Acid scarification as a potent treatment for an in vitro germination of mature endozoochorous Vanilla planifolia seeds.

BACKGROUND: Vanilla planifolia is the most widely cultivated species of vanilla with high economic importance. However, seed germination under artificial conditions is difficult and yields low germination percentages. The seeds are adapted to endozoochorous dispersal, and we therefore tried to simulate the conditions in the digestive tract by acid scarification of seeds. RESULTS: Immature seeds lacking dormancy, used as a control, showed the highest germination percentage. Among the treatments tested for mature seeds, the hydrochloric acid treatments were significantly the best in breaking dormancy and inducing germination, irrespective of the acid concentration and the presence of pepsin. Conventional treatment with a hypochlorite solution induced much lower germination percentage. Sulphuric acid at concentration 50% was too strong and caused damage to the seeds. Important factor is also high cultivation temperature 30 °C as there was nearly no germination at 25 °C. CONCLUSIONS: Our protocol significantly improves the efficiency of generative propagation of vanilla and allows for significantly higher germination percentages than previously described. The strongly positive effect of hydrochloric acid may be related to the adaptation of seeds to endozoochorous dispersal.

Integrative Study Supports the Role of Trehalose in Carbon Transfer From Fungi to Mycotrophic Orchid.

Orchids rely on mycorrhizal symbiosis, especially in the stage of mycoheterotrophic protocorms, which depend on carbon and energy supply from fungi. The transfer of carbon from fungi to orchids is well-documented, but the identity of compounds ensuring this transfer remains elusive. Some evidence has been obtained for the role of amino acids, but there is also vague and neglected evidence for the role of soluble carbohydrates, probably trehalose, which is an abundant fungal carbohydrate. We therefore focused on the possible role of trehalose in carbon and energy transfer. We investigated the common marsh orchid (Dactylorhiza majalis) and its symbiotic fungus Ceratobasidium sp. using a combination of cultivation approaches, high-performance liquid chromatography, application of a specific inhibitor of the enzyme trehalase, and histochemical localization of trehalase activity. We found that axenically grown orchid protocorms possess an efficient, trehalase-dependent, metabolic pathway for utilizing exogenous trehalose, which can be as good a source of carbon and energy as their major endogenous soluble carbohydrates. This is in contrast to non-orchid plants that cannot utilize trehalose to such an extent. In symbiotically grown protocorms and roots of adult orchids, trehalase activity was tightly colocalized with mycorrhizal structures indicating its pronounced role in the mycorrhizal interface. Inhibition of trehalase activity arrested the growth of both symbiotically grown protocorms and trehalose-supported axenic protocorms. Since trehalose constitutes only an inconsiderable part of the endogenous saccharide spectrum of orchids, degradation of fungal trehalose likely takes place in orchid mycorrhiza. Our results strongly support the neglected view of the fungal trehalose, or the glucose produced by its cleavage as compounds transported from fungi to orchids to ensure carbon and energy flow. Therefore, we suggest that not only amino acids, but also soluble carbohydrates are transported. We may propose that the soluble carbohydrates would be a better source of energy for plant metabolism than amino acids, which is partially supported by our finding of the essential role of trehalase.

Integrative Study of Genotypic and Phenotypic Diversity in the Eurasian Orchid Genus Neotinea.

