Phytochrome controls the water permeability in Mougeotia

Botanisches Institut der Universitat Erlangen-Niirnberg, W.-Germany

Phytochrome controls the Water Permeability in Mougeotia M. H.

WEISENSEEL

and E.

SMEIBIDL

With 1 figure Received May 15, 1973

Zusammenfassung Phytochrom kontrolliert die Wasserpermeabilitat bei Mougeotia Wir beobachteten die Plasmolyse von Mougeotiazellen nach hellroter oder dunkelroter Vorbelichtung, somit bei hohem bzw. niedrigem Pfr-Spiegel. In Osmotika, die vorwiegend Mannit enthalten, beschleunigt HeUrot den Beginn und die Geschwindigkeit, und erhoht den Endwert der Plasmolyse. In gleicher Weise werden Beginn und Geschwindigkeit der Deplasmolyse beeinfluik Zugabe von KCI beschleunigt den Beginn der Plasmolyse nach Dunkelrotbestrahlung. Die Differenz zu hell rot bestrahlten Zellen wird daher verringert. Zugabe von NaCl wirkt ebenso, beschleunigt aber zusatzlich die Geschwindigkeit und erhoht den Endwert der Plasmolyse in dunkelrot belichteten Zellen bis nahe auf den Hellrot-Wert. Na+ scheint somit Hellrot zu ersetzen. Hellrotes und dunkelrotes Licht beeinflussen weder das elektrische Potential der Zellen, noch die elektrische Lcitfahigkeit im Medium in signifikanter Weise. (Vorlaufige Ergebnisse.) Aus diesen Ergebnissen laGt sich der SchluG ziehen, daG P lr die Wasserpermeabilitat im Plasmalemma von .Mougeotiazellen erhoht.

Summary In the cells of the green alga Mougeotia the onset of plasmolysis in various osmotic a is controlled by phytochrome. In osmotic a of low ionic strength, viz. culture medium supplemented with mannitol, the onset of plasmolysis is more than twice as fast after red light than after far red light. The effect of red light is reversible by far red light. Addition of KCl or NaCI to the osmoticum accelerates the onset of protoplast contraction in cells after far red irradiation and thus it decreases the phytochrome dependent difference. Phytochrome also controls rate and amount of plasmolysis in mannitol or mannitol plus KCI osmotica. During the first minutes of plasmolysis the rate of contraction is 25 % to 50 % higher in cells with a high concentration of P lr compared to cells with a low Pfr-Ievel. The final value of shortening of the protoplasts is about 10 Ofo greater in cells with a high P h.CO:lcentration than in the other ones. Na+ added to the osmoticum abolishes the difference in rate and amount of contraction by substituting for P lr . Onset and rate of protoplast expansion after plasmolysis are also affected by the level of Pfr- The initiation time is shorter and the rate of deplasmolysis is faster in cells which have a high level of P lr . Red light and far red light have no immediate effect on the electrical potential of punctured Z. Pflanzenphysiol. Bd. 70. S. 420-431. 1973.

Phytochrome and Water Permeability

421

cells, nor do they, or the process of protoplast contraction itself, change the electrical conductivity of the surrounding osmoticum appreciably. (Preliminary results.) These results suggest that phytochrome controls the water permeability of the outer plasmamembrane in Mougeotia cells to some extent.

