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Somatic embryogenesis in Coffea spp. is important for mass production and genetic engineering. Coffee is an exceptional case, as somatic embryogenesis can be induced by applying a cytokinin such as 2iP as the only plant growth regulator. 1-(2-(2-Hydroxyethyl)phenyl)-3-(3-(trifluoromethoxy)phenyl)urea (3TFM-2HE) and 1-(3-bromo-5-(trifluoromethoxy)phenyl)-3-(2-(2-hydroxyethyl)phenyl)urea (3TFM,5Br-2HE) are two newly designed cytokinin oxidase/dehydrogenase inhibitors (CKX) from the diphenylurea group. We used a Coffea arabica leaf disks bioassay to demonstrate the indirect somatic embryo induction potential of these compounds. The leaf disks were incubated on modified Murashige and Skoog (MS) semi-solid medium in which 3TFM-2HE or 3TFM,5Br-2HE were combined with N6-(2-isopentenyl)adenine (2iP). Although these compounds do not possess intrinsic cytokinin activity, they enhanced the activity of 2iP, resulting in direct somatic embryogenesis after seven weeks. The best results were obtained with 1 µM 3TFM-2HE and 5 or 10 µM 2iP. Maturation of somatic embryos into fully developed plants took place on medium supplemented with 0.5 µM kinetin and the somatic embryos developed true leaves and a root system.

Foliar water uptake (FWU), the direct uptake of water into leaves, is a global phenomenon, having been observed in an increasing number of plant species. Despite the growing recognition of its functional relevance, our understanding of how FWU occurs and which foliar surface structures are implicated, is limited. In the present study, fluorescent and ionic tracers, as well as microcomputed tomography, were used to assess potential pathways for water entry in leaves of beech, a widely distributed tree species from European temperate regions. Although none of the tracers entered the leaf through the stomatal pores, small amounts of silver precipitation were observed in some epidermal cells, indicating moderate cuticular uptake. Trichomes, however, were shown to absorb and redistribute considerable amounts of ionic and fluorescent tracers. Moreover, microcomputed tomography indicated that 72% of empty trichomes refilled during leaf surface wetting and microscopic investigations revealed that trichomes do not have a cuticle but are covered with a pectin-rich cell wall layer. Taken together, our findings demonstrate that foliar trichomes, which exhibit strong hygroscopic properties as a result of their structural and chemical design, constitute a major FWU pathway in beech.

This work describes the development of a novel method for quantitative mapping of Hg and Se in mushroom fruit body tissues with laser ablation coupled to inductively coupled plasma-mass spectrometry (LA-ICP-MS). Different parameters of the protocol for preparation of the standards used for quantification via external calibration were assessed, e.g., the dissolution temperature of gelatin standards and the addition of chitosan and l-cysteine as additives to the gelatin-based calibration droplets to better match the sample matrix. While chitosan was not suited for this purpose, the presence of l-cysteine considerably improved the figures of merit of the calibration, leading to limits of detection of 0.006 and 0.3 µg g−1 for Hg and Se, respectively, at a pixel size of 20 × 20 µm. Further, an in-house reference material, ideally suited for the validation of the method for application to mushroom samples, was successfully prepared from a paste of Boletus edulis. The newly developed method was used to investigate the distribution of Hg and Se in tissue sections of five porcini mushroom individuals of three different species (Boletus edulis, Boletus aereus, and Boletus pinophilus) and one sample of a parasol mushroom (Macrolepiota procera). For one sample, additional areas were ablated at higher spatial resolution, with a laser spot size down to 5 µm, which allows a detailed investigation of the spatial distribution of Hg and Se in mushrooms. Graphical abstract

Hereditary symbioses have the potential to drive transgenerational effects, yet the mechanisms responsible for transmission of heritable plant symbionts are still poorly understood. The leaf symbiosis between Dioscorea sansibarensis and the bacterium Orrella dioscoreae offers an appealing model system to study how heritable bacteria are transmitted to the next generation. Here, we demonstrate that inoculation of apical buds with a bacterial suspension is sufficient to colonize newly-formed leaves and propagules, and to ensure transmission to the next plant generation. Flagellar motility is not required for movement inside the plant, but is important for the colonization of new hosts. Further, stringent tissue-specific regulation of putative symbiotic functions highlight the presence of two distinct subpopulations of bacteria in the leaf gland and at the shoot meristem. We propose that bacteria in the leaf gland dedicate resources to symbiotic functions, while dividing bacteria in the shoot tip ensure successful colonization of meristematic tissue, glands and propagules. Compartmentalization of intra-host populations, together with tissue-specific regulation may serve as a robust mechanism for the maintenance of mutualism in leaf symbiosis.ImportanceSeveral plant species form associations with bacteria in their leaves, called leaf symbiosis. These associations are highly specific, but the mechanisms responsible for symbiont transmission are poorly understood. Using the association between the yam species Dioscorea sansibarensis and Orrella dioscoreae as a model leaf symbiosis, we provide experimental evidence that bacteria are transmitted vertically and distributed to specific leaf structures via association with shoot meristems. Flagellar motility is required for initial infection, but does not contribute to spread within host tissue. We also provide evidence that bacterial subpopulations at the meristem or in the symbiotic leaf gland differentially express key symbiotic genes. We argue that this separation of functional symbiont populations, coupled to tight control over bacterial infection and transmission, explain the evolutionary robustness of leaf symbiosis. These findings may provide insights into how plants may recruit and maintain beneficial symbionts at the leaf surface.

