Research Theme within School of Biosciences: Plant Genetics and Cell Biology
Targets and mechanisms involved in the self-incompatibility response in Papaver rhoeas pollen
Sexual reproduction in higher plants involves pollination, involving specific interactions between pollen and pistil. A key mechanism to prevent inbreeding is self-incompatibility (SI), which is a controlled by a single, multi-allelic S-locus. Incompatible ("self") pollen is rejected and compatible ("non-self") pollen is allowed to fertilize the plant.
My lab is investigating the signalling cascades, targets and mechanisms regulating pollen tube tip growth inhibition induced by SI in Papaver. The "self" interaction triggers a Ca2+-dependent signalling network, resulting in incompatible pollen tube tip growth being inhibited and programmed cell death being induced. Our long-term goal is to establish how the different components integrate and interact in what has turned out to be a complex signalling network. Currently research falls into several areas:
1. Investigations into programmed cell death in the SI response.
Programmed cell death (PCD) is an important mechanism responsible for the controlled death of targeted cells in animal and plant cells. We demonstrated that PCD is triggered in incompatible pollen and identified several caspase-like activities (a VEIDase and a LEVDase) that are activated by an incompatible SI response. We identified increases in reactive oxygen species (ROS) and nitric oxide (NO) during SI-PCD and are currently investigating their role in incompatible pollen using a mass spectrometry approach. We are exploring further mechanisms involved in PCD with Dr Maurice Bosch (IBERS).
2. Alterations in the actin cytoskeleton
In many cells alterations to the cytoskeleton are the first visible response to extracellular stimuli. We have shown that both the actin and microtubule cytoskeleton are important target for SI signals, and recently obtained evidence for crosstalk between actin and microtubules. In animal cells sustained actin depolymerization can stimulate apoptosis via caspases. We have shown that actin depolymerization can trigger PCD in pollen and propose that the formation of highly stable punctate actin foci is integral to SI-PCD. The actin-binding proteins cyclase-associated protein (CAP) and actin depolymerizing factor (ADF) key downstream targets of SI-PCD and co-localize to the foci.
3. Analysis of phosphorylation of soluble inorganic pyrophosphatases (sPPases)
We previously identified soluble inorganic pyrophosphatases (sPPases) as a novel target for phosphorylation, as Pr-p26.1a/b was phosphorylated after SI.We are analysing the phosphorylation sites on Pr-p26.1a/b using mass spectrometry and using site-directed mutagenesis to attempt to establish those involved in sPPase phospho-regulation. We are also analysing the CDPK(s) involved in SI-induced phosphorylation of Pr-p26.1a/b.
4. Analysis of SI-induced acidification
We previously observed a major acidification of the pollen cytosol during PCD in incompatible pollen. We recently measured temporal alterations in cytosolic pH ([pH]cyt); they were surprisingly rapid, reaching pH 6.4 within 10 min of SI induction, stabilizing by 60 min at pH 5.5. Exploring the role of this, we found that the [pH]cyt acidification is an integral and essential event for SI-induced PCD. We demonstrated that [pH]cyt acidification is necessary and sufficient for triggering several key hallmark features of the SI-PCD signalling pathway, notably the activation of a DEVDase/caspase-3-like activity and formation of SI-induced punctate actin foci.
5. Integrating signals and targets involved in mediating SI-PCD in incompatible pollen It has become evident that many of the PCD events are intertwined. Current focus is on understanding the mechanisms involved. The long-term goal is to establish how the different components integrate and interact in what has turned out to be a complex signalling network.
6. Investigating the functionality of Papaver S-determinants in heterologous systems
We recently showed that the pollen S-determinant, PrpS could be expressed in compatible Arabidopsis thaliana, a self-compatible model plant. Exposing transgenic A. thaliana pollen to recombinant Papaver PrsS protein triggered remarkably similar responses to those observed in incompatible Papaver pollen. This demonstrated that PrpS is functional in a species with no SI system that diverged ~140 million years ago, and suggests that the Papaver SI system uses cellular targets that are, perhaps, common to all eudicots and that endogenous signaling components can be recruited to elicit a response that most likely never operated in this species. We are currently exploring if the Papaver S-determinants are functional in other plant species/genera and also if they work in other cell types.
7. We are pursuing possible commercial aspects of using SI, by investigating whether poppy SI can be transferred to other species in collaboration with Dr Barend de Graaf (Cardiff) and Dr Wendy Harwood (JIC). This follows on from filing a patent in 2008. This is a very exciting opportunity; LES College KT Development funding (£20,000) allowed us to pursue obtaining further data to consolidate the patent. Links have been made with PBL, who are currently marketing this application, which could be of immense value to plant breeders in the production of F1 hybrids.
Katie A. Wilkins, Maurice Bosch, Tamanna Haque, Nianjun Teng, Natalie Poulter and Vernonica E. Franklin-Tong (2015). Self-Incompatibility-induced Programmed Cell Death in Papaver pollen involves dramatic acidification of the incompatible pollen tube cytosol. Plant Physiology January 2015 pp.114.252742 doi: http://dx.doi.org/10.1104/pp.114.252742
Wilkins KA, Poulter NS, Franklin-Tong VE (2014) Taking one for the team: self -recognition and cell suicide in pollen.J Exp Bot. 65 : 1331-1342.