Knowledge of population variation across species' ranges is a prerequisite for correctly assessing the overall variability of any group of organisms and provides an invaluable basis for unraveling evolutionary history, optimizing taxonomy and devising effective conservation strategies. Here, we examine the genus Neotinea, which represents a relatively recently delimited monophyletic genus of orchids, for which a detailed study of its overall variability was lacking. We applied a suite of biosystematic methods, consisting of flow cytometry, multivariate and geometric morphometrics, and analysis of genomic SNP data, to identify phylogenetic lineages within the genus, to delineate phenotypic variation relevant to these lineages, and to identify potential cryptic taxa within lineages. We found clear differentiation into four major lineages corresponding to the groups usually recognized within the genus: Neotinea maculata as a distinct and separate taxon, the Neotinea lactea group comprising two Mediterranean taxa N. lactea and Neotinea conica, the Neotinea ustulata group comprising two phenologically distinct varieties, and the rather complex Neotinea tridentata group comprising two major lineages and various minor lineages of unclear taxonomic value. N. conica constitutes both a monophyletic group within N. lactea and a distinct phenotype within the genus and merits its proposed subspecies-level recognition. By contrast, the spring and summer flowering forms of N. ustulata (var. ustulata and var. aestivalis) were confirmed to be distinct only morphologically, not phylogenetically. The most complex pattern emerged in the N. tridentata group, which splits into two main clades, one containing lineages from the Balkans and eastern Mediterranean and the other consisting of plants from Central Europe and the central Mediterranean. These individual lineages differ in genome size and show moderate degrees of morphological divergence. The tetraploid Neotinea commutata is closely related to the N. tridentata group, but our evidence points to an auto- rather than an allopolyploid origin. Our broad methodological approach proved effective in recognizing cryptic lineages among the orchids, and we propose the joint analysis of flow cytometric data on genome size and endopolyploidy as a useful and beneficial marker for delineating orchid species with partial endoreplication.

Repeat proliferation and partial endoreplication jointly shape the patterns of genome size evolution in orchids.

Although the evolutionary drivers of genome size change are known, the general patterns and mechanisms of plant genome size evolution are yet to be established. Here we aim to assess the relative importance of proliferation of repetitive DNA, chromosomal variation (including polyploidy), and the type of endoreplication for genome size evolution of the Pleurothallidinae, the most species-rich orchid lineage. Phylogenetic relationships between 341 Pleurothallidinae representatives were refined using a target enrichment hybrid capture combined with high-throughput sequencing approach. Genome size and the type of endoreplication were assessed using flow cytometry supplemented with karyological analysis and low-coverage Illumina sequencing for repeatome analysis on a subset of samples. Data were analyzed using phylogeny-based models. Genome size diversity (0.2-5.1 Gbp) was mostly independent of profound chromosome count variation (2n = 12-90) but tightly linked with the overall content of repetitive DNA elements. Species with partial endoreplication (PE) had significantly greater genome sizes, and genomic repeat content was tightly correlated with the size of the non-endoreplicated part of the genome. In PE species, repetitive DNA is preferentially accumulated in the non-endoreplicated parts of their genomes. Our results demonstrate that proliferation of repetitive DNA elements and PE together shape the patterns of genome size diversity in orchids.

Fungal symbionts may modulate nitrate inhibitory effect on orchid seed germination.

Many orchid species are threatened, while some disappear from their natural habitats without obvious reasons. Eutrophication has been suggested as a possible factor and nitrate, which is able to suppress non-symbiotic orchid seed germination even at very low concentrations, and could pose a serious threat for natural orchid populations. Early ontogenesis of all orchids entirely depends on orchid mycorrhizal symbiosis, and at this initial mycoheterotrophic stage, many terrestrial green orchids associate with polyphyletic fungal symbionts (i.e., mycobionts), collectively called "rhizoctonias." We asked whether these fungi might also have some non-nutritional roles, i.e., whether they might confer resistance to eutrophication. To test this hypothesis, we co-cultivated seeds of the terrestrial orchid Dactylorhiza majalis with five rhizoctonias (two Tulasnella, two Ceratobasidium and one Serendipita isolate) at various ecologically meaningful nitrate concentrations (0 to 100 mg/L). With the exception of one Tulasnella isolate, all mycobionts supported the growth of protocorms and formed orchid mycorrhiza, i.e., intracellular hyphal pelotons, in the protocorms. Nitrate suppressed asymbiotic, as well as symbiotic, seed germination in all but one fungal treatment; the seeds co-cultivated with one of the Ceratobasidium isolates were indeed insensitive to nitrate. We conclude that nitrates also negatively affect symbiotic orchid germination, depending on the available compatible mycobionts. Thus, eutrophication with nitrate may decrease the number of orchid mycobionts capable of supporting seed germination.

