Action and interaction of red and far-red radiation on lipoxidase metabolism of squash seedlings

Lipoxidase, in the cotyledons of squash (Cucurbita moscata) seedlings grown in the dark, reached its peak activity on the fifth day and then declined to its lowest activity on the eighth day. Under continuous irradiation, the rate of enzyme disappearance was accelerated by red (655 mμ) and was retarded by far-red (735 mμ) radiation. Acceleration of enzyme disappearance caused by red light was reversed repeatedly by far-red light in seedlings that received an initial exposure to red radiation. These responses were independent of the duration of irradiation at each of the alternating wavebands. No change was observed when the white light was administered either 24 hours before or 24 hours after the red, far-red treatment.

The lipoxidase system of the seedlings given an initial exposure to far-red radiation also responded reversibly to alternating far-red, red extended exposures, but it failed to respond reversibly when short exposures were employed. Similarly, no change occurred in these seedlings when either pre- or post-treatment with the white light was applied.

These results demonstrate that the capacity of lipoxidase to act reversibly depends primarily on the duration of exposure and on the kind of light (red or far-red) to which the seedlings were exposed initially. In spite of these variations, lipoxidase metabolism can be considered an additional biochemical manifestation of red, far-red reaction that operates in the photomorphogenesis of plants.

     

Effects of Light Intensity on Photosynthetic Carboxylative Phase Enzymes and Chlorophyll Synthesis in Greening Leaves of Hordeum vulgare L

The effects of various light intensities on in vivo increases in activities of phosphoriboisomerase, phosphoribulokinase and ribulose-1, 5-diP carboxylase and on synthesis of chlorophyll were studied in greening leaves of Hordeum vulgare L.

Each enzyme was already present in dark-grown plants, but further increases in activities required both a light treatment of the intact plant and a favorable temperature. The amount of enzymatic activity and chlorophyll developed was governed by light intensity.

Measured activities of phosphoriboisomerase and ribulose 1,5-diP carboxylase were highly correlated with synthesis of chlorophyll at all intensities studied. Measured activity of phosphoribulokinase was correlated with synthesis of chlorophyll only at saturating or near saturating light intensities. At decreasing light intensities the response curves of this enzyme differed from those of chlorophyll and of phosphoriboisomerase and ribulose-1, 5-diP carboxylase. A lag period of phosphoribulokinase increased with decreasing light intensity. After the lag period a rapid rate of increase occurred which did not level off during 48 hours of illumination. Thus, a different control mechanism may be operative in inducing increased activity of this enzyme.

     

Control of chlorophyll production in rapidly greening bean leaves

The possible involvement of nucleic acid and protein synthesis in light-regulated chlorophyll formation by rapidly greening leaves has been studied.

Removing leaves from illumination during the phase of rapid greening results in a reduction in the rate of pigment synthesis; cessation occurs within 2 to 4 hours. Etiolated leaves which exhibit a lag in pigment synthesis when first placed in the light do not show another lag after a 4 hour interruption of illumination during the phase of rapid greening.

Actinomycin D, chloramphenicol, and puromycin inhibit chlorophyll synthesis when applied before or during the phase of rapid greening. Application of δ-amino-levulinic acid partially relieves the inhibition by chloramphenicol.

It is suggested that light regulates chlorophyll synthesis by controlling the availability of δ-aminolevulinic acid, possibly by mediating the formation of an enzyme of δ-aminolevulinate synthesis. This process may result from gene activation or derepression; the involvement of RNA synthesis of some sort is suggested by the inhibitory effect of actinomycin D on chlorophyll production by rapidly greening leaves.

     

Development of Enzymes Involved in Photosynthetic Carbon Assimilation in Greening Seedlings of Maize (Zea mays)

