Self-Incompatibility-Induced Programmed Cell Death in Field Poppy Pollen Involves Dramatic Acidification of the Incompatible Pollen Tube Cytosol  

Abstract

Self-incompatibility (SI) is an important genetically controlled mechanism to prevent inbreeding in higher plants. SI involves highly specific interactions during pollination, resulting in the rejection of incompatible (self) pollen. Programmed cell death (PCD) is an important mechanism for destroying cells in a precisely regulated manner. SI in field poppy (Papaver rhoeas) triggers PCD in incompatible pollen. During SI-induced PCD, we previously observed a major acidification of the pollen cytosol. Here, we present measurements of temporal alterations in cytosolic pH ([pH] cyt ); they were surprisingly rapid, reaching pH 6.4 within 10 min of SI induction and stabilizing by 60 min at pH 5.5. By manipulating the [pH] cyt of the pollen tubes in vivo, we show that [pH] cyt acidification is an integral and essential event for SI-induced PCD. Here, we provide evidence showing the physiological relevance of the cytosolic acidification and identify key targets of this major physiological alteration. A small drop in [pH] cyt inhibits the activity of a soluble inorganic pyrophosphatase required for pollen tube growth. We also show that [pH] cyt acidification is necessary and sufficient for triggering several key hallmark features of the SI PCD signaling pathway, notably activation of a DEVDase/caspase-3-like activity and formation of SI-induced punctate actin foci. Importantly, the actin binding proteins Cyclase-Associated Protein and ActinDepolymerizing Factor are identified as key downstream targets. Thus, we have shown the biological relevance of an extreme but physiologically relevant alteration in [pH] cyt and its effect on several components in the context of SI-induced events and PCD.

Identification of Phosphorylation Sites Altering Pollen Soluble Inorganic Pyrophosphatase Activity

“…We constructed further phosphomutants with five and seven substituted residues mapped from in vitro phosphorylations with recombinant CPKs (Supplemental Table S4, A and B): p26a(5E), p26b(59E), p26a(7E), and p26b(79E). There was no difference in activity between the wild-type p26a/b and phosphomimic/phosphonull mutants at pH 7 ( We previously demonstrated that SI triggers rapid, dramatic cytosolic acidification of incompatible pollen, with cytosolic pH ([pH] cyt ) decreasing from pH 7 to 6.8 within 10 min and to pH 5.5 within 60 min (Wilkins et al, 2015). Both p26a and p26b had identical pH profiles and displayed a pH-dependent attenuation typical of other Family I sPPases (Cooperman et al, 1992;Wilkins et al, 2015).…”

“…There was no difference in activity between the wild-type p26a/b and phosphomimic/phosphonull mutants at pH 7 ( We previously demonstrated that SI triggers rapid, dramatic cytosolic acidification of incompatible pollen, with cytosolic pH ([pH] cyt ) decreasing from pH 7 to 6.8 within 10 min and to pH 5.5 within 60 min (Wilkins et al, 2015). Both p26a and p26b had identical pH profiles and displayed a pH-dependent attenuation typical of other Family I sPPases (Cooperman et al, 1992;Wilkins et al, 2015). Examining if pH affected the PPase activity of the phosphomimic mutants, we found that their activities were more sensitive to decreases in pH (Fig.…”

“…Downstream, SI triggers transient increases in reactive oxygen species (ROS) and nitric oxide, which participate in signaling alterations to the actin cytoskeleton and programmed cell death (PCD; Wilkins et al, 2011Wilkins et al, , 2014. SI also triggers dramatic acidification of the cytosol, which is necessary and sufficient to trigger PCD, involving the activation of a DEVDase/ caspase-3-like activity (Bosch and Franklin-Tong, 2007;Wilkins et al, 2015). However, one of the earliest targets of the SI-mediated Ca 2+ signals are a pair of pollen-expressed Family I sPPases, Pr-p26.1a and Pr-p26.1b (hereafter called p26a and p26b), that are rapidly phosphorylated in a Ca 2+ -dependent manner in incompatible pollen after SI (Rudd et al, 1996).…”

Pollen-Pistil Interactions and Their Role in Mate Selection

“…In vitro systems have been used successfully to elucidate pollen tube cell biology and pollen tube attraction (Cheung et al, 1995;Palanivelu and Preuss, 2006;Okuda et al, 2009;Kanaoka and Higashiyama, 2015), but P. rhoeas is unique among pollen rejection systems because the SI response also can be reproduced faithfully. The addition of incompatible Prs-S protein to pollen tube growth medium rapidly initiates a sequence of events, including cessation of growth, changes in calcium currents and pH, and fragmentation of the actin cytoskeleton in pollen tubes (Franklin-Tong, 2008;Wilkins et al, 2014Wilkins et al, , 2015. Over longer time periods, a programmed cell death response is initiated, and pollen tube growth inhibition is irreversible Franklin-Tong, 2007, 2008).…”

“…non-S-locus genes that are required for full function) on either the pollen or the pistil side, or both, Papaver SI appears to be simpler. The initial signaling to Prp-S in incompatible pollen tubes may directly affect intracellular calcium, leading to a complex but conserved series of events with cell death as the end point (Wilkins et al, 2014(Wilkins et al, , 2015.…”