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.

     

The effect of red and far red light on the desaturation of Fatty acids in barley leaves

Barley (Hordeum vulgare) leaf tissue was either (a) exposed to continuous red light or (b) exposed to red, far red, or red followed by far red light. The fatty acid composition and incorporation of acetate-2-¹⁴C into linolenate were determined. Changes occurred in the fatty acid composition of dark-grown barley leaves regardless of whether the plants were subsequently exposed to red light or whether the tissue remained in the dark. Measurements were also made of the fatty acids of the coleoptile. Red light treatment did not reduce the lag period for the synthesis of linolenate when chlorophyll synthesis was inhibited. It appears that the desaturation process per se in the synthesis of linolenate is not phytochrome-mediated but may appear to be phytochrome mediated if, possibly, galactolipid and chlorophyll syntheses occur concomitantly.     

Light Mediated Changes in the Chloroplasts of the Liverwort, Sphaerocarpos donnellii Aust

Dark-grown plants of Sphaerocarpos, incubated in a liquid medium containing sucrose and mineral salts, have a much lower chlorophyll and nitrogen content than do light-grown plants. Two minutes of red light per 12 hours is about two-thirds as effective in increasing chlorophyll and nitrogen content as is continuous white light. These red light-induced increases are mediated by phytochrome, as they are reversible by alternating exposures to red and far-red light. They appear to be related to differences in the ultrastructure of the chloroplasts. Plastids from dark-grown plants are full of starch and develop few lamellae, while light-grown plastids contain little starch and have many lamellae. The ultrastructural studies are supported by starch determinations which revealed a phytochrome-mediated decrease in starch content. The effect of white light in increasing the chlorophyll and nitrogen content above the level attained in red light-treated plants is not mediated by photosynthetic activity. These results are related to similar responses in other archegoniates and angiosperm seedlings.     

Kinetics of Accumulation of Ribulose-1,5-bisphosphate Carboxylase during Greening in Euglena gracilis: Photoregulation

The preparation of a rabbit antibody to ribulose-1,5-bisphosphate carboxylase (RuBPCase) from Euglena gracilis and its use to quantitate RuBPCase in dark- and light-grown cells and during light-induced chloroplast development (greening) are described. Light-grown Euglena have at least 36 times more RuBPCase than dark-grown Euglena. Light is required for both the initiation and continued increase in net synthesis of RuBPCase over the dark level: brief illumination 12 hours before exposure to continuous light eliminates the lags in the accumulation and increase in activity of RuBPCase (as well as in chlorophyll accumulation); net synthesis is blocked in greening cells returned to the dark or exposed to 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Streptomycin or cycloheximide prevents RuBPCase accumulation when added at the beginning of greening but only partially blocks accumulation when added after 25 hours of greening. After 24 hours of greening, the activity of RuBPCase per milligram chlorophyll continues to increase slowly while concentration of the enzyme per milligram chlorophyll remains constant. This increased activity may be due to activation of the enzyme as well as to net synthesis.     

Control of delta-Aminolevulinic Acid and Chlorophyll Accumulation in Greening Maize Leaves upon Light-Dark Transitions

The accumulation of δ-aminolevulinic acid (ALA) was studied in greening maize (Zea mays) leaves which were transferred to darkness and reilluminated after various periods of time. The system synthesizing ALA decays in the dark with a half-life of about 80 minutes. The onset of enzyme decay after transfer to darkness shows a 40-minute lag. The accumulation of ALA in the presence of levulinic acid in leaves transferred to darkness corresponds to that expected from the estimated half-life of the enzyme synthesizing ALA. On the other hand, the accumulation of protochlorophyll upon transfer to darkness in the absence of levulinic acid stops much earlier. It is suggested that a control point exists in the pathway between ALA and protochlorophyll, preventing utilization of the accumulated ALA upon transfer of greening leaves to darkness. This is supported by the observed effects of low intensities of monochromatic light (648 nm) on ALA and chlorophyll accumulation.     