Introduction Within the range of our current research on the role of cytoplasmic membranes in cell differentiation and growth we are investigating the function of pigments which are associated with these membranes and which are significant for development. The most important of these pigments is phytochrome. It controls a wide variety of developmental events (MOHR, 1972; BORTHWICK, 1972) and it has been argued for its localization in plasmamembranes (HENDRICKS and BORTHWICK, 1967; JAFFE and GALSTON, 1967; JAFFE, 1968). In the green alga Mougeotia this could even be demonstrated experimentally (HAUPT, 1968, 1970, 1972). Several investigators have therefore attempted to reveal changes in membrane permeabilities related to the photoconversion of phytochrome. So far, to our knowledge, a Pir-dependent change in the potassium flux in the pulvinule motor cells of Albizzia julibrissin (SATTER et al., 1970; SATTER and GALSTON, 1971 a, b) and a H+-efflux from mung bean roots (JAFFE, 1970) could be found. There have also been reports of phytochrome-mediated changes in the surf:o:.ce charge of mung bean root tips (TANADA, 1968; JAFFE, 1968, 1970; RACUSEN and MILLER, 1972) and effects on bioelectric potentials of oat coleoptiles (NEWMAN and BRIGGS, 1972). All these phenomena might be related to changes in membrane properties but their origin remains unclear. We therefore thought it worth the effort to probe further and more directly into the problem of phytochrome controlled membrane changes. In our opinion there is probably only one key response of phytochrome eventually related to plasmamembranes (see BORTHWICK, 1972). This view receives some stimulation from recent findings of a control of DNA-synthesis by the outer plasma membrane (McDONALD et al., 1972). To attack the problem of early events in phytochrome action more directly, measurements on individual cells are needed. We decided to work with cells of the filamentous green alga M ougeotia. The cylindrical cells of this species contain active, membrane-bound phytochrome (HAUPT, 1968, 1970, 1972) which controls chloroplast movements (HAUPT, 1959) and morphogenic events (NEUSCHELER-WIRTH, 1970 a). Mougeotia is an aquatic organism whose environment can easily be controlled and rapidly changed. The long threads of cells facilitate fastening and observation of defined cells. Individual cells are still large enough for intracellular measurements with microelectrodes. The experiments reported below were performed to test the hypothesis that phytochrome might modify the water permeability of the plasmalemma. This hypothesis was greatly stimulated by the observation of a phytochrome controlled water exchange in epidermal cells of Taraxacum (CARCELLER and SANCHEZ, 1972).

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Material and Methods The fresh water alga Mougeotia spec. was grown in petridishes at 16° C and 220 lux white fluorescent light applied for 12 h a day. The culture medium (ENS) contained in 1 I: 90 mg NaN0 3, 90 mg K 2HP0 4 and 0.1 I earthdecoct, a watery extract from rotten leaves; pH 6.7 to 6.9; osmolarity about 10 mosmollkg. For fastening filaments and observing individual cells during plasmolysis and deplasmolysis, a lump of about 0.05 ml of algal threads was sucked into one end of a glass tube whose middle part had been pulled to capillary size of roughly 1 mm in outside diameter and 20 mm in length. The section where the algae stuck was lined with a piece of nylon net of 80 ,urn wide meshes (Zurich Bolting Cloth Mfg). This lining proved essential in preventing clogging and allowing rapid medium exchange. There were always a few threads of algae protruding from the lump into the thin part of the tube so that up to 10 cells could be observed clearly. These threads hardly moved during medium change. One to two such tubes were filled with algae and about 0.25 ml ENS and clamped in a pIe xi glass holder which was mounted on a microscope slide. Then, regardless of the type of experiment, the tubes were illuminated for 30 min with dim white fluorescent light from above, to orient the chloroplasts in face position, followed by 10 min of far red light of about 3 W . m- 2 (Schott & Gen. RG 9/2 mm, 15 W incandescent lamp). Afterwards, the tubes were placed in darkness for 45 min. Manipulations during the dark period and all observations were carried out under green safelight (Schott & Gen. BG 18/4 mm + GG 495/2 mm + KG 1/3 mm). All experiments were performed in a temperature controlled room at 20 ± 1° C. For observation of plasmolysis, the holder was mounted on a microscope stage and the glass tube was fitted to a syringe, filled with osmoticum, leaving an air gap between both solutions. The algae were then irradiated with either 5 min red light (Schott & Gen. interference filter AL 664 nm, about 0.6 W . m- 2 ), 5 min far red light (Schott & Gen. interference filter AL 724 nm, about 0.4 W . m- 2 ) or 5 min green safelight (about 0.3 W . m- 2 in most experiments and about 1 W' m-2 in some early experiments1» or 5 min red light followed by 10 min of far red light and green safelight, respectively. As a light source served the regular 6 Vj20 W microscope lamp in connection with the condensor. A heat absorbing filter (KG 1/2 mm) was always in the light path. After irradiation the solution was changed by forcing 1 to 2 ml of osmoticum past the algal threads. It averagely took 3.5 s for ENS supplemented with 0.5 M mannitol and 3.9 s for ENS supplemented with 1.0 M mannitol to reach the cells under observation. When we investigated onset and rate of deplasmolysis, the ENS in the tubes was first replaced by 0.5 M mannitol-ENS after 20 min of the initial dark period had elapsed. Towards the end of the 45 min dark period the tubes were irradiated with the same light regimes as mentioned above, i.e. the cells were illuminated while plasmolyzed. The onset of the irradiation was shifted as to make the total time of plasmolysis 25 min in all experiments. The length of the protoplast was measured at the onset and at the end of each irradiation. Immediately after the light treatment, the osmotic urn was replaced by ENS. The relatively short time in osmoticum and the moderate amount of protoplast contraction had no adverse effects on the cells. Deplasmolysis was always total. All osmotica were prepared by solving the appropriate quantities of mannitol and salts in ENS. The pH of the osmotica varied between 6.8 and 7.4. The osmotic values of aliquots of the solutions were measured with a Knauer osmometer in mosmollkg 2) and are presented in the tables 1 and 4. All observations were performed in groups using the same batch of osmoticum for the experiments with the different light pretreatments.

