A clonal analysis of anthocyanin accumulation by cell cultures of wild carrot

The accumulation of anthocyanin by clones and subclones from a cell suspension culture of wild carrot (Daucus carota L.) has been measured under standard conditions. Clones which accumulate low amounts of anthocyanin were shown, by recloning after maintenance by serial passage, to have become heterogenous and to contain cells with increased accumulation of anthocyanin. There appears to be a maximum amount of anthocyanin that clones can accumulate. Clones which accumulate the maximum amount of anthocyanin were shown by recloning after maintenance by serial passaging, to have become heterogenous and to contain many cells which accumulate less than the maximum possible amount of anthocyanin. When clones which accumulate the maximum amount of anthocyanin are maintained by serial passage, the decline in anthocyanin accumulation is different in different media. The results indicate that the changes in the ability of cells to accumulate anthocyanin involve no qualitative change in the genetic information of the cells, i.e., the changes are not the consequence of mutations.     

Sinapic acid stimulator of anthocyanin accumulation in carrot cell cultures

In clones of wild carrot (Daucus carota L.) cells which accumulate anthocyanin, exogenously supplied sinapic acid increases their anthocyanin accumulation in the presence or absence of dihydroquercetin which is a known precursor of cyanidin. The exogenously supplied sinapic acid was not converted into malvidin by the cells. The cells accumulate anthocyanin with cyanidin as the only chromophore in the presence or absence of sinapic acid. Sinapic acid feeding did not initiate anthocyanin accumulation in clones which were not anthocyanin accumulating.     

The stability of accumulated anthocyanin in suspension cultures of the parental line and high and low accumulating subclones of wild carrot

Abhibition of the accumulation of anthocyanin but not of growth in wild carrot cultures was achieved using 2-amino-oxyphenylpropionic acid or phenylpropiolic acid. The concentration of inhibitor required to achieve this differential effect increased with increased culture density.By several additions of inhibitor at differential times, anthocyanin accumulation in the parental culture and in high-and low-acculmulating subclones could be completely inhibited. Under these conditions the level of anthocyanin in the cultures remained unchanged. These data show that the accumulated anthocyanin is not metabolized and that differences in the rate of degradation of anthocyanin does not account for the differences in anthocyanin accumulation by the subclones examined.     

Fitness effects of two facultative endosymbiotic bacteria on the pea aphid,Acyrthosiphon pisum,and the blue alfalfa aphid,A. kondoi

The effects of two bacterial endosymbionts, designated PASS and PAR, were evaluated on the pea aphid, Acyrthosiphon pisum (Harris) (Hemiptera:Aphididae), in which they occur facultatively, and on the blue alfalfa aphid, A. kondoi Shinji, in which these bacteria have not been found in natural populations. Subclones of pea aphids and blue alfalfa aphids, derived from parent aphid clones that did not contain PASS or PAR, were infected with one or both bacteria, generating PASS- and/or PAR-positive subclones with minimal genetic differences from the parent clones. Under laboratory conditions at 20 °C, PAR consistently reduced the fecundity (by between 19 and 60%) of subclones derived from three different parent pea aphid clones on bur clover, Medicago hispida Gaertn. PAR had intermediate effects on pea aphids reared on sweet pea, Lathyrus odoratus L., and had no significant effect on pea aphids on alfalfa, Medicago sativa L. The effect of PASS was either neutral or negative, depending on parent clone as well as host plant. Also at 20 °C, PASS reduced fecundity (70–77%) and longevity (40–48%), and increased the age of first reproduction (by up to 1.5 days) of blue alfalfa aphid reared on alfalfa and clover. PAR had a less dramatic effect (e.g., 30–39% reduction in fecundity) on these traits of blue alfalfa aphid. In contrast, PAR and PASS increased the fitness of pea aphid subclones of one parent clone reared for three generations at 25 °C on each of the three test plants. Without facultative bacteria, fecundity of the parent clone was reduced to a mean total of < 6 offspring per adult at this elevated temperature, but with PASS or PAR, mean total fecundity of its subclones was > 35. However, this ameliorative effect of facultative bacteria at 25 °C was not found for two other sets of parent clones and their derived subclones. Alate production in pea aphids was significantly increased in large populations of two PASS- and PAR-positive subclones relative to their parent clones. Attempts to transmit PASS or PAR horizontally, i.e., from aphid to aphid via feeding on host plants (bur clover), were unsuccessful.     

