Showing papers in "Sexual Plant Reproduction in 2012"

New insights into the variability of reproduction modes in European populations of Rubus subgen. Rubus: how sexual are polyploid brambles?

Abstract: Rubus subgen. Rubus includes common European species with highly complicated taxonomy, ongoing hybridisation and facultative apomixis. Out of approximately 750 species recognised in Europe, only 3 diploid sexual species are known, along with numerous apomictic brambles that are highly connected to polyploidy. One exception of a tetraploid taxon is R. ser. Glandulosi, which is known for prevalent sexuality. This taxon highly hybridises with tetraploid members of R. ser. Discolores and leads to the origin of many hybridogenous populations and individuals. In this study, we verify reproduction modes in different diploid, triploid and tetraploid species of subgen. Rubus, with focus on taxa putatively involved in such hybridisation by applying flow cytometric seed screen analysis. We found 100 % sexuality of diploid species, whereas triploid species had obligate unreduced embryo sac development. In contrast, tetraploid plants had varying degrees of sexuality. Additionally, we discovered that R. bifrons has the ability to undergo a reproduction mode switch as a reaction to environmental conditions. These results provide insight into reproductive modes of European brambles and shed light on their reticulate evolution and speciation.

Wide-scale screening of T-DNA lines for transcription factor genes affecting male gametophyte development in Arabidopsis

Abstract: Male gametophyte development leading to the formation of a mature pollen grain is precisely controlled at various levels, including transcriptional, post-transcriptional and post-translational, during its whole progression. Transcriptomic studies exploiting genome-wide microarray technologies revealed the uniqueness of pollen transcriptome and the dynamics of early and late successive global gene expression programs. However, the knowledge of transcription regulation is still very limited. In this study, we focused on the identification of pollen-expressed transcription factor (TF) genes involved in the regulation of male gametophyte development. To achieve this, the reverse genetic approach was used. Seventy-four T-DNA insertion lines were screened, representing 49 genes of 21 TF families active in either early or late pollen development. In the screen, ten phenotype categories were distinguished, affecting various structural or functional aspects, including pollen abortion, presence of inclusions, variable pollen grain size, disrupted cell wall structure, cell cycle defects, and male germ unit organization. Thirteen lines were not confirmed to contain the T-DNA insertion. Among 61 confirmed lines, about half (29 lines) showed strong phenotypic changes (i.e., ≥25% aberrant pollen) including four lines that produced a remarkably high proportion (70–100%) of disturbed pollen. However, the remaining 32 lines exhibited mild defects or resembled wild-type appearance. There was no significant bias toward any phenotype category among early and late TF genes, nor, interestingly, within individual TF families. Presented results have a potential to serve as a basal information resource for future research on the importance of respective TFs in male gametophyte development.

Differential effects of polyploidy and diploidy on fitness of apomictic Boechera.

Abstract: The co-occurrence of apomixis (asexual reproduction) and polyploidy in plants has been the subject of debate in regard to the origin and evolution of asexuality. In recent years, polyploidy has been postulated as a maintenance and stabilization factor rather than as a source of apomixis origin. It is assumed polyploidy facilitates the compensation for mutation accumulation, and hence, the rare occurrence of diploid apomixis indirectly supports this finding. Nevertheless, diploid apomicts exist and are successful, especially in the genus Boechera. While comparing phenotypic traits, fitness-related traits and apomixis penetrance between both diploid and triploid apomicts in the genus Boechera, it was expected to find trait variance that can be attributed to ploidy. Surprisingly, little trait variation could be assigned to ploidy, but rather trait variations were mainly genotype-specific. Additionally, it is shown that paternal contribution is very important for trait success, even though all offspring are genetically identical to the mother plant. This harbors implications for the introduction of apomixis into crop plants, considering the effects of paternal contribution during asexual reproduction. Nevertheless, polyploidy is an efficient way to buffer deleterious mutations, but the flexibility of diploid apomicts of the genus Boechera for rare sexual events contributes to their success in nature.

Pollen performance, cell number, and physiological state in the early-divergent angiosperm Annona cherimola Mill. (Annonaceae) are related to environmental conditions during the final stages of pollen development.

Abstract: Pollen performance is an important determinant for fertilization success, but high variability in pollen behavior both between and within species occurs in different years and under varying environmental conditions. Annona cherimola, an early-divergent angiosperm, is a species that releases a variable ratio of bicellular and tricellular hydrated pollen at anther dehiscence depending on temperature. The presence of both bi- and tricellular types of pollen is an uncommon characteristic in angiosperms and makes Annona cherimola an interesting model to study the effect of varying environmental conditions on subsequent pollen performance during the final stages of pollen development. In this work, we study the influence of changes in temperature and humidity during the final stages of pollen development on subsequent pollen performance, evaluating pollen germination, presence of carbohydrates, number of nuclei, and water content. At 25 °C, which is the average field temperature during the flowering period of this species, pollen had a viability of 60–70 %, starch hydrolyzed just prior to shedding, and pollen mitosis II was taking place, resulting in a mixture of bi- and tricellular pollen. This activity may be related to the pollen retaining 70 % water content at shedding. Temperatures above 30 °C resulted in a decrease in pollen germination, whereas lower temperatures did not have a clear influence on pollen germination, although they did have a clear effect on starch hydrolysis. On the other hand, slightly higher dehydration accelerated mitosis II, whereas strong dehydration arrested starch hydrolysis and reduced pollen germination. These results show a significant influence of environmental conditions on myriad pollen characteristics during the final stages of pollen development modifying subsequent pollen behavior and contributing to our understanding of the variability observed in pollen tube performance.

