Hydrated Pollen Grains Research Articles

Generating images of hydrated pollen grains using deep learning

Pollen grains dehydrate during their development and following their departure from the host stigma. Since the size and shape of a pollen grain can be dependent on environmental conditions, being able to predict both of these factors for hydrated pollen grains from their dehydrated state could be beneficial in the fields of climate science, agriculture, and palynology. Here, we use deep learning to transform images of dehydrated Ranunculus pollen grains into images of hydrated Ranunculus pollen grains. We also then use a deep learning neural network that was trained on experimental images of different genera of pollen grains to identify the hydrated pollen grains from the generated transformed images, to test the accuracy of the image generation neural network. This pilot work demonstrates the first steps needed towards creating a general deep learning-based rehydration model that could be useful in understanding and predicting pollen morphology.

Arabidopsis annexin 5 is involved in maintenance of pollen membrane integrity and permeability.

In Arabidopsis, a dry stigma surface enables a gradual hydration of pollen grains by a controlled release of water. Occasionally the grains may be exposed to extreme precipitations that cause rapid water influx and swelling, eventually leading to pollen membrane rupture. In metazoans, calcium- and phospholipid-binding proteins, referred to as annexins, participate in the repair of plasma membrane damages. It remains unclear, however, how this process is conducted in plants. Here, we examined whether plant annexin 5 (ANN5), the most abundant member of the annexin family in pollen, is involved in the restoration of pollen membrane integrity. We analyzed the cellular dynamics of ANN5 in pollen grains undergoing hydration in favorable or stress conditions. We observed a transient association of ANN5 with the pollen membrane during in vitro hydration that did not occur in the pollen grains being hydrated on the stigma. To simulate a rainfall, we performed spraying of the pollinated stigma with deionized water that induced ANN5 accumulation at the pollen membrane. Interestingly, calcium or magnesium application affected pollen membrane properties differently, causing rupture or shrinkage of pollen membrane, respectively. Both treatments, however, induced ANN5 recruitment to the pollen membrane. Our data suggest a model in which ANN5 is involved in the maintenance of membrane integrity in pollen grains exposed to osmotic or ionic imbalances.

Mechanism of Pollen Germination and Pollen Tubes’ Growth in Common Juniper (Juniper communis L.) In Vitro

Pollen tubes are popular objects in the study of physiological-biochemical and structural-dynamic processes in plant cells. Most of such studies are devoted to pollen tubes of flowering plants with a short reproductive cycle. Much less attention is paid to the study of pollen tubes of gymnosperms. The article discusses the processes of hydration of pollen grains and the development of pollen tubes in juniper. The mature juniper pollen grain is in a dehydrated state and contains no more than 2–3% free moisture. Rehydration leads to activation of metabolism, pollen germination, and growth of pollen tubes. Hydration of the juniper pollen grain, which leads to the formation of a hydrophilic capsule, opening, and dumping of the exina is a sequence of events controlled, most likely, by the proteins of the outer nuclear membrane of the microspore. An unusually high content of mom-inositol was found in the orbiculae of juniper pollen grains, which is probably necessary for rapid water diffusion into the middle layer of the intina and the formation of a hydrophilic capsule. Two phytosteroids, which stimulate the germination of pollen and growth of pollen tubes, beta-sitosterol and campesterol, are found in pollen tubes of juniper. Vesicles, moving in the hyaloplasm of the pollen tube, are accumulated near its tip. Vesicles with their membranes are embedded in the membrane of the tip of the tube, releasing their contents. The highest concentration of mitochondria in growing pollen tubes is observed near both nuclei and along the periphery of the protoplast associated with the tube cell nucleus. There is a high probability of the existence of a redox signaling connection channel between the tube cell nucleus and the generative nucleus in the pollen tubes of common juniper. It is possible that this connection can be activated by proteins of the outer membranes of both or one of the nuclei.

