A simple and efficient protocol for isolation of functional RNA from plant tissues rich in secondary metabolites

A protocol is described for rapid RNA isolation from various plant species and tissues rich in polyphenolics and polysaccharides. The method is based on the Nucleon PhytoPure^™ system without the use of phenol. The procedure can be completed within 1 h and many samples can be processed at the same time. The yield ranged from 240 μg up to 3 mg per gram of tissue with an average purity measured as A_260/280 of 1.85. The RNA was of sufficient quality for use in RT-PCR reactions. Quantitation of single-stranded cDNA was carried out with the RiboGreen^™ reagent and of PCR products with the PicoGreen^™ reagent.     

RNA extraction from plant tissues

Several protocols and commercial kits are used for the extraction of nucleic acids from different plant tissues. Although there are several procedures available to remove sugars, which hinder the extraction of clean genomic DNA, there are few to assist with extraction of RNA. Those presently used include precipitations with ethylene glycol monobutyl ether or lithium chloride (LiCl), or centrifugation in cesium chloride (CsCl) gradients, but these generally either do not allow high recovery of RNA, are time consuming, rely on hazardous chemicals or need special equipment. Here we present the use of the simple cation, Ca²⁺, which has been tested and shown to be very efficient for the precipitation of high molecular weight pectic sugars during RNA extraction. Results are presented for different plant tissues, especially tissues of peach and apple fruits at varying ripening stages.     

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 omicron-dihydroxy groups react with Os-KI solution more rapidly than other structurally different phenols. Therefore, omicron-dihydroxy units in an aromatic ring seem to function as primary sites of reactivity with the osmium-iodide complexes.     

Isolation of microbodies from plant tissues

Specialized microbodies have previously been isolated and characterized from fatty seedling tissues (glyoxysomes) and leaves (leaf peroxisomes). We have now examined 11 other plant tissues, including tubers, fruits, roots, shoots, and petals, and find that all contain particulate catalase, a distinctive common enzyme component of microbodies. On linear sucrose gradients the catalase activity peaks sharply at a higher equilibrium density (1.20 to 1.25 gram per cm³ in the various tissues) than the mitochondria (1.17 to 1.20). Only small amounts of protein are recovered in the fractions containing catalase, although a definite band is visible in preparations from some tissues, e.g., potato. As in the preparations from castor bean endosperm and spinach leaves for which comparable data are provided, the distribution of glycolate oxidase and uricase follows closely that of catalase on the gradients. The preparations from potato lack glyoxylate reductase and the transaminases, typical enzymes of leaf peroxisomes, and the distinctive enzymes of glyoxysomes are missing. Nonspecialized microbodies with limited enzyme composition can thus be isolated from a variety of plant tissues.     

Factors affecting the extraction of intact ribonucleic Acid from plant tissues containing interfering phenolic compounds

Using conventional methods it is impossible to extract RNA as uncomplexed intact molecules from the leaves of grapevines (Vitis vinifera L.) and from a number of woody perennial species that contain high levels of reactive phenolic compounds. A procedure involving the use of high concentrations of the chaotropic agent sodium perchlorate prevents the binding of phenolic compounds to RNA during extraction. Analyses of the phenolics present in plant tissues used in these experiments indicate that there is a poor correlation between the total phenolic content and the complexing of RNA. However, qualitative analyses suggest that proanthocyanidins are involved in the tanning of RNA during conventional extractions.     

Isolation of functional RNA from small amounts of different grape and apple tissues

An efficient, simple, and small-scale procedure for isolating functional ribonucleic acid (RNA) was successfully applied to many different tissues of grape and apple. These woody plants are rich in polyphenolic compounds and polysaccharides that could impair the RNA extraction. The method chosen is based on the use of hot borate buffer at alkaline pH supplemented with several adjuvants and followed by selective precipitations. Starting with only 0.4 g of fresh tissue and working with small tubes (2 mL), we were able to obtain good yields of high-quality RNA suitable for further applications. The procedure can be proposed for many applications, and it is particularly highly recommended when isolating RNA from a large number of samples.     

Isolation of functional RNA from cactus fruit

Isolating RNA from cactus fruit is notoriously difficult because the fruit contains high amounts of secondary metabolites and polysaccharides. These form insoluble complexes with nucleic acids during extraction and can inhibit enzyme action. Our procedure allows for the extraction of RNA from finely ground tissue. The RNA we isolated was of high quality and undegraded, as gauged by spectrophotometry and electrophoresis in agarose gels. Quality was further assessed through use of the RNA in RT-PCR and northern blot analysis, indicating that it could be used to construct cDNA libraries. Using this modified protocol, 90μg of RNA was routinely obtained from 1 g of dried cactus fruit. Isolating RNA from other polysaccharide-rich fruits was also possible.     

Isolation of intact plastids from a range of plant tissues

A technique for the isolation of intact plastids from spinach (Spinacia oleracea) and pea (Pisum sativum) leaves, pea roots and castor bean (Ricinus communis) endosperm is described. This technique involves brief centrifugation of whole homogenates on density gradients. Intact plastids were located in the gradient by assaying for triose phosphate isomerase activity. Contamination of the plastic peak with mitochondria and microbodies was estimated by measurement of cytochrome oxidase and catalase, respectively. For three of the four tissues the level of contamination of the plastids by these organelles was 2% or less. The sedimentation behavior of microbodies from different tissues is discussed.     

Isolation of bacteria of the family enterobacteriaceae from plant tissues

Bacteria of the family Enterobacteriaceae were isolated from the tissues of a number of wild and cultivated plants. All the cultures isolated had a broad spectrum of resistance to antibiotics and were highly adhesive to human erythrocytes. The studies conducted indicate the possibility of concentration of microorganisms pathogenic for humans in plant tissues.     

Uptake of phenolic compounds from plant foods in human intestinal Caco-2 cells

In continuation of our studies on the bioaccessibility of phenolic compounds from food grains as influenced by domestic processing, we examined the uptake of phenolics from native/sprouted finger millet (Eleucine coracana) and green gram (Vigna radiata) and native/heat-processed onion (Allium cepa) in human Caco-2 cells. Absorption of pure phenolic compounds, as well as the uptake of phenolic compounds from finger millet, green gram, and onion, was investigated in Caco-2 monolayer model. Transport of individual phenolic compounds from apical compartment to the basolateral compartment across Caco-2 monolayer was also investigated. Sprouting enhanced the uptake of syringic acid from both these grains. Open-pan boiling reduced the uptake of quercetin from the onion. Among pure phenolic compounds, syringic acid was maximally absorbed, while the flavonoid isovitexin was least absorbed. Apparent permeability coefficient P_(app) of phenolic compounds from their standard solutions was 2.02 × 10^?6 cm/s to 8.94 × 10^?6 cm/s. Sprouting of grains enhanced the uptake of syringic acid by the Caco-2 cells. Open-pan boiling drastically reduced the uptake of quercetin from the onion. The permeability of phenolic acids across Caco-2 monolayer was higher than those of flavonoids.     

Improved method for the isolation of RNA from plant tissues

A fast and efficient method for the isolation of RNA from plant tissues is described. Tuber tissue is homogenized in a guanidine hydrochloride-containing buffer followed by direct extraction with phenol/chloroform. The RNA is precipitated from the aqueous phase, washed with 3 M sodium acetate and 70% ethanol, and finally dissolved in water. The yield of RNA is up to 500 micrograms/g of tissue and several tests indicate intact and nondegraded RNA. This method can be adapted to a small-scale version by the use of 1.5-ml tubes, allowing rapid isolation of RNA from a larger number of samples. Finally, this method is of particular use for isolating RNA from tissues with a high polysaccharide and nuclease content such as wounded potato tubers.     

Isolation of detergent-resistant membranes from plant photosynthetic and non-photosynthetic tissues

Microdomains, or lipid rafts, are transient membrane regions enriched in sphingolipids and sterols that have only recently, but intensively, been studied in plants. In this work, we report a detailed, easy-to-follow, and fast procedure to isolate detergent-resistant membranes (DRMs) from purified plasma membranes (PMs) that was used to obtain DRMs from Phaseolus vulgaris and Nicotiana tabacum leaves and germinating Zea mays embryos. Characterized according to yield, ultrastructure, and sterol composition, these DRM preparations showed similarities to analogous preparations from other eukaryotic cells. Isolation of DRMs from germinating maize embryos reveals the presence of microdomains at very early developmental stages of plants.     

Biodepollution of wastewater containing phenolic compounds from leather industry by plant peroxidases

This study deals with the use of peroxidases (POXs) from Allium sativum, Ipomoea batatas, Raphanus sativus and Sorghum bicolor to catalyze the degradation of free phenolic compounds as well as phenolic compounds contained in wastewater from leather industry. Secretory plant POXs were able to catalyze the oxidation of gallic acid, ferulic acid, 4-hydroxybenzoic acid, pyrogallol and 1,4-tyrosol prepared in ethanol 2% (v:v). Efficiency of peroxidase catalysis depends strongly on the chemical nature of phenolic substrates and on the botanical source of the enzymes. It appeared that POX from Raphanus sativus had the highest efficiency. Results show that POXs can also remove phenolic compounds present in industrial wastewater such as leather industry. Removal of phenolic compounds in wastewater from leather industry by POX was significantly enhanced by polyethylene glycol.