Turgor-mediated Leaf Movements in Analogy With Stomatal Function and Under the General Aspect of Water Flux Through the Plant I. Microautoradiographic Localization of 86Rb and 43K in the Laminar Pulvinus of Phaseolus vulgaris L.

UTA MAmR-MAERCKER*)

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

Received October 28, 1983 . Accepted March 20, 1984

Summary

Microautoradiographic methods applied to the pulvinar region of Phaseolus vulgaris L. re­vealed specific patterns of ion distribution according to the phases of upward or downward movements of the leaves:

When feeding was taking place during the dark phase of the diurnal cycle, the main source of radioactivity was the dorsal groove of the vascular core on the flexor side. During the phase of leaf opening, 8CXb and 43K were mainly found within the parenchymatous bundle sheath. As an additional similarity to the guard cell physiology, toxic metal ions were accumulated in the cells of the bundle sheath when introduced into the plant during the light phase. Additional experi­ments on the influence of osmotic gradients supported the view that the characteristic distribu­tion of ions during the two phases of the diurnal cycle is the result of regulatory processes con­trolling water and solute flux from the sites of storage to the evaporating surfaces.

The conclusion was that in light the cells of the bundle sheath removed the ions from the flexor sites and that this process is substantially controlled by transpiration rates. Findings of other authors were discussed in view of the hypothesis that K+ movements in the pulvinar re­gion depend on regulatory systems other than active ion pumps. Among the processes prob­ably involved in the initiation of primary water flux changes are the import of carbohydrates into the cells of the bundle sheath and starch hydrolysis.

Key words: Phaseolus vulgaris L., ion transport, microautoradiography f 6Rb, 43K), nyctinastic leaf movements.

Introduction

Movements leading to reversible changes in the angle between leaf blade and petiole or rhachis and the opening and closing movements of stomata have certain features in common. Both result from differential changes in the turgor, the vacuolar volume and the shape of motor cells. Antagonistic changes in guard and subsidiary cells occur during stomatal movements while leaf movements are the result of changes in motor

*) Present address: Lehrstuhl fiir Forstbotanik der Universitat Miinchen, Amalienstr. 52, D-8000 Munchen 40, F.R.G.

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406 UTA MAIER-MAERCKER

cells which are situated on opposite sides of the vascular tissue of the pulvinus at the base of the leaf or leaflet blade.

The flux of ions (particularly of K+) is a crucial event leading to an antagonistic pat­tern of ion distribution which is clearly correlated with these turgor-controlled movements. The potassium ion is therefore generally regarded as the «effector» in the turgor-regulatory systems.

The general view holds that active K+ transport plays the fundamental role in the stomatal mechanism. However, measurements of electrical potentials in stomatal com­plexes have revealed nonspecific migration of monovalent cations along their elec­trochemical gradients (Saftner and Raschke, 1981), and there is also evidence that ion fluxes during stomatal movements are largely attributable to water potential gradients (Maier-Maercker, 1983 b). In addition, the vacuole of the guard cell maintains a contin­uous import of ions via steepened local concentration gradients between evaporating cell wall and vacuole (Maier-Maercker, 1979, 1980, 1981, 1983 a). This view is supported by the finding of large phase shifts between the kinetics of stomatal opening and ion ac­cumulation within the guard cells and of the fact that accumulation proceeds during the maintenance of maximum stomatal aperture (Laffray et aI., 1982).

Similarly, disagreement also seems to exist as to the mode of ion flux during the nyctinastic movements of leaves (d. Satter, 1979; Satter and Galston, 1981). On the one hand, the assumption was brought forward that active hyperpolarizing by active electrogenic pump mechanisms is coupled with the loss of osmotic substances from the symplast to the apoplast in extensor and flexor cells of the bean pulvinus (Freudling et all., 1980). However, there were reasons to conclude that no direct causal relationships exist between the electrogenic pump and the turgor changes (Mayer, 1981). In Trifolium - on the other hand - the time course of changes in mem­brane potentials corresponded quantitatively to what would have been expected if the membrane potential is determined principally by passive diffusion of K+ (Scott and Gulline, 1975).

