Transcriptomics of manually isolated Amborella trichopoda egg apparatus cells.

KEY MESSAGE: A protocol for the isolation of egg apparatus cells from the basal angiosperm Amborella trichopoda to generate RNA-seq data for evolutionary studies of fertilization-associated genes. Sexual reproduction is particularly complex in flowering plants (angiosperms). Studies in eudicot and monocot model species have significantly contributed to our knowledge on cell fate specification of gametophytic cells and on the numerous cellular communication events necessary to deliver the two sperm cells into the embryo sac and to accomplish double fertilization. However, for a deeper understanding of the evolution of these processes, morphological, genomic and gene expression studies in extant basal angiosperms are inevitable. The basal angiosperm Amborella trichopoda is of special importance for evolutionary studies, as it is likely sister to all other living angiosperms. Here, we report about a method to isolate Amborella egg apparatus cells and on genome-wide gene expression profiles in these cells. Our transcriptomics data revealed Amborella-specific genes and genes conserved in eudicots and monocots. Gene products include secreted proteins, such as small cysteine-rich proteins previously reported to act as extracellular signaling molecules with important roles during double fertilization. The detection of transcripts encoding EGG CELL 1 (EC1) and related prolamin-like family proteins in Amborella egg cells demonstrates the potential of the generated data set to study conserved molecular mechanisms and the evolution of fertilization-related genes and their encoded proteins.

The autonomous cell fate specification of basal cell lineage: the initial round of cell fate specification occurs at the two-celled proembryo stage.

In angiosperms, the first zygotic division usually gives rise to two daughter cells with distinct morphologies and developmental fates, which is critical for embryo pattern formation; however, it is still unclear when and how these distinct cell fates are specified, and whether the cell specification is related to cytoplasmic localization or polarity. Here, we demonstrated that when isolated from both maternal tissues and the apical cell, a single basal cell could only develop into a typical suspensor, but never into an embryo in vitro. Morphological, cytological and gene expression analyses confirmed that the resulting suspensor in vitro is highly similar to its undisturbed in vivo counterpart. We also demonstrated that the isolated apical cell could develop into a small globular embryo, both in vivo and in vitro, after artificial dysfunction of the basal cell; however, these growing apical cell lineages could never generate a new suspensor. These findings suggest that the initial round of cell fate specification occurs at the two-celled proembryo stage, and that the basal cell lineage is autonomously specified towards the suspensor, implying a polar distribution of cytoplasmic contents in the zygote. The cell fate transition of the basal cell lineage to the embryo in vivo is actually a conditional cell specification process, depending on the developmental signals from both the apical cell lineage and maternal tissues connected to the basal cell lineage.

Frequent chloroplast RNA editing in early-branching flowering plants: pilot studies on angiosperm-wide coexistence of editing sites and their nuclear specificity factors.

BACKGROUND: RNA editing by cytidine-to-uridine conversions is an essential step of RNA maturation in plant organelles. Some 30-50 sites of C-to-U RNA editing exist in chloroplasts of flowering plant models like Arabidopsis, rice or tobacco. We now predicted significantly more RNA editing in chloroplasts of early-branching angiosperm genera like Amborella, Calycanthus, Ceratophyllum, Chloranthus, Illicium, Liriodendron, Magnolia, Nuphar and Zingiber. Nuclear-encoded RNA-binding pentatricopeptide repeat (PPR) proteins are key editing factors expected to coevolve with their cognate RNA editing sites in the organelles. RESULTS: With an extensive chloroplast transcriptome study we identified 138 sites of RNA editing in Amborella trichopoda, approximately the 3- to 4-fold of cp editing in Arabidopsis thaliana or Oryza sativa. Selected cDNA studies in the other early-branching flowering plant taxa furthermore reveal a high diversity of early angiosperm RNA editomes. Many of the now identified editing sites in Amborella have orthologues in ferns, lycophytes or hornworts. We investigated the evolution of CRR28 and RARE1, two known Arabidopsis RNA editing factors responsible for cp editing events ndhBeU467PL, ndhDeU878SL and accDeU794SL, respectively, all of which we now found conserved in Amborella. In a phylogenetically wide sampling of 65 angiosperm genomes we find evidence for only one single loss of CRR28 in chickpea but several independent losses of RARE1, perfectly congruent with the presence of their cognate editing sites in the respective cpDNAs. CONCLUSION: Chloroplast RNA editing is much more abundant in early-branching than in widely investigated model flowering plants. RNA editing specificity factors can be traced back for more than 120 million years of angiosperm evolution and show highly divergent patterns of evolutionary losses, matching the presence of their target editing events.

