A novel method for efficient in vitro germination and tube growth of Arabidopsis thaliana pollen.

In addition to its importance in studies of plant reproduction and fertility, pollen is as widely employed as a model system of cell growth and development. This work demands robust, reproducible methods to induce pollen germination and morphologically normal growth of pollen tubes in vitro. Despite numerous advantages of Arabidopsis thaliana as a model plant, such experiments on pollen germination and pollen tube growth have often proved challenging. Our new method employs a physical cellulosic membrane, overlying an agarose substrate. By modulating the substrate composition, we provide important insights into the mechanisms promoting pollen growth both in vitro and in vivo. This effective new technical approach to A. thaliana pollen germination and tube growth results in swift, consistent and unprecedented levels of germination to over 90%. It can also promote rapid growth of long, morphologically normal pollen tubes. This technical development demonstrates that exogenous spermidine and a cellulosic substrate are key factors in stimulating germination. It has potential to greatly assist the study of reproduction in A. thaliana and its closest relatives, not only for the study of germination levels and pollen tube growth dynamics by microscopy, but also for biochemical and molecular analysis of germinating pollen.

Small RNA activity and function in angiosperm gametophytes.

Small non-coding RNAs are key post-transcriptional and transcriptional regulators of plant gene expression in angiosperm sporophytes. In recent years, gametophytic small RNAs have also been investigated, predominantly in Arabidopsis male gametophytes, revealing features in common with the sporophyte as well as some surprising differences. Transcriptomic and deep-sequencing studies confirm that multiple small RNA pathways operate in male gametophytes, with over 100 miRNAs detected throughout development. Trans-acting siRNA pathways that are associated with novel phased transcripts in pollen, and the nat-siRNA pathway have important roles in pollen maturation and gamete function. Moreover, a role for siRNA-triggered silencing of transposable elements in male and female germ cells has been established, a feature in common with the role of piRNAs in animal germlines. Current evidence supports an integral role for small RNAs in angiosperm gametophyte development and it can be anticipated that novel small RNAs with significant roles in germline development and genome integrity await discovery.

Molecular mechanisms of self-incompatibility in flowering plants and fungi - different means to the same end.

Hermaphrodite flowering plants and fungi face the same sexual dilemma - how to avoid self-fertilization. Both have evolved ingenious recognition systems that reduce or eliminate the possibility of selfing. These self-incompatibility (SI) systems offer unique opportunities to study recognition and signalling in non-animal cells and also represent model systems for studying the evolution of breeding systems at a molecular level. In this review, the authors discuss recent molecular data that predict an astonishing diversity in the cellular mechanisms of SI operating in flowering plants and fungi.

Pollen stigma interactions: so near yet so far.

Getting a firm grip on the 'S' (incompatibility)-loci, which encourage outbreeding in many flowering plants, continues to be a frustrating exercise. Only last year it seemed that all the male and female S-locus factors that regulate self-incompatibility in a key group of plants - Brassica - had at last been characterized. However, it now appears that the first S-locus product to be identified, does not, after all, play a part in determining S-specificity.

The epigenetic basis of gender in flowering plants and mammals.

What makes a sperm male or an egg female, and how can we tell? A gamete's gender could be defined in many ways, such as the sex of the individual or organ that produced it, its cellular morphology, or its behaviour at fertilization. In flowering plants and mammals, however, there is an extra dimension to the gender of a gamete--due to parental imprinting, some of the genes it contributes to the next generation will have different expression patterns depending on whether they were maternally or paternally transmitted. The non-equivalence of gamete genomes, along with natural and experimental modification of imprinting, reveal a level of sexual identity that we describe as 'epigender'. In this paper, we explore epigender in the life history of plants and animals, and its significance for reproduction and development.

GermOnline, a cross-species community knowledgebase on germ cell differentiation.

GermOnline provides information and microarray expression data for genes involved in mitosis and meiosis, gamete formation and germ line development across species. The database has been developed, and is being curated and updated, by life scientists in cooperation with bioinformaticists. Information is contributed through an online form using free text, images and the controlled vocabulary developed by the GeneOntology Consortium. Authors provide up to three references in support of their contribution. The database is governed by an international board of scientists to ensure a standardized data format and the highest quality of GermOnline's information content. Release 2.0 provides exclusive access to microarray expression data from Saccharomyces cerevisiae and Rattus norvegicus, as well as curated information on approximately 700 genes from various organisms. The locus report pages include links to external databases that contain relevant annotation, microarray expression and proteome data. Conversely, the Saccharomyces Genome Database (SGD), S.cerevisiae GeneDB and Swiss-Prot link to the budding yeast section of GermOnline from their respective locus pages. GermOnline, a fully operational prototype subject-oriented knowledgebase designed for community annotation and array data visualization, is accessible at http://www.germonline.org. The target audience includes researchers who work on mitotic cell division, meiosis, gametogenesis, germ line development, human reproductive health and comparative genomics.

