Effects of Inhibitors of Protein Serine/Threonine Phosphatases on Pollination in Brassica

We have examined the effect of the protein phosphatase inhibitors okadaic acid and microcystin on pollen-pistil interactions in Brassica. Inhibitor-treated flowers or floral buds were pollinated with untreated pollen and examined for pollen tube growth by fluorescence microscopy. Our results show that type 1 or type 2A serine/threonine phosphatases play a crucial role in the pollination responses of Brassica. We observed two distinct effects of protein phosphatase inhibitors on pollination: (a) the inhibition of pollen tube growth during cross-pollination in flowers, and (b) the break-down of self-incompatibility or promotion of pollen tube growth during self-pollination in flower buds just prior to anthesis. Thus, treatment of flower pistils with protein phosphatase inhibitors resulted in the inhibition of pollen tube growth at the surface of the papillar cells of the stigma in crosses between different self-incompatible Brassica oleracea strains, in an interspecific cross between B. oleracea and Brassica campestris, and in self-pollinations of a self-fertile Brassica napus cultivar. With four different self-incompatibility genotypes, treatment of mature flowers with protein phosphatase inhibitors had no effect on self-pollination response. In contrast, treatment of flower buds just prior to the anthesis stage allowed self-pollen tube invasion of papillar cells. However, the magnitude of this effect was genotype dependent, being most pronounced in the S22 genotype. The data support the conclusion that pollinations in Brassica are controlled in part by the presence of phosphorylated proteins in the papillar cells of the stigma, and that the quantity of these proteins or their levels of phosphorylation changes during stigma development.     

Neither compatible nor self-incompatible pollinations of Brassica napus involve reorganization of the papillar cytoskeleton

Compatible pollination of Brassica napus necessitates pollen hydration, pollen germination and growth of the pollen tube through the loosened walls of stigmatic papillar cells, whereas self-incompatible (SI) pollinations fail at one of these stages. Analyses of the early stages of pollination show that at high (but not low) relative humidities, both compatible and SI pollen hydrates, but SI germination is reduced and the rare pollen tubes generally fail to penetrate the papillar walls, although there is some wall loosening. Inside the papillae, both compatible and SI interactions may induce the formation of callose, but there is no evidence for a major accumulation of cytoplasm or secretory vesicles in the vicinity of the pollen tubes and neither microtubule nor F-actin patterns re-arrange in this zone. These observations indicate that the source of the wall-loosening enzymes is probably the pollen tube or pollen coat, and that the common cellular responses of plants to attempted invasions have become suppressed in the papilla–pollen tube interaction.     

Pollen-stigma adhesion in Arabidopsis: a species-specific interaction mediated by lipophilic molecules in the pollen exine.

To investigate the nature and role of cell adhesion in plants, we analyzed the initial step of pollination in Arabidopsis: the binding of pollen grains to female stigma cells. Here we show this interaction occurs within seconds of pollination. Because it takes place prior to pollen hydration, it also requires adhesion molecules that can act in a virtually dry environment. We developed assays that monitored adhesion of populations of pollen grains and individual cells. Adhesion between pollen and stigma cells is highly selective - Arabidopsis pollen binds with high affinity to Arabidopsis stigmas, while pollen from other species fails to adhere. Initial binding is independent of the extracellular pollen coat (tryphine), indicating that adhesion molecules reside elsewhere on the pollen surface, most likely within the exine walls. Immediately after pollination, the stigma surface becomes altered at the interface, acquiring a pattern that interlocks with the exine; this pattern is evident only with pollen from Arabidopsis and its close relatives. Purified exine fragments bind to stigma cells, and biochemical analyses indicate that this specific, rapid and anhydrous adhesion event is mediated by lipophilic interactions.     

Genetic Ablation of Floral Cells in Arabidopsis

A chimeric toxic gene consisting of the diphtheria toxin A chain gene fused to a promoter previously shown to direct pistil- and anther-specific expression was used to genetically target cell killing in transgenic Arabidopsis. Flowers of Arabidopsis transformants that carried the toxic gene fusion had distinct structural defects. The papillar cells at the stigma surface were stunted and were biosynthetically inactive. Anther development was also impaired by toxic gene expression, leading to abnormalities in anther dehiscence, pollen morphology, and pollen germination. The combined defects of pistil and anther rendered transformants that carried the toxic gene fusion self-sterile. However, the transformants were cross-fertile with untransformed plants: the viable pollen of ablated plants was rescued by wild-type stigmas, and, strikingly, the ablated papillar cells allowed the growth of wild-type pollen.     

Peroxynitrite mediates programmed cell death both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.)