Agave tequilana MADS genes show novel expression patterns in meristems, developing bulbils and floral organs

Abstract: Agave tequilana is a monocarpic perennial species that flowers after 5–8 years of vegetative growth signaling the end of the plant’s life cycle. When fertilization is unsuccessful, vegetative bulbils are induced on the umbels of the inflorescence near the bracteoles from newly formed meristems. Although the regulation of inflorescence and flower development has been described in detail for monocarpic annuals and polycarpic species, little is known at the molecular level for these processes in monocarpic perennials, and few studies have been carried out on bulbils. Histological samples revealed the early induction of umbel meristems soon after the initiation of the vegetative to inflorescence transition in A. tequilana. To identify candidate genes involved in the regulation of floral induction, a search for MADS-box transcription factor ESTs was conducted using an A. tequilana transcriptome database. Seven different MIKC MADS genes classified into 6 different types were identified based on previously characterized A. thaliana and O. sativa MADS genes and sequences from non-grass monocotyledons. Quantitative real-time PCR analysis of the seven candidate MADS genes in vegetative, inflorescence, bulbil and floral tissues uncovered novel patterns of expression for some of the genes in comparison with orthologous genes characterized in other species. In situ hybridization studies using two different genes showed expression in specific tissues of vegetative meristems and floral buds. Distinct MADS gene regulatory patterns in A. tequilana may be related to the specific reproductive strategies employed by this species.

Mechanism of seedlessness in a new lemon cultivar ‘Xiangshui’ [Citrus limon (L.) Burm. F.]

Abstract: Seedlessness is an important economic trait of lemon. Understanding the cellular and molecular mechanisms of seedlessness in ‘Xiangshui’ lemon requires detailed data on pollen and embryo sac fertility, embryo development and compatibility mechanisms governing self- and cross-pollination. The results of the current study indicate that the fertility of pollen and mature embryo sac remains normal. When flowers were self- or cross-pollinated, pollen grains of ‘Xiangshui’ were able to germinate on the stigma. In the case of self-pollination, pollen tubes became twisted, tube tips enlarged and tubes ruptured in the bottom of stigma. Following cross-pollination, tubes were able to grow normally in the style and ovary and enter the embryo sac, where double fertilization took place. Embryonic development resulting from cross-pollination was normal. After cross-pollination, the zygote began to divide at 2 weeks post-pollination, with early globular embryos observed after 3 weeks, globular and heart-shaped embryos at 4 weeks, torpedo-shaped embryos at 5 weeks, cotyledonary embryos at 6 weeks and thereafter germinable seeds. After self-pollination, however, ovules began to abort at 2 weeks post-pollination, with ovules disappearing at 5 weeks, ultimately producing seedless fruits. Emasculated unpollinated flowers also developed into seedless fruits, indicating that seedlessness contributes to parthenocarpy. However, gametophytic self-incompatibility has a major role in seedlessness in ‘Xiangshui’ lemon by blocking fertilization at the bottom of the stigma.

Morphological, histological and ultrastructural changes in the olive pistil during flowering

Abstract: Sexual reproduction is essential for the propagation of higher plants. From an agronomical point of view, this is a particularly key process because fertilization guarantees fruit formation in most cultivated fruit species. In the olive, however, in spite of its agricultural importance, little attention has been paid to the study of sexual reproduction. In order to investigate the cellular and molecular mechanisms that regulate pollen–pistil interactions in the olive during the progamic phase, it is essential to first have a good knowledge of the reproductive structures involved in such interactions. This study characterizes the anatomical and ultrastructural changes in the olive pistil, beginning from the young pistil developing within the bud until the time of petal loss and visible stigma senescence. We have correlated changes in the pistil with a series of defined floral developmental stages and determined that olive pistil structures cannot be considered completely mature and ready to be pollinated and fertilized until the onset of anthesis. Our results clearly show histological and ultrastructural variation during the diverse flowering events. We discuss whether the changes observed might influence or result from pollen–pistil interactions during the progamic phase.

Characterization of callase (β-1,3-D-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum.

