Correlation of betacyanin synthesis with cell division in cell suspension cultures of Phytolacca americana

In suspension cultures of Phytolacca americana , betacyanin accumulation was reduced when cell division was inhibited by treatment with various inhibitors of DNA synthesis or anti-microtubule drugs. Aphidicolin (APC), an inhibitor of DNA synthesis, reduced the incorporation of radioactivity from labeled tyrosine into betacyanin, but the incorporation of radioactivity from labeled 3,4-dihydroxyphenylalanine (DOPA) into betacyanin was not affected by similar treatments. Propyzamide, another anti-microtubule drug, reduced incorporation of radioactivity from tyrosine and DOPA into betacyanin. However, the rate of incorporation from DOPA was higher than that from tyrosine. The results suggest that inhibition of betacyanin accumulation in Phytolacca americana cells by APC and propyzamide is due to suppression of the reaction converting tyrosine to DOPA, which may be closely related to cell division.     

Effects of sucrose on betacyanin accumulation and growth in suspension cultures of Phytolacca americana

The effects of sucrose on betacyanin accumulation and growth in suspension cultures of Phytolacca americana L. were investigated. Maximal betacyanin accumulation was observed at 88 m M sucrose on cell number basis and at 175 m M sucrose on fresh weight basis. This is because cell size decreased as the initial sucrose concentration was increased. Supplementary studies using mannitol indicated that sucrose itself caused increased cell number and that cell size was affected by both sucrose concentration and water potential. Betacyanin accumulation per cell and per fresh weight at a constant concentration of sucrose (88 m M ) decreased with decreasing water potential. When sucrose concentration increased at a constant water potential (–0.7 MPa), betacyanin accumulation per fresh weight increased up to 88 m M and remained at constant level at higher concentrations, while betacyanin accumulation per cell decreased remarkably, due to a dramatic increase in cell number.     

Growth regulator-induced betacyanin accumulation and dopa-4,5-dioxygenase (<Emphasis Type="Italic">DODA</Emphasis>) gene expression in euhalophyte <Emphasis Type="Italic">Suaeda salsa</Emphasis> calli

Suaeda salsa calluses cultured in darkness for 28 d were transferred to Murashige and Skoog (MS) media containing various growth regulators under white light conditions for 10 d to investigate cell growth, betacyanin accumulation, and expression of dopa-4,5-dioxygenase (DODA). Callus growth was markedly promoted when 0.2 mg·L⁻¹ 2,4-D and 0.5 mg·L⁻¹ 6-BA were added to the MS medium. Surprisingly, of the auxins tested, IAA had no effect on betacyanin content, but 2,4-D strongly decreased betacyanin content. Betacyanin content was positively correlated with 6-BA concentrations in the range of 0.1–2.0 mg·L⁻¹. DODA mRNA levels were consistent with the response of betacyanin content to exogenous growth regulators. These results suggest that betacyanin metabolism in S. salsa calluses is regulated under white light conditions by growth regulators through the regulation of genes such as DODA that are involved in betacyanin synthesis.     

Regulatory mechanisms of biosynthesis of betacyanin and anthocyanin in relation to cell division activity in suspension cultures

Regulatory mechanisms of betacyanin biosynthesis in suspension cultures of Phytolacca americana and anthocyanin in Vitis sp. were investigated in relation to cell division activity.Betacyanin biosynthesis in Phytolacca cells clearly shows a positive correlation with cell division, as the peak of betacyanin accumulation was observed at the log phase of batch cultures. Incorporation of radioactivity from labelled tyrosine into betacyanin also showed a peak at early log phase. Aphidicolin, an inhibitor of DNA synthesis, and propyzamide, an antimicrotubule drug, reduced betacyanin accumulation and inhibited the incorporation of radioactivity from labelled tyrosine into betacyanin at concentrations which were inhibitory to cell division. Both inhibitors reduced the incorporation of radioactivity from labelled tyrosine to 3,4-dihydroxyphenylalanine (DOPA), but the incorporation of labelled DOPA into betacyanin was not affected. These results suggest that the conversion of tyrosine to DOPA is coupled with cell division activity.In contrast, the anthocyanin accumulation in Vitis cells showed a negative correlation with cell division. Accumulation occurred at the stationary phase in batch cultures when cell division ceased. Aphidicolin or reduced phosphate concentration induced a substantial increase in anthocyanin accumulation as well as the inhibition of cell division. Chalcone synthase (CHS) activity increased at the time of anthocyanin accumulation. Northern blotting analysis indicated that changes in CHS mRNA levels corresponded to similar changes in enzymatic activity. The pool size of endogenous phenylalanine was low during active cell division, but increased before anthocyanin began to accumulate and concomitantly with increasing levels of CHS mRNA. Exogenous supply of phenylalanine at the time of low endogenous levels induced the elevation of CHS mRNA and anthocyanin accumulation. These results indicate that the elevation of endogenous phenylalanine levels, when cell division ceases, may cause the increase in CHS mRNA levels, resulting in increased CHS activity and subsequently in anthocyanin accumulation in Vitis suspension cultures.Abbreviations CHS chalcone synthase - CHFI chalcone flavanone isomerase - DOPA 3,4-dihydroxyphenylalanine - PAL phenylalanine ammonia lyase     

