Regulation of phenylalanine ammonia-lyase genes in carrot suspension cultured cells

Induction of anthocyanin synthesis occurs during metabolic differentiation in carrot suspension cultured cells grown in medium lacking 2,4-dichlorophenoxyacetic acid (2,4-D), and is closely correlated with embryogenesis. Anthocyanin synthesis may also be induced by light-irradiation under different culture conditions. The phenylalanine ammonia-lyase (PAL) gene (TRN-PAL), which was transiently induced by the transfer effect, was also rapidly induced after light-irradiation. However, TRN-PAL was not involved in anthocyanin synthesis. A second PAL gene, ANT-PAL, was involved in anthocyanin synthesis. ANT-PAL was induced during metabolic differentiation in medium lacking 2,4-D parallel with the induction of chalcone synthase (CHS). PAL genes in the carrot genome are expressed differentially depending on the nature of the environmental stimulus, e.g. transfer effect and light, and other parameters which also affect anthocyanin synthesis.Abbreviations CHS chalcone synthase - 2,4-D 2,4-dichlorophenoxyacetic acid - GUS -glucuronidase - Luc firefly luciferase - PAL phenylalanine ammonia-lyase - UV ultraviolet     

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.     

Differential regulation of phenylalanine ammonia-lyase genes during anthocyanin synthesis and by transfer effect in carrot cell suspension cultures

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) catalyses the first step in the phenylpropanoid pathway and is induced during differentiation and by various stimuli. In carrot ( Daucus carota L. cv. Kurodagasun) suspension culture cells, PAL is slowly induced during anthocyanin synthesis which occurs in a medium lacking 2,4-dichlo-rophenoxyacetic acid and is also induced rapidly and transiently by transferring and diluting cells to fresh medium. Analyses of nucleotide sequences derived from PAL cDNAs revealed that the PAL mRNAs induced by transfer were transcribed from different carrot PAL genes than the PAL mRNAs induced during anthocyanin synthesis. Northern blotting using probes derived from 3'non-coding regions for PAL cDNAs confirmed that different PAL genes were induced during anthocyanin synthesis and after transfer. Induction of different PAL genes occurs in response to differences in the induction trigger.     

Light-induced Synthesis of Anthocyanin in Carrot Cells in Suspension: II EFFECTS OF LIGHT AND 2, 4-D ON INDUCTION AND REDUCTION OF ENZYME ACTIVITIES RELATED TO ANTHOCYANIN SYNTHESIS

Time-courses of light-induced activities of enzymes relatingto anthocyanin formation were studied. Phenylalanine ammonia-lyase(PAL), 4-cumarate CoA ligase (4CL) and cinnamate-4-hydroxylase(C4H) (group 1 enzymes) and chalcone synthase (CHS) and chalcone-flavanoneisomerase (CHFI) (group 2 enzymes), were studied in carrot suspensioncells which were irradiated 5 d after transfer to a 2,4-dichlorophenoxyaceticacid (2,4-D)-free medium in the dark. Time-courses of group1 enzymes showed two peaks (fast and slow) with the slow peakincreasing almost parallel to anthocyanin accumulation. Time-coursesof group 2 enzymes showed one peak corresponding to the slowpeak of group 1. From the inhibitor experiment, the fast peakalso corresponded to the activity of the newly synthesized enzyme.From the initial phase of the time-courses, enzymes belongingto group 1 always induced more rapidly than those of group 2,and their induction was co-operative. However, once anthocyanin synthesis was induced by light, neitheraddition of 2,4-D nor transfer to darkness could prohibit anthocyaninsynthesis completely. Addition of 2,4-D in the dark completelysuppressed anthocyanin synthesis within 1 d and the activityof CHS also disappeared within 1 d. These results are explainedby a previous hypothesis (Takeda, 1988) that 2,4-D induces thestate change of cells. Key words: Anthocyanin, co-ordinate induction, Daucus carota, 2,4-dichlorophenoxyacetic acid, light-triggered     

Changes in Activities of Enzymes Involved in General Phenylpropanoid Metabolism during the Induction and Reduction of Anthocyanin Synthesis in a Carrot Suspension Culture as Regulated by 2,4-D

