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Mineral components of plant cell walls.

Plant cell walls that are secondarily thickened contain silicon and metal cations. The silicon occurs predominantly as silica (SiO2.nH2O) deposited in intimate association with the organic components of the walls and, according to recent evidence, as an integral constituent of polyuronides. Relatively large amounts of deposited (i.e., solid) silica are found in rice and other cereals and in grasses. When ingested by ruminant animals, practically all the solid silica may be recovered in the feces. However, microscopic particles of silica from plants are, to a small extent, absorbed as such through the gastrointestinal wall in both man and ruminant animals. It has now been shown that silicon is essential for animals, and that it is a constituent of certain mucopolysaccharides, thereby contribution to the architecture of connective tissues. The acidic silanol group of solid silica in plant cell walls may be involved in binding metal cations, but carboxyl and phenolic hydroxyl groups of the organic components of the walls are probably mainly responsible. Binding of metal cations by these components of plant cell walls, and possibly by silica, is likely to reduce availability of the cations for intestinal absorption.

Animal Nutritional Physiological Phenomena

Production of biologicals by plant cell cultures.

The biologicals currently produced from plants are generally low molecular weight chemicals such as drug compounds rather than high molecular weight compounds such as proteins. The reasons for considering a fermentation technology based on plant cell cultures have been described. The state of technology of plant cell cultures is sufficiently advanced to suggest that the major questions remaining are economic rather than primarily technical. Limited analysis of the economics of commercial production of biologicals by plant cell cultures shows that a commercial process is certainly feasible.

Biological Products

Inducing effect of plant cells on nitrogenase activity by Spirillum and Rhizobium in vitro.

Eleven different plant cell tissue cultures of both legume and non-legume origin have been grown in direct association, and in separate but close proximal association with both Spirillum lipoferum and Rhizobium sp. 32H1. Basic similarities were found in the nutritional requirement for the induction of nitrogenase activity (C2H2) in both organisms. In the absence of plant cell cultures both organisms need to be provided with a pentose sugar and a tricarboxylic acid to induce high levels of nitrogen-fixing activity. Plant cell callus tissue appears only capable of supplying the tricarboxylic acid to induce high levels of nitrogen-fixing activity. Plant cell callus tissue appears only capable of supplying the tricarboxylic acids needed but not the sugar component. The plant tissue, however, seems able to activate certain carbohydrates, which in themselves are incapable of substituting for the pentose additive.

Enzyme Induction

Transfer, maintenance, and expression of bacterial Ti-plasmid DNA in plant cells transformed with A. tumefaciens.

The mechanism of induction of the plant cancer crown gall by Agrobacterium tumefaciens has been briefly described. The salient points are as follows. 1. Large plasmids of molecular weight (100 to 150) S 10(6), called Ti-plasmids, are essential to the transformation process. 2. Ti-plasmids carry a DNA segment that can be transferred to, and maintained and expressed in, transformed plant cells. 3. This DNA segment has been identified both by direct hybridization experiments between Ti DNA fragments and crown gall DNA and by the study of a deletion mutant of a Ti-plasmid. 4. Indirect evidence suggests that genes involved in the synthesis of abnormal amino acids (such as octopine and nopaline) by crown gall cells, and known to be carried on the Ti-plasmids, are in fact located on the DNA segment that is transferred to the plant cells. 5. Ti-plasmids are efficient conjugative plasmids, since they can promote their own transfer by conjugation to various plasmid-free bacterial strains. Their conjugative properties may be involved also in the Ti DNA transfer from Agrobacterium to plant cells. 6. Preliminary evidence indicates that the transferable segment of the Ti-plasmid has the structure of a transposon, since it appears to be flanked by a sequence exhibiting most of the properties of the sequences that border the known bacterial drug-resistance gene transposons.

Cell Transformation, Neoplastic

Differential staining of tannin in sections of epoxy-embedded plant cells.

A staining procedure is described for the light microscopic localization of ergastic tannins in epoxy sections of plant cells embedded for study by transmission electron microscopy. Callus and cell suspensions of Pseudotsuga menziesii and Pinus taeda fixed in glutaraldehyde:acrolein and then OsO4, followed by epoxy embedding, were sectioned 0.5 mum thick, stained on a glass slide with ethanolic Sudan black B at 60 C as described by Bronner, and then mounted in Karo syrup. Tannin deposits stained brownish-orange and were easily distinguished from lipid bodies of similar size, which stained dark blue to black, and from starch grains, which were unstained. The significance of this differential polychromasia was confirmed by transmission electron microscopy. This staining procedure should prove valuable in the cytoplasmic evaluation of the plant cell ergastics (especially tannins) via light microscopy whether or not electroc microscopic examination is intended.