Programmed cell death (PCD) has been found to be induced after pollination both in papillar cells and in self-incompatible pollen in the olive (Olea europaea L.). Reactive oxygen species (ROS) and nitric oxide (NO) are known to be produced in the pistil and pollen during pollination but their contribution to PCD has so far remained elusive. The possible role of ROS and NO was investigated in olive pollen-pistil interaction during free and controlled pollination and it was found that bidirectional interaction appears to exist between the pollen and the stigma, which seems to regulate ROS and NO production. Biochemical evidence strongly suggesting that both O(2)(-) and NO are essential for triggering PCD in self-incompatibility processes was also obtained. It was observed for the first time that peroxynitrite, a powerful oxidizing and nitrating agent generated during a rapid reaction between O(2)(-) and NO, is produced during pollination and that this is related to an increase in protein nitration which, in turn, is strongly associated with PCD. It may be concluded that peroxynitrite mediates PCD during pollen-pistil interaction in Olea europaea L. both in self-incompatible pollen and papillar cells.     

Plantacyanin plays a role in reproduction in Arabidopsis

Plantacyanins belong to the phytocyanin family of blue copper proteins. In the Arabidopsis (Arabidopsis thaliana) genome, only one gene encodes plantacyanin. The T-DNA-tagged mutant is a knockdown mutant that shows no visible phenotype. We used both promoter-beta-glucuronidase transgenic plants and immunolocalization to show that Arabidopsis plantacyanin is expressed most highly in the inflorescence and, specifically, in the transmitting tract of the pistil. Protein levels show a steep gradient in expression from the stigma into the style and ovary. Overexpression plants were generated using cauliflower mosaic virus 35S, and protein levels in the pistil were examined as well as the pollination process. Seed set in these plants is highly reduced mainly due to a lack of anther dehiscence, which is caused by degeneration of the endothecium. Callose deposits occur on the pollen walls in plants that overexpress plantacyanin, and a small percentage of these pollen grains germinate in the closed anthers. When wild-type pollen was used on the overexpression stigma, seed set was still decreased compared to the control pollinations. We detected an increase in plantacyanin levels in the overexpression pistil, including the transmitting tract. Guidance of the wild-type pollen tube on the overexpression stigma is disrupted as evidenced by the growth behavior of pollen tubes after they penetrate the papillar cell. Normally, pollen tubes travel down the papilla cell and into the style. Wild-type pollen tubes on the overexpression stigma made numerous turns around the papilla cell before growing toward the style. In some rare cases, pollen tubes circled up the papilla cell away from the style and were arrested there. We propose that when plantacyanin levels in the stigma are increased, pollen tube guidance into the style is disrupted.     

High humidity partially rescues the Arabidopsis thaliana exo70A1 stigmatic defect for accepting compatible pollen

We have previously proposed that Exo70A1 is required in the Brassicaceae stigma to control the early stages of pollen hydration and pollen tube penetration through the stigmatic surface, following compatible pollination. However, recent work has raised questions regarding Arabidopsis thaliana Exo70A1’s expression in the stigma and its role in stigma receptivity to compatible pollen. Here, we verified the expression of Exo70A1 in stigmas from three Brassicaceae species and carefully re-examined Exo70A1’s function in the stigmatic papillae. With previous studies showing that high relative humidity can rescue some pollination defects, essentially bypassing the control of pollen hydration by the Brassicaceae dry stigma, the effect of high humidity was investigated on pollinations with the Arabidopsis exo70A1-1 mutant. Pollinations under low relative humidity resulted in a complete failure of wild-type compatible pollen acceptance by the exo70A1-1 mutant stigma as we had previously seen. However, high relative humidity resulted in a partial rescue of the exo70A1-1 stigmatic papillar defect resulting is some wild-type compatible pollen acceptance and seed set. Thus, these results reaffirmed Exo70A1’s proposed role in the stigma regulating compatible pollen hydration and pollen tube entry and demonstrate that high relative humidity can partially bypass these functions.     

The S-locus specific glycoproteins of Brassica accumulate in the cell wall of developing stigma papillae

Self-incompatibility in Brassica oleracea is now viewed as a cellular interaction between pollen and the papillar cells of the stigma surface. In this species, the inhibition of self-pollen occurs at the stigma surface under the influence of S-locus specific glycoproteins (SLSG). We used antibodies specific for a protein epitope of SLSG to study the subcellular distribution of these molecules in the stigmatic papillae. The antibodies have uncovered an interesting epitope polymorphism in SLSG encoded by subsets of S-alleles, thus providing us with useful genetic controls to directly verify the specificity of the immunolocalization data. Examination of thin sections of Brassica stigmas following indirect immunogold labeling showed that SLSG accumulate in the papillar cell wall, at the site where inhibition of self-pollen tube development has been shown to occur. In addition, the absence of gold particles over the papillar cell walls in the immature stigmas of very young buds, and the intense labeling of these walls in the stigmas of mature buds and open flowers, correlates well with the acquisition of the self-incompatibility response by the developing stigma.     