The timing of abiotic stress elicitors on wood formation largely affects xylem traits that determine xylem efficiency and vulnerability. Nonetheless, seasonal variability of elevated CO2 (eCO2) effects on tree functioning under drought remains largely unknown. To address this knowledge gap, 1-year-old aspen (Populus tremula L.) trees were grown under ambient (±445 ppm) and elevated (±700 ppm) CO2 and exposed to an early (spring/summer 2019) or late (summer/autumn 2018) season drought event. Stomatal conductance and stem shrinkage were monitored in vivo as xylem water potential decreased. Additional trees were harvested for characterization of wood anatomical traits and to determine vulnerability and desorption curves via bench dehydration. The abundance of narrow vessels decreased under eCO2 only during the early season. At this time, xylem vulnerability to embolism formation and hydraulic capacitance during severe drought increased under eCO2. Contrastingly, stomatal closure was delayed during the late season, while hydraulic vulnerability and capacitance remained unaffected under eCO2. Independently of the CO2 treatment, elastic, and inelastic water pools depleted simultaneously after 50% of complete stomatal closure. Our results suggest that the effect of eCO2 on drought physiology and wood traits are small and variable during the growing season and question a sequential capacitive water release from elastic and inelastic pools as drought proceeds.

In order to achieve a significant size reduction to get ultrasmall upconverting nanoparticles (UCNPs) following a thermal coprecipitation pathway, we identified two critical points: the UCNP precursor mixing and high-temperature heating steps. Significant differences could be observed according to the way the inorganic sodium and fluoride sources were mixed to the rare-earth oleate before the high-temperature heating step. More interestingly, accurate monitoring of the high-temperature heating step using microwave (MW) dielectric heating yielded major improvement toward ultrasmall UCNPs. Thus, hexagonal, Tm-doped sub-5-nm UCNPs with an unusual Na(Yb-Gd)F4 matrix with 53% Yb were produced, displaying satisfactory luminescence. Noticeably, MW heating was achieved in a weakly MW-absorbing oleic acid (OA)/octadecene mixture, and the influence of the OA content composition on the MW heating efficiency is discussed in this report.

About 90% of all land plants form mycorrhiza to facilitate the acquisition of essential nutrients such as phosphorus, nitrogen, and sometimes carbon. Based on the morphology of the interaction and the identity of the interacting plants and fungi, four major mycorrhizal types have been distinguished: arbuscular mycorrhiza (AM), ectomycorrhizal (EcM), ericoid mycorrhiza, and orchid mycorrhiza. Although most plants are assumed to form only one type of mycorrhiza, some species simultaneously form associations with two mycorrhizal types within a single root system. However, the dual-mycorrhizal status of many species is under discussion and in some plant species the simultaneous association with two mycorrhizal types varies in space or time or depends on the ecological context. Here, we assessed the mycorrhizal communities associating with common hawthorn (Crataegus monogyna), a small tree that commonly associates with AM fungi, and investigated the potential factors that underlie variation in mycorrhizal community composition. Histological staining of C. monogyna roots showed the presence of a Hartig net and hyphal sheaths in and around the roots, demonstrating the capacity of C. monogyna to form EcM. Meta-barcoding of soil and root samples of C. monogyna collected in AM-dominated grassland vegetation and in mixed AM + EcM forest vegetation showed a much higher number of EcM sequences and OTUs in root and soil samples from mixed AM + EcM vegetation than in samples from pure AM vegetation. We conclude that C. monogyna is able to form both AM and EcM, but that the extent to which it does depends on the environmental context, i.e., the mycorrhizal type of the surrounding vegetation.

A vibrating microtome is widely used to produce good-quality sections of plant organs or tissues. This method allows for an improved preservation of antigenicity and structure and is compatible with most (immuno)cytochemical staining procedures.

Background: Plant reproductive output is determined by a complex set of factors including resource availability, pollination success and pre-dispersal predation. Disentangling the relative ...

Background and aims A key structural adaptation of vascular plants was the evolution of specialized vascular and mechanical tissues, innovations likely to have generated novel cell wall architectures. While collenchyma is a strengthening tissue typically found in growing organs of angiosperms, a similar tissue occurs in the petiole of the fern Asplenium rutifolium. Methods The in situ cell wall (ultra)structure and composition of this tissue was investigated and characterized mechanically as well as structurally through nano-indentation and wide-angle X-ray diffraction, respectively. Key results Structurally the mechanical tissue resembles sclerenchyma, while its biomechanical properties and molecular composition both share more characteristics with angiosperm collenchyma. Cell wall thickening only occurs late during cell expansion or after cell expansion has ceased. Conclusions If the term collenchyma is reserved for walls that thicken during expansive growth, the mechanical tissue in A. rutifolium represents sclerenchyma that mimics the properties of collenchyma and has the ability to modify its mechanical properties through sclerification. These results support the view that collenchyma does not occur in ferns and most probably evolved in angiosperms.

