Plant Physiol. (1981) 68, 1135-11430032-0889/81/68/1 135/09/$00.50/0

Water Relations of Leaf Epidermal Cells of Tradescantiavirginiana"2

Received for publication February 11, 1981 and in revised form May 27, 1981

ALUN DERI TOMOS, ERNST STEUDLE, ULRICH ZIMMERMANN, AND ERNST-DETLEV SCHULZEArbeitsgruppe Membranforschung am Institutfur Medizin der Kernforschungsanlage Julich, Postfach 1913,5170 Julich, Federal Republic of Germany (A.D. T., E.S., U.Z.); and Lehrstuhlffur Pflanzenokologie,Universitat Bayreuth, Universitatsstrasse 30, 8580 Bayreuth, Federal Republic of Germany (E.-D.S.)

ABSTRACT

Water-relation parameters (ceil turgor pressure [PI, volumetrc elasticmodulus [el and hydraulic conductivity ILpI) of individual leaf epidermalcells of Tradewscaia vigiaiana have been determined with the pressure-probe technique. Turgor was 4.5 ± 2.1 1411 bar (mean ± SD; In bracketsthe number of cells) and ranged from 0.9 to 9.6 bar. By vacuum infiltrationwith nutrient solution, it was raised to 7.5 ± 1.5 151 bar (range: 5.3-8.8bar). There was a large variability in the absolute value of e of individualcells. e ranged from 40 to 360 bar, mean ± SD: 135 ± 83 bar; n - 50 cells.e values of individual cells seemed to be rather independent of changes incell turgor. A critical assessment of the errors incurred in determining e bythe technique is included. The half-times of water exchange of individualcells ranged from 1 to 35 seconds, which gave values of 0.2 to 11 x 10-'centimeters per second per bar for Lp (mean ± SD: 3.1 ± 2.3 x 10-6centimeters per second per bar, n - 39 cells). The large range in Lp ande Is believed to be due to the difficulties in determining the effective surfacearea of water exchange of the cells. Lp is not influenced by active saltpumping driven by respiration energy inasmuch as it was not altered by 0.1m_liolar KCN. The temprature dependence of Lp (T1/2) was measuredfor the first time in individual higher-plant cells. Lp increased by a factorof 2 to 4, when the temperature was increased by 10 C. The activationenergy of water exchange was found to be between 50 and 186 kilojoulesper mole. Within the large range of variation it was found that T1/2, Ip,and e did not change under various experimental conditions (intact andexcised tissue, water content and turgidity, age, etc.). Similar results wereobtained for the epidermal cells of Tradescaxda anderoiana. The mea-surements suggest that the entire epidermis would respond very rapidly(Lc. with a half-time of 1 to 30 s) to a demand for water from the stomata.

Lp,3 of cell membranes, the volumetric elastic modulus, E, ofindividual cells and their half-times [rate constants] of waterexchange). Recently, these water transport parameters have beenmeasured for mesophyll, subsidiary and epidermal cells of Tra-descantia virginiana (26). It has been shown that the half-times ofwater exchange for epidermal and subsidiary cells of this specieswere rather short (a few to some tens of seconds). The half-timesof the mesophyll cells were, on average, significantly larger thanthose for epidermal cells. However, we found (for intact leaves aswell as for leaf slices) that there was a rather large variability inthe half-times and Lp values of epidermal cells. This could havebeen due either to a biological variation in the plant material (e.g.caused by water status [turgidity], growth conditions, transpira-tional stress, or temperature) or to difficulties in the determinationof cell volumes and surface areas. These have to be known for theaccurate evaluation of e and Lp of higher-plant cells from turgorpressure changes and turgor-pressure relaxation processes, respec-tively.The absolute values of the hydraulic conductivities of the

epidermal cells and of the apoplast as well as the conducting areasfor the cell-to-cell and apoplasmic pathway determine the waterconductance of the epidermis. These parameters are important forcontrolling the proportion of water that flows in the mesophylland in the epidermis during transpiration. A sufficiently highwater conductance (i.e. Lpx conducting area) of epidermal cellscould result in a preferred transport of water in the epidermis. Theconventional picture of the mesophyll as the major site of tran-spiration has been questioned (3, 15, 16). Therefore, it seemedimportant to examine the water relations of the epidermal cells inmore detail.

In this communication, possible effects of leaf hydration (tur-gidity), of cell volume (size), and experimental conditions on cellwater-relation parameters have been studied in an attempt toidentify the reasons for the variability mentioned above.

The evaluation of transport models for the transpiration fromleaves requires data on the hydraulic resistances of the differentsites of water transport and evaporation, ie. of the stomatalapparatus, the epidermis, and the mesophyll. For a correct descrip-tion, it is also necessary to separate the apoplasmic from the cell-to-cell pathway of water in the leaf. The latter includes watertransport across cell membranes as well as through plasmodes-mata. In principle, the miniaturized pressure-probe (8, 23, 24, 28,29) can be used to measure most of the parameters controllingwater transport at the cellular level (i.e. the hydraulic conductivity,

' Supported by a grant from the Deutsche Forschungsgemeinschaft, Zi99/7, and by a research fellowship to A. D. T. from the Royal Society(U.K.).

2Dedicated to the memory of Nod Higinbotham.

MATERIALS AND METHODS

Materials. Seeds and plants of T. virginiana were obtained fromE. Brinckmann, University of Bayreuth. These together with rootcuttings of the original plants were grown in potting soil up to aheight of 10 to 60 cm for 3 to 6 months prior to the experiments.At least 4 weeks before the plants were used, they were transferredto a climatic chamber at a 14 h/10 h (18 C/12 C) light/darkrhythm at a humidity corresponding to a constant dew point ofair humidity of 4 C. Irradiance of 50 to 80 ,uE m-2 s-1 at the level

3Abbreviations: Lp, hydraulic conductivity; e, volumetric elastic mod-ulus; P, turgor pressure; V, cell volume; A, cell surface area; T,,2, half-timeof water exchange of individual cell; k, rate constant of water exchange ofindividual cell; E., activation energy of water flow across cell membrane.

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Plant Physiol. Vol. 68, 1981

of the plants was provided by Osram Fluora lamps.Most of the experiments were performed on excised leaves

dipped in nutrient solution (see below) with the cut end, or onleaves of intact plants, with the leaf held in air in a Perspex holder(26). Additional measurements were also performed on leaves(intact or excised) submerged in a nutrient solution (half-strengthHoagland solution [6] or Johnson's solution [in 5]). In severalcases, the excised leaves were vacuum-infiltrated with Hoaglandsolution before measurement to observe whether turgidity andhydration had an effect on the water-relation parameters (Lp, E).For infiltration, the margins of the leaf slices were cut off, theslices submerged in nutrient solution, and vacuum applied 3 timesfor about 30 min, with occasional shaking to remove air bubbles.

