Differences in the Occurrence of Cell Wall Components between Distinct Cell Types in Glands of Drosophyllum lusitanicum.

Carnivorous plants are mixotrophs that have developed the ability to lure, trap, and digest small organisms and utilize components of the digested bodies. Leaves of Drosophyllum lusitanicum have two kinds of glands (emergences): stalked mucilage glands and sessile digestive glands. The stalked mucilage glands perform the primary role in prey lure and trapping. Apart from their role in carnivory, they absorb water condensed from oceanic fog; thus, plants can survive in arid conditions. To better understand the function of carnivorous plant emergences, the molecular composition of their cell walls was investigated using immunocytochemical methods. In this research, Drosophyllum lusitanicum was used as a study system to determine whether cell wall immunocytochemistry differs between the mucilage and digestive glands of other carnivorous plant species. Light and electron microscopy were used to observe gland structure. Fluorescence microscopy revealed the localization of carbohydrate epitopes associated with the major cell wall polysaccharides and glycoproteins. The mucilage gland (emergence) consists of a glandular head, a connecting neck zone, and stalk. The gland head is formed by an outer and inner layer of glandular (secretory) cells and supported by a layer of endodermoid (barrier) cells. The endodermoid cells have contact with a core of spongy tracheids with spiral-shaped thickenings. Lateral tracheids are surrounded by epidermal and parenchymal neck cells. Different patterns of cell wall components were found in the various cell types of the glands. Cell walls of glandular cells generally are poor in both low and highly esterified homogalacturonans (HGs) but enriched with hemicelluloses. Cell walls of inner glandular cells are especially rich in arabinogalactan proteins (AGPs). The cell wall ingrowths in glandular cells are significantly enriched with hemicelluloses and AGPs. In the case of cell wall components, the glandular cells of Drosophyllum lusitanicum mucilage glands are similar to the glandular cells of the digestive glands of Aldrovanda vesiculosa and Dionaea muscipula.

Quantification of Protein Uptake by Endocytosis in Carnivorous Nepenthales.

Carnivorous plants adsorb prey-derived nutrients partly by endocytosis. This study quantifies endocytosis in Drosophyllum lusitanicum, Drosera capensis, Drosera roseana, Dionaea muscipula and Nepenthes × ventrata. Traps were exposed to 1% fluorescent-labeled albumin (FITC-BSA), and uptake was quantified repeatedly for 64 h. Formation of vesicles started after ≤1 h in adhesive traps, but only after 16 h in species with temporary stomach (D. muscipula and N. × ventrata). In general, there are similarities in the observed species, especially in the beginning stages of endocytosis. Nonetheless, further intracellular processing of endocytotic vesicles seems to be widely different between species. Endocytotic vesicle size increased significantly over time in all species except in D. capensis. Fluorescence intensity of the endocytotic vesicles increased in all species except D. muscipula. After 64 h, estimates for FITC-BSA absorption per gland ranged from 5.9 ± 6.3 ng in D. roseana to 47.8 ± 44.3 ng in N. × ventrata, demonstrating that endocytosis substantially contributes to the adsorption of prey-derived nutrients.

Gland cell responses to feeding in Drosera capensis, a carnivorous plant.

Glands of Drosera absorb and transport nutrients from captured prey, but the mechanism and dynamics remain unclear. In this study, we offered animal proteins in the form of fluorescent albumin (FITC-BSA) and observed the reactions of the glands by live cell imaging and fluorescence microscopy. The ultrastructure of these highly dynamic processes was also assessed in high-pressure frozen and freeze substituted (HPF-FS) cells. HPF-FS yielded excellent preservation of the cytoplasm of all cell types, although the cytosol looked different in gland cells as compared to endodermoid and stalk cells. Especially prominent were the ER and its contacts with the plasma membrane, plasmodesmata, and other organelles as well as continuities between organelles. Also distinct were actin microfilaments in association with ER and organelles. Application of FITC-BSA to glands caused the formation of fluorescent endosomes that pinched off the plasma membrane. Endosomes fused to larger aggregates, and accumulated in the bulk cytoplasm around the nucleus. They did not fuse with the cell sap vacuole but remained for at least three days; in addition, fluorescent vesicles also proceeded through endodermoid and transfer cells to the epidermal and parenchymal cells of the tentacle stalk.

