Amaranthin Synthesis Research Articles

Isolation of amaranthin synthetase from Chenopodium quinoa and construction of an amaranthin production system using suspension-cultured tobacco BY-2 cells.

SummaryBetalains are plant pigments primarily produced by plants of the order Caryophyllales. Because betalain possesses anti‐inflammatory and anticancer activities, it may be useful as a pharmaceutical agent and dietary supplement. Recent studies have identified the genes involved in the betalain biosynthesis of betanin. Amaranthin and celosianin II are abundant in the quinoa (Chenopodium quinoa Willd.) hypocotyl, and amaranthin comprises glucuronic acid bound to betanin; therefore, this suggests the existence of a glucuronyltransferase involved in the synthesis of amaranthin in the quinoa hypocotyl. To identify the gene involved in amaranthin biosynthesis, we performed a BLAST analysis and phylogenetic tree analysis based on sequences homologous to flavonoid glycosyltransferase, followed by expression analysis on the quinoa hypocotyl to obtain three candidate proteins. Production of amaranthin in a transient Nicotiana benthamiana expression system was evaluated for these candidates and one was identified as having the ability to produce amaranthin. The gene encoding this protein was quinoa amaranthin synthetase 1 (CqAmaSy1). We also created a transgenic tobacco bright yellow‐2 (BY‐2) cell line wherein four betalain biosynthesis genes were introduced to facilitate amaranthin production. This transgenic cell line produced 13.67 ± 4.13 μm (mean ± SEM) amaranthin and 26.60 ± 1.53 μm betanin, whereas the production of isoamaranthin and isobetanin could not be detected. Tests confirmed the ability of amaranthin and betanin to slightly suppress cancer cell viability. Furthermore, amaranthin was shown to significantly inhibit HIV‐1 protease activity, whereas betanin did not.

Antagonistic effect of indole-3-acetic acid on kinetin-stimulated amaranthin accumulation in the cotyledons of Amaranthus mangostanus seedlings

ABSTRACTLight triggered the initiation of amaranthin biosynthesis in cotyledons of Amaranthus mangostanus L. seedlings. Cytokinin induced amaranthin synthesis in the dark and increased the accumulation of amaranthin under light irradiation. No studies have explored whether indole-3-acetic acid (IAA) can affect kinetin-induced amaranthin accumulation in seedlings of A. mangostanus L. In this study, we found that IAA inhibited both the kinetin- and light-induced synthesis of amaranthin. In the dark, 10.0 mg l−1 IAA caused a 68% reduction in amaranthin production after induction by 5.0 mg l−1 kinetin. In the presence of light, 10.0 mg l−1 IAA resulted in a 50% decrease in amaranthin synthesis following induction by 5.0 mg l−1 kinetin. In addition, IAA could reverse kinetin-induced amaranthin accumulation under red, blue, or far-red light conditions. Our results suggest that IAA had an antagonistic effect on the light-induced or cytokinin-stimulated accumulation of amaranthin in the cotyledons of A. mangostanus L. seedlings.

Effect of rare earth element europium on amaranthin synthesis in Amarathus caudatus seedlings.

Considering the resemblances between Eu3+ and Ca2+ in their atomic radius and structures of the valence electron, the effects of Eu3+ on amaramthin synthesis in Amarathus caudatus seedling were studied. Eu3+ had both promoting and inhibiting effects on amaramthin synthesis. The optimum promoting concentration and half inhibiting concentration of Eu3+ to synthesis of amaranthin were 0.4 mmol/L and 2.5 mmol/L, respectively. In the dark, A23187 (ions carrier) could carry Eu3+ into cells through the Ca2+ channel. When Ca2+ was chelated with EGTA, the synthesis of amaranthin could be partly retrieved by Eu3+. Eu3+ treatment could also activate Ca2+- ATPase on plasma membrane. Moreover, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of total proteins from the plants treated by Eu3+ and Ca2+ were similar but slightly different in the contents. It suggested that the effects of Eu3+ and Ca2+ on amaranthin synthesis were similar. After being treated by Eu3+ or Ca2+, the outside Ca2+ could enter into cells to promote synthesis of amaranthin. The results above indicated that Eu3+ might replace Ca2+ in the calcium/calmidulindependent phytochrome signal transduction system and play important roles in plant development by promoting calcium transportation across plasma membrane.

Research of the entry of rare earth elements Eu3+ and La3+ into plant cell.