For some noninfiltrated leaves, the osmotic pressure of the leafsap (i.e. cell sap and solution in the vessels) was determinedcryoscopically with a Knauer osmometer. Cell sap was obtainedby squeezing the leaf between two metal plates and collecting thesap with a capillary. The osmotic pressure of this sap was about7 bar. This corresponded to the turgor pressure measured on cellsof vacuum-infiltrated tissue. Under these conditions, where theextracellular osmotic pressure was low, the turgor approached theinternal osmotic pressure of the cells.Methods.Pressure Probe Technique. Cell turgor, volumetric elastic moduli,

and pressure relaxation curves were measured by the pressure-probe technique. This technique has been extensively described inseveral previous papers (e.g. 2, 8, 18) and reviews (e.g. 4, 23, 24,28, 29). The reader is referred to these papers for experimentaldetails. The intact or excised leaf, with the lower epidermisupwards, was mounted in a Perspex holder in a Plexiglas chamber(26) where the tissue could also, if required, be submerged in anutrient solution of a defined temperature (21-23 C unless other-wise mentioned). The tip of the pressure probe was introducedinto an epidermal cell under the stereomicroscope (magnification70-130x). The technique permits the continuous measurement ofturgor and also the manipulation of cell volume and turgor toinduce volume flows. The volumetric elastic modulus (e) may beevaluated by instantaneously changing cell volume and measuringthe corresponding change in turgor (AP). E is given by (cf. 4, 28):

dP APVdV A (1)

The hydraulic conductivity (Lp) of the cell membrane may becalculated by measuring the relaxation of turgor pressure afterinducing an instantaneous change in cell volume (and turgor).Provided that there is a linear relationship between water flowand the driving forces (i.e. the differences of the chemical potentialof water between cell and surroundings [4, 28]), Lp is estimatedfrom the half-time of pressure relaxation (T1/2) by (cf. 4, 28):

V.ln2P AT112(E + Ti) (2)

where V = cell volume, A = cell surface area and vi = osmoticpressure of the cell sap.Changes in cell volume (A V) were measured by following,

under the microscope, the movement of an oil/cell-sap meniscuswithin the tip of the probe. This procedure avoids errors due tothe compressibility of the equipment, to leakages, and to thermalexpansion (8, 28). In the pressure probe technique, the meniscusis held constant in a desired position and is then moved by adefined amount and is again fixed in the new position. Theposition of the meniscus can be controlled electronically (8) ormanually (28). In this paper, manual control was preferred, be-cause the half-times of water exchange were rather short andbecause some of the measurements have been performed with theleaf or leaf slice in air. The measurement requires tips that aresufficiently fine (2-5 ,um for Tradescantia cells) but in which the

meniscus size and shift are both sufficiently large to enable A V tobe estimated with accuracy. The hydraulic resistance of the tipmust be much smaller than that of the cell. This was checkedbefore each experiment. In all cases it was ensured that the half-time of water exchange across the tip was much smaller than thatof the cell and that the ratio AP:A Vwithout a cell was at least oneorder of magnitude smaller than when the tip was in a cell.

Evaluation ofe- Values. Provided that the ranges for AP are nottoo large (see below), E is practically constant and the plots APversus A Vwil yield straightlines. A complication of the measure-ments (especially of E) may arise if the cells measured have shorthalf-times of water exchange. This is generally the case with theleaf epidermal cells of Tradescantia. If the time required by theequipment to bring about the volume change is of the same orderas the half-time of water exchange across the cell membrane, Ewil be underestimated, i.e. the A V measured from the meniscusshift in the tip of the probe will overestimate the actual A V of thecell (see equation [1]). The resulting AP/A V-curves will be sig-moid, i.e. they will bend towards the positive and negative A V-axes (18). It has been shown (18) that for cells with rapid waterexchange, E can be estimated for a certain pressure interval fromAP/A V-curves by an extrapolation to zero AV. Provided that ecan be taken as constant in the given pressure range it holds (18)for either positive or negative AP and AV that:

AP = B(1-ebV) (3)or

B - APln =-b.AVB (3a)

where B = e J'VaPP/ V - k (J VaPP = water movement in the tip ofthe probe in cm3 s-'; k = In 2/T1/2 is the rate constant of waterexchange and b = k/l VaPP). It is evident that for A V- 0 equations(3) and (3a) become identical with equation (1).

It should be noted that the correction for water flows can bemade only if the assumption of a constant e in the given pressurerange is justified. If e were not constant but increased withincreasing pressure, it would result in an additional complicationdue to a superposition of both effects. In this case, the sigmoidcurves bend asymmetrically with respect to the origin. For AV,AP < 0 (i.e. for a decrease in cell turgor) the bending towards thenegative A V-axis will be increased, whereas for A V, AP> 0 (i.e.for an increase in turgor) the effect would be opposite.The AP/A V-curves measured in this paper were usually sym-

metrically bent, which points to a rather small dependence of E oncell turgor, and in these cases a constant e was assumed over thewhole range of pressure. For those AP/A V-curves which wereasymmetric, it has been assumed that e was constant for the rangesof high (i.e. AP> 0) and low (i.e. AP < 0) pressures, respectively.To evaluate e from bent (exponential) AP/A V-curves, the mea-

sured data of AP and AV were fitted according to equation (3a)by a least-squares fit to obtain b and B.4 The volumetric elasticmodulus can then be calculated if the cell volume is known, sincee= b.B. V.Cell volumes (V) and cell surface areas (A) were calculated

from measurements of the cell dimensions. For the calculation,the cells were assumed to be shaped like rectangular columns ofeither cylindrical or square cross section, since tissue sections (bycourtesy of Dr. D. Kramer, Darmstadt, and Dr. E. Brinckmann,Bayreuth) showed that the epidermal cells were as deep as wide(Fig. 1). A discussion of the errors in e and Lp resulting from thedifferent experimental parameters (i.e. V, A, A V, AP, T1/2) is given

4 It should be noted that equation (3) does not describe the entiresigmoid curve, but only either the positive or negative part of it. For thosesymmetrical cases where both positive and negative values were takentogether, the absolute values of AP and A V were used.

