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Polyploidization is a fundamental process in plant evolution. One of the biggest challenges faced by a new polyploid is meiosis, particularly discriminating between multiple related chromosomes so that only homologous chromosomes synapse and recombine to ensure regular chromosome segregation and balanced gametes. Despite its large genome size, high DNA repetitive content and similarity between homoeologous chromosomes, hexaploid wheat completes meiosis in a shorter period than diploid species with a much smaller genome. Therefore, during wheat meiosis, mechanisms additional to the classical model based on DNA sequence homology, must facilitate more efficient homologous recognition. One such mechanism could involve exploitation of differences in chromosome structure between homologues and homoeologues at the onset of meiosis. In turn, these chromatin changes, can be expected to be linked to transcriptional gene activity. In this study, we present an extensive analysis of a large RNA-Seq data derived from six different genotypes: wheat, wheat-rye hybrids and newly synthesized octoploid triticale, both in the presence and absence of thePh1locus. Plant material was collected at early prophase, at the transition leptotene-zygotene, when the telomere bouquet is forming and synapsis between homologues is beginning. The six genotypes exhibit different levels of synapsis and chromatin structure at this stage; therefore, recombination and consequently segregation, are also different. Unexpectedly, our study reveals that neither synapsis, whole genome duplication nor the absence of thePh1locus are associated with major changes in gene expression levels during early meiotic prophase. Overall wheat transcription at this meiotic stage is therefore highly resilient to such alterations, even in the presence of major chromatin structural changes. This suggests that post-transcriptional and post-translational processes are likely to be more important. Thus, further studies will be required to reveal whether these observations are specific to wheat meiosis, and whether there are significant changes in post-transcriptional and post-translational modifications in wheat and other polyploid species associated with their polyploidisation.

Wild relatives provide an important source of useful traits in wheat breeding. Wheat and wild relative hybrids have been widely used in breeding programs to introduce such traits into wheat. However, successful introgression is limited by the low frequency of homoeologous crossover (CO) between wheat and wild relative chromosomes. Hybrids between wheat carrying a 70 Mb deletion on chromosome 5B (ph1b) and wild relatives, have been exploited to increase the level of homoeologous CO, allowing chromosome exchange between their chromosomes. In ph1b-rye hybrids, CO number increases from a mean of 1 CO to 7 COs per cell. CO number can be further increased up to a mean of 12 COs per cell in these ph1b hybrids by treating the plants with Hoagland solution. More recently, it was shown that the major meiotic crossover gene ZIP4 on chromosome 5B (TaZIP4-B2) within the 70 Mb deletion, was responsible for the restriction of homoeologous COs in wheat-wild relative hybrids, confirming the ph1b phenotype as a complete Tazip4-B2 deletion mutant (Tazip4-B2 ph1b). In this study, we have identified the particular Hoagland solution constituent responsible for the increased chiasma frequency in Tazip4-B2 ph1b mutant-rye hybrids and extended the analysis to Tazip4-B2 TILLING and CRISPR mutant-Ae variabilis hybrids. Chiasma frequency at meiotic metaphase I, in the absence of each Hoagland solution macronutrient (NH4 H2PO4, KNO3, Ca (NO3)2·4H2O or Mg SO4·7H2O) was analyzed. A significant decrease in homoeologous CO frequency was observed when the Mg2+ ion was absent. A significant increase of homoeologous CO frequency was observed in all analyzed hybrids, when plants were irrigated with a 1 mM Mg2+ solution. These observations suggest a role for magnesium supplementation in improving the success of genetic material introgression from wild relatives into wheat.

Here we present data on a study of the species composition and structure of borokotoh, a habitat associated with central Amazonian seasonally black-water flooded forests. Borokotoh appears never to have been specifically studied before, being mentioned only once or twice in the literature (and then not under this name). This is the first report on the phytosociology of this vegetation association. Borokotoh is, on average, only some 20 m wide, but very species rich (107 species in 60 genera from 34 families in one 0.25-ha plot). It consists of a series of rounded hummocks of fine-grained mud, each 6–12 m in length and 2–3 m in height, separated by narrow sloughs. Hummocks are seasonally inundated almost to their flat tops, which remained dry in the years of the survey. Species are strongly stratified within the borokotoh, with over 78% of identified species being recorded in just one of the three hummock sub-divisions: top, side or in between-hummock slough. This specificity, almost certainly linked to inundation duration and waterlogging tolerance, may explain the high species diversity recorded. The study is significant in demonstrating that undescribed habitats still exist in Amazonia, and that biodiversity there may be structured in ways that are highly localized and habitat-specific. There is clearly a need for more detailed work on borokotoh and similar vegetation associations where high biodiversity may be associated with very specific local features.

The mechanism whereby the same genome can give rise to different cell types with different gene expression profiles is a fundamental problem in biology. Chromatin organization and dynamics have been shown to vary with altered gene expression in different cultured animal cell types, but there is little evidence yet from whole organisms linking chromatin dynamics with development. Here, we used both fluorescence recovery after photobleaching and two-photon photoactivation to show that in stem cells from Arabidopsis thaliana roots the mobility of the core histone H2B, as judged by exchange dynamics, is lower than in the surrounding cells of the meristem. However, as cells progress from meristematic to fully differentiated, core histones again become less mobile and more strongly bound to chromatin. We show that these transitions are largely mediated by changes in histone acetylation. We further show that altering histone acetylation levels, either in a mutant or by drug treatment, alters both the histone mobility and markers of development and differentiation. We propose that plant stem cells have relatively inactive chromatin, but they keep the potential to divide and differentiate into more dynamic states, and that these states are at least in part determined by histone acetylation levels.

