Transformations of the Pleiomorphic Plant MTOC during Sporogenesis in the Hepatic Marchantia polymorpha

Sporogenesis in the hepatic Marchantia polymorpha L. provides an outstanding example of the pleiomorphic nature of the plant microtubule organizing center （MTOC）. Microtubules are nucleated from γ-tubuUn in MTOCs that change form during mitosis and meiosis. Following entry of cells into the reproductive pathway of sporogenesis, successive rounds of mitosis give rise to packets of 4-16 sporocytes. Mitotic spindles are organized at discrete polar organizers （POs）, a type of MTOC that is unique to this group of early divergent land plants. An abrupt and radical transformation in microtubule organization occurs when sporocytes enter meiosis： POs are lost and γ-tubulin is closely associated with surfaces of two large elongated plastids that subsequently divide into four. Migration of the four plastid MTOCs into a tetrahedral arrangement establishes the future spore domains and the division polarity of meiosis. As is typical of many bryophytes, cones of microtubules from the four plastid MTOCs initiate a quadripolar microtubule system （QMS） in meiotic prophase. At this point a transformation in the organization of the MTOCs occurs. The γ-tubulin detaches from plastids and forms a diffuse spheroidal pole in each of the spore domains. The plastids, which are no longer MTOCs, continue to divide. The diffuse MTOCs continue to nucleate cones of microtubules during transformation of the QMS to a bipolar spindle. Following meiosis I, γ-tubulin is associated with nuclear envelopes, and the spindles of meiosis II are organized from diffuse MTOCs at the tetrad poles. At simultaneous cytokinesis, radial microtubule systems are organized at nuclear envelope MTOCs in each of the tetrad members.     

Diversity in meiotic spindle origin and determination of cytokinetic planes in sporogenesis of complex thalloid liverworts (Marchantiopsida)

As the earliest divergent land plants, bryophytes (mosses, hornworts, and liverworts) provide insight into the evolution of the unique plant process of sporogenesis by which meiosis results in heavy walled spores. New immunohistochemical data on microtubules and γ-tubulin in four genera of complex thalloid liverworts combined with previously published data on another four genera demonstrate grades in the evolution of spindle organization in meiosis. We have discovered that all recognized forms of microtubule organizing centers (MTOCs) in plant cells (plastid MTOCs, spheroid cytoplasmic MTOCs, polar organizers, and nuclear envelope MTOCs) occur in organization of the meiotic spindle of complex thalloid liverworts. In addition, all aspects of pre-meiotic preparation for quadripartitioning of the sporocyte into a tetrad of spores occur, with the exception of pre-meiotic wall precursors found in certain simple thalloids. The preparation includes morphogenetic plastid migration, cortical bands of microtubules that mark future cytokinetic planes in pre-meiosis, quadrilobing of the cytoplasm during meiotic prophase, and quadripolar microtubule systems that are transformed into functionally bipolar metaphase I spindles. Quadripolar spindle origin is typical of bryophyte sporogenesis even though the MTOCs involved may differ. However, in certain crown taxa of complex thalloids the spindle develops with no traces of quadripolarity and placement of intersporal walls is determined after meiosis, as is typical of higher plants.     

Polar organizers and girdling bands of microtubules are associated with γ-tubulin and act in establishment of meiotic quadripolarity in the hepatic Aneura pinguis (Bryophyta)

