Relation between Paramylum Content and the Length of the Lag Period of Chlorophyll Synthesis during Greening of Dark-grown Euglena gracilis

Euglena cells, strains Z and bacillaris, were grown in the dark under various nutritional deficiencies. After 3 days of nondivision, cells were transferred to the light, and the following parameters were measured: the paramylum content at the time of illumination (zero time), the rate of paramylum consumption during the first 10 hours of greening, and the length of the lag phase of chlorophyll synthesis. Similar results were obtained with both strains and can be summarized as follows. (a) The use of various nutritional deficiencies allows the control, to a certain extent, of the amount of paramylum present at zero time. (b) The rate of paramylum consumption is proportional to the cellular paramylum content for values in excess of 50 picograms/cell. (c) The length of the lag phase increases rapidly when the cellular content of paramylum decreases below 50 picograms. This period can be greatly diminished by the addition of an exogenous organíc carbon source. (d) The amount of paramylum (rate of paramylum consumption × length of lag phase) consumed during the lag phase is around 5 to 10 picograms/cell for cells which contain less than 50 picograms of paramylum/cell. It increases when the cellular paramylum content increases, this increment being more rapid for bacillaris than for Z cells.     

Events Surrounding the Early Development of Euglena Chloroplasts: I. Induction by Preillumination 1

Preillumination, followed by a dark period prior to exposure of dark-grown nondividing cells of Euglena gracilis var. bacillaris to normal lighting conditions for chloroplast development, results in potentiation, or abolishment of the usual lag in chlorophyll accumulation. The degree of potentiation is a function of the length of the preillumination period, the intensity of preilluminating light, and the length of the dark period interposed before re-exposure to continuous light for development. The optimal conditions are found to be: 90 minutes of preillumination with white light at an intensity greater than 30 microwatts per square centimeter (14 foot candles) followed by a dark period of at least 12 hours. Reciprocity is not found between duration and intensity of preilluminating light. Preillumination with blue light and red light was found to be the most effective in promoting potentiation, and the ratio of effectiveness of blue to green to red is consistent with protochlorophyll-(ide) being the photoreceptor. Although red light is effective, there is no reversal by far red light, and these facts, taken together with the effectiveness of blue light, suggest that the phytochrome system is not involved. The amount of chlorophyll formed at the end of preillumination is proportional to the resulting potentiation, suggesting that the amount of protochlorophyll(ide) removed or chlorophyll(ide) formed regulates this phenomenon. Potentiated and nonpotentiated cells show comparable rates of protochlorophyll(ide) resynthesis, suggesting that this is not the limiting factor in nonpotentiated cells. Although light is required for protochlorophyll(ide) conversion in chlorophyll synthesis, a brief preillumination seems also to initiate the production of components in the subsequent dark period which, in nonpotentiated cells, are ordinarily synthesized during the lag period under continuous illumination. These components are necessary to sustain maximal rates of subsequent chlorophyll accumulation.     

Events Surrounding the Early Development of Euglena Chloroplasts: VI. Action Spectra for the Formation of Chlorophyll, Lag Elimination in Chlorophyll Synthesis, and Appearance of TPN-dependent Triose Phosphate Dehydrogenase and Alkaline DNase Activities

Action spectra derived from dose-response curves measured for various processes associated with chloroplast development in Euglena gracilis var. bacillaris are presented. The action spectrum for chlorophyll synthesis during the first 36 hours of continuous illumination of dark-grown resting cells resembles the absorption spectrum of protochlorophyll(ide). The action spectrum for the preillumination phase of potentiation, during which preillumination followed by a dark period brings about lag elimination in chlorophyll synthesis when the cells are subsequently exposed to postilluminating light, shows a high peak in the blue region (at about 433 nm) with a small peak in the yellow-orange region (at about 597 nm); the postillumination phase yields an action spectrum very similar to that obtained for chlorophyll synthesis in continuous light in normal, unpotentiated cells, with peaks at 433 and 631 nm. Alkaline DNase and TPN-linked triose phosphate dehydrogenase, two plastid enzymes which are synthesized outside the chloroplast, yield action spectra which are consistent with protochlorophyll(ide) being the major light receptor. The action spectra which implicate pigments resembling protochlorophyll(ide) holochrome have blue to red peak ratios in the vicinity of 5:1 as does the absorption spectrum of the protochlorophyllide holochrome from beans; the action spectrum is not identical with the holochrome spectrum indicating that the Euglena holochrome may differ from the bean pigment in details of its absorption spectrum. The action spectrum for preillumination, shows a ratio of the blue peak to the red effectiveness of about 24:1. This suggests that preillumination is controlled by a photoreceptor different from the protochlorophyll(ide) holochrome.     

