Vacuolar Deposits Research Articles

Combined morphological and multi-omics analyses to reveal the developmental mechanism of Zanthoxylum bungeanum prickles.

Zanthoxylum bungeanum Maxim. as an important economic forest, its epidermis bears prickles which complicate the harvesting process and increase the labor costs. To explore the developmental mechanism of prickles, three varieties of Zanthoxylum bungeanum (PZB, SZB, GSZB) were selected for morphological and multi-omics analyses. The absorption spectra of prickles and stems were detected using Fourier-transform infrared spectroscopy (FTIR), and they were found different at 1617, 1110, 3319, and 1999 cm–1. The morphology of prickles and stems were observed using light microscopy and transmission electron microscopy (TEM). The growth direction of cells on the prickle side and stem side were perpendicular to each other, and there was a resembling abscission zone (RAZ) between them. The vacuolar deposits of prickle cells were much more than stem cells, indicating that the lignification degree of prickles was higher than stems. In addition, 9 candidate genes (ZbYABBY2, ZbYABBY1, ZbYABBY5, ZbWRKY, ZbLOG5, ZbAZG2, ZbGh16, ZbIAA33, and ZbGh16X1) were screened out and validated base on transcriptome and qRT-PCA. As well as, 30 key metabolites were found related to prickle development base on metabolome analysis. Among them, 4-hydroxy-2-oxopentanoate, trans-2-hydroxy-cinnamate, trans-cinnamate, polyhydroxy-fatty acid, 10,16-dihydroxypalmitate, cinnamic acid were related to the biosynthesis of cutin, suberine and wax. Indole-3-acetate, tryptamine, anthranilate, fromylanthranilate, N6-(delta2-isopentenyl)-adenine were related to plant hormone signal transduction. Generally, this is the first study to reveal the developmental mechanism of prickles. The results of this study lay the foundation for the breeding of non-prickle Zanthoxylum bungeanum.

Endocytosis is a significant contributor to uranium(VI) uptake in tobacco (Nicotiana tabacum) BY-2 cells in phosphate-deficient culture

Endocytosis of metals in plants is a growing field of study involving metal uptake from the rhizosphere. Uranium, which is naturally and artificially released into the rhizosphere, is known to be taken up by certain species of plant, such as Nicotiana tabacum, and we hypothesize that endocytosis contributes to the uptake of uranium in tobacco. The endocytic uptake of uranium was investigated in tobacco BY-2 cells using an optimized setup of culture in phosphate-deficient medium. A combination of methods in biochemistry, microscopy and spectroscopy, supplemented by proteomics, were used to study the interaction of uranium and the plant cell. We found that under environmentally relevant uranium concentrations, endocytosis remained active and contributed to 14% of the total uranium bioassociation. Proteomics analyses revealed that uranium induced a change in expression of the clathrin heavy chain variant, signifying a shift in the type of endocytosis taking place. However, the rate of endocytosis remained largely unaltered. Electron microscopy and energy-dispersive X-ray spectroscopy showed an adsorption of uranium to cell surfaces and deposition in vacuoles. Our results demonstrate that endocytosis constitutes a considerable proportion of uranium uptake in BY-2 cells, and that endocytosed uranium is likely targeted to the vacuole for sequestration, providing a physiologically safer route for the plant than uranium transported through the cytosol.

Morphology, ultrastructure and mineral uptake is affected by copper toxicity in young plants of Inga subnuda subs. luschnathiana (Benth.) T.D. Penn.

