4 Notes
1. The amount of plant material required depends on the desired
amount of protein needed for subsequent analysis. As an example, using 25 g of fully expanded grapevine leaves (around
30 leaves) yields 12–30 mL of APF with a protein concentration between 0.6 and 0.9 μg/μL. Both the amount of apoplast
and the protein concentration vary with the status of the leaves,
plant species, and cultivar.
2. It is best to use fully expanded 3–7 leaves from the shoot apex
for grapevine as younger leaves may be more susceptible to
mechanical damage that may lead to cell leakage and apoplast
contamination.
3. L-Ascorbic acid (or sodium ascorbate) is used as an antioxidant;
potassium chloride is used to maintain the ionic strength of the
medium and increase the solubility of the salt soluble proteins.
4. Mercaptoethanol is used to inhibit the oxidation of free sulfhydryl residues and hence maintains protein activity; it acts as a
biological antioxidant by scavenging hydroxyl radicals
(amongst others). It is widely used because the hydroxyl
group confers solubility in water and lowers the volatility.
Due to its diminished vapor pressure, its unpleasant odor, and
its toxicity, caution must be taken when using it (read safety
instruction of the product).
5. CHAPS is a nondenaturing, zwitterionic sulfobetaine detergent. It is mild and solubilizes membrane proteins and receptors. It is useful for breaking protein–protein interactions,
increasing the extraction of soluble proteins.
6. Sulfite is the most commonly employed oxygen scavenger used
to prevent oxidation (avoiding browning reactions).
7. To reach 25 g of fresh weight, around 30 randomized grapevine leaves were needed. To reach around 10 g of fresh weight,
around 16 randomized coffee leaves were needed.
8. During this procedure, keep the leaf pieces at 4
C by maintaining the beaker on ice to minimize the stress induced to the
leaf tissue (Fig. 4).
9. Leaf infiltration of woody plants as grapevine and coffee is
difficult since leaves are not very pulpy or present hairs or waxes.
The middle and lateral veins were removed, and leaves were cut
into pieces to increase the accessibility of the infiltration buffer,
promoting higher infiltration efficiency. Infiltration buffers
should be optimized for different woody plants. Buffer composition for other woody plants like prunus was already
described. Briefly, for prunus, the infiltration buffer should be
prepared as 50 mM TRIS-acetate buffer (pH 6.0); add about
100 mL of water to a 1-L graduated glass beaker, weigh 60.5 g
Extraction of Apoplastic Fluid from Woody Plants
55
1. The amount of plant material required depends on the desired
amount of protein needed for subsequent analysis. As an example, using 25 g of fully expanded grapevine leaves (around
30 leaves) yields 12–30 mL of APF with a protein concentration between 0.6 and 0.9 μg/μL. Both the amount of apoplast
and the protein concentration vary with the status of the leaves,
plant species, and cultivar.
2. It is best to use fully expanded 3–7 leaves from the shoot apex
for grapevine as younger leaves may be more susceptible to
mechanical damage that may lead to cell leakage and apoplast
contamination.
3. L-Ascorbic acid (or sodium ascorbate) is used as an antioxidant;
potassium chloride is used to maintain the ionic strength of the
medium and increase the solubility of the salt soluble proteins.
4. Mercaptoethanol is used to inhibit the oxidation of free sulfhydryl residues and hence maintains protein activity; it acts as a
biological antioxidant by scavenging hydroxyl radicals
(amongst others). It is widely used because the hydroxyl
group confers solubility in water and lowers the volatility.
Due to its diminished vapor pressure, its unpleasant odor, and
its toxicity, caution must be taken when using it (read safety
instruction of the product).
5. CHAPS is a nondenaturing, zwitterionic sulfobetaine detergent. It is mild and solubilizes membrane proteins and receptors. It is useful for breaking protein–protein interactions,
increasing the extraction of soluble proteins.
6. Sulfite is the most commonly employed oxygen scavenger used
to prevent oxidation (avoiding browning reactions).
7. To reach 25 g of fresh weight, around 30 randomized grapevine leaves were needed. To reach around 10 g of fresh weight,
around 16 randomized coffee leaves were needed.
8. During this procedure, keep the leaf pieces at 4
C by maintaining the beaker on ice to minimize the stress induced to the
leaf tissue (Fig. 4).
9. Leaf infiltration of woody plants as grapevine and coffee is
difficult since leaves are not very pulpy or present hairs or waxes.
The middle and lateral veins were removed, and leaves were cut
into pieces to increase the accessibility of the infiltration buffer,
promoting higher infiltration efficiency. Infiltration buffers
should be optimized for different woody plants. Buffer composition for other woody plants like prunus was already
described. Briefly, for prunus, the infiltration buffer should be
prepared as 50 mM TRIS-acetate buffer (pH 6.0); add about
100 mL of water to a 1-L graduated glass beaker, weigh 60.5 g
Extraction of Apoplastic Fluid from Woody Plants
55