Incompatible Pollen Research Articles

Evidence for DNA fragmentation triggered in the self-incompatibility response in pollen of Papaver rhoeas.

Studies of the molecular and biochemical basis of self-incompatibility (SI) in Papaver rhoeas have revealed much about the signalling pathways triggered in pollen early in this response. The aim of the current investigation was to begin to study downstream events in order to elucidate some of the later cellular responses involved in the SI response and identification of the mechanisms controlling the irreversible inhibition of pollen tube growth. We have used the FragEL assay to investigate if there is any evidence for DNA fragmentation stimulated in pollen of P. rhoeas in an S-specific manner. Our data clearly demonstrate that S proteins are responsible for triggering this, specifically in incompatible, and not compatible, pollen. DNA fragmentation was first detected in incompatible pollen tubes 4 h after challenge with S proteins, and continued to increase for a further 10 h. This provides the first evidence, to our knowledge, that this phenomenon is associated with the SI response. We also demonstrate that mastoparan, which increases [Ca2+]i, also triggers DNA fragmentation in these pollen tubes, thereby implicating an involvement of Ca2+ signalling in this process. Together, our data represent a significant breakthrough in understanding of the SI response in Papaver pollen.

Effect of pollen competition and stigmatic receptivity on seed set in white clover (Trifolium repens L.)

The effect of competition between incompatible and compatible pollen grains on the seed production of white clover was studied. Stigmatic receptivity was also studied. A selection line of white clover that has red leaves was used as a pollen donor and as a genetic marker to allow determination of the pollen donor responsible for ovule fertilisation. Results show that incompatible pollen did not inhibit compatible pollen grains from fertilising ovules and producing seeds, although it did slightly impair seed yield. At temperatures of 20/10°C (day/night) stigmas of white clover remained receptive to additional pollination up to 32 h after an initial compatible pollination and 40 h after an incompatible pollination. The results imply that factors other than inadequate pollination and the self-incompatibility system are responsible for failure of white clover to attain its potential seed yield.

Alterations in the actin cytoskeleton of pollen tubes are induced by the self-incompatibility reaction in Papaver rhoeas.

Self-incompatibility (SI) is a genetically controlled process used to prevent self-pollination. In Papaver rhoeas, the induction of SI is triggered by a Ca(2)+-dependent signaling pathway that results in the rapid and S allele-specific inhibition of pollen tube tip growth. Tip growth of cells is dependent on a functioning actin cytoskeleton. We have investigated the effect of self-incompatibility (S) proteins on the actin cytoskeleton in poppy pollen tubes. Here, we report that the actin cytoskeleton of incompatible pollen tubes is rapidly and dramatically rearranged during the SI response, not only in our in vitro SI system but also in vivo. We demonstrate that nonspecific inhibition of growth does not result in similar actin rearrangements. Because the SI-induced alterations are not observed if growth stops, this clearly demonstrates that these alterations are triggered by the SI signaling cascade rather than merely resulting from the consequent inhibition of growth. We establish a detailed time course of events and discuss the mechanisms that might be involved. Our data strongly implicate a role for the actin cytoskeleton as a target for signaling pathways involved in the SI response of P. rhoeas.

Signalling and the Cytoskeleton of Pollen Tubes of Papaver rhoeas

Self-incompatibility (SI) is a genetically controlled system used by many flowering plants to prevent self-pollination, often by the inhibition of pollen tube growth. The importance of cytosolic free calcium, [Ca2+]i, for the regulation of pollen tube growth is well known. We have established, using calcium imaging, that the SI response in Papaver rhoeas L. pollen involves a calcium-mediated intracellular signalling pathway. Tip growth of cells is dependent upon a typical configuration of the actin cytoskeleton, which is controlled by actin binding proteins. In animal cells, the actin-binding protein, profilin, is thought to act as a key intermediate between signalling pathways and actin rearrangements. Profilin is an abundant component of pollen. To better understand the signalling cascades that modulate pollen tip growth and actin dynamics, we are investigating a possible signalling role for profilin. We have demonstrated that profilin modulates the phosphorylation of pollen proteins in vitro. This implicates a role for profilin in altering protein kinase or phosphatase activity. Furthermore, we demonstrate for the first time that profilin from pollen can be phosphorylated in vitro. This provides compelling evidence that profilin interacts with signalling pathways in angiosperms. Finally, we demonstrate that in the SI response, the actin cytoskeleton of incompatible pollen tubes is dramatically rearranged. Our data strongly support a role for the cytoskeleton and actin-binding proteins interacting with signalling pathways involved in the regulation of pollen tube growth.