Toxic effects of copper (Cu) were analyzed in young plants of Inga subnuda subs. luschnathiana, a species that is highly tolerant to flooding and found in Brazil in wetlands contaminated with Cu. Plants were cultivated in fully nutritive solution, containing different concentrations of Cu (from 0.08 μmol to 0.47 mmol L(-1)). Symptoms of Cu toxicity were observed in both leaves and roots of plants cultivated from 0.16 mmol Cu L(-1). In the leaves, Cu clearly induced alterations in the thickness of the epidermis, mesophyll, palisade parenchyma, and intercellular space of the lacunose parenchyma. Also, this metal induced disorganization in thylakoid membranes, internal and external membrane rupture in chloroplasts, mitochondrial alterations, and electrodense material deposition in vacuoles of the parenchyma and cell walls. The starch grains disappeared; however, an increase of plastoglobule numbers was observed according to Cu toxicity. In the roots, destruction of the epidermis, reduction of the intercellular space, and modifications in the format of initial cells of the external cortex were evident. Cell walls and endoderm had been broken, invaginations of tonoplast and vacuole retractions were found, and, again, electrodense material was observed in these sites. Mineral nutrient analysis revealed higher Cu accumulation in the roots and greater macro- and micronutrients accumulation into shoots. Thus, root morphological and ultrastructural changes induced differential nutrients uptake and their translocations from root toward shoots, and this was related to membrane and endoderm ruptures caused by Cu toxicity.

Ultrastructure of Gladiolus x hybridus root endodermis

<em>Gladiolus</em>.x <em>hybridus</em> Van Houtte roots had primary, secondary and tertiary endodermis depending on the distance from the root apex. Free calcium ions were localized with the method of Slocum and Roux (1982). Electron microscopic observations revealed that electron dense calcium antimonite deposits were localized in some endodermis cells. Endodermis cells situated above xylem elements were rich in calcium antimonate deposits in contrary to those situated above the phloem elements and passage cells. The cells rich in calcium ions were present both in endodermis 11 and III stage of development. Localization of calcium deposits was on plasma membrane and tonoplast. There were also numerous calcium antimonate deposits in vacuoles. Dictiosome's cisterns and Golgi derived vesicles show presence of calcium ions. Calcium rich endodermis cells had plastids with antimonate deposits present in their tylacoids lumen. Plasmode-smata looked the same in all endodermis cells, but those from calcium rich endodermis cells had calcium antimonate deposits in the lumen of plasmodesmata.

Host/patogen interactions between Scots pine seedlings (Pinus sylvestris L.) and the p-strains of Heterobasidion annosum (Fr.) Bref. in pure culture

<em>Pinus sylvestris</em> L. seedlings representing half-sib families of several trees of known resistance in field conditions were inoculated with three strains of <em>Heterobasidion annosum</em> (Fr.) Bref. representing the P intersterility group. Standardized inoculation procedure and uniform in vitro conditions allowed for evaluation of the genetically controlled host/pathogen interactions. Early and advanced stages of infection after prolonged exposure to the fungus were studied with the help of light and transmission electron microscopy. Abundance of osmiophilic material in vacuoles, necrotic reaction of cortical cells and secondary thickenings of cell walls in root regions in proximity to external mycelium were responsible for transient stagnation of pathogen spread in the cortex of resistant seedlings. Immobilized and degenerating hyphae inside vacuolar deposits in cortical cells of short roots of resistant plants are indicative of elevated toxicity of the protective materials at the initial stage of infection. Structural changes of vacuolar deposits and their subsequent disappearance, which coincided with heavy colonization of cortical cells of moderately susceptible seedlings, preceded hyphal penetration into the stele within 2-6-months after inoculation. Despite severe damage to root caps, there was no further spread of the fungus into meristems of growing roots and short dormant roots. , In susceptible seedlings, lack of a hypersensitive reaction and apparently low levels of osmiophilic material resulted in quick colonization of cortical cells, breakdown of endodermis and root abortion after entering the stele.

Cadmium tolerance and removal from Cunninghamella elegans related to the polyphosphate metabolism.