Diversity in genome size and GC content shows adaptive potential in orchids and is closely linked to partial endoreplication, plant life-history traits and climatic conditions.

In angiosperms, genome size and nucleobase composition (GC content) exhibit pronounced variation with possible adaptive consequences. The hyperdiverse orchid family possessing the unique phenomenon of partial endoreplication (PE) provides a great opportunity to search for interactions of both genomic traits with the evolutionary history of the family. Using flow cytometry, we report values of both genomic traits and the type of endoreplication for 149 orchid species and compare these with a suite of life-history traits and climatic niche data using phylogeny-based statistics. The evolution of genomic traits was further studied using the Brownian motion (BM) and Ornstein-Uhlenbeck (OU) models to access their adaptive potential. Pronounced variation in genome size (341-54 878 Mb), and especially in GC content (23.9-50.5%), was detected among orchids. Diversity in both genomic traits was closely related to the type of endoreplication, plant growth form and climatic conditions. GC content was also associated with the type of dormancy. In all tested scenarios, OU models always outperformed BM models. Unparalleled GC content variation was discovered in orchids, setting new limits for plants. Our study indicates that diversity in both genome size and GC content has adaptive consequences and is tightly linked with evolutionary transitions to PE.

In vitro axenic germination and cultivation of mixotrophic Pyroloideae (Ericaceae) and their post-germination ontogenetic development.

BACKGROUND AND AIMS: Pyroloids, forest sub-shrubs of the Ericaceae family, are an important model for their mixotrophic nutrition, which mixes carbon from photosynthesis and from their mycorrhizal fungi. They have medical uses but are difficult to cultivate ex situ; in particular, their dust seeds contain undifferentiated, few-celled embryos, whose germination is normally fully supported by fungal partners. Their germination and early ontogenesis thus remain elusive. METHODS: An optimized in vitro cultivation system of five representatives from the subfamily Pyroloideae was developed to study the strength of seed dormancy and the effect of different media and conditions (including light, gibberellins and soluble saccharides) on germination. The obtained plants were analysed for morphological, anatomical and histochemical development. KEY RESULTS: Thanks to this novel cultivation method, which breaks dormancy and achieved up to 100 % germination, leafy shoots were obtained in vitro for representatives of all pyroloid genera (Moneses, Orthilia, Pyrola and Chimaphila). In all cases, the first post-germination stage is an undifferentiated structure, from which a root meristem later emerges, well before formation of an adventive shoot. CONCLUSIONS: This cultivation method can be used for further research or for ex situ conservation of pyroloid species. After strong seed dormancy is broken, the tiny globular embryo of pyroloids germinates into an intermediary zone, which is functionally convergent with the protocorm of other plants with dust seeds such as orchids. Like the orchid protocorm, this intermediary zone produces a single meristem: however, unlike orchids, which produce a shoot meristem, pyroloids first generate a root meristem.

The Low Molecular Weight Heparin Tinzaparin Attenuates Platelet Activation in Terms of Metastatic Niche Formation by Coagulation-Dependent and Independent Pathways.

An intimate interplay with platelets is an initial key issue for tumor cells in terms of hematogenous metastasis. Tumor cells activate platelets by different pathways and receive, upon forming a platelet cloak, protection from immune surveillance and support in metastatic niche creation. Therapeutic intervention with this early interaction is promising to antagonize the whole metastatic cascade. Here we aimed to investigate the capability of low molecular weight heparin (LMWH), unfractionated heparin (UFH), and a non-anticoagulant heparin derivative or FXa inhibitor fondaparinux to interfere with platelet activation by tumor cells. Coagulation-dependent and independent pathways of platelet activation by three tumor cell lines, and interference therewith were analyzed by fluorigenic thrombin formation assay, platelet aggregometry, ATP and VEGF release and endothelial tube formation assay. LMWH and UFH were found to repress various routes of platelet activation, reflected by attenuated endothelial tube formation. This confirms the duality of anti-coagulative and anti-adhesive properties of heparin. While non-anticoagulative heparin (RO-heparin) depressed platelets' ATP and VEGF release by contact inhibition sufficiently, fondaparinux just attenuated tissue factor mediated thrombin generation. Concluding, these data suggest that LMWH as a guideline-based drug for anticoagulative strategies in oncology is promising to provide additional benefit for interference with metastatic activities.

