Sites of self-pollen tube inhibition in Papaveraceae (sensu lato)

Papaver rhoeas (Papaveraceae) has a well-characterized gametophytic self-incompatibility system in which self-pollen tube growth ceases either just before, or just after, emergence from the copal aperture. Papaver flowers are unusual, however, in having flat stigmatic rays sitting directly on top of the hard ovary and no style. Immediate self-pollen arrest might be required with this floral architecture. There is much variation in floral architecture among Papaveraceae and self-incompatibility is widespread. However, there are no reports of the site of self-pollen tube inhibition in Papaveraceae other than P. rhoeas. We examined the site of self-pollen tube inhibition in four species (Argemone munita, Lamprocapnos spectabilis, Eschscholzia californica, and Platystemon californicus) representing a broad phylogenetic and morphological sample of Papaveraceae. Squash preparation was used for species with soft stigmas whereas woody tissue was sectioned with a cryostat and images were stitched into a mosaic to visualize pollen tubes on whole stigmas. For three species, self-pollen tube inhibition appeared similar to that described for P. rhoeas. Self-pollen tubes were arrested before any substantial penetration of female tissue and usually did not grow longer than 100?μm. In the fourth species, A. munita, self-pollen tubes grew up to 500?μm in length. However, self-pollen tubes appeared to grow along the stigmatic spines, and growth ceased once tubes contacted the stigma surface. Despite variation in floral architecture, rapid arrest of self-pollen tubes occurred before or just after penetration of female tissue in all species, consistent with the hypothesis that members of the family share the same incompatibility mechanism.     

Self-incompatibility in Papaver: A MAP kinase signals to trigger programmed cell death

Self-incompatibility (SI) in higher plants prevents inbreeding through specific recognition and rejection of incompatible (“self”) pollen. In Papaver rhoeas, S proteins encoded by the pistil component of the S-locus interact with incompatible pollen, triggering a Ca²⁺-dependent signaling network resulting in programmed cell death (PCD). We recently showed that a mitogen-activated protein kinase (MAPK) is involved in loss of pollen viability, stimulation of caspase-3-like (DEVDase) activity and later DNA fragmentation in incompatible pollen. As p56 appears to be the only MAPK activated by SI, our data suggest that p56 could be the MAPK responsible for mediating SI-induced PCD.Key words: MAPK, self-incompatibility, PCD, caspase-3-like activity, Papaver rhoeas     

Proteins implicated in mediating self-incompatibility-induced alterations to the actin cytoskeleton of Papaver pollen

Background and Aims

Sexual reproduction in angiosperms involves a network of signalling and interactions between pollen and pistil. To promote out-breeding, an additional layer of interactions, involving self-incompatibility (SI), is used to prevent self-fertilization. SI is generally controlled by the S-locus, and comprises allelic pollen and pistil S-determinants. This provides the basis of recognition, and consequent rejection, of incompatible pollen. In Papaver rhoeas, SI involves interaction of pistil PrsS and pollen PrpS, triggering a Ca²⁺-dependent signalling network. This results in rapid and distinctive alterations to both the actin and microtubule cytoskeleton being triggered in ‘self’ pollen. Some of these alterations are implicated in mediating programmed cell death, involving activation of several caspase-like proteases.

Scope

Here we review and discuss our current understanding of the cytoskeletal alterations induced in incompatible pollen during SI and their relationship with programmed cell death. We focus on data relating to the formation of F-actin punctate foci, which have, to date, not been well characterized. The identification of two actin-binding proteins that interact with these structures are reviewed. Using an approach that enriched for F-actin from SI-induced pollen tubes using affinity purification followed by mass spectrometry, further proteins were identified as putative interactors with the F-actin foci in an SI situation.

Key Results

Previously two important actin-binding proteins, CAP and ADF, had been identified whose localization altered with SI, both showing co-localization with the F-actin punctate foci based on immunolocalization studies. Further analysis has identified differences between proteins associated with F-actin from SI-induced pollen samples and those associated with F-actin in untreated pollen. This provides candidate proteins implicated in either the formation or stabilization of the punctate actin structures formed during SI.

Conclusions

This review brings together for the first time, our current understanding of proteins and events involved in SI-induced signalling to the actin cytoskeleton in incompatible Papaver pollen.     

