (fig. 3b). Common celldivision was still evident in the mesophyll layers. SEM of sec-tioned pitchers clearly showed a gradient of gland maturity(fig. 3c): glands at the base of the pitcher were fully matureand were separated from the surrounding epidermal surface(fig. 3e), while less-developed glands were present at the topof pitcher and were obviously still forming in epidermal de-pressions (fig. 3d).

Stage 3. In general, most digestive glands of stage 3pitchers appeared to be fully mature. Periclinal and anticlinalcell divisions formed mature glands, consisting of three orfour cell layers. Sectioned gland head cells appeared vertically

Fig. 5 Stage 4 pitcher anatomy. a, Transverse section of opened Nepenthes tobaica pitcher gland showing darkly stained gland head cells withthick cell walls and cytoplasm. b, Gland structure of N. ventricosa appears different from that of the other two Nepenthes species. Gland head

cells appear flattened and squarer, lying level with epidermal surface. A significant network of vascular vessels can be seen adjacent to digestive

gland. c, Vascular traces and glands of N. tobaica stained with chlorazol black E. d, SEM, N. alata opened pitcher showing a range of development

of digestive glands. e, At higher magnification, material was seen on digestive glands (possibly animal detritus or microfloral colonies). Bar¼ 50 mmin a, b, and e; 100 mm in c; 200 mm in d.




elongated and rectangular in shape in Nepenthes alata andNepenthes tobaica and smaller and more cuboidal in N. ven-tricosa (fig. 4a, 4b). Subtending these cells were layers of hori-zontally flattened gland cells. Lip-shaped epidermal cellspartially covered digestive glands. Vascular vessels lying in themesophyll layer that had terminal ends feeding directly into thebottom cell layer of digestive glands were seen in N. tobaicasections. Most glands appeared mature (fig. 4c). At highermagnification (fig. 4d), glands of N. ventricosa appeared as aslight protrusion above the epidermal surface, but gland delin-eation was less obvious when compared with the other twoNepenthes species.

Stage 4. Adult pitchers of N. alata and N. tobaica exhibitedglands that were distinctly cup shaped, although N. tobaicaglands were distinguishably more rounded (fig. 5a). In con-trast, mature digestive glands of N. ventricosa were long, flat-tened disks, ca. 20 cells in length (fig. 5b). In many glands,direct connection with underlying vascular bundles were ob-served. The network of vascular bundles was also signifiedby protoxylem and phloem connections. In serial sections (se-quential 2-mm sections), vascular connectivity with digestiveglands appeared in only three or four sequential sections,

suggesting that such connections are localized and only twoto three cell layers thick. Staining of whole-tissue pieces withchlorazol black E revealed that connectivity between vascularbundles and overlying glands is very common (fig. 5c). Thevascular network connecting glands appears to be complexwith much branching occurring. SEM showed a uniformmaturity of digestive glands (fig. 5d). Delineation of glandsfrom the pitcher epidermis was observed in mature glandsof N. alata and N. tobaica (fig. 5e) but was not obvious inN. ventricosa.

Enzyme Cytochemistry

APase and esterase. Enzyme staining of tissue from openedpitchers resulted in strong positive reactions in two Nepenthesspecies (N. alata and N. tobaica; figs. 6c, 7c) but was onlymoderately strong in N. ventricosa. Within the digestive glands,cells showed a very strong reaction in the peripheral areas ofthe cell wall. Unopened pitchers also exhibited positive stain-ing reactions for APase and esterase, but the intensity of stain-ing was noticeably weaker in comparison to open pitcherassays (figs. 6a, 7a). Closed pitchers stained more strongly inN. alata than in the other two species. Occasionally, we noted

Fig. 6 Nepenthes tobaica APase assays. a, Stage 3 APase test showing patchy positive staining in some glands. b, No enzyme staining in a stage

3 APase control heat-inactivated sample. c, Stage 4 APase test showing strong staining activity occurring in cytoplasm of glands cells. d, A stage 4APase control test, indicating no enzyme activity. Bar ¼ 100 mm.




partial APase staining of glands, which exhibited patchy de-posits of pale red-orange stain (fig. 6a). This may be an indica-tion that individual digestive glands cells begin synthesizingenzymes independently rather than starting production con-currently. In contrast, the heat-inactivated tissue had virtuallyno staining in both unopened and opened pitcher assays (fig.6b, 6d; fig. 7b, 7d).