OsAPT1 a Pollen Preferentially Expressed Gene Is Essential for Pollen Tube Germination and Elongation in Rice

Pollen tube germination and elongation are important processes for double fertilization in flowering plants. However, little is known about the regulatory mechanism of pollen germination and tube growth in rice. Here, we reported that OsAPT1, a pollen-high expression gene and encoding a putative APT1 protein in Oryza sativa, regulating pollen tube germination and elongation in rice. Real-time quantitative PCR (qRT-PCR) and in situ hybridization analysis showed high expression level of OsAPT1 at late stage of microsporogenesis including late uninucleate stage, binucleate pollen stage, and mature pollen grains. OsAPT1 RNA interference (OsAPT1-RNAi) lines showed that the pollen tube germination and elongation rate were decreased. Compared with wild type (WT), the integrity and assembly of actin microfilaments were abnormal in hydrated pollen grains of OsAPT1-RNAi plants, and the fluorescence signals of actin microfilaments were altered in the pollen tube of OsAPT1-RNAi plants. GO analysis of the co-expressing genes of OsAPT1 showed that the GO terms such as pollination, cell growth, cellular cytoskeleton organization, and motor activity were enriched. In addition, the qRT-PCR results showed variations in the expression of some genes related to pollen tube growth. Overall, our results indicated that OsAPT1 is required for pollen tube germination and elongation in rice.

Gene-expression profile of developing pollen tube of Pyrus bretschneideri

Pollen is an ideal model system for investigation of cell growth. In order to better understand the molecular biology mechanisms of the process of pear pollen tube development, RNA sequencing (RNA-Seq) technology was used to characterize the expression of genes during four development stages of pear pollen, including mature pollen grains (MP), hydrated pollen grains (HP), growing pollen tubes (PT) and stopped-growth pollen tubes (SPT). The four libraries generated a total of 47,072,151 clean reads that were mapped and assembled into 21,394 genes. Transcripts from the four stages were classified into 38 functional subcategories. Between MP and HP, 305 genes were differentially expressed, and 502 genes were differentially expressed between HP and PT. More importantly, we have observed that 2208 genes were differentially expressed between PT and SPT, and this is the first report of the gene expression comparison between the two development stages. Eight of the differentially expressed genes were randomly selected to confirm the RNA-Seq results by quantitative real-time PCR (qRT-PCR). Taken together, this research provides a platform for future research on pear pollen tube growth and growth cessation.

Glutathione-s-transferase is a minor allergen in birch pollen because of restricted release from hydrated pollen grains

Methods bGST was expressed in Escherichia coli, purified and characterized by mass spectrometry. BALB/c mice were immunized with bGST or Bet v 1. Antibody and T cell responses were assessed. 217 sera from birch pollen-allergic patients were tested for IgE-reactivity to bGST by ELISA. The allergenicity of bGST was evaluated with IgE-loaded rat basophil leukaemia cells (RBL) expressing the a-chain of the human receptor FceRI. Cross-reactivity of IgE between bGST and GST from house dust mite, Der p 8, was assessed with murine and human sera in ELISA. The release kinetics of bGST and Bet v 1 from birch pollen upon hydration were studied by immunoblotting.

Antibodies against ROS-producing ragweed pollen proteins may prevent allergic sensitization (P6375)

Abstract We have previously described that hydrated pollen grains and their allergenic extracts possess intrinsic NAD(P)H oxidase activity, which catalyzes the production of reactive oxygen species (ROS) and induces oxidative stress in the lung or conjunctiva. This oxidative insult promotes the development of adaptive immune responses against innocuous pollen proteins and augments allergic airway inflammation induced by pollen antigens. In this study we investigated the ROS-producing activity of short ragweed (Ambrosia artemisiifolia) pollen extract (RWE) in detail. First, we found that in the presence of NADPH, RWE produces more ROS than after addition of NADH. An in situ nitroblue tetrazolium assay demonstrated that at least two distinct enzymes are responsible for ROS generation in RWE. The first one can use both NADPH and NADH as substrates, while the second one produces ROS only in the presence of NADPH. We observed that despite the fact that sera from non-allergic individuals have lower non-enzymatic antioxidant capacity, they exhibit higher efficacy in inhibition of ROS generation by RWE compared to sera from allergic patients. Western blot analysis revealed that antibodies against some pollen proteins associated with ROS production of RWE are present only in the sera of non-allergic subjects. Our novel findings raise the possibility that these antibodies may be able to block the ROS production by pollen enzymes hereby prevent the sensitization of non-allergic individuals.

