Original Papers

Accumulation of 86Rb and 43K Ions in the Cells Surrounding the Air Pores of Conocephalum conicum

UTA MAIER-MAERCKER

Physik Department der Technischen Universitat Miinchen, D-8046 Garching bei Miinchen, James-Franck-Stral1e, F.R.G.

Received September 3,1981 . Accepted November 12, 1981

Summary 86Rb and 43K were introduced into the thallus of Conocephalum conicum through the rhizoids

and were finally localized by micro autoradiography in the cells around the air pores. Parallel experiments with T12S0.led to massive accumulation ofTl+ in the same sites.

Due to the spatial separation of the air pore apparatus from the rest of the thallus, evapora­tion must be strong around the pore. In accordance with the conditions of peristomatal trans­piration in vascular plants, ion concentration in the apoplast around the pore must increase. As in the cells lining the pore the activity of B-glycerophosphatase was high, it was thought that carbohydrate transport must take place there, and that this process helped to pull water and ions from the apoplast into the cell.

Kry words: Conocephalum conicum, air pores of thall~ B·glycerophosphatase, microautoradio· graphy, peristomatal transpiration.

Introduction

The Marchantiaceae are peculiar among liverworts in posessing an assimilatory tissue with air spaces in many ways comparable with those of a leaf. Conspicuous air chambers communicate with the external atmosphere via air pores in the upper epidermis. These pores do not show the subtle responses to light or atmospheric humid­ity which are characteristic of the stomata in vascular plants but they have a tendency to close when the plant is "uffering from severe water deficit (Raven, 1977 a).

Goebel (1882) appears to be the first to describe movements of marchantiaceous air pores. He supposed that the mechanism resembled that of a stoma and that partial closure of the pores was likely to cause diminution of the rate of transpiration. Kamerling (1897) discovered the pore of some genera of the Marchantiaceae being capable of closing reactions due to turgor changes in a manner which recalls the mechanism of stomata. In any case, the biological advantage of a free path for the dif­fusion of gasses to and from the interior cells of the tissue is obvious. The position of the cells surrounding the pore is similar to that of the guard cells of stomata namely «in the gas-diffusion part of the water transport pathway» (Raven, 1977 a). This suggested that these cells might accumulate ions in the same way as do the guard cells of stomata (Maier-Maercker, 1979, 1980, 1981 a; Maier-Maercker and Jahnke, 1980).

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Material and Methods 86RbCl was purchased from Amersham and Buchler, HKCl was produced with the compact

cyclotron at the Faculty of Physics of the Technische Universitat, Miinchen, using a 7 MeV triton beam (d. Wegmann et aI., 1981).

Thalli of Conocephalum conicum were placed on cotton wool in 3 em diameter petridishes soaked with 20 mM solutions with a label activity of 296 . 10' Bq . ml- 1 for 86Rb and 444 . 10' Bq . ml- 1 for '3K respectively. The plants were under continuous illumination (100 W . m-2

)

from a mercury vapour lamp (HQIE, 400 W) for 48 h (86Rb) and 24 h (43K). To keep humidity high, the petridishes were kept closed.

Following the technique with leaves of vascular plants (Maier-Maercker, 1981 a) the thalli were finally pressed with their upper epidermis on to pre-cooled stripping film (Kodak, AR 10) and kept for five days of exposure at -16°C. After the tissue had been removed, the film was processed and examined under the microscope. Chemography was excluded by control experi­ments.

In parallel experiments thalli were treated with an aqueous solution of TlzSO, (1 %). For localization of the Tl+ the thalli were immersed in a methanolic solution of NaCl as described by Maercker (1965 a).

Results

In Conocephalum conicum each air chamber is covered by the upper epidermal layer with a single pore in its center. Like the stomata of vascular plants the pore is surrounded by kidney-shaped edge cells, 6 to 10 in number. Together with the other cells of the pore surroundings these edge cells form a complex (d. the scanning electron micrograph in Schonherr and Ziegler, 1975) very much like stomatal com­plexes found in several species of vascular plants. Because of numerous subsidiary cells in Bergenia cordi/olia (d. Maercker, 1965 b) the likeness is particularly great with this species.

