The association between Dioscorea sansibarensis and Orrella dioscoreae as a model for hereditary leaf symbiosis.

Hereditary, or vertically-transmitted, symbioses affect a large number of animal species and some plants. The precise mechanisms underlying transmission of functions of these associations are often difficult to describe, due to the difficulty in separating the symbiotic partners. This is especially the case for plant-bacteria hereditary symbioses, which lack experimentally tractable model systems. Here, we demonstrate the potential of the leaf symbiosis between the wild yam Dioscorea sansibarensis and the bacterium Orrella dioscoreae (O. dioscoreae) as a model system for hereditary symbiosis. O. dioscoreae is easy to grow and genetically manipulate, which is unusual for hereditary symbionts. These properties allowed us to design an effective antimicrobial treatment to rid plants of bacteria and generate whole aposymbiotic plants, which can later be re-inoculated with bacterial cultures. Aposymbiotic plants did not differ morphologically from symbiotic plants and the leaf forerunner tip containing the symbiotic glands formed normally even in the absence of bacteria, but microscopic differences between symbiotic and aposymbiotic glands highlight the influence of bacteria on the development of trichomes and secretion of mucilage. This is to our knowledge the first leaf symbiosis where both host and symbiont can be grown separately and where the symbiont can be genetically altered and reintroduced to the host.

Motility-Independent Vertical Transmission of Bacteria in Leaf Symbiosis.

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, tissue-specific regulation of putative symbiotic functions highlights 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 intrahost populations together with tissue-specific regulation may serve as a robust mechanism for the maintenance of mutualism in leaf symbiosis. IMPORTANCE Hereditary symbioses with bacteria are common in the animal kingdom, but relatively unexplored in plants. Several plant species form associations with bacteria in their leaves, which is 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 show that bacteria are 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 with 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.

Quantitative mapping of mercury and selenium in mushroom fruit bodies with laser ablation-inductively coupled plasma-mass spectrometry.

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.

Limited plasticity of anatomical and hydraulic traits in aspen trees under elevated CO2 and seasonal drought.

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.

Acoustic Vulnerability, Hydraulic Capacitance, and Xylem Anatomy Determine Drought Response of Small Grain Cereals.

Selection of high-yielding traits in cereal plants led to a continuous increase in productivity. However, less effort was made to select on adaptive traits, favorable in adverse and harsh environments. Under current climate change conditions and the knowledge that cereals are staple foods for people worldwide, it is highly important to shift focus to the selection of traits related to drought tolerance, and to evaluate new tools for efficient selection. Here, we explore the possibility to use vulnerability to drought-induced xylem embolism of wheat cultivars Excalibur and Hartog (Triticum aestivum L.), rye cultivar Duiker Max (Secale cereale L.), and triticale cultivars Dublet and US2014 (x Triticosecale Wittmack) as a proxy for their drought tolerance. Multiple techniques were combined to underpin this hypothesis. During bench-top dehydration experiments, acoustic emissions (AEs) produced by formation of air emboli were detected, and hydraulic capacitances quantified. By only looking at the AE50 values, one would classify wheat cultivar Excalibur as most tolerant and triticale cultivar Dublet as most vulnerable to drought-induced xylem embolism, though Dublet had significantly higher hydraulic capacitances, which are essential in terms of internal water storage to temporarily buffer or delay water shortage. In addition, xylem anatomical traits revealed that both cultivars have a contrasting trade-off between hydraulic safety and efficiency. This paper emphasizes the importance of including a cultivar's hydraulic capacitance when evaluating its drought response and vulnerability to drought-induced xylem embolism, instead of relying on the AE50 as the one parameter.

The Effect of Surrounding Vegetation on the Mycorrhizal Fungal Communities of the Temperate Tree Crataegus monogyna Jacq.

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.

Vibratome Sectioning of Plant Materials for (Immuno)cytochemical Staining.

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.

Identifying the pathways for foliar water uptake in beech (Fagus sylvatica L.): a major role for trichomes.

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.

Physiological responses and aquaporin expression upon drought and osmotic stress in a conservative vs prodigal Fragaria x ananassa cultivar.

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.

cis-Cinnamic acid is a natural plant growth-promoting compound.

Agrochemicals provide vast potential to improve plant productivity, because they are easy to implement at low cost while not being restricted by species barriers as compared with breeding strategies. Despite the general interest, only a few compounds with growth-promoting activity have been described so far. Here, we add cis-cinnamic acid (c-CA) to the small portfolio of existing plant growth stimulators. When applied at low micromolar concentrations to Arabidopsis roots, c-CA stimulates both cell division and cell expansion in leaves. Our data support a model explaining the increase in shoot biomass as the consequence of a larger root system, which allows the plant to explore larger areas for resources. The requirement of the cis-configuration for the growth-promoting activity of CA was validated by implementing stable structural analogs of both cis- and trans-CA in this study. In a complementary approach, we used specific light conditions to prevent cis/trans-isomerization of CA during the experiment. In both cases, the cis-form stimulated plant growth, whereas the trans-form was inactive. Based on these data, we conclude that c-CA is an appealing lead compound representing a novel class of growth-promoting agrochemicals. Unraveling the underlying molecular mechanism could lead to the development of innovative strategies for boosting plant biomass.