Identification and cloning of related self-incompatibility S-genes in Papaver rhoeas and Papaver nudicaule

 The primary goal of this study was to identify, clone and analyse new S-gene sequences in order to provide a basis for identifying amino acid residues that confer S-allele specificity. Three new putative S-alleles from Papaver rhoeas and Papaver nudicaule were identified using immunological and PCR methods. cDNAs encoding full-length open reading frames of the P. rhoeas S ₈ and P. nudicaule Sn ₁ genes were isolated. Nucleotide sequencing of these cDNAs, together with the partial S ₇ sequence obtained by PCR, was used to derive the corresponding amino acid sequences. It is of interest that the P. nudicaule Sn₁ sequence, which is the first S-allele isolated from another species of Papaver, shares a closer sequence identity to the P. rhoeas S₃ amino acid sequence than S₃ does to S₁ from P. rhoeas. The identity of the S₈ allele was confirmed by expressing the coding region in Escherichia coli and demonstrating that the recombinant protein, designated S₈e, specifically inhibited S ₈ pollen in an in vitro bioassay. Information from sequence analysis of the S₈, Sn₁ and partial S₇ amino acid sequences revealed important information about Papaver S-proteins. It confirmed previous observations based on only two S-alleles, that whilst exhibiting a high degree of amino acid sequence polymorphism ranging from 51.3% to 63.7%, these molecules probably share very similar secondary structures. These studies also revealed that, in contrast to the S-proteins from the Solanaceae and Brassica, amino acid sequence variation is not found in hypervariable blocks, but instead, is found throughout the S-proteins, interspersed with numerous short strictly conserved segments. Received: 16 March 1998 / Revision accepted: 19 May 1998     

3属4种罂粟科植物幼苗初生维管系统的结构

罂粟科属间演化关系一直存在争议。本文以罂粟科罂粟属的虞美人（Papaver rhoeas）和东方罂粟（Papaver orientale）、白屈菜属的白屈菜（Chelidonium majus）以及紫堇属的地丁草（Corydalis bungeana）为材料,应用整体透明和石蜡连续切片的方法,在明场显微镜和偏光显微镜下对其幼苗期的结构及发育进行了观察。结果表明,3属植物幼苗子叶节区下部存在的单中柱类型各异：罂粟属为外始式二原型圆形单中柱,白屈菜属为外始式二原型近圆形单中柱,紫堇属为外始式二原型“S”形单中柱。3属植物的子叶脉序也存在区别：虞美人和东方罂粟具单一叶脉,无叶隙;白屈菜为环结曲行羽状脉;地丁草为基出完全顶聚脉。根据上述幼苗初生维管系统的结构,认为3属中罂粟属较为原始,白屈菜属较为进化,紫堇属更为进化。     

Self-sterility in flowering plants: preventing self-fertilization increases family diversification rates

Background and Scope

New data are presented on the distribution and frequency of self-sterility (SS) – predominantly pre-zygotic self-incompatibility (SI) systems – in flowering plants and the hypothesis is tested that families with self-sterile taxa have higher net diversification rates (DRs) than those with exclusively self-compatible taxa using both absolute and relative rate tests.

Key Results

Three major forms of SI systems (where pollen is rejected at the stigmatic, stylar or ovarian interface) are found to occur in the oldest families of flowering plants, with times of divergence >100 million years before the present (mybp), while post-fertilization SS and heterostyly appear in families with crown ages of 81 and 87 mybp, respectively. It is also founnd that many (22) angiosperm families exhibit >1 SI phenotype and that the distribution of different types of SS does not show strong phylogenetic clustering, collectively suggesting that SS and SI systems have evolved repeatedly de novo in angiosperm history. Families bearing self-sterile taxa have higher absolute DRs using all available calibrations of the angiosperm tree, and this affect is caused mostly by the high DR of families with homomorphic SI systems (in particular stigmatic SI) or those in which multiple SS/SI phenotypes have been observed (polymorphic). Lastly, using sister comparisons, it is further demonstrated that in 29 of 38 sister pairs (including 95 families), the self-sterile sister group had higher species richness and DR than its self-compatible sister based on either the total number of taxa in the clade with SS or only the estimated fraction to harbour SS based on literature surveys.

Conclusions

Collectively, these analyses point to the importance of SS, particularly pre-zygotic SI in the evolution of flowering plants.     

S-RNase-based self-incompatibility in Petunia inflata

Background

For the Solanaceae-type self-incompatibility, also possessed by Rosaceae and Plantaginaceae, the specificity of self/non-self interactions between pollen and pistil is controlled by two polymorphic genes at the S-locus: the S-locus F-box gene (SLF or SFB) controls pollen specificity and the S-RNase gene controls pistil specificity.

Scope

This review focuses on the work from the authors'' laboratory using Petunia inflata (Solanaceae) as a model. Here, recent results on the identification and functional studies of S-RNase and SLF are summarized and a protein-degradation model is proposed to explain the biochemical mechanism for specific rejection of self-pollen tubes by the pistil.