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WATER RELATIONS OF LEAF EPIDERMIS

FIG. 1. A, Cross-section of a leaf of Tradescantia virginiana (mcation: 160x). B, Surface view on lower epidermis (magnification:

under "Results".Temperature Dependence of T1/2; Effect ofKCN. The ten

ture dependence of T1/2 and e was measured with tissuewhich were vacuum-infiltrated and submerged in Hoaglailution. The solution was maintained at temperatures betweand 25.8 C. The vacuum-infiltration prevented the suddcpearance of leakages in punctured cells that occurred 4changes of temperature of untreated slices. These leakagesto have been due to tissue movement as a result of tUexpansion of the intercellular air spaces. Between each nevperature regime, control experiments were performed attemperature to ensure that all the changes observed were r(ble. Measurements of e were made both before and after tideterminations at each temperature point. It should be notethe temperature dependence of T1/2 (Lp) was measured oIvidual cells. Therefore, errors due to a variation in V, A, orcell to cell should only play a role for the evaluation 4absolute value of Lp (see equation 2) but not for the richanges with temperature, i.e. they should not affect the evalof activation energies.

Several experiments were performed in which the tissubeen vacuum-infiltrated with solutions of KCN. This wasfor both 1 mm and 0.1 mm KCN solutions and measurementperformed from 15 to 90 min after poisoning the cells. Consi4intracellular air spaces, the actual KCN concentration in theafter this procedure was estimated to be 10 ,UM to 0.1 mm.

RESULTS

Elastic Moduli. Figure 2 shows typical examples of AJcurves from which e has been determined. Usually the curve

* symmetrically (i.e. E was practically constant) (Fig. 2, A and B),although the ranges for cell turgor during the measurement of ewere (for experimental reasons) rather large (e.g. P = 1 to 5 bar orP = 9 to 15 bar [Table I]). Sometimes, also asymmetrically shapedcurves (Fig. 2C) and straight lines (Fig. 2D) were observed. Asexpected from the theory, straight lines occurred whenever thehalf-times of water exchanges of the cells were long.

oF As described under "Materials and Methods," the data pointsof Figure 2, A through C were fitted according to equation (3) byan exponential least squares fit to yield b and B. Replotting of the

B - APdata in the form of In B against AV I (equation 3a) yieldedlinear relationships (see insets in Fig. 2, A-C), which indicate thatthe assumption made on constant E holds in practice for thepressure ranges given.The e values found for the epidermal cells (Table I) were rather

large (E= 40360 bar; mean ± SD: 135 ± 83 bar; n = 50 cells) incomparison with other higher-plant cells (see 28, 29). They are ofthe same order as those given previously for these cells (E = 40-240 bar [26]).

In Table I the E values and the corresponding total errors (seebelow) are listed for all cells together with the ranges of turgorpressure for which they had been determined. The ranges for thecell volume changes are also given. It can be seen that AV wasonly up to 7% of the cell volume.

I J The e values of Table I show that there is a large variation inE from cell to cell and that (within the limits of accuracy) E is notinfluenced by cell turgor and cell volume (Table II, Fig. 3). Thisfinding is in agreement with the fact that few of the AP/A Vcurveswere found to be significantly asymmetric with respect to the

W ,, origin (see above). However, we have to admit that the pressurechanges for measuring e were fairly large, so that a small pressuredependence of e could have been overlooked due to the largevariation of e from cell to cell. Therefore, the apparent absence ofa pressure and volume dependence of e found for many other

i fi plant cells (28, 29) must be treated with caution. Although, in the1agnif case reported here, the volume was varied by more than one order

x) of magnitude (Table I).Error Considerations. Quantitative measurements of water re-

npera- lation parameters on the cellular level are subject to differentslices sources of error, (a) because the measured quantities (volume,hdso- force changes, surface areas, etc.) are rather small and (b) becauseen 8.4 some of the parameters such as E are not directly measured, buten ap- are calculated from other quantities. They could, thus, accumulateduring different errors. This is independent of the method used fors seem determining water relation parameters.iermal The basis for the evaluation of accumulative errors in E isv tem- GauB's law of error propagation (see textbooks of physics androom statistics, e.g. [9]), which has been used in this study.

eversi- For the nonlinear AP/A V curves, e is given by e = b *B * V and,ie T1/2 thus, errors in estimating b, B, and V have been considered. Thed that errors in b and B can be estimated from the semilog plots of Ina

frodm B P versus IA V I (see insets of Fig. 2, A-C). Errors in b are theof theelativeuation

ie hads doneLs werederingtissue

P/A V--s bent

errors in determining the regression coefficients of the straightlines in these plots, while the errors in B are given by the ordinateintersections. In this paper these two errors have been determinedfor a 0.95 confidence interval of the regression coefficient. Theyadd up with the error in estimating the cell volume (20-50%) togive the accumulative error in e.' Furthermore, errors in measuring

5Since e = b-B. V, the accumulative error in e(aE) is given by:

(FE= ±4E (y+ (C)B + (c)

according to GauB3's law of error propagation, ob, aB, and av representerrors in determining b, B, and V, respectively.

Plant Physiol. Vol. 68, 1981 1137

. ..r--

le; -1t'..j14 I.,

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Plant Physiol. Vol. 68, 1981

1cs.0I-

C0

-0.5 Av [pi1-10 Cett No: 42

P=3.8 to7.2 bar-1 5 V=1760pt

lj, 161-35.5 s

FIG. 2. AP/A V curves for the determination of E in epidermal leaf cells of T. virginiana as obtained by the pressure probe. Due to rapid water flows,the AP/AV curves can be shaped sigmoidally (A-C). For constant e these curves are symmetrical with respect to the origin (A and B), whereas for eincreasing with increasing turgor they become assymmetrical (C). If the rates of water exchange during measurement are sufficiently small and econstant, straight lines are obtained (D). In the insets of A through C the data are replotted according to equation (3a) to correct for water flows (seetext). Dotted lines in the insets of A through D represent the 0.95 confidence interval for the regression coefficient of the straight lines. Ranges ofabsolute cell turgor (P), cell volumes (V) and TI/2 are also given.

A V have to be considered, i.e. abscissa errors denoted by horizon-tal bars in Figure 2, A through D. They result from uncertaintiesin measuring the diameter and shift ofthe meniscus in the capillaryand range from 0 to 40%o. These errors have to be added to theaccumulative error to give the total error in e.The linear AP/A V curves (Fig. 2D) have been similarly ana-

lyzed. The errors in their regression coefficients have also beenestimated for 0.95 confidence intervals and the errors in V andA V considered as for the nonlinear curves. The e values of TableI are given with the corresponding total errors in e.The errors in e mainly originate from uncertainties in the

estimation of the cell volume (shape, dimensions) and not fromerrors in the measurement of turgor and cell volume changeswhich are determined by the pressure probe.

