Sequential control of phytochrome-mediated synthesis de novo of β-amylase in the cotyledons of mustard (Sinapis alba L.) seedlings

In the cotyledons of mustard (Sinapis alba L.) seedlings irradiated from the time of sowing with continuous red light, the photoreversibility of the phytochrome-mediated increase in -amylase activity (EC 3.2.1.2) is lost 36 h after sowing (coupling point). However, the induced increase of -amylase activity cannot be detected before 46 h after sowing (starting point). Density labeling with deuterium oxide shows that the increase of enzyme activity in light and darkness coincides precisely with the synthesis of -amylase protein. Thus, phytochrome mediates an increase of -amylase synthesis de novo. Since there is no turnover detectable by density labeling, it is concluded that -amylase of mustard cotyledons is a physiologically stable enzyme (half-life >5 d). The 10-h time gap between loss of photoreversibility and onset of light-induced -amylase synthesis points to a relatively stable regulatory element within the signal chain (transmitter) which links -amylase synthesis to the primary action of phytochrome. A 12-h lag between the cessation of phytochrome action and the cessation of induced -amylase synthesis indicates a limited lifetime of the transmitter (about 12 h). The effect of this result on the interpretation of the coupling point is discussed.Abbreviations P_r, P_fr red and far-red absorbing forms of phytochrome     

Blue Light Mediated Regulation of {beta}-Amylase Activity in Mustard (Sinapis alba L.) Cotyledons

In the cotyledons of mustard (Sinapis alba L.) seedlings grownunder continuous blue light, ß-amylase activity increasedbetween 42–96 h from sowing and thereafter the ß-amylaseactivity abruptly declined. Preirradiation with blue light didnot increase the responsivity of the subsequent phytochrome-mediatedß-amylase increase in the cotyledons. The run-offkinetics of ß-amylase increase in seedlings transferredfrom blue light to darkness indicated that the components ofthe blue light-triggered signal chain are kinetically identicalto those of the phytochrome-mediated signal chain. Far-red reversibilityexperiments showed that the above blue light response is eithermediated by phytochrome directly or the blue light photoreceptorrequires the coaction of phytochrome. (Received November 11, 1987; Accepted March 23, 1988)     

Time Dependence of Phytochrome-mediated Carotenoid and Chlorophyll Synthesis in Sorghum bicolor L

In 5-day-old etiolated Sorghum seedlings, red light irradiationfor 1 s enhanced carotenoid and chlorophyll accumulation, and5 min of red light treatment saturated the photoresponse. Thedegree of red/far-red photoreversibility of carotenoid accumulationwas dependent on the age of the plant. No significant escapefrom far-red reversibility was observed up to 30 min after theonset of a saturating red light pulse in 5-day-old etiolatedseedlings. Thereafter, the escape was relatively fast and completedwithin 180 min. Sorghum bicolor L, carotenogenesis, phytochrome, time dependence, chlorophyll synthesis     

Phytochrome appearance and distribution in the embryonic axis and seedling of Alaska peas

Summary Apparent synthesis of phytochrome has been observed in the embryonic axis of germinating Alaska pea and in the young seedling. The onset of photoreversibility can be delayed, and the amount of phytochrome appearing in a plant of a given size somewhat decreased, by treatment with cycloheximide in appropriate concentrations. Similar concentrations of cycloheximide also reduce elongation. Soaking seeds in water at 4° for 24 hr also prevents the onset of photoreversibility in the embryonic axis. The pattern of phytochrome appearance, and the distribution of relative amounts of phytochrome, suggest that synthesis may be closely related to general protein synthesis during elongation.This work was supported in part by National Science Foundation Grant GB-2846 and GB-6683, and a grant from Research Corporation to W. R. Briggs. It represents a portion of the dissertation of J. A. McArthur submitted in partial fulfillment of the requirements for the degree Doctor of Philosophy to Stanford University in 1969.     