When 86Rb or 43K were introduced into the thalli of Conocephalum conicum, both isotopes were discovered in the cells around the air pore (Figs. 1 a, b).

Because the epidermal roof of each air chamber protrudes, one might argue that due to intimate contact only the top cells form an image on the stripping film. This may apply to HC or tritium with comparatively low radiation energy (0,2 MeV for HC; 0.002 MeV for tritium) but not to 86Rb and 43K with the much higher radiation energy of 1.8 MeV (86Rb) and 0.8 MeV ('3K). The low energy fraction of ~-rays is recorded in close proximity to the film while the high energy portion traverses the film without leaving a trace. Although resolution decreases, the chance that ~-rays affect the emulsion increases therefore with the distance of the Rb or K source.

Experiments using T12S04 as tracer substance led to deposits in the same sites around the pore (Fig. 2). This is additional evidence that the microautoradiographic record reflects the true pattern of ion distribution. As with stomata, the TICI pre­cipitate was not strictly confined to the interior of the cell; it was often found adher­ing to the outside walls around the pore as well. This makes the apoplast the most likely site of transport from where the ions may enter the cells.

In response to water stress the dome-shaped epidermal roof collapsed as the epi-

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Accumulation of 86Rb and 43K in air pore cells of Conocephalum 99

Fig. 1: Conocephalum conicum: Microautoradiographs of the pore surroundings. (a) showing 86Rb, (b) showing 4JK. Fig. 2: Massive deposition of Tl + in the pore cells. Fig. 3: Localization of p-glycerophosphatase in the pore surrounding.

dermal cells shrivelled up. The pore size did not change significantly. When detached epidermal strips were treated with osmotic agents, the loss of water led to general cell deformation; however, the edge cells, bordering the pore suffered most. Shrinkage made them assume the shape of a sickle.

Employing the Gomory-reaction adapted to the study of guard cells (Maercker, 1965 b) l3-glycerophosphatase activity was found to be high in the pore cells of Conocephalum conicum (Fig. 3).

Discussion

In Conocephalum conicum the accumulation of 86Rb and 4JK occurred in the neigh­bourhood of the pore. In vascular plants both isotopes were found at the same place, and while the pore stayed open, the guard cells were preferential sites of deposition (Maier-Maercker, 1981 a; Maier-Maercker and Jahnke, 1980).

Besides K +, dyes and heavy metal ions were shown to accumulate within the guard cells when stomata are open (Maier-Maercker, 1979). As deposition of lanthanum

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ions preferentially occurred in the apoplast of the same epidermal areas (Maier­Maercker, 1980), it was therefore concluded that peristomatal transpiration must be the driving force and the main cause of ion accumulation in the guard cells.

Peristomatal transpiration was defined as the cuticular loss of water to the atmosphere from subsidiary and guard cells (Maercker, 1965 a). The process involves the external surfaces of the leaf and is also taking place around the substomatal cavity (Maier-Maercker, 1980) although there may be reasons to make a distinction between evaporative loss from outside and from inside the stomatal throat. Peristomatal trans­piration thus depends on the two conditions, cuticular penetrability and/or large eva­porating surface areas (Maier-Maercker, 1980). As to the Marchantiaceae, Walker and Pennington (1939) have suggested that the pore cells of Preissia quadrata were readily permeable to water and therefore able to change their shape and the size of the pore accordingly. As in the Marchantiaceae the epidermal roof containing the pore is sep­arated from the parenchyma during air pore development, the second of the above conditions is fulfilled. The spatial separation of the epidermis inevitably leads to large surfaces in the area around the pore. In accordance with the model of the substomatal cavity proposed by Meidner (1976), water must also escape from the internal surfaces of the pore cells, when the water vapour concentration in the bulk air is not in equi­librium with the wet cell walls.