Unfractionated and Low Molecular Weight Heparin Reduce Platelet Induced Epithelial-Mesenchymal Transition in Pancreatic and Prostate Cancer Cells.

The interaction with platelets is of crucial importance for tumor cells passing through hematogenous metastasis. Platelets protect cancer cells from immune surveillance and exhibit many other prometastatic effects. Notably, platelets can change the epithelial tumor phenotype, a process termed epithelial-mesenchymal transition (EMT), which confers stem cell-like properties onto tumor cells associated with an increased motility and drug resistance. The aim of the study is to investigate the impact of heparin on the platelet induced EMT program in pancreatic and prostate tumor cells. Platelet activation and interaction with cancer cells were determined by static adhesion assays. Applying ELISAs, the platelet release of EMT inducing mediators was quantified. EMT marker protein expression by tumor cells was explored by western blot and qPCR. Our data show that different tumor cell entities have different platelet binding capacities and also that a weak interaction is sufficient to change tumor cell phenotype. Additionally, unfractionated heparin (UFH) as well as low molecular weight heparin (LMWH) reduced tumor cell platelet interaction. Subsequently, attenuated platelet-derived mediator release resulted in reduced EMT marker protein and transcription factor expression by the cancer cells and decreased cell migration. These data suggest that heparin reduces platelet induced EMT program and prevents the formation of cancer cells with stem cell-like properties. This additional mechanism argues for the use of heparin in oncological applications.

The mechanisms how heparin affects the tumor cell induced VEGF and chemokine release from platelets to attenuate the early metastatic niche formation.

Metastasis is responsible for the majority of cancer associated fatalities. Tumor cells leaving the primary tumor and entering the blood flow immediately interact with platelets. Activated platelets contribute in different ways to cancer cell survival and proliferation, e.g. in formation of the early metastatic niche by release of different growth factors and chemokines. Here we show that a direct interaction between platelets and MV3 melanoma or MCF7 breast cancer cells induces platelet activation and a VEGF release in citrated plasma that cannot be further elevated by the coagulation cascade and generated thrombin. In contrast, the release of platelet-derived chemokines CXCL5 and CXCL7 depends on both, a thrombin-mediated platelet activation and a direct interaction between tumor cells and platelets. Preincubation of platelets with therapeutic concentrations of unfractionated heparin reduces the tumor cell initiated VEGF release from platelets. In contrast, tumor cell induced CXCL5 and CXCL7 release from platelets was not impacted by heparin pretreatment in citrated plasma. In defibrinated, recalcified plasma, on the contrary, heparin is able to reduce CXCL5 and CXCL7 release from platelets by thrombin inhibition. Our data indicate that different chemokines and growth factors in diverse platelet granules are released in tightly regulated processes by various trigger mechanisms. We show for the first time that heparin is able to reduce the mediator release induced by different tumor cells both in a contact and coagulation dependent manner.

The Enigma of Progressively Partial Endoreplication: New Insights Provided by Flow Cytometry and Next-Generation Sequencing.