Phytochrome Regulation of Greening in Pisum: Chlorophyll Accumulation and Abundance of mRNA for the Light-Harvesting Chlorophyll a/b Binding Proteins

A brief pulse of red light eliminates or reduces the lag in chlorophyll accumulation that occurs when dark-grown pea seedlings are transferred to continuous white light. The red light pulse also induces the accumulation of specific mRNAs. We compared time courses, escape from reversal by far-red light, and fluence-response behavior for induction of mRNA for the light-harvesting chlorophyll a/b binding proteins (Cab mRNA) with those for induction of rapid chlorophyll accumulation in seedlings of Pisum sativum cv Alaska. In both cases the time courses of low fluence and very low fluence responses diverged from each other in a similar fashion: the low fluence responses continued to increase for at least 24 hours, while the very low fluence responses reached saturation by 8 to 16 hours. Both responses escaped from reversibility by far-red slowly, approaching the red control level after 16 hours. The fluence-response curve for the Cab mRNA increase, on the other hand, showed threshold and saturation at fluences 10-fold lower than threshold and saturation values for the greening response. Therefore, the level of Cab mRNA, as measured by the presence of sequences hybridizing to a cDNA probe, does not limit the rate of chlorophyll accumulation after transfer of pea seedlings to white light. The Cab mRNA level in the buds of seedlings grown under continuous red light remained high even when the red fluence rate was too low to allow significant greening. In this case also, abundance of Cab mRNA cannot be what limits chlorophyll accumulation.     

Events Surrounding the Early Development of Euglena Chloroplasts: 14. Biosynthesis of Cytochrome c-552 in Wild Type and Mutant Cells

Lack of a suitable assay has thwarted attempts to measure cytochrome c-552 in dark-grown wild type cells of Euglena gracilis var. bacillaris in mutants and in other situations where the concentrations are low. Purification methods are described based on electrofocusing which provide a cytochrome c-552 preparation homogeneous enough to elicit a single reactive antibody in rabbits; this antibody is then used as a specific and sensitive assay for cytochrome c-552. Dark-grown cells of wild type and of mutants O₁BS, O₂BX, G₁BU and P₁BXL (which make normal sized chloroplasts with abnormal internal structure in the light) have 0.02 to 0.1 × 10⁻¹¹ micromoles of cytochrome c-552 per cell, 10 to 150 times less than light-grown cells. Light-grown cells of these mutants and of wild type show a ratio of chlorophyll to cytochrome of about 300 (mole to mole). Cytochrome c-552 is undetectable in dark-grown Y₁BXD, Y₃BUD, and W₃₄ZUD which cannot carry plastid development beyond the proplastid in light; the light-grown cells of these mutants have levels of cytochrome similar to or lower than dark-grown wild type cells. Cytochrome c-552 is undetectable in light- and dark-grown mutants in which plastid DNA is undetectable (such as Y₂BUL, W₃BUL, W₈BHL, and W₁₀BSmL) consistent with the view, but not proving, that this molecule may be coded, at least in part, in plastid DNA. During light-induced chloroplast development in resting cells, cytochrome c-552 formation behaves in all respects like chlorophyll except that the dark-grown cells contain low amounts of the cytochrome c-552 but lack chlorophyll. Thus, both cytochrome c-552 and chlorophyll show the same lag period even when the length is changed by nutritional manipulation; preillumination largely eliminates the lag in the formation of both molecules, cycloheximide and streptomycin both inhibit the biosynthesis of chlorophyll and cytochrome c-552 in the same manner, and the formation of both during chloroplast development is strictly light-dependent. It is shown that chloroplasts isolated from Euglena by methods thought to give intact organelles, lack 95% of the cytochrome c-552; this and the loss of similar molecules may explain why these isolated chloroplasts are not photosynthetically active.     

