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.     

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.     

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     

Elicitation of anthocyanin production in callus cultures of <Emphasis Type="Italic">Daucus carota</Emphasis> and the involvement of methyl jasmonate and salicylic acid

The effect of methyl jasmonate (MeJA) and salicylic acid (SA) on the anthocyanin accumulation, endogenous titres of polyamines and ethylene production in callus cultures of Daucus carota were studied. The interaction of these signaling molecules with elicitors from Aspergillus niger was investigated and the involvement of MeJA was elucidated through the use of the jasmonic acid (JA) biosynthetic inhibitor ibuprofen. MeJA and SA were both found to stimulate the anthocyanin production in the callus cultures. The highest levels of anthocyanin was observed in the cultures treated with 200 μM SA 0.36 % and 0.01 μM MeJA 0.37 %. The MeJA and SA treatments were also found to result in higher activity of Ca²⁺ ATPase suggesting that the enhancement of anthocyanin by SA and MeJA could be mediated through the involvement of the calcium channel. The treatment of the callus cultures with SA was found to result in marginally higher titres of endogenous polyamines (PAs) whereas MeJA resulted in lower levels of PAs as compared to the control. The SA treatment was found to result in lower ethylene production and the treatment with MeJA stimulated the ethylene production. These results suggest that the stimulation of anthocyanin production by MeJA and SA in callus cultures of D. carota is not related to the ethylene production.     

Effects of gibberellic acid and uniconazole on the activities of some enzymes of anthocyanin biosynthesis in carrot cell cultures

Gibberellic acid (GA₃) inhibition of anthocyanin accumulation by carrot cell-suspension cultures was reversed by supplying dihydroquercitin or naringenin to the culture and not by supplying 4-coumaric acid or malonic acid. This suggested that gibberellic acid was inhibiting chalcone synthase, chalcone isomerase, or acetyl CoA carboxylase. Acetyl-CoA-carboxylase specific activity was the same in GA₃-treated and untreated cultures and was not detected in cultures treated with uniconazole, an inhibitor of gibberellic acid biosynthesis. Chalcone-isomerase specific activity was lower in GA₃-treated cultures than in untreated cultures and was lower in uniconazole-treated cultures than in the GA₃-treated cultures. The total chalcone synthase activity in extracts from GA₃- and from uniconazole-treated cells was not significantly different from that in extracts of untreated tissue. When these extracts were chromatographed on a Mono Q column, three peaks of chalcone synthase activity were found in extracts of nontreated cells, whereas only two of these peaks were detected in extracts of GA₃-treated cells. The extracts from GA₃-treated cells did not contain the peak of chalcone synthase activity that, in untreated cells, preceded the main peak. The correlation between the absence of this peak and the inhibition of anthocyanin accumulation suggests that this form of chalcone synthase is responsible for anthocyanin synthesis and that GA₃ prevents this form from appearing in the cells.     

Effects of surfactants on cell growth and pigment production in suspension cultures ofPerilla frutescens

The effects of surfactants, adecanol LG-294 and silicone A, on anthocyanin accumulation and the growth ofPerilla frutescens cells in suspension cultures were studied. Production of the red pigment was remarkably reduced from about 1.9 g/l to 0.4 g/l by adecanol LG-294 at 0.06 ml/l but not by silicone A up to 0.4 ml/l. Several repeated shake-flask cultures also demonstrated no adverse effects of silicone A on the metabolite accumulation by the suspended cells. Furthermore, the addition of silicone A to a culture in a stirred bioreactor produced a three-fold higher growth rate and a seven-fold increase in anthocyanin compared with surfactant-free cultures. The improvement was due to the substantial reduction or prevention of foaming and of cell adhesion to the bioreactor wall.     

