The nature of the stimuli causing digestive juice secretion in Dionaea ...

Planta

Planta (Berl,) 128, 263-265 (1976)

9 by Springer-Verlag 1976

SHORT COMMUNICATIONS The Nature of the Stimuli Causing Digestive Juice Secretion in Dionaea muscipula Ellis (Venus's Flytrap) R.J. Robins Botany School, University of Oxford, South Parks Road, Oxford, OX13RA, U.K.

Summary. An investigation into the stimuli of the secretory system in the carnivorous plant Dionaea muscipula is presented. Secretion of fluid and protein are both stimulated by various nitrogenous small molecules. These secretions are studied as a function of time. A new method is described for the collection of secreted juice. Significant differences are found between the quantities of fluid and protein produced in response to different stimuli. The results are discussed in comparison to the mammalian gastro-intesfinal secretory systems.

draw secretion, This capillary was introduced into the trap only when collection was required. Thus no interference with natural trap closure occurred, and loss of liquid by evaporation was avoided. Volume was obtained directly as the fraction of capillary length containing liquid and each vial could be individually stored at - 15~ C, avoiding wastage of material. Protein content was measured by the method of Lowry et al. (1951). All reagents were of "Analar" standard, and obtained from one of the following sources: British Drug House Ltd., Poole, England; May and Baker Ltd., Dagenham, England; Harrington Brothers Ltd., London, England; Fisons Scientific Apparatus Ltd., Loughborough, England.

Results

Very little is known about the stimulus for secretion in any of the insectivorous plants. In Dionaea muscipula Ellis it is distinct from that for trap closure, which is mechanical (Lloyd, 1942). Darwin (1875) showed that plants secreted digestive fluid in response to nitrogenous material only and suggested that this resulted from the absorption of soluble nitrogenous matter. The secretory system of Dionaea has been studied with respect to the quantities of fluid and protein produced as a function of time following stimulation by nitrogenous small molecules, using a new method for the collection of juice.

Maximum fluid and protein secretion occur within 4 to 7 days and cease within 8 to 10 days after stimulation (Fig. 1). Uric acid, ammonia and glutamine cause fluid secretion as effectively as feeding with whole flies (Calliphora sp.), which were used as a control. Phenylalanine and urea are poor secretogogues of fluid. Protein secretion on the other hand shows a more varied response. The extent of both fluid and protein secretion is dependent on the load administered (Fig. 2). Uric acid and ammonia show sharp peaks of secretion when exhibited at loads of approximately 10-6 and 10.5 mol respectively. Glutamine displays a linear response over the range tested, while urea shows an enhancement at a load of about 10- 6 mol.

Materials and Methods

Discussion

Plant material was grown at 25 ~ C, with a 12 h photoperiod at 1001x from fluorescent lighting, in equal parts acid leaf mould, peat and Sphagnum moss, and at high humidity. Specimens were kept healthy by periodic feeding, in which the traps were stimulated to close over the prey. All experiments were conducted on leaves one to three weeks old and each leaf was used only once. A 20 ~1 microcapillary (Microcap, Camlab, Cambridge) was used to with-

It appears that the volume of secretion and the protein content may each be subject to separate control mechanisms (Table 1). Thus, although glutamine elicited a strong volume response, it caused the secretion of comparatively little protein, whereas uric acid was effective at causing the secretion of both fluid and

Introduction

R.J. Robins : Digestive Juice Secretion in Dionaea

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DAYS Fig. 1. Cumulative time-course of the volume and protein content of digestive fluid produced in response to stimuli. There was no significant difference on a "Sign Test" (Siegal, 1960) between the responses of individual plants fed with live Calliphora sp. and hence in all subsequent treatments the results from several different plants were combined. Between seven and eleven lobes were stimulated in each of the following cases: (a) Control=live Calliphora sp. sealed into dialysis membrane (6.3 ram, Medicell International) ( A - - A ) ; (b) Uric acid=loads of 10, 20, 40, 100, 200 and 400lag given on days 0, 1, 2, 3, 4 and 7 respectively ( o - - o ) ; (c) Ammonia =loads of 10, 20, 40, 100, 200 and 400 gg given on days 0, 1, 2, 3, 4 and 7 respectively ( u - - n ) ; (d) Glutamine=loads of 10, 20, 40 and 100 gg given on days 0, 1, 2 and 3 respectively (zx--A); (e) U r e a = l o a d s of 10, 20, 40 and 100 ~tg given on days 0, 1, 2 and 3 respectively ( 9169 (0 Phenylalanine=loads of 10, 20, 40, 100, 200, 400 and 800~tg given on days 0, 1, 2, 3, 4, 5 and 7 respectively ( • Administration was by microcapillary and fluid was removed daily before further stimulant was given

protein. With urea both the volume and the quantity of protein secreted were low. Ammonia, however, proved slightly more effective than uric acid, but induced a much smaller secretion of protein. The relatively greater quantity of protein observed to be secreted in the control may represent an additive effect of individual chemical stimuli on protein release as distinct from the volume release mechanism. Uric acid, the principal constituent of insect excreta (Altmann, 1961), proved the most effective secretogogue. Flies excrete profusely when trapped and thus uric acid reaches the leaf surface within a few minutes of capture. Its effectiveness at activating both mechanisms indicates an important role in the wild. Glutamine, one of the major amino acids of insect hemolymph (Wyatt, 1961), will be released