Despite possessing related ancestral genomes, hexaploid wheat behaves as a diploid during meiosis. The wheat Ph1 locus promotes accurate synapsis and crossover of homologous chromosomes. Interspecific hybrids between wheat and wild relatives are exploited by breeders to introgress important traits from wild relatives into wheat, although in hybrids between hexaploid wheat and wild relatives, which possess only homoeologues, crossovers do not take place during meiosis at metaphase I. However, in hybrids between Ph1 deletion mutants and wild relatives, crossovers do take place. A single Ph1 deletion (ph1b) mutant has been exploited for the last 40 years for this activity. We show here that selection of chemical induced mutant lines possessing mutations in TaZIP4-B2 exhibit high levels of homoeologous crossovers when crossed with a wild relative. Exploitation of Tazip4-B2 mutants rather than mutants with whole Ph1 locus deletions may improve introgression of wild relative chromosome segments into wheat. Such mutant lines may be more stable over multiple generations, as multivalents causing accumulation of chromosome translocations are less frequent.Key messageExploiting the ZIP4 homologue within the wheat Ph1 locus has identified two wheat mutants through a non-GM route, which can be exploited as an alternative to the Chinese Spring ph1b mutant in wheat introgression strategies.

Despite possessing multiple sets of related (homoeologous) chromosomes, hexaploid wheat (Triticum aestivum) restricts pairing to just true homologs at meiosis. Deletion of a single major locus, Pairing homoeologous1 (Ph1), allows pairing of homoeologs. How can the same chromosomes be processed as homologs instead of being treated as nonhomologs? Ph1 was recently defined to a cluster of defective cyclin-dependent kinase (Cdk)-like genes showing some similarity to mammalian Cdk2. We reasoned that the cluster might suppress Cdk2-type activity and therefore affect replication and histone H1 phosphorylation. Our study does indeed reveal such effects, suggesting that Cdk2-type phosphorylation has a major role in determining chromosome specificity during meiosis.

Eukaryotic chromosomes occupy distinct territories within interphase nuclei. The arrangement of chromosome territories (CTs) is important for replication, transcription, repair and recombination processes. Our knowledge about interphase chromatin arrangement is mainly based on results from in situ labeling approaches. The phylogenetic affiliation of a species, cell cycle, differentiation status and environmental factors are all likely to influence interphase nuclear architecture. In this review we survey current data about relative positioning of CTs, somatic pairing of homologs, and sister chromatid alignment in meristematic and differentiated tissues, using data derived mainly from Arabidopsis thaliana, wheat (Triticum aestivum) and their relatives. We discuss morphological constraints and epigenetic impacts on nuclear architecture, the evolutionary stability of CT arrangements, and alterations of nuclear architecture during transcription and repair.

The current revolution in proteomics has been generated by the combination of very sensitive mass spectrometers coupled to microcapillary liquid chromatography, specific proteolysis of protein mixtures and software that is capable of searching vast numbers of mass measurements against predicted peptides from sequenced genomes. The challenges of post‐genomic plant biology include characterization of protein function, post‐translational modifications and composition of protein complexes as well as deciphering protein complements in intracellular compartments – proteomes of cell organelles. In this review we summarize the current mass spectrometry methods currently being used in plant proteomics and discuss the various tagging strategies that are being used for purification and proteomic analysis of plant protein complexes. Abbreviations: BCCD, biotin carboxyl carrier protein domain; CBP, calmodulin‐binding protein; CID, collision‐induced dissociation; ESI, electrospray ionization; EST, expressed se...

AtTRB1, 2 and 3 are members of the SMH (single Myb histone) protein family, which comprises double-stranded DNA-binding proteins that are specific to higher plants. They are structurally conserved, containing a Myb domain at the N-terminus, a central H1/H5-like domain and a C-terminally located coiled-coil domain. AtTRB1, 2 and 3 interact through their Myb domain specifically with telomeric double-stranded DNA in vitro, while the central H1/H5-like domain interacts non-specifically with DNA sequences and mediates protein-protein interactions. Here we show that AtTRB1, 2 and 3 preferentially localize to the nucleus and nucleolus during interphase. Both the central H1/H5-like domain and the Myb domain from AtTRB1 can direct a GFP fusion protein to the nucleus and nucleolus. AtTRB1-GFP localization is cell cycle-regulated, as the level of nuclear-associated GFP diminishes during mitotic entry and GFP progressively re-associates with chromatin during anaphase/telophase. Using fluorescence recovery after photobleaching and fluorescence loss in photobleaching, we determined the dynamics of AtTRB1 interactions in vivo. The results reveal that AtTRB1 interaction with chromatin is regulated at two levels at least, one of which is coupled with cell-cycle progression, with the other involving rapid exchange.

Transcription-related chromatin decondensation has been studied in mammals for clusters of structurally and/or functionally related genes that are coordinately regulated (e.g., the homeobox locus in mice and the major histocompatability complex locus in humans). Plant genes have generally been considered to be randomly distributed throughout the genome, although several examples of metabolic gene clusters for synthesis of plant defense compounds have recently been discovered. Clustering provides for genetic linkage of genes that together confer a selective advantage and may also facilitate coordinate regulation of gene expression by enabling localized changes in chromatin structure. Here, we use cytological methods to investigate components of a metabolic gene cluster for synthesis of developmentally regulated defense compounds (avenacins) in diploid oat (Avena strigosa). Our experiments reveal that expression of the avenacin gene cluster is associated with cell type–specific chromatin decondensation, providing new insights into regulation of gene clusters in plants. Importantly, chromatin decondensation could be visualized not only at the large-scale level but down to the single gene level. We further show that the avenacin and sterol pathways are likely to be inversely regulated at the level of transcription.

