RNA synthesis in newly isolated and cultivated mesophyll protoplasts

Tobacco leaf mesophyll protoplasts exhibit low incorporation of [3H]uridine and 32P into RNA, up to 12 h of cultivation, irrespective of the presence of phytohormones. After 24 h of cultivation a dramatic increase in RNA synthesis is observed; it is the highest in the heterogeneous nucleoplasmic RNA fraction. The protoplasts cultivated in the absence of phytohormones show lower incorporation of precursors.     

RNA synthesis in whole cells and protoplasts of centaurea: a comparison

Protoplasts enzymically isolated from suspension cultures of Centaurea cyanus L. incorporate radioactive precursors into RNA with kinetics similar to that of whole cells. There are differences, however, in several other aspects of RNA metabolism. The proportion of total RNA that contains poly(A) sequences (25 to 30%) is similar in both freshly isolated protoplasts and whole cells after a 20-minute pulse with [³H]adenosine. After a 4-hour pulse, however, poly(A)-containing RNA makes up 30% of the total RNA in protoplasts whereas it drops to 8% in whole cells. There appears to be a faulty processing of ribosomal precursor into the mature ribosomal species, as the precursor seems to accumulate to higher levels relative to the mature 18S and 25S rRNAs in protoplasts as compared to whole cells. Additional differences are seen in the size distributions of poly(A)-containing RNA, although the length of the poly(A) segment is similar in both protoplasts and whole cells. Within 24 hours protoplasts appear to have resumed a pattern of RNA synthesis similar to that of whole cells.     

Hormonal control of tobacco protoplast nucleic acid metabolism during in vitro culture

Tobacco mesophyll protoplasts cultivated in vitro do not synthesize a measurable quantity of chloroplastic ribosomal RNA, but actively synthesize cytoplasmic ribosomal RNA, polyadenylated RNA, and proteins. These syntheses are essentially independent of the presence of hormones in the culture medium and are thus related to the ageing phenomenon induced by isolation from the plant and in-vitro culture. At all stages of culture and in all culture media, protoplasts incorporate low levels of thymidine into their DNA. However, the incorporation of considerable quantities of thymidine, indicative of the S phase, only takes place after 25–30 h and requires the presence of auxin and cytokinin.Abbreviations 6-BA 6-benzyladenine - 2,4-D 2,4 dichlorophenoxyacetic acid - DPC diethylpyrocarbonate - OD optical density; oligo-dT cellulose-oligothymidylic acid-cellulose - poly A⁺ RNA polyadenylated RNA - poly A^- RNA non-polyadenylated RNA - tRNA ribosomal RNA - SDS sodium dodecyl sulphate - TCA trichloroacetic acid - Tris buffer Tris (hydroxymethyl)aminomethane - tRNA transfer RNA     

Sedimentation characteristics of virion RNA of Machupo virus reproducing in the presence of actinomycin D

Actinomycin D treatment (0.005-05 g/ml) of Vero and BHK-21 cells infected with Machupo virus suppressed the synthesis of ribosomal RNAs but did not affect the production of infectious Machupo virus. Virion RNAs contained 3 high molecular weight RNA species: 28-31 S, 22-24 S and 18 S. In the presence of actinomycin D [3H]-uridine incorporated only in 30-31 S and 22-24 S RNA species. The data are supported by previous results which show that Machupo virus genome contains two RNA species: "large" (30-31 S) and "small" (22-24 S).     

Purine metabolism in mesophyll protoplasts of tobacco (Nicotiana tabacum) leaves.

The overall metabolism of purines was studied in tobacco (Nicotiana tabacum) mesophyll protoplasts. Metabolic pathways were studied by measuring the conversion of radioactive adenine, adenosine, hypoxanthine and guanine into purine ribonucleotides, ribonucleosides, bases and nucleic acid constituents. Adenine was extensively deaminated to hypoxanthine, whereupon it was also converted into AMP and incorporated into nucleic acids. Adenosine was mainly hydrolysed to adenine. Inosinate formed from hypoxanthine was converted into AMP and GMP, which were then catabolized to adenine and guanosine respectively. Guanine was mainly deaminated to xanthine and also incorporated into nucleic acids via GTP. Increased RNA synthesis in the protoplasts resulted in enhanced incorporation of adenine and guanine, but not of hypoxanthine and adenosine, into the nucleic acid fraction. The overall pattern of purine-nucleotide metabolic pathways in protoplasts of tobacco leaf mesophyll is proposed.     