Plant meiosis: the means to 1N.

Meiosis is pivotal in the life history of plants. In addition to providing an opportunity for genetic reassortment, it marks the transition from diploid sporophyte to haploid gametophyte. Recent molecular data suggest that, like animals, plants possess a common set of genes (also conserved in eukaryotic microorganisms) responsible for meiotic recombination and chromosome segregation. However, unlike animals, plant meiocytes do not differentiate from a pool of primordial germ cells, but rather arise de novo from a germline formed from sub-epidermal cells in the anthers and ovules. Mutants defective in the specification of these reproductive cell lines and disrupted in different aspects of the meiotic process are beginning to reveal many features unique to plant meiosis.

An evaluation of Coomassie Brilliant Blue as a stain for quantitative microdensitometry of protein in section.

The specificity and stoichiometry of the binding of Coomassie Brilliant Blue (CBB) to protein in section has been examined using both frozen protein matrices and plant material. The maximum adsorbance of the stain, bound and in solution, was found to be 620 nm although variation in the results at this wavelength necessitated measurements to be made at 600 nm. After enzyme treatments of sectioned plant material embedded in resin, all CBB-binding biological material was shown to be sensitive to non-specific protease. The relationship between optical density at 600 nm and section thickness was tested statistically against the Lambert-Beer law, using microdensitometry of cryostat-sectioned, frozen genatine solution. The analyses showed conclusively that, under these conditions, CBB adheres strongly to the Lambert-Beer relationship. CBB may thus be considered as a very specific protein stain, eminently suited both to cytological observation and quantitative microdensitometry.

Identification of pollen components regulating pollination-specific responses in the stigmatic papillae of Brassica oleracea.

When components of the mature pollen grain of Brassica oleracea are applied to the cuticularised surfaces of the stigmatic papilla, a number of changes can take place in the architecture of the subjacent cell wall. Treatment of the papillar surface with isolated pollen coating evokes a rapid and extensive expansion of the outer of the two stigmatic wall layers. This response occurs within 4 h but is restricted to those regions where no callose is formed and where the coating exhibits a characteristic increase in electron opacity. This study establishes that the coating alone is responsible for initiating the expansion of the outer wall, which is considered to be an essential step preceding penetration of the stigma surface by the pollen tube. Vesicle-like inclusions, some staining intensely, occasionally occur in regions of the wall expanded by isolated coating, sometimes fusing to form a subcuticular 'boundary layer'. These structures are not observed under compatible grains in vivo and their presence is regarded as artefaetual. However, close examination of the plasma membrane at sites beneath areas of expanded cell wall reveals membrane-bound structures resembling the vesicles being generated by the cytoplasm and moving into the apoplast. These data indicate that signals from the coating initiate the loosening of the wall matrix by stimulating an unusual form of localized secretion which appears to be an essential prerequisite for stigmatic penetration. Isolated pollen coating contaminated with fragments of the grain itself engender a very different response, including the formation of densely-staining vesicles accompanied by extensive accumulations of callose; wall expansion never occurs under these circumstances. Self-pollinations in B. oleracea are frequently accompanied by the synthesis of stigmatic callose, the presence of which has, in the past, been interpreted as forming a structural barrier to incompatible pollen tubes. However, callose elicited both by self-pollinations and coating supplemented by killed grains is independent of protein synthesis and occurs in the presence of the phosphatase inhibitor, okadaic acid. Since both these inhibitors also overcome self-incompatibility (SI) in B. oleracea, these data strongly suggest that callose synthesis is unrelated to the operation of SI, and it is proposed that callose, when formed, is elicited by molecules released from the necrotic pollen protoplast. Interestingly, isolated pollen coating of self- and cross-genotypes does not readily elicit callose. The significance of these changes in the stigmatic wall is discussed in the perspective of current views on pollination and self-incompatibility in Brassica.

Anther dehiscence in Lycopersicon esculentum: II. Water relations.

Water tracers have been used to demonstrate the continued functioning of the anther filament during dehiscence in Lycopersicon esculentum Mill. Since Lycopersicon possesses neither nectar nor nectaries dehiscence cannot be related to sugar secretion, as has been demonstrated for other plants. The anthers seem structurally adapted for water conservation and the only transpirational loss of the flower appears to be through the petals. Transpiration from the anthers themselves thus appears not to be involved in regulating dehiscence. Anther opening is preceded by dehydration of the locule and circumstantial evidence indicates it to be an active process, with water being exported through the filaments to the petals along an osmotic gradient generated by starch/sugar interconversion. Measurement of water potential differentials between the petals and anthers points to the mechanism driving this export of water from the anthers. Interestingly, turgor pressure is maintained in the majority of the anther tissues until senescence, remaining remarkably constant against a background of dramatic changes in osmotic potential. These data, combined with the observation that the hydration level of the anthers falls only to 70% at senescence, indicates that dehiscence cannot primarily be a desiccatory process. Some domains within the anther do desiccate, but these are strictly localized. Thus, while hydration levels of living tissues remain independent of the environment, those observed to undergo protoplast degeneration and wall collapse exhibit environmentally-linked hygroscopic absorption. Dehiscence thus emerges as an orchestrated programme of structural and physiological events leading to the desiccation of specific domains of the anthers. These findings are discussed in terms of current models for anther dehiscence in flowering plants.