1) This difference had no significant effects on the parameters under observation. 2) Measurements were kindly performed at the Universitats-Krankenhaus. Z. P/lanzenphysiol. Bd. 70. S. 420-431. 1973.

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For measurements of the electrical potentials of individual cells the threads of algae were fastend to stretched nylon nets with vaseline and pretreated as described. Single cells were penetrated with KCI-filled micropipettes, which were selected for tip potentials below 10 m V and resistances of 40 to 60 MQ in ENS. The cells were punctured in green safelight and then kept in darkness. After 5 min they were irradiated with far red light and then with red light. This light regime was repeated if possible. The intracellular potentials were recorded on an oscilloscope (Tektronix 5031) and a chart recorder. The puncturing and recording equipment is essentially the same as described earlier (WEISENSEEL and JAFFE, 1972). The electrical conductivity in the tubes was measured with agar bridges and a Keithley electrometer (Type 600 B). Further details will be described in another paper.

Results

The onset of protoplast contraction in osmotica

From the data in Tab. 1 it is obvious that the onset of plasmolysis, which is measured by observing the retraction of the protoplast from the cross walls, is controlled by phytochrome. It starts earlier when the P lr level in the cells is high. In osmotic a of low ionic strength (left part in Tab. 1) the onset of plasmolyses after 5 min of red light is more than twice as fast than after 5 min far red. The effect of 5 min red light is reversible by 10 min far red irradiation. Phytochrome is controlling the onset of plasmolysis even if it starts quite rapidly like in 1.0 M mannitol-ENS. We also found some evidence that the effect of P lr on the initiation of plasmolysis is more intense in shorter, i. e. younger cells than in older ones. This fact is illustrated in Tab. 2 where data compiled in Tab. 1 (left column) have been rearranged and grouped according to cell length. Still another fact becomes apparent by comparing the left side of Tab. 1 with the Table 1: Lag-phase of the onset of plasmolysis in various osmotic a after irradiation with different light regimes. The osmotic a were applied after the irradiations. The average times (± standard error of the mean = S.E.) are those which elapsed between advent of the osmoticum at the cells under observation and retraction of the protoplasts from the cross walls. Each value is the average of about 40-60 cells. Abbreviations: R = red light (664 nm), FR = far red light (724 nm), GS = green safelight, ENS = culture medium.