Summary. Meiosis in Aneura pinguis is preceded by extensive cytoplasmic preparation for quadripartitioning of the diploid sporocyte into a tetrad of haploid spores. In early prophase the four future spore domains are defined by lobing of the cytoplasm and development of a quadripolar prophase spindle focused at polar organizers (POs) centered in the lobes. Cells entering the reproductive phase become isolated and, instead of hooplike cortical microtubules, have endoplasmic microtubule systems centered on POs. These archesporial cells proliferate by mitosis before entering meiosis. In prophase of each mitosis, POs containing a distinct concentration of γ-tubulin appear de novo at tips of nuclei and initiate the bipolar spindle. Cells entering meiosis become transformed into quadrilobed sporocytes with four POs, one in each lobe. This transition is a complex process encompassing assembly of two opposite POs which subsequently disperse into intersecting bands of microtubules that form around the central nucleus. The girdling bands define the future planes of cytokinesis and the cytoplasm protrudes through the restrictive bands becoming quadrilobed. Two large POs reappear in opposite cleavage furrows. Each divides and the resulting POs migrate into the tetrahedral lobes of cytoplasm. Cones of microtubules emanating from the four POs interact to form a quadripolar microtubule system (QMS) that surrounds the nucleus in meiotic prophase. The QMS is subsequently transformed into a functionally bipolar metaphase spindle by migration of poles in pairs to opposite cleavage furrows. These findings contribute to knowledge of microtubule organization and the role of microtubules in spatial regulation of cytokinesis in plants. Correspondence and reprints: Department of Biology, University of Louisiana-Lafayette, Lafayette, LA 70504-2451, U.S.A.     

{gamma}-Tubulin and microtubule organization during meiosis in the liverwort Ricciocarpus natans (Ricciaceae)

Extant liverworts are "living fossils" considered sister to all other plants and as such provide clues to the evolution of the microtubule organizing center (MTOC) in anastral cells. This report is the first on microtubule arrays and their γ-tubulin-nucleating sites during meiosis in a member of the Ricciales, a specialized, species-rich group of complex thalloid (marchantioid) liverworts. In meiotic prophase, γ-tubulin becomes concentrated at several sites adjacent to the nuclear envelope. Microtubules organized at these foci give rise to a multipolar prometaphase spindle. By metaphase I, the spindle has matured into a bipolar structure with truncated poles. In both first and second meiosis, γ-tubulin forms box-like caps at the spindle poles. γ-Tubulin moves from spindle poles to the proximal surfaces of telophase chromosomes where interzonal microtubules are nucleated. Although a phragmoplast is organized, no cell plate is deposited, and second division occurs simultaneously in the undivided sporocyte. γ-Tubulin surrounds each of the tetrad nuclei, and phragmoplasts initiated between both sister and nonsister nuclei direct simultaneous cytokinesis. The overall pattern of meiosis (unlobed polyplastidic sporocytes, nuclear envelope MTOC, multipolar spindle origin, spindles with box-like poles, and simultaneous cytokinesis) more closely resembles that of Conocephalum than other marchantiod liverworts.     

The quadripolar microtubule system in lower land plants

The quadripolar microtubule system (QMS) is a complex array that is associated with predivision establishment of quadripolarity in sporocytes of lower plants (bryophytes and lycopsids). The QMS unerringly predicts the polarity of the two meiotic divisions and plays a central role in development of both the mitotic apparatus (MA) and cytokinetic apparatus (CA) which together accomplish quadripartitioning of the sporocyte into four haploid spores. The QMS is typically, but not exclusively, associated with monoplastidy and precocious quadrilobing of the cytoplasm. In early meiotic prophase the single plastid divides and the resultant plastids migrate so that either the tips of two plastids or the four plastids resulting from a second division are located in the future spore domains. Microtubules that emanate from the plastid tips or from individual plastids in the spore domains interact in the future planes of cytokinesis and give rise to the QMS. The QMS, which encages the prophase nucleus, consists of at least four and usually six (when spore domains are in tetrahedral arrangement) bipolar spindle-like arrays of microtubules presumably with minus ends at plastids in spore domains and plus ends interacting in the future plane of cytokinesis. Each of the six arrays is essentially like the single axial microtubule system (AMS) that intersects the division site and is transformed into the spindle in monoplastidic mitosis in hornworts. As comparative data accumulate, it appears that the AMS is not unique to monoplastidic cell division but instead represents a basic microtubule arrangement that survives as spindle and phragmoplast in cell division of higher plants.     

