Literature Review

Part II: Digestive Activities of Carnivorous Plants

From the 1975 Masters Thesis: Bacteriological and ecological observations on the northern pitcher plant, Sarracenia purpurea. John A. Lindquist. Department of Bacteriology, University of Wisconsin, Madison, WI.

A. Plant Glands and Leachates

Plant glands are specific cells or tissues which function in the synthesis and release of enzymes or other physiological compounds. Glands have been extensively reviewed by Luttge (1971) for plants in general. A variety of specific substances has been shown to be released by glands (Luttge, 1971; Sakai, 1974):

Physiological products may be transported from the plant tissue into the environment by various means. Nectar sugars, for example, depending on the type of plant, may not only be released from the external membrane of gland cells, which show a high degree of specialization, but also from ruptured or decomposing cells and from open stomata "connected" to sieve tubes by unspecialized parenchyma (Luttge, 1971). Malic acid, noted previously in the discussion on the acidity of Sarracenia purpurea pitcher fluid, is secreted by the glandular hairs of certain non-carnivorous plants (Blakeman, 1971). Released plant products are classified according to their metabolic origins (Luttge, 1971):

A variety of substances can be leached from plants into drops or films of water upon their leaf surfaces, originating from rain, dew, mist or fog. Under proper conditions (including age and health factors), perhaps anything soluble can be leached from the leaves. Observed leachates have included all essential minerals, all plant amino acids, free sugars (especially sucrose), pectic substances, sugar alcohols, many organic acids, gibberellins, vitamins, alkaloids and phenolic substances (Tukey, 1971). Leachates may thus serve as nutrients for the growth of epiphytic and pathogenic bacteria and fungi on leaf surfaces.

Stomata are not believed to be the primary pathway of nutrient loss, as leaching can also occur through the cuticle. Channels appear to exist in the cuticles of some plants through which wax can pass to the exterior, and exudates may also pass through (Blakeman, 1971). A highly-developed waxy cuticle tends to waterproof the leaf and thus decrease leaching (Holloway, 1971).

B. Sarracenia and Darlingtonia – Digestion and Absorption

B1. Early Studies

Digestive and absorptive processes in the North American pitcher plants, Sarracenia and Darlingtonia, were intensively studied in the late nineteenth and early twentieth centuries; very little work has been done more recently. Comprehensive reviews of early work have been made by Hepburn (1927), Lloyd (1942) and Plummer and Kethley (1964).

In 1829 Burnett (cited in McKee, 1962) speculated on the pitchers of Sarracenia: "The water in these receptacles, impregnated by the half-decomposing animal matter, doubtless affords a highly nutritive and invigorating diet to the plant, for it is well known that the drainings of dunghills give a powerful stimulus to vegetables, as the rainwater that percolates there-through dissolves and carries with it, in solution, much of the nutritious and more subtle ingredients of manure; and as the food of plants is chiefly, if not wholly, absorbed in the fluid state, the more soluble manures are ever the best conducive to their growth. Nor must the nitrogen thus afforded to the prehensile plants be overlooked in the account, when we know how potent an excitant ammonia is to the vegetable frame." That ammonia is readily absorbed by S. purpurea pitcher leaves was shown by Higley (1885).

Batalin (1880), working with greenhouse plants of Sarracenia and Darlingtonia, noted an exfoliation of the cuticle of cells in contact with insects. His observations for S. flava, for example, were made in the lowermost zone of the pitcher which, below the detentive hairs, appears to consist of a smooth area without glands, somewhat equivalent to zone 5 in S. purpurea but about six centimeters in length in a fully grown leaf (Lloyd, 1942). When an insect adhered to the epidermal cells, the cuticle was cast off from parts of the cells in contact, apparently being assisted in their removal by yellowish bubbles of a secreted substance. Similar results were seen in the lower area of S. purpurea pitchers. Batalin concluded that the detached cuticle, which normally resists penetration of substances into the cell, permits the entrance of substances into the plant. Batalin incorrectly stated glands to be absent from Sarracenia and did no experiments to prove digestion actually did take place. He apparently did his experiments on dry pitchers, uncommon in the field.

Schimper (1882) studied wild S. purpurea which contained water. Bacterial action was definitely associated with the decomposition of insects and meat. The epidermal and subepidermal cells of the detentive-absorptive zone in "fed" pitchers were observed to lose the chlorophyll granules. The protoplasm, acquiring a greater power of imbibition, withdrew water from the vacuolar sap, leaving the colloidal tannin in the now strongly-refractile cell vacuole. Imbibition of extracellular organic matter from insect decomposition was therefore suspected.