Structural traits of leaf epidermis correspond to metal tolerance in Rumex acetosella populations growing on metal-contaminated soils.

The pseudometallophyte Rumex acetosella L. occupies habitats with normal and high soil concentrations of zinc (Zn), lead (Pb), and copper (Cu). It remains unclear if the plants respond to the toxic metals by altering their morphology and increasing the resilience of their cells. We compared plants growing on soils contaminated with Zn/Pb (populations Terézia, Lintich), or Cu (populations Spania Dolina, Staré Hory), with those from non-contaminated soil (Dúbravka) in Slovakia, and analysed leaf structure, physiology, and metal contents by light and electron microscopy, element localization by energy-dispersive X-ray analysis (EDX) in scanning electron microscope, and by specific fluorescence dyes. In control population, the epidermis of the amphistomatic leaves of R. acetosella contained capitate glandular trichomes, consisting of four head (secretory), two stalk, and two basal cells. The ultrastructure of secretory cells revealed fine wall ingrowths bordered by plasma membrane protruding into the cytoplasm. The metallicolous populations had higher contents of Zn and Cu in the epidermal and glandular cells, and a higher density of both stomata and trichomes. Extensive cell wall labyrinth was present in the trichome secretory cells. Their abnormal number and elevated metal contents might indicate effects of heavy metals, especially of Cu, on mitosis and cell plate formation. Differences in leaf physiology were indicated by significantly higher cytoplasmic tolerance to Zn and Cu in metallicolous populations and by structural properties of glandular heads suggesting secretion of toxic metals. Our findings are suggestive of plant reactions to metal stress, which facilitate the populations to occupy the metal-contaminated sites.

The effect of endophytic fungi on growth and nickel accumulation in Noccaea hyperaccumulators.

The role of endophytic fungi isolated from different populations of European Ni hyperaccumulators was investigated in regard to the microorganisms' ability to enhance the hyperaccumulation of Ni in Noccaea caerulescens. Effects of particular species of endophytic fungi on adaptation of N. caerulescens to excess Ni were tested by co-cultivation with single strains of the fungi. Seven of these had a positive effect on plant biomass production, whereas two of the tested species inhibited plant growth; biomass production of inoculated plants was significantly different compared to non-inoculated control. Inoculation with six fungal strains: Embellisia thlaspis, Pyrenochaeta cava, Phomopsis columnaris, Plectosphaerella cucumerina, Cladosporium cladosporioides and Alternaria sp. stimulated the plant to uptake and accumulate more Ni in both roots and shoots, compared to non-inoculated control. P. columnaris was isolated from all plant species sampled. Strains isolated from Noccaea caerulescens and Noccaea goesingensis increased Ni root and shoot accumulation of their native hosts (compared to non-inoculated control). Inoculation of different populations of Noccaea with P. columnaris of foreign origin did not cause its host to accumulate more Ni, with the exception of the Ni-unadapted ecotype of N. goesingensis. Inoculation with P. columnaris from N. caerulescens significantly improved Ni uptake, but the effect of the fungus was not as prominent as in the case of N. caerulescens. By comparing the transcriptomes of N. caerulescens and N. goesingensis from Flatz inoculated with P. columnaris, we showed that enhanced uptake and accumulation of Ni in the plants is accompanied by an upregulation of several genes mainly involved in plant stress protection and metal uptake and compartmentation.

The response of the accumulator plants Noccaea caerulescens, Noccaea goesingense and Plantago major towards the uranium.