Mobilisation of NADPH-glutamate dehydrogenase mRNA in regreening cultures of Euglena gracilis

Exposure of dark-grown resting Euglena gracilis Klebs strain Z Pringsheim to light results in a transient increase in the specific activity of NADPH-glutamate dehydrogenase. NADPH-glutamate dehydrogenase antibody was used to detect NADPH glutamate dehydrogenase resulting from the translation of total polyadenylated RNA and polysomal RNA from Euglena in a cell-free rabbit reticulocyte lysate system. NADPH-glutamate dehydrogenase mRNA was present in cells at all stages of development and present on polysomes from dark-grown and regreening cells but not on polysomes from dark-grown resting cells. These results indicate that the light-induced increase in NADPH-glutamate dehydrogenase in dark-grown resting cells represent an increase in the rate enzyme synthesis resulting from the mobilisation of NADPH-glutamate dehydrogenase mRNA onto polysomes.     

Effect of carbon dioxide, osmotic potential of nutrient solution, and light intensity on transpiration and resistance to flow of water in pepper plants

The rate of transpiration, temperature of the leaves, and relative water content of leaves of pepper plants were measured in a small chamber in which the temperature, relative humidity, and carbon dioxide concentration of recirculated air were controlled and measured. The data reported were obtained by noting the response of pepper plants to all combinations of the following treatments: high light, 1.5 × 10⁶ ergs per square centimeter per second; low light, 3.0 × 10⁴ ergs per square centimeter per second; three levels of CO₂: 50, 268, and 730 parts per million; nutrient solution osmotic potentials of −0.5, −5.0, −7.5, and −9.5 bars.     

Ethylene is not involved in the blue light-induced growth inhibition of red light-grown peas

Although the growth of intact plants is inhibited by irradiation with blue light, the growth rate of isolated stem segments is largely unaffected by blue light. We hypothesized that this loss of responsiveness was a result of ethylene production as part of the wounding response. However, we found no interaction between ethylene- and blue light-induced growth inhibition in dark- or red light-grown seedlings of pea (Pisum sativum L.). Inhibition of growth begins in dark-grown seedlings exposed to blue light within 3 min of the onset of blue light, as was known for red light-grown seedlings. By contrast, ethylene-induced inhibition of growth occurs only after a lag of 20 to 30 min or more (dark-grown seedlings) or 60 min (red light-grown seedlings). Also, the inhibition response of red light-grown seedlings is the same whether ethylene is present from the onset of continuous blue-light treatment or not. Finally the spatial distribution of inhibition following blue light was different from that following ethylene treatment.     

Biosynthesis of phytoene from isopentenyl pyrophosphate by a Neurospora enzyme system.

An enzyme system catalyzing the conversion of isopentenyl pyrophosphate to phytoene has been isolated from Neurospora crassa mycelia. This enzyme system shows an absolute requirement for Mg^?, but no other cofactors. Cultures of N. crassa exhibit a low level of phytoene synthesizing activity when grown in the dark. A 2-min in vivo blue light irradiation results in a ninefold increase in activity after 24 h. This increase is dependent on the duration of the light treatment and is inhibited by cycloheximide. A similar blue light-induced elevation of phytoene synthesizing activity was demonstrated in an albino-1 mutant. This enzyme activity was not found in either dark-grown or irradiated cultures of an albino-2 or an albino-3 mutant.     

Genetic analysis of the role of gibberellin in the red light inhibition of stem elongation in etiolated seedlings

Red light causes a reduction in the extension growth of dark-grown seedlings. The involvement of gibberellin in this process was tested by screening a number of gibberellin synthesis and gibberellin response mutants of Pisum sativum L. for the kinetic response of stem growth inhibition by red light. Gibberellin deficient dwarfs, produced by mutant alleles at the Le, Na, and Ls loci, and gibberellin response mutants produced by mutant alleles at the La and Cry², Lka, and Lkb loci were tested. Extension growth of expanding third internodes of dark-grown seedlings was recorded with high resolution using angular position transducers. Seedlings were treated with red light at a fluence rate of 4 micromoles per square meter per second either continuously or for 75 seconds, and the response was measured over 9 hours. With certain small exceptions, the response to the red light treatments was similar in all the mutants and wild types examined. The lag time for the response was approximately 1 hour and a minimum in growth rate was reached by 3 to 4 hours after the onset of the light treatment. Growth rate depression at this point was about 80%. Seedlings treated with 75 seconds red light recovered growth to a certain extent. Red/far-red treatments indicated that the response was mediated largely by phytochrome. The similar responses to red light among these wild-type and mutant genotypes suggest that the short-term (i.e. 9 hour) response to red light is not mediated by either a reduction in the level of gibberellin or a reduction in the level or affinity of a gibberellin receptor.     