Results implying apoplastic migration of ions to flexor and extensor sites (Satter et aI., 1980, Campbell et aI., 1981; Satter et al., 1982) throw a new light onto the whole subject. Special transfer cells at the periphery of the vascular system seem to remove the ions from the flexor cells during the phases of upward movement of the leaf (Sat­ter and Galston, 1981; Satter et al., 1982). This paper presents evidence in favour of this view and is further intended to show that the transpiration stream is involved in the transport process, the way and whether the process is active or passive remaining open to question.

Material and Methods Bean plants (Phaseolus vulgaris L.) were 'grown from seeds in hydroponic solutions (Lewatit

HD 5, Bayer, Leverkusen, for nutrition). They were raised in the greenhouse under a 12 h light/12h dark cycle. The plants were used for the experiments when the primary leaves had just expanded to full size.

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1. Secondary pulvini of primary leaves were detached under water. On one side of the pulvinus were a few millimeters of the petiole and on the other side a portion of the leaf midrib with the blade tissue removed. The pulvinus was then passed through solutions of increasing os­molality. The experiments were carried out in the light (20W· m-2

) and at a temperature of 25°C. The solutions were changed hourly. The osmolality of the solutions was increased with either mannitol or KCl and was determined immediately prior to each experiment using a Wescor micro-osmometer (Model 5100 B). Because distilled water as well as certain buffers (e.g. phosphate- and tris-maleate buffers) seemed to have additional effects on the bending movements of the pulvini, the media were made up with tap water which had been decalcified by boiling. The bending angle was recorded photographically at regular intervals while the tissue was lying within the incubation medium.

2. For the microautoradiographic studies, the bean seedlings were cut under water at the hypo­cotyl. Feedin§ occurred through the cut end. 86RBCl was purchased from Amersham and Buchler and 4 KCl was produced with the compact cyclotron at the Technische Universitat Miinchen (Wegmann et aI., 1981). Load solutions were 10 mM in concentration. In the case of 86Rb, label activity was 296.104 Bq· ml-1

• The activity of 43K was 750.104 Bq· ml- 1•

Feeding occurred inside an environmental chamber. During the natural dark period of the cycle, maximum dark position of the leaves was sustained by keeping the temperature below 20°C. The relative humidity of the chamber air was not allowed to exceed 50 %. Loading started after the maximum dark position had been reached. During the light treatments, os­cillations of the leaf were quelled by keeping the temperature at 23°C and the relative humidity at a minimum of 75 %. Light was provided by a mercury vapour lamp (HQIE); total radiation inside the chamber being 100 W· m -2.

3. The pulvinus was cut from leaf and petiole, immediately placed onto the precooled table of a Leitz Cryomat, and was enclosed by polyethylene glycol 1000 which was kept just above its melting point. Together with the tissue it was then rapidly frozen by pourring liquid nitro­gen or Freon 11 over it. The polyethylene glycol provided good support and assured smooth sectioning. It was possible to handle the thick sections (40 I'm at the least) using a cooled pair of forceps. While still frozen, the longitudinal or cross sections were transferred to precooled stripping film (Kodak, AR 10) mounted on slides. The samples were stored on a metal plate in a polystyrene box containing liquid nitrogen. Each mount was covered with one half of a small petri dish to avoid condensation on the cold surface. The box was finally put into the freezer (-16 0e). Exposure of the film took place from the fresh frozen tissue for up to 24 hours (for 43K) or 5 days (for 86Rb). While processing the film, the plant sections generally were pealed off and removed from the developer for comparison with the microautoradiog­raphic images. Unloaded tissue produced no trace of blackening on the film.

4. Ion accumulation was also studied by feeding plants with either ThS04 (20 mM) or AgN03 (100 mM). The cations were localized with methanolic solutions of NaCI (Maercker, 1965; Maier-Maercker, 1979). For structural analysis, the tissue was fixed in 3 % glutaraldehyde followed by a treatment with 2 % OS04. Tissue samples were embedded in polyethylene glycol or Araldite and sectioned using a Leitz Cryomat or a LKB Ultra­microtome respectively. Ultrathin sections were stained with lead citrate.

Results

L «Passive» movements of the bean pulvinus

For the first series of experiments, the secondary pulvini of bean leaves were taken from illuminated plants. Each pulvinus was passed through a full series of media of increasing osmolalities (for details see the chapter on material and methods). The pul­vinus responded immediately to each increase in osmolality, and within 5 minutes it

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408 UTA MAIER-MAERCKER

, , I , , ; , t.