The aim of the present work was to study the cadmium effects on growth, ultrastructure and polyphosphate metabolism, as well as to evaluate the metal removal and accumulation by Cunninghamella elegans (IFM 46109) growing in culture medium. The presence of cadmium reduced growth, and a longer lag phase was observed. However, the phosphate uptake from the culture medium increased 15% when compared to the control. Moreover, C. elegans removed 70%–81% of the cadmium added to the culture medium during its growth. The C. elegans mycelia showed a removal efficiency of 280 mg/g at a cadmium concentration of 22.10 mg/L, and the removal velocity of cadmium was 0.107 mg/h. Additionally, it was observed that cadmium induced vacuolization, the presence of electron dense deposits in vacuoles, cytoplasm and cell membranes, as well as the distinct behavior of polyphosphate fractions. The results obtained with C. elegans suggest that precipitation, vacuolization and polyphosphate fractions were associated to cadmium tolerance, and this species demonstrated a higher potential for bioremediation of heavy metals.

Glomerular Lipidosis Accompanied by Renal Tubular Oxalosis in Wild and Laboratory-Reared Japanese Rock Ptarmigans (Lagopus mutus japonicus)

Glomerular lipidosis is a disease characterized by lipid accumulation in mesangial cells but that has not been fully investigated in avian species. We examined four wild and two laboratory-reared Japanese rock ptarmigans (Lagopus mutus japonicus)--an endangered avian species--presenting vacuolar deposits in the glomeruli. All cases had vacuolar deposits in the glomeruli. In the wild cases, fewer than 30% of all glomeruli were affected, compared with more than 90% in the laboratory-reared cases. In the wild cases, most deposits were mild and restricted to the mesangial areas of glomeruli. In the laboratory-reared cases, nearly all of the deposits covered entire glomeruli. Electron microscopy of mild deposits revealed vacuoles in the cytoplasm of mesangial cells. These vacuoles were positive for Sudan III, Sudan black B, oil red O, Nile blue, periodic acid-Schiff, Schultz test, and digitonin stain and were negative for performaric acid-Schiff stains. Based on these results, we diagnosed the glomerular lesion as glomerular lipidosis caused by uptake of low-density lipoprotein in mesangial cells. Except for one wild case, all cases exhibited renal tubular oxalosis. The severity of tubular oxalosis tended to be related to the severity of glomerular lipidosis: In cases of mild glomerular lipidosis, tubular oxalosis was also mild or absent. We therefore diagnosed the primary lesion as glomerular lipidosis accompanied by tubular oxalosis. The four wild cases came from different zones and therefore had no opportunities to interbreed and no common relatives. We believe these data support the hypothesis that glomerular lipidosis is a disease of the general population ofJapanese rock ptarmigans. This is the first report of glomerular lipidosis accompanied by renal tubular oxalosis in an avian species.

Structural and ultrastructural study of Capsicum annuum leaves after treatment with Uncaria tomentosa bark extracts

<p>The influence of an <em>Uncaria tomentosa </em>extract on the development of <em>Capsicum </em>plants grown in green-house conditions was examined. The effect of the treatment was investigated with microscopic techniques (light and electron microscope) in leaves from three levels of control plants and plants after treatment with the extract added to the soil in doses of 0.4 and 16 mg/ml (200 ml per pot/plant). In control leaves, changes typical of the subsequent phases of normal development were observed: nuclear chromatin became slightly condensed, plastoglobuli of chloroplasts increased in number and size, intragranal thylakoids were somewhat dilatated. In addition to such commonly occurring changes, some symptoms typical of pepper were observed in the ontogenesis of the examined plant: an increased number of spherical electron-dense deposits in vacuoles, an increased number of peroxisomes, the occurrence of numerous paracrystalline structures in chloroplasts of mature leaves, and, starting in mature leaves, expulsion of plastoglobuli from chloroplasts.</p><p>After the treatment, most of those changes, leading to ageing, occurred much earlier and were more distinct. Chloroplasts, already in the youngest examined leaves, showed dilatation of intergranal thylakoids, which intensified with aging of the leaves and degradation of grana in the oldest leaves. Starch grains decreased in size and number and plastoglobuli became large. Vesiculation of ground cytoplasm in all leaves was stronger than in the control. No paracrystalline structures in chloroplasts or expulsion of plastoglobuli were observed. Another unusual phenomenon was the disappearance of spherical electron-dense deposits in the central vacuoles of cells.</p><p>Those observations suggested that <em>U. tomentosa </em>extract enhanced the natural ontogenesis of <em>Capsicum annuum </em>leaves, by accelerating and enhancing the typical characteristics of ageing, and, additionally, it changed the structure and physiology of cells.</p>