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.     

Desaturation of Oleic and Linoleic Acids by Leaves of Dark- and Light-grown Maize Seedlings

Oleate and linoleate desaturation in leaves of maize seedlings was largely independent of previous light treatment of the seedlings; there was no evidence of light-induced desaturase activities. These results are in sharp contrast to those observed with developing cucumber cotyledons in which pronounced increase in desaturation occurs after exposure of tissue to light. The rates of desaturation of oleate were about four times those of linoleate in both etiolated and 16-hour greened maize leaves. In both etiolated and greened tissues, about two-thirds of the label from oleate was esterified after 4 hours, half of which was in phosphatidylcholine. Phosphatidylcholine and diglyceride contained large proportions of [¹⁴C]linoleate formed from [¹⁴C]oleate but not [¹⁴C]linolenate. In monogalactolipid, about two-thirds of the labeled fatty acids were linolenate. In vivo desaturase activity was present in tissue of widely different levels of differentiation and chlorophyll content obtained from light-grown maize seedlings.     

Functional and Structural Organization of Chlorophyll in the Developing Photosynthetic Membranes of Euglena gracilis Z: II. CHARACTERISTICS OF THE LATE FORMATION OF ACTIVE PHOTOSYSTEM II REACTION CENTERS DURING FIRST STAGES OF GREENING

During light-induced greening of dark-grown, nondividing Euglena gracilis Z, there is a delay of about 10 hours in the formation of active photosystem II (PSII) reaction centers compared to chlorophyll synthesis. Experiments with greening under different light intensities rule out the possibility that this delay results from a late induction of active PSII reaction center formation when a definite amount of chlorophyll is attained in the early greened cells. Experiments on greening after preillumination show that this delay does not originate in a long, light-induced formation of specific synthesizing machinery for reaction center components. Experiments with greening in the presence of streptomycin show that, when this inhibitor of protein synthesis by chloroplastic ribosomes is added to dark-grown, preilluminated cells or to cells already greened for 24 hours, the formation of active PSII reaction centers is inhibited after a time which depends on the light intensity used for greening. Under very low light intensity (150 lux), the addition of streptomycin to 24-hour greened cells does not prevent further development of functional chloroplasts. These observations lead to the conclusion that streptomycin-insensitive chloro-plast development occurs due to syntheses of cytoplasmic origin and of light-induced pools of components synthesized early by chloroplastic ribo-somes. Conformational changes requiring time may allow the insertion of components necessary for the reorganization of PSII reaction centers in the developing thylakoid after synthesis. This hypothesis accounts for the observed delay in PSII reaction center formation compared to chlorophyll synthesis.     

Accumulation of delta-Aminolevulinic Acid and Its Relation to Chlorophyll Synthesis and Development of Plastid Structure in Greening Leaves

Levulinic acid inhibited the greening of etiolated maize (Zea mays) and bean (Phaseolus vulgaris) leaves and caused accumulation of δ-aminolevulinic acid (ALA). ALA accumulation in maize was equivalent to the decrease in chlorophyll, over a wide range of experimental conditions. It was saturated at low light intensities and was not limited by the supply of substrates during the early hours of greening. During 20 hours in light, levulinic acid had little effect on the structural development of thylakoids in bundle sheath chloroplasts but significantly reduced the number and size of thylakoids in grana of mesophyll chloroplasts. Recrystallization of prolamellar bodies and their reformation was inhibited. Mitochondria appeared not to be affected.     

