Biology Reference
In-Depth Information
It is still far from clear to which extend the aromatic domain of
suberin is identical or similar to lignin and how to distinguish
those. Abovementioned staining can be considered as indication
but not as a proof of lignifi cations. There are several others histori-
cally established methods of detection of lignifi cation.
The most frequently used is Wiesner's reaction [
28
] using
phloroglucinol condensation with cinnamic aldehydes (coniferyl
aldehyde) in acidic environment and formation of cherry red prod-
uct [
29
,
30
]. There is potential cross-reactivity with other aliphatic
and aromatic aldehydes [
31
], but in standard conditions the speci-
fi city can be considered rather high. Alternatively aniline sulphate
[
28
,
32
] is proposed. The output of the reaction and localization
seems to be very similar to phloroglucinol reaction, but with lower
contrast of resulting yellow coloration.
Another often-used lignin test is Mäule's reaction [
33
].
Syringyl moieties of lignin are considered to be the reaction target
[
29
,
30
]. The lignin composition related difference in detection,
comparing to phloroglucinol can be strongly pronounced during
development [
30
,
34
] as well as in between taxonomic groups
[
35
]. Schiff 's reagent staining might be also used for detection of
aldehydes of lignin [
25
].
There is wide spectrum of acidophilic dyes that have some
affi nity to lignifi ed cell walls (PI, DAPI, Hoechst, basic Fuchsine,
etc.). However, because of dependence on staining conditions
and low specifi city of such staining, it should be considered as
informative and further confi rmation of lignifi cation is recom-
mended. Autofl uorescence of aromatic compounds is another
very useful approach to follow phenolic compounds within the
cell walls [
36
,
37
].
1.4 Detection
of Enzyme Activities
Apoplastic plant peroxidases play a key role in various metabolic pro-
cesses—e.g., lignin and suberin formation, cross-linking of cell wall
components, auxin metabolism, and metabolism of reactive oxygen
species [
38
]. Peroxidase enzymatic activity might be probed with
various co-substrates in presence of H
2
O
2
. The most common is
diaminobenzidine (DAB), which yields upon oxidation brownish
polymer [
39
,
40
] and tetramethylbenzidine (TMB)—chromogen
which yields a blue reaction product upon oxidation [
41
,
42
].
Substrate does not have specifi c selectivity for particular heme pro-
tein, and therefore distinction of catalase and peroxidase is based on
their different pH optimum. Peroxidase has its optimum at neutral
range (pH ~6.5) while for catalase it is above pH 10 (
see
ref. [
43
]). To
optimize the reaction progress, higher temperature (37 °C) is recom-
mended which increases the enzyme activity, and adequately reduced
exposure decreases spontaneous precipitation of DAB in presence of
H
2
O
2
. Precipitation is further decreased if the reaction proceeds in
dark as light induces spontaneous decomposition of H
2
O
2
. It is very
important to include suitable controls (e.g., reaction mixture without
H
2
O
2
and sections where peroxidase activity was inhibited) [
7
].
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