The mechanisms of long-term adaptation to low oxygen environment are quite well studied, but little is known about the sensing of oxygen shortage, the signal transduction and the shortterm effects of hypoxia in plant cells. We have found that an RNA helicase eIF4A-III, a putative component of the Exon Junction Complex, rapidly changes its pattern of localisation in the plant nucleus under hypoxic conditions. In normal cell growth conditions GFPeIF4A-III was mainly nucleoplasmic, but in hypoxia stress conditions it moved to the nucleolus and splicing speckles. This transition occurred within 15–20 min in Arabidopsis culture cells and seedling root cells, but took more than 2 h in tobacco BY-2 culture cells. Inhibition of respiration, transcription or phosphorylation in cells and ethanol treatment had similar effects to hypoxia. The most likely consequence is that a certain mRNA population will remain bound to the eIF4A-III and other mRNA processing proteins, rather than being transported from the nucleus to the cytoplasm, and thus its translation will be suspended.

Here, we identify the Arabidopsis thaliana ortholog of the mammalian DEAD box helicase, eIF4A-III, the putative anchor protein of exon junction complex (EJC) on mRNA. Arabidopsis eIF4A-III interacts with an ortholog of the core EJC component, ALY/Ref, and colocalizes with other EJC components, such as Mago, Y14, and RNPS1, suggesting a similar function in EJC assembly to animal eIF4A-III. A green fluorescent protein (GFP)-eIF4A-III fusion protein showed localization to several subnuclear domains: to the nucleoplasm during normal growth and to the nucleolus and splicing speckles in response to hypoxia. Treatment with the respiratory inhibitor sodium azide produced an identical response to the hypoxia stress. Treatment with the proteasome inhibitor MG132 led to accumulation of GFP-eIF4A-III mainly in the nucleolus, suggesting that transition of eIF4A-III between subnuclear domains and/or accumulation in nuclear speckles is controlled by proteolysis-labile factors. As revealed by fluorescence recovery after photobleaching analysis, the nucleoplasmic fraction was highly mobile, while the speckles were the least mobile fractions, and the nucleolar fraction had an intermediate mobility. Sequestration of eIF4A-III into nuclear pools with different mobility is likely to reflect the transcriptional and mRNA processing state of the cell.

Cyclin-dependent kinases (CDKs) play key regulatory roles in diverse cellular functions, including cell-cycle progression, transcription and translation. In plants, CDKs have been classified into several groups, named A through to G, but the functions of most are poorly characterized. CDKCs are known to phosphorylate the C-terminal domain (CTD) of RNA polymerase II (RNAP II), and therefore the CDKC-cyclinT (CycT) complex may have a role similar to the animal CDK9-CycT complex of the positive transcription elongation factor b (P-TEFb). However, we found that the predicted structure of the Arabidopsis CDKC2 protein is more similar to the mammalian cdc2-related kinase, CRK7, than to CDK9. CRK7 is proposed to link transcription with splicing, and CDKC2 contains all the structural features of CRK7 that make the latter distinct from CDK9. Consistent with this, we show that GFP-CDKC2 fusion proteins co-localize with spliceosomal components, that the expression of CDKC2 modifies the location of these components, and that co-localization was dependent on the transcriptional status of the cells and on CDKC2-kinase activity. We propose, therefore, that the Arabidopsis CDKC2 combines the functions of both CRK7 and CDK9, and could also couple splicing with transcription.

The expression pattern of genes can identify the cells in which the respective proteins are active during development. As a step towards defining the genetic network that controls the development of roots, a high-throughput method of whole-mount in situ hybridization has been developed that does not require expensive equipment and allows the definition of the expression patterns of 137 transcription factor genes in young developing roots. Of the 137 transcription factors, 81.8% were expressed in the root while 18.2% showed no detectable expression. In all three proximal distal zones (meristem, elongation, and differentiation) of the root, 52.6% were expressed whereas 21.2% were expressed in only two zones. Eight percent of the genes were expressed in a single proximal distal zone. Cell-specific gene expression patterns were also detected. This rapid approach identified potential key regulators of cell differentiation and provides important spatial information for the expression patterns of a large number of transcriptional regulators that function during root development.

Background Recent developments in both microscopy and fluorescent protein technologies have made live imaging a powerful tool for the study of plant cells. However, the complications of keeping plant material alive during a long duration experiment while maintaining maximum resolution has limited the use of these methods. Results Here, we describe an imaging chamber designed to overcome these limitations, which is flexible enough to support a range of sizes of plant materials. We were able use confocal microscopy to follow growth and development of plant cells and tissues over several days. The chamber design is based on a perfusion system, so that the addition of drugs and other experimental treatments are also possible. Conclusions In this article we present a design of imaging chamber that makes it possible to image plant material with high resolution for extended periods of time. Electronic supplementary material The online version of this article (doi:10.1186/s13007-015-0065-7) contains supplementary material, which is available to authorized users.

SUMMARY As part of a long-term open-air fumigation experiment, Scots pine was exposed continuously to SO2 pollution following a predetermined pattern of hourly mean values based upon monitoring data from a site in central England. Although annual mean concentrations did not exceed 22 ppb, up to 200% of trees developed foliar necrosis in three consecutive growing seasons similar to published descriptions of acute SO2 damage. The number of trees affected increased with S02 concentration, and there was a significant tendency for the same trees to be affected in consecutive years, implying that sensitivity to these levels was genetically based. As a group, trees showing necrotic symptoms were not significantly retarded in their growth, although their 1990 budburst was significantly delayed and the few trees which lost over 50 % of their foliage were visibly stunted. Fumigation of potted trees for short periods in chambers with up to 1000 ppb SO2 failed to produce any comparable effects. Regression analyses between the number of plants affected each year and a range of gas concentration parameters showed mean concentrations during a critical period in May to be most closely related to the development of symptoms, although no effects became visible until approximately 5 wk later. The start date for the critical period differed between years, and for the two years for which budburst data were available it coincided with the start of needle elongation. The duration of the critical period lay in the range 6-14 d. Linear extrapolation from the data suggested the threshold mean S02 concentration for visible injury to be in the range 6-8 ppb (measured during the critical period during needle expansion) although this could underestimate the true value.

