Demonstration of Phenolic Compounds in Plant Tissues by An Osmium-Iodide Postfixation Procedure

A simple procedure to stain phenols in plant tissues is described, Postfixation with an aqueous solution prepared by mixing 2 cc of 2% osmium tetroxide and 8 cc of 3% potassium iodide yields brilliant visualization of phenol-containing vacuoles in different tissues of plants (e.g. coffee, oak, tobacco and spruce) bearing high concentration of phenolic compounds. Areas bearing phenols become dark gray to black. Chemical experiments demonstrate that osmium-potassium iodide (Os-KI) mixture reacts rapidly with several naturally occurring plant phenols, developing black solutions from which black solids precipitate. Phenols containing o-dihydroxy groups react with Os-KI solution more rapidly than other structurally different phenols. Therefore, o-dihydroxy units in an aromatic ring seem to function as primary sites of reactivity with the osmium-iodide complexes.     

Lipids and Lipoprotein Complex in Photosynthetic Tissues

The lipids and pigments of corn and soybean leaves grown in light and in dark were examined. The green leaf contained appreciably greater amounts of the two galactolipids, sulfolipid, phosphatidyl glycerol and diphosphatidyl glycerol, while the etiolated leaf had relatively more cerebroside, sterol glucoside, phosphatidyl ethanolamine and lecithin. The prominent component of glycerides was 1,2-diglyceride in green leaf, and triglyceride in etiolated leaf. The green leaf lipids showed a remarkable specificity of component fatty acids, but those of etiolated leaf only at lower rate. From the results, possible functions of the lipids were discussed.     

Lipids and Lipoprotein Complex in Photosynthetic Tissues

Anacystis nidulans cells were fractionated, and characterized. The Hill reaction activity of the lamellar fractions depended at a considerable degree on the media used in cell fractionations and on the freshness of the cells. Analytical results suggest that two types of lamellae are present in the cells. One existing in the cell in a large amount, showed higher content of chlorophyll and lower carotenoids. The other which seems to be located near cell membrane, had carotenoids in relatively higher concentration, and contained probably a chlorophyll absorbing far-red light in addition to a common chlorophyll a.     

Lipids and Lipoprotein Complex in Photosynthetic Tissues

Lipids and pigments of photosynthetic bacteria, Rhodospirillum rubrum and Rhodopseudomonas capsulatus were examined. Common and prominent lipids in both bacteria were phosphatidyl ethanolamine and phosphatidyl glycerol. Rhodospirillum rubrum contained a special lipid containing ornithine. Their component fatty acids were straight chain saturated and monoenoic acids. No glycolipids were found in both bacteria. Ubiquinone-50 was detected in large amounts in both bacteria, and a new quinone and rhodoquinone were found in Rhodospirillum rubrum. The major carotenoids were spirilloxanthin, lycopene, and probably rhodopin. The results were compared with those of spinach and Anacystis, and discussed.     

Localization of Plant Lipids for Light Microscopy Using P-Phenylenediamine in Tissues of Arachis Hypogaea L.

p-Phenylenediamine (pPD) can be used en bloc to preserve and differentiate cell lipids in aldehyde-fixed peanut plant tissues treated with osmium tetroxide during dehydration in 70% ethanol. Semithin plastic sections for light microscopy need o further staining and can be mounted in Histoclad after drying on a slide. Brown staining above background differentiates lipid-containing structures. Nonspecific staining can be distinguished in control preparations extracted en bloc with lipid solvents.     

Membrane Lipids in Senescing Green Tissues2

At a late stage in sensescence cucumber cotuledons lose freshweight rapidly; at the same time there is an increase in apparentfree space and large quantities of electrolyte leak out whendiscs of cotyledon tissue are floated on water. It is concludedthat tonoplast and plasma membrane become leaky at this time. Phosphatidyl choline, the major phospholipid present, beginsto disappear once the cotyledons reach maximum fresh weight;by the time rapid water loss starts, 56 per cent has gone, andphosphatidyl ethanolamine and inositol start to disappear. Onlyat maturity is there enough of these phospholipids to furnishmore than two complete membranes around each cell; it is suggestedthat the decline in phospholipid level at senescence destroysmembrane integrity and allows leakage. The glyohpids begin to disappear at the same time as chlorophyll,2 weeks before weight loss starts. The minor lipid phosphatidyl glycerol is the first to disappearfrom the cotyledons.     

Mordanting Plant Tissues

Selected current ideas on the mordanting of plant tissues are presented in the hope that they will be of practical assistance, especially to the beginner. The nature of the mordanting process and the results which may be expected following mordanting are briefly described. Specific technics are presented for the mordanting of natural dyes, synthetic dyes generally, the basic synthetic dyes and the acid synthetic dyes.     

Histochemical Demonstration of Acid Phosphatase in Hard Tissues

The action of the following decalcifying solutions for the demonstration of acid phosphatase has been studied: buffer solution acetic-acetate 0.05 M, pH 5; 2, 5, 10, 20, and 50% formic acid and 20% sodium citrate in equal parts (pH 2.6, 3, 3.8, 4.2, and 5); 0.5 M citric acid-NaOH, pH 4.2; Versene solution, 5%, pH 7. A comparative study of fixatives has been made also (neutral formalin, 10-20%; formalin-chloral hydrate (Fishman), acetone and 80% alcohol). The best results were obtained with fixation at 4°C in 10-20% neutral formalin or formalin-chloral hydrate, for a period of 24 hr, and decalcification with 20% sodium citrate, 5% formic acid, in equal parts, pH 4.2, which can act on both specimens or sections for a period up to 2 wk with very little loss of enzyme. It is not necessary to reactivate the enzyme after decalcification; frozen sections should be used and should be washed in distilled water before proceeding with the demonstration of the enzyme (Gomori's method or azo dye coupling). Other fixatives (acetone and alcohol) and paraffin embedding produce a greater loss in enzymes and very irregular results.     