[Length of Meristematic and Fully Elongated Root Cells Related to Haploid DNA Content].

The lengths of meristematic (l(m)) and fully-elongated cells (l(e)) were measured in the roots of 118 monocot and dicot species of herbaceous plants from 20 angiosperm families. The results were analyzed using the data on haploid DNA content (C(val)) for the same species from the website (http://data.kew.org/cvalues). The distribution range of lm, le, and C(val) was wider in monocot plants compared to dicots. Values of l(m) and l(e) in monocot and lm in dicot species correlated positively with C(val). Dependence of lm and le on C(val) was similar in diploid and polyploid species, both monocots and dicots. The average length of root cells differed less than the root length.

Cytotype diversity and genome size variation in Knautia (Caprifoliaceae, Dipsacoideae).

BACKGROUND: Polyploidisation is one of the most important mechanisms in the evolution of angiosperms. As in many other genera, formation of polyploids has significantly contributed to diversification and radiation of Knautia (Caprifoliaceae, Dipsacoideae). Comprehensive studies of fine- and broad-scale patterns of ploidy and genome size (GS) variation are, however, still limited to relatively few genera and little is known about the geographic distribution of ploidy levels within these genera. Here, we explore ploidy and GS variation in Knautia based on a near-complete taxonomic and comprehensive geographic sampling. RESULTS: Genome size is a reliable indicator of ploidy level in Knautia, even if monoploid genome downsizing is observed in the polyploid cytotypes. Twenty-four species studied are diploid, 16 tetraploid and two hexaploid, whereas ten species possess two, and two species possess three ploidy levels. Di- and tetraploids are distributed across most of the distribution area of Knautia, while hexaploids were sampled in the Balkan and Iberian Peninsulas and the Alps. CONCLUSIONS: We show that the frequency of polyploidisation is unevenly distributed in Knautia both in a geographic and phylogenetic context. Monoploid GS varies considerably among three evolutionary lineages (sections) of Knautia, but also within sections Trichera and Tricheroides, as well as within some of the species. Although the exact causes of this variation remain elusive, we demonstrate that monoploid GS increases significantly towards the limits of the genus' distribution.

Sporoderm and tapetum development in Eupomatia laurina (Eupomatiaceae). An interpretation.

For the first time, the developmental events in the course of exine structure establishment have been traced in detail with TEM in Eupomatia, with the addition of cytochemical tests. A new look at unfolding events is suggested using our recent hypothesis on self-assembling micellar mesophases. The process proved to be unusual and includes "ghost" stages. The first units observed in the periplasmic space are spherical ones (= normal spherical micelles). These accumulate, resulting in a granular layer up to middle tetrad stage. Sporopollenin precursor accumulation on these units makes the ectexine layer looking as homogenous at late tetrad stage. Simultaneously, the columns of globules are added in the periplasmic space, which reminds an attempt to form columellae; but, the process failed. Instead, a fimbrillate endexine layer of compressed globules appears. The latter augments via additional globules, appearing in the periplasmic space in the free microspore period. The endexine formation is double-stepped spatially and temporally. The second, lamellate endexine layer (laminate micelles) appears late in development, when the channeled intine-I is already established-a very unusual feature. Moreover, a "fenestrated" stage comes unexpectedly at vacuolate stage, when hitherto amorphous ectexine appears pierced by cavernae-the results of reversal of normal spherical micelles (constituents of ectexine) to reverse the ones that open their cores for the entrance of hydrophilic nutrients from tapetum and give them over to the microspore cytoplasm by exchanging their solubilizates.

