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.

Self-incompatibility in Papaver pollen: programmed cell death in an acidic environment.

Self-incompatibility (SI) is a genetically controlled mechanism that prevents self-fertilization and thus encourages outbreeding and genetic diversity. During pollination, most SI systems utilize 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-localized 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 to identify 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.

Improved UAV Opium Poppy Detection Using an Updated YOLOv3 Model.

Rapid detection of illicit opium poppy plants using UAV (unmanned aerial vehicle) imagery has become an important means to prevent and combat crimes related to drug cultivation. However, current methods rely on time-consuming visual image interpretation. Here, the You Only Look Once version 3 (YOLOv3) network structure was used to assess the influence that different backbone networks have on the average precision and detection speed of an UAV-derived dataset of poppy imagery, with MobileNetv2 (MN) selected as the most suitable backbone network. A Spatial Pyramid Pooling (SPP) unit was introduced and Generalized Intersection over Union (GIoU) was used to calculate the coordinate loss. The resulting SPP-GIoU-YOLOv3-MN model improved the average precision by 1.62% (from 94.75% to 96.37%) without decreasing speed and achieved an average precision of 96.37%, with a detection speed of 29 FPS using an RTX 2080Ti platform. The sliding window method was used for detection in complete UAV images, which took approximately 2.2 sec/image, approximately 10× faster than visual interpretation. The proposed technique significantly improved the efficiency of poppy detection in UAV images while also maintaining a high detection accuracy. The proposed method is thus suitable for the rapid detection of illicit opium poppy cultivation in residential areas and farmland where UAVs with ordinary visible light cameras can be operated at low altitudes (relative height < 200 m).

MAP Kinase PrMPK9-1 Contributes to the Self-Incompatibility Response.

Mitogen-activated protein kinases (MAPKs) form important signaling modules for a variety of cellular responses in eukaryotic cells. In plants, MAPKs play key roles in growth and development as well as in immunity/stress responses. Pollen-pistil interactions are critical early events regulating pollination and fertilization and involve many signaling events. Self-incompatibility (SI) is an important mechanism to prevent self-fertilization and inbreeding in higher plants and also is known to utilize signaling to achieve incompatible pollen rejection. Although several pollen-expressed MAPKs exist, very little is known about their function. We previously identified a pollen-expressed MAPK (p56) from Papaver rhoeas that was rapidly activated during SI; several studies implicated its role in signaling to SI-induced programmed cell death involving a DEVDase. However, to date, the identity of the MAPK involved has been unknown. Here, we have identified and cloned a pollen-expressed P. rhoeas threonine-aspartate-tyrosine (TDY) MAPK, PrMPK9-1 Rather few data relating to the function of TDY MAPKs in plants currently exist. We provide evidence that PrMPK9-1 has a cell type-specific function, with a distinct role from AtMPK9 To our knowledge, this is the first study implicating a function for a TDY MAPK in pollen. We show that PrMPK9-1 corresponds to p56 and demonstrate that it is functionally involved in mediating SI in P. rhoeas pollen: PrMPK9-1 is a key regulator for SI in pollen and acts upstream of programmed cell death involving actin and activation of a DEVDase. Our study provides an important advance in elucidating functional roles for this class of MAPKs.

Endophytes of opium poppy differentially modulate host plant productivity and genes for the biosynthetic pathway of benzylisoquinoline alkaloids.