Abstract: We examined callase activity in anthers of sterile Allium sativum (garlic) and fertile Allium atropurpureum. In A. sativum, a species that produces sterile pollen and propagates only vegetatively, callase was extracted from the thick walls of A. sativum microspore tetrads exhibited maximum activity at pH 4.8, and the corresponding in vivo values ranged from 4.5 to 5.0. Once microspores were released, in vitro callase activity peaked at three distinct pH values, reflecting the presence of three callase isoforms. One isoform, which was previously identified in the tetrad stage, displayed maximum activity at pH 4.8, and the remaining two isoforms, which were novel, were most active at pH 6.0 and 7.3. The corresponding in vivo values ranged from pH 4.75 to 6.0. In contrast, in A. atropurpureum, a sexually propagating species, three callase isoforms, active at pH 4.8-5.2, 6.1, and 7.3, were identified in samples of microsporangia that had released their microspores. The corresponding in vivo value for this plant was 5.9. The callose wall persists around A. sativum meiotic cells, whereas only one callase isoform, with an optimum activity of pH 4.8, is active in the acidic environment of the microsporangium. However, this isoform is degraded when the pH rises to 6.0 and two other callase isoforms, maximally active at pH 6.0 and 7.3, appear. Thus, factors that alter the pH of the microsporangium may indirectly affect the male gametophyte development by modulating the activity of callase and thereby regulating the degradation of the callose wall.

Comparative proteomic analyses reveal the changes of metabolic features in soybean (Glycine max) pistils upon pollination.

Abstract: Siphonogamy is a critical process in plant reproductive growth, during which numerous cell–cell interaction events occur between pistil and pollen. Previous studies in Solanaceae, Papaveraceae, and Brassicaceae focusing on pollen–stigma recognition in self-incompatible systems have provided many important views. In this study, we profiled the proteome in soybean mature pistils before and after pollination. Comparative analyses of two-dimensional gel electrophoresis maps from un-pollinated and pollinated pistils were conducted. The results showed that 22 proteins were increased and 36 proteins decreased after pollination. Functional categorization showed that most of them were metabolism- and redox-related proteins. The enhancement of primary metabolism, biosynthesis of pollen tube guidance compounds, and adjustment of redox homeostasis system might be helpful for a successful pollination. Quantitative reverse transcript-polymerase chain reaction analysis implied that the regulation of gene expression might happen at both transcriptional and posttranscriptional levels during pollination. This study will enhance our understanding of pollen–stigma interaction in plant sexual reproductive growth.

The breeding systems of diploid and neoautotetraploid clones of Acacia mangium Willd. in a synthetic sympatric population in Vietnam

Abstract: Colchicine-induced neoautotetraploid genotypes of Acacia mangium were cloned and planted in mixture with a set of diploid clones in an orchard in southern Vietnam. Following good general flowering, open-pollinated seed was collected from trees of both cytotypes and microsatellite markers were used to determine the breeding system as characterised by the proportion of outcrosses in young seedling progeny. As predicted from the literature, the progeny of diploid clones were predominantly outcrossed (t m = 0.97). In contrast, the progeny of the tetraploid clones were almost entirely selfs (t m = 0.02; 3 of 161 seedlings assayed were tetraploid outcrosses and there were no triploids). Segregation at loci heterozygous in the tetraploid mothers followed expected ratios, indicating sexual reproduction rather than apomixis. Post-zygotic factors are primarily responsible for divergence of the breeding systems. Commonly, less than 1 % of Acacia flowers mature as a pod, and after mixed pollination, diploid outcrossed seed normally develops at the expense of selfs. Selfs of the tetraploid trees appear to express less genetic load and have a higher probability of maturing. However, this does not fully explain the observed deficiency of outcross tetraploid progeny. Presumably, there are cytogenetic reasons which remain to be investigated. In nature, selfing would increase the probability of establishment of neotetraploids irrespective of cytotype frequency in the population. Breeders need to review their open-pollinated breeding and seed production strategies. It remains to be seen whether this is an ephemeral problem, with strong fertility selection restoring potential for outcrossing over generations.

Male fertility versus sterility, cytotype, and DNA quantitative variation in seed production in diploid and tetraploid sea lavenders (Limonium sp., Plumbaginaceae) reveal diversity in reproduction modes.

Abstract: The genus Limonium Miller, a complex taxonomic group, comprises annuals and perennials that can produce sexual and/or asexual seeds (apomixis). In this study, we used diverse cytogenetic and cytometric approaches to analyze male sporogenesis and gametogenesis for characterizing male reproductive output on seed production in Limonium ovalifolium and Limonium multiflorum. We showed here that the first species is mostly composed of diploid cytotypes with 2n = 16 chromosomes and the latter species by tetraploid cytotypes with 2n = 32, 34, 35, 36 chromosomes and had a genome roughly twice as big as the former one. In both species, euploid and aneuploid cytotypes with large metacentric chromosomes having decondensed interstitial sites were found within and among populations, possibly involved in chromosomal reconstructions. L. ovalifolium diploids showed regular meiosis resulting in normal tetrads, while diverse chromosome pairing and segregation irregularities leading to the formation of abnormal meiotic products are found in balanced and non-balanced L. multiflorum tetraploids. Before anther dehiscence, the characteristic unicellular, bicellular, or tricellular pollen grains showing the typical Limonium micro- or macro-reticulate exine ornamentation patterns were observed in L. ovalifolium using scanning electron microscopy. Most of these grains were viable and able to produce pollen tubes in vitro. In both balanced and unbalanced L. multiflorum tetraploids, microspores only developed until the “ring-vacuolate stage” with a collapsed morphology without the typical exine patterns, pointing to a sporophytic defect. These microspores were unviable and therefore never germinated in vitro. L. ovalifolium individuals presented larger pollen grains than those of L. multiflorum, indicating that pollen size and ploidy levels are not correlated in the Limonium system. Cytohistological studies in mature seeds from both species revealed that an embryo and a residual endosperm were present in each seed. Flow cytometric seed screens using such mature seeds showed quantitative variations in seeds ploidy level. It is concluded that male function seems to play an important role in the reproduction modes of Limonium diploids and tetraploids.