Pollen morphology of the genus Iris L. (Iridaceae) from Croatia and surrounding area: taxonomic and phylogenetic implications

In Croatia and the surrounding area, the genus Iris is represented with about 20 Alpine-Dinaric, Mediterranean and Pannonian taxa from the subgenera Iris and Limniris. We researched pollen morphology of all taxa by using scanning electron microscopy. All pollen grains are sulcate with few palynological features, which could have taxonomic importance on different classification levels: shape of dry pollen grains; shape, outline and approximate size of hydrated pollen grains, approximate size of the sulcus, and ornamentation of the sulcus membrane and of the exine. At least four pollen types were recognised and taxonomically delimited to the series level: two characterise the subgenus Iris, section Iris (series Elatae and Pumilae), and two the subgenus Limniris, section Limniris (series Laevigatae, Sibiricae and Spuriae). Taxonomic implications of pollen morphology of their critical groups and taxa have been evaluated. Possible phylogenetic implications of pollen morphology of the genus Iris on the Southern European area were suggested: the subgenus Iris is more advanced than the subgenus Limniris; I. graminea seems to be the most primitive member of the subgenus Limniris on the Southern European territory; and pollen morphology of the population of the subspecies I. sibirica subsp. erirrhiza from the Mountain Bjelolasica could represent a link between the subgenera Limniris and Iris in the territory of Southern Europe. The possible pathway of evolution of the genus Iris on the territory of Southern Europe was suggested: from the subgenus Limniris, through the “linked taxon” I. sibirica subsp. erirrhiza from the Bjelolasica Mountain, to the subgenus Iris, series Pumilae, and finally to the series Elatae.

A comparative analysis of the distribution and composition of lipidic constituents and associated enzymes in pollen and stigma of sunflower

Spatial distribution and compositional analyses of the lipidic constituents in pollen and stigma of sunflower (Helianthus annuus L. cv. Morden) were conducted using ultrastructural, histochemical, and biochemical analysis. Detection of secretions at the base of stigmatic papillae and neutral lipid accumulations on the surface of stigmatic papillae and between adjacent pseudopapillae demonstrates the semidry nature of stigma surface in sunflower. Pollen coat is richer in lipids (8%) than stigma (2.2%) on fresh weight basis. Nile Red-fluorescing neutral lipids are preferentially localized in the pollen coat. Neutral esters and triacylglycerols (TAGs) are the major lipidic constituents in pollen grains and stigma, respectively. Lignoceric acid (24:0) and cis-11-eicosenoic acid (20:1) are specifically expressed only in the pollen coat. Similar long-chain fatty acids have earlier been demonstrated to play a significant role during the initial signaling mechanism leading to hydration of pollen grains on the stigma surface. Lipase (EC 3.1.1.3) activity is expressed both in pollen grains and stigma. Stigma exhibits a better expression of acyl-ester hydrolase (EC 3.1.1.1) activity than that of observed in both the pollen fractions. Expression of two acyl-ester hydrolases (41 and 38 kDa) has been found to be specific to pollen coat. Specific expression of lignoceric acid (24:0) in pollen coat and localization of lipase in pollen and stigma have been discussed to assign possible roles that they might play during pollen-stigma interaction.

Transcriptome analyses show changes in gene expression to accompany pollen germination and tube growth in Arabidopsis.