Osmoticum: Osmolarity (mosmol . kg- 1 ) ~

.8 ... 5 min R

:.0
.= Q)

R+10 min GS R +10 min FR FR GS

Onset of plasmolysis (s) 1.0 M 0.5 M 0.5 M mannitol + 0.5 M mannitol + mannitol-ENS mannitol-ENS 0,1 M KCl-ENS 0.1 M NaCl-ENS 535

± 1010

1100

± 9010

760

± 10010

760

± 10 Ofo

13.7 13.8 27.7 33.9 32.4

± 0.64 ± 1.02 ± 1.16 ± 1.72 ± 2.16

3.5

± 0.14

5.8

± 0.14

6.9

± 0.20

7.3

± 0.22

8.6

± 0.40

8.1 7.8 9.8

± 0.30 ± 0.22 ± 0.10

8.4 9.1 9.7

± 0.30 ± 0.28 ± 0.20

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M. H. WEISENSEEL and E. SMEIBIDL

Table 2: Correlation of lag-phase of the onset of plasmolysis (± S.E.) and cell length. The data were taken from Tab. 1 left column and have been arranged according to cell length into two different groups. (For details see Tab. 1.) Onset of plasmolysis in 0.5 M mannitol-ENS (s) Cell length Cell length 85 to 135 ,urn 180 to 270 ,urn c

.g tI!

:.0tI!

... .~ ...'" ~

5minR 5 min R + 10 min GS 5 min R +10 min FR 5 min FR 5 min GS

12.0 ± 0.68 12.4 ± 0.81 29.0 ± 1.67 34.0 ± 2.71 35.0 ± 2.83

16.2

± 1.61

22.8 29.1

± 2.95 ± 1.97

right one. While in osmotica which contain much KCI or NaCI the onset of plasmolysis after red light is more or less corresponding with the strength of these osmotica, the response in cells with low levels of P lr is not. When the osmoticum contains 0.1 M of KCI or NaCI the time for the onset of protoplast contraction after far red light or green safelight is reduced. Application of 0.05 M KCl-ENS or 0.05 M NaCl-ENS, 10 min before irradiation, effects the onset of plasmolysis in a way similar to that one with KCI- or NaClosmotica (Tab. 4). A phytochrome-dependent difference in initiation time can hardly be recognized. The kinetics and amount of protoplast contraction The data presented in Fig. 1 and Tab. 3 clearly indicate that phytochrome controls the initial rate and the total amount of protoplast contraction in osmotica with mannitol and mannitol plus KCl. The rate of plasmolysis and the amount of contraction are increased by a high level of P lr i. e. if the irradiation preceding application of the osmoticum is red. The Pir-dependent difference of the amount of protoplast contraction which is about 10 % after 20 min, is reached to about 80 % to 90 % within the first 5 min of plasmolysis. Afterwards the protoplast contraction proceeds with about the same rate irrespective of the light pretreatment (considering the same osmoticum). The rate of the early plasmolysis after red irradiation increases by a factor of about 1.5 when the strength of the osmoticum is doubled (0.5 M to 1.0 M mannitol-ENS), but rises by a factor of about 1.8 in cells with a low level of P lr . This indicates that at high rates of plasmolysis factors besides the concentration of P lr can become limiting. The response is quite different when NaCI is present in the osmoticum (Fig. 1 b). Rate and amount of protoplast contraction are no more different with respect to the light pretreatment. All values fall quite close to the data obtained with mannitol plus KCI-osmoticum after red irradiation. This effect is similar to observations on Z. Pjlanzenphysiol. Bd. 70. S. 420-431. 1973.