Conclusions

The protein-degradation model invokes specific degradation of non-self S-RNases in the pollen tube mediated by an SLF, and can explain compatible versus incompatible pollination and the phenomenon of competitive interaction, where SI breaks down in pollen carrying two different S-alleles. In Solanaceae, Plantaginaceae and subfamily Maloideae of Rosaceae, there also exist multiple S-locus-linked SLF/SFB-like genes that potentially function as the pollen S-gene. To date, only three such genes, all in P. inflata, have been examined, and they do not function as the pollen S-gene in the S-genotype backgrounds tested. Interestingly, subfamily Prunoideae of Rosaceae appears to possess only a single SLF/SFB gene, and competitive interaction, observed in Solanaceae, Plantaginaceae and subfamily Maloideae, has not been observed. Thus, although the cytotoxic function of S-RNase is an integral part of SI in Solanaceae, Plantaginaceae and Rosaceae, the function of SLF/SFB may have diverged. This highlights the complexity of the S-RNase-based SI mechanism. The review concludes by discussing some key experiments that will further advance our understanding of this self/non-self discrimination mechanism.     

Effect of variation in self-incompatibility on pollen limitation and inbreeding depression in Flourensia cernua (Asteraceae) scrubs of contrasting density

Background and Aims

Selection may favour a partial or complete loss of self-incompatibility (SI) if it increases the reproductive output of individuals in the presence of low mate availability. The reproductive output of individuals varying in their strength of SI may also be affected by population density via its affect on the spatial structuring and number of S-alleles in populations. Modifiers increasing levels of self-compatibility can be selected when self-compatible individuals receive reproductive compensation by, for example, increasing seed set and/or when they become associated with high fitness genotypes.

Methods

The effect of variation in the strength of SI and scrub density (low versus high) on seed set, seed germination and inbreeding depression in seed germination (δ_germ) was investigated in the partially self-incompatible species Flourensia cernua by analysing data from self-, cross- and open-pollinated florets.

Key Results

Examination of 100 plants in both high and low scrub densities revealed that 51% of plants were strongly self-incompatible and 49 % varied from being self-incompatible to self-compatible. Seed set after hand cross-pollination was higher than after open-pollination for self-incompatible, partially self-incompatible and self-compatible plants but was uniformly low for strongly self-incompatible plants. Strongly self-incompatible and self-incompatible plants exhibited lower seed set, seed germination and multiplicative female fitness (floral display × seed set × seed germination) in open-pollinated florets compared with partially self-incompatible and self-compatible plants. Scrub density also had an effect on seed set and inbreeding depression: in low-density scrubs seed set was higher after open-pollination and δ_germ was lower.

Conclusions

These data suggest that (a) plants suffered outcross pollen limitation, (b) female fitness in partially self-incompatible and self-compatible plants is enhanced by increased mate-compatibility and (c) plants in low-density scrubs received higher quality pollen via open-pollination than plants in high-density scrubs.Key words: Flourensia cernua, population density, seed set, seed germination, female fitness, partial self-incompatibility, Mapimí Biosphere Reserve     

Adlumidiceine and adlumiceine: New alkaloids of narceine type

The structures of new alkaloids of narceine type, adlumidiceine (II), adlumiceine (III), and enol lactone of adlumidiceine (IV), isolated from Papaver rhoeas L. and Corydalis sempervirens (L.) Pers. (Papaveraceae), were determined by PMR, MS and IR spectra.     

Origin,loss, and regain of self-incompatibility in angiosperms

The self-incompatibility (SI) system with the broadest taxonomic distribution in angiosperms is based on multiple S-locus F-box genes (SLFs) tightly linked to an S-RNase termed type-1. Multiple SLFs collaborate to detoxify nonself S-RNases while being unable to detoxify self S-RNases. However, it is unclear how such a system evolved, because in an ancestral system with a single SLF, many nonself S-RNases would not be detoxified, giving low cross-fertilization rates. In addition, how the system has been maintained in the face of whole-genome duplications (WGDs) or lost in other lineages remains unclear. Here we show that SLFs from a broad range of species can detoxify S-RNases from Petunia with a high detoxification probability, suggestive of an ancestral feature enabling cross-fertilization and subsequently modified as additional SLFs evolved. We further show, based on its genomic signatures, that type-1 was likely maintained in many lineages, despite WGD, through deletion of duplicate S-loci. In other lineages, SI was lost either through S-locus deletions or by retaining duplications. Two deletion lineages regained SI through type-2 (Brassicaceae) or type-4 (Primulaceae), and one duplication lineage through type-3 (Papaveraceae) mechanisms. Thus, our results reveal a highly dynamic process behind the origin, maintenance, loss, and regain of SI.

