A constitutively expressed Myc-like gene involved in anthocyanin biosynthesis from Perilla frutescens: molecular characterization,heterologous expression in transgenic plants and transactivation in yeast cells

The coordinate expression of anthocyanin biosynthetic genes in leaves and stems of a red forma of Perilla frutescens is presumably controlled by regulatory gene(s). A Myc-like gene (Myc-rp) was isolated from a cDNA library prepared from the leaves of red P. frutescens, and its deduced amino acid sequence shows 64% identity with that of delila from snapdragon. The Myc-rp gene was expressed in leaves and roots of both red and green P. frutescens equally. Comparison of deduced amino acid sequence of Myc-rp with that of Myc-gp, the second allele isolated from a green forma of P. frutescens, indicates that the 132nd amino acid, alanine, existing in MYC-RP was changed to serine in MYC-GP. The heterologous expression of these two alleles of Myc-like gene in tobacco and tomato resulted in an increase of the anthocyanin contents in flowers of tobacco and vegetative tissues and flowers of tomato. However, the flowers of transgenic tobacco expressing the fragment with a partial deletion (encoding 1–115 amino acids deleted) of Myc-gp gave no change in anthocyanin accumulation, but some morphological changes of the flower were observed. In yeast, the MYC-RP/GP and Delila protein exhibited transactivation activity on the GAL-1 promoter from yeast and the promoter of dihydroflavonol 4-reductase (DFR) gene from P. frutescens. A transactivation domain of MYC-RP/GP and Delila could be located in the region between the 193rd and the 420th amino acid of MYC-RP/GP proteins. Our data indicate that this Myc-like gene presumably functions in the regulation of anthocyanin biosynthesis similarly in different tissues of dicot plants.     

Nitrogen metabolism in plant cell suspension cultures: I. Effect of amino acids on growth

Certain amino acids inhibit growth of tobacco (Nicotiana tabacum L. var. xanthi), tomato (Lycopersicon esculentum) carrot (Daucus carota), and soybean (Glycerine max L. co. Mandarin) cell cultures when nitrate or urea are the nitrogen sources but not when ammonia is the nitrogen source. These amino acids also inhibit development of nitrate reductase activity (NADH:nitrate oxidoreductase EC 1.6.6.1) in tobacco and tomato cultures. Threonine, the most inhibitory amino acid, also inhibits nitrate uptake in tobacco cells. Arginine, and some other amino acids, abolish the inhibition effects caused by other amino acids. We suggest that amino acids inhibit assimilation of intracellular ammonium into amino acids in cells grown on nitrate or urea.     

Primary culture of adrenal medullary chromaffin cells in a chemically defined medium

Primary cultures of bovine adrenal medullary chromaffin cells have been maintained in the absence of serum for up to 3 weeks. Chromaffin cell catecholamine and protein contents were maintained, after an initial loss at the time of plating, as were the functional properties of the cells, including nicotine-evoked, calcium-dependent catecholamine secretion. Important factors in the maintenance of chromaffin cells included the cell plating density, frequency of medium replacement, and extent of medium replacement, suggesting ‘conditioning’ of the culture medium. Initially, serum was used for the first 48 h of culture, but pretreatment of the tissue culture plates with fibronectin allows complete elimination of serum from the plating medium. The establishment of serum-free culture conditions for chromaffin cells should facilitate the study of their cell biology and biochemistry.     