Phytochrome and Water Permeability

425

Ui

. ~ 70

o

·0

---

Co 60

c~

50

.... '"' ''Q.. ~

- ~ -o

()

.... " ""O R

smlnR

5 min FA SminA+,OmlnFA SminGS

5

- O.5 M mannitol+o.lM KCI · EN$ - ---0.5 M mannitoh·O.IM N,CI - ENS

~··-1.0 Mm l"n"ol-E NS

10

15

20

min

10

5

15

20

Fig. 1: Time course of protoplast length contraction in various osmotica after irradiation with different light regimes (see insets) prior to application of the osmotica. The standard errors are small and have been omitted for clarity. (For standard errors at 20 min see Tab. 3.) Table 3: Percent protoplast length (± S.E.) at 20 min in various osmotica after different light pretreatments. (For details see Tab. 1.)

Osmoticum: to:

.g
:.0
...

.~

"...

p...

5 min 5 min 5 min 5 min 5 min

R R + 10 min GS R + 10 min FR FR GS

Ofo protoplast length after 20 min 1.0 M 0.5 M mannitol + 0.5 M mannitol + 0.5 M mannitol-ENS mannitol-ENS 0.1 M KCl-ENS 0.1 M NaCI-ENS 66.9 67.6 75.8 80.2 75.1

± 0.77 ± 0.93 ± 0.86 ± 0.76 ± 0.88

51.3

± 1.23

58.6

± 0.72

61.2

± 0.98

63.2 59.9 61.3

± 1.93 ± 1.43 ± 1.64

68.5 68.3 70.6

± 0.77 ± 0.88 ± 0.73

61.0 61.9 62.2

± 0.75 ± 0.80 ± 0.71

root tips (TANADA, 1972) where Na+ can substitute for Pfr in the adhesion response. Different responses with KCI and NaCI were also obtained when 0.05 M of these salts in ENS were applied 10 min before irradiation (Tab. 4, right part). However, in contrast, in these experiments the amount of protoplast contraction in NaCI-osmoticum is always identical with that in cells of low P lr level plasmolyzed in KCI-osmoticum. This can be explained with the assumption that the cells accumulate NaCI during the incubation time in 0.05 M NaCI-ENS, a process which is probably passive since the gradient of the electrochemical potential for Na+ is directed inward (see below). We also made measurements of the changes in diameter of protoplasts contracted in 0.5 M mannitol-ENS. We found the average change in diameter near 15 Ofo and independent of the P lr level. From this and the change in length the average change

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M. H. WEISENSEEL and E. SMEIBIDL

Table 4: Lag-phase of the onset of plasmolysis (± S.E.) and protoplast length (± S.E.) in two different osmotica. The cells have been incubated in O.OS M KCl-ENS or O.OS M NaCl-ENS, prior to irradiation and application of osmotica. D = darkness. (For details see Tab. 1.).

Ofo protoplast length after 20 min

Onset of plasmolysis (s) 2S min in Osmoticum

I

I

O.OS M KCl-ENS O.OS M NaCl-ENS O.OS M KCl-ENS O.OS M NaCl-ENS O.S M mannitol + O.S M mannitol + O.S M mannitol + O.S M mannitol + O.OS M KCl-ENS O.OS M NaCl-ENS O.OS M KCl-ENS O.OS M NaCl-ENS

Osmolarity (mosmol . kg-1j

640 ± 10 Ofo

660 ± S Ofo

SminR +10 min D c:: .g Smin R :.a +10min FR ...... S min FR >-< +10min D S min GS +10min D

9.7 ± 0.48

10.6 ± 0.22

61.4 ± 0.81

69.1 ± 1.01

10.S ± 0.14

11.9 ± 0.28

71.4 ± 0.S6

71.8 ± 0.70

11.8 ± 0.36

11.1±0.14

71.0 ± 0.47

70.7 ± 0.77

11.9 ± 0.14

12.9 ± 0.22

70.2 ± 0.97

73.2 ± 0.72

'" '"

of the volume was calculated, assuming cylindrical shape of the protoplast. In 0.5 M mannitol-ENS the protoplast volume decreases within 20 min to 57 % after red irradiation and to 65 % after far red. The onset and rate of deplasmolysis

The measurements of onset and rate of deplasmolysis were carried out separately from those of plasmolysis since preliminary results had shown that an exposure time to osmotica exceeding about 30 min can affect the protoplast expansion adversely. Starting with comparable values of protoplast contraction, the onset and rate of protoplast expansion in ENS is obviously modified by phytochrome (Tab. 5). A high Table s: Onset and rate of deplasmolysis (± S.E.) in ENS after a total of 2S min in O.S M mannitol-ENS. Note that in the column «Ofo protoplast length before irradiation» the exposure times in osmotic a differ up to 10 min.

