Biology Reference
In-Depth Information
3. Azide-sensitive mitochondrial H
+
ATPase (F-ATPase): 25 mM
K
2
SO
4
, 50 mM Tris/MES pH 8.5, 0.1 mM EDTA, 4 mM
MgSO
4
, 100
M Na-molybdate, 0.005 % Triton X100; add
directly to cuvettes 100 nM bafi lomycin, 200
μ
μ
M sodium
vanadate, ±1 mM sodium azide.
2.7 Substrate Stock
Solution
100 mM sodium ATP: Prepare the ATP stock solution by dissolving
sodium ATP in a buffer of 25 mM BTP of pH 8.0 (
see
Note 9
).
3
Methods
In general, no differences in germination frequency, tube length,
or tube morphology can be observed between fresh and frozen
pollen grains which are stored under optimal conditions. However,
some pollen species need a gentle “warm-up” when taken from the
−80 °C freezer and a few minute incubation in a humid chamber
(imbibition) helps to obtain high germination frequencies. For
some pollen species seasonal changes in the germination frequency
and the tube length can be observed. In lily pollen, the seasonal
variation in their germination capability during the year is still con-
served in the frozen state at −80 °C (Fig.
1a
). Lily pollen grains
collected and aliquots frozen in June/July germinate quite reason-
able in July, August, and September when incubated in culture
medium. The germination rate declines during the winter months
and, surprisingly, increases again from April to June! Furthermore,
in the winter months no reproducible pattern of the expression of
the plasma membrane H
+
ATPase could be observed. The variations
of the time-dependent expression were really high (Fig.
1b
) and
during these months no reproducible data could be obtained neither
by immunodetection nor by identifi cation of peptides via mass
spectrometry analysis.
3.1
Pollen Culture
1. Take fresh or frozen lily pollen grains (
L. longifl orum
) from
one anther and resuspend in 6-12 ml lily pollen germination
medium (
see
Note 10
).
2. Incubate for up to 4 h. Usually, pollen tubes emerge after
40-60 min (Fig.
2
). Incubation for longer times than 4 h may
yield in a high amount of bursting pollen tubes which will
falsify the results (
see
Note 11
).
3.1.1
Lily Pollen Culture
1. Collect fl owers from Arabidopsis plants and dehydrate for
1-2 h at room temperature [
4
,
6
].
2. Imbibe anthers, entire fl owers, or frozen pollen grains for
30 min at RT in a humid chamber.
3. Dip fl owers onto the solidifi ed agar germination medium or dust
pollen grains onto the agar surface. In case of liquid cultures
resuspend pollen grains in germination medium (40 fl owers
3.1.2 Arabidopsis Pollen
Culture