The effect of 2n gametes on sex ratios in Actinidia

Abstract: Sex can sometimes lead to complications. In some crops, 2n gametes have been exploited by plant breeders to transfer genetic variation between taxa of different ploidy levels. However, their role and use in dioecious genera have received relatively little attention. In the dioecious genus Actinidia (kiwifruit), seedling populations usually segregate equally for females and males as sex is determined by an XX female/XY male system. While fertilization involving 2n egg cells is not expected to affect the sex ratios of progenies, fertilization involving 2n pollen is likely to produce progenies with excess males. The extent of sex ratio distortion will depend on the relative contributions of first and second division restitution, and the frequency and location of cross-overs in meiosis. In this study, seedlings recovered from crosses between females of hexaploid Actinidia deliciosa and males of two diploid species, Actinidia chinensis and Actinidia eriantha, included a proportion of pentaploid hybrids presumably derived from fertilization involving 2n pollen. Most of these pentaploids were male, and a proportion of them were likely to be carrying two Y chromosomes. If used as parents in further crosses, males with multiple Y chromosomes are likely to cause distorted sex ratios in their immediate progenies. In dioecious genera such as Actinidia, the effects on sex ratios of different mechanisms of ploidy change need to be taken into account when considering the evolution of polyploidy and the design of breeding strategies involving ploidy manipulation.

Pollen tube development in two species of Trithuria (Hydatellaceae) with contrasting breeding systems

Abstract: Trithuria (Hydatellaceae; Nymphaeales) is unique among early-divergent angiosperms in that its species are extremely small and most have exceptionally short, annual life histories. Given the evolution of these extremes of size and development, we sought to understand whether post-pollination processes still varied predictably with breeding system in Trithuria. To address this question, we studied two Western Australian species, Trithuria austinensis (dioecious, obligately outcrossing) and Trithuria submersa (bisexual, highly selfing). To document developmental timing, carpels were hand-pollinated, collected at sequential time points, and examined with light and fluorescence microscopy. In both species, pollen tubes first entered ovules <1 h after pollination, but the pollen tube pathway of outcrossing T. austinensis was almost four times longer and its pollen tube growth rates were up to six times faster (≤2,166 vs. 321 μm/h) than those of T. submersa. T. austinensis also exhibited greater male investment, slower pollen germination, and greater pollen tube attrition. These differences in male gametophyte development are predicted for outcrossers versus selfers in phylogenetically derived angiosperms. These new data for Hydatellaceae reinforce the idea that an acceleration of pollen tube development occurred in the Nymphaeales stem lineage, before the origin of Hydatellaceae. We infer that a recent evolutionary transition to selfing in T. submersa has been accompanied by predictable modifications to reproductive development, which, because of the ancient relationship between Hydatellaceae and all other angiosperms, suggests that traits underlying the lability of flowering plant post-pollination biology were present early in their history.

Zygospore formation between homothallic and heterothallic strains of Closterium.

Abstract: Zygospore formation in different strains of the Closterium peracerosum-strigosum-littorale complex was examined in this unicellular isogamous charophycean alga to shed light on gametic mating strains in this taxon, which is believed to share a close phylogenetic relationship with land plants. Zygospores typically form as a result of conjugation between mating-type plus (mt+) and mating-type minus (mt−) cells during sexual reproduction in the heterothallic strain, similar to Chlamydomonas. However, within clonal cells, zygospores are formed within homothallic strains, and the majority of these zygospores originate as a result of conjugation of two recently divided sister gametangial cells derived from one vegetative cell. In this study, we analyzed conjugation of homothallic cells in the presence of phylogenetically closely related heterothallic cells to characterize the reproductive function of homothallic sister gametangial cells. The relative ratio of non-sister zygospores to sister zygospores increased in the presence of heterothallic mt+ cells, compared with that in the homothallic strain alone and in a coculture with mt− cells. Heterothallic cells were surface labeled with calcofluor white, permitting fusions with homothallic cells to be identified and confirming the formation of hybrid zygospores between the homothallic cells and heterothallic mt+ cells. These results show that at least some of the homothallic gametangial cells possess heterothallic mt−-like characters. This finding supports speculation that division of one vegetative cell into two sister gametangial cells is a segregative process capable of producing complementary mating types.