TRV-GFP: a modified Tobacco rattle virus vector for efficient and visualizable analysis of gene function.

Virus-induced gene silencing (VIGS) is a useful tool for functional characterization of genes in plants. Unfortunately, the efficiency of infection by Tobacco rattle virus (TRV) is relatively low for some non-Solanaceae plants, which are economically important, such as rose (Rosa sp.). Here, to generate an easy traceable TRV vector, a green fluorescent protein (GFP) gene was tagged to the 3' terminus of the coat protein gene in the original TRV2 vector, and the silencing efficiency of the modified TRV-GFP vector was tested in several plants, including Nicotiana benthamiana, Arabidopsis thaliana, rose, strawberry (Fragaria ananassa), and chrysanthemum (Dendranthema grandiflorum). The results showed that the efficiency of infection by TRV-GFP was equal to that of the original TRV vector in each tested plant. Spread of the modified TRV virus was easy to monitor by using fluorescent microscopy and a hand-held UV lamp. When TRV-GFP was used to silence the endogenous phytoene desaturase (PDS) gene in rose cuttings and seedlings, the typical photobleached phenotype was observed in 75-80% plants which were identified as GFP positive by UV lamp. In addition, the abundance of GFP protein, which represented the concentration of TRV virus, was proved to correlate negatively with the level of the PDS gene, suggesting that GFP could be used as an indicator of the degree of silencing of a target gene. Taken together, this work provides a visualizable and efficient tool to predict positive gene silencing plants, which is valuable for research into gene function in plants, especially for non-Solanaceae plants.

The CEP family in land plants: evolutionary analyses, expression studies, and role in Arabidopsis shoot development.

In Arabidopsis, more than 1000 putative small signalling peptides have been predicted, but very few have been functionally characterized. One class of small post-translationally modified signalling peptides is the C-TERMINALLY ENCODED PEPTIDE (CEP) family, of which one member has been shown to be involved in regulating root architecture. This work applied a bioinformatics approach to identify more members of the CEP family. It identified 10 additional members and revealed that this family only emerged in flowering plants and was absent from extant members of more primitive plants. The data suggest that the CEP proteins form two subgroups according to the CEP domain. This study further provides an overview of specific CEP expression patterns that offers a comprehensive framework to study the role of the CEP signalling peptides in plant development. For example, expression patterns point to a role in aboveground tissues which was corroborated by the analysis of transgenic lines with perturbed CEP levels. These results form the basis for further exploration of the mechanisms underlying this family of peptides and suggest their putative roles in distinct developmental events of higher plants.

Volume-based pollen size analysis: an advanced method to assess somatic and gametophytic ploidy in flowering plants.

Pollen size is often used as a biological parameter to estimate the ploidy and viability of mature pollen grains. In general, pollen size quantification is performed one- or two-dimensionally using image-based diameter measurements. As these approaches are elaborate and time consuming, alternative approaches that enable a quick, reliable analysis of pollen size are highly relevant for plant research. In this study, we present the volume-based particle size analysis technique as an alternative method to characterize mature pollen. Based on a comparative assay using different plant species (including tomato, oilseed rape, kiwifruit, clover, among others), we found that volume-based pollen size measurements are not biased by the pollen shape or position and substantially reduce non-biological variation, allowing a more accurate determination of the actual pollen size. As such, volume-based particle size techniques have a strong discriminative power in detecting pollen size differences caused by alterations in the gametophytic ploidy level and therefore allow for a quick and reliable estimation of the somatic ploidy level. Based on observations in Arabidopsis thaliana gametophytic mutants and differentially reproducing Boechera polyantha lines, we additionally found that volume-based pollen size analysis provides quantitative and qualitative data about alterations in male sporogenesis, including aneuploid and diploid gamete formation. Volume-based pollen size analysis therefore not only provides a quick and easy methodology to determine the somatic ploidy level of flowering plants, but can also be used to determine the mode of reproduction and to quantify the level of diplogamete formation.