Uranium (U) is a naturally occurring metal; its environmental levels can be increased due to processes in the nuclear industry and fertilizer production. The transfer of U in the food chain from plants is associated with deleterious chemical and radiation effects. To date, limited information is available about U toxicity on plant physiology. This study investigates the responses of metal-accumulating plants to different concentrations of U. The plants Noccaea caerulescens and Noccaea goesingense are known as metal hyperaccumulators and therefore could serve as candidates for the phytoremediation of radioactive hotspots; Plantago major is a widely used pharmaceutical plant that pioneers polluted grounds and therefore should not contain high concentrations of toxic elements. The experimental plants were grown hydroponically at U concentrations between 1 µM and 10 mM. The content of U and essential elements was analyzed in roots and leaves by ICP-MS. The amount of accumulated U was influenced by its concentration in the hydroponics. Roots contained most of the metal, whereas less was transported up to the leaves, with the exception of N. goesingense in a medium concentration of U. U also influenced the nutrient profile of the plants. We localized the U in plant tissues using EDX in the SEM. U was evenly distributed in roots and leaves of Noccaea species, with one exception in the roots of N. goesingense, where the central cylinder contained more U than the cortex. The toxicity of U was assessed by measuring growth and photosynthetic parameters. While root biomass of N. caerulescens was not affected by U, root biomass of N. goesingense decreased significantly at high U concentrations of 0.1 and 10 mM and root biomass of P. major decreased at 10 mM U. Dry weight of leaves was decreased at different U concentrations in the three plant species; a promotive effect was observed in N. caerulescens at lowest concentration offered. Chlorophyll a fluorescence was not affected or negatively affected by U in both Noccaea species, whereas in Plantago also positive effects were observed. Our results show that the impact of U on Plantago and Noccaea relates to its external concentration and to the plant species. When growing in contaminated areas, P. major should not be used for medicinal purpose. Noccaea species and P. major could immobilize U in their rhizosphere in hotspots contaminated by U, and they could extract limited amounts of U into their leaves.

A new insight on structural and some functional aspects of peri-endodermal thickenings, a specific layer in Noccaea caerulescens roots.

BACKGROUND AND AIMS: Cell walls of the peri-endodermis, a layer adjacent to the endodermis in alpine pennycress (Noccaea caerulescens) roots, form C-shaped peri-endodermal thickenings (PETs). Despite its specific position close to the endodermis, the assumed similarity of PETs to phi thickenings in many other species, and the fact that N. caerulescens is a well-studied heavy-metal-hyperaccumulating plant, the PET as a root trait is still not understood. METHODS: Here, we characterized PET cell walls by histochemical techniques, Raman spectroscopy, immunolabelling and electron microscopy. Moreover, a role of PETs in solute transport was tested and compared with Arabidopsis thaliana plants, which do not form PETs in roots. KEY RESULTS: Cell walls with PETs have a structured relief mainly composed of cellulose and lignin. Suberin, typical of endodermal cells, is missing but pectins are present on the inner surface of the PET. Penetrating dyes are not able to cross PETs either by the apoplasmic or the symplasmic pathway, and a significantly higher content of metals is found in root tissues outside of PETs than in innermost tissues. CONCLUSIONS: Based on their development and chemical composition, PETs are different from the endodermis and closely resemble phi thickenings. Contrarily, the different structure and dye impermeability of PETs, not known in the case of phi thickenings, point to an additional barrier function which makes the peri-endodermis with PETs a unique and rare layer.

Response of Arabidopsis halleri to cesium and strontium in hydroponics: Extraction potential and effects on morphology and physiology.