Gene expression associated with apogamy commitment in Ceratopteris richardii

Abstract: Apogamy is a phenomenon in which a sporophyte develops asexually, directly from a cell or cells of a gametophyte. It is a phenomenon described mainly in lower plants, but shares certain aspects with apomixis in angiosperms. The genes involved in apogamy commitment in ferns are unknown. We hypothesize that the mechanism of asexual reproduction is controlled in lower and higher plants by overlapping sets of genes. To this end, we created a normalized subtracted cDNA library that represents genes with increased expression during apogamy commitment in the fern Ceratopteris richardii. The cDNA library consists of 170 unique sequences. Compared to the mature gametophyte transcriptome of the fern Pteridium aquilinum, the apogamy library is enriched in plant GO-Slim terms that are associated with stress and metabolism. In silico expression analyses of the closest Arabidopsis homologs of the apogamy library revealed many genes that display preferential expression in seed and flower tissues, structures that are absent in ferns. This apogamy library provides a rich resource for investigations into the genetic control of apogamy in ferns and comparisons with the asexual processes of higher plants.

Confocal observations of late-acting self-incompatibility in Theobroma cacao L.

Abstract: Cocoa (Theobroma cacao) has an idiosyncratic form of late-acting self-incompatibility that operates through the non-fusion of incompatible gametes. Here, we used high-resolution confocal microscopy to define fine level changes to the embryo sac of the strongly self-incompatible cocoa genotype SCA 24 in the absence of pollination, and following compatible and incompatible pollination. All sperm nuclei had fused with the female nuclei by 48 h following compatible pollinations. However, following incompatible pollinations, we observed divergence in the behaviour of sperm nuclei following release into the embryo sac. Incomplete sperm nucleus migration occurred in approximately half of the embryo sacs, where the sperm nuclei had so far failed to reach the female gamete nuclei. Sperm nuclei reached but did not fuse with the female gamete nuclei in the residual cases. We argue that the cellular mechanisms governing sperm nucleus migration to the egg nucleus and those controlling subsequent nuclear fusion are likely to differ and should be considered independently. Accordingly, we recommend that future efforts to characterise the genetic basis of LSI in cocoa should take care to differentiate between these two events, both of which contribute to failed karyogamy. Implications of these results for continuing efforts to gain better understanding of the genetic control of LSI in cocoa are discussed.

Different regulatory processes control pollen hydration and germination in Arabidopsis

Abstract: To elucidate the functional differences in how Arabidopsis stigmas regulate pollen hydration and germination, we analyzed receptivity of stigmas, epidermal surfaces (leaves, stems of inflorescence bolts, and floral organs), and an abiotic surface (cover glass) for pollen hydration and germination. Using 65% relative humidity (RH), we found that mature pollen grains were able to hydrate and germinate on stigmas at flower developmental stages 9–13, but not on the distal end of pistils at stage 8, epidermal surfaces, or glass. Furthermore, under 100% RH, pollen grains could hydrate on all tested surfaces, but pollen germination was observed only on the young floral organs (stages 9–12) and the stigmas at stages 9–13. The distal ends of pistils at stage 8, the epidermal surfaces, and the cover glass did not support pollen germination even under 100% RH. Our results indicate that pistil factors regulating pollen hydration and germination are synthesized at stage 9 when stigmatic papillar cells begin to develop. Although pistil factors involved in pollen hydration may only be present on the stigma, the factors involved in pollen germination may localize on both the stigma and surfaces of unopened floral organs.

The intriguing complexity of parthenogenesis inheritance in Pilosella rubra (Asteraceae, Lactuceae).

Abstract: Neither the genetic basis nor the inheritance of apomixis is fully understood in plants. The present study is focused on the inheritance of parthenogenesis, one of the basic elements of apomixis, in Pilosella (Asteraceae). A complex pattern of inheritance was recorded in the segregating F1 progeny recovered from reciprocal crosses between the facultatively apomictic hexaploid P. rubra and the sexual tetraploid P. officinarum. Although both female and male reduced gametes of P. rubra transmitted parthenogenesis at the same rate in the reciprocal crosses, the resulting segregating F1 progeny inherited parthenogenesis at different rates. The actual transmission rates of parthenogenesis were significantly correlated with the mode of origin of the respective F1 progeny class. The inheritance of parthenogenesis was significantly reduced in F1n + n hybrid progeny from the cross where parthenogenesis was transmitted by female gametes. In F1n + 0 polyhaploid progeny from the same cross, however, the transmission rate of parthenogenesis was high; all fertile polyhaploids were parthenogenetic. It appeared that reduced female gametes transmitting parthenogenesis preferentially developed parthenogenetically and only rarely were fertilized in P. rubra. The fact that the determinant for parthenogenesis acts gametophytically in Pilosella and the precocious embryogenesis in parthenogenesis-transmitting megagametophytes was suggested as the most probable explanations for this observation. Furthermore, we observed the different expression of complete apomixis in the non-segregating F1 2n + n hybrids as compared to their apomictic maternal parent P. rubra. We suggest that this difference is a result of unspecified interactions between the parental genomes.

