Dihydrofolate reductase from Daucus carota cell suspension cultures: purification,molecular and kinetic characterization

Summary The purification of dihydrofolate reductase (5, 6, 7, 8 tetrahydrofolate: NADP⁺ oxidoreductase, E.C.: 1.5.1.3) from Daucus carota to apparent homogeneity, is described. The enzyme is a soluble protein with a molecular weight of 183 000±2 500, composed of identical subunits of 58 400±1 000. The enzyme is only weakly recognized by antibodies against human DHFR. The carrot DHFR is characterized by a pH optimum of 5.9, Km values for dihydrofolate and NADPH of 3.7 M and 2.2 M, respectively and a turnover number of 4 750 or 1 500 when referring to the 183 K form or the 58 K monomer, respectively. Molecular and kinetic properties are remarkably different from those reported for the soybean enzyme. Sensitivity to methotrexate is similar to that of bacterial and mammalian enzymes while sensitivity to trimethoprim and dihydrotriazine is intermediate between the two groups of organisms.     

Anthocyanins from cell suspension cultures of Daucus carota.

Six anthocyanins were isolated from cell suspension cultures of an Afghan cultivar of Daucus carota by PC or HPLC. The structures of these compounds were elucidated by spectroscopic methods as cyanidin 3-O-lathyroside, cyanidin 3-O-(2'-O-beta-D-xylopyranosyl-6'-O-beta-D-glucopyranosyl-beta-D- galactopyranoside), and the latter acylated with 4-coumaric, ferulic, 4-hydroxybenzoic or sinapic acid. Unusual 1H NMR chemical shifts and 1H NOE data indicate an intramolecular copigmentation of the aglycone with these aromatic residues.     

Chalcone synthase from cell suspension cultures of Daucus carota L

Chalcone synthase (CHS) has been partially purified about 35-fold. Withdrawal of 2-mercaptoethanol after precipitation with ammonium sulfate led to higher stability during further purification steps. In order to determine CHS activity, two procedures [according to Schr?der et al. (1979) Plant Sci. Lett. 14, 281-286] were applied. The radioactivity extracted with ethyl acetate from the assay mixture (total products) was compared to 14C-labeled flavanone purified by TLC. The activity of CHS increased with bovine serum albumin (BSA) or 2-mercaptoethanol in the assay. Both effects were synergistic, but BSA did not promote "side products" as 2-mercaptoethanol did. BSA (10 mg ml-1) and 2-mercaptoethanol (1.4 mM) were components of the standard assay. Under these conditions, the CHS from Daucus carota had different pH optima for naringenin formation (7.9) and eriodictyol formation (6.8). The apparent Km values were 0.6 microM for 4-coumaroyl-CoA (pH 7.9), 7.7 microM for caffeoyl-CoA (pH 6.8), and 3.0 microM for malonyl-CoA (pH 7.9). Substrate inhibition was observed with 4-coumaroyl-CoA (greater than 10 microM) and malonyl-CoA (greater than 50 microM). The inhibitory activity of various flavonoids and related compounds (100 microM) was investigated. Naringenin and naringenin-chalcone inhibited eriodictyol formation totally and naringenin formation by 50%. In contrast, eriodictyol and eriodictyol-chalcone inhibited only eriodictyol formation by 40%. It was shown that the inhibition with naringenin was fully uncompetitive. These in vitro data support the view that the true substrate of CHS in D. carota is 4-coumaroyl-CoA.     

Regulation of glutamate dehydrogenase activity by manipulation of nucleotide supply in Daucus carota suspension cultures