Stable isotopes of cesium (Cs) and strontium (Sr) as well as their radioactive isotopes are of serious environmental concern. The pollution of the biosphere, particularly soil and water has received considerable attention for removal of these contaminants in recent years. Arabidopsis halleri (A. halleri) is a hyperaccumulator plant species able to take up large amounts of several metals into its above ground organs without showing significant signs of toxicity. Therefore, we investigated responses, metal accumulation and element distribution in roots and leaves of A. halleri after treatment with stable Cs and Sr. Plants were hydroponically grown in different concentrations of cesium sulfate (between 0.002 and 20 mM) and strontium nitrate (between 0.001 and 100 mM). Uptake of Cs and Sr into leaves was analyzed from extracts by inductively coupled plasma mass spectrometry (ICP-MS). Although internal concentration of Cs and Sr increased with rising external concentrations, the amount of accumulated metal in relation to available metal decreased. Therefore, the potential of the plant to effectively transfer metals from growth medium to leaves occurred at low and moderate concentrations, whereas after that when the concentration of metal increased further the transfer factors were decreased. A. halleri accumulated Sr more efficiently than Cs. The transfer factors were higher for Sr (up to 184) than for Cs (up to 16). The results indicate positive correlation of Cs and Sr accumulation to K and Ca transport to leaves. The toxicity of Cs and Sr was assessed by measuring photosynthetic efficiency and growth parameters. In leaves, Cs and Sr affected the chlorophyll fluorescence at their low and high concentrations. Significant reduction of plant growth (dry weight of roots and leaves) was observed at Sr concentrations >0.01 mM. Cs-treated plants exhibited only decreased length of leaves at concentrations>0.02 mM. The distribution of the elements within the different tissues of leaves and roots was investigated by using Energy Dispersive X-Ray microanalysis (EDX) with a scanning electron microscope (SEM). EDX revealed that Cs and Sr were accumulated differently in root and leaf tissues. The hydroponic experiment showed a potential for A. halleri to treat hotspots with radioactive Cs and Sr.

Response of Plantago major to cesium and strontium in hydroponics: Absorption and effects on morphology, physiology and photosynthesis.

Human activities lead to increasing concentration of the stable elements cesium (Cs) and strontium (Sr) and their radioactive isotopes in the food chain, where plants play an important part. Here we investigated Plantago major under the influence of long-term exposure to stable Cs and Sr. The plants were cultivated hydroponically in different concentrations of cesium sulfate (between 0.002 and 20 mM) and strontium nitrate (between 0.001 and 100 mM). Uptake of Cs and Sr into leaves was analyzed from extracts by inductively coupled plasma mass spectrometry (ICP-MS). It was increased with increasing external Cs and Sr concentrations. However, the efficiency of Cs and Sr transfer from solution to plants was higher for low external concentrations. Highest transfer factors were 6.78 for Cs and 71.13 for Sr. Accumulation of Sr was accompanied by a slight decrease of potassium (K) and calcium (Ca) in leaves, whereas the presence of Cs in the medium affected only uptake of K. The toxic effects of Cs and Sr were estimated from photosynthetic reactions and plant growth. In leaves, Cs and Sr affected the chlorophyll fluorescence even at their low concentrations. Low and high concentrations of both ions reduced dry weight and length of roots and leaves. The distribution of the elements between the different tissues of leaves and roots was investigated using Energy Dispersive X-Ray microanalysis (EDX) with scanning electron microscope (SEM). Overall, observations suggested differential patterns in accumulating Cs and Sr within the roots and leaves. When present in higher concentrations the amount of Cs and Sr transferred from environment to plants was sufficient to affect some physiological processes. The experimental model showed a potential for P. major to study the influence of radioactive contaminants and their removal from hotspots.

Silicon Uptake and Localisation in Date Palm (Phoenix dactylifera) - A Unique Association With Sclerenchyma.

