Anthocyanin yields of clonal wild carrot cell cultures

The anthocyanin yields in clonal populations of wild carrot suspension cultures were measured after four patterns of cloning and selection. These patterns were:

Phenotypic differences in anthocyanin accumulation among clonally related cultured cells of carrot

Subclones from a wild carrot cell culture have been examined for their anthocyanin accumulation in the absence and presence of DMSO and 4-coumaric acid, naringenin, dihydroquercetin or leucocyanidin. Subclones that accumulate no or extremely low levels of anthocyanin do not increase their anthocyanin accumulation when treated with DMSO or intermediates. These compounds increased the anthocyanin accumulation in subclones which produce detectable anthocyanin in their absence.Chalcone synthase was shown to be present in clones and the activity showed no correlation with the amount of anthocyanin accumulated. This suggests that the enzymes of anthocyanin biosynthesis are not coordinately repressed in the subclones which accumulate little or no anthocyanin. Dihydroquercetin and catechin were present in subclones with little or no anthocyanin but no procyanidin was detected which suggests that these subclones biosynthesize leucocyanidin but do not convert it into colorless procyanidins as a major alternative metabolic pathway to anthocyanin biosynthesis. The possibility that some clones are not anthocyanin accumulating because they have impaired transport of the sinapoylated anthocyanin into the vacuole is discussed.     

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.     

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     

Differential regulation in tobacco cell suspensions of genes involved in plant-bacteria interactions by pathogen-related signals

Six cDNA clones whose corresponding mRNAs accumulate early during the hypersensitive reaction in tobacco leaves have been classified into 2 groups according to their maximum levels of accumulation in an incompatible versus a compatible interaction withPseudomonas solanacearum. We present evidence that, at least in the first stages of the interaction, tobacco cell suspensions retain the ability to respond differentially to compatible and incompatible isolates ofP. solanacearum.In addition, studies on the effect of a fungal elicitor on the accumulation of the mRNAs corresponding to the cDNA clones in cell suspensions indicate that only one group of genes responds to this treatment.     

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.     

Anthocyanin pathway in rice (Oryza sativa L): identification of a mutant showing dominant inhibition of anthocyanins in leaf and accumulation of proanthocyanidins in pericarp

The present study has surveyed a collection of indica rice (Oryza sativa) lines for tissue-specific anthocyanin pigmentation pattern, which has also been used for a genetically meaningful classification. This classification helped predict probable genotypes of rice lines and, in the process, a leaf blade-specific dominant inhibitor of pigmentation (Ilb) was predicted and its presence later confirmed in two lines. We ascribe most tissue-specific accumulation of anthocyanins to the presence of a different set of Pl alleles. Cyanidin, as a major pigment, and peonidin, as a minor pigment, were detected in purple-pigmented tissues. Further, the floral organ-derived tissues always contained a higher level of anthocyanins and, correspondingly, a relatively increased proportion of peonidin. One line, N22B, with a brown pericarp was identified and shown to accumulate proanthocyanidins, but with no anthocyanins, in the pericarp. We propose that the accumulation of proanthocyanidins is due to a block in the anthocyanin biosynthetic pathway in rice at the anthocyanidin synthase-mediated conversion of leucoanthocyanidin to anthocyanidin.     

Studies on the Persistent Infection with Measles Virus in HeLa Cells

HeLa/MV cells which have previously been found to contain measles antigen in more than 90% of cells were very similar to normal HeLa cells in their morphology and growth. Although almost all of HeLa/MV cells may be infected, only 10% of them released infectious virus particles. The hemadsorption test, however, showed that most of the infected cells produced by hemagglutinin. Two kinds of clones were obtained by cloning HeLa/MV cells in the presence of anti-measles serum. One was the virus-releasing clone and the other the uninfected clone which did not contain any measles antigen. The proportions of virus-releasing clones to all clones varied between 20 to 54%, and did not show any increase even after recloning of the virus-releasing clones. The susceptibility of the uninfected clones to the standard measles virus was not different from that of normal HeLa cells.     