Half-times of Pressure Relaxation and Lp. The e values for theepidermal cells have been used to evaluate the hydraulic conduc-tivity of the cell membranes from pressure-relaxation curves ac-cording to equation (2). In each case the relaxation curves weremeasured within the same pressure range as the corresponding evalues. The results are summarized in Table III. Lp ranged from0.19 to 10.8. 10-6 cm S-1 bar-' (mean ± SD: 3.1 ± 2.3. 1-6 cm.s-1 bar-'; n = 39 cells) and thus varied by almost two orders ofmagnitude. The absolute values as well as the range for Lp weresimilar to those reported previously (0.26 to 5.5.10-6 cm S-1bar-' [261). These values are in the upper part of the range of Lpvalues reported for higher-plant cells in the literature (28, 29). The

relaxation curves have been measured under various conditions inorder to identify the cause of the variability in the half-times ofthe epidermal cells of Tradescantia. Most of the measurementswere performed on excised leaves in air or submerged in Hoaglandsolution, but also intact leaves (still attached to the plant) wereused. In some cases, leaf slices were vacuum-filtrated with nutrientsolution to see whether turgidity, water content, or size of inter-cellular air spaces play a role in the variability of half-times. Theincrease in weight after this procedure was 24 to 27% of the freshweight, indicating large intercellular spaces in the mesophyll. Afterinfiltration, cell turgor was substantially higher (P = 7.5 ± 1.5 [5]bar (mean ± SD) than in epidermal cells of untreated tissue (P =4.5 ± 2.1(41] bar). However, the half-times were not changed bythe variation of the water content and ranged between I and 35 s(see Table III). These half-times of water exchange of the leafepidermal cells of Tradescantia are rather short. Although thevalues in this paper are of the same order as the majority of thevalues reported previously (Tl/2 = 4-98 s [26]), they are on theaverage somewhat smaller.

Effect of Temperature and KCN on T,/2, Lp, and c. Theinvestigation of the temperature dependence of T,/2 (Lp) wasperformed on several individual cells to avoid variations in e andLp from cell to cell. The mean half-times were observed to beincreased some 2 to 4 times by a temperature decrease of 10degrees between 22 and 12 C. Since there was no indication of atemperature dependence of e for temperatures between 8.4 and

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Plant Physiol. Vol. 68, 1981 WATER RELATIONS OF LEAF EPIDERMIS

Table l: Volumetric elastic moduli (t) of leaf epidermal cells ot Tradescantia virginiana. E-values refer to the pressure ranges and to the ranges of absolute cell volumechange (IAVI) given. The errors in f include the errors in b. B. V and JAVI (see Eqs. (3) and (3a) and Results). The errors in b and B have been determined byregression analysis (n = number of data points; confidence interval = 0.95) according to semilog plots such as those shown in Fig. 2.

Cell CellCl volume.

V [p1]

240

320

380

390

430

440

440

460

470

470

520

570

620

630

710

710

710

750

790

830

900

920

940

950

970

n

4

444

5364

141410

4

12

8

344

99548

6

12

5

441098664161412

8

Pressurerangefor t

[bar)

2.0- 4.7

6.7- 10.76.7- 3.43.3- 5.6

5.4 - 10.75.4 - 0.78.8 - 14.88.8- 4.05.9- 11.15.9 - 2.35.3- 9.1

6.8 - 11.3

2.5- 5.0

4.7- 7.4

2.3- 3.82.3- 1.04.7- 6.9

9.6 - 11.69.6- 7.42.2- 4.52.2- 0.88.1 - 11.2

2.2- 4.1

4.9- 7.8

6.3- 8.1

5.8- 7.85.8- 4.15.1- 6.25.1- 3.55.7- 7.75.7- 3.55.4- 7.75.4- 3.12.2- 3.72.2- 0.88.7- 11.8

32- 6.1

MeanPressureP [bar]

3.4

8.75.14.4

8.13.1

11.86.48.54.17.2

9.1

3.8

6.1

3.11.75.8

10.68.53.41.59.7

3.2

6.4

7.2

6.85.05.74.36.74.66.64.33.01.5

10.3

47

Range ofIAVI in [pi]and 0/0 of V(in brackets)

[[bar)

17.0(7.1) 46± 32

8.5 (2.7)7.3 (2.3)

13.2 (3.5)

7.4 (1.9)17.4 (1.9)!8.5 (2.0)17.3 (1.7)!9.7 (2.2)9.7 (2.2)

23.1 (5.3)

26.9 (5.9)

25.9 (5.6)

23.1 (5.0)

10.9 (2.1)10.9 (2.1)23.1 (4.1)

18.7 (3 0)18.7 (3.0)9.1 (1.5)9.1 (1.5)

34.5 (4.9)

20.4 (2.9)

10.9 (1.5)

27.3 (3 7)

21.1 (2.7))16.9 (2.1)5.4 (0.7)6.5 (0.8)

25.2 (2.8)25.2 (2.8)25.2 (2.7)25.2 (2.7)16.5 (1 8)16.5 (1.8)34.5 (3.6)

34.5 (3 6)

212 ± 140234 ± 15591 ± 53

233 ± 153

305 ± 142

229 ± 147235 ± 14374 ± 30

77 t 51

63 ± 29

144 ± 72

97 ± 77

73 ± 31

125 ± 88136 t 80169 ± 121192 ± 20267 ± 52

62 ± 22

315 + 250

72 + 31

74 ± 44

315 ± 256191 + 11094 + 4680 + 45

111 + 5995 t 86109 + 4372 + 30145 t 78

96 + 34

Cell CellNo volume.No V [pi]

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

1090

1090

1110

1150

1150

1210

1240

1320

1370

1370

1390

1460

1480

1540

1640

1710

1760

1770

1950

1970

2050

2150

2530

2750

5640

n

739494738

1345

11815

55864

9

4

10

16

10814

18

13

12

14

8

8

8

Pressurerangefor a(bar]

5.9- 9.65.9- 3.97.0- 8 77.0- 5.44.8- 7.04.8- 3.53.2- 7.73.2 - 0.94.7- 7.8

5.3- 7.55.3- 3.35.2- 7.0

1.8- 2.61.8- 1.06.1 - 12.2

5.1- 7.15.1- 3.29.2- 11.59.2- 6.42.6- 4.2

0.9- 2.6

1.3- 3.1

3.5- 6.5

3.4- 7.8

5.2- 7.25.2- 3.87. - 10.2

3.4- 5.1

7.8 - 11.2

3.2- 6.9

4.4- 9.6

5.0- 8.6

8.5- 10.3

5.3- 7.3

MeanPressureP [bar]