The role of the exine coating in pollen-stigma interactions in Brassica oleracea L.

When the dehydrated pollen grain of Brassica oleracea L. alights on a receptive stigma the pollen coat flows out from the exine to form an appresoria-like 'foot' and, within a matter of some 30 min, gross ultrastructural changes become visible both within the protoplast and in the foot itself. These changes are interpreted as reflecting the limited movement of water, and presumably other materials, from the stigma to the grain. The compatible pollen grain then continues to take up water, whilst undergoing other cytoplasmic changes and eventually producing the pollen tube. The tube grows from the colpus towards the point of contact with the stigma, beneath which the outer layer of the papillar wall has become more loosely packed. The pollen tube enters the wall at this point and, as a consequence of its rapid extension, the grain is frequently lifted away from the papilla. The tube then grows between two layers of the pectocellulosic papillar wall into the stigmatic parenchyma, where it follows an intercellular route. These events are discussed in terms of current views of the relationship between male and female cells at these early stages of the pollen stigma interaction.

Pollination in species with dry stigmas: the nature of the early stigmatic response and the pathway taken by pollen tubes.

Interaction between the pollen grain, pollen tube and the stigma surface has been studied in five species regarded as possessing dry stigma surfaces; Brassica oleracea L., Arabidopsis thaliana (L.), Heynh, Papaver rhoeas L., Cosmos bipinnatus Cav. and Helianthus annuus L. In B. oleracea and A. thaliana, stigmatic response to pollination includes events in the papillar cytoplasm and changes to the stigmatic surface beneath the grain. In particular a specialized outer element of the cell wall expands prior to pollen tube penetration. The pollen tube, which enters through a 'foot' of pollen coating, grows in a space generated between an inner and the outer element of the wall and extends to the base of the papilla where it enters the middle lamellae of the subjacent cell layer. However, in A. thaliana tubes frequently were seen to penetrate all components of the stigmatic cell wall, an event only previously recorded in immature stigmas of B. oleracea. In self pollinations of self-incompatible B. oleracea involving strong S (incompatibility) alleles no changes take place in the papillar cell wall. In P. rhoeas the stigmatic surface responds to self and cross pollination by the secretion of electron-lucent material beneath the cuticle, causing it to become detached from the outer surface of the stigmatic cell wall. The pollen tubes then penetrate the cuticle and grow towards the base of the papilla in the space thus generated. The tubes continue to grow intercellularly in the transmitting tissue which lies horizontally in rays beneath the papillae. In members of the Compositae, C. bipinnatus and H. annuus, both stigma and pollen respond to pollination by producing copious quantities of an electron-opaque matrix, which frequently causes individual papillae to adhere together. Pollen tubes, which are formed following both compatible and incompatible intraspecific pollinations, grow into this matrix and toward the base of the papillae. There, in common with the other plants studied, they grow intercellularly and enter the transmitting tissue of the style. These findings are discussed in the light of current views of the mechanisms operating during angiosperm pollination and of the significance of the stigmatic response to the functioning of self-incompatibility mechanisms.

MicroRNA misregulation: an overlooked factor generating somaclonal variation?

Somaclonal variation is an important phenomenon that can be observed at high levels in plant tissue culture. Although known to science since plant cell culture techniques were first developed, its origins remain mysterious. Here, we propose that misregulation of microRNAs and small RNA pathways can make a significant contribution to the phenomenon. For many reasons, microRNAs and related small RNAs appear ideal candidates. Their mode of action gives them disproportionate influence over the transcriptome, proteome and epigenome. They regulate important developmental and physiological events such as meristem formation, phase changes and hormone responses. However, the genomic locations of microRNA genes and their unique biogenesis might make them unusually susceptible to aberrant regulation in vitro.

Epigenetics and its implications for plant biology 2. The 'epigenetic epiphany': epigenetics, evolution and beyond.

