Diploid Generations Research Articles

Characterization of three species of aquatic mosses in axenic culture for biomonitoring and biotechnological applications

Bryophytes are known bioindicators and are also emerging as effective tools for bioremediation. In vitro culture of bryophytes is an important tool for the implementation of several research and industrial applications but it is a poorly explored technology. In this study, we characterize in sterile conditions three aquatic moss species largely used all over the world for decoration but poorly studied: Leptodictyum riparium, Vesicularia montagnei and Taxiphyllum barbieri. They share interesting morphological traits that suggest their use as natural biofilters. Results include protocols for the establishment of axenic in vitro cultures, different for the different species because of their sensitivity to treatments, on which the morphological characters of the three species were described. The sporophytic generation was observed in L. riparium and V. montagnei but not in T. barbieri that may be unable to develop the diploid generation. The effect of plant growth regulators on gametophyte fragments was described applying 6-benzylaminopurine as cytokinin and α-naphthalene acetic acid as auxin. The absorption of several trace elements was measured in a mixed solution simulating environmental pollution, evidencing specie specificity toward the different elements. The possible applications for these mosses are not only in the field of bioindication but also in bioremediation and environmental restoration. Our study produced widely applicable protocols and basic information for further applications.

Protocol: an improved method for inducing sporophyte generation in the model moss Physcomitrium patens under nitrogen starvation

BackgroundLand plants exhibit a haplodiplontic life cycle, whereby multicellular bodies develop in both the haploid and diploid generations. The early-diverging land plants, known as bryophytes, have a haploid-dominant life cycle, in which a short-lived multicellular body in the diploid generation, known as the sporophyte, develops on the maternal haploid gametophyte tissues. The moss Physcomitrium (Physcomitrella) patens has become one of the most powerful model systems in evolutionary plant developmental studies. To induce diploid sporophytes of P. patens, several protocols are implemented. One of the conventional approaches is to grow approximately one-month-old gametophores for another month on Jiffy-7 pellets made from the peat moss that is difficult to fully sterilize. A more efficient method to obtain all tissues throughout the life cycle should accelerate studies of P. patens.ResultsHere, we investigated the effect of nitrogen conditions on the growth and development of P. patens. We provide an improved protocol for the sporophyte induction of P. patens using a BCD-based solid culture medium without Jiffy-7 pellets, based on the finding that the formation of gametangia and subsequent sporophytes is promoted by nitrogen-free growth conditions. The protocol consists of two steps; first, culture the protonemata and gametophores on nitrogen-rich medium under continuous light at 25 °C, and then transfer the gametophores onto nitrogen-free medium under short-day and at 15 °C for sporophyte induction. The protocol enables to shorten the induction period and reduce the culture space.ConclusionsOur more efficient and shortened protocol for inducing the formation of sporophytes will contribute to future studies into the fertilization or the diploid sporophyte generation of P. patens.

The origin of a land flora.

The origin of a land flora fundamentally shifted the course of evolution of life on earth, facilitating terrestrialization of other eukaryotic lineages and altering the planet's geology, from changing atmospheric and hydrological cycles to transforming continental erosion processes. Despite algal lineages inhabiting the terrestrial environment for a considerable preceding period, they failed to evolve complex multicellularity necessary to conquer the land. About 470 million years ago, one lineage of charophycean alga evolved complex multicellularity via developmental innovations in both haploid and diploid generations and became land plants (embryophytes), which rapidly diversified to dominate most terrestrial habitats. Genome sequences have provided unprecedented insights into the genetic and genomic bases for embryophyte origins, with some embryophyte-specific genes being associated with the evolution of key developmental or physiological attributes, such as meristems, rhizoids and the ability to form mycorrhizal associations. However, based on the fossil record, the evolution of the defining feature of embryophytes, the embryo, and consequently the sporangium that provided a reproductive advantage, may have been most critical in their rise to dominance. The long timeframe and singularity of a land flora were perhaps due to the stepwise assembly of a large constellation of genetic innovations required to conquer the terrestrial environment.

