P-fluorophenylalanine Research Articles

Attempts to induce haploids in anther cultures of sugar, fodder and wild species of beet

In the present investigation, aimed at obtaining beet haploids from anthers, the effect of mineral media, potato and sugar beet extract and p-fluorophenylalanine (PFP) in combination with growth substances was tested. Nutrient-starved plants as anther-donors, anther-starvation, cold treatment and photoperiod were also analysed. On all mineral media the anthers produced callus and roots; however, the percentage depended on the combination of growth substances used. The best medium for differentiation was that of Linsmaier and Skoog with 25 µM zeatin or 6-(3-methyl-2-butenylamino)purine with 5 µM naphthalene-l-acetic acid (25.5%). The addition of PFP caused an increase in the percentage of anther differentiation (41.6%). Besides callus and roots on one of the anthers (in ca. 140000 tested), vegetative buds were formed from which numerous plants were obtained (2n). Plant and anther nutrient starvation did not improve the anther response to differentiation, nor did it induce haploid development, similarly as cold treatment of inflorescences or isolated anthers. The anthers of wild species showed lower ability to differentiate than those of sugar or fodder beets. Cytological analyses showed formation of multicellular structures until ca. the 12-th day of anther culture; afterwards, they degenerated.

Shikonin production by p-fluorophenylalanine resistant cells of Lithospermum erythrorhizon

Studies were conducted with a BK-39 callus culture of Lithospermum erythrorhizon, which produced seven shikonin derivatives (acetylshikonin, propionylshikonin, isobutyrylshikonin, β,β-dimethylacrylshikonin, isovalerylshikonin, β-hydroxyisovalerylshikonin and α-methyl- n-butyrylshikonin). A selection of cell aggregates of BK-39 culture on a medium containing p-fluorophenylalanine (PFP) yields a cell line possessing a higher resistance to the inhibitor than the initial culture. Selected BK-39F cultures produced almost the same profile of shikonin naphthoquinones as the initial culture. The shikonin derivative content of PFP-resistant culture was approximately two times higher than that of the control, reaching 12.6% of DW cell biomass.

Selection for Hyoscyamine and Cinnamoyl Putrescine Overproduction in Cell and Root Cultures of Hyoscyamus muticus

Hairy root cultures of Hyoscyamus muticus have been shown to produce stable levels of tropane alkaloids comparable to those found in whole plants. In contrast, cell cultures of this and other solanaceous species produce only trace amounts of alkaloids but can be used for selection of metabolic variants. We have taken advantage of both systems and the ability to convert between them in vitro in an effort to select for increased production of the tropane alkaloid hyoscyamine. Hairy roots were converted into cell suspensions by addition of 1 mg/L 2,4-dichlorophenoxyacetic acid to Murashige-Skoog medium (T. Murashige and F. Skoog [1962] Physiol Plant 15: 473-497) and screened for resistance to the amino acid analog p-fluorophenylalanine (PFP). Cells that could grow in media containing 400 [mu]M PFP were selected and cloned from single cells. The resistant cells accumulated high levels of cinnamoyl putrescines, which share the same biosynthetic precursors as hyoscyamine. Hairy root cultures were regenerated from both PFP-sensitive and PFP-resistant cells by removing 2,4-dichlorophenoxyacetic acid from the medium. Resistance to PFP continued to be expressed in regenerated roots. Higher levels of hyoscyamine were found in hairy roots regenerated from PFP-resistant cells than were found in controls. We suggest that the precursors overproduced by the PFP-resistant cells can be diverted into the hyoscyamine pathway upon the regeneration of root cultures.

L‐Phenylalanine production by double auxotrophic analogue resistant mutants of Arthrobacter globiformis

AbstractA number of tryptophan plus tyrosine double auxotrophic mutants isolated by the NTG treatment of a glutamate producing strain of Arthrobacter globiformis were found to excrete phenylalanine in a mineral salt medium. By controlling the pH of the medium to near neutrality, the active growth period could be extended up to 72 h and more phenylalanine was accumulated compared to the unregulated culture where the growth period took up to 48 h. Under optimum culture conditions, the best double auxotroph (TT‐39) produced 3 g phenylalanine/l. Further improvement of phenylalanine production has been achieved by the step‐by‐step isolation of a mutant resistant to the phenylalanine analogues p‐fluorophenylalanine (PFP) and β‐2‐thienylalanine (TA) from the TT‐39 strain. Under optimum culture conditions, the best double auxotrophic analogue resistant mutant TT‐39 PTr‐21 yielded 8.7 g/l phenylalanine.