Plant Cells

Ribonuclease H activity in cultured plant cells.

Ribonuclease H (RNAase H) was extracted from cultured plant cells, strain GD-2 and characterized. RNAase H activity in logarithmical growing cells is much higher than that of stationary cells, and the response of RNAase H activity was very similar to that of DNA polymerase after culture. The activities of RNAase, DNAase, phosphodiesterase and alkaline phosphatase decrease parallel with the increase in growth, and increase to stationary phase, contrasting with those of DNA polymerase and RNAase H.

Cell Division

The lignin fraction of plant cell walls.

Methods are discussed for determining lignin in plant cell walls. The increase in apparent lignin content that may occur as a result of artifacts produced during food preparation is also discussed. The phenolic components, including lignin, of cell walls separated from 12 vegetable, fruit, and cereal foods are determined. Wheat bran, on a fresh weight basis, had a high cell wall content (48.6%) compared with the vegetables and fruit whose contents ranged from 1.0 to 6.0%. Wheat bran and spinach cell walls had the highest lignin contents (11.4 and 4.4%, respectively) and the lowest degradabilities (35.8 and 39.6%, respectively) as determined by a cellulase technique. Cell walls of spinach, beetroot, sweet corn, pineapple, and wheat bran contained the phenolic acids (ferulic, p-coumaric, and diferulic) bound to polysaccharide components. Cell walls of cabbage, Brussels sprout, celery, cauliflower, green bean, carrot, and pea contained only traces of these acids.

Cell Wall

Plant cell lines.

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Cell Line

Action of bleomycin on proliferating plant cells.

Bleomycin (10-(6) M) has been tested in Allium cepa L. meristems which are formed by a proliferating cell population growing under steady state conditions. Chromosome breaks were apparently induced by the antibiotic in cells in G2 period since anaphases with chromatid breaks were formed at a time shorter than G2 + prophase duration. Stimulation of entrance of G2 cells into mitosis is suggested both by an increase in the frequency of early prophases and by the study of waves of prophases in a synchronous subpopulation labelled by caffeine. Progression of other mitotic phases was unaffected. Nucleologenesis rate was increased by the antibiotic in a fashion resembling protein synthesis inhibitors. Protein synthesis is inhibited by 10-(6) M bleomycin to the same extent as 4 X 10(-6) M anisomycin. Both facts suggest that bleomycin has a direct inhibitory effect on protein synthesis in meristems. Given the nucleologenesis sensitivity to nucleolar RNA inhibition it is suggested that the antibiotic activity on nucleolar transcription is mediated through DNA.

Bleomycin

A simple method for the separation of tissue-cultured plant cells from unbound bacteria: a demonstration of acquired affinity for bacteria by cells of soy bean.

Tissue-cultured cells of soy bean, grown in liquid medium and then transferred to an agar substratum in so-called tissue chambers, showed an affinity for the binding of applied bacteria (Klebsiella pneumoniae). This binding, which became greater as the plant cells remained on the agar for up to 10 days, was manifested as an increasing resistance of the bacteria to be washed from the plant cells in a standard, gentle and reproducible procedure devised for this purpose. The number of bacteria firmly bound to the cells was determined by the use of 32P-labelled bacteria and was largely independent of the concentration of bacteria in the suspension applied to the tissue-cultured cells.

Adsorption

Plant cell wall fractionation and structural analysis.

The structure of the major plant polysaccharides contributing to dietary fiber, their organization within the plant cell wall, and the problems associated with their analysis are briefly reviewed. Alkaline, acidic, and detergent fractionation methods are described and considered in relation to their specificity. The analysis of pectin, often ignored as a component of dietary fiber, is examined in detail. The solubility of pectins from a number of common plant foods was determined, indicating that a considerable portion of pectin polyuronide is lost to many of the commonly used analytical procedures.

Cell Wall

Alternative localization of HEME OXYGENASE 1 in plant cells regulates cytosolic heme catabolism.