Change in sensitivity to far-red irradiation on anthocyanin biosynthesis in red cabbage seedlings

Abstract. Anthocyanin biosynthesis in red cabbage seedlings has been shown to be under a red/far-red (R/FR) reversible phytochrome control. The 'escape' of anthocyanin synthesis from FR photoreversibility occurred between 12 and 20 h. However, a similarity exists between the escape curve and the temporal change in sensitivity to FR alone, which suggests that some of the 'escape' may be due to the promotive effect of FR. Some escape times may not therefore be a true measure of the duration of Pfr action, contrary to the traditional view.     

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.     

Phytochrome-mediated regulation of β-amylase mRNA level in mustard (Sinapis alba L.) cotyledons

Phytochrome, activated by continuous red light, increases the amount of total polyadenylated RNA during photomorphogenesis of mustard (Sinapis alba L.) cotyledons. In-vitro translation of total polyadenylated RNA in a reticulocyte translation system has shown that the activity of translatable -amylase mRNA is increased by phytochrome about threefold in the 3-d-old cotyledons, based on equal amounts of polyadenylated RNA, and about eightfold on a per-cotyledon basis. Cordycepin prevents the accumulation of translatable -amylase mRNA. It is concluded that the phytochrome-mediated control of -amylase synthesis is exerted on the level of mRNA synthesis. During seedling development in continuous red light, a phytochrome-dependent increase of -amylase mRNA can be observed at least 6 h before the onset of -amylase synthesis. If, after a period of enzyme synthesis, phytochrome action is interrupted by long-wavelength far-red light followed by darkness, -amylase mRNA as well as -amylase synthesis remain at a high level for 8–10 h and then decline sharply. It is concluded that -amylase mRNA, having an apparent lifetime of the order of 8–10 h, can be formed under the influence of phytochrome during early seedling development but it activates -amylase synthesis only after a lag-phase of about 8 h, when the cotyledons acquire competence to synthesize the enzyme. The consequences of these findings for the signal-transduction chain of phytochrome are discussed.Abbreviations EDTA Na₂-ethylenediaminotetraacetic acid - PAGE polyacrylamide gel electrophoresis - poly(A)⁺RNA polyadenylated mRNA - P_r, P_fr red- and far-red-absorbing forms of phytochrome - SDS sodium dodecyl sulfate - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Temporal Pattern of Gene Expression in Cotyledons of Mustard (Sinapis alba L.) Seedlings

The time course of appearance of competence to phytochrome (P_fr) was studied in cotyledons of mustard (Sinapis alba L.) with regard to the light-mediated accumulation of mRNAs encoding for SSU, CAB and the 23 kDa protein of the oxygen evolving complex of photosystem II (OEC). For each gene family a specific starting point of P_fr-induced mRNA accumulation was observed (SSU: 42 h; CAB: 36 h; OEC: 30 h). An increase of SSU-mRNA levels can be detected 24 h after sowing in dark-grown seedlings whereas for OEC the time points for the increase of mRNA are the same whether the seedlings are kept in darkness or induced by light via P_fr. For all gene families a responsiveness to P_fr (coupling point) could be demonstrated before the starting points. The coupling points are also gene specific (SSU: ca. 12 h; CAB and 23 kDa peptide of OEC: ca. 24 h). The responsiveness to light before the starting point indicates that the light-induced signal must be stored.     

Phytochrome Levels in Light-Grown Avena Change in Response to End-of-Day Irradiations

The effect of 15-minute end-of-day irradiations on photoreversible phytochrome levels in light-grown oat (Avena sativa L., cv Garry) seedlings was investigated. Oat seedlings were grown in a cycle of 8 hours of natural daylight and 16 hours of complete darkness, from sowing until harvest at day 10. The level of extractable, photoreversible phytochrome per unit fresh weight was 60% higher after end-of-day far-red irradiation than after either end-of-day red irradiation or end-of-day far-red followed by end-of-day red. Seedlings irradiated with end-of-day far-red also exhibited a small but significant increase in shoot height and fresh weight per seedling. Extracts of seedlings given each of these end-of-day treatments were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electroblotted, and immunostained with monoclonal antibodies specific to different phytochromes. Regardless of end-of-day light treatment, phytochrome that is abundant in etiolated tissue was below the limit of detection, indicating that one or more of the phytochromes predominating in green tissue changes in abundance.     