Phenylalanine ammonia‐lyase activity and capsaicin‐precursor compounds in p‐fluorophenylalanine‐resistant and ‐sensitive variant cells of chili pepper (Capsicum annuum)

Cell suspension cultures of chili pepper (Capsicum annuum L. cv. Tampiqueño 74) displaying differences in their resistance to p‐fluorophenylalanine (PFP) and in their contents of capsaicin (the compound which is responsible for the hot taste of chili pepper fruits) were characterized in relation to the activity of phenylalanine ammonia‐lyase (PAL; EC 4.3.1.5), the levels of free l‐phenylalanine, phenolics and the phenylpropanoid acids involved in capsaicin biosynthesis. A nonselected cell line, a sensitive line (CA‐02), a moderately resistant cell line (CA‐29) and two resistant cell lines (CA‐04 and CA‐16) were studied. Higher PAL activities and higher levels of phenylalanine and phenolics were found in the PFP‐resistant cells even after a minimum of 9 subcultures (15 days each) in the absence of the analog, indicating that the selected trait was stable. PFP‐resistant chili pepper cells accumulated higher amounts of capsaicin precursors (cinnamic, caffeic and ferulic acids) than either the nonselected cells or the sensitive cell line. p‐Coumaric acid was not detected at significant levels in any of the cell cultures. Overall, accumulation of free phenyl‐alanine correlated well with PAL activity, phenolics, phenylpropanoids and capsaicin levels, suggesting an active flow through the phenylpropanoid pathway in PFP‐resistant cells of chili pepper.

Increased capsaicin content in PFP-resistant cells of chili pepper (Capsicum annuum L.)

Cell suspensions of chili pepper (Capsicum annuum L.) were subjected to a selection process on semisolid medium containing the amino acid analog p-fluorophenylalanine (PFP). Four cell lines with different degrees of resistance were selected and suspension cultures were established from each of them. Resistance was retained even after 75 days of culture in the absence of PFP. PFP-resistant cell lines accumu lated higher levels of capsaicin than sensitive lines even after prolonged culture in PFP-free medium. Capsaicin production in non-selected cells was only 26.8% of that found in one cell line resistant to 500 μM PFP. The capsaicin content in the non-selected cell suspension and in one of the resis tant cell lines was 6.7% and 24.9% respectively, that of fruits.

Defective regulation of the phenylalanine biosynthetic operon in mutants of the phenylalanyl-tRNA synthetase operon

Among mutants of Escherichia coli resistant to p-fluorophenylalanine (PFP) were some with constitutive expression of the phenylalanine biosynthetic operon (the pheA operon). This operon is repressed in the wild type by phenylalanine. The mutation in three of these mutants mapped in the aroH-aroD region of the E. coli chromosome at 37 min. A plasmid bearing wild-type DNA from this region restored p-fluorophenylalanine sensitivity and wild-type repression of the pheA operon. Analysis of subclones of this plasmid and comparison of its restriction map with published maps indicated that the mutations affecting regulation of the pheA operon lie in the structural genes for phenylalanyl-tRNA synthetase, pheST, probably in pheS. Thus, the pheST operon has a role in the regulation of phenylalanine biosynthesis, the most likely being that wild-type phenylalanyl-tRNA synthetase maintains a sufficient intracellular concentration of Phe-tRNA(Phe) for attenuation of the pheA operon in the presence of phenylalanine. A revised gene order for the 37-min region of the chromosome is reported. Read clockwise, the order is aroD, aroH, pheT, and pheS.

Variation in the Chromosome number of cultured shoot tip of Vitis spp. by p-fluorophenylalanine treatment.

The effect of p-fluorophenylalanine (PFP) on the induction of variations in the chromosome number was investigated in cultured shoot tips of Vitis clones, including triploid and tetraploid cultivars and interspecific hybrids. A higher concentration (400 μM) of PFP inhibited the growth of the shoot tips. The abnormal mitoses and the variations in the chromosome number including the induction of haploid and aneuploid cells were observed in the shoot tip cells treated with PFP. Variations in the chromosome number were also observed in the root tip cells of regenerated plants after PFP treatment. However, most of cells with abnormal chromosome number co-existed with normal cells. The average perc6ntage of the anormal cells was about 1.0% among total observed cells of all the clones examined. In most of the cases the abnormal chromosome number observed in the roots was euploids, regardless of the ploidy level of the clones examined. There were fewer aneuploids in root cells than in the shoot tip cells. In all the experiments, two variants (about 0.3% of the plants checked) had large sectors with reduced numbers of chromosomes in the shoots. One was a triploid-tetraploid variant from the tetraploid cultivar Kyoho, and the other was a diploid-triploid variant from the triploid clone KC-1.