Events surrounding the early development of euglena chloroplasts: v. Control of paramylum degradation

The degradation of the storage carbohydrate, paramylum, is induced by light in wild-type Euglena gracilis Klebs var. bacillaris Pringsheim and in a mutant, W₃BUL, which lacks detectable plastid DNA. Treatment of wild type with cycloheximide in the dark produces 60% as much paramylum breakdown as light, whereas treatment with levulinic acid in the dark yields a slightly greater response than light. Both cycloheximide and levulinic acid produce a greater paramylum breakdown in the light than they do in the dark. Treatment of W₃BUL with levulinic acid in darkness produces a larger paramylum degradation than light, with values similar to wild type in the light. Treatment of W₃BUL with cycloheximide induces paramylum degradation in darkness, and as with wild type, light is slightly stimulatory in the presence of both cycloheximide or levulinic acid. Streptomycin brings about only a very small amount of paramylum breakdown in the dark and only slightly inhibits breakdown in the light. Thus paramylum breakdown induced by light does not require the synthesis of proteins on cytoplasmic or plastid ribosomes. A model which explains these results postulates the existence of a protein which inhibits paramylum breakdown. When the synthesis of this protein is prevented either by light, cycloheximide, or by levulinic acid acting as a regulatory analog of delta amino levulinic acid, paramylum breakdown takes place. Because levulinic acid is a better inducer than light in W₃BUL, W₃BUL may not be able to form as much delta amino levulinic acid in light as wild type. The small amount of induction by streptomycin is viewed as a secondary regulatory effect attributable to interference with plastid protein synthesis which affects regulatory signals from the plastid to the rest of the cell.     

Evidence for light-dependent reversible conformational change in spinach chloroplast CF1

We have investigated an inhibition of photophosphorylation which occurs during preillumination of isolated spinach chloroplasts. Preillumination for 4–6 min in the absence of a complete set of components required for ATP synthesis inhibits photophosphorylation to a maximum of 25–40%; no inhibition occurs if all components for phosphorylation are present from the time illumination begins. The inhibition is about 40% recoverable by imposing a dark (“rebound”) period after the preillumination. Photoinhibition is accompanied by an increased leakiness of the thylakoid membrane to protons and is prevented by the presence of FCCP during the preillumination. Several lines of evidence implicate changes in conformation of chloroplast coupling factor (CF₁) as the cause of both photoinhibition and dark rebound. Conditions which result in photoinhibition also result in a loss of Mg²⁺-dependent ATPase activity which can be elicited from chloroplasts. Both photoinhibition and dark rebound are accompanied by changes in the K_m of CF₁ for both ADP and P_i. Photoinhibition precludes further inhibition of phosphorylation by light plus N-ethylamleimide (NEM) while phosphorylating activity regained by dark rebound is sensitive to subsequent inhibition by light plus NEM. The results are consistent with the conformational coupling hypothesis in indicating that CF₁ may be able to store energy in a conformational state which can be released by the reversal of that state. The photoinhibition we observe may represent conformational changes in CF₁ which are related to conformational coupling but which lead to photoinhibition under our conditions of preillumination.     

Effect of oxygen and pentachlorophenol on the induction lag of photosynthesis in Chlamydomonas reinhardi

Following dark anaerobic incubation Chlamydomonas reinhardi exhibit a pronounced induction lag in photosynthesis; in contrast, dark aerobic incubation caused only a minimal induction lag. Addition of a low concentration of the uncoupler, pentachlorophenol (PCP), to respiring cells in darkness extended the induction period. Far red light preillumination partially overcame the induction lag resulting from dark anaerobic treatment or uncoupling with PCP in aerobic medium. The induction lag in photosynthesis is explained in terms of intracellular energy reserve and its effects on the level of phosphorylated intermediates of the Calvin cycle e.g., RuDP, in the dark. Further, evidence is presented which demonstrates that: (a) PCP, at concentrations below 10 μm, preferentially uncouples oxidative phosphorylation in vivo and (b) photophosphorylation, both in in vivo and in vitro, is much more resistant.     

