Time course of signal transduction in phytochrome-mediated anthocyanin synthesis in mustard cotyledons

Abstract. The effectiveness of phytochrome (Pfr) in mediating anthocyanin synthesis in epidermal cells of mustard ( Sinupis alba L.) cotyledons is strongly increased by a light pretreatment of the seedling prior to competence (Mohr et al. , 1979; Johnson, 1980). Here we test the hypothesis that the observed 'sensitivity amplification' is related to a change in the time course of signal transduction from phytochrome to the responsive cell function. It was found that the time course of escape from photoreversibility—considered to reflect the time course of signal transduction—is not changed by a light pretreatment, although the rate of signal transduction increases with age and depends strongly on the level of Pfr.     

Behaviour of phenylalanine ammonia-lyase in carrot cells in suspension cultures

The high PAL activity in carrot cells in suspension culture was found at the linear and early stationary phases, with concomitant increases in phenylal     

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     

Time-dependent changes in the responsiveness to light of phytochrome-mediated anthocyanin synthesis

Abstract. It is well established that seedlings of mustard ( Sinapis alba L.) synthesize juvenile anthocyanin only if treated with light pulses or continuous light. The light effects are considered to be due to the operation of phytochrome. Here we show that the responsiveness of anthocyanin synthesis to a saturating red light pulse or to continuous far-red light varies as a function of time and is strongly influenced by a light pretreatment prior to competence. Competence appears approximately 25 h after sowing. The starting point of anthocyanin synthesis, which is 27 h after sowing, and the lag- phase of this response, which is 2 h, are not affected by light pretreatments prior to competence. It is concluded that quantitative interpretations of phytochrome responses based entirely on properties of phytochrome can no longer be considered adequate.     

The effect of light pretreatments on phytochrome-mediated induction of anthocyanin and of phenylalanine ammonia-lyase

Induction by light of phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and of anthocyanin in cotyledons of the mustard (Sinapis alba L.) seedling is strongly affected by a light pretreatment which operates through phytochrome. If PAL or anthocyanin is induced by a light pulse, the effectiveness of phytochrome (P_fr) is strongly increased by a light pretreatment; however, if the increase of the PAL level or synthesis of anthocyanin is elicited by continuous far-red light (operating via phytochrome in the High Irradiance Response), effectiveness of light is strongly reduced by the same light pretreatment. This reduction of effectiveness is correlated with a decrease of total phytochrome (P_tot) caused by the light pretreatment. It is argued that the observations are compatible only with the open phytochrome-receptor model as suggested by Schäfer (J. Mathem. Biol. 2, 41–56, 1975). The peaks of the time courses of the PAL levels under continous far-red light are located at 48 h after sowing and do not depend on the original level of phytochrome. The decrease of the PAL levels beyond 48 h after sowing takes place independently of phytochrome and of the actual level of PAL.Abbreviations P_r red absorbing form of phytochrome - P_fr far-red absorbing form of phytochrome - P_tot total phytochrome (P_r+P_fr) - {ie369-1} [P_fr] /[P_tot], photoequilibrium of phytochrome at wavelength - HIR High Irradiance Response - PAL phenylalanine ammonialyase (EC 4.3.1.5)     

Photosensitivity of seedlings differing in their potential to synthesize anthocyanin

In the present report the suggestion (Paech, K. 1950. Biochemie und Physiologie der sekundären Pflanzenstoffe. - Springer, Berlin, pp. 201–203) was tested that the photosynthetic apparatus requires light protection during the early phase of its development and that this is the reason (in a teleonomic sense) for the transient formation of large amounts of juvenile anthocyanin in outer tissue layers of seedlings and young leaves of deciduous trees and shrubs. Seedlings of two species ( Sinapis alba L. and Sesamum indicum L.) which differ in their potential to produce anthocyanin were compared under identical light conditions. The results obtained are compatible with the idea that juvenile anthocyanins are involved in photoprotection. However, the experimental results also indicate that full photostability of the plastid is attained - irrespective of the presence or absence of anthocyanin - once a certain amount of chlorophyll has been accumulated. Thus, photosensitivity of a seedling under natural light conditions is restricted to an early phase of development prior to intense greening.     

Evidence that a mustard seedling responds to the amount of Pfr and not to the Pfr/Ptotratio

Abstract Phytochrome-mediated anthocyanin synthesis of the mustard seedling (Sinapis alba L.) was investigated. Light pre-treated and dark-grown seedlings differing in responsiveness and level of phytochrome (P_tot) were compared. The data obtained support the traditional view that a seedling measures the amount of P_fr. The alternative view that a plant measures the P_fr/P_tot ratio does not seem to be compatible with the data obtained with the mustard seedling.     