The effects of sodium chloride-salinity upon growth, nodulation, and root nodule structure of pea (Pisum sativum L.) plants

Pea (Pisum sativum L.) plants inoculated with Rhizobium leguminosarum bv. viciae effective strain 248 were irrigated with nitrogen-free medium supplemented with 0, 25, 50 or 75 mM NaCl. The inhibitory effect of salinity on the growth of pea plants treated with 25 mM NaCl occurred 6 weeks post inoculation. In case of 75 mM NaCl treatment, the same effect was observed 2 weeks post inoculation. In contrast to investigations described in the literature our results clearly indicated that 25 mM NaCl stimulated nodule formation, however, in contrast to control nodules (the medium without NaCl), the nodules were considerably smaller. Remaining variants of salt treatment reduced plant growth, nodulation, and total nodule volume calculated per plant. Microscopic observations showed that salinity: (1) caused the loss of turgor of the nodule peripheral cells, (2) changed nodule zonation, (3) stimulated infection thread enlargement and expansion, (4) caused disturbances in bacterial release from the infection threads, and (5) induced synthesis of electron dense material (EDM) and its deposition in vacuoles. It was also found that cisternae of RER were involved in the formation of special cytoplasmic compartments responsible for synthesis of EDM. Autofluorescence study revealed that salinity increased accumulation of phenolics in pea nodules, as well.

Specific intra-tissue responses to manganese in the floating lamina of Trapa natans L.

Plant tolerance to heavy metals requires morpho-physiological mechanisms that are still poorly understood, especially in hydrophytes. This study focuses on the young floating lamina of the rhyzophyte Trapa natans exposed for 10 d to 130 microM Mn. The lamina has the ability to bioaccumulate Mn (> 3000 microg g(-1)). X-ray microanalysis of Mn cellular distribution revealed accumulation in the upper epidermis, in the first palisade layer, and in the idioblasts of the spongy tissue, which were shown with electron microscopy to contain osmiophilic vacuolar deposits, also observed to a minor extent in the control leaves. On the basis of biochemical and histochemical tests, these deposits were attributed to phenolic compounds that were probably able to chelate Mn. Net photosynthesis, photosynthetic pigments, room temperature microspectrofluorimetric analyses, and ultrastructural studies of plastids were performed to evaluate the status of the photosynthetic apparatus. A greater development of thylakoid membranes was observed in plastids of the second palisade and spongy tissue, which, however, did not accumulate Mn. Only the spongy tissue experienced inadequate assembly of PS II, but this did not significantly influence the photosynthetic yield of the whole lamina. It was concluded that T. natans can optimise productivity in the presence of Mn by means of specific intra-tissue responses within the framework of the floating lamina.

Ultrastructural changes in Chlamydomonas acidophila (Chlorophyta) induced by heavy metals and polyphosphate metabolism

Ultrastructural changes induced by heavy metals (cadmium, zinc, and copper) and polyphosphate metabolism were studied in Chlamydomonas acidophila. Transmission electron microscopy indicated that cadmium led to the most drastic morphometric changes. An increase in number and volume of starch grains and vacuoles as well as the presence of electron dense deposits in vacuole and membrane whorls were observed. Energy-dispersive X-ray analysis revealed that vacuolar deposits inside cells treated with cadmium contained phosphate and cadmium. These ultrastructural changes were accompanied by a change in the intracellular polyphosphate level, as shown by in vivo 31P-nuclear magnetic resonance. It was also observed that cadmium treatment caused polyphosphate degradation and increased vacuolar short-chains and orthophosphates.

Developing seeds of Arabidopsis store different minerals in two types of vacuoles and in the endoplasmic reticulum.

