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Fig. 6.3.
A caspase-like activity is triggered during SI-induced programmed cell death (
PCD
).
a
DNA fragmentation was assayed in pollen tubes following SI induction, using terminal
deoxynucleotide transferase dUTP nick end labelling (
TUNEL
). In untreated pollen (con-
trol) and pollen challenged with compatible S proteins (
black bars
), low levels of DNA
fragmentation were observed. In incompatible pollen (SI,
white bar
)highlevelsofDNA
fragmentation levels were observed. This was reduced by pre-treatment of pollen with the
peptide DEVD (SI + DEVD,
white bar
), whereas YVAD (SI + YVAD,
white bar
)gaveonly
a small decrease in DNA fragmentation.
Bars
are the mean
the standard error of the mean
(
SEM
).
b
Poly(ADP-ribose) polymerase (
PARP
) cleavage activity in pollen protein extracts
was assayed by adding bovine PARP and performing Western blotting. Incompatible pollen
protein extracts 5 h after SI induction (SI,
white bar
) showed high PARP cleavage activity.
This activity was reduced in the presence of DEVD (SI + DEVD,
grey bar
), whereas YVAD
(SI+YVAD,
black bar
)hadnoeffect.
Bars
are the mean
±
±
the SEM
SI stimulates a caspase-3 like cleavage activity.
We e x a m i n e d m o r e d i -
rectlywhetherSIstimulatedacaspase-3likeactivitybytestingwhether
incompatible pollen had an activity that could cleave PARP. Using bovine
PARP as a substrate, we demonstrated that protein extracts from incom-
patiblepollentubeshadanactivitythatgenerateda24-kDaPARPcleavage
product (Thomas and Franklin-Tong 2004). Extracts from untreated and
compatible pollen did not have a PARP-cleavage activity, demonstrating
S-specificity. Pre-treatment of extracts with DEVD gave 60% less 24-kDa
PARP cleavage product, whilst no effect of YVAD pre-treatment was ob-
served(Fig.6.3b).Thisclearlyimplicatestheinvolvementofacaspase-3
like activity, induced in incompatible pollen by SI, in the generation of the
24-kDa PARP cleavage fragment.
Increases in
Ca
2+
stimulate PCD in
Papaver
pollen.
Since [Ca
2+
]
i
acts as
a second messenger for the SI response, we investigated if increases in
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