MAIN CONCLUSION: Endophytes reside in different parts of the poppy plant and perform the tissue-specific functions. Most leaf endophytes modulate photosynthetic efficiency, plant growth, and productivity while capsule endophytes modulate alkaloid biosynthesis. Endophytes promote plant growth, provide protection from environmental stresses and are the source of important secondary metabolites. Here, we established that the endophytes of opium poppy Papaver somniferum L. may play a role in the modulation of plant productivity and benzylisoquinoline alkaloid (BIA) biosynthesis. A total of 22 endophytes isolated from leaves, roots, capsules and seeds of the poppy plants were identified. Isolated endophytes were used to inoculate the endophytes free poppy seeds and screened for their ability to improve plant productivity and BIA production. It was evident that the endophytes from leaf were involved in improving photosynthetic efficiency, and thus crop growth and yield and the endophytes from capsule were involved in enhancing BIA biosynthesis. Capsule endophytes of alkaloid-rich P. somniferum cv. Sampada enhanced BIA production even in alkaloid-less cv. Sujata. Expression study of the genes involved in BIA biosynthesis conferred the differential regulation of their expression in the presence of capsule endophytes. The capsule endophyte SM1B (Acinetobacter) upregulated the expression of the key genes for the BIA biosynthesis except thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM). On the other hand, another capsule endophyte SM3B (Marmoricola sp.) could upregulate both T6ODM and CODM. Colonization of poppy plant by endophytes isolated from leaves, roots and capsules found to be higher in their respective plant parts confirmed their tissue-specific role. Overall, the results demonstrate the specific role of endophytes in the modulation of host plant productivity and BIA production.

PsAP2 an AP2/ERF family transcription factor from Papaver somniferum enhances abiotic and biotic stress tolerance in transgenic tobacco.

The AP2/ERFs are one of the most important family of transcription factors which regulate multiple responses like stress, metabolism and development in plants. We isolated PsAP2 a novel AP2/ERF from Papaver somniferum which was highly upregulated in response to wounding followed by ethylene, methyl jasmonate and ABA treatment. PsAP2 showed specific binding with both DRE and GCC box elements and it was able to transactivate the reporter genes in yeast. PsAP2 overexpressing transgenic tobacco plants exhibited enhanced tolerance towards both abiotic and biotic stresses . Real time transcript expression analysis showed constitutive upregulation of tobacco Alternative oxidase1a and Myo-inositol-1-phosphate synthase in PsAP2 overexpressing tobacco plants. Further, PsAP2 showed interaction with NtAOX1a promoter in vitro, it also specifically activated the NtAOX1a promoter in yeast and tobacco BY2 cells. The silencing of PsAP2 using VIGS lead to significant reduction in the AOX1 level in P. somniferum. Taken together PsAP2 can directly bind and transcriptionally activate NtAOX1a and its overexpression in tobacco imparted increased tolerance towards both abiotic and biotic stress.

Phenological mismatch with abiotic conditions implications for flowering in Arctic plants.

Although many studies have examined the phenological mismatches between interacting organisms, few have addressed the potential for mismatches between phenology and seasonal weather conditions. In the Arctic, rapid phenological changes in many taxa are occurring in association with earlier snowmelt. The timing of snowmelt is jointly affected by the size of the late winter snowpack and the temperature during the spring thaw. Increased winter snowpack results in delayed snowmelt, whereas higher air temperatures and faster snowmelt advance the timing of snowmelt. Where interannual variation in snowpack is substantial, changes in the timing of snowmelt can be largely uncoupled from changes in air temperature. Using detailed, long-term data on the flowering phenology of four arctic plant species from Zackenberg, Greenland, we investigate whether there is a phenological component to the temperature conditions experienced prior to and during flowering. In particular, we assess the role of timing of flowering in determining pre-flowering exposure to freezing temperatures and to the temperatures-experienced prior to flowering. We then examine the implications of flowering phenology for flower abundance. Earlier snowmelt resulted in greater exposure to freezing conditions, suggesting an increased potential for a mismatch between the timing of flowering and seasonal weather conditions and an increased potential for negative consequences, such as freezing 'damage. We also found a parabolic relationship between the timing of flowering and the temperature experienced during flowering after taking interannual temperature effects into account. If timing of flowering advances to a cooler period of the growing season, this may moderate the effects of a general warming trend across years. Flower abundance was quadratically associated with the timing of flowering, such that both early and late flowering led to lower flower abundance than did intermediate flowering. Our results indicate that shifting the timing of flowering affects the temperature experienced during flower development and flowering beyond that imposed by interannual variations in climate. We also found that phenological timing may affect flower abundance, and hence, fitness. These findings suggest that plant population responses to future climate change will be shaped not only by extrinsic climate forcing, but also by species' phenological responses.

Identification of the pollen self-incompatibility determinant in Papaver rhoeas.

