Salmonella enterica strains belonging to O serogroup 1,3,19 induce chlorosis and wilting of Arabidopsis thaliana leaves

The number of outbreaks and illness linked to the consumption of contaminated salad leaves have increased dramatically in the last decade. Escherichia coli and Salmonella enterica are the most common food-borne pathogens linked to consumption of fresh produce. Different serovars of S. enterica subspecies enterica have been shown to bind the surface of salad leaves, to exhibit tropism towards the stomata and to invade leaves and reach the underlying mesophyll. However the consequences of leaf invasion are not known. Here we show that following infiltration, serovars Typhimurium, Enteritidis, Heidelberg and Agona, as well as strains of S. enterica subspecies arizonae and diarizonae, survive in the mesophyll of Arabidopsis thaliana leaves but induce neither leaf chlorosis nor wilting. In contrast, S. Senftenberg induced strong leaf wilting 4 days post infiltration in A. thaliana accession Col-0 but not in accession Ws-0. Dead S. Senftenberg and bacterial lysates also induced leaf wilting. We found that mutations in the Arabidopsis pathogen associated molecular pattern (PAMP) recognition receptors (PRRs) FLS2, which recognizes flagellin, and EFR, which recognizes the bacterial elongation factor EF-Tu, had no effect on the wilting response of A. thaliana to S. Senftenberg. Infiltration of A. thaliana leaves with serovars Cannstatt, Krefeld and Liverpool, which like Senftenberg belong to Salmonella serogroup E(4) (O:1,3,19), also resulted in rapid leaf wilting, while all tested rough S. Senftenberg strains (lacking the O antigen) failed to elicit leaf wilting. These results suggest that the Salmonella O antigen 1,3,19 specifically triggers leaf chlorosis and wilting in A. thaliana.     

Bemisia tabaci （Hemiptera： Aleyrodidae） nymphal feeding in cotton （Gossypium hirsutum） leaves

We used brightfield electron microscopy （BEM）, differential interference contrast microscopy （DICM）, scanning electron microscopy （SEM）, transmission electron microscopy （TEM）, and confocal laser scanning microscopy （CLSM） to investigate the stylet pathways of Bemisia tabaci during nymphal feeding behavior in cotton leaves beginning with penetration of the abaxial leaf surface and ending with stylets in sieve tubes in phloem tissues. Most nymphal stylets within salivary sheaths penetrating leaf tissues made complex turns and developed more than one salivary sheath branch before ending in sieve tubes. The external morphology of the salivary sheaths and their routes between and through leaf cells are described during the present study. Results showed the presence of the stylet within the sieve tubes. B. tabaci nymphs may remove stylets and feed in different sieve tubes. Ten short movies showing the progression of the stylet penetrations from adaxial surface to the sieve tubes are attached to Figures 8-15. The report and movies can be viewed from the internet. Download the movies to a local drive in your computer first for fast upload. The movies are posted on the website http：//www.ars.usda.gov/Services/docs.htm？docid=14629. The movies can be used as a teaching aid in biology classes.     

Leaf age as a risk factor in contamination of lettuce with Escherichia coli O157:H7 and Salmonella enterica

Outbreaks of Escherichia coli O157:H7 infections have been linked increasingly to leafy greens, particularly to lettuce. We present here the first evidence that this enteric pathogen can multiply on the leaves of romaine lettuce plants. The increases in population size of E. coli O157:H7 in the phyllosphere of young lettuce plants ranged from 16- to 100-fold under conditions of warm temperature and the presence of free water on the leaves and varied significantly with leaf age. The population size was consistently ca. 10-fold higher on the young (inner) leaves than on the middle leaves. The growth rates of Salmonella enterica and of the natural bacterial microflora were similarly leaf age dependent. Both enteric pathogens also achieved higher population sizes on young leaves than on middle leaves harvested from mature lettuce heads, suggesting that leaf age affects preharvest as well as postharvest colonization. Elemental analysis of the exudates collected from the surfaces of leaves of different ages revealed that young-leaf exudates were 2.9 and 1.5 times richer in total nitrogen and carbon, respectively, than middle-leaf exudates. This trend mirrored the nitrogen and carbon content of the leaf tissue. Application of ammonium nitrate, but not glucose, to middle leaves enhanced the growth of E. coli O157:H7 significantly, suggesting that low nitrogen limits its growth on these leaves. Our results indicate that leaf age and nitrogen content contribute to shaping the bacterial communities of preharvest and postharvest lettuce and that young lettuce leaves may be associated with a greater risk of contamination with E. coli O157:H7.     