The activities of enzymes involved in general phenylpropanoidmetabolism were followed in a carrot suspension culture duringthe induction and reduction of anthocyanin synthesis regulatedby 2,4-D. When no anthocyanin synthesis occurred in a mediumcontaining 2,4-D (+2,4-D medium), the activities of phenylalanineammonia-lyase (PAL) and 4-coumarate:CoA ligase (4CL) increased1 day after transfer due to the transfer effect, but subsequentlydecreased and remained at a low level. Cinnamate-4-hydroxylase(C4H) activity showed a low level throughout culture. When cellswere transferred to a medium lacking 2,4-D (–2,4-D medium),the activities of PAL, C4H and 4CL increased and maximum activitiesof these enzymes were observed 6–7 days after transfer,when anthocyanin was most rapidly synthesized. When cells were cultured in the –2,4-D medium, the additionof 2,4-D immediately reduced the induced activity of PAL. PALactivity was super-induced by the transfer effect, while anthocyaninsynthesis decreased. The addition of intermediates of generalphenylpropanoid metabolism, with 2,4-D, to the medium 6 daysafter transfer to the –2,4-D medium did not promote anthocyaninsynthesis, whereas dihydroquercetin did promote it. Regulationof anthocyanin synthesis by 2,4-D is discussed in relation tochanges in enzyme activities involved in general phenylpropanoidmetabolism. ¹ Present address: Cell Science and Technology Division, FermentationResearch Institute, Agency of Industrial Science and Technology,Yatabe-machi, Ibaraki 305, Japan. ² Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai 980, Japan.     

Regulation of Flavonoid Biosynthetic Genes in Germinating Arabidopsis Seedlings

Many higher plants, including Arabidopsis, transiently display purple anthocyanin pigments just after seed germination. We observed that steady state levels of mRNAs encoded by four flavonoid biosynthetic genes, PAL1 (encoding phenylalanine ammonia-lyase 1), CHS (encoding chalcone synthase), CHI (encoding chalcone isomerase), and DFR (encoding dihydroflavonol reductase), were temporally regulated, peaking in 3-day-old seedlings grown in continuous white light. Except for the case of PAL1 mRNA, mRNA levels for these flavonoid genes were very low in seedlings grown in darkness. Light induction studies using seedlings grown in darkness showed that PAL1 mRNA began to accumulate before CHS and CHI mRNAs, which, in turn, began to accumulate before DFR mRNA. This order of induction is the same as the order of the biosynthetic steps in flavonoid biosynthesis. Our results suggest that the flavonoid biosynthetic pathway is coordinately regulated by a developmental timing mechanism during germination. Blue light and UVB light induction experiments using red light- and dark-grown seedlings showed that the flavonoid biosynthetic genes are induced most effectively by UVB light and that blue light induction is mediated by a specific blue light receptor.     

Analysis of the Expression of Anthocyanin Pathway Genes in Developing Vitis vinifera L. cv Shiraz Grape Berries and the Implications for Pathway Regulation

Anthocyanin synthesis in Vitis vinifera L. cv Shiraz grape berries began 10 weeks postflowering and continued throughout berry ripening. Expression of seven genes of the anthocyanin biosynthetic pathway (phenylalanine ammonia lyase [PAL], chalcone synthase [CHS], chalcone isomerase [CHI], flavanone-3-hydroxylase [F3H], dihydroflavonol 4-reductase [DFR], leucoanthocyanidin dioxygen-ase [LDOX], and UDP glucose-flavonoid 3-o-glucosyl transferase [UFGT]) was determined. In flowers and grape berry skins, expression of all of the genes, except UFGT, was detected up to 4 weeks postflowering, followed by a reduction in this expression 6 to 8 weeks postflowering. Expression of CHS, CHI, F3H, DFR, LDOX, and UFGT then increased 10 weeks postflowering, coinciding with the onset of anthocyanin synthesis. In grape berry flesh, no PAL or UFGT expression was detected at any stage of development, but CHS, CHI, F3H, DFR, and LDOX were expressed up to 4 weeks postflowering. These results indicate that the onset of anthocyanin synthesis in ripening grape berry skins coincides with a coordinated increase in expression of a number of genes in the anthocyanin biosynthetic pathway, suggesting the involvement of regulatory genes. UFGT is regulated independently of the other genes, suggesting that in grapes the major control point in this pathway is later than that observed in maize, petunia, and snapdragon.     