Polysaccharide elicitors enhance anthocyanin and phenolic acid accumulation in cell suspension cultures of <Emphasis Type="Italic">Vitis vinifera</Emphasis>

The effects of yeast extract and selected polysaccharide elicitors on secondary metabolite production, particularly of anthocyanin and phenolic acid, in cell suspension cultures of Vitis vinifera were investigated. All elicitors either maintained or promoted cell growth in culture. Overall, secondary metabolite production in V. vinifera cell suspension cultures responded differently to different elicitors. Chitosan, pectin, and alginate enhanced production of anthocyanin within 13 days of culture with levels of 2.5-, 2.5-, and 2.6-fold increase, respectively, over that of control. Chitosan, alginate, and gum arabic significantly promoted accumulation of phenolic acids, particularly 3-O-glucosyl-resveratrol, in V. vinifera cultures, as well as in the culture medium. Intracellular phenolic acid production was significantly enhanced by alginate and chitosan, with 1.7- and 1.5-fold levels, respectively, of that of control. Extracellular phenolic acid production was also significantly increased in the presence of chitosan and gum arabic, with levels of 3.3- and 1.7-fold higher, respectively, than those of control. In addition, DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity was enhanced in the presence of elicitors, and this was positively correlated with increased accumulation of anthocyanin in V. vinifera cell suspension cultures.     

Improved anthraquinone accumulation in cell cultures of Cinchona `Robusta' by feeding of biosynthetic precursors and inhibitors

The effects of feeding of biosynthetic precursors and pathway specific inhibitors on anthraquinone (AQ) accumulation in fungal elicited cell cultures of Cinchona`Robusta' were studied. Addition of glyceraldehyde (1 mM), the initial precursor in the methyl-d-erythritol 4-phosphate (MEP) pathway, did not increase AQ accumulation, suggesting that the endogenous level of this precursor is not a limiting factor of AQ flux. It is proposed that AQs in Cinchona might be derived from the phenylpropanoid pathway, e.g. from caffeic acid. Addition of ferulic acid (1 mM) did not stimulate AQ accumulation, while addition of caffeic acid increased AQ accumulation by 48% compared to the control. The stimulating effect of feeding caffeic acid on AQ accumulation might be due to activation of other pathways. Addition of tectoquinone (2-methyl-anthraquinone) did not change the AQ patterns nor the shifts between AQs in control and tectoquinone-treated cell cultures. Addition of lovastatin, a specific inhibitor of the mevalonic acid pathway, did not inhibit the AQ accumulation. Clomazone, an inhibitor in the MEP pathway, inhibited the AQ accumulation, however. The simultaneous addition of lovastatin and clomazone inhibited both cell growth and AQ accumulation. These results further support the finding that isopentenyl diphosphate, which constitutes ring C of AQs in Cinchona `Robusta', is derived from the MEP pathway, and not from the mevalonic acid pathway.     

Abilities of organic acids to support growth and anthocyanin accumulation by suspension cultures of wild carrot cells using ammonium as the sole nitrogen source

Summary Fifteen organic acids were examined for their abilities to support the growth and anthocyanin accumulation by suspension cultures of wild carrot (Daucus carota L.) using ammonium as the sole nitrogen source. Glutarate, adipate, pimelate, azelate, cinnamate, and phthalate were toxic to the culture. They prevented growth and anthocyanin accumulation at 5 mM or less in media that were otherwise adequate for growth. Succinate, fumarate, malate, α-ketoglutarate, glutamate, maleate, malonate, tartarate, and citrate all supported growth and anthocyanin accumulation but in varying amounts. The growth achieved in medium containing 20 mM acid was higher at an initial pH of 5.5 than at an initial pH of 4.5. The growth achieved was dependent on the organic acid used, its concentration, and the initial pH of the medium. When growth occurred the final pH was higher than the initial pH with most of the acids. Anthocyanin accumulation was greatest with succinate at 14 to 20 mM at an initial pH of 4.2 or 4.3 and declined when the initial pH was above 4.3. These studies were supported by grants from General Foods Corporation and the W. Alton Jones Foundation.     