Pre-meiotic bands and novel meiotic spindle ontogeny in quadrilobed sporocytes of leafy liverworts (Jungermannidae, Bryophyta)

Indirect immunofluorescence and confocal microscopy were used to study the nucleation and organization of microtubules during meiosis in two species of leafy liverworts, Cephalozia macrostachya and Telaranea longifolia. This is the first such study of sporogenesis in the largest group of liverworts important as living representatives of some of the first land plant lineages. These studies show that cytoplasmic quadrilobing of pre-meiotic sporocytes into future spore domains is initiated by girdling bands of γ-tubulin and microtubules similar to those recently described in lobed sporocytes of simple thalloid liverworts. However, spindle ontogeny is not like other liverworts studied and is, in fact, probably unique among bryophytes. Following the establishment of quadrilobing, numerous microtubules diverge from the bands and extend into the enlarging lobes. The bands disappear and are replaced by microtubules that arise from γ-tubulin associated with the nuclear envelope. This microtubule system extends into the four lobes and is gradually reorganized into a quadripolar spindle, each half spindle consisting of a pair of poles straddling opposite cleavage furrows. Chromosomes move on this spindle to the polar cleavage furrows. The reniform daughter nuclei, each curved over a cleavage furrow, immediately enter second meiotic division with spindles now terminating in the lobes. Phragmoplasts that develop in the interzones among the haploid tetrad nuclei guide deposition of cell plates that join with the pre-meiotic furrows resulting in cleavage of the tetrad of spores. These observations document a significant variation in the innovative process of sporogenesis evolved in early land plants.     

Mosses share mitochondrial group II introns with flowering plants, not with liverworts

Extant bryophytes are regarded as the closest living relatives of the first land plants, but relationships among the bryophyte classes (mosses, liverworts and hornworts) and between them and other embryophytes have remained unclear. We have recently found that plant mitochondrial genes with positionally stable introns are well suited for addressing questions of plant phylogeny at a deep level. To explore further data sets we have chosen to investigate the mitochondrial genes nad4 and nad7, which are particularly rich in intron sequences. Surprisingly, we find that in these genes mosses share three group II introns with flowering plants, but none with the liverwort Marchantia polymorpha or other liverworts investigated here. In mitochondria of Marchantia, nad7 is a pseudogene containing stop codons, but nad7 appears as a functional mitochondrial gene in mosses, including the isolated genus Takakia. We observe the necessity for strikingly frequent C-to-U RNA editing to reconstitute conserved codons in Takakia when compared to other mosses. The findings underline the great evolutionary distances among the bryophytes as the presumptive oldest division of land plants. A scenario involving differential intron gains from fungal sources in what are perhaps the two earliest diverging land plant lineages, liverworts and other embryophytes, is discussed. With their positionally stable introns, nad4 and nad7 represent novel marker genes that may permit a detailed phylogenetic resolution of early clades of land plants.     

Vegetative and reproductive innovations of early land plants: implications for a unified phylogeny

