Reactive Oxygen Species and Nitric Oxide Mediate Actin Reorganization and Programmed Cell Death in the Self-Incompatibility Response of Papaver      

Abstract

Pollen-pistil interactions are critical early events regulating pollination and fertilization. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization and inbreeding in higher plants. Although data implicate the involvement of reactive oxygen species (ROS) and nitric oxide (NO) in pollen-pistil interactions and the regulation of pollen tube growth, there has been a lack of studies investigating ROS and NO signaling in pollen tubes in response to defined, physiologically relevant stimuli. We have used live-cell imaging to visualize ROS and NO in growing Papaver rhoeas pollen tubes using chloromethyl-2#7#-dichlorodihydrofluorescein diacetate acetyl ester and 4-amino-5-methylamino-2#,7#-difluorofluorescein diacetate and demonstrate that SI induces relatively rapid and transient increases in ROS and NO, with each showing a distinctive "signature" within incompatible pollen tubes. Investigating how these signals integrate with the SI responses, we show that Ca 2+ increases are upstream of ROS and NO. As ROS/NO scavengers alleviated both the formation of SI-induced actin punctate foci and also the activation of a DEVDase/caspase-3-like activity, this demonstrates that ROS and NO act upstream of these key SI markers and suggests that they signal to these SI events. These data represent, to our knowledge, the first steps in understanding ROS/NO signaling triggered by this receptor-ligand interaction in pollen tubes.Pollen-pistil interactions in flowering plants are involved in pivotal events regulating pollination and fertilization. An important mechanism that operates during pollination to prevent inbreeding and its consequential debilitating effects is self-incompatibility (SI). Three distinct SI systems have been identified to date at the molecular level, which suggests that SI has evolved independently several times (for recent reviews, see Takayama and Isogai, 2005;Franklin-Tong, 2008). These systems use a variety of mechanisms to prevent self-fertilization. Despite being controlled by different genes, all the SI systems use an S-locus. Selffertilization is prevented by use of a specific recognition system, which results in "self" pollen (i.e. incompatible pollen) being rejected at some point in the pollination process, while compatible pollen is allowed to grow freely. The S-locus is multiallelic, which allows for different specificities to be generated, and combinations of different haplotypes allow discrimination between self and nonself. When pollen S-and pistil S-haplotypes match, this creates an incompatible combination and incompatible pollen is rejected.

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

“…These feed into later suicide signaling events involved in commitment to PCD through the activation of caspase-3-like/DEVDase activities. Events include activation of the p56 MAPK Li et al, 2007), increases in ROS (Wilkins et al, 2011), and formation of F-actin foci (Poulter et al, , 2011Wilkins et al, 2011; for review, see Wilkins et al, 2014). Collectively, these signals trigger suicide signaling events leading to a gateway, around 10 min, that incompatible pollen must pass to become irreversibly inhibited and killed.…”

“…In field poppy (Papaver rhoeas), the S-determinants are a 14-kD signaling ligand field poppy stigma S (PrsS) and a unique transmembrane protein field poppy pollen S (PrpS; Foote et al, 1994;Wheeler et al, 2010). These interact in an S-specific manner, and increases in cytosolic free calcium ([Ca 2+ ] cyt ) are triggered in incompatible pollen tubes , resulting in phosphorylation of soluble inorganic pyrophosphatases (sPPases; Rudd et al, 1996;de Graaf et al, 2006), activation of a Mitogen-Activated Protein Kinase (MAPK; , and increases in reactive oxygen species (ROS) and nitric oxide (Wilkins et al, 2011(Wilkins et al, , 2014. Most of these components are integrated into a signaling network leading to PCD (Bosch et al, 2008;Wilkins et al, 2014).…”

Supergenes and their role in evolution

“…In this system, the female S locus gene encodes a secreted 15-kDa protein, which is thought to interact with a Sencoded, pollen tube-expressed transmembrane receptor (Foote and Ride, 1994;Wheeler et al, 2009). Interaction of non-compatible S proteins leads to disruption of pollen-tube growth through calciumdependent depolymerisation of the pollen-tube cytoskeleton and apoptosis (Wilkins et al, 2011).…”

Papaver rhoeas S-Determinants and the Signaling Networks They Trigger

“…Investigating how these signals integrate with the SI responses, using ROS/NO scavengers, revealed the alleviation of both the formation of SI-induced actin punctate foci and the activation of a DEVDase/caspase-3-like activity, providing evidence that ROS and NO act upstream of these key SI markers and suggesting they signal to these SI events (Wilkins et al 2011 ) (see Fig. 23.1b ).…”

“…It has recently been shown, using live-cell imaging to visualize reactive oxygen species (ROS) and nitric oxide (NO) in growing Papaver pollen tubes, that SI induces relatively rapid and transient increases in ROS and NO, which have distinct temporal "signatures" in incompatible pollen tubes (Wilkins et al 2011 ). Investigating how these signals integrate with the SI responses, using ROS/NO scavengers, revealed the alleviation of both the formation of SI-induced actin punctate foci and the activation of a DEVDase/caspase-3-like activity, providing evidence that ROS and NO act upstream of these key SI markers and suggesting they signal to these SI events (Wilkins et al 2011 ) (see Fig.…”