Upon illumination of dark-grown maize seedlings (5 days old) with incandescent light, there occurred a nearly simultaneous increase, after a certain lag period, in the activities of enzymes engaged in the C₄ pathway and the Calvin-Benson cycle. The light-induced biosynthesis of chlorophyll (a and b) precedes the increase in enzyme activities and proceeds without lag phase. A diphasic feature in the elevation of enzyme activities as a function of the intensities of light provided was observed; the increase in enzyme activities was enhanced by light intensities greater than 10³ ergs per square centimeter per second in comparison with light of lower intensities. Under light intensities greater than 10³ ergs per square centimeter per second, the simultaneous addition of levulinic acid, which inhibited chlorophyll formation, markedly reduced the increase of enzyme activities. However, neither the diphasic light effect nor the inhibitory effect of levulinic acid was observed with ribulose-1,5-bisphosphate carboxylase. The enzyme activities in the dark-grown maize seedlings were enhanced by a brief irradiation with the red light and the red light effect was reversed by the following far red light treatment. The red light-induced increase in the enzyme activities did not accompany chlorophyll synthesis, and was completely inhibited by cycloheximide, indicating that enzyme synthesis rather than activation might be involved. Light may play a dual role in enzyme induction; one is as an energy source through the photosystems at high intensities and the other is presumably as a signal mediated by phytochrome at low intensities.     

Alternative Routes for the Synthesis of 5-Aminolevulinic Acid in Maize Leaves : II. Formation from Glutamate

Intact plastids from greening maize (Zea mays L.) leaves converted [¹⁴C]glutamate and [¹⁴C]2-ketoglutarate (KG) to [¹⁴C]5-aminolevulinic acid (ALA). Glutamate appeared to be the immediate precursor of ALA, while KG was first converted to glutamate, as shown by the effect of various inhibitors of amino acid metabolism. Plastids from greening leaves contained markedly higher activity as compared with etioplasts or chloroplasts. The synthesis of ALA by intact plastids was light dependent. The enzyme system resides in the stroma of plastids or may be lightly bound to membranes. The solubilized system showed maximal activity around pH 7.9 and required Mg²⁺, ATP, and NADPH although dependence on the latter was not clear-cut. A relatively high level of activity could be extracted from etioplasts. Maximal activity was obtained from plastids of leaves which had been illuminated for 90 minutes, after which activity declined sharply. The enzyme system solubilized from plastids also catalyzed the conversion of putative glutamate 1-semialdehyde to ALA in a reaction which was not dependent on the addition of an amino donor.

The system in maize greatly resembled the one which had been reported from barley. It is suggested that this system is the one responsible for the biosynthesis of ALA destined for chlorophyll formation.

     

delta-Aminolevulinic Acid Synthase of Euglena gracilis: Regulation of Activity

δ-Aminolevulinic acid (ALA), a key precursor of the tetrapyrroles heme and chlorophyll, is capable of being synthesized by two different routes in cells of the unicellular green alga Euglena gracilis: from the intact carbon skeleton of glutamate, and via the condensation of glycine and succinyl CoA, mediated by the enzyme ALA synthase. The regulatory properties of ALA synthase were examined in order to establish its role in Euglena.

Partially purified Euglena ALA synthase, unlike the case with the bacterial or animal-derived enzyme, does not exhibit allosteric inhibition by the tetrapyrrole pathway products heme, protoporphyrin IX, and porphobilinogen, at concentrations up to 100 micromolar.

In aplastidic mutant cells, extractable ALA synthase activity is constant during exponential growth, and decreases to low levels as the cells reach the stationary state. Rapid exponential decline of ALA synthase (t_1/2 = 55 min) occurs after administration of 43 micromolar cycloheximide, but not 6.2 millimolar chloramphenicol. These results suggest that, as in other eukaryotic cells, ALA synthase is synthesized on cytoplasmic ribosomes and is subject to rapid turnover in vivo.

Extractable ALA synthase activity increases 2.5-fold within 6 hours after administration of 100 millimolar ethanol, a stimulator of mitochondrial development, and 4.5-fold within 12 hours after administration of 1 millimolar 4,6-dioxoheptanoic acid, which blocks ALA utilization, suggesting that activity is controlled in vivo by a feedback induction-repression mechanism, coupled with rapid enzyme turnover.

In heterotrophically grown wild-type cells, low levels of ALA synthase rapidly increase 4.5-fold within 12 hours after cells are transferred from the light to the dark, and decrease exponentially (t_1/2 = 75 min) when cells are transferred from the dark to light. The dark levels are equal to those in light- or dark-grown aplastidic mutant cells. The low level occurring in light-grown wild-type cells is not altered by the presence of 10 micromolar 3-(3,4-dichlorophenyl)-1,1-dimethylurea, which blocks photosynthetic O₂ production. The decrease that occurs on dark-to-light transfer can be diminished by 12- or 24-hour prior incubation with 6.2 millimolar chloramphenicol, which also retards chlorophyll synthesis after the transfer to light.