The pathway of betalain biosynthesis: Effect of cytokinin on enzymic oxidation and hydroxylation of tyrosine in Amaranthus tricolor seedlings

The effect of exogenously added tyrosine or l -3-(3,4-dihydroxyphenyl) alanine on the accumulation of betacyanin in response to cytokinin in Amaranthus tricolor (L.) var. tricolor half-seedlings depends on the age of the seedlings and the treatment of the seedlings prior to induction of pigment by benzyladenine. Neither extracted polyphenol oxidase, peroxidase or tyrosine hydroxylase activity, nor in vivo tyrosine hydroxylation is increased in response to exposure of seedlings to cytokinin. However a small percentage of the polyphenol oxidase activated or unmasked by Triton X-100 treatment of membrane fractions is increased after cytokinin treatment of half-seedlings for 22 h. It is concluded that cytokinin control may be on a multi-enzyme membrane-located complex involving part of the polyphenol oxidase activity of the tissue (possibly an isoenzyme), and that the majority of the polyphenol oxidase activity in Amaranthus is a constitutive membrane enzyme which is not involved in betacyanin synthesis. Although cytokinins do not affect in vivo tyrosine hydroxylation, this activity follows closely the accumulation of betacyanin which is first detectable about 6.5 h after the application of cytokinin. Only a very low level of in vivo hydroxylation can be demonstrated in half-seedlings treated for 6 h either with or without cytokinin but it begins to increase shortly after this time. A large increase in this activity by 16 h (independent of cytokinin) can be almost completely (79%) prevented by chloramphenicol (300 μM) although the drug increases accumulation of betacyanin. At this concentration about 30% of the protein synthesis in inhibited. In vitro tyrosine hydroxylation is, however, not reduced in half-seedlings treated with chloramphenicol during 16 h induction nor is extractable polyphenol oxidase reduced. It is concluded that chloramphenicol is inhibiting the synthesis of some protein essential for in vivo hydroxylation other than the activity measured during in vitro hydroxylation and that the inhibition of synthesis of 79% in vivo hydroxylation still leaves enough activity for maximum betacyanin synthesis.     

Metabolism of 2,4-dichlorophenoxyacetic Acid in soybean root callus and differentiated soybean root cultures as a function of concentration and tissue age

The metabolism of [1-¹⁴C]2,4-dichlorophenoxyacetic acid (2,4-D) in soybean (Glycine max [L.] Merrill var. Amsoy) root callus and in differentiated soybean root cultures was investigated as a function of pesticide concentration and age of tissue. The chronological age of the tissue was found to be correlated with the mitotic index which reached a peak at 2 weeks and then declined. The metabolism of 2,4-D changed with age of the root callus tissue. The amount of free 2,4-D found in 3-week-old root callus tissue rapidly increased as the concentration of 2,4-D in the medium was increased from 10⁻⁶ to 10⁻⁵ molar, whereas the low level of aqueous (glycosides) and ether soluble metabolites (2,4-D amino acid conjugates) increased slowly. With 9-week-old root callus tissue, the amount of free 2,4-D remained at a relatively low, constant level (saturation level) as the concentration of 2,4-D in the medium increased. Under these conditions the aqueous metabolites increased only slightly but the ether fraction (2,4-D amino acid conjugates) rapidly increased. Thus, the older root callus tissue appeared to regulate the level of free 2,4-D at about 4 nanomoles per gram by converting any excess 2,4-D into amino acid conjugates.

In 3-week-old, differentiated root cultures the metabolism of 2,4-D closely paralleled the metabolism found in the older 9-week-old callus tissue. The saturation level of free 2,4-D found in this tissue was only about 1 to 2 nanomoles per gram.