Date palm (Phoenix dactylifera) can accumulate as much as 1% silicon (Si), but not much is known about the mechanisms inherent to this process. Here, we investigated in detail the uptake, accumulation and distribution of Si in date palms, and the phylogeny of Si transporter genes in plants. We characterized the PdNIP2 transporter following heterologous expression in Xenopus oocytes and used qPCR to determine the relative expression of Si transporter genes. Silicon accumulation and distribution was investigated by light microscopy, scanning electron microscopy coupled with X-ray microanalysis and Raman microspectroscopy. We proved that PdNIP2-1 codes for a functional Si-permeable protein and demonstrated that PdNIP2 transporter genes were constitutively expressed in date palm. Silicon aggregates/phytoliths were found in specific stegmata cells present in roots, stems and leaves and their surfaces were composed of pure silica. Stegmata were organized on the outer surface of the sclerenchyma bundles or associated with the sclerenchyma of the vascular bundles. Phylogenetic analysis clustered NIP2 transporters of the Arecaceae in a sister position to those of the Poaceae. It is suggested, that Si uptake in date palm is mediated by a constitutively expressed Si influx transporter and accumulated as Si aggregates in stegmata cells abundant in the outer surface of the sclerenchyma bundles (fibers).

Symbiotic microbes of Saxifraga stellaris ssp. alpigena from the copper creek of Schwarzwand (Austrian Alps) enhance plant tolerance to copper.

Saxifraga stellaris var. alpigena grows as one of the very few higher plants in the copper rich area of the "blue creek" in Austria. Two endophytes were isolated from it: Mortierella sp. (fungus), and Stenotrophomonas maltophilia (bacterium). Both microbes were practically inseparable due to resistance of the bacteria to the antibiotics tested. On PDA media, the fungus showed higher tolerance to copper than the bacterium, which disappeared from both the media and the surface of the fungus in the presence of 150 µM of Cu. However, at this Cu concentration, the bacteria were still detectable inside the mycelium and reappeared on the outside when transferred to media of lower Cu concentration. Microscopic studies of in vitro cultivated plants showed that the fungus was present in both, the roots and shoots of the plant. The effects of endophytes on plant performance were assessed in rhizoboxes filled with Cu-rich substratum; plants inoculated with both microbes showed better growth, survival and photosynthesis performance than the non-inoculated controls. The results of this study prove the beneficial influence of the isolated endophytes on the Cu tolerance of S. stellaris, and indicate the ecological potential of applying microbial consortia to plants under extreme environmental conditions.

Strontium in the environment: Review about reactions of plants towards stable and radioactive strontium isotopes.

Radiostrontium is released to the environment from routine and accidental discharge and acts on living organisms either from external sources or after absorption. When incorporated by plants, it enters the food chain and causes primary threat to human health and the environment. Understanding the mechanisms of plants for strontium uptake and retention is therefore essential for decision making concerning agriculture: are uptake rates low enough so that plants can serve as food? Or is radiostrontium accumulated so that plants should not be eaten but could be probably used for extracting strontium from water and soil in hot spots of pollution? The review presents a summary of studies about the origin of stable and radioactive strontium in the environment and effects coming from both internal and external exposure of plants. Mobility and availability of strontium to plant roots in soil are controlled by external factors such as chemical composition of the soil and pH, temperature and agricultural soil cultivation as well as soil biological networks built by microbial communities. Plant surfaces may receive input of strontium from deposition induced by atmospheric pollution or by acquisition from water through the whole immersed surface. Cells have entry mechanisms for strontium such as plasma membrane transporters for calcium and potassium. Part of absorbed strontium can be lost via processes discussed in this review. We give examples on strontium transfer factors for 149 plants to estimate plant absorption capacity for strontium from soil, water and air. Uptake efficiency of terrestrial and aquatic plants is deciding about their remediation potential to either remove radiostrontium by accumulation and rhizofiltration or to retain it in roots or aerial parts. Data of strontium content in soils after fallout and edible plants from long-term monitoring support the evaluation of the potential hazards posed by strontium input to the food chain.

Adaptive responses of mature giant chloroplasts in the deep-shade lycopod Selaginella erythropus to prolonged light and dark periods.

