Quantitative analysis of plant ER architecture and dynamics.

The endoplasmic reticulum (ER) is a highly dynamic polygonal membrane network composed of interconnected tubules and sheets (cisternae) that forms the first compartment in the secretory pathway involved in protein translocation, folding, glycosylation, quality control, lipid synthesis, calcium signalling, and metabolon formation. Despite its central role in this plethora of biosynthetic, metabolic and physiological processes, there is little quantitative information on ER structure, morphology or dynamics. Here we describe a software package (AnalyzER) to automatically extract ER tubules and cisternae from multi-dimensional fluorescence images of plant ER. The structure, topology, protein-localisation patterns, and dynamics are automatically quantified using spatial, intensity and graph-theoretic metrics. We validate the method against manually-traced ground-truth networks, and calibrate the sub-resolution width estimates against ER profiles identified in serial block-face SEM images. We apply the approach to quantify the effects on ER morphology of drug treatments, abiotic stress and over-expression of ER tubule-shaping and cisternal-modifying proteins.

Viewing oxidative stress through the lens of oxidative signalling rather than damage.

Concepts of the roles of reactive oxygen species (ROS) in plants and animals have shifted in recent years from focusing on oxidative damage effects to the current view of ROS as universal signalling metabolites. Rather than having two opposing activities, i.e. damage and signalling, the emerging concept is that all types of oxidative modification/damage are involved in signalling, not least in the induction of repair processes. Examining the multifaceted roles of ROS as crucial cellular signals, we highlight as an example the loss of photosystem II function called photoinhibition, where photoprotection has classically been conflated with oxidative damage.

Scanning Electron Microscopy (SEM) Protocols for Problematic Plant, Oomycete, and Fungal Samples.

Common problems in the processing of biological samples for observations with the scanning electron microscope (SEM) include cell collapse, treatment of samples from wet microenvironments and cell destruction. Using young floral tissues, oomycete cysts, and fungi spores (Agaricales) as examples, specific protocols to process delicate samples are described here that overcome some of the main challenges in sample treatment for image capture under the SEM. Floral meristems fixed with FAA (Formalin-Acetic-Alcohol) and processed with the Critical Point Dryer (CPD) did not display collapsed cellular walls or distorted organs. These results are crucial for the reconstruction of floral development. A similar CPD-based treatment of samples from wet microenvironments, such as the glutaraldehyde-fixed oomycete cysts, is optimal to test the differential growth of diagnostic characteristics (e.g., the cyst spines) on different types of substrates. Destruction of nurse cells attached to fungi spores was avoided after rehydration, dehydration, and the CPD treatment, an important step for further functional studies of these cells. The protocols detailed here represent low-cost and rapid alternatives for the acquisition of good-quality images to reconstruct growth processes and to study diagnostic characteristics.

Preventing False Negatives for Histochemical Detection of Phenolics and Lignins in PEG-Embedded Plant Tissues.

Polyethylene glycol (PEG) is a low-cost and advantageous embedding medium, which maintains the majority of cell contents unaltered during the embedding process. Some hard or complex plant materials are better embedded in PEG than in other usual embedding media. However, the histochemical tests for phenolics and lignins in PEG-embedded plant tissues commonly result in false negatives. We hypothesize that these false negatives should be prevented by the use of distinct fixatives, which should avoid the bonds between PEG and phenols. Novel protocols for phenolics and flavanols detection are efficiently tested, with fixation of the samples in ferrous sulfate and formalin or in caffeine and sodium benzoate, respectively. The differentiation of lignin types is possible in safranin-stained sections observed under fluorescence. The Maule's test faultlessly distinguishes syringyl-rich from guaiacyl- and hydroxyphenyl-rich lignins in PEG-embedded material under light microscopy. Current hypothesis is corroborated, that is, the adequate fixation solves the false-negative results, and the new proposed protocols fill up some gaps on the detection of phenolics and lignins.

Imaging plant nuclei and membrane-associated cytoskeleton by field emission scanning electron microscopy.