Higher plants produce seed through pollination, using specific interactions between pollen and pistil. Self-incompatibility is an important mechanism used in many species to prevent inbreeding; it is controlled by a multi-allelic S locus. 'Self' (incompatible) pollen is discriminated from 'non-self' (compatible) pollen by interaction of pollen and pistil S locus components, and is subsequently inhibited. In Papaver rhoeas, the pistil S locus product is a small protein that interacts with incompatible pollen, triggering a Ca(2+)-dependent signalling network, resulting in pollen inhibition and programmed cell death. Here we have cloned three alleles of a highly polymorphic pollen-expressed gene, PrpS (Papaver rhoeas pollen S), from Papaver and provide evidence that this encodes the pollen S locus determinant. PrpS is a single-copy gene linked to the pistil S gene (currently called S, but referred to hereafter as PrsS for Papaver rhoeas stigma S determinant). Sequence analysis indicates that PrsS and PrpS are equally ancient and probably co-evolved. PrpS encodes a novel approximately 20-kDa protein. Consistent with predictions that it is a transmembrane protein, PrpS is associated with the plasma membrane. We show that a predicted extracellular loop segment of PrpS interacts with PrsS and, using PrpS antisense oligonucleotides, we demonstrate that PrpS is involved in S-specific inhibition of incompatible pollen. Identification of PrpS represents a major advance in our understanding of the Papaver self-incompatibility system. As a novel cell-cell recognition determinant it contributes to the available information concerning the origins and evolution of cell-cell recognition systems involved in discrimination between self and non-self, which also include histocompatibility systems in primitive chordates and vertebrates.

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 Ca²âº-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.

Taking one for the team: self-recognition and cell suicide in pollen.

Self-incompatibility (SI) is an important genetically controlled mechanism used by many angiosperms to prevent self-fertilization and inbreeding. A multiallelic S-locus allows discrimination between 'self' (incompatible) pollen from 'nonself' pollen at the pistil. Interaction of matching pollen and pistil S-determinants allows 'self' recognition and triggers rejection of incompatible pollen. The S-determinants for Papaver rhoeas (poppy) are PrsS and PrpS. PrsS is a small secreted protein that acts as a signalling ligand to interact with its cognate pollen S-determinant PrpS, a small novel transmembrane protein. Interaction of PrsS with incompatible pollen stimulates increases in cytosolic free Ca(2+) and involves influx of Ca(2+) and K(+). Data implicate involvement of reactive oxygen species and nitric oxide signalling in the SI response. Downstream targets include the cytoskeleton, a soluble inorganic pyrophosphatase, Pr-p26.1, and a MAP kinase, PrMPK9-1. A major focus for SI-induced signalling is to initiate programmed cell death (PCD). In this review we provide an overview of our understanding of SI, with focus on how the signals and components are integrated, in particular, how reactive oxygen species, nitric oxide, and the actin cytoskeleton feed into a PCD network. We also discuss our recent functional expression of PrpS in Arabidopsis thaliana pollen in the context of understanding how PCD signalling systems may have evolved.

Self-incompatibility in Papaver rhoeas activates nonspecific cation conductance permeable to Ca2+ and K+.

Cellular responses rely on signaling. In plant cells, cytosolic free calcium is a major second messenger, and ion channels play a key role in mediating physiological responses. Self-incompatibility (SI) is an important genetically controlled mechanism to prevent self-fertilization. It uses interaction of matching S-determinants from the pistil and pollen to allow "self" recognition, which triggers rejection of incompatible pollen. In Papaver rhoeas, the S-determinants are PrsS and PrpS. PrsS is a small novel cysteine-rich protein; PrpS is a small novel transmembrane protein. Interaction of PrsS with incompatible pollen stimulates S-specific increases in cytosolic free calcium and alterations in the actin cytoskeleton, resulting in programmed cell death in incompatible but not compatible pollen. Here, we have used whole-cell patch clamping of pollen protoplasts to show that PrsS stimulates SI-specific activation of pollen grain plasma membrane conductance in incompatible but not compatible pollen grain protoplasts. The SI-activated conductance does not require voltage activation, but it is voltage sensitive. It is permeable to divalent cations (Ba(2+) ≥ Ca(2+) > Mg(2+)) and the monovalent ions K(+) and NH(4)(+) and is enhanced at voltages negative to -100 mV. The Ca(2+) conductance is blocked by La(3+) but not by verapamil; the K(+) currents are tetraethylammonium chloride insensitive and do not require Ca(2+). We propose that the SI-stimulated conductance may represent a nonspecific cation channel or possibly two conductances, permeable to monovalent and divalent cations. Our data provide insights into signal-response coupling involving a biologically important response. PrsS provides a rare example of a protein triggering alterations in ion channel activity.