Inhibition of Self-incompatible Pollen in Papaver rhoeas Involves a Complex Series of Cellular Events

Self-incompatibility (SI) in Papaver rhoeas involves allele-specific recognition between stigmatic S proteins and pollen which results in the rapid inhibition of incompatible pollen. In this article we discuss current understanding of the components and mechanisms involved in this reaction. Analysis of cDNA sequences encoding several stigmatic S alleles has allowed us to establish, using site-directed mutagenesis, that certain amino acids are essential for S-specific inhibitory activity. An S protein binding protein (SBP) identified in pollen plasma membrane extracts has been proposed to act as a receptor. Study of S1 gene mutants defective in SBP binding has confirmed the important role of SBP in the SI reaction, however the exact nature of this remains to be defined. Although it was previously thought that the Papaver stigmatic S gene was unique, detailed analysis of Arabidopsis thaliana genomic sequences has revealed the presence of a large number of open reading frames with homology to the S gene. We have named these potential genes SPH (S-protein homologues). Studies of the molecular and biochemical basis of self-incompatibility (SI) in P. rhoeas have revealed much about the early signals triggered in pollen in this response. We have established that a Ca2+-dependent signal transduction pathway mediates the inhibition of incompatible pollen. Rapid increases in cytosolic free Ca2+ and the rapid loss of the pollen apical Ca2+ gradient are accompanied by the hyperphosphorylation of p26 and p68, two soluble pollen phosphoproteins. Studies reveal that p26 is a soluble inorganic pyrophosphatase. Recent evidence has been obtained that suggests irreversible pollen tube growth and cell death involves a programmed cell death (PCD) pathway.

Pollination system and evolution of dioecy from distyly in Mussaenda parviflora (Rubiaceae)

AbstractThe floral morphology and reproductive ecology of a scandent shrub species, Mussaenda parviflora Miq. (Rubiaceae), were studied on Iriomote and Amami Ohshima Islands, Japan. The morphological study demonstrated that M. parviflora was gynodioecious. Crossing experiments, however, revealed that it was functionally dioecious. The flowers were visited mainly by butterflies in the daytime and mainly by crepuscular hawkmoths in the evening. Dioecy in this species is thought to have evolved from distyly. Dioecy of this species, which is pollinated by long‐tongued Lepidoptera, is unusual as the evolution of dioecy from distyly in plants with long corolla tubes is thought to have been triggered by a pollinator shift from long‐ to short‐tongued insects. In this species, however, two floral morphological features may have caused the evolution. The first feature is the presence of many upward‐facing hairs covering the inner surface of the upper part of the corolla tube; these hairs appear to have impeded pollen flow from long‐ to short‐styled flowers. The second feature is the overlap in anther height between the two types of flowers, which may have reduced seed set by clogging stigma with incompatible pollen grains. Accordingly, male sterility in long‐styled plants may have evolved.

S-protein mutants indicate a functional role for SBP in the self-incompatibility reaction of Papaver rhoeas.