Uptake of Lucifer yellow CH in leaves ofCommelina communis is mediated by endocytosis

Summary Lucifer yellow CH (LY) uptake into intact leaves ofCommelina communis has been studied with conventional fluorescence microscopy as well as confocal laser scanning microscopy. LY, a highly fluorescent tracer for apoplastic transport in plants and fluid phase endocytosis in animal cells, accumulates in the vacuole of leaf cells. However, considerable differences in the ability to take up LY were observed among the various cell types. Mesophyll cells take up large amounts of the dye whereas epidermal cells, including guard and subsidiary cells, showed no fluorescence in their vacuoles. An exception to this are trichome cells which show considerable accumulation of LY. When introduced into the cytoplasm of mesophyll protoplasts ofC. communis by means of a patch-clamp pipette, LY does not enter the vacuole. This supports the contention that exogenous LY can only gain access to the vacuole via endocytosis. Differences in the capacity for LY uptake may therefore reflect differences in endocytotic activity.Abbreviations CLSM Confocal laser scanning microscopy - DIC differential interference contrast - LY Lucifer yellow CH - PM plasma membrane     

Quantitative and qualitative shifts in defensive metabolites define chemical defense investment during leaf development in Inga,a genus of tropical trees

Selective pressures imposed by herbivores are often positively correlated with investments that plants make in defense. Research based on the framework of an evolutionary arms race has improved our understanding of why the amount and types of defenses differ between plant species. However, plant species are exposed to different selective pressures during the life of a leaf, such that expanding leaves suffer more damage from herbivores and pathogens than mature leaves. We hypothesize that this differential selective pressure may result in contrasting quantitative and qualitative defense investment in plants exposed to natural selective pressures in the field. To characterize shifts in chemical defenses, we chose six species of Inga, a speciose Neotropical tree genus. Focal species represent diverse chemical, morphological, and developmental defense traits and were collected from a single site in the Amazonian rainforest. Chemical defenses were measured gravimetrically and by characterizing the metabolome of expanding and mature leaves. Quantitative investment in phenolics plus saponins, the major classes of chemical defenses identified in Inga, was greater for expanding than mature leaves (46% and 24% of dry weight, respectively). This supports the theory that, because expanding leaves are under greater selective pressure from herbivores, they rely more upon chemical defense as an antiherbivore strategy than do mature leaves. Qualitatively, mature and expanding leaves were distinct and mature leaves contained more total and unique metabolites. Intraspecific variation was greater for mature leaves than expanding leaves, suggesting that leaf development is canalized. This study provides a snapshot of chemical defense investment in a speciose genus of tropical trees during the short, few‐week period of leaf development. Exploring the metabolome through quantitative and qualitative profiling enables a more comprehensive examination of foliar chemical defense investment.     

THE USE OF INTERFERENCE REFLECTION CONTRAST IN THE EXAMINATION OF DIATOM VALVES

Even though scanning electron microscopy (SEM) is now needed to identify some species of diatoms, the majority of identifications and quantification of these organisms in ecological works is accomplished with a light microscope, using transmitted light optical methods. In this paper we demonstrate the use of interference reflection contrast (incident light) for the examination of diatoms, a method that significantly improves the resolution of structural detail, and therefore, identification of diatom taxa with light microscopy. Using incident light we were routinely able to distinguish between structures that were close to the theoretical limit of resolution for visible light, and that were not resolvable with such standard transmitted light techniques as phase contrast and differential interference contrast (DIC). Light microscopes with epi-illumination light paths can be easily and inexpensively outfitted to use this simple technique.
Abbreviations:  DIC, differential interference contrast; IRC, interference reflection contrast; LM, light microscopy     