Regulation of gene expression involved in flavonol and anthocyanin biosynthesis during petal development in lisianthus (Eustoma grandiflorum)

To elucidate gene regulation of flower colour formation, the gene expressions of the enzymes involved in flavonoid biosynthesis were investigated in correlation with their product during floral development in lisianthus. Full-length cDNA clones of major responsible genes in the central flavonoid biosynthetic pathway, including chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3',5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), and flavonol synthase (FLS), were isolated and characterized. In lisianthus, the stage of the accumulation of flavonols and anthocyanins was shown to be divided clearly. The flavonol content increased prior to anthocyanin accumulation during floral development and declined when anthocyanin began to accumulate. CHS, CHI, and F3H were necessary for both flavonol and anthocyanin biosynthesis and were coordinately expressed throughout all stages of floral development; their expressions were activated independently at the stages corresponding to flavonol accumulation and anthocyanin accumulation, respectively. Consistent with flavonol and anthocyanin accumulation patterns, FLS, a key enzyme in flavonol biosynthesis, was expressed prior to the expression of the genes involved in anthocyanin biosynthesis. The genes encoding F3'5'H, DFR, and ANS were expressed at later stages, just before pigmentation. The genes responsible for the flavonoid pathways branching to anthocyanins and flavonols were strictly regulated and were coordinated temporally to correspond to the biosynthetic order of their respective enzymes in the pathways, as well as in specific organs. In lisianthus, FLS and DFR, at the position of branching to flavonols and anthocyanins, were supposed to play a critical role in regulation of each biosynthesis.     

Clones from a shooty tobacco crown gall tumor II: irregular T-DNA structures and organization,T-DNA methylation and conditional expression of opine genes

Summary Transformed clones from a shooty tobacco crown gall tumor, induced byAgrobacterium tumefaciens strain LBA1501, having the auxin locus of the TL-region inactivated by a Tn1831 insertion, were investigated for their T-DNA structure and expression. It has been described previously (28) that in addition to clones with an expected phenotype (phytohormone independent growth in tissue culture (Aut⁺), shoot regeneration (Reg⁺) and octopine synthesis (Ocs⁺)), clones were obtained with an aberrant phenotype. One of these clones, TSO38, is Aut⁺Reg⁺ but shows little or no octopine synthesis activity (Ocs^-). Subclones of TSO38, however, are either Ocs^- or Ocs⁺. Ocs^- shoots become Ocs⁺ under certain states of differentiation, indicating that the octopine synthase gene is present. The fact that in the Ocs^- subclones the octopine synthase gene is not expressed, is probably due to DNA methylation (29). The present paper describes that shoots derived from both an Ocs⁺ and an Ocs^- subclone of TSO38, which were negative for the presence of mannopine (Mas^-) and agropine (Ags^-), became Mas⁺Ags⁺ after culturing on medium containing the hypomethylating agent 5-azacytidine. This means that both in the Ocs^- line and in the Ocs⁺ line expression of TR-DNA opine genes most likely was hampered by DNA methylation. The T-DNA structures of an Ocs^- and an Ocs⁺ TSO38 subclone proved to be identical and surprisingly complex. No intact copy of Tn1831 was present. TL-DNA and TR-DNA segments, present in high copy numbers, were truncated; several T-DNA segments existed in tandem arrangements. When DNA from an Ocs⁺ and an Ocs^- subclone of TSO38 were compared for cleavability by the methylation sensitive restriction enzymes HpaII and MspII, differences were detected, but it became also clear that both lines contained methylated T-DNA segments. This indicates that the Ocs^- and the Ocs⁺ TSO38 subclones differ only quantitatively in respect to degree of T-DNA methylation.     

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 FUSCA genes of Arabidopsis: negative regulators of light responses

More than 200 fusca mutants of Arabidopsis have been isolated and characterised, defining 14 complementation groups. Mutations in at least nine FUSCA genes cause light-dependent phenotypic changes in the absence of light: high levels of anthocyanin accumulation in both the embryo and the seedling, inhibition of hypocotyl elongation, apical hook opening, and unfolding of cotyledons. In double mutants, the fusca phenotype is epistatic to the hy phytochromedeficiency phenotype, indicating that the FUSCA genes act downstream of phytochrome. By contrast, the accumulation of anthocyanin is suppressed by mutations in TT and TTG genes, which affect the biosynthesis of anthocyanin, placing the FUSCA genes upstream of those genes. Regardless of the presence or absence of anthocyanin, fusca mutations limit cell expansion and cause seedling lethality. In somatic sectors, mutant fus1 cells are viable; expressing tissue-specific phenotypes: reduced cell expansion and accumulation of anthocyanin in subepidermal tissue, formation of ectopic trichomes but no reduced cell expansion in epidermal tissue. Our results suggest a model of FUSCA gene action in light-induced signal transduction.     

