Sections Of Plant Tissues Research Articles

Detection of cytokinins and auxin in plant tissues using histochemistry and immunocytochemistry

We report a new method for histochemical localization of cytokinins (CKs) in plant tissues based on bromophenol blue/silver nitrate staining. The method was validated by immunohistochemistry using anti-trans-zeatin riboside antibody. Indole-3-acetic acid (auxin, IAA) was localized by anti-IAA antibody in plant tissues as a proof for IAA histolocalization. We used root sections, because they are major sites of CKs synthesis, and insect galls of Piptadenia gonoacantha that accumulate IAA. Immunostaining confirmed the presence of zeatin and sites of accumulation of IAA indicated by histochemistry. The colors developed by histochemical reactions in free-hand sections of plant tissues were similar to those obtained by thin layer chromatography (TLC), which reinforced the reactive sites of zeatin. The histochemical method for detecting CKs is useful for galls and roots, whereas IAA detection is more efficient for gall tissues. Therefore, galls constitute a useful model for validating histochemical techniques due to their rapid cell cycles and relatively high accumulation of plant hormones.

Tracing the fate and transport of secondary plant metabolites in a laboratory mesocosm experiment by employing mass spectrometric imaging

Mass spectrometric imaging (MSI) has received considerable attention in recent years, since it allows the molecular mapping of various compound classes, such as proteins, peptides, glycans, secondary metabolites, lipids, and drugs in animal, human, or plant tissue sections. In the present study, the application of laser-based MSI analysis of secondary plant metabolites to monitor their transport from the grass leaves of Dactylis glomerata, over the crop of the grasshopper Chorthippus dorsatus to its excrements, and finally in the soil solution is described. This plant-herbivore-soil pathway was investigated under controlled conditions by using laboratory mesocosms. From six targeted secondary plant metabolites (dehydroquinic acid, quinic acid, apigenin, luteolin, tricin, and rosmarinic acid), only quinic acid, and dehydroquinic acid, an in-source-decay (ISD) product of quinic acid, could be traced in nearly all compartments. The tentative identification of secondary plant metabolites was performed by MS/MS analysis of methanol extracts prepared from the investigated compartments, in both the positive and negative ion mode, and subsequently compared with the results generated from the reference standards. Except for tricin, all secondary metabolites could be tentatively identified by this approach. Additional liquid-chromatography mass spectrometry (LC-MS) experiments were carried out to verify the MSI results and revealed the presence of quinic acid only in grass and chewed grass, whereas apigenin-hexoside-pentoside and luteolin-hexoisde-pentoside could be traced in the grasshopper body and excrement extracts. In summary, the MSI technique shows a trade-off between sensitivity and spatial resolution.Graphical abstractMonitoring quinic acid in a mesocosm experiment by mass spectrometric imaging (MSI).

In situ hybridization for the detection of rust fungi in paraffin embedded plant tissue sections.

BackgroundRust fungi are obligate pathogens with multiple life stages often including different spore types and multiple plant hosts. While individual rust pathogens are often associated with specific plants, a wide range of plant species are infected with rust fungi. To study the interactions between these important pathogenic fungi and their host plants, one must be able to differentiate fungal tissue from plant tissue. This can be accomplished using the In situ hybridization (ISH) protocol described here.ResultsTo validate reproducibility using the ISH protocol, samples of Chrysanthemum × morifolium infected with Puccinia horiana, Gladiolus × hortulanus infected with Uromyces transversalis and Glycine max infected with Phakopsora pachyrhizi were tested alongside uninfected leaf tissue samples. The results of these tests show that this technique clearly distinguishes between rust pathogens and their respective host plant tissues.ConclusionsThis ISH protocol is applicable to rust fungi and potentially other plant pathogenic fungi as well. It has been shown here that this protocol can be applied to pathogens from different genera of rust fungi with no background staining of plant tissue. We encourage the use of this protocol for the study of plant pathogenic fungi in paraffin embedded sections of host plant tissue.

