Sunlight-induced anthocyanin pigmentation in maize vegetative tissues

Although, in maize, sunlight-regulated anthocyanin formation in vegetative tissues is observed only in the cultivars harbouring homozygous recessive pl loci, the identity of the photoreceptor mediating this process is not yet fully established. In this study the nature of photoreceptor(s) mediating this response was examined using an Indian hybrid maize cultivar (Kanchan-521). The etiolated maize seedlings of this cultivar on exposure to sunlight formed anthocyanin in all vegetative organs. Sunlight elicited photoinduction of anthocyanin with a slow increase between 4-16 h after the sunlight exposure, followed by a rapid increase between 16-24 h. The photoinduction of anthocyanin was primarily mediated by the UV-B component of sunlight and could be elicited by exposure to an artificial UV-B light source. The sunlight-mediated induction of anthocyanin was reduced if the sunlight exposure was terminated with a far-red pulse before transfer to darkness, indicating a coaction of phytochrome in this photoresponse. Exposure to sunlight also stimulated phenylalanine ammonia lyase (PAL) activity in all organs with two temporally separated peaks. The first peak of PAL between 4-12 h was induced by phytochrome, and the second peak of PAL between 12-24 h was induced by UV-B light. These results indicate that the photoinduction of anthocyanin in maize is mediated by a coaction of UV-B light and phytochrome.     

Der Einfluß des Plasmotypus auf die Regulation der Aktivität der L-Phenylalanin-Ammonium-Lyase (Untersuchungen an Raimannia-Oenotheren)

Summary The regulation of the activity of L-phenylalanine ammonia-lyase (PAL) in seedlings of the complex heterocygotes Oenothera berteriana (with the chromosome complexes B and I) and Oenothera odorata (with the complexes v and I) and of their progeny was investigated. The following main results were obtained:The plasmotype (plasmon) has a significant effect upon the amount of increase of the activity of PAL during illumination with continuous far-red light (Figs. 2-5). Combined with the genotypes v.I, B.I and l.v the plasmotype of Oenothera berteriana effects a higher maximal activity after 20 hrs than the plasmotype of Oenothera odorata. This influence of the plasmon is constantly inherited.The influence of the chromosome complexes is characterized by the order I-v-l-B; the I-complex causes the highest, the B-complex the lowest increase in PAL-activity.Cytoplasm and plastids have no apparent influence either on the PAL-activity in darkgrown seedlings or on the amount of increase of PAL-activity which is brought about by wounding (Fig. 7). Therefore it can be concluded that the plasmotype affects very specifically the light-induced PAL-synthesis.There is no evidence so far for the existence of isoenzymes of PAL in the seedlings of Oenothera forms.Because of various correlations between light-induced PAL-activity and anthocyanin formation (Table 2), it can be supposed that the plasmon takes part in the regulation of anthocyanin formation by directing the PAL-activity. By the technique of simultaneous irradiation it is demonstrated that anthocyanin formation under continuous illumination with far-red is mediated by phytochrome (Fig. 6).The possibility of explaining the influence of the plasmotype of Oenothera forms on various phytochrome dependent reactions on the basis of a pleiotropic mechanism is discussed.     

Production of Anthocyanins by Vitis Cells in Suspension Culture

A large amount of anthocyanin was accumulated in the callus tissues of Vitis hybrids without light irradiation.

Culture conditions for the production of anthocyanins by Vitis cells in suspension cultures were investigated. High sucrose and low phosphate concentrations brought about a marked increase of anthocyanin formation, while high concentrations of nitrate, phosphate, and 2, 4-D repressed the pigment formation. The effects of these nutrients depended on the concentrations of coexistent ones.

Regulation of the aeration rate was important for anthocyanin formation in submerged aerated cultures and light irradiation enhanced anthocyanin formation in cultured cells.     

Phytochrome-induced flavonoid biosynthesis in mustard (Sinapis alba L.) cotyledons. Enzymic control and differential regulation of anthocyanin and quercetin formation

Phytochrome-induced increases in enzyme activities for phenylalanine ammonia-lyase (EC 4.3.1.5) and chalcone isomerase (EC 5.5.1.6), and in amounts of the related end products, anthocyanin and the flavonol, quercetin, were measured in cotyledons of mustard (Sinapis alba L.). There was no correlation between the activities of these enzymes and the rate of anthocyanin accumulation; however, some correlation was found with the quercetin accumulation rate. Since anthocyanin and flavonol accumulation is spatially separated in mustard (flavonols in the upper epidermis, anthocyanin in the lower epidermis), it was possible to measure anthocyanin-associated phenylalanine ammonia-lyase independently. This activity correlated well with the accumulation rate for anthocyanin during the first few hours after induction. The phytochrome effect on anthocyanin formation differed from that on quercetin formation: anthocyanin was strongly induced by continuous far-red light and by both continuous red light and red light pulses, whereas quercetin was only effectively induced by continuous far-red light.Abbreviations CHI chalcone isomerase - PAL phenylalanine ammonia-lyase     