Due to the permanent loss of water, the ions are trapped in the apoplast around the pore. Provided the cell membrane is permeable to these ions, they eventually follow the local concentration gradient that builds up between cell wall and cell vacuole. The pore cells of Marchantiaceae and the guard cells of the vascular plants as well, are both terminals of the transpiration pathway and must therefore accumulate mobile ions.

This interpretation may be correct in its fundamentals, but to explain fully the mas­sive deposition of ions in the cell vacuole it must be assumed that metabolites are involved to steepen the gradient between apoplast and vacuole of the cell further. As the pore cells of Conocephalum evidently lack chloroplasts, a dependency must exist on resources in the thallus parenchyma. Import of carbohydrates in the pore cells can be postulated. The presence of acid phosphatases in the pore cells may support this vIew.

Also in the guard cells of vascular plants the activity of acid phosphatases is particularly high (Maercker, 1965 b). Recent developments indicate an intense transfer of assimilates, in particu­lar sugars, from the mesophyll to the guard cells (Dittrich and Raschke, 1977; Will mer and Firth, 1980) and the ability of guard cells to absorb sugars (Dittrich and Raschke, 1977). Follow­ing Lloyd (1908) it was even concluded that guard cells derive all of their assimilates from the underlying tissue rather than through the photosynthetic activity of their own plastids. It appears that the photosynthates are transferred through the apoplast (Dittrich and Raschke, 1977). Outlaw et al. (1977, 1979) suggest that massive import of organic material is taking place on stomatal opening.

As sugars pass into the cell, a pOSItIVe tendency to take in water supervenes (Spanner, 1975). This osmotic entry of water reduces the concentration of ions and

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Accumulation of 86Rb and 4JK in air pore cells of Conocephalum 101

this stimulates ion flux from the apoplast into the cell vacuole. By contibuting to the osmotic pressure, the ions assist further inflow (Raven, 1977 b). In algae accumulated organic compounds participate in controlling internal osmotic potential (Kirst and Bisson, 1979). It had been suggested by these authors that a large water movement caused rapid concentration changes and that these changes masked regulation by active transport processes.

The uptake of metabolites provides a regulatory possibility for passive ion trans­port (Raven, 1977 b). Their influx into the cells is regulated by negative feedback loops related to assimilation and storage (Cram, 1976; Raven, 1977 b). According to

the source-sink-relationship influx of metabolized solutes maintains net water influx and ion influx in consequence. The ion flux rate is thus matched with the rate of metabolism (Raven, 1977 b) and accumulation is dependent on environmental factors. There is no need to resort to active ion transport as sole processes involved, not even if the cells are devoid of starch. Where starch is present, however, its hydrolysis may increase the amount of soluble carbohydrates and provide an addi­tional signal for osmotic entry of water into the cell. Water uptake occurring in response to starch hydrolysis is changing the ion concentration gradient between cell wall and cell vacuole; ion influx is thus stimulated (Maier-Maercker, 1979, 1981 a, b). This is supposed a superior regulatory possibility for the control of passive flux in relation to changes in the environment, particularly those from dark to light.

High ion concentrations in the apoplast certainly reinforce net solute influx into the cell. Since the apoplastic pool is replenished via the transpiration stream, varia­tions in the evaporation rate must be of dominating influence in the control of pas­sive ion fluxes. This applies to pore cells of Conocephalum conicum as well as to the guard cells of vascular plants.

Acknowledgements The author is greatly indepted to Prof. Dr. H. Morinaga and to H. Muthig. This work has

been supported by the Deutsche Forschungsgemeinschaft.

References CRAM, W. J.: Negative feedback regulation of transport in cells. The maintenance of turgor,

volume and nutrient supply. In: LOTTGE, D., PITMAN, M. G. (Eds.): Transport in Plants II, Encyclopedia of Plant Physio!. New Series, Vol 2 A, 284- 316. Springer, Berlin, 1976.