Pollen germination, along with pollen tube growth, is an essential process for the reproduction of flowering plants. The germinating pollen with tip-growth characteristics provides an ideal model system for the study of cell growth and morphogenesis. As an essential step toward a detailed understanding of this important process, the objective of this study was to comprehensively analyze the transcriptome changes during pollen germination and pollen tube growth. Using Affymetrix Arabidopsis (Arabidopsis thaliana) ATH1 Genome Arrays, this study is, to our knowledge, the first to show the changes in the transcriptome from desiccated mature pollen grains to hydrated pollen grains and then to pollen tubes of Arabidopsis. The number of expressed genes, either for total expressed genes or for specifically expressed genes, increased significantly from desiccated mature pollen to hydrated pollen and again to growing pollen tubes, which is consistent with the finding that pollen germination and tube growth were significantly inhibited in vitro by a transcriptional inhibitor. The results of Gene Ontology analyses showed that expression of genes related to cell rescue, transcription, signal transduction, and cellular transport was significantly changed, especially for up-regulation, during pollen germination and tube growth. In particular, genes of the calmodulin/calmodulin-like protein, cation/hydrogen exchanger, and heat shock protein families showed the most significant changes during pollen germination and tube growth. These results demonstrate that the overall transcription of genes, both in the number of expressed genes and in the levels of transcription, was increased. Furthermore, the appearance of many novel transcripts during pollen germination as well as tube growth indicates that these newly expressed genes may function in this complex process.

Gene Family Analysis of the Arabidopsis Pollen Transcriptome Reveals Biological Implications for Cell Growth, Division Control, and Gene Expression Regulation

Upon germination, pollen forms a tube that elongates dramatically through female tissues to reach and fertilize ovules. While essential for the life cycle of higher plants, the genetic basis underlying most of the process is not well understood. We previously used a combination of flow cytometry sorting of viable hydrated pollen grains and GeneChip array analysis of one-third of the Arabidopsis (Arabidopsis thaliana) genome to define a first overview of the pollen transcriptome. We now extend that study to approximately 80% of the genome of Arabidopsis by using Affymetrix Arabidopsis ATH1 arrays and perform comparative analysis of gene family and gene ontology representation in the transcriptome of pollen and vegetative tissues. Pollen grains have a smaller and overall unique transcriptome (6,587 genes expressed) with greater proportions of selectively expressed (11%) and enriched (26%) genes than any vegetative tissue. Relative gene ontology category representations in pollen and vegetative tissues reveal a functional skew of the pollen transcriptome toward signaling, vesicle transport, and the cytoskeleton, suggestive of a commitment to germination and tube growth. Cell cycle analysis reveals an accumulation of G2/M-associated factors that may play a role in the first mitotic division of the zygote. Despite the relative underrepresentation of transcription-associated transcripts, nonclassical MADS box genes emerge as a class with putative unique roles in pollen. The singularity of gene expression control in mature pollen grains is further highlighted by the apparent absence of small RNA pathway components.

The roles of calmodulin polar distribution during pollen hydration and germination

Polarity patterning of pollen germination is a vital process for angiosperm fertilization. In our study a new method was employed to investigate the real-time distribution of calmodulin (CaM) in living pollen grains and pollen tubes. The CaM–GFP fusion gene was constructed under the control of the pollen-specific promoter LAT52-7 and transformed into Nicotiana tabacum L. Through confocal laser scanning microscopy, high levels of CaM were observed to accumulate in the three germinal apertures, and a tip–base gradient of CaM was detected in elongating pollen tubes. During pollen-grain hydration and germination, one of the three germinal apertures aggregated a much higher level of CaM than the other two. In addition, CaM showed a directional migration from the cytoplasm to this germinal aperture, where the pollen tube would emerge. Interestingly, CaM was not detected in the reproductive nucleus of either pollen grains or pollen tubes. Our findings indicated that the directional migration of CaM existed during pollen hydration and germination, and this movement may play a crucial role in the normal polarity establishment of pollen germination.Key words: calmodulin, polarity, pollen grain, Nicotiana tabacum.