     

Einfluss möglicher phosphodiesterase-inhibitoren und cAMP auf die betacyan-akkumulation

The influence of cyclic AMP, theophylline, papaverine and NH₄NO₃ on the accumulation of betacyanin in callus of Portulaca grandiflora, var. JR, were studied in relation to amounts of dihydroxyphenylalanine (DOPA), dopamine, phenylalanine, tyrosine, protein, ‘lipid’ and the nucleotides CMP, AMP, GMP and UMP present. Inhibition of betacyanin formation is characterized by reduced amounts of DOPA and dopamine and a constant rise of GMP and CMP (GMP/CMP = 8). Protein accumulation is also inhibited. The increase in pigment accumulation due to theophylline and NH₄NO₃ is characterized by a raised DOPA and protein concentration and a lower GMP/CMP ratio (=3). The increase in betacyanin accumulation is due to de novo synthesis of enzymes. Inhibition is probably due to the regulation of the callus phosphodiesterase by papaverine and theophylline (≦ 10^?5 M/1) which triggers a change in concentration of nucleotides, which eventually regulates tyrosinase biosynthesis.     

Studies on the Growth in Culture of Plant Cells: XXIV. EFFECTS OF 2, 4-DICHLOROPHENOXYACETIC ACID AND LIGHT ON THE GROWTH AND METABOLISM OF ACER PSEUDOPLATANUS L. SUSPENSION CULTURES

Omission of 2,4-dichlorophenoxyacetic acid (2,4-D) from batchcultures of sycamore (AcerpseudoplatanusL.) caused growth cessationafter an initial period of exponential growth. The presenceof white light reduced the amount of growth after 2,4-D withdrawal.Growth cessation, in the absence of 2,4-D, was accompanied bythe accumulation of the free amino acid, serine. This accumulationbegan before the cessation of growth and was rapidly reversedby the re-addition of 2,4-D.     

The effect of plant growth regulators on growth, morphology and condensed tannin accumulation in transformed root cultures of Lotus corniculatus

This study concerns the effects of four different classes of plant growth regulators on root morphology, patterns of growth and condensed tannin accumulation in transgenic root cultures of Lotus corniculatus L. (Bird's-foot trefoil). Growth of transformed roots in 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in decreased tannin levels relative to controls at concentrations of 10^-6 M and above, while gibberellic acid (GA₃) inhibited tannin accumulation at concentrations of 10^-7 M and above. Benzyladenine (BA) had little effect at low concentrations (10^-7 M and below) but resulted in an increase in tannin levels at 10^-6 M. Abscisic acid had little effect on levels of condensed tannins at any of the concentrations used. Experiments involving growth regulator addition and medium transfer demonstrated that 2,4-D inhibition of tannin accumulation could be reversed by GA₃ and BA, while GA₃ downregulation could only be reversed by the addition of 2,4-D. Although 2,4-D inhibited tannin accumulation, addition of 2,4-D to root cultures grown for 14 or 28 days in the absence of plant growth regulators stimulated both growth and tannin biosynthesis. Characteristic alterations in root morphologies accompanied growth regulator-mediated modulation of tannin biosynthesis. Growth in 2,4-D resulted in partially de-differentiated root cultures while growth in GA₃ produced roots with an elongated phenotype. Restoration of tannin biosynthesis in 2,4-D-treated roots was accompanied by root re-differentiation and the production of new lateral roots.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid 3 - FW fresh weight     

Studies on the Growth in Culture of Plant Cells: XX. UTILIZATION OF 2,4-DICHLOROPHENOXYACETIC ACID BY STEADY-STATE CELL CULTURES OF ACER PSEUDOPLATANUS L

Omission of 2,4-dichlorophenoxyacetic acid (2,4-D) from batchcultures of sycamore produced an immediate reduction in ratesof cell division and eventually in rates of biomass accumulation.The sequential responses of a chemostat and of turbidostat culturessubjected to gradual withdrawal of 2,4-P were: (i) a transientincrease in biomass accumulation, (ii) increased accumulationof p-coumaric acid, flavonoids, and lignin, (iii) increasedcell aggregation, (iv) reduced rates of cell division, and (v)death. During stepwise reduction of 2,4-D supplied to turbidostatcultures, rates of 2,4-D uptake were reduced when the spentmedium concentration fell to 3?5–1?0 ? 10^–7 M. Underthese conditions the 2,4-D concentration in soluble and insolublecell fractions declined. The growth responses were correlatedwith the spent-medium 2,4-D concentration but not with its concentrationin the intracellular fractions.     

Effects of nitrogen source on betacyanin accumulation and growth in suspension cultures of Phytolacca americana

In suspension cultures of Phytolacca americana L., betacyanin accumulation per cell increased with increasing total nitrogen concentration (initial NH⁺₄:NO⁻₃ ratio 1:2) in the range 0–40 m M and then remained almost constant in the range 40–80 m M . Increasing ammonium increased growth while betacyanin accumulation was reduced. On the other hand, betacyanin accumulation increased when nitrate was increased while growth was almost constant in the concentration range examined. A time-course study of ammonium and nitrate concentration changes in the medium showed that betacyanin accumulation was associated with nitrate uptake.     

