Functional transfer of the papaver si system into self-compatible a. thaliana and investigating the role of the proteasome in the papaver si response

Self-incompatibility is adopted by many flowering plants to prevent inbreeding, and is controlled by a multi-allelic $S$-locus. In $Papaver$ $rhoeas$, the pistil $S$-determinant is PrsS (a small secreted protein); the pollen $S$-determinant is PrpS (a novel transmembrane protein). Cognate PrpS-PrsS interaction induces DEVDase-mediated programmed cell death of incompatible pollen. Here, we examined the role of proteasome during the $Papaver$ SI response and showed that the proteasome is a target of the $Papaver$ SI response, and is distinct from the SI-induced DEVDase activity. Our main focus here is translational work, attempting to move the Papaver SI system into $A$. $thaliana$. We previously demonstrated that PrpS:GFP expressed in $A$. $thaliana$ pollen was functional $in$ $vitro$. Here, we expressed the female $S$-determinant, PrsS, in $A$. $thaliana$ and investigated function $in$ $vivo$. We present data demonstrating that transgenic A. thaliana stigmas expressing PrsS pollinated with $A$. $thaliana$ pollen expressing PrpS:GFP inhibited pollen tube growth in an S-specific manner, and virtually no seed was set. Transformation of both $PrpS$$:$$GFP$ and $PrsS$ into $A$. $thaliana$ generated self-incompatible plants that set no self-seed. This demonstrates that transfer of the $Papaver$ SI system into a highly diverged self-compatible species can result in a fully functional SI system

Exo84c-regulated degradation is involved in the normal self-incompatible response in Brassicaceae

The self-incompatibility system evolves in angiosperms to promote cross-pollination by rejecting self-pollination. Here, we show the involvement of Exo84c in the SI response of both Brassica napus and Arabidopsis. The expression of Exo84c is specifically elevated in stigma during the SI response. Knocking out Exo84c in B.napus and SI Arabidopsis partially breaks down the SI response. The SI response inhibits both the protein secretion in papillae and the recruitment of the exocyst complex to the pollen-pistil contact sites. Interestingly, these processes can be partially restored in exo84c SI Arabidopsis. After incompatible pollination, the turnover of the exocyst-labeled compartment is enhanced in papillae. However, this process is perturbed in exo84c SI Arabidopsis. Taken together, our results suggest that Exo84c regulates the exocyst complex vacuolar degradation during the SI response. This process is likely independent of the known SI pathway in Brassicaceae to secure the SI response. [Abstract copyright: Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.

Self-incompatibility in Papaver Pollen:Programmed Cell Death in an Acidic Environment

Self-Incompatibility (SI) is a genetically controlled mechanism that prevents self-fertilisation and thus encourages outbreeding and genetic diversity. During pollination, most SI systems utilise cell-cell recognition to reject incompatible pollen. Mechanistically, one of the best-studied SI systems is that of Papaver rhoeas (poppy), which involves the interaction between the two S-determinants, a stigma-expressed secreted protein (PrsS) and a pollen-expressed plasma-membrane localised protein (PrpS). This interaction is the critical step in determining acceptance of compatible pollen or rejection of incompatible pollen. Cognate PrpS-PrsS interaction triggers a signalling network causing rapid growth arrest and eventually programmed cell death (PCD) in incompatible pollen. In this review, we provide an overview of recent advances in our understanding of the major components involved in the SI-induced PCD (SI-PCD). In particular, we focus on the importance of SI-induced intracellular acidification and consequences for protein function, and the regulation of soluble inorganic pyrophosphatase (Pr-p26.1) activity by post-translational modification. We also discuss attempts at the identification of protease(s) involved in the SI-PCD process. Finally, we outline future opportunities made possible by the functional transfer of the P. rhoeas SI system to Arabidopsis

Ectopic expression of a self-incompatibility module triggers growth arrest and cell death in vegetative cells