Fitness of Salmonella enterica serovar Thompson in the cilantro phyllosphere

The epiphytic fitness of Salmonella enterica was assessed on cilantro plants by using a strain of S. enterica serovar Thompson that was linked to an outbreak resulting from cilantro. Salmonella serovar Thompson had the ability to colonize the surface of cilantro leaves, where it was detected by confocal laser scanning microscopy (CLSM) at high densities on the veins and in natural lesions. The population sizes of two common colonizers of plant surfaces, Pantoea agglomerans and Pseudomonas chlororaphis, were 10-fold higher than that of the human pathogen on cilantro incubated at 22 degrees C. However, Salmonella serovar Thompson achieved significantly higher population levels and accounted for a higher proportion of the total culturable bacterial flora on cilantro leaves when the plants were incubated at warm temperatures, such as 30 degrees C, after inoculation, indicating that the higher growth rates exhibited by Salmonella serovar Thompson at warm temperatures may increase the competitiveness of this organism in the phyllosphere. The tolerance of Salmonella serovar Thompson to dry conditions on plants at 60% relative humidity was at least equal to that of P. agglomerans and P. chlororaphis. Moreover, after exposure to low humidity on cilantro, Salmonella serovar Thompson recovered under high humidity to achieve its maximum population size in the cilantro phyllosphere. Visualization by CLSM of green fluorescent protein-tagged Salmonella serovar Thompson and dsRed-tagged P. agglomerans inoculated onto cilantro revealed that the human pathogen and the bacterial epiphyte formed large heterogeneous aggregates on the leaf surface. Our studies support the hypothesis that preharvest contamination of crops by S. enterica plays a role in outbreaks linked to fresh fruits and vegetables.     

Are green islands red herrings? Significance of green islands in plant interactions with pathogens and pests

The term green island was first used to describe an area of living, green tissue surrounding a site of infection by an obligately biotrophic fungal pathogen, differentiated from neighbouring yellowing, senescent tissue. However, it has now been used to describe symptoms formed in response to necrotrophic fungal pathogens, virus infection and infestation by certain insects. In leaves infected by obligate biotrophs such as rust and powdery mildew pathogens, green islands are areas where senescence is retarded, photosynthetic activity is maintained and polyamines accumulate. We propose such areas, in which both host and pathogen cells are alive, be termed green bionissia. By contrast, we propose that green areas associated with leaf damage caused by toxins produced by necrotrophic fungal pathogens be termed green necronissia. A range of biotrophic/hemibiotrophic fungi and leaf-mining insects produce cytokinins and it has been suggested that this cytokinin secretion may be responsible for the green island formation. Indeed, localised cytokinin accumulation may be a common mechanism responsible for green island formation in interactions of plants with biotrophic fungi, viruses and insects. Models have been developed to study if green island formation is pathogen-mediated or host-mediated. They suggest that green bionissia on leaves infected by biotrophic fungal pathogens represent zones of host tissue, altered physiologically to allow the pathogen maximum access to nutrients early in the interaction, thus supporting early sporulation and increasing pathogen fitness. They lead to the suggestion that green islands are 'red herrings', representing no more than the consequence of the infection process and discrete changes in leaf senescence.     

Interactions of Salmonella enterica with lettuce leaves

Aims:  To investigate the interactions of Salmonella enterica with abiotic and plant surfaces and their effect on the tolerance of the pathogen to various stressors.
Methods and Results:  Salmonella strains were tested for their ability to form biofilm in various growth media using a polystyrene plate model. Strong biofilm producers were found to attach better to intact Romaine lettuce leaf tissue compared to weak producers. Confocal microscopy and viable count studies revealed preferential attachment of Salmonella to cut-regions of the leaf after 2 h at 25°C, but not for 18 h at 4°C. Storage of intact lettuce pieces contaminated with Salmonella for 9 days at 4°C resulted only in small changes in population size. Exposure of lettuce-associated Salmonella cells to acidic conditions (pH 3·0) revealed increased tolerance of the attached vs planktonic bacteria.
Conclusions:  Biofilm formation on polystyrene may provide a suitable model to predict the initial interaction of Salmonella with cut Romaine lettuce leaves. Association of the pathogen with lettuce leaves facilitates its persistence during storage and enhances its acid tolerance.
Significance and Impact of the Study:  Understanding the interactions between foodborne pathogens and lettuce might be useful in developing new approaches to prevent fresh produce-associated outbreaks.     