Use of 19F NMR spectroscopy for study of the different steps of transamination model reactions

The reaction products of pyridoxal-5′-phosphate (PLP) and p-fluorophenyl alanine were observed versus time using 1H noise decoupled 19F NMR signal in the 4–8 pD range, in D 2O at room temperature. Several signals were observed whose chemical shifts are pH dependant. Two of them are seen at early times and regularly decreased, then an other signal appears, goes through a maximum and then disappears in the same time that two (one of them being splitted in two for a small pH range) increases and are finally the only signals for long reaction times. This behaviour can be interpreted with the following equations: ▪ and where the underlined symbols contains aromatic fluorine. In the cited conditions Ket bas too low a concentration to be observed (it was nevertheless identified from KA and PMP). Equilibrium 1 and 3 are fast at the NMR time scale. From measurements of interesting of signals k 2(k - 2 being neglected) and k 4 can be measured versus pH. Comparison of k 2 values with those obtained from ordinary UV measurements are made. Some more applications of these 19F NMR measurements which are actually in progress will be presented.

Effect of p-fluorophenylalanine and amino acid derivatives on variation of somatic chromosome number in grape root tip cells.

To analyze the pattern of variations in chromosome number of root tip cells of grape seedlings induced by p-fluorophenylalanine (PFP), the effectiveness of treatment with phenylalanine derivatives and amino acid analogs during germination period was investigated. The frequency of cells showing variations in the chromosome number was significantly correlated with the frequency of occurrence of abnormal mitosis, but not with seed germination, seedling growth nor mitotic activity in the root tips. Compounds closely related to phenylalanines, such as those obtained by substitution on the phenyl ring, the replacement of the phenyl ring by a thienyl ring, or the methylation on the side chain, were effective, though less than PFP. A comparison of the stereoisomers of PFP showed that L-PFP induced a reduction of the chromosome number, unlike the D-enantoimer. N-acetyl-PFP, which was a modification of the amino group of PFP, did not induce chromosome variation. Among eight analogs of other amino acids, only m-fluorotyrosine and 5-fluorotryptophan induced variation in the chromosome number to the same extent as PFP.

Isolation, Protoplast Culture, and Plant Regeneration of PFP-Resistant Variants of Nicotiana tabacum Sulsu

Summary p-Fluorophenylalanine (PFP) resistant variants of Nicotiana tabacum Sulsu were routinely isolated from rapidly growing suspension cultures by plating in the presence of 0.5 mM PFP. The PFP-resistant phenotypes of about 10% of the variants isolated were stable, and calli have retained resistance to PFP after more than one year in culture. Resistance was retained whether the cells were grown in the presence or absence of PFP. Calli of PFP-resistant cell lines were induced to regenerate shoots on MS medium with 5 µM 6-benzylaminopurine. In some PFP-resistant lines shoots were regenerated in the presence of 0.5 mM PFP. Shoot regeneration from PFP-resistant cell lines was only achieved shortly after their isolation. Although shoots could be rooted, the albino Su mutantion precluded transfer of the plants to soil. Suspension cultures of PFP-resistant cell lines retained the same chromosome number (2n = 48) of intact plants, even after several months in liquid culture. Protoplasts (isolated from suspension cultures established from calli of PFP-resistant cell lines) were cultured in the presence of 0.125 mM PFP, a concentration sufficient to inhibit cell wall regeneration and cell division of normal Sulsu protoplasts. Suitable conditions are therefore defined for the use of PFP resistance as a selective marker in recognition of somatic cell hybrids accomplished via protoplasts fusion.