Is the bacterial leaf nodule symbiosis obligate for Psychotria umbellata? The development of a Burkholderia-free host plant.

BACKGROUND & AIMS: The bacterial leaf nodule symbiosis is an interaction where bacteria are housed in specialised structures in the leaves of their plant host. In the Rubiaceae plant family, host plants interact with Burkholderia bacteria. This interaction might play a role in the host plant defence system. It is unique due to its high specificity; the vertical transmission of the endophyte to the next generation of the host plant; and its supposedly obligatory character. Although previous attempts have been made to investigate this obligatory character by developing Burkholderia-free plants, none have succeeded and nodulating plants were still produced. In order to investigate the obligatory character of this endosymbiosis, our aims were to develop Burkholderia-free Psychotria umbellata plants and to investigate the effect of the absence of the endophytes on the host in a controlled environment. METHODS: The Burkholderia-free plants were obtained via embryo culture, a plant cultivation technique. In order to analyse the endophyte-free status, we screened the plants morphologically, microscopically and molecularly over a period of three years. To characterise the phenotype and growth of the in vitro aposymbiotic plants, we compared the growth of the Burkholderia-free plants to the nodulating plants under the same in vitro conditions. KEY RESULTS: All the developed plants were Burkholderia-free and survived in a sterile in vitro environment. The growth analysis showed that plants without endophytes had a slower development. CONCLUSIONS: Embryo culture is a cultivation technique with a high success rate for the development of Burkholderia-free plants of P. umbellata. The increased growth rate in vitro when the specific endophyte is present cannot be explained by possible benefits put forward in previous studies. This might indicate that the benefits of the endosymbiosis are not yet completely understood.

Importance of the Mixing and High-Temperature Heating Steps in the Controlled Thermal Coprecipitation Synthesis of Sub-5-nm Na(Gd-Yb)F4:Tm.

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.

Detection of Burkholderia in the seeds of Psychotria punctata (Rubiaceae) - Microscopic evidence for vertical transmission in the leaf nodule symbiosis.

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.

Comparative in situ analysis reveals the dynamic nature of sclerenchyma cell walls of the fern Asplenium rutifolium.

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.

Post-veraison irreversible stem shrinkage in grapevine (Vitis vinifera) is caused by periderm formation.

Grapevines are characterized by a period of irreversible stem shrinkage around the onset of ripening of the grape berries. Since this shrinkage is unrelated to meteorological conditions or drought, it is often suggested that it is caused by the increased sink strength of the grape berries during this period. However, no studies so far have experimentally investigated the mechanisms underlying this irreversible stem shrinkage. We therefore combined continuous measurements of stem diameter variations and histology of potted 2-year-old grapevines (Vitis vinifera L. 'Boskoop Glory'). Sink strength was altered by pruning all grape clusters (treatment P), while non-pruned grapevines served as control (treatment C). Unexpectedly, our results showed irreversible post-veraison stem shrinkage in both treatments, suggesting that the shrinkage is not linked to grape berry sink strength. Anatomical analysis indicated that the shrinkage is the result of the formation of successive concentric periderm layers, and the subsequent dehydration and compression of the older bark tissues, an anatomical feature that is characteristic of Vitis stems. Stem shrinkage is hence unrelated to grape berry development, in contrast to what has been previously suggested.

Dihydrofolate Reductase/Thymidylate Synthase Fine-Tunes the Folate Status and Controls Redox Homeostasis in Plants.

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.

Heat girdling does not affect xylem integrity: an in vivo magnetic resonance imaging study in the tomato peduncle.

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.

Mitochondrial Defects Confer Tolerance against Cellulose Deficiency.

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.

Antibody-based screening of cell wall matrix glycans in ferns reveals taxon, tissue and cell-type specific distribution patterns.

BACKGROUND: While it is kno3wn that complex tissues with specialized functions emerged during land plant evolution, it is not clear how cell wall polymers and their structural variants are associated with specific tissues or cell types. Moreover, due to the economic importance of many flowering plants, ferns have been largely neglected in cell wall comparative studies. RESULTS: To explore fern cell wall diversity sets of monoclonal antibodies directed to matrix glycans of angiosperm cell walls have been used in glycan microarray and in situ analyses with 76 fern species and four species of lycophytes. All major matrix glycans were present as indicated by epitope detection with some variations in abundance. Pectic HG epitopes were of low abundance in lycophytes and the CCRC-M1 fucosylated xyloglucan epitope was largely absent from the Aspleniaceae. The LM15 XXXG epitope was detected widely across the ferns and specifically associated with phloem cell walls and similarly the LM11 xylan epitope was associated with xylem cell walls. The LM5 galactan and LM6 arabinan epitopes, linked to pectic supramolecules in angiosperms, were associated with vascular structures with only limited detection in ground tissues. Mannan epitopes were found to be associated with the development of mechanical tissues. We provided the first evidence for the presence of MLG in leptosporangiate ferns. CONCLUSIONS: The data sets indicate that cell wall diversity in land plants is multifaceted and that matrix glycan epitopes display complex spatio-temporal and phylogenetic distribution patterns that are likely to relate to the evolution of land plant body plans.