Molecular dissection of Salmonella-induced membrane ruffling versus invasion

Type III secretion system-mediated injection of a cocktail of bacterial proteins drives actin rearrangements, frequently adopting the shape of prominent protuberances of ruffling membrane, and culminating in host cell invasion of Gram-negative pathogens like Salmonella typhimurium . Different Salmonella effectors are able to bind actin and activate Rho-family GTPases, which have previously been implicated in mediating actin-dependent Salmonella entry by interacting with N-WASP or WAVE-complex, well-established activators of the actin nucleation machine Arp2/3-complex. Using genetic deletion and RNA interference studies, we show here that neither individual nor collective removal of these Arp2/3- complex activators affected host cell invasion as efficiently as Arp2/3-complex knock-down, although the latter was also not essential. However, interference with WAVE-complex function abrogated Salmonella -induced membrane ruffling without significantly affecting entry efficiency, actin or Arp2/3-complex accumulation. In addition, scanning electron microscopy images captured entry events in the absence of prominent membrane ruffles. Finally, localization and RNA interference studies indicated a relevant function in Salmonella entry for the novel Arp2/3-complex regulator WASH. These data establish for the first time that Salmonella invasion is separable from bacteria-induced membrane ruffling, and uncover an additional Arp2/3-complex activator as well as an Arp2/3-complex-independent actin assembly activity that contribute to Salmonella invasion.     

Deciduous leaf drop reduces insect herbivory

Deciduous leaf fall is thought to be an adaptation that allows plants living in seasonal environments to reduce water loss and damage during unfavorable periods while increasing photosynthetic rates during favorable periods. Observations of natural variation in leaf shedding suggest that deciduous leaf fall may also allow plants to reduce herbivory. I tested this hypothesis by experimentally manipulating leaf retention for Quercus lobata and observing natural rates of herbivory. Quercus lobata is primarily deciduous although individuals show considerable natural variation in leaf retention. Oak saplings with no leaves through winter experienced reduced attack by cynipid gall makers the following spring. This pattern was consistent with the positive correlation between natural leaf persistence and gall numbers. These cynipids do not overwinter on the leaves that trees retain through winter, although they appear to use persistent leaves as oviposition cues. If these results are general for woody plants in continental temperate habitats, they suggest that an important and unrecognized consequence of deciduous leaf shedding may be a reduction in herbivore damage, and that this effect should be included in models of deciduous and evergreen behavior.     

Methods for Observing Microbial Biofilms Directly on Leaf Surfaces and Recovering Them for Isolation of Culturable Microorganisms

Epifluorescence microscopy, scanning electron microscopy, and confocal laser scanning microscopy were used to observe microbial biofilms directly on leaf surfaces. Biofilms were observed on leaves of all species sampled (spinach, lettuce, Chinese cabbage, celery, leeks, basil, parsley, and broad-leaved endive), although the epifluorescent images were clearest when pale green tissue or cuticle pieces were used. With these techniques, biofilms were observed that were about 20 (mu)m in depth and up to 1 mm in length and that contained copious exopolymeric matrices, diverse morphotypes of microorganisms, and debris. The epifluorescence techniques described here can be used to rapidly determine the abundance and localization of biofilms on leaves. An additional technique was developed to recover individual biofilms or portions of single biofilms from leaves and to disintegrate them for isolation of the culturable microorganisms they contained. Nineteen biofilms from broad-leaved endive, spinach, parsley, and olive leaves were thus isolated and characterized to illustrate the applications of this technique.     