Correlation between far-red absorbing phytochrome and response in phytochrome-mediated anthocyanin synthesis

Abstract Mustard seedlings were light treated at 24 h after sowing (25°C) to induce phytochrome-mediated anthocyanin synthesis in cotyledons and hypocotylar hook. All light treatments were performed within the range of the reciprocity law. The in situ photoconversion kinetics of phytochrome (P_r→ P_fr) were measured under the same light treatment. It was found that between 0.4 and 1.0 relative P_fr level the amount of anthocyanin extracted from the organs at 52 h after sowing was linearily correlated with the amount of P_fr produced at 24 h in cotyledons and hypocotylar hook. It is concluded that an explanation of the fluence response function for red light mediated anthocyanin synthesis in the mustard seedling does not require the concept of active vs. bulk phytochrome.     

Phytochrome control mechanisms in leaf expansion of Phaseolus vulgaris cv. Limburg.

Abstract. The effectiveness of a red-light pulse acting through phytochrome in inducing primary leaf expansion in 9-d-old etiolated bean ( Phaseolus vulgaris L. ev. Limburg) seedlings is strongly increased by a continuous far-red light (CFR) pretreatment. This increase in effectiveness of a red pulse is positively correlated with the time and the fluence rate of the CFR pretreatment. Escape from photoreversibility of this red pulse after the CFR pretreatment is extremely slow (more than 3 d). When a dark period is interposed between the end of the CFR pretreatment and the inductive red pulse the photoreversible part of the response to this pulse is highly dependent upon the photostationary state of phytochrome at the onset of the dark period.
The results give strong evidence for the synergistic activity of two components of phytochrome action during leaf growth induction, one of them acting via a very stable Pfr fraction.     

Regulation of enzyme levels by phytochrome in mustard cotyledons: Multiple mechanisms?

Phytochrome controls the appearance of many enzymes in the mustard (Sinapis alba L.) cotyledons. The problem has been whether the effect of phytochrome on the appearance of enzymes in this organ is due to a common initial action of P_fr, e.g. due to the liberation of a second messenger. We have compared the modulation by light (phytochrome) of the appearance of phenylalanine ammonia lyase (PAL)⁺ and ribulosebisphosphate carboxylase (Carboxylase)⁺. PAL becomes detectable in the mustard cotyledons at 27 h after sowing while Carboxylase starts to appear only at 42 h after sowing (starting points, 25° C). The starting points cannot be shifted by light. As a major result, in the case of PAL the inductive effect of continuous red light (given from the time of sowing) remains fully reversible by 756 nm-light up to the starting point (27 h after sowing) while with Carboxylase full reversibility in continuous red light is lost at approximately 15 h after sowing. While the induction of Carboxylase is already saturated at a very low level of P_fr (e.g. continuous 756 nm-light saturates the response) and does not depend on irradiance (e.g. continuous 675 mW m^-2 red light and 67.5 mW m^-2 red light lead to the same time course), PAL induction is a graded response over a wide range of P_fr doses and depends strongly on the fluence rate (high irradiance response, HIR). It is concluded that PAL induction and Carboxylase induction are not only separated in time but differ in every regard except that both responses are mediated by phytochrome.The present data support the previous conclusion that the specification of the temporal and spatial pattern of development is independent of phytochrome even though the realization of the pattern of development can only occur in the presence of phytochrome (P_fr). It seems that there is no feedback from pattern realization to pattern specification.Abbreviations P_fr the far-red absorbing, physiologically active form of phytochrome - P_r the red absorbing physiologically inactive form of phytochrome - P_total [P_r]+[P_fr] - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - Carboxylase ribulosebisphosphate carboxylase (EC 4.1.1.39)     

Temporal separation of two components of phytochrome action

Abstract In germinating seedlings of Sinapis alba nitrate reductase activity as assayed in vivo becomes accessible to phytochrome control between 15 and 17 h after sowing. Phytochrome operates via the high irradiance reaction to control nitrate reductase activity in the period 15 to 20 h after sowing. Both continuous red light and far-red light elicit this response with a strong fluence rate dependency being apparent in each case. The induction of nitrate reductase activity by light pulses at 20 h after sowing is greatly influenced by red light pre-treatments (operating through phytochrome) given between 0 and 15 h after sowing. Low fluence rate pre-treatments reduce the effectiveness of a subsequent pulse to below the level of a dark control whilst high fluence rate pre-treatments greatly increase the effectiveness of a subsequent pulse.     