Reactive oxygen species and nitric oxide mediate actin reorganization and programmed cell death in the self-incompatibility response of papaver.

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.

Self-incompatibility in Papaver targets soluble inorganic pyrophosphatases in pollen.

In higher plants, sexual reproduction involves interactions between pollen and pistil. A key mechanism to prevent inbreeding is self-incompatibility through rejection of incompatible ('self') pollen. In Papaver rhoeas, S proteins encoded by the stigma interact with incompatible pollen, triggering a Ca2+-dependent signalling network resulting in pollen tube inhibition and programmed cell death. The cytosolic phosphoprotein p26.1, which has been identified in incompatible pollen, shows rapid, self-incompatibility-induced Ca2+-dependent hyperphosphorylation in vivo. Here we show that p26.1 comprises two proteins, Pr-p26.1a and Pr-p26.1b, which are soluble inorganic pyrophosphatases (sPPases). These proteins have classic Mg2+-dependent sPPase activity, which is inhibited by Ca2+, and unexpectedly can be phosphorylated in vitro. We show that phosphorylation inhibits sPPase activity, establishing a previously unknown mechanism for regulating eukaryotic sPPases. Reduced sPPase activity is predicted to result in the inhibition of many biosynthetic pathways, suggesting that there may be additional mechanisms of self-incompatibility-mediated pollen tube inhibition. We provide evidence that sPPases are required for growth and that self-incompatibility results in an increase in inorganic pyrophosphate, implying a functional role for Pr-p26.1.

Actin-binding proteins implicated in the formation of the punctate actin foci stimulated by the self-incompatibility response in Papaver.

The actin cytoskeleton is a key target for signaling networks and plays a central role in translating signals into cellular responses in eukaryotic cells. Self-incompatibility (SI) is an important mechanism responsible for preventing self-fertilization. The SI system of Papaver rhoeas pollen involves a Ca(2+)-dependent signaling network, including massive actin depolymerization as one of the earliest cellular responses, followed by the formation of large actin foci. However, no analysis of these structures, which appear to be aggregates of filamentous (F-)actin based on phalloidin staining, has been carried out to date. Here, we characterize and quantify the formation of F-actin foci in incompatible Papaver pollen tubes over time. The F-actin foci increase in size over time, and we provide evidence that their formation requires actin polymerization. Once formed, these SI-induced structures are unusually stable, being resistant to treatments with latrunculin B. Furthermore, their formation is associated with changes in the intracellular localization of two actin-binding proteins, cyclase-associated protein and actin-depolymerizing factor. Two other regulators of actin dynamics, profilin and fimbrin, do not associate with the F-actin foci. This study provides, to our knowledge, the first insights into the actin-binding proteins and mechanisms involved in the formation of these intriguing structures, which appear to be actively formed during the SI response.

Self-incompatibility in Papaver: identification of the pollen S-determinant PrpS.

Many flowering plants are hermaphrodite, posing the problem of self-fertilization and the subsequent loss of the genetic fitness of the offspring. To prevent this, many plants have developed a genetically controlled mechanism called self-incompatibility (SI). When the male and female S-determinants match, self (incompatible) pollen is recognized and rejected before fertilization can occur. In poppy (Papaver rhoeas), the pistil S-determinant (PrsS) is a small secreted protein that interacts with incompatible pollen, initiating a Ca(2+)-dependent signalling network. SI triggers several downstream events, including depolymerization of the cytoskeleton, phosphorylation of two soluble inorganic pyrophosphatases and an MAPK (mitogen-activated protein kinase). This culminates in PCD (programmed cell death) involving several caspase-like activities. The recent discovery of the Papaver pollen S-determinant PrpS marks a significant step forward in the understanding of the Papaver SI system. PrpS encodes a ~20 kDa predicted transmembrane protein which has no homology with known proteins. It is specifically expressed in pollen, linked to the pistil S-determinant, and displays the high polymorphism expected of an S-locus determinant. The present review focuses on the discovery and characterization of PrpS which strongly support the hypothesis that Papaver SI is triggered by the interaction of PrsS and PrpS.