Self-incompatibility (SI) is used by many angiosperms to reject 'self' pollen and avoid inbreeding. In field poppy (Papaver rhoeas), SI recognition and rejection of 'self' pollen is facilitated by a female S-determinant, PrsS, and a male S-determinant, PrpS. PrsS belongs to the cysteine-rich peptide (CRP) family, whose members activate diverse signaling networks involved in plant growth, defense and reproduction. PrsS and PrpS are tightly regulated and expressed solely in pistil and pollen cells, respectively. Interaction of cognate PrsS and PrpS triggers pollen tube growth inhibition and programmed cell death (PCD) of 'self' pollen. We previously demonstrated functional intergeneric transfer of PrpS and PrsS to Arabidopsis (Arabidopsis thaliana) pollen and pistil. Here we show that PrpS and PrsS, when expressed ectopically, act as a bipartite module to trigger a 'self-recognition:self-destruct' response in A. thaliana independently of its reproductive context, in vegetative cells. Addition of recombinant PrsS to seedling roots expressing the cognate PrpS resulted in hallmark features of the Papaver SI response, including S-specific growth inhibition and PCD of root cells. Moreover, inducible expression of PrsS in PrpS-expressing seedlings resulted in rapid death of the entire seedling. This demonstrates that, besides specifying SI, the bipartite PrpS-PrsS module can trigger growth arrest and cell death in vegetative cells. Heterologous, ectopic expression of a plant bipartite signaling module in plants has not been shown previously and, by extrapolation, our findings suggest that CRPs diversified for a variety of specialized functions, including regulation of growth and PCD

Self-incompatibility triggers irreversible oxidative modification of proteins in incompatible pollen

Self-incompatibility (SI) is used by many angiosperms to prevent self-fertilization and inbreeding. In common poppy (Papaver rhoeas), interaction of cognate pollen and pistil S-determinants triggers programmed cell death (PCD) of incompatible pollen. We previously identified that reactive oxygen species (ROS) signal to SI-PCD. ROS-induced oxidative posttranslational modifications (oxPTMs) can regulate protein structure and function. Here, we have identified and mapped oxPTMs triggered by SI in incompatible pollen. Notably, SI-induced pollen had numerous irreversible oxidative modifications, while untreated pollen had virtually none. Our data provide a valuable analysis of the protein targets of ROS in the context of SI-induction and comprise a benchmark because currently there are few reports of irreversible oxPTMs in plants. Strikingly, cytoskeletal proteins and enzymes involved in energy metabolism are a prominent target of ROS. Oxidative modifications to a phosphomimic form of a pyrophosphatase result in a reduction of its activity. Therefore, our results demonstrate irreversible oxidation of pollen proteins during SI and provide evidence that this modification can affect protein function. We suggest that this reduction in cellular activity could lead to PCD

ATP depletion plays a pivotal role in self-incompatibility, revealing a link between cellular energy status, cytosolic acidification and actin remodelling in pollen tubes

Self-incompatibility (SI) involves specific interactions during pollination to reject incompatible ('self') pollen, preventing inbreeding in angiosperms. A key event observed in pollen undergoing the Papaver rhoeas SI response is the formation of punctate F-actin foci. Pollen tube growth is heavily energy-dependent, yet ATP levels in pollen tubes have not been directly measured during SI. Here we used transgenic Arabidopsis lines expressing the Papaver pollen S-determinant to investigate a possible link between ATP levels, cytosolic pH ([pH]cyt ) and alterations to the actin cytoskeleton. We identify for the first time that SI triggers a rapid and significant ATP depletion in pollen tubes. Artificial depletion of ATP triggered cytosolic acidification and formation of actin aggregates. We also identify in vivo, evidence for a threshold [pH]cyt of 5.8 for actin foci formation. Imaging revealed that SI stimulates acidic cytosolic patches adjacent to the plasma membrane. In conclusion, this study provides evidence that ATP depletion plays a pivotal role in SI upstream of programmed cell death and reveals a link between the cellular energy status, cytosolic acidification and alterations to the actin cytoskeleton in regulating Papaver SI in pollen tubes

New opportunities and insights into Papaver selfincompatibility by imaging engineered Arabidopsis pollen

