Effect of exogenous amino acids on the intracellular content of proline and other amino acids in Daucus carota cells

The effect of tryptophan on the biosynthesis of proline has been investigated. Cells of Daucus carota grown in B5 medium supplemented with 5×10^–4M tryptophan acquired the ability to grow in the presence of inhibitory concentrations of azetidine-2-carboxylic acid, an analog of proline. When trp was added to carrot cell cultures at sub-growth inhibiting concentrations, overproduction of intracellular free proline was observed. An increase was also observed for lys, his, ala, leu and phe. Likewise, the addition of asparagine, glutamic acid and phenylalanine to the medium stimulated the intracellular increase of free proline and other amino acids.Abbreviations A2CA azetidine-2-carboxylic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 5MT 5-methyltryptophan - P5C pyrroline-5-carboxylic acid - f.wt. fresh weight - d.wt. dry weight     

Detection,characterization, and phytotoxic activity of the nucleoside antibiotics,Blasticidin S and 5-Hydroxylmethyl-Blasticidin S

Screening for herbicidal compounds carried out on culture broths of Streptomyces strains isolated from soil resulted in the detection of potent phytotoxic activity. The active principles were isolated, and identified as the nucleoside antibiotics Blasticidin S (Bl-S) and 5-Hydroxymethyl-Blasticidin S (H-M-Bl-S). Bl-S was more active than H-M-Bl-S on seedling germination in petri dishes and in postemergence greenhouse tests. Moreover both antibiotics were more phytotoxic to dicotyledonous than to monocotyledonous plants. The increased sensitivity of dicots was confirmed in carrot and rice cell cultures. Both compounds also inhibited [¹⁴C]amino acid incorporation into proteins of rice and carrot cell cultures. Protein synthesis was more affected in carrot than rice.     

Biochemical differences between carrot inbreds differing in plant regeneration potential

Cultures derived from domestic carrot (Daucus carota L.) inbreds were found to vary with respect to regeneration potential as measured by the production of somatic embryos in suspension cultures. A number of biochemical parameters previously reported to distinguish embryogenic from non-embryogenic cultures of other species were measured in these carrot cell lines. Ethylene production was found to be inversely related to regeneration potential. The cell line producing the greatest number of somatic embryos exhibited the lowest rate of ethylene biosynthesis, even when grown on 2, 4-D-containing maintenance medium. A specific isozyme of acid phosphatase was associated with embryogenic calli. Proteins visualized by SDS-PAGE did not discriminate between embryo-forming and proliferating calli in all inbreds.     

Daucus carota Cells Contain Specific DNA Methyltransferase Inhibitors that Interfere with Somatic Embryogenesis

Abstract: Results reported in this paper show that carrot cells contain a thermostable inhibiting activity for cytosine‐5‐DNA methyltransferase that, upon filtration chromatography, can be resolved into three major peaks. Inhibiting activity was found in all plant species tested, though at a concentration lower than in carrot. These inhibiting activities differ in size, sensitivity to various hydrolytic treatments, specificity for DNA METases of eukaryotic and bacterial origin and kinetics of inhibition. Results of chemical analyses indicate that the inhibitors differ from lipidic inhibitors described in Escherichia coli and Streptomyces sp. and, given their sensitivity to proteinase K, appear to have a proteinaceous nature. The addition of these inhibitors (Sephadex G25 peak II and peak III) to actively growing suspension rice cells reduced the rate of in vivo DNA methylation without interfering with DNA synthesis. Peak II also induced a general demethylation effect in carrot cell suspension, even if weaker than that caused by 5‐azacytidine. Interestingly, inhibitors suppressed carrot embryogenesis but did not prevent undifferentiated cell proliferation of suspension cultures.     