As the oldest extant lineages of land plants, bryophytes provide a living laboratory in which to evaluate morphological adaptations associated with early land existence. In this paper we examine reproductive and structural innovations in the gametophyte and sporophyte generations of hornworts, liverworts, mosses and basal pteridophytes. Reproductive features relating to spermatogenesis and the architecture of motile male gametes are overviewed and evaluated from an evolutionary perspective. Phylogenetic analyses of a data set derived from spermatogenesis and one derived from comprehensive morphogenetic data are compared with a molecular analysis of nuclear and mitochondrial small subunit rDNA sequences. Although relatively small because of a reliance on water for sexual reproduction, gametophytes of bryophytes are the most elaborate of those produced by any land plant. Phenotypic variability in gametophytic habit ranges from leafy to thalloid forms with the greatest diversity exhibited by hepatics. Appendages, including leaves, slime papillae and hairs, predominate in liverworts and mosses, while hornwort gametophytes are strictly thalloid with no organized external structures. Internalization of reproductive and vegetative structures within mucilage-filled spaces is an adaptive strategy exhibited by hornworts. The formative stages of gametangial development are similar in the three bryophyte groups, with the exception that in mosses apical growth is intercalated into early organogenesis, a feature echoed in moss sporophyte ontogeny. A monosporangiate, unbranched sporophyte typifies bryophytes, but developmental and structural innovations suggest the three bryophyte groups diverged prior to elaboration of this generation. Sporophyte morphogenesis in hornworts involves non-synchronized sporogenesis and the continued elongation of the single sporangium, features unique among archegoniates. In hepatics, elongation of the sporophyte seta and archegoniophore is rapid and requires instantaneous wall expandability and hydrostatic support. Unicellular, spiralled elaters and capsule dehiscence through the formation of four regular valves are autapomorphies of liverworts. Sporophytic sophistications in the moss clade include conducting tissue, stomata, an assimilative layer and an elaborate peristome for extended spore dispersal. Characters such as stomata and conducting cells that are shared among sporophvtes of mosses, hornworts and pteridophytes are interpreted as parallelisms and not homologies. Our phylogenetic analysis of three different data sets is the most comprehensive to date and points to a single phylogenetic solution for the evolution of basal embryophytes. Hornworts are supported as the earliest divergent embryophyte clade with a moss/liverwort clade sister to tracheophytes. Among pteridophytes, lycophytes are monophyletic and an assemblage containing ferns, Equisetum and psilophytes is sister to seed plants. Congruence between morphological and molecular hypotheses indicates that these data sets are tracking the same phylogenetic signal and reinforces our phylogenetic conclusions. It appears that total evidence approaches are valuable in resolving ancient radiations such as those characterizing the evolution of early embryophytes. More information on land plant phylogeny can be found at: http: //www.science.siu.edu/ landplants/index.html.     

Evolution of apolar sporocytes in marchantialean liverworts: implications from molecular phylogeny

In meiosis of basal land plants, meiotic division planes are typically predicted by quadri-lobing of the cytoplasm and/or quadri-partitioning of plastids prior to nuclear divisions. However, sporocytes of several marchantialean liverworts display no indication of premeiotic establishment of quadripolarity, as is observed in flowering plants. In these cases, the shape of sporocytes remains spherical or elliptical and numerous plastids are distributed randomly in the cytoplasm during meiosis. Through a survey of sporocyte morphology in marchantialean liverworts, we newly report the occurrence of apolar sporocytes in Sauteria japonica and Athalamia nana (Cleveaceae; Marchantiales). Molecular phylogenetic analyses revealed that the quadri-lobing of cytoplasm and quadri-partitioning of plastids were lost independently several times during the evolution of marchantialean liverworts. In addition, our phylogenetic analyses indicate that the simplified sporophytes of several marchantialean liverworts are not a primitive condition but rather represent the result of reductive evolution. The loss of the quadripolarity of sporocytes appears to correlate with the evolutionary trend of the sporophyte towards reductions. Through the evolution of the simplified sporophytes, suppression of mitotic divisions of sporogenous cells might had caused not only the modification of sporophyte ontogeny but also the drastic cytological change of sporocyte.     

THE QUADRIPOLAR MICROTUBULE SYSTEM AND MEIOTIC SPINDLE ONTOGENY IN HORNWORTS (BRYOPHYTA: ANTHOCEROTAE)

Ontogeny of the meiotic spindle in hornworts was studied by light microscopy of live materials, transmission electron microscopy, and indirect immunofluorescence microscopy. As in monoplastidic meiosis of mosses and Isoetes, the single plastid divides twice, and the four resultant plastids migrate into the future spore domains where they organize a quadripolar microtubule system (QMS). Additionally, a unique axial microtubule system (AMS) was found to parallel the plastid isthmus at each division in meiosis, much as in the single plastid division of mitosis. This finding is used to make a novel comparison of mitotic and meiotic spindle development. The AMS contributes directly to development of the mitotic spindle, whereas ontogeny of the meiotic spindle is more complex. Nuclear division in meiosis is delayed until after the second plastid division; the first AMS disappears without spindle formation, and the two AMSs of the second plastid division contribute to development of the QMS. Proliferation of microtubules at each plastid results in the QMS consisting of four cones of microtubules interconnecting the plastids and surrounding the nucleus. The QMS contributes to the development of a functionally bipolar spindle. The meiotic spindle is comparable to a merger of two mitotic spindles. However, the first division spindle does not terminate in what would be the poles of mitosis; instead the poles converge to orient the spindle axis midway between pairs of non-sister plastids.     