Effect of Oxygen on the Light-enhanced Dark Carbon Dioxide Fixation in Chlorella Cells

With Chlorella ellipsoidea cells, the effect of oxygen was investigated on the products of enhanced dark ¹⁴CO₂ fixation immediately following preillumination in the absence of CO₂. When the reaction mixture was made aerobic by bubbling air (CO₂-free) throughout preillumination and the following dark ¹⁴CO₂ fixation periods, the initial fixation product was mainly 3-phosphoglyceric acid. When nitrogen gas had been used instead of air, only about one-half of the total radioactivity in the initial fixation products was in 3-phosphoglyceric acid and the rest in aspartic, phosphoenolpyruvic, and malic acids. The percentage distribution of radioactivity incorporated in these initial products rapidly decreased during the rest of the dark period. Concurrent with the decrease in the initial ¹⁴CO₂ fixation products, some increase was observed in the radioactivities of the sugar phosphates. The maximal radioactivity incorporated in sugar mono- and diphosphates accounted for only 10% of total ¹⁴C, under either the aerobic or anaerobic conditions. Under anaerobic conditions most of the ¹⁴C incorporated was eventually transferred to alanine, whereas the main end products under aerobic conditions were aspartate and glutamate. The pattern of ¹⁴CO₂ fixation products was unaffected by the atmospheric condition during the period of preillumination. The preferential flow of the fixed carbon atom to alanine or aspartate depended on the presence or absence of oxygen during the period of dark CO₂ fixation.     

Analysis of photoernzymatic repair of UV lesions in DNA by single light flashes. XI. Light-induced activation of the yeast photoreactivating enzyme

Photoreactivating enzyme (PRE) from yeast (as semi-crude extract, or in highly purified form) shows increased activity if its is illuminated with near UV or short wavelength visible light prior to its use for photoenzymatic repair of UV-induced pyrimidine dimers in transforming DNA in vitro. This effect results from an alternation in PRE molecules changing those with low activity in the light-dependent step of the reaction to a higher activity. Light-induced activation of PRE preparations is slowly lost by dark storage for several hours to 1 day (faster at 23°C than at 5°C), but can be recovered repeatedly by renewed preillumination. The action spectrum for these preillumination effects generally resembles that for the photoenzymatic repair reaction itself, having its maximum in the same 355–385 nm region as the latter, but light of somewhat longer wavelengths (546 nm) is still effective. Preilluminated PRE is also more stable to thermal inactivation (65°C) than untreated enzyme.     

Aspects of clock resetting in flowering of xanthium

Flowering is induced in Xanthium strumarium by a single dark period exceeding about 8.3 hours in length (the critical night). To study the mechanism which measures this dark period, plants were placed in growth chambers for about 2 days under constant light and temperature, given a phasing dark period terminated by an intervening light period (1 min to several hrs in duration), and finally a test dark period long enough normally to induce flowering. In some experiments, light interruptions during the test dark period were given to establish the time of maximum sensitivity.

If the phasing dark period was less than 5 hours long, its termination by a light flash only broadened the subsequent time of maximum sensitivity to a light flash, but the critical night was delayed. In causing the delay, the end of the intervening light period was acting like the dusk signal which initiated time measurement at the beginning of the phasing dark period.

If the phasing dark period was 6 hours or longer, time of maximum sensitivity during the subsequent test dark period was shifted by as much as 10 to 14 hours. In this case the light terminating the phasing dark period acted as a rephaser or a dawn signal.

Following a 7.5-hour phasing dark period, intervening light periods of 1 minute to 5 hours did not shift the subsequent time of maximum sensitivity, but with intervening light periods longer than 5 hours, termination of the light acts clearly like a dusk signal. The clock appears to be suspended during intervening light periods longer than 5 to 15 hours. It is restarted by a dusk signal. There is an anomaly with intervening light periods of 10 to 13 hours, following which time of maximum sensitivity is actually less than the usual 8 hours after dusk.

Ability of the clock in Xanthium to be rephased, suspended, restarted, or delayed, depending always upon conditions of the experiment, is characteristic of an oscillating timer and may confer upon this plant its ability to respond to a single inductive cycle. It is suggested that phytochrome may influence only the phase of the clock and not other aspects of flowering such as synthesis of flowering hormone.