Loss of callose in the stigma papillae does not affect the Brassica self-incompatibility phenotype

As part of the Brassicaceae self-incompatibility response, callose is deposited in the stigma papillar cells. To determine if callose plays an important role in the rejection of incompatible pollen by the stigma, transgenic Brassica napus. L. plants were produced which express the tobacco β-1,3-glucanase cDNA (the enzyme which degrades callose) in the stigma papillae. Using aniline blue fluorescence, little or no callose was detected in the papillar cells of transgenic stigmas. However, the self-incompatibility system appeared to be unaffected based on the lack of pollen tube growth and the subsequent lack of seed set. The transgene had no effect on compatible pollinations. Thus, while callose deposition is associated with the B. napus self-incompatibility response, it is not required for the rejection of incompatible pollen. Received: 14 March 1997 / Accepted: 15 April 1997     

Transgenic GFP as a molecular marker for approaches to quantify pollination mechanism and gene flow in Arabidopsis thaliana

Arabidopsis thaliana is commonly regarded as a self-pollinated plant. We observed that the stigma in each flower of A. thaliana cannot be pollinated by its own pollen in the early phases of the flowering process, when the anthers had dehisced but the filaments were still too short for the pollen to be deposited on the stigma. In the later stages, after elongation of the filaments, self-pollination can occur. After artificial pollination of the flower of a wild plant with GFP transgenic pollen grains in earlier stages of flowering, GFP expressed within epidermal cells was detected in some of the offspring (26.1-57.1 %). Wind-mediated pollen dispersal was poor but is likely to exist in natural habitats, while insects were observed visiting flowers of A. thaliana in natural and experimental populations. We constructed an experimental population consisting of 28 GFP transgenic plants and 240 wild plants and examined gene flow in the population. The result was that the distance of gene flow was limited to 0.5 m. 22 offspring with expressed GFP were found in 28,299 filial individuals examined, which suggested a relatively low outcrossing rate (0.74%). We conclude that outcrossing in populations of A. thaliana is mainly due to insect pollination. The data on gene flow could be useful to assess the ecological hazards of experimental transgene combinations.     

Secretory Activity Is Rapidly Induced in Stigmatic Papillae by Compatible Pollen,but Inhibited for Self-Incompatible Pollen in the Brassicaceae

[In the Brassicaceae, targeted exocytosis to the stigmatic papillar plasma membrane under the compatible pollen grain is hypothesized to be essential for pollen hydration and pollen tube penetration. In contrast, polarized secretion is proposed to be inhibited in the stigmatic papillae during the rejection of self-incompatible pollen. Using transmission electron microscopy (TEM), we performed a detailed time-course of post-pollination events to view the cytological responses of the stigmatic papillae to compatible and self-incompatible pollinations. For compatible pollinations in Arabidopsis thaliana and Arabidopsis lyrata, vesicle secretion was observed at the stigmatic papillar plasma membrane under the pollen grain while Brassica napus stigmatic papillae appeared to use multivesicular bodies (MVBs) for secretion. Exo70A1, a component of the exocyst complex, has been previously implicated in the compatible pollen responses, and disruption of Exo70A1 in both A. thaliana and B. napus resulted in a loss of secretory vesicles/MVBs at the stigmatic papillar plasma membrane. Similarly, for self-incompatible pollinations, secretory vesicles/MVBs were absent from the stigmatic papillar plasma membrane in A. lyrata and B. napus; and furthermore, autophagy appeared to be induced to direct vesicles/MVBs to the vacuole for degradation. Thus, these findings support a model where the basal pollen recognition pathway in the stigmatic papilla promotes exocytosis to accept compatible pollen, and the basal pollen recognition pathway is overridden by the self-incompatibility pathway to prevent exocytosis and reject self-pollen.     