Serologically-Related Anionic Peroxidases from Petunia and Cucumber can Substitute Flavonoid Antioxidants

In this study we have investigated whether naturally occurring flavonoid-deficient mutant Red Star of Petunia hybrida is capable of metabolizing H₂O₂ by invoking other antioxidant enzyme system. We demonstrated that reduced flower pigmentation due to a reduction in the chalcone synthase mRNA expression results in strong H₂O₂ accumulation accompanied by the induction of a specific set of anionic peroxidase (PRX), serologically-related to main cucumber srPRX. We found correlation between rate of H₂O₂ accumulation and qualitative, as well as quantitative changes in the srPRX expression which seems to be determined by flower phenotype. In detached flower buds cultured in vitro both abscisic acid and anther extirpation prevented anthocyanin pigmentation, and thus flavonoid biosynthesis, resulting in a marked accumulation of immunoprecipitable srPRX. In contrast, pigmented flowers cultivated under the same conditions did not accumulate corresponding srPRX. The results suggest that a specific set of anionic PRX can substitute for the absence of flavonoid antioxidants.     

Gibberellic acid decreases anthocyanin accumulation in wild carrot cell suspension cultures but does not alter 3'-nucleotidase activity

Gibberellic acid (GA₃) applied at different times during the growth of wild carrot ( Daucus carota ssp. Carota ) cell suspension cultures inhibited anthocyanin accumulation. Application of 3 × 10^–6 M GA₃ to cultures on day 0 or day 4 gave, respectively, 10 or 35% of anthocyanin accumulation relative to levels occurring when GA₃ was applied at the end of the growth period. Endogenous GAs were separated by high pressure liquid chromatography, and identified and quantified by gas chromatography-selected ion monitoring. Gibberellins GA₁, GA₃ and traces of GA₈. GA₁₉ and GA₂₀ were identified in carrot cell suspension cultures of both high and low anthocyanin-accumulating clones. The concentrations of GA₁. GA₃ and GA₈ in the two clones were similar and were not significantly different after the application of uniconazole which promoted anthocyanin accumulation. This suggests that these endogenous GAs are not the sole factors controlling the accumulation of anthocyanin in these different clones. Exogenous GA₃ and uniconazole had no effect on 3'-nucleotidase and 5'-nucleotidase activity in the carrot cell suspension cultures. Thus 3'-nucleotidase does not appear to play a role in the inhibition of anthocyanin accumulation by exogenous GA₃.     

A common precursor for CD4+ T cells producing IL-2 or IL-4.

To investigate whether CD4+ T cells are predetermined to produce a given pattern of lymphokines, we have used a culture system that allows the controlled induction of either IL-2- or IL-4-producing CD4+ T cells. Single, freshly isolated murine CD4+ T cells were activated with Con A, rIL-2, and APC; the developing clones were split and then cultured for an additional 14 days with either rIL-2 alone or with rIL-2 and anti-CD3 stimulation. Subclones expanded in the presence of rIL-2 alone produced predominantly IL-2, although subclones derived from the same precursor and expanded in the presence of rIL-2 and a mitogenic antibody to CD3 released predominantly IL-4. Subclones expanded for 2 wk in the presence of rIL-2 plus a mitogenic mAb to CD3 released up to 60 times more IL-4 but only 1/90 the amount of IL-2 released by subclones derived from the same precursor cell and expanded with rIL-2. Both phenotypes can be derived from IL-2-producing precursor cells. These results demonstrate that IL-2-producing clones can be derived from the same cells as IL-4-producing clones and are most consistent with the view that the IL-2-producing Th1 or the IL-4-producing Th2 phenotype of a T cell clone is acquired during T cell differentiation and is not secondary to the expansion of distinct subpopulations that are predetermined to produce a specific cytokine pattern.     