     

Enhanced Dark CO(2) Fixation by Preilluminated Chlorella pyrenoidosa and Anacystis nidulans

The products of short time photosynthesis and of enhanced dark ¹⁴CO₂ fixation (illumination in helium prior to addition of ¹⁴CO₂ in dark) by Chlorella pyrenoidosa and Anacystis nidulans were compared. Glycerate 3-phosphate, phosphoenolpyruvate, alanine, and aspartate accounted for the bulk of the ¹⁴C assimilated during enhanced dark fixation while hexose and pentose phosphates accounted for the largest fraction of isotope assimilated during photosynthesis. During the enhanced dark fixation period, glycerate 3-phosphate is carboxyl labeled and glucose 6-phosphate is predominantly labeled in carbon atom 4 with lesser amounts in the upper half of the C₆ chain and traces in carbon atoms 5 and 6. Tracer spread throughout all the carbon atoms of photosynthetically synthesized glycerate 3-phosphate and glucose 6-phosphate. During the enhanced dark fixation period, there was a slow formation of sugar phosphates which subsequently continued at 5 times the initial rate long after the cessation of ¹⁴CO₂ uptake. To explain the kinetics of changes in the labelling patterns and in the limited formation of the sugar phosphates during enhanced dark CO₂ fixation, the suggestion is made that most of the reductant mediating these effects did not have its origin in the preillumination phase.

It is concluded that a complete photosynthetic carbon reduction cycle operates to a limited extent, if at all, in the dark period subsequent to preillumination.

     

Adaptation to CO(2) Level and Changes in the Phosphorylation of Thylakoid Proteins during the Cell Cycle of Chlamydomonas reinhardtii

The photosynthetic performance of synchronously grown Chlamydomonas reinhardtii alternated rhythmically during the cell cycle. The activity of the “CO₂ concentrating mechanism” including the ability to accumulate CO₂ internally and the activity of carbonic anhydrase peaked after 6 to 9 hours of light and reached minimum after 6 to 9 hours of dark. Consequently, the apparent photosynthetic affinity to extracellular CO₂ alternated rhythmically. At the end of the dark period the cells behaved as if they were adapted to high CO₂ even though they were continuously aerated with air. Results from experiments in which the light or dark periods were extended bear on the interaction between the internal (cell cycle or biological clock) and the external (light) signal. The observed rhythmical alterations in photosynthetic V_max may result from changes in PSII activity. The latter may be partly explained by the capacity for phosphorylation of thylakoid proteins, which reached maximum after 9 hours of light and decreased toward the dark period.     

Enhanced Dark Carbon Dioxide Fixation in Maize: Effect of the Oxygen Concentration during Preillumination on CO(2) Uptake and the Intramolecular Labeling Pattern of Malate and Aspartate

The enhanced dark CO₂ uptake after a preillumination period under varying O₂ concentrations has been measured with maize, a C₄ plant. For comparison the same study has been conducted with tomato, a C₃ plant. Increasing the O₂ concentration during preillumination inhibits by 70% the subsequent dark CO₂ uptake in tomato but stimulates 2-fold this CO₂ uptake in maize. The O₂ enhancement of CO₂ uptake in maize is due to the enhancement of malate and aspartate synthesis. The percentages of radioactivity incorporated in the C-4 of malate and aspartate vary from 74 to 87% when O₂ concentration during preillumination is increased from 0 to 100%.     

Enhancement of repair of UV-irradiated plasmids in cultured fish cells by fluorescent light preillumination and growth arrest.

The UV-irradiated plasmid pBSCATSV, which could express chloramphenicol acetyltransferase (CAT) in the presence of SV40 early promoter, was transfected into RBCF-1 cells derived from the goldfish (Carassius auratus). The cells were incubated in the dark for 24 h and then the CAT activity was measured. CAT expression relative to non-irradiated control was calculated. The CAT expression of the exponentially growing cells transfected with UV-irradiated plasmid was enhanced by fluorescent light (FL) preillumination of the cells 8 h before transfection. The efficiency of photorepair (PR) measured by CAT expression was also enhanced by the same FL preillumination. This suggests that FL preillumination enhances both photorepair and dark repair of RBCF-1 cells for UV-damaged plasmid transfected into the cells. The enhancement of repair of UV damage by FL preillumination was also observed in survival assays. When the UV-irradiated pBSCATSV was transfected into growth-arrested cells in confluent culture, CAT expression was less sensitive to UV irradiation, and FL preillumination was much less effective in enhancing photorepair and dark repair.     