Diploid-dominant life cycles characterize the early evolution of Fungi

Most of the described species in kingdom Fungi are contained in two phyla, the Ascomycota and the Basidiomycota (subkingdom Dikarya). As a result, our understanding of the biology of the kingdom is heavily influenced by traits observed in Dikarya, such as aerial spore dispersal and life cycles dominated by mitosis of haploid nuclei. We now appreciate that Fungi comprises numerous phylum-level lineages in addition to those of Dikarya, but the phylogeny and genetic characteristics of most of these lineages are poorly understood due to limited genome sampling. Here, we addressed major evolutionary trends in the non-Dikarya fungi by phylogenomic analysis of 69 newly generated draft genome sequences of the zoosporic (flagellated) lineages of true fungi. Our phylogeny indicated five lineages of zoosporic fungi and placed Blastocladiomycota, which has an alternation of haploid and diploid generations, as branching closer to the Dikarya than to the Chytridiomyceta. Our estimates of heterozygosity based on genome sequence data indicate that the zoosporic lineages plus the Zoopagomycota are frequently characterized by diploid-dominant life cycles. We mapped additional traits, such as ancestral cell-cycle regulators, cell-membrane- and cell-wall-associated genes, and the use of the amino acid selenocysteine on the phylogeny and found that these ancestral traits that are shared with Metazoa have been subject to extensive parallel loss across zoosporic lineages. Together, our results indicate a gradual transition in the genetics and cell biology of fungi from their ancestor and caution against assuming that traits measured in Dikarya are typical of other fungal lineages.

Gametophyte genome activation occurs at pollen mitosis I in maize.

Flowering plants alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. Pollen actively transcribes its haploid genome, providing phenotypic diversity even among pollen grains from a single plant. In this study, we used allele-specific RNA sequencing of single pollen precursors to follow the shift to haploid expression in maize pollen. We observed widespread biallelic expression for 11 days after meiosis, indicating that transcripts synthesized by the diploid sporophyte persist long into the haploid phase. Subsequently, there was a rapid and global conversion to monoallelic expression at pollen mitosis I, driven by active new transcription from the haploid genome. Genes showed evidence of increased purifying selection if they were expressed after (but not before) pollen mitosis I. This work establishes the timing during which haploid selection may act in pollen.

The DME demethylase regulates sporophyte gene expression, cell proliferation, differentiation, and meristem resurrection

The flowering plant life cycle consists of alternating haploid (gametophyte) and diploid (sporophyte) generations, where the sporophytic generation begins with fertilization of haploid gametes. In Arabidopsis, genome-wide DNA demethylation is required for normal development, catalyzed by the DEMETER (DME) DNA demethylase in the gamete companion cells of male and female gametophytes. In the sporophyte, postembryonic growth and development are largely dependent on the activity of numerous stem cell niches, or meristems. Analyzing Arabidopsis plants homozygous for a loss-of-function dme-2 allele, we show that DME influences many aspects of sporophytic growth and development. dme-2 mutants exhibited delayed seed germination, variable root hair growth, aberrant cellular proliferation and differentiation followed by enhanced de novo shoot formation, dysregulation of root quiescence and stomatal precursor cells, and inflorescence meristem (IM) resurrection. We also show that sporophytic DME activity exerts a profound effect on the transcriptome of developing Arabidopsis plants, including discrete groups of regulatory genes that are misregulated in dme-2 mutant tissues, allowing us to potentially link phenotypes to changes in specific gene expression pathways. These results show that DME plays a key role in sporophytic development and suggest that DME-mediated active DNA demethylation may be involved in the maintenance of stem cell activities during the sporophytic life cycle in Arabidopsis.

