Skip Navigation


JXB Advance Access originally published online on May 21, 2004
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
55/401/1411    most recent
erh150v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (11)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Santrucek, J.
Right arrow Articles by Schreiber, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Santrucek, J.
Right arrow Articles by Schreiber, L.
Agricola
Right arrow Articles by Santrucek, J.
Right arrow Articles by Schreiber, L.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Journal of Experimental Botany, Vol. 55, No. 401, pp. 1411-1422, June 1, 2004
© 2004 Oxford University Press

RESEARCH PAPER

A new technique for measurement of water permeability of stomatous cuticular membranes isolated from Hedera helix leaves

Received 3 December 2003; Accepted 18 February 2004

J. Santrucek1,2,*, Eva Simánová3, Jana Karbulková2, Marie Simková1 and Lukas Schreiber3

1 Institute of Plant Molecular Biology, AS CR, Branisovská 31, CZ-37005 Ceské Budejovice, Czech Republic
2 The University of South Bohemia, Faculty of Biology, Branisovská 31, CZ-370 05, Ceské Budejovice, Czech Republic
3 Institute of Cellular and Molecular Botany, University of Bonn, Kirschallee 1, D-53115, Bonn, Germany

* To whom correspondence should be addressed. Fax: +420 38 777 2371. E-mail: jsan{at}umbr.cas.cz
Abbreviations: CM, isolated cuticular membrane; MX, polymer matrix; 14C-2,4-DB, 14C-labelled 2,4-dichlorophenoxybutyric acid; F, diffusion flux; D, diffusion coefficient; g, diffusive conductance; indexes: t, total; c, cuticular; s, stomatal; a, astomatal; N, nitrogen; H, helium; w, water.


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Discussion
 Conclusions
 Appendix
 References
 
Transpiration of cuticular membranes isolated from the lower stomatous surface of Hedera helix (ivy) leaves was measured using a novel approach which allowed a distinction to be made between gas phase diffusion (through stomatal pores) and solid phase diffusion (transport through the polymer matrix membrane and cuticular waxes) of water molecules. This approach is based on the principle that the diffusivity of water vapour in the gas phase can be manipulated by using different gases (helium, nitrogen, or carbon dioxide) while diffusivity of water in the solid phase is not affected. This approach allowed the flow of water across stomatal pores (‘stomatal transpiration’) to be calculated separately from the flow across the cuticle (cuticular transpiration) on the stomatous leaf surface. As expected, water flux across the cuticle isolated from the astomatous leaf surface was not affected by the gas composition since there are no gas-filled pores. Resistance to flux of water through the solid cuticle on the stomatous leaf surface was about 11 times lower than cuticular resistance on the astomatous leaf surface, indicating pronounced differences in barrier properties between cuticles isolated from both leaf surfaces. In order to check whether this difference in resistance was due to different barrier properties of cuticular waxes on both leaf sides, mobility of 14C-labelled 2,4-dichlorophenoxy-butyric acid (14C-2,4-DB) in reconstituted cuticular wax isolated from both leaf surfaces was measured separately. However, mobility of 14C-2,4-DB in reconstituted wax isolated from the lower leaf surface was 2.6 times lower compared with the upper leaf side. The significantly higher permeability of the ivy cuticle on the lower stomatous leaf surface compared with the astomatous surface might result from lateral heterogeneity in permeability of the cuticle covering normal epidermal cells compared with the cuticle covering the stomatal cell surface.

Key words: Cuticular transpiration, diffusion, helium, Knudsen diffusion, plant cuticle, pores, stomatal transpiration.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Discussion
 Conclusions
 Appendix
 References
 
The plant cuticle forms the outermost layer of leaves and fruits, separating the living cells from the atmosphere. The development of the ‘waterproof’ cuticle was a prerequisite for the colonization of dry land, because the permanent water deficit in the atmosphere causes a constant loss of water from the plant surface into the atmosphere. However, a reduced permeability of the leaf surface for water simultaneously reduced CO2 uptake. This dilemma between carbon uptake and water loss was solved by the evolution of stomata, often located only on the shaded lower (abaxial) leaf side. In hypostomatous leaves the upper (adaxial) surface is covered with a continuous cuticle. On leaf surfaces carrying stomata, the cuticle covers the outer epidermal cell walls and periclinal walls of guard cells, and it extends down the ventral walls forming the stomatal pore to the substomatal cavity (Appleby and Davies, 1983a; Wullschleger and Oosterhuis, 1989; Osborn and Taylor, 1990; Pesacreta and Hasenstein, 1999).

Under favourable environmental conditions, 94–99.7% of the gas exchange occurs through stomatal pores (Körner, 1994). However, under drought stress with stomata closed, the survival of plants depends on the barrier properties of the cuticle, which effectively control water loss. Consequently, cuticles of plant species adapted to dry conditions have, by one to two orders of magnitude, lower permeances for water than cuticles of mesophytes from more humid climates (Schreiber and Riederer, 1996). The transport properties of cuticles could also play a significant physiological role in the short-term control of leaf water loss via humidity-sensing stomata (Kerstiens, 1996a, b). The direct response of stomata to air humidity assumes a higher permeance of the cuticle covering the guard cells walls and facing the stomatal pores (Appleby and Davies, 1983a, b).

Present knowledge of the transport properties of cuticles is based exclusively on results obtained with astomatous leaf surfaces (Holmgren et al., 1965; Kerstiens, 1995; Boyer et al., 1997; Kirsch et al., 1997; Kerstiens, 1997) or astomatous cuticular membranes (Schönherr and Lendzian, 1981; Schreiber and Riederer, 1996, 1997). The investigation of cuticular water permeability of stomatous leaf cuticles is difficult, because stomata may contribute to ‘cuticular transpiration’ due to an imperfect stomatal closure (Hygen, 1951; Slavík, 1958; Hoad et al., 1996; Kerstiens, 1996a, b) or due to limited possibilities in elimination or evaluation of diffusion through open stomata (Moreshet, 1970; Santrucek and Slavík, 1990a, b; Santrucek, 1991). In both cases, the residual water diffusing through stomata leads to an overestimation of cuticular permeability.

A newly developed approach is presented here for measuring transpiration across isolated stomatous cuticles, by separating quantitatively the diffusion of water across the gas-filled stomatal pores from water diffusion through the solid cuticle polymer. By applying this technique it was possible for the first time to measure the water permeabilities of cuticles from stomatous leaf surfaces and to compare them with water permeabilities of cuticles from astomatous leaf surfaces.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Discussion
 Conclusions
 Appendix
 References
 
Isolation of cuticular membranes
Ivy (Hedera helix L.) leaves were collected in the Botanical Garden in Würzburg (Germany) in autumn 1999. Cuticular membranes (CM) were isolated as described in detail by Schönherr and Riederer (1986) by immersing leaf discs in a mixture of 2% (v:v) cellulase (Celluclast, Novo Nordisk, Bagsvaerd, Denmark) and 2% (v:v) pectinase (Trenolin Super DF, Erbslöh, Geisenheim, Germany) dissolved in citric buffer (pH=3.0, adjusted with KOH). 1 mol m–3 sodium azide (NaN3, Fluka, Neu-Ulm, Germany) was added to prevent growth of micro-organisms. Isolated cuticles (CM) from upper and lower leaf surfaces were separated, carefully washed with deionized water, air-dried, and stored at room temperature.