Protease. Protease tests produced the same consistent re-sults as APase and esterase tests. Digestion of two layers ofthe film substrate occurred in unopened pitchers (fig. 8a).Opened pitcher film tests showed complete digestion of filmlayers solely by glands (fig. 8c) and partial digestion of thesubstrate layers by surrounding areas. Heat-inactivated con-trol test pitchers exhibited no significant digestion of thelayers over the gland areas (fig. 8b, 8d). Some slight changeswere occasionally observed in the film representing generalpitcher areas. This may have been due to mechanical abra-sion or leaching of incompletely denatured enzyme. However,the gland areas remained significantly darker relative to thesurrounding area, indicating that heat inactivation was suc-

cessful. These results indicated the presence of protease inNepenthes pitchers and that activity begins before pitcherlids open.

Discussion

The pitchers of Nepenthes are modified leaf structuresused to attract and trap animals, predominately arthropods,and then digest and absorb these prey for nutritional gain.This study focused on two facets of carnivory in Nepenthes,digestive glands and enzymes and specifically their appear-ance during pitcher development.

This study indicated that glands do indeed form from thedivision of epidermal cells, in conjunction with the initial in-flation of the pitcher cavity. Our study shows a gradient ofgland development evident in stage 2 pitchers, with matureglands appearing first at the base of the pitcher. Owen andLennon (1999) suggested that larger-sized cells less intenselystained with toluidine blue indicated the site of future glanddevelopment. Toluidine blue–stained sections in our study

Fig. 7 Nepenthes alata esterase assays. a, Stage 3 esterase assay, indicating weak positive staining in digestive gland cell peripheral regions and

no staining of epidermal surface. b, No staining occurs in glands of heat-inactivated stage 3 sample. c, Positive dark brown esterase staining occurs

in gland cell peripheral and cytoplasmic areas of a stage 4 pitcher. d, Stage 4 esterase control shows weak positive staining of gland cell peripheral.

Bar ¼ 50 mm in a and b; 100 mm in c and d.




revealed epidermal cells of uniform size and staining intensity,and no cells were observed to support Owen and Lennon’ssuggestion.

Direct connections between vascular bundles and digestiveglands were observed in sections of both unopened and openpitchers. Direct contact between glands and vascular bundleswas not observed in every gland sectioned, but staining ofwhole stage 4 tissue pieces showed that each digestive glandis indeed located on or connected to a vascular bundle, indi-cating a clear pathway for fluid and nutrient transfer. Giventhe small diameter of vascular cells when sectioned longitudi-nally, the likelihood of sectioning through such connectionsis minimal, and thus only a small number of such connectionswere observed in sections. Previous studies have mentionedclose association between digestive glands and vascular bun-dles. Two studies, by Owen and Lennon (1999) and Owenet al. (1999), showed that the terminal ends of xylem tracesended beneath the gland base, but our work indicates thatvascular bundles connect directly to digestive glands. The con-nection suggests a likely source and pathway of fluid that ac-cumulates in unopened pitchers. This fluid has been shown to

be sterile in previous studies (Jentsch 1972; Tokes et al.1974). The use of glands and their vascular connections intransporting nutrients back into the plant after prey digestionis also likely, as was previously suggested by Owen et al.(1999), who observed nutrient loading from digestive glandsinto vascular strands. Schulze et al. (1999) searched for feed-ing pathways between glands and vascular bundles using fluo-rescent dyes and clearly demonstrated apoplastic transportthrough the xylem to glands and symplastic transport fromthe gland into subtending cells. Venugopal (1999) proposedthat there were both digestive and absorptive glands found inpits formed by epidermal lips that participate in the digestionof food materials. The simple cytochemical dyes used in ourstudy could not show the entire transport pathway used byNepenthes pitchers. This does not mean that such a pathwaydoes not exist, but because the dye was not loaded into thepathway by specific membrane transporters, it was not ob-servable. Further investigations on the direct connections be-tween glands and vascular bundles may reveal the pathwaythat Nepenthes pitchers use to transport nutrients back intothe plant.