Control of anthocyanin biosynthesis pathway gene expression by eutypine,a toxin from Eutypa lata,in grape cell tissue cultures

Eutypine, 4-hydroxy-3-(3-methyl-3-butene-1-ynyl) benzaldehyde, is a toxin produced by Eutypa lata, the causal agent of Eutypa dieback in grapevine. The effect of the toxin on anthocyanin synthesis has been investigated in Vitis vinifera cv. Gamay cell cultures. At concentrations higher than 200 micromol/L, eutypine reduced anthocyanin accumulation in cells. The reduction in anthocyanin accumulation was proportional to the eutypine concentrations and HPLC analysis showed that eutypine affected the levels of all anthocyanins. The effect of eutypine application on the expression of five genes of the anthocyanin biosynthesis pathway, including chalcone synthase (CHS), flavonone-3-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), and UDP glucose-flavonoid 3-O-glucosyl transferase (UFGT) was determined. Expression of CHS, F3H, DFR and LDOXwas not affected by the addition of eutypine to grapevine cell cultures. In contrast, expression of the UFGT gene was dramatically inhibited by the toxin. These results suggest that in grapevine cell cultures, eutypine strongly affects anthocyanin accumulation by inhibiting UFGT gene expression. The mechanism of action of eutypine is discussed.     

Axillary shoot proliferation of blue honeysuckle

Callus cultures of Ajuga reptans flowers produced a complex mixture of cyanidin- and delphinidin-based pigments, of which more than 90% were acylated. The anthocyanin composition varied little during one growth period. During a time span of 5 years no new anthocyanin classes appeared. Quantitative differences in anthocyanin composition between the callus lines and during a 5 year time span were more pronounced. In general, the accumulation of delphinidin-based anthocyanins decreased. The percentage of acylated anthocyanins was stable in time. The accumulation of metabolically evolved anthocyanins (5′-substituted and acylated) decreased during passage from solid culture to liquid culture. The accumulation of acylated anthocyanins was influenced by the type of aeration in liquid cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

The effect of hormones on anthocyanin accumulation in cell cultures of Haplopappus gracilis

Summary Suspension cultures of Haplopappus gracilis accumulated anthocyanin when grown in defined media with 4.5×10^-6M 2,4-D. Transfer of cells to media with 10^-5M kinetin or benzyladenine and no auxin or 10^-7M NAA for 6 days resulted in increased anthocyanin concentration of the cells but the total amount of pigment was unaffected due to differences in growth rates. The cultures yielded up to 35 mg pigment per gram dry weight.Cells grown in batch culture in media with 10^-5M kinetin and with 10^-7 M NAA or 5×10^-5M NAA sampled and analyzed daily grew at the same rate. The concentration of anthocyanin differed, being lower in cells at 5×10^-5M NAA. After 6 days there was a rapid increase in pigment formation, and by 14 days the concentration of anthocyanin in cells in the two media were the same.When the cells were cultured in 3.5-1 phytostats and 600 ml culture was replaced daily with 600 ml medium, anthocyanins accumulated when the NAA concentration was 10^-7M but not at 10^-6M. At 10^-7M NAA the cultures remained pigmented and anthocyanin accumulation could be restored after a temporary loss of pigmentation due to an earlier, higher auxin concentration. The changes in concentration of phenylalanine ammonia-lyase did not correspond to changes in the rate of anthocyanin accumulation. The enzyme showed a maximum 4–8 h after inoculation of cells to fresh media. Cells grown on agar plates and rich in anthocyanin were observed to divide without loss of pigmentation, demonstrating that cells differentiated with respect to anthocyanin production undergo mitosis.Issued as NRCC No. 11388.Abbreviations used: 2,4-D=2,4-dichlorophenoxyacetic acid, NAA + -naphthaleneacetic acid.     