Para-Fluorophenylalanine Resistant Cell Lines of Tobacco

Thirty-one p-fluorophenylalanine (PFP)-resistant cell lines of tobacco cell cultures ( Nicotiana tabacum L. cv. Xanthi, Nicotiana tabacum L. cv. Maryland Mammoth and Nicotiana glauca ) were selected in liquid media and on agar plates. Three of these cell lines were also resistant to 5-methyltryptophan (5-MT) indicating a resistance due to decreased uptake of amino acids and their analogs. Three lines of Nicotiana tabacum L. cv. Maryland Mammoth and three of Nicotiana glauca accumulated higher levels of phenylalanine and tyrosine and became likely resistant to PFP due to the overproduction of the corresponding natural amino acids. All five lines of Nicotiana tabacum L. cv. Xanthi, one of Nicotiana tabacum L. cv. Maryland Mammoth and five of Nicotiana glauca accumulated higher levels of phenolics. Decreased uptake of an analog or dilution of the toxic antimetabolite by enlargement of the pool size of the corresponding natural amino acid are reasonable explanations for acquired resistance. The often observed concomitant occurrence of PFP-resistance with increased levels of phenolics is discussed.

Effects of α-Aminooxy-β-phenylpropionic Acid on Phenylalanine Metabolism in p-Fluorophenylalanine Sensitive and Resistant Tobacco Cells

A p-fluorophenylalanine (PFP) resistant cell line with high phenylalanine ammonia lyase (PAL) activity and wild type cells with low PAL activity were compared in their responses to PAL inhibition by alpha-aminooxy-beta-phenylpropionic acid (AOP). Inhibition of PAL reduced the levels of the main phenolic compounds to 30% of the controls. Free phenylalanine pools increased 17 fold in the resistant line and 6 fold in the sensitive line, respectively. The accumulation of phenylalanine did not reduce the flow of labeled shikimic acid into the aromatic amino acids tyrosine and phenylalanine. The results are discussed with respect to the feedback inhibition of chorismate mutase activity by phenylalanine and tyrosine in both cell lines.

Mutational synergism between p-fluorophenylalanine and UV in Coprinus lagopus

Previous studies have shown that the amino acid analogue p-fluorophenyl-alanine (PFP) is mutagenic to Coprinus lagopus due to its incorporation into proteins [32]. Spontaneous mutations, PFP and UV mutagenesis and PFP/UV synergism have been studied in a UV resistant strain and in two complementing UV sensitive mutant strains. By comparison to the UV resistant strain, one UV sensitive strain shows normal spontaneous mutations, 1.4% PFP-induced mutations and 50-fold UV mutagenesis. The second UV sensitive strain has 19-fold spontaneous mutation frequency, 8% PFP induced mutations ans slightly elevated UV mutagenesis. In all 3 strains the PFP/UV synergism is comparable (4–5 times the arithmetic expected). The results indicate that PFP mutagenesis is due to the incorporation of PFP into enzymes normally functioning in the organism but which also participate in UV repair mechanisms. A model is proposed for UV repair which is based on a PFP sensitive excision repair system of at least two enzymes, an alternative “error proof” pathway which is not susceptible to PFP and an “error prone” pathway which is responsible for UV mutagenesis and is suscetible to PFP as shown by the PFP/UV synergism. Because PFP is given before UV treatment, this implies a UV inducible cofactor and a PFP sensitive enzyme which only functions after UV activation.

Correlation between Phenylalanine Ammonia Lyase Activity and Phenolic Biosynthesis in p-Fluorophenylalanine-sensitive and -resistant Tobacco and Carrot Tissue Cultures

Phenylalanine ammonia lyase (PAL) activity was measured in p-fluorophenylalanine (PFP)-sensitive and -resistant tobacco (Nicotiana tabacum L.) and carrot (Daucus carota L.) cell lines which are known to oversynthesize phenylalanine. A correlation between phenolic levels and PAL activities was detected. The phenylalanine analog-resistant and -sensitive carrot cells showed no differences in the accumulation of phenolic compounds and PAL activities. The PFP-resistant tobacco cells, however, had 10 times higher levels of phenolics and also 10 to 20 times higher PAL activities than the PFP-sensitive line. The PAL activity in the resistant tobacco line increased dramatically after inoculation of the cells into fresh medium. Conditions affecting this increase were characterized.A comparison of the responses of PAL activity to light treatment and changes in the 2,4-dichlorophenoxyacetic acid concentration in the medium revealed no differences for both tobacco lines. After inoculation of the cells into fresh medium, the rates of nitrate uptake from the medium were similar for both lines as were the rates of protein synthesis. The reason for the increased PAL levels in the resistant tobacco cells remains unknown.

Linkage analysis of developmental mutations in aggregation-deficient mutants of Dictyostelium discoideum.