" b

86 mOs·kg-1

... ---- ..

I

.'

86 mOs.kg-1

-J

d

'. , ,

112 mOs·kg-1

Fig. 1: Selected stages of a bean pulvinus incubated in mannitol solutions of increasing osmolali­ties; each group comparing present stage (solid line) with the preceeding stage (dashed): (la) 66mOs-kg-1 with control in tap water (58mOs·kg- I); (lb) 86mOs-kg-1 with 66mOs·kg- l

; (lc) 112mOs·kg-1 with 86mOs·kg- l; (ld) 184mOs·kg-1 with

112mOs' kg-I.

, I

I I ,

I I

I

, , , ,

(",- ................. 1

a

62 mOs. kg-l

74 mOs·kg-1

b

, •• / 62

181

74 mOs' kg-l 108mOs·kg-1

Fig. 2: Changes occurring in KCl solutions of increasing osmolalities (2 a) 62 mOs' kg-I (com­pared with tap water of 52mOs-kg-I); (2b) 74mOs-kg- l

; (2 c) 108mOs·kg- l; (2 d)

181 mOs' kg-I.

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Leaf movements and localization of 86Rb and 43K 409

had settled at an angle which remained nearly constant for several hours when the os­molality remained constant. At each step the given osmolality produced a bending angle characteristic of the individual pulvini. At low ranges of osmolality, the pul­vinus responded to increasing osmolality with heavy bending while the process was reversed when osmolality was further increased (Fig. 1 a-d). The bending always reached its maximum within the range of 80 and 90 mOs' kg -1 when mannitol was the chosen osmoticum. When stretching in the high osmolality range, the pulvinus seemed to decrease in size. Shrinkage was significant at the upper adaxial portion which is the flexor in this species. At about 180 mOs' kg -1, the pulvinus assumed the shape of a saddle (Fig. 1 d). Similar results were obtained when KCI instead of man­nitol was used to increase the osmolalities of the incubation media (Fig. 2 a-d).

IL Microautoradiographic record and corresponding structure

The asymmetry of the vascular bundle, which was always clearly reproduced by the stripping film, allowed one to readily identify dorsal and ventral portions of the pulvinar tissue. Following the common trend, the first aim was to discern characteris­tic differences in loading capacity of the two opposite sides of the pulvinus depending on the phases of the diurnal cycle. In fact, some microautoradiographs seemed to sug­gest an antagonistic pattern of the blackened areas. the upper side corresponding to the flexor region appeared comparatively dense after feeding the plant during the dark phase of the cycle while light treatment seemed to stimulate the impregnation of the lower portion of the section with particular dense silver granulation. However, not too much emphasis can be placed on these casual results because (1) the radio­active isotope could have been displaced from disrupted cells during sectioning, and (2) these images were not at all the rule. In most cases the radioactivity seemed to ex­pand equally from the central vascular bundle with a clear front reaching more and more towards the periphery with prolonged feeding time. Neither the whole dark pe­riod nor the light period of a cycle was long enough to allow the central core of deep blackening to expand to the total size of the cross section through the pulvinus.

Instead of the expected bipolar distribution of the ions, usually a dense ring ap­peared on the film when feeding had occurred during the light phase (Fig. 3 a). It en­closed the central bundle and reproduced even details of its outline. Cross sections through the pulvinus show that the bundle tissue is rolled-up leaving a more or less wide dorsal fissure (Fig. 4 a). Where this dorsal groove is deep, there the outline of the «ring» becomes kidney-shaped accordingly. Longitudinal sections produced deep black bands on either side of the vascular tissue (Fig. 3 c) implicating that this zone of heavy ion accumulation is corresponding to a tubular sheath around the bundle.