Mineral-accumulating compartments in developing seeds of Arabidopsis were studied using high-pressure-frozen/freeze-substituted samples. Developing seeds store minerals in three locations: in the protein storage vacuoles of the embryo, and transiently in the endoplasmic reticulum (ER) and vacuolar compartments of the chalazal endosperm. Energy dispersive x-ray spectroscopy and enzyme treatments suggest that the minerals are stored as phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate) salts in all three compartments, although they differ in cation composition. Whereas embryo globoids contain Mg, K, and Ca as cations, the chalazal ER deposits show high levels of Mn, and the chalazal vacuolar deposits show high levels of Zn. The appearance of the first Zn-phytate crystals coincides with the formation of network-like extensions of the chalazal vacuoles. The core of these networks consists of a branched network of tubular ER membranes, which are separated from the delineating tonoplast membranes by a layer of cytosolic material. Degradation of the networks starts with the loss of the cytosol and is followed by the retraction of the ER, generating a network of collapsed tonoplast membranes that are resorbed. Studies of fertilized fis2 seeds, which hyperaccumulate Zn-phytate crystals in the chalazal vacuolar compartments, suggest that only the intact network is active in mineral sequestration. Mineral determination analysis and structural observations showed that Zn and Mn are mobilized from the endosperm to the embryo at different developmental stages. Thus, Zn appears to be removed from the endosperm at the late globular stage, and Mn stores appear to be removed at the late bent-cotyledon stage of embryo development. The disappearance of the Mn-phytate from the endosperm coincides with the accumulation of two major Mn binding proteins in the embryo, the 33-kD protein from the oxygen-evolving complex of photosystem II and the Mn superoxide dismutase. The possible functions of transient heavy metal storage in the chalazal endosperm are discussed. A model showing how phytic acid, a potentially cytotoxic molecule, is transported from its site of synthesis, the ER, to the different mineral storage sites is presented.

Detoxification of cadmium in tobacco plants: formation and active excretion of crystals containing cadmium and calcium through trichomes.

In tobacco (Nicotiana tabacum L.), long and short trichomes can be distinguished morphologically. The established function of long trichomes is to exude a sticky gum containing diterpenes, whereas that of short trichomes is not known. When tobacco seedlings were exposed to toxic levels of cadmium (Cd), growth was retarded, but trichome number was increased up to 2-fold in comparison with untreated samples. Observation by variable-pressure scanning electron microscopy (VP-SEM) indicated that large crystals of 150 microm in size were formed on head cells of both short and long trichomes. An energy-dispersive X-ray analysis system fitted with VP-SEM revealed the crystals to contain amounts of Cd and calcium (Ca) at much higher concentrations than in the head cells themselves. Transmission electron microscopy demonstrated crystal formation in amorphous osmiophilic deposits in vacuoles. When seedlings were treated with Cd in the presence of Ca, tolerance was increased in proportion to the increase in Ca concentration. These results indicate that tobacco plants actively exclude toxic Cd by forming and excreting Cd/Ca-containing crystals through the head cells of trichomes.

The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat endothelium.

Phenolic compounds that are present in the testa interfere with the physiology of seed dormancy and germination. We isolated a recessive Arabidopsis mutant with pale brown seeds, transparent testa12 (tt12), from a reduced seed dormancy screen. Microscopic analysis of tt12 developing and mature testas revealed a strong reduction of proanthocyanidin deposition in vacuoles of endothelial cells. Double mutants with tt12 and other testa pigmentation mutants were constructed, and their phenotypes confirmed that tt12 was affected at the level of the flavonoid biosynthetic pathway. The TT12 gene was cloned and found to encode a protein with similarity to prokaryotic and eukaryotic secondary transporters with 12 transmembrane segments, belonging to the MATE (multidrug and toxic compound extrusion) family. TT12 is expressed specifically in ovules and developing seeds. In situ hybridization localized its transcript in the endothelium layer, as expected from the effect of the tt12 mutation on testa flavonoid pigmentation. The phenotype of the mutant and the nature of the gene suggest that TT12 may control the vacuolar sequestration of flavonoids in the seed coat endothelium.