The self-incompatibility response involves S-allele specific recognition between stigmatic S proteins and incompatible pollen, resulting in S-specific pollen inhibition. In Papaver rhoeas, the pollen S gene product is predicted to be a receptor that interacts with the stigmatic S protein in an S specific manner. We recently identified an S protein binding protein (SBP) in pollen that binds stigmatic S proteins, although apparently not in an S-allele-specific manner. In order to investigate the functional significance of the interaction between S proteins and SBP, we constructed mutant derivatives of the S1 protein and tested their SBP-binding activity and their biological activity. Here we present an evaluation of nine mutant derivatives of the S1 protein. Western ligand blotting was used to show that mutations to amino acid residues in predicted loops 2 and 6 of the S1 protein cause significant reductions in their SBP-binding activity. These same mutants show a concomitant reduction in their ability to inhibit incompatible pollen. This establishes a direct link between SBP binding and inhibition of incompatible pollen and implicates SBP as a pollen component playing a key role in the self-incompatibility reaction. We discuss the possible nature of the contribution of SBP in the S-specific rejection of incompatible pollen.

Commonalities between pollen/stigma and host/pathogen interactions: calcium accumulation during stigmatic penetration by Brassica oleracea pollen tubes

Parallels have been explored between the early stages of stigmatic penetration by pollen tubes and the infection of epidermal cells by fungal pathogens. In a striking resemblence to events following the infection of Hordeum sp. by Erysiphe graminis, X-ray microanalysis has revealed the accumulation of calcium at the stigmatic surface following pollinations in Brassica oleracea. X-ray mapping strongly indicates the calcium to be localised at the points at which either the pollen grain or its tube makes contact with the surface of the stigmatic papilla. No definitive measures were made of the concentration of calcium at these sites, but controls indicated the levels to be well in excess of those found in the cytosol. X-ray microanalysis at the pollen/stigma interface failed to detect the presence of silicon, an element frequently accumulated by epidermal cells in response to pathogenic challenge. The phenylpropanoid biosynthesis pathway is activated by many plant hosts following infection by fungal pathogens, and the accumulation of autofluorescent material in the stigma 24 h after contact with self pollen strongly indicates this pathway also to be activated after pollination. The timing of this response, however, suggests that phenolic products are unlikely to be involved in the rejection of self pollen. These data are discussed in the perspective of current views of defence systems present in angiosperm epidermal cells, and why these mechanisms fail to identify and reject incompatible pollen tubes.

ENHANCEMENT OF POLLEN GERMINATION BY PROMOTIVE FACTORS IN INCOMPATIBLE POLLEN

Cultivated and wild almonds are self-incompatible and thus require outcrossing by insect pollinators to produce viable seed. In commercial production, considerable efforts are directed towards placement and selection of cultivars for cross-pollination. However, since honeybees do not distinguish between the different cultivars, stigmas are usually covered by a mixture of both compatible and incompatible pollen. Using in vitro assays, we demonstrated that pollen extracts promoted germination in self pollen with no inhibitory effects observed. Elemental analyses of pollen extracts showed that enhanced levels of Ca, Mg, K, Na, and P were eluted from the grains. From this, we raise the question of possible interaction between compatible and incompatible pollen, and speculate that incompatible pollen grains may support and enhance germination of adjacent compatible pollen.

The intracellular events triggered by the self-incompatibility response inPapaver rhoeas

In recent years self-incompatibility (SI) has come to be recognised as an important model system for studying cell-cell interactions and signalling in flowering plants. In this article we discuss the intracellular events associated with the SI response in the field poppy,Papaver rhoeas. The SI response inP. rhoeas is known to involve a Ca2+-based signalling pathway, activated following molecular interactions on the surface of incompatible pollen tubes. Evidence demonstrates that, following a transient increase in the concentration of cytosolic free Ca2+ ([Ca2+];) initiated by the SI response, phosphorylation of certain cytosolic proteins occurs, followed by activation of pollen gene expression. The magnitude of this transient Ca2+ wave and the localisation of cytosolic [Ca2+]i following the SI response are discussed. We also describe the character of the proteins specifically phosphorylated in the SI response and the nature of the protein kinases involved in their phosphorylation. Finally, we consider the possibility that the end result of the SI response inP. rhoeas may be analogous to programmed-cell-death mechanisms such as those seen in developmental processes and defence responses in various plant cells.