Heme, an organometallic tetrapyrrole, is widely engaged in oxygen transport, electron delivery, enzymatic reactions, and signal transduction. In plants, it is also involved in photomorphogenesis and photosynthesis. HEME OXYGENASE 1 (HO1) initiates the first committed step in heme catabolism, and it has generally been thought that this reaction takes place in chloroplasts. Here, we show that HO1 in both Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) has 2 transcription start sites (TSSs), producing long (HO1L) and short (HO1S) transcripts. Their products localize to the chloroplast and the cytosol, respectively. During early development or de-etiolation, the HO1L/HO1S ratio gradually increases. Light perception via phytochromes (Phys) and cryptochromes elevates the HO1L/HO1S ratio in the whole seedling through the functions of ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOG and through the suppression of DE-ETIOLATED 1, CONSTITUTIVE PHOTOMORPHOGENESIS 1, and PHYTOCHROME INTERACTING FACTORs. HO1L introduction complements the HO1-deficient mutant; surprisingly, HO1S expression also restores the short hypocotyl phenotype and high pigment content and helps the mutant recover from the genomes uncoupled (gun) phenotype. This indicates the assembly of functional Phys within these lines. Furthermore, our findings support the hypothesis that a mobile heme signal is involved in retrograde signaling from the chloroplast. Altogether, our work clarifies the molecular mechanism of HO1 TSS regulation and highlights the presence of a cytosolic bypass for heme catabolism in plant cells.

Heme

Messenger ribonucleoprotein particles from plant cell cultures.

Messenger ribonucleoprotein particles were isolated from the polysomes of logarithmically growing plant cell cultures, pulse-labeled with [3H] adenosine for 30 min. More than 80% of the labeled RNP was present in particles sedimenting between 80 S and 30 S on sucrose density gradients, but was not associated with ribosomal subunits. The size distribution differs from those reported for polysomal mRNP particles to date. After fixation with glutaraldehyde the labeled RNP particles had a buoyant density of 1.38 g/cm3 in CsCl gradients. Radioactively labeled RNA extracted from the RNP particles showed a heterodisperse size distribution and contained poly (A) stretches as determined by affinity chromatography and ribonuclease digestion experiments.

Cells, Cultured

The molecular weight of rRNA precursor molecules and their processing in higher plant cells.

Actively dividing callus cells of higher plants (Petroselinum crispum, Daucus carota, Acer pseudoplatanus) were used to detect the primary gene product of rDNA in vivo. Parsley and carrot cells were labelled with [32P]orthophosphate. Under non-denaturing conditions, in both cases only one high molecular weight rRNA precursor was present on polyacrylamide gels. Its molecular weight did not exceed 2.5 x 10(6) dalton. Under denaturing conditions, 2.0--2.1 x 10(6) dalton were determined on formamide gels. This rRNA precursor was already present after a labelling period of 5--10 min. In parsley cells labelled mature rRNA (25S and 18S) arrived in the cytoplasm 45 min after onset of incubation. In Acer pseudoplatanus incubated with [3H]uridine two rapidly labelled components did emerge from polyacrylamide gels without formamide; their molecular weights were 2.3 and 3.2--3.4 x 10(6) dalton. After electrophoresis in formamide, the larger component disappeared, thus indicating that it would be an intermolecular aggregate of different RNAs. From these results we have no evidence for the existence of rRNA precursors exceeding the molecular weight of 2.5 x 10(6) dalton.

Cells, Cultured

Properties and subunit composition of RNA polymerase II from plant cell cultures.

The purification of DNA-dependent RNA polymerase II (EC 2.7.7.6) from plant cell cultures of Petroselinum (parsley) is described. The procedure during which enzyme I is eliminated includes initial precipitation with (NH4)2SO4, an ultracentrifugation step, gel filtration on Sepharose 4B, chromatography on DEAE-cellulose, DNA-agarose and DEAE-Sephadex. The enzyme purified almost to homogeneity exhibits maximal activity with denatured DNA, and is activated preferentially by Mn2+; alpha-amanitin acts as a strong inhibitor. Electrophoresis of the enzyme in the presence of dodecylsulphate indicates that it is composed of seven subunits with mol. wts of 200 000, 180 000, 140 000, 43 000, 26 000, 25 000 and 16 000. The results of molecular weight and molar ratio determinations suggest that Petroselinum RNA polymerase II may exist in two active forms differing only in the composition of their high molecular weight subunits.

Amanitins