Moderately increased constitutive proline does not alter osmotic stress tolerance

Proline-overproducing carrot cell lines were isolated by selection in medium containing hydroxyproline, a toxic analogue of proline. During growth of the cells in culture, length of lag phase, doubling time, and maximum fresh weight were the same for the hydroxyproline-resistant cell line (HP) and the wild-type cell line (JW). Proline content and resistance to hydroxyproline in the HP and JW lines were not strictly correlated indicating that another reason besides the constitutive level of proline is involved in hydroxyproline resistance. Tolerance to polyethylene glycol-induced desiccation stress was not different between the two lines except perhaps at the early stages of culture growth when the proline levels of the two cell lines were nearly the same. The complexity of the relationship between proline accumulation and osmotolerance is discussed and strategies to achieve constitutive high levels of proline accumulation in plants are proposed.     

Lipids of Daucus carota cell suspension cultures

In carrot cell suspension cultures greater amounts of phospholipid were detected than in carrot root material, but the major phospholipid classes were the same in both materials, and their fatty acid composition was very similar. In contrast to the cultured cells, no significant amounts of free fatty acids and monoglycerides, as well as diglycerides, could be detected in the carrot root. The fatty acid composition of the major lipid classes from cell cultures is reported for the first time in this report. The degree of unsaturation was higher in triglycerides and phospholipids than in free fatty acids. In a study of phospholipid biosynthesis, [³H]-glycerol was shown to be incorporated into 4-phospholipids (PE, PC, PG, PS⁷) to different extents. The highest specific activity was observed in PC and PG. Five molecular species were isolated from each of the 4 phospholipids and analyzed by GC-MS and LSC.     

Hydroxyproline in the major capsid protein VP1 of polyomavirus.

Amino acid analysis of [3H]proline-labeled polyomavirus major capsid protein VP1 by two-dimensional paper chromatography of the acid-hydrolyzed protein revealed the presence of 3H-labeled hydroxyproline. Addition of the proline analog L-azetidine-2-carboxylic acid to infected mouse kidney cell cultures prevented or greatly reduced hydroxylation of proline in VP1. Immunofluorescence analysis performed on infected cells over a time course of analog addition revealed that virus proteins were synthesized but that transport from the cytoplasm to the nucleus was impeded. A reduction in the assembly of progeny virions demonstrated by CsCl gradient purification of virus from [35S]methionine-labeled infected cell cultures was found to correlate with the time of analog addition. These results suggest that incorporation of this proline analog into VP1, accompanied by reduction of the hydroxyproline content of the protein, influences the amount of virus progeny produced by affecting transport of VP1 to the cell nucleus for assembly into virus particles.     

Elicited Teucrium chamaedrys cell cultures produce high amounts of teucrioside,but not the hepatotoxic neo-clerodane diterpenoids

Teucrium chamaedrys, one of the most common and investigated species of the genus Teucrium, has been used for centuries in traditional medicine for many purposes. Its phytochemical components comprise, among others, phenylethanoid glycosides (PGs) and neo-clerodane diterpenoids. Several reports have demonstrated a wide range of beneficial biological and pharmacological activities of the phenylethanoid components, while the diterpenes were shown to be strongly hepatotoxic. In this work, in vitro cultures were established from leaf explants of T. chamaedrys. Both solid (callus) and liquid (cell suspension) cultures maintained the capacity to produce PGs, with teucrioside (TS) representing the most abundant one. Cell suspensions had a lower TS content than that found in leaf extracts, but higher than that of calli. An NMR-based metabolomics approach was used to compare the product profile of intact plants vs. cell suspension cultures, and results showed that neo-clerodane diterpenes, present in the intact plant, were not detected in cell cultures. Several elicitors were supplied to cell cultures with the aim of increasing TS production, and elicitation was tested at different growth phases and by exposing cells for different periods. Methyl jasmonate and fungal mycelia from Trichoderma viridae and Fusarium moniliforme were able to significantly increase TS production if supplied at the early-exponential growth phase for 24 h. Based on the proposed link between proline and the phenylpropanoid pathways, proline accumulation in cell cultures was followed throughout a 14-day culture period, showing that it strictly reflected that of TS production. Moreover, exogenously supplied proline, and its analogue hydroxyproline, turned out to be very effective in increasing teucrioside production.     