Phytochrome control of two low-irradiance responses in etiolated oat seedlings

Light-induced coleoptile stimulation and mesocotyl suppression in etiolated Avena sativa (cv. Lodi) has been quantitated. Etiolated seedlings showed the greatest response to light when they were illuminated 48 to 56 hours after imbibition. Two low-irradiance photoresponses for each tissue have been described. Red light was 10 times more effective than green and 1,000 times more effective than far red light in evoking these responses. The first response, which resulted in a 45% mesocotyl suppression and 30% coleoptile stimulation, had a threshold at 10⁻¹⁴ einsteins per square centimeter and was saturated at 3.0 × 10⁻¹² einsteins per square centimeter of red light. This very low-irradiance response could be induced by red, green, or far red light and was not photoreversible. Reciprocity failed if the duration of the red illumination exceeded 10 minutes. The low-irradiance response which resulted in 80% mesocotyl suppression and 60% coleoptile stimulation, had a threshold at 10⁻¹⁰ einsteins per square centimeter and was saturated at 3.0 × 10⁻⁸ einsteins per square centimeter of red light. A complete low-irradiance response could be induced by either red or green light but not by far red light. This response could be reversed by a far red dose 30 times greater than that of the initial red dose for both coleoptiles and mesocotyls. Reciprocity failed if the duration of the red illumination exceeded 170 minutes. Both of these responses can be explained by the action of phytochrome.     

Photoinhibition of Growth in Acanthamoeba castellanii Cultures

Light from 350 to 680 nm at intensities up to 1.62 × 10⁵ ergs per sec per cm² slowed exponential growth and lowered the maximum yield in axenic cultures of Acanthamoeba castellanii. Photoinhibition was a linear function of light intensity up to 1.25 × 10⁵ ergs per sec per cm². At higher intensities, growth was too slow to be measured accurately. A photochemical change occurring in the growth medium on irradiation was a function of light dosage and not intensity per se. Light in dosages which appreciably changed the growth-supporting properties of the medium exceeded the dosages received by exponentially growing cultures during irradiation. Consequently, photoinhibition of growth was attributed to a direct effect of light on the amoebae, not to photodegradation of the medium. The growth-supporting properties of irradiated media could be restored by the addition of yeast extract and Proteose peptone. The reduced growth rate in the light was not due to cyst formation or induction of multinuclearity. Light affected the amoebae either through absorption by intracellular pigment(s) or through binding to the amoebae of a photosensitizing compound in the medium.     

Events Surrounding the Early Development of Euglena Chloroplasts: 7. Inhibition of Carotenoid Biosynthesis by the Herbicide SAN 9789 (4-Chloro-5-(methylamino)-2-(alpha,alpha,alpha,-trifluoro-m-tolyl)-3-(2H)pyridazinone) and Its Developmental Consequences

The herbicide SAN 9789 (4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl-3- (2H)pyridazinone) blocks carotenoid synthesis in growing and resting cells of Euglena at concentrations of 20 to 100 μg/ml without affecting cell viability. Although the inhibition is immediate and complete, in resting cells no decrease in already synthesized carotenoids is found indicating a lack of turnover. In cells growing in the dark, carotenoids are diluted out as the cells divide. Cells dividing in the light in the presence of SAN 9789, eventually lose viability, presumably because of photooxidations usually prevented by carotenoids. During 72 hours of light-induced plastid development in dark-grown resting cells, none of the usual carotenoids increase while phytoene accumulates, indicating that SAN 9789 blocks carotenoid synthesis at this point. Chlorophyll synthesis and membrane formation are also blocked by the herbicide, but these inhibitions appear to be secondary to the inhibition of carotenoid synthesis. That carotenoid levels are strongly correlated with and may control the synthesis of chlorophyll and the formation of plastid membranes is suggested by the following data. (a) If dark-grown dividing cells are placed in the presence of the herbicide for various periods, rested and exposed to light in the presence of the drug, different amounts of carotenoids remain in the cells and the amount of chlorophyll finally synthesized is proportional to the amount of carotenoids present. (b) Photodestruction of chlorophyll is excluded, since the same amounts of chlorophyll are formed at intensities of 10 to 100 foot-candles of light. (c) Photoconversion of protochlorophyll(ide) to chlorophyll(ide) in dark-grown cells is not blocked by the herbicide. (d) Initial rates of chlorophyll synthesis are the same in treated and nontreated cells. (e) The extent of membrane formation appears to parallel the amount of carotenoids present as judged by electron microscopy.