Severe photobiont injuries of lichens are strongly associated with air pollution.

The photobiont ultrastructure of the epiphytic lichens Bryoria fuscescens and Bryoria fremontii was studied along the pollution gradient from two Cu-Ni smelters in Nikel and Monchegorsk in northern Finland and north-western Russia. The relationship between ultrastructural characteristics of B. fuscescens and environmental factors (i.e. climate, atmospheric SO2 and bark element concentrations) was studied by using a principal component analysis (PCA) aiming to assess the air quality in a northern environment. Based on PCA, increased plasmolysis and mitochondrial changes in the Trebouxia photobiont were significantly correlated with elevated pollutant concentrations. Degenerated cells, showing altered chloroplasts and electron-translucent pyrenoglobuli, occurred in lichens growing 35-50 km from the Monchegorsk smelter. Cell wall and cytoplasmic lipid volumes, and size of pyrenoglobuli, positively correlated with the distance from the Monchegorsk smelter. Vacuoles and electron-opaque vacuolar deposits were significantly increased at the Finnish site in the vicinity of a pulp mill. Swelling of mitochondrial cristae and thylakoids showed little correlation with environmental factors, but indicated of initial stage of injuries and were observed at several slightly polluted sites in northern Finland and north-western Russia. The results suggest that the severe photobiont injuries of lichens are strongly associated with poor air quality.

Anatomical study of zygotic and somatic embryos of Tilia cordata

A comparative anatomical study was carried out on zygotic and somatic embryos of Tilia cordata Mill. to evaluate the effect of growth conditions on their development. Zygotic embryos (heart-shaped, torpedo, cotyledonary), collected during two autumn periods, were examined to investigate the effect of growing season on embryo development. In comparison, the influence of growth conditions on the development of somatic embryos in vitro was also studied. Treatment with abscisic acid (ABA) and polyethylene glycol-4000 induced the development of somatic cotyledonary embryos similar to zygotic embryos with respect to morphology and anatomy, as illustrated by the differentiation of the apical meristems and of the procambium. The pattern of accumulation of starch and protein was also similar in these embryos. Somatic cotyledonary embryos that developed spontaneously without ABA showed defective accumulation of storage material and a general failure to form the shoot apical meristem, leading to very low germination rates. Vacuolar phenolic deposits were observed along the procambium of both zygotic and somatic embryos regardless of the maturation stage. Tracheid formation was observed only in somatic embryos formed without ABA in the medium and in precociously germinated somatic embryos. Phenolic vacuolar inclusions were frequently observed in epidermal cells of these embryos.

Cyclopiazonic acid, an inhibitor of calcium-dependent ATPases, induces exit from metaphase I arrest in growing pig oocytes.

Calcium plays an important role in the regulation of meiotic maturation in mammalian oocytes. In the present study, mycotoxin cyclopiazonic acid (CPA), an inhibitor of calcium-dependent ATPases, was used to mobilize intracellular calcium deposits in growing pig oocytes, which had not attained full meiotic competence and in which maturation is thus spontaneously blocked at the metaphase I stage. CPA treatment significantly increased the ratio of growing oocytes that are able to overcome the spontaneously occurring metaphase I block to complete their maturation at the metaphase II stage. CPA treatment of a least 2 hours' duration is necessary to overcome the metaphase I block in growing oocytes. A similar effect upon release from the spontaneous meiotic block at the metaphase I stage was observed after treatment of growing pig oocytes with thapsigargin, another inhibitor of endogenous calcium-dependent ATPases. Numerous calcium deposits in vacuoles, the mitochondria and on the surface of yolk granules in growing pig oocytes were observed. CPA treatment is able to mobilize calcium from the mitochondria, but deposits in vacuoles and deposits on the surface of yolk granules seem to remain intact after CPA treatment. A microinjection of heparin, which is known to bind with the inositol trisphosphate receptors, significantly decreased the ratio of CPA-treated growing oocytes overcoming the block at the metaphase I stage. This indicates that CPA might mobilize calcium in growing pig oocytes through inositol trisphosphate receptors. On the other hand, a microinjection of procaine or a microinjection of ruthenium red, both inhibitors of ryanodine receptors, did not prevent the overcoming of the metaphase I block, induced by CPA treatment. The calcium channel blocker, verapamil, significantly reduces the proportion of CPA-treated growing oocytes that overcome the metaphase I block. This indicates that the influx of calcium from extracellular sources is necessary to overcome the metaphase I block. The calmodulin inhibitors ophiobolin A and W7 also reduce the proportion of CPA-treated growing oocytes overcoming the metaphase I block.