HAG1 and SWI3A/B control of male germ line development in P. patens suggests conservation of epigenetic reproductive control across land plants

Key messageBryophytes as models to study the male germ line: loss-of-function mutants of epigenetic regulators HAG1 and SWI3a/b demonstrate conserved function in sexual reproduction.With the water-to-land transition, land plants evolved a peculiar haplodiplontic life cycle in which both the haploid gametophyte and the diploid sporophyte are multicellular. The switch between these phases was coined alternation of generations. Several key regulators that control the bauplan of either generation are already known. Analyses of such regulators in flowering plants are difficult due to the highly reduced gametophytic generation, and the fact that loss of function of such genes often is embryo lethal in homozygous plants. Here we set out to determine gene function and conservation via studies in bryophytes. Bryophytes are sister to vascular plants and hence allow evolutionary inferences. Moreover, embryo lethal mutants can be grown and vegetatively propagated due to the dominance of the bryophyte gametophytic generation. We determined candidates by selecting single copy orthologs that are involved in transcriptional control, and of which flowering plant mutants show defects during sexual reproduction, with a focus on the under-studied male germ line. We selected two orthologs, SWI3a/b and HAG1, and analyzed loss-of-function mutants in the moss P. patens. In both mutants, due to lack of fertile spermatozoids, fertilization and hence the switch to the diploid generation do not occur. Pphag1 additionally shows arrested male and impaired female gametangia development. We analyzed HAG1 in the dioecious liverwort M. polymorpha and found that in Mphag1 the development of gametangiophores is impaired. Taken together, we find that involvement of both regulators in sexual reproduction is conserved since the earliest divergence of land plants.

Signalling Pinpointed to the Tip: The Complex Regulatory Network That Allows Pollen Tube Growth.

Plants display a complex life cycle, alternating between haploid and diploid generations. During fertilisation, the haploid sperm cells are delivered to the female gametophyte by pollen tubes, specialised structures elongating by tip growth, which is based on an equilibrium between cell wall-reinforcing processes and turgor-driven expansion. One important factor of this equilibrium is the rate of pectin secretion mediated and regulated by factors including the exocyst complex and small G proteins. Critically important are also non-proteinaceous molecules comprising protons, calcium ions, reactive oxygen species (ROS), and signalling lipids. Among the latter, phosphatidylinositol 4,5-bisphosphate and the kinases involved in its formation have been assigned important functions. The negatively charged headgroup of this lipid serves as an interaction point at the apical plasma membrane for partners such as the exocyst complex, thereby polarising the cell and its secretion processes. Another important signalling lipid is phosphatidic acid (PA), that can either be formed by the combination of phospholipases C and diacylglycerol kinases or by phospholipases D. It further fine-tunes pollen tube growth, for example by regulating ROS formation. How the individual signalling cues are intertwined or how external guidance cues are integrated to facilitate directional growth remain open questions.

Induction of Multichotomous Branching by CLAVATA Peptide in Marchantia polymorpha

A key innovation in land plants was the evolution of meristems with stem cells possessing multiple cutting faces (division planes) from which three-dimensional growth is derived in both haploid (gametophyte) and diploid (sporophyte) generations [1-3]. Within each meristem exists a pool of stem cells that must be maintained at a relatively constant size for development to occur appropriately [4-6]. In flowering plants, stem cells of the diploid generation are maintained by CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) peptide signaling [7, 8]. In the liverwort Marchantia polymorpha, the haploid body undergoes dichotomous branching, an ancestral characteristic of growth derived from the meristem, in which two equivalent body axes are developed via stem cell division, regulated by unknown molecular mechanisms. We show here that in M.polymorpha, treatment with MpCLE2/CLAVATA3 (CLV3) peptide resulted in the accumulation of undifferentiated cells, marked by MpYUC2 expression, in the apical meristem. Removal of MpCLE2 peptide resulted in multichotomous branching from the accumulated cells. Genetic analysis demonstrated that the CLAVATA1 (MpCLV1) receptor, but not the WUSCHEL-related HOMEOBOX (MpWOX) transcription factor, is responsible for MpCLE2 peptide signaling. In the apical meristem, MpCLV1 was expressed broadly in the central region, including the MpYUC2-positive area, whereas MpCLE2 was expressed in a largely complementary manner compared to MpYUC2, suggesting MpCLE2 mediates local cell-to-cell communication. CLV3/CLE peptide, a negative regulator of diploid stem cells in flowering plants, acts as a haploid stem cell-promoting signal in M.polymorpha, implicating a critical role for this pathway in the evolution of body plan in land plants.