Replication of endogenous avian retrovirus in permissive and nonpermissive chicken embryo fibroblasts.

Clones of chicken embryo fibroblasts exogenously infected with the endogenous avian retrovirus were analyzed to examine the replication of this virus in permissive (Gr+) and nonpermissive (Gr-) cells. The results demonstrate that the endogenous virus was capable of infecting both Gr+ and Gr- cells with equal efficiency. Infected clones of Gr+ and Gr- cells differed, however, in two significant ways. At the time of their initial characterization, the Gr+ clones produced 100- to 1,000-fold more virus than the Gr- clones. Further, the amount of virus produced by Gr+ clones did not change significantly during serial passage of the cells. In contrast, continued passage of the infected Gr- clones resulted in a gradual increase in the amount of virus produced. Individual clones of infected Gr- cells produced infectious virus at rates that, initially, differed by a factor of more than 10(4). The large differences in the production of virus by these clones could not be explained by equally large differences in the number of infected cells within the clonal populations. Greater than 80% of the clonal populations examined ultimately produced virus at rates that were not significantly different from the rates observed in infected Gr+ cells. Virus produced by these infected Gr- cells exhibited the same restricted replication upon establishing a new infection in nonpermissive cells. Analysis of the appearance of free and integrated viral DNA sequences during endogenous virus infection of Gr+ and Gr- cells demonstrated that, after an initial delay in the synthesis of free viral DNA in Gr- cells, the nonpermissive cells ultimately acquired as many integrated viral DNA sequences as were found in infected Gr+ cells. These results indicate that a majority of the infectious particles of the endogenous virus are capable of establishing infection in a Gr- cell and, ultimately, of producing virus at a rate that is not significantly different from that produced by infected Gr+ cells. The virus produced from the Gr- cells is not a stable genetic variant of the original endogenous virus that is capable of unrestricted replication in nonpermissive cells. The reduced efficiency with which the endogenous virus initially replicates in nonpermissive cells and the increased length of time required for infected Gr- cells to produce maximal virus titers suggest that the endogenous virus may utilize a different mechanism of replication in Gr+ and Gr- fibroblasts.     

Heterogeneity withinFrankia sp. LDAgpl studied among clones and reisolates

Summary Frankia sp. LDAgpl, an isolate from spore positive nodules ofAlnus glutinosa, only slowly infects its host plant. Reisolates obtained from occasional nodules caused by infection with LDAgpl, are capable of infecting the alder much more rapidly. A variability analysis of LDAgpl has been performed to obtain more insight into the question whether these reisolates constitute a different genotype within LDAgpl and if the plant is exerting an influence during plant passage. High dilutions of mildly sonicatedFrankia suspensions were plated to obtain genetically homogeneous colonies. Clones thus generated showed differences in growth pattern, sporulation and C₂H₂-reduction on media containing propionic acid as sole C-source (P-medium). Differences in sporulation on P-medium indicate that LDAgpl was a highly heterogeneous strain. Comparisons of sporulation on several different media gave evidence that the differences in sporulation between LDAgpl clones are the result of differences in efficiency of propionic acid utilization.The differences observed between the reisolates and LDAgpl clones indicate that the reisolates constitute a different genotype, which could be selected for by the plant during the infection process. Comparison with similar changes in phenotype occuring in a spore negative type strain fromA. glutinosa is discussed.     