EFFECT OF CHROMATIC LIGHTS ON PHYCOBILIN FORMATION IN A BLUE-GREEN ALGA, TOLYPOTHRIX TENUIS

1. The formation of phycobilin pigments in a blue-green alga Tolypothrixtenuis was investigated with special reference to the effectsof preillumination with colored lights.
2. It was discoveredthat the algal cells are capable of formingphycobilin pigmentsin the dark, if they have been previouslyilluminated for severalhours in the presence of CO₂.
3. The color of light applied inthe later period of preillumination(chromatic illumination)was found to affect the ratio of phycoerythrinto phycocyaninformed in the subsequent dark period. A greenlight acceleratesthe dark-formation of phycoerythrin, a redlight that of phycocyanin,and the two lights counteractingwith each other in their effects.
4. These directive effects of the "chromatic illumination" canbe accomplished within a very short period, for instance, in3 minutes if it is preceded by sufficient "preillumination"with an incandescent or day light fluorescent light. The reactionsoccurring during the period of chromatic illumination does notrequire the presence of CO₂ and the aerobic condition.
5. Thealga can be grown heterotrophically when supplied with casaminoacids and glucose. Under such a condition the alga forms phycocyanintogether with chlorophyll and carotenoids, but not phycoerythrin.
6. On the basis of the results obtained, a tentative scheme forthe biosynthesis of phycobilin pigments in the alga was proposed,assuming the light-induced formation of unknown precursors whichare converted into phycocyanin and phycoerythrin in the subsequentdark period.

(Received July 4, 1960; )     

Photosynthetic Genes of Petunia (Mitchell) Are Differentially Expressed during the Diurnal Cycle

The petunia (Petunia [Mitchell]) chloroplast proteins, the chlorophyll a/b-binding (Cab) proteins, and the small subunit of ribulose bisphosphate carboxylase (RbcS) are encoded by nuclear genes that are expressed in a light-dependent manner. The steady-state concentrations of five cab mRNAs vary with a dramatic circadian rhythm in plants grown under a constant diurnal cycle (10 hours light, 14 hours dark). cab mRNA levels reach their maximum during the light period, but begin to drop prior to the dark period. These RNAs fall to their minimum concentration during the dark period and then begin to increase again in anticipation of the light. Within this general pattern, there are variations in expression among specific classes of cab genes. The light harvesting complex of photosystem II LHCII-type 1 cab mRNAs rise to a well-defined maximum at 2 hours prior to the dark period. All but one of these genes are expressed in anticipation of the light period. The LHCII type 2 cab mRNA and the LHC of photosystem I cab mRNA are expressed at more constant levels throughout the light period. The expression of these genes anticipates the light more than does the expression of the LHCII type 1 genes. The steady state mRNA levels for the petunia rbcS genes show no significant diurnal fluctuation.     

Correlation between the activity of membrane-bound ATPase and the decay rate of flash-induced 515-nm absorbance change in chloroplasts in intact leaves,assayed by means of rapid isolation of chloroplasts

(1) The relationship between activation of the membrane-bound ATPase and the stimulation of dissipation of the flash-induced membrane potential by preillumination was studied in intact spinach leaves by measuring the ATPase activity of rapidly isolated chloroplasts and the decay of the flash-induced 515-nm absorbance change (ΔA₅₁₅) in intact leaves. (2) The decay of ΔA₅₁₅ was accelerated by preillumination. The ΔA₅₁₅ decay in leaves treated with N,N′-dicyclohexylcarbodiimide (DCCD) became slower and was not accelerated by preillumination. However, treatment with DCCD did not lower the intensity of delayed fluorescence. (3) Membrane-bound ATPase of chloroplasts which were rapidly isolated from the preilluminated leaves (90 s preparation time) showed a higher activity (over 200 μmol P_i/mg chlorophyll per h in the case of 2-min preillumination) than that of chloroplasts isolated from dark-adapted leaves. (4) The acceleration of ΔA₅₁₅ decay and the activation of ATPase showed similar dependences on illumination time in intact leaves. 3-(3′,4′-Dichlorophenyl)-1,1-dimethylurea, carbonyl cyanide p-chlorophenylhydrazone and DCCD inhibited the activation of ATPase and the acceleration of the ΔA₅₁₅ decay by preillumination. (5) The ATPase activity of chloroplasts isolated from illuminated leaves showed a single exponential decay (‘dark inactivation in vitro’). The ATPase activity induced by illuminating the leaves became lower as the dark interval between illumination and the isolation of chloroplasts was increased (‘dark inactivation in vivo’). The time course of the decay of activity had a lag and showed a sigmoidal curve when plotted semilogarithmically. The decay had an apparent half-time of 25 min. (6) The recovery of the accelerated ΔA₅₁₅ decay in preilluminated leaves to the original slow rate showed a sigmoidal decay similar to that of the activity of ATPase in intact leaves with a half-time of about 23 min in the dark. (7) It was concluded that the decay rate of ΔA₅₁₅ reflected the chloroplast ATPase activity in intact leaves and that the ion conductance of thylakoid membrane was mainly determined by the H⁺ flux through the ATPase, the activity of which was increased after the formation of the high-energy state.     