Scanning electron microscopy (SEM) is a powerful technique that can image exposed surfaces in 3D. Modern scanning electron microscopes, with field emission electron sources and in-lens specimen chambers, achieve resolutions of better than 0.5 nm and thus offer views of ultrastructural details of subcellular structures or even macromolecular complexes. Obtaining a reliable image is, however, dependent on sample preparation methods that robustly but accurately preserve biological structures. In plants, exposing the object of interest may be difficult due to the existence of a cell wall. This protocol shows how to isolate plant nuclei for SEM imaging of the nuclear envelope and associated structures from both sides of the nuclear envelope in cultured cells as well as in leaf or root cells. Further, it provides a method for uncovering membrane-associated cytoskeletal structures.

Calcium and silicon mineralization in land plants: transport, structure and function.

Plant biomineralization involves calcium and silicon transport and mineralization. Respective analytical methods and case studies are listed. Calcium carbonate is deposited in cystoliths, calcium oxalate in idioblasts. Silicon is deposited in phytoliths. Biomineralization is a coordinated process.

Subcellular distribution of ascorbate in plants.

The compartment specific distribution of ascorbate in plants is of great importance for plant development, growth and defense as this multifunctional metabolite plays important roles in the detoxification of reactive oxygen species (ROS), redox signaling, modulation of gene expression and is important for the regulation of enzymatic activities. Even though changes in ascorbate contents during plant growth and various stress conditions are well documented and the roles of ascorbate in plant defense during abiotic stress conditions are well established, still too little is known about its compartment specific roles during plant development and defense. This mini-review focuses on the subcellular distribution of ascorbate in plants and describes different methods that are currently used to study its compartment specific distribution. Finally, it will also briefly discuss data available on compartment specific changes of ascorbate during some abiotic stress conditions such as high light conditions and exposure to ozone.

Morphology and ultrastructure of megaspores and microspores of Isoetes sehnemii Fuchs (Lycophyta)

The morphology and wall ultrastructure of megaspores and microspores of Isoetes sehnemii that grows in Brazil were analyzed as part of the study of the Isoetaceae present in Southern South America. The observations were performed with light, scanning and transmission electron microscopes. The megaspores are trilete, 350-450μm in equatorial diameter. The surface is reticulate. In section, the sporoderm is 100μm thick including the ornamentation. The wall is composed of a siliceous perispore, which consists of short fused flatten, elements forming a three-dimensional mesh. The exospore has two zones of different structure. The endospore is fibrillar. The microspores are monolete, 21-27μm in equatorial diameter. The sporoderm is composed of a sporopollinic rugulate perispore. A space between the paraexospore and the exospore is evident. The exospore is compact. The endospore is fibrillar. The ultrastructural analysis akes hoologies evident concerning structure and organization of the layers belo the perispore in both spore types. A possible similarity and stability in the ultrustructure of the present spores and fossils could be also inferred. In addition, there would be a correlation among the plant habitat, the spore ornamentation and the geographic distribution.

A morfologia e a ultraestrutura da parede de megasporos e microspores de Isoetes sehnemii que crescem no Brasil foram analisados como parte do estudo de Isoetaceae presente no sul da América do Sul. As observações foram realizadas com microscopias de luz e eletrônicas de transmissão e varredura. Os megasporos são triletes com 350-450μm de diâmetro equatorial. A superfície é reticulada. Em secção o esporoderma possui 100μm de espessura incluindo ornamentação. A parede é composta de um perisporo silicoso que consiste de elementos fusionados curtos e achatados formando uma rede tridimensional. O exosporo tem duas zonas com diferentes estruturas. O endosporo é fibrilar. Os microsporos são monoletes, 21-27μm de diâmetro equatorial. A esporoderme é composta por um perisporo esporopolínico rugulado. Um espaço entre o para-exosporo e o exosporo é evidente. O exosporo é compacto. O endosporo é fibrilar. A análise ultraestrutural torna evidente homologias relativas a estrutura e organização das camadas abaixo do perisporo em ambos os tipos de esporos. Uma possível similaridade e estabilidade na ultraestrutura do presente esporo e fósseis pode também ser inferida. Além disso, haveria uma correlação entre o habitat da planta, a ornamentação do esporo e a distribuição geográfica.