Comparing the silver pattern on the film with the thick section used for microauto­radiography, the regular ring of dense silver granulation on the film can be attributed to a 1-2 layered ring of specialized cells around the vascular system. This sheath of cells is well delineated and clearly separated from the vascular tissue by the adjacent

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410 UTA MAIER-MAERCKER

Fig.3: The laminar pulvinus of the bean primary leaf: Microautoradiographs of the central bundle; adaxial side on top. The isotopes had been introduced into the transiration stream: (3 a) during the early light phase of the cycle, 6 hours feeding time, 43K (750 ·10 Bq· ml-1

); (3 b) during the entire dark phase of the cycle, 86Rb (296 .104 Bq· ml-1

); activity mainly in the groo­ve area; (3 c) corresponding area from the longitudinal section, seedling loaded during the light phase of the diurnal cycle, 86Rb (296 .104 Bq· ml-1).

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Fig. 4: Cryosections of bean pulvini: (4 a) structural features of material fixed in glutaraldehyde and OS04; note the ring of small heavily stained parenchymatous cells; (4 b) plants fed with ThS04 and treated with methanolic solutions of NaC!. The cells of the continuous bundle sheath accumulate the Tl+ ions; (4 c) longitudinal section through pulvinar tissue fixed in glut­araldehyde and OS04; near the vascular strand flexor cells with thick walls and cell content stained.

collenchyma cells surrounding the phloem layer. In semithin sections the small cells of the parenchymatous bundle sheath stain heavily with OS04, and there can be no doubt that they close on top of the vascular tissue to form a continuous jacket (Fig. 4 a).

In parallel experiments when ThS04 or AgN03 had been introduced into the transpiration stream of the seedlings, these cells of the bundle sheath accumulated TI+ and Ag+ provided that feeding had taken place during the phase of light (Fig. 4 b).

Features of these cells which supposedly bear relevance to the present problem are as follows (Figs. 5 a-d):

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412 UTA MAIER-MAERCKER

Fig. 5: Features of the bundle sheath: (5 a) chloroplasts with huge starch grains; dense cyto­plasm; sheath cell bordering vacuolated cell of the cortex; thin common wall, the wall is thick where the cell faces an intercellular space; (5 b) cell of the bundle sheath with two chloroplasts and pit fields in the thin wall portion; (5 c) for comparison, chloroplast and narrow cytoplasmic layer of a parenchyma cell of the inner cortex; (5 d) intermediate type of chloroplast from the bundle sheath; grana structures prevail in one half while the other one is filled with two huge starch grains; note the wall area bordering the intercellular space (top).

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Leaf movements and localization of 8~b and 43K 413

(1) Their cytoplasm is particularly dense. (2) They border highly vacuolated cells of the cortical parenchyma, and many plasmodesmata assembled to pit field cross the thin walls of these cells. (3) Intercellular spaces prevail between the compact sheath and the rounded cells of the cortex. The wall areas separating the cells from these intercellular spaces are tick and stain heavily. (4) Chloroplasts are numerous and con­tain huge starch grains while lacking grana structures of normal chloroplasts such as the few seen in the narrow cytoplasmatic layers of the cortical cells (Fig. 5 c). It seems that the chloroplasts are centrifugally arranged during the dark phase while during the light phase they were more or less randomly dispersed within the cell, sometimes preferring the radial walls.

In contrast to the light phase of the diurnal cycle, neither dense bands (longitudinal sections) nor rings (cross sections) were found on the autoradiographs when feeding had taken place during the period of darkness. However, particular dense silver de­posits marked the site of the dorsal groove above the vascular core (Fig. 3 b). The flexor cells immediately above the vascular strands are peculiar, they are small in size, have irregular arrangement and have comparatively thick walls which stain heavily with osmium. The brownish tint of their cell content may be given by phenolic com­pounds (see the longitudinal section in Fig. 4 c).

In some cases feeding in the dark was extended beyond the normal period of the cycle and diurnal rhythm caused leaf opening without the effect of light. Again the microautoradiographic ring pattern was observed. Representation of the bundle sheath as a regular black ring was particularly distinct when plants had been loaded during the entire dark phase of the cycle and were thereafter illuminated until reach­ing maximum day position; this may take 1 hour or less. Then the signs of the ring appeared first on the extensor side while at the flexor side blackening remained dif­fuse.

Discussion

Microautoradiography has been applied to the problem of ion movement during stomatal movements (Maier-Maercker, 1981; Maier-Maercker and Jahnke, 1980). The method has proven that it can, on a comparative basis, give valuable information concerning the distribution of ions although, owing to the high radiation energy of 86Rb (1.8 MeV) and 43K (0.8 MeV), it is not suitable for the quantitative approach.