Knowledge of the organization of transcription, RNA processing and transport, and the assembly of complexes such as the ribosome, spliceosome and other RNPs is essential to understanding gene expression. Over several years, the nucleolus and Cajal bodies have been examined in plants, and recently, various other sub-nuclear domains that are involved in RNA metabolism and hormonal responses have been discovered. These novel domains illustrate the complexity and subtlety of expression control and herald a new era of research on the molecular and cell biology of plant nuclei.

We describe a streamlined and systematic method for cloning green fluorescent protein (GFP)-open reading frame (ORF) fusions and assessing their subcellular localization in Arabidopsis thaliana cells. The sequencing of the Arabidopsis genome has made it feasible to undertake genome-based approaches to determine the function of each protein and define its subcellular localization. This is an essential step towards full functional analysis. The approach described here allows the economical handling of hundreds of expressed plant proteins in a timely fashion. We have integrated recombinational cloning of full-length trimmed ORF clones (available from the SSP consortium) with high-efficiency transient transformation of Arabidopsis cell cultures by a hypervirulent strain of Agrobacterium. To demonstrate its utility, we have used a selection of trimmed ORFs, representing a variety of key cellular processes and have defined the localization patterns of 155 fusion proteins. These patterns have been classified into five main categories, including cytoplasmic, nuclear, nucleolar, organellar and endomembrane compartments. Several genes annotated in GenBank as unknown have been ascribed a protein localization pattern. We also demonstrate the application of flow cytometry to estimate the transformation efficiency and cell cycle phase of the GFP-positive cells. This approach can be extended to functional studies, including the precise cellular localization and the prediction of the role of unknown proteins, the confirmation of bioinformatic predictions and proteomic experiments, such as the determination of protein interactions in vivo, and therefore has numerous applications in the post-genomic analysis of protein function.

We report a two year survey of saprotrophic fungi colonising woodchip mulch at two contrasting sites in Surrey, a formal garden and a roadside planting scheme. Two alien species ( Stropharia aurantiaca and Psilocybe cyanescens ) occurred at both sites, along with other notable species including Psilocybe percevalii, Agrocybe putaminum and Collybia biformis . Very little fruiting occurred on areas mulched with bark chippings. In the formal garden there was virtually no repeat fruiting in successive years. At the roadside site most species fruited profusely in the first season after deposition, then decreased greatly in numbers in the second year. It is concluded that ornamental woodchips, but not bark chippings, can support diverse fungal communities including unusual species, although these assemblages are short-lived.

We have developed a novel pre-embedding in situ hybridization labelling method for electron microscopy which has given much greater sensitivity and higher labelling levels than have been achieved previously, together with good ultrastructural preservation. Vibratome sections of plant tissue were labelled throughout their thickness with 1 nm gold antibodies and then silver enhanced, embedded in resin and sectioned for electron microscopy. Because the labelling extends throughout the depth of the specimen, this method permits the study of the 3D arrangement of the labelling at the electron microscope level by either stereo-pair recording, tomographic reconstruction or 3D reconstruction from serial sections. In this paper we describe the application of this method to study the organization of rDNA in pea root tissue.

The rDNA transcription units are enormous macromolecular structures located in the nucleolus and containing 50-100 RNA polymerases together with the nascent pre-rRNA attached to the rDNA. It has not previously been possible to visualize nucleolar transcription units directly in intact nucleoli, although highly spread preparations in the electron microscope have been imaged as "Christmas trees" 2-3 microm long. Here we determine the relative conformation of individual transcription units in Pisum sativum plant nucleoli using a novel labelling technique. Nascent transcripts were detected by a highly sensitive silver-enhanced 1 nm gold procedure, followed by 3D electron microscopy of entire nucleoli. Individual transcription units are seen as conical, elongated clusters approximately 300 nm in length and 130 nm in width at the thickest end. We further show that there were approximately 300 active ribosomal genes in the nucleoli examined. The underlying chromatin structure of the transcribing rDNA was directly visualized by applying a novel limited extraction procedure to fixed specimens in order to wash out the proteins and RNA, thus specifically revealing DNA strands after uranyl acetate staining. Using this technique, followed by post-embedding in situ hybridization, we observed that the nucleolar rDNA fibres are not extended but show a coiled, thread-like appearance. Our results show for the first time that native rDNA transcription units are linear, compacted Christmas trees.

Soil N transformations were studied at Ironhill, near Liphook, UK as part of a forest fumigation experiment. Nitrification potential was measured in a humoferric podzol soil, of pH 3 (in 0.01 M CaCl2). An initial experiment into nitrogen mineralisation potential indicated that nitrification was linked strongly to the species of coniferous tree growing in the soil. Transfer of soil solution between soils had no influence on mineralisation potential and allelopathic effects of the trees were not demonstrated. The initial finding was attributed subsequently to the type of ground vegetation and its management. Attempts to reproduce soil conditions, which promoted nitrification, were partially successful.