Deep-shade plants have adapted to low-light conditions by varying morphology and physiology of cells and chloroplasts, but it still remains unclear, if prolonged periods of high-light or darkness induce additional modifications in chloroplasts' anatomy and pigment patterns. We studied giant chloroplasts (bizonoplasts) of the deep-shade lycopod Selaginella erythropus in epidermal cells of mature fully developed microphylls and subjected them to prolonged darkness and high-light conditions. Chloroplast size and ultrastructure were investigated by light and electron microscopy. Physiological traits were studied by pigment analyses, photosynthetic performance of photosystem II, and formation of reactive oxygen species. Results show that (a) thylakoid patterns and shape of mature bizonoplasts vary in response to light and dark conditions. (b) Prolonged darkness induces transitory formation of prolamellar bodies, which so far have not been described in mature chloroplasts. (c) Photosynthetic activity is linked to structural responses of chloroplasts. (d) Photosystem II is less active in the upper zone of bizonoplasts and more efficient in the grana region. (e) Formation of reactive oxygen species reflects the stress level caused by high-light. We conclude that during prolonged darkness, chlorophyll persists and even increases; prolamellar bodies form de novo in mature chloroplasts; bizonoplasts have spatial heterogeneity of photosynthetic performance.

Stable and radioactive cesium: A review about distribution in the environment, uptake and translocation in plants, plant reactions and plants' potential for bioremediation.

Radiocesium in water, soil, and air represents a severe threat to human health and the environment. It either acts directly on living organisms from external sources, or it becomes incorporated through the food chain, or both. Plants are at the base of the food chain; it is therefore essential to understand the mechanisms of plants for cesium retention and uptake. In this review we summarize investigations about sources of stable and radioactive cesium in the environment and harmful effects caused by internal and external exposure of plants to radiocesium. Uptake of cesium into cells occurs through molecular mechanisms such as potassium and calcium transporters in the plasma membrane. In soil, bioavailability of cesium depends on the chemical composition of the soil and physical factors such as pH, temperature and tilling as well as on environmental factors such as soil microorganisms. Uptake of cesium occurs also from air through interception and absorption on leaves and from water through the whole submerged surface. We reviewed information about reducing cesium in the vegetation by loss processes, and we extracted transfer factors from the available literature and give an overview over the uptake capacities of 72 plants for cesium from the substratum to the biomass. Plants with high uptake potential could be used to remediate soil and water from radiocesium by accumulation and rhizofiltration. Inside plants, cesium distributes fast between the different plant organs and cells, but cesium in soil is extremely stable and remains for decades in the rhizosphere. Monitoring of contaminated soil therefore has to continue for many decades, and edible plants grown on such soil must continuously be monitored.

Expression of Genes for Si Uptake, Accumulation, and Correlation of Si with Other Elements in Ionome of Maize Kernel.

The mineral composition of cells, tissues, and organs is decisive for the functioning of the organisms, and is at the same time an indicator for understanding of physiological processes. We measured the composition of the ionome in the different tissues of maize kernels by element microanalysis, with special emphasis on silicon (Si). We therefore also measured the expression levels of the Si transporter genes ZmLsi1, ZmLsi2 and ZmLsi6, responsible for Si uptake and accumulation. Two weeks after pollination ZmLsi1 and ZmLsi6 genes were expressed, and expression continued until the final developmental stage of the kernels, while ZmLsi2 was not expressed. These results suggest that exclusively ZmLsi1 and ZmLsi6 are responsible for Si transport in various stages of kernel development. Expression level of ZmLsi genes was consistent with Si accumulation within kernel tissues. Silicon was mainly accumulated in pericarp and embryo proper and the lowest Si content was detected in soft endosperm and the scutellum. Correlation linkages between the distribution of Si and some other elements (macroelements Mg, P, S, N, P, and Ca and microelements Cl, Zn, and Fe) were found. The relation of Si with Mg was detected in all kernel tissues. The Si linkage with other elements was rather specific and found only in certain kernel tissues of maize. These relations may have effect on nutrient uptake and accumulation.