Summary Heat girdling is a method to estimate the relative contribution of phloem vs xylem water flow to fruit growth. The heat girdling process is assumed to destroy all living tissues, including the phloem, without affecting xylem conductivity. However, to date, the assumption that xylem is not affected by heat girdling remains unproven. In this study, we used in vivo magnetic resonance imaging (MRI) velocimetry to test if heat girdling can cause xylem vessels to embolize or affect xylem water flow characteristics in the peduncle of tomato (Solanum lycopersicum cv Dirk). Anatomical and MRI data indicated that, at the site of girdling, all living tissues were disrupted, but that the functionality of the xylem remained unchanged. MRI velocimetry showed that the volume flow through the secondary xylem was not impeded by heat girdling in either the short or the long term (up to 91 h after girdling). This study provides support for the hypothesis that in the tomato peduncle the integrity and functionality of the xylem remain unaffected by heat girdling. It therefore confirms the validity of the heat girdling technique as a means to estimate relative contributions of xylem and phloem water flow to fruit growth.

A vibrating microtome is widely used to produce good-quality sections of plant organs or tissues. This method allows for an improved preservation of antigenicity and structure and is compatible with most (immuno)cytochemical staining procedures.

In order to improve the understanding of plant water relations under drought stress, the water use behavior of two Fragaria x ananassa Duch. cultivars, contrasting in their drought stress phenotype, is identified. Under drought, stomatal closure is gradual in Figaro. Based on this, we associate Figaro with conservative water use behavior. Contrarily, drought stress causes a sudden and steep decrease in stomatal conductance in Flair, leading to the identification of Flair as a prodigal water use behavior cultivar. Responses to progressive drought on the one hand and an osmotic shock on the other hand are compared between these two cultivars. Tonoplast intrinsic protein mRNA levels are shown to be upregulated under progressive drought in the roots of Figaro only. Otherwise, aquaporin expression upon drought or osmotic stress is similar between both cultivars, i.e. plasma membrane intrinsic proteins are downregulated under progressive drought in leaves and under short term osmotic shock in roots. In response to osmotic shock, root hydraulic conductivity did not change significantly and stomatal closure is equal in both cultivars. De novo abscisic acid biosynthesis is upregulated in the roots of both cultivars under progressive drought.

Plants are known to respond to warming temperatures. Few studies, however, have included the temperature experienced by the parent plant in the experimental design, in spite of the importance of this factor for population dynamics. We investigated the phenological and growth responses of seedlings of two key temperate tree species (Fagus sylvatica and Quercus robur) to spatiotemporal temperature variation during the reproductive period (parental generation) and experimental warming of the offspring. To this end, we sampled oak and beech seedlings of different ages (1–5 years) from isolated mother trees and planted the seedlings in a common garden. Warming of the seedlings advanced bud burst in both species. In oak seedlings, higher temperatures experienced by mother trees during the reproductive period delayed bud burst in control conditions, but advanced bud burst in heated seedlings. In beech seedlings, bud burst timing advanced both with increasing temperatures during the reproductive period of the parents and with experimental warming of the seedlings. Relative diameter growth was enhanced in control oak seedlings but decreased with warming when the mother plant experienced higher temperatures during the reproductive period. Overall, oak displayed more plastic responses to temperatures than beech. Our results emphasise that temperature during the reproductive period can be a potential determinant of tree responses to climate change.

Background and aims The bacterial leaf nodule symbiosis is a close interaction between endophytes and their plant hosts, mainly within the coffee family. The interaction between Rubiaceae species and Burkholderia bacteria is unique due to its obligate nature, high specificity, and predominantly vertical transmission of the endophytes to the next generation of host plants. This vertical transmission is intriguing since it is the basis for the uniqueness of the symbiosis. However, unequivocal evidence of the location of the endophytes in the seeds is lacking. The aim of this paper is therefore to demonstrate the presence of the host specific endophyte in the seeds of Psychotria punctata and confirm its precise location. In addition, the suggested location of the endophyte in other parts of the host plant is investigated. Methods To identify and locate the endophyte in Psychotria punctata, a two-level approach was adopted using both a molecular screening method and fluorescent in situ hybridisation microscopy. Key results The endophytes, molecularly identified as Candidatus Burkholderia kirkii, were detected in the leaves, vegetative and flower buds, anthers, gynoecium, embryos, and young twigs. In addition, they were in situ localised in leaves, flowers and shoot apical meristems, and, for the first time, in between the cotyledons of the embryos. Conclusions Both independent techniques detected the host specific endophyte in close proximity to the shoot apical meristem of the embryo, which confirms for the first time the exact location of the endophytes in the seeds. This study provides reliable proof that the endophytes are maintained throughout the growth and development of the host plant and are transmitted vertically to the offspring.

At present, the importance of sample preparation equipment for electron microscopy represents the driving force behind major breakthroughs in microscopy and cell biology. In this paper we present an introduction to the most commonly used cryo-fixation techniques, with special attention paid towards high-pressure freezing followed by freeze substitution. Techniques associated with cryo-fixation, such as immunolocalisation, cryo-sectioning, and correlative light and electron microscopy, are also highlighted. For studies that do not require high resolution, high quality results, or the immediate arrest of certain processes, conventional methods will provide answers to many questions. For some applications, such as immunocytochemistry, three-dimensional reconstruction of serial sections or electron tomography, improved preservation of the ultrastructure is required. This review of nematode cryo-fixation highlights that cryo-fixation not only results in a superior preservation of fine structural details, but also underlines the fact that some observations based on results solely obtained through conventional fixation approaches were either incorrect, or otherwise had severe limitations. Although the use of cryo-fixation has hitherto been largely restricted to model organisms, the advantages of cryo-fixation are sufficiently self-evident that we must conclude that the cryo-fixation method is highly likely to become the standard for nematode fixation in the near future.