Ironhill, near Liphook, UK, was the site of a forest fumigation experiment. Nitrogen cycling within the humoferric podzol soil was a component of the study into the impacts of sulphur dioxide and ozone on coniferous trees. Variation in total soil N and N mineralisation was too great to determine impacts from the fumigant gases. Differences in the nitrogen mineralisation potential of the soils were unrelated to the initial levels of mineral or total N, or to pH. Mineralisation potential was affected by temperature and a Q10 of approximately 3 was demonstrated. Mineralisation potential was reduced in very dry soils, but the wetting of these dry soils did not result in enhanced mineralisation, relative to fresh samples of equivalent moisture content. Nitrification potential was detected in this forest soil of pH 3 (in 0.01 m CaCl2).

The kinases responsible for phosphorylation of inositol-containing lipids are essential for many aspects of normal eukaryotic cell function. Genetic and biochemical studies have established that the phosphatidylinositol (PtdIns) 3-kinase encoded by the yeast VPS34 gene is essential for the efficient sorting and delivery of proteins to the vacuole; the kinase encoded by the human VPS34 homolog has been equally implicated in the control of intracellular vesicle traffic. The plant VPS34 homolog also is required for normal growth and development, and although a role for PtdIns 3-kinase in vesicle trafficking is likely, it has not been established. In this study, we have shown that considerable PtdIns 3-kinase activity is associated with the internal matrix of nuclei isolated from carrot suspension cells. Immunocytochemical and confocal laser scanning microscopy studies using the monoclonal antibody JIM135 (John Innes Monoclonal 135), raised against a truncated version of the soybean PtdIns 3-kinase, SPI3K-5p, revealed that this kinase appears to have a distinct and punctate distribution within the plant nucleus and nucleolus. Dual probing of root sections with JIM135 and anti-bromo-UTP antibodies, after in vitro transcription had been allowed to proceed in the presence of bromo-UTP, showed that SPI3K-5p associates with active nuclear and nucleolar transcription sites. These findings suggest a possible link between PtdIns 3-kinase activity and nuclear transcription in plants.

Morphometric data were collected from Dactylorhiza growing on two types of industrial waste (pulverized fuel ash or PFA, and Leblanc process waste) during the summer of 1997. Three species grew on PFA ( D. fuchsii , D. incarnata , D. praetermissa ). The same species plus D. purpurella grew on the Leblanc site, although on both substrates the majority of plants failed to correspond precisely with published descriptions, introducing an element of subjectivity into the field identifications. Principal Components Analysis and Detrended Correspondence Analysis ordinations confirmed that textbook species descriptions corresponded to extremes of multivariate space. Cluster Analysis failed to produce a useful resolution of the data. Discriminant Functions Analysis initially gave the misleading result that any plant with spotted leaves was D. fuchsii , but produced useful results after leaf spotting was removed from the analysis. On PFA sites hybrids appeared to be mainly D. praetermissa×D. fuchsii (= D. grandis ) or D. praetermissa×D. incarnata (= D. wintoni ). The identity of hybrids on the Leblanc site was unclear, perhaps reflecting the greater age of this site which may have allowed extensive introgression.

Summary We have developed a method using vibratome sectioning coupled with confocal microscopy to analyse pre-meiotic and meiotic events in developing wheat anthers. This report describes the application of fluorescence in situ hybridization to three-dimensionally preserved tissue sections from intact florets. We show that excellent imaging of all the stages of meiosis up to telophase I can be achieved, and show centromere positions and pre-meiotic homologous pairing both in meiocytes and tapetum cells. These clear results using intact tissue sections are contrasted with conventional spreading methods for analysis of meiosis in plants, which we show can produce artefactual or uninterpretable results.

Incorporation by RNA polymerases of BrUTP into both plant root tissue and isolated plant nuclei as a method for localization of the sites of transcription has been used. In this paper pea root tissue was used, and under the conditions employed, nearly all the incorporation occurs in the nucleolus, and thus must be catalysed by RNA polymerase I. Immunofluorescence and confocal microscopy shows that incorporation occurs in a pattern consisting of many small foci distributed widely through the dense fibrillar component of the nucleoli. Immunogold labelling using silver-enhanced Nanogold probe at the electron microscopic level confirms the sites of transcription as small foci approximately 200nm in diameter. Simultaneous fluorescence in situ hybridization with a probe to the external transcribed spacer (ETS) region of the pre-rRNA shows that the structures revealed by this probe and the BrUTP immunofluorescence labelling are very similar. A probe to the transcribed portion of the rDNA (18S) also shows a good correlation to the sites of BrUTP incorporation within the nucleolus. On the other hand a probe to the non-transcribed intergenic spacer region (NTS) shows very little coincidence with the sites of BrUTP incorporation, and double fluorescence in situ labelling with both 18S and NTS probes confirms this difference in localization. These results suggest that most BrUTP foci correspond to single transcribed genes.

Summary By microinjecting rhodamine-labelled tubulin into living plant cells, it is possible to observe microtubules (MTs) directly and to see how the cortical array reorganizes itself. The validity of the conclusions drawn from such observations depends upon the assumption that most, if not all, of the native MTs are dynamic and incorporate labelled tubulin. However, if arrays also contain MTs that are not exchanging tubulin subunits, such MTs will remain unlabelled, and the labelled MT population will be under-representative of the whole array. To address this potential problem, we microinjected pea epidermal cells with rhodamine-labelled tubulin, then fixed the cells and used fluorescein-conjugated antibodies against tubulin to detect the entire MT array. The two fluorescent patterns corresponded well, confirming that the MTs labelled with exogenous tubulin were evenly distributed throughout the entire array. Also, by comparing the MT image before and after aldehyde fixation, we observed that, although some of the MTs were lost in the procedure, the fixation was able to preserve the arrangement of MTs seen in the living cell. We conclude that fluorescence analogue cytochemistry provides a valid representation of the entire cortical MT array.