Ribosomal ribonucleic acid maturation in synchronous strain l cells

Summary The incorporation of [³H]-5-uridine into cytoplasmic 18S and 28S ribosomal ribonucleic acid (rRNA) was examined in Colcemid-synchronized strain L cells during G1 and S phases of the cell cycle in the presence of 5×10⁻⁵ m uridine, which was determined to be the saturating concentration for this system. The data show that in S phase a significant increase occurs in the level of [³H]-5-uridine incorporation into each rRNA species. During a 90-min exposure period, S phase cells incorporate 3.4 times as much [³H]-5-uridine into 18S rRNA and 1.9 times as much into 28S rRNA as do G1 cells. The time required for maturation of the ribosomal RNA species during G1 and during S phase is the same, with 18S rRNA appearing in the cytoplasm in 20 min and 28S rRNA in 40 min.     

Comparative inhibition of nuclear RNA synthesis in cultured mouse leukemia L1210 cells by adriamycin and 4'-epi-adriamycin

Adriamycin and 4'-epi-adriamycin were compared as to their effect on nRNA synthesis. 4'-Epi-adriamycin was a more effective inhibitor than the parent compound of RNA synthesis as measured by incorporation of [3H]-uridine. Adriamycin inhibited all three species of nRNA (ribosomal, non-poly(A)hnRNA, poly(A)hnRNA) to approximately the same extent. 4'-Epi-adriamycin on the other hand inhibited the nRNA species in the following order: non-poly(A)hnRNA greater than ribosomal RNA greater than poly(A)hnRNA. The inhibitory effects of both drugs on incorporation of uridine into RNA were reversible at low concentrations (5 microgram/ml).     

An Evaluation of Some Parameters Required for the Enzymatic Isolation of Cells and Protoplasts with CO2 Fixation Capacity from C3 and C4 Grasses

Variable factors affecting the enzymatic isolation of mesophyll protoplasts from Triticum aestivum (wheat), a C₃ gras, and mesophyll protoplasts and bundle sheath strands from Digitaria sanguinalis (crabgrass), a C₄ grass, have been examined with respect to yields and also photosynthetic capacity after isolation. Preparations with high yields and high photosynthetic capacity were obtained when small transverse leaf segments were incubated in enzyme medium in the light at 30°C, without mechanical shaking and without prior vacuum infiltration. Best results were obtained with an enzyme medium that included 0.5 M sorbitol, 1 mM MgCl₂, 1 mM KH₂PO₄, 2% cellulase and 0.1% pectinase at pH 5.5. In gerneral, leaf age and leaf segment size were important factors, with highest yields and photosynthetic capacities obtained from young leaves cut into segments less than 0.8 mm. To facilitate the cutting of such small segments, a mechanical leaf cutter is described that uniformly (± 0.05 mm) cuts leaf tissue into transverse segments of variable size (0.4–2 mm). Isolations that required more than roughly 4 h gave poor yields with reduced photosynthetic capacity; however, using the optimum conditions described, functional preparations could be roughly 2 h. High rates of light dependent CO₂ fixation by the C₄ mesophyll protoplasts required the addition of pyruvate and low levels of oxalacetate, while isolated bundle sheath strands and C₃ mesophyll protoplasts supported CO₂ fixation without added substrates. Rates of CO₂ fixation by isolated wheat protoplasts generally exceeded the reported rates of whole leaf photosynthesis. Wheat mesophyll protoplasts and crabgrass bundle sheath strands were stable when stored at 4°C while C₄ mesophyll protoplasts were stable when stored at 25°C.     

Autoradiographic detection of the earliest stage of [3H]-uridine incorporation into the cow embryo

Cow embryos, obtained from superovulated heifers on days 3 and 4 after oestrus, were cultured for 20 min in Ménézo's complete culture medium (B2), enriched with 200 microCi/ml of 5-[3H]-uridine. Semi-thin Epon sections of this material were investigated by autoradiography for sites of RNA synthesis. It was found that 5-[3H]-uridine was incorporated into the nucleoplasm and nucleoli only at the end of the 8-cell stage. This suggested that synthesis of hnRNA and rRNA occurred from this stage onwards. Ultrastructural studies were performed on these embryos as well as on other non-incubated 4-cell embryos recovered on day 2. The transformation of dense fibrillar primary nucleoli into functional reticulated nucleoli appeared sooner in the development of cow embryos than in other mammalian species hitherto studied and took place generally during the 8-cell stage. An unusual step in this transformation was represented by the development of a single vacuole in nucleoli at the beginning of this stage (day 3 post-oestrus).     

