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Self-incompatibility in Papaver targets soluble inorganic pyrophosphatases in pollen

Abstract

In higher plants, sexual reproduction involves interactions between pollen and pistil. A key mechanism to prevent inbreeding is self-incompatibility through rejection of incompatible (‘self’) pollen1. In Papaver rhoeas, S proteins encoded by the stigma interact with incompatible pollen, triggering a Ca2+-dependent signalling network2,3,4,5 resulting in pollen tube inhibition and programmed cell death6. The cytosolic phosphoprotein p26.1, which has been identified in incompatible pollen, shows rapid, self-incompatibility-induced Ca2+-dependent hyperphosphorylation in vivo3. Here we show that p26.1 comprises two proteins, Pr-p26.1a and Pr-p26.1b, which are soluble inorganic pyrophosphatases (sPPases). These proteins have classic Mg2+-dependent sPPase activity, which is inhibited by Ca2+, and unexpectedly can be phosphorylated in vitro. We show that phosphorylation inhibits sPPase activity, establishing a previously unknown mechanism for regulating eukaryotic sPPases. Reduced sPPase activity is predicted to result in the inhibition of many biosynthetic pathways, suggesting that there may be additional mechanisms of self-incompatibility-mediated pollen tube inhibition. We provide evidence that sPPases are required for growth and that self-incompatibility results in an increase in inorganic pyrophosphate, implying a functional role for Pr-p26.1.

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Figure 1: Expression analysis of Pr-p26.1a and Pr-p26.1b sPPases.
Figure 2: Pr-p26.1a and Pr-p26.1b show sPPase activity and are phosphorylated, and phosphorylation affects sPPase activity.
Figure 3: Functional evidence for Pr-p26.1 and sPPase involvement in modulating pollen tube growth.

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Acknowledgements

We thank G. Wullems for the ntp303 pollen promoter; G. Jones for help and advice on statistical analysis; and E. Sanchez-Moran for help with imaging. Work in the laboratories of F.C.H.F. and V.E.F-T. is funded by the UK Biotechnology and Biological Sciences Research Council. Author Information Sequences have been deposited in the EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/) under accession codes AM162550 and AM162551. The authors declare no competing financial interests.

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Correspondence to Vernonica E. Franklin-Tong.

Supplementary information

Supplementary Figure Legends

This file contains text to accompany Supplementary Figures 1–4. (DOC 32 kb)

Supplementary Figure 1

Model for sPPase phosphorylation, effect on activity and biological consequences. (PDF 63 kb)

Supplementary Figure 2

Deduced amino acid sequences of the Pr-p26.1a/b cDNAs. (PDF 138 kb)

Supplementary Figure 3

sPPase activities in different tissues from Papaver rhoeas (PDF 66 kb)

Supplementary Figure 4

Antisense oligonucleotides show that Pr-p26.1a/b play a key role in pollen tube growth (PDF 144 kb)

Supplementary Methods

This file contains additional details on the methods used in this study. (DOC 51 kb)

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de Graaf, B., Rudd, J., Wheeler, M. et al. Self-incompatibility in Papaver targets soluble inorganic pyrophosphatases in pollen. Nature 444, 490–493 (2006). https://doi.org/10.1038/nature05311

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