Auxin transport at cellular level: new insights supported by mathematical modelling

The molecular basis of cellular auxin transport is still not fully understood. Although a number of carriers have been identified and proved to be involved in auxin transport, their regulation and possible activity of as yet unknown transporters remain unclear. Nevertheless, using single-cell-based systems it is possible to track the course of auxin accumulation inside cells and to specify and quantify some auxin transport parameters. The synthetic auxins 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene-1-acetic acid (NAA) are generally considered to be suitable tools for auxin transport studies because they are transported specifically via either auxin influx or efflux carriers, respectively. Our results indicate that NAA can be metabolized rapidly in tobacco BY-2 cells. The predominant metabolite has been identified as NAA glucosyl ester and it is shown that all NAA metabolites were retained inside the cells. This implies that the transport efficiency of auxin efflux transporters is higher than previously assumed. By contrast, the metabolism of 2,4-D remained fairly weak. Moreover, using data on the accumulation of 2,4-D measured in the presence of auxin transport inhibitors, it is shown that 2,4-D is also transported by efflux carriers. These results suggest that 2,4-D is a promising tool for determining both auxin influx and efflux activities. Based on the accumulation data, a mathematical model of 2,4-D transport at a single-cell level is proposed. Optimization of the model provides estimates of crucial transport parameters and, together with its validation by successfully predicting the course of 2,4-D accumulation, it confirms the consistency of the present concept of cellular auxin transport.     

Metabolism of 2,4-dichlorophenoxyacetic Acid in 2,4-dichlorophenoxyacetic Acid-resistant soybean callus tissue

Three 2,4-dichlorophenoxyacetic acid (2,4-D) -resistant root callus tissue lines of Glycine max L. Merrill var. Acme were derived by culturing callus tissue 2 to 6 months on 40 milligrams per liter 2,4-D and designated 40R, 40B, and 40C. Tissue line 40R had a lower level of 2,4-D uptake in 2-week-old tissue which disappeared in 3.5-week-old tissue and less free 2,4-D following incubation for 24 hours with [1-¹⁴C]2,4-D. This tissue line accumulated more hydroxylated glycosides of 2,4-D than did nonresistant tissue. Tissue line 40B showed no difference in uptake of 2,4-D when compared to nonresistant tissue but it did contain less free 2,4-D and more hydroxylated glycosides. The metabolism of 2,4-D in the 40C tissue line did not differ significantly from nonresistant tissue although uptake was less. The 40R line reverted to the same 2,4-D sensitivity as Acme root callus following six transfers on 10 micromolar naphthaleneacetic acid medium. The altered 2,4-D uptake and metabolism characteristic of 40R were also lost. The levels of amino acid conjugates of 2,4-D in the resistant root callus tissue lines were either lower or not significantly different from the Acme tissue lines. Therefore, variations in uptake and metabolism of 2,4-D do not wholly explain the resistance of the derived tissue lines, and perhaps modification of the active site or compartmentation is involved.     

Analysis of variability in the amaranthus betacyanin assay for cytokinins: effects of "aging" excised cotyledons

“Aging” of excised cotyledons plus the top part of the hypocotyl of Amaranthus tricolor seedlings was carried out by washing in distilled H₂O for varying periods. This led to increased betacyanin accumulation during the subsequent 24-hour induction period in the presence of tyrosine and Na⁺ + K⁺ phosphate. Endogenous accumulation as well as that dependent on added benzyladenine and on added fusicoccin was stimulated. This stimulation could not be due to a carryover of a wound-induced burst of ethylene since 2-chloroethylphosphonic acid (Ethrel) was shown to be extremely inhibitory to betacyanin synthesis if present during the induction process. It is possible that a wound-induced burst of ethylene could give rise to increased betacyanin synthesis as an after effect. The procedure for obtaining good induction with the most reproducible results is described.     