7.84.97.96.25.94.25.52.16.3

6.44.36.1

2.21.49.2

6.14.2

10.47.83.4

1.8

2.2

5.0

5.6

6.24.58.9

4.3

9.5

5.1

7.0

6.8

9.4

6.3

Range ofJAVI in [pi]and 00 of V(in brackets)

30.6 (2.8)122.4 (2.1) !16.5 (1.5)l16.5 (1 5)141.1 (3.7) l20.6 (1.9) 156.4 (4.9) 141.4 (3.6) 121.8 (1.9)

31.6 (2.6)37.9 (3 1)16.5 (1 3)

16.9 (1.3)14.5 (1.1)97.0 (7.1)

12.1 (09)l12.1 (09)120.2 (1.5)25.2 (1.8)38.2 (2.6)

39.6 (2.7)

38.8 (2.5)

42.9 (2.6)

53.8 (3.1)

64.7 (3.7)38.8 (2.2)52.6 (3.0)

60.6 (3.1)

43.1 (2.2)

67.3 (3.3)

74.0 (3 5)

56.9 (2.3)

49 1 (1 8)

48.5 (0.9)

[bar]

97 + 53

213 ± 101

89 ± 38

78 ± 35

174 ± 58

83 ± 4298 + 56170 t 91

93 ± 4168 t 26151 ± 65

364 + 236

243 t 147236 + 15264 + 17

95 t 58

40 t 28

165 + 117

73 ± 21

71 + 2269 ± 20140 ± 59

66 + 18

164 + 47

50 + 13

71 + 18

211 + 66

96 + 34

356 ± 92

Table I: Volumetic lastic modulus (e) Of ea epideml cell of Tr,adscantisvgirga of difbrng mmn turgor presu (P5). e-vak are given ± SDwith t number of cell in bracket. E is independent of pressr for thegimn prure inWrvas at a probabit lvel of p = 0.05.

Range of meancell turgorpressr.P[bar]

Voluericelabic

mxok*n,d[be]

Cell volume

p1]

1 - 3 96± 45 (8) 520- 15403 - 5 101 ± 56 (14) 240 - 19505- 7 166 101 (21) 320-56407- 9 154 ± 70 (9) 320- 21509 - 11 153 ± 78 (9) 430 -2750

5. Because of the difficulty of maintaining the cells free of leaksfor a sufficiently long period oftime (up to 6 h) for the experiment,a satisfactory number of measurements were performed on sevencells only. Estimates for the activation energy of water transport,EW, were calculated from the slopes of Arrhenius plots obtainedfrom each cell (such as those illustrated in Fig. 5). These rangedbetween 50 and 186 kJ.molF' with a mean value of 106.9 ± 55.1kJ mol-' (expressed ± SD for seven cells).The Lp (T1/2) values obtained for cells of leaf slices infiltrated

with KCN solution were not different from those obtained fromnonpoisoned tissue. Lp values of 1.34 ± 0.64. 10-6 cm s-' bar-'were obtained from measurements on five cells. This figure is inthe range given for the Lp of untreated cells (Lp = 3.1 ± 2.3-10-6 cm s-' bar-) and within the given variability it is notdifferent from the Lp of nonpoisoned cells. The e values werethose expected from infiltrated tissue and the AP/AV curvesshowed all the characteristics described for nonpoisoned cells.

1139

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

25.8 C (Fig. 4) (the range within which the experiments wereperformed) the values ofLp were accordingly considerably smallerat lower temperatures. This is exemplified for two cells in Figure

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Plant Physiol. Vol. 68, 1981

x

x

x

x

xx

P= 2 to 4bor

x

xx

500 1000 1500 2000 2500

x Cell volume, V [pI]P =6to 8bor

x

x

x

xx

x

x x x X

U W mWUCell volume, VlplJ

FIG. 3. Volumetric elastic modulus (e) as a function of cell volume (V)for two ranges of average cell turgor (P). Within the limits of the variationof e from cell to cell (each point represents a different cell) and the limitsof accuracy for the estimation of e (see "Results"), there is no dependenceof e on cell volume.

18-83bar Pc-n 200-E

* U

al

* * V

>

I I .

0 10 20

Temperature, t [ C ]

P=19-6 8bar

**s* ,S

0 10 20

Temperature, t [ C

0

FIG. 4. Effect of temperature on the volumetric elastic modulus of twodifferent leaf epidermal cells (A and B) of T. virginiana. Within the limitsof accuracy there is no effect of temperature on e.

To determine whether there might be a difference between theLp and e values of different Tradescantia hybrids, several experi-ments were performed on Tradescantia andersoniana (obtainedfrom Kayser and Seibert, Rol3dorf/Darmstadt, FRG). Here againno significant deviation from the values obtained for T virginiana

was observed. The Lp value was 4.7 ± 1.3. 10-6 cm s-1 bar-' forfour measured cells. e ranged from 46 to 138 bar and the AP/A Vcurves also showed all the characteristics of those described for T.virginiana.

DISCUSSION

The results show a large variability in e and T1/2 and an even

larger variation in Lp. The data indicate that, at the present stateof knowledge, differences in the leaf water content, turgidity, cellvolume, and experimental conditions have (within the limits ofaccuracy) no effect on the variability in T1/2 and Lp and are

unlikely to cause the variations observed. The variability betweenepidermal cells of T. virginiana is of the same order as betweencells of this species and a hybrid (T. andersoniana). Furthermore,the respiratory status of the leaf cannot be contributing to thevariability in Lp, as shown by the inhibition experiment withKCN. This latter point suggests that interactions ofwater transportwith solute transport driven by respiratory energy are not involved.A coupling between solute and water flow has been suggested tobe the reason for a strong pressure dependence of Lp in lowpressure ranges for giant algae such as Valonia (27).

Variations in T1/2 and Lp may arise from the fact that cells maynot behave like isolated cells placed in a well-stirred medium, asassumed in the relaxation experiments. Furthermore, unstirredlayer effects (sweep away effects [4J) and back pressures fromadjacent cells may be significant. However, these effects shouldbe negligible, as the amounts of water moved across the cellmembranes are small (see discussion in ref. 18). Experimentalevidence that they are insignificant comes from probe experimentson suspension culture cells of Chenopodium rubrum (1, 2) whichshow that T1/2, Lp, and e of these cells were the same as that ofcells in the intact tissue.