Extraction of cuticular wax and desorption experiments
Cuticles isolated from the upper and lower sides of 50 ivy leaves were extracted separately in chloroform at 40 °C yielding 99 mg and 76 mg wax, respectively. The bulk solutions in chloroform (Roth, Karlsruhe, Germany) were vacuum evaporated (Büchi B124, B481, B168, Germany) to a final wax concentration of 50 mg ml–1. Waxes extracted from the upper and lower leaf surfaces were used for measuring diffusion coefficients of 14C-labelled 2,4-dichlorophenoxy-butyric acid (14C-2,4-DB; specific activity: 3.3 MBq in 1000 ml of Toluol) in reconstituted cuticular wax as described in detail in Schreiber and Schönherr (1993). Desorption of 14C-2,4-DB into borax buffer (10–2 mol l–1, pH 9.0) was measured at time intervals of 5, 15, 30, 60, and 90 min. Radioactivity was determined by liquid scintillation counting (Wallac counter, model 1409, Turku, Finland). Diffusion coefficients were obtained from plots of relative amounts of 14C-2,4-DB desorbed from the wax versus the square root of time (Schreiber and Schönherr, 1993). As it was shown that cuticular permeability was correlated with diffusion in reconstituted wax (Kirsch et al., 1997), this method can also be used for comparing permeability between adaxial and abaxial leaf sides.

Diffusion through the isolated stomatous cuticles
Theory: The diffusion of water vapour through nitrogen-filled pores (stomata) of a membrane (stomatous cuticle) is defined as Fs N and diffusion through the cuticle itself is defined as Fc. The total flux Ft N of water through the membrane in a nitrogen atmosphere is given by:

Ft N=Fs N+Fc(1)

Water flux Ft H through the same membrane surrounded by helium is given by:

Ft H=Fs H+Fc(2)

provided that the solid membrane and Fc are not affected by the gas species. Steady-state diffusion of water along the two defined pathways depends on the concentration gradient and on the diffusivity of the substance. The diffusion coefficient, D, as a measure of diffusivity, is a function of the mean free path of the molecule (here water) in the gas phase. It is affected by the mass of both the diffusing substance and the surrounding molecules, by temperature, and by the pressure in the gas system. Thus, the flux of water vapour (solute) at a constant concentration gradient across pores of invariable geometry will depend only on the molecular characteristics of the gas (solvent). The ratio of water fluxes across the pores filled with helium and with nitrogen is given by the following equation

where Dw H and Dw N are binary diffusion coefficients of water in helium and in nitrogen, respectively. The diffusion of water vapour in helium should be 3.6 times faster than that in nitrogen (Dw N=2.56x10–5 m2 s–1 at 34 °C and Dw H=9.08x10–5 m2 s–1 at 25 °C; Cussler, 1987). The ratio Dw H/Dw N will be called R and the parameter k=R/(R–1) can be defined. Substituting Fs H in equation (2) by equation (3) and combining equations (1) and (2) the following equation describing the water flux through the solid-phase of the stomatous cuticle is obtained

Fc=kxFt N–(k–1)xFt H(4)

Equation 4 states that when the fluxes measured in helium and in nitrogen have the same values (Ft H=Ft N), then the total flux of water Ft N is realised only across the solid cuticle (Ft N=Fc) and there is no diffusion across pores. This situation is expected in an astomatous cuticle. At the opposite extreme, the flux through the solid cuticle, Fc, is zero when the Ft H/Ft N ratio approaches the R value (Ft H/Ft N=k/(k–1) when Fc=0 in equation 4). This might be the case with flux through aluminium foil into which holes have been punched, for example. The relative proportions of ‘cuticular water loss’, Fc, and ‘stomatal water loss’, Fs N, are

and

The lines in Fig. 1 show the complementarity of the relative cuticular and stomatal conductances when the helium/nitrogen water flux ratio varies between its limits 1 and R.



View larger version (9K):
[in this window]
[in a new window]
  		Fig. 1. Relative flux of water through the solid cuticle, Fc/Ft N, and through stomatal pores, Fs/Ft N, as a function of the ratio of total diffusive fluxes in helium and nitrogen, Ft H/Ft N. Simulation by using equations 5a and 5b.

 
The binary diffusion coefficients Dw H, Dw N and the gas-phase fluxes involved in the above equations require that the water molecules interact predominantly with molecules of the gas phase (helium or nitrogen), but not with boundary structures of the diffusion path. This may not be the case with a substance diffusing through narrow gas-filled pores and the Knudsen diffusion should have been taken into account (Cussler, 1987). However, the diffusion coefficients are employed here as the ratio R only; fluxes are also in the form of ratios. Thus, the effect of Knudsen diffusion is largely eliminated provided it is equal for both water in nitrogen and water in helium. This situation will be discussed later in this paper. Another important point should be mentioned here and will be solved later. Equations 3–5 are valid only for binary diffusion, that is, for a system having two components: one solvent (gas) and one solute (water vapour). When, for example, a radiolabelled probe is used, correction for ‘tracer diffusion’ has to be taken into account (Cussler, 1987).

Transport experiments in helium and nitrogen
Stomatous (abaxial) and astomatous (adaxial) cuticular membranes (CM) isolated from ivy leaves have been used. Wax-free polymer matrix membranes (MX) were prepared by extracting single CM with chloroform (20 ml) for 1 h at room temperature. For permeability measurements in helium and nitrogen, four different types of cuticle samples have been used: stomatous CM (CMs), stomatous MX (MXs), astomatous CM (CMa), and astomatous MX (MXa). Water flux across the membranes was measured using a newly developed transport chamber (Fig. 2) and two different ways of measuring water transport: (i) measurements with radiolabelled water (‘3H2O’; specific activity: 925 MBq g–1; Hartmann Analytic, Braunschweig, Germany); and (ii) a gravimetric method.



View larger version (16K):
[in this window]
[in a new window]
  		Fig. 2. Diagram of the chamber used for measurements of water vapour diffusion through porous membranes in different gases. Donor represents 3H2O water and receiver is a paper disc saturated with non-labelled water. See the text for more details.

 
When using 3H2O, membranes were mounted between two compartments, a donor compartment with the 3H2O donor solution and a receiver compartment with water-saturated filter paper (Fig. 2). The concentration gradient of 3H2O between donor and receiver compartments across the membrane represents the driving force of 3H2O diffusion across the membrane. Amounts of radioactivity accumulating in the receiver during a certain period of time are proportional to the water flux, which can be used to calculate the permeance of the membrane (see Appendix for details). High-vacuum silicon grease (Wacker Chemie, Burghausen, Germany) was used to mount and seal the membrane on the top of the donor compartment. Usually, the physiological inner side of CMa or MXa was in contact with the donor solution and the physiological outer side of the membrane was oriented towards an air-filled receiver compartment (Schreiber et al., 2001). The arrangement is modified here. In order to avoid penetration of the liquid donor solution through the pores of CMs or MXs and to allow measurements in a particular gas atmosphere, a chamber as shown in Fig. 2 has been used. The membrane is surrounded by a gas medium on both donor and receiver sides and the receiver compartment can be flushed with the proper gas. The receiver, which was a disc of filter paper, 10 mm in diameter, soaked with 12 µl of deionized water, was fixed to a Teflon plug on a sealed revolving rod (Fig. 2). This allowed sampling and replacement of the receiver without a substantial contamination of the atmosphere inside the chamber. Nevertheless, after each sampling and before the next measurement of 3H2O flux started, the receiver compartment was flushed for 30 s with the appropriate gas (helium or nitrogen with purity better than 99.9%).