Fig. 8 Nepenthes ventricosa protease film tests. a, Stage 3 protease film shows varied digestion of layers by glands. Note digestion halo around

some glands. b, Partial digestion of top film layer of a stage 3 control film. Heat-inactivated glands show no digestion activity. c, Stage 4 proteasefilm shows complete digestion of all substrate layers by glands. Red areas indicate digestion of top film layer. d, Stage 4 protease control film shows

some color change, but gland activity appears minimal, most appearing as black dots. Bar ¼ 100 mm.




A preliminary study of five different Nepenthes species (N.ampullaria, N. bicalcarata, N. gracilis, N. rafflesiana, and N.reiwardtiana) proposed that each species had a unique glandstructure and shape (Nishida and Takahashi 2001). Structureranged from highly stacked gland cells with large digestivehead cells, sunken into the surrounding epidermal layer (N.reinwardtiana), to greatly flattened digestive gland cells thatappeared level with the surrounding epidermal cells (N. grac-ilis). The results from our study confirmed that each speciesdoes have a unique gland formation and structure. The cellu-lar construction of glands for N. alata and N. tobaica appearedsimilar, both having three or four cell layers in a depressionof the epidermis; however, head cells of the glands appearedslightly different, creating a notable difference in gland shape.The digestive glands of the other species studied, N. ventricosa,had a more distinctive structure, the glands appearing greatlyflattened in a shallow epidermal depression, lying almost co-planar with the surrounding epidermal cells. Individual glandcells appeared flattened, and the head cells appeared smallerand cuboidal in shape compared with those of the other twospecies. Unique gland structure may have some unknownfunctional role in prey trapping and could also be used as adiagnostic feature in taxonomy, although the steps required toachieve sections of digestive glands make it a time-consumingcharacter to describe.

Cytochemical tests performed on the three Nepenthes spe-cies demonstrated that each plant is capable of enzyme pro-duction and that these enzymes are localized to, and thereforeprobably produced by, the digestive glands. Previous studies

concentrating on the contents of the pitcher fluid have showna wide array of digestive enzymes, including phosphatases,proteases, and chitanase (Amagase et al. 1972; Jentsch 1972;Tokes et al. 1974; Higashi et al. 1993), while direct stainingof glands has also shown enzyme presence (Parkes 1980; Anet al. 2001, 2002). However, analysis of enzyme fluid fromopen pitchers can be questionable as any enzymes identifiedmay have an exogenous origin, for example, by bacteria orfungi. We studied three generic enzyme groups more closelywith respect to their production in the developing pitcher. Ourresults support the assertion by Heslop-Harrison (1978) thatthe gland is the site of production of digestive enzyme. This isunequivocally demonstrated by the presence of gland enzymesin both open and unopened pitchers. Heat-inactivated tissuewas used to confirm the cytochemistry assay. Higashi et al.(1993) suggested that enzyme activity is a response to prey ac-cumulation in pitchers, but this is strongly refuted by our ob-servations of enzymes in unopened pitchers. That the enzymesare restricted to gland cells in unopened pitchers and then in-crease (stronger cytochemical staining) in opened pitchers,where they are still restricted to gland cells, allows us to con-clude that the enzymes are indeed of plant origin and are pro-duced in increasing amounts during the maturation of thepitcher.

Acknowledgments

We would like to thank the two anonymous reviewers fortheir comments on a previous version of this manuscript.

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Documents

The development of the digestive glands and enzymes in the pitchers of three Nepenthes species: N. alata, N. tobaica, and N. ventricosa (Nepenthaceae)