International audience; Folates (B9 vitamins) are essential cofactors in one-carbon metabolism. Since C1 transfer reactions are involved in synthesis of nucleic acids, proteins, lipids, and other biomolecules, as well as in epigenetic control, folates are vital for all living organisms. This work presents a complete study of a plant DHFR-TS (dihydrofolate reductase-thymidylate synthase) gene family that implements the penultimate step in folate biosynthesis. We demonstrate that one of the DHFR-TS isoforms (DHFR-TS3) operates as an inhibitor of its two homologs, thus regulating DHFR and TS activities and, as a consequence, folate abundance. In addition, a novel function of folate metabolism in plants is proposed, i.e., maintenance of the redox balance by contributing to NADPH production through the reaction catalyzed by methylenetetrahydrofolate dehydrogenase, thus allowing plants to cope with oxidative stress.

Background and Aims: Innovations in vegetative and reproductive characters were key factors in the evolutionary history of land plants and most of these transformations, including dramatic changes in life cycle structure and strategy, necessarily involved cell-wall modifications. To provide more insight into the role of cell walls in effecting changes in plant structure and function, and in particular their role in the generation of vascularization, an antibody-based approach was implemented to compare the presence and distribution of cell-wall glycan epitopes between (free-living) gametophytes and sporophytes of Ceratopteris richardii 'C-Fern', a widely used model system for ferns. Methods: Microarrays of sequential diamino-cyclohexane-tetraacetic acid(CDTA) and NaOH extractions of gametophytes, spores and different organs of 'C-Fern' sporophytes were probed with glycan-directed monoclonal antibodies. The same probes were employed to investigate the tissue-and cell-specific distribution of glycan epitopes. Key Results: While monoclonal antibodies against pectic homogalacturonan, mannan and xyloglucan widely labelled gametophytic and sporophytic tissues, xylans were only detected in secondary cell walls of the sporophyte. The LM5 pectic galactan epitope was restricted to sporophytic phloem tissue. Rhizoids and root hairs showed similarities in arabinogalactan protein (AGP) and xyloglucan epitope distribution patterns. Conclusions: The differences and similarities in glycan cell-wall composition between 'C-Fern' gametophytes and sporophytes indicate that the molecular design of cell walls reflects functional specialization rather than genetic origin. Glycan epitopes that were not detected in gametophytes were associated with cell walls of specialized tissues in the sporophyte.

The majority of microbial mediated soil processes depend on availability of organic matter (OM), water and air. Because of its ability to visualize the 3D architecture of soil non-destructively, X-ray computed tomography (CT) is becoming a widespread tool for studying soil pore network structure. However, phase determination of pore space, soil OM, soil mineral matter (MM) and water is often limited even with the latest technological and software advances, allowing high resolution and better quality imaging. Contrast agents commonly used in histology enable enhancement of X-ray attenuation of targeted structures or compounds. Here we report on the first systematic investigation of the use of such X-ray contrast agents for soil research. An evaluation procedure as well as a method to apply the agents to soil samples was developed and applied on reference soil samples. The effectiveness and selectivity of the contrast agents was evaluated for soil organic matter (SOM), MM and water. Several products were found to selectively increase the attenuation of water or SOM. The four agents with the best OM-staining capabilities (phosphomolybdenic acid (PMA), silver nitrate, lead nitrate and lead acetate) were further tested on an OM-MM mixture and all showed an increased of the SOM attenuation coefficient above the MM values.

† Background and Aims Environmental change is increasingly impacting ecosystems worldwide. However, our knowledge about the interacting effects of various drivers of global change on sexual reproduction of plants, one of their key mechanisms to cope with change, is limited. This study examines populations of poorly regenerating and threatened common juniper (Juniperus communis) to determine the influence of four drivers of global change (rising temperatures, nitrogen deposition, potentially acidifying deposition and altering precipitation patterns) on two key developmental phases during sexual reproduction, gametogenesis and fertilization (seed phase two, SP2) and embryo development (seed phase three, SP3), and on the ripening time of seeds. † Methods In 42 populations throughout the distribution range of common juniper in Europe, 11 943 seeds of two developmental phases were sampled. Seed viability was determined using seed dissection and related to accumulated temperature (expressed as growing degree-days), nitrogen and potentially acidifying deposition (nitrogen plus sulfur), and precipitation data. † Key Results Precipitation had no influence on the viability of the seeds or on the ripening time. Increasing temperatures had a negative impact on the viability of SP2 and SP3 seeds and decreased the ripening time. Potentially acidifying depositions negatively influenced SP3 seed viability, while enhanced nitrogen deposition led to lower ripening times. † Conclusions Higher temperatures and atmospheric deposition affected SP3 seeds more than SP2 seeds. However, this is possibly a delayed effect as juniper seeds develop practically independently, due to the absence of vascular communication with the parent plant from shortly after fertilization. It is proposed that the failure of natural regeneration in many European juniper populations might be attributed to climate warming as well as enhanced atmospheric deposition of nitrogen and sulfur.