Reactive oxygen species generated by pollen grain's NAD(P)H oxidase augment allergic ocular symptoms

Rationale We have discovered that all tested pollens important in allergic diseases contain NAD(P)H oxidases. These oxidases induce profound oxidative stress in the lungs independent of adaptive immunity, and play a critical role in induction of allergic airway inflammation (Boldogh et al). In this study we tested the role of pollen NAD(P)H oxidases in induction of allergic conjunctivitis. Methods ROS generation by hydrated pollen grains were measured by using H 2DCF-DA, NBT reduction test and Amplex Red assay. Their impact on intracellular ROS levels was studied in normal human bronchial epithelial and A549 cells, as well as in conjunctival epithelium in sensitized and non-sensitized groups of BALB/c mice. Their role in induction of allergic ocular symptoms was investigated in a murine model of allergic conjunctivitis. Results Intact, hydrated pollen grains generate ROS due to intrinsic NAD(P)H oxidase in them. These radicals increase intracellular ROS levels in cultured epithelial cells and ocular mucous membranes of sensitized mice. A similar phenomenon was observed in naïve mice, indicating it is independent of adaptive immunity. Inactivation of pollens' ROS-producing activity notably decreases the immediate-type response and inflammatory cell infiltration into the conjunctiva in murine allergic conjunctivitis. Conclusions Our data indicate that ROS generated by NAD(P)H oxidase activity in pollen grains contributes to development of allergic conjunctivitis.

Transcriptional profiling of Arabidopsis tissues reveals the unique characteristics of the pollen transcriptome.

Pollen tubes are a good model for the study of cell growth and morphogenesis because of their extreme elongation without cell division. Yet, knowledge about the genetic basis of pollen germination and tube growth is still lagging behind advances in pollen physiology and biochemistry. In an effort to reduce this gap, we have developed a new method to obtain highly purified, hydrated pollen grains of Arabidopsis through flowcytometric sorting, and we used GeneChips (Affymetrix, Santa Clara, CA; representing approximately 8,200 genes) to compare the transcriptional profile of sorted pollen with those of four vegetative tissues (seedlings, leaves, roots, and siliques). We present a new graphical tool allowing genomic scale visualization of the unique transcriptional profile of pollen. The 1,584 genes expressed in pollen showed a 90% overlap with genes expressed in these vegetative tissues, whereas one-third of the genes constitutively expressed in the vegetative tissues were not expressed in pollen. Among the 469 genes enriched in pollen, 162 were selectively expressed, and most of these had not been associated previously with pollen. Their functional classification reveals several new candidate genes, mainly in the categories of signal transduction and cell wall biosynthesis and regulation. Thus, the results presented improve our knowledge of the molecular mechanisms underlying pollen germination and tube growth and provide new directions for deciphering their genetic basis. Because pollen expresses about one-third of the number of genes expressed on average in other organs, it may constitute an ideal system to study fundamental mechanisms of cell biology and, by omission, of cell division.

Immunogold Electron Microscopic Localization of the Cross–Reactive Two–EF–Hand Calcium–Binding Birch Pollen Allergen Bet v 4 in Dry and Rehydrated Birch Pollen

Background: Recently, a novel family of low–molecular–weight (8–9 kD), two–EF–hand calcium–binding proteins has been described as allergens in plant pollens. Approximately 10% of pollen–allergic patients have IgE antibodies which cross–react with the two–EF–hand allergens in tree, grass and weed pollens. The aim of the present study was to localize Bet v 4, the two–EF–hand allergen from birch, in mature, dry pollen and to study the release of this allergen after hydration of the pollen by immunogold electron microscopy. Methods: Using completely anhydrous fixation techniques in combination with immunogold electron microscopy, we localized Bet v 4 and, for control purposes, the major birch pollen allergen Bet v 1, in dry birch pollen as well as in pollen grains after different periods of hydration. Parallel with these morphological studies, we monitored the release of Bet v 4 and Bet v 1 into aqueous supernatants of hydrated birch pollen grains by immunoblotting. Results: Bet v 4 was found in the electron–dense cytosol, in particular between the vesicles and cisternae of the endoplasmic reticulum, inside mitochondria and in the vegetative as well as in the generative nucleus. Bet v 1 was localized in similar cellular compartments except for the mitochondria. After 30 s to 1 min of hydration, Bet v 4 migrated into the pollen exine and into the aqueous supernatants. Bet v 1 also moved out of the pollen grain, though not as quickly as Bet v 4. Conclusion: Bet v 4 represents an intracellular pollen protein which, following hydration of pollen grains, rapidly migrates to the pollen surface (exine) and is washed out. This behavior explains how Bet v 4, being primarily an intracellular pollen protein, becomes available to sensitize patients.