Faithful editing of a tomato-specific mRNA editing site in transgenic tobacco chloroplasts

RNA editing sites and their site-specific trans-acting recognition factors are thought to have coevolved. Hence, evolutionary loss of an editing site by a genomic mutation is normally followed by the loss of the specific recognition factor for this site, due to the absence of selective pressure for its maintenance. Here, we have tested this scenario for the only tomato-specific plastid RNA editing site. A single C-to-U editing site in the tomato rps12 gene is absent from the tobacco and nightshade plastid genomes, where the presence of a genomic T nucleotide obviates the need for editing of the rps12 mRNA. We have introduced the tomato editing site into the tobacco rps12 gene by plastid transformation and find that, surprisingly, this heterologous site is efficiently edited in the transplastomic plants. This suggests that the trans-acting recognition factor for the rps12 editing site has been maintained, presumably because it serves another function in tobacco plastids. Bioinformatics analyses identified an editing site in the rpoB gene of tobacco and tomato whose sequence context exhibits striking similarity to that of the tomato rps12 editing site. This may suggest that requirement for rpoB editing resulted in maintenance of the rps12 editing activity or, alternatively, the pre-existing rpoB editing activity facilitated the evolution of a novel editing site in rps12.     

Introns control expression of sucrose transporter LeSUT1 in trichomes,companion cells and in guard cells

In solanaceous plants such as tomato and tobacco, the sucrose transporter SUT1 is crucial for phloem loading. Using GUS as a reporter, the promoter and other regulatory cis elements required for the tomato LeSUT1 expression were analyzed by heterologous expression of translational chimeric constructs in tobacco. Although LeSUT1 is highly expressed at the RNA level, GUS expression under the control of a 1.8 kb LeSUT1 promoter resulted in few plants expressing GUS. In GUS-positive transformants, expression levels were low and limited to leaf phloem. Increasing or decreasing the length of LeSUT1 promoter did not lead to a significant increase in positive transformants or higher expression levels. Translational fusion of GUS to the LeSUT1 C-terminus in a construct containing all exons and introns and the 3'-UTR led to a higher number of positive transformants and many plants with high GUS activity. LeSUT1 expression was detected in ab- and adaxial phloem companion cells, trichomes and guard cells. The role of individual introns in LeSUT1 expression was further analyzed by placing each LeSUT1 intron into the 5'-UTR within the 2.3 kb LeSUT1 promoter construct. Results showed remarkable functions for the three introns for SUT1 expression in trichomes, guard cells and phloem cells. Intron 3 is responsible for expression in trichomes, whereas intron 2 is necessary for expression in companion cells and guard cells. The combination of all introns is required for the full expression pattern in phloem, guard cells and trichomes.     

High plating efficiency with plant cell cultures

In this paper we describe a simple method to improve the plating efficiency in plant cell cultures.Two-stage plating is used; in the first stage the cells are inoculated at high density in 0.2% agarized culture medium for ten days to facilitate growth; under this condition, each cell produces a single micro-colony trapped in the agar network. In the second stage the colonies are plated at different densities in 1% agarized medium.These colonies are self-sufficient and able to improve the cell growth by conditioning the medium.Abbreviations 6-BAP 6-Benzyl-aminopurine - 2,4-D 2,4-Dichlorophenoxyacetic acid     

Nutritional requirements of protoplast-derived,haploid tobacco cells grown at low cell densities in liquid medium

Preliminary attempts to define a completely synthetic medium able to support divisions of haploid tobacco mesophyll protoplasts at low initial densities have failed. High protoplast concentrations together with large amounts of naphtaleneacetic acid in the medium (3 mg l^-1 NAA) were required for maximal induction of protoplast division. However, cell suspensions derived from haploid protoplasts after four days of preculture at high initial cell densities could be diluted to densities as low as 1–4 cells ml^-1, provided the concentration of NAA in the medium was lowered to below 0.3 mg l^-1. The optimal NAA supply for low cell density growth was affected by the nature of the nitrogen source.A simple minimal medium which supports the growth of these haploid cells with a plating efficiency of 30–40%, independent of the cell density in the range of 1–4 to 3·10⁴ cells ml^-1, has been established. In this medium inositol was the only vitamin stringently required for growth.Growth of cells at low densities was also possible in a medium initially containing 3 mg l^-1 NAA, provided it was conditioned by the growth of protoplasts at high densities. Preliminary experiments with [¹⁴C]NAA showed that the amount of free NAA remaining in the medium after preincubation at high densities was drastically reduced. Simultaneously, NAA conjugates accumulated in the medium. The implications of these results are discussed.Abbreviations BA 6-benzyladenine - EDTA ethylene diaminetetraacetic acid - NAA naphtaleneacetic acid     