Mangrove trees are remarkably well adapted to the tropical intertidal environment. Aerial roots and salt excluding mechanisms at the root and leaf level are one of the important mechanisms that help them surviving the highly dynamic mangrove environment. The formation of viviparous hydrochorous seedlings is another adaptation. All mangrove species belonging to the pantropical family Rhizophoraceae are viviparous. This means that their seeds germinate without a dormancy period, while they are still attached to the parent tree. The resulting mangrove seedlings form the mangrove dispersal units or propagules. Like most plants, adult mangrove trees exhibit diurnal reversible stem diameter patterns, which are caused by diurnal fluctuations in internal water storage tissues. During on-tree development, viviparous Rhizophoraceae mangrove seedlings receive water, minerals and carbohydrates from the parent trees. Yet, little is known on the diameter dynamics of these developing conspicuous structures (reflecting fluctuations in internal water content), let alone on the same plants after abscission during establishment. We registered the diameter dynamics of on-tree developing and post-abscission establishing Bruguiera gymnorrhiza (L.) Sav. seedlings at high spatial (micrometer) and temporal (minutes) resolution by use of point dendrometers, and simultaneously recorded the variation in environmental conditions. The developing and maturing seedlings elongated, but lost in diameter in a stepwise manner towards abscission. Complementary anatomical observations, based on the combination of several techniques, provided context for understanding the seedling diameter fluctuations and contribution of the seedling to its own carbon balance. Establishing seedlings started exhibiting diurnal diameter fluctuations, typical of adult trees, upon the opening of their first leaf pair.

Because the plant cell wall provides the first line of defense against biotic and abiotic assaults, its functional integrity needs to be maintained under stress conditions. Through a phenotype-based compound screening approach, we identified a novel cellulose synthase inhibitor, designated C17. C17 administration depletes cellulose synthase complexes from the plasma membrane in Arabidopsis thaliana, resulting in anisotropic cell elongation and a weak cell wall. Surprisingly, in addition to mutations in CELLULOSE SYNTHASE1 (CESA1) and CESA3, a forward genetic screen identified two independent defective genes encoding pentatricopeptide repeat (PPR)-like proteins (CELL WALL MAINTAINER1 [CWM1] and CWM2) as conferring tolerance to C17. Functional analysis revealed that mutations in these PPR proteins resulted in defective cytochrome c maturation and activation of mitochondrial retrograde signaling, as evidenced by the induction of an alternative oxidase. These mitochondrial perturbations increased tolerance to cell wall damage induced by cellulose deficiency. Likewise, administration of antimycin A, an inhibitor of mitochondrial complex III, resulted in tolerance toward C17. The C17 tolerance of cwm2 was partially lost upon depletion of the mitochondrial retrograde regulator ANAC017, demonstrating that ANAC017 links mitochondrial dysfunction with the cell wall. In view of mitochondria being a major target of a variety of stresses, our data indicate that plant cells might modulate mitochondrial activity to maintain a functional cell wall when subjected to stresses.

Common juniper (Juniperus communis L.) populations in northwest European lowlands are currently declining in size and number. An important cause of this decline is a lack of natural regeneration. Low seed viability seems to be one of the main bottlenecks in this process. Previous research revealed a negative relation between seed viability and both temperature and nitrogen deposition. Additionally, the seeds of common juniper have a variable ripening time, which possibly influences seed viability. However, the underlying mechanisms remain unresolved. In order to elucidate this puzzle, it is important to understand in which phases of seed production the main defects are situated, together with the influence of ripening time. In this study, we compared seed viability of populations with and without successful recruitment. We examined three seed phases: (i) gamete development; (ii) fertilisation and early-embryo development; and (iii) late-embryo development. After the first two phases, we found no difference in the percentage viable seeds between populations with or without recruitment. After late-embryo development, populations without recruitment showed a significantly lower percentage of viable seeds. These results suggest that late-embryo development is a bottleneck in seed development. However, the complex interaction between seed viability and ripening time suggest that the causes should be in the second seed phase, as the accelerated development of male and female gametophytes may disturb the male-female synchrony for successful mating.

Background and aims – In Cyperaceae, the single-ovuled, usually triangular gynoecia are widely considered to have a basic number of three carpels, often reduced to two, resulting in dimerous pistils. However, laterally flattened dimerous pistils cannot be explained by any existing carpel reduction theories, because a single stigma in median position replaces the two adaxial stigmata. This paper presents a comparative study of the ontogenetic and anatomical adaptations facilitating the origin of new pistil forms in Cyperoideae, focusing on modified gynoecia. It includes a re-evaluation of Blaser’s (1941) anatomical studies in Cyperaceae. We aim to test Blaser’s hypothesis that is based on an acropetal developmental model of the floral vasculature and the general Cyperoid ontogenetic model of Vrijdaghs et al. (2009), which states that cyperoid ovaries originate from an annular primordium. Methods – SEM, dark field and phase contrast microscopy. Key Results – All cyperoid pistils studied develop according to a cyperoid floral ontogenetic pattern, in which carpel primordia are congenitally fused. In Pycreus sanguinolentus (and other species), separate procambial initiation zones were observed in both the flower receptacle and separate floral primordia, which connect (or not) at later developmental stages. Conclusions – The presence of an annular ovary primordium instead of individual carpel primordia, combined with the bidirectional development of the pistil vasculature liberate the developing gynoecium from the structural constraints proper to a typical carpellate organisation. Procambial initiation zones in the receptacular vascular plexus and in individual floral primordia constitute the basis for the formation of a flexible vascular system in cyperoid flowers. Moreover the development of the ovary and ovule are decoupled. Consequently, in Cyperoideae the acquired developmental freedom of the pistil resulted in various adaptations.