Genetic, functional, phylogenomic, and ancestral state reconstruction studies reveal a highly dynamic process behind the origin, maintenance, loss, and regain of self-incompatibility in angiosperms.     

The effect of sheltered load on reproduction in Solanum carolinense, a species with variable self-incompatibility

In previous studies, we have investigated the strength of self-incompatibility (SI) in Solanum carolinense, a highly successful weed with a fully functional SI system that inhabits early successional and other disturbed habitats. We have found that the SI response in S. carolinense is a plastic trait—its strength being affected by the age of the flowers, and the presence of developing fruits and that there are genetic differences among families in their self-fertility. However, in species with a fully functional SI response, selfing would not be that common. As a result, deleterious recessives scattered though the genome of horsenettle are only occasionally exposed to selection. It has been suggested that deleterious recessives accumulate near S-alleles in strong SI species because the S-locus is located in a non-recombining region of the genome and because strong S-alleles are never in the homozygous state, thus sheltering some of the genetic load near the S-locus from selection. We performed a series of laboratory and greenhouse experiments to determine the extent to which sheltered load adds to the overall magnitude of inbreeding depression in horsenettle. Specifically, we amplified and sequenced the S-alleles from 16 genets collected from a large population in Pennsylvania and performed a series of controlled self-pollinations. We then grew the selfed progeny in the greenhouse; recorded various measures of growth and reproductive output; and amplified and sequenced their S-allele(s). We found that the heterozygous progeny of self-pollinations produce more flowers and have a greater ability to set both self and cross seed than S-homozygous progeny. We also found evidence of variation in the magnitude of load among S-alleles. These results suggest that sheltered load might slow the fixation of weak (partially compatible) S-alleles in this population, thus adding to the maintenance of a mixed mating system rather than leading to the fixation of the selfing alleles.     

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

Summary 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.     

Meconic acid as a chemotaxonomic marker in the papaveraceae

Of 48 species of Papaveraceae examined representing 12 genera, 24 were shown to contain meconic acid and of these, 21 were Papaver species. Meconopsis cambrica was also found to contain meconic acid, in contrast to other species of Meconopsis examined. One recent rearrangement of the Papaveraceae includes a grouping of Papaver, Roemeria and Stylomecon together with M. cambrica and our further discovery of meconic acid in two Roemaria species examined leads to the conclusion that meconic acid may be a distinctive taxonomic feature of this new grouping. Some attempt has also been made to correlate meconic acid occurrence with that of certain types of alkaloids, and of chelidonic acid.     

Breeding systems,hybridization and continuing evolution in Avon Gorge Sorbus

Background and Aims

Interspecific hybridization and polyploidy are key processes in plant evolution and are responsible for ongoing genetic diversification in the genus Sorbus (Rosaceae). The Avon Gorge, Bristol, UK, is a world ‘hotspot’ for Sorbus diversity and home to diploid sexual species and polyploid apomictic species. This research investigated how mating system variation, hybridization and polyploidy interact to generate this biological diversity.

Methods

Mating systems of diploid, triploid and tetraploid Sorbus taxa were analysed using pollen tube growth and seed set assays from controlled pollinations, and parent–offspring genotyping of progeny from open and manual pollinations.

Key Results

Diploid Sorbus are outcrossing and self-incompatible (SI). Triploid taxa are pseudogamous apomicts and genetically invariable, but because they also display self-incompatibility, apomictic seed set requires pollen from other Sorbus taxa – a phenomenon which offers direct opportunities for hybridization. In contrast tetraploid taxa are pseudogamous but self-compatible, so do not have the same obligate requirement for intertaxon pollination.

Conclusions

The mating inter-relationships among Avon Gorge Sorbus taxa are complex and are the driving force for hybridization and ongoing genetic diversification. In particular, the presence of self-incompatibility in triploid pseudogamous apomicts imposes a requirement for interspecific cross-pollination, thereby facilitating continuing diversification and evolution through rare sexual hybridization events. This is the first report of naturally occurring pseudogamous apomictic SI plant populations, and we suggest that interspecific pollination, in combination with a relaxed endosperm balance requirement, is the most likely route to the persistence of these populations. We propose that Avon Gorge Sorbus represents a model system for studying the establishment and persistence of SI apomicts in natural populations.     