High ploidy diversity and distinct patterns of cytotype distribution in a widespread species of Oxalis in the Greater Cape Floristic Region.

BACKGROUND AND AIMS: Genome duplication is widely acknowledged as a major force in the evolution of angiosperms, although the incidence of polyploidy in different floras may differ dramatically. The Greater Cape Floristic Region of southern Africa is one of the world's biodiversity hotspots and is considered depauperate in polyploids. To test this assumption, ploidy variation was assessed in a widespread member of the largest geophytic genus in the Cape flora: Oxalis obtusa. METHODS: DNA flow cytometry complemented by confirmatory chromosome counts was used to determine ploidy levels in 355 populations of O. obtusa (1014 individuals) across its entire distribution range. Ecological differentiation among cytotypes was tested by comparing sets of vegetation and climatic variables extracted for each locality. KEY RESULTS: Three majority (2x, 4x, 6x) and three minority (3x, 5x, 8x) cytotypes were detected in situ, in addition to a heptaploid individual originating from a botanical garden. While single-cytotype populations predominate, 12 mixed-ploidy populations were also found. The overall pattern of ploidy level distribution is quite complex, but some ecological segregation was observed. Hexaploids are the most common cytotype and prevail in the Fynbos biome. In contrast, tetraploids dominate in the Succulent Karoo biome. Precipitation parameters were identified as the most important climatic variables associated with cytotype distribution. CONCLUSIONS: Although it would be premature to make generalizations regarding the role of genome duplication in the genesis of hyperdiversity of the Cape flora, the substantial and unexpected ploidy diversity in Oxalis obtusa is unparalleled in comparison with any other cytologically known native Cape plant species. The results suggest that ploidy variation in the Greater Cape Floristic Region may be much greater than currently assumed, which, given the documented role of polyploidy in speciation, has direct implications for radiation hypotheses in this biodiversity hotspot.

Reorganization of the plant cortical microtubule array.

The interphase microtubule arrays in flowering plant cells assemble at the cell cortex into patterns that affect cellular morphogenesis. A decade of live cell imaging studies has provided significant information about the in vivo properties of the microtubule polymers. Efforts to extrapolate individual properties to larger roles in organizing or patterning the microtubule array have produced models focused on self-organization and local levels of biological control. Recent studies looking at cortical microtubule arrays as they transition from an existing pattern to a new pattern have re-emerged as a testbed for examining these models and the molecular hypotheses underpinning them. The evidence suggests that microtubule patterning is locally controlled on the scale of a cell face, using or circumventing self-organizating properties as necessary.

[Genesis of cells of apical meristems and realization of gametophytic apomixis in flowering plants].

Based on our own and literature data on peculiarities of caryotypical variability, we concluded that gametophytic apomixis is naturally accompanied with phenomena of poly-, aneu-, and mixoploidy and that apomicts have genome instability manifesting at the level of meristematic somatic cells. In this connection, a hypothesis is substantiated that realization of this mode of seed reproduction in flowering plants is caused by modification of systems of cell cycle control, following after acts of hybridogenesis and/or polyploidization. It is concluded that instability of the seed reproduction system by gametophytic apomixis manifests not only at the stage of choice of a seed reproduction pathway (apomeiosis-euspory; apozygosis-zygosis) but also in all the cycles of reproduction of the cells of a germ line in plant ontogenesis.

Nickel(II)-induced excimer formation of a naphthalene-based fluorescent probe for living cell imaging.

Ni(2+)-induced intramolecular excimer formation of a naphthalene-based novel fluorescent probe, 1-[(naphthalen-3-yl)methylthio]-2-[(naphthalen-6-yl)methylthio]ethane (L), has been investigated for the first time and nicely demonstrated by excitation spectra, a fluorescence lifetime experiment, and (1)H NMR titration. The addition of Ni(2+) to a solution of L (DMSO:water = 1:1, v/v; λ(em) = 345 nm, λ(ex) = 280 nm) quenched its monomer emission, with subsequent enhancement of the excimer intensity (at 430 nm) with an isoemissive point at 381 nm. The fluorescence lifetime of free L (0.3912 ns) is much lower than that of the nickel(2+) complex (1.1329 ns). L could detect Ni(2+) as low as 1 × 10(-6) M with a fairly strong binding constant, 2.0 × 10(4) M(-1). Ni(2+)-contaminated living cells of plant origin could be imaged using a fluorescence microscope.

