References

86 S.G. Thomas et al.
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 (control)
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 examined more directlywhetherSIstimulatedacaspase-3likeactivitybytestingwhether
incompatible pollen had an activity that could cleave PARP. Using bovine
PARP as a substrate, we demonstrated that protein extracts from incompatiblepollentubeshadanactivitythatgenerateda24-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 observed(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 Ca2+ stimulate PCD in Papaver pollen. Since [Ca2+ ]i acts as
a second messenger for the SI response, we investigated if increases in