The enzyme glutamate dehydrogenase (GDH, EC 1.4.1.2) is ubiquitous in plant species. It is now generally accepted that the primary role of this enzyme is not assimilation of ammonium and it has been suggested that GDH may be important in provision of carbon skeletons under conditions of carbon limitation. In carrot ( Daucus carota L. Chantenay) cell suspension cultures carbon starvation results in de-repression of GDH activity. The regulation of this de-repression has not been investigated. This paper examines the possibility that the availability of adenosine nucleotides is instrumental in the regulation of GDH activity. In repressed cultures the adenosine nucleotides cAMP (0.2 m M ), AMP (0.2 m M ) and ADP (0.4 m M ) caused an increase in GDH activity of 61, 33 and 7%, respectively. ATP (0.2 m M ) had the opposite effect in maintaining repression of GDH. Under de-repressed conditions only cAMP (0.2 m M ) enhanced GDH activity (14%). Inhibition of oxidative phosphorylation using a range of inhibitors resulted in de-repression of GDH and stimulation of respiration. The results from this work indicate that exogenously applied adenosine nucleotides and electron transport inhibitors alter the GDH repression/de-repression status. Addition of these compounds alters or disrupts ATP levels, mimicking carbon depletion. This causes an increase in GDH activity, supporting the idea that GDH may provide carbon skeletons for carbon metabolism and suggesting that ATP status is important in regulation of the enzyme activity.     

Morphogenetic effects of Brefeldin A on embryogenic cell cultures of Daucus carota L.

Brefeldin A, an inhibitor of protein secretion, caused typical alterations to the endomembrane system with limited effects on viability when given to unorganized carrot cells growing in suspension. When given to the same cells during particular stages of embryogenesis, it caused similar endomembrane lesions and an almost complete arrest of the embryogenic process. Addition of conditioned medium containing extracellular secreted proteins to the embryos during treatment with Brefeldin A allowed acquisition of polarity and the continuation of a quasi-normal embryogenic process. Received: 4 December 1996 / Accepted: 4 March 1997     

AC electrokinetic characterisation and separation of cells with high and low embryogenic potential in suspension cultures of carrot (Daucus carota)

Suspension cultures of Daucus carota L. were established, and cells with embryogenic potential were separated from those without by density gradient centrifugation in Ficoll at different stages in the growth curve. In order to obtain information about the electrical properties of individual cells, electrorotation spectra of single plant cells from different fractions were measured before and after induction of embryogenesis. The data were analysed using models based on Maxwell–Wagner's theories of interfacial polarisation. It was found that the denser cells had a higher embryogenic potential, a darker appearance and a higher internal conductivity (>1 S m^–1) than the less dense cells, which had less or no embryogenic potential and a lower internal conductivity (<1 S m^–1). Modelling the dielectrophoretic (DEP) response on the basis of the electrorotation data suggested that separation of cells with high embryogenic potential may be achievable in the frequency range 1–10 MHz. Actual dielectrophoretic separation of cells with high embryogenic potential from suspensions in which embryogenesis had not yet been induced was achieved using steric as well as hyperlayer dielectrophoretic Field-Flow Fractionation (DEP-FFF).     

Development and characterization of embryogenic suspension cultures of pecan

A method for the establishment and proliferation of developmentally stable, embryogenic suspension cultures in pecan is described, and the growth and development of cultures characterized. Suspension cultures were generated from somatic embryos derived from zygotic embryo cotyledon explants induced on a solidified medium with naphthaleneacetic acid. Cultures were repetitively embryogenic and proliferated in growth-regulator-free medium. The suspensions consisted of a mixture of globular stage embryo-aggregates, freely suspended globular embryos and pre-globular stage embryo masses. Culture growth and proembryo production were evaluated with respect to several liquid media and pH conditions. Significant differences in growth and productivity were observed between cultures. Pre-globular stage embryo masses collected on filter paper and overlaid on solidified medium continued ontological development and converted into plants. Thus a method has been developed for pecan suspension culture, which presents a major improvement in embryogenic tissue culture within the Juglandaceae. This revised version was published online in June 2006 with corrections to the Cover Date.     

Purification and characterization of a soluble beta-fructofuranosidase from Daucus carota.

