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Many plants contain ribosome-inactivating proteins (RIPs) which are either single enzymatically active polypeptides (type-1 RIPs) or heterodimers (type-2 RIPs) composed of an A-chain, functionally equivalent to a type-1 RIP, which is disulphide bonded to a sugar-binding B-chain. Much attention has focused on the use of RIPs as components of immunotoxins or, more recently, as antiviral agents. In contrast, relatively little is known about either the synthesis and targeting of RIPs or their role within plants. In this study the cellular and subcellular distributions of saporins, the type-1 RIPs from soapwort, have been determined in seeds using immunogold labelling. Saporins were present in the seed storage tissue (perisperm), but are not synthesised in the developing embryo, demonstrating that the expression of saporin genes is subject to tissue-specific control. Within the perisperm, saporin was found in extracellular spaces, in the paramural region between the primary wall and plasmalemma and within the vacuole. In addition, saporin was localised in leaf intercellular spaces. This dual localisation, both vacuolar and extracellular, is significantly different from the localisation of ricin, a type-2 RIP found in castor beans, which is targeted to endosperm protein bodies, and to pokeweed antiviral protein which accumulates in the cell wall matrix of leaf mesophyll cells.

The expression of extA, an extensin gene from Brassica napus L. (oilseed rape) was examined in transgenic Nicotiana tabacum L. (tobacco) and untransformed Brassica juncea L. and B. napus tissues. Northern analysis showed that this gene maintained its normal pattern of expression when transferred to tobacco. In transgenic tobacco plants containing an extA promoter/β-glucuronidase coding sequence fusion, expression of extA was detected in the external and internal phloem of the main stem. High expression levels were seen in cortical parenchyma cells at the point where the axillary flowering branch joined the main stem. Expression was greatest in regions where the maximum tensile stress would seem to be exerted on the main stem by the weight of the axillary branch. It was confirmed that this expression pattern was due to tensile stress by using weights to induce expression of the fusion gene in axillary flowering stalks. In B. juncea pods, in-situ hybridisation studies showed that the extensin gene was strongly expressed in cells of the carpel walls within which considerable tensile stresses develop.

Following a zinc iodine-osmium tetroxide fixation, nuclear pore distribution was studied in 0.3-μm sections from cotyledons of developing Vicia faba L. Localised absence of nuclear pores was found to be associated with proximity of organelles to the nucleus. Golgi cisternae and mitochondria are associated with areas of pore absence while cisternal endoplasmic reticulum and tubular endoplasmic reticulum are linked with areas showing reduction in pore density. Pores were seen in the nuclear membrane adjacent to vacuoles. Pattern analysis of pore distribution indicated possible clustering within an overall regularity.

The ultrastructure of the endoplasmic reticulum (ER) in storage parenchyma cells in the cotyledons of mung beans (Vigna radiata L.) was examined during germination and seedling growth. Two different methods were used to visualize the ER: thin (0.08 μm) sections of tissue fixed in formaldehyde and glutaraldehyde and post-fixed with osmium tetroxide, and thick (1 μm) sections of tissue fixed in buffered aldehyde and post-fixed with zinc iodide-osmium tetroxide (ZIO). Changes in relative amounts of ER were quantified by morphometry (stereology).The ER occurs in two forms: a cisternal form with associated ribosomes which can be seen at all stages from imbibition to cotyledon senescence, and a tubular form which initially has associated ribosomes. Stereoscopic images of thick sections of cotyledons of 2-day-old seedlings show that the tubular ER consists of a three-dimensional array of interconnecting tubules which have numerous connections with the cisternal ER. The network of tubules and cisternae extends throughout the cytoplasm enveloping the protein bodies. Germination and seedling growth are accompanied by a reduction in the total volume occupied by the ER. This reduction is the result of a preferential loss of tubular ER and occurs largely before protein mobilization. Cisternal ER decreases during the first 48 h of imbibition and seedling growth, but storage cells subsequently show an increase in cisternal ER just prior to and during the period of protein mobilization. Cisternal ER remains conspicuous during the last phase of reserve mobilization when starch is broken down and the cells are starting autophagy.

The changes in endoplasmic reticulum (ER) morphology during seed development have been followed using a thick section electron microscope technique. The tissues were stained with a zinc iodineosmium tetroxide complex which preferentially accumulated in the lumen between double membranes. Sections up to 2 μm in thickness were examined in a high voltage electron microscope (HVEM) with tilt facility to produce stereo pairs. The micrographs from HVEM showed an increase in the extent of interconnecting tubular and cisternal ER during the protein deposition phase of seed maturation with subsequent degeneration of the cisternae to a reticular form during the final seed maturation phase. No evidence of cisternal ER vesicles was found, instead our work suggests that such structures are artefacts of thin sectioning with the so-called vesicles representing the interconnection of cisternal and tubular ER. The results are discussed with reference to the transport of storage protein from its site of synthesis, the rough cisternal ER, to that of accumulation, the vacuolar protein bodies.

