Projects per year
Angiosperms perform sexual reproduction using pollination, utilizing specific interactions between pollen (male) and pistil (female) tissues. Many angiosperms use self-incompatibility (SI) to prevent self-fertilization and inbreeding. These genetically controlled systems trigger rejection of “self” (incompatible) pollen. Common poppy (Papaver rhoeas) uses a SI system involving the female S-determinant (PrsS) protein, a ligand secreted by the pistil (Foote et al., 1994) and the male S-determinant protein (PrpS; Wheeler et al., 2009). SI also triggers programmed cell death (PCD), involving the activation of a DEVDase/caspase-3-like activity (Bosch and Franklin-Tong, 2007). A MAP kinase, p56, is involved in signaling to SI-PCD (Rudd et al., 2003; Li et al., 2007; Chai et al., 2017). The actin cytoskeleton is an early target of the SI signaling cascade in P. rhoeas pollen (Geitmann et al., 2000; Snowman et al., 2002) beginning with actin depolymerization and later formation of punctate F-actin foci (Geitmann et al., 2000; Snowman et al., 2002; Poulter et al., 2010). SI also triggers increases in reactive oxygen species (ROS) and nitric oxide (NO; Wilkins et al., 2011). Live-cell imaging of ROS in growing P. rhoeas pollen tubes, using chloromethyl- 2′7 ′-dichlorodihydrofluorescein oxidation, showed that SI induces relatively rapid increases in ROS, as early as 2 min after SI in some incompatible pollen tubes. A link between SI-induced ROS and PCD was identified using ROS scavengers, which revealed alleviation of SI-induced events, including formation of actin punctate foci and the activation of a DEVDase/caspase-3-like activity (Wilkins et al., 2011). These data provided evidence that ROS increases are upstream of these key SI markers and are required for SI-PCD (Wilkins et al., 2011) and represented the first steps in understanding ROS signaling in this system.
Exactly how ROS mediate SI-induced events is an important question that needs to be addressed. One possibility is that oxidative posttranslational modifications to proteins (oxPTMs) are involved. These include reversible modifications to Cys (e.g. sulfenylation, disulphide bonds, S-glutathionylation) and Met (Met sulfoxide) as well as a range of irreversible oxPTMs (Møller et al., 2007). In the case of Cys, reversible oxPTMs mediate signaling or changes in protein function (Waszczak et al., 2014, 2015; Akter et al., 2015a). NO produced during SI (Wilkins et al., 2011) also provides the possibility of a role for Cys S-nitrosylation. Although we had previously identified ROS as a signal to SI-PCD (Wilkins et al., 2011), earlier studies did not extend to identifying the protein targets of oxidation.
In this study, we aimed to identify and map oxPTMs on pollen proteins triggered by SI and H2O2 using liquid chromatography tandem mass spectrometry (LC-MS/MS). We analyzed the protein targets of ROS in the context of SI induction and identified and mapped oxidative modifications. Our data reveal that irreversible oxidation is likely an important mechanism involved in SI events in incompatible P. rhoeas pollen and provide a link between irreversible oxPTMs and a ROS-mediated physiological process.
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- 2 Finished
1/08/17 → 31/12/20
Project: Research Councils
16/02/09 → 15/05/13
Project: Research Councils