细胞均一性对葡萄细胞生长和花青素合成的影响

通过色差筛选法建立了一个相对均一的葡萄细胞悬浮系E,其细胞团较小,在长期继代培养过程中花青素合成能力的变异系数为8.7%,重复摇瓶实验的变异系数为5%。以E为实验材料进行的各组前体饲喂、诱导子添加、光照等联合作用实验,其生物量和花青素合成的变异系数均可控制在12%以内,充分说明了培养体系的均一性对维持稳定生产的重要性;黑暗条件下添加30μmol/L苯丙氨酸(Phe)和218μmol/L茉莉酸甲酯(MeJA)可使单位细胞花青素含量达到对照组的5.89倍,花青素产量为对照组的4.30倍,且连续5次继代培养过程中生物量和花青素合成的变异系数均比对照组降低。     

Hormonal regulation of ripening in the strawberry,a non-climacteric fruit

Anthocyanin accumulation is one measure of ripening in the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit. Neither aminoethoxyvinylglycine, an inhibitor of 1-aminocyclopropane carboxylic acid synthase, nor inhibitors of ethylene action (silver, norbornadiene) affected anthocyanin accumulation in ripening fruit. When the achenes were removed from one half of an unripe fruit there was an accelerated accumulation of anthocyanin and induction of phenylalanine ammonia lyase on the de-achened portion of the ripening fruit. These effects of achene removal could be prevented by the application of the synthetic auxins 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid to the de-achened surface. The introduction of 1-naphthalene acetic acid into intact unripe strawberry fruit through the peduncle delayed their subsequent ripening, as measured by the accumulation of anthocyanin, loss of chlorophyll and decrease in firmness. These findings suggest that the decline in the concentration of auxin in the achenes as strawberry fruit mature modulates the rate of fruit ripening.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - NAA 1-naphthaleneacetic acid - PA1 phenylalanine ammonia-lyase - POA phenoxyacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Cyanidin 3-glucoside accumulation in plane tree (Platanus acerifolia) cell-suspension cultures

The accumulation of only one anthocyanin, cyanidin 3-glucoside, in cell-suspension cultures of plane tree (Platanus aceriflia) is reported for the first time. During a time span of 6 years, no new anthocyanin was detected and cyanidin 3-glucoside was maintained at about 35 mg l^–1 cell culture medium. This stable cell culture system could therefore be used for the biotechnological production of cyanidin 3-glucoside.     

Changes in activities of enzymes involved in flavonoid metabolism during the initiation and suppression of anthocyanin synthesis in carrot suspension cultures regulated by 2,4-dichlorophenoxyacetic acid

When anthocyanin synthesis was induced in cell suspension cultures of carrot ( Daucus carota L. cv. Kurodagosun) by transfer to medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), chalcone synthase (CHS, EC 6.-.-.-), and chalcone-flavanone isomerase (CHFI, EC 5.5.1.6) activities appeared, reaching maxima 6–7 days after transfer. The maximum specific activity of CHS was much lower than that of PAL or CHFI. In a medium containing 2,4-D, no anthocyanin was synthesized, PAL and CHFI activities were suppressed and CHS activity could not be detected at all. The activities of PAL and CHS in cells cultured without 2,4-D for 6 days began to decrease within 3–6 h of 2,4-D addition. CHS activity was completely repressed 24–36 h after the addition, but CHFI activity was almost unchanged at this time. After culture without 2,4-D for 6 days, cell suspensions were transferred to fresh media either lacking or containing 2,4-D. After transfer, PAL increased in both media within 3 h, whereas CHS activity and anthocyanin accumulation were coordinated and both were completely regulated by 2,4-D. Changes in CHS activity rather than PAL activity correlate with changes in anthocyanin accumulation under various culture conditions.     

Increase in anthocyanin yield from wild-carrot cell cultures by a selection system based on cell-aggregate size

Wild-carrot (Daucus carota L.) cell cultures were screened to yield small (less than 63 m) or large (greater than 170 m) cell aggregates which were then subcultured. Cultures of the small-size class had a higher, those of the large-size class a lower anthocyanin yield than the unscreened culture. This relationship became accentuated with an increasing number of passages with screening prior to subculture. At the end of six months (12 passages), the pigment yield of the small-size class was triple that of the unscreened cells. Following this selection period, the tendency of the small-size fraction to increase in clump size when subcultured without screening was much less than that of freshly isolated cell aggregates of the same size. These observations may be explainable on the basis of a differential distribution of cytokinin between aggregates of different sizes. High levels of cytokinin inhibit anthocyanin accumulation and inhibit cell separation; these effects result in large cell aggregates having low levels of anthocyanin. In support of this hypothesis, it is shown that addition of kinetin to cultures of small cell aggregates causes an increase in the size of cell aggregates and a parallel decrease in anthocyanin yield.     