Two ANGUSTIFOLIA genes regulate gametophore and sporophyte development in Physcomitrella patens.

In Arabidopsis thaliana the ANGUSTIFOLIA (AN) gene regulates the width of leaves by controlling the diffuse growth of leaf cells in the medio-lateral direction. In the genome of the moss Physcomitrella patens, we found two normal ANs (PpAN1-1 and 1-2). Both PpAN1 genes complemented the A.thaliana an-1 mutant phenotypes. An analysis of spatiotemporal promoter activity of each PpAN1 gene, using transgenic lines that contained each PpAN1-promoter- uidA (GUS) gene, showed that both promoters are mainly active in the stems of haploid gametophores and in the middle to basal region of the young sporophyte that develops into the seta and foot. Analyses of the knockout lines for PpAN1-1 and PpAN1-2 genes suggested that these genes have partially redundant functions and regulate gametophore height by controlling diffuse cell growth in gametophore stems. In addition, the seta and foot were shorter and thicker in diploid sporophytes, suggesting that cell elongation was reduced in the longitudinal direction, whereas no defects were detected in tip-growing protonemata. These results indicate that both PpAN1 genes in P.patens function in diffuse growth of the haploid and diploid generations but not in tip growth. To visualize microtubule distribution in gametophore cells of P.patens, transformed lines expressing P.patens α-tubulin fused to sGFP were generated. Contrary to expectations, the orientation of microtubules in the tips of gametophores in the PpAN1-1/1-2 double-knockout lines was unchanged. The relationships among diffuse cell growth, cortical microtubules and AN proteins are discussed.

Evolution, Initiation, and Diversity in Early Plant Embryogenesis.

During embryogenesis in plants, cell identities are specified de novo, starting from a single cell. By combining imaging, genomic profiling, and genetics, principles of early plant development have been unraveled in the dicotyledonous plant Arabidopsis. A central emerging question, however, is how well zygotic embryogenesis in Arabidopsis reflects homologous processes in other plant species, including early diverging, non-flowering, and non-seed plants. Here, we consider plant embryogenesis with an emphasis on its evolutionary history, the diverse modes of its initiation, and the concepts in pattern formation among morphologically distinct plant groups. Furthermore, we explore challenges and future directions in plant embryogenesis research.

Nuclear behavior and roles indicate that Codiolum phase is a sporophyte in Monostroma angicava (Ulotrichales, Ulvophyceae).

The life-cycle system of Ulotrichales, a major order of Ulvophyceae, remains controversial because it is unclear whether the Codiolum phase, a characteristic unicellular diploid generation in ulotrichalean algae, is a zygote or a sporophyte. This controversy inhibits the understanding of the diversified life cycles in Ulvophyceae. To distinguish between zygotes and sporophytes, we have to examine not only whether diploid generations function as sporophytes, but also whether mitosis occurs before meiosis in diploid generations. However, the nuclear behavior in the Codiolum phases is largely unknown, probably because no suitable methods are available. Using fluorescent microscopy with ethidium bromide and transmission electron microscopy of cell-wall-dissected specimens, we report the nuclear behavior in the Codiolum phases of an ulotrichalean alga with a representative life cycle, Monostroma angicava. Each vegetative Codiolum phase had a single polyploid nucleus due to endoreduplication, a type of mitosis without nuclear division. During zoosporogenesis, the nucleus had a structure that would be a meiosis-specific complex. We quantitatively showed that Codiolum phases grew extremely large and produced numerous zoospores. Our results suggest that an event comparable to mitosis occurs before meiosis in the Codiolum phase of M.angicava. This nuclear behavior and the functions (growth and zoospore production abilities) correspond to those of sporophytes. Therefore, the life-cycle system of M.angicava is a heteromorphic haplo-diplontic cycle. This system appears to be widely adopted among other ulotrichalean algae.