Corn starch as an alternative gelling agent for plant tissue culture

Growth and differentiation of plant cell cultures was increased when media were gelled with corn starch instead of agar. Dry weight of tobacco and wild carrot cell cultures on media gelled with starch was more than three times that of cultures on media gelled with agar. Higher yield of anthocyanin and dry weight of embryos were found in wild carrot cultures grown on media gelled with corn starch. The starch-mediated increase in growth and differentiation of wild carrot cells was accompanied by an increase in density of the cultures shown by higher dry weight/fresh weight ratios.     

Influence of copper ions on growth,lipid peroxidation,and proline and polyamines content in carrot rosettes obtained from anther culture

Effect of Cu (0.1, 1, 10, and 100 μM) on the regeneration of carrot (Daucus carota L.) androgenic embryos of var. Feria and 1014 breeding line as well as on polyamines (PAs), proline contents, lipid peroxidation and Cu accumulation after 16 and 24 weeks was studied. Generally, growth of Feria rosettes was better than that of the 1014 line. Significant increase in Cu content in tissues was observed in both cultures grown at the highest Cu concentration (100 μM). The dose-dependent increase in proline in the 16-week-old culture of Feria was observed, while in 1014 its level increased only at the highest applied Cu concentration. On the contrary, in the 24-week-old culture, significant increase in the proline content were observed at 100 and 10 μM Cu in Feria and in 1014 breeding lines, respectively. The decline in proline content and decrease in embryogenic ability in the line 1014 grown in the presence of the highest Cu concentration for 24 weeks may indicate that a certain threshold of intracellular Cu was crossed. Both in Feria and 1014 line, putrescine and spermidine were the most abundant free PAs. The increased content of proline and higher contents of the constitutive free putrescine and spermidine in Feria cultivated for 24 weeks at the highest Cu concentration point to better protection of this cultivar. Thus, it seems that the higher tolerance of Feria to oxidative stress (characterized by lower thiobarbituric acid reactive substances value) may result from higher constitutive level of PAs. These data confirm the suggestion that variations in PA levels depend not only on the concentrations of metals tested, but also on plant species and cultivars. The role of PAs and proline in the carrot cultures treated with Cu is discussed.     

Isopropyl m-Chlorocarbanilate and Its Hydroxylated Metabolites: Their Effects on Cell Suspensions and Cell Divison in Soybean and Carrot

Hydroxylated metabolites of isopropyl m-chlorocarbanilate (chlorpropham) are found in intact soybean plants (Glycine max Merr.). The metabolites are isopropyl 2-hydroxy-5-chloro-carbanilate (2OH) and isopropyl 3-chloro-4-hydroxycarbanilate (4OH). The phytotoxicity of these metabolites and chlorpropham was tested in cell suspensions and roots of intact soybean seedlings and cell cultures of carrot (Daucus carota L.). The growth of soybean cell suspensions was inhibited with 50 μM chlorpropham. Ten μM chlorpropham usually slowed initial growth of the cultures while 5 μM and 0.1 μM chlorpropham had no effect. The 2OH and 4OH metabolites had no significant effect on dry weight over the same concentration range. Some metabolism of chlorpropham, 2OH and 4OH occurred during 6 and 48 h of incubation with soybean cells. The results are interpreted to mean that all three analogs penetrated into the cells, were metabolized, and some of the metabolites excreted back into the medium. Mitotic index studies of intact 3-day-old soybean roots showed that 2OH inhibited mitosis to a greater extent than chlorpropham, whereas 4OH produced only a slight and insignificant reduction compared to controls. Chlorpropham, 2OH and 4OH (at 50 μM) all reduced the growth of wild carrot cultures grown in the presence or absence of 2,4-D. Therefore, hydroxylation of chlorpropham at the 2′ or 4′ positions of the 5′ chlorinated benzene ring is not sufficient to render the compound nonphytotoxic in all plant systems.     

