Biology Reference
In-Depth Information
mol photons/m
2
s in the case of
Arabidopsis
) [
13
]. In some cases, the starch content of the
leaves may decrease during incubation in water overnight.
Most of the monocotyledons including rice and wheat are of
the saccharophyll type. Since the starch is accumulated in the
leaf sheath (the culm in the case of rice), the mixing of starches
can be suppressed if the leaf blades are used. Incubation in
darkness for approximately 3 days before extraction is also
effective in the preparation of rice chloroplasts [
7
].
2. The formation of crystallized inorganic substances (phytoliths)
causes a decrease in the effi ciency of extraction of intact chlo-
roplasts. This is because the crystals physically destroy the
enveloping membrane of the plastids. Such crystals, which are
known to be formed in various plants, include silicic acid (rice,
cactus,
Solanaceae
,
Osmundaceae
, pteridophyte plants), cal-
cium carbonate (
Urticaceae
,
Moraceae
,
Ulmaceae
), and cal-
cium oxalate (bitter vegetables). To successfully isolate intact
plastids from such plants, the most effective approach is to
increase the amount of sample.
3. High contents of polyphenols and water-soluble proteins cause
the chloroplasts and their membranes to aggregate, thus
impeding solubilization by sodium dodecyl sulfate (SDS). It
should be noted that tobacco contains a large amount of poly-
phenols [
7
].
4. Solutions that contain HEPES must be kept in darkness to
prevent the formation of hydrogen peroxide [
14
].
5. A high concentration of EDTA is important to keep the chlo-
roplasts intact and active [
15
].
6. In rice, the effi ciency of plastid extraction may increase when it
is performed with a high concentration of sucrose (0.6 M).
7. Sodium ascorbate inhibits myrosinase, an enzyme that hydro-
lyzes glucosinolates, which cause cell-organelle damage. It is
necessary to note, however, that myrosinase is active at low
concentrations (0.1-10 mM) of sodium ascorbate [
10
].
8. Mg
2+
is crucial for the stability of biological membranes.
However, thylakoid membranes from broken chloroplasts are
easily aggregated under a high concentration of Mg
2+
.
9. The addition of cetyltrimethylammonium bromide (CTAB)
improves the effi ciency of protein extraction from starchy tis-
sues (plastids) by decreasing the viscosity of the extract.
10. For example, rosette leaves harvested from
Arabidopsis
plants
grown for 10-14 days under a short-day regime (10-h
light/14-h darkness or 8-h light/16-h darkness) to suppress
fl ower stalk development (bolting) [
10
,
15
].
or weak light (100-150
μ