几种植物原生质体的扫描电镜观察

扫描电镜观察表明,分离自马铃薯、萱草。甘蔗、木薯和落花生等不同植物和组织的原生质体表面呈现不同程度的凹凸不平。马铃薯叶肉原生质体表面较粗糙,其余四种植物叶肉、幼茎或子叶原生质体稍光滑。有的原生质体显现不同程度的凹陷现象。有的原生质体表面尚残留有未完全水解的胞壁碎片。在木薯幼茎原生质体制备物中见有呈“裂片”状的球形结构。原生质体表面扫描图象的差异似与不同种植物有关,与组织源不同更有密切关系。 原生质体镀膜前,涂布于已镀膜的盖玻片支持物上的原生质体很少或无凹陷现象,涂布于已镀膜的双面胶支持物上的原生质体凹陷严重。     

A rapid procedure for plant regeneration from protoplasts isolated from suspension cultures and leaf mesophyll cells of wild Solanum species and Lycopersicon pennellii

A rapid procedure for protoplast isolation, culture and plant regeneration has been developed for two Solanum species (S. lycoperisicoides and S. verrucosum) and Lycopersicon pennellii. Freshly isolated protoplasts were initially cultured in liquid Solanum Culture Medium (SCM), containing 2,4-dichlorophenoxy acetic acid (2,4-D). Subsequent dilution with fresh culture medium without auxins appeared to be essential to obtain rapid regeneration medium later on. The resulting micro calli were first grown in a culture medium containing 0.5 mg/l 6-BAP and 0.05 mg/l NAA and 0.2 M mannitol and 7.3 mM sucrose to induce greening, at a lower osmolarity (300 mOsm · kg⁻¹). Then, the green micro calli were transferred to shoot induction medium, containing 2 mg/l zeatin, 0.1 mg/l IAA and 2% sucrose (150 mOsm · kg⁻¹). In this way plants could be regenerated from leaf mesophyll protoplasts and suspension cell-derived protoplasts of L. pennellii and S. lycopersicoides within 2 months. Shoot regeneration from leaf mesophyll protoplasts of the two lines of S. verrucosum could be obtained 3 months after protoplast isolation.     

The Generation of Active Oxygen Species Differs in Tobacco and Grapevine Mesophyll Protoplasts

Our previous results have shown that oxidative stress may reduce the regeneration potential of protoplasts, but only protoplasts that are able to supply extracellularly H(2)O(2) can actually divide (C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1993] Physiol Plant 87: 263-270; C.I. Siminis, A.K. Kanellis, K.A. Roubelakis-Angelakis [1994] Plant Physiol 1105: 1375-1383; A. de Marco, K.A. Roubelakis-Angelakis [1996a] Plant Physiol 110: 137-145; A. de Marco, K.A. Roubelakis-Angelakis [1996b] J Plant Physiol 149: 109-114). In the present study we have attempted to break down the oxidative burst response into the individual active oxygen species (AOS) superoxide (O(2)(*-)) and H(2)O(2), and into individual AOS-generating systems during the isolation of regenerating tobacco (Nicotiana tabacum L.) and non-regenerating grape (Vitis vinifera L. ) mesophyll protoplasts. Wounding leaf tissue or applying purified cellulase did not elicit AOS production. However, the application of non-purified cellulase during maceration induced a burst of O(2)(*-) and H(2)O(2) accumulation in tobacco leaf, while in grape significantly lower levels of both AOS accumulated. AOS were also generated when protoplasts isolated with purified cellulase were treated with non-purified cellulase. The response was rapid: after 5 min, AOS began to accumulate in the culture medium, with significant quantitative differences between the two species. In tobacco protoplasts and plasma membrane vesicles, two different AOS synthase activities were revealed, one that showed specificity to NADPH and sensitivity to diphenyleneiodonium (DPI) and was responsible for O(2)(*-) production, and a second NAD(P)H activity that was sensitive to KCN and NaN(3), contributing to the production of both AOS. The first activity probably corresponds to a mammalian-like NADPH oxidase and the second to a NAD(P)H oxidase-peroxidase. In grape, only one AOS-generating activity was detected, which corresponded to a NAD(P)H oxidase-peroxidase responsible for the generation of both AOS.     

Compartmentation and transport of zinc in barley primary leaves as basic mechanisms involved in zinc tolerance

The heavy metal zinc was administered to barley seedlings by increasing its concentration in the hydroponic medium. The most dramatic effect was a severe inhibition of root elongation with little effect on root biomass production. The growth of primary leaves was little affected although the zinc content of the primary leaves increased several-fold. A detailed compartment analysis was performed for 10-d-old barley primary leaves. Under low zinc nutrition (2mmol m ⁻³), highest zinc contents were observed in the cytoplasm of mesophyll protoplasts. At inhibitory zinc concentrations in the hydroponic medium (400 μmol m ⁻³), zinc levels dramatically and preferentially increased in the apoplastic space. Elevated zinc levels were also observed in the epidermal cells, and to a lesser extent, in mesophyll vacuoles. The cytoplasmic content of mesophyll protoplasts was unchanged, indicating perfect zinc homeostasis within the leaf. In order to understand the transport mechanisms underlying the steady-state distribution profile, we used ⁶⁵Zn to conduct uptake experiments with leaves whose lower epidermis had been stripped. The leaves were placed on zinc solutions of varying concentrations containing ⁶⁵Zn for 5 min to 6 h. After the incubation, the leaves were fractionated into mesophyll and epidermis protoplasts and residue, the latter mainly representing cell wall. Adsorption of Zn to the extracellular matrix was 100 times faster than Zn uptake into the cells. By far the largest portion taken up into the mesophyll protoplasts rapidly appeared in the vacuolar compartment. These results demonstrate the importance of compartmentation and transport as homeostatic mechanisms within the leaves to handle high, possibly toxic, zinc levels in the shoot.     