Phytohormone Profiling across the Bryophytes

BackgroundBryophytes represent a very diverse group of non-vascular plants such as mosses, liverworts and hornworts and the oldest extant lineage of land plants. Determination of endogenous phytohormone profiles in bryophytes can provide substantial information about early land plant evolution. In this study, we screened thirty bryophyte species including six liverworts and twenty-four mosses for their phytohormone profiles in order to relate the hormonome with phylogeny in the plant kingdom.MethodologySamples belonging to nine orders (Pelliales, Jungermanniales, Porellales, Sphagnales, Tetraphidales, Polytrichales, Dicranales, Bryales, Hypnales) were collected in Central and Northern Bohemia. The phytohormone content was analysed with a high performance liquid chromatography electrospray tandem-mass spectrometry (HPLC-ESI-MS/MS).ConclusionThe apparent differences in conjugation and/or degradation strategies of growth hormones between liverworts and mosses might potentially show a hidden link between vascular plants and liverworts. On the other hand, the complement of stress hormones in bryophytes probably correlate rather with prevailing environmental conditions and plant survival strategy than with plant evolution.     

Slow molecular evolution in 18S rDNA, rbcL and nad5 genes of mosses compared with higher plants

The evolutionary potential of bryophytes (mosses, liverworts and hornworts) has been debated for decades. Fossil record and biogeographical distribution patterns suggest very slow morphological evolution and the retainment of several ancient traits since the split with vascular plants some 450 million years ago. Many have argued that bryophytes may evolve as rapidly as higher plants on the molecular level, but this hypothesis has not been tested so far. Here, it is shown that mosses have experienced significantly lower rates of molecular evolution than higher plants within 18S rDNA (nuclear), rbcL (chloroplast) and nad5 (mitochondrial) genes. Mosses are on an average evolving 2-3 times slower than ferns, gymnosperms and angiosperms; and also green algae seem to be evolving faster than nonvascular plants. These results support the observation of a general correlation between morphological and molecular evolutionary rates in plants and also show that mosses are 'evolutionary sphinxes' regarding both morphological and molecular evolutionary potential.     

Centromere pairing precedes meiotic chromosome pairing in plants

Meiosis is a specialized eukaryotic cell division, in which diploid cells undergo a single round of DNA replication and two rounds of nuclear division to produce haploid gametes. In most eukaryotes, the core events of meiotic prophase I are chromosomal pairing,synapsis and recombination. To ensure accurate chromosomal segregation, homologs have to identify and align along each other at the onset of meiosis. Although much progress has been made in elucidating meiotic processes, information on the mechanisms underlying chromosome pairing is limited in contrast to the meiotic recombination and synapsis events. Recent research in many organisms indicated that centromere interactions during early meiotic prophase facilitate homologous chromosome pairing, and functional centromere is a prerequisite for centromere pairing such as in maize. Here, we summarize the recent achievements of chromosome pairing research on plants and other organisms, and outline centromere interactions, nuclear chromosome orientation,and meiotic cohesin, as main determinants of chromosome pairing in early meiotic prophase.     

Divergent intron conservation in the mitochondrial nad2 gene: signatures for the three bryophyte classes (mosses,liverworts, and hornworts) and the lycophytes