The copper spoil heap Knappenberg, Austria, as a model for metal habitats - Vegetation, substrate and contamination.

Historic mining in the Eastern Alps has left us with a legacy of numerous spoil heaps hosting specific, metal tolerant vegetation. Such habitats are characterized by elevated concentrations of toxic elements but also by high irradiation, a poorly developed substrate or extreme pH of the soil. This study investigates the distribution of vascular plants, mosses and lichens on a copper spoil heap on the ore bearing Knappenberg formed by Prebichl Layers and Werfener Schist in Lower Austria. It serves as a model for discriminating between various ecological traits and their effects on vegetation. Five distinct clusters were distinguished: (1) The bare, metal rich Central Spoil Heap was only colonised by highly resistant specialists. (2) The Northern and (3) Southern Peripheries contained less copper; the contrasting vegetation was best explained by the different microclimate. (4) A forest over acidic bedrock hosted a vegetation overlapping with the periphery of the spoil heap. (5) A forest over calcareous bedrock was similar to the spoil heap with regard to pH and humus content but hosted a vegetation differing strongly to all other habitats. Among the multiple toxic elements at the spoil heap, only Cu seems to exert a crucial influence on the vegetation pattern. Besides metal concentrations, irradiation, humidity, humus, pH and grain size distribution are important for the establishment of a metal tolerant vegetation. The difference between the species poor Northern and the diverse Southern Periphery can be explained by the microclimate rather than by the substrate. All plant species penetrating from the forest into the periphery of the spoil heap originate from the acidic but not from the calcareous bedrock.

Live microscopy analysis of endosomes and vesicles in tip-growing root hairs.

Tip growth is one of the most preferable models in the study of plant cell polarity; cell wall deposition is restricted mainly to a certain area of the cell, and cell expansion at this specific area leads to the development of tubular outgrowth. Tip-growing root hairs are well-established systems for such studies, because their lateral position within the root makes them easily accessible for experimental approaches and microscopic observations. Fundamental structural and molecular processes driving tip growth are exocytosis, endocytosis, and all aspects of vesicular and endosomal dynamic trafficking, as related to targeted membrane flow. Study of vesicles and endosomes in living root hairs, however, is rather difficult, due to their small size and due to the resolution limits of conventional light microscopes. Here we present noninvasive approaches for visualizing vesicular and endosomal compartments in the tip of growing root hairs using electronic light microscopy, contrast-enhanced video light microscopy, and confocal laser scanning microscopy (CLSM). These methods allow utilizing the maximum resolution of the light microscope. Together with protocols for appropriate preparation of living plant samples, the described methods should help improve our understanding on how tiny vesicles and endosomes support the process of tip growth in root hairs.

Salt-induced subcellular kinase relocation and seedling susceptibility caused by overexpression of Medicago SIMKK in Arabidopsis.