SCOPE: In the second part of a two-part review, the ubiquity and universality of epigenetic systems is emphasized, and attention is drawn to the key roles they play, ranging from transducing environmental signals to altering gene expression, genomic architecture and defence. KEY ISSUES: The importance of transience versus heritability in epigenetic marks is examined, as are the potential for stable epigenetic marks to contribute to plant evolution, and the mechanisms generating novel epigenetic variation, such as stress and interspecific hybridization. FUTURE PROSPECTS: It is suggested that the ramifications of epigenetics in plant biology are immense, yet unappreciated. In contrast to the ease with which the DNA sequence can be studied, studying the complex patterns inherent in epigenetics poses many problems. Greater knowledge of patterns of epigenetic variation may be informative in taxonomy and systematics, as well as population biology and conservation.

Characterization of the three Arabidopsis thaliana RAD21 cohesins reveals differential responses to ionizing radiation.

The RAD21/REC8 gene family has been implicated in sister chromatid cohesion and DNA repair in several organisms. Unlike most eukaryotes, Arabidopsis thaliana has three RAD21 gene homologues, and their cloning and characterization are reported here. All three genes, AtRAD21.1, AtRAD21.2, and AtRAD21.3, are expressed in tissues rich in cells undergoing cell division, and AtRAD21.3 shows the highest relative level of expression. An increase in steady-state levels of AtRAD21.1 transcript was also observed, specifically after the induction of DNA damage. Phenotypic analysis of the atrad21.1 and atrad21.3 mutants revealed that neither of the single mutants was lethal, probably due to the redundancy in function of the AtRAD21 genes. However, AtRAD21.1 plays a critical role in recovery from DNA damage during seed imbibition, prior to germination, as atrad21.1 mutant seeds are hypersensitive to radiation damage.

Epigenetics and its implications for plant biology. 1. The epigenetic network in plants.

BACKGROUND: Epigenetics has rapidly evolved in the past decade to form an exciting new branch of biology. In modern terms, 'epigenetics' studies molecular pathways regulating how the genes are packaged in the chromosome and expressed, with effects that are heritable between cell divisions and even across generations. CONTEXT: Epigenetic mechanisms often conflict with Mendelian models of genetics, and many components of the epigenetic systems in plants appeared anomalous. However, it is now clear that these systems govern how the entire genome operates and evolves. SCOPE: In the first part of a two-part review, how epigenetic systems in plants were elucidated is addressed. Also there is a discussion on how the different components of the epigenetic system--regulating DNA methylation, histones and their post-translational modification, and pathways recognizing aberrant transcripts--may work together.

The structure and chemistry of plastid differentiation during male meiosis in Lilium henryi.

The unusual content of the plastids characteristic of male meiosis and microsporogenesis in Lilium henryi has been investigated using enzymic digestion of material prepared for electron microscopy. Apart from membranous tubules and osmiophilic droplets, commonly regarded as normal constituents of the stroma of undifferentiated plastids, these organelles contained a single conspicuous association between membrane and particulate material and wefts of filaments. Enzymic digestion revealed the membrane-particle association (MPA) to contain RNA and some protein, and the filaments to be entirely proteinaceous. The use of DNase on this material proved both unreliable and, on occasions, unspecific, so no new information has emerged as to the disposition of DNA within these organelles. Many of the plastids divide immediately after meiosis and the MPAs normally divide in step with the organelles. The activities of the MPA and those of the filaments are discussed in terms of changes in the organelle population as a whole, and also in consideration of the fundamental events occurring elsewhere in the cells during the alternation from the diploid sporophyte to the haploid gametophyte.

Pollen-stigma interactions in Brassica. IV. Structural reorganization in the pollen grains during hydration.

With the aid of osmium tetroxide vapour, dry pollen and pollen at various stages of hydration has been fixed anhydrously for examination with the transmission electron microscope (TEM). In addition to establishing features characteristic of grains at different states of hydration, this technique has enabled the detection of a superficial layer investing both the exine and the pollen coating. This layer, some 10 nm in depth, binds both lanthanum and Alcian Blue and is shown to be the first component of the pollen grain to make contact with the stigmatic pellicle. The use of vapour fixation has also rendered it possible to chart the passage of water into the pollen grains with great accuracy, for each level of hydration displays a strikingly different cytoplasmic organization. For example, dry pollen is characterized by the presence of unusual structures at the protoplast surface and large numbers of spherical fibrillar bodies, whilst the protoplast of hydrating pollen is conspicuously stratified and contains a peripheral layer of membranous cisternae, subjacent to which is a fibrillar matrix derived from the spherical bodies found in the dry grains. Vapour-fixed, fully hydrated pollen resembles conventionally fixed grains. The pollen coating appears electron-translucent after anhydrous fixation and contains discrete, slightly rounded bodies some 50 nm in diameter. The uptake of water by grains on the stigma is accompanied by conspicuous structural changes in this layer for, after a short period in contact with the papillar surface, the spherical bodies rapidly disappear and the coat becomes electron-opaque. Close examination of this 'converted' coating reveals the presence of membranous vesicles and other structural components.