Control by light of hypocotyl growth in de-etiolated mustard seedlings

After sowing, mustard (Sinapis alba L.) seedlings were grown for 48 h in white light (25°C). These fully de-etiolated, green seedlings were used as experimental material between 48 and 72 (84) h after sowing. The question researched was to what extent control by light of hypocotyl elongation is due to phytochrome in these seedlings. It was found that the light effect on hypocotyl growth is very probably exerted through phytochrome only. In particular, we found no indication for the involvement of a specific blue light photoreceptor pigment.Abbreviations HIR high irradiance reaction - P_fr far-red absorbing, physiologically active form of phytochrome - P_r red absorbing, physiologically inactive form of phytochrome - Pot total phytochrome, i.e. [P_r]+[P_fr] - [P_fr]/[P_tot] - red red light - fr far-red light - wl white light - bl blue light - di dichromatic irradiation - l hypocotyl length     

Induction of amylase in mustard seedlings by phytochrome

Summary In the cotyledons of mustard seedlings (Sinapis alba L.) amylase activity can be induced by phytochrome. In the dark amylase activity remains low. Gibberellic acid (GA₃) does not stimulate an increase of amylase activity in this system. Inhibitors of RNA and protein synthesis strongly inhibit the increase of amylase activity mediated by phytochrome. In gel electrophoresis amylase from mustard seedlings reveals 3 bands. The electrophoretic pattern is the same for extracts from dark-grown and from irradiated seedlings. When mustard amylases were incubated with starch the pattern of products was similar to that produced by commercially available barley -amylase and not similar to that produced by Bacillus subtilis -amylase.     

Differential expression of two β-amylase genes of rye during seed development

The expression of two β-amylase loci was analysed in the developing seeds of two inbred lines of rye (Secale cereale L.), one of which was a β-amylase deficient mutant. Enzymatic activity and the contents of enzymatic protein and mRNA specific for each of an endosperm-characteristic and ubiquitous β-amylase were determined throughout the course of caryopsis development. Both loci were expressed in the developing normal line caryopses according to different temporal and quantitative patterns. The ubiquitous enzyme-specific locus β-Amy 2 was expressed earlier; both mRNA and enzymatic protein accumulated to a maximum extent at 10 to 15 days after pollination. In contrast, the highest content of mRNA for endosperm β-amylase (encoded by the β-Amy I locus) was found 20 days after pollination, and the corresponding enzymatic protein accumulated throughout seed development. The expression of the β-Amy I locus was 30- to 40-fold higher than that of the β-Amy 2 locus in terms of maximum specific mRNA accumulation. The expression product of only the β-Amy 2 locus was found in the developing mutant line caryopses. The expression pattern of this locus was similar in the developing normal and mutant line seeds in terms of the temporal accumulation of mRNA and enzymatic protein. However, an approximately 4-fold higher level of ubiquitous β-amylase-specific mRNA was found in the mutant than in the normal line caryopses, and the content of ubiquitous β-amylase protein decreased to near zero at seed maturity in the mutant line, but not in the normal line, caryopses. The enzymatic activities of both β-amylases appeared to be regulated at the level of accumulated enzymatic protein.     

Phytochrome action on chlorophyll synthesis-a study of the escape from photoreversibility

A brief red light pretreatment (pulse), operating through phytochrome, stimulates the synthesis of chlorophyll a and b in Sorghum vulgare shoots that are placed in continuous saturating white light. The red light effect is fully reversible by a far-red (756 nanometers) light pulse for 45 minutes. Thereafter, escape from reversibility is fast, being completed within 2 hours. It is shown here that physiologically active phytochrome (Pfr) is required continuously during these first 45 minutes if the onset of the loss of photoreversibility is to begin 45 minutes after the red light treatment. Thus, the initial action of Pfr consists of two distinct processes: the first process is to overcome the lag prior to escape from photoreversibility; the second process is the actual stimulation of chlorophyll synthesis by Pfr. The duration of the lag prior to escape from photoreversibility depends on the level of Pfr established by the light pulse. The duration increases with increasing Pfr levels from nondetectable to 45 minutes. Above approximately 15% Pfr (Pfr/P_lot ≈ 0.15), the duration of the lag prior to escape from photoreversibility remains constant at 45 minutes.     