Background and aims – Recent molecular studies validate a broad definition of Cyperus (Cyperaceae) uniting genera previously scattered in Cyperoideae. First indication of their affinity with Cyperus was obtained through embryography. Cyperus consists of a paraphyletic C3 Cyperus and monophyletic C4 Cyperus. In this study, we aim to check and clarify the putative positions of the segregate genera in C3 Cyperus. Additional information is given and remarks are made on the position of some as yet unplaced species or sections in the C3 Cyperus phylogeny. Methods – Embryos of Cyperus constanzae and C. gardneri were cleared and drawn. Inflorescences of selected C3 Cyperus species were investigated using scanning electron and light microscopy. Histochemical tests were performed to assess the presence of suberin in the ‘corky’ tissue of the nutlets of Cyperus pectinatus. Key results – Embryography not only supports tribal classification in Cyperoideae, it is also phylogenetically informative in C3 Cyperus. Morphology and ontogeny support molecular phylogenetic results suggesting the inclusion of the segregate genera in C3 Cyperus as new sections or in established sections, and confirm the need to broaden the circumscription of some of these sections. Conclusion – Although less diverse than C4 Cyperus, C3 Cyperus includes clades which evolved an exceptional morphological diversity compared to its limited species numbers. The segregate genera Courtoisina (deciduous spikelets), Kyllingiella (spirally-arranged glumes) and Oxycaryum (spirally- arranged glumes and dorsiventrally flattened dimerous gynoecia), and the taxon Anosporum (recognised at sectional, subgeneric or generic level) are here included in C3 Cyperus (= Cyperus subg. Anosporum) as sections or included in an existing section (Kyllingiella is included in Cyperus sect. Leucocephali). A formal taxonomic revision is presented with relevant new names and combinations, synonyms, diagnoses and identification keys.

The anatomy of Equisetum stems is characterized by the occurrence of vallecular and carinal canals. Previous studies on the carinal canals in several Equisetum species suggest that they convey water from one node to another.Cell wall composition and ultrastructure have been studied using immunocytochemistry and electron microscopy, respectively. Serial sectioning and X-ray computed tomography were employed to examine the internode-node-internode transition of Equisetum ramosissimum.The distribution of the LM1 and JIM20 extensin epitopes is restricted to the lining of carinal canals. The monoclonal antibodies JIM5 and LM19 directed against homogalacturonan with a low degree of methyl esterification and the CBM3a probe recognizing crystalline cellulose also bound to this lining. The xyloglucan epitopes recognized by LM15 and CCRC-M1 were only detected in this lining after pectate lyase treatment. The carinal canals, connecting consecutive rings of nodal xylem, are formed by the disruption and dissolution of protoxylem elements during elongation of the internodes. Their inner surface appears smooth compared with that of vallecular canals.The carinal canals in E. ramosissimum have a distinctive lining containing pectic homogalacturonan, cellulose, xyloglucan and extensin. These canals might function as water-conducting channels which would be especially important during the elongation of the internodes when protoxylem is disrupted and the metaxylem is not yet differentiated. How the molecularly distinct lining relates to the proposed water-conducting function of the carinal canals requires further study. Efforts to elucidate the spatial and temporal distribution of cell wall polymers in a taxonomically broad range of plants will probably provide more insight into the structural-functional relationships of individual cell wall components or of specific configurations of cell wall polymers.

In the current model of gravitropism, negative gravitropic (upward) growth of the shoot of a dicotyledonous plant involves sedimentation of starch-containing plastids (statoliths) in the endodermis (starch sheath). Here we show that high levels of brassinosteroids (BRs) counteract gravitropic growth, while BR deficiency enhances gravitropism irrespective of the presence of a functional starch sheath. These data support a role for BRs as negative regulators of the shoot graviresponse.

How the diverse polysaccharides present in plant cell walls are assembled and interlinked into functional composites is not known in detail. Here, using two novel monoclonal antibodies and a carbohydrate-binding module directed against the mannan group of hemicellulose cell wall polysaccharides, we show that molecular recognition of mannan polysaccharides present in intact cell walls is severely restricted. In secondary cell walls, mannan esterification can prevent probe recognition of epitopes/ligands, and detection of mannans in primary cell walls can be effectively blocked by the presence of pectic homogalacturonan. Masking by pectic homogalacturonan is shown to be a widespread phenomenon in parenchyma systems, and masked mannan was found to be a feature of cell wall regions at pit fields. Direct fluorescence imaging using a mannan-specific carbohydrate-binding module and sequential enzyme treatments with an endo-β-mannanase confirmed the presence of cryptic epitopes and that the masking of primary cell wall mannan by pectin is a potential mechanism for controlling cell wall micro-environments.

† Background and Aims ‘Loxoscaphoid’ Asplenium species are morphologically a remarkably distinct group of Aspleniaceae. Except for two preliminary chromosome counts of Asplenium theciferum, the cytology of this group of species has, however, been largely unstudied. † Methods Chromosome counts were obtained by acetocarmine squash preparations of one mitotic cell and several meiotic cells. Relative DNA content of gametophytic and sporophytic cells was determined by flow cytometry. The phylogenetic placement of A. loxoscaphoides, A. rutifolium s.l. and A. theciferum s.l. was investigated through an analysis of rbcL sequences. † Key Results The dysploid base number is reported to be x ¼ 35 in Asplenium centrafricanum, A. loxoscaphoides, A. sertularioides and A. theciferum. Analysis of rbcL sequences confirms that ‘loxoscaphoids’ nest robustly within Asplenium. Several high ploidy levels exceeding the tetraploid level were found in A. theciferum s.l. and A. rutifolium s.l. All taxa proved to be sexual. † Conclusions Four base numbers are known at present for Aspleniaceae: x ¼ 39, 38, 36 and 35. The dysploid base number x ¼ 35 found in the ‘loxoscaphoid’ Asplenium spp. sheds a novel light on the cytoevolution of the whole family. We postulate a recurrent descending dysploid evolution within Aspleniaceae, leading to speciation at the (sub)generic and species/group level.