Whether rare earth elements can enter into plant cells remains controversial. This article discusses the ultracellular structural localization of lanthanum (La(3+)) and europium (Eu(3+)) in the intact plant cells fed by rare earth elements Eu(3+) and La(3+). Eu-TTA fluorescence analysis of the plasmalemma, cytoplast, and mitochondria showed that Eu(3+) fluorescence intensities in such structures significantly increased. Eu(3+) can directly enter or be carried by the artificial ion carrier A23187 into plant cells through the calcium ion (Ca(2+)) channel and then partially resume the synthesis of amaranthin in the Amaranthus caudatus growing in the dark. Locations of rare earth elements La(3+) and Eu(3+) in all kinds of components of cytoplasmatic organelles were determined with transmission electron microscope, scanning electron microscope, and energy-dispersive X-ray microanalysis. The results of energy-dispersive X-ray microanalysis indicated that Eu(3+) and La(3+) can be absorbed into plant cells and bind to the membranes of protoplasm, chloroplast, mitochondrion, cytoplast, and karyon. These results provide experimental evidence that rare earth elements can be absorbed into plant cells, which would be the basis for interpreting physiological and biochemical effects of rare earth elements on plant cells.

Effects of G protein and cGMP on phytochrome-mediated amaranthin synthesis inAmaranthus caudatus seedlings

The effects of G protein and cGMP on phytochrome-mediated amaranthin biosynthesis inAmaranthus caudatus seedlings were studied. It was shown that G protein agonist cholera toxin induced amarathin synthesis in darkness, whereas G protein antagonist pertussis toxin inhibited red light-induced amaranthin synthesis. Amaranthin synthesis was also induced by exogenous cGMP, while the amaranthin biosynthesis induced by cholera toxin, red light and exogenous cGMP was inhibited by genistein. L Y-83583, an inhibitor of guanylyl cyclase, inhibited the amarenthin synthesis induced both by red light and cholera toxin, while it was not able to inhibit the amaranthin synthesis induced by exogenous cGMP. These results suggest that G protein, guanylyl cyclase and cGMP were the candidates in phytochrone signal transduction chain for red light-induced amaranthin biosynthesis and the red light signal transduction chain might be as follows: red light --> phytochrome --> G protein --> guanylyl cyclase --> cGMP.

THE EFFECTS OF LIGHT QUALITY ON ENDOGENOUS CYTOKININ LEVELS IN IPT-TRANSGENIC TOBACCO

Similarities exist between the effects of phytochrome and cytokinins on plant growth and development (e.g., chloroplast development, amaranthin synthesis, seed germination). It is unclear, however, if and how these two systems interact. The coaction between phytochrome and cytokinins was investigated by using Nicotiana plumbaginifolia plants transformed with the isopentenyl transferase (ipt) cytokinin gene and treated with end-of-day (EOD) red (R) and far-red (FR) light. The ipt gene was under control of either a constitutive cauliflower mosaic virus promoter (35S-plants) or an inducible, heat shock promoter (HS-plants). When treated with EOD FR light, whole plants were characterized by decreased chlorophyll concentrations and increased fresh weights. When treated with EOD R light, 35S-plants contained high concentrations of zeatin riboside (ZR) compared to plants treated with EOD FR light. When treated with EOD FR light, HS-plants contained high concentrations of ZR compared to plants treated with EOD R light. Both cytokinin responses were photoreversible. The reasons for the differences between the 35S- and HS-plant responses are not known. Results appear to implicate interactions between phytochrome and cytokinins in plant growth and development.

IPTASE SYNTHESIS AND LIGHT MEDIATION IN TRANSGENIC TOBACCO

Similarities exist between the effects of phytochrome and cytokinins on plant growth and development (e.g., chloroplast development, amaranthin synthesis. seed germination, photomorphogenesis). It is unclear, however, if and how these two systems interact. As a beginning step to determine cytokinin-phytochrome interactions, we developed a strategy utilizing ipt -transgenic tobacco in phytochrome/light treatment investigations. The sour-cc of the ipt gene was Agrobacterium tumefaciens Ti plasmid 15955. This gene encodes for isopentenyl transferase which is an enzyme active in cytokinin biosynthesis. Ipt -transgenic tobacco cultures (grown on MS medium supplemented with kanamycin but no plant growth regulators) were treated with end-of-day red or far-red light for 15 minutes. After 30 days of treatment, the plant tissue was harvested and either homogenized for SDS-PAGE or fixed for transmission electron microscopic analysis. Results from immuno-gold labelling using polyclonal antibodies specific to iptase will he used to Indicate the influence of phytochrome on cytokinin activity. Also, structural changes at the ultra-cellular level will be determined.