Electrorotation of isolated generative and vegetative cells, and of intact pollen grains of Lilium longiflorum.

The dielectric structure of mature pollen of the angiosperm Lilium longiflorum was studied by means of single-cell electrorotation. The use of a microstructured four-electrode chamber allowed the measurements to be performed over a wide range of medium conductivity from 3 to 500 mS m-1. The rotation spectra of hydrated pollen grains exhibited at least three well-resolved peaks in the kHz-MHz frequency range, which obviously arise due to the multilayered structure of pollen grains. The three-shell model can explain the complex rotational behavior of pollen grains in terms of conductivities, permittivities and thicknesses of the following compartments: the exine and intine of the pollen grain wall as well as the membrane and cytoplasm of the vegetative cell. However, the number of unknown parameters (more than 8) was too large to allow unambiguous values to be assigned to any of them. Therefore, to facilitate the evaluation of the pollen grain parameters, additional rotational measurements were made on isolated vegetative and generative cells. The rotation spectra of these cells could be fitted very accurately on the basis of the single-shell model by assuming a dispersion of the cytoplasm. The data on the membrane and cytoplasmic properties of isolated vegetative cells were then used for modeling the rotation spectra of pollen grains. This greatly facilitated the fitting of the theoretical model to the experimental data and allowed the dielectric properties of the major structural units to be determined. The dielectric characterization of pollen is of enormous interest for plant biotechnology, where pollen and isolated germ cells are successfully used for production of transgenic crop and drug plants of economic importance by means of electromanipulation techniques.

Actin Organization and Distribution during Hydration, Activation and Germination of Pollen in Amaryllis vittata Ait. Using TRITC-phalloidin as a Probe.

Dynamics of actin organization and distribution during hydration, activation and germination of pollen in Amaryllis vittata Ait. was examined after TRITC-phalloidin staining. Fresh pollen grains and pollen tubes without fixation were treated and observed in a staining buffer contained dimethylsulphoxide (DMSO) as a permeabilising agent. Among the mature pollen grains, two organizational patterns of actin were confirmed in the cytoplasm of the vegetative cell. Those were fusiform bodies or a network constructed by filamentous fibres. Five min after incubation in a germination medium, the hydrated pollen grains appeared in various sizes representing the different degrees of hydration. In the larger-sized pollen grains, actin filaments confined to one end of the germ furrow. While in the smaller-sized pollen grains, actin appeared as needle-like bodies together with filamentous fibres. The results proved that the figuration of actin changed from fusiform bodies to a network of fibres accompanied the progress of hydration. The pollen grains activated 10-20 min after incubation. Actin in the vegetative cells of the activated pollen grains became highly polarized. After 30-40 min of incubation, the pollen grains began to germinate. Actin was observed in the protruding region (s). During pollen tube growing, actin filaments were transfered to the pollen tube and reached the tip. Actin in the ungerminated pollen grains remained in the storage form, which was very similar to that in the mature pollen grains. Cytochalasin D inhibited pollen germination. These results showed that the developmental and physiological states of the pollen cells could be different and be directly reflected by the organization patterns of actin.

S-RNase gene of Nicotiana alata is expressed in developing pollen.

In the solanaceous plant Nicotiana alata, self-incompatibility is controlled by a single, multiallelic locus (S locus) expressed in both pollen and pistil. Previously, we have shown cosegregation between alleles of the S locus and alleles of a gene that encodes a glycoprotein with ribonuclease activity (S-RNase). Furthermore, expression of the S-RNase gene is apparently confined to the pistil and is correlated with the onset of self-incompatibility. In this paper, we report that the S-RNase gene is also expressed at low levels in developing pollen. A transcript in developing pollen hybridized to a cDNA encoding the S2-RNase allele of the parent plant and did not hybridize to cDNAs encoding other S-RNase alleles. Two cDNAs for the S2-RNase were cloned from a library derived from anthers of a plant homozygous for the S2 allele and both corresponded to the coding sequence of the S2-RNase. The product of the S-RNase gene was detected by immunocytochemistry in the intine of mature, hydrated pollen grains. These results are interpreted in the light of current knowledge of the structure of the S locus.