Response to chilling of tomato mesophyll protoplasts

Freshly isolated protoplasts from tomato leaves show two completely different responses to a chilling treatment of 12 h at 7° C prior to culture at 29° C, depending on the presence or absence of glucose in the medium. In the culture medium with glucose as osmoticum, where the rate of cell divisions under optimal culture conditions is relatively high (about 20% plating efficiency), protoplasts were drastically injured by the chilling procedure and died. In the medium with mannitol as the osmoticum instead of glucose, where the plating efficiency even under optimal conditions is rather low (about 8%), protoplasts withstand the chilling procedure. More-over, after the chilling treatment when the protoplasts were transferred to the optimal culture temperature of 29° C, the plating efficiency was raised to about 20%, which is the same level as in the glucose-containing medium without chilling. This effect was not observed when the medium in which the protoplasts were suspended during the chilling period was replaced with fresh medium. This suggests that under these conditions tomato protoplasts produce and excrete a factor in the cold that improves the vitality of the cells or stimulates cell division. The possible relationship between chilling sensitivity of tomato protoplasts and their ability to divide will be discussed.     

Arabinogalactan-protein epitopes in somatic embryogenesis of Daucus carota L.

Two monoclonal antibodies (ZUM 15 and ZUM 18) directed against carrot (Daucus carota L.) seed arabinogalactan proteins (AGPs) were used to isolate specific AGP fractions. For both carrot and tomato (Lycopersicon esculentum Mill.) seed AGPs analyzed by crossedelectrophoresis, the ZUM 15 and ZUM 18 AGP fractions showed one identical peak. However, the Rf values for the two species were different: 0.82 for carrot seed AGPs and 0.52 for tomato seed AGPs. When the fractionated AGPs (carrot or tomato) were added to carrot cell lines they had a dramatic effect on the culture. One AGP fraction (ZUM 15 AGPs) was able to induce vacuolation of embryogenic cells. Those cells failed to produce embryos. The other AGP fraction (ZUM 18 AGPs) increased the percentage of embryognic cells from about 40% up to 80% within one week and this subsequently resulted in the formation of more embryos on hormone-free medium. This activity was higher than that of unfractionated carrot seed AGPs, while the optimum concentration was 50-fold lower. Since both ZUM 18 AGPs (carrot or tomato) yielded identical responses it can be concluded that neither the Rf value nor the source are essential for biological activity. The dose-response curve of ZUM 18 AGPs showed a sharp optimum. When the AGPs that also bound to the antibody ZUM 15 were removed, the dose-response curve of the remaining AGPs (containing only the ZUM 18 epitope, not the ZUM 15 epitope) resembled a saturation curve. Regardless of its concentration, the fraction in which AGP molecules contained both epitopes showed no appreciable embryogenesis-promoting activity. The biological activity of AGPs was therefore determined by the presence of embryogenesis-enhancing and-inhibiting epitopes. The inhibiting and enhancing epitopes can be located on separate molecules or one single AGP molecule.     

Neural induction from ES cells portrays default commitment but instructive maturation