Self-incompatibility triggers programmed cell death in Papaver pollen.

Sexual reproduction in many angiosperm plants involves self-incompatibility (SI), which is one of the most important mechanisms to prevent inbreeding. SI is genetically controlled by the S-locus, and involves highly specific interactions during pollination between pollen and the pistil on which it lands. This results in the rejection of incompatible ('self') pollen, whereas compatible ('non-self') pollen is allowed to fertilize the plant. In Papaver rhoeas, S-proteins encoded by the stigma component of the S-locus interact with incompatible pollen, triggering a Ca2+-dependent signalling network, resulting in the inhibition of pollen-tube growth. Programmed cell death (PCD) is a mechanism used by many organisms to destroy unwanted cells in a precisely regulated manner. Here we show that PCD is triggered by SI in an S-specific manner in incompatible pollen. This provides a demonstration of a SI system using PCD, revealing a novel mechanism to prevent self-fertilization. Furthermore, our data reveal that the response is biphasic; rapid inhibition of pollen-tube growth is followed by PCD, which is involved in a later 'decision-making' phase, making inhibition irreversible.

Pollen-pistil signaling in self-incompatible poppy: does it allow more efficient resource allocation in the pistil?

In the process of pollination, haploid pollen germinates on the stigma surface and a pollen tube grows through the diploid tissues of the pistil toward the ovary. The pistil has two basic functions: to prevent unwanted pollen from gaining access to the ovary and to support the growth of desirable pollen. Pollen-pistil signaling allows these different types of pollen to be distinguished. Self-incompatibility (SI) systems, controlled by the S locus, are the best-understood pollen-pistil signaling systems. Other SI systems have been investigated at the molecular level, but the physiology of pollen tube rejection is best understood in the field poppy, Papaver rhoeas. This species has a gametophytic SI system: Pollen is rejected when its S haplotype is the same as either of the two S haplotypes expressed in the diploid pistil. Recent advances reveal new ways that SI controls pollen tube metabolism. A soluble pyrophosphatase is down-regulated as part of the rapid SI response, and, over the long term, perturbations of the actin cytoskeleton lead to programmed cell death in incompatible pollen tubes. Manipulating incompatible pollen tube metabolism in this way may leave more resources available for supporting the growth of compatible pollen tubes, the complementary function of the pistil.

Self-incompatibility in Papaver: signalling to trigger PCD in incompatible pollen.

Sexual reproduction in higher plants uses pollination, involving interactions between pollen and pistil. Self-incompatibility (SI) prevents self-fertilization, providing an important mechanism to promote outbreeding. SI is controlled by the S-locus; discrimination occurs between incompatible pollen, which is rejected, while compatible pollen can achieve fertilization. In Papaver rhoeas, S proteins encoded by the pistil part of the S-locus interact with incompatible pollen to effect rapid inhibition of tip growth. This self-incompatible interaction triggers a Ca(2+)-dependent signalling cascade. SI-specific events triggered in incompatible pollen include rapid depolymerization of the actin cytoskeleton; phosphorylation of soluble inorganic pyrophosphatases, and activation of a MAPK. It has recently been shown that programmed cell death (PCD) is triggered by SI. This provides a precise mechanism for the specific destruction of 'self' pollen. Recent data providing evidence for SI-induced caspase-3-like protease activity, and the involvement of actin depolymerization and MAPK activation in SI-mediated PCD will be discussed. These studies not only significantly advance our understanding of the mechanisms involved in SI, but also contribute to our understanding of functional links between signalling components and initiation of PCD in a plant cell. Recent data demonstrating SI-mediated modification of soluble inorganic pyrophosphatases are also described.