Direct analysis of elemental biodistribution in pea seedlings by LA-ICP-MS, EDX and confocal microscopy: Imaging and quantification

Qualitative and quantitative methods were developed for cadmium, lead, cooper and zinc mapping and quantification in root of pea Pisum sativum L. Plants were cultivated hydroponically in a Hoagland medium with the addition of 50μM of: CdCl2, Pb(NO3)2, CuSO4, or ZnSO4. After 48h, parts of roots were cut and analysed. LA-ICP-MS was used for in vivo quantitative imaging of the distribution of metals in thin sections of plant tissues. Calibration curve was obtained using standard addition method NIST SRM 1515 Apple Leaves spiked with standard and application of sulfur isotope as an internal standard. Energy Dispersive X-ray microanalysis Spectroscopy (EDX) was used in order to confirm results obtained by LA-ICP-MS and identify the exact localization of metals deposits in root tissue. In the electron-microscopic photographs we observed deposits of metals in the cell wall, cell membrane, vacuoles, cytoplasm and organelles, such as: mitochondria and peroxisomes. Formation and localization of O2− and H2O2 in cross-sections of root tips was confirmed by confocal microscopy. The presence of metals induced ROS (reactive oxygen species: O2−, H2O2) production in areas corresponding to patterns of metals accumulation.

Synchronization-free all-solid-state laser system for stimulated Raman scattering microscopy.

We introduce an extremely simple and highly stable system for stimulated Raman scattering (SRS) microscopy. An 8-W, 450-fs Yb:KGW bulk oscillator with 41 MHz repetition rate pumps an optical parametric amplifier, which is seeded by a cw tunable external cavity diode laser. The output radiation is frequency doubled in a long PPLN crystal and generates 1.5-ps long narrowband pump pulses that are tunable between 760 and 820 nm with >50 mW average power. Part of the oscillator output is sent through an etalon and creates Stokes pulses with 100 mW average power and 1.7 ps duration. We demonstrate SRS microscopy at a 30-μs pixel dwell time with high chemical contrast, signal-to-noise ratio in excess of 45 and no need for balanced detection, thanks to the favorable noise properties of the bulk solid-state system. Cw seeding intrinsically ensures low spectral drift. We discuss its application to chemical contrast microscopy of freshly prepared plant tissue sections at different vibrational bands.

Multiplex MALDI-MS imaging of plant metabolites using a hybrid MS system.

Plant tissues present intriguing systems for study by mass spectrometry imaging, as they exhibit a complex metabolism and a high degree of spatial localization. This chapter presents a methodology for preparation of plant tissue sections for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) analysis and the use of a hybrid mass spectrometer for "multiplex" imaging. The multiplex method described here provides a wide range of analytical information, including high-resolution, accurate mass imaging and tandem MS scans for structural information, all within a single experiment. While this procedure was developed for plant tissues, it can be readily adapted for analysis of other sample types.

A microautoradiographic method for fresh-frozen sections to reveal the distribution of radionuclides at the cellular level in plants.

Microautoradiography (MAR) is a conventional imaging method based on the daguerreotype. The technique is used to visualize the distribution of radionuclide-labeled compounds within a tissue section. However, application of the classical MAR method to plant tissue sections is associated with several difficulties. In this study, we report an MAR method applicable to fresh-frozen plant sections. Our method had two features: (i) the sample was kept frozen from plant tissue collection to radioisotope detection, making it possible to fix solutes without solvent exchange; and (ii) 1.2 µm thick polyphenylene sulfide film was inserted between the fresh-frozen plant section and the photosensitive nuclear emulsion to separate the section from the emulsion before autoradiography was conducted, which significantly improved the quality of the section until microscopic detection, the quality of the MAR image and the success rate. Then, the passage of cadmium (Cd) through vegetative rice stem tissue after 24 h of (109)Cd absorption was described for the first time using the MAR method. MAR clearly revealed the distribution of (109)Cd at the tissue level with high resolution. The (109)Cd concentration in phloem cells was found to be particularly high, whereas the xylem cells contained only small amounts of (109)Cd. The MAR method was also applicable for detecting (109)Cd and [(33)P]phosphate in roots. The MAR method developed here is expected to provide distribution images for a variety of compounds and ions in plant tissue.