Three sesquiterpene compounds biosynthesised from artemisinic acid using suspension-cultured cells of Averrhoa carambola (Oxalidaceae).

A new sesquiterpene glycoside, artemisinic acid 3-ß-O-ß-D-glucopyranoside (3, 31.24%) and other two biotransformation products, 3-ß-hydroxyartemisinic acid (2, 36.69%) and 3-ß-hydroxyartemisinic acid ß-D-glucopyranosyl ester (4, 7.03%), were biosynthesised after artemisinic acid (1) was administered to the cultured cells of Averrhoa carambola. The three biotransformation products were obtained for the first time by using the suspension-cultured cells of A. carambola as a new biocatalyst system, and their structures were identified on the basis of the physico-chemical properties, NMR and mass spectral analyses. The results indicate that the cultured cells of A. carambola have the abilities to hydroxylate and glycosylate sesquiterpene compounds in a regio- and stereoselective manner. Furthermore, the anti-tumour activity of compounds 3 and 4 was evaluated against K562 and HeLa cell lines. Compound 4 showed strong activity against HeLa cell line, with the IC50 value of 0.56 µmol mL⁻¹.

Sporogenesis and development of gametophytes in an endangered plant, Tetracentron sinense Oliv

The sporogenesis and development of gametophytes in Tetracentron sinense Oliv. were studied with light microscopy. The anther has four microsporangia; its primary anther wall consists of an epidermis, an endothecium, one or two middle layers and one glandular tapetum. Simultaneous cytokinesis follows meiosis, forming a tetrahedral tetrad. Mature pollen grains are two-celled at the time of anther dehiscence. Its ovule is anatropous, bitegmic and crassinucellate; the development of the female gametophyte is of the monosporic 8-nucleate Polygonum type. Significantly, some striking features were first found in T. sinense: (1) anther dehiscence occurs soon after the endothecium fibrously thickens and the intersporangial septum degenerates; (2) tapetum degeneration is retarded, persisting up to the stage of two-celled pollen grain; (3) a few cellular events such as the vacuolization and the contraction and deformation of the pollen mother cell (PMC) and microspore are not normal at the PMC, dyad and tetrad stages. The abnormalities during male reproduction might be one of important factors resulting in the poor natural regeneration of T. sinense.

Primary and secondary thickening in the stem of Cordyline fruticosa (Agavaceae)

The growth in thickness of monocotyledon stems can be either primary, or primary and secondary. Most of the authors consider this thickening as a result of the PTM (Primary Thickening Meristem) and the STM (Secondary Thickening Meristem) activity. There are differences in the interpretation of which meristem would be responsible for primary thickening. In Cordyline fruticosa the procambium forms two types of vascular bundles: collateral leaf traces (with proto and metaxylem and proto and metaphloem), and concentric cauline bundles (with metaxylem and metaphloem). The procambium also forms the pericycle, the outermost layer of the vascular cylinder consisting of smaller and less intensely colored cells that are divided irregularly to form new vascular bundles. The pericycle continues the procambial activity, but only produces concentric cauline bundles. It was possible to conclude that the pericycle is responsible for the primary thickening of this species. Further away from the apex, the pericyclic cells undergo periclinal divisions and produce a meristematic layer: the secondary thickening meristem. The analysis of serial sections shows that the pericycle and STM are continuous in this species, and it is clear that the STM originates in the pericycle.The endodermis is acknowledged only as the innermost layer of the cortex.