Although changes in the cell surroundings should be small, it isnot clear which part of the cell surface is effectively conductingduring the pressure relaxation experiments. Compared with otherspecies, large epidermal cells (Fig. 1) are a typical feature ofTradescantia and, thus, the conducting area for the lateral watertransport from cell to cell is large compared with the conductingarea for apoplasmic transport. Cutinized anticlinal walls (whichare found in other species) are not present in the epidermis ofTradescantia (E. Brinckmann, University of Bayreuth, personalcommunication). The outer cell wall (which is covered by thecuticle) and the inner cell walls (which are in contact with themesophyll) may not completely take part in the water exchangebecause water would have to flow tangentially over long distancesin these walls and, thus, they could offer a rather large resistanceto water flow (cf. 17). This may be one of the reasons for the largevariation in Lp from cell to cell (Table III), because Lp has beencalculated using the cell surface area and not the effectivelyconducting area of the cell. Another reason for the variation in Lpmay result from the fact that in using the cell surface area it hasbeen assumed that the plasma membrane of the cells is planar, i.e.that invaginations or folding do not occur which may differ fromcell to cell. Invaginations of plasma membranes have been re-ported for several plant cells, e.g. under conditions of osmoticstress (22, cf. 25). The linear relationships between water flow anddriving forces are not valid for folded membranes and the Lpvalues of these membranes may be changed substantially (13).Both hypotheses are plausible, but the present results do not

allow us to test them. This would require ultrastructural investi-gations of the epidermis as well as measurements of Lp on isolatedcells of Tradescantia. These have not been performed up to now.We may assume that the E. for the diffusion of water in the

walls (or cytoplasm) of epidermal cells is similar to that for theself-diffusion of water in aqueous solutions (abot 19.3 kJ.mol-l;cJf 7). With this assumption, the view of membrane-controlledwater flow across the conducting cell surface area (see above) inthe relaxation experiments is supported by the large activationenergy of 50 to 186 kJ mol-' found for Lp (T0/2) in this paper.There are only a few attempts in the literature to measure activa-tion energies of water transport in higher plant tissue. Weigl(21) found an Ea of 26 kJ-mol-1 for the water uptake into cornroots measuring the temperature dependence of tne D20 exchange.More recently, Tyree and Cheung (19) have used the pressurebomb to get the same value for beech leaves. In both cases, theinterpretation of the experimental data is not straightforwardbecause of the influence of unstirred layers (D20 exchange) andbecause of uncertainties in the identification of the hydraulic

200-

x

-Z

100-

V~w'D oL

6-tna300a1)

Eo 200

1oo[

P=1-0

W'I

1200-

0

0w 100-

>-

0111

1140 TOMOS ET AL.

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Table IlIl: Hydraulic conductivity (Lp) of leaf epidermal cells of Tradescantia virginiana. Lp-values have been calculated from half-times of water exchange (T,,2) using thee-values of Table I. which were determined for the same ranges of cell turgor. Values of inwardly directed endosmotic (en) and exomotic (ex) water flow are givenseparately. Forthe calculation of Lp an intemal osmotic pressure (or') of about 7 barwas estimated from cryoscopic measurements of expressed cell sap. For someexperiments only T,,2 and not Lp is given since no E was measured in these cases. Values are given ± SD.

CellNo ex

/en

I28

30

31

33

34

36

37

39

40

41

42

43

44

45

46

47

48

49

50

Pressurerangefor Lp[bar]

ex 4.7- 7.5en 5.0- 2.7ex 4.6- 7.7enex 5.7- 7.5enex 1.5- 2.7

en 1.7- 0.5

ex 6.0- 12.1en 6.3- 2.8ex 9.0 - 11.8en 9.3- 6.4ex 2.7- 4.3enex 1.4- 2.4en 1.5- 0.8ex 3.0- 4.7en 3.0- 1.3ex 3.5- 5.9en 3.9- 0.6ex 5.2- 6.1en 5.1- 3.7ex 7.3 - 11.2en 7.6- 4.7ex 3.8- 5.6en 4.1 - 2.5ex 8.1 - 10.9en 8.3- 5.5ex 3.4- 4.5en 3.6- 2.5ex 4.6- 6.4en 4.8- 3.4ex 5.2- 8.7en 5.3- 2.3ex 8.6 - 10.6en 9.0- 7.2ex 5.6- 7.7en 5.7- 3.5

Half-timesfor waterexchangeT l2[1]

3.7. 10.34.7. 5.822.9

7.5 8.4.4.6

2.7 4.1. 3.9. 5.75.8. 5.4. 5.7 5.64.3. 3.8. 3.8. 7.37.6. 7.5. 5.1. 9.9

2.5 6.35.8. 4.6

2.53.4. 3.0. 3.9

3.6. 3.9

7.37.79.2

15.0. 13.24.4. 6.0

6.1. 5.2 6.416.1. 34.8. 33.2

212. 35.52.64.54.07.3

2.9. 2.92.7. 2.9

5.58.0

7.1. 8.710.6. 11.6

10.316.510.414.0

2.3. 4.22.8. 3.2

Hydraulicconductivity.

Lp 1 o6[cm s-' bar'l

2.6 ± 1.71.8 ± 0.3

0.28

2.1 ±0.7

2.7 ± 0.9

3.0 1.1

3.6 + 2.2

2.21.7 + 0.25.2 t 0.3

4.1

* 0.92

3.7 ± 0.8

0.76 + 0.360.73 t 0.26

3.9

5.2

3.0 + 0.0

4.9

2.5 ± 0.3

0.7

1.6

2.0 + 0.8

Experi-mentalcondi-tions

(2)(2)

(3)

(8)

(8)(2)

(3)(2)

(2)

(3)(2)

(2)

(2)

(2)

(3)

(2)

(3)

(1)

(7)

(3)

(3)

(3)

(3.8)

(3)

(7)

(3)

(3)

(6)

(3)

(3)

(3)

(5)

(3)

(1) Intact plant. Lower epidermis. Measured in air.(2) Intact plant. Lower epidermis. Measured under Johnson's solution.(3) Excised leaf. Lower epidermis. Measured in air.(4) Excised leaf. Lower epidermis. Measured under Hoagland's solution.(5) Leaf slice. Lower epidermis. Infiltrated with Hoagland's solution under vacuum. Measured under Hoagland's solution.(6) Excised leaf. Lower epidermis. Hydrated with Hoagland's solution via cut end for 1 8h Measured under Hoagland's solution.(7) Detached leaf. Upper epidermis. Hydrated with Hoagland's solution via petiole for 18 h. Measured under Hoagland's solution.(8) 18 month old plant taken directly from greenhouse.(9) 5 month old plant taken direcfly from greenhouse

resistances which had been actually measured (pressure bomb).The pressure probe avoids these difficulties because with thistechnique hydraulic resistances of individual cells in a tissue aredetermined. Perhaps, the differences between the E. values foundfor Tradescantia and those reported previously are due to this fact.