Like the stomatal apparatus, the pulvinus is intercalated into the soil-plantat­mosphere continuum; water and solutes are passing through it on their way from stem to blade. Even parts of the plant include evaporating surfaces and sites of storage and are thus able to maintain a permanent transpirational flow. This basic knowledge is probably the reason why more and more attention is recently given to the interac­tions of water and solutes flowing together in the transpiration stream (cf. Molz and Ferrrier, 1982). Potassium is very mobile between the vascular system and the sur­rounding tissues of stem and petiole (Grange and Peel, 1978). This ion can penetrate a

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414 UTA MAlliR-MAERCKER

cell (reflection coefficient u< 1); in the vacuole it alters the osmotic driving force for water within a tissue (Fiscus, 1975). On the other hand, ion content in the cells adja­cent to the main water pathway is under the control of mass flow and is likely to be reduced by a sudden increase in transpiration rate (Newman, 1974).

Water moves within the apoplast in response to evaporative demand, and the cells of the various tissues compete for the water. During periods of intense transpiration cells may not develop turgor pressures of the magnitude the osmotic potentials of their sap would permit in the absence of transpiration. E.g., with the onset of light, transpiration rates suddenly increase and water supply cannot keep pace with the amount of water being lost. Flexor cells are likely to be most affected by this water loss because their osmotic pressure at any leaf position is lower than that of the ex­tensor cells (d. Mayer, 1977). Accordingly, the flexor cells were the first to shrink on account of osmotic agents, thus lifting up the leaf by increasing the angle at the ab­axial side of the pulvinus.

Under natural conditions, the cells of the bundle sheath may act as gauges. Accord­ing to environmental conditions they may draw water from surrounding tissues ei­ther by metabolic processes or because their vacuoles are much smaller than in any other cell; increasing solute concentrations owing to water stress have a much higher osmotic effect on small cells than on larger ones (Cutler et aI., 1977). As soon as trans­piration rates decline, the cells of the flexor may seek to replenish their water contents. When the cells of the bundle sheath have adjusted their demand for water to a lower rate, water may even move preferentially towards the cells of the cortex and there again towards the smallest ones. Experiments with pulvini bending in solutions of comparatively low osmolalities can be interpreted in this sense.

Within the apoplast, ions are dragged along by the pressure-induced water flow (Newman, 1976). Owing to the tendency of the vacuoles to adjust their water po­tentials to the tension in their wall area, local ion concentration gradients between apoplast and vacuoles are built up by the uptake of water. Within the flexor portion of the pulvinus ion accumulation was particularly heavy at the site close to the vascu­lar bundle. There the comparatively thick cell walls which stain heavily with OS04 and the osmiophilic cell content (Fig. 4 c) suggest that potassium may be bound to negative charges in the walls or ion diffusion may here be faciliated by phenolic com­pounds in the cell vacuoles. For the corresponding area in Samanea saman, Satter et al. (1974) have described high accumulation rates; ions seemed to be impeded in dimi­nishing from this site (Satter et al., 1974).

In the absence of transpiration from the leaf surface proper, water movement to­wards the periphery of the pulvinus may, in addition, preferentially occur toward hairs and hydathodes which maintain evaporation and which abound at the upper side of the bean pulvinus (see also Brauner, 1933). The lateral flanks of the cortex being deionized during the night (Mayer, 1977; Schrempf and Mayer, 1980) may thus be subject to mass flow while during the day water and solutes preferentially flow to those parts of the pulvinus which are close to the tub-shaped bundle.

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The following points are in keeping with the hypothesis that solute flux within the pulvinus follows the transpirational water flow:

(1) The adaxial/abaxial flux ratio is significantly greater or less than unity (Scott and Gulline, 1975) indicating that there is not a 1: 1 shuttle of ions between flexor and extensor cells (Schrempf et al., 1976; Mayer, 1977). For quantitative reason, it is thus clear that some of the K+ entering the extensor during the day phase do not come from the opposite motor tissue but from other sources, and the vascular system has been suggested as the most likely one (Satter, 1970; Satter and Galston, 1981).

