Short and Long Term Effects of Salicylic Acid on Protection to Phytoplasma Associated Stress in Potato Plants - Salicylic Acid: Plant Growth and Development - page 315

2.4.1 Determination of H 2 O 2 Content
Leaf tissue (0.25 g) frozen in liquid nitrogen was powdered, extracted in 1.2 mL
ice-cold 5 % (v/w) trichloroacetic acid (TCA) and clarified by centrifugation
(15 min at 11,000 g) at 4 C. Samples (0.5 mL) of supernatants were passed
through 0.5 g of Dowex-1 resin (Dow Chemical Company, Midland, MI) followed
with 3.5 mL of 5 % TCA. H 2 O 2 content was measured in the eluates using the
luminol-dependent chemiluminescence method of Mora-Herrera et al. ( 2005 ):
0.5 mL of eluates was added to 0.5 mL luminol, the volume was made up to
4.5 mL with 0.2 M NH 4 OH (pH 9), and 450 mL of this mixture was analyzed
using a Optocomp P luminometer (MGM Instruments, USA). Chemiluminescence
was initiated by injecting 50 mL of 0.5 mM potassium ferricyanide in 0.2 M of
NH 4 OH, and emitted photons were counted over 5 s. A parallel sample of each
initial extract was processed after addition of a known concentration of H 2 O 2 to
provide a recovery correction factor b.
2.4.2 Peroxidase Activity
Leaf tissue (0.5 g) was crushed into fine powder in a mortar and pestle under liquid
nitrogen. Soluble protein was extracted by homogenizing the powder in 2 mL of
50 mM potassium phosphate buffer (pH 7.2) containing 5 mM DTT, 1 mM EDTA
and 1 % PVP (Anderson et al. 1995 ). Insoluble materials were removed by cen-
trifugation at 11,000 g for 15 min at 4 C. Peroxidase activity was determinated
according to the Mora-Herrera and López-Delgado ( 2007 ) method. The total
reaction mixture (3 mL) contained 50 mM sodium phosphate (pH 7.0), 3.33 mM
guaiacol and 4 mM H 2 O 2 . The reaction was initiated by the addition of 20 mL of
extract. Progress of the reaction was measured directly by the increment in
absorbance at 470 nm (extinction coefficient 2.6 mM -1 cm -1 ) at 30 s intervals for
3 min at 22 C. Protein content was determinated using a NanoDrop 1,000 spec-
trophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA).
2.4.3 Ascorbate Content
Leaf tissue (100 mg) was powdered using liquid nitrogen and extracted in 1 M
perchloric acid (HClO 4 ). The samples were ground continuously until completely
thawed. The homogenates were then clarified by centrifugation for 10 min at
17,000 g. The supernatant (0.5 mL) was transferred to fresh microtubes containing
120 mM sodium phosphate buffer (0.1 mL, pH 7.6). The pH of each sample was
adjusted to 5.0 with 80 + lL of 2.5 M K 2 CO 3 . The insoluble KClO produced
during neutralization was removed by centrifugation at 17,000 g. The pellet was
discarded and the supernatant used for determining the oxidized and reduced
ascorbate content of each tissue. The supernatant was then assayed for AA
quantification as described by Foyer et al. ( 1983 ).