Cytological features of oogenesis and their evolutionary significance in the fern Osmunda japonica

Abstract: The development of the egg and canal cells in the fern Osmunda japonica Thunb. was studied during oogenesis by transmission electron microscopy. The mature egg possesses no fertilization pore and no typical egg envelope. In addition, an extra wall formed around the canal cells during oogenesis and apparently blocked protoplasmic connections between the egg and the canal cells. The periodic acid Schiff (PAS) reaction revealed that the extra wall was most likely composed of polysaccharides. Maturation of the egg was accompanied by the formation of a separation cavity above the egg and by some changes in the morphology of the nucleus and cytoplasmic organelles. The chromatin of the nucleus becomes condensed and the upper surface of the nucleus becomes closely associated with the plasmalemma. Amyloplasts in the egg cytoplasm were numerous and conspicuous, with most in close proximity to the nucleus. Finally, the cytoplasm on one side of the egg became vesiculated and the overlying plasmalemma was easily disrupted. These cytological features of the egg and the canal cells during oogenesis in O. japonica are markedly different from those of the leptosporangiate ferns and suggest a significant evolutionary divergence in reproductive cellular features between Osmundaceae and leptosporangiate ferns.

Gamete formation via meiotic nuclear restitution generates fertile amphiploid F1 (oat×maize) plants.

Abstract: Hybrid (oat × maize) zygotes developed into euhaploid plants with complete oat chromosome complements without maize chromosomes and into aneuhaploid plants with complete oat chromosome complements and different numbers of retained individual maize chromosomes. The elimination of maize chromosomes in the hybrid embryo is caused by uniparental genome loss during early steps of embryogenesis. Some of these haploid plants set seed in up to 50% of their self-pollinated spikelets. The high fertility was found to be mainly caused by formation of numerically unreduced female and male gametes (n unreduced = 3x + 0 … 3 = 21 … 24 chromosomes). Gamete formation involves meiotic nuclear restitution. The restitution process is caused by an alternative type of meiosis. It follows the model of levigatum-type semi-heterotypic divisions, but with a formation of the nuclear membrane at the transition from telophase I to interkinesis, which resembles the model of pygaera-type pseudo-homotypic divisions. We propose the name haploid meiotic restitution for this particular process combination. We discuss the use and implications of the specific process of gamete formation in F1 (oat × maize) plants.

A novel pollen tube growth assay utilizing a transmitting tract-ablated Nicotiana tabacum style

Abstract: Sexual plant reproduction requires multiple pollen–pistil interactions from the stigma (pollen adhesion, hydration, and germination) to the ovary (fertilization). Understanding the factors that regulate pollen tube growth is critical to understanding the processes essential to sexual reproduction. Many pollen tube growth assays (PTGAs) have shorter and slower pollen tube growth when compared to pollen tube growth through the style. The identification and study of factors that regulate pollen tube growth have been impeded by a lack of an efficient and reproducible PTGA. The objective of this research is to develop a robust assay for Nicotiana tabacum pollen tube growth in an environment that supports sustained and normal growth yet is amenable to testing the effects of specific factors. In this paper, we introduce a novel PTGA, which uses pistils from N. tabacum that lack a mature transmitting tract (TT) due to tissue-specific ablation. The TT-ablated style supports normal pollen tube growth and the hollow structure of the style allows modification of the growth environment by direct injection of test material. This PTGA is robust and allows for rapid and accurate measurement of pollen tube length and pollen tube morphology, supporting pollen tube growth from 20 to 35°C and at pH ranging from 4.8 to 7.6. Use of the ablated style for a PTGA is a novel method for the culture of pollen tubes with sustained growth in vivo while permitting the application of treatments to the growing pollen tubes.

Post-pollination prefertilization drops affect germination rates of heterospecific pollen in larch and Douglas-fir

Abstract: Pollen of larch (Larix × marschlinsii) and Douglas-fir (Pseudotsuga menziesii) was used in homospecific and heterospecific crosses. Germination of heterospecific pollen in ovulo was reduced in post-pollination prefertilization drops. This provides evidence of selection against foreign pollen by open-pollinated exposed ovules in these two sister taxa, which share the same type of pollination mechanism. Of the other prezygotic stages in pollen–ovule interactions, uptake of pollen by stigmatic hairs did not show any selection. Pollen tube penetration of the nucellus was similar for hetero- and homospecific pollen tubes, but heterospecific tubes only delivered gametes in one cross. To test for differences in the post-pollination prefertilization drops of each species, drops were gathered and analysed. Glucose and fructose were present in similar amounts in Douglas-fir and larch, while sucrose was found in larch only. Other carbohydrates such as xylose and melezitose were species-specific. In P. menziesii, sucrose is absent due to its conversion to glucose and fructose by apoplastic invertases. In contrast, Larix × marschlinsii drops have sucrose because they lack apoplastic invertases. The presence of invertase activity shows that the composition of gymnosperm post-pollination prefertilization drops is not static but dynamic. Drops of these two species also differed in their calcium concentrations.