Foliar elements were analysed in Scots pine, Sitka spruce and Norway spruce over a 6 year period before and during continuous exposure to SO 2 and O 3 in an open-air fumigation experiment. Sulphur dioxide treatment elevated foliar sulphur concentration in all species, and there were increases in foliar nitrogen in the two spruce species but not in pine. The concentrations of cations were frequently increased by SO 2 treatment, but there was no correlation between the sulphur concentration of needles and their total cation charge. SO 2 -related elevations of foliar magnesium were correlated with the concentration of this element in soil solution, but the mechanism by which other cations were enhanced remains unclear. The only consistent effects on nutrient ratios were for SO 2 treatments to increase sulphur/cation ratios

In most eukaryotes that have been studied, the telomeres cluster into a bouquet early in meiosis, and in wheat and its relatives and in Arabidopsis the centromeres pair at the same time. In Arabidopsis, the telomeres do not cluster as a typical telomere bouquet on the nuclear membrane but are associated with the nucleolus both somatically and at the onset of meiosis. We therefore assessed whether Brachypodium distachyon, a monocot species related to cereals and whose genome is approximately twice the size of Arabidopsis thaliana, also exhibited an atypical telomere bouquet and centromere pairing. In order to investigate the occurrence of a bouquet and centromere pairing in B distachyon, we first had to establish protocols for studying meiosis in this species. This enabled us to visualize chromosome behaviour in meiocytes derived from young B distachyon spikelets in three-dimensions by fluorescent in situ hybridization (FISH), and accurately to stage meiosis based on chromatin morphology in relation to spikelet size and the timing of sample collection. Surprisingly, this study revealed that the centromeres clustered as a single site at the same time as the telomeres also formed a bouquet or single cluster.

rDNA Structure The nucleus is a highly organized structure. However, unlike cytoplasmic organelles, nuclear substructures are not bounded by membranes, but rather are held together by interactions between their component proteins and nucleic acids, and are thus probably best regarded as giant

The work described below was performed in an open air fumigation experiment in which young conifers were exposed to controlled levels of the pollutant gases sulphur dioxide (SO 2 ) and ozone (O 3 ). The mycorrhizal community was analysed both by fruitbody counts and root harvests for morphotype analysis throughout the 6 yr of the experiment. Above ground there was a classical fruit-body succession from nursery species to a mixed community dominated by Suillus spp. under Scots pine and Cortinarius spp. under Norway spruce. Below ground there were changes in the morphotype community structure with time, but no similarity between the morphotype and fruit-body data. There were no effects attributable to SO 2 or O 3 on the mycorrhizal community above or below ground, but multivariate analyses suggested that the fruit-body succession was more advanced in plots with low levels of available phosphate.

The large unicellular flagellate,Gloeomonas kupfferi, has recently been used as an important tool in chlamydomonad cell biology research, especially in studies dealing with the structure and function of the endomembrane system. However, little is known about the main secretory product, the cell wall. This study presents structural, chemical and immunological information about this wall. This 850–900 nm thick matrix is highly elaborate and consists of three distinct layers: an inner stratum (325 nm thick) consisting of tightly interwoven fibers, a medial crystalline layer consisting of 22–23 nm subunits and an outer wall layer (500 nm thick) of outwardlyradiating fibrils. Rapid freeze-deep etch analysis reveals that the 35–40 nm fibers of the outer layer form a quasi-lattice of 160 nm subunits. The outer wall can be removed from whole pellets using the chelator, CDTA. The medial wall complex can be solubilized by perchlorate. SDS-gel electrophoresis reveals that the perchlorate soluble-material consists of five high molecular weight glycoproteins and five major low molecular weight glycoproteins. The electrophoretic profile is roughly similar to that ofChlamydomonas reinhardtii. Antibodies were successfully raised against the outer wall component and were shown to label the outer wall layer.

The unicellular green algal flagellate,Mesostigma viride, is characterized by an extracellular matrix of multiple layers of scales. These scales are processed within the Golgi apparatus (GA). The GA consists of 11–13 closely stacked cisternae. The cis cisternae are highly fenestrated and grow via vesicles from adjacent transition ER. Medial-trans cisternae are plate-like with swollen peripheries. The calcified basket scales are produced in the peripheries of GA cisternae, usually first observable in the medial zone of the cisternal stack. Cisternal membrane closely conforms to the precise architecture of the developing scale. Antimonate labeling reveals that a population of smooth cytoplasmic vacuoles situated near the GA contains a store of calcium, perhaps used for scale processing. Vesicles carry calcium from these vacuoles to the cisternal loci where basket scale ontogenesis is occurring. The smaller scale types are produced within the central areas of the GA. A discussion concerning membrane flow through the GA is provided.

The eukaryotic nucleolus is multifunctional and involved in the metabolism and assembly of many different RNAs and ribonucleoprotein particles as well as in cellular functions, such as cell division and transcriptional silencing in plants. We previously showed that Arabidopsis thaliana exon junction complex proteins associate with the nucleolus, suggesting a role for the nucleolus in mRNA production. Here, we report that the plant nucleolus contains mRNAs, including fully spliced, aberrantly spliced, and single exon gene transcripts. Aberrant mRNAs are much more abundant in nucleolar fractions, while fully spliced products are more abundant in nucleoplasmic fractions. The majority of the aberrant transcripts contain premature termination codons and have characteristics of nonsense-mediated decay (NMD) substrates. A direct link between NMD and the nucleolus is shown by increased levels of the same aberrant transcripts in both the nucleolus and in Up-frameshift (upf) mutants impaired in NMD. In addition, the NMD factors UPF3 and UPF2 localize to the nucleolus, suggesting that the Arabidopsis nucleolus is therefore involved in identifying aberrant mRNAs and NMD.

