Water and Solute Permeability of Plant Cuticles: Measurement and ...

260 9 General Methods, Sources of Errors, and Limitations in Data Analysis
and initially we isolated cuticles according to his directions (Schönherr and
Bukovac 1973). Initially, we used crude preparations of pectinase and cellulase
of fungal origin, which can be obtained from suppliers of biochemicals. As these
enzymes are rather expensive, we later changed to pectinases used by the fruit and
wine industry (Erbslöh, Geisenheim, Germany). The results were identical.
In an attempt to find optimum conditions, Schönherr and Riederer (1986) used
fungal pectinase (2–6,841) and cellulase (catalogue number 2–3,056) obtained from
Roth (Germany). Leaf discs (15 mm in diameter) were punched from young but fully
expanded rubber-tree leaves (Ficus elastica var. decora). Effects of enzyme concentration,
pH and temperature on time needed to detach CM were studied. Based on
this investigation and some 30 years of experience, some basic rules for successful
isolation of cuticles have evolved.
Best results are obtained with young but fully expanded leaves. With increasing
age it takes much longer to detach cuticles from the epidermal wall, and often some
cellular remnants cannot be removed. With some species (e.g. Populus canescens,
Liriodendron tulipifera, Juglans regia), isolation of old cuticles becomes more and
more difficult or they cannot be isolated at all. Integrity of young leaves is superior,
because they have not been exposed to pests and diseases and damage by wind
and storm. However, there are many species from which astomatous cuticles cannot
be isolated enzymatically. With hypostomatous leaves, cuticles from the abaxial
surface are perforated by stomatal pores, and they cannot be used for transport measurements.
They can easily be sorted out if they are stained with a water-insoluble
marker. We have used waterproof felt pens.
The activity of our pectinases was optimum between pH 3 to 4. Cuticles could not
be isolated when pH was 5 or 6 and, we usually used a 0.01mol l −1 citric acid buffer
adjusted with KOH. NaN3 at a final concentration of 10 −3 mol l −1 must be added
to suppress growth of micro-organisms. Proteins can spoil glass electrodes, and for
this reason it is better to add the enzyme to the buffer rather than adjusting the pH
of the enzyme solution. Time needed to detach cuticles decreases with increasing
enzyme concentration, but in most cases 1–2% worked well. Crude pectinases often
have some cellulase activity, and adding cellulase was not necessary nor beneficial.
Pectinase molecules cannot penetrate cuticles, and for this reason leaf discs
must be vacuum-infiltrated, with the pectinase solution entering the leaf from the
cut edges. Infiltrated leaves appear dark green and glassy. If this step is omitted,
the enzyme can work only at the cut edges, and it may take weeks before it has
reached the centre of the leaf disc. During disintegration of the leaf tissue, phenolic
compounds are often liberated which inhibit enzyme activity. If the enzyme
solution turns brown, it is advantageous to decant it and add fresh enzyme solution
periodically. The bottles with the leaf discs and the enzyme solution should never
be shaken or agitated in any other way, as this will damage many cuticles, especially
when they are thin and fragile. Enzymes inside the intercellular space digest
the middle lamellae and pectins in the outer epidermal wall. They do not benefit
from shaking external solutions. Enzyme activity depends on temperature, and we
obtained best results at 30–40 ◦ C. As all cuticles tested so far underwent a phase
transition between 35 and 50 ◦ C, temperatures higher than 30 ◦ C must be avoided.