(2) Extensor and flexor were not 1800 out of phase with each other (Schrempf et al., 1976), and K+ changes continued for several hours after the leaf was fully opened or closed (Scott and Gulline, 1975; Scott et aI., 1977). Repeated determinations of K+ changes during a diurnal cycle (Mayer, 1977) revealed that the drastic K+ loss from the flexor wich occurred at the beginning of the light period did not correspond to a concomitant K+ gain of the extensor. After an initial period, K+ contents of flexor and extensor increased in parallel (see Fig. 3 in Mayer, 1977) thus suggesting supply from the oncoming transpiration stream.

(3) Scintillations per minute in the cells of the Samanea extensor region differed by 1500% during the diurnal cycle and differences in K+ content per mg protein in ex­tracts from extensor halves of the Phaseolus pulvinus amount to 60 %. These differen­ces shrink to an 8 % change when the fresh weight is made the basis of comparison (Satter and Galston, 1981). Therefore, changes in K+ content must not be equated with concentration changes.

(4) Cl-, N03 - and the anions of organic acids accompany potassium during its movement from the upper to the lower portion of the pulvinus and vice versa (Kiyo­sawa, 1979).

(5) More energy is required during the phases of leaf opening than for the reverse process (Satter et 1., 1979). This fact is easier to understand when the movements are related to the varying activity of the cells of the bundle sheath, rather than assuming that every cell in the cortex of the pulvinus has its own circadian clock, each with dif­ferent phase relations relative to the others (d. Schrempf and Mayer, 1980). Energy is required for more processes than metabolic processes modulating transmembrane ion transport in the sense of chemiosmosis (d. Zeiger et al., 1978). Other questions to be raised in this connection are why stomatal subsidiary cells manage to take up consid­erable amounts of ions in the absence of these transport processes and during condi­tions of low energy (d. Maercker, 1965; Maier-Maercker, 1979, 1981). As the uptake of thallium and silver occurs under conditions of considerable toxicity it appears highly unlikely that active processes are involved (d. Barber, 1974). Within the bundle sheath cells, other energy processes wich initiate changes in the direction of water movement may be favoured under the conditions of leaf opening. One of the processes that cause osmotic water entry and therefore must stimulate ion fluxes into the cells of the vascular bundle sheath is the import of organic substances which are very rapidly translocated from assimilating leaf areas to the petiole of Phaseo/us (d.

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416 UTA MAffiR-MAERCKER

Zucconi et al., 1980). Phosphatases in the bundle sheath (Bauer, 1953) may aid their import. Another such process would be the hydrolysis of starch within specialized chloroplasts within the bundle sheath (Fig. 5) and the inner cortical cells of Samanea (Morse and Satter, 1979). They resemble chloroplasts of stomatal guard cells. Like in guard cells, significant changes may occur at the beginning of the light regime (d. Donkin and Martin, 1980). A rapid phosphorolysis (d. Heldt et al., 1977) and the stimulation of this reaction by ATP and perhaps phytochrome may explain the fast light-dependent trigger which must be situated within the pulvinus (Satter and Galston, 1971).

Conclusion

It seems unlikely that highly toxic metal ions are being incorporated in large quantities without disturbing the mechanism of an active transport system. The main aspects - however - embraced in this paper are reasons listed which can be inter­preted in the sense that direction and control of ion fluxes are largely dependent on transpiration rates. Earlier experiments concerning stomatal movements had implied that solute flux is always directed towards the guard cells in response to evaporative demand of the guard cell wall (Maier-Maercker, 1983 a). While ions may be excreted there, subsidiary cells were suggested to maintain a continuous import of ions by new supply from the transpiration stream (Maier-Maercker, 1979, 1981). In analogy to the guard cells, the cells of the bundle sheath are the controlling gauges for water and solute flux between the vascular system and the surrounding tissues. When these cells take up the ions in response to light, they probably do not transfer them from flexor to extensor cells but rather from the sites of storage within the flexor region to the xyleme. From there they eventually reach the evaporating surfaces of the leaf blade. The ion pool within the extensor is replenished out of the oncoming transpiration stream.

Acknowledgements

The author is greatly indebted to Professor H. Morinaga and to Dr. H. Muthig. This work has been supported by the Deutsche Forschungsgemeinschaft.

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