The positive relationship of ALA synthase activity to degree of mitochondrial expression, and the inverse relationship to plastid development and chlorophyll synthesis, suggests that ALA synthase functions to provide precursors to nonplastid tetrapyrroles in Euglena. In light-grown, wild-type cells, the diminished levels of ALA synthase may be due to the ability of developing plastids to export heme or a heme precursor to other cellular regions, which thereby supplants the necessity for ALA formation via the ALA synthase route.

     

Endogenous Rhythms in Photosynthesis, Sucrose Phosphate Synthase Activity, and Stomatal Resistance in Leaves of Soybean (Glycine max [L.] Merr.)

Experiments were conducted with soybean (Glycine max [L.] Merr. cv `Ransom') plants to determine if diurnal rhythms in net carbon dioxide exchange rate (CER), stomatal resistance, and sucrose-phosphate synthase (SPS) activity persisted in constant environmental conditions (constant light, LL; constant dark DD) and to assess the importance of these rhythms to the production of nonstructural carbohydrates (starch, sucrose, and hexose). Rhythms in CER, stomatal resistance, and SPS activity were observed in constant environmental conditions but the rhythms differed in period length, amplitude, and phase. The results indicated that these photosynthetic parameters are not controlled in a coordinated manner. The activity of UDPG pyrophosphorylase, another enzyme involved in sucrose formation, did not fluctuate rhythmically in constant conditions but increased with time in plants in LL. In LL, the rhythm in CER was correlated positively with fluctuations in total chlorophyll (r = 0.810) and chlorophyll a (r = 0.791) concentrations which suggested that changes in pigment concentration were associated with, but not necessarily the underlying mechanism of, the rhythm in photosynthetic rate. Assimilate export rate, net starch accumulation rate, and leaf sucrose concentration also fluctuated in constant light. No single photosynthetic parameter was closely correlated with fluctuations in assimilate export during LL; thus, assimilate export may have been controlled by interactions among the endogenous rhythms in CER, SPS activity, or other metabolic factors which were not measured in the present study.     

Growth, Pigment Synthesis, and Ultrastructural Responses of Phaseolus vulgaris L. cv. Blue Lake to Intermittent and Flashing Light

Growing bean plants (Phaseolus vulgaris L. cv. Blue Lake) on cycles of 1 minute light-1 minute dark or 5 minutes light-5 minutes dark, providing an integrated 12 hours light-12 hours dark per day for each set of plants, led to production after 21 days of new leaves low or lacking in chloroplast pigments. Subsequently, dry weight increase was sharply cut. Leaf area was affected by the light regimes after the second week of growth. By the fourth week, plants on the 1 minute light-1 minute dark cycle showed about one-half the leaf area of the controls. Shoot growth was favored over root growth to the greatest degree on the 1 minute light-1 minute dark regimes. Chlorophyll a/b ratios were close to 3.0 in all of the intermittent light regimes, but the total amounts of chlorophyll in milligrams per primary leaf were higher from day 9 to day 23 for the 12 hour light-12 hours dark controls than for other plants.

Although they produced chlorophyll, the plants receiving 1 or 2 milliseconds per second of light continued to lose weight at the same rate as the dark controls; thus, it is assumed there was no net photosynthesis. Plants receiving flashing light allocated significantly more food reserves from the seed to roots than did dark controls. Total chlorophyll formation was significantly accelerated by 2 milliseconds per second light. With 1 millisecond per second light, it took 5 days longer to achieve the same level of chlorophyll. After the 18th day, there was a steady decline in chlorophyll, b degrading more rapidly than a.

It is thought that several light-driven reactions are involved in the observed pigment synthesis, photosynthesis, food allocation, and growth of bean. Some of these reactions may be cyclic and others linear. Collectively, they must reach a harmonic point for normal metabolism and development to occur. Because time courses for each of these reactions are different, the intermittent and flashing light technique offers the possibility of individually studying some of the key light-driven reactions.

     

Inhibition of the formation of photosynthetic enzymes by inhibitors of photosynthesis

It has been shown previously that an increase in ribulose diphosphate carboxylase activity occurs upon brief illumination of leaves of dark-grown Zea mays plants; an increase in ribose 5-phosphate isomerase occurs after prolonged illumination. Both of these responses to illumination are inhibited by chloramphenicol.