Fenner (1904) added flies to the lowermost part of a pitcher of Sarracenia flava without added water and noticed a mucilaginous secretion pouring out of the pitcher lining and digesting the insect bodies in contact with the lining in the course of several hours. The mass of insects became saturated with the fluid, and putrefaction was entirely absent until this "absorptive" zone was filled and additional insects began collecting in the hairy "detentive" zone. Thus S. flava was shown to be a truly insectivorous plant with a digestive enzyme. The action of glands, later shown in S. flava to lie above the "detentive" zone, was not indicated and was perhaps not suspected at the time.

B2. Studies of Hepburn, Jones and St. John – Secretion and Absorption

The most comprehensive studies of digestion and absorption in the pitcher plants Sarracenia and Darlingtonia were done by Hepburn, Jones and St. John (1927).

The authors made exhaustive studies on secretion and absorption in pitchers of S. alata, S. flava, S. leucophylla and D. californica. Mechanical stimulation, including the dangling of glass beads and gentle brushing on the cavity walls, failed to produce any secretion by the pitchers. However, there was a marked increase in the amount of pitcher fluid upon stimulation by milk, beef broth and some inorganic salt solutions, especially potassium ferrocyanide. Stimulation with cubes of raw beef, but not cooked beef, was successful. The nature of the additional liquid was not investigated. Upon the addition of dilute acid (hydrochloric or acetic) or dilute alkali (sodium hydroxide) a net loss of fluid by absorption was generally seen for the Sarracenia species but not for Darlingtonia. Several days after the addition of the acid or alkali, the fluids of the open pitchers of all four species returned to their normal reaction: acidic for S. flava and neutral for the other species (Jones and Hepburn, 1927). Apparently no water control was run for comparison at the same time. In separate experiments with these species, water, by itself, underwent absorption (Hepburn, St. John and Jones, 1927).

Thus it appeared that nutritive substances, especially in solution, would cause a net increase in the amount of fluid, possibly due to "digestive juices." Also, marked acidic or basic reactions seen during bacterial decomposition of insects or other food could stimulate absorption by the pitcher. S. purpurea was unfortunately omitted from these studies.

The three Sarracenia species cited above plus S. purpurea were used by the authors in the study of other specific substances absorbed by the pitchers. In these species, nitrogenous compounds (e.g. urea, ammonium chloride, peptone and egg albumin) in aqueous solutions were seen to be absorbed. The nitrogenous compounds usually moved into the pitchers faster than the water. When a neutral phosphate buffer, used to prevent the escape of volatile nitrogenous compounds, was used for S. purpurea in addition to the nitrogenous solution, the nitrogenous compound was seen to be absorbed into the pitcher, while there was a net increase in the volume of the liquid. When only the phosphate solution was added to S. purpurea pitchers, the water was absorbed at a faster rate than the phosphate. Lithium ion was absorbed from a neutral lithium citrate solution introduced into S. purpurea pitchers.

From these studies it was indicated that products formed by proteolysis of the prey, as well as phosphates and probably other inorganic materials, are absorbed by the pitchers and utilized by the plant (Hepburn, St. John and Jones, 1927).

B3. Studies of Hepburn, Jones and St. John – Digestive Enzymes

Hepburn and Jones (1927a) examined the possibility of specific digestive enzymes occurring in the pitcher cavities, especially protease; however, no mention was made of the "digestive" glands. Species of Sarracenia (except S. oreophila) and Darlingtonia californica were studied. In vitro tests were made on the naturally-occurring fluid from both opened and unopened pitchers, with and without the addition of weak solutions of hydrochloric acid or sodium carbonate (both used as pH-related activators for the enzymes) and always in the presence of the bactericide tricresol. The protein substrates used were carmine fibrin, edestin, casein and coagulated egg albumin. Careful note was made of the amounts of substrate and solution used; no artificial stimulation for extra secretion was made.

A plant-produced protease was found in the fluid of closed pitchers of five of the species as follows: An acidic protease was found in S. flava and S. minor, and an alkaline protease was found in S. oreophila, S. alata and S. rubra (Hepburn and Jones, 1927a). As closed pitcher leaves are internally sterile, there was no contribution of enzymes by bacteria.