Two stainless-steal chambers were used in parallel for the experiments. One was filled with nitrogen and the other with helium. Five samplings at intervals of between 10 and 20 min were carried out with each chamber, then the chambers were reconnected to the other gas, thoroughly flushed (20 min) with the new gas and again sampled five times. The gas cylinders and other equipment were in thermal equilibrium with the laboratory atmosphere ranging from 21–24 °C. At each sampling, the paper disc from the receiver compartment was quickly transferred to a scintillation vial, 2 ml of scintillation cocktail was added, and the amount of radioactivity was counted in a scintillation counter (Wallac Counter, Model 1409, Turku, Finland). In this way, 3H2O flux through every CM or MX investigated was measured five times in nitrogen and five times in helium under otherwise identical conditions (geometry, temperature, pressure). In order to estimate the ratio of the diffusion coefficients (R) in different gases, 3H2O-diffusion in empty chambers without membranes were measured. Such R was typical for the geometry of this experimental set-up. In addition, estimating diffusion coefficients of 3H2O in empty chambers was also necessary to address the problem of tracer diffusion (Cussler, 1987), which is related to the fact that water flux measured using radiolabelled water as a tracer does not necessarily correspond to the water flux measured using non-radiolabelled water. This problem is considered in the Appendix.

Alternatively to radiolabelled water, water fluxes in two different gases (helium and carbon dioxide) were measured by gravimetry. The gravimetric technique is less sensitive than the radioactive technique, but it has an advantage in avoiding the corrections for tracer diffusion (see Appendix). To increase the sensitivity, carbon dioxide has been used instead of nitrogen, because CO2 is a ‘heavier’ gas with a lower diffusion coefficient. Aluminium caps were fixed to the receiver compartments of the revolving rod instead of the Teflon plugs (Fig. 2). Inside the aluminium cap was a paper disc soaked with a saturated solution of water-free lithium chloride dried at 120 °C. During sampling, the aluminium cap was quickly removed and regularly weighed at predetermined time intervals on a microbalance (Mettler MT5, Toledo) with an accuracy of 1 µg. The flux of water was calculated from the kinetic of water accumulation in the receiver using equation A1 (see Appendix). The slope s was proportional to the flux of water and the parameter a was set to 1 in the case of non-labelled water.

Conductance of pores and the solid phase of cuticle for water
The diffusive flux of water vapour through a barrier depends on the gradient (or difference {Delta}c) of water vapour and on the transport properties of the barrier. The conductance for the total water transported in this chamber across CMs or MXs (reciprocal value of total resistance rt) can be calculated from the simple flux resistance–driving force relationship 1/rt=Ft N/{Delta}cw. Both, the total flux in nitrogen Ft N and the difference of water vapour concentration between donor and receiver {Delta}cw (driving force) can be assessed. In a similar way, this approach can be used for the solid phase of CMs or MXs cuticles. As the flux across the solid cuticle is known from equation 4, the relationship 1/rc=Fc/{Delta}cw can be used. However, this simple method is not suitable for the estimation of the resistance of pores (rs). Therefore, the following approach for the estimation of rs has been used. The resistance of the porous membranes (CMs or MXs), rm, is composed of two parallel resistances: the resistance of the pores rs and the resistance of the solid cuticle rc (1/rm=1/rs+1/rc). In addition, the membrane is arranged in series with the boundary layer of the unstirred gas in the receiver compartment with the resistance rb. Therefore, the total resistance for the water diffusion in the chamber is rt=rm+rb. The parallel and serial arrangement of these resistances leads to the following equation:

where the composite term on the right hand side is the resistance rm. Rearranging equation 6 leads to:

The boundary layer resistance is defined as rb=l/D, where l is the distance between the cuticle and the receiver and D is the diffusion coefficient of water in nitrogen. As the distance between the mounted cuticle and the receiver was 12 mm in experiments with radiolabelled water and 18 mm when measuring water flux gravimetrically, rb was 510 or 594 s m–1, respectively. These values were used for calculating permeances.

Electron microscopic observations
The possible structural effects of wax extraction on the stomatal pores and on the cutin matrix were investigated by scanning electron microscopy (JEOL 6300 SEM, Japan). Lengths and widths of antestomatal chambers (the throat between the external ledges) were measured and images of the cuticle surfaces were taken.

Statistics and units used
Water transport using radiolabelled water was measured in helium and in nitrogen using 6 CMs and 9 MXs. Using the gravimetric method, 6 CMs and 9 MXs were investigated. As the results obtained by radiometry and gravimetry did not differ significantly, they were pooled. In order to check the premise of the method that the transmembrane fluxes through the solid phase in helium and nitrogen are equal, astomatous membranes were also measured: water transport in He and N2 through 4 CMa and 3 MXa and four parafilm discs. Due to small fluxes and the lower sensitivity of gravimetry, only radiometry was used for astomatous membranes. Results are given as means and 95% confidence intervals (CI). Desorption experiments measuring diffusion coefficients of 14C-2,4-DB in reconstituted cuticular wax were conducted by investigating five replicates of each leaf side. Results are given as means and 95% CI. For 30–60 randomly selected stomata, the length and width of their antestomatal chambers were measured and given as means and 95% CI.

For the sake of clarity, the transport properties of the gas and solid phase are initially presented in terms of fluxes and then as resistances or their reciprocals, conductances, as appropriate.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Discussion
 Conclusions
 Appendix
 References
 
Water diffusion in chambers without membranes (‘empty’ chambers)
The kinetics of 3H2O accumulation in the receiver of empty chambers filled with nitrogen or with helium were linear, with correlation coefficients greater than 0.99 (Fig. 3a). The average flux of tritiated water was 2.1 times higher in helium than in nitrogen (see F*t H and Ft N in Table 1). Diffusion coefficients of tritiated water in nitrogen and helium were D*w N = 2.36x10–5 and D*w H = 4.87x10–5 m2 s–1, respectively, when calculated from the experimentally estimated fluxes. The relevant handbook-tabulated binary diffusion coefficients of water in nitrogen and helium are Dw N=2.42x10–5 and Dw H=8.81x10–5 m2 s–1, respectively. Based on this, all fluxes measured in helium were corrected using the ratio Dw H / D*w H = 1.809 (see equation A3 in the Appendix).



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 3. Examples of cumulative kinetic of 3H2O transfer between the donor and receiver compartments filled with nitrogen or helium. Specific activities a of the donor water are given. The donor–receiver pathway was continuous (gas) (a) or separated with parafilm membrane (b), stomatous cuticular membrane (c), or stomatous cuticular matrix (d). Slopes s of the regression lines were used in equation A1 for calculation of apparent water fluxes.

