Whole-Cell Electron Tomography Analysis of Vacuole Biogenesis.

Vacuoles in plant cells are the most prominent organelles that harbor distinctive features, including lytic function, storage of proteins and sugars, balance of cell volume, and defense responses. Despite their dominant size and functional versatility, the nature and biogenesis of vacuoles in plants per se remain elusive and several models have been proposed. Recently, we used the whole-cell 3D electron tomography (ET) technique to study vacuole formation and distribution at nanometer resolution and demonstrated that small vacuoles are derived from multivesicular body maturation and fusion. Good sample preparation is a critical step to get high-quality electron tomography images. In this chapter, we provide detailed sample preparation methods for high-resolution ET in Arabidopsis thaliana root cells, including high-pressure freezing, subsequent freeze-substitution fixation, embedding, and serial sectioning.

Histochemical and ultrastructural localization of triterpene saponins in Medicago truncatula.

In the Medicago genus, saponins are complex mixtures of triterpene pentacyclic glycosides extensively studied for their different and economically relevant biological and pharmaceutical properties. This research is aimed at determining for the first time the tissue and cellular localization of triterpene saponins in vegetative organs of Medicago truncatula, a model plant species for legumes, by histochemistry and transmission electron microscopy. The results showed that saponins are present mainly in the palisade mesophyll layer of leaves, whereas in stems they are mostly located in the primary phloem and the subepidermal cells of cortical parenchyma. In root tissue, saponins occur in the secondary phloem region. Transmission electron microscopy revealed prominent saponin accumulation within the leaf and stem chloroplasts, while in the roots the saponins are found in the vesicular structures. Our results demonstrate the feasibility of using histochemistry and transmission electron microscopy to localize M. truncatula saponins at tissue and cellular levels and provide important information for further studies on biosynthesis and regulation of valuable bioactive saponins on agronomic relevant Medicago spp., such as alfalfa (Medicago sativa L.). RESEARCH HIGHLIGHTS: The Medicago genus represents a valuable rich source of saponins, one of the most interesting groups of secondary plant metabolites, which possess relevant biological and pharmacological properties. Plant tissue and cellular localization of saponins is of great importance to better understand their biological functions, biosynthetic pathway, and regulatory mechanisms. We elucidate the localization of saponins in Medicago truncatula with histochemical and transmission electron microscopy studies.

Anatomical features of ecological importance and taxonomic value revealed by scanning electron microscopy and light microscopy in Camonea umbellata (L.) A.R. Simões & Staples (Convolvulaceae).

This article describes detailed and novel data on the anatomy and histochemistry of leaves, stems, and roots of Camonea umbellata (L.) A.R.Simões & Staples in different environments for the identification of characters with taxonomical value and of ecological importance, with provision of light and scanning electron microscopy images. To analyze the characters, we collected samples of the vegetative organs of three individuals in each of three populations, which were in a grazing area, an urban environment, and a biological reserve. The main diagnostic anatomical markers for the identification of C. umbellata include amphistomatic leaves, tetracytic and brachyparatetracytic stomata, peltate trichomes, long simple trichomes, epidermis with striated cuticle ornamentation, mesophyll with acute borders, presence of druses, secretory channels, angular collenchyma, fibrous pericycle in the stem, intraxylary phloem in the vegetative organs, oil bodies throughout the midrib, petiole, stem and root, and epicuticular waxes of the crust and coiled rodlet types. Since the characters above did not show variation in the environments evaluated, we consider these characters taxonomically useful for the identification of C. umbellata. RESEARCH HIGHLIGHTS: The anatomy of the aerial vegetative organs of Camonnea umbellata retains common Convolvulaceae characters. The sinuosity of the epidermal cell walls and the density of trichomes in the epidermis of the petiole were visually variable characters among the analyzed individuals. Amphistomatic leaves, tetracytic and brachyparatetracytic stomata, peltate trichomes, epidermis with striated cuticle ornamentation, dorsiventral mesophyll with border acute, presence of druses, secretory structures, angular collenchyma, fibrous pericycle in the stem, intraxillary phloem, presence of oil bodies in all organs, and epicuticular waxes of the crust type and coiled rods were considered important anatomical markers for the recognition and correct identification of Camonea umbellata.

Biology of Callistethus plagiicollis (Coleoptera: Scarabaeoidea: Rutelinae) and comparative morphology of its larvae.