181 AN ULTRACELLULAR LOOK AT LIGHT-TREATED IPT-TRANSGENIC TOBACCO

Similarities exist between the effects of phytochrome and cytokinins on plant growth and development (e.g., chloroplast development. amaranthin synthesis, seed germination, photomorphogenesis). It is unclear. however, if and how these two systems interact. To determine the effects of phytochrome activity on cytokinin synthesis and ultracellular plant development, we utilized tobacco transformed with the Agrobacterium tumefaciens isopentenyl transferase (ipt) gene. This gene encodes for isopentenyl transferase (iptase) which is an enzyme active in cytokinin biosysthesis. Ipt-transgenic tobacco cultures were treated with end-of-day red or far-red light for 15 minutes. After 15-30 days of treatment, the plant tissue was harvested and ipt expression was verified by SDS-PAGE and western blot analysis. Polyclonal antibodies specific to iptase were used as a primary antibody. Colloidal gold conjugated to goat. anti-rabbit antiserum served as an electron dense, secondary antibody and a probe to light-influenced iptase synthesis and distribution within the cell. A Hitachi 600AB transmission electron microscope was used to determine the influence of phytochrome/light treatments on the ultrastructure of ipr-transgenic cells.

Influence of calcium and calcium and calmodulin antagonists on the cytokinin-induced amaranthin accumulation inAmaranthus tricolor

The concentration of kinetin and kinetinriboside plays an essential role in the induction of amaranthin accumulation in cotyledons ofAmaranthus tricolor during germination. The dose/effect ratio shows that kinetin induced 3- to 3.5-fold more amaranthin than kinetinriboside at the same molecular concentration. Various concentrations of exogenous Ca2+ did not influence the effects of kinetin on the betacyanin synthesis. However, when Ca2+ was applied together with kinetinriboside, the amaranthin production was stimulated. Time-course experiments show a lag phase of 16 h starting from the incubation with kinetin and a distinct increase of amaranthin thereafter. If the seedlings were treated simultaneously with kinetin and Ca2+, the increase of amaranthin started after 12 h. At 16 h of incubation in kinetin/Ca2+, the amount of amaranthin increased significantly compared to controls incubated with kinetin alone. If Ca2+ ions (16 h kinetin/Ca2+ incubation) were removed from the medium after 2 h, 4 h, and up to 14 h, the amaranthin content was enhanced compared to controls without Ca2+. The stimulating effect was highest in the presence of Ca2+ for 8 h. These data show that exogenous Ca2+ stimulated the amaranthin synthesis mainly during the first 12 h of incubation. The Ca2+ antagonists EGTA, chlorotetracycline, and CoCl2 reduced the amaranthin content up to 80%. The calmodulin antagonists chloropromazine and trifluoperazine inhibited the betacyanin accumulation up to 97% when applied at the beginning of the incubation. Neither Co2+ nor trifluoperazine after 12 h of preincubation in kinetin had inhibiting effects on the amaranthin production. Therefore, we presume that a specific period of competence is required for calmodulin-mediated Ca2+ effects on the accumulation of amaranthin induced by cytokinins in the seedlings ofAmaranthus tricolor.

Use of 5,6-dichloro-l-?-D-ribofuranosylbenzimidazole to distinguish light stimulation from cytokinin stimulation of amaranthin synthesis in Amaranthus tricolor

In Amaranthus tricolor seedlings, amaranthin synthesis can be induced by cytokinins and (or) by light. It was found that 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) was a specific inhibitor of the cytokinin response whereas the light response was unchanged. DRB also inhibited the negative response of pigment production to an anticytokinin, 7-(pentylamino)-3-methylpyrazolo(4,3-d) pyrimidine (PAMPP). DRB can be considered as a specific inhibitor of the physiological responses to cytokinins. The evolution of the inhibition percentage versus cytokinin or anticytokinin concentration shows that DRB is not a `competitor' of cytokinins. The experiments suggest that cytokinins or anticytokinins act as modulators of a fundamental light activated reaction of amaranthin synthesis.