Stimulation of growth of culturedNicotiana tabacum W 38 pollen tubes by poly(ethylene glycol) and Cu(II) salts

Growth of pollen tubes ofNicotiana tabacum W 38 in a defined liquid medium buffered at pH 5.9 and containing sucrose, amino-acids, boric acid, salts and an antibacterial agent was stimulated by the addition of poly(ethylene glycol) 6000 (PEG-6000) and Cu(II) salts. In the absence of both these supplements, up to 50% of the hydrated pollen grains did not develop further, and the germinated tubes were slow-growing and abnormal, with thickened walls, kinked growth, and fragile, swollen tips containing granular cytoplasm. Addition of 10–15% (w/v) purified PEG-6000 increased germination to 80–90% and prevented the progressive bursting of pollen grains and tube tips, but growth was still slow and kinked and tips remained swollen. Addition of 30 μM CuSO4 did not stimulate germination or prevent tip bursting, but produced straight-growing tubes with smooth-sided tips resembling the tips of tubes growing through stylar tissue; the free Cu2+ concentration under these conditions was about 1.0 μM due to chelation by amino-acids, and similar tube morphologies were obtained with 1.0–1.5 μM added CuSO4 when NH4Cl replaced the amino-acids. When the medium containing amino-acids was supplemented with both 12.5% PEG-6000 and 30 μM CuSO4, long-term (48 h) growth of straight pollen tubes with smooth-sided tips, thin walls and long ladders of callose plugs was observed; growth occurred at 250 μm/h, approximately 30–40% of the rate observed in the style. Although omission of CuSO4 from this complete medium severely affected tube growth and callose plug deposition, it did not alter the timing of generative-nucleus division, and thus the different parameters associated with the second phase of pollen-tube growth can be uncoupled in culture. High levels of FeSO4 (300 μM) had a similar morphogenetic effect to CuSO4, but addition of 300 μM L-ascorbate or D-iso-ascorbate was required to prevent precipitation of Fe(III) oxide and prolong the stimulation of pollen-tube growth; EDTA removed the morphogenetic effect of both CuSO4 and FeSO4. Further, an impure grade of PEG-4000 was contaminated with an organic morphogen that allowed continued slow growth of pollen tubes with smooth, straight-sided tips in the absence of added CuSO4 or FeSO4, with tube morphology unaffected by ascorbate or EDTA. However, the long-term morphogenetic effect of trace levels of CuSO4 suggests that Cu(II) salts play an important role in pollen-tube development in at least this species ofNicotiana.

Localization of adenylate cyclase activity in Populus: its relation to pollen-pistil recognition and incompatibility

Adenylate cyclase has been localized cytochemically in female and male parents as well as during the pollen-stigma interaction with an original technique employing strontium as the capture ion and adenyl imidodiphosphate as the specific substrate. The specificity of the reaction was checked by using several controls. No final specific reaction product was detected in unpollinated P. deltoides stigmas or in the P. deltoides or P. alba pollen grains used for compatible and incompatible pollinations. In the compatible cross between P. deltoides × P. deltoides, fine dense precipitates were observed in the dictyosomes and the plasma membrane and exterior to the exine of hydrated pollen grains adhering to the stigma surface. Labeling of the stigmatic pellicle was also observed after pollen adhesion and hydration. This was accompanied by a strong reactivity of the cell wall and plasmalemma of the stigma papillae at the sites of pollen tube germination on the stigma surface and at the sites of penetration of pollen tubes between adjacent papillae. In the incompatible cross between P. deltoides x P. alba, adenylate cyclase activity was still present but reduced at the stigma surface following adhesion, hydration, and germination of P. alba pollen. This activity was completely abolished after the penetration of pollen tubes between stigma papillae. These findings suggest that in Populus, adenylate cyclase activity is correlated to pollen adhesion, hydration, and germination at the stigma surface, and that the abolition of this enzyme activity could be one of the cellular events governing the gametophytic phenotype of incompatibility in the cross between P. deltoides and P. alba.