Barry Martin

High Resolution Secondary Ion Mass Spectrometry Reveals the Contrasting Subcellular Distribution of Arsenic and Silicon in Rice Roots

Rice (Oryza sativa) takes up arsenite mainly through the silicic acid transport pathway. Understanding the uptake and sequestration of arsenic (As) into the rice plant is important for developing strategies to reduce As concentration in rice grain. In this study, the cellular and subcellular distributions of As and silicon (Si) in rice roots were investigated using high-pressure freezing, high-resolution secondary ion mass spectrometry, and transmission electron microscopy. Rice plants, both the lsi2 mutant lacking the Si/arsenite efflux transporter Lsi2 and its wild-type cultivar, with or without an iron plaque, were treated with arsenate or arsenite. The formation of iron plaque on the root surface resulted in strong accumulation of As and phosphorous on the epidermis. The lsi2 mutant showed stronger As accumulation in the endodermal vacuoles, where the Lsi2 transporter is located in the plasma membranes, than the wild-type line. As also accumulated in the vacuoles of some xylem parenchyma cells and in some pericycle cells, particularly in the wild-type mature root zone. Vacuolar accumulation of As is associated with sulfur, suggesting that As may be stored as arsenite-phytochelatin complexes. Si was localized in the cell walls of the endodermal cells with little apparent effect of the Lsi2 mutation on its distribution. This study reveals the vacuolar sequestration of As in rice roots and contrasting patterns of As and Si subcellular localization, despite both being transported across the plasma membranes by the same transporters.

Transport of virally expressed green fluorescent protein through the secretory pathway in tobacco leaves is inhibited by cold shock and brefeldin A

Abstract. Potato virus X (PVX) has been used as an expression vector to target the green fluorescent protein (GFP) from the jellyfish Aequorea victoria to the endoplasmic reticulum (ER) of tobacco (Nicotiana clevelandii L.) leaves. Expression of free GFP resulted in strong cytoplasmic fluorescence with organelles being imaged in negative contrast. Translocation of GFP into the lumen of the ER was mediated by the use of the sporamin signal peptide. Retention of GFP in the ER was facilitated by the splicing of the ER retrieval/retention tetrapeptide, KDEL to the carboxy terminus of GFP. Fluorescence of GFP was restricted to a labile cortical network of ER tubules with occasional small lamellae and to streaming trans-vacuolar strands. Secretion of ER-targeted GFP was inhibited both by cold shock and low concentrations of the secretory inhibitor brefeldin A. However, both prolonged cold and prolonged incubation in brefeldin A resulted in the recovery of secretory capability. In leaves infected with the GFP-KDEL construct, high concentrations of brefeldin A induced the tubular network of cortical ER to transform into large lamellae or sheets which reverted to the tubular network on removal of the drug.

Trafficking of storage proteins in developing grain of wheat

The processing properties of the wheat flour are largely determined by the structures and interactions of the grain storage proteins (also called gluten proteins) which form a continuous visco-elastic network in dough. Wheat gluten proteins are classically divided into two groups, the monomeric gliadins and the polymeric glutenins, with the latter being further classified into low molecular weight (LMW) and high molecular weight (HMW) subunits. The synthesis, folding and deposition of the gluten proteins take place within the endomembrane system of the plant cell. However, determination of the precise routes of trafficking and deposition of individual gluten proteins in developing wheat grain has been limited in the past by the difficulty of developing monospecific antibodies. To overcome this limitation, a single gluten protein (a LMW subunit) was expressed in transgenic wheat with a C-terminal epitope tag, allowing the protein to be located in the cells of the developing grain using highly specific antibodies. This approach was also combined with the use of wider specificity antibodies to compare the trafficking and deposition of different gluten protein groups within the same endosperm cells. These studies are in agreement with previous suggestions that two trafficking pathways occur in wheat, with the proteins either being transported via the Golgi apparatus into the vacuole or accumulating directly within the lumen of the ER. They also suggest that the same individual protein could be trafficked by either pathway, possibly depending on the stage of development, and that segregation of gluten proteins both between and within protein bodies may occur.

Plant cell wall architecture is revealed by rapid-freezing and deep-etching

SummaryThis paper reports on preliminary investigations into the structure of cell walls of varying complexity as revealed by the rapidfreeze deep-etch technique. Three cell types from different species were examined in order to compare the three-dimensional arrangement of random, polylamellate and helicoidal walls. Each cell type displayed a distinctive level of organisation with respect to the cellulose microfibrils and the matrix material. In polylamellated walls, the microfibrils within each layer were linked to each other by 16–20 nm long side chains regularly spaced along the length of the microfibril. In helicoidal walls, the shifting of the microfibrils could cleary be seen, yet no recognisable structures were observed which could mediate this movement.

Monoclonal antibodies for the detection of spoilage fungi

Monoclonal antibodies (M Abs) have proved useful in the development of immunoassays to detect the mycelium of specific fungi in foods and feeds. Development of M Ab-ELISA (enzyme-linked immunosorbent assay) and ‘user-friendly’ dip-stick assays for the detection of Humicola lanuginosa and Penicillium islandicum, involved in undesirable post-harvest discoloration of rice grains, is described briefly, as is a M Ab-ELISA for the detection of Botrytis cinerea in extracts from infected strawberries and grapes. These assays are sensitive, rapid, easily replicated and do not require surface sterilisation. The antigens, which are located on the surfaces and within the walls of the hyphae, are not present in the ungerminated spores of P. islandicum and are masked in aleurospores of H. lanuginosa, thus immunoassays for these fungi have the added advantage of detecting only mycelial growth. The most sensitive fungal M Abs are those that recognise heat-stable carbohydrate epitopes on carbohydrate or glycoprotein molecules. Difficulties in raising species-specific M Abs to fungi and particularly those related to choice of immunogen, are discussed.

Freeze-fracture deep-etch methods.

Publisher Summary Freeze-fracture deep-etch methods avoids the use of both chemical fixatives and cryoprotectants and results in the production of a three-dimensional replica of the etched surface of the specimen. Samples are ultra-rapidly frozen and fractured, the ice is sublimed under vacuum from the fracture surface, and the specimen is replicated. Using this technique, great advances have been made in the understanding of the structure and molecular make up of animal cell cytoplasm. The one main advantage of the rapid-freeze deep-etch technique is that little prior preparation of material is needed other than that required by the experiments that are being carried out. Therefore, most of the material requirements are in the instrumentation, and accessability to the necessary freezing and fracturing equipment is often the major limitation in the application of this technique. The procedure can be split into two separate events: freezing, followed by the fracturing, etching, and replication of the frozen material. Specimens can be frozen and stored under liquid nitrogen for any length of time prior to the fracturing procedure.