Isoetes pedersenii, a new species from Southern South America

The name Isoetes pedersenii H.P. Fuchs (Lycophyta), a species known only from the Mburucuyá National Park, Corrientes, Argentina, is validated. Observations were carried out on herbarium material with stereoscopic, light and scanning electron microscopes. The species is described and typified. A diagnosis and discussion about its distribution and its relationship with the morphology of other species of Isoetes are provided.

O nome Isoetes pedersenii H.P. Fuchs (Lycophyta) foi validado para a espécie identificada apenas no Parque Nacional de Mburucuyá, em Corrientes na Argentina. Material preservado em herbário foi avaliado com microscópios estereoscó pico, de luz branca e eletrônico de varredura. A espécie foi descrita e tipificada. Um diagnóstico e uma discussão sobre a distribuição e relação com a morfologia de outras espécies de Isoetes são relatados.

The plant endoplasmic reticulum: a cell-wide web.

The ER (endoplasmic reticulum) in higher plants forms a pleomorphic web of membrane tubules and small cisternae that pervade the cytoplasm, but in particular form a polygonal network at the cortex of the cell which may be anchored to the plasma membrane. The network is associated with the actin cytoskeleton and demonstrates extensive mobility, which is most likely to be dependent on myosin motors. The ER is characterized by a number of domains which may be associated with specific functions such as protein storage, or with direct interaction with other organelles such as the Golgi apparatus, peroxisomes and plastids. In the present review we discuss the nature of the network, the role of shape-forming molecules such as the recently described reticulon family of proteins and the function of some of the major domains within the ER network.

Harnessing plant trichome biochemistry for the production of useful compounds.

Plant trichomes come in a variety of shapes, sizes and cellular composition. Some types, commonly called glandular trichomes, produce large amounts of specialized (secondary) metabolites of diverse classes. Trichomes are implicated in a variety of adaptive processes, including defense against herbivores and micro-organisms as well as in ion homeostasis. Because trichomes protrude from the epidermis and can often be easily separated from it and harvested, the mRNAs, proteins and small molecules that they contain are unusually accessible to analysis. This property makes them excellent experimental systems for identification of the enzymes and pathways responsible for the synthesis of the specialized metabolites found in these structures and sometimes elsewhere in the plant. We review the literature on the biochemistry of trichomes and consider the attributes that might make them highly useful targets for plant metabolic engineering.

Enhanced separation of membranes during free flow zonal electrophoresis in plants.

Free flow zonal electrophoresis (FFZE) is a versatile technique that allows for the separation of cells, organelles, membranes, and proteins based on net surface charge during laminar flow through a thin aqueous layer. We have been optimizing the FFZE technique to enhance separation of plant vacuolar membranes (tonoplast) from other endomembranes to pursue a directed proteomics approach to identify novel tonoplast transporters. Addition of ATP to a mixture of endomembranes selectively enhanced electrophoretic mobility of acidic vesicular compartments during FFZE toward the positive electrode. This has been attributed to activation of the V-ATPase generating a more negative membrane potential outside the vesicles, resulting in enhanced migration of acidic vesicles, including tonoplast, to the anode (Morré, D. J.; Lawrence, J.; Safranski, K.; Hammond, T.; Morré, D. M. J. Chromatogr., A 1994, 668, 201-213). We confirm that ATP does induce a redistribution of membranes during FFZE of microsomal membranes isolated from several plant species, including Arabidopsis thaliana, Thellungiella halophila, Mesembryanthemum crystallinum, and Ananas comosus. However, we demonstrate, using V-ATPase-specific inhibitors, nonhydrolyzable ATP analogs, and ionophores to dissipate membrane potential, that the ATP-dependent migrational shift of membranes under FFZE is not due to activation of the V-ATPase. Addition of EDTA to chelate Mg2+, leading to the production of the tetravalent anionic form of ATP, resulted in a further enhancement of membrane migration toward the anode, and manipulation of cell surface charge by addition of polycations also influenced the ATP-dependent migration of membranes. We propose that ATP enhances the mobility of endomembranes by screening positive surface charges on the membrane surface.