Soluble beta-fructofuranosidase with an intracellular location and an isoelectric point of 3.8 (isoenzyme I) was purified and characterized from dry seeds and seedlings of carrot (Daucus carota). The enzyme hydrolyzed sucrose with a Km of 5 mM and a broad pH optimum around 5.0. The purified protein, which was N-glycosylated with high-mannose-containing and high-xylose-containing complex glycans, eluted as a monomeric polypeptide with a molecular mass of 68,000 from a gel-filtration column. On SDS/PAGE, the protein separated in the presence of SDS and 2-mercaptoethanol into three polypeptides with molecular masses of 68, 43 and 25 kDa. The amount of the 68-kDa polypeptide was highest in dry seeds and decreased with increasing age of carrot seedlings. Amino acid sequence analysis and immunological studies showed that the 43-kDa and 25-kDa polypeptides were N-terminal and C-terminal proteolytic fragments of the 68-kDa polypeptide. A comparison of partial amino acid sequences of the soluble beta-fructofuranosidase with the complete sequence of carrot cell-wall beta-fructofuranosidase showed that their N-terminal sequences were different, whereas some of the internal tryptic peptide sequences were up to 70% identical.     

Inhibition of flavonoid biosynthesis by gibberellic acid in cell suspension cultures of Daucus carota L.

In carrot cells (Daucus carota L.), cultured in the presence of gibberellic acid, anthocyanin synthesis is blocked at the level of chalcone synthase. By feeding suitable precursors for anthocyanins (naringenin, eriodictyol, dihydroquercetin) biosynthesis of cyanidin glycosides can be restored. After addition of these substrates to the culture medium in the presence of gibberellic acid, the activity of chalcone synthase remained as low as in the control without precursors. The highest increase in anthocyanin content was achieved using dihydroquercetin as the added precursor. The time course of this supplementation showed a rapid response; within 4 h a substantial increase in anthocyanin could be observed. In contranst, the flavonol quercetin is not a precursor for cyanidin. The fact that naringenin was also accepted for cyanidin synthesis leads to the conclusion that hydroxylation in 3-position of ring B in Daucus carota takes place at the flavonoid stage.Abbreviations CHI Chalcone isomerase - CHS chalcone synthase - DMSO dimethylsulfoxide - GA₃ gibberellic acid - PAL phenylalanine ammonia-lyase     

Spontaneous and induced apoptosis in embryogenic cell cultures of carrot (Daucus carota L.) in different physiological states

Programmed cell death (apoptosis) in plants - and plant cells in culture - has received much less attention than its animal counterpart. In the present work, using agents producing biotic or abiotic stress on cultivated cells from carrot - and, in a few experiments, Arabidopsis -, we show that DNA fragmentation, random or oligonucleosomal, can be induced by different treatments. Moreover, we demonstrate that the same cultures may or may not respond to the inducing signal according to their physiological state. In particular, stationary cells are more responsive to the inducing signal than actively proliferating ones, and cells growing in an unorganized way are more responsive than cells carrying out the embryogenic programme. Senescent cells in culture also appear to die by apoptosis, but healthy cells can also be induced to die apoptotically if exposed to the medium conditioned by senescent cells of the same or different species.     

Establishment and characterization of long-term embryogenic maize callus and cell suspension cultures

Friable callus (type 2) was selected from three genotypes (A188, hybrid A188 × B73, and hybrid B73 × A188) of Zea mays L. The three genotypes of type 2 callus doubled in fresh weight after 1 week, and growth was better on N6 than on Murashige-Skoog (MS) medium. Type 2 callus of hybrid B73 × A188 was maintained in culture longer than A188 type 2 callus, and it regenerated higher numbers of plants than the other two genotypes. Type 2 callus of the hybrid B73 × A188 was used to establish cell suspensions. Suspension cells initially grew better on N6 than on MS medium, but after several months of subculture, cells in either N6 or MS medium grew at similar rates. Suspension cells were in mid-log phase by 5–7 days and in stationary phase by about 10 days depending on inoculum density. Growth rate was optimal when cells were transferred at mid-low phase and dry weight of the suspension cells increased at least 10-fold during a 10-day period. Suspension cells from 9-month-old cultures plated on solid medium regenerated plants at an efficiency similar to that of the friable type 2 callus but with more phenotypic abnormalities. Thus, cell suspensions derived from type 2 B73 × A188 callus, in culture for over 1 year, were capable of regenerating plants when 9-months old.     