Top‐down mass spectrometry imaging of intact proteins by laser ablation ESI FT‐ICR MS

Laser ablation ESI (LAESI) is a recent development in MS imaging. It has been shown that lipids and small metabolites can be imaged in various samples such as plant material, tissue sections or bacterial colonies without any sample pretreatment. Further, LAESI has been shown to produce multiply charged protein ions from liquids or solid surfaces. This presents a means to address one of the biggest challenges in MS imaging; the identification of proteins directly from biological tissue surfaces. Such identification is hindered by the lack of multiply charged proteins in common MALDI ion sources and the difficulty of performing tandem MS on such large, singly charged ions. We present here top-down identification of intact proteins from tissue with a LAESI ion source combined with a hybrid ion-trap FT-ICR mass spectrometer. The performance of the system was first tested with a standard protein with electron capture dissociation and infrared multiphoton dissociation fragmentation to prove the viability of LAESI FT-ICR for top-down proteomics. Finally, the imaging of a tissue section was performed, where a number of intact proteins were measured and the hemoglobin α chain was identified directly from tissue using CID and infrared multiphoton dissociation fragmentation.

A Cryohistological Protocol for Preparation of Large Plant Tissue Sections for Screening Intracellular Fluorescent Protein Expression

In this study, we have developed a robust cryohistological method that allows imaging of virtually any type of plant cell or tissue while preserving fluorescent protein signals and maintaining excellent cellular and subcellular morphology. This method involves modified fixation of plant tissues (i.e., leaves, stems, and petioles), infiltration in a sucrose gradient, freezing, and collection of cryosections directly onto a cryoadhesive tape. Using this method followed by microscopic analysis, we demonstrated a localized accumulation of green fluorescent protein (GFP) in Nicotiana benthamiana plants agroinfiltrated with the movement-incompetent tobacco mosaic virus-based vector and systemic accumulation of GFP in plants infiltrated with the movement-competent vector. Overall, this simple cryohistological procedure reduced sample preparation time and allowed processing of tissue sections for high-resolution imaging of targeted fluorescent proteins in all plant tissues.

Feeding behavior and spatial distribution of two planthoppers, Megamelus scutellaris (Delphacidae) and Taosa longula (Dictyopharidae), on water hyacinth

Megamelus scutellaris Berg (Delphacidae) and Taosa (Cuernavaca) longula Remes Lenicov (Dictyopharidae) are specialist planthoppers that feed and reproduce on the invasive aquatic weed, Eichhornia crassipes (Martius) Solms-Laubach (Pontederiaceae). They overlap geographically in several regions of South America and may, therefore, interact and compete for food and microhabitat. Preliminary observations indicated that both species do not feed on the same part of the plant. We hypothesized that they partition the resource; hence, we studied (1) the feeding mechanism at the tissue level and (2) the spatial distribution of both species on the water hyacinth plant. Salivary sheaths were detected through histological sections of plant tissues using light microscopy. The location of either planthopper species on the plant was recorded when in the presence or absence of the other species. Both species produced true salivary sheaths, mostly branched (M. scutellaris: 82%; T. longula: 84%), ending in phloem (M. scutellaris: 56%; T. longula: 52%), and xylem tissues (M. scutellaris: 24%; T. longula: 28%). They resided on different parts of the water hyacinth plant even when they did not coexist; nymphs of T. longula occurred primarily on the back side of the leaf laminas, while nymphs of M. scutellaris occupied the basal zone of the petioles. This study shows that these planthoppers complement each other and could be used in combination as control agents for water hyacinth. Further experimental studies and field observations are necessary to quantify interactions.

Analysis and modification of surfaces using molecular ions in the ambient environment

The analysis and modification of surfaces in their native conditions can be performed using new mass spectrometric methods. Ambient ionization sources, including desorption electrospray ionization (DESI), have been implemented for the rapid analysis of unmodified biological surfaces including whole plant material, tissue sections, algae, and bacterial colonies. Recent advances have shown promise for in vivo and high-throughput clinical analysis. Additionally, the recent development of ambient ion soft landing (SL) allows polyatomic ions to be deposited onto surfaces in open air. Ambient SL offers speed, control, and flexibility for surface reactions and modification.

Atomic Force Microscope Observation on Ultrastructures in Plant Cells

AFM is being applied in increasingly wide research fields and extracting more biochemical/biophysical information that is beyond the capability of traditional SEM and TEM. Due to its inherent features, AFM is rarely used to observe the subcellular details within cells. Although subcellular features were recently observed on thin sections of plant tissues using AFM, this method might introduce unexpected artifacts during sample processing. Here we try to observe plant cells still embedded in resin block. This modified method minimizes the possibility of artifacts. The comparison among outcomes of AFM, SEM, TEM and LM on the same single cell suggest that this modified method is a good, applicable, efficient and faithful way applying AFM on biological materials.