Ofo protoplast length before irradiation

:.a'" ......'" >-<

S min S min S min S min S min

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P/lanzenphysiol. Bd. 70. S. 420-431. 1973.

c::

.g

R R+I0 min GS R +10 min FR FR GS

Ofo protoplast Onset of pro- Completion of L1 time length after top last expan- protoplast ex(s) irradiation sion in ENS (s) pans ion (s) i.e. at onset of deplasm.

68.9 73.1 73.6 69.6 72.6

± ± ± ± ±

0.76 0.71 0.89 0.81 O.7S

67.9 72.3 70.7 68.S 72.0

± ± ± ± ±

0.83 0.90 0.91 0.81 0.72

19.7 22.S 28.7 30.0 32.0

± ± ± ± ±

0.79 0.48 0.87 0.36 0.78

21.7 24.7 31.S 32.9 3S.1

± ± ± ± ±

0.79 0.48 0.96 0.36 0.81

2.0 2.2 2.8 2.9 3.1

Phytochrome and Water Permeability

427

level of P lr reduces the initiation time of deplasmolysis and increases the rate of the expansion process. The effect of red light is reversible by far red light. As already mentioned, the expansion of the protoplast was 100 10 110 in all experiments. The slower onset of deplasmolysis compared to plasmolysis is probably caused by the space seperating cell walls and protoplast and thus delaying the medium exchange. Some preliminary results from electrical measurements

When Mougeotia cells which had been treated with 30 min white light, 10 min far red light, and at least 45 min darkness, were punctured with microelectrodes, a potential difference between ENS and inside of up to 69 mY and an average value of 51 mY (inside negative) was observed. This potential difference appears instantaneously upon penetration. Normally, the potential depolarizes slowly during puncturing. Irradiation of an impaled cell with far red or red light had no detectable effect on the potential within several minutes. We also punctured several cells which had been plasmolyzed in darkness in 0.5 M mannitol-ENS. Again, we found no change in potential upon shining far red or red light on the cells. The average potential of plasmolyzed cells seems to be larger than in normal cells. Furthermore, we measured the electrical resistance in the tubes, filled with the usual amount of algae, searching for a possible decrease in resistance when the protoplasts contract. So far, we found no major change in the tube resistance. It stays near 10 6 Q for 0.5 M mannitol-ENS irrespective of plasmolysis of the cells and of the light conditions. If there would be a loss of about 25 mM dissociable salts from the cells during plasmolysis after red irradiation (see discussion), the tube resistance should dercease by at least 50 0/0. Discussion Plasmolysis and deplasmolysis in a vacuolated plant cell, like a M ougeotia cell, are rather complex phenomena. However, they are mainly dependent on the flux of water across the barriers involved (cell wall, plasmalemma, tonoplast). They are also affected to some extent by a possible flux of solutes and by forces acting between plasmalemma and cell wall. Assuming the more general case of water and solute flux and their interdependency, we may write as an approximation for the rate of plasmolysis -dY/dt i.e. the rate of volume change (cm 3 . S-l), the following equation: (1)

Jv is the net volume-flux, i. e. the rate of movement of water and solute volumes across unit area of membrane (cm 3 • cm- 2 • S-l); a net flux from inside to outside is, by convention, considered to be negative. Vj is the molar volume of solute species, j (cm 3 • mole-1 ). Jw denotes the net volume-flux of water (cm 3 • cm- 2 • S-l) and Jj the net solute-flux (mole· cm- 2 • S-l). Z. P/lanzenphysiol. Bd. 70. S. 420-431. 1973.