Putative cis-elements in the promoter region of the carrot phenylalanine ammonia-lyase gene induced during anthocyanin synthesis

Deletion mutants of the carrot phenylalanine ammonia-lyase gene promoter were used to survey cis-elements for their effect on expression of promoter activity by transient expression. Two putative cis-elements were required to give full activity, but a third might be the most important in regulation of the promoter by 2,4-dichlorophenoxyacetic acid. Electronic Publication     

The effect of growth retardants on anthocyanin production in carrot cell suspension cultures

The effect of growth retardants on anthocyanin production was studied in wild carrot (Daucus carota) cell suspension cultures. Paclobutrazol [(2RS,3RS) — 1 — (4-chlorophenyl) — 4,4 —dimethyl-2-(1,2,4-triazol-1-yl) pentan-3-ol], uniconazole [(E)-1-(4-chlorophenyl-4,4 —) dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol], tetcyclacis [5-(4-chloro-phenyl) -3,4,5,9,10-pentaaza-tetracyclo-5, 4, 10^2,6, O^8,11 — dodeca-3, 9-diene], ancymidol [-cyclopropyl — 4 — methoxy-(pyrimidine-5-yl)benzyl alcohol] and CCC (2-chloro-ethyltrimethylammonium chloride) increased anthocyanin accumulation. AMO-1618 [(2-isopropyl-5-methyl-4-trimethyl-ammonium-chloride)-phenyl-1-piperidinium carboxylate] did not increase anthocyanin accumulation in the first passage but did increase it during the second passage on medium for improved anthocyanin accumulation. Prohexadione (3,5-dioxo-4-propionylcyclohexane carboxylic acid) decreased anthocyanin accumulation by 10%–12.5%.The inhibitory effect of gibberellin on anthocyanin accumulation was reversed by paclobutrazol. Paclobutrazol together with 10^–6M GA₃ increased anthocyanin level from 33% of control in GA₃ treated cell suspension to 76%. These results are consistent growth retardants increasing anthocyanin accumulation in carrot cell suspension cultures by inhibiting gibberellin biosynthesis.     

The 'end-of-day' phytochrome control of internode elongation in mustard: kinetics, interaction with the previous fluence rate, and ecological implications

Abstract The ‘end-of-day’ phytochrome control of internode growth was characterized in Sinapis alba, seedlings previously grown under continuous white light for 13 d. The transition from white light to darkness caused a reduction in internode extension rate with a lag of less than 10 min. Following this, extension rate remained almost constant for at least 48 h. i.e. ‘re-etiolation’ was not noticed. The phytochorme controlling the growth processes was stable in the Pfr form. The growth rate of plants receiving a red light pulse, and the growth promotion caused by a far-red light pulse, increased with increasing fluence rate of the previous white light treatment. In far-red treated plants a first growth rate acceleration peaked at 20–30 min after the end of white light, followed by a transient deceleration which led to a growth rate minimum at 40–60 min, followed by a final growth rate recovery yielding a more-or-less steady elevated rate. Pulses establishing different Pfr/P modified the extent, but not the early kinetics, of the growth response. The relative promotion of growth caused by low Pfr/P was limited by darkness as follows: (a), The growth promotion caused by far-red directed to the internode alone was transient. (b), The promotion caused by a reduction of Pfr/P in the whole shoot persisted in darkness for at least 48 h and also persisted if, after a 3–9 h dark period, the plants were returned to continuous white light. In darkness, however, the magnitude of this growth rate promotion decreased with time, particularly when the previous white light fluence rate was low, or the pulse preceding darkness provided the lowest Pfr/P. (c), When compared over the same period in darkness, growth rate was higher in those seedlings in which Pfr/P was reduced during the continuous white light pretreatment than in those ones in which the Pfr/P was only reduced immediately before darkness. It is proposed that in the natural environment, red/far-red signals could be more effective when provided during daytime than at the end of the photoperiod, as both the background growth rate and the relative promotion caused by low Pfr/P are reduced by darkness.     