Larvae of the beetle subfamily Rutelinae are poorly described in the literature. Notably, the morphology of the larvae of Callistethus plagiicollis Fairmaire has not previously been analyzed. Here, we report for the first time that these larvae feed on the tubers and roots of Gastrodia elata Blume, an important traditional Chinese herbal medicine, which causes a reduction in the yield and economic value of G. elata. We employed scanning electron microscopy and light microscopy to investigate the morphology and occurrence regularity of egg, larvae, pupae, and adult specimens of C. plagiicollis collected from the G. elata planting base in Guizhou Province, China, with a focus on the ultrastructure of mature larvae. The results revealed one generation of C. plagiicollis per year in the study area and three instar stages of larvae. Mature larvae were identified by the following characteristics: raster without palidia with a large number of hamate setae, antennal apex containing seven sensilla basiconica, larval haptomerum containing eight sensilla styloconica and four enlarged heli, and seven longitudinally arranged stridulatory teeth on the stipes of the maxilla. The combination of scanning electron and light microscopy effectively revealed the difference between membranous and sclerotized structures, ensuring accurate identification of C. plagiicollis larvae. By determining the feeding characteristics and occurrence regularity of C. plagiicollis, this study has implications for improved pest management in G. elata crops. RESEARCH HIGHLIGHTS: We identified C. plagiicollis as a new pest of G. elata, a traditional Chinese medicine Scanning electron and light microscopy were combined to analyze the morphology of the mature larvae of C. plagiicollis for the first time We determined the feeding characteristics and occurrence regularity of C. plagiicollis, which can be used to develop effective pest management strategies.

Arbuscular mycorrhizal infection in two morphological root types of Araucaria araucana (Molina) K. Koch / Infección por micorrizas arbusculares en dos tipos de raíces de Araucaria araucana (Molina) K. Koch

Araucaria araucana (Molina) K. Koch is a conifer distributed in the Andean-Patagonian forests in the south of Argentina and Chile. The main objective of this work was to relate the different root classes appearing in A. araucana to mycorrhizal behavior. Samples were collected in three different sites in the Lanín National Park (NW Patagonia, Argentina). Two different root classes were present in A. araucana: longitudinal fine roots (LFR) and globular short roots (GSR). Both had extensive mycorrhizal arbuscular symbiosis (AM) and presented abundant hyphae and coils in root cells, a characteristic of the anatomical Paris-type. Dark septate fungal endophytes were also observed. Values of total AM colonization were high, with similar partial AM% values for each root class. Seasonal differences were found for total and partial colonization, with higher values in spring compared to autumn. Regarding the percentage of fungal structures between root classes, values were similar for vesicles and arbuscules, but higher coil percentages were observed in GSR compared to LFR. The percentages of vesicles increased in autumn, whereas the arbuscule percentages increased in spring, coinciding with the plant growth peak. Results show that both root classes of A. araucana in Andean-Patagonian forests are associated with AM fungi, which may have ecological relevance in terms of the importance of this symbiosis, in response to soil nutrient-deficiencies, especially high P-retention.

La conífera Araucaria araucana (Molina) K. Koch se encuentra distribuida en los bosques Andino-Patagónicos de Argentina y Chile. En este trabajo se relacionaron las diferentes clases morfológicas de raíces presentes en la especie con el comportamiento micorrícico. Las muestras fueron tomadas en tres sitios del Parque Nacional Lanín (NO de Patagonia, Argentina). Se observaron dos clases diferentes de raíces: raíces finas longitudinales (RFL) y raíces cortas globulares (RCG). Ambas clases presentaron una importante simbiosis micorrícico-arbuscular (MA), con abundante desarrollo de hifas intracelulares y rulos, característico del tipo anatómico Paris. También se observaron hifas marrones septadas. Los valores totales de colonización MA fueron altos, con porcentajes parciales similares en cada clase radicular. Se encontraron diferencias estacionales en los porcentajes de infección (totales y parciales), con valores mayores en primavera comparados con los de otoño. Los porcentajes de estructuras fúngicas presentes en cada clase radicular fueron similares para vesículas y arbúsculos, pero se observaron mayores valores de rulos en las RCG que en las RFL. Los porcentajes de vesículas aumentaron en otoño, mientras que los de arbúsculos lo hicieron en primavera, en coincidencia con el pico de crecimiento vegetativo de los árboles. Los resultados muestran una fuerte asociación simbiótica MA con A. araucana y su relevancia en términos ecológicos en respuesta a la deficiencia de nutrientes en el suelo, en especial, a la alta retención de fósforo.

Microscopic and transcriptome analyses of early colonization of tomato roots by Trichoderma harzianum