Direct sample preparation methods for the detection of Plum pox virus by real-time RT-PCR. Validation and practice parameters

Direct systems to process plant materials allowed high-throughput testing of Plum pox virus (PPV) by real-time reverse transcription (RT)-PCR without nucleic acids purification. Crude plant extracts were diluted in buffer or spotted on membranes to be used as templates. Alternatively, immobilized PPV targets were amplified from fresh sections of plant tissues printed or squashed onto the same supports, without extract preparation. Spot real-time RT-PCR was validated as a PPV diagnostic method in samples collected during the dormancy period and showed high sensitivity (93.6%), specificity (98.0%), and post-test probability (97.9%) towards sharka disease. In an analysis of 2919 Prunus samples by spot real-time RT-PCR and DASI-ELISA 90.8% of the results coincided, demonstrating high agreement (k = 0.77 ± 0.01) between the two techniques. These results validate the use of immobilized PPV targets and spot real-time RT-PCR as screening method for largescale analyses. [Int Microbiol 2009; 12(1):1-6].

A method for obtaining high quality RNA from paraffin sections of plant tissues by laser microdissection

Laser microdissection (LM) combined with microarray analysis or next-generation sequencing of cDNA is a powerful tool for understanding molecular events in individual cell types of plants as well as animals. Obtaining high quality RNA is essential for this approach. For plant tissues, paraffin-embedded sections better preserve cell structure than do frozen sections. However, the conventional method for preparing paraffin sections is a lengthy process involving embedding the tissue and floating and drying the sections, during which time RNA degradation occurs. Here, we describe a method for preparing serial sections that greatly reduces RNA degradation: we reduced (1) the embedding time from 4-6days to about 5h by using a recently developed microwave method; (2) the time of floating sections from ~10min to less than 5min, (3) the drying time from ~12 to 1h; and (4) the drying temperature from 42 to 4°C. With this method, we were able to isolate higher integrity RNA from many kinds of plant tissues than is typically obtained by the conventional paraffin preparation method. The improvement in RNA quality and yield removes a major obstacle to the widespread use of LM with high-throughput technologies for plants.

The crystal structure of XG‐34, an evolved xyloglucan‐specific carbohydrate‐binding module

Proteins: Structure, Function, and BioinformaticsVolume 78, Issue 3 p. 785-789 Structure Note The crystal structure of XG-34, an evolved xyloglucan-specific carbohydrate-binding module Fredrika Gullfot, Fredrika Gullfot Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, Stockholm, SwedenSearch for more papers by this authorTien-Chye Tan, Tien-Chye Tan Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, Stockholm, SwedenSearch for more papers by this authorLaura von Schantz, Laura von Schantz Department of Immunotechnology, Lund University, Lund, SwedenSearch for more papers by this authorEva Nordberg Karlsson, Eva Nordberg Karlsson Department of Biotechnology, Lund University, Lund, SwedenSearch for more papers by this authorMats Ohlin, Mats Ohlin Department of Immunotechnology, Lund University, Lund, SwedenSearch for more papers by this authorHarry Brumer, Harry Brumer Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, Stockholm, SwedenSearch for more papers by this authorChristina Divne, Corresponding Author Christina Divne divne@biotech.kth.se Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, Stockholm, SwedenSchool of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, S-106 91 Stockholm, Sweden===Search for more papers by this author Fredrika Gullfot, Fredrika Gullfot Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, Stockholm, SwedenSearch for more papers by this authorTien-Chye Tan, Tien-Chye Tan Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, Stockholm, SwedenSearch for more papers by this authorLaura von Schantz, Laura von Schantz Department of Immunotechnology, Lund University, Lund, SwedenSearch for more papers by this authorEva Nordberg Karlsson, Eva Nordberg Karlsson Department of Biotechnology, Lund University, Lund, SwedenSearch for more papers by this authorMats Ohlin, Mats Ohlin Department of Immunotechnology, Lund University, Lund, SwedenSearch for more papers by this authorHarry Brumer, Harry Brumer Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, Stockholm, SwedenSearch for more papers by this authorChristina Divne, Corresponding Author Christina Divne divne@biotech.kth.se Division of Glycoscience, School of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, Stockholm, SwedenSchool of Biotechnology, Royal Institute of Technology (KTH), AlbaNova University Centre, S-106 91 Stockholm, Sweden===Search for more papers by this author First published: 26 October 2009 https://doi.org/10.1002/prot.22642Citations: 4Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume78, Issue315 February 2010Pages 785-789 RelatedInformation

Direct sample preparation methods for the detection of Plum pox virus by real-time RT-PCR.