Elicitor-induced changes of phenylalanine ammonia-lyase activity in barley cell suspension cultures

Suspension-cultured barley cells responded to treatments with crude yeast extract and purified glucan preparation by rapidly and transiently (4 h postelicitation) inducing L-phenylalanine ammonia-lyase activity. Similarly, treatment of cell cultures with chitosan resulted in increased phenylalanine ammonia-lyase activity 2–4 h after elicitation, whereas a mycelium preparation of a fungal pathogen, Bipolaris sorokiniana, and purified chitin caused a more delayed induction of phenylalanine ammonia-lyase (8 h postelicitation). The most abundant of the plant cell wall degrading enzymes produced by Bipolaris sorokiniana, β-1,4-xylanase, had only a weak elicitor activity in barley cells suggesting that fungal cell wall components rather than the hydrolytic enzymes secreted by the fungus function as recognizable components that cause barley cells to induce defences. Treatment of the elicited cells with a phenylalanine ammonia-lyase inhibitor, α-aminooxy-β-phenylpropionic acid, resulted in the superinduction of the enzyme indicating the blocking of the feedback regulation mechanisms, whereas in the presence of 1 mM trans-cinnamic acid the elicitor-induction of phenylalanine ammonia-lyase was completely inhibited. Elicitor treatments increased the accumulation of wall-bound phenolics as evidenced by phloroglucinol-HCl staining and thioglycolic acid methods. However, α-aminooxy-β-phenylpropionic acid applied in combination with the elicitor did not prevent the accumulation of phenolics in barley cell walls. This suggested that phenylalanine ammonia-lyase might not play an important role in the synthesis wall-bound phenolic compounds in barley. However, cinnamic acid, whether applied alone or together with the elicitor, increased the amount of wall-bound phenolics in suspension-cultured barley cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

Phenolic synthesis and phenylalanine ammonia-lyase activity in suspension cultures of Acer pseudoplatanus L.

Summary Phenolic metabolism is influenced by the levels of sucrose, nitrogen and 2,4 dichlorophenoxyacetic acid (2,4-D) in the growth medium. Chromatographic evidence suggests that the principle products are polymers of leucocyanin, (-) epicatechin and (+) catechin, constituting condensed tannins. Comparison of ethanolic cell extracts with extracts from plant organs shows that although these compounds are present in parts of the plant they are not the major phenolics.Cells maintained in a modified Heller's medium containing 9.0×10^–7 M 2,4-D produce increased levels of tannins from mid passage (day 12) onwards. The presence of 2,4-D at 9.0×10^–6 M supresses this response and increased initial sucrose levels cause the amount of tannins to be greater. At the period when tannin levels increase the standard medium is exhaused of its nitrogen sources, urea and nitrate. Increased initial nitrogen levels delay the beginning of increased tannin production and the addition of urea or 2,4-D to cultures already containing high levels of tannins causes the tannin content per gram fresh weight and per culture to decline. These results indicate an antagonism between tannin synthesis and nitrogen metabolism. The activity of phenylalanine ammonia-lyase EC 4.1.1.5. (PAL) estimated by a spectrophotometric method in acetone powders derived from Acer cells increases three to four fold at the onset of increased tannin synthesis and then declines sharply. The phase of high PAL activity correlates with the exhausion of the medium nitrogen sources.Abbreviation 2,4-D 2,4-dichlorophenoxyacetic acid One of the authors (R.J.W.) was supported by a Science Research Council Studentship during the course of this work.     

Ammonium toxicity: description of the syndrome in Sinapis alba and the search for its causation

In many plant species, prolonged application of ammonium (NH₄⁺) as a source of nitrogen results in physiological and morphological disorders (‘ammonium toxicity’). In the mustard (Sinapis alba L.) seedling we have previously observed particularly severe symptoms of ammonium toxicity in the absence of external nitrate (NO₃^-) or with increasing NH₄⁺/NO₃^- ratios. In the present investigation we have studied the symptoms of this ‘toxicity’in more depth, i.e. at the morphological, plastidic, enzyme and mRNA levels, in an effort to elucidate the causation of the syndrome. It could be confirmed that the syndrome is specific for ammonium and is not caused by a surplus of nitrogen. The syndrome is caused neither by pH changes in the medium nor by non-specific osmotic effects. Furthermore, the syndrome is not causally related to the fact that nitrate reductase (NR; EC 1.6.6.1.) is induced by ammonium. Development of the syndrome requires neither photosynthesis nor intact plastids. Nevertheless, the plastids are severely affected by ammonium application as is anthocyanin synthesis. Enzymes are differently affected. Among the plastidic enzymes, levels of ribulose-1,5-bisphosphate carboxylase (RuBPCase; EC 4.1.1.39) and NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (NADP-GPD; EC 1.2.1.13) are strongly reduced and abundance of translatable mRNA of the small subunit of RuBPCase is decreased, whereas nitrite reductase (NIR; EC 1.7.7.1) is not affected. Among extraplastidic enzymes, the level of chalcone synthase (CHS; EC 2.3.1.74) is strongly reduced, the NAD-dependent glyceraldehyde-3-phosphate dehydrogenase (NADGPD; EC 1.2.1.12) level is unaffected, whereas the isocitrate lyase (ICL; EC 4.1.3.1) level is strongly promoted. The fat → carbohydrate transformation seems to be impaired by ammonium: fat degradation is reduced, starch accumulation is strongly inhibited and the levels of glucose and fructose are decreased. It appears from the present data and from results obtained in a companion study (U. Hecht and H. Mohr, in preparation) that the ammonium toxicity syndrome is detectable as soon as ammonium accumulation occurs in the plant. However, the actual mechanism through which the excess ammonium affects metabolism remains unclear at present.     