Identification of residues in a hydrophilic loop of the Papaver rhoeas S protein that play a crucial role in recognition of incompatible pollen.

The self-incompatibility response involves S allele-specific recognition between stigmatic S proteins and incompatible pollen. This response results in pollen inhibition. Defining the amino acid residues within the stigmatic S proteins that participate in S allele-specific inhibition of incompatible pollen is essential for the elucidation of the molecular basis of the self-incompatibility response. We have constructed mutant derivatives of the S1 protein from Papaver rhoeas by using site-directed mutagenesis and have tested their biological activity. This has enabled us to identify amino acid residues in the stigmatic S proteins of P. rhoeas that are required for S-specific inhibition of incompatible pollen. We report here the identification of several amino acid residues in the predicted hydrophilic loop 6 of the P. rhoeas stigmatic S1 protein that are involved in the inhibition of S1 pollen. Mutation of the only hypervariable amino acid, which is situated in this loop, resulted in the complete loss of ability of the S protein to inhibit S1 pollen. This clearly demonstrates that this residue plays a crucial role in pollen recognition and may also participate in defining allelic specificity. We have also established the importance of highly conserved amino acids adjacent to this hypervariable site. Our studies demonstrate that both variable and conserved amino acids in the region of the S protein corresponding to surface loop 6 are key elements that play a role in the recognition and inhibition of incompatible pollen in the pollen-pistil self-incompatibility reaction.

Identification of Residues in a Hydrophilic Loop of the Papaver rhoeas S Protein That Play a Crucial Role in Recognition of Incompatible Pollen

Identification of Residues in a Hydrophilic Loop of the Papaver rhoeas S Protein That Play a Crucial Role in Recognition of Incompatible Pollen

Fused organs in the adherent1 mutation in maize show altered epidermal walls with no perturbations in tissue identities

In the absence of wounding, the epidermis is only rarely involved in cell or organ fusion events; in fact, intact epidermal layers prevent graft unions. In Zea mays L. the mutation adherent1 (ad1) shows abnormal fusions between cells and organs. Fusions involve epidermal cells of vegetative and floral organs and occur early in the ontogeny of organs. Even so, epidermal cell types differentiate normally in the fused regions and internal tissue identities are maintained. In contrast, the extracellular matrix (cell wall and cuticle) of the epidermal cells is perturbed. Epidermal cell walls in adherent leaves are thicker than normal. Epicuticular wax particles appear reduced in size and number and altered in shape in mutant leaves. In addition, the outer epidermal cell walls of adherent leaves fluoresce when stained with aniline blue, a reagent that binds to callose. Immunolocalizations to specific cell wall epitopes suggest that pectins but not arabinogalactans may have a role in the fusion events. Taken together, these results suggest that the ad1 mutation results in cell-wall and epicuticular-wax defects similar to responses seen in wounding, pollination by incompatible pollen, or pathogen attack. Since cell wall components and epicuticular waxes are extracellular secreted products, the ad1 mutation may disrupt normal functioning and/or composition of the secretory pathway and its cargo.

PCP-A1, a defensin-like Brassica pollen coat protein that binds the S locus glycoprotein, is the product of gametophytic gene expression.

Self-incompatibility (SI) in Brassica species is controlled by a single polymorphic locus (S) with multiple specificities. Two stigmatically expressed genes that have been cloned from this region encode the S locus glycoprotein (SLG) and S receptor kinase (SRK). Both appear to be essential for the operation of SI. It is believed that rejection of incompatible pollen grains is effected by recognition events between an as yet unidentified S locus-encoded pollen coating-borne protein and the SLG/SRK. We previously identified a small pollen coat protein PCP7 (renamed here PCP-A1, for pollen coat protein, class A, 1) that binds with high affinity to SLGs irrespective of S genotype. Here, we report the cloning of PCP-A1 from Brassica oleracea and demonstrate that it is unlinked to the S locus. In situ localization of PCP-A1 transcripts revealed that they accumulate specifically in pollen at the late binucleate/trinucleate stage of development rather than in the tapetum, which previously was taken to be the principal source of the pollen coat. PCP-A1 is characterized by the presence of a structurally important motif consisting of eight cysteine residues shared by the plant defensins. Based on the presence of this motif and other data, homology modeling has been used to produce a putative structure for PCP-A1. Protein-protein interaction analyses demonstrate that SLG exists in monomeric and dimeric forms, both of which bind PCP-A1. Evidence is also presented for the existence of putative membrane-associated PCP-A1 binding proteins in stigmatic tissue.