In open pitchers, an acidic protease was found in S. flava, and an alkaline protease was found in S. leucophylla and S. alata. Protease active on either acidic or alkaline substrates was observed for S. minor (Hepburn and Jones, 1927a). Possible contribution of pre-existing protease by bacteria was not mentioned.

Fluids from closed pitchers of S. psitticina and S. purpurea were not studied, as the small amounts of these fluids prohibited their recovery. To each of fifty open S. psitticina pitchers (free of captures) 0.5 ml of water was added. After 4 hours, the water was withdrawn from the pitchers, pooled and tested in vitro for protease activity. No conclusive protease activity could be detected even after prolonged incubation of the test for 4 months (Hepburn and Jones, 1927a).

Open pitchers of S. purpurea were washed out completely, emptied and filled with 10 to 15 ml of water. After 10 or 15 days the water was withdrawn, pooled and tested. Protease activity was found under alkaline but not acidic or neutral conditions (Hepburn and Jones, 1927a). A major criticism of this experiment can be raised in that 10 or 15 days is plenty of time for bacteria to arise in a washed-out pitcher, and during this time extracellular bacterial proteases could be produced which could be detected in the in vitro test for protease.

Fluids from field pitchers of S. purpurea were also tested, and a proteolytic enzyme was found which was most active in alkaline conditions (Hepburn and Jones, 1927a). As before, bacterial enzymes were not considered and would certainly be present.

Bacteriology textbooks of this time (the 1920's) recognized extracellular bacterial "ferments" (enzymes), but Hepburn and Jones did not consider them worthy of discussion in their work on pitcher plant-associated proteases except for Darlingtonia californica. D. californica was tested with a wide variety of substrates, and no enzyme could be shown in the fluid from closed, plugged or open pitchers except for a few samples from open pitchers. In these exceptions, where digestion was seen upon prolonged incubation of the protease test, the possibility of bacterial enzymes contributing to the pitcher fluid was recognized. Reference was made to earlier researchers who remarked on the offensive, putrid odors emanating from clusters of D. californica which were actively entrapping and digesting insects (Hepburn and Jones, l927a; Hepburn, 1927). Since these studies, the absence of glands in Darlingtonia has been definitively proven (Lloyd, 1942).

When various substrates (casein, egg white, beef) were added to closed, plugged and open pitchers of D. californica, no change was observed upon observation one to twelve days later (Hepburn and Jones, 1927a). That there was no change in the open pitchers seems surprising, but apparently most of the tests on open pitchers were done on those which had recently opened, and perhaps not enough bacteria were present to cause any noticeable change.

B4. Studies of Hepburn, Jones and St. John – Immobilization of Insects

Samples of fluid from closed and open pitchers of S. alata, S. flava and S. leucophylla were seen to cause a rapid cessation of movement of insects dropped into the fluid; water was used as the control. The fluid from open S. flava pitchers was also found to have a considerably lower surface tension than water; thus, insects would be expected to drown faster in the pitcher fluid. However, the fluid from D. californica did not differ from water with respect to halting the notions of insects or to surface tension (Jones and Hepburn, 1927).

B5. Recent Studies

No detailed studies have been made since those of Hepburn, Jones and St. John on the digestion and absorption of Sarracenia and Darlingtonia, until the studies of Plummer and Jackson (1963) and Plummer and Kethley (1964) on S. flava.

Foliar absorption of amino acids and mineral elements was proven by Plummer and Kethley (1964) on S. flava. With auto-radiography, an uptake of radioactive elements – zinc, iodine, cobalt, sulfur, calcium and phosphorous – was observed. Ants which had been fed radioactive phosphorous and sulfur were fed to pitchers, and these elements were found to migrate into the plant tissues from the decaying ants.

By the use of chromatography of the fluid, leaves and roots, an increase in the number of free amino acids was detected in the plant tissues after insects or solutions of amino acids were fed to the pitchers. Twenty of the twenty-four amino acids used were completely removed from the fluid within two days; the possibility that bacteria consumed some of the amino acids was considered. A decrease in the types of free amino acids from the plant tissues at the end of two weeks was thought to be due to metabolism by the plant (Plummer and Kethley, 1964).