Bacterial colonization and biofilm development on minimally processed vegetables

Bacterial biofilms have been observed and reported on food and food-processing surfaces and can contribute to increased risks for product quality and food safety. The colonization of fruit and vegetables by pectynolitic bacteria like Pseudonomas fluorescens attributable to conditions such as soft rot, can also manifest as biofilms. A developed biofilm structure can provide a protective environment for pathogens such as Listeria monocytogenes reducing the effectiveness of sanitisers and other inhibitory agents. Understanding the colonization of bacteria on leaf surfaces is essential to the development of a better understanding of the leaf ecology of vegetable products. Studies of microbial colonization of leaf surfaces have been conducted using SEM and more recently using confocal microsocpy techniques. In the current study, a Leica TCS NT laser scanning confocal microscope was used to investigate biofilm formation using vital fluorescence staining on intact vegetable leaves. Reflection contrast and fluorescence three-dimensional imaging successfully delineated bacterial and biofilm morphology without disturbing the bacterial or leaf surface structure. The results demonstrate the presence and development of biofilm on the surface of lettuce. The biofilms appeared to originate on the cuticle in distinct micro-environments such as in the natural depression of the stomata, or in the intercellular junction. Bacteria also adhered to and developed biofilm colonies within an hour of contact and with clean stainless steel surfaces. Our study investigates the progression of biofilm formation from leaf colonization, and will assist in characterising the critical mechanisms of plant/host interaction and facilitate the development of improved preservation, sanitising and packaging strategies for minimally processed vegetable products.     

Fungal species diversity in juvenile and adult leaves of Eucalyptus globulus from plantations affected by Mycosphaerella leaf disease

In recent years, Mycosphaerella leaf disease (MLD) has become very common in Eucalyptus globulus plantations in Galicia, northwest Spain. The aetiology of MLD is complex and is associated with several species of Mycosphaerella and Teratosphaeria. A survey of the fungal mycobiota associated with juvenile and adult leaves and with leaf litter of the same trees in MLD‐affected plantations was made. The goal was to identify pathogens and endophytes, to determine whether the mycobiota of each leaf type differed and whether leaf litter might be a reservoir of MLD inoculum. Fungi belonging to 113 different species were isolated from the leaves of juvenile and adult trees sampled at 10 locations; 81 species occurred in juvenile and 65 in adult leaves. The average number of species obtained from juvenile leaves was significantly greater (P > 0.01) compared to adult leaves. This difference suggested that juvenile leaves are not only more susceptible to a group of pathogens, but to a wide range of fungi. Therefore, a general resistance mechanism might be lacking or be less effective in juvenile than in adult leaves. Several pathogenic species were identified in both leaf types. Leaf litter and living leaf mycobiotas were very different. However, some of the species they shared were MLD pathogens, suggesting that leaf litter could contribute to the inoculum of MLD.     

The role of leaf lobation in elongation responses to shade in the rosette-forming forb Serratula tinctoria (Asteraceae)

BACKGROUND AND AIMS: Lobed leaves are considered selectively advantageous in conditions of high irradiance. However, most studies have involved woody species, with only a few considering the role of leaf lobation in herbaceous rosette species. In this study, it is hypothesized that, in addition to its adaptive value in high light, leaf lobation may add to the function of petioles as vertical spacers in herbaceous species in conditions of strong competition for light. METHODS: To test this hypothesis, leaf development was examined under seasonally changing natural light conditions and a field experiment was conducted in which light climate was manipulated in a wooded meadow population of Serratula tinctoria. KEY RESULTS: No changes in leaf lobation were observed in response to experimental shading or different natural light conditions. However, in tall herbaceous vegetation, plants with highly lobed leaves achieved significantly greater vertical elongation than plants with less-lobed leaves. In contrast to herbaceous shade, tree shade had no effect on leaf elongation, suggesting differential responsiveness to competition from neighbouring herbs versus overhead shade. In shading treatments, imposed shade could only be responded to by the elongation of leaves that were produced late in development. CONCLUSIONS: The results show that extensive leaf lobation can enable greater leaf elongation in response to shade from surrounding herbaceous vegetation. The different morphological responses displayed by Serratula tinctoria to different types of shade demonstrate the importance of critically assessing experimental designs when investigating phenotypic plasticity in response to shade.     

山东产蹄盖蕨科2属植物形态解剖学的研究

采用光镜和扫描电镜对山东分布的蹄盖蕨科2属(蹄盖蕨属和假蹄盖蕨属)7种植物的根、根茎、叶柄、叶轴、叶表皮、表皮毛和孢子囊进行了形态解剖学的系统研究.结果表明,在形态解剖学方面2属植物的共同特征为:根均为无髓中柱;叶柄基部的双柱型维管束向上渐靠近联合形成1个周韧型维管束;叶上下表皮垂周壁均呈波状;气孔主要为胞环型、周胞型或极附型.2属植物的不同特征是:蹄盖蕨属植物体无毛;而假蹄盖蕨属植物叶片和叶轴上均生有腺毛;蹄盖蕨属植物根皮层外侧为薄壁细胞,假蹄盖蕨属则为棕色厚壁细胞环.研究结果表明蹄盖蕨科为一个自然分类群,并支持假蹄盖蕨属的成立.     