Interrelationships between Cytoplasmic Ca2+ Peaks, Pollen Hydration and Plasma Membrane Conductances during Compatible and Incompatible Pollinations of Brassica napus Papillae

We have investigated Ca²⁺-involving cell signaling, plasma membranepotentials and conductances and callose formation during earlystages of pollination of papillae of Brassica napus. Using fluorescenceimaging of calcium green-1, we found that application of a rangeof pollen types and controls all rapidly produced small localizedpeaks in papillar cytoplasmic [Ca²⁺]. This response was morefrequent in compatible than incompatible interactions and wascorrelated with subsequent hydration of the applied pollen grains,indicating that it may be a differential prerequisite of thecompatible signaling pathway leading to successful pollinations.In contrast, a slight trend to increased plasma membrane conductance(but with no indications of action potential-like responses)and also callose deposition in papillae adjacent to pollen grainsfollowed pollination in both SC and SI interactions, indicatingthat alterations in plasma membrane permeability and callosedeposition during early phases of pollination are not primarydeterminants of the fate of attempted pollinations. ²On leave of absence from A.N. Bach Institute of Biochemistry,Russian Academy of Sciences, Leninsky Pr., 33, Moscow, Russia,117071     

The Impact of the Invasive Alien Plant,Impatiens glandulifera,on Pollen Transfer Networks

Biological invasions are a threat to the maintenance of ecological processes, including pollination. Plant-flower visitor networks are traditionally used as a surrogated for pollination at the community level, despite they do not represent the pollination process, which takes place at the stigma of plants where pollen grains are deposited. Here we investigated whether the invasion of the alien plant Impatiens glandulifera (Balsaminaceae) affects pollen transfer at the community level. We asked whether more alien pollen is deposited on the stigmas of plants on invaded sites, whether deposition is affected by stigma type (dry, semidry and wet) and whether the invasion of I. glandulifera changes the structure of the resulting pollen transfer networks. We sampled stigmas of plants on 10 sites invaded by I. glandulifera (hereafter, balsam) and 10 non-invaded control sites. All 20 networks had interactions with balsam pollen, although significantly more balsam pollen was found on plants with dry stigmas in invaded areas. Balsam pollen deposition was restricted to a small subset of plant species, which is surprising because pollinators are known to carry high loads of balsam pollen. Balsam invasion did not affect the loading of native pollen, nor did it affect pollen transfer network properties; networks were modular and poorly nested, both of which are likely to be related to the specificity of pollen transfer interactions. Our results indicate that pollination networks become more specialized when moving from the flower visitation to the level of pollen transfer networks. Therefore, caution is needed when inferring pollination from patterns of insect visitation or insect pollen loads as the relationship between these and pollen deposition is not straightforward.     

The effect of wild radish floral morphology on pollination efficiency by four taxa of pollinators

The effects of floral morphology on rates of pollen removal and deposition by different pollinators in generalist plant species are not well known. We studied pollination dynamics in wild radish, Raphanus raphanistrum, a plant visited by four groups of pollinators: honey bees, small native bees, butterflies, and syrphyd flies. The effects of anther position and other factors on pollen removal during single visits by all four pollinator taxa were measured. Flowers with high anther exsertion (i.e., anthers placed higher above the opening of the corolla tube) tended to have the highest numbers of pollen grains removed, but this effect was strongest for honey bees and butterflies. For all pollinator taxa, pollen removal increased with the number of pollen grains available on a flower and whowed a positive, decelerating relationship with the duration of the visit. The effects of stigma position and other factors on pollen deposition during single visits by honey bees and butterflies were also studied. The nectar-feeding butterflies had a higher pollination efficiency (percentage of pollen grains removed from anthers that were subsequently deposited on a stigma) than the nectar- and pollen-feeding honey bees. Flowers with intermediate stigma exsertion had the highest numbers of pollen grains deposited on their stigmas by butterflies, but stigma exsertion had no effect on deposition by honey bees. For both butterflies and honey bees, pollen deposition on the recipient flower increased with the amount of pollen removed from the donor flower, and there was a positive, decelerating relationship between deposition and time spent at the flower; these results are analogous to those for pollen removal. The effects of anther and stigma exsertion on pollen removal and denosition did not fit predictions based on patterns of floral correlations, but results for morphology, pollen availability, time spent per visit, and pollinator efficiency are in broad agreement with previous studies, suggesting the possible emergence of some general rules of pollen transfer.     

Studies on flower longevity in Digitalis

Flower lifespan was terminated by corolla abscission 5–6 days after stigma opening in the unpollinated flower. Increased pollen loads produced increased seed set and reduced flower longevity progressively to a minimum of one day after pollination with pure pollen. Weakening of the abscission zone was detectable 8 h after pollination, whilst the pollen tubes were still within the stigmatic zone, suggesting that a stimulus, moving at 4 mm h^–1 minimum, was transmitted through the style and ovary. Soon after pollination removal of the stigma prevented the pollination-induced corolla abscission. Later it was necessary to remove the stigma and upper style, and later still the whole style to delay abscission. The progressive induction of the stigma and style took place at a rate of 1.5 mm h^–1, in advance of the pollen tubes which grew at 0.75 mm h^–1. It was not possible to reproduce the pollination effects by application of indoleacetic acid (IAA) to the stigma or the style.Abbreviation IAA Indoleacetic acid