Since the development of X-ray computed tomography as a medical diagnostic tool, it was adapted and extended for many scientific applications, including plant structure research. As for many biological studies, sample preparation is of major importance to obtain good-quality images. Therefore, we present a new preparation method for fresh material which includes critical point drying and heavy metal staining. This technique enhances the contrast of fresh tissues, prevents artefacts such as tissue compression, and requires no embedding.

The use of computers and digital media in unlawful activities has increased so dramatically that investigation of any criminal activity may nowadays produce electronic evidence. However, the rapid growth in the number of criminal cases involving electronic evidence has all‐too‐often found law enforcement and the judiciary badly prepared to deal with the new issues created by this evidence. The gathering, conservation, communication and presentation of the computer‐derived evidence must fulfil legal requirements with regard to the admissibility of the evidence. Electronic evidence that was gathered in a way that was not in accordance with the law will be declared inadmissible and be ruled out of court. This report aims to briefly present the core principles of the law when handling electronic evidence. Therefore, this paper examines the conditions of admissibility of evidence in four European countries. In order to be complete and to give an interesting pan‐European view on the question, the English law sy...

Plants are very well adapted to growth in ultraviolet-B (UV-B) containing light. In Arabidopsis thaliana, many of these adaptations are mediated by the UV-B receptor UV RESISTANCE LOCUS 8 (UVR8). Using small amounts of supplementary UV-B light, we observed changes in the shape of rosette leaf blades. Wild type plants show more pronounced epinasty of the blade edges, while this is not the case in uvr8 mutant plants. The UVR8 effect thus mimics the effect of phytochrome (phy) B in red light. In addition, a meta-analysis of transcriptome data indicates that the UVR8 and phyB signaling pathways have over 70% of gene regulation in common. Moreover, in low levels of supplementary UV-B light, mutant analysis revealed that phyB signaling is necessary for epinasty of the blade edges. Analysis of auxin levels and the auxin signal reporter DR5:GUS suggest that the epinasty relies on altered auxin distribution, keeping auxin at the leaf blade edges in the presence of UV-B. Together, our results suggest a co-action of phyB and UVR8 signaling, with auxin as a downstream factor.

The ground ice contained in screes with various rock debris (“rock glacier”) is the most common form of permafrost currently encountered in the Alps between 2500 and 3200 m asl. This altitudinal range is exactly the one which is concerned with the development of major ski resorts. In the manner of glaciers, rock glaciers will move on the slopes as a result of creep of the ice. However, because of the presence of rocky debris, the internal friction slows the movement. Confirming the presence of ice in the ground can be based at first on ground temperature measurements. But regarding the distribution with depth, in the absence of soundings technically and economically difficult to achieve, the geophysical methods provide good results. In the current context of climatic change, the problem of foundations of structures is increasingly taken into account. The observed warming in the Alps causes a slow melting of permafrost; settlements and collapses are observed. Geotechnical studies to illustrate this behavior are of the two following types: (i) feasibility study upstream projects, in order to define areas of permafrost and to avoid them for optimizing the implementation of the works; (ii) diagnostic studies giving solutions for reinforcement works after disorders due to the evolution of permafrost. Two specific examples are given: (i) the choice to draw a line for a chairlift, and (ii) the reinforcement of a ski-lift station with differential settlements.

Background and Aims Parasitic plants obtain nutrients from their hosts through organs called haustoria. The hyaline body is a specialized parenchymatous tissue occupying the central parts of haustoria in many Orobanchaceae species. The structure and functions of hyaline bodies are poorly understood despite their apparent necessity for the proper functioning of haustoria. Reported here is a cell wall-focused immunohistochemical study of the hyaline bodies of three species from the ecologically important clade of rhinanthoid Orobanchaceae. Methods Haustoria collected from laboratory-grown and field-collected plants of Rhinanthus minor, Odontites vernus and Melampyrum pratense attached to various hosts were immunolabelled for cell wall matrix glycans and glycoproteins using specific monoclonal antibodies (mAbs). Key Results Hyaline body cell wall architecture differed from that of the surrounding parenchyma in all species investigated. Enrichment in arabinogalactan protein (AGP) epitopes labelled with mAbs LM2, JIM8, JIM13, JIM14 and CCRC-M7 was prominent and coincided with reduced labelling of de-esterified homogalacturonan with mAbs JIM5, LM18 and LM19. Furthermore, paramural bodies, intercellular deposits and globular ergastic bodies composed of pectins, xyloglucans, extensins and AGPs were common. In Rhinanthus they were particularly abundant in pairings with legume hosts. Hyaline body cells were not in direct contact with haustorial xylem, which was surrounded by a single layer of paratracheal parenchyma with thickened cell walls abutting the xylem. Conclusions The distinctive anatomy and cell wall architecture indicate hyaline body specialization. Altered proportions of AGPs and pectins may affect the mechanical properties of hyaline body cell walls. This and the association with a transfer-like type of paratracheal parenchyma suggest a role in nutrient translocation. Organelle-rich protoplasts and the presence of exceptionally profuse intra-and intercellular wall materials when attached to a nitrogen-fixing host suggest subsequent processing and transient storage of nutrients. AGPs might therefore be implicated in nutrient transfer and metabolism in haustoria.