 

View this table:
[in this window]
[in a new window]
  		Table 1. Total (stomatal plus cuticular) water flux (Ft) measured in nitrogen (Ft N) and in helium (Ft H) using radiolabelled water and gravimetry Ft partitioning into cuticular (solid phase) and stomatal (gaseous phase) portions, Fc N and Fs N, respectively, normalized to Ft N using equation 5. Four different types of cuticle-derived membranes, parafilm, and empty chamber (without a membrane) were investigated. Diffusive resistance of solid cuticle rc and of stomatal pores rs was calculated from equations 5 and 6. Mean values and 95% confidence intervals (CI) are shown.
 
Diffusion coefficients of non-labelled water in helium and in CO2 measured by gravimetry were close to those tabulated by Cussler (1987) (9.08x10–5 and 2.02x10–5 m2 s–1, for Dw H at 25 °C and Dw C at 34 °C, respectively). The mean value of R measured in gravimetry was 3.9, which was 87% of the theoretical value (4.5).

Water diffusion through membranes
Astomatous and stomatous cuticular membranes (CMa and CMs, respectively), extracted membranes (MXa, MXs), and parafilm showed linear transport kinetics with correlation coefficients greater than 0.99 (Fig. 3b–d). The difference between the slopes of transport kinetics in nitrogen and helium corresponded with the presence (Fig. 3a) or absence (Fig. 3b) of gas-filled pores between the donor and receiver compartments. The total water flux through CMs was 25 times higher than the flux through CMa when measured in nitrogen. Wax extraction increased the flux through MXs 7.5 times when compared to CMs (Fig. 4). The ratios of fluxes in helium and nitrogen were 1.21, 1.14, 1.93, 1.91, and 1.94 for parafilm, CMa, CMs, MXa, and MXs, respectively (Table 1). From these ratios the relative fractions of the total flux along the solid cuticle and pores can be calculated using equations 5a and 5b. On average, 35% of water diffused along gaseous pores in CMs and this proportion remained the same in MXs. A non-significant portion of total flux passed apparent pores in CMa and parafilm (5% and 8%) with 95% confidence intervals exceeding 2–3 times these values and thus including zero. However in MXa, one-third of the total flux passed through gaseous pores. Wax extraction in CMs increased water flux across both the solid cuticle and across pores by a factor of 7.5.



View larger version (19K):
[in this window]
[in a new window]
  		Fig. 4. Apparent (3H2O-estimated) water fluxes through parafilm membrane (PF), astomatous cuticle (CMa), stomatous cuticle (CMs), stomatous (MXa), and astomatous cuticular matrix (MXs) in nitrogen and in helium atmosphere saturated with water vapour. For nitrogen, fluxes calculated from equation A1 are given. The fluxes in helium were found from equation A1 and corrected for tracer diffusion using equation A3. The 3H2O-estimated fluxes across CMs and MXs were pooled with those estimated gravimetricly in CO2 and He atmosphere using slopes of transport kinetics and equation A1. The fluxes were measured at 21±2 °C. Upper halves of the 95% confidence intervals are shown.

 
In terms of resistances, the solid phase of CMs had an 11.4 times lower resistance than the CMa (Table 1). Wax extraction decreased the resistance of the solid stomatous cuticle by a factor of 9. Diffusive resistance of pores in CMs was even higher than that of the solid cuticle and dropped 15.5 times after the wax had been extracted. All measurements in terms of conductances g (inverse values of resistance, also called permeance P) are summarized in Fig. 5. Conductance of the apparent gas phase in CMa and parafilm was several orders of magnitude lower than conductance of the solid phase in these membranes. In each of the other three types of membranes including MXa, the conductances of pores and of the solid membrane were in a similar range. The conductance of the solid phase increased by approximately one order of magnitude each time between parafilm, CMa, CMs, and MXs. Conductance of MXa was in the same range as conductance of CMs.



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 5. Conductance of a solid body (black bars) and gas-filled pores (white bars) in presumably non-porous membranes (parafilm PF, astomatous cuticular membrane CMa, astomatous cuticular matrix MXa) and in porous membranes (stomatous cuticle CMs, stomatous matrix MXs). The solid and gas-phase conductances were calculated from flux partitioned by equations 5a, and 5b as an inverse value of resistance rc and resistance rs found from equation 7. Upper halves of the 95% confidence intervals are shown.

 
Scanning electron microscopic investigations
Electron microscopic investigation of stomatal pores in isolated cuticles showed that the internal pore between the cuticles of guard cells was very small (tenths of micrometers), difficult to estimate reliably or even absent (Fig. 6a, b). Wax extraction had no pronounced effect on the opening of the antestomatal chamber (the cross-sectional area of the throat between the external ledges) (Table 2).



View larger version (125K):
[in this window]
[in a new window]
  		Fig. 6. SEM micrographs of stomatal pores in cuticular membrane isolated from the abaxial surface of a H. helix leaf. The pore viewed from the physiologically outer (a) and inner (b) side of the cuticle.

 

View this table:
[in this window]
[in a new window]
  		Table 2. Dimensions of external opening of stomatal antechamber in H. helix measured on the isolated cuticular membrane (CM) and after cuticular wax extraction (MX) Means, and 95% confidence intervals (CI) are shown.
 
Desorption experiments
Desorption kinetics of 14C-2,4-DB in reconstituted wax isolated from astomatous (adaxial) and stomatous (abaxial) leaf surfaces were linear, with a correlation coefficient greater than 0.99. The diffusion coefficient of 14C-2,4-DB was 2.6 times higher in adaxial wax than in wax isolated from the abaxial leaf surface (Table 3).


View this table:
[in this window]
[in a new window]
  		Table 3. Diffusion coefficients for 14C-2,4-DB in reconstituted wax isolated from stomatous and astomatous leaf surfaces of Hedera helix Means, and 95% confidence intervals (CI) are shown.
 

   Discussion
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
Conclusions
 Appendix
 References
 
It was shown that water flux across isolated stomatous cuticles of H. helix was about 25 times higher than water flux across astomatous cuticles (Table 1). This is obviously due to the presence of stomatal pores in isolated stomatous cuticles. However, surprisingly, a substantial part of the water flux (about 65%) passed the solid non-porous phase of the stomatous cuticle (Table 1). Furthermore, the diffusive resistance of the solid cuticle on the stomatous leaf surface was about 11 times lower than the resistance of the astomatous cuticle. These results look strange at first sight and if they are true this would have important physiological implications for the gas exchange of intact leaves. However, before discussing these points any further, three important aspects concerning the applied technique itself and its reliability need to be mentioned. (i) Why is the diffusion coefficient of tritiated water in helium in this system only slightly more than half of that tabulated for non-labelled water in handbooks? (ii) Why is the flux of water through stomatous cuticles measured here distinctly lower than that expected from gas exchange measurements with intact illuminated leaves (open stomata)? (iii) How far is the flux fractionation into solid and gaseous pathways in stomatous cuticles affected by the dimensions and boundaries of the pores?