JM47, a cyclic tetrapeptide HC-toxin analogue from a marine Fusarium species

The known metabolite, enniatin B, and a cyclic tetrapeptide, JM47, which is a new natural product, were extracted from brown rice cultures of a marine fungus, identified as a Fusarium species, isolated from the marine alga Codium fragile. NMR studies, including 15N HMQC and 15N HMBC, established the structure of JM47 as cyclo(Ala-Ala-Aoh-Pro), where Aoh is the amino acid, (2S,9S)-2-amino-8-oxo-9-hydroxydecanoic acid. The absolute stereochemistry of the Aoh side chain carbinol centre was determined using Mosher ester methodology. Analysis of NOESY data assisted by molecular modelling revealed an alternating L-, D-, L-, D-configuration for the tetrapeptide core. The absolute stereochemistry of the core was determined by acidic hydrolysis and chiral TLC analysis of the proline residue. JM47 belongs to the HC-toxin family of cyclic tetrapeptides which possess a 2-amino-8-oxo-9,10-epoxydecanoic acid residue in place of the Aoh unit. This is the first report of an analogue of HC-toxin from a marine Fusarium species.     

Purification from conditioned medium and chemical identification of a factor that inhibits somatic embryogenesis in carrot

Somatic embryogenesis is strongly inhibited in cultures of carrot (Daucus carota L.) cells when the cell density is high. The inhibition is caused by factors that are released by cells into the medium of such cultures. In this study, we purified and identified one of the inhibitory factors found in the medium of high-cell-density cultures of carrot cells. The inhibitory factor with the strongest apparent activity was purified by fractionation with ethylacetate, chromatography on an octadecylsilyl (ODS) silica gel-column and HPLC. The inhibitory factor had a single peak of absorbance at 280 nm and was identified as 4-hydroxybenzyl alcohol by mass spectrometry and 1H- and 13C-NMR spectroscopy. Authentic 4-hydroxybenzyl alcohol strongly inhibited the formation of somatic embryos at a concentration equal to that in high-cell-density cultures. These results suggest that 4-hydroxybenzyl alcohol is a major factor that accumulates in high-cell-density cultures of carrot cells and inhibits somatic embryogenesis.     

Resistance to Azetidine-2-carboxylic Acid and Sodium Chloride Tolerance in Carrot Cell Cultures and Spirulina platensis

Mutants of Spirulina platensis and of Daucus carota resistantto azetidine-2-carboxylic acid were tested for NaCl tolerance.A positive correlation was found between proline overproductionand osmotolerance. In carrot lines proline overproduction wasnot strictly proportional to NaCl tolerance insofar as cellscharacterized by differences in proline overproduction showedsimilar osmotolerance, suggesting that other factors could beinvolved. (Received March 17, 1983; Accepted June 14, 1983)     

Purification, properties and comparative specificities of the enzyme prolyl-transfer ribonucleic acid synthetase from Phaseolus aureus and Polygonatum multiflorum