Definitive enzymatic assays in polyphosphate analysis.

Inorganic polyphosphate (polyP), an anhydride-linked linear polymer of orthophosphate, is present in a wide variety of organisms from bacteria to mammals (Kulaev 1979; Kulaev and Vegabov 1983; Wood and Clark 1988). Despite the prominence of polyP (as in the vacuolar deposits in yeast that may represent 10 to 20% of cellular dry weight), this molecule has received scant attention. Studies by Kulaev, Wood and a few others (Kulaev et al. 1987; Wood and Clark 1988; Kornberg 1995) disclosed the ubiquity of polyP and identified a few related enzyme activities. Yet, polyP has remained a largely forgotten polymer. Among the reasons is the lack of evidence describing its metabolic role, due in measure to the inadequacy of specific quantitative methods for polyP analysis.

Specialized phloem parenchyma cells in Norway spruce (Pinaceae) bark are an important site of defense reactions

The bark anatomy of Norway spruce clones that were resistant or susceptible to Ceratocystis polonica, a bark-beetle-vectored fungal pathogen, was compared. The major difference concerned the axial parenchyma cells, called polyphenolic parenchyma (PP cells) because of their vacuolar deposits. The phenolic nature of the deposits was indicated by autofluorescence under blue light, and immunocytochemical studies demonstrating PP cells are enriched in phenylalanine ammonia lyase (EC 4.3.1.5), a key enzyme in phenolic synthesis. Susceptible-clone PP cells occurred as single rows filled with dense deposits. The resistant clone had 40% more PP cells, which occurred in rows two cells thick plus as individual cells scattered among the sieve cells and had lighter deposits. Trees inoculated with fungus were analyzed but a distinct fungal response could not be separated from the general wound response. In the resistant clone, phenolic bodies were reduced in size and density or disappeared completely 12 d after wounding, and PP cell size increased. The susceptible-clone phenolics and cell size changed only slightly. These data show that PP cells are active in synthesis, storage, and modification of phenolics in response to wounding, providing an important site of constitutive and inducible defenses.

Prion protein immunocytochemistry – UK five centre consensus report

Creutzfeldt-Jakob disease (CJD) and other prion diseases are associated with the deposition of insoluble prion protein (PrPCJD) in the central nervous system (CNS). Antibodies raised against PrPCJD also react with its precursor protein, a soluble form of PrP (PrPC), which is widely distributed in the normal CNS. This cross-reactivity has in the past raised doubts as to the specificity and diagnostic reliability of PrP immunolocalization, especially in familial cases which are atypical clinically and which lack characteristic pathology findings. Following an MRC-funded workshop which focused on this problem, a multicentre prospective study was set up to identify a reliable protocol for PrPCJD immunocytochemistry. Five UK centres took part in this study and demonstrated consistent staining of plaques, vacuolar deposits in severe spongiform change, and perineuronal deposits using a variety of antibodies and enhancement procedures. A protocol using formic acid, guanidine thiocyanate, and hydrated autoclaving pre-treatment in conjunction with a monoclonal PrPCJD antibody produced the clearest immunochemical results and is presented as the consensus UK recommendation for PrPCJD immunocytochemical procedures.