Methodological aspects of the flux partitioning technique
Tracer diffusion: The binary diffusion of water in helium is described by the binary diffusion coefficient Dw H which is independent of molar fractions of water and helium. However, using labelled water as a tracer of water transport, the diffusion coefficient of the tracer, D*w H, will be lower than Dw H and it will vary with the molar fraction of water. This is due to additional interactions of tritiated water with non-labelled water molecules. This type of diffusion is called ‘tracer diffusion’ (Cussler, 1987). Because the molar fraction of water in water-saturated nitrogen is very low and because the diffusivity of water in nitrogen is also fairly low, the interactions between 3H-labelled water and non-labelled water have low frequency in nitrogen. Therefore, the tracer diffusion coefficient in nitrogen is very close to the binary diffusion coefficient of water. However, water diffusivity is much higher in helium, and here the tracer diffusion coefficient decreases significantly. Cussler (1987) suggests how the diffusion coefficient of tritiated water in a water vapour-saturated atmosphere of helium can be calculated. However, for this calculation, data on the diffusivity of tritiated water in water vapour (self-diffusion) and data on the molar fraction of water in helium at different degrees of water saturation are required. As these data were not available, D*w H was estimated empirically in this experimental system and it was related to tabulated Dw H. This D*w H was 55% of Dw H. In addition to tracer diffusion, there are probably other factors contributing to the reduction of D value. The same factors probably caused the 14% drop in Dw H when it was measured gravimetrically. However, when the empirical value of D*w H or R used is constant (geometry, leakage, etc. of the chamber are invariant with the gas species), the flux partitioning is not affected. This is demonstrated by the consistency of the results obtained with tritiated water and by gravimetry (Fig. 4).

Stomatous water flux in intact leaves compared to isolated stomatous cuticles: Water flux across astomatous cuticular membranes measured here was in the same range compared to published values obtained with isolated astomatous cuticles of H. helix measured at saturated air humidity (Kerstiens, 1996a; Schreiber et al., 2001). Furthermore, the values of the flux measured with isolated stomatous ivy cuticles correspond to the data for isolated stomatous cuticles of Prunus laurocerasus L. (Schreiber and Riederer, 1996). However, the flux across isolated stomatous CM (CMs), on average 0.08 mmol m–2 s–1, was between 1.3–31 times lower than transpiration rates obtained with intact leaves of H. helix. Transpiration rates of attached irradiated (1 mmol (photons) m–2 s–1) leaves ranged from 0.1–2.5 mmol (H2O) m–2 s–1 measured in a ventilated LI-6400 leaf chamber. Both leaf sides were measured and transpiration was calculated on a projected leaf area basis. Leaf–air vapour pressure differences ranged from 1.5 to 4.1 kPa. Total leaf resistances of intact leaves varied from 2.0x104 s m–1 (with stomata closed) to 2.4x102 s m–1 (with stomata opened). These figures roughly correspond to conductances ranging from 0.002–0.17 mol m–2 s–1. The average total resistance of the isolated stomatous CM was 1.8x104 s m–1 (1/(1/rc+1/rs)). This is a surprising result, as one should expect water permeability of isolated stomatous cuticles to be close to that of intact leaves with stomata opened. Two factors could contribute to this discrepancy. First, gas exchange measurements are normally performed in a well-mixed air atmosphere with a thickness of the boundary layer in the order of tenths of millimetres (Nobel, 1991). The boundary layer conductance of 2.8 mol m–2 s–1 in the ventilated leaf chamber of LI-6400 is equivalent to a resistance of 15 s m–1 and this corresponds to a thickness of the boundary layer of 0.36 mm. In this study with isolated cuticles, the thickness of the unstirred air layer between cuticle surface and receiver was about 12 mm, which corresponds to a 33 times thicker boundary layer. However, since the total resistance (rc and rs arranged in parallel) of CMs mounted in the chamber was 1.8x104 s m–1, the additional boundary resistance of 5.1x102 s m–1 only accounts for 2.4% of the total. Thus, the fairly thick boundary layer cannot explain the low flux across stomatous cuticles observed here. Alternatively, it is more reasonable to assume that the aperture of the stomatal pores in isolated stomatous cuticles may be significantly smaller than the aperture of stomatal pores in the intact leaves. This explanation is confirmed by the SEM observations of stomatal pores of isolated cuticles, which all looked fairly closed (Fig. 6). Moreover, the leaf internal cuticle, which remains intact after the enzymatic isolation (Pesacreta and Hasenstein, 1999), seems to occlude the stomatal pores of isolated cuticles.

Effect of the pore dimension: In the extreme case of very narrow pores where the collisions between water molecules and the pore wall dominate over the collisions between the gas molecules, the difference between water flux in helium and in nitrogen will be drastically reduced. This situation is called the Knudsen diffusion and it is of significance in porous membranes with pore diameters below the mean free paths of the diffusing molecules (Cussler, 1987). However, provided that the pores reduce free diffusion in helium and in nitrogen to a similar extent, the ratio of diffusion rates (and diffusion coefficients) should not change substantially. Because the ratio of fluxes and ratio of D is considered (equations 5a, b), the helium–nitrogen method of flux partitioning should yield correct numbers even for the pores with diameters well below the mean free path of molecular motion. Nevertheless, there might be a particular pore diameter where water diffusion in helium is affected, but water diffusion in nitrogen much less so. For example, in air at room temperature, the mean free path of water is about 60 nm long, whereas it is 130 nm in helium. Consequently, diffusion through pores with diameters of 0.1 µm obeys Knudsen D in helium, but ordinary D in nitrogen. This leads to depression of Ft H relative to Ft N. As a result, Fc may be overestimated (equation 5a). However, there is no sharp transition between Knudsen and regular diffusion and the pore diameter probably has a wide distribution. Hence, it is reasonable to assume that R does not change dramatically with the pore diameter and that the flux through the solid cuticle is not significantly overestimated in these measurements.

In support of the flux partitioning in stomatous cuticles shown here, there is substantial indirect evidence that the cuticle of the stomatous leaf surface is more permeable than the cuticle of the astomatous leaf surface. For example, permeance for 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) across adaxial and abaxial surfaces of intact H. helix leaves was measured using chlorophyll fluorescence imaging. DCMU is a photosynthetic herbicide blocking electron transport in photosystem II and, therefore, increasing dissipation of radiation energy in chlorophyll fluorescence. The kinetics of the fluorescence increase can be used for distinguishing between less and more permeable cuticle in intact leaves. The results show a 6.3 times higher permeability of the stomatous leaf surface than the astomatous surface (J Santrucek and L Schreiber, unpublished results). The corresponding permeability ratio estimated here by the nitrogen-helium method was 11.4. Qualitatively similar differences between cuticular permeabilities of intact leaves have been reported independently several times (Sargent and Blackman, 1962; Schönherr and Bukovac, 1978; Schreiber, 1990). Up to now there are no further data available comparing the permeability of the solid stomatous to the astomatous cuticle, except for the indication of increased water permeance of stomatous guard cells (peristomatal transpiration) shown by Maier-Maercker (1983).