Circumventing senescence is associated with stem cell properties and metformin sensitivity

Most cancers arise in old individuals, which also accumulate senescent cells. Cellular senescence can be experimentally induced by expression of oncogenes or telomere shortening during serial passage in culture. In vivo, precursor lesions of several cancer types accumulate senescent cells, which are thought to represent a barrier to malignant progression and a response to the aberrant activation of growth signaling pathways by oncogenes (oncogene toxicity). Here, we sought to define gene expression changes associated with cells that bypass senescence induced by oncogenic RAS. In the context of pancreatic ductal adenocarcinoma (PDAC), oncogenic KRAS induces benign pancreatic intraepithelial neoplasias (PanINs), which exhibit features of oncogene‐induced senescence. We found that the bypass of senescence in PanINs leads to malignant PDAC cells characterized by gene signatures of epithelial‐mesenchymal transition, stem cells, and mitochondria. Stem cell properties were similarly acquired in PanIN cells treated with LPS, and in primary fibroblasts and mammary epithelial cells that bypassed Ras‐induced senescence after reduction of ERK signaling. Intriguingly, maintenance of cells that circumvented senescence and acquired stem cell properties was blocked by metformin, an inhibitor of complex I of the electron transport chain or depletion of STAT3, a protein required for mitochondrial functions and stemness. Thus, our studies link bypass of senescence in premalignant lesions to loss of differentiation, acquisition of stemness features, and increased reliance on mitochondrial functions.     

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.     

The effect of light and 2,4-D on anthocyanin production in Oxalis reclinata callus

In vitro cultures of O. reclinata accumulate red anthocyanin pigments. Two callus lines were established from O. reclinata, one red and the other non-pigmented. The red callus accumulated cyanidin-3-glucoside as a major pigment. Light irradiation induced anthocyanin synthesis in white callus, resulting in a heterogenous red callus line being formed. The incubation of red and white callus cultures in the dark or at low-light resulted in the repression of red pigment accumulation. The application of 2,4-D (1.0 mg l^-1) inhibited pigment production in the white callus and decreased anthocyanin accumulation in the red callus. The polypeptide composition of the red and white callus lines from O. reclinata were compared using two-dimensional electrophoresis. The red callus had a larger subset of neutral and acidic polypeptides.     

Growth is required for cell competition in the imaginal discs ofDrosophila melanogaster

Summary Imaginal wing discs from late third-instar larvae were gammairradiated to induce clones of rapidly growingMinute ^– cells in a background of slowly growingMinute cells and culturedin vivo for periods up to 18 days. Clones in discs cultured for 16 to 18 days did not grow significantly larger than clones in uncultured controls, indicating that competition between populations of cells having potentially different mitotic rates does not occur in imaginal discs after their growth is completed.     

Improved culture conditions forCowdria ruminantium (Rickettsiales), the agent of heartwater disease of domestic ruminants

The causal agent of heartwater disease of domestic ruminants,Cowdria ruminantium, can, with difficulty, be isolated and passaged in lines of bovine endothelial cells grown in the presence of the Glasgow modification of Eagle's minimal essential medium. However, when Leibovitz's L-15 medium supplemented with 0.45% glucose at pH 6.0–6.5 is used as maintenance medium for these cells, isolation and serial passage may routinely be achieved.     

Anthocyanin production in selected cell lines of grape (Vitis vinifera L.)

Summary Anthocyanin production of two lines ofVitis vinifera cell cultures, i.e., 5.4 and 13.1, which were obtained from the same starting material after 20 and 37 mo. of clonal selection, respectively, was investigated. Cell suspension cultures of lines 5.4 and 13.1 maintained an anthocyanin content of 0.44 ± 0.15 and 1.02 ± 0.31 mg·g⁻¹ fresh weight during 50 and 32 weekly maintenance subcultures, respectively. Under anthocyanin-promoting culture conditions, both lines showed an enhancement of their anthocyanin level by approximately fourfold. While line 5.4 accumulated peonidin 3-glucoside and cyanidin 3-glucoside in decreasing order, line 13.1 accumulated primarily peonidin 3-p-coumaroylglucoside with lesser amounts of malvidin monoglucoside. Results show that while the anthocyanin content was improved during the course of repeated selections, the anthocyanin composition was modified markedly favoring the accumulation of more metabolically-advanced anthocyanins.