Table 1. Effects of secretogogues in comparison to control values Stimulus given

Cumulative mean a protein secretion

Cumulative mean a volume secretion

Maximum value attained (Mp)

Maximum value attained (My)

Mp % of control

(~g) Control Uric acid Ammonia Glutamine Urea Phenylalanine

89.6 56.1 38.6 18.5 8.0 -

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% of control

1.22 0.72 0.44 0.24 0.41 -

100 51 36 20 34 -

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(~1) 100 63 44 20 9 -

73.5 78.5 88.8 68.7 19.6 11.5

100 107 121 94 27 16

For experimental details see Fig. 1. "Cumulative M e a n " defined as the mean daily secretion per secreting lobe summated over the total experimental period.

R.J. Robins : Digestive Juice Secretion in Dionaea

when traps close tightly, crushing the prey (Batalin, 1877). It might therefore be expected that taurine, proline, and asparagine, the other major buffers of hemolymph (Wyatt, 1961), will also be effective secretogogues. Glutamine may act partly by degradation yielding ammonia which is rapidly released on exposure of hemolymph to air (Florkin et al., 1940). Urea and phenylalanine, both poor stimulants, are found only in trace amounts in hemolymph, although traces of urea occur in excreta (Altman, 1961). Phenylalanine is the most potent secretogogue for pancreatic secretion in dogs (Grossman et al,, 1972). The secretory response in Dionaea thus seems to depend upon a mechanism that specifically recognises those compounds to which it is exposed in the natural state. Nitrogenous substances occurring in insects in trace amounts only are relatively ineffective. Several parallels may be drawn between the system reported here and the mammalian intestinal secretory systems. In mammals the volume and protein content of the pancreatic exocrine juice are differentially controlled via the hormones secretin and cholecystokinin (PZ-CCK) respectively (Dockray, 1972; Wang et al., 1951). These are released in a toad-dependent manner only in response to the presence of certain secretogogues in the duodenum (Grossman et al., 1972). Acid causes secretin release, which acts at the pancreas to secrete fluid of low protein content. Phenylalanine, tryptophan, and leucine, releasing PZCCK, cause much greater protein secretion with considerable fluid as well (Dockray, 1972). Other amino acids, including glutamine, are ineffective in dogs (Thomas et al., 1940). Load-dependence, differential control of fluid and protein, and specifity to certain compounds are characteristics also found in the Dionaea system.

265 I am extremely grateful to my supervisor, Dr. D.S. Parsons (Department of Biochemistry, Oxford) for his advice and encouragement during these investigations. The work was conducted as part of the Part II Biochemistry examination.

References Altman, P.L. (ed.) : Blood and other body fluids. Washington D.C. : Publ. Fed. Amer. Soc. Exp. Biol. 1961 Batalin, A.: Mechanik der Bewegung der insektfressenden Pflanzen. Flora 35, 54-58 ; 105-111 ; 129-154 (1877) Darwin, C. : Insectivorous plants. London: Murray 1875 Dockray, G.T. : The action of secretin, chotecystokinin-pancreozyrain and caerulein on pancreatic secretion in the rat. J. Physiol. 225, 679-692 (1972) Florkin, M., Frappez, Gh.: La concentration de l'ammoniaque in vivo et in vitro dans le milieu int~rieur des invert6br6s. Arch. int. Physiol. 50, 197502 (1940) Grossman, M.I., Meyer, J.H. : Release of secretin and cholecystokinin. In: Gastrointestinal hormones, pp. 43-55. Ed. : L. Demling, Stuttgart: Erlangen. 1972 Lloyd, F.E.: The carnivorous plants. Waltham, Mass.: Chronica Botanica Co. 1942 Lowry, O.H., Roseborough, N.J., Farr, S.L., Randall, R.J.: Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265 275 (1951) Siegal, S.: Nonparametric statistics for the behavioral sciences. New York:McGraw-Hill 1960 Thomas, J.E., Crider, J.O.: A quantitative study of acid in the intestine as a stimulus for the pancreas. Amer. J. Physiol. 131, 349-356 (1940) Wang, C.C., Grossman, M.I.: Physiological determination of release of secretin and pancreozymin from intestine of dogs with transplanted pancreas. Amer. J. Physiol. 164, 527-545 (1951) Wyatt, G.R.: The biochemistry of insect hemolymph. Ann. Rev. Entomol. 6, 75-102 (1961)

Received 4 October," accepted 14 October 1975