The slow-evolving mitochondrial DNAs of plants have potentially conserved information on the phylogenetic branching of the earliest land plants. We present the nad2 gene structures in hornworts and liverworts and in the presumptive earliest-branching vascular land plant clade, the Lycopodiopsida. Taken together with the recently obtained nad2 data for mosses, each class of bryophytes presents another pattern of angiosperm-type introns conserved in nad2: intron nad2i1 in mosses; intron nad2i3 in liverworts; and both introns, nad2i3 and nad2i4, in hornworts. The lycopods Isoetes and Lycopodium show diverging intron conservation and feature a unique novel intron, termed nad2i3b. Hence, mitochondrial introns in general are positionally stable in the bryophytes and provide significant intraclade phylogenetic information, but the nad2 introns, in particular, cannot resolve the interclade relationships of the bryophyte classes and to the tracheophytes. The necessity for RNA editing to reconstitute conserved codon entities in nad2 is obvious for all clades except the marchantiid liverworts. Finally, we find that particularly small group II introns appear as a general feature of the Isoetes chondriome. Plant mitochondrial peculiarities such as RNA editing frequency, U-to-C type of RNA editing, and small group II introns appear to be genus-specific rather than gene-specific features.     

Phylogeny and diversification of bryophytes

The bryophytes comprise three phyla of embryophytes that are well established to occupy the first nodes among extant lineages in the land-plant tree of life. The three bryophyte groups (hornworts, liverworts, mosses) may not form a monophyletic clade, but they share life history features including dominant free-living gametophytes and matrotrophic monosporangiate sporophytes. Because of their unique vegetative and reproductive innovations and their critical position in embryophyte phylogeny, studies of bryophytes are crucial to understanding the evolution of land plant morphology and genomes. This review focuses on phylogenetic relationships within each of the three divisions of bryophytes and relates morphological diversity to new insights about those relationships. Most previous work has been on the mosses, but progress on understanding the phylogeny of hornworts and liverworts is advancing at a rapid pace. Multilocus multigenome studies have been successful at resolving deep relationships within the mosses and liverworts, whereas single-gene analyses have advanced understanding of hornwort evolution.     

Evidence for the most basal split in land plants dividing bryophyte and tracheophyte lineages

The problem of relationships among the major basal living groups of land plants is long standing, yet the uncertainty as to the phylogenetic affinity of these lines persists in the literature. Molecular and modern cladistic studies of the phylogenetic relationships of the above groups resulted in a large number of conflicting topologies. However, with the exception of the cladistic analyses of spermatogenesis, suggesting monophyly of extant bryophytes, these studies agree the paraphyletic bryophyte grade is basal within the embryophyte tree. Here we would like to present analyses on the basis of the concatenated datasets of nucleotide and amino-acid sequences of 57 protein-coding genes common to 17 chloroplast genomes of land plants and a charophyte alga Chaetosphaeridium globosum. Character-wise, these are the largest datasets currently available to address the problem of basal relationships within embryophytes. Main lineages of bryophytes, i.e liverworts, hornworts and mosses are represented in our alignments with a single taxon, whereas 14 taxa represent the tracheophytes. With our data, phylogeny with liverwort basal appears to be and artifact related to high and unequal A+T contents among the sequences analysed. Reducing this compositional bias and applying methods developed to counter it, we recovered an alternative, strongly supported topology wherein both bryophytes and tracheophytes are monophyletic. Within bryophytes, hornworts are basal and liverworts are sister to mosses.     

Division polarity,development and configuration of microtubule arrays in Bryophyte meiosis

Summary Immunofluorescence and TEM studies of meiosis in two mosses (Bryophyta) provide evidence that the prophasic tetrahedral system of microtubules contributes directly to the metaphase I spindle. Intense staining of tubulin, conspicuously absent around the nuclear envelope, is first seen associated with plastids. By mid-prophase, microtubules radiate from the plastids to the nuclear envelope and become organized into six bands that interconnect the four plastids, forming a tetrahedral cytoskeleton surrounding the nucleus. During transition of prophase to metaphase, the four poles of the tetrahedral microtubule system converge in pairs toward opposite cleavage furrows. Opposite furrows occupy mutually perpendicular planes and the pair of microtubule focal points straddling one furrow lies at right angles to the pair straddling the opposite furrow. Additional microtubules terminate in numerous small clusters in the concave polar regions arching over the cleavage furrows. By early anaphase, the microtubule focal points lie very close to the division axis. We conclude that microtubules recruited from the prophasic quadripolar system are incorporated into the mature metaphase I spindle and the two principal focal points at each pole are those derived from poles of the prophasic quadripolar system.