Simple parasexual genetic techniques have been employed to extend the linkage analysis initiated in an earlier study (Coukell, 1975) of developmental mutations (agg mutations) in 40 independently isolated aggregation-deficient mutants of Dictyostelium discoideum. Using these techniques, agg mutations in 28 of the 40 mutants have been assigned to 4 linkage groups: 16 in group II, 1 in group III, 10 in group IV, and 1 in group VI. None of the agg mutations analyzed appear to map in linkage group I. In addition, a new temperature-sensitive growth locus, designated tsgJ, was mapped in group III. It was also found that diploid strains of D. discoideum are readily induced to undergo haploidization when grown on 0.1% p-fluorophenylalanine (PFP) at 25.5 degrees C. Growth of diploid strains on PFP had no effect on the type of segregant classes obtained (i.e. PFP does not induce mitotic crossing-over), the subsequent growth and/or development of the segregants, or the ability of the segregants to reform stable diploids.

The Dynamics of Cell Proliferation in Haploid and Diploid Tissues of Nicotiana tabacum

Summary Haploid plants of Nicotiana tabacum were obtained by culturing anthers at the microspore stage. Explants of pith tissue from haploid as well as diploid plants were grown in vitro, and changes in cell number, cell size, and fresh weight were determined over a 16-day period. In the tissue from diploid plants a decrease in cell size was seen after the eighth day in culture, but the tissues from haploid plants exhibited a slight increase in cell size. Spectrophotometric determination of deoxyribonucleic acid (DNA) was made to investigate the time course of DNA synthesis during cell proliferation and culture. A wide range of ploidy levels was seen in the fresh pith tissues of both haploid and diploid .plants. Culture of these tissues resulted in increased levels of ploidy in a large proportion of the cells. In general, cells with lower DNA content per nucleus entered the cell cycle sooner than those with a higher amount. After an extended period of time in culture both haploid and diploid cxplants showed a variety of polyploid and aneuploid cells. The effects of pfluorophenylalanine (PFP) on explant growth and ploidy were tested. PFP was inhibitory to the growth of all cells, but haploid cells were inhibited slightly less than those of higher ploidy. Spectrophotometric data are presented to demonstrate that PFP can be used to maintain - or even increase - the population of haploid cells in tissue cultures derived from haploid plants of tobacco.

Effects of p-fluorophenylalanine on the induction of mutations in bacteriophage T4 II. Nitrous acid mutagenesis

The effects of p -fluorophenylalanine (PFPA), an analogue of phenylalanine (PHE), on the potency and the specificity of nitrous acid (NA) mutagenesis in the r system of bacteriophage T4 were measured. 1. (a) Forward mutation The frequencies of NA-induced r mutants were approximately doubled when mutagenized phage infected E. coli B in the presence of the analogue compared to controls where PHE was substituted for PFPA, or where no amino acid was added. The spontaneous forward mutation frequency was not affected by the analogue, nor was the specificity of the NA mutagenesis. 2. (b) Reverse mutation The frequency of spontaneous and NA-induced reversion of an rII transition mutant was unaffected by PFPA if the phage were plated direct on the restrictive host after mutagenesis. However, if mutagenized phage were first allowed a cycle of growth in the presence of PFPA the induced reversion frequency was increased about 3-fold compared to the control.

Effects of p-fluorophenylalanine on the induction of mutations in bacteriophage T4 I. 5-bromouracil mutagenesis

The amino acid analogue p -fluorophenylalanine (PFPA) was found to have no mutagenic activity in the r system of bacteriophage T4. However, under standard conditions for 5-bromouracil (5-BU) mutagenesis, PFPA depressed the induced frequencies for both forward and reverse mutations. When the folate antagonist sulphanilamide (SU) was omitted from the mutagenic treatment medium or when it was replaced by Trimethoprim (TM), another folate antagonist, this depressive effect was abolished. It was proposed that PFPA alleviated the inhibitory action of SU.

Effects of p-fluorophenylalanine (PFP) on the growth of cell lines differing in ploidy and derived from Nicotiana sylvestris

The growth inhibitory activity of p-fluorophenylalanine (PFP) (concentration range 25–75 μg/ml) has been tested on callus initiation and early callus growth and on suspension cultures derived from one diploid and three haploid plants of Nicotiana sylvestris. All the culture lines contained cells at more than one level of ploidy; the cultures of haploid origin contained a high level of haploid cells. Under conditions of partial growth inhibition by PFP there was, despite the reduced sensitivity of the cultures of haploid origin, no preferential growth of haploid cells in these cultures. Genotype not ploidy level determined sensitivity to growth inhibition by PFP.