Induction of phenylalanine ammonia-lyase in hypocotyls of sunflower seedlings by light, excision and sucrose

Light, excision and sucrose increased extractable phenylalanine ammonia-lyase (PAL) activity from hypocotyl tissue of sunflower ( Helianthus annuus L. cv. Peredovik) to 2–6 times the basal level. Intact sunflower seedlings or whole hypocotyls incubated in water or 0.1 M sucrose exhibited, in continuous light, a pattern in which PAL peaked 4 and 28 h after the beginning of the illumination. When 0.5 cm long hypocotyl segments were incubated in water or 0.1 M sucrose, they exhibited, both in continuous light and in the dark, a pattern in which PAL rose during an initial period of 10 h (assay in sucrose and light) to 48 h (assay in water and dark) and then remained nearly constant at a high value for at least the next 10 h. When whole hypocotyls were incubated in 0.1 M sucrose, a third pattern in PAL activity was found in which PAL peaked after 28 h and subsequently declined. In all the above systems the increase in PAL activity was significantly reduced by cycloheximide. Furthermore, the subsequent decay of PAL activity following illumination was prevented by delayed transfer to cycloheximide. It is suggested that the results can be explained on the basis of a turnover mechanism involving continued de novo enzyme synthesis and subsequent synthesis of a PAL-inactivating system.     

Induction of Phenylalanine Ammonia-lyase in Strawberry Leaf Disks: Action Spectra and Effects of Wounding, Sucrose, and Light

The increase in phenylalanine ammonia-lyase (PAL) activity in strawberry (Fragaria vesca var. WSU-1232) leaf disks required wounding, sucrose, and light and was cycloheximide-sensitive. In injured leaves and in leaf disks, the highest PAL activity was detected nearest the wounded tissues. Without wounding, no increase in activity was observed when leaves were cultured in sucrose and light.     

Distribution of phenylalanine ammonia-lyase in etiolated and far-red irradiated radish seedlings

Summary In dark-grown Raphanus seedlings, most of the PAL activity is found in roots where it increases sigmoidally during organ development. In hypocotyls, the dark increase of enzyme activity is linear with time. In cotyledons and hooks, dark activity is very low and remains constant. After onset of continuous far-red irradiation, an activity increase is observed in all parts of the seedling. In cotyledons and hooks, the increase is followed by a decrease. This is comparable to light-induced PAL activity described in other materials. In roots and hypocotyls, the initial increase is not followed by a decrease. In dark-grown roots and hypocotyls PAL activity is correlated to fresh weight augmentation. In no part of the seedling could a correlation be found between light-induced PAL activity and anthocyanin formation.     

Anomalous effects of cycloheximide on phenylalanine ammonia-lyase: role of synthesis and inactivation in leaf disks of helianthus annuus*

The activity of phenylalanine ammonia-lyase (PAL) increases dramatically in leaf disks of sunflower (Helianthus annuus) cultured on 0.1 M sucrose in the dark. If disks are subsequently transferred to water, PAL activity decays rapidly. After inactivation the level of PAL can be increased again by transferring the tissue back to sucrose. The initial increase in PAL activity appears to involve an increase in the rate of PAL formation and the appearance is inhibited by cycloheximide. Inactivation of the enzyme is also inhibited by cycloheximide. A comparison of cycloheximide inhibition at different concentrations showed that inactivation was much more sensitive to the inhibitor than PAL formation. The rate of PAL inactivation was very low in fresh disks placed directly on water (t 1/2 = > 1 day) but increased greatly after culture on sucrose (t_1/2 = 2 to 4 hr). Therefore, culture appears to increase PAL inactivation as well as PAL formation. Reappearance of PAL activity after inactivation is stimulated rather than inhibited by cycloheximide. The change in effect of cycloheximide from inhibition to apparent stimulation can best be explained by the observation that (1) the turnover of PAL, both formation and inactivation, increases greatly as a result of culture on sucrose and (2) inactivation is more sensitive to cycloheximide than formation. Thus, even where an anomalous cycloheximide insensitive appearance of PAL activity occurs, a mechanism other than reactivation of the enzyme may be involved.     

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.     