Eaves DJ, Flores-Ortiz C, Haque T, Lin Z, Teng N, Franklin-Tong VE. (2014) Self-Incompatibility in Papaver: Advances in integrating the signalling network. Biochermical Transactions 42:370-6. doi: 10.1042/BST20130248
Barend H. J. de Graaf, Sabina Vatovec, Javier Andrés Juárez-Díaz, Lijun Chai, Kreepa Kooblall, Katie A. Wilkins, Huawen Zou, F. Christopher H. Franklin and Vernonica E. Franklin-Tong. The Papaver self-incompatibility pollen S-determinant, PrpS, functions in Arabidopsis thaliana. Current Biology 22 (2), 154-159. doi:10.1016/j.cub.2011.12.006
Katie A. Wilkins, James Bancroft, Maurice Bosch, Jennifer Ings, Nicholas Smirnoff and Vernonica E. Franklin-Tong (2011). ROS and NO mediate actin reorganization and programmed cell death in the Self-Incompatibility response of Papaver. Plant Physiology 156, 404-416. DOI: 10.1104/pp.110.167510.
Smertenko, A. & Franklin-Tong, VE (2011). Organization & Regulation of the cytoskeleton in plant programmed cell death. Cell Death & Differentiation 18, 1263-1270. doi:10.1038/cdd.2011.39
Wouter G. van Doorn, Eric P. Beers, Jeffery L. Dangl, Vernonica E. Franklin-Tong, Hiroo Fukuda, Patrick Gallois, Jean Greenberg, Ikuko Hara-Nishimura, Alan M. Jones, Maki Kawai-Yamada, Eric Lam11, John Mundy12, Luis Mur, Morten Petersen, Andrei Smertenko, Michael Taliansky, Frank van Breusegem16, Thomas Wolpert, Ernst Woltering, Boris Zhivotovsky, Peter V. Bozhkov(2011). Morphological classification of plant cell deaths. Cell Death & Differentiation 18, 8, 1241-1246.doi:10.1038/cdd.2011.36
Noni Franklin-Tong (2011). Self-fertilization: article in: Brenner’s Encyclopedia of Genetics.
V.E. Franklin-Tong (ed.) Self-Incompatibility in Flowering Plants – Evolution, Diversity, and Mechanisms. Publ. Springer-Verlag Berlin Heidelberg 2008. This is the first monograph on this topic for 30 years.
Natalie S. Poulter, Maurice Bosch, &Vernonica E. Franklin-Tong (2011) Proteins implicated in mediating Self-Incompatibility-induced alterations to the actin cytoskeleton of Papaver pollen. Annals of Botany: doi:10.1093/aob/mcr022.
Juyou Wu, Su Wang, Yuchun Gu, Shaoling Zhang, Stephen J Publicover and V. E. Franklin-Tong (2011) Self-incompatibility in Papaver rhoeasactivates non-specific cation conductance(s) permeable to Ca2+and K+. Plant Physiology 155: 963-973.
Maurice Bosch, Natalie S. Poulter, Ruth M. Perry, Katie Wilkins and V. E. Franklin-Tong (2010). Characterization of a legumain/vacuolar processing enzyme and YVADase activity in Papaver pollen. Plant Molecular Biology 74 (4), 381-393. DOI: 10.1007/s11103-010-9681-9.
Natalie S. Poulter, Christopher J. Staiger, Joshua Z. Rappoport, and Vernonica E. Franklin-Tong (2010). Actin-Binding Proteins Implicated in the Formation of the Punctate Actin Foci Stimulated by the Self-Incompatibility Response in Papaver. Plant Physiol. 152: 1274-1283.
Wheeler, M.J., de Graaf, B.H.J., Hadjiosif, N.E., Perry, R.M., Poulter, N.S., Osman, K., Vatovec, S., Harper, A., Franklin, F.C.H & Franklin-Tong, V.E. (2009). Identification of the pollen self-incompatibility determinant in Papaver rhoeas. Nature 459, 992-995.
Poulter, N. S., Vatovec, S. and Franklin-Tong VE. (2008). Microtubules Are a Target for Self-Incompatibility Signaling in Papaver Pollen. Plant Physiol. 146, 1358-1367.
Bosch M. & Franklin-Tong VE. (2007). Temporal and spatial activation of caspase-like enzymes induced by self-incompatibility in Papaver pollen. Proc. Natl. Acad. Sci. USA. 104 (46) 18327-18332.
S. Li, J. Samaj & V. E. Franklin-Tong. (2007). A MAP kinase signals to Programmed Cell Death induced by Self-Incompatibility in Papaver pollen. Plant Physiol 145, 236-245.
B.H.J. de Graaf, J.J. Rudd, M. J. Wheeler, R.M. Perry, E. M. Bell, K. Osman, F. C.H. Franklin & V. E. Franklin-Tong (2006). Self-incompatibility in Papavertargets soluble inorganic pyrophosphatases in pollen. Nature 444, 490-493. Selected for "Editor’s Choice" in Nature.
Thomas, S. Huang , S. Li, C.J. Staiger and V.E. Franklin-Tong. (2006). Actin depolymerization is sufficient to induce programmed cell death in self-incompatible pollen. S.G. Journal of Cell Biology 174, 221-229. This article was selected for a highlight JCB "In This Issue" section and by Cell in a "Leading Edge"; Aug 11th 2006 issue of Cell).
Huang, S., Blanchoin, L., Chaudhry, F., Franklin-Tong, V.E. and Staiger, C.J. (2004) A gelsolin-like protein from Papaver rhoeaspollen (PrABP80) stimulates calcium-regulated severing and depolymerization of actin filaments. Journal of Biological Chemistry 279, 23364-23375.
Thomas, S.G. & Franklin-Tong, VE. (2004) Programmed Cell Death is triggered by self-incompatibility in Papaver pollen. Nature 429, 305-309.