1. A prolyl-s-RNA synthetase (prolyl-transfer RNA synthetase) has been purified about 250-fold from seed of Phaseolus aureus (mung bean), a species not producing azetidine-2-carboxylic acid, and more than 10-fold from rhizome apices of Polygonatum multiflorum, a liliaceous species containing azetidine-2-carboxylic acid. The latter enzyme was unstable during ammonium sulphate fractionation. 2. The enzymes exhibited different substrate specificities towards the analogue. That from Phaseolus, when assayed by the ATP-PP(i) exchange, showed azetidine-2-carboxylic acid activation at about one-third the rate with proline. Both labelled imino acids gave rise to a labelled aminoacyl-s-RNA. The enzyme from Polygonatum, however, activated only proline. 3. The enzyme from Polygonatum also formed a labelled prolyl-s-RNA with Phaseolus s-RNA but at a lower rate than when the Phaseolus enzyme was used. No reaction occurred when the Phaseolus enzyme was coupled with Polygonatum s-RNA, and only a very slight one was observed when both enzyme and s-RNA came from Polygonatum. 4. Protein preparations from seeds of Pisum sativum, another species not producing azetidine-2-carboxylic acid, also activated the analogue in addition to proline, whereas those from rhizome and seeds of Convallaria, the species from which the analogue was originally isolated, failed to activate it. However, a liliaceous species not producing the analogue, Asparagus officinalis, activated it. 5. Of the other proline analogues investigated, only 3,4-dehydro-dl-proline and l-thiazolidine-4-carboxylic acid were active with the enzyme preparation from Phaseolus. 6. pH optima of 7.9 and 8.4 were established for the enzymes from Phaseolus and Polygonatum respectively. 7. The Phaseolus enzyme was specific for ATP and PP(i). Mn(2+) partially replaced the requirement for Mg(2+) as cofactor. Preincubation with p-chloromercuribenzoate at a concentration of 0.5mm or higher produced over 99% inhibition of the Phaseolus enzyme. One-half the enzymic activity was destroyed by preheating for 5min. at 62 degrees in tris-hydrochloric acid buffer, pH7.9. 8. All experimental evidence supports the hypothesis that azetidine-2-carboxylic acid and proline are activated by the same enzyme in Phaseolus preparations, whereas the analogue was inactive in all Polygonatum preparations. The possible nature of this different substrate behaviour is discussed.     

Stress‐driven increase in proline levels,and not proline levels themselves,correlates with the ability to withstand excess salt in a group of 17 Italian rice genotypes

• In most plant species, a rapid increase in free proline content occurs following exposure to hyperosmotic stress conditions. However, inconsistent results were reported concerning the role of such an increase on the plant response to water shortage or excess salt. Therefore, the possibility that proline accumulation may help the cell to withstand stress conditions, or that it simply represents a stress marker, is still a matter of debate. A possible relationship between proline accumulation and salt tolerance was investigated in a set of 17 Italian rice varieties.
• Rice seedlings were exposed to increasing salt concentrations during germination and early growth. The resulting levels of free proline were measured separately in shoots and roots and compared to those in untreated controls. Results were related to the corresponding ability of a given genotype to tolerate stress conditions.
• Neither absolute proline levels in untreated or in salt‐stressed seedlings showed a straightforward relationship to the relative tolerance to salt, estimated as conductivity values able to reduce growth by 10 or 50%. Conversely, a highly significant correlation was found between the increase in proline levels in shoots and the ability to withstand stress.
• The results strengthen a recent hypothesis suggesting than an increase in proline metabolic rates, more than the resulting proline content, may help the cell to counteract the effects of abiotic stress conditions.

     

Influence of dietary carrot on cytostatic drug activity of cyclophosphamide and its main directly acting metabolite: induction of sister-chromatid exchanges in normal human lymphocytes, Chinese hamster ovary cells, and their DNA repair-deficient cell lines