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For deplasmolysis, the same equation is valid: (2)

A parameter of some importance especially for the initiation time but also the rate of plasmolysis and deplasmolysis is the «force of attraction» between plasmalemma and cell wall. The greater the area which adheres and the stronger the attraction, the larger becomes the influence of this parameter (which we shall symbolize with the letter q). It is conceivable that red light can cause an increase or decrease in q, which means that the membrane would become more, respectively, less attracted to the wall e. g. by a possible change in surface charges. Now, a decrease in q, mediated by Pin would accelerate the onset and rate of plasmolysis, but slow down the initiation and the rate of deplasmolysis. As a high level of P lr accelerates the onset and rate of both responses and as it seems unlikely that P lr can affect the same parameter in two opposite ways, we may exclude q as a major target of phytochrome action. In the following discussion we shall consider the various parameters which are included in the term J,. trying to elucidate the Ph·-dependence in each case. Moreover, this discussion focuses on the plasmalemma since HAUPT (1972) has shown that phytochrome, active in chloroplast movement, is associated with this membrane. The first parameter to discuss is the volume flux of water Ow) which is proportional to the difference in water potential (,po_Pi) and therefore also to the concentration difference of osmotically active solutes: (3)

The parameter for the hydrostatic pressure outside has been omitted, because it is zero in our experiments. Lw is the water conductivity coefficient. n denotes the osmotic and P the hydrostatic pressure. 0 and i indicate outside and inside, respectively. Now, can P lr affect pi and in this way modify the rate of plasmolysis and deplasmolysis? To accelerate the rate of plasmolysis, pi has to increase. Such an increase would require a substantial power of constriction of the plasmamembranes. (The cell walls can be excluded from this discussion because of their rigid structure and their lack of phytochrome.) We don't know if the plasmalemma can exert pressure on the cell sap by constriction, but we think it rather improbable. To increase the rate of deplasmolysis would mean to decrease pi below zero since the hydrostatic pressure in plasmolyzed cells is supposedly zero. The development of a negative pressure in the protoplast of a M ougeotia cell is quite unlikely. For these reasons, viz. improbability of changes in pi and requirement for opposite effects of P lr on pi, it seems justified to exclude that P lr acts by affecting the inside hydrostatic pressure. The next question to ask is, if the osmotic pressure inside (ni) can change within minutes under the influence of P lr . To cause the observed effect on plasmolysis, ni has to decrease during red irradiation. Now, ni is proportional to the inside concentrations, ci, of solute particles (ni <::S! R T .2' jCj i). Therefore, solutes have to leave the cell or

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Phytochrome and Water Permeability

