Identification of SI-induced Ca2+ signalling led to investigation of downstream events, including phosphorylation of pollen proteins. Calmodulin and Ca2+-dependent protein kinases frequently transduce Ca2+-signals via protein phosphorylation. Labelling pollen with 32 P ortho-phosphate enabled identification of several cytosolic pollen phosphoproteins exhibiting increases in phosphorylation specifically in incompatible pollen after SI (Rudd et al. 1996).
p26 was identified as a 26 kDa cytosolic pollen protein that exhibits SI-specific increases in phosphorylation within 90 seconds after interaction with incompatible S-proteins. Kinase inhibitor studies suggest that a CDPK (calcium dependent protein kinase) is involved (Rudd et al. 1996). p26 comprises two proteins, Pr-p26a and Pr-p26b, which are soluble inorganic py-rophosphatase (sPPase) homologues (de Graaf et al., personal communication). sPPases catalyze hydrolysis of pyrophosphate (PPi) to orthophosphate (2Pi). Their activity is essential for making biosynthetic processes thermody-namically favourable (Cooperman et al. 1992). Therefore, changes in sPPase activity and the PPi/Pi equilibrium can have a dramatic effect on metabolism. Because pollen tubes are fast growing cells, p26 sPPase may function in pollen tubes by affecting the rate of long chain polymer biosynthesis involved in tip growth. As increases in [Ca + ]c reduce Pr-p26a/b sPPase activities, this suggests a possible functional involvement for sPPase in the inhibition of incompatible pollen tube growth, as sPPase activity is predicted to be essential for growth.
MAPKs (mitogen activated protein kinases) are often associated with stress responses in plants (Innes 2001). As SI might be regarded as a stress response, the possible involvement of MAPKs in SI was investigated. A pollen phosphoprotein, p56-MAPK, which exhibited increased MAPK activity in incompatible, but not compatible, pollen tubes, peaking at 10 minutes after SI induction, was identified (Rudd et al. 2003). An intriguing feature of the p56-MAPK activation is its timing. Its activity peaks several minutes after the arrest of pollen tube growth, which means that p56-MAPK is unlikely to play a role in the initial rapid arrest of tube growth. However, because MAPKs can induce programmed cell death (PCD) in plants (Yang et al. 2001), p56 MAPK may be involved in a PCD signalling cascade in incompatible pollen (Sect. 5.4).
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