Eleocharis R.Br. (Cyperaceae) no Estado do Rio de Janeiro, Brasil / Eleocharis R. Br. (Cyperaceae) in Rio de Janeiro State, Brasil

Foi realizado o levantamento das espécies de Eleocharis (Cyperaceae) no Estado do Rio de Janeiro - Brasil, a partir de exsicatas depositadas nos principais herbários do Estado (FCAB, GUA, HB, HUNI, R, RB, RBR, RFA, RUSU), assim como coletadas em excursões a ecossistemas aquáticos na área de estudo, entre novembro de 1998 a setembro de 2003. O gênero Eleocharis está representado no Estado do Rio de Janeiro por 19 espécies: Eleocharis acutangula (Roxb.) Schult., E. debilis Kunth, E. elongata Chapm., E. equisetoides (Elliott) Torr., E. filiculmis Kunth, E. flavescens (Poir.) Urb., E. geniculata (L.) Roem. & Schult., E. interstincta (Vahl) Roem. & Schult., E. maculosa (Vahl) Roem. & Schult., E. minarum Boeck.*, E. minima Kunth, E. montana (Kunth) Roem. & Schult., E. mutata (L.) Roem. & Schult., E. nana Kunth, E. pachystyla (C. Wright) C. B. Clarke*, E. radicans (Poir.) Kunth*, E. sellowiana Kunth, E. squamigera Svenson e E. subarticulata (Nees) Boeck (*espécies raras no Estado do Rio de Janeiro). São apresentadas chave de identificação, descrições, ilustrações, informações adicionais de natureza ecológica e distribuição geográfica das espécies de Eleocharis do Estado do Rio de Janeiro.

A floristic inventory of the species of Eleocharis (Cyperaceae) from Rio de Janeiro State - Brazil was made. This research included material from most important Herbaria of Rio de Janeiro (FCAB, GUA, HB, HUNI, R, RB, RBR, RFA, RUSU) as well as plants collected during expeditions to aquatic ecosystems of Rio de Janeiro, between November 1998 and September 2003. The genus Eleocharis is represented in the area by 19 species: Eleocharis acutangula (Roxb.) Schult., E. debilis Kunth, E. elongata Chapm., E. equisetoides (Elliott) Torr., E. filiculmis Kunth, E. flavescens (Poir.) Urb., E. geniculata (L.) Roem. & Schult., E. interstincta (Vahl) Roem. & Schult., E. maculosa (Vahl) Roem. & Schult., E. minarum Boeck.*, E. minima Kunth, E. montana (Kunth) Roem. & Schult., E. mutata (L.) Roem. & Schult., E. nana Kunth, E. pachystyla (C. Wright) C. B. Clarke*, E. radicans (Poir.) Kunth*, E. sellowiana Kunth, E. squamigera Svenson, and E. subarticulata (Nees) Boeck. (*species rare in Rio de Janeiro state). A key of identification, descriptions of species, illustrations, and notes on ecological features and geographical distribution are presented.

[Mobile reticular organization of plastids and mitochondria in plant cells].

Results of confocal, fluorescent and video microscopy of plant cell organelles and of stromule network uniting them are reviewed. The vast information on the structure of stromules, their mobility, proposed functions and development has been analyzed, in addition to factors stimulating and suppressing this development. Structural similarity between the network of stromules in living cells, observed by confocal fluorescence microscopy, and the endoplasmic reticulum, seen on micrographs of preparations fixed for electron microscopy is discussed. As a result of this discussion, a conclusion is made with regard to the identity of these endomembranous networks. The intercellular symplastic organization is shown for both networks in plant tissues. The existence of a common transport and trophic compartment is proposed that includes organelles, intercellular endoplasmic reticulum and its derivatives, phloem and xylem. The trophic system development might have been induced in the course of endosymbiogenesis with some bacterial precursors of organelles.