The capacity of the fungus Trichoderma harzianum CECT 2413 to colonize roots and stimulate plant growth was analyzed. Tobacco seedlings (Nicotiana benthamiana) transferred to Petri dishes inoculated with T. harzianum conidia showed increased plant fresh weight (140%) and foliar area (300%), as well as the proliferation of secondary roots (300%) and true leaves (140%). The interaction between strain CECT 2413 and the tomato-root system was also studied during the early stages of root colonization by the fungus. When T. harzianum conidia were inoculated into the liquid medium of hydroponically grown tomato plants (Lycopersicum esculentum), profuse adhesion of hyphae to the plant roots as well as colonization of the root epidermis and cortex were observed. Confocal microscopy of a T. harzianum transformant that expressed the green fluorescent protein (GFP) revealed intercellular hyphal growth and the formation of plant-induced papilla-like hyphal tips. Analysis of the T. harzianum-tomato interaction in soil indicated that the contact between T. harzianum and the roots persisted over a long period of time. This interaction was characterized by the presence of yeast-like cells, a novel and previously undescribed developmental change. To study the molecular mechanism underlying fungal ability to colonize the tomato-root system, the T. harzianum transcriptome was analyzed during the early stages of the plant-fungus interaction. The expression of fungal genes related to redox reactions, lipid metabolism, detoxification, and sugar or amino-acid transport increased when T. harzianum colonized tomato roots. These observations are similar to those regarding the interactions of mycorrhiza and pathogenic fungi with plants (AU)

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Subterranean structures and mycotrophy of the achlorophyllous Dictyostega orobanchoides (Burmanniaceae)

Plants of Dictyostega orobanchoides arise from about 1 mm thick rhizomes, which are densely covered by sessile, imbricate, peltate scale leaves. The resulting interfoliar spaces are inhabited by fungal hyphae up to 6 microns thick, often developing vesicle-like bladders. The fungus also colonizes the tissue of the scale leaves, inter- as well as intracellularly, forming vesicles but no arbuscules, and it even penetrates the vascular bundles of the leaves. The rhizome itself does not become infected. The 200 microns thick roots emerge from the rhizome and have a 2-layered cortex and voluminous rhizodermis, which both are delicate and often disrupted or missing. In contrast, the strongly reinforced, tertiary endodermis and the central cylinder are durable and have a considerable tensile strength. Although the roots grow through the hyphal masses in the interfoliar spaces when emerging from the rhizome, they only become infected from the rhizosphere. A collar of rhizomogenous tissue hinders the interfoliar hyphae from direct contact to the roots. Only within the rhizodermis, the mycorrhizal fungus builds coils of heteromorphic hyphae, arbuscule-like structures, and vesicles. Hence, the mycorrhiza in D. orobanchoides is assigned to the arbuscular mycorrhiza. It is hypothezised, that the ephemeral mycorrhizal tissue combined with the durable vascular system of the roots is a strategy to avoid the high costs of protecting the large rhizodermal surface area. The rhizomogenous collar is explained as an extra protection to the tender, young roots, when emerging from the rhizome. The necessity to include other subterranean plant organs along with the roots in future mycorrhizal studies is emphasized.

The laticifer system of Chamaesyce thymifolia: a closed host environment for plant trypanosomatids

Specimens of Chamaesyce thymifolia (Euphorbiaceae) infected and uninfected by Phytomonas sp., a parasite of the Trypanosomatidae family, were anatomically and ultrastructurally analyzed with special emphasis on the laticifer system. C. thymifolia presents branched non-articulated laticifers and was heavily infected by Phytomonas sp. in all collection sites. Infection was often observed in the initial stages inside the vacuole, when the latex particles could be seen. In intermediary stages of laticifer differentiation, Phytomonas sp. were found free in the cytoplasm, inside small vacuoles or in the central vacuole. In differentiated laticifers that had only the plasma membrane, Phytomonas sp. were free in the latex and close to the cell membrane. Infected and uninfected plants showed identical anatomy and ultrastructure and the starch grain numbers in the latex were not reduced in the presence of this flagellate. Biochemical analysis of the latex of infected and uninfected plants presented similar levels of protein, carbohydrate and beta-1,3-glucanase, suggesting that this species is not pathogenic for the host. Besides, all infected plants complete its life cycle. Plants infected with Phytomonas presented occasionally virus like particles and bacteria inside the laticifer tubes.

The laticifer system of Chamaesyce thymifolia: a closed host environment for plant trypanosomatids

Specimens of Chamaesyce thymifolia (Euphorbiaceae) infected and uninfected by Phytomonas sp., a parasite of the Trypanosomatidae family, were anatomically and ultrastructurally analyzed with special emphasis on the laticifer system. C. thymifolia presents branched non-articulated laticifers and was heavily infected by Phytomonas sp. in all collection sites. Infection was often observed in the initial stages inside the vacuole, when the latex particles could be seen. In intermediary stages of laticifer differentiation, Phytomonas sp. were found free in the cytoplasm, inside small vacuoles or in the central vacuole. In differentiated laticifers that had only the plasma membrane, Phytomonas sp. were free in the latex and close to the cell membrane. Infected and uninfected plants showed identical anatomy and ultrastructure and the starch grain numbers in the latex were not reduced in the presence of this flagellate. Biochemical analysis of the latex of infected and uninfected plants presented similar levels of protein, carbohydrate and beta-1,3-glucanase, suggesting that this species is not pathogenic for the host. Besides, all infected plants complete its life cycle. Plants infected with Phytomonas presented occasionally virus like particles and bacteria inside the laticifer tubes.(AU)