Physiology and biochemistry of theAmaranthus cytokinin bioassay and its applications

The influence of some substances on the amaranthin biosynthesis inAmaranthus-seedlings was investigated with regard to the accuracy and sensitivity of theAmaranthus cytokinin bioassay. 1) The herbicide glyphosate inhibited plgment formation in a concentration of 10-4 mol 1-1 in most cases only slightly (approximately 10–25 %). 2) Shikimic acid does not seem to be a precursor, in many experiments it was slightly inhibitory. 3) Jasmonic acid promoted slightly the plgment formation with a distinct maximum at about 5 x 10-4 mol 11-. 4) The oxidation product of zeatin (with KMnO4) is not active in theAmaranthus bioassay; only weak activity was recorded at very high concentrations. 5) Glucosides, extracted fromGinkgo biloba leaves, stimulate amaranthin synthesis, as we could conclude from applications of this bioassay to senescing, abscisingGinkgo leaves.

Changes in the Growth Pattern of Eichhornia crassipes (Mart.) Solms. Leaves Caused by Growth Regulators

Summary Changes in leaf morphology of water-hyacinth ( Eichhornia crassipes ) caused by the environment could occur through changes in the levels of endogenous gibberellins and cytokinins. Gibberellic acid enhances petiole elongation. Kinetin although ineffective, when in the presence of gibberellic acid stimulates leaf elongation even higher than gibberellic acid treatment alone. Significant increases of gibberellin and cytokinin-like activities (as detected by inhibition and stimulation of amaranthin synthesis respectively) were detected in root extracts of long-leaf plants relative to short-leaf plants.

Effect of a cytokinin antagonist on cytokinin and light-dependent amaranthin synthesis inAmaranthus Tricolor seedlings

InAmaranthus tricolor seedlings, amaranthin synthesis can be induced under the effect either of a cytokinin or of white light. The 3-methyl-7-(n-pentylamino)pyrazolo(4,3-d)pyrimidine (PAMPP), a cytokinin analog that strongly inhibits the growth of tobacco callus, antagonizes the stimulating effect of cytokinin as well as stimulation by light. In dose-response terms, the inhibitory effect of PAMPP was described as competitive with respect to N6-benzyladenine (b6Ade) or light. The inhibition by PAMPP of the b6Ade amaranthin test response or the inhibition by this cytokinin analog of the light amaranthin test response were both reversed by either subsequent light or b6Ade treatment.

An analysis of the action of light on betalain synthesis in the seedling of Amaranthus caudatus, var. viridis

Accumulation of betalain (amaranthin) in the seedling of Amaranthus caudatus, var. viridis, is inducible by light. Since the apparent lag-phase of amaranthin accumulation after the onset of light is of the order of 3 h, light induction experiments could be performed up to 3 h after the onset of light without interference with actual synthesis. The intricate induction phenomena can be explained as follows: The inductive light operates through phytochrome and through a blue/UV photoreceptor ('cryptochrome'). A phytochrome-dependent 'High Irradiance Reaction' is of minor importance. However, there is a strong, specific interaction between the light effects mediated through phytochrome and cryptochrome in the sense that the 'extent of the reversible response' - (response obtained with a particular light treatment terminated with a saturating red light pulse) minus (response obtained with the same light treatment when terminated with a saturating 756 nm light pulse) -increases with increasing Pfr level and total fluence rate during the induction period. It is concluded that light induced amaranthin synthesis is, in fact, a convenient biochemical model system of photomorphogenesis in the case when phytochrome and cryptochrome operate simultaneously in mediating photomorphogenesis.

Significance of Cyclic Nucleotides in Amaranthin Synthesis by Amaranthus caudatus Seedlings

Explants of 72–76 h old Amaranthus caudatus seedlings synthesize the betalain pigment amaranthin in response to light. Light can be replaced with a cytokinin or a cyclic nucleotide with an N6-substituent. Cyclic 3′5′-AMP shows only weak activity and that only at high unphysiological concentrations. Even cyclic 2′3′-AMP, which docs not act as a ‘second messenger’, induces amaranthin synthesis to a greater degree than cyclic 3′,5′-AMP. But N6-monobutyryl-cyclic 3′,5′-AMP and N6-2′-O-dibutyryl-cyclicAMP show high activity, higher even than kinetin at its optimum concentration of 10−5 M. 2′-O-Monobutyryl-cyclicAMP, on the other hand, is considerably less active, suggesting that N6-substitution of the adenine ring is responsible for the enhanced activity. N6-Propionyl, butyryl and valeryladenines are all highly active, indicating that the cyclic monophosphate moiety is unnecessary for this response. All the compounds tested, including cyclic 3′,5′-AMP, show additive effects, but there is no amplification of the response, typical of second messenger action. Inhibition of amaranthin synthesis imposed by hadacidin, is relieved by kinetin, DBc AMP, N6-monobutyryl-cAMP and N6-butyryl adenine. Cyclic 3′,5′-AMP is weakly active in this regard. As natural cytokinins are N6-substituted adenine compounds, and as only N6-substituted cyclic nucleotides are able to mimic the effect of cytokinin, it is concluded that these cyclic nucleotides function as cytokinin analogues and not as ‘second messengers’'.