A comparison of 2,4-dichlorophenoxyacetic Acid metabolism in cultured soybean cells and in embryogenic carrot cells

The removal or reduction in concentration of auxin is often a successful method for obtaining morphogenesis in cell cultures of higher plants, such as carrot, but not for soybean. For this reason, the metabolism of one auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), was compared in both carrot and soybean cells. Whereas soybean cells conjugated a high percentage of their 2,4-D to amino acids, carrot cells contained primarily free 2,4-D. Moreover, after long-term exposure to 2,4-D, carrot cells released much more 2,4-D upon transfer to 2,4-D-free (embryogenic) medium than did soybean cells. It appears that the retention of 2,4-D by soybean cells might interfere with subsequent morphogenesis. Because no impairment of 2,4-D efflux was found with short-term exposure to radiolabeled 2,4-D, it was concluded that 2,4-D retention in soybean cells might be due to a time-dependent, metabolic process. The conjugation of 2,4-D to amino acids was shown to be one such time-dependent process. Additionally, the release of 2,4-D from the cells was shown to be due primarily to a loss of free 2,4-D and not 2,4-D-amino acid conjugates. It seems that the greater retention of 2,4-D by soybean cells upon transfer to 2,4-D-free medium is due to greater formation of 2,4-D-amino acid conjugates.     

Auxin-induced rapid changes in translatable mRNAs in tobacco cell suspension

When 2,4-dichlorophenoxyacetic acid (2,4-D)-dependent tobacco cell suspensions, one normal and one transformed by Agrobacterium tumefaciens, were subcultured on hormone-lacking medium the stationary phase of the cell cycle was reached earlier than on medium containing 2,4-D. Addition of the auxin 2,4-D could restore cell division activity within 10–12 h for the most rapidly reacting cell line. The cell-division response was characterized as being auxin-specific and optimal with 2,4-D at 2.2 10^-6 M. Although the cell lines used showed different characteristics, both reacted with a rapid increase in at least three mRNA species within 1 or 2 h after 2,4-D application. Two, 2,4-D-induced protein spots, seen after in-vitro translation, had the same characteristics (MWs 35 kilodaltons (kDa) and 25 kDa with isoelectric points of 7.1 and 6.3, respectively) in both cell lines. Water-treated controls did not show alterations in the translatable mRNA populations. This indicates that the accumulation of the corresponding mRNAs is an early hormone-induced event. Since cell division is the only measurable reaction found after auxin application, cell systems as described here offer excellent possibilities for studying early auxin-induced changes at the molecular level preceding mitosis.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - kDa kilodalton     

The influence of some inhibitors on the formation of caffeic acid in cultures ofPerilla cell suspensions

A cell suspension culture, prepared fromPerilla frutescens var.crispa callus induced by Murashige and Skoog (1962) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 ml/l) and kinetin (0.1 mg/l), contained caffeic acid derivatives as the phenolic components. Fresh and dry weights of the cells increased exponentially for about 11 days after transfer to a fresh medium. The contents of caffeic acid and protein also reached a maximum on the 11th day, but α-amino nitrogen phenylalanine and tyrosine continued to increase in amount until the 20th to 23rd day. Caffeic acid formation in the cells was increased by lowering the concentration of 2,4-D. The administration ofl-2-aminooxy-3-phenylpropionic acid (l-AOPP), 2-aminooxyacetic acid (AOA) andN-(phosphonomethyl)glycine (glyphosate) to the cells inhibited caffeic acid formation to a large extent. An 80% inhibition of caffeic acid formation was caused by 10⁻⁴Ml-AOPP whereas phenylalanine and tyrosine contents of the cells became 7.5 and 2.3 times higher at thisl-AOPP concentration than those in the control. An 85% inhibition of caffeic acid formation was achieved at 10⁻³M glyphosate concentration, while 10⁻³M AOA inhibited caffeic acid formation by 95% and also growth rate by 80%. The influence of inhibitors on caffeic acid formation is discussed in relation to the level of α-amino nitrogen, particularly aromatic amino acids, in the cell suspension cultures.     

Increased Copper Content of the Medium Improves Plant Regeneration from Immature Embryo Derived Callus of Wheat (Triticum aestivum)

The effect of various concentrations of CuSO₄ on the induction and regeneration of embryogenic callus from immature embryos of wheat was investigated. Immature embryos of wheat cvs C-306 and R-3777 were cultured on MS medium supplemented with 2,4-D (11.3 µM) and different levels of cupric sulphate, i.e. 0, 0.1 (MS level), 0.5, 1 and 5 µM. Relatively high induction frequency of callus was obtained on MS medium supplemented with 2,4-D (11.3 µM) and 0.5 µM CuSO₄. The compact, nodular, embryogenic callus was maintained on the medium having 2,4-D (11.3 µM) and proline (86.8 µM) by regular subculturing. Plant regeneration from the embryogenic callus occurred on MS medium supplemented with NAA (1.07 µM) and BAP (44.4 µM). Regenerated plantlets were rooted on MSmedium supplemented with IAA (2.85 µM). The average number of regenerated plantlets produced from primary callus induced on 2,4-D (11.3 µM) and 5x CuSO₄ was significantly higher.