Assessment of leaf micromorphology after full desiccation of resurrection plants

Resurrection plants are unique among higher plants because of their ability to withstand long periods of dehydration without damages. In this study, leaf epidermis and palisade mesophyll of three resurrection species, Haberlea rodopensis, Ramonda serbica and Ramonda myconi, grown under full desiccation and benign conditions, were analyzed by differential interference contrast microscopy. Detailed investigation of adaxial and abaxial leaf surfaces revealed species-specific differences in the size and number of epidermal cells and stomatal density. The applied full desiccation did not cause any significant deviations of these parameters from the controls. There were no changes in the size and number of mesophyll cells as well. Analysis of stomatal patterning displayed essentially hypostomatic leaves, having stomata mainly abaxially positioned. The most significant change detected in the leaves of dehydration-treated plants was the increased formation of adaxially positioned trichomes. This increase was very high in R. myconi, where the adaxial leaf surface was fully covered by trichomes. Despite the existence of small species-specific differences, the results showed uniform desiccation-related responses of the studied resurrection species. The quantified leaf epidermal and mesophyll features are discussed with respect to their possible contribution to the desiccation tolerance of resurrection species.     

Infection Process of Burkholderia glumae Before Booting Stage of Rice

Burkholderia glumae is a well‐known pathogen for causing bacterial panicle blight of rice. In this study, the infection process of B. glumae in rice plants at different growing stages was tracked by means of real‐time fluorescence quantitative PCR. Burkholderia glumae tended to colonize at the growing point of rice plants, and the biomass of population was 10⁴ to 10⁸ CFU/g. The most intensive colonization was detected in the upmost leaf in the two‐leaf period. However, after the two‐leaf period, the population of pathogens decreased significantly, and they successfully recovered in the booting stage and broke out in panicles. We also illustrated the incubation location of B. glumae by presenting the infection pattern in the seedling and tillering stage of rice. Under fluorescent microscopy, the gfp‐labelled pathogens were first found in the vascular bundle of lateral roots, taproots and injured cells, then they were observed in the root hairs, epidermal cells and main root cap. The pathogens in the vascular bundle laterally dispersed towards the epidermal cells. By spray application of a bacterial suspension, the pathogens landed on the leaf sheaths and leaves, colonized in the epidermal hairs and leaf hairs, or invaded into the cells through the stomas. At the same time, the pathogens from the vascular bundle of the roots spread into the vascular bundle of leaf sheaths and leaves, which caused the leaves to curl and wilt, beginning from the tip.     

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.     

Al(3+) and Zn(2+)-induced structural changes and localization in a fully-hydrated live eukaryotic cellular model.

This study combined techniques that did not require preparation protocols that were potentially harmful to the cell, making it possible to investigate cells at, or close to, their natural physiological state. We used the freshwater protozoon Chilomonas paramecium as a eukaryotic cellular model to locate sites of Al(3+) or Zn(2+) accumulation and quantify the associated structural changes. Cells were fully hydrated throughout the study, which used a combination of differential interference contrast light microscopy, confocal laser scanning microscopy and transmission X-ray microscopy. The latter technique allowed high resolution (50 nm) and high contrast imaging of live cells in solution. For confocal laser scanning microscopy the relatively new fluorochrome Newport Green was used. This made fluorescent complexes with intracellular Al(3+) and Zn(2+), allowing localisation of metal-containing granules and vesicles. After long term exposure a previously unreported annular-shaped site of metal accumulation was found, signifying a vesicle with metal accumulated in the periphery only. After exposure to Al(3+) and Zn(2+), the cell pellicle was thinner and the majority of rounded-up cells had a concentric layering of organelles. By combining a variety of techniques it was possible to gain high resolution structural and chemical information on cells minimally exposed to potentially artefact-inducing procedures.