Pollen tube growth is essential for plant reproduction. Their rapid extension using polarized tip growth provides an exciting system for studying this specialized type of growth. Self-incompatibility (SI) is a genetically controlled mechanism to prevent self-fertilization. Mechanistically, one of the best-studied SI systems is that of Papaver rhoeas (poppy). This utilizes two S-determinants: stigma-expressed PrsS and pollen-expressed PrpS. Interaction of cognate PrpS–PrsS triggers a signalling network, causing rapid growth arrest and programmed cell death (PCD) in incompatible pollen. We previously demonstrated that transgenic Arabidopsis thaliana pollen expressing PrpS–green fluorescent protein (GFP) can respond to Papaver PrsS with remarkably similar responses to those observed in incompatible Papaver pollen. Here we describe recent advances using these transgenic plants combined with genetically encoded fluorescent probes to monitor SI-induced cellular alterations, including cytosolic calcium, pH, the actin cytoskeleton, clathrin-mediated endocytosis (CME), and the vacuole. This approach has allowed us to study the SI response in depth, using multiparameter live-cell imaging approaches that were not possible in Papaver. This lays the foundations for new opportunities to elucidate key mechanisms involved in SI. Here we establish that CME is disrupted in self-incompatible pollen. Moreover, we reveal new detailed information about F-actin remodelling in pollen tubes after SI

Self-incompatibility in Papaver:advances in integrating the signalling network

Self-fertilization, which results in reduced fitness of offspring, is a common problem in hermaphrodite angiosperms. To prevent this, many plants utilize SI (self-incompatibility), which is determined by the multi-allelic S-locus, that allows discrimination between self (incompatible) and non-self (compatible) pollen by the pistil. In poppy (Papaver rhoeas), the pistil S-determinant (PrsS) is a small secreted protein which interacts with the pollen S-determinant PrpS, a ~20 kDa novel transmembrane protein. Interaction of matching pollen and pistil S-determinants results in self-recognition, initiating a Ca2+-dependent signalling network in incompatible pollen. This triggers several downstream events, including alterations to the cytoskeleton, phosphorylation of sPPases (soluble inorganic pyrophosphatases) and an MAPK (mitogen-activated protein kinase), increases in ROS (reactive oxygen species) and nitric oxide (NO), and activation of several caspase-like activities. This results in the inhibition of pollen tube growth, prevention of self-fertilization and ultimately PCD (programmed cell death) in incompatible pollen. The present review focuses on our current understanding of the integration of these signals with their targets in the SI/PCD network. We also discuss our recent functional expression of PrpS in Arabidopsis thaliana pollen.</jats:p

The Papaver rhoeas S determinants confer self-incompatibility to Arabidopsis thaliana in planta

Distant relatives can share gene function The plants Arabidopsis thaliana and Papaver rhoeas (poppy) shared a common ancestor approximately 140 million years ago. Because of this evolutionary distance, although many of their genes share function, the mechanisms that allow these genes to function are expected to have diverged. However, Z. Lin et al. found that a pair of genes that prevent self-fertilization in poppy can confer the same trait when expressed in Arabidopsis. This incompatibility was much more like that of poppy than that of incompatible close relatives of Arabidopsis. Thus, similar long-distance transfer of incompatibility, a trait of interest for plant breeding, may be useful between other distantly related species. Science , this issue p. 684 </jats:p

KIRA1 and ORESARA1 terminate flower receptivity by promoting cell death in the stigma of Arabidopsis

Flowers have a species-specific functional life span that determines the time window in which pollination, fertilization and seed set can occur. The stigma tissue plays a key role in flower receptivity by intercepting pollen and initiating pollen tube growth toward the ovary. In this article, we show that a developmentally controlled cell death programme terminates the functional life span of stigma cells in Arabidopsis. We identified the leaf senescence regulator ORESARA1 (also known as ANAC092) and the previously uncharacterized KIRA1 (also known as ANAC074) as partially redundant transcription factors that modulate stigma longevity by controlling the expression of programmed cell death-associated genes. KIRA1 expression is sufficient to induce cell death and terminate floral receptivity, whereas lack of both KIRA1 and ORESARA1 substantially increases stigma life span. Surprisingly, the extension of stigma longevity is accompanied by only a moderate extension of flower receptivity, suggesting that additional processes participate in the control of the flower's receptive life span