Anthocyanin accumulation and PAL activity in a suspension culture of Daucus carota L.

Cells of Daucus carota grown in a liquid medium produced large amounts of cyanidin as the only flavonoid aglycon. After inoculation in fresh medium a maximum activity of phenylalanine ammonia lyase (PAL; EC 4.3.1.5) was observed within 24 h. L--aminooxy--phenylpropionic acid (L-AOPP), thought to be a competitive inhibitor of PAL, inhibited cyanidin accumulation up to 80%. In order to study the regulatory role of PAL, the effects of L-AOPP and t-cinnamic acid, the product of the deamination of phenylalanine, were investigated. Cinnamic acid, applied in vivo (10^-4 M), was not able to compensate for the inhibition of cyanidin production caused by L-AOPP (10^-4 M) in the same sample. Carrot cells treated with L-AOPP exhibited a super-induction of PAL already described for gherkin hypocotyls (Amrhein and Gerhardt 1979). This effect was not influenced by t-cinnamic acid. L-AOPP seems to be a very specific inhibitor since it affected neither growth nor soluble protein content, whereas t-cinnamic acid inhibited both. Investigations on the content of soluble amino acids in L-AOPP-treated cells revealed a specific accumulation of soluble phenylalanine, whereas treatment with t-cinnamic acid led to an increase of amino acids in general, thus indicating that the latter compound has a rather unspecific effect on cellular metabolism. In vitro studies with PAL isolated from Daucus carota revealed that L-AOPP inhibited the enzyme at very low doses (K _I=2.4·10^-9), whereas t-cinnamic acid, by comparison, affected the enzyme at high concentrations (K _I=1.8·10^-4).Abbreviations PAL phenylalanine ammonia lyase - L-AOPP L--aminooxy--phenylpropionic acid     

Influence of 2-deoxy-D-glucose upon growth and invertase activity of carrot (Daucus carota L.) cell suspension cultures

Carrot (Daucus carota L.) cell suspension cultures grew well when provided with glucose, fructose, sucrose or raffinose. Galactose and melibiose supported less growth unless supplemented with glucose or fructose. In combination with ten different sugar mixtures, 2-deoxy-D-glucose (dGlc) inhibited culture growth. Inhibitory effects of dGlc were more marked with fructose, melibiose, raffinose or mixtures of these sugars in the culture medium. The presence of glucose or galactose reduced the inhibitory effects of dGlc on culture growth. Experiments with radioactive labelled sugars demonstrated that dGLc uptake was greater in the presence of fructose than glucose, and that growth inhibition of dGlc coincided with its uptake. Reduced protein content was also associated with the inhibitory effects of dGlc. Cultured cells contained lower levels of invertase (EC 3.2.1.26) activity during the active phase of culture growth (up to 25 days after subculture) than when growth had peaked and subsequently declined. Acid and alkaline invertase activities were not greatly reduced by exogenous hexoses. Invertase activity was greatest during periods of low protein content in all cultures and was inhibited by dGlc during the latter phases of the culture period. Free intracellular sugars throughout the culture period consisted mainly of glucose and fructose.     

Partial purification and characterization of dihydrobenzophenanthridine oxidase from Eschscholtzia californica cell suspension cultures

The observation that upon elicitation cell suspension cultures of Eschscholtzia california showed a decrease of dihydromacarpine with a concomittant increase of macarpine led to the discovery of a novel enzyme which catalyzes the oxidation of dihydrobenzophenanthridines in the presence of oxygen. The enzyme was enriched approx. 70-fold. It has a pH-optimum of 7.0, an isoelectric point at pH 8.8, molecular weight of 56 kD and shows a high degree of substrate specificity. The enzyme obviously catalyzes the terminal step in the formation of benzophenanthridine alkaloids containing methylene dioxy substitutions in rings A and D.     