Genetic engineering of commercially important crops has become routine in many laboratories. However, the inability to predict where a transgene will integrate and to efficiently select plants with stable levels of transgenic expression remains a limitation of this technology. Fluorescence in situ hybridization (FISH) is a powerful technique that can be used to visualize transgene integration sites and provide a better understanding of transgene behavior. Studies using FISH to characterize transgene integration have focused primarily on metaphase chromosomes, because the number and position of integration sites on the chromosomes are more easily determined at this stage. However gene (and transgene) expression occurs mainly during interphase. In order to accurately predict the activity of a transgene, it is critical to understand its location and dynamics in the three-dimensional interphase nucleus. We and others have developed in situ methods to visualize transgenes (including single copy genes) and their transcripts during interphase from different tissues and plant species. These techniques reduce the time necessary for characterization of transgene integration by eliminating the need for time-consuming segregation analysis, and extend characterization to the interphase nucleus, thus increasing the likelihood of accurate prediction of transgene activity. Furthermore, this approach is useful for studying nuclear organization and the dynamics of genes and chromatin.

We have developed methods, based on confocal microscopy and three-dimensional (3D) modelling, for the analysis of complex tissues and individual nuclei. These methods were used to study the development of early wheat (Triticum aestivum) endosperm as a whole and of endosperm nuclei undergoing polyploidization. Fixed sections of immature caryopses were either stained with SYTOX Green or used for fluorescence in situ hybridization (FISH) to visualize centromeres, telomeres and a rye chromosome arm substitution. Each section was imaged as a confocal image stack. By using Amira 3.0 for computer image processing, rendered models were produced of the whole endosperm and of individual nuclei. We followed endosperm development up to the formation of a complete syncytium, which develops via a dorsal and a ventral plate of nuclei in the central cell. Modelling of nuclei showed that wheat chromosomes are not anchored to the nuclear membrane and become more randomly positioned in endoreduplicated nuclei. This analysis produced a precise description of the positioning of nuclei throughout the developing endosperm and of chromosomal domains in single nuclei.

The cereal caryopsis is a complex tissue in which maternal and endosperm tissues follow distinct but coordinated developmental programs. Because of the hexaploid genome in wheat (Triticum aestivum), the identification of genes involved in key developmental processes by genetic approaches has been difficult. To bypass this limitation, we surveyed 888 genes that are expressed during caryopsis development using a novel high-throughput mRNA in situ hybridization method. This survey revealed novel distinct spatial expression patterns that either reflected the ontogeny of the developing caryopsis or indicated specialized cellular functions. We have identified both known and novel genes whose expression is cell cycle–dependent. We have identified the crease region as important in setting up the developmental patterning, because the transition from proliferation to differentiation spreads from this region to the rest of the endosperm. A comparison of this set of genes with the rice (Oryza sativa) genome shows that approximately two-thirds have rice counterparts but also suggests considerable divergence with regard to proteins involved in grain filling. We found that the wheat genes had significant homology with 350 Arabidopsis thaliana genes. At least 25 of these are already known to be essential for seed development in Arabidopsis, but many others remain to be characterized.

Yields of Tuber melanosporum fruit bodies from a commercial truffle plantation are examined in relation to time, soil conditions and basal diameter of host tree. Fruiting started 10 yr after planting, and the number of trees producing truffles increased steadily for the following 8 yr. The best predictor of truffle production was tree basal diameter.

A putative G1 cyclin gene, Antma;CycD1;1 (CycD1), from Antirrhinum majus is known to be expressed throughout the cell cycle in the meristem and other actively proliferating cells. To test its role in cell cycle progression, we examined the effect of CycD1 expression in the tobacco (Nicotiana tabacum) cell suspension culture BY-2. Green fluorescent protein:CycD1 is located in the nucleus throughout interphase. Using epitope-tagged CycD1, we show that it interacts in vivo with CDKA, a cyclin dependent protein kinase that acts at both the G1/S and the G2/M boundaries. We examined the effect of induced expression at different stages of the cell cycle. Expression in G0 cells accelerated entry into both S-phase and mitosis, whereas expression during S-phase accelerated entry into mitosis. Consistent with acceleration of both transitions, the CycD1-associated cyclin dependent kinase can phosphorylate both histone H1 and Rb proteins. The expression of cyclinD1 led to the early activation of total CDK activity, consistent with accelerated cell cycle progression. Continuous expression of CycD1 led to moderate increases in growth rate. Therefore, in contrast with animal D cyclins, CycD1 can promote both G0/G1/S and S/G2/M progression. This indicates that D cyclin function may have diverged between plants and animals.

A new Arabidopsis meiotic mutant has been isolated. Homozygous ahp2-1 (Arabidopsis homologue pairing 2) plants were sterile because of failure of both male and female gametophyte development. Fluorescent in situ hybridisation showed that in ahp2-1 male meiocytes, chromosomes did not form bivalents during prophase I and instead seemed to associate indiscriminately. Chromosome fragmentation, chromatin bridges and unbalanced segregation were seen in anaphase I and anaphase II. The ahp2-1 mutation was caused by a T-DNA insertion in an Arabidopsis homologue of meu13 + , which has been implicated in homologous chromosome pairing during meiosis in Schizosaccharomyces pombe. Our results suggest that meu13 + function is conserved in higher eukaryotes and support the idea that Arabidopsis, yeast and mouse share a pairing pathway that is not present in Drosophila melanogaster and Caenorhabditis elegans.