Ion-Stimulated Stomatal Opening Induced by Preillumination in Epidermal Strips of Commelina communis

The effects of preillumination were investigated on ion-stimulated stomatal opening of epidermal strips isolated from Commelina communis L. leaves, which are dark-starved 24 hours or more. The rate and the extent of ion-stimulated stomatal openings were increased by preexposure of epidermal strips to light. The evidences are interpreted as indicating that the energy induced by preillumination can be conserved in guard cells for considerable time periods and then used for a delayed stomatal opening in the presence of higher concentration of potassium or sodium ions. Action spectrum showed two peaks, one in blue and one in the red light region. The ratio of the blue peak to the red peak is 1.2; which is the smallest reported value in action spectra of stomatal movements. 3-(4-chlorophenyl)-1,-1-Dimethylurea suppressed the ion-stimulated stomatal opening induced by the preillumination. We conclude that the photosynthetic electron transport system, containing photosystem II, in guard cell chloroplasts is a basic system of energy acquirement for stomatal opening.     

The reconstitution of photophosphorylation and the effect of inorganic phosphate at different pH's on the phenazine methosulfate photoinhibition of Rhodospirillum rubrum chromatophores

Preillumination of R. rubrum membranes in the presence of 50M phenazine methosulfate produces an inhibition of their photophosphorylating capacity.At low pH's phosphate protects against this photo-inhibition, whereas increasing the pH eliminates this protective effect. The inhibition can be produced not only by preillumination at high pH in the presence of phenazine methosulfate, but also by preillumination at low pH (5.0) and then shifting the pH to 8.0 in the dark. We have also measured the effect of preillumination in the presence of an uncoupler such as carbonylcyanide p-trifluoromethoxyphenylhydrazone, and found that at normal pH's while photophosphorylation was protected, the uncoupler activated ATPase was inhibited pointing to a clear difference for both reactions and perhaps different structural requirements for their activities.The purified coupling factor protein isolated from either normal or photoinactivated membrane will reconstitute normal photophosphorylation in previously uncoupled membranes but not in uncoupled membranes which were inactivated by preillumination with phenazine methosulfate prior to uncoupling.     

Phototropism of Pellia: Evidence for Mediation by Auxin-stimulated Acid Efflux

Flash induced 685 nm fluorescence emission of preilluminated and dark kept Chlamydobotrys stellata has been measured under conditions of CO₂-deprivation. The difference in fluorescence intensity between dark kept and preilluminated cells is taken as a measure for the reduced state of the primary stable electron acceptor of photosystem II, Q, at the given intensity of preillumination. CO₂ removal from growing cultures of this alga for 15 min diminishes photosynthetic electron transport at the oxidizing side of this photosystem. Prolonged CO₂-absence influences also its reducing side. Measurements of flash induced oxygen yields support the conclusion that both sides of photosystem II are affected in the absence of bicarbonate.     

Metabolism of Separated Leaf Cells: II. Uptake and Incorporation of Protein and Ribonucleic Acid Precursors by Tobacco Cells

Enzymatically separated tobacco leaf cells took up amino acids, uracil, and uridine from the incubation medium and incorporated them into proteins and RNA, respectively, at a linear rate for approximately 30 hours. Both uptake and incorporation were light-dependent, although cells prepared from preilluminated plants or preillumination of cells allowed some uptake and incorporation to occur in the dark. The light was necessary to satisfy a photosynthetic requirement, but could be replaced in part by ATP in the medium.