Cellular test for RNA-synthesis in rat bone marrow and its use in testing cryoprotective and toxic agents]

A system for the measurement of the RNA-synthesis of bone marrow cells of the rat has been developed and the incorporation of [3H]-uridine into the cellular RNA has been standardized with respect to the time of incubation, the concentration of [3H]-uridine and the number of cells. A plateau of the incorporation of [3H]-uridine into the RNA is reached after 20 min of incubation and is interpreted as the expression of a steady state in synthesis and degradation of the cellular RNA. A constant labelling of the RNA is reached above 8.3 with 10(-6)M [3H]-uridine. The optimal cell number in the 500 mul standard assay is 4 with 10(6). Actinomycin D inhibits the RNA-synthesis to 94% in a concentration of 1.2 with 10(2) mug/ml. The cryoprotectants dimethylsulfoxide, polyethylene-oxide and glycerol and the potential haematotoxic substances dichlorodiphenyltrichloroethane and gamma-hexane were tested in this system. 5% dimethylsulfoxide and 10% polyethylen-oxide in Eagle's-medium with ethylendiamintetra-acetate do not influence the RNA-synthesis. 5% glycerol reduces the incorporation of [3H]-uridine into the cellular RNA to about 30%.     

Developmental changes in ribosomal ribonucleic Acid and fraction I protein in wheat leaves

In light-grown wheat (Triticum aestivum L.) seedlings, the amount of chloroplast and cytoplasmic ribosomal RNA increased to a maximum in the first leaf near the end of its growth and declined by about 60% in the following 3 days. While total ribosomal RNA was declining, labeled uracil was still incorporated into cytoplasmic ribosomal RNA, but the rate of incorporation into chloroplast ribosomal RNA fell by more than 80%, as did the incorporation of labeled leucine into fraction I protein. Either there is greater replacement of cytoplasmic ribosomal RNA than chloroplast ribosomal RNA in mature leaves, or chloroplasts are able to repress the incorporation of exogenous precursor when there is no net synthesis of RNA.     

Ribonucleic acid synthesis and loss of viability in pea seed

Summary RNA synthesis and protein synthesis in embryonic axis tissue of viable pea (Pisum arvense L. var. N.Z. maple) seed commences during the first hour of germination. Protein synthesis in axis tissue of non-viable pea seed is barely detectable during the first 24 h after the start of imbibition. Nonviable axis tissue incorporates significant levels of [³H]uridine into RNA during this period but the level of incorporation does not increase significantly over the first 24 h of imbibition. In axis tissue of non-viable seed during the first hour of imbibition most of the [³H]uridine was incorporated into low molecular weight material migrating in advance of the 4S and 5S RNA species in polyacrylamide gels but some radioactivity was incorporated into a discrete species of RNA having a molecular weight of 2.7×10⁶. After 24 h, non-viable axis tissue incorporates [³H]uridine into ribosomal RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and a heterogeneous RNA species of molecular weight ranging from 2.2×10⁶ to 2.7×10⁶. No 4S or 5S RNA synthesis is detectable after 24 h of imbibition in non-viable axis tissue. Axis tissue of viable pea seed synthesises rRNA, 4S and 5S RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and the rRNA precursor species at both periods of germination studied. Loss of viability in pea seed appears to be accompanied by the appearance of lesions in the processing of rRNA precursor species and a significant loss of RNA synthesising activity.Abbreviations rRNA ribosomal RNA - TCA trichloroacetic acid - SLS sodium lauryl sulphate - PPO 2,5 Diphenyloxazole - POPOP 1,4-Bis-2-(4-methyl-5-penyloxazolyl)-benzene     

Deoxyribonucleic Acid and Ribonucleic Acid Synthesis during the Cell Expansion Phase of Cotyledon Development in Vicia faba L

In Vicia faba L., the tissue specific proteins, legumin and vicilin, are synthesized during the cell expansion phase of cotyledon development. During this growth period, RNA and nuclear DNA increase 8- to 10-fold. ³H-Uridine and ³H-adenosine are incorporated into ribosomal RNA, both 25S and 18S, and into transfer RNA. DNA isolated from cotyledons in the cell division phase of growth has been compared with DNA isolated from cotyledons undergoing expansion growth. Results indicate that the DNA increase involves replication of the whole genome (endoreduplication).