Hexaploid wheat possesses 42 chromosomes derived from its three ancestral genomes. The 21 pairs of chromosomes can be further divided into seven groups of six chromosomes (one chromosome pair being derived from each of the three ancestral genomes), based on the similarity of their gene order. Previous studies have revealed that, during anther development, the chromosomes associate in 21 pairs via their centromeres. The present study reveals that, as a prelude to meiosis, these 21 chromosome pairs in hexaploid (and tetraploid) wheat associate via the centromeres into seven groups as the telomeres begin to cluster. This results in the association of multiple chromosomes, which then need to be resolved as meiosis progresses. The formation of the seven chromosome clusters now explains the occasional occurrence of remnants of multiple associations, which have been reported at later stages of meiosis in hexaploid (and tetraploid) wheat. Importantly, the chromosomes have the opportunity to be resorted via these multiple interactions. As meiosis progresses, such interactions are resolved through the action of loci such as Ph1, leaving chromosomes as homologous pairs.

It has long been known that many forest trees depend on a specialised group offungi which form a dense sheath enveloping the feeder roots, and act as a functional extension of the root system in exchange for sugars from their host. These symbioses are collectively known as the sheathing or ecto-mycorrhizas. Most species become visible in autumn when thei r fruitbodies (toadstools) emerge. Ectomycorrhizal fungi include well known species such as the fly agaric Amanita muscaria, death cap A. phalloides, penny bun Boletus edulis, and the chanterelle Cantharellus cibarius . Mycologists have repeatedly observed that the mycorrhizal fungi found under young forest trees change from year to year. A few are typically associated with very young trees these 'nursery species' include the deceiver Laccaria laccata and the roll rim Paxillus involutus, which occur under a wide range of hosts. As trees mature the fungal community diversifies, with the composition depending on the age of the host tree, its species and other factors such as soil conditions . (Last et al., 1984; Mason et al. , 1984; Dighton, Poskitt & Howard , 1986; Last, Dighton & Mason, 1987). Curiously, although some workers have found equivalent changes in the mycorrhizal infections below-ground (Deacon, Donaldson & Last, 1984; Mason, Wilson & Last, 1984), others have failed to find any relationships between fruitbodies and mycorrhizas (Meier et al. , 1990; Shaw et al ., 1992). Although there are a number of theories about factors driving thi s type of succession , no final verdict has been reached. It has been observed that later stage fungi are associated with increased levels of soil organic matter, and that they require higher sugar supplies than 'nursery' fungi (Dighton & Mason, 1985; Last et al. , 1987). The difficulty in studying this phenomenon in the field is the need for an extended time scale (> 5 years). Studies on chronosequences using stands of different ages on different sites may be complicated by other site factors such as differences in their soils. We report below some results from an eightyear study ofmycorrhizal fruitbodies under homogenous, even-aged stands of Scots pine (Pinus sylvestris). Data for the first five years have been published by Shaw et al . (1992), which was concerned with testing whether air pollutant effects could be detected. No pollutant effects were found, but these data suggested that succession was most advanced in plots with low soil phosphate. This result was based on soil water data from only two locations per plot, and could not be regarded as definitive. New fruitbody data have been combined with data on soil chemistry to investigate further factors causing site-specific differences in the fruitbody succession.

Summary DNA helicases catalyse the transient opening of duplex DNA during nucleic acid transactions. Here we report the isolation of a second nuclear DNA helicase (65 kDa) from Pisum sativum (pea) designated pea DNA helicase 65 (PDH65). The enzyme was immunoaffinity purified using an antihuman DNA helicase I (HDH I) antibody column. The purified PDH65 showed ATP- and Mg2+ -dependent DNA and RNA unwinding activities, as well as ssDNA-dependent ATPase activity. The direction of DNA unwinding was 3¢ to 5¢ along the bound strand. Antibodies against HDH I recognized the purified PDH65, and immunodepletion with these antibodies removed the DNA and RNA unwinding and ATPase activities from purified preparations of PDH65. The DNA and RNA unwinding activities were upregulated after phosphorylation of PDH65 with CK2 and cdc2 protein kinases. By incorporation of BrUTP into pea root tissue, followed by double immunofluorescence labelling and confocal microscopy, PDH65 was shown to be localized within the dense fibrillar component of pea root nucleoli in the regions around the rDNA transcription sites. These observations suggest that PDH65 may be involved both in rDNA transcription and in the early stages of pre-rRNA processing.

We compared the subcellular distribution of native and artificial reticuloplasmins in endosperm, callus, and leaf tissues of transgenic rice (Oryza sativa) to determine the distribution of these proteins among endoplasmic reticulum (ER) and post-ER compartments. The native reticuloplasmin was calreticulin. The artificial reticuloplasmin was a recombinant single-chain antibody (scFv), expressed with an N-terminal signal peptide and the C-terminal KDEL sequence for retrieval to the ER (scFvT84.66-KDEL). We found that both molecules were distributed in the same manner. In endosperm, each accumulated in ER-derived prolamine protein bodies, but also in glutelin protein storage vacuoles, even though glutelins are known to pass through the Golgi apparatus en route to these organelles. This finding may suggest that similar mechanisms are involved in the sorting of reticuloplasmins and rice seed storage proteins. However, the presence of reticuloplasmins in protein storage vacuoles could also be due to simple dispersal into these compartments during protein storage vacuole biogenesis, before glutelin deposition. In callus and leaf mesophyll cells, both reticuloplasmins accumulated in ribosome-coated vesicles probably derived directly from the rough ER.

The application of molecular and modern cell biological techniques is beginning to show that the classical picture of the meiotic process is an oversimplification. The comparison of different species and analysis of mutants have recently demonstrated that many of the features previously thought to be an integral part of meiosis can be altered in their timing or even entirely dispensed with. In plants, differences in the meiotic pathway have been observed, by using methods for 3-D optical imaging, between the polyploids maize and wheat. The fact that two such closely related species differ may be the result of different mechanisms for dealing with polyploidy or may be species differences, but shows that a detailed understanding of the process in the particular species of interest is necessary.