Pollination and late-acting self-incompatibility in Cyrtanthus breviflorus (Amaryllidaceae): implications for seed production

Background and Aims

Animal pollination is typically an uncertain process that interacts with self-incompatibility status to determine reproductive success. Seed set is often pollen-limited, but species with late-acting self-incompatibility (SI) may be particularly vulnerable, if self-pollen deposition results in ovule discounting. Pollination is examined and the occurrence of late-acting SI and ovule discounting assessed in Cyrtanthus breviflorus.

Methods

The pollination system was characterized by observing floral visitors and assessing nectar production and spectral reflectance of flowers. To assess late-acting SI and ovule discounting, growth of self- and cross-pollen tubes, and seed set following open pollination or hand pollination with varying proportions of self- and cross-pollen, were examined.

Key Results

Native honeybees Apis mellifera scutellata pollinated flowers as they actively collected pollen. Most flowers (≥70 %) did not contain nectar, while the rest produced minute volumes of dilute nectar. The flowers which are yellow to humans are visually conspicuous to bees with a strong contrast between UV-reflecting tepals and UV-absorbing anthers and pollen. Plants were self-incompatible, but self-rejection was late-acting and both self- and cross-pollen tubes penetrated ovules. Seed set of open-pollinated flowers was pollen-limited, despite pollen deposition exceeding ovule number by 6-fold. Open-pollinated seed set was similar to that of the cross + self-pollen treatment, but was less than that of the cross-pollen-only treatment.

Conclusions

Flowers of C. breviflorus are pollinated primarily by pollen-collecting bees and possess a late-acting SI system, previously unknown in this clade of the Amaryllidaceae. Pollinators of C. breviflorus deposit mixtures of cross- and self-pollen and, because SI is late-acting, self-pollen disables ovules, reducing female fertility. This study thus contributes to growing evidence that seed production in plants with late-acting SI systems is frequently limited by pollen quality, even when pollinators are abundant.     

Cloning and sequencing of cDNAs encoding two self-incompatibility associated proteins in Solanum chacoense

Summary We have isolated and sequenced cDNAs for S₂- and S₃-alleles of the self-incompatibility locus (S-locus) in Solanum chacoense Bitt., a wild potato species displaying gametophytic self-incompatibility. The S₂-and S₃-alleles encode pistil-specific proteins of 30 kDa and 31 kDa, respectively, which were previously identified based on cosegregation with their respective alleles in genetic crosses. The amino acid sequence homology between the S₂- and S₃-proteins is 41.5%. This high degree of sequence variability between alleles is a distinctive feature of the S-gene system. Of the 31 amino acid residues which were previously found to be conserved among three Nicotiana alata S-proteins (S₂, S₃, and S₆) and two fungal ribonucleases (R Nase T₂ and R Nase Rh), 27 are also conserved in the S₂- and S₃-proteins of S. chacoense. These residues include two histidines implicated in the active site of the R Nase T₂, six cysteines, four of which form disulfide bonds in R Nase T₂, and hydrophobic residues which might form the core structure of the protein. The finding that these residues are conserved among S-proteins with very divergent sequences suggests a functional role for the ribonuclease activity of the S-protein in gametophytic self-incompatibility.     

Characterization of a legumain/vacuolar processing enzyme and YVADase activity in Papaver pollen

Legumains, also known as Vacuolar Processing Enzymes (VPEs) have received considerable attention recently, as they share structural properties with mammalian caspase-1 and exhibit YVADase/caspase-1-like cleavage activity. Although many legumains have been cloned, knowledge about their detailed characteristics and intracellular localization is relatively limited. We previously identified several caspase-like activities activated by self-incompatibility (SI) in pollen; a DEVDase was required for programmed cell death (PCD), but YVADase was not (Bosch and Franklin-Tong in Proc Natl Acad Sci USA 104:18327–18332, 2007; Thomas and Franklin-Tong in Nature 429:305–309, 2004). Here we report identification of a legumain/VPE from Papaver rhoeas pollen (PrVPE1) that binds to the DEVD tetrapeptide, a signature substrate for caspase-3. A detailed characterization of the recombinant PrVPE1 cleavage activity revealed that, like other VPEs, it has YVADase activity and requires an acidic pH for activity. Unlike other legumain/VPEs, it also exhibits DEVDase and IETDase activities and apparently does not require processing for activity. The pollen-expressed PrVPE1 localizes to a reticulate compartment resembling the vacuole. Examination of YVADase activity using live-cell imaging of pollen tubes revealed YVADase activity in mitochondria of growing pollen tubes. The unexpected features of PrVPE1, together with evidence for YVADase activity in plant mitochondria, indicate that VPEs, YVADases, their localization and functions in plant cells merit further investigation.