Growth and anthocyanin accumulation rates of carrot suspension cultures grown with excess nutrients after semicontinuous culture with different limiting nutrients at several dilution rates, pHs, and temperatures

When carrot cell cultures, after growth in semicontinuous culture, were transferred to media containing excess nutrients, they grew at different rates. The growth rates were generally higher after semicontinuous culture at higher dilution rates. There appears to be a limit on dilution rate above which growth rate does not increase. These changes were also displayed by clones from the parental culture. The possibility that these changes in growth rate reflect a need for the cultures to adapt to their new conditions is discussed. The growth rates of the cultures is markedly increased at 25 degrees C compared with 22 degrees C with little further increase at 28 degrees C. Growth rate is altered by less than 20% when pH is changed from 4.5 to either 5.5 or 4.2. The rates of anthocyanin accumulation by the cultures were similar under all conditions tested except at 22 degrees C. They were larger than the rates of dry weight accumulation. In contrast, the amounts of anthocyanin accumulated in the clones and in the parental cultures grown at pH 5.5 instead of pH 4.5 were very different. The observations were interpreted as showing that the clones differ in the rate of metabolism but not in the rate of synthesis of anthocyanins and that at pH 5.5 the rate of metabolism of anthocyanins but not the rate of synthesis is higher than it is at pH 4.5. The use of semicontinuous cultures as a source of inoculum for batch cultures rather than as a source of biomass for extraction of chemicals is discussed.     

Anthocyanin accumulation and changes in activities of phenylalanine ammonia-lyase and chalcone synthase in roselle (Hibiscus sabdariffa L.) callus cultures

Time-course changes in anthocyanin accumulation, phenylalanine ammonia-lyase activity and chalcone synthase activity were examined in roselle callus tissues incubated under different culture conditions. Phenylalanine ammonia-lyase activity was not affected by either the kind of auxin supplemented to the medium or light regime. In contrast, chalcone synthase activity was markedly suppressed when the callus was cultured with a medium containing indole-3-acetic acid instead of 2,4-dichlorophenoxyacetic acid (2,4-D) or in the dark. The results imply that in roselle callus cultures chalcone synthase plays a more important role in anthocyanin biosynthesis regulated by 2,4-D and light irradiation than phenylalanine ammonialyase.Abbreviations LS Linsmaier and Skoog - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - PAL phenylalanine ammonia-lyase - CHS chalcone synthase     

Manipulating anthocyanin composition in Vitis vinifera suspension cultures by elicitation with jasmonic acid and light irradiation

Jasmonic acid altered the accumulation of major anthocyanins in Vitis vinifera cell culture. Peonidin 3-glucoside content at day three was increased from 0.3 to 1.7 mg g^–1 dry cell wt while other major anthocyanins were increased by smaller increments. By day 14, the content of methylated and acylated anthocyanins (peonidin 3-p-coumaroylglucoside and malvidin 3-p-coumaroylglucoside) was 6.3 mg g^–1 DCW, in response to treatment with jasmonic acid, and comprising 45% (w/w) of total anthocyanins. In comparison, the untreated control culture contained 1.2 mg g^–1 DCW which made up 32% (w/w) of total anthocyanins. Light further enhanced anthocyanin accumulation induced by jasmonic acid elicitation. The content of peonidin 3-glucoside at day 3 was 6.6 mg g^–1 DCW, 22-fold higher than control cultures while the content in response to light irradiation alone was 0.6 mg g^–1 DCW. When a highly pigmented cell line was elicited with jasmonic acid total anthocyanins increased from 9.2 to 20.7 mg g^–1 DCW, but there was no change in the anthocyanin composition.