Inhibition of anthocyanin synthesis and phenylalanine ammonialyase activity by Co2+ in leaf disks of Terminalia catappa

Treatment with sucrose induced anthocyanin synthesis and phenylalanine ammonialyase (PAL, EC 4. 3. 1. 5) activity in leaf disks of Indian almond ( Terminalia catappa L. Duthie). Co²⁺, an inhibitor of ethylene biosynthesis, inhibited anthocyanin synthesis and PAL activity when given together with sucrose. Ethephon (an exogenous source of ethylene) given along with sucrose, promoted anthocyanin synthesis and PAL activity, but in the presence of Co²⁺ its effectiveness decreased. In an attempt to understand the inhibitory action of Co²⁺ in the presence of ethephon, the effect of Co²⁺ on PAL activity was studied in vitro. A kinetic study showed an uncompetitive type of inhibition of PAL by Co²⁺, which was not time dependent. Addition of 2-mercaptoethanol, cysteine or glutathione overcame the in vitro effect of Co²⁺, and 2-mercaptoethanol also restored the activity of PAL extracted from Co²⁺-treated leaf disks. It is suggested that sulfhydryl group(s) might be involved in the inactivation of PAL by Co²⁺. The effects of N-ethylmaleimide (NEM) and HgCl₂ (other sulfhydryl reagents) were also studied. Both NEM and Hg²⁺ competitively inhibited PAL activity in vitro, and the inhibition could be reversed by sulfhydryl compounds.     

Induktion der Phenylalanin-Ammonium-Lyase in Gewebekulturen vonHaplopappus gracilis

Zusammenfassung In Zellkulturen vonHaplopappus gracilis, die nur im Licht Anthocyan synthetisierten, wurde die PAL-Aktivität untersucht. Diese Kulturen wiesen auch im Dunkeln, abhängig vom Zeitpunkt der letzten Übertragung auf frisches Nährmedium, deutliche Unterschiede in der Höhe der PAL-Aktivität auf. Blaulicht führte in jedem Falle zu einer Erhöhung der PAL-Aktivität und zu anschließender Anthocyansynthese. Den größten Einfluß auf die Aktivität des Enzyms hatte Blaulicht auf 3 Wochen alten Kulturen. Nach 48stündiger Bestrahlung betrug der Anstieg der Enzymaktivität 400%, dann folgte ein allmählicher Rückgang. Der Proteingehalt und die MDH-Aktivität der Kulturen blieben während dieser Zeit konstant. Ein PAL-inaktivierendes System konnte in denHaplopappuskulturen nicht nachgewiesen werden. Aber Actinomycin D und Puromycin hemmten die Blaulicht-induzierte PAL-Synthese. Rotlicht hatte in allen Versuchen keinen Effekt auf die Höhe der Enzymaktivität.

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Induction of phenylalanine ammonia-lyase in tissue cultures ofHaplopappus gracilis

Summary The PAL activity of cell cultures ofHaplopappus gracilis was investigated. These showed anthocyanin synthesis only after exposure to light. Blue light always led to an increase of PAL activity and subsequent anthocyanin production. But also in the dark, only depending on the time after the passage to fresh medium significant changes in PAL activity have been detected. The highest effect of blue light on the activity of the enzyme was observed in the cultures three weeks after passage to fresh medium. Irradiation for 48 hours with blue light then resulted in a 400% rise of PAL activity followed by a gradual decline. In these experiments no changes of total protein content or MDH activity were found. There also was no evidence for a PAL-inactivating system. Actinomycin D and Puromycin inhibited the blue light induced increase of PAL activity. Irradiation with red resp. farred light alone or given after induction with blue light had no effect on the enzyme synthesis.

     

Lag-phase and rate of synthesis in light-mediated anthocyanin synthesis

Mustard (Sinapis alba L.) seedlings were irradiated with continuous far-red light either with or without a pretreatment with 3 or 6 h of the same far-red light, separated by a 15 h dark period. The pretreatment increases the initial rate of anthocyanin accumulation — as caused by the 2nd light treatment — at least 6-fold but leads to an earlier cessation of anthocyanin accumulation. Moreover, the pretreatment seems to shorten the apparent lag-phase of anthocyanin accumulation considerably but it does not eliminate the lag. If the pretreatment with far-red light is terminated before the seedling reaches competence (with regard to phytochrome and anthocyanin synthesis) the pretreatment has no effect on the apparent lag-phase even though the future capacity of anthocyanin biogenesis is considerably stimulated by the pretreatment. The time course of induction of anthocyanin and that of phenylalanine ammonia-lyase (PAL) (Acton et al. 1980, Fig. 1) is in line with the concept that induction of PAL by light is a prerequisite for the onset of light-mediated anthocyanin synthesis.Abbreviation PAL phenylalanine ammonia-lyase     