Dual-specificity mitogen-activated protein kinases kinases (MAPKKs) are the immediate upstream activators of MAPKs. They simultaneously phosphorylate the TXY motif within the activation loop of MAPKs, allowing them to interact with and regulate multiple substrates. Often, the activation of MAPKs triggers their nuclear translocation. However, the spatiotemporal dynamics and the physiological consequences of the activation of MAPKs, particularly in plants, are still poorly understood. Here, we studied the activation and localization of the Medicago sativa stress-induced MAPKK (SIMKK)-SIMK module after salt stress. In the inactive state, SIMKK and SIMK co-localized in the cytoplasm and in the nucleus. Upon salt stress, however, a substantial part of the nuclear pool of both SIMKK and SIMK relocated to cytoplasmic compartments. The course of nucleocytoplasmic shuttling of SIMK correlated temporally with the dual phosphorylation of the pTEpY motif. SIMKK function was further studied in Arabidopsis plants overexpressing SIMKK-yellow fluorescent protein (YFP) fusions. SIMKK-YFP plants showed enhanced activation of Arabidopsis MPK3 and MPK6 kinases upon salt treatment and exhibited high sensitivity against salt stress at the seedling stage, although they were salt insensitive during seed germination. Proteomic analysis of SIMKK-YFP overexpressors indicated the differential regulation of proteins directly or indirectly involved in salt stress responses. These proteins included catalase, peroxiredoxin, glutathione S-transferase, nucleoside diphosphate kinase 1, endoplasmic reticulum luminal-binding protein 2, and finally plasma membrane aquaporins. In conclusion, Arabidopsis seedlings overexpressing SIMKK-YFP exhibited higher salt sensitivity consistent with their proteome composition and with the presumptive MPK3/MPK6 hijacking of the salt response pathway.

NanoSIMS combined with fluorescence microscopy as a tool for subcellular imaging of isotopically labeled platinum-based anticancer drugs.

Multi-elemental, isotope selective nano-scale secondary ion mass spectrometry (NanoSIMS) combined with confocal laser-scanning microscopy was used to characterize the subcellular distribution of 15N-labeled cisplatin in human colon cancer cells. These analyses indicated predominant cisplatin colocalisation with sulfur-rich structures in both the nucleus and cytoplasm. Furthermore, colocalisation of platinum with phosphorus-rich chromatin regions was observed, which is consistent with its binding affinity to DNA as the generally accepted crucial target of the drug. Application of 15N-labeled cisplatin and subsequent measurement of the nitrogen isotopic composition and determination of the relative intensities of platinum and nitrogen associated secondary ion signals in different cellular compartments with NanoSIMS suggested partial dissociation of Pt-N bonds during the accumulation process, in particular within nucleoli at elevated cisplatin concentrations. This finding raises the question as to whether the observed intracellular dissociation of the drug has implications for the mechanism of action of cisplatin. Within the cytoplasm, platinum mainly accumulated in acidic organelles, as demonstrated by a direct combination of specific fluorescent staining, confocal laser scanning microscopy and NanoSIMS. Different processing of platinum drugs in acidic organelles might be relevant for their detoxification, as well as for their mode of action.

Cell-specific analysis of the tomato pollen proteome from pollen mother cell to mature pollen provides evidence for developmental priming.

Tomato is a globally important crop grown and consumed worldwide. Its reproductive activity is highly sensitive to environmental fluctuations, for instance temperature and drought. Here, pollen development is one of the most decisive processes. The present study aims for the identification of cell-specific proteins during pollen developmental stages of tomato. We have setup a protocol for stage-specific pollen isolation including microsporocytes (pollen mother cells), tetrads, microspores, polarized microspores, and mature pollen. Proteins were extracted using phenol and prefractionated using SDS-PAGE followed by protein digestion, peptide extraction, and desalting. Identification and quantification of proteins were performed using nanoHPLC coupled to LTQ-Orbitrap-MS. In total, 1821 proteins were identified. Most of these proteins were classified based on their homology and designated functions of orthologs. Cluster and principal components analysis revealed stage-specific proteins and demonstrated that pollen development of tomato is a highly controlled sequential process at the proteome level. Intermediate stages such as tetrad and polarized microspore are clearly distinguished by different functionality compared to other stages. From the predicted functions, energy-related proteins are increased during the later stages of development, which indicates that pollen germination depends upon presynthesized proteins in mature pollen. In contrast, heat stress-related proteins are highly abundant in very early developmental stages, suggesting a dominant role in stress protection. Taken together, the data provide a first cell-specific protein reference set for tomato pollen development from pollen mother cells to the mature pollen and give evidence for developmentally controlled processes that might help to prepare the cells for specific developmental programs and environmental stresses.