The Effect of Potassium on Amaranthin Synthesis in Seedlings of Amaranthus caudatus L.

Summary Potassium, as KCl or KNO3, enhanced amaranthin and chlorophyll production in seedlings of Amaranthus caudatus. Potassium chloride and KN03 induced a different response in the relative levels of amaranthin synthesised in seedlings given a light treatment and those kept completely in the dark. Kinetin in combination with KCl (and to a lesser extent with KN03) induced a relatively large amaranthin content in dark-grown seedlings. The results indicate that KCl and KN03 stimulate amaranthin formation by increasing active pool tyrosine (and DOPA). This was confirmed by the specific radioactivity of amaranthin purified from «salt» treated seedlings incubated with 14C-tyrosine.

The hormonal control of betacyanin synthesis in Amaranthus caudatus

Gibberellic acid (GA 3) inhibits amaranthin synthesis whereas the growth retardant, phosphon D, enhances pigment levels in A. caudatus seedlings exposed to light. No effect was observed on chlorophyll and carotenoid synthesis. Radioactive tyrosine and DOPA were incorporated into amaranthin. The specific activity of amaranthin synthesised in the presence of 14C-tyrosine or 14C-DOPA in seedlings treated with GA 3 is higher than water controls. The specific activity of pigment from phosphon D treated tissue is relatively low. GA 3 treated tissue has lower active tyrosine and DOPA pools compared to phosphon treated seedlings. Tyrosine and DOPA-oxidase activity increases in GA 3 treated and H 2O control seedlings exposed to light. Kinetin stimulates the synthesis of amaranthin in dark-grown seedlings and this is not overcome by simultaneous GA 3 application. Dark-grown seedlings treated with different kinetin concentrations and incubated in 14C-tyrosine synthesise radioactive amaranthin of similar specific activity. Kinetin treatment of dark-grown seedlings brings about an increased tyrosine and DOPA-oxidase activity. The results indicate that GA 3 controls the production and/or availability of tyrosine whereas kinetin can mimic light treatment and controls the utilisation of tyrosine probably by bringing about the synthesis or activation of tyrosine and DOPA-oxidase protein.

Effects of light and kinetin on amaranthin synthesis induced by cAMP

The effects of cyclic 3′,5′-adenosine monophosphate ( cAMP) on amaranthin synthesis in the dark, or in the presence of kinetin or light were investigated in isolated cotyledons of Amaranthus tricolor and A. caudatus. The results suggest that sites or modes of action of cAMP and kinetin are not separated and differ from those of light and that the nucleotide cannot be considered a messenger involved in amaranthin formation stimulated by kinetin or by light.

Light control of amaranthin synthesis in isolated Amaranthus cotyledons

The effect of light on the amaranthin synthesis stimulated by exogenous precursors has been studied in isolated cotyledons of Amaranthus tricolor and A. caudatus. The results indicate that light acts at the level of the formation of the dihydropyridine moiety of the pigment.

Kinetik der durch Inhibitoren des Protein- und Nukleinsäurestoffwechsels bewirkten Hemmung der Amaranthinbildung bei Amaranthus caudatus-Keimlingen

The synthesis of amaranthin in Amaranthus caudatus Seedlings is inhibited by inhibitors of protein (chloramphenicol, puromycin) and nucleic acid synthesis (2-thiouracil). The degree of inhibition depends on the time of application. Already 8 h after onset of irradiation the effect of chloramphenicol is diminished and after 12 h this antibiotic does not inhibit pigment formation. Puromycin, however, shows after 12 h an inhibition of about 15 per cent. Only after 16 h 2-thiouracil has no inhibiting effect on amaranthin synthesis. The action of 2-thiouracil seems to be competitive, because the effect of 2-thiouracil can be overcome by simultaneous application of higher concentrations of uracil.