We have utilized an in vivo drug metabolism technique (i.e. injecting the chemical into rat and isolating plasma with metabolites from blood) for detecting the genotoxicity of indirectly acting cyclophosphamide and its directly acting metabolite phosphoramide mustard in cultures of human peripheral blood lymphocytes of normal individuals, Fanconi's anaemia (FA) and aplastic anaemia (AA) patients, wild-type Chinese hamster ovary cells (CHO) and its DNA repair-deficient mutant 43-3B cells. In addition, the influence of dietary carrot on the clastogenic activity of these 2 chemicals in all the different cell types was studied. The genotoxicity was assessed by the ability of the metabolites of these agents to induce sister-chromatid exchanges in the treated cells. A dose-dependent increase in the frequencies of sister-chromatid exchanges was observed in all cell strains following treatment with activated metabolites of cyclophosphamide or phosphoramide mustard. The sensitivity of lymphocytes from normal donors, FA and AA patients to these 2 chemicals was similar. In CHO cell lines the induced frequency of sister-chromatid exchanges was slightly higher after treatment with the metabolites of cyclophosphamide than with phosphoramide mustard. The mutant 43-3B cells responded with higher frequencies of SCEs when compared to the wild-type CHO cells, about 1.5-2-fold, at low doses. Pretreating of rats with fresh carrot juice effectively inhibited the increase in the frequencies of sister-chromatid exchanges induced by cyclophosphamide in wild-type and mutant CHO cells (P less than 0.01), and to a lesser extent in human lymphocytes (p less than 0.05). In contrast, no inhibitory effect was observed in any of these cell types in combination of dietary carrot for direct acting phosphoramide mustard on the frequency of induced sister-chromatid exchanges. The possibility that dietary carrot exerts its antimutagenic effect by affecting the processes of enzymatic activation of cyclophosphamide is discussed.     

The Incorporation of 14C into the Protein of Particles Isolated from Plant Cells

The incorporation of ¹⁴C from labelled fructose, succinate,urea, and proline, by particulate preparations from dormantand tissue-cultured carrot cells, is examined. It is shown that¹⁴C is incorporated readily from proline, and less readily fromfructose. No significant incorporation occurs from succinateor urea. No differences are noted between the two kinds of preparation.It is concluded that the incorporation of ¹⁴C does not dependon prior transfer of the label to carbon dioxide followed byfixation of carbon dioxide, since the particles do not incorporate¹⁴C from supplied carbon dioxide. Incorporation of ¹⁴C by various fractions of dormant carrottissue is examined, and it is established that the greatestincorporation per mg. nitrogen occurs in particles isolatedat 10,000 g. A total cell homogenate fails completely to incorporate¹⁴C from proline into protein, and this may be due to suppressionof the activity of the particles by a constituent of the supernatantliquid. The presence of coconut milk reduces the incorporationof ¹⁴C from proline by particles sedimented at 10,000 g, andaddition of a protein hydrolysate reduces it further. Hydroxy-prolinedoes not appear to compete with proline for incorporation, andin this respect the paniculate preparations contrast with wholecells. Particles from carrot tissue are shown to be more active inincorporating ¹⁴C from proline than are particles extractedby the same procedure from red beet roots, potato tubers, andskunk cabbage inflorescences. They are, however, considerablyless active than a mitochondrial preparation from rat liver. It is demonstrated by paper chromatography that the bulk ofthe ¹⁴C incorporated in the particles from carrot cells remainsin proline and there is little or no conversion of proline tohydroxyproline in the preparations. The nature of the particlesemployed in this investigation is discussed, and their metabolismconsidered, in relation to the structure and activity of wholecells.     

Effect of anti-microtubular drug amiprophos-methyl on somatic embryogenesis and DNA ploidy levels in alfalfa and carrot cell suspension cultures

A short treatment with the anti-microtubular drug amiprophos-methyl (APM) blocked somatic embryogenesis in alfalfa(Medicago sativa L.) and carrot (Daucus carota L.). The interruption was temporary and restoration of somatic embryogenesis was observed in long-term cultures. In addition to the effect on somatic embryogenesis, APM treatment induced polyploidization the extent of which was concentration dependent. In long-term alfalfa cultures, APM-induced loss of somatic embryogenesis led to ploidy instability and to a shift to DNA aneuploidy. Critical stages of somatic embryogenesis sensitive to disruption of microtubule-mediated processes were determined in carrot cell cultures. Complete embryogenic arrest occurred when APM was added within the first 5 d of embryogenesis from single cells. The role of the cytoskeleton in the first events of somatic embryogenesis and the relation between totipotency and ploidy stabilityin vitro is discussed.