DITTRICH, P. and K. RASCHKE: Dptake and metabolism of carbohydrates by epidermal tissue. Plant a 134, 83 - 90 (1977).

GOEBEL, K.: Handbuch der Botanik. 9. Lieferung Berlin (1882). KAMERLING, Z.: Zur Biologie und Physiologie der Marchantiaceae. Flora 84, 1-173 (1897). KIRST, G. O. and M. A. BISSON: Regulation of turgor pressure in marine algae: Ions and low­

molecular-weight organic compounds. Aust. J. Plant Physio!. 6, 539-556 (1979). MAERCKER, U.: Zur Kenntnis der Transpiration der SchlieBzellen. Protoplasm a 60, 61-78

(1965 a). - Beitrage zur Histochemie der SchlieBzellen. Protoplasma 60, 173 - 191 ( 1965 b). MAIER-MAERCKER, D.: «Peristomatal transpiration» and stomatal movement: A controversial view.

IV. Ion accumulation by peristomatal transpiration. Z. Pflanzenphysiol. 91, 239 - 254 (1979).

Z. Pjlanzenphysiol. Ed. 105. S. 97-102. 1982.

102 UTAMAIER-MAERCKER

- V. Rubidium-86 in the epidermal transpiration stream. Z. Pflanzenphysiol. 100, 447-459 (1981 a).

- VI. Lanthanum deposits in the epidermal apoplast. Z. Pflanzenphysiol. 100, 121-130 {1980}. - VIII. Stomatal control by conditions of water supply and peristomatal transpiration. Z.

Pflanzenphysiol. 103, 15-25 {1981 b}. MAIER-MAERCKER, U. and A. JAHNKE: Microautoradiography with 43K: A method for the

reliable tracing of ion transport in stomata. Z. Pflanzenphysiol. 100, 35-42 {1980}. MEIDNER, H.: Water vapour loss from a physical model of a substomatal cavity. J. Exp. Bot. 27,

691-637 {1976}. OUTLAW, W. H., Jr. and o. L. LOWRY: Organic acid and potassium accumulation in guard cells

during stomatal opening. Proc. Natl. Acad. Sci. U.S.A. 74, 4434-4438 {1977}. OUTLAW, W. H., Jr. and J. MANCHESTER: Guard cell starch concentration quantitatively related

to stomatal aperture. Plant Physiol. 64, 79-82 {1979}. RAVEN, J. A.: The evolution of vascular land plants in relation to supracellular transport pro­

cesses. Advances in Botanical Research. Vol. 5, 153 - 219 {1977 a}. - Regulation of solute transport at the cell level. Symp. Soc. Exp. BioI. 31, 73 -99 {1977 b}. SCHONHERR, J. and H. ZIEGLER: Hydrophobic cuticular ledges prevent water entering the air

pores of liverwort thalli. Planta 124, 51- 60 {1975}. SPANNER, D. c.: Electroosmotic flow. In: M. H. Zimmermann, J. A. Milburn {Eds.}: Transport

in Plants I, Encyclopedia of Plant Physiol. New Series Vol. 1, 301-327. Springer, Berlin, 1975.

WALKER, R. and W. PENNINGTON: The movements of the air pores of Preissia quadrata {Scop}. New Phytologist 38,62-68 {1939}.

WEGMANN, H., H. HUENGES, H. MUTHIG, and H. MORINAGA: Acceleration of tritons with a compact cyclotron. Nuclear Instruments and Methods 179, 217 - 222 {1981}.

WILLMER, C. and P. FIRTH: Carbon isotope discrimination of epidermal tissue and mesophyll tissue from the leaves of various plants. J. Exp. Bot. 31, 1- 5 {1980}.

Z. Pjlanzenphysiol. Bd. 105. S. 97-102. 1982.