Ribosomes in incompatible pollen tubes in the Solanaceae

Some members of the Solanaceae have a self‐incompatibility mechanism preventing self‐fertilization. Stylar ribonucleases (S‐RNases) are responsible for growth inhibition of self‐pollen tubes. A prevalent model postulates that the S‐RNases act as intracellular cytotoxins that degrade ribosomal RNA, and possibly also messenger RNA, in the incompatible pollen tubes. Since ribosomes and polysomes are easily noticed with the electron microscope, it should be possible to confirm disintegration of these structures. However, our inspection by electron microscopy revealed the presence of ribosomes and polysomes in pollen tubes formed after self‐pollination of the self‐sterile species Brugmansia (Datura) suaveolens and Nicotiana alata. There was no decrease over time in the number of bound ribosomes per unit of rough endoplasmic reticulum (RER) membrane. The results indicate that the inhibition of tube growth is not due to a general degradation of ribosomal and messenger RNA. Therefore, the substrate for S‐RNases presumably is very specific.

Abscisic acid signaling through cyclic ADP-ribose in plants : Wu Y, Kuzma J, Marechal E, Graeff R, Lee HC, Foster R, Chua N-H: Science 1997, 278:2126–2130

Many angiosperms use specific interactions between pollen and pistil proteins as “self” recognition and/or rejection mechanisms to prevent self-fertilization. Self-incompatibility (SI) is encoded by a multiallelic S locus, comprising pollen and pistil S-determinants [1, 2]. In Papaver rhoeas, cognate pistil and pollen S-determinants, PrpS, a pollen-expressed transmembrane protein, and PrsS, a pistil-expressed secreted protein [3, 4], interact to trigger a Ca2+-dependent signaling network [5, 6, 7, 8, 9, 10], resulting in inhibition of pollen tube growth, cytoskeletal alterations [11, 12, 13], and programmed cell death (PCD) [14, 15] in incompatible pollen. We introduced the PrpS gene into Arabidopsis thaliana, a self-compatible model plant. Exposing transgenic A. thaliana pollen to recombinant Papaver PrsS protein triggered remarkably similar responses to those observed in incompatible Papaver pollen: S-specific inhibition and hallmark features of Papaver SI [11, 12, 13, 14, 15]. Our findings demonstrate that Papaver PrpS is functional in a species with no SI system that diverged ∼140 million years ago [16]. This suggests that the Papaver SI system uses cellular targets that are, perhaps, common to all eudicots and that endogenous signaling components can be recruited to elicit a response that most likely never operated in this species. This will be of interest to biologists interested in the evolution of signaling networks in higher plants.

Patterns and consequences of self‐pollen deposition on stigmas in heterostylous Persicaria japonica (Polygonaceae)

In order to test the "anti-interference" hypothesis for adaptive significance of reciprocal herkogamy, patterns of illegitimate pollination and effects of self-pollen on the legitimate pollination and fertility were investigated in a naturally pollinated experimental population of distylous Persicaria japonica. Pollen deposition was compared among the emasculation treatments, i.e., emasculation of a single flower from individual inflorescences, emasculation of all the flowers of individual inflorescences, and no emasculation control. In both morphs, considerable illegitimate pollination was found to occur in all the treatments, and there was no significant difference in incompatible pollen load among the treatments. Therefore, it is suggested that herkogamy of P. japonica can sufficiently reduce both intraflower and intra-inflorescence self-pollinations, but not interinflorescence geitonogamous and-or interclonal illegitimate pollination. Measurements of pollen load after the repetitive pollen addition showed that space on the stigma surface may not limit the legitimate pollination under natural pollination conditions. Seed sets after legitimate pollination following prior self-pollination did not differ from the controls without self-pollination. Therefore, there is little possibility that incompatible pollen load interferes with either pollination or fertilization by compatible pollen, suggesting that "anti-interference" is unimportant for adaptive significance of reciprocal herkogamy at least in P. japonica.