二乔玉兰开花过程中花色变化的生理生化机制

以4年生二乔玉兰不同花期外层花瓣为试材,测定其在开花过程中花瓣色度值、花色苷、类黄酮、可溶性糖含量、细胞pH值以及相关酶活性的变化,以探讨二乔玉兰花色呈色机理。结果显示:(1)随着花期的推移,苯丙氨酸解胺酶(PAL)和查尔酮异构酶(CHI)活性逐渐减弱,细胞pH值逐渐变大,可溶性糖、花色苷、类黄酮含量不断降低,而花瓣明亮度增强,红色度以及彩色度减弱,且不同花期各参数值之间差异显著。(2)花瓣可溶性糖含量、PAL和CHI的活性与其花色素苷、类黄酮含量变化之间呈显著正相关关系,花瓣pH值的变化、明亮度L*值与花色素苷、类黄酮含量之间呈显著负相关,色相值a*与花色苷含量的变化呈显著正相关。研究表明,二乔玉兰花瓣花色苷和类黄酮含量的高低可以影响其花色的深浅,可溶性糖含量、PAL和CHI活性、细胞pH通过参与一定的生理代谢来调节花色素的形成,进而引起二乔玉兰花色色调的改变。     

Mechanism of phytochrome action in the control of biosynthesis of anthocyanin in Brassica oleracea

-The synthesis of anthocyanin in red-cabbage is very sensitive to control by light, R/FR reversibility being effected by exposures of 5 min duration, The demonstration of this control does not depend upon a preceding irradiation of high-intensity light but depends upon the duration of incubation in darkness subsequent to irradiation. R/FR reversibility is well shown in seedlings kept in darkness for 48 hr after exposure but after 120 hr this reversibility is no longer evident. This is due to the fact that a further synthesis of anthocyanin occurs in unexposed seedlings and in FR and R/FR treated material in the period from 48 to 120 hr but does not occur in the R treatment after 48 hr. Reagents such as n-propanol which are believed to increase membrane permeability, greatly increase anthocyanin synthesis in dark grown material. n-PrOH also reverses the effect of 5 min FR irradiation but, by contrast with R light, does not promote PAL activity. It is concluded that the limitation to synthesis in material unexposed to light is substrate availability at the site of flavonoid biosynthesis, rather than the level of PAL activity. The evidence presented supports the hypothesis that R/FR reversible phytochrome action involves the control of the passage of substrate through a membrane to the site of anthocyanin biosynthesis.     

Regulation of phenylalanine ammonia-lyase activity and growth in lettuce by light and gibberellic acid

Abstract The effects of light and gibberellic acid (GA3) on growth and phenylalanine ammonia-lyase (PAL) activity were studied in seedlings of lettuce (Lactuca sativa L.). Using an in vivo assay for PAL it was shown that wounding caused by excising hypocotyls results in an increase in PAL activity with time that can mask the effect of light on the activity of this enzyme. When hypocotyl sections were excised from light-treated seedlings immediately prior to the in vivo assay of PAL, light was shown to cause a marked increase in PAL activity. Experiments with an inhibitor of PAL activity, α-aminooxy-β-phenylpropionic acid (AOPP), confirmed that the volatile radioactive products measured in the in vivo assay resulted from the activity of PAL. Gibberellic acid suppresses the light-induced increase in PAL activity and there is an inverse relationship between GA₃-induced growth and the activity of PAL. Over a wide range of GA₃ concentrations, the activity of PAL is also inversely correlated with growth rate along the length of the hypocotyl section; the upper halves of sections elongate more rapidly and have lower levels of PAL than the lower halves. Despite the strong correlation between growth and PAL activity, experiments with AOPP and t-cinnamic acid show that it is unlikely that elongation is regulated directly by products of PAL activity.     

Ultraviolet-B-Responsive Anthocyanin Production in a Rice Cultivar Is Associated with a Specific Phase of Phenylalanine Ammonia Lyase Biosynthesis

Seedlings of 17 rice (Oryza sativa L.) cultivars were classified on the basis of anthocyanin pigmentation into three groups: an acyanic group with 9 cultivars, a moderately cyanic group with 5 cultivars, and a cyanic group with 3 cultivars. Seedlings of the cyanic group were deep purple in color, possessing copious amounts of anthocyanin in shoots. Sunlight (SL)-mediated anthocyanin and phenylalanine ammonia lyase (PAL) induction in a cyanic cultivar, purple puttu, was compared with an acyanic cultivar, black puttu. A brief exposure of dark-grown purple puttu seedlings to SL induced anthocyanin formation during a subsequent dark period with a peak at 24 h. The magnitude of SL-mediated anthocyanin induction is age dependent, the 4-d-old seedlings being the most responsive to SL. The anthocyanin induction in purple puttu seedlings is mediated exclusively by the ultraviolet-B (UV-B) component of SL. The SL-triggered anthocyanin induction was reduced by about 30% by a terminal far-red light pulse and was restored by a red light pulse, indicating the role of phytochrome in modulation of anthocyanin level. The SL-mediated induction of PAL showed two peaks, one at 4 h and the other at 12 h. Whereas the first PAL peak (4 h) was induced by phytochrome and was seen in both cultivars, the second PAL peak (12 h) was inducible by UV-B only in the cyanic purple puttu cultivar.