Freeze-Sectioning of Plant Tissues

Techniques are described for freeze-sectioning a wide range of both fresh and fixed plant tissues. Gelatin-antifreeze media are used to support but not infiltrate the tissue during sectioning. At cryostat temperatures of -10 to -15 C, 15% gelatin (w/v) containing 0.8% dimethyl sulfoxide (DMSO), or 1.5% ethanediol (ethylene glycol), or 2% glycerol is used. Lower concentrations of gelatin and higher concentrations of antifreezes are required for sectioning at -24 C. Petri plates of media are stored at 2 C, and used by simply melting a hole in the medium. Fresh tissues can be placed directly in the hole, or prefrozen at temperature of liquid nitrogen, or equilibrated in antifreeze solution, before freeze-sectioning in the gelatin antifreeze medium. Many plant tissues have highly vacuolated cells and need equilibration in antifreeze solutions prior to freeze-sectioning. Fixed tissues are rehydrated and washed in water or buffer for 15-24 hr before equilibrating in a 10% solution of either DMSO, ethanediol or glycerol (named in order of rapidity of equilibration). Pretreatment in 10% DMSO is usually for 1-6 hr at 2 C for histochemical studies; or in 10% ethanediol or glycerol for 15-24 hr at either room temperature or 37 C for morphological studies. These methods permit serial cryostat sections free from freezing and thawing artifacts to be cut as thin as 2 μ.     

Metabolic Engineering of Plant Lipids

Lipid metabolism in plants provides uncommon opportunities for genetic engineering to produce plant oils suited to a variety of end-uses. These opportunities include improvement of food and nutritional value, creating specialty lipids and feedstocks for high-value products and designing custom-made materials for industry. Genetic engineering intervention for production of novel transgenic plants which elaborate the desired product has graduated from academic exercise to commercial possibilities. It is now realized that transgenic crops can serve as biological factories for upscaling production of premium lipids via molecular farming. This review is an attempt at analyzing the status in this field.     

Ethanolamine Metabolism in Plant Tissues

Ethanolamine is readily metabolized by oat, pea, wheat, apple and carrot tissue preparations. Ethanolamine-1,2 (14)C was incorporated into the lipid fraction, and (14)C activity was distributed in the organic acid, sugar, acid volatile, carbon dioxide and insoluble residue fractions. The distribution varied with the particular tissue. Incorporation into the lipid fraction occurred in tissue homogenates in the absence of ATP by a Ca(++) activated system similar to that reported for animal preparations. The initial step in ethanolamine oxidation involves an amine oxidase. Glycolaldehyde and glyoxylic acid are metabolic intermediates, the former in the conversion of ethanolamine to carbon dioxide. No evidence was obtained for the operation of an ethanolamine transaminase or for the involvement of phosphorylated intermediates in the conversion of ethanolamine to carbon dioxide.     

Electroosmotic Phenomena in Plant Tissues

The effect of a direct electric current on electrolyte transport through plant tissues was studied by applying it to 10-mm segments of the mesocotyls of etiolated maize seedlings, similar segments of one-year linden shoots with the normal conducting system and without vascular bundles, and isolated elements of the xylem and cell wall segments. At current densities of 9–38 A/mm² (10–20 V), electrolyte solutions in plant tissues always moved toward the cathode. The results suggest that electroosmosis is one of the factors responsible for changes in solution transport through the conductive plant tissues that occur under the effect of electric current.     

Histological Methods for the Demonstration of Gold in Tissues

Two methods for the demonstration of gold in tissues are described. The tissue is fixed in neutral formalin, embedded in paraffin, sectioned and run down to water. In the SnCl₂ method, modified from that of Christeller, it is then incubated for 24 hours at 56° C. in a mixture of ten parts of 5% SnCl₂·2H₂O and one part of concentrated HCl. The interpretation of the results obtained by this method is frequently difficult because of the presence of accessory precipitates and the presence of the normal pigments of the tissue. This has led to the development of a new method, in which the sections are incubated for periods varying from 24 hours to 6 days at 37° C. in 3% H₂O₂. The gold is reduced to the metallic state, the interfering tissue pigments are bleached, and, since no metallic ions have been added, accessory precipitates do not occur. After both methods, the sections are washed thoroughly, run up, and mounted in damar.     

Softening Paraffin-Embedded Plant Tissues

Soaking paraffin-embedded plant specimens 2-3 days at 37°C. in a mixture of glycerol, 10 ml., Dreft, 1 g., and water 90 ml. is an effective means of softening them prior to sectioning. One side of the paraffin block must be pared away to expose the tissue before immersion in the softening solution.     

类脂对植物生长和发育的作用

简述了类脂在植物生长和发育中的作用,特别是类脂中脂肪酸的饱和度对植物生长发育的影响,植物固醇对植物的表型和在低温下的生长、胚胎的发育以及可育性中的作用.     

Freezing-Sectioning of Plant Tissues1

An improved and simple method is described by which serial 10µ frozen sections of plant tissues may be obtained. Thevalue of this method for use in plant histochemistry is discussed.