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     

Induction of anthocyanin synthesis in relation to embryogenesis in a carrot suspension culture: Correlation of metabolic differentiation with morphological differentiation

A system in which anthocyanin synthesis could be induced under a defined condition, was established in a carrot suspension culture. A cell suspension culture of carrot ( Daucus carota L. cv. Kurodagosun) was subcultured for more than a year in a medium containing 5 × 10⁻⁷ M 2,4-dichlorophenoxyacetic acid (2,4-D). At every subculture the cultures were sieved through nylon screens and the cells and cell clusters collected in the size range of 31–81 μm were transferred to a fresh medium. When the cells were transferred to a medium without auxin, synthesis of anthocyanin was induced. Zeatin promoted anthocyanin synthesis in a medium lacking auxin, with maximum yields of anthocyanin obtained at 10⁻⁷ to 10⁻⁸ M zeatin, 2,4-D at higher concentrations than 10⁻⁷ M inhibited anthocyanin synthesis completely. The sieved cells were fractionated by Ficoll density gradient centrifugation. Somatic embryos were formed in the fraction of higher density (>14% of Ficoll) in a medium containing 10⁻⁷ M zeatin but lacking auxin, while synthesis of anthocyanin was hardly observed. On the other hand, cells in the fraction of lower density (<12% of Ficoll) synthesized anthocyanin in the same medium, but formed few embryos. Forty to fifty percent of the total cells in this lighter cell fraction synthesized anthocyanin at a maximum. The similarity between anthocyanin synthesis and embryogenesis was observed in the time course as well as in the effects of growth regulators. The correlation between metabolic and morphological differentiation is discussed.     

Intracellular location of NADP+-linked malic enzyme in C3 plants

Ultraviolet light induces anthocyanin biosynthesis in cell cultures of an Afghan cultivar of Daucus carota (Daucus carota L. ssp. sativus). Simultaneous treatment with a fungal elicitor from Pythium aphanidermatum results in an inhibition of the catalytic activity of chalcone synthase (CHS), which in turn correlates with an inhibition of anthocyanin biosynthesis. On immunoblots, one isoenzyme (40 kDa) of CHS disappears upon elicitor treatment. On an mRNA level, only the mRNA for the 40-kDa-CHS is active after treatment with ultraviolet light. After inhibition of anthocyanin biosynthesis by the elicitor the enzyme protein disappears and the CHS mRNA is strongly diminished. This inhibition depends on the concentration of the elicitor. In addition, elicitor treatment leads to an induction of the general phenylpropanoid pathway as well as to the accumulation of 4-hydroxybenzoic acid which is covalently bound to wall polysaccharides of the carrot cells. The possible function of phenylalanine ammonia-lyase in providing precursors for 4-hydroxybenzoic acid is discussed.Abbreviations CHI chalcone isomerase - CHS chalcone synthase - PAL phenylalanine ammonia-lyase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor K. Hahlbrock (Max-Planck-Institut für Züchtungsforschung, Köln, FRG) for providing us with antisera to CHS and PAL, respectively. This work was supported by a grant from the Deutsche Forschungsgemeinschaft and scholarships from the Friedrich-Ebert-Stiftung (J. G.), the Landesgraduierten-förderungsgesetz Baden-Württemberg (J.-P. S) and the Gerhard-Rösch-Stiftung (D. S.). We thank R. Hofmann for her excellent technical assistance.     