Physiological significance
These results show that 35% of water crossed stomatal pores and 65% the solid phase of the CMs membrane. Measurement of flux across the astomatous CM (CMa) leads to the conclusion that cuticular transpiration of the stomatous leaf surface was about 11 times higher than that of the astomatous leaf surface. There is a long-standing discrepancy between cuticular conductance measured with intact leaves using gas exchange equipment and those measured with isolated cuticles. This difference amounts, on average, to one order of magnitude (Kerstiens, 1996a). It is usually argued that this discrepancy is caused by an incomplete stomatal closure in intact leaves compared with isolated astomatous cuticles. Alternatively, accepting the finding that the permeability of the solid cuticle on the lower (stomatous) leaf surface is significantly higher than the permeability of the astomatous cuticle and keeping in mind that the opposite is true for diffusion in waxes comparing lower and upper leaf surfaces, a lateral non-uniformity of cuticular resistance would be the most likely explanation for this observation. There are indeed some reports showing that the cuticle on guard cells lack the amorphous cuticle proper, that it is thinner than the cuticle on periclinal walls of the other non-stomatal epidermal cells and that, like subsidiary cells, guard cells may have a reticulate cuticle (Osborn and Taylor, 1990). This could indicate a lower resistance of the guard cell cuticle and, at least in part, provide an explanation for the apparently lower resistance of the stomatous cuticle reported here. Water loss from guard and/or subsidiary cells, the so-called ‘peristomatal transpiration’, has been hypothesized as a prerequisite for the direct stomatal response to air humidity (Maier-Maercker, 1983; Kappen and Haeger, 1991), but this is still a matter of controversy.

Effect of wax extraction on permeability of solid versus gas phase in stomatous cuticles
From the nitrogen–helium experiments it was found that both stomatal and cuticular fluxes increased by approximately the same factor after wax extraction. This is surprising, since one would expect only cuticular flux to increase after wax extraction. Porous fluxes should not be affected by wax extraction and their relative proportion should, therefore, decrease. While the flux partitioning in CMs raised doubts about overestimation of the cuticular flux, the partitioning in MXs seems to overestimate the flux through pores.

In the following, it will be judged what the effect of wax extraction on the pores in CMs could be. Starting with the equations introduced earlier (see Materials and methods), flux partitioning in CMs and MXs can be expressed as

Ft N CMs = Fs N CMs + Fc CMs(8a)

Ft N MXs = Fs N MXs + Fc MXs(8b)

Ft H CMs = Fs H CMs + Fc CMs(9a)

Ft H MXs = Fs H MXs + Fc MXs(9b)

It is suggested that the wax extraction did not change dimensions of stomatal pores and thus water fluxes through the pores in the particular gas remain the same in both CMs and MXs. It follows that Fs N CMs = Fs N MXs and Fs H CMs = Fs H MXs. When Fs N MXs is expressed through equation 8b and substituted for Fs N CMs in equation 8a, and the same is done in equations 9a and 9b, stomatal flows from equations 8a and 9a are eliminated. Both equations contain the terms Fc MXs and Fc CMs and can be condensed into:

Ft H MXs – Ft H CMs = Ft N MXs – Ft N CMs (10)

which is only another expression for the ‘null-hypothesis’ that stomatal pores (and stomatal fluxes) did not change during the wax extraction. However, now the hypothesis is testable as all four terms in equation 10 are estimated directly in the experiments. Equation 10 states that the whole increment in flux caused by wax extraction is due to an enhanced transport in the solid cuticle and, therefore, must be the same in helium and in nitrogen. The average difference (MXs–CMs) of total fluxes in helium was 0.99 mmol m–2 s–1 and that in nitrogen was 0.49 mmol m–2 s–1 (see Table 1 for stomatous CM and stomatous MX) and, therefore, the null- hypothesis must be rejected. In conclusion, the gas-filled pores and flux across the pores changed due to the wax extraction. From this it is not possible to say whether wax extraction resulted in enlarged stomatal pores or if it created new pores.

However, the presence of pores in the matrix, whether they are an artefact of wax extraction or naturally present and filled with wax in the intact cuticle, was demonstrated in astomatous MXa as well (Fig. 5; Table 1). While this seems rather surprising it corresponds to previous observations that MXa of several plant species were easily penetrated by dead bacteria (Knoll, 1998), indicating the presence of pores in the range between 0.5–1 µm. CMa were not penetrated by dead bacteria. Additional experiments using special transpiration chambers with 30 cm long glass capillaries fixed gas-tight to an opening at the rear of the transpiration chambers strongly confirmed these conclusions. These chambers were turned upside down and a pressure gradient across the cuticle was induced by placing a 1 cm column of mercury at the top of the capillary. With CMa (n=8) the mercury column did not move downwards at all within 1 h, indicating the absence of gas-filled pores (only with two CMa the mercury did move downwards within 1 h by 1–2 mm). However, when MXa (n=10) was used, the mercury column rapidly moved downwards (on average 2–4 cm min–1), which is direct proof of continuous gas-filled pores traversing right through the entire wax-free MXa. Thus, the pores that formed in MXa membranes upon wax extraction must have been occluded by cuticular wax in CMa membranes. A mechanistic model of plant cuticles containing wax-filled pores was also described in studies analysing the optical properties of cuticles (Meyer, 1938; Roelofsen, 1952). The nitrogen–helium technique proved the absence of significant free diffusion in gas-filled pores in CMa (Fig. 5). An attempt was made to check the presence of pores in MXa by scanning electron microscopy. There were no open pores or cracks visible on the upper side of MXa, but narrow cracks were observed on the physiologically inner side of MXa (Fig. 7b).



View larger version (147K):
[in this window]
[in a new window]
  		Fig. 7. Cuticular matrix isolated from the astomatous cuticular membrane of a H. helix leaf. No pores were detected on the physiologically outer side (a); the inner side (b) shows cracks.

 

   Conclusions
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Conclusions
Appendix
 References
 
Data are presented indicating that the solid phase of the abaxial stomatous cuticular membrane (CMs) has a lower resistance to the diffusion of water than the adaxial astomatous CM (CMa) isolated from the same leaf of Hedera helix. A new method allowing the estimation of transport resistance of the solid cuticle in the presence of pores was employed. An attempt to explain the difference in resistances by a difference in transport properties of wax isolated from opposite leaf surfaces surprisingly showed that the diffusion coefficient for 2,4-DB C14 in adaxial wax was 2–3 times higher than that in abaxial wax. It is possible to explain the discrepancy by lateral heterogeneity of cuticular conductance, for example, by differences in transport properties of epidermal, subsidiary, and guard cell cuticle. Finally, there were pores allowing gas-phase diffusion in astomatous wax-free matrix (MXa), but not in intact CMa.


   Acknowledgements
 
The authors are indebted to Dr Gerhard Kerstiens (Lancaster University, UK) for critically reviewing an earlier version of this manuscript. This work was supported by grants from the Academy of Sciences of the Czech Republic (A6051002 to JS), by the Czech Ministry of Education (ME 368, MSM 123100004, LN00A141 to MS, ES, and JK), the Bundesministerium für Bildung und Forschung (TSR-001-99), and the Deutsche Forschungsgemeinschaft (DFG).