The neural induction has remained a debatable issue pertaining to whether it is a mere default process or it involves precise instructive cues. We have chosen the embryonic stem (ES) cell model to address this issue. In a devised monoculture strategy, the cell-cell interaction availed through optimum cell plating density could define the niche for the attainment of efficient in vitro neurogenesis from the ES cells. The medium plating density was found ideal in generating optimum number of progenitors and also yielded about 80% mature neurons in a serum free culture set up barring any exogenous inducers. We could also demarcate and quantify the neural stem cells/progenitors among the heterogeneous cell population of differentiating ES cells using nestin intron II driven EGFP expression as a tool. The one week post-plating was determined to be the critical time window for optimum neural progenitor generation from ES cells that helped us further in purifying these cells and in demonstrating their proliferation and multipotent differentiation potential. Seeding cells at varying densities, we could decipher an interesting paradoxical scenario that interlinked both commitment and maturation with the initial plating density having a vital influence on neuronal maturation but not specification and the secretory factors were apparently playing a key role during this process. Thus it was comprehended that, the neural specification was a default process independent of exogenous factors and cellular interaction. Conversely, a defined number of cells at the specification stage itself seemed critical to provide an auto-/paracrine means of signaling threshold for the maturation process to materialize.     

Monoclonal antibody to intermediate filament antigen cross-reacts with higher plant cells

The monoclonal antibody (anti-IFA) raised (Pruss et al., 1981, Cell 27:419-428) against an intermediate filament antigen, which is widespread throughout phylogeny, has been shown here to cross-react with higher plants. On immunoblotting, anti-IFA cross-reacted with proteins in homogenates of carrot suspension cells and of meristematic cells from onion root tips. A 50-kD cross-reactive protein was enriched in a fraction that consisted of detergent-insoluble bundles of 7-nm fibrils from carrot protoplasts (Powell et al., 1982, J. Cell Sci. 56:319-335). By use of indirect immunofluorescence, anti-IFA stained formaldehyde-fixed onion meristematic cells and carrot protoplasts in patterns approximating those obtained with monoclonal anti-tubulins. That anti-IFA was not recognizing plant tubulins was established by use of immunoblots of two-dimensional gels on which the proteins that comprised isolated fibrillar bundles and taxol-purified carrot tubulins had been separated. The two groups of proteins had different positional coordinates: anti-IFA recognized the fibrillar bundle proteins, and anti-tubulins recognized plant microtubule proteins with no cross-reaction to the heterologous proteins. Likewise, formaldehyde-fixed taxol microtubules from carrot cells could be stained with anti-tubulin but not with anti-IFA. It is concluded that an epitope common to intermediate filaments from animals co-distributes with microtubules in higher plant cells.     

Spontaneous neoplastic evolution of Swiss mouse embryo cells (PMS-14) in culture

Some biological properties related to transformation have been studied to characterize a spontaneously transformed mouse embryonic fibroblast cell line, PMS-14. Low population doubling time with moderately high plating efficiency and saturation density were noted. These cells showed reduced adhesion to substratum, enhanced agglutinability by concanavalin A, anchorage independent growth in soft agar and tumorigenicity in mice.     

Effect of glyphosate on carrot and tobacco cells

The growth of suspension-cultured carrot (Daucus carota L.) and tobacco (Nicotiana tabacum L. cv. Xanthi) cells was inhibited by glyphosate (N-[phosphonomethyl]glycine). This inhibition was reversed by adding combinations of phenylalanine, tyrosine, and tryptophan or casein hydrolysate. Casein hydrolysate and phenylalanine + tyrosine + tryptophan were the most effective treatments. Reversal of glyphosate-induced inhibition occurred only if the aromatic amino acids were added during the first 8 days of glyphosate incubation. Glyphosate uptake was not reduced when the aromatic amino acids or casein hydrolysate were added.     

Nutritional and karyotypic characterization of a haploid cell culture ofDaucus carota

Summary The purpose of this study was to optimize growth conditions for a strain of haploid carrot callus and to follow its karyotypic changes in a long span of time. The strain has been maintained in liquid suspension since September 1977. It has remained predominantly haploid in its karyotype since that time. The original explant was initiated and subsequently subcultured in Gamborg's B5 medium. The components of the B5 medium were omitted one at a time and sequentially added back to determine their minimum, optimum, and maximum nontoxic concentrations. These changes were made in the original formula: the addition of an organic buffering agent and an increase in the iron and other micronutrient concentrations. Using this slightly modified B5 medium, we assessed the effect on growth by single additions of amino acids, different carbon sources, growth regulators, and vitamins. No improvement in plating efficiency resulted from addition of any of these compounds. We conclude that there are factors limiting the plating efficiency of the haploid cells other than these tested, or that single additions will not make a discernible difference, or that growth promoting factors cannot be exogenously supplemented to cultured cells.     