Floral rewards in the tribe Sisyrinchieae (Iridaceae): oil as an alternative to pollen and nectar?

Abstract: Iridaceae is one of the few families in which floral oils are produced and collected by pollinators as a resource. Perigonal nectaries and trichomal elaiophores are highly unusual within the tribe Sisyrinchieae. Both structures occur mainly on the staminal column, while they are usually distributed on the tepals in the other tribes of the subfamily Iridoideae. Sisyrinchieae is the largest tribe of Iridaceae present on the American continent, and the diversity observed may be related to the exceptional development of trichomal elaiophores within the genus Sisyrinchium, but knowledge concerning the other types of nuptial glandular structures within the tribe is still limited, preventing us from estimating their implication for species diversity. Structural observations and histochemical tests were performed to identify and characterize glandular structures and pollen rewards within the flowers of the genera Orthrosanthus, Sisyrinchium and Solenomelus. Perigonal nectaries were detected only in Solenomelus segethi, and trichomal elaiophores were characterized only within Sisyrinchium. All species showed large amounts of additional resources available for pollinators in the form of pollenkitt and polysaccharides present in the cytoplasm of the pollen grains. The results are discussed in a phylogenetic context, with regard to pollinators and floral rewards reported for the tribe Sisyrinchieae.

Ultrastructural and cytochemical studies on oogenesis of the fern Pteridium aquilinum

Abstract: Egg development in Pteridium aquilinum var. latiusculum was studied using ultrastructural and cytochemical methods to examine structural features influencing fertilization in leptosporangiate ferns. Ultrastructural observations indicate a separation cavity is first formed above the egg during oogenesis with a pore region persistently connecting the egg and the ventral canal cell. The egg envelope is formed by deposition of amorphous materials in the separation cavity on the outer surface of plasmalemma. The egg envelope was not formed across the pore region; instead, a fertilization pore was formed. During oogenesis, the egg nucleus produced extensive evaginations containing osmiophilic bodies. Cytochemical experiments revealed that the egg envelope displays strong periodic acid-Schiff reaction indicative of polysaccharides, with negligible Sudan black B staining for lipids, suggesting that the egg envelope is composed principally of polysaccharides, and not lipids. The present manuscript provides new insights into egg structure and development of Pteridium, including discovery and characterization of the fertilization pore and observations on the chemical nature of the egg envelope, thus contributing to the understanding of the cytological mechanism of the sexual reproduction of ferns.

A time course of GFP expression and mRNA stability in pollen tubes following compatible and incompatible pollinations in Solanum chacoense

Abstract: The self-incompatibility (SI) reaction in the Solanaceae involves molecular recognition of stylar haplotypes by pollen and is mediated by the S-locus from which a stylar-localized S-RNase and several pollen-localized F-box proteins are expressed. S-RNase activity has been previously shown to be essential for the SI reaction, leading to the hypothesis that pollen rejection in incompatible crosses is due to degradation of pollen RNA. We used pollen expressing the fluorescent marker GFP, driven by the LAT52 promoter, to monitor the accumulation of mRNA and protein in pollen after compatible and incompatible pollinations. We find that GFP mRNA and protein gradually accumulate in pollen tubes until at least 18-h post-pollination and, up to this time, are only slightly more abundant in compatible compared with incompatible crosses. However, between 18- and 24-h post-pollination, pollen tube GFP mRNA and protein levels show a dramatic increase in compatible crosses and either remain constant or decrease in incompatible crosses. In contrast to these molecular correlates, the growth rates of compatible and incompatible pollen tubes begin to differ after 6-h post-pollination. We interpret the changes in growth rate at 6-h post-pollination as the previously described transition from autotrophic to heterotrophic growth. Thus, while pollen rejection is generally considered to result from the cytotoxic effects of S-RNase activity, this time course reveals that a difference in the growth rate of compatible and incompatible pollen appears prior to any marked effects on at least some types of pollen RNA.

Female parthenogenetic apomixis and androsporogenetic parthenogenesis in embryonal cells of Araucaria angustifolia : interpolation of progenesis and asexual heterospory in an artificial sporangium