Induction of chalcone synthase in cell suspension cultures of carrot (Daucus carota L. spp. sativus) by ultraviolet light: evidence for two different forms of chalcone synthase

Two cell lines of carrot (Daucus carota L. spp. sativus), grown as cell-suspension cultures in the dark, were irradiated with ultraviolet light (315–420 nm) 10 d after the onset of cultivation. Chalcone synthase (CHS) enzyme activity was induced in both cell lines. Anthocyanin synthesis was only stimulated in the anthocyanin-containing cell line DCb. Parallel to the increase in CHS activity there was an increase with time in the amount of one CHS form with an isoelectric point of 6.5 and a molecular weight of 40 kilodaltons (kDa) per subunit. Whereas the anthocyanin-free cell line DCs failed to accumulate anthocyanin, it did stimulate another CHS form with an isoelectric point at pH 5.5 and a molecular weight of 43 kDa per subunit. Both enzyme activities could be separated by isoelectric focusing and stabilized using sodium hydrosulfite as an oxidation protectant. In carrot plants, CHS was restricted to the dark purple petals of the inflorescence (40 kDa) and to the leaves (43 kDa).Abbreviations BSA bovine serum albumin - CHS chalcone synthase - IEF isoelectric focusing - kDa kilodaltons - KP_i potassium phosphate buffer - PAL phenylalanine ammonialyase - pI isoelectric point - UV ultraviolet     

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     

Ultraviolet A-specific induction of anthocyanin biosynthesis in the swollen hypocotyls of turnip (Brassica rapa)

Ultraviolet A (UV-A)-mediated regulation of anthocyanin biosynthesis was investigated in swollen hypocotyls of the red turnip 'Tsuda'. The shaded swollen hypocotyls which contained negligible anthocyanin were exposed to artificial light sources including low fluence UV-B, UV-A, blue, red, far-red, red plus UV-A, far-red plus UV-A, and blue plus red. Among these lights, only UV-A induced anthocyanin biosynthesis and co-irradiation of red or far-red with UV-A did not affect the extent of UV-A-induced anthocyanin accumulation. The expression of phenylalanine ammonia lyase (PAL; EC 4.3.1.5), chalcone synthase (CHS; EC 2.3.1.74), flavanone 3-hydroxylase (F3H; EC 1.14.11.9), dihydroflavonol 4-reductase (DFR; EC 1.1.1.219), and anthocyanidin synthase (ANS; EC 1.14.11.19) genes was increased with time during a 24 h exposure to UV-A. In contrast, irradiation with red, blue, UV-B, and a combination of blue with red failed to induce CHS expression. Microarray analysis showed that only a few genes, including CHS and F3H, were induced significantly by UV-A, while a separate set of many genes was induced by low fluence UV-B. The UV-A-specific induction of anthocyanin biosynthesis and the unique gene expression profile upon UV-A irradiation as compared with blue and UV-B demonstrated that the observed induction of anthocyanin biosynthesis in red turnips was mediated by a distinct UV-A-specific photoreceptor, but not by phytochromes, UV-A/blue photoreceptors, or UV-B photoreceptors.     

Identification and characterization of genes induced for anthocyanin synthesis and chlorophyll degradation in regenerated torenia shoots using suppression subtractive hybridization, cDNA microarrays, and RNAi techniques

Anthocyanin synthesis and chlorophyll degradation in regenerated torenia (Torenia fournieri Linden ex Fourn.) shoots induced by osmotic stress with 7% sucrose were examined to identify the genes regulating the underlying molecular mechanism. To achieve this, suppression subtractive hybridization was performed to enrich the cDNAs of genes induced in anthocyanin-synthesizing and chlorophyll-degrading regenerated shoots. The nucleotide sequences of 1,388 random cDNAs were determined, and these were used in the preparation of cDNA microarrays for high-throughput screening. From 1,056 cDNAs analyzed in the microarrays, 116 nonredundant genes were identified, which were up regulated by 7% sucrose to induce anthocyanin synthesis and chlorophyll degradation in regenerated shoots. Of these, eight genes were selected and RNAi transformants prepared, six of which exhibited anthocyanin synthesis inhibition and/or chlorophyll degradation in their leaf discs. Notably, the RNAi transformants of the glucose 6-phosphate/phosphate translocator gene displayed inhibition both of anthocyanin synthesis and chlorophyll degradation in both leaf discs and regenerated shoots. There was also less accumulation of anthocyanin in the petals, and flowering time was shortened. The genes we identified as being up-regulated in the regenerated torenia shoots may help further elucidate the molecular mechanism underlying the induction of anthocyanin synthesis and chlorophyll degradation.