胡萝卜培养细胞的盐适应蛋白

近年来植物离体培养细胞盐适应蛋白的发现以及盐影响下植物信使 RNA 体外转译产物的研究,为耐盐分子机理的研究和耐盐基因的鉴定和克隆开辟了道路。烟草细胞在适应1—2.5%NaCl 之后,细胞蛋白质合成发生变化,特别是26kD 的蛋白质大量积累。体内标记实验表明,细胞从不加 NaCl 的培养基转到加有1%     

Peritoneal exudate cells. I. Growth requirement of cells capable of forming colonies in soft agar

Mouse peritoneal exudate cells induced by thioglycollate medium can form colonies in soft agar with a plating efficiency of about 5% (0.6%–10%). Cells from an unstimulated peritoneal cavity form no colonies or have a plating efficiency of less than 0.001 %. These colony-forming cells from the peritoneal exudate are similar to bone marrow colony-forming cells in vitro in that they both require a substance(s) present in conditioned medium from L-cells or mouse embryo fibroblasts or the serum from endotoxin-treated mice for the initiation and the continuation of their growth. However, peritoneal exudate colony-forming cells have a much longer initial lag period (10–14 days) and can survive longer in the absence of L-cell conditioned medium than bone marrow colony-forming cells. Only mononuclear cells, presumably macrophages, are observed in peritoneal exudate colonies, whereas bone marrow cell colonies contain both polymorphonuclear cells and macrophages.     

Microcallus formation from Hevea brasiliensis protoplasts isolated from embryogenic callus

Conditions for successful culture of rubber tree (Hevea brasiliensis) protoplasts were investigated. Protoplasts, derived from embryogenic callus, regenerated cell walls then underwent division when embedded in alginate and cultivated on a modified Murashige and Sook medium (9 M 2,4-dichlorophenoxyacetic acid, 0.6 M glucose, 0.93 M kinetin, NH ₄ ⁺ reduced by half) in the presence of nurse cells (tobacco feeder cell layer). The presence of nurse cells was essential to maintain viability and sustain protoplast division. Several parameters which influenced the plating efficiency were analysed, such as the density of feeder cells and the duration of contact of the feeder layer.Abbreviations BSA Bovine Serum Albumin - CPW Cell and Protoplast Washing medium (Frearson et al. 1973) - 2,4-D 2,4-dichlorophenoxyacetic acid - 3,4-D 3,4-dichlorophenoxy-acetic acid - PDA Fluorescein diacetate - FW Fresh weight - KIN Kinetin - MES [2N-morpholino] ethane sulfonic acid - MS medium Murashige and Skoog (1962) medium - PE plating efficiency - SAB South American Leaf Blight - WPM] Woody Plant Medium (Russel and Mc Cown 1986)     

High plating efficiency and plant regeneration frequency in low density protoplast cultures derived from an embryogenic Brassica napus cell suspension

Protoplasts were isolated from an embryogenic cell suspension culture derived from microspores of Brassica napus cv. Jet Neuf. Protoplast yield varied with the cell suspension growth medium. Optimization of protoplast plating density, manipulation of culture medium, carbon source and medium matrix, and inclusion of Ficoll resulted in protoplast plating efficiencies close to 30%. Placement of the protoplasts close to the gas interface contributed greatly to the elevated plating efficiency. Low density cultures could be induced to regenerate calli at optimum plating efficiencies if grown in the presence of nurse culture. This is of great advantage for manipulation of individual protoplasts or for microinjection. Plants were regenerated directly from the cell suspension or from the protoplast cultures.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid