Safer DNA extraction from plant tissues using sucrose buffer and glass fiber filter

For some plant species, DNA extraction and downstream experiments are inhibited by various chemicals such as polysaccharides and polyphenols. This short communication proposed an organic-solvent free (except for ethanol) extraction method. This method consists of an initial washing step with STE buffer (0.25?M sucrose, 0.03?M Tris, 0.05?M EDTA), followed by DNA extraction using a piece of glass fiber filter. The advantages of this method are its safety and low cost. The purity of the DNA solution obtained using this method is not necessarily as high as that obtained using the STE/CTAB method, but it is sufficient for PCR experiments. These points were demonstrated empirically with two species, Japanese speedwell and common dandelion, for which DNA has proven difficult to amplify via PCR in past studies.     

Inhibition of sucrose phosphatase by sucrose

1. Partially purified sucrose phosphatase from immature stem tissue of sugarcane is inhibited by sucrose. The enzyme was also inhibited by maltose, melezitose and 6-kestose but not by eight other sugars, including glucose and fructose. 2. The relative effectiveness of sucrose, maltose and melezitose as inhibitors is different for sucrose phosphatase from different plants. 3. The inhibition of the sugar-cane enzyme by sucrose was shown to be partially competitive. The K(i) for sucrose is about 10mm. 4. Melezitose is also a partially competitive inhibitor of the enzyme but the inhibition by maltose is probably mixed. 5. The possibility that sucrose controls both the rate of accumulation of sucrose in stems of sugar-cane and sucrose synthesis in leaves by inhibiting sucrose phosphatase is discussed.     

A specific alkaline phosphatase from Saccharomyces cerevisiae with protein phosphatase activity

In this paper, specific PHO13 alkaline phosphatase from Saccharomyces cerevisiae was demonstrated to possess phosphoprotein phosphatase activity on the phosphoseryl proteins histone II-A and casein. The enzyme is a monomeric protein with molecular mass of 60 kDa and hydrolyzes p-nitrophenyl phosphate with maximal activity at pH 8.2 with strong dependence on Mg²⁺ ions and an apparent K_m of 3.6×10⁻⁵ M. No other substrates tested except phosphorylated histone II-A and casein were hydrolyzed at any significant rate. These data suggest that the physiological role of the p-nitrophenyl phosphate-specific phosphatase may involve participation in reversible protein phosphorylation.     

Pyrophosphate-dependent sucrose metabolism and its activation by fructose 2,6-bisphosphate in sucrose importing plant tissues

In the presence of pyrophosphate and uridine diphosphate, sucrose was cleaved to form glucose 1-phosphate and fructose with soluble extracts from sucrose importing plant tissues. The glucose 1-phosphate then was converted through glycolysis to triose phosphates in a pyrophosphate-dependent pathway which was activated by fructose 2,6-bisphosphate. Much less activity, less than 5%, was found in sucrose exporting tissue extracts from the same plants. These findings suggest that imported sucrose is metabolized in the cytoplasm of plant tissues by utilizing pyrophosphate and that sucrose metabolism is partially regulated by fructose 2,6-bisphosphate.     

Enzymic assay of 10 to 10 moles of sucrose in plant tissues

Procedures are described for measuring sucrose in plant extracts or freeze-dried tissue in the range between 10⁻⁷ and 10⁻¹⁴ moles. The method is based on the destruction of pre-existing glucose and fructose, followed by the hydrolysis of sucrose and reduction of NADP⁺ by a series of coupled enzymic reactions. Depending on the sensitivity required, the NADPH is determined directly with a spectrophotometer or a fluorometer, or is amplified as much as 30,000 times before fluorometric assay. The procedures suggested for the macro level are simpler than current methods, and those suggested for microanalysis are several orders of magnitude more sensitive.     

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.     

Production of galactinol from sucrose by plant enzymes

Galactinol, 1-O-(alpha-D-galactopyranosyl)-myo-inositol, was produced from sucrose as a starting material. UDP-Glc was prepared with sucrose and UDP using sucrose synthase partially purified from sweet potato roots. Then, the UDP-Glc was converted to UDP-Gal using yeast UDP-Gal 4-epimerase from a commercial source. Finally, galactinol was produced from the UDP-Gal and myo-inositol using galactinol synthase partially purified from cucumber leaves. The product was identified as galactinol by the retention times of HPLC, alpha-galactosidase digestion, and NMR spectrometry.     

A sensitive and specific assay for cystathionine: cystathionine content of several plant tissues

A sensitive and specific method for measuring cystathionine is described. The method utilizes the ability of a crude enzyme extract from Bacillus subtilis to catalyze the exchange of [¹⁴C]cysteine into cystathionine. The [¹⁴C]cystathionine thus formed is isolated and its radioactivity determined. The exchange reaction was shown to be specific, and proportional to the amount of cystathionine present in the reaction, within the range from 0.025 to 1 nmole. Tissue extracts to be assayed were purified by ion exchange chromatography, electrophoresis, and paper chromatography, to concentrate the cystathionine and to remove interfering compounds.Application of the method to plant tissues showed that cystathionine is a widespread plant constituent. It is present in plants covering a wide phylogenetic range, as well as in various tissues, at concentrations ranging from 0.04 to 2.6 nmole/g wet wt.     

A highly specific phosphatase from Saccharomyces cerevisiae implicated in tRNA splicing.

We identified and partially purified a phosphatase from crude extracts of Saccharomyces cerevisiae cells that can catalyze the last step of tRNA splicing in vitro. This phosphatase can remove the 2'-phosphate left over at the splice junction after endonuclease has removed the intron and ligase has joined together the two half-molecules. We suggest that this phosphatase is responsible for the completion of tRNA splicing in vivo, based primarily on its specificity for the 2'-phosphate of spliced tRNA and on the resistance of the splice junction 2'-phosphate to a nonspecific phosphatase. Removal of the splice junction 2'-phosphate from the residue adjacent to the anticodon is likely necessary for efficient expression of spliced tRNA. The phosphatase appears to be composed of at least two components which, together with endonuclease and ligase, can be used to reconstitute the entire tRNA-splicing reaction.     

Light-induced biophotonic emission from plant tissues

The emission of biophotons in the visible range, following a delay time of 2-200 seconds after exposure to light, has been measured in germinating seeds, roots, flowers, leaves, and cells. It was found that the biophotonic signals are reproducible and light-induced. The observed signals from germinating seeds of Phaseolus aures and decaying leaves of Eucalyptus are presented to show that the signals have characteristic kinetics and intensity. The kinetics of the signal was found to be independent of the stage of growth or decay, though its intensity varied with biological factors. The kinetics in the first minute is characterized by a single exponential decay term while that in the region t ≤ 200 s is characterized by two exponentials. The variation in the intensity of the signal with mass, state of hydration, and growth, and the effect of inhibitors in various systems (e.g. leaves, lichen, Chlorella) are reported.     

A diffusion approach to the electrolyte leakage from plant tissues

The exchange of ions between plant tissues and the external solution in vitro exhibits prominent biphasic kinetics. This is generally ascribed to the different contribution of the two compartments – apoplast and symplast – involved in the process. In this regard, an electro-diffusion model of the leakage is proposed in the paper. On the basis of the balance of fluxes through the plasmalemma and the cell wall, a system of differential equations describing the ion concentration in the outer solution is found. For a wide range of the system's coefficients, its behaviour is well approximated by a previously obtained analytical function. The values of the function's parameters, derived from the fit with experimental data, correlate adequately with the water deficit conditions of the samples. Hence, these parameters may be used to characterize the physiological status of the investigated plants.     

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.     

A fully automatable enzymatic method for DNA extraction from plant tissues

Background  

DNA extraction from plant tissues, unlike DNA isolation from mammalian tissues, remains difficult due to the presence of a rigid cell wall around the plant cells. Currently used methods inevitably require a laborious mechanical grinding step, necessary to disrupt the cell wall for the release of DNA.     

A method for two-dimensional electrophoresis of proteins from green plant tissues

A method of extraction of proteins from green plant tissues for two-dimensional electrophoresis is presented. The method is demonstrated on barley and potato leaves and on spruce needles for use in isoelectric focusing as well as in nonequilibrium pH gradient electrophoresis. The following three-step procedure was used: (i) The tissue was ground in liquid nitrogen and then in a pH 5.0 buffer with thiourea added to inhibit phenoloxidase and polyvinylpyrrolidone to bind phenolic compounds. (ii) The proteins were precipitated with acetone at -20 degrees C. (iii) The proteins were dialyzed. Protease activity was generally not a problem during this procedure.     

Co-purification of type I alkaline phosphatase and type I phosphoprotein phosphatase from various animal tissues

A purification procedure, which included ethanol treatment as a step for dissociating the large molecular forms of type I phosphoprotein phosphatase, was employed for the studies of the alkaline phosphatase and phosphoprotein phosphatase activities in bovine brain, heart, spleen, kidney, and uterus, rabbit skeletal muscle and liver, and lobster tail muscle. The results indicate that the major phosphoprotein phosphatase (phosphorylase a as a substrate) and alkaline phosphatase (p-nitrophenyl phosphate as a substrate; Mg²⁺ and dithiothreitol as activators) activities in the extracts of all tissues studied were copurified as an entity of M_r = 35,000. The purified enzymes from different tissues exhibit similar physical and catalytic properties with respect to either the phosphoprotein phosphatase or the alkaline phosphatase activity. The present findings indicate that (a) the M_r = 35,000 species, which represents a catalytic entity of the large molecular forms of type I phosphoprotein phosphatase, is widespread in animal tissues, indicating that it is a multifunctional phosphatase; (b) the association of type I alkaline phosphatase activity with type I phosphoprotein phosphatase is a general phenomenon.