The administration of p-chlorophenyldimethylurea, an inhibitor of photosynthesis, to etiolated maize does not affect the normal early rise in ribulose diphosphate carboxylase activity when the leaves are illuminated but does block the increase in ribose 5-phosphate isomerase. This pattern of response suggests that photosynthetic activity is required for the increase in isomerase—perhaps products of photosynthesis induce isomerase synthesis—but that the level of ribulose diphosphate carboxylase is controlled by other processes. Chlorophyll formation (as has been shown by others) is slightly suppressed by the inhibitor; levels of total soluble leaf protein appear to be unaffected.

Salicylaldoxime, which is a more general inhibitor of metabolism than p-chlorophenyldimethylurea, arrests the normally observed increases of ribulose diphosphate carboxylase, ribose 5-phosphate isomerase, and chlorophyll during illumination of dark-grown maize. The level of soluble leaf protein is also lower in leaves treated with this compound.

     

Light-stimulation of alanine aminotransferase activity in dark-grown leaves of Lolium temulentum L. as related to chlorophyll formation

Summary The activity of alanine aminotransferase (=glutamate-pyruvate transaminase, GPT) in dark-grown first leaves of Lolium temulentum L. was increased, after an initial lag-phase of 4–6 hr, by more than 130% during the first 24 hr of light-exposure. In comparison, aspartate aminotransferase (=glutamateoxalacetate transaminase, GOT) activity rose by only 18%. Red light treatments of up to 60 min duration produced subsequent increases in GPT activity but the effects were too small to indicate a phytochrome-mediated response. The amounts of enzyme formed were equivalent to those obtained with similar incident intensities of white light. Retuern to darkness after light exposure resulted in an arrestation of the light-stimulated GPT increase. Pre-treatment with cycloheximide caused either stimulatory or inhibitory effects depending upon the concentration applied but, in general, chlorophyll formation and GPT activity responded in a similar manner, whilst GOT showed virtually no response. Chloramphenicol at 6x10^-3 M depressed chlorophyll and Fraction 1 protein synthesis but stimulated GPT activity.The data are discussed in relation to the possible roles of GPT in the leaf. It is suggested that the enzyme, as determined, may be a complex of forms and that at least part of the activity may be involved in the early stages of chlorophyll biosynthesis.     

Light Activation of NADP-Malate Dehydrogenase in Guard Cell Protoplasts from Vicia faba L

Light-induced swelling of guard cell protoplasts (GCP) from Vicia faba was accompanied by increases in content of K⁺ and malate. DCMU inhibited the increase of K⁺ and malate, and consequently swelling.

Effect of light on the activity of selected enzymes that take part in malate formation was studied. When isolated GCP were illuminated, NADP-malate dehydrogenase (NADP-MDH) was activated, and the activity reached a maximum within 5 minutes. The enzyme activity underwent 5- to 6-fold increase in the light. Upon turning off the light, the enzyme was inactivated in 5 minutes NAD-MDH and phosphoenolpyruvate carboxylase (PEPC) were not influenced by light. The rapid light activation of NADP-MDH was inhibited by DCMU, suggesting that the enzyme was activated by reductants from the linear electron transport in chloroplasts. An enzyme localization study by differential centrifugation indicates that NADP-MDH is located in the chloroplasts, NAD-MDH in the cytosol and mitochondria, and PEPC in the cytosol. After light activation, the activity of NADP-MDH in guard cells was 10 times that in mesophyll cells on a chlorophyll basis. The physiological significance of light-dependent activation of NADP-MDH in guard cells is discussed in relation to stomatal movement.

     

Overexpression of HEMA1 encoding glutamyl-tRNA reductase

5-Aminolevulinic acid (ALA) synthesis has been shown to be the rate limiting step of tetrapyrrole biosynthesis. Glutamyl-tRNA reductase (GluTR) is the first committed enzyme of plant ALA synthesis and is controlled by interacting regulators, such as heme and the FLU protein. Induced inactivation of the HEMA1 gene encoding GluTR by RNAi expression in tobacco resulted in a reduced activity of Mg chelatase and Fe chelatase indicating a feed-forward regulatory mechanism that links ALA synthesis posttranslationally with late enzymes of tetrapyrrole biosynthesis (Hedtke et al., 2007). Here, the regulatory impact of GluTR was investigated by overexpression of AtHEMA1 in Arabidopsis and tobacco plants. Light-dependent ALA synthesis cannot benefit from an up to 7-fold induced expression of GluTR in Arabidopsis. While constitutive AtHEMA1 overexpression in tobacco stimulates ALA synthesis by 50-90% during light-exposed growth of seedlings, no increase in heme and chlorophyll contents is observed. HEMA1 overexpression in etiolated and dark-grown Arabidopsis and tobacco seedlings leads to additional accumulation of protochlorophyllide. As excessive accumulation of GluTR does not correlate with increased ALA formation, it is hypothesized that ALA synthesis is additionally limited by other effectors that balance the allocation of ALA with the activity of enzymes of chlorophyll and heme biosynthesis.     