   Appendix
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Conclusions
 Appendix
References
 
Measurements of water diffusion in a gas using 3H2O as a tracer
By sequential sampling and scintillation counting of the receiver, a cumulative kinetic of 3H activity in the receiver is obtained. The kinetic yields a linear increase with the slope s proportional to the total 3H2O flux across the membrane at the particular gas composition (Ft H for helium and Ft N for nitrogen). The examples of the kinetic are shown in Fig. 3. The activity a of the donor solution (dpm µl–1) was adjusted for CM, MX, and for empty chambers, in order to obtain a good final resolution. The apparent flux of non-labelled water in mmol (H2O) m–2 s–1 was calculated as

where s is the slope of the kinetic of 3H2O activity in the receiver in dpm, a is specific activity of donor solution in dpm µl–1, and A is area of exposed cuticle in cm2; in the chambers used here (12 mm diameter) it was 1.13 cm2. (Note that there is no net flux of non-labelled water in this radiometric system with the gas at the donor and receiver saturated with water. The accumulation of activity in the receiver shows the net flux of labelled water, which is constant with time if the receiver activity is negligibly small when compared with the donor activity. This is why the flux of non-labelled water calculated from equation A1 is called an ‘apparent’ flux of water.) While this formula yields sufficiently accurate results for the apparent flux of water vapour in nitrogen, it fails for water flux in helium. This is because 3H2O does not truly trace water vapour in the presence of non-labelled water in helium. 3H2O molecules are significantly slower than 1H2O in helium (but not in nitrogen) due to the phenomenon called tracer diffusion (Cussler, 1987). A system is considered as having three physically different substances even if two of them are chemically identical: H2O, 3H2O, and He or N2. In this case the diffusion coefficient of 3H2O can be substantially different from the binary diffusion coefficient of water especially at the exceptional mobility of helium (Cussler, 1987). A tracer, which can be, for example, a radiolabelled substance or an optical isomer added in small quantity to a chemically identical substance, collides with the identical counterpart molecules in addition to its collisions with the solvent (gas medium). Diffusion coefficient (and flux) of the tracer (D*) is lower than those of the other non-labelled molecules (D) which still behave like a substance in a binary system. While D does not depend on concentration of the substance in question, D* changes with its molar fraction. For small molar fractions and dense media like for fraction of water in water-saturated nitrogen or air (close to 0.03) and nitrogen, respectively, the difference between D and D* is small. However, when diffusion of 3H2O in helium saturated with water vapour is considered, D*w H can be significantly smaller than Dw H (see Cussler, 1987, for the calculation of D*). Therefore, the apparent flux of non-labelled water calculated from the accumulation of tracer activity, F*t H, will be smaller than the flux of non-labelled water in helium measured, for example, by a tracer-independent gravimetric method, Ft H. The correction factor is Dw H / D*w H:

This correction is valid only when all water is transported in continuous helium (for example, water diffusing in the chamber filled with helium without a membrane septum). With a porous membrane, the Dw H / D*w H factor has to be applied only to the flux of tritiated water passing the helium-filled pores, F*s H; not to the whole F*t H. How can the F*s H flux be assessed? It was assumed in equations 1 and 2 that Fc H=Fc N=Fc. Then, when subtracting equations 2 and 1 written for labelled water (F*t H – Ft N)=(F*s H – Fs N) provided F*x N = Fx N. The fluxes across the pores filled with nitrogen and helium can be related as Fs N = F*s H / R*. The parameter R* is the ratio of flows of tritiated water in an empty chamber filled with helium and nitrogen, which is the same as D*w H / Dw N (see equation 3 written for labelled water). Substitution for Fs N into the (equation 2–equation 1) relation given above yields F*s H=(F*t H – Ft N)R* / (R* – 1). F*s H is the portion of the total flux which has to be removed (subtracted) first, then multiplied with the Dw H / D*w H factor, and added again to obtain the flux of non-labelled water: Ft H=F*t H – F*s H + F*s HxDw H / D*w H. When (F*t H – Ft N)R* / (R* – 1) is substituted for F*s H into the last relation, the total apparent flux of non-labelled water across a porous membrane in helium is

where the fraction containing R* is F*s H. With non-stomatous cuticle, where F*s H is zero and the fluxes in helium and in nitrogen are the same, it follows from equation A3 that Ft H=F*t H. In an opposite extreme when there is no solid membrane and F*s H=F*t H, then equation A3 becomes identical to equation A2. The flux estimated from equation A1 by scintillation counting of the samples treated in helium is F*t H and the flux needed in equation 5 is Ft H. Therefore, it is necessary to know the ratio Dw H / D*w H to recalculate the measured F*t H and obtain Ft H from equation A3. The estimation of Dw H / D*w H is described below.

Estimation of diffusion coefficient of 3H2O water in helium saturated with water, D*w H
As explained above, diffusion coefficients for labelled water vapour in nitrogen, D*w N, and for labelled water in helium, D*w H, were experimentally estimated. The measurements were carried out with the chambers shown in Fig. 2, but without membranes between the donor and receiver compartments. The distance l in the gas phase between the donor surface and receiver was 16 mm. The gas at the surface of the donor solution was saturated with water containing significant amounts of 3H2O molecules; the receiver had a concentration of 3H2O molecules close to zero. Thus, the donor–receiver concentration difference of 3H2O was equivalent to a water concentration difference of 0.961 mol m–3 at 20 °C (Nobel, 1991). Under these conditions, it was estimated that the average flow of water vapour in nitrogen was 1.416 mmol m–2 s–1. If the diffusion coefficient is calculated as flux multiplied by length l (16 mm) and divided by the concentration difference (all in the appropriate units), it is obtained for D*w N = 2.36x10–5 m2 s–1. Average flux of tritiated water in helium was 2.922 mmol m–2 s–1 which from a similar calculation yields D*w H = 4.87x10–5 m2 s–1. The value of Dw air at 20 °C is 2.42x10–5 m2 s–1 (Nobel, 1991) which is close to the estimated value of D*w N = 2.36x10–5 m2 s–1 and, for nitrogen, it is assumed that D*w N = Dw N. So, in nitrogen, the tracer (tritiated water) directly measures transport of non-labelled water. For helium, however, the tabulated value of the binary diffusion of water is Dw H=9.08x10–5 m2 s–1 at 25 °C (Cussler, 1987) and, with 3% correction for a temperature difference of 5 °C, Dw H8.81 at 20 °C. The estimate of the tracer diffusion coefficient here is D*w H = 4.87x10–5 m2 s–1. Therefore, to obtain the real flux of water in equation A3, the factors Dw H / D*w H = 1.809 and R* = D*w H / Dw N = 2.064 have been used. Now, the R ratio can be evaluated as Dw H/Dw N=0.881/0.236=3.733. This R value was used in equations 5a, and 5b for all calculations of relative Fc and Fs.



View larger version (163K):
[in this window]
[in a new window]
 
 


View larger version (134K):
[in this window]
[in a new window]
 
 

   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 Conclusions
 Appendix
 References
 
Appleby RF, Davies WJ. 1983a. The structure and orientation of guard cells in plants showing stomatal response to changing vapour pressure difference. Annals of Botany 52, 459–468.[Abstract/Free Full Text]

Appleby RF, Davies WJ. 1983b. A possible evaporation site in the guard cell wall and the influence of leaf structure on the humidity response by stomata of woody plants. Oecologia 56, 30–40.

Boyer JS, Wong SC, Farquhar GD. 1997. CO2 and water vapour exchange across leaf cuticle (epidermis) at various water potentials. Plant Physiology 114, 185–191.[Abstract]

Cussler EL. 1987. Diffusion. Mass transfer in fluid systems. Cambridge: Cambridge University Press.

Hoad SP, Grace J, Jeffree CE. 1996. A leaf disc method for measuring cuticular conductance. Journal of Experimental Botany 47, 431–437.[Abstract/Free Full Text]

Holmgren P, Jarvis PG, Jarvis MS. 1965. Resistance to carbon dioxide and water vapour transfer in leaves of different plant species. Physiologia Plantarum 18, 557–573.[CrossRef]

Hygen G. 1951. Studies in plant transpiration. I. Physiologia Plantarum 4, 57–183.