Abstract: Cell fate, development timing and occurrence of reproductive versus apomictic development in gymnosperms are shown to be influenced by culture conditions in vitro. In this study, female parthenogenetic apomixis (fPA), androsporogenetic parthenogenesis (mAP) and progenesis were demonstrated using embryonal initials of Araucaria angustifolia in scaled-up cell suspensions passing through a single-cell bottleneck in darkness and in an artificial sporangium (AS). Expression was based on defined nutrition, hormones and feedforward-adaptive feedback process controls at 23–25 °C and in darkness. In fPA, the nucleus of an embryonal initial undergoes endomitosis and amitosis, forming a diploid egg-equivalent and an apoptotic ventral canal nucleus in a transdifferentiated archegonial tube. Discharge of egg-equivalent cells as parthenospores and their dispersal into the aqueous culture medium were followed by free-nuclear conifer-type proembryogenesis. This replaced the plesiomorphic and central features of proembryogenesis in Araucariaceae. Protoplasmic fusions of embryonal initials were used to reconstruct heterokaryotic expressions of fPA in multiwell plates. In mAP, restitutional meiosis (automixis) was responsible for androsporogenesis and the discharge of monads, dyads, tetrads and polyads. In a display of progenesis, reproductive development was brought to an earlier ontogenetic stage and expressed by embryonal initials. Colchicine increased polyploidy, but androspore formation became aberrant and fragmented. Aberrant automixis led to the formation of chromosomal bouquets, which contributed to genomic silencing in embryonal initials, cytomixis and the formation of pycnotic micronucleated cells. Dispersal of female and male parthenospores displayed heteromorphic asexual heterospory in an aqueous environment.

Generative cell–specific activation of the histone gH2A gene promoter of Lilium longiflorum in tobacco

Abstract: The Lilium longiflorum gH2A promoter is active exclusively in the generative cells of mature pollen in transgenic tobacco expressing the gH2A promoter::GUS (β-glucuronidase) construct as a reporter gene. Temporal and spatial aspects of gH2A promoter activity examined during pollen development in transgenic tobacco reveal that GUS reporter activity was not detected until developing pollen entered the early bicellular developmental stage. Activity was first detected in generative cells at early-mid stages and gradually increased to maximum levels at mid-bicellular stages. The patterns of appearance and longevity of GUS activity in tobacco were very similar to those of gH2A mRNA during pollen development in Lilium. Exogenous treatment with colchicine, a well-known microtubule depolymerize, blocked microspore mitosis and inhibited generative cell differentiation. No GUS signal was detected in the resulting anomalous pollen, which lacked generative cell differentiation. These data strongly suggest that normal generative cell development is essential for activation of the gH2A promoter. Furthermore, these results indicate that common transcriptional activator(s) of the gH2A promoter may be present in both Lilium and Nicotiana, and that such putative factor(s) activates the gH2A promoter only when generative cells undergo normal development.

Pollen factors controlling self-incompatibility strength in Japanese pear.

Abstract: Japanese pear has a genetically controlled self-incompatibility system, but both the pollen-tube growth in a semi in vivo assay and fruit set after self-pollination differ considerably among cultivars. The percentage of styles in which pollen tubes have reached the base ranges from 0 to 36 %, a value determined by culture of styles in vitro, and fruit set ranges from 0.6 to 15.2 %. Based on these data, we have assigned a value for the self-incompatibility weakness to each cultivar. Here, we showed that pollen factors control the degree of self-incompatibility. When the pollen-tube growth of 13 cultivars was compared in a completely compatible ‘Hougetsu’ (S1S7) style, it differed a fair amount among cultivars and showed a significantly positive relation to self-incompatibility weakness (r = 0.707). The degree of self-incompatibility of pear is, therefore, determined by pollen factor(s) unrelated to the S-locus. Although the fruit set and fruit growth of ‘Hougetsu’ were not affected by the pollen donor, a positive relationship was also observed between seed number and self-incompatibility weakness (r = 0.972). However, in a style with no S-RNase production (genotype: S4smS4sm), the relationship disappeared (r = 0.341) and pollen-tube growth was promoted by 12–36 % except in one cultivar. These results suggest that S-RNase functions as a cytotoxin on compatible pollen in a cultivar-dependent manner, and that the degree of inhibition is determined by pollen factor(s) unrelated to the S-locus. The pollen factor also functions on S-RNase in incompatible styles, resulting in a different degree of self-incompatibility.

Sexual fusion of protoplasts in a marine green alga, Bryopsis plumosa.

Abstract: We isolated protoplasts from male and female gametophytes of a strictly dioecious strain of the coenocytic marine green alga Bryopsis plumosa. The protoplasts successfully developed into macrothalli. These in turn produced swimming cells, which appeared similar to biflagellated gametes even when the mixed protoplasts were comprised of protoplasm from male and female gametophytes. We found that swimming cell sizes depended on the male/female protoplasm ratio; macrothalli successfully produced swimming cells with male/female protoplasm ratios of 10:0; 9:1; 7:3; 5:5; 1:9; and 0:10. In male/female protoplasm ratios ranging from equal to strongly female biased (5:5; 3:7; 1:9), swimming cells exhibited normal behaviors of gametes and resultant zygotes, displaying positive and negative phototaxis, respectively. Negatively phototactic swimming cells were quadriflagellated and had two nuclei, apparently as a result of fusion, but never developed into microthalli. Thus, these swimming cells might lack functionality essential for normal gametes. Our findings suggested that natural monoecy observed in this genus did not originate from hybridization of protoplasm between the sexes.