Rapid turnover of life-cycle-related genes in the brown algae

BackgroundSexual life cycles in eukaryotes involve a cyclic alternation between haploid and diploid phases. While most animals possess a diploid life cycle, many plants and algae alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. In many algae, gametophytes and sporophytes are independent and free-living and may present dramatic phenotypic differences. The same shared genome can therefore be subject to different, even conflicting, selection pressures during each of the life cycle generations. Here, we analyze the nature and extent of genome-wide, generation-biased gene expression in four species of brown algae with contrasting levels of dimorphism between life cycle generations.ResultsWe show that the proportion of the transcriptome that is generation-specific is broadly associated with the level of phenotypic dimorphism between the life cycle stages. Importantly, our data reveals a remarkably high turnover rate for life-cycle-related gene sets across the brown algae and highlights the importance not only of co-option of regulatory programs from one generation to the other but also of a role for newly emerged, lineage-specific gene expression patterns in the evolution of the gametophyte and sporophyte developmental programs in this major eukaryotic group. Moreover, we show that generation-biased genes display distinct evolutionary modes, with gametophyte-biased genes evolving rapidly at the coding sequence level whereas sporophyte-biased genes tend to exhibit changes in their patterns of expression.ConclusionOur analysis uncovers the characteristics, expression patterns, and evolution of generation-biased genes and underlines the selective forces that shape this previously underappreciated source of phenotypic diversity.

Haploid females in the isomorphic biphasic life-cycle of Gracilaria chilensis excel in survival

BackgroundConditional differentiation is one of the most fundamental drivers of biodiversity. Competitive entities (usually species) differ in environmental or ecological niche enabling them to co-exist. Conditional differentiation of haploid and diploid generations is considered to be a requirement for the evolutionary stability of isomorphic biphasic life-cycles and the cause for the natural occurrence of both phases at uneven abundances. Theoretically, stage dependent survival rates are the most efficient way to explain conditional differentiation.ResultsWe tested for conditional differentiation in survival rates among life stages (haploid males, haploid females, and diploids) of Gracilaria chilensis, an intertidal red alga occurring along the Chilean shores. Therefore, the fate of individuals was followed periodically for 3 years in five intertidal pools and, for the first time in isomorphic red algae, a composite model of the instantaneous survival rates was applied. The results showed the survival dependency on density (both competition and Allee effects), fertility, age, size, season and location, as well as the differentiation among stages for the survival dependencies of these factors. The young haploid females survived more than the young of the other stages under Allee effects during the environmentally stressful season at the more exposed locations, and under self-thinning during the active growth season. Furthermore, fertile haploid females had a higher survival than fertile haploid males or fertile diploids.ConclusionsHere, we show a survival advantage of haploids over diploids. The haploid females probably optimize their resource management targeting structural and physiological adaptations that significantly enhance survival under harsher conditions. In a companion paper we demonstrate a fertility advantage of diploids over haploids. Together, the survival and fertility differentiation support the evolution and prevalence of biphasic life-cycles.

Sporophytes of polysporangiate land plants from the early Silurian period may have been photosynthetically autonomous.

The colonization of land by vascular plants is an extremely important phase in Earth's life history. This key evolutionary process is thought to have begun during the Middle Cambrian 1 period and culminated in the Silurian/Early Devonian period (interval about 509-393 million years ago (Ma)), and is documented primarily by microfossils (that is, by dispersed spores, phytodebris including fragments of algae, tissues, sporangia and cuticles), tubes and rare megafossils 2 . A newly recognized fossil cooksonioid plant with in situ spores from the Barrandian area, Czech Republic, is of the highest importance because it represents extremely ancient megafossil evidence of land plant diploid generation: sporophytes (~432 Ma). The robust size of this plant places it among the largest known early polysporangiate land plants and it is probable that it attained adequate size for both aeration and effective photosynthetic competence. This would mean not only that sporophytes were photosynthetically autonomous but also that the they might have been able to sustain a relatively gametophyte-independent existence.

Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M.polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.

De novo transcriptome assembly of a fern, Lygodium japonicum, and a web resource database, Ljtrans DB.