Tubular evaginations of the plasmalemma of the scutellar epithelial cells of barley are described. The evaginations are similar to those present at other sites where solute flux occurs for a limited period only and wall development of the transfer-cell form has not occured. Differential uptake of the fluorescent dyes fluorescein, which moves into the symplast, and 8-anilino-1-naphthalene sulphonic acid, which remains in the apoplast only, indicates that the scutellar epithelial cells contain the boundary between the apoplast and symplast. We suggest that i) the plasmalemma evaginations, which have a specific form and localisation, may be referred to as plasmatubules rather than by the general term plasmalemmasome, and that ii) the plasmatubules may act in membrane amplification in a short-term structural modification which is an alternative to transfer cells.

Plasmatubules are tubular evaginations of the plasmalemma. They have previously been found at sites where high solute flux between apoplast and symplast occurs for a short period and where wall proliferations of the transfer cell type have not been developed (Harris et al. 1982, Planta 156, 461-465). In this paper we describe the distribution of plasmatubules in transfer cells of the leaf minor veins of Pisum sativum L. Transfer cells are found in these veins associated both with phloem sieve elements and with xylem vessels. Plasmatubules were found, in both types of transfer cell and it is suggested that the specific distribution of the plasmatubules may reflect further membrane amplification within the transfer cell for uptake of solute from apoplast into symplast.

Some of the physiological and biochemical changes which were found to occur during paraquat (1,1'-dimethyl-4,4'-bipyridylium ion) treatment of cotyledon leaves of Linium usitatissimum, are reported. Results showed an inhibition of photosynthetic CO2 uptake and electron flow of isolated chloroplasts. An increase in membrane permeability, changes in the level of the major lipid components and of malondialdehyde. These are correlated with ultrastructural changes and the discussion includes a proposed mode of action for the herbicides.

Plasmatubules are tubular evaginations of the plasmalemma associated with sites where high solute flux occurs between apoplast and symplast. Plasmatubules of the scutellar epithelial cells of germinating barley (Hordeum vulgare L.) have been examined following a variety of fixation methods. Of the aqueous fixations, primary aldehyde fixation with osmium post-fixation and osmium as the primary fixative gave comparable images, whilst potassium permanganate resulted in some distortion of the tissue in general including dilation of the tubular evaginations of the plasmalemma. Freeze-fixation and substitution with acetone and acetone-osmium gave images of the plasmalemma comparable to those obtained by the aqueous aldehyde and osmium methods. The similarity of structure with aldehyde or osmium and freezing as the primary fixation is taken to indicate that plasmatubules are real and not artefacts resulting from the fixation procedure.

Ultrastructural aspects of the breakdown of starch grains during the mobilisation of reserves in Phaseolus aureus Roxb. seed germination are described. The starch grains show erosion from within leading to the formation of a hollow shell. The erosion is accompanied by intrusion of cytoplasm into the shell. No evidence of a vesicular transport system to or from the eroding face was found.

A transient plasmolysis/deplasmolysis (plasmolytic cycle) of onion epidermal cells has been shown to induce the formation of fluid-phase endocytic vesicles. Plasmolysis in the presence of the membrane-impermeant fluorescent probes Lucifer Yellow CH (LYCH) and Cascade Blue hydrazide resulted in the uptake of these probes by fluid-phase endocytosis. Following deplasmolysis, many of the dye-containing vesicles left their parietal positions within the cell and underwent vigorous streaming in the cytoplasm. Vesicles were observed to move within transvacuolar strands and their movements were recorded over several hours by video-microscopy. Within 2 h of deplasmolysis several of the larger endocytic vesicles had clustered around the nuclear membrane, apparently lodged in the narrow zone of cytoplams surrounding the nucleus. In further experiments LYCH was endocytically loaded into the cells during the first plasmolytic cycle and Cascade Blue subsequently loaded during a second plasmolytic cycle. This resulted in the introduction of two populations of endocytic vesicles into the cells, each containing a different probe. Both sets of vesicles underwent cytoplasmic streaming. The data are discussed in the light of previous observations of fluid-phase endocytosis in plant cells.

The major albumin protein in storage parenchyma tissue of developing peas has been localised at an ultrastructural level by immunocytochemistry. Tissue was fixed in buffered aldehyde and embedded in LR White resin which was polymerised by addition of catalyst. Sections were labelled by the indirect method of absorption of Protein A-gold to specifically bound antibodies. This method gives high levels of specific labelling on sections which retain good ultrastructural preservation and have high contrast after conventional staining. The albumin is located throughout the cytoplasm although no labelling was found associated with the endoplasmic reticulum, Golgi apparatus, vacuoles-protein bodies or other organelles.

Changes in the fine structure of flax (Linum usitatissimum) cotyledon leaf cells treated with the herbicide paraquat (1,1′-dimethyl-4,4′-bipyridylium ion.) were investigated. After 6 h treatment under constant illumination, tonoplast breakdown was evident. This was followed by a rapid and progressive deterioration of cell contents including the disruption of mitochondria, and the breakdown of chloroplast thylakoid membrane structure with the accumulation of osmiophilic plastoglobuli. There was no apparent deterioration after 18 h paraquat treatment in darkness but by 30 h there was a limited breakdown of chloroplast membranes.