Development of Photosynthetic Activity following Anaerobic Germination in Rice-Mimic Grass (Echinochloa crus-galli var oryzicola)

Shoots of anaerobically germinated Echinochloa crus-galli var oryzicola are nonpigmented whether germinated in light or dark, and chlorophyll synthesis is minimal for the first 12 to 18 hours of greening after exposure to ambient conditions. When chlorophyll development is compared between greening anoxic and etiolated shoots, there is a 100-fold difference in chlorophyll levels at 8 hours, an 8-fold difference at 24 hours, but roughly equal amounts at 60 hours. The chlorophyll a/b ratio approaches 3 earlier in greening anoxic shoots than in greening etiolated shoots, relative to total chlorophyll. The long lag in chlorophyll synthesis can be shortened by giving dark-grown anoxic shoots a 24-hour midtreatment of air before light.

Development of photosynthetic activity in etiolated shoots, determined by CO₂ gas exchange, ¹⁴CO₂ uptake, and activity of carboxylating enzymes closely parallels development of chlorophylls. However, development of photosynthetic capability in greening anoxic shoots does not parallel chlorophyll development; ability to fix carbon lags behind chlorophyll synthesis. A reason for this lag is the very low activity of RuBP carboxylase during the first 36 hours of greening in anoxic shoots. The activity of phosphoenolpyruvate carboxylase is also delayed, but its kinetics more closely match those of chlorophyll development.

     

Formation of chlorophyll B, and the fluorescence properties and photochemical activities of isolated plastids from greening pea seedlings

Chlorophyll b was first detectable after 10 minutes of illumination of etiolated pea seedlings (Pisum sativum L. var Greenfeast) with continuous white light. The chlorophyll a/b ratio decreased from 300 at 10 minutes to 15 after 1 hour. There was little change in the chlorophyll a/b ratio between 1 and 2 hours, and it declined to 3 between 2 and 5 hours of illumination. In red light, the time courses of total chlorophyll synthesis and chlorophyll a/b ratio were similar to those in white light for the first 5 hours of illumination. But with increasing time of illumination with red light, there was an increase in the chlorophyll a/b ratio to 7 after 30 hours. Illumination with white light of very low intensity also gave high chlorophyll a/b ratios. Seedlings which had been illuminated for varying periods and then returned to darkness always showed an increase in chlorophyll a/b ratio during the dark period. It is concluded that the synthesis of chlorophyll b is controlled by light.     

Polypeptides of chloroplastic and cytoplastic origin required for development of Photosystem II activity,and chlorophyll-protein complexes,in Euglena gracilis Z chloroplast membranes

The development of photosynthetic activity and synthesis of chloroplast membrane polypeptides was studied during greening of Euglena gracilis Z in alternate light-dark-light cycles. The results show: (a) The development of both Photosystem II and Photosystem I can be dissociated from chlorophyll synthesis. (b) Most of the polypeptides required for development of Photosystem I are already synthesized during the initial light period (10–12 h); the further rise in Photosystem I activity in the dark is not inhibited by cycloheximide nor by chloramphenicol. (c) The development of Photosystem II requires continuous de novo synthesis of polypeptides and is inhibited by chloramphenicol. The water-splitting activity already present at the end of the first light period decays in the presence of chloramphenicol while that of 1,5-diphenylcarbazide oxidation is only partially retained. The activity can be repaired in the absence of chlorophyll synthesis and is correlated with the de novo synthesis of polypeptides of 50 000–60 000 daltons. The synthesis of these polypeptides and associated repair of Photosystem II activity is not inhibited by cycloheximide. (d) The chloroplast membranes can be resolved into about 40 distinct polypeptides, among them several in the molecular weight range 50 000–60 000, 20 000–35 000 and 10 000–15 000, which are major membrane constitutents. (e) The synthesis of two major polypeptides (M_r = 20 000–30 000) required for the formation of chlorophyll-protein complex(es) containing chlorophyll a and traces of chlorophyll b (CPII?) is light-dependent and cycloheximide-inhibited. It is concluded that the synthesis and addition to the growing membrane of chlorophyll and polypeptides required for the formation of Photosystem II and Photosystem I complexes can be dissociated in time. The H₂O-splitting enzyme(s) and possibly other components of Photosystem II complex are of chloroplastic origin and turn over in the dark while at least some of the chlorophyll binding polypeptides are of cytoplastic origin and their synthesis is light-controlled.     

