Tillandsia stricta Sol (Bromeliaceae) leaves as monitors of airborne particulate matter-A comparative SEM methods evaluation: Unveiling an accurate and odd HP-SEM method.

Airborne particulate matter (PM) has been included among the most important air pollutants by governmental environment agencies and academy researchers. The use of terrestrial plants for monitoring PM has been widely accepted, particularly when it is coupled with SEM/EDS. Herein, Tillandsia stricta leaves were used as monitors of PM, focusing on a comparative evaluation of Environmental SEM (ESEM) and High-Pressure SEM (HPSEM). In addition, specimens air-dried at formaldehyde atmosphere (AD/FA) were introduced as an SEM procedure. Hydrated specimen observation by ESEM was the best way to get information from T. stricta leaves. If any artifacts were introduced by AD/FA, they were indiscernible from those caused by CPD. Leaf anatomy was always well preserved. PM density was determined on adaxial and abaxial leaf epidermis for each of the SEM proceedings. When compared with ESEM, particle extraction varied from 0 to 20% in air-dried leaves while 23-78% of particles deposited on leaves surfaces were extracted by CPD procedures. ESEM was obviously the best choice over other methods but morphological artifacts increased in function of operation time while HPSEM operation time was without limit. AD/FA avoided the shrinkage observed in the air-dried leaves and particle extraction was low when compared with CPD. Structural and particle density results suggest AD/FA as an important methodological approach to air pollution biomonitoring that can be widely used in all electron microscopy labs. Otherwise, previous PM assessments using terrestrial plants as biomonitors and performed by conventional SEM could have underestimated airborne particulate matter concentration.

Sugarcane cell wall structure and lignin distribution investigated by confocal and electron microscopy.

Lignocellulosic plant cell wall is considered a potential source for second generation biofuels. The plant cell wall is a highly complex structure mainly composed of cellulose, hemicelluloses, and lignin that form a network of crosslinked fibers. The structural organization of the sugarcane cell wall has not been previously analyzed in detail, and this analysis is a prerequisite for further studies on the recalcitrance and deconstruction of its biomass. In this work, cellulose and lignin localization were investigated by confocal laser scanning microscopy. In addition, the internode sugarcane cell wall structural organization was analyzed by electron microscopy. Internode stem anatomy showed a typical monocot structure consisting of epidermis, hypoderm, and vascular bundles scattered throughout ground parenchyma tissue and surrounded by sclerenchyma fibers. Confocal images of safranin labeled sugarcane showed that lignin distribution was predominant in the vessel elements, cell wall corners (CC), and middle lamella (ML), while cellulose-rich cell walls were randomly distributed in the ML and organized in the other cell wall layers. KMnO4 cytochemistry revealed that lignin was predominantly distributed in secondary cell walls, ML and CC. Cell wall sublayers (S1, S2, and S3) were identified and measured by transmission electron microscopy. Our results provide insights that may help further understanding of sugarcane cell wall organization, which is crucial for the research and technology of plant-based biofuel production.

A new protocol to detect light elements in estuarine sediments by X-ray microanalysis (SEM/EDS).

The analytical scanning electron microscope (SEM) has been used to determine the presence and distribution of atomic elements in mineralogy. However, the detection of light elements such as carbon is difficult to obtain with standard energy-dispersive X-ray spectrometry (EDS) and usual proceedings for SEM. This study proposes a new protocol to detect calcium carbonate by SEM/EDS using sediments from the Jaguaribe River estuary, NE Brazil, as a model. Handmade gold mounting discs (Au stubs) were used as sample support and samples were adhered with inexpensive glue (Loctite Super_Bonder) or directly disposed on the Au stubs. CaCO(3) and NaCl for chemical analysis were used as control and counterproof to the carbon adhesive tape. Control salts EDS analyses indicate that the method was efficient to detect light elements. Sediments obtained from different depths in the core sampled at the Jaguaribe River estuary consist of particles and aggregates with diverse morphology that covers a wide range of particle or aggregate size. Morphology and dimensions were similar for all core depths. Analysis of samples disposed on gold mounting disc without glue showed that sediment bulk particles usually presented small particles adhering on the surface. Clay minerals were predominant but silica was also often identified. Calcium was a trace element in a small number of sediment bulk particles. Biological and non-biological calcium carbonates, including nanoparticles, were identified in all core depths. X-ray emitted from Au stub did not interfere in the CaCO(3) EDS analysis. Calcium carbonate particles from sediments were identified using this novel approach.