Induction versus modulation in phytochrome-regulated biochemical processes

The time course of appearance of competence towards phytochrome (P_fr) was studied in cotyledons of mustard (Sinapis alba L.) with regard to the light-mediated formation of anthocyanin (aglycone cyanidin) and NADP-dependent plastidal glyceraldehyde-3-phosphate dehydrogenase (GPD, EC 1.2.1.13). The experiments were performed to answer the following question: Does phytochrome act to turn responses on (induction), or — as an alternative — does phytochrome cause an amplification of processes already occurring in absolute darkness albeit at low rates once competence is reached (modulation)? The data show that in the case of GPD, phytochrome causes an amplification of the rate of synthesis once the competence point is reached at approximately 36 h after sowing at 25° C. In the case of anthocyanin, it was found that two distinct points of competence exist (26 h and 39 h after sowing, 25° C). In the case of ‘early anthocyanin’ (competence point at 26 h), synthesis does not occur in darkness without P_fr, while in the case of ‘late anthocyanin’ (competence point at 39 h), phytochrome causes an amplification of a process occurring in complete darkness albeit at a very low rate. It is concluded that in phytochrome-mediated photomorphogenesis, modulation as well as induction of biosynthetic processes plays a role.     

Action of light,nitrate and ammonium on the levels of NADH- and ferredoxin-dependent glutamate synthases in the cotyledons of mustard seedlings

In mustard (Sinapis alba L.) cotyledons, NADH-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14) is only detectable during early seedling development with a peak of enzyme activity occurring between 2 and 2.5 d after sowing. With the beginning of plastidogenesis at approximately 2 d after sowing, ferredoxindependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) appears while NADH-GOGAT drops to a very low level. The enzymes were separated by anion exchange chromatography. Both enzymes are stimulated by light operating through phytochrome. However, the extent of induction is much higher in the case of Fd-GOGAT than in the case of NADH-GOGAT. Moreover, NADH-GOGAT is inducible predominantly by red light pulses, while the light induction of Fd-GOGAT operates predominantly via the high irradiance response of phytochrome. The NADH-GOGAT level is strongly increased if mustard seedlings are grown in the presence of nitrate (15 mM KNO₃,15 mM NH₄NO₃) while the Fd-GOGAT level is only slightly affected by these treatments. No effect on NADH-GOGAT level was observed by growing the seedlings in the presence of ammonium (15 mM NH₄Cl) instead of water, whereas the level of Fd-GOGAT was considerably reduced when seedlings were grown in the presence of NH₄Cl. Inducibility of NADH-GOGAT by treatment with red light pulses or by transferring water-grown seedlings to NO ₃ ^- -containing medium follows a temporal pattern of competence. The very low Fd-GOGAT level in mustard seedlings grown under red light in the presence of the herbicide Norflurazon, which leads to photooxidative destruction of the plastids, indicates that the enzyme is located in the plastids. The NADH-GOGAT level is, in contrast, completely independent of plastid integrity which indicates that its location is cytosolic. It is concluded that NADH-GOGAT in the early seedling development is mainly concerned with metabolizing stored glutamine whereas Fd-GOGAT is involved in ammonium assimilation.Abbreviations and symbols c continuous - D darkness - Fd-GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - FR far-red light (3.5 W·m^-2) - NADH-GOGAT NADH-dependent glutamate synthase (EC 1.4.1.14) - Pfr far-red absorbing form of phytochrome - Ptot total phytochrome - R red light (6.8 W· m^-2) - RG9-light long wavelength FR (10 W·m^-2, _RG9<0.01) - () Pfr/Ptot=wavelength-dependent photoequilibrium of the phytochrome system