Light-induced changes of enzyme activities in parsley cell suspension cultures: Increased rate of synthesis of phenylalanine ammonia-lyase

When dark-grown cell suspension cultures of parsley (Petroselinum hortense) were illuminated for increasing periods of time, increasing amounts of phenylalanine ammonialyase activity were obtained 5 hr after the onset of light.Pulses of [³⁵S]methionine of varying duration from 1 to 150 min were given to cell cultures in the dark period subsequent to a light period of 2.5 hr. The cells were harvested 5 hr after the onset of light. Analysis of the soluble proteins by polyacrylamide gel electrophoresis revealed a distinct peak of radioactivity coinciding with the activity of phenylalanine ammonia-lyase. The results of experiments in which radioactive methionine was administered for 10 min to dark-grown or light-induced cells at different times after the light period were compared. An efficient incorporation of radioactivity into the fractions possessing the enzyme activity was observed 5 hr after induction, while no significant labeling was detected either after 1.5 or 25 hr, or in extracts from nonilluminated cells. The radioactive fractions containing the enzyme activity were further analyzed by sodium dodecyl sulfate-disc gel electrophoresis. Significant amounts of radioactivity at the molecular weight of the subunits of phenylalanine ammonia-lyase (84,000) were found only in the extracts from cells which had been labeled 5 hr after induction. These results suggest that the light-induced increase in phenylalanine ammonia-lyase activity is due to de novo synthesis, but not to an activation of preformed, inactive enzyme.     

Nutrient utilization during biomass and anthocyanin accumulation in suspension cultures of wild carrot cells

The medium used for the growth of anthocyanin-accumulating wild carrot (D. carota) suspension cultures contained ammonia as a sole nitrogen source and was buffered with succinate. Ammonia was the first nutrient to be completely utilized.The uptake of carbohydrate, phosphate and succinate continued after ammonia depletion. Biomass accumulation was faster and greater when sucrose was initially present in the medium than when glucose was present. When sucrose was provided in the medium it was rapidly hydrolysed to glucose and fructose and the fructose was used preferentially to glucose. Anthocyanin accumulation was rapid after ammonia fell below 3 mM and until the pH of the medium rose from 4.5 to 5.1 or 5.2.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Regulation of anthocyanin synthesis in suspension cultures of strawberry cell by pH

Within the initial medium pH range of 3.7-8.7, anthocyanin synthesis in strawberry cell suspension cultures was influenced significantly and gave an irregular trend. No evident influence was observed for cell growth due to the strong pH buffering capacity of strawberry cells. The maximum content (0.81 mg/g FCW) and production (143 mg/l) of antho-cyanin were achieved at pH 8.7 on day 3 and day 9, respectively. Measurement of the ratio of pigmented cells suggested that the variation in pH changed the accumulation of anthocyanin inside the pigmented cells only.     

Different effects of supplied ammonium on glutamine synthetase activity in mustard (Sinapis alba) and pine (Pinus sylvestris) seedlings

The activity of glutamine synthetase (GS) in mustard ( Sinapis alba L.) and Scots pine ( Pinus sylvestris L.) seedlings was used as an index to evaluate the capacity to cope with excessive ammonium supply. In these 2 species GS activity was differently affected by the application of nitrogen compounds (NH₄⁺ or NO₃⁻). Mustard seedlings older than 5 days showed a considerable increase in GS activity after NH₄⁺ or NO₃⁻ application. This response was independent of the energy flux, but GS activity in general was positively affected by light. Endogenous NH₄⁺ did not accumulate greatly after nitrogen supply. In contrast, seedlings of Scots pine accumulated NH₄⁺ in cotyledons and roots and showed no stimulation of GS activity after the application of ammonium. In addition, root growth was drastically reduced. Thus, the pine seedlings seem to have insufficient capacity to assimilate exogenously supplied ammonium. NO₃⁻, however, did not lead to any harmful effects.