Despite substantial variation, the values of T1/2, Lp, and e ofthe epidermal cells of Tradescantia should fall within the limitsgiven in this paper. These results allow two conclusions about thestationary and dynamic (transient) water relations ofthe epidermisof Tradescantia. These are also important for the water relationsof the whole leaf. (a) It cannot be excluded that under stationaryconditions (e.g. during transpiration) water flow in the epidermiscould be a cell-to-cell (membrane-controlled) transport over shortdistances. (b) Propagation of changes of the chemical potential of

water in the epidermis is a fast process.The first conclusion may be drawn from the large absolute

value of Lp (which would be even larger if the conducting area ofthe cells [see above] would have been taken into account) andfrom the large cross-sectional area for the lateral water transportin the epidermis (see above). However, membrane-controlled wa-ter transport in the epidermis would be substantial (as comparedwith transport in vessels) only if there were not too many mem-brane barriers in series, ie. over distances of only a few cell layers.To decide whether or not the epidermis of Tradescantia (and

perhaps also of other higher plants) is a preferred path for tran-spirational water flow, the resistances of cell-to-cell and apo-plasmic pathways in the mesophyll and epidermis have to beknown. At the present, only the Lp values of epidermal and

CellNo ex/

Pressurerangefor Lp[bar]

2.1 - 4.22.3- 0.66.4 - 10.36.9- 3.58.6- 12.49.0- 4.86.0- 10.96.1 - 2.26.0- 8.85.3- 2.62.8- 4.42.7- 1.54.7- 6.95.0- 3.34.7- 6.44.8- 3.4

9.6- 6.38.7-11.18.7- 6.42.0- 4.0

4.9- 8.04.9- 1.56.4- 8.86.5- 4.25.7- 9.25.9- 2.85.4 - 6.75.3- 3.65.7- 7.55.7- 3.95.0- 7.25.4 - 3.22.2- 6.02.4- 1.13.4- 6.23.7- 1.87.0- 9.77.0- 4.9

1 exen

2 exen

5 exen

6 exen

7 exen

9 exen

10 exen

12 exen

13 exen

15 exen

16 exen

17 exen

18 exen

19 exen

20 exen

21 exen

22 exen

23 exen

25 exen

27 exen

Half-timesfor waterexchangeT,,2[sJ

5.4, 17.013.47.311.9

12.3, 12.315.1

2.7, 4.93.2

4.9, 8.75.3, 9.0

5.1. 1.7, 2.54.3. 4.1. 5.3

1.01.52.31.9

4.41.7.1.21.6.1.66.7

2.53.2

3.2. 2.13.6. 3.9

5.97.22.8

5.2. 3.1. 7.35.1

4.1. 6.92.0

5.4. 6.6. 7.01.7. 1.9. 2.22.9. 2.5. 2.3

6.7. 6.710.9. 9.71.5. 1.61.7. 2.2

Hydraulicconductivity,

Lp 1 o6[cm -s- barII

0.93 ± 0.68 (2)

0.410.23

0.19 0.0 (2)

0.70 + 0.28 (2)0.74

1.7 0.7 (2)

5.3 + 2.7 (3)

5.2

5.2

1.59.3 ± 2.0 (2)

2.0

1.31.2

4.0 1.2 (2)

2.3

10.810.8 ± 4.6 (3)

2.22.5 ± 1.0 (2)

5.11.9 0.3 (3)7.5 1.0 (3)3.8 ± 0.5 (3)1.6 t 0.0 (2)

36 0.1 (2)

Experi-mentalcondi-tions

(1)

(5)

(5)

(4)

(5)

(1)

(1)

(1)

(3)

(5)

(3)

(3)

(7)

(3)

(3.9)

(3)

(3)

(1)

(1)

(7)

I

Plant Physiol. Vol. 68, 1981 WATER RELATIONS OF LEAF EPIDERMIS 1 141

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Plant Physiol. Vol. 68, 1981

m 1.0-

-CL

r_0.0 -

-1.0-

- 2.0 -

3.50 360

)Xi03 K-1T

330

30.0

E.0

*Li

0-

'D0

0

*\\ \3 40 3.50 360

Yx10 [ K ]

FIG. 5. Temperature dependence of the Lp of two different leaf epidermal cells (cells A and B of Fig. 4) of T. virginiana (T = absolute temperature).From the slope of the Arrhenius plot, the E. are calculated. Cell A: Ea = 68.9 + 10.1 (14.6%) kJ mol-'. Cell B: E. = 109.8 19.4 (17.6%) kJ mol-'.Errors of the regression coefficients in the plots (- - -) have been calculated for 95% confidence interval for n = 44 (cell A) and n = 28 (cell B) datapoints. In the inset, Lp is plotted against temperature; vertical bars denote SD of Lp measurements at a given temperature (n = 4-9).

mesophyll cells (Lp = 3 6. 10-7 cm- s-' bar-' [26]) are known.Measurements of the apoplasmic resistances could be performedusing the pressure probe technique. They require a more sophis-ticated approach from the experimental as well as from thetheoretical point of view. For example, two or more pressureprobes could be introduced in a tissue and the propagation ofpressure signals could be followed. Since the rate of propagationdepends on the conductance (diffusivity) of the signal in both theapoplast and cell-to-cell path, the apoplasmic conductance can beevaluated if the conductance of the cell-to-cell path is known.A preference of the epidermal path as compared with the

mesophyll during transpiration has been suggested by Sheriff andMeidner (15, 16), who measured water transport in excised epi-dermal and mesophyll tissue of T. virginiana and Hedera helix.From their measurements, they concluded that the epidermis isan important path for transpirational water from the veins tostomata and, furthermore, that water moves in the wall space ofthe epidermis. However, the values of Sheriff and Meidner for theconductance of the entire epidermis seem to be too large. Theyhave been criticized and may be caused mainly by a transport ofwater on the surface of the excised tissue (20).