Dihydroflavonol Reductase Activity in Relation to Differential Anthocyanin Accumulation in Juvenile and Mature Phase Hedera helix L

Juvenile phase English ivy (Hedera helix L.) plants accumulate anthocyanin pigment in the hypodermis of stems and petioles, whereas genetically identical plants of the mature phase do not. The objective of this work was to assess which enzyme(s) might limit anthocyanin accumulation in mature phase ivy. Leaf discs of both juvenile and mature phase ivy accumulated comparable levels of the flavonols kaempferol and quercetin, whereas only juvenile phase discs accumulated anthocyanin. The accumulation of quercetin, but lack of accumulation of leucocyanidin or anthocyanin in mature phase discs, suggested that mature discs lacked dihydroflavonol reductase activity. There was no detectable dihydroflavonol reductase activity in mature phase discs, whereas there was an induction of activity in juvenile phase discs in response to sucrose, or photosynthetically fixed carbon, and light as a photomorphogenic signal. Phenylalanine ammonia-lyase, an enzyme early in the anthocyanin biosynthetic pathway, was induced above its basal level by sucrose and light in discs of both phases of ivy, with greater activity in mature phase discs. Phenylpropanoids, a class of compounds that are precursors to flavonoids, accumulated in leaf discs of both phases, with greater levels in mature phase discs. These results indicate that the lack of dihydroflavonol reductase activity limits the accumulation of anthocyanin in mature phase tissue.     

DMSO increases tyrosine residue phosphorylation in membranes from murine erythroleukemia cells

Phosphorylation of membranes from murine erythroleukemia cells was performed in the presence and absence of the polar solvent dimethyl sulfoxide. Quantitation of the phosphoamino acid content revealed that DMSO stimulated phosphotyrosine accumulation by three-fold; serine and threonine phosphorylation decreased significantly. We had previously shown that DMSO stimulated tyrosine residue phosphorylation of the hepatic epidermal growth factor receptor. EGF had little effect in MEL membranes; therefore, DMSO results in accumulation of phosphotyrosine in cell membranes that do not exhibit significant EGF-dependent phosphorylation.     

The role of heme in the regulation of the late program of Friend cell erythroid differentiation.

The addition of a chemical inducer, such as dimethylsulfoxide (DMSO), to cultures of mouse Friend erythroleukemic cells results in the induction of a number of late erythroid events, including the accumulation of globin mRNA, the inducation of hemoglobin synthesis, the appearance of erythrocyte membrane antigens (EMA), and the cessation of cell division. The experiments presented in this study demonstrate that heme is necessary but not sufficient for the loss of proliferative capacity associated with DMSO-induced Friend cell differentiation, whereas the accumulation of globin mRNA and EMA can occur in the absence of heme synthesis or heme itself. These conclusions were reached by selectively inhibiting heme synthesis in DMSO-treated cells in two independent ways: (i) Inducible cells were treated with 3-amino-1,2,4-triazole (AT), a drug which inhibits the induction of heme synthesis in Friend cells in a dose-dependent manner. Treatment of inducible Friend cells with 1.5% DMSO for five days caused the plating efficiency in methyl cellulose to decrease to 1% of that in untreated cultures. However, treatment of the cells with DMSO plus AT almost totally prevented this decrease in plating efficiency. The addition of exogenous hemin, which alone had no significant effect on plating efficiency, largely reversed the effect of AT in DMSO-treated cells, reducing the plating efficiency to below 5%. In contrast to the marked effects of AT on the proliferative capacity of differentiating Friend cells, the levels of globin mRNA and EMA were only partially decreased in cells treated with DMSO plus AT, compared to cells treated with DMSO alone. (ii) The relationship between heme synthesis, terminal cell division, and the induction of globin mRNA was investigated further through the use of non-inducible Friend cell variant clones. One such non-inducible clone, M18, appears to be a phenotypic analog of inducible cells treated with DMSO plus AT. Clone M18 did not accumulate heme or hemoglobin, as detected by benzidine staining, nor lose its proliferative capacity in response to DMSO. However, globin mRNA was induced by DMSO in this clone. Treatment of clone M18 with DMSO plus hemin overcame the block in hemoglobin accumulation suggesting that M18 has a defect in the induction of heme biosynthesis. In addition, exposure of M18 cells to DMSO plus hemin caused a gradual decrease in plating efficiency which was not due to non-specific toxicity. Prior incubation of M18 cells in DMSO for three to five days was necessary before hemin caused a rapid loss of proliferative capacity. Thus, these results, in agreement with the AT studies on inducible Friend cells and previous studies on the induction of EMA in clone M18, indicate that there may be both heme-dependent and heme-independent events in the program of Friend cell differentiation.