Self incompatibility and temperature affect pollen tube growth in ‘Sundrop’apricot (Prunus armeniaca L.)

SummaryComparison of fruit set after controlled self and cross pollination in two New Zealand regions confirmed that ‘Sundrop’ apricot was effectively self sterile (fruit set <2%). Fluorescence microscopy showed that tubes from ‘Sundrop’ pollen were self-incompatible and generally aborted in the upper part of the style. The strength of the inhibition reaction varied so that the tubes occasionally reached the ovary but none penetrated ovules. Measurement of tube growth in ‘Sundrop’ styles at 5°, 10°, 15°, 20° and 25°C showed that temperature affected the rate with which self incompatible pollen tubes were inhibited. Self tube penetration was strongest at 10° and 15°C and weaker at higher temperatures. However, fertilization by ‘Sundrop’ pollen did not occur at any temperature and so the need to cross pollinate this cultivar is not an interaction with low temperatures during bloom.

Gametophyte-Sporophyte Interactions in Pollen-Pistil System after Compatible and Incompatible Pollination.

The gametophyte-sporophyte interactions govern the reproductive strategy of higher plants by forming different barriers to self-pollination in pollen-pistil system. Self-incompatibility, barrier was studied in the experimental system including 2 clones of petunia : (a) self-compatible (self-pollination results in adhesion, hydration and germination of pollen grains, growth of pollen tubes and fertilization), (b) self-incompatible (with the S-gene of incompatibility, the expression of which inhibits pollen tube growth in sporophyte style tissues). Thus, our experimental model permitted us to study signal systems capable of transmitting not only positive information favoring the growth of compatible pollen tubes to the ovary, but also a negative information preventing the penetration of incompatible pollen tubes. A concept of multilevel system controlling gametophyte-sporophyte interactions was put forward. Here, we provide evidence for the participation of sporophytic proteins and ethylene in the regulation of male gametophyte growth and development. The protein specificity in pollen-pistil system during floral organogenesis is characterized by common antigens to male and female systems, as well as by pollen-and pistil-specific antigens, which are synthesized at the late stages of flower development (including a unique S-antigen in sporophyte tissue of pistil). The pattern of lectin activity observed in pistil transmitting tissue is related to the lectin molecule functioning in the interactions between the cells of male gametophyte and those of pistil sporophytic tissues. We have shown that two enzymes are related to the operation of the gametophyte-sporophyte interactions : style RNase takes part in the gametophytic selfincompatibility mechanism, whereas cholinesterase is the most likely candidate for the specific recognition and signal transduction in pollen-pistil system. It is demonstrated that the sporophyte stigma tissues are the source of ethylene production during pollen tube growth in the pistil in the case of both compatible and incompatible pollination.

Hormonal Changes after Compatible and Incompatible Pollination in Theobroma cacao L.

In order to characterize the self-incompatibility system in Theobroma cacao, the levels of ethylene, indole-3-acetic acid (IAA), and abscisic acid (ABA) were determined after pollination with compatible and incompatible pollen and in unpollinated flowers. Pollen tube growth rates after incompatible and compatible pollinations were identical, and the majority of the pollen tubes reached the ovules between 12 and 20 hours after pollination. ABA levels rose in incompatibly pollinated flowers, and fell in compatibly pollinated flowers, prior to pollen tube—ovule contact. Ethylene evolution remained stable in compatibly pollinated flowers and rose in incompatibly pollinated flowers. IAA concentrations increased in compatibly pollinated flowers, and remained stable in incompatibly pollinated flowers after pollination and subsequent to pollen tube—ovule contact.