Direct systems to process plant materials allowed high-throughput testing of Plum pox virus (PPV) by real-time reverse transcription (RT)-PCR without nucleic acids purification. Crude plant extracts were diluted in buffer or spotted on membranes to be used as templates. Alternatively, immobilized PPV targets were amplified from fresh sections of plant tissues printed or squashed onto the same supports, without extract preparation. Spot real-time RT-PCR was validated as a PPV diagnostic method in samples collected during the dormancy period and showed high sensitivity (93.6%), specificity (98.0%), and post-test probability (97.9%) towards sharka disease. In an analysis of 2919 Prunus samples by spot real-time RT-PCR and DASI-ELISA 90.8% of the results coincided, demonstrating high agreement (k = 0.77 +/- 0.01) between the two techniques. These results validate the use of immobilized PPV targets and spot real-time RT-PCR as screening method for largescale analyses.

Elaboration of the method for immunocytochemical studying cytokinin-binding protein CBP70 localization in maize seedling tissues

Some approaches for the elaboration of the method for immunocytochemical detection of labile protein on sections prepared from maize (Zea mays L.) seedling tissues are outlined. The effects of severe chemical reagents applied during tissue pretreatment (fixatives, inhibitors of endogenous enzymes, solutions blocking nonspecific antibody binding) on labile protein immunoreactivity in extracts were assessed in preliminary experiments. The procedure was elaborated with a triple control, which provided for the lowest background staining on plant tissue sections and the high level of antibody specific binding.

Nonradioactive In Situ Hybridization of RNA Probes to Sections of Plant Tissues

INTRODUCTIONBoth radioactive and nonradioactive probes can be used to detect RNA in situ. Radioactive in situ hybridizations are believed by many to be more sensitive, but the localization of signal is considerably less precise than for nonradioactive in situs, and each experiment takes much longer. Thus, nonradioactive in situ hybridization is generally preferred. Antisense RNA is typically labeled using digoxygenin (DIG), which is subsequently detected with an anti-DIG antibody coupled to alkaline phosphatase. This protocol describes in situ hybridization of a DIG-labeled probe to paraffin-embedded sections of plant tissue. Double labeling using both DIG- and fluorescein isothiocyanate (FITC)-conjugated ribonucleotides is also described.

Immunohistochemistry on Sections of Plant Tissues Using Enzyme-Coupled Avidin-Biotin Complex

INTRODUCTIONLike RNA, proteins can be localized in situ on tissue sections. In situ localization or immunohistochemistry provides information on the cellular and subcellular distribution of proteins in different tissues. The technique described in this protocol employs a biotinylated secondary antibody in conjunction with a preformed Avidin:Biotinylated enzyme Complex (ABC) for immunohistochemistry. It allows for sensitive detection of antigens while avoiding fluorescent secondary antibodies, which produce a much less stable signal. Formation of the complex is achieved by mixing avidin and biotinylated enzyme in dilute solution and in defined amounts prior to use. The complex is then allowed to bind to biotinylated secondary antibodies, thus linking the antigen via primary and secondary antibodies to the enzyme used to generate the signal. Kits for this procedure are available from Vector Laboratories. The VECTASTAIN ABC kits are of three types, using the enzymes horseradish peroxidase, alkaline phosphatase, or glucose oxidase. This protocol assumes use of the peroxidase kit, but can be easily modified for the other kits.

Immunohistochemistry on Sections of Plant Tissues Using Alkaline-Phosphatase-Coupled Secondary Antibody

INTRODUCTIONLike RNA, proteins can be localized in situ on tissue sections. In situ localization or immunohistochemistry provides information on the cellular and subcellular distribution of proteins in different tissues. This protocol describes an alternative to the use of ABC (a preformed Avidin:Biotinylated enzyme Complex used in conjunction with a biotinylated secondary antibody) for non-fluorescent detection in immunohistochemistry. Here, an alkaline-phosphatase-coupled secondary antibody is used with colorimetric substrates for antigen detection.