O crescimento em espessura do caule de monocotiledônea pode ser primário, ou primário e secundário. A maioria dos autores consideram o espessamento resultante do MEP (Meristema de Espessamento Primário) e do MES (Meristema de Espessamento Secundário). Há divergências de qual seria o meristema responsável pelo espessamento primário. Em Cordyline fruticosa o procâmbio forma feixes vasculares de dois tipos: traços foliares colaterais (com proto e metaxilema e proto e metafloema), e feixes caulinares concêntricos (com metaxilema e metafloema). O procâmbio também forma o periciclo, a camada mais externa do cilindro vascular, constituída por células menores e menos coradas que se dividem irregularmente, formando novos feixes vasculares. O periciclo dá continuidade à atividade procambial, originando somente feixes concêntricos. Concluiu-se ser o periciclo responsável pelo espessamento primário desta espécie. Mais distante do ápice as células pericíclicas passam a sofrer divisões periclinais originando o Meristema de Espessamento Secundário. A análise dos cortes seriados mostra que o periciclo e o MES são contínuos nesta espécie, ficando claro que o periciclo origina oMES. A endoderme é reconhecida, apenas, como a camada mais interna do córtex.

Protecting cell walls from binding aluminum by organic acids contributes to aluminum resistance.

Aluminum-induced secretion of organic acids from the root apex has been demonstrated to be one major Al resistance mechanism in plants. However, whether the organic acid concentration is high enough to detoxify Al in the growth medium is frequently questioned. The genotypes of Al-resistant wheat, Cassia tora L. and buckwheat secrete malate, citrate and oxalate, respectively. In the present study we found that at a 35% inhibition of root elongation, the Al activities in the solution were 10, 20, and 50 muM with the corresponding malate, citrate, and oxalate exudation at the rates of 15, 20 and 21 nmol/cm(2) per 12 h, respectively, for the above three plant species. When exogenous organic acids were added to ameliorate Al toxicity, twofold and eightfold higher oxalate and malate concentrations were required to produce the equal effect by citrate. After the root apical cell walls were isolated and preincubated in 1 mM malate, oxalate or citrate solution overnight, the total amount of Al adsorbed to the cell walls all decreased significantly to a similar level, implying that these organic acids own an equal ability to protect the cell walls from binding Al. These findings suggest that protection of cell walls from binding Al by organic acids may contribute significantly to Al resistance.

Defensin-like polypeptide LUREs are pollen tube attractants secreted from synergid cells.

For more than 140 years, pollen tube guidance in flowering plants has been thought to be mediated by chemoattractants derived from target ovules. However, there has been no convincing evidence of any particular molecule being the true attractant that actually controls the navigation of pollen tubes towards ovules. Emerging data indicate that two synergid cells on the side of the egg cell emit a diffusible, species-specific signal to attract the pollen tube at the last step of pollen tube guidance. Here we report that secreted, cysteine-rich polypeptides (CRPs) in a subgroup of defensin-like proteins are attractants derived from the synergid cells. We isolated synergid cells of Torenia fournieri, a unique plant with a protruding embryo sac, to identify transcripts encoding secreted proteins as candidate molecules for the chemoattractant(s). We found two CRPs, abundantly and predominantly expressed in the synergid cell, which are secreted to the surface of the egg apparatus. Moreover, they showed activity in vitro to attract competent pollen tubes of their own species and were named as LUREs. Injection of morpholino antisense oligomers against the LUREs impaired pollen tube attraction, supporting the finding that LUREs are the attractants derived from the synergid cells of T. fournieri.

Got milk? The secret life of laticifers.

Laticifers are specialized cells that occur in over 20 plant families in several unrelated angiosperm orders. Although laticifers are likely to be of polyphyletic origin, their occurrence is considered a morphological indicator of relatedness among species. The classification of laticifers is based on developmental patterns and overall morphology. The cytoplasmic latex exuded in response to damage often includes specialized metabolites, such as cardenolides, alkaloids and natural rubber. Laticifers provide an effective location to store defense metabolites, although not all latex-bearing plants accumulate bioactive natural products. Ecophysiological studies have shown that latex and its associated metabolites are vital for the defense of plants against insects. The anatomy, development and physiology of laticifers are discussed with a focus on evolutionary and ecological perspectives.