In many plant species, somatic cell differentiation is accompanied by endoreduplication, a process during which cells undergo one or more rounds of DNA replication cycles in the absence of mitosis, resulting in nuclei with multiples of 2C DNA amounts (4C, 8C, 16C, etc.). In some orchids, a disproportionate increase in nuclear DNA contents has been observed, where successive endoreduplication cycles result in DNA amounts 2C + P, 2C + 3P, 2C + 7P, etc., where P is the DNA content of the replicated part of the 2C nuclear genome. This unique phenomenon was termed "progressively partial endoreplication" (PPE). We investigated processes behind the PPE in Ludisia discolor using flow cytometry (FCM) and Illumina sequencing. In particular, we wanted to determine whether chromatin elimination or incomplete genome duplication was involved, and to identify types of DNA sequences that were affected. Cell cycle analysis of root tip cell nuclei pulse-labeled with EdU revealed two cell cycles, one ending above the population of nuclei with 2C + P content, and the other with a typical "horseshoe" pattern of S-phase nuclei ranging from 2C to 4C DNA contents. The process leading to nuclei with 2C + P amounts therefore involves incomplete genome replication. Subsequent Illumina sequencing of flow-sorted 2C and 2C + P nuclei showed that all types of repetitive DNA sequences were affected during PPE; a complete elimination of any specific type of repetitive DNA was not observed. We hypothesize that PPE is part of a highly controlled transition mechanism from proliferation phase to differentiation phase of plant tissue development.

A New Species of Cleisostoma (Orchidaceae) from the Hon Ba Nature Reserve in Vietnam: A Multidisciplinary Assessment.

A new species, Cleisostoma yersinii J. Ponert & Vuong, is described and illustrated based on the material collected in the Hon Ba Nature Reserve in southern Vietnam. In addition to conventional (macro)morphological examination we comparatively investigated root and leaf anatomy (using light and fluorescent microscopy), assessed nectar characteristics (using HPLC analysis), determined nuclear genome size (using DNA flow cytometry) and reconstructed phylogenetic relationships (using nrITS sequences). Cleisostoma yersinii differs from its putative closest relative C. birmanicum in wider and shorter leaves, larger flowers, distinct lip with S-shaped tip of the mid-lobe, and a shallow spur with two large nectar sacks separated by prominent calli and septum. Nectar is sucrose-dominant and very rich in sugars. Stomata are developed on both sides of the leaf and have prominent hyperstomatal chambers and substomatal cavities. Roots with well-developed exodermis and tracheoidal idioblasts are covered by a two-layer Vanda-type velamen. Chloroplasts occur not only in the cortex but are also abundant in the stele. Mean 1C-value was estimated to 2.57 pg DNA. An updated identification key is provided for SE Asian sections and all Vietnamese species of Cleisostoma.

Challenges of flow-cytometric estimation of nuclear genome size in orchids, a plant group with both whole-genome and progressively partial endoreplication.

Nuclear genome size is an inherited quantitative trait of eukaryotic organisms with both practical and biological consequences. A detailed analysis of major families is a promising approach to fully understand the biological meaning of the extensive variation in genome size in plants. Although Orchidaceae accounts for ∼10% of the angiosperm diversity, the knowledge of patterns and dynamics of their genome size is limited, in part due to difficulties in flow cytometric analyses. Cells in various somatic tissues of orchids undergo extensive endoreplication, either whole-genome or partial, and the G1-phase nuclei with 2C DNA amounts may be lacking, resulting in overestimated genome size values. Interpretation of DNA content histograms is particularly challenging in species with progressively partial endoreplication, in which the ratios between the positions of two neighboring DNA peaks are lower than two. In order to assess distributions of nuclear DNA amounts and identify tissue suitable for reliable estimation of nuclear DNA content, we analyzed six different tissue types in 48 orchid species belonging to all recognized subfamilies. Although traditionally used leaves may provide incorrect C-values, particularly in species with progressively partial endoreplication, young ovaries and pollinaria consistently yield 2C and 1C peaks of their G1-phase nuclei, respectively, and are, therefore, the most suitable parts for genome size studies in orchids. We also provide new DNA C-values for 22 orchid genera and 42 species. Adhering to the proposed methodology would allow for reliable genome size estimates in this largest plant family. Although our research was limited to orchids, the need to find a suitable tissue with dominant 2C peak of G1-phase nuclei applies to all endopolyploid species.