Differential gene expression in embryogenic and non-embryogenic clusters from cell suspension cultures ofCoffea arabica

Somatic embryogenesis (SE) is a very useful system for studying the differentiation process in plants and involves gene regulation at several levels. During SE induction in Coffea arabica cv. Catura Rojo two types of cell clusters, embryogenic (EC) and non-embryogenic (NEC), were observed. The goal of this work was to compare the most relevant characteristics between EC and NEC for a better understanding of the mechanism driving SE. Morphohistological observations indicated a correlation between the morphological features of clusters and their embryogenic competence. On the other hand, no variation at the DNA level, studied by AFLP, were found to explain the disparity in embryogenic competence of clusters, but gene expression, observed by RNA differential display, and SDS-PAGE showed differences that can explain that disparity. Our results lead us to propose that differential gene expression can modulate the embryogenic capacity of coffee cells and that the number of genes turned off in somatic cells to allow for the change from a somatic to an embryogenic state, is higher than those genes that are turned on.     

Population and biomass kinetics in fed-batch cultures of Daucus carota L. somatic embryos

The population dynamics of developing somatic embryos of carrot (Daucur carota L.) was investigated in batch and fed-batch cultures using modified Murashige and Skoog medium. These substrate limitations coincided not only with stoppage of biomass increase, but also with the increase in total concentration of embryos as well as the advancement of the embryo into a more mature stage. Both glucose and ammonium were depleted from the culture. Restoring either glucose, or ammonium and nitrate, as to approximately initial concentrations in fed-batch experiments, did not result in a significant increase of the total normal embryo concentration. On the other hand, medium replacement led to increase in biomass concentration, total embryo number, and improved embryo maturity. The addition of a mixture of glucose, ammonium, and nitrate to the spent medium resulted in variable increases in biomass and embryo number, but always less than those resulting from media replacement. Although the total number of embryos was higher after medium replacement, the fraction of embryos reaching torpedo stage was still only 50%. The need for a better means of population characterization for further kinetic studies is discussed. (c) 1993 Wiley & Sons, Inc.     

Chitinase profiles in mature carrot (Daucus carota) roots and purification and characterization of a novel isoform

The profile of chaitinases (EC 3.2.1.14) in mature carrot ( Daucus carota L. cv. Eagle) roots was studied. Multiple chitinase bands (8–10) were observed in native and sodium dodecylsulfate-denaturing polyacrylamide gels. The molecular masses of these chitinases were estimated to be from 20 000 to 40 000. One major chitinase was purified and found to be an acidic protein with pI at 4.3 and a molecular mass of 39 500. The optimum pH for enzymatic activity was around 5 and the optimum temperature was 25°C. The enzyme was stable at pHs below 8 and temperatures below 60°C. The protein did not have a chitin-binding domain, but showed some similarity to the amino acid composition of tobacco class I chitinase. The N-terminal amino acid sequence did not resemble any of the described classes of chitimases. This chitinase did not possess lysozyme activity and showed antifungal activity when tested against Trichoderma sp.     

Somatic embryogenesis and plant regeneration from embryogenic suspension cultures of perennial ryegrass

Summary Embryogenic callus induced from mature caryopses of perennial ryegrass (Lolium perenne L.) were placed in liquid half-strength Murashige and Skoog (MS) basal medium and supplemented with 6.0 mg/l 2,4-dichlorophenoxy acetic acid (2,4-D), 3 g/l (w/v) casein hydrolysate (CH), and B5 vitamins, to initiate fast-growing highly embryogenic cell suspension cultures. Newly initiated suspension cultures contained a high level of large non-embryogenic cells (NE) with relatively few embryogenic (E) cells. Cell types were separated by discontinuous Percolls gradients or by filtering the newly initiated cultures through 31-μm nylon mesh. The growth conditions of the E cell were optimized by testing various media components including 2,4-D and sucrose, and subculture diluton ratio. Optimal shoot formation occurred after pretreatment of the embryogenic cells on solidified callus maintenance medium supplemented with 60 mg/l cefotaxime for 4 weeks prior to transfer to regeneration medium Regeneration media consisted of half-strength MS basal medium supplemented with B5 vitamins, 0.5 mg/l fluridone, and 0.5 mg/l BA. Most plants regenerated were albino with only a few green plants. Journal Paper number MAES 2959 of the Massachusetts Agricultural Experiment Station.