Kappen L, Haeger S. 1991. Stomatal response of Tradescantia albiflora to changing air humidity in light and in darkness. Journal of Experimental Botany 42, 979–986.[Abstract/Free Full Text]

Kerstiens G. 1995. Cuticular water permeance of european trees and shrubs grown in polluted and unpolluted atmospheres, and its relation to stomatal response to humidity in beech (Fagus sylvatica L.). New Phytologist 129, 495–503.[CrossRef]

Kerstiens G. 1996a. Cuticular water permeability and its physiological significance. Journal of Experimental Botany 47, 1813–1832.[Abstract/Free Full Text]

Kerstiens G. 1996b. Diffusion of water vapour and gases across cuticles and through stomatal pores presumed closed. In: Kerstiens G, ed. Plant cuticles; an integrated functional approach. Oxford: Bios, 121–133.

Kerstiens G. 1997. In vivo manipulation of cuticular water permeance and its effect on stomatal response to air humidity. New Phytologist 137, 473–480.[CrossRef][Web of Science]

Kirsch T, Kaffarnik F, Riederer M, Schreiber L. 1997. Cuticular permeability of the three species Prunus laurocerasus L. Ginkgo biloba L. and Juglans regia L.: comparative investigation of the transport properties of intact leaves, isolated cuticles and reconstituted cuticular waxes. Journal of Experimental Botany 48, 1035–1045.[Abstract/Free Full Text]

Knoll D. 1998. Die Bedeutung der Kutikula bei der Interaktion zwischen epiphyllen Mikroorganismen und Blattoberflächen. PhD thesis, University of Würzburg, Germany.

Körner C. 1994. Scaling from species to vegetation: the usefulness of functional groups. In: Schulze ED, Mooney HA, eds. Biodiversity and Ecosystem Function. Berlin: Springer, 117–140.

Maier-Maercker U. 1983. The role of peristomatal transpiration in the mechanism of stomatal movement. Plant, Cell and Environment 6, 369–380.

Meyer M. 1938. Die Submikroskopishe Struktur der kutinisierten Zellmembranen. Protoplasma 29, 552–586.[CrossRef]

Moreshet S. 1970. Effect of environmental factors on cuticular transport resistance. Plant Physiology 46, 815–818.[Abstract/Free Full Text]

Nobel PS. 1991. Physicochemical and environmental plant physiology. San Diego: Academic Press.

Osborn JM, Taylor TN. 1990. Morphological and ultrastructural studies of plant cuticular membranes. I. Sun and shade leaves of Quercus velutina (Fagaceae). Botanical Gazette 151, 465–476.[CrossRef]

Pesacreta TC, Hasenstein KH. 1999. The internal cuticle of Cirsium horridulum (Asteraceae) leaves. American Journal of Botany 86, 923–928.[Abstract/Free Full Text]

Roelofsen PA. 1952. On the submicroscopic structure of cuticular cell walls. Acta Botanica Neerlandica 1, 99–114.

Sargent JA, Blackman GE. 1962. Studies on foliar penetration. 1. Factors controlling the entry of 2,4-dichlorophenoxyacetic acid. Journal of Experimental Botany 13, 348–368.[Abstract/Free Full Text]

Santrucek J. 1991. Transpiration efficiency and apparent cuticular transpiration in some C3 and C4 plants. Biologia Plantarum 33, 192–199.

Santrucek J, Slavík B. 1990a. A method for separating cuticular and stomatal components of gas exchange by amphistomatous leaves. I. Model solution. Journal of Experimental Botany 41, 795–802.[Abstract/Free Full Text]

Santrucek J, Slavík B. 1990b. A method for separating cuticular and stomatal components of gas exchange by amphistomatous leaves. II. Experimental solution. Journal of Experimental Botany 41, 803–813.[Abstract/Free Full Text]

Schönherr J, Bukovac MJ. 1978. Foliar penetration of succinic acid-2,2-dimethylhydrazide: mechanism and rate limiting step. Physiologia Plantarum 42, 243–257.[CrossRef]

Schönherr J, Lendzian KJ. 1981. A simple and inexpensive method of measuring water permeability of isolated plant cuticular membranes. Zeitschrift fuer Pflanzenphysiologie 102, 321–327.

Schönherr J, Riederer M. 1986. Plant cuticles sorb lipophilic compounds during enzymatic isolation. Plant, Cell and Environment 9, 459–466.[CrossRef]

Schreiber L. 1990. Untersuchungen zur Schadstoffaufnahme in Blätter. PhD thesis, Technical University Munich, Germany.

Schreiber L, Riederer M. 1996. Ecophysiology of cuticular transpiration: comparative investigation of cuticular water permeability of plant species from different habitats. Oecologia 107, 426–432.[CrossRef][Web of Science]

Schreiber L, Schönherr J. 1993. Mobilities of organic compounds in reconstituted cuticular wax of barley leaves: determination of diffusion coefficients. Pesticide Science 38, 353–361.[CrossRef][Web of Science]

Schreiber L, Skrabs M, Hartmann KD, Diamantopoulos P, Simánová E, Santrucek J. 2001. Effect of humidity on cuticular transpiration of isolated cuticular membranes and leaf disks. Planta 214, 274–282.[Web of Science][Medline]

Slavík B. 1958. The influence of water deficit on transpiration. Physiologia Plantarum 11, 524–536.[CrossRef]

Wullschleger SD, Oosterhuis DM. 1989. The occurrence of an internal cuticle in cotton (Gossypium hirsutum L.) leaf stomates. Environmental and Experimental Botany 29, 229–235.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J Exp BotHome page
J. Karbulkova, L. Schreiber, P. Macek, and J. Santrucek
Differences between water permeability of astomatous and stomatous cuticular membranes: effects of air humidity in two species of contrasting drought-resistance strategy
J. Exp. Bot., October 3, 2008; (2008) ern238v1.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Bot.Home page
R. J. Carpenter, G. J. Jordan, A. Leigh, and T. J. Brodribb
Giant cuticular pores in Eidothea zoexylocarya (Proteaceae) leaves
Am. J. Botany, August 1, 2007; 94(8): 1282 - 1288.
[Abstract] [Full Text] [PDF]

Home page
 Plant Physiol.Home page
A. R. Howard and L. A. Donovan
Helianthus Nighttime Conductance and Transpiration Respond to Soil Water But Not Nutrient Availability
Plant Physiology, January 1, 2007; 143(1): 145 - 155.
[Abstract] [Full Text] [PDF]

Home page
 J Exp BotHome page
G. Kerstiens, L. Schreiber, and K. J. Lendzian
Quantification of cuticular permeability in genetically modified plants
J. Exp. Bot., August 1, 2006; 57(11): 2547 - 2552.
[Abstract] [Full Text] [PDF]

Home page
 J Exp BotHome page
G. Kerstiens
Water transport in plant cuticles: an update
J. Exp. Bot., August 1, 2006; 57(11): 2493 - 2499.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
55/401/1411    most recent
erh150v1
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (11)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Santrucek, J.
Right arrow Articles by Schreiber, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Santrucek, J.
Right arrow Articles by Schreiber, L.
Agricola
Right arrow Articles by Santrucek, J.
Right arrow Articles by Schreiber, L.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?