During plant evolution, ferns originally evolved as a major vascular plant with a distinctive life cycle in which the haploid and diploid generations are completely separated. However, the low level of genetic resources has limited studies of their physiological events, as well as hindering research on the evolutionary history of land plants. In this study, to identify a comprehensive catalog of transcripts and characterize their expression traits in the fern Lygodium japonicum, nine different RNA samples isolated from prothalli, trophophylls, rhizomes and sporophylls were sequenced using Roche 454 GS-FLX and Illumina HiSeq sequencers. The hybrid assembly of the high-quality 454 GS-FLX and Illumina HiSeq reads generated a set of 37,830 isoforms with an average length of 1,444 bp. Using four open reading frame (ORF) predictors, 38,142 representative ORFs were identified from a total of 37,830 transcript isoforms and 95 contigs, which were annotated by searching against several public databases. Furthermore, an orthoMCL analysis using the protein sequences of L. japonicum and five model plants revealed various sets of lineage-specific genes, including those detected among land plant lineages and those detected in only L. japonicum. We have also examined the expression patterns of all contigs/isoforms, along with the life cycle of L. japonicum, and identified the tissue-specific transcripts using statistical expression analyses. Finally, we developed a public web resource, the L. japonicum transcriptome database at http://bioinf.mind.meiji.ac.jp/kanikusa/, which provides important opportunities to accelerate molecular research in ferns.

A Lallzyme MMX‐based rapid method for fission yeast protoplast preparation

Fungal cells including yeasts are surrounded by cell wall that counteracts turgor pressure and prevents cell lysis. Many yeast experiments, including genetic manipulation of sterile strains, morphogenesis studies, nucleic acid isolation and many others, require mechanical breakage or enzymatic removal of the cell wall. Some of these experiments require the generation of live cells lacking cell walls, called protoplasts, that can be maintained in osmostabilized medium. Enzymatic digestion of cell wall proteoglycans is a commonly used method of protoplast preparation. Currently existing protocols for fission yeast cell wall digestion are time consuming and not very efficient. We developed a new rapid method for fission yeast protoplast preparation that relies on digesting cell walls with Lallzyme MMX commercial enzyme mix, which produces protoplasts from all cells in less than 10 min. We demonstrate that these protoplasts can be utilized in three commonly used fission yeast protocols. Thus, we provide the fission yeast community with a robust and efficient plasmid extraction method, a new protocol for diploid generation and an assay for protoplast recovery that should be useful for studies of morphogenesis. Our method is potentially applicable to other yeasts and fungi.

Maternal control of PIN1 is required for female gametophyte development in Arabidopsis.

Land plants are characterised by haplo-diploid life cycles, and developing ovules are the organs in which the haploid and diploid generations coexist. Recently it has been shown that hormones such as auxin and cytokinins play important roles in ovule development and patterning. The establishment and regulation of auxin levels in cells is predominantly determined by the activity of the auxin efflux carrier proteins PIN-FORMED (PIN). To study the roles of PIN1 and PIN3 during ovule development we have used mutant alleles of both genes and also perturbed PIN1 and PIN3 expression using micro-RNAs controlled by the ovule specific DEFH9 (DEFIFICENS Homologue 9) promoter. PIN1 down-regulation and pin1-5 mutation severely affect female gametophyte development since embryo sacs arrest at the mono- and/or bi-nuclear stages (FG1 and FG3 stage). PIN3 function is not required for ovule development in wild-type or PIN1-silenced plants. We show that sporophytically expressed PIN1 is required for megagametogenesis, suggesting that sporophytic auxin flux might control the early stages of female gametophyte development, although auxin response is not visible in developing embryo sacs.

Mirror, Mirror on the Wall: A Role for AGP18 in Functional Megaspore Selection

The life cycle of plants alternates between diploid sporophyte and haploid gametophyte generations. Most Angiosperms are monosporic; one haploid megaspore per ovule primordium is selected as the functional megaspore, which gives rise to the megagametophyte, and the remaining three megaspores perish