For dynamic (transient) processes in the leaf the T1/2 valuesreported in this communication allow an estimation of the lowerlimit of the speed of propagation of a change of the chemicalpotential of water (e.g. at the stomata). If we assume that (a) thehydraulic contact between mesophyll and epidermis is small incomparison with the lateral hydraulic contact between epidermalcells and that (b) apoplasmic water transport is negligible, we cancalculate the average half-time for a change in the chemicalpotential of water within the epidermis in response to a demandof water from the stomata. Using Philip's theory (12) and consid-ering that neighboring stomata are only 2 to 4 cells (165-184 ,Am)apart (Fig. 1B), the average half-time for the shrinking of thecylindrical surrounding of a stoma at a demand of water from theatmosphere would be only 1 to 30 s, i.e. of the same order as T,/2.This half-time would also be the overall half-time of the entirelower epidermis following an even stomatal demand. Any contri-bution of apoplasmic water flow would only serve to shorten this

time if possible.This example shows that the epidermis of Tradescantia could

be well suited to transmit changes of either the water status of theleaf (i.e. of the mesophyll or xylem) or of air humidity (viacuticular transpiration; cf. 10, 11, 14) to the stomata. The signifi-cance of these processes would depend on the hydraulic resistancebetween mesophyll and epidermis and on the hydraulic resistanceof the cuticle which have not been measured up to now.

Acknowledgments-We are grateful to Dr. D. Kramer, Technische HochschuleDarmstadt, and to Dr. E. Brinckmann, University of Bayreuth, for preparing thelight micrographs used in this study and toJ. Zillikens for skillful technical assistance.We appreciate the use of data processing programs prepared by Diplom-Physiker S.Wendler, and thank Drs. D. Cosgrove, K. F. A. Julich, and J. A. C. Smith, TechnischeHochschule Darmstadt, for critical reading of the manuscript.

LITERATURE CITED

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2. BUCHNER K-H, U ZImRMRmANN, F-W BENTRup 1980 Turgor pressure and watertransport properties of suspension-cultured cells of Chenopodium rubrum L.Planta 151: 95-102

3. COWAN IR 1977 Stomatal behaviour and environment. Adv Bot Res 5: 117-2284. DAINTY J 1976 Water relations of plant cells. In U Luttge, MG Pitman, eds,

Encyclopedia of Plant Physiology, New Series, Vol 2, Part A. Springer-Verlag,Berlin, pp 12-35

5. EPSTEIN E 1972 Mineral Nutrition of Plants: Principles and Perspectives. Wiley,New York

6. HOAGLAND DR, DI ARNON 1950 The water culture method of growing plantswithout soil. Univ Calif Berkeley Coil Agric Circ 347

7. HouSE CR 1974 Water Transport in Cells and Tissues. Edward Arnold, London8. HUsCEN D, E STEuDLE, U Zm aRmANN 1978 Pressure probe technique for

measuring water relations of celLs in higher plants. Plant Physiol 61: 158-1639. KREYSZIG E 1977 Statistische Methoden und ihre Anwendungen. Vandenhoeck

and Ruprecht, Gottingen10. LANGE OL, R LOSCH 1979 Plant water relations. In H Ellenberg, K Esser, K

Kubitzki, E Schnepf, H Ziegler, eds, Progress in Botany, Vol 41. Springer-Verlag, Berlin, pp 10-43

11. LANGE OL, R LOSCH, E-D SCHULiE, L KAPPEN 1971 Responses of stomata tochanges in humidity. Planta 100: 76-86

12. PHLIP JR 1958 Osmosis and diffusion in tissue: Half-times and internal gradients.Plant Physiol 33: 275-278

-

C.-JC

t (cI

II I s-

1142 TOMOS ET AL.

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WATER RELATIONS OF LEAF EPIDERMIS

13. RICHARDSON IW, V LICKo, E BARTOLI 1973 The nature of passive flows throughtightly folded membranes. J Membrane Biol 11: 293-308

14. SCHULZE E-D, OL LANGE, U BUSCHBOM, L KAPPEN, M EvENARI 1972 Stomatalresponses to changes in humidity in plants growing in the desert. Planta 108:259-270

15. SHERIFF DW, H MEIDNER 1974 Water pathways in leaves of Hedera helix L andTradescantia virginiana L. J Exp Bot 25: 1147-1156

16. SHERIFF DW, H MEIDNER 1975 Water movement into and through Tradescantiavirginiana L leaves. J Exp Bot 26: 897-902

17. STEuDLE E, U LUiTGE, U ZIMSERMANN 1975 Water relations of the epidermalbladder cells of the halophytic species Mesembryanthemum crystailmum: directmeasurement of hydrostatic pressure and hydraulic conductivity. Planta 126:229-246.

18. STEuDLE E, JAC SMITH, U LuTrGE 1980 Water relation parameters ofindividualmesophyll cells of the CAM plant Kalanchoe daigremontiana Plant Physiol 66:1155-1163

19. TyREE MT, YNS CHEUNG 1977 Resistance to water flow in Fagus grandifolialeaves. Can J Bot 55: 2591-2599

20. TYREE MT, P YINOULIS 1980 The site of water evaporation from sub-stomatalcavities, liquid path resistances and hydroactive stomatal closure. Ann Bot 46;175-193

21. WEIGL J 1967 Wasserstruktur und Permeation: Die Aktivierungsenergie und dermolekulare Mechanismus der Wasserpermeation. Z Naturforschung 22b: 885-

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tischen Regulation bei der marinen Rotalge Porphyra umbilicalis (L.) Kutz f.umbilicalis. PhD Thesis, University of Hannover, FRG

23. Zim1RmANN U 1978 Physics of turgor and osmoregulation. Annu Rev PlantPhysiol 29: 121-148

24. ZIMMERMANN U 1980 Pressure mediated osmoregulatory processes and pressuresensing mechanisms. In R Gilles, ed, Animals and Environmental Fitness.Pergamon Press, Oxford and New York, pp 441-459

25. ZIMMRMANN U, R BENZ, H KOCH 1981 A new electronic method for thedetermination of the true membrane area and the individual membraneresistances in plant cells. Planta. In press

26. ZIMMERMANN U, D HUsKEN, E-D SCHULZE 1980 Direct turgor pressure mea-surements in individual leaf cells of Tradescantia virginiana Planta 149:445-453

27. ZimIMRMANN U, E STEUDLE 1974 The pressure-dependence of the hydraulicconductivity, the cell membrane resistance and the membrane potential duringturgor pressure regulation in Valonia utricularis. J Membrane Biol 16: 331-352

28. ZIMMERMAN U, E STEuDLE 1978 Physical aspects of water relations of plant cells.Adv Bot Res 6: 45-117

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Plant Physiol. Vol. 68, 1981

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