Surface replicas of normal and vitrified leaves of Datura insignis, Barb Rodr.

Vitrification is a morphological and physiological disorder affecting plants during their in vitro vegetative propagation. Vitrified plants have a poor survival rate when transferred from in vitro to greenhouse conditions, a fact mainly due to water loss and dissecation. It has been shown that normal and vitreous leaves of Datura insignis differ in the frequency of normal and abnormal stomata. The purpose of this work was to compare the surface of normal and vitreous leaves of D. insignis, using a modification of the platinum/carbon replica method. Adaxial and abaxial leaf surfaces of normal plantlets have a smooth and homogenous cuticle. A granular aspect, probably due to leaf age, rarely occurs at the periphery of the epidermal cells. Both adaxial and abaxial leaf surfaces of vitrified plantlets show discontinuities in the cuticle, occurring at several regions of the outer periclinal cell walls. However, such discontinuities are most noticeable in the region between adjacent epidermal cells. Fibrils 20-30 nm thick show a random arrangement or an oriented pattern in cuticular discontinuities. In D. insignis vitrified plantlets, adaxial and abaxial leaf cuticle has discontinuities or gaps which may cause an increase in cuticular transpiration contributing to the low survival rate of vitrified plantlets.

Herbaspirillum seropedicae and sugarcane endophytic interaction investigated by using high pressure freezing electron microscopy

A interação entre plântulas de cana-de-açúcar e H. seropedicae foi investigada pelo uso da técnica de congelamento por alta pressão seguida de criosubstituição. Observações microscópicas evidenciaram diferenças marcantes entre esta técnica e preparações convencionais, especialmente relacionadas a ultraestrutura da bactéria e às estruturas envolvidas na adesão à superfície da parede celular da planta hospedeira.

Herbaspirillum seropedicae and sugarcane endophytic interaction investigated by using high pressure freezing electron microscopy

The interaction between sugar cane plantlets and H. seropedicae was investigated using High Pressure Freezing followed by Freeze Substitution. Microscopical observation showed consistent differences between this approaches when compared with the conventional preparation, specially related to appearance of the bacteria cell and the endophytic attachment to the host cell wall.

A interação entre plântulas de cana-de-açúcar e H. seropedicae foi investigada pelo uso da técnica de congelamento por alta pressão seguida de criosubstituição. Observações microscópicas evidenciaram diferenças marcantes entre esta técnica e preparações convencionais, especialmente relacionadas a ultraestrutura da bactéria e às estruturas envolvidas na adesão à superfície da parede celular da planta hospedeira.

Expression and immunolocalization of a Boophilus microplus cathepsin L-like enzyme.

Efforts are being undertaken to control tick infestations that cause important economic losses. A cathepsin L-like endopeptidase of Boophilus microplus was expressed in Escherichia coli; the recombinant enzyme was capable of hydrolysing gelatin, tick vitellin and bovine haemoglobin. In this paper we focus on the expression and local of synthesis of this enzyme in the tick. RT-PCR experiments showed that this endopeptidase is transcribed in the gut of partially engorged tick females. In immunoblotting, polyclonal antibodies against the recombinant enzyme reacted with proteins of larvae older than 5 days, of fully and partially engorged female gut. In immunolocalization experiments the enzyme was localized in probable secretory cells of the gut. Based on our findings we postulate that BmCL1 may be involved in haemoglobin degradation in the B. microplus gut. This enzyme may be used as target for the control of this parasite.

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)