Plant cell walls are essential for most aspects of plant growth, development, and survival, including cell division, expansive cell growth, cell-cell communication, biomechanical properties, and stress responses. Therefore, characterizing cell wall diversity contributes to our overall understanding of plant evolution and development. Recent biochemical analyses, concomitantly with whole genome sequencing of plants located at pivotal points in plant phylogeny, have helped distinguish between homologous characters and those which might be more derived. Most plant lineages now have at least one fully sequenced representative and although genome sequences for fern species are in progress they are not yet available for this group. Ferns offer key advantages for the study of developmental processes leading to vascularisation and complex organs as well as the specific differences between diploid sporophyte tissues and haploid gametophyte tissues and the interplay between them. Ceratopteris richardii has been well investigated building a body of knowledge which combined with the genomic and biochemical information available for other plants will progress our understanding of wall diversity and its impact on evolution and development. Olivier Leroux is supported by an IRCSET EMPOWER award (PD/2011/2326). A grant from Research Foundation – Flanders, Belgium (F.W.O. – Vlaanderen; Research stay K209012N) supported Sharon Eeckhout during a research visit to NUI Galway (June 2012). peer-reviewed

Laminae of Adiantum raddianum Presl., a fern belonging to the family Pteridaceae, are characterised by the presence of epidermal fibre-like cells under the vascular bundles. These cells were thought to contain silica bodies, but their thickened walls leave no space for intracellular silica suggesting it may actually be deposited within their walls. Using advanced electron microscopy in conjunction with energy dispersive X-ray microanalysis we showed the presence of silica in the cell walls of the fibre-like idioblasts. However, it was specifically localised to the outer layers of the periclinal wall facing the leaf surface, with the thick secondary wall being devoid of silica. Immunocytochemical experiments were performed to ascertain the respective localisation of silica deposition and glycan polymers. Epitopes characteristic for pectic homogalacturonan and the hemicelluloses xyloglucan and mannan were detected in most epidermal walls, including the silica-rich cell wall layers. The monoclonal antibody, LM6, raised against pectic arabinan, labelled the silica-rich primary wall of the epidermal fibre-like cells and the guard cell walls, which were also shown to contain silica. We hypothesise that the silicified outer wall layers of the epidermal fibre-like cells support the lamina during cell expansion prior to secondary wall formation. This implies that silicification does not impede cell elongation. Although our results suggest that pectic arabinan may be implicated in silica deposition, further detailed analyses are needed to confirm this. The combinatorial approach presented here, which allows correlative screening and in situ localisation of silicon and cell wall polysaccharide distribution, shows great potential for future studies.

International audience; Background and Aims Extraxylary helical cell wall thickenings in vascular plants are not well documented, except for those in orchid velamen tissues which have been studied extensively. Reports on their occurrence in ferns exist, but detailed information is missing. The aim of this study is to focus on the broad patterns of structure and composition and to study the taxonomic occurrence of helical cell wall thickenings in the fern family Aspleniaceae. Methods Structural and compositional aspects of roots have been examined by means of light, electron, epifluorescence and laser scanning confocal microscopy. To assess the taxonomical distribution of helical cell wall thickenings a molecular phylogenetic analysis based on rbcL sequences of 64 taxa was performed. Key Results The helical cell wall thickenings of all examined species showed considerable uniformity of design. The pattern consists of helical, regularly bifurcating and anastomosing strands. Compositionally, the cell wall thickenings were found to be rich in homogalacturonan, cellulose, mannan and xyloglucan. Thioacidolysis confirmed our negative phloroglucinol staining tests, demonstrating the absence of lignins in the root cortex. All taxa with helical cell wall thickenings formed a monophyletic group supported by a 100% bootstrap value and composed of mainly epiphytic species. Conclusions This is the first report of non-lignified pectin-rich secondary cell walls in ferns. Based on our molecular analysis, we reject the hypothesis of parallel evolution of helical cell wall thickenings in Aspleniaceae. Helical cell wall thickenings can mechanically stabilize the cortex tissue, allowing maximal uptake of water and nutrients during rainfall events. In addition, it can also act as a boundary layer increasing the diffusive pathway towards the atmosphere, preventing desiccation of the stele of epiphytic growing species.

Cell wall appositions (CWAs), formed by the deposition of extra wall material at the contact site with microbial organisms, are an integral part of the response of plants to microbial challenge. Detailed histological studies of CWAs in fern roots do not exist. Using light and electron microscopy we examined the (ultra)structure of CWAs in the outer layers of roots of Asplenium species. All cell walls studded with CWAs were impregnated with yellow-brown pigments. CWAs had different shapes, ranging from warts to elongated branched structures, as observed with scanning and transmission electron microscopy. Ultrastructural study further showed that infecting fungi grow intramurally and that they are immobilized by CWAs when attempting to penetrate intracellularly. Immunolabelling experiments using monoclonal antibodies indicated pectic homogalacturonan, xyloglucan, mannan and cellulose in the CWAs, but tests for lignins and callose were negative. We conclude that these appositions are defense-related structures made of a non-lignified polysaccharide matrix on which phenolic compounds are deposited in order to create a barrier protecting the root against infections.