429

become rapidly bound or polymerized. Large scale polymerization orland binding is improbable because the change in concentration has mainly to take place in the vacuole since the amount of cytoplasm in a M ougeotia cell is only 2-3 Vol. Ofo (K. Foos, pers. comm.). Then, considering the different changes of volume during plasmolysis in 0.5 M mannitol-ENS, a loss of solutes of about 50 mosmol· kg- 1 is to be expected from cells with a high level of P lr . We know that the total amount of solutes inside a cell is about 260 mosmol· kg-t, as determined by incipient plasmolysis. We also know that the inside concentration of Na+, K+ and CI- is about 50 mM each (WAGNER, pers. comm.). Therefore, we can assume that if 50 mosmol· kg- 1 of solutes are lost, a certain part has to be ions, since other likely cell constituents, as e. g. sugars, are not readily permeable. So far, we could find no indication of a substantial flux of ions. It has also been shown with radio isotopes that the fluxes of K+ and Cl- do not change with respect to the level of P lr (WAGNER, pers. comm.). There is even some evidence in our experiments on deplasmolysis that major solute fluxes do not occur. The tendency of the protoplast towards further contraction in 0.5 M mannitol-ENS (Tab. 5) indicates that solutes are not accumulated by the cells. These arguments make it unlikely that major Ph·-dependent solute fluxes are involved in plasmolysis and deplasmolysis in our experiments. (We can therefore omit the term for solute fluxes from our discussion.) But we cannot exclude that some solutes especially small ions, are forced to move by the drag of the water flux. Incidentally, such an effect might bring about changes in surface-potentials. The discussion has now to focus on the coefficient of water conductivity (Lw) which describes the water permeability of the barriers per unit pressure. The first question to ask is, if there is any restriction to the water movement due to membranes at all? There is ample evidence that in living cells such restrictions exist. E. g. the facts that growing plant cells like Nitella (NOBEL, 1970), or cells of germinating lettuce seeds (NABORS and LANG, 1971 a, b) can maintain a difference in water potential, or that the protozoon Opalina (HILDEBRAND and STIEVE, 1972) and erythrocytes (COOK, 1961) take up water after membrane damage by UV, or that the activation energy for water permeation through membranes of oat roots is about 40 % higher than in water (WEIGL, 1967), indicate limitations on the water flux in plasmamembranes. In M ougeotia, a difference in water potential can be inferred from the high growthrate which is about 15 % per day (NEUSCHELER-WIRTH, 1970 b). We may now ask, what the consequences of a Plr-mediated increase in Lw would be. An increase in Lw does mean an increase in the water flux together with a decrease in the water potential difference. This is exactly what has been found in our experiments. The higher rate of plasmolysis and deplasmolysis is evidence for an increase in ]w. The difference in water potential after 20 min in 0.5 M mannitol-ENS is less in those cells with a high level of Plr; Ll'l' in cells after red light is equivalent to about 80 mosmol . kg- 1 and to about 130 mosmol . kg- 1 in cells after far red light. (These values are calculated from ci = 260 mosmol . kg- 1 in normal cells and the appropriate volumes after 20 min plasmolysis, viz. 57 Ofo and 65 Ofo, respectively; pi = po = 0 and CO = 535 mosmol . kg- 1 ).

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Our results therefore suggest that PI,. increases the water permeability in the plasmalemma of M ougeotia. They also indicate that this control is limited since it does not abolish the difference of the water potential. The efficiency of the control probably depends on the number of sites in the membrane occupied by phytochrome. It should not be left unmentioned that in epidermal cells of Taraxacum P r seems to increase the water permeabilty (CARCELLER and SANCHEZ, 1972). One major problem with these experiments seems to be that in tissues, normally not submersed, the physiological medium surrounding the cells is not known. That it is not distilled water, seems obvious. In M ougeotia, we have already some evidence that incubation in distilled water and plasmolysis in 0.5 M mannitol solution can give results opposite to those in culture medium (Details will be communicated later). We like to thank Prof. W. HAUPT and Dr. K. HARTMANN for critical reading of the manuscript and Mrs. CHWOJKA for technical assistance. With support from the Deutsche Forschungsgemeinschaft.

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(1972). MOHR, H.: Lectures on Photomorphogenesis. Springer-Verlag, Berlin 1972. NABORS, M. W., and A. LANG: Planta 101,1-25 (1971 a). - - Planta 101, 26-42 (1971 b). NEUSCHELER-WIRTH, H.: Z. Pflanzenphysio!. 63,238-260 (1970 a). - Z. Pflanzenphysio!. 63, 352-369 (1970 b). NEWMAN, I. A., and W. R. BRIGGS: Plant Physio!. 50, 687-693 (1972). NOBEL, P. S.: Plant Cell Physiology. A Physicochemical Approach. W. H. Freemann and Company, San Francisco 1970. RACUSEN, R., and K. MILLER: Plant Physio!. 49, 654-655 (1972). SATTER, R. L., and A. W. GALSTON: Plant Physio!. 48, 740-746 (1971 b). - - Science 174, 518-520 (1971 a). SATTER, R. L., P. MARINOFF and A. W. GALSTON: Amer.]. Bot. 57, 916-926 (1970). TANADA, T.: Plant Physio!. 43, 2070 (1968).

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