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.     

Phenylalanine ammonia-lyase,chalcone synthase and polyphenolic compounds in adult and rejuvenated hybrid walnut tree

Summary During the first growth phase of walnut (Juglans sp.) stump shoots, the concentrations of the two major phenolic compounds are not correlated with an increasing rate of shoot growth. The concentration of hydrojuglone glucoside (naphtoquinone) decreases as shoot growth rate increases, whereas the concentration of myricitrin (flavonol) remains constant. In contrast, phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity is proportional to the growth rate of shoots. Rejuvenation, which induces a higher growth rate and vegetative propagation ability, results in an increase of both PAL and chalcone synthase (CHS, EC 2.3.1.74) activities and hydrojuglone glucoside/myricitrin ratio. Moreover, physiological ageing is characterized by an accelerated functioning of polyphenolic metabolism. Fluctuations in PAL activity are associated with changes in shoot growth rate and with rejuvenation, but PAL does not directly control the accumulation of flavonoid compounds during rejuvenation. On the contrary, mathematical correlation of CHS activity and flavonoid accumulation during annual shoot growth of both adult and rejuvenated trees, indicates that CHS is the rate-limiting enzyme of the pathway.     

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     

Distribution of Phenylalanine Ammonia Lyase and Chalcone Synthase within Trunks of Robinia pseudoacacia L.

During heartwood formation, a kind of apoptosis in the inner parts of woody axes, phenolic substances are accumulated by in situ biosynthesis. In Robinia pseudoacacia L, these compounds are mainly flavonoids. In the present work, we performed a study to show if there is a correlation between measurable activities and detectable protein levels of phenylalanine ammonia lyase (PAL; EC 4.3.1.5) and chalcone synthase (CHS; EC 2.3.1.74), key enzymes of general phenylpropanoid metabolism and flavonoid biosynthesis, respectively. After separation of total protein extracts by one-dimensional micro-gel electrophoresis, newly emerging polypeptides were detectable within the sapwood-heartwood transition zone, pointing toward a transient activation of metabolism shortly before cell death occurs. Most prominent was a polypeptide around 46 kDa. By immunoblotting, this band was identified as a CHS subunit. Thus, the exclusive presence of both enzyme protein and extractable enzyme activity of CHS in the heartwood bordering tissue was shown. In contrast, levels of PAL protein were similar in all xylem tissues which contain living cells. PAL activity, however, was measurable only in the differentiating xylem and the sapwood-heartwood transition zone. From these results we conclude that during heartwood formation, CHS and PAL differ in their mode of regulation. It seems likely that CHS activity is regulated at the level of enzyme protein while PAL regulation is most probably post-translational.     

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.     

Anthocyanin accumulation in the hypocotyl of an ABA-over producing male-sterile tomato (Lycopersicon esculentum) mutant

Anthocyanin accumulation is known to be regulated by light and plant hormones but its occurrence varies with plant species and/or organ and tissue, and it has been negatively correlated with male sterility. In this study, we have examined the light responsive changes in anthocyanin in an abscisic acid (ABA) over-producer, male-sterile 7B-1 mutant and wild-type (WT) tomato hypocotyls. The results show that light-induced anthocyanin accumulation in the hypocotyl was more in WT compared with the 7B-1 mutant and more so under white light (W) compared with blue light (B) or red light (R). In contrast, the chlorophyll content was greater in the mutant than in WT. Exogenous ABA caused a transitory increase in anthocyanin content in WT but a reduction in 7B-1 , both in W and B. The high level of anthocyanin in WT under light conditions was not correlated with increased mRNA levels of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR), some of the anthocyanin biosynthetic genes. However, the activity of PAL (EC 4.3.1.5) was higher in the WT than in 7B-1 hypocotyls, and exogenous ABA caused an increase in PAL activity in the WT but a reduction in the mutant. The results presented show that high ABA negatively affects anthocyanin accumulation and that in the 7B-1 mutant it is related, in part, to reduced PAL activity. The results also support the view that the 7B-1 mutant has a defect in light perception and ABA sensitivity.     

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.     

Time courses for phytochrome-induced enzyme levels in phenylpropanoid metabolism (phenylalanine ammonia-lyase,naringenin-chalcone synthase) compared with time courses for phytochrome-mediated end-product accumulation (anthocyanin,quercetin)

Time course for changes in the levels of enzymes characteristic of general phenylpropanoid metabolism (phenylalanine ammonia-lyase, PAL; EC 4.3.1.5) and of the flavonoid-glycoside branch pathway (naringenin-chalcone synthase, CHS; EC 2.3.1.74) were measured in the cotyledons of mustard (Sinapis alba L.) seedlings and compared with the rates of accumulation of related end products (anthocyanin and quercetin). Induction of enzyme levels and of end-product accumulation was carried out with red and far-red (FR) light, operating via phytochrome. The data are compatible with the concept that the phytochrome-mediated appearance of enzymes such as PAL and CHS is indeed a prerequisite for the appearance of anthocyanins and flavonols. However, there is no close correlation between enzyme levels and the rates of synthesis of end products which could justify the identification of specific rate-limiting enzymes. Rather, the data indicate that there is a second phytochrome-dependent step, beyond enzyme induction, where the actual rate of flavonoid accumulation is determined. Anthocyanin and quercetin accumulation respond differently to light. However, the relative action of continuous FR, red light pulses and stored phytochrome signal is the same in both cases. This indicates that the mode of operation of phytochrome is the same in both cases. The two syntheses differ only in the degree of responsiveness towards phytochrome. The time course for changes in CHS levels in continuous FR, i.e. under conditions of phytochrome photosteady state, is similar to the time course for PAL levels whereas the time courses in darkness, following transfer from FR to darkness, are totally different. In the case of CHS, a transient rise is observed whereas, with PAL, an instantaneous drop in enzyme level occurs after transfer from FR to darkness. It is concluded that the stored phytochrome signal operates in darkness in the case of CHS but not in the case of PAL.Abbreviations c continuous - CHS naringenin-chalcone synthase (EC 2.3.1.74) - FR far-red light (3.5 W·m^-2) - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - Pfr phytochrome (far-red absorbing) - Pr phytochrome (red absorbing) - R red light (6.8 W·m^-2) - RG9-light long-wavelength far-red light obtained with RG9 glass filter - [Pfr]/[Ptot], whereby - Ptot total phytochrome (Pr+Pfr)