Four kinds of enzymatic activities involved in insect digestion were postulated by Plummer and Jackson (1963) for S. flava and similar species: (1) acid hydrolysis, (2) plant enzyme hydrolysis, (3) hydrolysis by extracellular bacterial enzymes and (4) autolysis of the insect tissue. Acid hydrolysis in the initial stages of digestion could be suspected as evidenced by the general finding of acidic conditions in the pitcher fluid; Higley's (1885) report of malic acid in this regard may be recalled. A plant-produced protease has been considered significant in S. flava and related species by Hepburn and Jones (1927a). Concerning the contribution of insect autolytic enzymes, most insect proteinases are active under neutral or alkaline conditions (Gilmour, 1961), in the range of active insect digestion found by early investigators.

C. Non-Pitcher Plants – Digestion and Absorption

In contrast to the North American pitcher plants, considerable recent work has been done on the glands and digestive enzymes of other carnivorous plants. Some of this research is summarized here.

Scala, et al. (1969) analyzed the products of the digestive glands of Dionaea, stimulating the leaf with cubes of gelatin. Secretion started within 24 hours and continued for several days. Enzymes with acidic optima were noted: proteinase (similar to papain), phosphatase, deoxyribonuclease and possibly amylase. Aseptic conditions with regard to the leaves and digestive fluid were maintained but were not detailed in the paper.

Heslop-Harrison and Knox (1971) studied the glands of Pinguicula. Stalked glands, bearing a muco-polysaccharide secretion, are concerned with insect capture and also contain an amylase. The sessile glands are specialized for protease secretion. Both types of glands produce acid phosphatase, esterase and ribonuclease. After stimulation by nitrogenous materials, the glands were observed to secrete fluid within one hour. Using autoradiography, digestive products were seen to enter the leaves within two hours and to start moving out of the leaves within twelve hours. The glands were observed to function not only in secretion but also resorption.

Harder (1967) studied the possibility that antiseptic substances were produced by carnivorous plants in order to decrease the interference of bacteria. Dionaea, Pinguicula, Drosera and Darlingtonia leaves were immersed in sterile media to which were added bacteria. No antiseptic action was noted.

According to Luttge (1971), Nepenthes glands, found in the lower third of the pitcher cavity, serve the dual function of secretion-excretion and absorption. Noted at the pitcher wall-cavity interface, as suggested by autoradiographic studies, were the following activities:

In a series of three papers by Amagase and associates (Nakayama and Amagase, 1968; Amagase, Nakayama and Tsugita, 1969; Amagase, 1972), secretion from closed and open pitchers of Nepenthes were studied as well as the secretion from Drosera. The secretion found in closed (sterile) Nepenthes pitchers was neutral and slightly proteolytic. That from open Nepenthes pitchers was acidic, containing a pepsin-like protease. Purified fractions of protease from open Nepenthes pitchers and Drosera were found to be similar in almost all of the physical and physiological properties investigated, a similarity which was considered "surprising" for such widely-divergent plants.

How the fluid from Nepenthes was obtained was not mentioned; the fluid from Drosera was collected by grinding the leaves with three parts of water. No mention was made of the associated bacteria and their possible enzymes in connection with the collection of the fluid, nor was the "cleanliness" of the Drosera leaves indicated. Extrinsic, microbial-contributed protease was discounted for Nepenthes, because of the electrophoretic similarities of the fluids from closed (sterile) and open pitchers.

In a fourth paper from this group (Amagase, Mori and Nakayama, 1972), ants were indicated as being generally associated with Nepenthes, both in nature and in greenhouses. It may be recalled that digestion of ants in open pitchers results in the release of formic acid (Wherry, 1929). When powdered ants were added to the crude protease derived from Nepenthes and Drosera, they were markedly digested. However, the powdered ants were not appreciably attacked by the purified protease fractions; the role of an additional enzyme, chitinase, was suspected to be necessary. A chitinase having a pH optimum of 4 was found in the crude Nepenthes secretion and an uncertainty of its origin was indicated; "symbiotic" bacteria were suspected.

From the studies of Amagase et al. (1972), Nepenthes and Drosera were found to possess an enzyme system which digests insects under strongly acidic conditions that appear to retard putrefaction. Certainly more work should be done on the origin (bacterial and/or plant) and roles of the digestive enzymes of these and other carnivorous plants.

Go on to Part III of the Literature Review.
Go back to Part I of the Literature Review.
Go to the References.
Return to the Pitcher Plant Project "Index" Page.

Page last modified on 1/9/98 at 9:45 AM, CST.
John Lindquist, Department of Bacteriology, University of Wisconsin – Madison