delta-Aminolevulinic Acid Transaminase in Chlorella vulgaris

An enzyme catalyzing the formation of δ-aminolevulinic acid by transamination of γ,δ-dioxovaleric acid with l-α-alanine, l-glutamic acid, or l-phenylalanine has been detected in extracts of Chlorella vulgaris. The activity of this enzyme does not appear to parallel changes in chlorophyll content in a Chlorella mutant which requires light for chlorophyll production. The role of this enzyme in δ-aminolevulinic acid metabolism in plants is not clearly understood.     

Development of the photosynthetic apparatus during light-dependent greening of a mutant of Chlorella fusca

The formation of chlorophyll, cytochrome f, P-700, ribulose bisphosphate carboxylase as well as photosynthesis and Hill reaction activities were tested during the light-dependent greening process of the Chlorella fusca mutant G 10. Neither chlorophyll nor protochlorophyllide was detected in the darkgrown cells. When transferred to light the mutant cells developed chlorophyll and established its photosynthetic capacity after a short lag phase. In the in vivo absorption spectra a spectral shift of the red absorption peak position from 674 to 680 nm was indicated during the first 3 h of greening. Cytochrome f was already present in the dark-grown cells, but during the greening phase a threefold increase in the cytochrome f content could be seen. At the early stages of greening a characteristic primary oscillation in the content of cytochrome f was observed. P-700 was lacking in the dark and during the first 30 min of illumination. From the first to the second h of light a forced synthesis of P-700 took place and the time-course curve for the ratios of P-700/chlorophyll rose to a sharp maximum. The synthesis of P-700 started together with photosystem I activity and showed similar kinetics. We found the simultaneous appearance of photosystem II, photosystem I, and photosynthetic activities 30 min after the beginning of the illumination. Based on chlorophyll content they attained maximum activity after 2 h of light, but at this time photosystem I capacity proved to be remarkably higher than photosynthetic and photosystem II activities. Highest carboxylase activity existed in darkgrown cells. During the greening process the activity of the enzyme decreased continuously. After 2 h of illumination chlorophyll synthesis partially served to increase the size of the photosynthetic unit, which consequently led to a decrease in the light energy needed to saturate photosynthesis and also to a decrease of photosynthetic rate based on chlorophyll content.Abbreviations Chl chlorophyll - Cyt f cytochrome f - DPIP 2,6-dichlorophenolindophenol - EDTA ethylenediaminetetraacetic acid - GSH glutathione - LH light-harvesting - PS photosystem - RuBP ribulose bisphosphate     

Changes in enzymatic activities in etiolated bean seedling leaves after a brief illumination

The phytochrome controlled increase in total protein in the primary leaf pair of etiolated bean (Phaseolus vulgaris var. Black Valentine) seedlings, which occurs during growth in the dark subsequent to a brief illumination, was investigated. Enzymes from the chloroplasts, the mitochondria, and the soluble cytoplasm all increase in total activity after the illumination.

The total protein and the ribulose carboxylase increases are not inhibited by FUdR, an inhibitor of DNA synthesis. Cycloheximide, an inhibitor of protein synthesis, applied at a time when the ribulose carboxylase activity increase has already commenced, blocks further increase. It was concluded that the total protein and the enzyme increases in the leaf are the result of increases in the per cell levels.

The initial brief illumination is saturating, but 40 minutes later the seedlings have acquired the ability to respond to a second brief illumination. The rate of increase in ribulose carboxylase activity in seedlings that have been illuminated twice is greater than the rate in seedlings that have been illuminated only once.

Far-red light prevents further increase in enzyme activity 48 hours after the initial illumination. There is a lag period interposed between the time of illumination with far-red light and the time at which the seedlings show the greatest effect of far-red light. It was concluded that the phytochrome influence on protein synthesis is not at the terminal steps.